# GENETIC KIDNEY DISEASES OF CHILDHOOD

EDITED BY : Max Liebau and Miriam Schmidts PUBLISHED IN : Frontiers in Pediatrics

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# GENETIC KIDNEY DISEASES OF CHILDHOOD

Topic Editors: Max Liebau, University of Cologne, Germany Miriam Schmidts, Radboud University, Netherlands; Freiburg University, Germany

Genetic conditions are amongst the most common causes of chronic renal disease and renal failure, especially in children and adolescents. Over the last years there has been outstanding progress in the knowledge about genetic kidney diseases, including the identification of multiple disease-associated genes and insights into the cellular pathophysiology. These developments have profoundly changed our understanding of genetic kidney diseases and our therapeutic approaches to (pediatric) patients suffering from these disorders. The aim of the research topic on Genetic Kidney Diseases of Childhood is to give the reader interested in pediatric nephrology a broad overview over the variety of genetic kidney diseases and recent developments in clinical fields, from a research point of view as well as from a patient's perspective.

Citation: Liebau, M., Schmidts, M., eds. (2019). Genetic Kidney Diseases of Childhood. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-860-8

# Table of Contents

*06 Editorial: Genetic Kidney Diseases of Childhood* Miriam Schmidts and Max C. Liebau

### GLOMERULAR DISEASE


Matthias C. Braunisch, Maike Büttner-Herold, Roman Günthner, Robin Satanovskij, Korbinian M. Riedhammer, Pierre-Maurice Herr, Hanns-Georg Klein, Dagmar Wahl, Claudius Küchle, Lutz Renders, Uwe Heemann, Christoph Schmaderer and Julia Hoefele


Henning Hagmann and Paul T. Brinkkoetter

*65 Using the* Drosophila *Nephrocyte to Model Podocyte Function and Disease*

Martin Helmstädter, Tobias B. Huber and Tobias Hermle

*74 Zebrafish as a Model for Drug Screening in Genetic Kidney Diseases* Jochen Gehrig, Gunjan Pandey and Jens H. Westhoff

### CILIOPATHIES AND CYSTIC KIDNEY DISEASES

*84 The Genetic and Cellular Basis of Autosomal Dominant Polycystic Kidney Disease—A Primer for Clinicians*

Adrián Cordido, Lara Besada-Cerecedo and Miguel A. García-González

*92 Is Autosomal Dominant Polycystic Kidney Disease Becoming a Pediatric Disorder?*

Stéphanie De Rechter, Luc Breysem and Djalila Mekahli


*122 Many Genes—One Disease? Genetics of Nephronophthisis (NPHP) and NPHP-Associated Disorders*

Shalabh Srivastava, Elisa Molinari, Shreya Raman and John A. Sayer


Jens Christian König, Andrea Titieni, Martin Konrad and The NEOCYST Consortium

*155 The KOUNCIL Consortium: From Genetic Defects to Therapeutic Development for Nephronophthisis*

Kirsten Y. Renkema, Rachel H. Giles, Marc R. Lilien, Philip L. Beales, Ronald Roepman, Machteld M. Oud, Heleen H. Arts and Nine V. A. M. Knoers

*162 Congenital Heart Defects and Ciliopathies Associated With Renal Phenotypes*

George C. Gabriel, Gregory J. Pazour and Cecilia W. Lo

*171 Cystic Kidney Diseases From the Adult Nephrologist's Point of View* Roman-Ulrich Müller and Thomas Benzing

### OTHER TUBULAR DISEASES

*179 Bartter Syndrome Type 3: Phenotype-Genotype Correlation and Favorable Response to Ibuprofen*

Xuejun Yang, Gaofu Zhang, Mo Wang, Haiping Yang and Qiu Li

*186 Nephropathic Cystinosis: Symptoms, Treatment, and Perspectives of a Systemic Disease*

Sören Bäumner and Lutz T. Weber


Jan Halbritter, Anna Seidel, Luise Müller, Ria Schönauer and Bernd Hoppe *214 Renal Cell Carcinoma in von Hippel–Lindau Disease—From Tumor Genetics to Novel Therapeutic Strategies*

Emily Kim and Stefan Zschiedrich

### VARIA

*223 Identification of a Novel Heterozygous* De Novo *7-bp Frameshift Deletion in* PBX1 *by Whole-Exome Sequencing Causing a Multi-Organ Syndrome Including Bilateral Dysplastic Kidneys and Hypoplastic Clavicles* Korbinian Maria Riedhammer, Corinna Siegel, Bader Alhaddad, Carmen Montoya, Reka Kovacs-Nagy, Matias Wagner, Thomas Meitinger and Julia Hoefele


Eva Nüsken, Jörg Dötsch, Lutz T. Weber and Kai-Dietrich Nüsken

## Editorial: Genetic Kidney Diseases of Childhood

Miriam Schmidts 1,2 \* and Max C. Liebau3,4,5 \*

*<sup>1</sup> Genome Research Division, Human Genetics Department, Radboud University Medical Center Nijmegen and Radboud Institute for Molecular Life Sciences, Nijmegen, Netherlands, <sup>2</sup> Center for Pediatrics and Adolescent Medicine, University Hospital Freiburg, Freiburg University Faculty of Medicine, Freiburg, Germany, <sup>3</sup> Department of Pediatrics, University Hospital of Cologne, Cologne, Germany, <sup>4</sup> Center for Molecular Medicine, University Hospital of Cologne, Cologne, Germany, <sup>5</sup> Nephrology Research Laboratory, Department II of Internal Medicine, University Hospital of Cologne, Cologne, Germany*

Keywords: ciliopathies, podocytes, nephrotic syndrome, cystic kidney disase, tubulopathies

**Editorial on the Research Topic**

#### **Genetic Kidney Diseases of Childhood**

Human kidneys are most spectacular organs. They have a busy life with filtering well over 100 liters of fluid every day and a few million liters over the course of a life. Not only is proper renal function essential for water and electrolyte balance and excretion of toxic substances in mammals, but our kidneys have amazing additional roles regarding blood pressure regulation and stimulation of red blood cell renewal. Those diverse functions are achieved by an array of different renal cell types, forming a complex tissue architecture. The "building plan" ensuring formation and maintenance of these amazing organs lies within our genes and tiny changes within this renal map will have devastating consequences within this fine-tuned building of blood vessels, glomeruli, and tubules. It therefore comes to no surprise that in children and adolescents, genetic defects are the most common cause for end stage renal disease.

Over the last years there has been outstanding progress in the knowledge about genetic kidney diseases, including the identification of multiple disease-associated genes and insights into the cellular pathophysiology. These developments have profoundly changed our understanding of genetic kidney diseases and our therapeutic approaches to (pediatric) patients suffering from these disorders. In this research topic on Genetic Kidney Diseases of Childhood published in Frontiers in Pediatrics we aim to give the reader interested in pediatric nephrology a broad overview over a variety of genetic kidney diseases and recent developments in clinical fields, from a research point of view as well as from a patient's perspective.

Amongst the most common heriditary renal disorders with childhood onset are glomerular diseases. The discovery of genetic origins of steroid resistant nephrotic syndrome has provided great diagnostic progress and dramatically influenced the therapeutic pathway for affected families, protecting affected children from unnecessary and unsuccessful immunosuppressive treatments (Kemper and Lemke). Further, genetic research in combination with cell biology and biochemistry approaches has revealed multiple novel components of the glomerular filtration barrier, greatly improving our biological understanding in general (Hagmann and Brinkkoetter). Novel experimental approaches including work on model organisms like Drosophila melanogaster or Danio rerio as described in two manuscripts of the research topic have further contributed to unravel the molecular mechanisms resulting in the clinical presentation of nephrotic syndrome and the histology of e.g., focal and segmental glomerulosclerosis (Helmstädter et al.; Gehrig et al.).

#### Edited and reviewed by:

*Michael L. Moritz, University of Pittsburgh, United States*

#### \*Correspondence:

*Miriam Schmidts miriam.schmidts@uniklinik-freiburg.de Max C. Liebau max.liebau@uk-koeln.de*

#### Specialty section:

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

Received: *04 December 2018* Accepted: *07 December 2018* Published: *19 December 2018*

#### Citation:

*Schmidts M and Liebau MC (2018) Editorial: Genetic Kidney Diseases of Childhood. Front. Pediatr. 6:409. doi: 10.3389/fped.2018.00409*

The observational Podonet-approach follows a large international cohort of patients and describes genotypephenotype correlations (Trautmann et al.). This study has interestingly revealed that changes in collagen genes may underly a substantial number of patients with focal and segmental glomerulosclerosis as also described by an independent group (Braunisch et al.). While podocyte-focussed research has received a lot of attention during the last years, genetic defects of the glomerular basement membrane thus represent a substantial cause for glomerular disease (Chew and Lennon).

Another rapidly-evolving field in genetic kidney diseases deals with cystic kidney diseases. This group of disorders is important for both children and adults and includes autosomal dominant and autosomal recessive polycystic kidney disease, nephronophthisis, and various rare syndromes presenting with cystic kidneys including e.g., Bardet-Biedl syndrome (BBS), Meckel Gruber syndrome (MKS), or von Hippel Lindau syndrome (VHL).

The management of pediatric patients with cystic kidney diseases remains a clinical challenge as no causative treatment options are available to date. The research topic at hand discusses some important aspects and presents state-of-the-art knowledge, including current and potential future ways of managing BBS (Hartill et al.), MKS (Forsythe et al.), or VHL (Kim and Zschiedrich), approaches to diagnosis and treatment of cystic kidney diseases in adults (Müller and Benzing), and current opinions on the use of gastrostomy tube insertion in children with ARPKD (Burgmaier et al.). A topic of ongoing debate is the question whether children of patients suffering from ADPKD should undergo early diagnosis or not. These children are at a 50% risk of having inherited the genetic variants responsible for ADPKD, a disorder that typically develops over decades. It has not yet been fully established whether children benefit from early diagnosis or whether the e.g., psychosocial burden of an early diagnosis outweighs benefits. Two manuscripts in the research topic deal with different aspects of ADPKD in children (De Rechter et al.; Harris). The genetic and cellular changes underlying ADPKD are summarized in an additional manuscript (Cordido et al.).

Over the last years a lot of attention has been paid to a specific cellular organelle, whose dysfunction has been linked to the development of cystic kidney disease. This organelle is the primary cilium, an antennae-like structure on the cellular surface that seems to be involved in the sensing of the extracellular environment. As cilia can be found on multiple cell types, it is plausible that cystic kidney diseases frequently present with extrarenal manifestations as in nephronophthisis and nephronophthisis-associated diseases. A manuscript of this research topic summarizes the molecular mechanisms and the genetic basis resulting in Nephronophthisis (Srivastava et al.). Two additional manuscripts describe current collaborative research consortia that aim to link the findings of genetics and cellular biology with a deep clinical phenotyping and biobanking in order to set the basis for evidence-based clinical recommendations and future translational research approaches for cystic kidney diseases (König et al.; Renkema et al.).

Likewise, an array of defective genes has been identified to date to cause renal tubular dysfunction, leading to diverse phenotypes in human such as Bartter syndrome (Yang et al.) Cystinosis (Bäumner and Weber), magnesium transport defects (Giménez-Mascarell et al.) or inherited disorders with kidney stone formation (Halbritter et al.). Genetic diagnosis has not only provided opportunities of highly specialized clinical care and genetic counseling of at-risk and carrier individuals but also offers for the first time causative treatment options such as for cystinosis where cysteamine therapy has now been implemented.

Inherited renal diseases with childhood onset can be manifestations of syndromal disease patterns with multiple organ systems involved such as in case of ciliopathies like BBS or MKS. Other examples include rare conditions affecting the skin and the kidneys (Reimer et al.), resulting from basal membrane defects or defects involving the heart and the kidneys (Gabriel et al.) The latter often but not always result from cilia dysfunction, however the precise underlying molecular mechanisms, e.g., cell signaling pathways defective, have not been understood. Last but not least, patients with congenital anomalies of the kidney and urinary tract (CAKUT) can present with complex syndromal appearances as described for individuals harboring PBX1 mutations (Riedhammer et al.) and kidney function seems to be regulated by programming events very early in life that likely result in long-term modulation of gene function in the kidney (Nüsken et al.).

This research topic provides a concise overview about current state-of-knowledge and outlook on future developments with respects to the diverse landscape of inherited childhood onset renal diseases.

### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### ACKNOWLEDGMENTS

ML acknowledges funding from the German Federal Ministry of Research and Education (BMBF grant 01GM1515E) and the Marga and Walter Boll-Foundation. MS acknowledges funding from Radboudumc and RIMLS Nijmegen (Hypatia tenure track fellowship), the Deutsche Forschungsgemeinschaft (DFG CRC1140 KIDGEM), and the European Research Council (ERC StG TREATCilia, grant No 716344).

**Conflict of Interest Statement:** ML has received honoraria for scientific lectures from Pfizer. Representing the University Hospital of Cologne, ML has been counseling Otsuka in an advisory board.

The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Schmidts and Liebau. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Basement Membrane Defects in Genetic Kidney Diseases

*Christine Chew1 and Rachel Lennon1,2\**

*<sup>1</sup> Faculty of Biology Medicine and Health, Wellcome Trust Centre for Cell-Matrix Research, Division of Cell Matrix Biology, School of Biological Sciences, University of Manchester, Manchester, United Kingdom, 2Department of Paediatric Nephrology, Royal Manchester Children's Hospital, Central Manchester University Hospitals NHS Foundation Trust, Manchester Academic Health Science Centre, Manchester, United Kingdom*

The glomerular basement membrane (GBM) is a specialized structure with a significant role in maintaining the glomerular filtration barrier. This GBM is formed from the fusion of two basement membranes during development and its function in the filtration barrier is achieved by key extracellular matrix components including type IV collagen, laminins, nidogens, and heparan sulfate proteoglycans. The characteristics of specific matrix isoforms such as laminin-521 (α5β2γ1) and the α3α4α5 chain of type IV collagen are essential for the formation of a mature GBM and the restricted tissue distribution of these isoforms makes the GBM a unique structure. Detailed investigation of the GBM has been driven by the identification of inherited abnormalities in matrix proteins and the need to understand pathogenic mechanisms causing severe glomerular disease. A well-described hereditary GBM disease is Alport syndrome, associated with a progressive glomerular disease, hearing loss, and lens defects due to mutations in the genes *COL4A3*, *COL4A4*, or *COL4A5*. Other proteins associated with inherited diseases of the GBM include laminin β2 in Pierson syndrome and *LMX1B* in nail patella syndrome. The knowledge of these genetic mutations associated with GBM defects has enhanced our understanding of cell–matrix signaling pathways affected in glomerular disease. This review will address current knowledge of GBM-associated abnormalities and related signaling pathways, as well as discussing the advances toward disease-targeted therapies for patients with glomerular disease.

Keywords: basement membrane, glomerulus, Alport syndrome, genetic variation, collagen IV, Pierson syndrome

## INTRODUCTION

The glomerular basement membrane (GBM) is an integral component of the glomerular filtration barrier; an important and highly complex capillary wall that is exposed to mechanical forces driven by capillary hydrostatic pressure. This barrier is permeable to water and small molecules, and selectively withholds cells and macromolecules such as albumin in the circulation (1). During glomerular development, the assembly of endothelial and podocyte layers generates the filtration barrier (2). The GBM separates endothelial cells and podocytes, and it represents a specialized extracellular matrix (ECM), which maintains barrier function (**Figure 1**). The GBM is formed during glomerulogenesis and is maintained by secreted components from both podocytes and endothelial cells (3–5). The mature, human GBM is relatively thick in comparison to other basement membranes and measures between 300 and 350 nm (4, 6). In addition to the cells of the filtration barrier, mesangial

#### *Edited by:*

*Max Christoph Liebau, Universitätsklinikum Köln, Germany*

#### *Reviewed by:*

*Kimberly Jean Reidy, Children's Hospital at Montefiore/ Albert Einstein College of Medicine, United States Francois Cachat, University Hospital of Bern, Switzerland Silviu Grisaru, University of Calgary, Canada*

### *\*Correspondence:*

*Rachel Lennon rachel.lennon@manchester.ac.uk*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 08 October 2017 Accepted: 11 January 2018 Published: 29 January 2018*

#### *Citation:*

*Chew C and Lennon R (2018) Basement Membrane Defects in Genetic Kidney Diseases. Front. Pediatr. 6:11. doi: 10.3389/fped.2018.00011*

**8**

cells (between adjacent capillary loops) and the parietal epithelial cells of Bowman's capsule are necessary for maintaining intact glomerular function.

The fusion of podocyte and endothelial basement membranes during the development of the GBM creates an intricate meshwork containing laminins, type IV collagen, nidogen, and heparan sulfate proteoglycans (HSPGs) (7–10). Our recent mass spectrometry-based proteomic analysis of human glomerular ECM *in vivo* revealed a more complex composition of 144 structural and regulatory matrix proteins supporting the unique organelle structure of the glomerulus (11). Not surprisingly collagen (types I, IV, and VI), and laminin isoforms were identified as the most abundant components (11). The secretion of matrix molecules into the GBM is likely to be facilitated by cross talk between podocytes and endothelial cells. Indeed, the proteomic investigation of cell-derived ECM isolated from glomerular cells in culture identified 35 highly connected matrix components and a number of these were differentially expressed in mono- versus coculture ECMs (12). Although a unique ECM niche, the GBM contains proteins that are found in other basement membranes; however, the specific arrangement of matrix isoforms in the GBM provides its distinctive composition and function.

Adhesion receptors such as integrins, syndecans, and dystroglycan connect cells to their associated ECM ligands in the extracellular space and to the cellular cytoskeleton inside the cell (**Figure 2**). The mature focal adhesion complexes that result from this cell–matrix interaction are vital in all aspects of normal cell development including growth, proliferation, signaling, differentiation, migration, and survival (13, 14). Furthermore, in addition to maintaining GBM structure, secreted growth factors support the ECM through organized cell–cell signaling (13).

The study of ECM components that maintain the integrity of the GBM has advanced the understanding of what constitutes a healthy glomerulus. However, disruption to this specialized ECM niche can alter the function of the filtration barrier and cause the leakage of albumin into the urine (albuminuria). Of the nine major proteins discovered in the GBM, genetic mutations in type IV collagen and laminin are reported to cause glomerular disease in humans (15, 16). Although these distinct genetic mutations have been defined, the pathogenesis of the majority of kidney diseases such as diabetic nephropathy are less clear, and it is thought that environmental influences may have a role. Not all conditions that present with proteinuria and glomerular disease have a genetic component, which is one of the main limitations in the diagnosis and treatment of these rare diseases. Animal models have been beneficial in deciphering pathogenic pathways of disease; however, targeted treatments for genetic diseases of the GBM currently do not exist. This review will cover key findings and recent discoveries of mechanisms that sustain a healthy GBM and known pathogenic pathways that lead to genetic kidney disease. In addition, recent advances and novel approaches in the field of ECM in glomerular health and disease will be discussed.

transient receptor potential cation channel-6; WASP, Wiskott–Aldrich syndrome protein.

## GBM BIOLOGY IN HEALTH AND DISEASE

### Type IV Collagen

In common with other basement membranes, type IV collagen forms a major structural component of the GBM and contributes significantly to its stability and assembly (17, 18). There are six collagen IV α-chains, α1(IV) to α6(IV), encoded by the genes *COLA4A1* and *COL4A2* on chromosome 13; *COL4A3* and *COL4A4* on chromosome 2, and *COL4A5* and *COL4A6* on the X chromosome. Each collagen IV α-chain is composed of an N-terminal 7S domain, Gly-X-Y repeats and a non-collagenous (NC1) domain at the C-terminus (**Figure 3**). It is the unique interrupted Gly-X-Y amino acid triplet repeats within these three domains that give collagen IV its flexible quality, enabling

domains to produce a tetramer. These complex interactions are important for the development of the type IV collagen scaffold, which are subsequently reinforced by suprastructural associations of collagenous domains.

it to successfully form basement membranes (19). The α-chains assemble in the endoplasmic reticulum forming the collagen IV triple helix or protomers, which are released into the extracellular space where they undergo polymerization *via* head-to-head NH2- and COOH-terminal domains that result in unique collagen IV hexameric networks (20).

The six collagen IV α-chains form three distinct networks comprising helical heterotrimers these are α1α1α2(IV), α3α4α5(IV), and α5α5α6(IV), and they are present in different glomerular compartments (20, 21). During glomerulogenesis, there is a shift in the composition of collagen IV α-chains involving a transition from α1α1α2(IV), predominantly found in the immature S-shaped and early capillary loop GBM, to the α3α4α5(IV) network, which defines the mature GBM (22). The α3α4α5(IV) isoform is primarily produced by podocytes, whereas the α1α1α2(IV) is present in all cell types including endothelial cells, podocytes, and mesangial cells (5). Type IV collagen networks differ between species and their expression is not solely restricted to the GBM, but these protomers have also been found in the mesangial matrix and Bowman's capsule (23, 24). Mechanisms underlying the transition between collagen IV isoforms during glomerular development are not fully understood. However, it is considered that the α3α4α5(IV) is less susceptible to proteolytic degradation than the α1α1α2(IV) isoform, which suggests that it may have a crucial role in maintaining the structural integrity of the GBM (25). The lateral links between the α3α4α5(IV) chains are also essential in fortifying networks within the GBM (26). The α1α1α2(IV) and α3α4α5(IV) networks are both present in the glomerular capillary tuft, whereas the α5α5α6(IV) is localized to the basement membrane of Bowman's capsule. Overall, the role of type IV collagen networks in supporting the GBM are crucial, and the absence or reduction of the α3α4α5(IV) chains reduces the quantity of protomers secreted, leading to thin basement membranes and Alport syndrome (AS) (27, 28).

#### Alport Syndrome

#### *Clinical Features*

A diagnosis of AS should be suspected in individuals who present with glomerular hematuria and a family history of AS or renal failure without other identified causes and these individuals should undergo further investigations (29). The diagnosis of Alport was previously confirmed by the presence of lamellated GBM or abnormal deposition of α3α4α5(IV) and α5(IV) collagen chains from the GBM and skin, respectively, the latter of which reported moderate sensitivity in 80% males and high specificity (30). More recently, genetic testing has been a preferred diagnostic method, as it is less invasive than skin or kidney biopsy and has a diagnostic specificity of 95% (29). Sanger sequencing was the gold standard for the diagnosis of AS where other techniques were unsuccessful; however, next-generation sequencing to screen genes corresponding to α3, α4, and α5 chains of type IV collagen has now succeeded this method (31).

The clinical phenotype in AS also includes ocular abnormalities and sensorineural hearing loss. Hearing loss was more likely to occur in 60% of patients with missense mutations before 30 years of age, which is lower than the 90% risk in individuals with other mutations. In the majority of patients with AS, the auditory deficit affects the higher sound frequencies, and it usually progresses to hearing loss within conversational range. The ocular defects in X-linked and autosomal recessive AS include anterior lenticonus, perimacular retinal flecks, posterior corneal vesicles, and recurrent corneal erosions (32).

#### *Etiology and Pathogenesis*

The significant role of type IV collagen in the maintaining the GBM has been highlighted by mutations in the α3, α4, and α5 chains of type IV collagen encoded by *COL4A3*, *COL4A4*, and *COL4A5* genes, respectively, which cause AS (15, 33). There are two main modes of inheritance in AS: X-linked and autosomal recessive (30, 34, 35). The X-linked form of AS occurs in around 80% of patients and involves mutations in the *COL4A5* gene encoding the α5 chain of type IV collagen. All affected males with the X-linked AS will ultimately develop end-stage renal disease (ESRD) requiring dialysis or kidney transplantation by at least 60 years of age (30). Female patients with X-linked AS also develop proteinuria; however, only around 30% develop ESRD at 60 years (36). Approximately 15% of patients with AS have the autosomal recessive form of disease arising from mutations in *COL4A3* or *COL4A4*, and the phenotype of these patients is similar to those with X-linked AS. A final small group of patients have heterozygous mutations in *COL4A3* or *COL4A4* and manifest with an AS phenotype.

Genetic abnormalities in the *COL4A5* gene have a significant influence on the rate of progression to ESRD in males with X-linked AS (30, 37). Female carriers of XLAS syndrome are known to experience milder disease than affected males, and X-chromosome inactivation may be a major determinant of the difference in phenotype (36, 38). All affected males and around 95% of females experience microscopic hematuria from early childhood and episodes of macroscopic hematuria may present episodically throughout life (30, 36). In a large cohort study of families with X-linked AS, the type of genetic mutation determined the risk of progression to ESRD and hearing loss (30). Male patients with large deletions, nonsense or small mutations carried a significant 90% probability of developing ESRD, whereas individuals with a missense or a splice mutation had a 50–70% risk of this complication.

In AS, there is a defect in the switch from the α1α1α2(IV) to the α3α4α5(IV) networks during glomerulogenesis and instead the α1α1α2(IV) network predominates in the mature GBM (25, 39). Changes in type IV collagen networks may influence signaling *via* discoidin domain receptor 1 (DDR1) and integrin α2β1 (40, 41), which may in turn alter podocyte function. These extracellular receptors, DDR1 and DDR2, belong to a family of receptor tyrosine kinases, which are important for cellular regulation (42). Receptor tyrosine kinases are typically activated by peptide-like growth factors; however, activation of DDRs is mediated by collagens, which act as DDR ligands (43–45). Mice deficient in α2 integrin do not have a renal phenotype; however, DDR1-knockout mice display thickening of the GBM, podocyte foot effacement, and proteinuria (46, 47). Mice with AS and DDR1 deficiency or haploinsufficiency had improved renal function and increased survival (48). These models suggest a potential role for DDR1 signaling in the pathogenesis of AS; however, exact mechanisms are not fully understood.

The structural components of the glomerulus including the capillary loops, mesangial cells, and matrix and podocytes may also undergo damage due to high-mechanical forces exerting biomechanical strain on glomerular cells. The effects of increased blood pressure were seen in Alport mice treated with l-NAME, which is an inhibitor of nitric oxide synthase and causes hypertension. These mice developed proteinuria and severe ultrastructural defects in the GBM (49). In contrast, the angiotensin-converting enzyme (ACE) inhibitor ramipril delayed the progression to proteinuria in the *COL4A3* knockout mice and has been shown to improve survival in both mouse models and human disease (50, 51). Recent work highlighted that biomechanical strain-sensitive activation of mesangial cell actin dynamics may be important in the pathogenesis of AS. This was demonstrated by the upregulation of endothelin A receptor expression in the glomeruli of Alport mouse models, which lead the formation of mesangial filopodia and the deposition laminin α2 (52, 53) in the GBM. The accumulation of mesangial proteins within the GBM led to the activation of focal adhesion kinase in podocytes and nuclear translocation of NF-κB, which induces a pro-inflammatory state and the release of cytokines, chemokines, and metalloproteinases (54). Interestingly, matrix metalloproteinases not only show increased expression in the glomeruli of Alport mouse models but pharmacological inhibition of these inflammatory mediators before the onset of proteinuria prevented disease progression and improved survival (55, 56). The inhibition of transforming growth factor-β1 was thought to slow disease progression and although this inhibitor prevented GBM thickening, it did not prevent foot process effacement (57, 58). Advances in GBM biology have enabled better understanding of pathogenic mechanisms in AS; however, more research is required to enable the development of therapeutic targets that may delay or stop the progression of the disease.

#### *Treatments*

Medical treatment using ACE inhibition has demonstrated significant benefits including the reduction of proteinuria and delay of ESRD in AS (51, 59); however, mechanisms of action of these drugs are not fully understood. In a retrospective study, the use of ACE inhibitors appeared to delay renal failure and improve life expectancy in three generations of Alport families (51). Findings resulting from the EARLY PRO-TECT Alport trial of the effects of RAS blockade on the progression of ESRD will be important in supporting the development of emerging candidate therapies (60, 61). At the point of requiring renal replacement therapy, renal transplantation is associated with good long-term outcomes (62). Appropriate donor selection is crucial as the risk of penetrance of *COL4A5* gene mutation is particular high within families, although transplantation among relatives has been reported (63, 64). This method is by no means suboptimal and important points need to be considered, which include the acceptance of donors without proteinuria and the counseling of families who may experience an increased risk of ESRD in the recipient (64).

A small proportion of around 3% of patients with AS suffer from severe post-transplant anti-GBM disease (63). This complication typically presents with hematuria, which progresses to graft rejection. The prognosis for survival in these patients who undergo subsequent transplantation is poor (65). The risk of posttransplant anti-GBM nephritis in patients with AS is increased if the underlying defect is due to a gene deletion (66). Antibodies in anti-GBM are generated against the α5(IV) and α3(IV) chains in patients with X-linked and autosomal recessive AS, respectively (67). Affected individuals display histological similarities to Goodpasture disease; however, post-transplant anti-GBM disease is different in that it does not occur spontaneously and it involves an alloimmune response against normal type IV collagen in the form of foreign antigenic epitopes.

Future alternative options to conventional therapy include gene replacement and several approaches have been employed, which may have broader implications for the development of therapeutic targets that aim to repair the GBM after pathological insult. The insertion of a full-length *COL4A3* under control of a tetracycline inducible promoter into *Col4a3<sup>−</sup>/<sup>−</sup>* mice significantly reduced proteinuria, increased survival, and restored missing collagen IV networks despite the persistence of ultrastructural damage within the GBM (68). Advances in stem cell technology have also explored the possibility of restoring type IV collagen network to the GBM in Alport AS. Replacement through the transplantation of wild-type bone marrow-derived stem cells into irradiated *Col4a3<sup>−</sup>/<sup>−</sup>* mice resulted in possible recruitment of podocytes and mesangial cells within damaged glomeruli and a partial re-expression of α3(IV) collagen chains (69). Histologically, these mice had improved glomerular architecture associated with a significant proteinuria compared with untreated *Col4a3<sup>−</sup>/<sup>−</sup>* mice or irradiated *Col4a3<sup>−</sup>/<sup>−</sup>* mice that had received bone marrow from adult *Col4a3<sup>−</sup>/<sup>−</sup>* mice. Stem cell therapies have highlighted novel approaches for the treatment of AS; however, these methods require further refinement before their application in human disease.

### Heterozygous Mutations in *COL4A3* and *COL4A4 Clinical Features*

Individuals with a single mutation in *COL4A3* or *COL4A4* present with microscopic hematuria in childhood and this is due to the diffuse thinning of the GBM. Patients with thin GBMs may have episodic macroscopic hematuria throughout life and are at risk of developing proteinuria and progressive CKD. Approximately 1% of the population is affected by thin GBMs and at least twothirds have another affected relative (70). These patients do not usually suffer from extrarenal manifestations such as hypertension or deteriorating renal function requiring dialysis or kidney transplantation.

#### *Etiology and Pathogenesis*

Heterozygous mutations in the *COL4A3* and *COL4A4* gene account for up to 40% of cases in families with thin GBMs and these mutations demonstrate linkage to the *COL4A3/COL4A4* locus, which is also affected in autosomal recessive AS (71, 72). The majority of these patients have non-progressive hematuria; however, a number of individuals will experience a deterioration in renal function with pathological changes, which include GBM thickening, proteinuria, nephrotic syndrome, and progressive CKD. Of the 82 patients from a cohort of patients with thin GBMs who had heterozygous mutations in *COL4A3* and *COL4A4*, around 37.8% developed CKD and 19.5% progressed to ESRD (73).

Patients with thin GBMs rarely require treatment, however, given the variation in presentation in a minority of individuals, long-term monitoring including blood pressure, proteinuria, and renal function is strongly recommended.

#### HANAC Syndrome

#### *Clinical Features*

Hereditary angiopathy, nephropathy, aneurysm, and cramps comprise HANAC syndrome. The disease phenotype in families is characterized by muscle cramps, mild cerebral small-vessel disease, retinal artery tortuosity, intracranial aneurysms, and renal disease associated with multicystic kidneys, occasional hematuria, and decreased glomerular filtration rate in older patients (74–76). Intrarenal structural abnormalities occur within Bowman's capsule, tubular basement membrane (TBM), and peritubular capillaries; however, the integrity of the GBM appears normal.

#### *Etiology and Pathogenesis*

Mutations in *COL4A1* affecting the α1α1α2(IV) network were first identified in families with porencephaly, a condition characterized by cystic and cerebral white matter lesions (77). Smallvessel disease affecting the brain and eye has also been described in a single family with *COL4A1* mutation (78). Missense mutations in the *COL4A1* gene, localized in exons 24 and 25, which affect glycine residues and interrupt the Gly-Xaa-Yaa amino acid repeat have been described in three families with autosomal dominant inheritance of the disease (79). In mice with the *Col4a1* G498V mutation used to model HANAC syndrome, there was evidence of delayed glomerulogenesis and podocyte differentiation without a reduction in nephron number (80). Given the extensive expression of the α1α1α2(IV) network as well as its importance in glomerular development and podocyte differentiation, it is not surprising that mutations in the *COL4A1* gene may lead to a systemic phenotype. Treatment for this condition is largely supportive, and distinct therapeutic targets have not yet been defined.

### Laminin

Laminins are large glycoproteins that produce α, β, and γ heterotrimers, which provide a structure for the attachment of matrix proteins within the ECM (81). In humans, there are at least 15 distinct laminin heterotrimers composed of an assembly of 4 α-, 4 β-, and 3 γ-chains (**Figure 4**) (82, 83). Laminin heterotrimers have tissue-specific expression depending on their αβγ composition. The predominant laminin heterotrimer secreted by endothelial cells and podocytes is α5β2γ1 or LM-521, which features in the mature GBM and replaces α1β1γ1 (LM-111) and α5β1γ1 (LM-511), which are present during glomerulogenesis (3, 84, 85). The majority of laminins form cross-shaped structures composed of one "long arm" formed by αβγ chains *via* coiled–coil interactions and disulfide bonding, and three "short arms" with NH2-terminal globular (LN) domains (86, 87). These LN domains in the short arms of cross-shaped structures mediate trimer polymerization, which has a significant role in basement membrane formation (88). Another unique feature of laminin is the α-chains that have C-terminal laminin globular (LG) domains at the distal end of the long arm, which binds the laminin trimer to laminin receptors (89).

The LG domain mediates interactions with cell surface receptors including integrins and dystroglycan (90, 91). Integrin α3β1 binds to LM-511 and LM-521 through the LG domain of the α5 chain (26). This integrin is a key surface receptor responsible for capillary loop formation and mesangial cell organization, and podocyte-specific deletion affects normal glomerular development (92, 93). Changes in expression of the α-dystroglycan subunit that binds to laminin occurs in glomerular disease; however, deletion in podocytes did not affect susceptibility to

injury or recovery from damage in the kidneys (94–98). In addition, glycosylation of α-dystroglycan which disrupts its binding with laminin resulted in mild podocyte foot process effacement without proteinuria (99).

#### Pierson Syndrome

#### *Clinical Features*

Pierson syndrome is a rare condition inherited in an autosomal recessive manner with phenotypic variability and characterized by congenital nephrotic syndrome which progresses to ESRD within the first year of life. In some cases, renal manifestations may present as early as the prenatal period with oligohydramios and kidney enlargement, which can be detected using ultrasonography (100, 101). Children with Pierson syndrome are affected by renal impairment soon after birth; however, with renal replacement therapy, some have lived for up to 2 years of age (102). There are currently no reports to date of adult patients with Pierson syndrome. Extrarenal manifestations include fixed pupil constriction (microcoria) but not all patients with Pierson syndrome have eye abnormalities (103, 104). Congenital muscle hypotonia and neurodevelopmental abnormalities may also present in some cases (105).

#### *Etiology and Pathogenesis*

Pierson syndrome is caused by the homozygous or compound heterozygous mutations in β2-laminin encoded by the *LAMB2* gene (106). Given the variable expression resulting from mutations in the *LAMB2* gene, there appears to be a broad spectrum of phenotypic presentations and screening of this gene is to be considered should there be no mutations found in *NPHS1*, *NPHS2*, or *WT1* (104). Patients with milder missense *LAMB2* mutations, including R246Q and C321R, present with a less severe renal and extrarenal phenotypes (16, 104). In Pierson syndrome, there appears to be a deficiency of the major laminin protomer LM-521 containing β2-laminin expression, which predominates in mature GBM. Mice with a null mutation in *LAMB2* which display the clinical features seen in Pierson syndrome are a good model for the study of this condition (107). The loss of LM-521 in the normal mature GBM is replaced by LM-511, which usually features in the developmental GBM. The generation of *LAMB2<sup>−</sup>/<sup>−</sup>* mice has been useful in that they recapitulate Pierson syndrome, and develop congenital albuminuria, podocyte foot process effacement, and are non-viable from 3 weeks of age due to neuromuscular defects and nephrotic syndrome (107, 108). In the *LAMB2−/−* mice, there was an ectopic deposition of several laminins, a possible a compensation for the loss of LM-521, and mislocalization of anionic sites despite a structurally normal GBM (109). Like in the human disease, proteinuria preceded structural abnormalities in the podocyte and slit diaphragm in these mice, which suggests the premature onset of pathological mechanisms before the onset of damage within the GBM.

Using the *Lamb2<sup>−</sup>/<sup>−</sup>* mice in the study of Pierson syndrome has its limitations in that they die at a young age, and therefore the long-term effects on the kidneys and neuromuscular junction are not known. Transgenic mice with varying podocyte expression levels of R246Q-mutant rat β2-laminin were subsequently developed to investigate the degree to which missense mutations in the *LAMB2* gene cause proteinuria. These mice were generated by crossing *LAMB2<sup>−</sup>/<sup>−</sup>* mice with transgenic mouse lines expressing rat laminin-β2 either in muscle or podocytes (110). In transgenic mice where laminin-β2 was restricted to the kidney, the degree of proteinuria was milder compared with non-transgenic *Lamb2−/−* mice, but severe kidney and neuromuscular defects were maintained. Where laminin-β2 was limited to muscle expression, synaptic function was restored; however, mice died from kidney disease at 1 month of age. Transgenic podocyte-specific Lamβ1 expression in *Lamb2−/−* mice successfully increased LM-511 trimer deposition, eliminated nephrotic syndrome, and extended survival, highlighting a potential new therapeutic approach (111). These findings implicate potential targets for future therapies and that targeting the kidneys alone may not be effective in Pierson syndrome. Treatment for Pierson syndrome is currently supportive.

### Nidogen

Also known as entactins, nidogen-1 and nidogen-2 are dumbbellshaped basement membrane proteins with three globular domains connected by two threads separating G-domains (112). Different genes encode nidogen-1 and nidogen-2, and nidogen-1 binds to the short arm of the laminin γ1 chain and type IV collagen, which is thought to bridge these separate networks in the basement membranes (113, 114). It is uncertain if nidogen may have an important role in basement membrane formation, as the deletion of either of the nidogen genes produce viable mice with a normal phenotype (115, 116). Interestingly, nidogen-2 knockout mice are more susceptible to the induction of renal injury compared with the wild-type mice, and these mice develop increased blood pressure, serum creatinine, and albumin excretion (117). Nidogen-2 may have a protective or reparative role in the GBM; however, mechanisms are not known. In mice with simultaneous deletion of both nidogen-1 and nidogen-2, basement membrane formation occurs normally; however, these mice die perinatally and develop subsequent basement membrane abnormalities in the lungs and heart (10). Despite a normal GBM appearance in double nidogen knockout mice, there was phenotypic variability and a subgroup developed renal dysgenesis or agenesis (unilateral or bilateral), hydronephrosis, and polycystic defects. To date, the effect of mutations in nidogen genes in the human kidney is not known; however, quantitative mass spectrometry proteomics have identified that the *NID1* gene, which encodes for nidogen-1, promotes lung metastases of breast cancer and melanoma (118).

### Heparan Sulfate Proteoglycans

The three major HSPGs identified in basement membranes include perlecan, collagen XVIII, and agrin. The commonest HSPGs in basement membranes during development are perlecan and collagen XVIII, whereas agrin predominates in the mature GBM (8, 119, 120). Perlecan binds to nidogen, laminin, and collagen IV through heparan sulfate chains in the immature GBM and after glomerulogenesis, a higher concentration of perlecan is found in the mesangial matrix (121–123). Perlecan mutant mice have normal kidneys; however, they appear to be more susceptible to proteinuria after albumin overload. Mice deficient in collagen XVIII have deranged creatinine levels and display mesangial matrix expansion, but did not display GBM abnormalities (124–126). Recent ultra-high resolution STORM imaging correlated with electron microscopy reveals differences in the distribution of agrin in mouse and human GBM (127). In mice, the C-terminal end of agrin is located adjacent to cell membranes and the N-terminus is found toward the center of the GBM, whereas in humans, agrin is mainly present in the subepithelial area where podocytes reside. Therefore, the phenotype seen in mouse models may not represent directly relevance to human mutations due to interspecies differences in the GBM distribution of HSPGs.

The heparan- and chondroitin-sulfate glycosaminoglycan side chains frequently undergo sulfation, resulting in a negatively charged proteoglycan, which is thought to contribute to charge selectivity within anionic sites in the GBM (9, 128). The anionic sites within the GBM can be detected with polyethyleneimine and cationised ferritin. Agrin has an N-terminal domain bound to the long arm of LM-521 and a C-terminus bound to integrins and dystroglycan, which implicates a potential role in charge selectivity (129). Mice with a podocyte-specific mutation of agrin had fewer negatively charged sites and a shorter length of protein; however, these defects did not result in proteinuria or GBM abnormalities after an albumin overload (9). The deletion of both perlecan and agrin in mice had no effect on permselectivity of the filtration barrier (130). Whether charge selectivity exists within the glomerular filtration barrier is not clear, and these studies confirm that HSPGs may not have a major role in this function.

### HSPGs in Glomerular Disease

Mutations in HSPGs in human glomerular disease are not well defined and remain limited to immunohistochemical staining in single case reports. Global or segmental loss of staining for heparan sulfate chains in the GBM is evident in lupus nephritis, membranous nephropathy, minimal change disease, and diabetic nephropathy but not in IgA nephropathy or AS (131). The loss of HSPG in the GBM is also observed in individual case reports of C3 glomerulopathy and Denys–Drash syndrome (132, 133). Similarly, animal models including the mouse model of lupus nephritis (MRL/lpr), rat model of active Heymann nephritis, or membranous nephropathy and rat model of streptozotocininduced diabetic nephropathy show reduced GBM heparan sulfate staining (134–136). A potential mechanism accounting for the reduction of heparan sulfate may be the increase in active heparanase in kidney disease, which shortens these chains through hydrolyzation of glycosidic bonds at specific sites (137). In mouse models of passive Heymann and anti-GBM nephritis, the increase in glomerular heparanase is accompanied by the loss of heparan sulfate, proteinuria, and complement activation. Treatment of these mice with anti-heparanase antibodies reverses proteinuria (138). Decreased expression of heparan sulfate may be due to these domains being masked by the deposition of autoantibodies and immune complexes in the GBM, which is seen in MRL/*lpr* mice and patients with systemic lupus erythematosus (SLE) (139). The reduction in heparan sulfate has been recently linked to complement activation. There appears to be a loss of complement regulator factor H in lupus nephritis and anti-GBM disease, which leads to inadequate complement activation and glomerular injury (140, 141). There is clearly a role for HSPGs in the regulation of the GBM and characterization of physiological networks is required to understand its function in health and disease.

### Fibronectin

Fibronectin is a large 270-kDa glycoprotein with diverse function, which binds to heterodimeric cell surface receptors such as integrins and other ECM components (142). There are three modular domains of fibronectin, which includes type I and II repeats maintained by disulfide bonds and type III repeats characterized by the absence of these bonds that enable it to undergo conformational changes (143–145). Fibronectin can occur in two forms. Plasma fibronectin is secreted into blood from hepatocytes in its soluble form and may integrate into the fibrillar matrix, a role previously attributed to cellular fibronectin (146, 147). Integrins are important in linking fibronectin to the actin cytoskeleton and enabling the process of matrix assembly; however, fibrin organization within fibrils is not known (148). Mechanical forces regulate interactions between fibronectin and collagen; however, these findings have been limited to *in vitro* fibroblasts, and the role of this novel mechanism *in vivo* is not known (149). Fibronectin is not an abundant component of the GBM; indeed, our proteomic analyses demonstrated a fivefold higher abundance of type IV collagen compared with fibronectin in healthy glomerular ECM (11). In disease, however, there appears to be a greater glomerular deposition of fibronectin, and this has been observed in a diabetic nephropathy model (150).

#### Fibronectin in Glomerular Disease

Fibronectin glomerulopathy is inherited in an autosomal dominant manner and is characterized by proteinuria, microscopic hematuria, hypertension, and abnormal renal function. Glomerular deposition of fibronectin can lead to progressive ESRD between the second and sixth decade of life (151). Light microscopy findings include enlarged glomeruli with minimal hypercellularity, and mesangial and subendothelial space deposits. Granular, fibrillary, or mixed deposits within the glomeruli can also be detected by electron microscopy. A significant finding in fibronectin glomerulopathy is the strong positive staining of fibronectin observed in the glomeruli. The immunoreactivity for immunoglobulins, complement proteins, laminin, and type IV collagen was absent or weak. Fibronectin deposition may potentially occur in other glomerular diseases, evident in mouse models of SLE and patients with lupus nephritis (152). However, recent proteomic analyses of laser-captured microdissected glomeruli comparing living-related healthy donors and patients with glomerular disease confirm that fibronectin specifically expressed in patients with fibronectin glomerulopathy (153). Mutations causing fibronectin glomerulopathy are not well studied; however, in a large Italian pedigree, linkage at the *FN1*

locus at 2q32 was detected and dominant mutations in this gene contributed to 40% of cases in this group (154).

### Transcription Factors

There are several transcription factors that have a role in regulating the GBM and ECM proteins. The LIM-homeodomain transcription factor encoded by the *LMX1B* gene is highly expressed in podocytes, with LIM domains at the N-terminus facilitating protein interactions and a central homeodomain enabling DNA binding. Mutations in the *LMX1B* gene result in the absence or inactivation of this central homeodomain, which is associated with nail patella syndrome (NPS) (155). In the *LMX1B* conditional knockout mouse, there is a decreased expression of *COL4A3* and *COL4A4* genes in the glomeruli (156). In addition, *LMX1B-*deficient podocytes displayed structural abnormalities including a reduced number of foot processes that were dysplastic and lacked slit diaphragms. The levels of CD2-associated protein and podocin expression were also reduced in *LMX1B-*deficient podocytes, which implicated a potential role for *LMX1B* in regulating these key proteins.

#### Nail Patella Syndrome

Nail patella syndrome, also known as hereditary osteoonychodysplasia, is a rare autosomal dominant disorder presenting with pleiotropic developmental abnormalities of dorsal limb structures involving hypoplasia or absence of the patellae, dystrophic nails, and dysplasia of the elbows and dorsal ilium (157). Phenotypic variability is present despite increased penetrance, and it appears that less than half of affected individuals with NPS develop nephropathy with proteinuria and hematuria (158, 159). Although the majority of patients experience a benign nephropathy, around 30% develop the risk of progression to renal failure.

Patients with NPS have over 140 heterozygous mutations in *LMX1B* with missense, splicing, insertion, deletion, and nonsense alterations (155). *LMX1B* mutations that occur in the LIMhomeodomain are associated with skeletal defects in NPS (160). Mutations affecting the central homeodomain are significantly associated with renal involvement and this pattern appears to cluster in families (161). The microscopic findings of structural renal abnormalities in NPS are fairly non-specific and they include irregular GBM thickening with a "moth-eaten" appearance (162). To date, mechanisms leading to human glomerular disease in NPS have not been characterized and no distinct therapeutic targets have been identified.

### GBM Disease Involving Mutations in Podocyte Cytoskeletal Genes

Myosins contribute to the cytoskeletal machinery of cells, hydrolyzing ATP, and interacting with actin to enable movement along muscle fibers. Non-muscle myosin heavy chain IIA (NMMHC-IIA) is encoded by *MYH9* and is expressed in glomerular podocytes, mesangial cells, and arteriolar and peritubullar capillaries. Mutations in the *MYH9* gene can result in variable phenotypic presentations, which include Epstein and Fechtner syndromes, May–Hegglin anomaly, and Sebastian syndrome. Both Epstein and Fechtner syndromes were once considered to be variants of AS due to similarities in clinical phenotype, which include hereditary nephritis, hearing loss, and ocular abnormalities; however, type IV collagen expression appears to be preserved (163, 164). Patients with Epstein and Fechtner syndromes may also develop thrombocytopenia, macrothrombocytopenia, and leukocyte inclusions (165).

Genotype–phenotype correlations have suggested that mutations in the amino-terminal domain, which binds myosin light chains interacts with actin and utilizes ATP, may cause more severe phenotypic manifestations including nephritis, thrombocytopenia, and deafness before 40 years of age compared with mutations in the carboxy-terminal tail (166). The effects of *MYH9* mutations on the GBM are not known, and microscopic findings have been fairly non-specific and likened to those observed in AS, with focal GBM thinning and foot process effacement (167). The expression of NMMHC-IIA in podocytes is reduced in *MYH9*-related disease (167, 168), but it is unclear whether these alterations affect GBM synthesis and organization. Our current knowledge on the long-term consequences of *MYH9* mutations on the GBM remains limited, and *MYH9* does not appear to be the only contributing factor to the spectra of conditions affecting the podocyte cytoskeleton.

### GBM Disease Caused by Abnormal Regulation of Adhesion Receptors

The GBM is composed of condensed sheets of ECM with a supramolecular assembly built around two major networks of laminin and collagen IV (169). The role of the GBM is to support its ECM components and provide a scaffold for adjacent endothelial cells and podocytes. Cell–matrix interactions are key to the integrity of the filtration barrier maintained by cells adhering to the ECM through adhesion receptors, which include integrins, syndecans, and dystroglycan (13).

Integrins are αβ-heterodimeric cell surface receptors that engage with the ECM to mediate cell–matrix signaling, and the α3β1 heterodimer is particularly important in linking the podocyte and GBM (170). The importance of the α3β1 heterodimer is evident in mice lacking the integrin α3 subunit, which are nonviable within the first day of life due to developmental defects in the kidneys and lungs, accompanied by the loss of specialized morphology and thickened irregular GBMs (93). The podocytespecific deletion of α3 subunit results in an abnormal renal phenotype manifesting as nephrotic syndrome and subsequent renal failure (171). Individuals homozygous for mutations in the integrin α3 gene, *ITGA3*, have disrupted basement membrane structures and compromised barrier functions. These patients develop congenital nephrotic syndrome, interstitial lung disease, and epidermolysis bullosa (172). Mice with the homozygous deletion of integrin β1 are not viable beyond the embryonic phase (173) and podocyte-specific deletion of the β1 subunit in mice causes early renal failure at 3 weeks of age, progressive podocyte apoptosis and capillary loop and mesangium degeneration (174). Mutations of the integrin β1 subunit in humans have not yet been described.

There is also a significant association between the tetraspanin CD151 and integrins in particular α3β1, which may have a role in cell migration (175). The global and podocyte-specific deletion of *CD151* gene in mice results in proteinuria accompanied by the focal glomerulosclerosis, disorganization of the GBM, and tubular cystic dilation (171). Interestingly, the phenotype of *CD151* knockouts significantly depends on the background of mouse used. *CD151*-deficient mice bred on an FVB background develop spontaneous and severe glomerular disease; however, this phenotype was not recapitulated in C57BL/6 knockout models (176). The *CD151* knockouts bred on a C57BL/6 background only display significant proteinuria when challenged with induced hypertension. Patients with a frameshift mutation in *CD151* develop a similar phenotype to mice, which includes focal thickening and irregularity of the GBM as well as combined hearing loss and skin defects (177).

### Genetic Disorders of the TBM

The TBM lies beneath the tubular epithelial cells and is in continuity with the interstitial connective tissue (178). Currently, there are no known genes exclusively linked to TBM abnormalities; however, these abnormalities are present in genetic disorders affecting the kidneys and often present alongside GBM or other extrarenal basement membrane phenotypes. The GBM is well preserved in HANAC syndrome, which is associated with mutations in the human *COL4A1* gene; however, splitting and thickening of the TBM is observed (79). Defects in laminin α5, a major GBM and TBM protein component encoded by *LAMA5* in mice manifest as GBM abnormalities, incomplete glomerular vascularization and polycystic kidneys, and defective interactions between tubular epithelial cells and the ECM may be the cause of cyst formations (179, 180). In AS, progressive disorganization of the GBM is followed by pro-fibrotic changes in the renal interstitium, which is characterized by abnormalities in the proximal renal tubular epithelium and defects in ECM turnover, leading to deterioration in renal function (49, 181, 182). These few examples demonstrate that abnormalities in the TBM appear to occur alongside GBM defects; however, genetic and pathogenic mechanisms causing disease are yet to be fully understood.

## SUMMARY OF RESEARCH ADVANCES

There have been significant research advances in glomerular disease since the early 1990s and the discovery of genetic disease associated with GBM defects and nephrotic syndrome. However, there remain unmet needs in the diagnosis, clinical care, and disease management. Importantly, there is currently a lack of targeted treatments across the spectrum of GBM conditions. Given the pivotal role of the GBM particularly in AS, a key area of focus is early diagnosis and treatment of disease, which has significant evidence in improving the long-term prognosis (51). The exponential discoveries through next-generation sequencing has enabled better understanding of the heterogeneity of GBM disease, which allows the integration of conventional therapies with a more stratified approach to treatment. Clinicians will therefore be able to avoid invasive biopsies and adjust tailor medications to a patient's needs more effectively. In addition, patients and families will have the opportunity to plan based on informed genetic counseling. The discovery of new therapeutics through the collaboration of the patients, clinicians, scientists, and industry is therefore necessary, to treat and even possibly prevent foot process effacement and GBM abnormalities (31). This in turn will generate a personalized diagnostic approach and potential therapeutic targets for the treatment of genetic kidney diseases affecting the basement membrane.

### PATIENT PERSPECTIVE

Living with kidney disease presents challenges for families and around 4 in 10 patients have experienced a delay in or lack of diagnosis (183), which may result in inappropriate management. Diseases of the GBM are considered to be rare diseases, which affect the minority of people among other more prevalent conditions in the general population. To date, between 5,000 and 8,000 distinct rare diseases have been reported and in the UK, around 1 in 17 people (approximately 3.5 million) will develop a rare disease at some point in their lives (184). Of the rare diseases, around 75% of children are affected and at least 80% have had a genetic component identified. Although clinical teams can provide their expertise in treating families with rare diseases, it is important to recognize the personal challenges that patients' encounter, which include pathways to getting an appropriate diagnosis, implementation of care, and patient support networks. In addition, there are international efforts working toward the development of 200 new therapies for rare diseases by 2020 (35).

Research in to rare diseases is urgently required to enable better prognoses and targeted treatments for patients and their families. To support people with rare disease, patients' advisory groups play an important role in helping families through websites, leaflets, and personal contact. For genetic diseases of the GBM, an example organization is Alport UK, which is a patientled organization dedicated to empowering people who live with AS through the provision of support and information for this condition (185). Alport UK works with a multi-disciplinary internatonal collaboration consisting of researchers, clinicians,

### REFERENCES


academics, pharmaceutical companies, and other patient groups. This valuable partnership enables families and clinicians to access up-to-date and accurate resources, which aids clinical management toward a better quality of life and understanding of the AS. Another role of patient organizations is to assist with recruitment into national patient registries. These efforts are crucial to improving understanding about the natural history rare diseases, which may also inform the causative pathways and improved management for other more common disorders.

### SUMMARY/CONCLUSION

Over the last two decades, important advances have been made in understanding the composition and function of the GBM, which is important for maintenance of filtration barrier integrity. Studies of core ECM components including laminin, collagen IV, HSPG, and nidogen in health and disease have highlighted the importance of these basement membrane proteins in enabling the GBM to function as the primary filtration barrier of the kidney. Significant progress in the application of global approaches including proteomics analyses has highlighted novel genotype–phenotype correlations, and thus enabling a broader range of molecular diagnoses for rare or unclassified glomerular diseases. In addition, these innovative approaches will enable the development of stratified therapeutic targets and guide the prognosis counseling of families with GBM diseases.

### AUTHOR CONTRIBUTIONS

CC and RL research the topic and wrote the review. CC prepared the figures.

### FUNDING

CC is a Clinical Research Training fellow funded by Arthritis Research UK (21370) and RL is supported by a Wellcome Trust Senior Fellowship award (202860/Z/16/Z).


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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Chew and Lennon. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

,

## Heterozygous COL4A3 Variants in Histologically Diagnosed Focal Segmental Glomerulosclerosis

Matthias C. Braunisch1,2, Maike Büttner-Herold<sup>3</sup> , Roman Günthner 1,2 , Robin Satanovskij 1,2, Korbinian M. Riedhammer 1,2, Pierre-Maurice Herr <sup>2</sup> , Hanns-Georg Klein<sup>4</sup> , Dagmar Wahl <sup>4</sup> , Claudius Küchle<sup>1</sup> , Lutz Renders <sup>1</sup> , Uwe Heemann<sup>1</sup> Christoph Schmaderer <sup>1</sup> and Julia Hoefele<sup>2</sup> \*

#### Edited by:

Max Christoph Liebau, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Köln, Germany

#### Reviewed by:

Rizaldy Paz Scott, Division of Nephrology & Hypertension, Feinberg School of Medicine, Northwestern University, United States Rachel Lennon, University of Manchester, United Kingdom

#### \*Correspondence:

Julia Hoefele julia.hoefele@tum.de

#### Specialty section:

This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics

Received: 25 March 2018 Accepted: 24 May 2018 Published: 12 June 2018

#### Citation:

Braunisch MC, Büttner-Herold M, Günthner R, Satanovskij R, Riedhammer KM, Herr PM, Klein HG, Wahl D, Küchle C, Renders L, Heemann U, Schmaderer C and Hoefele J (2018) Heterozygous COL4A3 Variants in Histologically Diagnosed Focal Segmental Glomerulosclerosis. Front. Pediatr. 6:171. doi: 10.3389/fped.2018.00171 <sup>1</sup> Department of Nephrology, Klinikum Rechts der Isar, Technical University of Munich, Munich, Germany, <sup>2</sup> Institute of Human Genetics, Klinikum Rechts der Isar, Technical University of Munich, Munich, Germany, <sup>3</sup> Department of Nephropathology, Institute of Pathology, Friedrich-Alexander-University, Erlangen, Germany, <sup>4</sup> Center for Human Genetics and Laboratory Diagnostics Dr. Klein, Dr. Rost and Colleagues, Martinsried, Germany

Introduction: Steroid-resistant nephrotic syndrome (SRNS) is one of the most frequent causes for chronic kidney disease in childhood. In ∼30% of these cases a genetic cause can be identified. The histological finding in SRNS is often focal segmental glomerulosclerosis (FSGS). In rare cases, however, pathogenic variants in genes associated with Alport syndrome can be identified in patients with the histological finding of FSGS.

Materials and Methods: Clinical information was collected out of clinical reports and medical history. Focused molecular genetic analysis included sequencing of COL4A5 and COL4A3 in the index patient. Segregation analysis of identified variants was performed in the parents and children of the index patient.

Results: The female index patient developed mild proteinuria and microscopic hematuria in childhood (12 years of age). The histological examination of the kidney biopsies performed at the age of 21, 28, and 32 years showed findings partly compatible with FSGS. However, immunosuppressive treatment of the index patient did not lead to a sufficient reduction of in part nephrotic-range proteinuria. After the patient developed hearing impairment at the age of 34 years and her daughter was diagnosed with microscopic hematuria at the age of 6 years, re-examination of the index's kidney biopsies by electron microscopy revealed textural changes of glomerular basement membrane compatible with Alport syndrome. Molecular genetic analysis identified two missense variants in COL4A3 in a compound heterozygous state with maternal and paternal inheritance. One of them is a novel variant that was also found in the 6 year old daughter of the index patient who presented with microscopic hematuria.

Discussion: We were able to show that a novel variant combined with a previously described variant in compound heterozygous state resulted in a phenotype that was

**24**

histologically associated with FSGS. Molecular genetic analysis therefore can be essential to solve difficult cases that show an unusual appearance and therefore improve diagnostic accuracy. Additionally, unnecessary and inefficient treatment with multiple side effects can be avoided.

Keywords: Alport syndrome, COL4A3, focal segmental glomerulosclerosis, FSGS, nephrotic syndrome, hearing impairment

### INTRODUCTION

Steroid-resistant nephrotic syndrome (SRNS) is one of the most frequent causes for chronic kidney disease especially in young adulthood (1, 2). In around 30% of cases a monogenetic cause can be identified (3). Histological findings in SRNS largely present as focal segmental glomerulosclerosis (FSGS) with progressive glomerular scarring (4). To date, more than 30 genes associated with SRNS have been identified placing the podocyte into the center of attention regarding the pathogenesis of SRNS and FSGS (5). Immunosuppressive treatment of proteinuria in hereditary FSGS often cannot induce remission and is also poorly tolerated (6, 7).

In rare cases, FSGS can be caused by variants in COL4A3 and COL4A4, both genes associated with Alport syndrome (AS) (8). AS typically occurs with the leading symptoms of progressive renal failure, sometimes associated with the extrarenal manifestations of hearing impairment (sensorineural deafness) and/or ocular abnormalities (anterior lenticonus, cataract) (9). In AS the first sign is microscopic hematuria in early childhood. Proteinuria increases as disease progresses. However, proteinuria commonly does not reach nephrotic range in contrast to FSGS or SRNS (10).

Variants in COL4A5 are the most frequent causes involved in the pathogenesis of AS and account for about 85% of cases (9). Due to the X-linked inheritance of COL4A5 mostly men are affected. In fewer cases, variants in COL4A3 and COL4A4 located at the long arm of chromosome 2 account for an autosomal recessive (14% of cases) or autosomal dominant (1% of cases) AS (11, 12). Male and female patients are equally affected.

Due to mainly economic reasons, genetic testing is often reluctantly used in clinical routine. Molecular genetic diagnosis can improve accuracy of disease classification, especially in phenotypes with multiple symptoms and unusual appearance and help to create personalized treatment options.

Here we report one novel COL4A3 variant in a compound heterozygous state in a 34 year old woman with hematuria and proteinuria who presented initially with histological findings compatible with FSGS. Only after hearing impairment occurred later in life, genetic testing of AS associated genes was initiated and the correct diagnosis was made.

### MATERIALS AND METHODS

### Clinical Case Information

The study was approved by the local Ethics Committee of the Technical University of Munich and performed according to the standard of the Helsinki Declaration of 1975. Written informed consent was obtained from the index patient and their relatives for publication. Clinical and phenotype information was gathered out of clinical reports and medical history.

### Histology

Histological examinations of the kidney biopsies at the age of 28 and 32 years were performed with light microscopy of sections stained with Periodic acid-Schiff (PAS) reaction and hematoxylin and eosin staining, immunohistochemistry and electron microscopy. Immunohistochemistry of the latest biopsy was performed with antibodies specific for IgA, IgG, IgM, C1q, and C3c (all polyclonal rabbit antibodies, Dako; Dilution and Code No. IgA 1:200000, A0262, IgG 1:150000, A0423, IgM 1:100000, A0425, C1q 1:100000, A0136, C3c 1:100000, A0062) after digestion with PronaseE for 45 min. For detection Envision Kit (Dako) was applied and DAB was used as a chromogen. For electron microscopy fixed renal biopsies were dehydrated and embedded in Epon. Semithin and ultrathin sections were prepared and stained with methylene blue or uranyl acetate/lead citrate, respectively. Ultrathin sections were then analyzed with a Zeiss electron microscope LEO EM 910 or LEO EM 912 (Zeiss, Oberkochen, Germany) at various magnifications. Histological and electron microscopic images were only available from the latest kidney biopsy.

### Genetics

Blood samples were collected after written informed consent. Genomic DNA was extracted from peripheral blood of the index patient, her parents, and her children using the Gentra Puregene Blood Kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. In the index patient exon 1 to 51 of COL4A5 were examined followed by exon 1 to 52 of COL4A3 using direct DNA sequencing on both strands applying the dideoxy chain termination method on an ABI capillary sequencer 3730 (Applied Biosystems, Foster City, USA). Primers were designed by Primer3 program (http://frodo.wi.mit. edu/primer3/input.htm). For segregation analysis, subsequent targeted sequencing was performed in both parents and children of the index patient in exon 26 and 52 of the COL4A3 gene. DNA alignment and sequence variant analysis were carried out using the Sequence PilotCE software (JSI Medical Systems GmbH, Kippenheim, Germany) and compared to EMBL (European

**Abbreviations:** ACE, angiotensin-converting enzyme; AS, Alport syndrome; ESRD, end-stage renal disease; FSGS, focal segmental glomerulosclerosis; GBM, glomerular basement membrane; MMF, mycophenolate mofetil; NC1, C-terminal globular non-collagenous domain; SRNS, steroid-resistant nephrotic syndrome; TBMN, thin basement membrane nephropathy.

Molecular Biology Laboratory) and GenBank databases as well as our in-house database. All variants were validated in a second independent DNA sample. Scaled gene structure was created with the Gene Structure Display Server version 2.0 (13).

### RESULTS

### Clinical Phenotype

### Index Patient

A 34 year old Caucasian woman of German ancestry presented in our department (**Figure 1A**, II-2). At the age of 12 years, mild proteinuria and microscopic hematuria was detected. This finding was, however, not followed.

At the age of 21 years, the index patient had, for the first-time, increased proteinuria (>1 g per day) and mild hypertension. The histology of a kidney biopsy showed, besides minimal mesangial cell proliferation, uncharacteristic findings. Therefore, no specific therapy was initiated. Unfortunately, no detailed report of the first kidney biopsy was available.

In the presence of persistent proteinuria of >1 g/d and a decrease of creatinine clearance from 83 to 65 mL/min/1.73 m2 re-biopsy of the kidney was performed at the age of 28 years. The diagnosis at that time was chronic kidney disease due to FSGS with secondary hypertension and proteinuria. The clinical presentation was normal, besides a slightly increased weight (body mass index 24.7 kg/m<sup>2</sup> ) possibly due to the discrete lower leg edema. Especially, there was no report of hearing impairment. Further laboratory work-up is shown in **Table 1.** Ultrasound examination showed morphologically normal kidneys. The histology of the kidney biopsy revealed focal global glomerulosclerosis with mild chronic tubulo-interstitial damage. Electron microscopy showed one scarred glomerulus and one glomerulus with clumsy podocyte foot processes with effacement compatible with the diagnosis of primary FSGS. Due to a gross proteinuria and the progressive decline of kidney function an angiotensin-converting enzyme (ACE) inhibitor and immunosuppressive therapy with cyclosporine for 7 months and prednisolone were initiated.

Four years later, at the age of 32 years, the patient experienced nephrotic range proteinuria (4.5 g per day). In the beginning, the patient responded well to immunosuppressive treatment with prednisolone (starting dose 60 mg, tapered to 7.5 mg once daily) and mycophenolate mofetil (MMF) (250 mg twice daily) with a reduction of the proteinuria to 1.7 g per day. To further reduce the proteinuria, MMF was increased to 500 mg twice daily. Nonetheless, proteinuria increased again to 4.3 g/d. Therefore, MMF was discontinued and the patient was admitted to our hospital for a kidney biopsy. Clinical presentation at that time was age-appropriate, besides discrete lower leg edema. Abnormal laboratory values are shown in **Table 1**.

Light microscopy of the third kidney biopsy revealed renal parenchyma with 11 glomeruli of which four were obliterated (**Figure 2A**). In one obliterated glomerulus fibrous thickening of the Bowman's capsule was present, indicating fibrous synechia (**Figure 2C**). Crescents were not identified. The remaining glomeruli were unremarkable (**Figure 2D**). No segmental sclerosis, extra- or intracapillary proliferation was seen. Patchy tubular atrophy and interstitial fibrosis was present in ∼15% (**Figures 2E,F**) and was accompanied by a mild lymphoplasmacytic interstitial infiltrate. Additionally, multiple foam cell nests were present in the interstitium (**Figure 2B**). Arterial vessel walls showed mild thickening (**Figure 2G**) and moderate arteriolar hyalinosis (**Figure 2H**). In immunohistochemical stainings with antisera against IgA, IgG, IgM, C1q, and C3c only mild mesangial staining for IgM was seen (**Figure 3**). Furthermore, electron microscopy showed that the GBM (glomerular basement membrane) was moderately thinned with extensive foot process effacement and microvillous transformation of podocytes going in line with the former report of primary FSGS (**Figure 4**). After an in-patient stay, an increase of MMF to 750 mg/d was recommended. Hypertension and proteinuria were treated with an ACE inhibitor and candesartan.

Two years later, at the age of 34 years, the patient developed moderate inner ear hearing impairment. Additionally, the 6 year old daughter of the index patient was diagnosed with microscopic hematuria. This new clinical symptomatology combined with a conspicuous family history led to a re-examination of electron microscopy of the kidney biopsy performed at the age of 32 years. In further electron microscopic analyses, segments with thinning and thickening of the GBM (170–700 nm) were found. Moreover, changes in the texture of the GBM were seen in some segments with mild lamellation and basket-woven texture. Moderate effacement of the foot processes was present (**Figure 4**). The re-evaluation of the ultra-thin sections revealed the beforehand described podocyte abnormalities in addition to structural changes of the GBM. Therefore, the changes in GBM structure in the context of the clinical findings and family history were suspicious of hereditary kidney disease and prompted a molecular genetic examination of genes involved in the pathogenesis of AS.

### Relatives

The daughter of the index patient presented with microscopic hematuria at the age of 6 years as already mentioned above. So far, the son of the index patient does not show any clinical symptoms of a kidney disease at the age of 9 years. Neither the mother nor the father of the index patient developed (chronic) kidney disease until the age of 54 and 58 years, respectively.

### Genetic Analysis

Pathogenic variants in COL4A5 could not be identified. The sequencing of COL4A3 (NM\_000091.4) revealed two missense variants in a compound heterozygous state in the index patient: c.1892G>T, p.Gly631Val (paternally inherited) and c.4981C>T, p.Arg1661Cys (maternally inherited) (**Figure 1A**, I-1 and I-2). The variants are located in exon 26 and 52, respectively (**Figures 1B,D**). Each parent is a heterozygous carrier for one variant (**Figure 1B**). The amino acid at position 631 is highly conserved in evolution (**Figure 1C**). It is located in the triple helical collagenous domain of the type IV collagen alpha-3 chain (**Figure 1D**). The variant has not been described in the literature before. The second variant, p.Arg1661Cys, has been described previously and is mostly associated to early development of end-stage renal disease (ESRD) (8, 12, 14). The encoded amino

acid is located in the cross-linking C-terminal globular noncollagenous domain (NC1) (14) (**Figure 1D**). The daughter of the index patient carries the newly discovered variant p.Gly631Val (**Figure 1A**, III-2). This variant was also present in the father of the index patient (**Figure 1A**, I-1). The son of the index patient carries the variant p.Arg1661Cys (**Figure 1A**, III-1).

### DISCUSSION

We identified a novel variant in COL4A3 combined with a previously described variant in a compound heterozygous state. In the context of the progressive proteinuria and the young age of the index patient the initial diagnosis was erroneously considered to be hereditary FSGS as it was the most probable differential diagnosis. The present study highlights the importance of including AS in the differential diagnosis of FSGS, especially when disease onset is early in life (<25 years of age). In these cases, it would be helpful to start a molecular genetic analysis promptly (i.e., already if mild proteinuria is present).

Several reports have linked variants in COL4A3 and COL4A4 to patients with nephrotic-range proteinuria and the histological findings of FSGS (8, 15).

In these cases, FSGS most likely occurs secondary to GBM pathology and therefore represents an independent entity different from primary FSGS with podocytopathy. Histologic diagnosis of FSGS depends on the size of the kidney biopsy as FSGS due to its local nature is prone to sampling error.

Using molecular diagnostics, we identified a novel missense variant in COL4A3. The variant p.Gly631Val is assumed to be disease-causing as it segregates within the family and since glycine is important for the spatial organization of the collagen triple helix as it resides at every third position of the collagen type IV chain. Using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph/) and MutationTaster (http://www.mutationtaster.org) to predict possible functional effects, the variant was classified as probably damaging (PolyPhen-2) and disease-causing (MutationTaster). The variant was not found in our in-house database and in the genome Aggregation Database (gnomAD).

The daughter of the index patient, who is a heterozygous carrier of the variant, already demonstrated microscopic hematuria in early childhood (6 years of age). This can be a first sign of thin basement membrane nephropathy (TBMN). However, the father of the index patient, who also carried this variant, showed no signs of (chronic) kidney disease going in line with the wide range of clinical phenotypes reported in individuals with single heterozygous variants (8, 12, 16).

The second variant, p.Arg1661Cys, has been identified both in a heterozygous and compound-heterozygous state in patients with autosomal recessive AS mostly at an early age at diagnosis of 10–18 years (in one case 44 years of age). In one case ESRD was reached at 19 years of age (8, 12). Previous cases presented with nephrotic range proteinuria (2–5 g/d), thickened GBM and foot process effacement. Hearing loss was reported in one of six cases. No ocular disease was reported (8, 12). However, so far, the variant was only reported in heterozygous form to be associated with ESRD (8) (family DUK6585, individual 1). Elsewhere, the variant was reported in a compound-heterozygous state, however, age of renal failure was not indicated (12).

fibrous synechia (arrow; magnification x400). (D) One unremarkable glomerulus (magnification x400). (E,F) Interstitial fibrosis and tubular atrophy with mild lymphoplasmacytic interstitial infiltrate (arrows) (E; magnification x200; F; magnification x400). (G) Arterial vessel walls showed mild thickening (arrow) and moderate arteriolar hyalinosis (H). All images were taken from sections stained with Periodic acid-Schiff (PAS) reaction.

#### TABLE 1 | Abnormal blood and urine laboratory values.


Erythrocyturia 0–3 per visual field - 4–10

GFR, glomerular filtration rate; MDRD, Modification of Diet in Renal Disease.

This exemplifies the wide range of clinical manifestation of heterozygous variants reaching from familial benign hematuria to AS with development of ESRD. The healthy son and the mother of the index patient carry the same variant and did not develop (chronic) kidney disease so far.

Because both parents and children of the index patient carry a heterozygous variant they should be monitored closely by a (pediatric) nephrologist if hypertension, proteinuria, or renal impairment is present (17). In this case, an ACE inhibitor therapy should be evaluated. Otherwise, in the absence of these symptoms regular examination every 1–2 years should be performed by a primary care physician (17). In patients with TBMN and isolated microscopic hematuria chronic renal failure is rare (16). However, if TBMN is accompanied by proteinuria, it has been associated with an increased risk to late onset FSGS with nephrotic syndrome (18, 19). Furthermore, in clinically

FIGURE 4 | Electron microscopy of the kidney biopsy. (A) In the ultrastructural analysis performed at the age of 32, podocytes showed strong foot process effacement with microvillous transformation and thinned glomerular basement membrane (GBM) with mild lamellation (white arrow) compatible with the preexisting diagnosis of primary FSGS (magnification x10,000). In additional ultrastructural analyses performed at the age of 34, segments with thinning and thickening of the GBM (B; magnification x16,000) with disrupted basket-woven texture (C, asterisk; magnification x16,000) were found. en, endothelium; po, podocyte; fp, foot process; fp eff, podocyte food process effacement.

affected patients with heterozygous variants in COL4A3, COL4A4 an increased risk of progression to chronic renal failure was observed in up to 38% and a progression to ESRD was observed in up to 20% (15). Age of ESRD onset was significantly earlier in untreated patients with heterozygous variants in COL4A3, COL4A4, and COL4A5 compared to those treated with blockade of the renin aldosterone system (20). Patients that progress to chronic renal failure and ESRD could have variants in hitherto unknown modifier genes.

The rapid course of disease in one family (DUK6696) with compound heterozygous variants in COL4A3 (p.Glu131fs<sup>∗</sup> 151 and p.Gln936<sup>∗</sup> ) and nephrotic range proteinuria and hematuria with ESRD at 8 to 12 years of age described by Malone et al. is comparable to our family (8). However, the severity and the more rapid course to ESRD in the previous report could be explained by the presence of two loss of function variants whereas our index patient has compound heterozygous missense variants. This is in line with a genotype-phenotype correlation postulated for COL4A3 as nonsense or larger rearrangement variants lead to a shortening of the protein and are associated with a younger age at renal failure (<20 years) (15, 21). The unusual appearance of AS with nephrotic range proteinuria in our index patient and the cases described by Malone et al. could be additionally modified by variants in FSGS genes that alter the podocyte in collagen IV-related kidney disease and therefore, could explain the high clinical variability. However, Malone et al. was not able to identify variants in genes associated with FSGS in his cohort (8).

Our index patient developed hearing impairment at the age of 34 years. In general, hearing loss seems to occur more often in cases with variants in COL4A3 as compared to COL4A4 (8, 12), but is not mandatory. In the study of Malone et al., hearing impairment was only diagnosed in all 8 of 15 individuals with variants in COL4A3 after genetic testing had already been done (8). Accordingly, in our case otologic examination was not performed at earlier age probably due to the absence of (severe) symptoms.

Several points may have led to the misdiagnosis of hereditary FSGS in our index patient. First, the quality of the kidney biopsy was low. The histological definition of FSGS comprises a broad spectrum of different pathologies together. So far it is not possible to definitely differentiate between subgroups of FSGS based on the morphology. To improve diagnostic accuracy ultrastructural analysis including measurements of the GBM in ultra-thin slices could be helpful to identify GBM defects. In most cases genetic testing has no therapeutic consequence except for the omission of immunosuppressive therapy and therefore avoidance of adverse side effects. Next generation sequencing would allow a much faster and more accurate diagnosis in clinically and genetically heterogeneous cases. Second, nephrotic proteinuria is unusual for AS (10). Third, classical AS symptoms (e.g., hearing impairment) occurred late in the medical history. Fourth, variants in COL4A3 show a highly variable clinical phenotype. Fifth, as classically men are affected by X-linked inherited AS female patients may be overseen.

Genetic implications are different for patients with autosomal recessive AS and could highlight caution in case of a kidney transplantation. The mode of inheritance is different compared to X-linked AS. A heterozygous (autosomal dominant) affection with microscopic hematuria and TBMN warrants a restrained selection as kidney donor. This is especially important if blood pressure is high or proteinuria is present (17). Therefore, the newly discovered p.Gly631Val variant with the clinical presentation of microscopic hematuria in the daughter indicates caution for selection as kidney donor.

In patients with AS and TBMN ACE inhibitors are recommended by the expert guidelines for the treatment of hypertension and proteinuria especially in individuals with genetic variants (17). Angiotensin-receptor blockers and aldosterone inhibitors could show additional effects on the reduction of proteinuria (22). To date, new insights for the treatment of AS are expected from the EARLY PRO-TECT Alport trail that is evaluating the effect of renin-angiotensin blockade on proteinuria and renal failure progression (23).

Some limitations of the present study have to be stated. We performed only targeted sequencing of COL4A3 and COL4A5 as this study was already performed several years ago. Examination of disease modifiers for collagen IV that could explain the high variability of phenotypes described before were not performed (8). Today panel diagnostics, whole exome or whole genome sequencing would be done in such a case. Also, we did not examine other genes or modifiers related to FSGS, which should be included in further studies to improve the understanding of the high variability of the clinical phenotype. Due to the absence of cryopreserved tissue of the kidney biopsies, it was not possible to perform immunofluorescence analysis with antibodies specific for alpha-3,4,5 subunits of collagen IV complex in the GBM and therefore directly document collagen dysfunction. Unfortunately, electron microscopic images for a re-examination were only available from the last kidney biopsy.

### CONCLUSION

We were able to show that a novel variant combined with a previously described variant in COL4A3 in compound heterozygous state can lead to a phenotype that was erroneously associated with hereditary FSGS. Finally, our study exemplifies the importance of molecular examination in the diagnosis of the renal phenotype to improve diagnostic accuracy and avoid unnecessary inefficient treatment with immunosuppression.

### AUTHOR CONTRIBUTIONS

MB and JH, analyzed and interpreted the patient data regarding the genetic and clinical findings and wrote the manuscript. MB-H conducted the histological examination of the third kidney biopsy, compiled histological, and electron microscopy figures and contributed to the writing of the manuscript. H-GK, DW, and JH performed the molecular diagnostics. CK, LR, UH, and CS conducted in-patient treatment. RG, RS, KR, P-MH, CK, LR, UH, and CS contributed important intellectual content during manuscript drafting and revision. All authors accept accountability for the overall work by ensuring that questions pertaining to the accuracy or integrity of any portion of the work are appropriately investigated and resolved. Text revision was performed by all authors. All authors read and approved the final manuscript.

### ACKNOWLEDGEMENTS

We thank the index patient and her family for their participation in the study. This work was supported by the German Research Foundation (DFG) and the Technical University of Munich (TUM) in the framework of the Open Access Publishing Program.

### REFERENCES


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Braunisch, Büttner-Herold, Günthner, Satanovskij, Riedhammer, Herr, Klein, Wahl, Küchle, Renders, Heemann, Schmaderer and Hoefele. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Exploring the Clinical and Genetic Spectrum of Steroid Resistant Nephrotic Syndrome: The PodoNet Registry

Agnes Trautmann<sup>1</sup> \*, Beata S. Lipska-Zi ˛etkiewicz <sup>2</sup> and Franz Schaefer <sup>1</sup> on behalf of the PodoNet Consortium

<sup>1</sup> Division of Pediatric Nephrology, University Center for Pediatrics and Adolescent Medicine, Heidelberg, Germany, <sup>2</sup> Clinical Genetics Unit, Department of Biology and Medical Genetics, Medical University of Gdansk, Gda ´ nsk, Poland ´

Background: Steroid resistant nephrotic syndrome (SRNS) is a rare condition, accounting for 10–15% of all children with idiopathic nephrotic syndrome. SRNS can be caused by genetic abnormalities or immune system dysfunction. The prognosis of SRNS varies from permanent remission to progression to end-stage kidney disease, and post-transplant recurrence is common.

#### Edited by:

Max Christoph Liebau, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Köln, Germany

#### Reviewed by:

Andrew Mallett, Royal Brisbane and Women's Hospital, Australia Jan Halbritter, Leipzig University, Germany

> \*Correspondence: Agnes Trautmann agnes.trautmann@ med.uni-heidelberg.de

#### Specialty section:

This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics

Received: 07 April 2018 Accepted: 25 June 2018 Published: 17 July 2018

#### Citation:

Trautmann A, Lipska-Zi ˛etkiewicz BS and Schaefer F (2018) Exploring the Clinical and Genetic Spectrum of Steroid Resistant Nephrotic Syndrome: The PodoNet Registry. Front. Pediatr. 6:200. doi: 10.3389/fped.2018.00200 Objectives: The PodoNet registry project aims to explore the demographics and phenotypes of immune-mediated and genetic forms of childhood SRNS, to assess genotype-phenotype correlations, to evaluate clinical management and long-term outcomes, and to search for novel genetic entities and diagnostic and prognostic biomarkers in SRNS.

Methods: In 2009, an international registry for SRNS was established to collect retro- and prospective information on renal and extrarenal disease manifestations, histopathological and genetic findings and information on family history, pharmacotherapy responsiveness and long-term outcomes. To date, more than 2,000 patients have been enrolled at 72 pediatric nephrology centers, constituting the largest pediatric SRNS cohort assembled to date.

Results: In the course of the project, traditional Sanger sequencing was replaced by NGS-based gene panel screening covering over 30 podocyte-related genes complemented by whole exome sequencing. These approaches allowed to establish genetic diagnoses in 24% of the patients screened, widened the spectrum of genetic disease entities presenting with SRNS phenotype (COL4A3-5, CLCN5), and contributed to the discovery of new disease causing genes (MYOE1, PTPRO). Forty two percent of patients responded to intensified immunosuppression with complete or partial remission of proteinuria, whereas 58% turned out multi-drug resistant. Medication responsiveness was highly predictive of a favorable long-term outcome, whereas the diagnosis of genetic disease was associated with a high risk to develop end-stage renal disease during childhood. Genetic SRNS forms were generally resistant to immunosuppressive treatment, justifying to avoid such pharmacotherapies altogether

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once a genetic diagnosis is established. Even symptomatic anti-proteinuric treatment with RAS antagonists seems to be challenging and of limited efficacy in genetic forms of SRNS. The risk of post-transplant disease recurrence was around 30% in non-genetic SRNS whereas it is negligible in genetic cases.

Conclusion: In summary, the PodoNet Registry has collected detailed clinical and genetic information in a large SRNS cohort and continues to generate fundamental insights regarding demographic and etiological disease aspects, genotype-phenotype associations, the efficacy of therapeutic strategies, and long-term patient and renal outcomes including post-transplant disease recurrence.

Keywords: SRNS, nephrotic syndrome, NPHS2, WT1, steroid resistant nephrotic syndrome

### INTRODUCING THE PODONET REGISTRY STUDY FOR STEROID RESISTANT NEPHROTIC SYNDROME

While most children with idiopathic nephrotic syndrome respond to oral glucocorticoid therapy and have a favorable long-term prognosis, ∼10–15% of children do not respond and are classified as steroid resistant nephrotic syndrome (SRNS). SRNS is a rare (incidence 3–4 per million person-years) and challenging clinical condition with heterogeneous etiology and highly variable disease courses and outcomes.

Whereas, a relevant fraction of SRNS children respond to intensified immunosuppressive therapy with temporary or persistent proteinuria remission, others exhibit primary multidrug resistance. Traditionally, the diagnostic and prognostic assessment of SRNS has been based on histopathological categorization. In recent years however, the understanding of SRNS pathophysiology has transformed fundamentally with the discovery of a rapidly growing number of genetic disorders of the podocytes. These novel insights have created a need to redefine diagnostic assessment, prognostic classification and therapeutic approaches in childhood-onset SRNS. However, clinical research in SRNS is compromised by the low incidence and heterogeneity of the disorder. To overcome the limitations related to the rarity of the disorder, the PodoNet Consortium set up an international web-based registry for childhood SRNS and CNS (www.podonet.org).

The objectives of the PodoNet registry project are to explore the demographics and phenotypes of immune-mediated and genetic forms of childhood SRNS, to evaluate genotypephenotype correlations, to evaluate clinical management and long-term outcomes, and to search for genetic entities and novel diagnostic and prognostic biomarkers of SRNS. Data collection is focused on the characteristics at first disease manifestation, genetic and histopathological findings, the responsiveness to pharmacological therapies, long-term renal survival, and posttransplant disease recurrence.

This manuscript reviews the accomplishments of the international PodoNet project since 2009, focusing on both genetic and clinical aspects of SRNS. The international PodoNet registry study with retro- and prospective data of currently more than 2,000 patients provides extended knowledge about SRNS demographics, SRNS genotype-phenotype correlations, therapeutic and prognostic outcomes and has discovered new genetic disorders. These results contribute to an improved understanding of the different SRNS entities and to a reclassification of SRNS based on molecular etiology and responsiveness to second-line treatment.

### PODONET STUDY COHORT: METHODS AND DEFINITIONS

The PodoNet registry is a web-based clinical database (www. podonet.org) for primary steroid resistant (SRNS) and congenital nephrotic syndrome (CNS). The registry follows patients with childhood-onset (age < 20 years) primary SRNS, CNS or persistent subnephrotic proteinuria with likely genetic disease, but not patients with secondary SRNS. Since August 2009, investigators from 72 clinical units in 28 countries have enrolled 2041 patients (**Figure 1**). The patients derive mainly from European countries (∼85%) but also from the Middle East and Latin America (15%) (1). Consanguinity and familial disease occurrence were most common in the Middle Eastern countries (**Figure 2**). Details on the structure of the PodoNet study cohort along with the registry study protocol and description were recently published (1).

Genetic screening was initially performed using Sanger sequencing of individual genes following a diagnostic screening algorithm based on age at presentation and extrarenal symptoms. From 2013 onward, next generation panel sequencing (NGS) of more than 30 podocytopathy-associated genes was systematically applied. Details of the diagnostic genetic screening studies and their completeness are described below.

Intensified immunosuppressive (IIS) therapies applied after confirmation of steroid resistance (persistent nephrotic-range proteinuria after 4 weeks oral prednisone at 60 mg/m<sup>2</sup> per day), included intravenous steroid pulses, calcineurin inhibitors (CNI), mycophenolate-mofetil (MMF), the combination of CNI and MMF, oral and intravenous cyclophosphamide (CPH), and rituximab.

The response to IIS (complete, partial, no remission) was assessed using a standardized set of criteria using changes in proteinuria and serum albumin, as previously defined (1, 2). To categorize IIS responsiveness as a basis for predicting longterm outcomes, the evaluation period was restricted to the first year of IIS treatment and disease. Patients with CNS were excluded. If patients were treated with more than one intensified immunosuppressive treatment in the first year, the most efficacious treatment and the best antiproteinuric response was identified. Patients who were unresponsive to any IIS applied were classified as multi-drug resistant.

End-stage kidney disease (ESKD) was defined by attainment of CKD stage 5 and/or start of renal replacement therapy.

All statistical analyses of the reported individual studies of the PodoNet registry were performed using SAS <sup>R</sup> Version 9.4 (Cary, USA). Statistical details can be obtained from the original articles (1–5).

### CLINICAL AND GENETIC SPECTRUM OF THE PODONET COHORT STUDY—RESULTS AND DISCUSSION

### Clinical Aspects of SRNS

#### First Manifestation

Among all patients reported to the PodoNet Registry, 6% presented with congenital nephrotic syndrome, 7% manifested as early infantile nephrotic syndrome (onset age 3–11 months), 51% at 1–5 years, 23% at 6–11 years, and 13% at age 12 years and older (**Figure 3**).

Hypoalbuminemia was most pronounced in congenital nephrotic syndrome (mean serum albumin 17 g/L) and least marked in adolescent-onset disease (26 g/L). Likewise, hypertension at diagnosis was most prevalent in adolescents (28%) as compared to 14% in infants. A small fraction of patients (8.7%) were diagnosed with non-nephrotic proteinuria, but progressed to full nephrotic syndrome during follow-up (1).

63.5% of children had a normal renal function (CKD stage 1), 23.5% a mildly impaired renal function at first presentation. Chronic kidney disease (CKD stage 3 and 4) was present in 13% (1). 7.4% of patients progressed to ESKD in the first year, whereas the median time to ESKD was 2.8 years.

Extrarenal disease manifestations pointing to a syndromic disorder most commonly included neurological symptoms (5.3% including brain anomalies, microcephaly and/or mental retardation), short stature (5.1%) and facial dysmorphism (2.2%).

#### Histopathological Findings

The predominant histopathological diagnosis was FSGS in 56% of all PodoNet patients, followed by minimal-change nephropathy (MCN) with 20% and mesangioproliferative GN (MesPGN) with 11% (**Figure 4**). The predominance of FSGS was also found in other SRNS populations (6–9), albeit with some variability likely related to variations of ethnic composition, biopsy indication policies, and disease duration at time of biopsy.

Twelve percent of the PodoNet patients received a second renal biopsy during the course of the disease. Two thirds of the re-biopsied patients previously diagnosed with MCN or MesGN showed FSGS in the second biopsy, and 10% of those with FSGS progressed to global glomerulosclerosis (GGS).

The children in our cohort who initially presented with FSGS, MCN, or MesPGN displayed similar severity of hypoalbuminemia (25–27 g/dl) and comparable prevalence of hypertension (15–18%). Patients with FSGS were on average slightly older (6 years vs. 4 years for MCN/MesPGN) and presented with a slightly lower eGFR than patients with MCN/MesPGN.

The other histopathological entities found in children with SRNS - diffuse mesangial sclerosis (DMS, 3%), membranoproliferative glomerulopathy (MPGN, 2%), membranous nephropathy (MN, 2%)—were much less common. Typically, patients with these diagnoses manifested with milder initial hypoalbuminemia but slightly higher prevalence of hypertension (23–26%) than patients with FSGS, MCN and MesPGN. Patients with DMS usually presented at earlier age (median age of 1.3 years), similar to the observations of the SRNS Study Group where DMS was found in 27% of infants (10) with established renal failure and an identified genetic cause in almost two thirds of cases (1). In patients with FSGS, the detection rate of genetic disease was lower with 22% and lowest with 12% in children with MCN (1).

### Genetic Aspects of SRNS

#### Diagnostic Screening Studies

To date, a total of 1,554 individual PodoNet patients were genetically screened for hereditary podocytopathies. Initially, the most commonly screened genes were NPHS2 and WT1 performed by Sanger sequencing whereas other podocyte genes were screened more selectively guided by age, histopathology, and/or syndromic features. These included WT1 disease (sex reversal/urogenital abnormalities and malignancies), mitochondrial dysfunction (myopathy, cardiomyopathy, impaired hearing), Pierson syndrome (impaired vision), and Schimke syndrome (osteodysplasia) (1).

Since 2014, next generation sequencing (NGS) gene panels were used to systematically screen for all known podocytopathy genes as part of the EURenOmics project (www.eurenomics.org). Identified mutations in the panel sequencing were confirmed by Sanger sequencing. To date, 539 unrelated patients with documented multi-drug resistance or unknown responsiveness to intensified immunosuppressive therapy were screened by NGS panel including more than 30 podocyte specific genes. These included 214 novel consecutively enrolled cases and 315 previously tested negative by conventional screening of NPHS2 and exons 8–9 of WT1.

Overall, since the beginning of the PodoNet Registry genetic diagnoses were established in 373 SRNS patients (24% of those tested; 19% of the entire cohort). This mutation detection rate is slightly lower compared to that recently reported in a study of 1783 SRNS families (29.5%) (10).

In line with other SRNS studies (9, 10), mutations in NPHS2, WT1 and NPHS1 represented the most common genetic SRNS causes, accounting for 42, 16, and 13% of cases, respectively (**Table 1**). Using the NGS gene panel, mutations were identified in 27% of novel consecutive patients and in 17% of patients previously found negative for NPHS2 and exons 8–9 of WT1 mutations. Hence, almost half of all genetic SRNS patients carry a mutation in NPHS2 or the zinc finger domains of WT1, which could provide a rationale for a cost-effective two-step mutational screening algorithm.

The mutation detection rate was highest in children with CNS and significantly lower with increasing age at first manifestation during the first 6 years of life (**Figure 3**), in

permission (1)].

keeping with observations by the SRNS Study Group (10) and the German Pediatric Nephrology Society (9). While genetic screening strategies for diagnosing childhood-onset nephrotic syndrome were generally established when the PodoNet registry was founded, screening strategies for adolescent-onset disease were not well-established. Utilizing our PodoNet cohort, 297 patients with non-syndromic SRNS and disease onset in the TABLE 1 | Mutation screening results.


Genetic diagnoses in 373 of 1,554 genetically screened children with hereditary SRNS (24%).

second decade of life were screened for mutations in NPHS2, WT1, TRPC6, ACTN4, and INF2 (3). Seventy-nine percent of adolescents had a sporadic disease occurrence, 21% familial disease occurrence (17% autosomal-recessive, 4% autosomaldominant forms). The overall mutation rate in the adolescent age group in those five genes was 11%, separated into 30% in the autosomal-dominant cases, 13% in the autosomal-recessive and 10% of the sporadic cases. NPHS2 mutations accounted for 7% of all adolescent cases. Based on the common involvement of p.R229Q in late-onset SRNS, an approach of a two-step screening algorithm (limiting full sequencing of the NPHS2 gene to carriers of the p.R229Q polymorphism) was suggested in previous studies (11, 12). As only 56% of adolescents with NPHS2-associated disease were compound-heterozygous for a mutation combined with the p.R229Q polymorphism in the PodoNet cohort, we suggested to rather screen the entire coding sequence of NPHS2 in all sporadic and autosomal-recessive cases of juvenile SRNS. The considerations of selective and limited genetic screening in adolescents subsequently became largely obsolete with the advent of next generation sequencing.

#### Gene Exploration Studies

One of the aims of the PodoNet Network is to foster the identification of new SRNS causing genes. Registered PodoNet patients were evaluated regarding familial disease occurrence and negative screening in the known SRNS genes and biomaterial was collected. This allows to contribute and to perform wholegenome linkage analysis and high-throughput sequencing in affected families. PodoNet Consortium partners have identified two new SRNS causing genes participation of SRNS families of the PodoNet cohort in 2011: PTPRO (13) and MYO1E (14).

### **PTPRO-associated SRNS**

The role of the protein tyrosine phosphatase receptor type O (PTPRO) gene, also known as GLEPP1 (glomerular epithelial protein 1) gene, is the regulation of glomerular pressure and permselectivity. The protein PTPRO is a tyrosine phosphatase expressed at the apical membrane of the podocyte foot processes. Thyrosine phosphorylation of tight junction proteins plays a major role in controlling paracellular permeability, cell signaling and actin cytoskeleton remodeling.

PTPRO mutations were identified in 5 of 29 affected children from two of 17 tested families with so far unknown genetic disease. The disease onset varied between 5 and 14 years, the initial serum albumin varied between 19 and 40 g/L at first manifestation. Apart from one affected child, none of the children had progressed to ESKD 2–5 years after first manifestation. Renal biopsy, performed in 2 children, revealed MCN as well as FSGS (13) and electron microscopy showed diffuse podocyte foot process fusion and extensive microvillus transformation of podocytes. The subsequent testing of almost 2000 SRNS cases of all age groups performed within the frames of the EuRenOmics project did not identify any further individuals with pathogenic biallelic PTPRO mutations, demonstrating its extremely rare character.

### **MYO1E-associated SRNS**

MYO1E encodes a non-muscle class I myosin (Myo1E) which is expressed mainly at the podocyte plasma membrane in the glomerulus. The role of Myo1E is maintaining the function of the glomerular filtration barrier and promoting the podocyte motility. Two mutations in MYO1E were identified in two families (3 siblings, 1 other child), which are closely associated with autosomal recessive SRNS/FSGS (14).

The age at disease onset of the index patient was 9 years; the siblings and the fourth child were diagnosed earlier (between age of 1–4 years). The histopathological diagnosis was FSGS in all children, electron microscopy showed thickening and disorganization of the glomerular basement membrane.

All four patients showed generally non-responsiveness to intensified immunosuppression. Cyclosporine A therapy was associated with a transient proteinuria reduction in 2 of 4 children, possibly related to a direct podocyte cytoskeleton stabilizing effect of calcineurin inhibition. One affected member progressed to ESKD at age of 13 years; the other affected siblings had milder disease courses, possibly related to earlier diagnosis and/or to effective antiproteinuric treatment. Three further unrelated individuals with MYO1E biallelic mutations were subsequently identified through NGS gene panel screening, all of them presented in early infancy.

### Genotype-Phenotype Association Studies **WT1-associated SRNS**

WT1 mutations are associated with a wide range of clinical phenotypes. The PodoNet consortium explored the phenotypic spectrum and analyzed potential genotype-phenotype correlations in 61 patients with WT1-associated SRNS, the largest cohort studied to date (4). Both renal and extrarenal phenotypes were found to be clearly associated with the type and location of the causative WT1 mutation.

55 of 61 patients (90%) carried mutations in the hot spot region (exons 8 and 9 and their intronic junctions). The mutations were categorized as exonic mutations (40/61 patients) including truncating mutations, DNA-binding-site mutations and other missense mutation and as intronic mutations (21/61 patients, KTS intron 9 and other intronic mutations). The two largest subgroups (70%) are exonic mutations affecting the nucleotides coding for DNA-binding residues and intronic (9) KTS mutations, the latter are classically associated with Denys-Drash- and Frasier-syndrome.

Patients with exonic mutations were significantly younger at diagnosis (1.1 vs. 4.5 years), presented with more severe proteinuria, edema, and hypertension and progressed more rapidly to ESKD (5-year renal survival from diagnosis 36 vs. 85%) and had a higher risk for nephroblastoma (73 vs. 19%) than patients with intronic mutations. Gonadoblastoma occurs less frequently than Wilms tumors and are more likely to develop in patients with sex reversal commonly associated with intronic mutation.

Missense mutations affecting the DNA-binding site were associated with diffuse mesangial sclerosis (74%), early steroidresistant nephrotic syndrome onset [0.9 (0.2–1.6) years] and rapid progression to ESKD. Truncating mutations implicated the highest Wilms tumor risk (78%) but had typically late-onset SRNS [12.3 (0.6–15.3) years]. Intronic (KTS) mutations were most likely to present as isolated SRNS (37%) with a median onset at the age of 4.5 (3.1–8.1) years, FSGS (67%) and slow progression (median ESKD age 13.6 years). All patients with isolated SRNS were genotypic and phenotypic females.

In contrast to the consistent associations of mutation type and clinical disease manifestation, histopathology showed high diagnostic and prognostic variability. Furthermore, genital and urinary tract abnormalities varied widely, from hypospadias and unilateral cryptorchidism to global penoscrotal hypoplasia. Genital malformations were detected in 50% of all patients. Male-to female sex reversal occurred exclusively in patients with intronic KTS mutation and exonic DNA-binding site mutation.

We concluded that establishing the genetic diagnosis and identifying the mutation type and localization is important to assess the associated features and complications. The important impact for the clinical decision is the consideration when is the appropriate timing to perform bilateral nephrectomy or gonadectomy to prevent the development of malignancies.

#### **SMARCAL1-associated SRNS**

Schimke immuno-osseous dysplasia (SIOD), caused by mutations in SMARCAL1, is a rare multisystem disorder characterized by the combination of progressive proteinuric glomerulopathy with spondyloepiphyseal dysplasia, growth retardation, dysmorphic features, episodic neutro-/lymphopenia and thrombocytopenia, defective T-cellular immunity, autoimmune disorders, abnormal skin pigmentation, and cerebral infarcts. Early mortality due to severe opportunistic infections or cerebral events is common.

The PodoNet consortium analyzed the renal and extrarenal phenotypic spectrum and genotype-phenotype associations in 34 PodoNet patients from 28 families—the largest SMARCAL1 associated nephropathy cohort to date (5). The diagnosis of SMARCAL1-associated disease was made either by the presence of typical syndromic feature or unexpectedly by NGS panel screening (11/34 children). All patients diagnosed incidentally through NGS screening were short in stature (height SDS −3.2 ± 1.5) but these patients developed a milder if any extrarenal phenotype during follow-up, with 88% 10-year patient survival as compared to 40% in the patients diagnosed by typical SIOD features.

The timing of proteinuria onset (median age 4.5 (IQR 3.2– 7.2) years, nephrotic-range in 69% of children) and the rate of progression to ESKD were similar in all patients. Median age at ESKD was 8.7 (IQR 5.6–10) years. Renal biopsy showed FSGS in 81.5% and MCN in the remaining patients. The extrarenal symptoms, but not the renal phenotype, correlated with the type of SMARCAL1 genetic mutation.

Since the study demonstrated that in a substantial number of patients SIOD initially presents as isolated SRNS with short stature, it was concluded that SMARCAL1 screening should be performed routinely also in non-syndromic SRNS and should be included into the SRNS gene panels.

#### **ADCK4-associated SRNS**

Mutations in ADCK4 (also known as COQ8) were recently identified as a novel autosomal-recessive cause of adolescentonset SRNS caused by defective Coenzyme Q<sup>10</sup> biosynthesis in podocytes. Hereditary defects of CoQ<sup>10</sup> biosynthesis can cause SRNS as part of multiorgan involvement or as isolated SRNS.

Systematic ADCK4 screening was performed in large cohort of 534 SRNS patients, including 202 patients of the PodoNet cohort (15). ADCK4 mutations were identified in 26 patients from 12 families with a mutation detection rate of 1.9%. The disease almost exclusively manifested during adolescence, typically with subnephrotic to nephrotic-range proteinuria with no or mild edema but with advanced CKD in 46% of patients progressing to ESKD within a median of 9 months after diagnosis. Renal biopsies revealed FSGS in all biopsied patients. ADCK4 mutations showed a mainly renal-limited phenotype, with mild neurological features in 5 of 26 patients (occasional seizures, mild mental retardation, retinitis pigmentosa).

ADCK4-associated glomerulopathy is the first hereditary form of SRNS with a potentially causative molecular therapy. Oral supplementation of CoQ<sup>10</sup> reduces proteinuria and stabilizes renal function when applied early in the course of disease (16). Therefore, ADCK4 mutation screening should be included into NGS screening of all patients with adolescent-onset proteinuric kidney disease.

#### **COL4A3-5-associated SRNS**

There is increasing evidence for overlapping phenotypes caused by abnormalities in the COL4 genes and those primarily associated with SRNS/FSGS (17–20).

Among 481 patients of the PodoNet Cohort screened in COL4A3-5, 11 carried pathogenic mutations in COL4A5, and one was homozygous for a mutation in COL4A3.

The detection rate of 2.5% is identical to a recent pediatric study from the UK, where disease-causing mutations in COL4A3- 5 genes were identified in six out of 255 individuals with an SRNS phenotype (19). Other studies found even higher rates of COL4 mutations particularly in adult SRNS cohorts, but these included heterozygous variants in COL4A3/4 which are unlikely to cause severe and progressive proteinuric kidney disease (17, 18). In line with other recent studies, all patients with COL4 gene mutations in our cohort showed FSGS on biopsy and no extrarenal disease manifestations were reported (17–19). These findings support the notion that genetic abnormalities collagen type IV formation may result in a phenotype of isolated nephrotic range proteinuria.

### **CLCN5-associated nephropathy**

A novel mutation in the CLCN5 gene (c.2000delC) was detected by exome sequencing in a family from the PodoNet cohort with apparently isolated nephrotic range proteinuria that had been classified as familial SRNS on clinical grounds. Both affected children were screened negative in 31 glomerulopathy-related genes. Mutations in CLCN5, which encodes a voltage-gated chloride ion channel expressed selectively in the renal tubule, are the major cause of Dent disease. Dent disease is an X-linked proximal renal tubular disorder; the key features include lowmolecular weight proteinuria, hypercalciuria, nephrocalcinosis, and/or nephrolithiasis.

The genetic finding triggered clinical investigation for a putative tubular defect, which revealed asymptomatic hypercalciuria in only one of two children studied. In the absence of nephrocalcinosis and nephrolithiasis, Dent disease was not considered as a potential differential diagnosis of nephrotic-range proteinuria before. The genetic classification of the disease has important therapeutic and prognostic implications for the affected children. The children had been (mis)-classified as familial SRNS based on the observed nephrotic range proteinuria (>1 g/m<sup>2</sup> per day). Our case illustrates the genetic and phenotypic heterogeneity of proteinuric kidney diseases. The remarkable phenotypic variability of mutations in CLCN5 suggests that this gene should be included in NGS panel screening for hereditary proteinuric disorders.

### TREATMENT AND LONG-TERM OUTCOME OF SRNS

### Responsiveness of SRNS Patients to Intensified Immunosuppression

Whereas, the initial treatment of idiopathic nephrotic syndrome with oral glucocorticoids is well-established, we observed a large variety of the intensified immunosuppressive protocols and algorithms applied following the diagnosis of primary steroid resistance (**Figure 5**). In an analysis of immunosuppressive therapies applied in 612 SRNS patients in the first year after diagnosis of steroid resistance (2), 62% of children were treated with a single treatment protocol, 28% with two, and 10% with

three or more different immunosuppressive drug combinations as a polypragmatic therapeutic approach. While approximately three quarters of patients received Calcineurin inhibitors as first line therapy, others were administered intravenous steroid pulses, mycophenolate mofetil, oral or intravenous cyclophoshamide, or Rituximab (**Figure 5**). More than 80% of patients were cotreated with oral steroids initially, and more than 70% with renin angiotensin aldosterone system (RAS) antagonists.

Applying a standardized set of criteria to define treatment response and remission, we observed only 41% of included 612 SRNS patients to respond to any intensified immunosuppression with a relevant proteinuria reduction to IIS (24% with complete, 17% with partial remission), whereas 59% of patients remained unresponsive—often to several therapeutic interventions (**Figure 6**). The highest rates of complete (30%) or partial (19%) remission were achieved with CNI based protocols. These response rates are in the lower range of previously reported treatment experience, where complete remission was observed in 31% to 89% and partial remission in 19 to 38% of patients (9, 21– 25). The variation of the response rates may be related to the selection and composition of the individual study cohorts and the chosen set of response criteria.

The efficacy of CNI-based therapies in the PodoNet cohort was by far superior to steroid pulses, cyclophosphamide, and MMF monotherapy, all of which did not show any therapeutic effect in around 85% of patients as first line therapy (**Figure 6**) and were completely non-efficacious as second- or third-line therapies in CNI resistant patients. These findings confirm previous studies (26–31) and provide strong evidence against the use of these therapeutics in SRNS. Notably, B-cell depleting therapy with Rituximab induced complete remission in 44%, and partial remission in 15% of patients (1).

RAS inhibition has been demonstrated to lower proteinuria by 40–50% in patients with SRNS (32, 33). In the PodoNet cohort, RAS inhibition alone was associated with partial proteinuria remission in 21% and even maintained complete remission in 27% of patients (1). Hence, the frequent co-administration of RAS antagonists with immunosuppressive agents is a major confounder to the efficacy analysis of the latter.

Among the 602 cohort patients available for pharmacotherapy analysis 82% had sporadic disease, 74 (12%) confirmed genetic disease and 36 (6%) familial disease with negative genetic screening in all known podocytopathy genes.

### Do Patients With Genetic SRNS Respond to Immunosuppression?

Anecdotal clinical observations in previous case series and small retrospective studies suggested that individual patients with pathogenic mutations in podocytopathy genes might show some responsiveness to intensified immunosuppression. In total, 11 hereditary SRNS patients reported in literature developed complete remission and 17 partial remission of proteinuria while on calcineurin inhibitor and usually concomitant RAS antagonist therapy (4, 34–38). A non-immunological antiproteinuric action

(green), others as multi-drug resistant (red).

(2)].

of Ciclosporin A (CsA) mediated by stabilization of the actin cytoskeleton has been suggested based on experimental findings (39).

The PodoNet registry allowed to evaluate the largest cohort of children with hereditary podocytopathies to date exposed to calcineurin inhibitor and other immunosuppressive therapy. Complete or partial proteinuria responsiveness to pharmacotherapy was observed in 45% of children with sporadic disease and in 47% of those with familial, gene screening negative disease, whereas only 13% of patients with hereditary podocytopathies displayed any, mostly transient responsiveness to pharmacotherapy (2).

Transient complete remission was documented in only two out of 74 patients (2.7%) diagnosed with genetic disease in whom extended treatment and response data were available: one child with a WT1 mutation reportedly achieved complete remission for 2 weeks after start of CsA, followed by subnephrotic range proteinuria for > 11 years. Another child with NPHS2 associated disease achieved complete remission for 4–6 weeks, followed by a relapse and subsequently persistent proteinuria with progression to ESKD within 4 years. Transient partial remission with reduction of proteinuria to the non-nephrotic range was observed in response to CsA in another 8 patients (10.8%) with genetic disease (4 NPHS2, 3 WT1, 1 COQ6). Five of these returned to nephrotic-range proteinuria within <2.5 years, and four progressed to CKD stage 3–5 within <5 years. Importantly, 4 of 8 patients were co-treated with RAS antagonists. It is impossible to differentiate whether the transient responsiveness was related to calcineurin inhibition, supportive antiproteinuric RAS antagonist therapy or the natural course of disease with diminishing proteinuria due to worsening renal function. Notably, almost all patients with genetic disease and apparent CsA responsiveness progressed to ESKD soon despite ongoing therapy.

Hence, the findings in the PodoNet cohort argue against a relevant nephroprotective effect of calcineurin inhibition—or other immunosuppressive therapies—in children with genetic forms of SRNS and support the notion that such patients should be spared immunosuppressant side effects. On the other hand, the knowledge about the heterogeneity of underlying genetic disease mechanisms might help to develop specific podocyteprotecting treatment strategies in order to minimize proteinuria. A promising example of an innovative gene specific treatment

option is successful use of CoQ10 in children with SRNS due to genetic defects leading to CoQ10 deficiency (15, 16, 40). In the light of these findings and given the progress in the speed, comprehensiveness and cost efficacy brought about by nextgeneration sequencing, genetic screening should be considered in all SRNS patients at the time of diagnosis of steroid resistance prior to starting intensified immunosuppressive treatment.

Finally, the efficacy of other supportive and/or preventive treatment strategies including antiproteinuric therapies and treatment of hyperlipidemia have not yet been studied in detail in the PodoNet or other cohorts and await exploration in future studies.

### Long-Term Outcome and Risk Factors for ESKD

Historically, the prognosis of SRNS was mainly staged according to histopathologic findings, with limited predictability of medium- and long-term disease outcomes (41–46). Providing comprehensive clinical and biochemical information with up to 15 years of follow-up, the PodoNet database allows to assess the prognostic effect of early treatment responsiveness in the context of genetic and histopathologic findings, which provide a rationale for a evidence-based reclassification of SRNS into sporadic immunosuppression-responsive (32%), sporadic multidrug-resistant (24%), genetic, and familial SRNS (30%)(**Figure 7**).

The average overall ESKD-free survival of patients with primary SRNS in the PodoNet cohort was 74% at 5 years, 58% at 10 years and 48% at 15 years, in keeping with previous cohort studies in which 5-year renal survival ranged from 65 to 92% and 15-year survival from 34 to 72% (43, 45, 47–49).

Responsiveness to intensified immunosuppression was highly predictive of long-term outcomes: Whereas complete remission during the first treatment year was associated with 94% 15 year renal survival, only 37% of the multidrug resistant patients were not in end-stage disease 15 years after disease onset (2). A fraction of patients achieved partial remission; it is controversial whether such intermediate response patterns reflect pharmacological effects on the immune system or non-specific proteinuria-lowering effects of RAS antagonists and possibly calcineurin inhibitors. Notably, partial proteinuria reduction was associated with superior long-term renal survival compared to patients with multidrug resistant proteinuria. The other key determinant of long-term renal outcome was the identification a genetic disease cause (2): Three quarters of patients diagnosed with a hereditary podocytopathy progressed to end-stage kidney disease within 15 years, as compared to only 4% of SRNS patients with sporadic disease and responsiveness to immunosuppressive

analysis [with copyright permission (2)].

therapy (**Figure 8**). Long-term renal outcomes were remarkably similar in children with different genetic disease entities. The 10-year renal survival rate was 72% for NPHS2-associated nephropathy, 77% for WT1-associated disease and 71% for the less common podocytopathies (2).

Remarkably, the outcome of children with proven genetic disease was also inferior compared to patients with multidrugresistant disease in whom no genetic diagnosis could be established. The prognostic values of genetic diagnosis and immunosuppression responsiveness were found to be independent by multivariate analysis: ESKD risk was increased by 150% in patients in whom a genetic diagnosis was established whereas complete remission reduced renal risk by 87% in patients who achieved complete remission and by 50% in those who responded partially to immunosuppressive therapy (2).

Traditionally, the diagnostic categorization and prognostic evaluation in SRNS was based on histopathologic diagnosis. As expected, we found strong associations between histopathologic findings at time of diagnosis and long-term renal survival: ESKDfree survival was significantly higher for MCN than FSGS (79% v. 37% at 15 years) (**Figure 9**), and the overall ESKD risk of children diagnosed with FSGS was 4-fold higher compared to MCN (2). Children diagnosed with DMS showed the poorest outcomes, with a 20-fold increase of ESKD risk relative to MCN with progression to ESKD in 80% within 5 years after initial SRNS manifestation. Importantly, the prognostic value of FSGS and DMS prevailed even when adjusting for CKD stage at diagnosis, responsiveness to intensified immunosuppression, and the presence or absence of a genetic diagnosis (2). For instance, a patient with a genetic podocyte disorder, multidrug resistance, and a given eGFR will still have a nearly threefold higher ESKD risk with the diagnosis of FSGS compared with MCN. Hence, the independence of genetic and histopathological findings in the multivariate risk analysis suggests that histopathological assessment is still relevant in the genetic era.

Additional independent risk factors of progression to ESKD were identified as age <1 year and older than 5 years at disease onset and advanced CKD at initial presentation (2).

### Which Lessons Have We Learned From the Podonet Registry So Far?

The understanding of the underlying molecular, genetic and pathophysiological mechanisms of SRNS has profoundly advanced during the past decade. Established in 2009, the PodoNet registry has grown to one of the largest pediatric SRNS cohorts with more than 2000 children feeding clinical and genetic research. While the large size of the cohort is a major asset, the incompleteness of data reporting and biosample availability has been a limitation to data analysis and interpretation. Also, in the early years of the Network technological limitations

and restricted resources hampered genetic screening. These limitations were partially overcome in later years thanks to improved funding and the central implementation of podocyte gene panel sequencing.

The discovery of numerous novel genetic podocytopathies and the progress in diagnostic screening technology have already changed clinical practice during the time span of the PodoNet project. Initially, children underwent a diagnostic algorithm and were selectively screened for individual SRNS genes by means of Sanger sequencing based on clinical criteria and depending on age at disease onset. With the advent of NGS technologies genetic screening has become much more rapid, comprehensive and cost-effective and consequently rapidly transgressed from an optional adjunct to a key component of the diagnostic workup in SRNS. This transformation is well-reflected in the output of PodoNet as well as other study consortia (10).

The PodoNet network has been an active player in this field by contributing to gene discoveries, developing and systematically applying podocytopathy gene panels, and analyzing phenotypes and outcomes by comprehensive assessment of clinical, genetic, and histopathological findings as well as responsiveness to pharmacotherapies.

Our studies allow concluding that genetic screening should be initiated as soon as the diagnosis of steroid resistance has been made, in parallel to renal biopsy (**Figure 10**).

We obtained evidence that genetic forms of SRNS are largely non-responsive to intensified immunosuppressive therapy, which, in consequence, should be avoided in patients with genetic disease. Future research will address in detail the short- and longterm usefulness of RAS inhibition and other supportive treatment strategies in genetic podocytopathies.

Our studies also have highlighted the lacking specificity of the most common histopathological diagnoses with respect to identifying underlying etiologies. On the other hand, the histopathological diagnosis was found to retain some prognostic value even when the genetic status of a patient is known.

In immune-mediated forms of SRNS, calcineurin inhibition was demonstrated to be the most efficacious second-line therapy following the diagnosis of steroid resistance. Children resistant to calcineurin inhibitors are usually also resistant to other immunosuppressive agents. Moreover, initial responsiveness to calcineurin inhibitors is uniquely predictive of long-term preservation of kidney function.

The insights obtained from the PodoNet cohort will facilitate the development of rational evidence based clinical practice recommendations for SRNS. In view of the major prognostic differences associated with genetic findings and pharmacotherapeutic responsiveness, we propose to sub-classify SRNS patients as sporadic immunosuppression-responsive, sporadic multidrug-resistant, genetic, and familial SRNS.

### AUTHOR CONTRIBUTIONS

AT, BL-Z, FS contributed substantially to the acquisition, analysis, and/or interpretation of the data, participated actively in preparing the manuscript and approved the submitted final version.

### PodoNet Collaborators

**Austria:** Dagmar Csaicsich; Chile: Marta Azocar, Santiago; Lily Quiroz, Santiago; **Colombia**: Lina Maria Serna Higuita, Medellín; **Czech Republic**: Jirí Dušek, Prague; **France**: Bruno Ranchin, Lyon; Michel Fischbach, Strasbourg; **Georgia**: Tinatin Davitaia, Tbilisi; **Germany**: Jutta Gellermann, Berlin; Sandra Habbig, Cologne; Jun Oh, Markus J. Kemper, Hamburg; Anette Melk, Hannover; Agnes Trautmann, Franz Schaefer, Heidelberg; Hagen Staude, Rostock; **Greece:** Nikoleta Printza, Thessaloniki;

### REFERENCES


**Hungary**: Peter Sallay, Budapest; **Iran:** Alaleh Gheissari, Isfahan; **Italy**: Marina Noris, Bergamo; Andrea Pasini, Bologna; Gian Marco Ghiggeri, Monica Bodria, Genova; Gianluigi Ardissino, Milano; Elisa Benetti, Padova; Francesco Emma, Rome; **Lebanon**: Bilal Aoun, Beirut; Pauline Abou-Jaoudé, Byblos; **Lithuania**: Augustina Jankauskiene, Vilnius; **Poland**: Anna Wasilewska, Bialystok; Ewa Gacka, Chorzow; Beata S. Lipska-Zi˛etkiewicz, Aleksandra Zurowska, Gdansk; Dorota Drozdz, Krakow; Marcin Tkaczyk, Małgorzata Stanczyk, Lodz; Halina Borzecka, Lublin; Magdalena Silska, Poznan; Tomasz Jarmolinski, Szczecin; Agnieszka Firszt-Adamczyk, Torun; Mieczyslaw Litwin, Elzbieta Kuzma-Mroczkowska, Hanna Szymanik-Grzelak, Warsaw; Anna Medynska, Wroclaw; Maria Szczepanska, Zabrze; **Portugal**: Alberto Caldas Afonso, Porto; Helena Jardim, Porto; **Romania**: Adrian Lungu, Bucharest; **Serbia:** Amira Peco-Antic Belgrade; Radovan Bogdanovic, Belgrade; **Sweden:** Rafael T. Krmar, Stockholm; **Switzerland**: Sybille Tschumi, Bern; **Syria:** Bassam Saeed, Damascus; **Turkey:** Ali Anarat, Adana; Ayse Balat, Gaziantep; Z. Esra Baskin, Ankara; Nilgun Cakar, Ankara; Ozlem Erdogan, Ankara; Birsin Özcakar, Ankara; Fatih Ozaltin, Ankara; Onur Sakallioglu, Ankara; Oguz Soylemezoglu, Ankara; Sema Akman, Antalya; Faysal Gok, Gulhane; Salim Caliskan, Istanbul; Cengiz Candan, Istanbul; Alev Yilmaz, Istanbul; Sevgi Mir, Izmir; Ipek Akil, Pelin Ertan, Manisa; Ozan Özkaya, Samsun; Mukaddes Kalyoncu, Trabzon; **United Arab Emirates:** Eva Simkova, Loai Akram Eid, Dubai; **Ukraine:** Svetlana Fomina, Kiev; Roman Sobko, Lviv.

### ACKNOWLEDGMENTS

The PodoNet project has been made possible by support received from E-Rare (German Ministry of Education and Research), the EU 7th Framework Programme (EURenOmics, grant 2012-305608), the Polish Ministry of Science and Education (grant N402631840), the German Research Foundation (Scha 477/11-1), and the Scientific and Technological Research Council of Turkey (TUBITAK) (grant 108S417).


multicentre trial by the Arbeitsgemeinschaft fur Padiatrische Nephrologie. Pediatr Nephrol. (2008) 23:1483–93. doi: 10.1007/s00467-008- 0794-1


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Trautmann, Lipska-Zi˛etkiewicz and Schaefer. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Treatment of Genetic Forms of Nephrotic Syndrome

#### *Markus J. Kemper1 and Anja Lemke2 \**

*1AK Nord Heidberg, Asklepios Medical School GmbH, Hamburg, Germany, 2Universitätsklinikum Hamburg-Eppendorf, Hamburg, Germany*

Idiopathic steroid-resistant nephrotic syndrome (SRNS) is most frequently characterized by focal segmental glomerulosclerosis (FSGS) but also other histological lesions, such as diffuse mesangial sclerosis. In the past two decades, a multitude of genetic causes of SRNS have been discovered raising the question of effective treatment in this cohort. Although no controlled studies are available, this review will discuss treatment options including pharmacologic interventions aiming at the attenuation of proteinuria in genetic causes of SRNS, such as inhibitors of the renin–angiotensin–aldosterone system and indomethacin. Also, the potential impact of other interventions to improve podocyte stability will be addressed. In this respect, the treatment with cyclosporine A (CsA) is of interest, since a podocyte stabilizing effect has been demonstrated in various experimental models. Although clinical response to CsA in children with genetic forms of SRNS is inferior to sporadic SRNS, some recent studies show that partial and even complete response can be achieved even in individual patients inherited forms of nephrotic syndrome. Ideally, improved pharmacologic and molecular approaches to induce partial or even complete remission will be available in the future, thus slowing or even preventing the progression toward end-stage renal disease.

Keywords: steroid-resistiant nephrotic syndrome, mutations, cyclosporine, treatment, congenital nephrotic

## INTRODUCTION

syndrome, Wilms tumor suppressor Gene 1, NPHS1, podocytes

For many years, steroid-resistant nephrotic syndrome (SRNS), especially focal segmental glomerulosclerosis (FSGS) was thought to be an immunological disorder. This concept was supported by the response to immunosuppression in many patients and by the fact that recurrence after renal transplantation occurred, possibly due to the presence of a humoral factor, e.g., produced by the immune system (1) However, it is now known that a significant proportion of patients with SRNS [FSGS, but also diffuse mesangial sclerosis (DMS)] have an inherited cause of the nephrotic syndrome (NS), e.g., affecting structural proteins such as nephrin or podocin (2). At first sight, immunosuppression in such patients with inherited structural defects of the podocytes makes no sense. Yet, clinical observations, typically made in patients who were treated with immunosuppression as the genetic result was not yet available, show that a subset of patients does achieve partial or complete remission associated with such treatment. This raises the issue of optimal treatment in this cohort. Should all patients receive immunosuppressive treatment and if so, for how long and in what form? Are their clinical characteristics that can guide treatment? In general, prospective studies addressing this issue have never been performed and evidence pro -and contra- have been generated by retrospective series. Also, treatment options beyond immunosuppression (supportive and pharmacologic) have never been addressed prospectively, which is not surprising due to the rarity, severity, and heterogeneity of the diseases involved.

*Rachel Lennon, University of Manchester, United Kingdom Frederick Jeffrey Kaskel, Children's Hospital at Montefiore, United States*

*Universitätsklinikum Köln, Germany*

#### *\*Correspondence:*

*Edited by: Max Christoph Liebau,* 

*Reviewed by:* 

*Anja Lemke lemke\_anja@gmx.de*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 01 November 2017 Accepted: 12 March 2018 Published: 26 March 2018*

#### *Citation:*

*Kemper MJ and Lemke A (2018) Treatment of Genetic Forms of Nephrotic Syndrome. Front. Pediatr. 6:72. doi: 10.3389/fped.2018.00072*

### General Considerations

Several factors have to be considered before choosing treatment for genetic causes of SRNS. First, the age of presentation and severity of initial symptoms is of utmost importance. In this respect, patients with congenital and infantile NS are probably the most problematic group, because children often present with severe symptoms, sometimes antenatally (3). Historically, the so called "Finnish" type of NS is a good example and aggressive treatment led to a dramatic improvement of survival and longterm-outcome (4). Although patients presenting with infantile NS (onset in the first year of life) have a high risk of carrying a monogenic mutation (5), it should be noted that some patients can have a good prognosis reaching remission with supportive treatment alone (6); individual patients may in fact have minimal change disease responding to steroids. Thus, not only genetic testing but also renal biopsy should be considered in this group.

Second, genetic testing results need to be considered for treatment. Although only mutations in few genes are frequent, there are now panels available testing for 30 or more genes and it can be assumed that there will be new genetic causes in the future. This implies a clinical heterogeneity, not only among identical but also between different genotypes. In this respect, it needs to be at least mentioned that there is often a delay in getting results of genetic testing, which often takes weeks, sometimes months (and sometimes years because new variants are to be detected). Some patients with a negative initial test result may have a yet undiscovered monogenetic cause, and this has implications in choosing a therapeutic approach.

The last problem arises from the fact that there is no universal consensus regarding the definition of treatment response. A (rapid) complete remission of proteinuria is the ideal situation and will generally be accepted without any discussion. However, the definition of partial remission is much more problematic. Fluctuations of proteinuria are difficult to evaluate as they can occur with and without treatment. Most authors would agree that a reduction of proteinuria, for instance, by 50% can be regarded as partial remission, others would demand a concomitant increase of serum-albumin with cessation of edema, which obviously is of greater clinical relevance. There is also no consensus on how to exactly assess the impact of treatment on glomerular filtration rate (GFR) and renal survival.

### Supportive and Non-Immunologic Treatment for Genetic Causes of NS Congenital and Infantile NS

The prognosis of congenital NS has improved substantially in recent years. No differences in mortality and transplant outcome between Finnish and non-Finnish patients with *NPHS1* mutations was noted in a recent registry report on 170 patients (4). Finnish patients started dialysis much earlier because of early bilateral nephrectomy, while in non-Finnish, many other interventions were performed (but not reported on in detail). Despite this, outcome of *NPHS1* patients on renal replacement therapy in fact compared to patients with congenital anomalies of the kidney and urinary tract (CAKUT). No details of treatment approaches and mortality prior end-stage renal disease are presented however. These may influence morbidity and mortality in patients with *NPHS1* and other genetic causes, however and include the following options.

#### *Albumin Infusions*

In severe forms of congenital but also infantile NS regular (mostly daily) albumin infusions have been recommended to decrease edema, increase urine output, and enhance nutrition (7). This strategy requires sufficient renal function; otherwise, fluid overload may occur with potentially severe consequences, such as cardiac failure or pulmonary edema. Since infusions have to be performed regularly, often daily, a central venous access is usually necessary with the associated risks of infection, thrombosis, and hospitalization. Of interest, a recent report by Reynolds et al. (7) showed that after adequate training, administration of albumin can be performed at home, which has an important impact on quality of life. Unfortunately, in reports on regular albumin infusions, other treatments have been used as well (see below). In a yet unpublished French study, 96% of patients received albumin infusions initially daily, with a subsequent reduction in frequency in many patients. It was even discontinued in 10 patients. However, in this report, data about concomitant drug treatment are not available (Berody et al. accepted by NDT, complete citation expected to be available in February).

#### *Nephrectomy*

Unilateral or even bilateral nephrectomy has been used as therapeutic option to decrease or stop proteinuria. Bilateral nephrectomy is probably the most aggressive approach, which will on the one hand completely stop proteinuria, normalize protein and lipid status, and improve nutritional state, but on the other, make (peritoneal) dialysis treatment inevitable (3). Unilateral nephrectomy has been advocated by some authors to reduce proteinuria in children with congenital NS, again often in addition with medical treatment (indomethacin and captopril). In one study (8), serum albumin (sAlb) increased from 11 to 18 g/l after 6–12 months and the number of albumin infusions could be reduced later. This series of five patients also documented an increase in height standard deviation score.

#### *Renin–Angiotensin–Aldosterone System (RAAS) Inhibitors (With/Without Indomethacin)*

A further more conservative approach is drug treatment in order to reduce GFR and thus decrease proteinuria. The value of inhibiting the RAAS by ACE inhibitors (ACEI) and angiotensin receptor blockers (ARBS) in proteinuric renal diseases has been established for many years (9), starting from studies of IgA nephritis. Therefore, in clinical practice, RAAS inhibitors are widely used frequently even in the absence of hypertension. The mechanisms of action relate to decreasing intraglomerular pressure as well as anti-TGFβ properties leading to deceleration of the progression of renal insufficiency. The Cochrane group (9) included RAAS inhibitors in their recommendations for treatment of SRNS but no large studies concerning their use in congenital or infantile NS are available. In one (8), RAAS inhibitors were combined with indomethacin and unilateral nephrectomy. Licht et al. (10) used a stepwise approach: five patients with different causes of congenital NS were treated with captopril and indomethacin serum protein and growth improved in four children. Unilateral nephrectomy was only deemed necessary in two patients during the subsequent course.

Although published evidence is limited, these studies support a stepwise approach in a clinically stable patient with congenital/ infantile NS starting with the use of RAAS inhibitors. In neonates and infants, captopril has been most frequently used and can be titrated best. In severe cases, a combination with indomethacin seems justified; if edema are controlled by this approach (unilateral), nephrectomy can be avoided.

#### Non-Immunologic Treatment in Pediatric and Adolescent Genetic SRNS

Presentation of SRNS after the neonatal period usually leads to a different approach, because the underlying genotype and histological lesions are typically different; *NPHS2* mutations are probably the most frequent single genetic cause (2). Bilateral or unilateral nephrectomy is only practiced in individual severe cases. Treatment with steroids usually has been initiated prior diagnosis and—per definition—failed. Since results of genetic testing are not available immediately in many patients, further treatment, e.g., with calcineurin-inhibitors will be considered and initiated, except maybe in syndromic SRNS. In most published registries (11–13), patients with genetic nephrotic syndrome had received immunosuppressive treatment.

As in congenital and infantile NS, treatment with RAAS inhibitors are a possibility to decrease proteinuria, slow progression of chronic kidney disease, and treat hypertension. RAAS inhibitors are often used in combination with immunosuppressants in glomerulonephrits and NS, although few systematic studies are available evaluating the isolated or combined treatment. As mentioned before, RAAS inhibitors are recommended by the Cochrane group (9) because of two studies. Yi et al. (14) treated SRNS patients with fosinopril and prednisone and compared to a group receiving prednisone alone. Proteinuria decreased significantly in both groups but more in the fosinopril-treated patients. Bagga et al. (15) studied the effect of enalapril at high and low doses showing that there is a dose-related reduction in proteinuria. The benefits of a combined treatment of ACEI and ARBS was suggested by small prospective study in eight patients with SRNS (16). One case report documented complete remission with captopril; regular albumin infusions could be stopped at the age of 15 months (17). Combined antiproteinuric therapy with RAAS inhibitors was also able to induce complete remission in a patient with Nail–Patella syndrome and NS (18). Although all these studies are small and data on genetic causes are not always provided, the long experience with RAAS inhibitors in children would probably be in favor for early use of these drugs in pediatric patients, especially since they are tolerated well and can also be used for associated hypertension. Nevertheless, more controlled data are desirable. Of interest, a current controlled study comparing sparsetan (a dual acting angiotensin receptor blocker and highly selective endotheline Type A receptor antagonist) with irbesartan has been initiated in FSGS to assess the impact also in genetic forms of NS (19).

### OTHER OPTIONS

In individual patients with FSGS (mainly with permeability factor associated NS), treatment with galactose had been reported to be beneficial. Trachtman et al. (20) evaluated treatment of FSGS with a TNF- α inhibiting antibody adalimumab and galactose, the latter being an intersting non-immunologic treatment option also for genetic forms of FSGS. In the cited study, 2 out of 7 patients with FSGS had a 50% reduction in proteinuria after galactose, confirming previous case reports. Data on genetic testing in patients are not available, so no definite conclusion about the utility of galactose in genetic SRNS can be drawn. Unfortunately, a controlled trial with a monoclonal anti-TGF-β antibody (fresolimumab) in SRNS did not lead to a significant reduction in proteinuria (21).

Similarly, the use of vitamin D analogs and stimulation of the calcium-sensing receptor has been assessed. Experimental studies have shown that stimulation of the calcium-sensing-receptor enhances podocyte stability and thus cinacalcet (or vitamin D) may be an option to improve proteinuria in NS (22). So far, data on cinacalcet are not available, but a recent meta-analysis in IgA nephropathy suggested an effect of vitamin D supplementation (23). However, a recent randomized controlled trial of vitamin D supplementation in steroid-sensitive NS did not reduce relapse rate, arguing against a direct podocyte stabilizing effect of vitamin D (24). Although definitive conclusions of these alternative approaches cannot be drawn, future studies into the field of podocyte stabilization by non-immunosuppressive drugs are warranted and could have an important impact on treatment in genetic SRNS.

### Immunosuppression in Hereditary SRNS Evidence from Experimental Studies

Several experimental studies have suggested that immunosuppressants have a direct glomerular effect leading to podocyte stabilization and thus have efficacy beyond their immunological actions. Some agents, such as cyclosporine also have a hemodynamic (nephrotoxic) effect leading, e.g., to reduction of GFR, thereby reducing proteinuria (25).

The initial studies confirming a stabilization of the glomerular cytoskeleton by cyclosporine independent of the immunosuppressive action was provided by Faul et al. (26) and confirmed by various other studies that will not be described in detail (27). Also, steroids, levamisole, mechanistic target of Rapamycin (mTOR) inhibitors, and even Rituximab have received attention in experimental studies although clinical data are virtually nonexistent. For instance, glucocorticoids have been shown to protect and enhance recovery of cultured murine podocytes *via* actin filament stabilization (28, 29). Levamisole, a drug that has never been used in SRNS, was able to induce expression of glucocorticoid receptor (GR) and to activate GR signaling and also protected against podocyte injury in a cell model (30). Also, low-dose rapamycin, an inhibitor of the mTOR, diminished disease progression in an experimental model of FSGS (31). Finally, rituximab, a B-cell-depleting antibody, may bind directly on sphingomyelin phosphodiesterase acid-like 3b protein (SMPDL3b), and thus could have an effect at the cellular level (32).

In summary, there is now emerging evidence that immunosuppressants, especially cyclosporine A (CsA), have a stabilizing effect at the podocyte levels aside from their immunological actions in experimental models and thus may be valuable therapeutic options in the treatment also of genetic forms of SRNS.

### CLINICAL DATA ON IMMUNOSUPPRESSANTS IN GENETIC NS

Most authors would agree that children with *NPHS2* mutations do not respond to treatment with steroids (33). However, in their first report on mutations in the *Plectin1* (*PLEC1) gene*, the authors mention two patients who responded to prednisolone treatment (34). This could not be confirmed from a French series (35); interestingly, the authors report on three unaffected and unrelated patients with homozygous PLCE1 mutations but without clinical disease. Thus, the natural history of SRNS with PLEC1 mutations may be different and modifier genes and environmental factors may play a role. There are no data, whether patients with SRNS and other mutations have a documented partial or complete remission after initial steroid treatment.

To our knowledge, no data on treatment with mycophenolate mofetil (MMF) are available in genetic NS. Unfortunately, in a randomized US study comparing MMF with cyclosporine, genetic testing was not performed (36). In contrast, data on treatment with calcineurin inhibitors (mainly CsA) in cohorts with genetic SRNS have been published; in most of these studies, genetic diagnosis was confirmed *post hoc*, sometimes after years. Unfortunately, response to treatment interventions is often not detailed and sometimes interpretations are superficial.

In two studies from Buscher et al. (12, 13), the vast majority of patients with genetic FSGS did not achieve remission with cyclosporine. In the first study on 91 patients, mutation in 1 of 6 genes studied were detected in 52% of patients. None of the patients with mutations showed a complete response to CsA, but two patients with Wilms tumor suppressor gene 1 (*WT1*) mutation showed partial remission. The response rate of CSA was significantly better without mutations in podocyte genes (68 vs. 17%, *p* < 0.005). In the second, larger retrospective multicenter study by Buscher et al., 131/231 patients with SRNS had an identified genetic diagnosis, including 60 patients with congenital NS; of these, 63 (48%) patients had received CSA. 2 patients (1 with congenital NS, CNS) entered complete and 5 partial remission (none with CNS). In summary, this series documents a partial or complete response to CSA in 19% of hereditary SRNS (with no details on genotype stated) and 1.7% of congenital NS.

In a recent report from the Podonet consortium, 906/1,354 patients with SRNS were treated with immunosuppressants (380 with one, 173 with two, and 59 with three or more different, respectively) (11). Among 74 patients with documented genetic diagnosis, two patients had complete and 8 partial remission, respectively. Another four patients entered partial remission with a combination of CSA and RAAS inhibitor. Thus, in this series, a total of 14/74 (19%) patients with genetic SRNS seemed to have a response to immunosuppression.

In our own retrospective single center study (37) of nine SRNS patients with an identified genetic diagnosis (excluding patients with CNS, *WT1* mutations, and syndromic NS), we observed a partial (2 patients with *NPHS2* mutations) or complete (one patient with compound heterozygous *NPHS1* and one with a dominant *ACTN4* mutation, respectively) remission. Thus 4/9 (44%) showed some response to CSA; **Figure 1** demonstrates evolution of serum-albumin levels in two patients with a complete and partial response, respectively.

Currently, no controlled data are available as to how long CSA treatment in genetic forms of SRNS should be continued, if no response is documented. The series by Klaassen et al. (12, 13, 37), however, show that most patients with complete remission responded after a median of 2 months, so that this treatment period seems to be a minimum. It is currently unknown, whether patients with distinct mutation, e.g., in *WT1* or *NPHS2* show a differential response.

### EXPERIENCES IN SPECIFIC GENETIC DISORDERS

### *WT1*-Mutations

The Wilms Tumor Suppressor Gene 1 (WT1) is a transcription factor with many functions, among them transcriptional as well as tumor-suppressor activities. WT1 plays a pivotal role in early urogenital and kidney development (38), in adults, it continues to be a key regulator of podocyte function (39). Mutations in *WT1* mostly occur as spontaneous heterozygous germline mutations, but familiar cases are also described. Type and location of specific mutations allow for limited prediction of clinical course, histology, and comorbidities (40). By far, not all patients show all symptoms of the classical syndromal descriptions associated with *WT1* mutation: Denys–Drash syndrome described in patients with missense mutations, it includes DMS, SRNS rapidly progressing to end-stage renal disease, XY disorder in sex development with complete gonadal dysgenesis, and a high risk of developing Wilms' tumor. Frazier Syndrome is typically caused by mutations affecting the canonic donor splice site of intron 9, patients present with streak gonads, and are at high risk of developing gonadoblastoma.

There is evidence from studies with small patient numbers that early initiation of treatment with CsA in combination with RAAS inhibitors can lead to favorable response of NS in patients with *WT1* mutations (41). Gellermann et al. report on three children with WT1 mutations and FSGS in whom long-term reduction of proteinuria could be achieved through treatment with CSA in combination with steroids and RAAS inhibitors while maintaining normal renal function (42). Wasilewska et al. describe a patient with *WT1* mutation and DMS on histology in whom nephrotic range proteinuria resolved after initiation of treatment with CSA and enalapril (43). Buscher et al. report two patients affected by NS due to mutations in *WT1* showing a partial response to CsA with a reduction of proteinuria and normalization of sAlb (13). Unfortunately, in their 2016 study, Buscher et al. do not give details on their patients with *WT1* mutations treated with CSA (12). Further studies are needed to define in more detail which patients with *WT1* mutations (type of mutation and histological changes) can benefit from immunosuppressive treatment with

CSA. Mechanisms mediating the benefits of CSA, other than stabilizing podocyte cytoskeleton, in patients with disturbed WT1 expression also need further elucidation (41). Collection of patients with *WT1*-mutations in a prospective registry has been initiated by the German Society of Pediatric Nephrology (GPN).

### Coenzyme Q10 (CoQ10)-Deficiency

CoQ10, also known as ubiquinone, is involved in many essential cellular processes, especially in the mitochondria. Its biosynthesis requires at least 15 genes. So far, mutations in eight of these genes have been found to cause primary CoQ10 deficiency, which results in diseases with variable age of onset. Associated clinical phenotypes are ranging from a multisystem disease to nephropathy or isolated central nervous system disease (myopathy or cerebellar ataxia) (44). Nephropathy can result in steroid resistant NS and loss of renal function in an isolated form (45) or in combination with sensorineural deafness (46) or other neurological symptoms (47).

In contrast to most mitochondrial respiratory chain disorders, for which there is no effective treatment, patients with primary CoQ10 deficiency partially respond to oral CoQ10 supplementation (48). *In vitro* data and first clinical experiences suggest that high-dose oral treatment with CoQ10 has the potential to stop the progression of encephalopathy, muscular symptoms, and NS and even induce remission, if initiated early enough. Heeringa et al. (46) describe proteinuria and hearing loss in patients with coenzyme Q10 biosynthesis monooxygenase (*COQ6)*-mutations. *In vitro*, apoptosis caused by *COQ6*-knockdown was partially reversed by CoQ10 treatment. The authors report a positive response to treatment with CoQ10 and RAAS inhibitors in two children with mild disease. Ashraf et al. (49) describe that mutations in the aarF domain containing kinase 4 gene (ADCK4) leads to CoQ10 deficiency causing SRNS. Knockdown of *ADCK4* in podocytes resulted in decreased migration, which was reversed by CoQ10 addition. Indeed, one individual with *ADCK4*-mutation was successfully treated with CoQ10 supplementation. Once severe kidney or neurological damage is established, this cannot be reversed (47, 48).

### Gene Therapy in Genetic Forms of SRNS

So far, no successful gene therapeutic approaches have been reported for hereditary forms of SRNS. However, a mouse model for NPHS2 has recently been reported (50) and may have important consequences also for the development of specific molecular treatment approaches. Other investigations have indicated a role of specific miRNAs in the etiology of FSGS, which may be amenable to specific treatment (51). Taken together, studies into the molecular biology of SRNS have a yet undiscovered potential to develop new treatment modalities and potentially a cure for certain genetic forms of SRNS.

### SUMMARY

Although no adequate systematic treatment studies have been performed in patients with genetic forms of NS, choice of treatment needs to consider clinical factors (e.g., severity of clinical presentation and age of presentation). In a clinically stable patient with congenital/infantile NS, use of an RAAS inhibitor could be

first choice. In neonates and infants, captopril has been most frequently used and can be titrated best. In severe cases, a combination of RAAS inhibitor with indomethacin seems appropriate in order to avoid (unilateral or even bilateral) nephrectomy, which is an effective option in severely affected individuals. In steroidresistant NS patients, most patients with Mendelian forms seem resistant to immunosuppressive treatment. Yet, several studies have shown that in some patients, a complete or partial remission with CSA can be achieved also in hereditary SRNS, including patients with infantile nephrotic syndrome (**Figure 2**). Therefore, in our opinion, a therapeutic trial with CSA in genetic SRNS is justified, since especially CSA has been shown to exert a relevant podocyte stabilizing effect, which may reduce proteinuria in some

### REFERENCES


patients. In the future, more clinical studies of optimal treatment of genetic SRNS are necessary, also evaluating the impact of confounding factors, such as genotype/phenotype correlations, impact of attenuation of proteinuria on rate of progression into ESRD, and others. Ultimately, gene therapy will hopefully be available in the future offering a specific cure of (some) genetic causes of steroid resistant NS.

### AUTHOR CONTRIBUTIONS

MK wrote the first draft of the manuscript; AL wrote sections of the manuscript. Both authors contributed to manuscript revision, read, and approved the submitted version.


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Kemper and Lemke. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Experimental Models to Study Podocyte Biology: Stock-Taking the Toolbox of Glomerular Research

Henning Hagmann and Paul T. Brinkkoetter\*

*Department II of Internal Medicine, Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany*

Diseases affecting the glomeruli of the kidney, the renal filtration units, are a leading cause of chronic kidney disease and end-stage renal failure. Despite recent advances in the understanding of glomerular biology, treatment of these disorders has remained extraordinarily challenging in many cases. The use of experimental models has proven invaluable to study renal, and in particular, glomerular biology and disease. Over the past 15 years, studies identified different and very distinct pathogenic mechanisms that result in damage, loss of glomerular visceral epithelial cells (podocytes) and progressive renal disease. However, animal studies and, in particular, mouse studies are often protracted and cumbersome due to the long reproductive cycle and high keeping costs. Transgenic and heterologous expression models have been speeded-up by novel gene editing techniques, yet they still take months. In addition, given the complex cellular biology of the filtration barrier, certain questions may not be directly addressed using mouse models due to the limited accessibility of podocytes for analysis and imaging. In this review, we will describe alternative models to study podocyte biology experimentally. We specifically discuss current podocyte cell culture models, their role in experimental strategies to analyze pathophysiologic mechanisms as well as limitations with regard to transferability of results. We introduce current models in *Caenorhabditis elegans, Drosophila melanogaster*, and *Danio rerio* that allow for analysis of protein interactions, and principle signaling pathways in functional biological structures, and enable high-throughput transgenic expression or compound screens in multicellular organisms.

#### Edited by:

*Miriam Schmidts, Radboud University Nijmegen, Netherlands*

#### Reviewed by:

*Bart Smeets, Radboud UMC Department of Pathology, Netherlands Jan Halbritter, Leipzig University, Germany*

\*Correspondence: *Paul T. Brinkkoetter paul.brinkkoetter@uk-koeln.de*

#### Specialty section:

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

Received: *14 March 2018* Accepted: *11 June 2018* Published: *13 July 2018*

#### Citation:

*Hagmann H and Brinkkoetter PT (2018) Experimental Models to Study Podocyte Biology: Stock-Taking the Toolbox of Glomerular Research. Front. Pediatr. 6:193. doi: 10.3389/fped.2018.00193* Keywords: model organism, kidney diseases, podocyte, mechanosensation, glomerular filtration barrier

### INTRODUCTION

Chronic kidney disease (CKD) is becoming an increasingly prevalent condition affecting almost 10% of the population in Western societies. The majority of kidney diseases that progress to end stage renal failure start in the glomerulus, the renal filtration unit, as a consequence of a very limited capacity of glomeruli for regeneration and the limited ability of terminally differentiated glomerular podocytes for self-renewal (1). The glomerular filtration barrier consists of three anatomic layers: fenestrated endothelial cells, the glomerular basement membrane and podocytes, post-mitotic epithelial cells located at the outer aspect of the capillary loops (**Figure 1B**). These cells enwrap the glomerular capillaries with their primary and secondary processes and form the outer layer of the filtration apparatus. All three layers contribute substantially to the glomerular filtration barrier and can be affected in human disease. Due to their exposed anatomic localization podocytes are constantly challenged not only by oxygen radicals, cytokines, immune complexes and inflammatory processes but also by mechanical forces. Podocyte damage plays a pivotal role in most, if not all, glomerular diseases that result in glomerulosclerosis (2). As podocyte loss cannot easily be compensated by cell proliferation the cells undergo hypertrophy, autophagy, and/or dedifferentiation depending on the injurious insult (3). Podocyte hypertrophy and the increase in cellular size and the covered area of the GBM represent protective measures to ascertain proper glomerular function. In contrast, dedifferentiation is considered to be maladaptive resulting in albuminuria and persistent podocyte loss. It has to be stressed, that the onset of albuminuria and even nephrotic range proteinuria does not require podocyte depletion. Mere cytoskeletal rearrangements, i.e., foot process effacement, are sufficient to cause massive albuminuria as often seen in patients with minimal change disease (MCD). A condition which lacks evidence of pathology in light microscopy but presents with vast foot-process effacement in electron microscopy. In the event of progressive or severe glomerular disease podocyte loss is the clue and patients develop massive albuminuria in combination with irreversible scarring, i.e., glomerulosclerosis (1, 4–7). Importantly, albuminuria and chronic kidney disease are independently associated with an increased risk for end stage renal failure and cardiovascular disease (8–10).

The finding that independent pathways and pathogenic principles contribute to the identical glomerular phenotype described as focal segmental glomerulosclerosis (FSGS) seems trivial but is of major importance. Podocytes are firmly attached to the underlying glomerular basement membrane and form a unique cell-cell contact to foot processes of neighboring podocytes, a cell junction called slit diaphragm. This specialized cell-cell contact is not only an integral part of the glomerular filtration barrier but also serves as signaling hub to regulate podocyte function (11). Over the past several years, various constituents of the podocyte slit diaphragm cell junction have been identified leading to the concept that the proteins at the slit diaphragm regulate podocyte biology through active signaling. The slit diaphragm bridges the distance between two adjacent foot processes, thus allowing formation of a filtration slit. In severe podocyte damage, the slit diaphragm disappears and podocytes simplify structure and shape due to cytoskeletal alterations, a process called foot process effacement (8, 10). Until recently, the function of the glomerular filtration barrier and the pathogenesis of proteinuria have not been well understood. This has changed with the identification of gene defects in (rare) human genetic diseases known to cause congenital or childhood steroid-resistant nephrotic syndrome and progressive glomerulosclerosis [for review see (9)]. These studies identified distinct deregulated pathways that independently contribute to podocyte injury and, potentially, loss of podocytes. Podocyte depletion has long been known to be the culprit of glomerulosclerosis and progressive loss of renal function (12–14). As multiple different pathogenic mechanisms result in proteinuria and FSGS lesions in kidney biopsies, it is not surprising that several clinical trials including all FSGS patients failed to provide new MCD/FSGS treatment options, e.g. the NIH has spent multimillion dollars on clinical trials that did not yield a single new drug for MCD/FSGS patients (15).

The advent of modern genetics with the development of animal models with cell specific gene manipulation including gene deletions and transgenic gene expression together with systems biology has deepened our understanding of the biology and physiology of the renal filtration barrier in states of health and disease. Despite our tremendous advances in understanding glomerular function and the contribution of the specific anatomic compartments to the renal filtration barrier, essential questions remain to be addressed as treatment of glomerular disorders is still unspecific and primarily based on various immunosuppressive regimens, including glucocorticoids or blockade of the renin-angiotensin-aldosterone system (15).

In the past, the use of experimental models has proven invaluable to study renal, and in particular, glomerular biology and disease. Even after the introduction of novel gene editing techniques, mouse models are time consuming. The relatively long reproductive cycle, high keeping cost, and not least the regulatory standards make these models less flexible. In this review, we will describe alternative models to study podocyte biology experimentally.

### PODOCYTE CELL CULTURE

Podocyte cell culture models were the first models to study podocyte biology and are still widely used (16, 17) as gene and protein expression as well as environmental cues can be easily manipulated in vitro for mechanistic analyses (18, 19). Multiple human, mouse, and rat podocyte cell lines have been generated in the past (16, 17, 20–22). Most groups rely on immortalized mouse or human podocyte cell lines which are cultured under proliferative (33◦C) and growth-restrictive (37◦C) conditions. These studies promoted our understanding of glomerular diseases (19) as well as the cytoskeletal regulation (23), cell cycle control (24), cell death mechanisms (25), signaling pathways (26, 27) as well as protein degradation (28).

However, certain limitations apply and have to be taken into account when translating experimental findings from cultured podocyte cell lines; podocytes in culture are cultivated on petri-dishes as a monolayer in the absence of mesangial and endothelial cells in close proximity. Podocytes in culture do not encounter mechanical stretch nor the flow of primary urine filtrate (29). Hence, it is not surprising that podocyte cell lines do not form secondary processes with slit diaphragms in-between neighboring cells and show only a very limited expression of specific marker proteins including nephrin (30), podocin (31), or transient receptor potential cation channel 6 (26, 32, 33).

In an attempt to further characterize these widely used work horses of podocyte research our group recently applied modern MS/MS technologies and created a comprehensive map at a depth of more than 7,000 proteins expressed in proliferating and differentiated cultured podocytes in vitro (26). To this

end, we examined an immortalized mouse podocyte cell line kindly provided by S. Shankland (Seattle, WA) as well as a conditionally immortalized human podocyte cell line obtained from M. Saleem (Bristol, UK), both are widely used cell-culture models generated by either isolation of primary podocytes from the immortomouse (34) or by retroviral transfection of primary human podocytes with a temperature-sensitive SV40 large T-cell antigen (35) (**Table 1**). The temperature shift from 33 to 37◦C induces in both cell lines a proteostatic shift. Undifferentiated podocytes express high abundance of proteasomal proteins while differentiated podocytes express high abundance of lysosomal proteins. Additional studies using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) and protein degradation assays determined protein dynamics and half-lives and revealed a globally increased stability of proteins in differentiated podocytes. Mitochondrial, cytoskeletal and membrane proteins were particularly stabilized in differentiated podocytes. However, the expression levels of socalled podocyte marker genes or podocytopathy gene products varied significantly as compared to primary cells. Highest levels were detected for Actin-regulating proteins comprising Myosin-9 (MYH9) (36), rho GDP-dissociation inhibitor 1 (ARHGDIA) (37) and alpha-actinin-4 (ACTN4) (5). Out of 15 podocytopathyassociated genes linked to cytoskeletal function 8 were expressed in the cultured cells (MYH9, ARHGDIA, ACTN4, anillin (ANLN), inverted formin-2 (INF2), unconventional myosin IE (MYO1E), synaptopodin (SYNPO), and podocalyxin (PODXL) (22, 38–41). With respect to basement membrane proteins 4 out of 8 were quantified in undifferentiated and differentiated human podocytes [CD151 antigen (CD151), integrin alpha-3 (ITGA3), integrin beta-4 (ITGB4), and laminin subunit beta-2 (LAMB2) (20, 42, 43). In contrast, only one out of six slit diaphragm proteins (CD2-associated protein (CD2AP) (44) could be detected.


*Respective proteins are either marked as expressed ("*+*") or not expressed ("–") in the two examined cell culture conditions (33*◦*C* = *undifferentiated, and 37*◦*C* = *differentiated cells). According to Schroeter et al. (45).*

### MODEL ORGANISMS TO STUDY PODOCYTE BIOLOGY

Caenorhabditis elegans with its short life cycle, completely established cell lineage including a neuronal map (connectome), compact and fully mapped genome, uncomplicated genetic modification by feeding of RNAi-expressing E. coli, and costeffective keeping is a prime model for cell biology (46). In addition and in contrast to other multicellular model organisms, (genetically modified) individuals can be easily frozen, stored for the longer term, and revived with immediate capability to reproduce.

These characteristics make the nematode an ideal model to study signaling pathways and functionality of proteins in a multicellular organism and represent an advantage as compared to mammalian models.

Principle signaling pathways like e.g., the insulin/mTOR signaling cascade are generally well-conserved across species (47). Along this line, the observation that the insulin/mTOR pathway is induced and refers damage in podocytes with mitochondrial dysfunction due to the loss of Prohibitin-2 (PHB-2) was substantiated in C. elegans using worm strains expressing fluorescence labeled DAF-16 (48). After heat-shock DAF-16 is pooled in the nuclear compartment. Redistribution of DAF-16 to the cytoplasm depends on the activity of the insulin pathway. Consistently with data from podocyte-specific PHB-2 knockout mice, phb-2-deficient worms showed accelerated recovery of the DAF-2 (insulin receptor) mediated cytosolic redistribution of DAF-16.

However—with regard to glomerular research—C. elegans does not contain a filtering excretory organ homolog to the mammalian glomerulum. Nevertheless, ortholog genes in analogs structures can be studied in the nematode to understand principle mechanisms of podocyte morphogenesis and podocyte slit-diaphragm function.

The mammalian slit diaphragm is composed of the transmembranous immunoglobulin family proteins nephrin and Neph1. Nephrin and Neph1 are lipid raft associated proteins that refer outside-in signals by tyrosin phosphorylation. Mutations in the nephrin encoding gene NPHS1 or lack of NEPH1 lead to defective assembly of the foot processes and loss of the slit diaphragm which becomes evident as (congenital) nephrotic syndrome (49, 50). The adhesion molecules nephrin and Neph1 are well conserved across species. In C. elegans orthologs of Neph1 and Nephrin are SYG-1 and SYG-2, respectively. SYG-1 and SYG-2 refer cell-cell recognition in synapse development between the hermaphrodite specific neuron (HSN) and specialized epithelial guidepost cells adjacent to the nematode's vulva muscle cells medially in the hermaphrodites' soma. The two HSN (HSNL and HSNR) localize to the lateral aspects in the middle of the nematode and protrude their axonal processes ventrally, where they innervate the vulvar muscle cells and provide the neuronal circuit required for egg laying. Interaction of SYG-1 on the HSN axon with SYG-2 expressed on guidepost cells initiates intracellular signaling processes in HSN to trigger synapse formation and maintenance (51, 52). Mutations in either syg-1 or syg-2 fail to exhibit functional synapses due to aberrant placement of presynaptic sites (53, 54). Interestingly, heterologous expression of mammalian nephrin or Neph 1, −2 or −3 can rescue phenotypes of mutant syg-1 or syg-2 (55, 56). The cytoplasmic tail of SYG-2 is required for subcellular trafficking of SYG-2 itself, whereas the cytoplasmic domain of SYG-1 is required for synapse formation but dispensable in later stages (57). Although elegant ultrastructural analyses in mammalian and avian glomeruli have challenged the concept of heterophilic nephrin and Neph1 interaction at the slit diaphragm, synapse formation at the HSN in C. elegans may represent a suitable model to study signaling mechanisms at the cytoplasmic domains of nephrin and Neph1 by visualization of synaptic vesicles in SNB-1::YFP transgenic worms.

Another example for the utility of C. elegans in glomerular research is based on the homology of mammalian podocin and C. elegans MEC-2. Podocin is an essential constituent of the mammalian slit diaphragm complex, whereas MEC-2 is part of the mechanosensory complex of C. elegans sensing gentle touch. Both stomatin-like proteins share a central stretch of hydrophobic amino acids which refers membrane association while the amino and the carboxy terminal ends face the cytoplasm. The highly conserved PHB domain mediates homophilic interactions and lipid binding via palmitoylation, creating the microenvironment that regulates signaling via the associated ion channel proteins TRPC6 in mammals and the DEG/ENaC channel MEC-4/MEC-10 in C. elegans (58–60). This regulatory role of MEC-2 can not only be assessed by quantification of mechanoreceptor channel currents but also in functional in vivo assays measuring sensitivity to gentle touch in adult hermaphrodites (61). In addition, regular localization of MEC-2 and other components of the mechanosensory complex in a characteristic punctate pattern on the six mechanosensory neurons of C. elegans can be evaluated by staining with MEC-2 specific antibodies or employing MEC-4::YFP transgenic worms (28, 62). Identifying co-localization of the primarily mitochondrial protein Prohibitin 2 (PHB-2) and MEC-4 in mechanosensory punctae of touch receptor neurons in C. elegans as well as partial loss of touch sensitivity in PHB-2 knock down worms helped to establish the role of PHB-2 as a slit-diaphragm protein (63). In a recent paper, the ubiquitin ligase Ubr4 has been shown to control podocin protein stability and conservation of this molecular mechanism could be confirmed for MEC-2 in C. elegans assays, where the loss of the Ubr4 ortholog C44E4.1 (ubr-4) resulted in a more dispersed staining pattern of MEC-2 positive punctae (28).

With regard to glomerular research, studying C. elegans is instrumental as a functional read out for protein interactions, trafficking and protein turnover as well as signaling of conserved pathways in a multicellular organism. This holds true especially for mammalian podocin, nephrin, Neph 1, and their orthologs. A weakness of the nematode as a model of conserved principles in mammalian (patho-)physiology as well as for compound screens is the lack of organs homolog to heart, liver, central nervous systems, and of course filtering organs like the kidney.

Drosophila melanogaster evolved as another high-capacity model organism for glomerular research. Cell type specific gene inactivation and editing make the fly a versatile, adaptable, and expedite model. The reproductive cycle is around 12 days.

Similar to the nematode the Drosophila model allows to study protein interactions in functional biologic structures. In the fly adhesion molecules ortholog to mammalian nephrin and Neph proteins called sticks'n stones/hibris (sns/hbs) and kirre/dumbfounded (duf) are involved in cell-cell recognition, and cellular signaling events to control adhesion, cell shaping, and programmed cell death during eye development in the fly. During embryonal development Drosophila's facet eyes develop from a single layered epithelium and remain undifferentiated during almost the entirety of larval stages. In the final differentiation steps heterophilic interactions of sns/hbs and duf determine cell fate specification and are required for patterning and separation of ommatidea (64, 65).

During the last decade, research explored Drosophila nephrocytes as a novel tool of cell biology. The term was initially coined by Bruntz and Kowalsky, who discovered ammonia carmin absorbing cells around the heart, the digestive organs, and the nervous system in arthropods (66, 67). Nephrocytes are specialized filtrating cells with high endocytic activity, that may have detoxifying and sequestration function. In the adult fly there are two populations of nephrocytes. The Garland nephrocytes, which are assembled along the esophagus, and the pericardial nephrocytes, which palisade the heart tube. Nephrocytes are large cells with a surface laced by invaginations, so called labyrinthine channels, and covered by a continuous basement membrane. Near the apical surface the invaginations are abridged by a slit diaphragm (**Figure 1A**). Loss of the nephrin ortholog sns or the Neph ortholog duf results in loss of slit diaphragm structures, smaller lacunae and thickening of the basement membrane (68). Filtration across the pericardial nephrocyte slit diaphragm is limited to substances smaller than 70 kDa (69). Molecules that get filtered into the labyrinthine channels are taken up by the nephrocyte via endocytosis (70). In this regard the nephrocyte differs significantly from mammalian podocytes as to our current understanding. Filtration across the nephrocyte slit diaphragm as well as endocytosis can be assessed in assays on explanted nephrocytes employing different tracers, like e.g., GFP-, labeled albumin, horseradish peroxidase or dextrans of variable sizes (68, 69, 71, 72).

A novel transgenic fly model expressing secreted atrial natriuretic factor labeled with red fluorescent protein (ANF-RFP) from muscle cells and nephrocyte-specific green fluorescent protein (GFP) combined with the option of nephrocyte specific gene manipulation via a nephyrocyte specific Dot-Gal4 driver has implemented an expedite and reliable screening tool for genes associated with human disease (69). This model was employed to screen Drosophila orthologs of human monogenic nephrotic disease. Reassuringly, most of the pathogenic alterations were conserved in the fly (71, 73). Loss of Coq2 or Rab GTPases e.g., leads to loss of labyrinthine channels and loss of function in nephrocytes (71, 74).

In addition, rescue and overexpression studies with human orthologs are possible in Drosophila knockout models. Both eye and nephrocyte development were evaluated to identify a conserved amino acid motif in mammalian Neph1 to functionally replace Drosophila duf, whereas neither Neph2 nor Neph3 showed similar effects (75).

In general, the fly is an ideal model organism for highthroughput transgenic screens and rescue experiments, when variants of ortholog human genes are expressed in the respective drosophila knock out. The in vivo filtration assay or eye pattern formation serve as reliable and expedite readouts.

Danio rerio, the zebrafish, provides a third model organism of glomerular filtration. In striking contrast to the nonvertebrate Drosophila and C. elegans models, the zebrafish forms vascularized glomeruli in the pronephrons and the mesonephros during development with the mesonephros maintained in adult life. At the pronephros-stage the zebrafish kidney consists of two fused glomeruli with connection to the aorta draining primary urine into the pronephric tubuli followed by the pronephric ducts (76). The pronephros glomeruli already contain fenestrated endothelium, mesangial cells and podocytes that form a functional slit diaphragm (77). In addition, genome conservation of 70% between human and Danio rerio as well as versatile morphlino techniques, CRSPR/Cas-based gene editing and cross-species rescue models make the fish a primary model to study glomerular disease. Loss of zebrafish nephrin and podocin, which are specifically expressed in pronephric podocytes, leads to the loss of slit-diaphragms early in development (78). As in human nephrotic syndrome, pericardial edema, periocular edema, and general edema develops. Likewise disruption of zNeph1 or zNeph2 showed similar phenotypes (55). Besides structural analyses functional assays of glomerular filtration have emerged. Early on, the integrity of the filtration barrier was monitored qualitatively on fixed tissue after injections of large molecular weight dextran, which was detected in tubular epithelial cells in case of disruption of the glomerular filter (78). By now, several assays to quantify glomerular filtration have been established. Amongst these are in vivo fluorescence measurement in the eye or in large vessels in time-laps experiments after injection of fluorescence-labeled 10-, 70-, and/or 500 kDa dextrans (79). Another approach employs transgenic fish expressing eGFP-labeled vitamin D-binding protein (eGFPDBP) of a molecular weight of 78 kDa in the liver, which is repelled from glomerular filtration in healthy fish but leaks into urine in states of glomerular damage and can be quantified in the eye (accumulation in control) and—as excreted protein—in the water surrounding the fish (80).

### ORGANOIDS

Generation of kidney organoids by differentiation of pluripotent stem cells (IPS cells) or re-aggregation of single cell suspensions of embryonic kidney cells in culture provided an important new tool for the study of kidney development and disease (81, 82).

However, the delicate morphology of the glomerular filter as well as the need for specific cellular interactions

and vascularization has hampered the study of glomerular biology on kidney organoids. In addition, recent single cell transcriptomic analysis of organoids has identified incomplete differentiation of all kidney organoid cell types, including podocyte progenitors (83).

In an elegant study, researchers generated organoids mixing murine embryonic kidney cells and implanted these organoids into nephrectomized athymic rats. They describe differentiation of morphologically and functionally intact glomeruli (84). In addition, the authors were able to integrate human amniotic fluid stem cells into chimeric organoids by mixing murine embryonic kidney cells and human amniotic fluid stem cells before in vitro organoid culture. Interestingly, also human amniotic fluid stem cells generated functional podocytes. Similar results were recently confirmed for human pluripotent stem cell derived organoids transplanted under the renal capsule of immunocompromised mice (85).

In conclusion, organoids will become a very important tool also in glomerular research. Again the complexity of glomerular structure and interaction of glomerular cell types is the major challenge to overcome.

### CONCLUSION

Research of the last two decades has boosted our understanding of podocyte cell biology and genetics and provides growing understanding of the composition of the renal filtration barrier and cellular interactions needed to maintain its function. Experimental work in podocyte cell culture models informed on expression, trafficking, interaction and turnover of essential proteins of podocyte function. However, cell culture studies in podocytes are limited due to the fact that podocytes in culture lose their characteristic features. Cultured podocytes lack the intricate foot process morphology, cell polarity is incompletely preserved, and most importantly, intercellular contacts are neither structurally nor functionally close to the slit-diaphragm found in vivo. It is very clear that in vivo models are needed to understand glomerular physiology and to address podocyte diseases experimentally. Besides indispensable rodent models, model organisms like C. elegans, Drosophila melanogaster, and the zebrafish have entered the stage of glomerular research and allow unparalleled functional analyses of inter-cellular interactions and morphogenesis, signaling mechanisms, cell polarity, and filtration in vivo. Kidney organoids may become an additional important tool in the future.

### REFERENCES


### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

### FUNDING

PB received grant support by the German Research Foundation (BR2955/6-1, KFO329 BR2955/8-1). HH was supported by the EKFS HH (2016-A62). HH and PB also received intramural funds from the Köln Fortune Program, University of Cologne.

### ACKNOWLEDGMENTS

The authors thank S. Koehler for providing Drosophila images.


garland cell nephrocyte. J Am Soc Nephrol. (2017) 28:1521–33. doi: 10.1681/ASN.2016050517


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Hagmann and Brinkkoetter. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Using the *Drosophila* Nephrocyte to Model Podocyte Function and Disease

#### *Martin Helmstädter 1,2, Tobias B. Huber <sup>3</sup> and Tobias Hermle1,2\**

*1Renal Division, University Medical Center Freiburg, Freiburg, Germany, 2 Faculty of Medicine, University of Freiburg, Freiburg, Germany, 3 III. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany*

Glomerular disorders are a major cause of end-stage renal disease and effective therapies are often lacking. Nephrocytes are considered to be part of the *Drosophila* excretory system and form slit diaphragms across cellular membrane invaginations. Nehphrocytes have been shown to share functional, morphological, and molecular features with podocytes, which form the glomerular filter in vertebrates. Here, we report the progress and the evolving tool-set of this model system. Combining a functional, accessible slit diaphragm with the power of the genetic tool-kit in *Drosophila*, the nephrocyte has the potential to greatly advance our understanding of the glomerular filtration barrier in health and disease.

#### *Edited by:*

*Miriam Schmidts, Radboud University Nijmegen, Netherlands*

#### *Reviewed by:*

*Andrew Mallett, Royal Brisbane and Women's Hospital, Australia Michal Malina, Charles University, Czechia Michael Peter Krahn, University Hospital Muenster, Germany*

#### *\*Correspondence:*

*Tobias Hermle tobias.hermle@uniklinikfreiburg.de*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 20 October 2017 Accepted: 24 November 2017 Published: 07 December 2017*

#### *Citation:*

*Helmstädter M, Huber TB and Hermle T (2017) Using the Drosophila Nephrocyte to Model Podocyte Function and Disease. Front. Pediatr. 5:262. doi: 10.3389/fped.2017.00262*

Keywords: nephrocyte, *Drosophila*, podocyte, glomerular disease, garland cell, endocytosis, renal disease

## INTRODUCTION

Disorders that affect the glomerulus are the predominant cause of end-stage renal disease (ESRD) (1). Beyond renal replacement therapy, potent therapeutic options are not available for most of the disorders from this heterogeneous group. This underscores the unmet need for valid model systems that serve to understand the mechanisms of glomerular diseases.

The most established model system for the glomerulus is currently the mouse. Despite being the undeniable gold standard, this model has its inherent limitations in costs, speed, and considerations of animal welfare. Complementary systems are thus desirable. An excellent alternative can be found in the zebrafish model. But the aforementioned limitations of the mouse model are alleviated only in parts in zebrafish, while additional obstacles like the shortcomings of the morpholino technology need to be considered. Significant insights were also derived from *in vitro* studies, mainly by using cultured podocytes (2). However, here other significant limitations are arising. This is mainly a consequence of the complex glomerular architecture. The glomerular filter is three-layered, including the fenestrated endothelium, the glomerular basement membrane, and the podocytes that form the slit diaphragm. Podocytes are characterized by their most intricate cell shape that is paralleled only by neuronal cells. Cultured podocytes lack crucial features of their *in vivo* counterparts, most notably the formation of slit diaphragms. The *Drosophila* anatomy contains no structure whose analogy to the mammalian kidney appears obvious. Thus, it was unexpected that the *Drosophila* nephrocyte was identified as an invertebrate model that has the potential to meet the needs for a complementary model for glomerular disease (3, 4).

Discovered more than 150 years ago (5) and identified as storage kidneys, the nephrocyte received limited attention until after the discovery that their auto-cellular junctions need to be considered as slit diaphragms that are formed by the orthologs of the mammalian slit diaphragm proteins nephrin (*sns*) and *NEPH1 (kirre)* (3, 4). Loss-of-function of slit diaphragm proteins results in a smooth

**65**

cell surface analogous to podocyte foot process effacement. Nephrocytes thus share molecular, ultrastructural, and functional features with podocytes (3, 4, 6, 7). This makes nephrocytes a unique model system with a functional slit diaphragm in a genetically highly tractable model organism. Now, nearly a decade after the introduction of the nephrocyte to a wider audience in the renal field, the purpose of this review is to examine the progress and the evolving tool-set of this model system for glomerular diseases that still has not reached its peak.

### NEPHROCYTE BIOLOGY

Regarding the history and basic principles of nephrocyte biology, we refer to detailed previous reviews (8–10) in order to focus on the more recent findings.

### Basic Concepts of Nephrocyte As an "Excretory" Organ

The two fundamental functional subunits of renal organs throughout vertebrate biology are the glomeruli that produce an ultrafiltrate and the tubules that process the ultrafiltrate further and finally feed the resulting urine into the disposal system. Together, they serve to eliminate toxins and waste products and maintain water, salt, and pH homeostasis. Following similar principles, *Drosophila* nephrocytes are considered to be part of the fly excretory system. The *Drosophila* renal system has two functional subunits as well: the nephrocytes, regarded as analogous to the glomeruli, and the Malpighian tubules, regarded as analogous to the renal tubular system. There are two distinct nephrocyte populations: the pericardial nephrocytes along the heart tube and the garland cell nephrocytes in a garland-like ring around

Filtration occurs from the blood through the filter into the urinary space. In *Drosophila*, nephrocytes function as individual cells that are clustered. The glomerular filter

is bi-layered, lacking the endothelium. Filtration occurs from the larval blood, the hemolymph, being destined for processing in endosomes.

the esophagus (**Figure 1A**). However, a substantial conceptual difference to the mammalian kidney is that nephrocytes have no connection to the tubular system of *Drosophila* that independently generates, modifies, and finally excretes urine into the intestinal lumen (8).

### Brief History of Nephrocyte Research

Despite the divergence of anatomy and functional concepts, the role of nephrocytes was already discovered in the mid-19th century. The garland cell nephrocytes were first described in 1864 by August Weismann in Freiburg. Studying larvae from *Musca vomitoria*, he noted a string of cells reminiscent of a garland that was floating in the larval body cavity. Accordingly, he termed them "Guirlandenzellen" (German *Girlande* means garland) (11). The function of these cells was an utter mystery to their discoverer but not much later, in 1886, Kowalevsky intended to stain the intestinal mucosa of muscid larvae by feeding them carmine, methylene blue or silver. However, he noted that only nephrocytes took up and stored these tracers (12). He correctly concluded from the tracer experiments that these cells function like a storage kidney to clear the larval blood. This concept was later expanded by Hollande (13). The elaborate ultrastructure of *Drosophila* nephrocytes was described in the sixties of the last century (14, 15) and few years later Crossley (16) showed the size-selectivity of uptake and already noted the analogy of this ultrastructure to podocytes: membrane invaginations called labyrinthine channels are bridged by an auto-cellular slit diaphragm that controls their entry. Nephrocytes are covered by a basement membrane. Thus, two of the three layers of the glomerular filter in vertebrates are found in *Drosophila* nephrocytes (**Figures 1B** and **2A**).

After the discovery of nephrin (17), Skaer (3), Abmayr and colleagues (4), finally put the pieces together and proved a molecular analogy of podocytes and nephrocytes. They noted that the orthologs are both expressed in nephrocytes and that their absence results in nephrocyte loss of the complex invaginations and a smoothing of the cell surface (3, 4). Interestingly, this disruption of nephrocyte ultrastructure also follows treatment with puromycine (18) and protamine sulfate (7), both of which have the same effect in podocytes. Mammalian NEPH proteins have been shown to be functional within this invertebrate system (19). A most recent and surprising discovery is that nephrocytes potentially exhibit apico-basal polarity (20). Crumbs, a marker of apical polarity, was observed along the membrane of the labyrinthine channels. The presence of integrins on the nephrocyte surface may be suggestive of a basolateral compartment (7). This raises the possibility that despite being spherical cells, nephrocytes might be polarized within their complex ultrastructural morphology. Consequently, analogous to mammalian podocytes, apico-basal polarity may be a potential prerequisite for nephrocyte morphology and function (20). However, at this point it has not been ruled out that the functional role of these proteins in nephrocytes is independent from polarity.

### Endocytosis As an Essential Nephrocyte Function

It is astounding that nephrocytes can exert their function as an "excretory" organ without a connection to the outside world. This feat is performed by endocytosis. The first step is endocytic uptake of material from the blood of the fly that is called hemolymph. Nephrocytes then sort the endocytic cargo for either degradation in the lysosome or recycling back to the hemolymph. Unwanted cargo that cannot be destroyed, will be stored intracellularly. In that way, nephrocytes detoxify the hemolymph and nephrocyte dysfunction reduces survival upon toxin exposure (3). Nephrocytes thus do not contribute to urine formation in any way but nevertheless the high endocytic activtity can be regarded as "excretory" activity (**Figure 1B**). To be optimized for that purpose, they increase their surface area by forming invaginations from the cell surface called labyrinthine channels. Endocytic uptake may occur from the cell surface but the major site of endocytosis is from within the labyrinthine channels (21, 22). These regularly spaced furrows cover the entire nephrocyte surface (4, 7). The high endocytic activity attracted researchers to use nephrocytes as a model to study mechanisms of endocytosis (22–26). Endocytic uptake is at least in part dependent on the ortholog of Cubilin (7, 27, 28). In face of this conceptual similarity to the proximal tubular function, it has been proposed that nephrocytes are also a model for the proximal tubule (28). Nephrocytes have been shown to regulate the hemolymph composition through their endocytic activity (29).

### Developmental Aspects

Like podocytes, both subsets of nephrocytes are derived from the mesoderm. Differentiation and maintenance of nephrocytes requires *dKLF15* as a nephrocyte-restricted growth factor (30). *dKLF15* is the ortholog of Krüppel-Like Factor 15 which plays a role in podocyte development (31). The expression of *sns* and *kirre* and the formation of slit diaphragms begin during mid-embryogenesis and are maintained through larval development into adulthood (3, 4, 7, 32). Garland cell nephrocytes undergo a fusion process between embryonic stage E13 and E18, resulting in binucleate cells (4). For unknown reasons, the cell number decreases for both, garland cell and pericardial nephrocytes throughout development. About a quarter of the 25 garland cell nephrocytes and 120 pericardial nephrocytes that are present at the end of embryogenesis are maintained during development into adulthood (4, 7, 8, 33). Adult garland cell nephrocytes exhibit tracer uptake and show regular ultrastructural morphology, supporting these to be functional cells (7).

### Frontiers of Nephrocyte Research

Despite the considerable interest nephrocytes have gained in the recent past, we do not fully understand the significance of nephrocytes within the adult fly organism. Ivy et al. (30) recorded the life-span of adult flies that lack nephrocytes due to the absence of *Klf15* in comparison with wild-type. In contrast to the observations in larval stages, the absence of nephrocytes did not affect survival of adult flies, not even under toxin stress. Further work will be required to determine the functional role of nephrocytes in the organism of adult flies.

A secretory function of nephrocytes that is suggested by the abundance of rough endoplasmic reticulum has been shown for lysozyme (16) but this functional aspect has not been further explored.

Regarding the two subsets of nephrocytes, i.e., pericardial and garland cell nephrocytes, it has become overwhelmingly clear that both cells share critical features. This includes gene expression patterns of the slit diaphragm proteins and other genes like *dKlf15, hand, rudhira*, or *Amnionless*. On the other hand, obvious differences define them as distinct cell lineages, beginning with the number of nuclei. These differences are still not explored in detail. Currently, garland cell and pericardial nephrocytes are best considered to be complementary.

### READOUTS AND STRATEGIES OF NEPHROCYTE RESEARCH

The functional tool-set for nephrocytes is still evolving. As a consequence, there is no defined optimal strategy or established gold standard. However, a general rule emerges that a functional and a morphological assay need to be combined. We will discuss the merits and pitfalls of different strategies to give an overview.

### Tracer Endocytosis As a Readout of Nephrocyte Function

Tracer endocytosis stood at the very beginning of nephrocyte research when they enabled Kowalevski to identify clearance of the larval circulation as their functional role. Until present time, virtually any manuscript regarding nephrocytes employs tracer uptake as a functional readout. This is based on the fact that the "excretory" function of nephrocytes occurs *via* endocytosis. An analogous effect has also been shown for endogenous proteins like *imaginal disk growth factor 2* (34).

A broad array of tracers has been used since Kowalevsky, who introduced AgNO3 that is still in use today. The most commonly used tracers are proteins like GFP-derivates (6), albumin (7), avidin (26), wheat germ agglutinin (22), or Horseradish peroxidase (HRP) (16). Another common approach uses polysaccharids like dextrans (3). Less commonly employed are colloidal substances like Coomassie Brilliant Blue (35). The choice of tracer may follow personal preference but it is important to consider the basic principles for these experiments that became clear through recent findings.

The broad range of possible tracer substances is astonishing. This reflects that the uptake of at least some tracers occurs *via* the scavenger receptor Cubilin (7), whose mammalian ortholog shows affinity for a broad range of ligands as well (36). Aside from the drastic reduction of tracer endocytosis upon silencing of Cubilin/Amnionless, this concept is supported by experiments that suggest that tracer uptake occurs receptor-mediated (7). The observation by Crossley (16) that Lanthanum-dioxide and HRP both enter the labyrinthine channels, while only the latter is taken up, indicates a certain extent of selectivity for the entry mechanism which most likely reflects the receptor affinity.

Being dependent on the scavenger receptor, measurements of nephrocyte tracer endocytosis for most tracers thus do not simply reflect the surface area of nephrocytes. The interpretation of experiments applying tracer endocytosis in nephrocytes should always take possible effects on receptor abundance, function, or specificity into account.

Filtration of tracers across the nephrocyte slit diaphragm is an important concept that directly relates to the functional analogy to podocytes and thus also underlines the significance of such experiments. Crossley (16) was able to show that in nephrocytes, tracers may be excluded size-dependently by the basement membrane and the slit membrane, thus defining a cut-off of about 12 nm. More recently, receptor competition experiments revealed that this size cut-off is comparable with the mammalian glomerulus around 70 kDa (7). Basement membrane thickening occurring upon loss of slit diaphragm proteins (3) mainly affects tracers with a high molecular weight while this effect is not directly related to the nephrocyte ultrastructure or filtration. This became obvious when it was shown using a rapid intervention like protamine treatment that perturbs the ultrastructure without thickening the basement membrane. This treatment has no effect on tracers with a high molecular weight while a tracer that is below the filtration cut-off is strongly reduced (7). In light of these findings, a tracer that is smaller than 70 kDa appears to be preferable. Another potential confounding effect comes with the saturability of tracer endocytosis (7). Choosing an excessive tracer concentration or incubation period thus may even blur the effect of silencing *sns* despite using tracers that are small enough to pass the slit diaphragm (3).

The two major alternative strategies are a pulsed uptake by exposing nephrocytes to tracers *ex vivo* or using an endogenous tracer *in vivo*. The latter strategy was first described by Han and colleagues (6). It employs a transgenic fusion protein of red fluorescent protein with the atrial natriuretic factor from rat. This transgenic tracer is expressed endogenously by muscle cells, secreted into the larval circulation followed by endocytosis and degradation by nephrocytes. The fluorescence intensity of larval pericardial nephrocytes can be observed by transcutaneous imaging. This elegant approach is fast and enabled large-scale genetic screens (6). Studying nephrocytes *in vivo*, this approach appears superior to *ex vivo* interventions. On the other hand, one has to be conscious of certain limitations that nevertheless may favor a decision for an *ex vivo* setting. Imaging fluorescence through the larval cuticle may entail weak and variable signal intensity. True *in vivo* imaging is complicated by larval movements that usually require measures to reduce larval motility. The continuous tracer expression furthermore inherently renders the net result from uptake and degradation as readout. A block in degradation and an increase in uptake thus may have the same outcome. Possible variations in tracer production, distribution, and alternative degradation are difficult to control. The *ex vivo* incubation on the other hand, is able to exclusively reflect rapid uptake under controlled conditions with direct imaging of the nephrocytes. These experiments do not require the presence of a transgene that controls the expression of the endogenous tracer and thus can be immediately applied in any genetic background that allows larval survival. This approach further allows the combination with additional interventions like drug exposure. The dissection of garland cell nephrocytes can be accomplished within few seconds which minimizes the impact of an *ex vivo* setting. An intermediate strategy by dissecting nephrocytes from transgenic animals that express a tracer endogenously may allow to combine some of the advantages of both approaches like speed and precision of direct imaging. Then again, this also retains disadvantages from both sides like the confounders entailed by continuous tracer production and the invasiveness of dissection. In summary, application of either strategy needs to be tailored to the experimental question and the available resources.

Finally, an important consideration for any uptake experiment is that it is hard to interpret without additional morphological studies. As we can learn from loss-of-function of endosomal proteins, reduced tracer uptake may occur while the slit diaphragms are maintained (25, 26) (or potentially also *vice versa* in different settings).

### Electron Microscopy As a Readout of Nephrocyte Morphology

The major advantage of nephrocytes lies in the formation of functional slit diaphragms that are easily accessible. These slit diaphragms can be observed directly by transmission electron microscopy (TEM) providing the most unequivocal evidence. That way the ultrastructural analysis is an important complement for the tracer studies whose interpretation can be more difficult but that still are indispensable as a rapid functional assay. Although the technique is well established after many decades of research, study of nephrocytes still holds challenges that entail the complex ultrastructural architecture of these cells. The labyrinthine channels are nearly parallel furrows that are observed across the whole nephrocyte surface (**Figure 2**). The slit diaphragms seal these invaginations like a ceiling. Tangential sections through slit diaphragms therefore impress as parallel lines (4, 7) (**Figure 2B**). The interpretation of equatorial cross sections is more difficult, as they are dependent on their orientation in regard to the lines formed by the slit diaphragm. A crosssection that cuts through the slit diaphragms at an approximately perpendicular angle renders the classical image (**Figure 2A**). However, if the section cuts through the parallel lines of the slit diaphragms at an angle that is too oblique (**Figure 2C**), the slit diaphragms and labyrinthine channels will be distorted and difficult to identify. For that reason, such a section may even be mistaken for a smoothing of the nephrocyte surface. However, these oblique sections are marked by elongated stretches of electron-density (**Figure 2C**) that clearly distinguish them from a true phenotype. Unfortunately, examples for this error can even be found in the literature.

Analogous to the observations regarding the mammalian slit diaphragm (37), one can assume that fixation and sample preparation may influence the resulting image.

Scanning electron microscopy (SEM) can be most useful to gather information regarding cell shape and number (4, 19), while this may be achieved by using approaches involving light microscopy as well. The assessment of the nephrocyte ultrastructure through this approach is complicated as the labyrinthine channels are concealed by the basement membrane. Specific protocols like protease treatments can overcome this limitation but need to be considered carefully to avoid artefact.

### Immunofluorescence (IF) As Complementary Strategy

Immunofluorescence of slit diaphragm proteins facilitated the discovery of nephrocytes as a model for glomerular diseases (3, 4). Still, this assay is regarded a complementary strategy that was omitted in a number of manuscripts. A significant recent observation concerns highly resolved tangential confocal sections of nephrocytes stained for Sns or Kirre. These stainings reveal a fingerprint-like pattern that correlates with the picture obtained in tangential sections in TEM (**Figure 2A** inset) (7). This not only implies that findings obtained with TEM can be backed up by stainings of the slit diaphragm proteins but also that significant information about ultrastructural aberrations can be obtained before applying TEM. Combined with the opportunity of co-IF, this allows a more comprehensive analysis for a number of questions. IF with 3D reconstruction also revealed that the entire cell body is covered by slit diaphragms (7). In a similar fashion, IF can be regarded as an alternative to SEM.

It remains to be seen, if super resolution imaging using stimulated emission depletion microscopy or stochastic optical reconstruction microscopy will be able to render additional information.

### Readouts of Mortality

Observing survival under the exposure to toxins like silver nitrate in larvae has been described as a possible assay that directly relates to nephrocyte function. That way it may be a suitable complement for tracer strategies that documents the effect on the larval physiology. Another assay that has been introduced more recently is to record adult life-span (38). This appears a logical addition to the spectrum of investigations. However, at this point in time, it is difficult to connect reduced adult survival with nephrocyte functions due to the findings by Hartley and colleagues (30). They did not observe an altered life-span when nephrocytes were even entirely absent entirely due to lack of *dKlf15*. Potential confounders for fly survival can result from expression in other tissues. The commonly used *GAL4*-drivers to control expression of RNAi do not follow anatomic structures but are dependent on gene expression patterns. It is very difficult to rule out that temporary or very limited expression in other tissues contributes to a broad compound readout like survival. At least until the role of nephrocytes in the physiology of the adult animal is better understood, in our opinion it appears prudent to rather make use of assays that are more specifically tailored to nephrocytes.

### Loss-of-Function Strategies

The virtually genome-wide availability of cost-effective, offthe-shelf RNAi libraries is a major advantage of *Drosophila* as a model system. Loss-of-function strategies that employ RNAi are inherently limited by potential off-targets or inefficient knockdown. This may result in contradicting results between different RNAi-transgenes. A common strategy in the *Drosophila* field consists in analyzing at least two independent RNAi stocks to confirm the findings. Further confirmation often remains desirable. This can be achieved by utilizing genetic null alleles. However, lethality frequently impedes this strategy like in the case of *sns*. Nephrocytes are not amenable to the classic mosaic analysis in *Drosophila* as they are non-dividing cells. The CRISPR/Cas9 technology allows to introduce a conditional loss-of-function that is not RNAi dependent (7). This can be accomplished by transgenic *Drosophila* that combine nephrocyte-restricted expression of Cas9 in conjunction with ubiquitous expression of a directed guide RNA. In this approach, individual cells may have divergent phenotypes dependent on individual CRISPR/ Cas-induced mutations. Possible draw backs of this strategy are off-targets, inefficient gene disruption, and persistence of mRNA. Therefore, it is reasonable to either use two independent guide RNAs or combine this with an RNAi approach. For both methods, RNAi and CRISPR/Cas, loss-of-function best is further verified, e.g., by IF using a specific antibody or rescue experiments with an RNAi/CRISPR-resistant transgene.

A number of different *GAL4*-driver lines have been utilized to control the expression of transgenes to induce loss-offunction in nephrocytes. These include *Dorothy*-*GAL4* (6, 39), *sns*-GCN-*GAL4* (4, 40), and *Hand*-*GAL4* (41). While these lines express *GAL4* in both subsets of nephrocytes, the equally common *prospero*-*GAL4* (3, 42) has predominantly been used for garland cell nephrocytes. A garland cell nephrocyterestricted expression has been described for *Aug21*-*GAL4* (43). All commonly used driver lines appear to exhibit robust expression during the larval stage. *G447.2-GAL4* (3) was used to direct expression in embryonic garland cell nephrocytes. *Dorothy*-*GAL4* (7, 44, 45), *Hand-GAL4* (46), and *sns-*GCN*-GAL4* (47) have been employed in nephrocytes dissected from adult animals. A systematic comparison of the available *GAL4* lines regarding onset, continuation, and intensity of transgene expression is currently lacking. The same is true for a characterization of the extra-nephrocytic expression that almost certainly occurs in all the *GAL4*-drivers.

## NEPHROCYTE AS DISEASE MODEL

### Monogenic Forms of Nephrotic Syndrome

*Drosophila* shows a surprising extent of conservation of disease genes and this model organism has proven to be appropriate for the investigation of monogenic diseases. These disorders are characterized by mutation of a single gene as the molecular cause of a disease. The role of nephrocytes as a model for genetic kidney disease has recently been reviewed in more detail (48). Nephrocytes are suitable to study the functional role of candidate genes that were identified in genetic studies and also elucidate the involved functional pathways which is facilitated by the low redundancy of the fly genome. The speed, cost-effectiveness, and reliability of this model make it well equipped to handle the considerable number of candidate variants that may result from contemporary sequencing efforts like whole exome sequencing. Beyond that, nephrocytes can also be applied to assess the significance of individual candidate mutations using transgenic rescue constructs. In the near future, *Drosophila* nephrocytes may further facilitate the development of targeted therapies and personalized medicine.

The most extensive work in *Drosophila* nephrocytes concerns steroid resistant nephrotic syndrome (SRNS). This disorder is characterized by childhood onset of edema, massive proteinuria, and progression to ESRD. SRNS is the second most frequent cause of ESRD within the first two decades of life and monogenic mutations explain a significant fraction of cases that manifest before 25 years of age (49). By now, more than 50 different genes have been implicated as a single gene cause for this disorder (50, 51) and more genes are likely to be discovered. The large number of known SRNS genes prompted systematic analyses that showed that about 60–80% of the studied orthologs of human disease genes resulted in a reduction of tracer uptake (7, 38) including genes that are involved in the slit diaphragm complex, actin regulation, CoQ10-biosynthesis, or interaction with the extracellular matrix. This bears testimony to the surprising extent of evolutionary conservation and underlines that central pathomechanisms of podocytopathies can be studied in nephrocytes. This is most obvious for *COQ2*-nephropathy (52) where findings in *Drosophila* indicate formation of reactive oxygen species as a critical event in the pathogenesis. Successful drug treatment of flies further provides evidence for Vanillic acid as a potential treatment (7). Importantly, these data further support that *COQ2*-nephropathy may be treatable (53) and that treatment strategies can be tested in nephrocytes. Some of these findings were confirmed in a recent manuscript that furthermore implicated autophagy and mitophagy as pathomechanistically relevant (54).

For the identification of several of the SRNS genes, studies in nephrocytes were instrumental by providing functional evidence supporting the genetic findings. This includes the KANK proteins (55), *ARHGDIA* (56), *ADCK4* (57), and most extensively *SGPL1* (58). For the ortholog of *SGPL1*, rescue constructs that reflect the mutations from SRNS patients were shown to be inefficient in contrast to a rescue with wild-type constructs. *Drosophila* nephrocytes also exhibited an altered lipid metabolism reflecting the observations in the SRNS patients.

### Diabetic Nephropathy

Diabetic nephropathy represents the single most significant cause of ESRD (1). Targeted therapies are unavailable and the current regimes are only sufficient to slow down the progress of disease. It is thus noteworthy that nephrocytes were found to be a model for diabetic nephropathy (59). High-glucose diet was shown to induce nephrocyte dysfunction and decrease the amount of Sns, which represents the ortholog of nephrin. The authors identified a transcriptional downregulation by a pathway that includes *Knot*, the ortholog of *EBF2*, to be responsible for the reduction of Sns in nephrocytes. Interestingly, the authors were able to validate their findings in *Drosophila* nephrocytes in mouse models of diabetic nephropathy.

### *APOL1-*Associated Nephropathies

Incidence of ESRD is nearly fourfold in African Americans compared with those of European descent. Much of the excess risk is attributable to two risk alleles of the *APOL1* gene. The study of *APOL1* is hampered by its poor evolutionary conservation as orthologs are lacking in most model organisms. Transgenic expression of the human *APOL1* in nephrocytes has been shown to result in a cellular toxicity that was more pronounced upon expression of the human *APOL1* risk alleles (44, 45). Nephrocyte dysfunction and loss was observed with increasing age of the flies, involving a mechanism connected to endosomal trafficking. More recently, the findings in *Drosophila* were confirmed by transgenic expression of the human gene in mouse (60). Essential features of the human disease were recapitulated in this model. It is intriguing, that the role of a common risk allele was studied successfully in *Drosophila* nephrocytes.

### Endocytosis and Proximal Tubular Diseases

Imerslund–Gräsbeck syndrome is characterized by Cobalamindeficiency and tubular proteinuria while renal function is usually preserved. Mutations of Cubilin (61) and Amnionless (62) have been shown to be causative for this disorder. The orthologs of both genes are expressed in *Drosophila* nephrocytes, exercising an analogous role for nephrocyte function (28). Megalin, that may cause Donnai–Barrow syndrome (63) that also involves tubular proteinuria in humans, does not seem to be required for nephrocyte function (7, 27). A megalin-indepdendent role of Cubilin/Amnionless has also been described in humans for the absorption of vitamin B12 in the terminal ileum (64).

### Organ Crosstalk

*Drosphila* is an appropriate model to study communication between organs. In an effort to identify renal cardiomodulatory factors, Hartley and colleagues induced nephrocyte disruption and observed signs of a cardiomyopathy in the fly. Analyzing the hemolymph composition, they were able to identify *secreted protein acidic and cysteine rich* (SPARC), whose circulating levels are downregulated by nephrocytes. Reducing the gene dosage of this factor independently, through a heterozygous null allele, ameliorated the cardiomyopathy. Interestingly, *SPARC* has been linked to cardiac aging and metabolic syndrome in humans.

### Translational Applications

The genetic tool-kit of Drosophila combined with the costeffectiveness and speed inherent to this model offer great opportunities for a translational approach. Only a glimmer of these opportunities can be recognized in the effect of Vanillic acid on defective CoQ10-biosynthesis (7). Screening platforms have been introduced that already enable large-scale genetic screens. These protocols need to be optimized to facilitate the discovery of treatment options for glomerular diseases.

### OUTLOOK

We have seen significant progress in the last decade, and the nephrocyte currently emerges from the phase of experimental establishment to a driving force of glomerular discovery research. Combining a functional, accessible slit diaphragm with the power of the genetic tool-kit in *Drosophila*, the nephrocyte as a complementary model system is well equipped to reveal mechanisms of podocyte function and glomerular diseases. Once the enormous potential for translational applications is unlocked, the nephrocyte will play its role in the identification of targeted therapies that are urgently needed in nephrology.

### REFERENCES


### AUTHOR CONTRIBUTIONS

The manuscript was written by TH with help from MH, MH designed the figures with help from TH, TBH critically reviewed and edited the manuscript and gave relevant additional insight. The title was suggested by TBH.

### ACKNOWLEDGMENTS

TH is supported by the Deutsche Forschungsgemeinschaft (DFG, HE 7456/1-1, CRC 1140/associated member) and the Faculty of Medicine of the University of Freiburg. TBH is supported by the DFG (CRC 1140, CRC 992, HU 1016/8-1), by the BMBF (01GM1518C), by the European Research Council—ERC grant 616891, and by the H2020-IMI2 consortium BEAt-DKD. We thank Maria Ericsson (Harvard Medical School Electron Microscopy Facility) for technical assistance.

is mutated in congenital nephrotic syndrome. *Mol Cell* (1998) 1:575–82. doi:10.1016/S1097-2765(00)80057-X


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Helmstädter, Huber and Hermle. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Zebrafish as a Model for Drug Screening in Genetic Kidney Diseases

#### Jochen Gehrig<sup>1</sup> , Gunjan Pandey 1,2 and Jens H. Westhoff <sup>2</sup> \*

<sup>1</sup> Acquifer is a Division of Ditabis, Digital Biomedical Imaging Systems AG, Pforzheim, Germany, <sup>2</sup> Department of Pediatrics I, University Children's Hospital Heidelberg, Heidelberg, Germany

Genetic disorders account for a wide range of renal diseases emerging during childhood and adolescence. Due to the utilization of modern biochemical and biomedical techniques, the number of identified disease-associated genes is increasing rapidly. Modeling of congenital human disease in animals is key to our understanding of the biological mechanism underlying pathological processes and thus developing novel potential treatment options. The zebrafish (Danio rerio) has been established as a versatile small vertebrate organism that is widely used for studying human inherited diseases. Genetic accessibility in combination with elegant experimental methods in zebrafish permit modeling of human genetic diseases and dissecting the perturbation of underlying cellular networks and physiological processes. Beyond its utility for genetic analysis and pathophysiological and mechanistic studies, zebrafish embryos, and larvae are amenable for phenotypic screening approaches employing high-content and high-throughput experiments using automated microscopy. This includes large-scale chemical screening experiments using genetic models for searching for disease-modulating compounds. Phenotype-based approaches of drug discovery have been successfully performed in diverse zebrafish-based screening applications with various phenotypic readouts. As a result, these can lead to the identification of candidate substances that are further examined in preclinical and clinical trials. In this review, we discuss zebrafish models for inherited kidney disease as well as requirements and considerations for the technical realization of drug screening experiments in zebrafish.

Keywords: zebrafish, drug screening, compound screening, genetic kidney disease, high-throughput, highcontent, automated microscopy

### INTRODUCTION

Modern genetic diagnostics allow the rapid discovery of human disease-associated mutations. Moreover, human genetic disorders can often be mimicked in animal models that can be exploited in large-scale chemical investigations for the search of modifiers of disease-associated phenotypes and potentially therapeutic compounds. The zebrafish (Danio rerio) has become an increasingly accepted vertebrate model organism for biomedical research (1, 2).

Despite being a member of the teleost class of fish species, there is great homology in development as well as cell- and organ-specific structural and physiological properties between zebrafish and humans. Furthermore, even with the evolutionary distance, > 80% of human disease-associated genes have orthologs in the zebrafish genome (3). The embryonic and larval

#### Edited by:

Miriam Schmidts, Radboud University Nijmegen, Netherlands

#### Reviewed by:

Ruxandra Bachmann-Gagescu, Universität Zürich, Switzerland Rachel Lennon, University of Manchester, United Kingdom Rebecca Ann Wingert, University of Notre Dame, United States

### \*Correspondence:

Jens H. Westhoff jens.westhoff@med.uni-heidelberg.de

#### Specialty section:

This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics

Received: 15 February 2018 Accepted: 04 June 2018 Published: 28 June 2018

#### Citation:

Gehrig J, Pandey G and Westhoff JH (2018) Zebrafish as a Model for Drug Screening in Genetic Kidney Diseases. Front. Pediatr. 6:183. doi: 10.3389/fped.2018.00183

**74**

characteristics of zebrafish include small size, ex utero development, optical transparency, and rapidity of organogenesis. In combination with the high fecundity of adult zebrafish and a relatively simple and cost-effective animal husbandry, this enables large-scale in vivo investigations. The zebrafish genome has been completely sequenced, thus facilitating genetic and genomic analysis and manipulation (3, 4). For instance, reverse genetics allow for precise investigation of associated phenotypes, by e.g., transient gene knockdown using antisense morpholino oligos or by genome-editing technologies like the CRISPR/Cas9 system (5, 6).

Due to the simplicity of the pronephros that can be readily studied in embryonic and larval stages, the zebrafish is an applicable experimental model system for the analysis of renal development and disease (7). The pronephros, as the earliest nephric stage, contains two nephrons sharing numerous genetic, structural, and functional aspects with the mammalian nephron (8). Phenotypic changes upon genetic alterations can be easily analyzed within intact live animals (9). Large-scale mutagenesis screens have identified various mutants affecting kidney development allowing the exploration of genetic and molecular mechanisms underlying pronephros development and function (10, 11). Moreover, reverse genetics approaches enable researchers to specifically alter orthologous genetic elements potentially associated with human disease. To date, major fields of research where such zebrafish models are being employed include glomerular (i.e., podocytopathies) and cystic renal disorders (i.e., ciliopathies).

Phenotype-based screening for drug discovery applications is increasingly employed in biomedical and pharmaceutical research. In contrast to target-based screening, phenotype-based approaches do not require exact knowledge of the therapeutic target (7). In addition, whole-organism in vivo approaches have the advantage that they can unravel toxic and other side-effects of drugs at a very early stage of the study. Over the last years, due to the versatility and power of the model, the zebrafish has emerged as the main vertebrate model system for high-throughput and high-content chemical screening experiments and large-scale phenotypic scoring (12, 13). Clear and scalable readouts for in vivo large-scale experiments can be readily established and a plethora of mutant and transgenic models expressing fluorescent proteins driven by tissue-specific promoters is available. In combination with automation technologies and dedicated sample handling workflows, this has led to various biomedical screening assays in fields such as genetics (14, 15), toxicology (16, 17), immunology and infection biology (18, 19), cardiovascular research (20, 21), drug discovery and safety (12, 13, 22, 23), personalized medicine (24), non-coding-genome analysis (25) as well as behavioral analysis (26, 27). Notably, several compounds that were identified in the zebrafish model have made it to preclinical and clinical trials, including new substance classes and repurposed drugs (12). For instance, in a chemical genetic screen testing 2.480 compounds, prostaglandin E2 (PGE2) was identified as an evolutionarily conserved regulator of hematopoietic stem cell (HSC) number in zebrafish embryos (28). Based on these results, a chemical derivative of PGE2 (Prohema), has been developed with the aim of improving the efficiency of HSC transplants using umbilical cord blood. Prohema has meanwhile advanced to Phase II clinical trials.

### GENETIC KIDNEY DISEASES IN ZEBRAFISH

Genetic kidney diseases can affect all parts of the kidney and its functions. To date, mutations in more than 150 genes have been identified that cause genetic human kidney diseases such as alterations of kidney development or specific glomerular and tubular diseases (29). Nephrogenesis in vertebrates is an intricate process that includes the successive formation of up to three kidneys depending on the species position in the phylogenetic tree: the pronephros, mesonephros, and metanephros (30). There is an increasing complexity with each successive kidney developing, however the structure and function of the basic renal units, the nephrons, remains largely unvaried across vertebrates (31).

In zebrafish, major vertebrate organ systems form within a few days after fertilization (32). The zebrafish pronephros is functional by 48 hpf (hours post fertilization) accomplishing the functions of blood filtration and osmoregulation (11, 33). It consists of two nephrons with a fused glomerulus at the midline (**Figure 1A**) (24). The tubular system consisting of the pronephric proximal and distal tubule and pronephric duct contains segment-specific conserved structural and physiological properties and spatio-temporal gene-expression patterns that are homologous to the human kidney (**Figure 1B**) (8, 35, 36). The zebrafish glomerulus is endowed with podocytes with extended and interdigitating foot processes and fenestrated endothelial cells forming a functional glomerular filtration barrier, analogous to the metanephric glomerulus of higher vertebrates (11).

At present, zebrafish is predominantly used as a genetic model for normal and abnormal kidney development, for hereditary glomerulopathies (i.e., podocytopathies) and for the study of ciliopathy-associated human cystic kidney diseases. These encompass polycystic kidney diseases and diseases of the nephronophthisis/medullary cystic kidney disease complex including more complex ciliopathies such as Joubert Syndrome, Meckel-Gruber Syndrome, and Bardet-Biedl-Syndrome (37). In this review, we focus on glomerulopathies and cystic kidney diseases.

### ZEBRAFISH AS A MODEL FOR HUMAN HEREDITARY GLOMERULOPATHIES

Zebrafish models can recapitulate human genetic glomerulopathies, i.e., a variety of podocytopathies that clinically often manifest by steroid-resistant nephrotic syndrome (SRNS) due to podocyte foot process effacement. SRNS is mostly therapy-resistant and leads to end-stage renal disease (ESRD) within a few years of onset. A growing number of SRNScausing mutations have been identified. For example, mutations in NPHS1, encoding Nephrin, cause congenital nephrotic syndrome of the Finnish type. Morpholino knockdown of nphs1 in zebrafish results in edema and loss of slit-diaphragms with

organization of each nephron into glomerulus (G), neck (N), proximal convoluted tubule (PCT), proximal straight tubule (PST), distal early (DE), corpuscle of Stannius (CS), distal late (DL), and pronephric duct (PD) that fuse

to the cloaca (C). Adapted from Wingert and Davidson (34).

abnormal podocyte foot processes (38, 39). Mutations in WT1 (Wilms' tumor gene 1) have been associated with syndromic disorders such as Denys-Drash syndrome and Frasier syndrome, but also with diffuse mesangial sclerosis and early-onset isolated nephrotic syndrome (40). In zebrafish, knockdown of wt1a results in defects in podocyte development leading to glomerular injury and nephrosis (41). Mutations in NPHS2, Podocin, are the most relevant cause of autosomal-recessive SRNS of childhood. Zebrafish nphs2 morphants display pronephric glomerular hypoplasia with pericardial edema and ultrastructural glomerular damage of the filtration barrier (38, 39). Mutations in PLCE1 (Nephrocystin-3, NPHS3) have been identified in patients with SRNS and disease onset in the first year of life with a rapid progression to ESRD (42, 43). Zebrafish plce1 morphants display an impairment of the kidney filtration barrier as measured by tubular uptake of filtered 500 kDa fluorescent dextran, accompanied by edema, and severe disorganization of slit diaphragms (43). Other rare human mutations that were mimicked in zebrafish include ADCK4 (AarF domain containing kinase 4 gene) (44), KANK1 (kidney ankyrin repeat-containing protein 1), KANK2, KANK4 (45), CRB2 (Crumbs homolog 2) (46), NUP107 (Nuclear Pore Complex Subunit 107) (47), and ARHGDIA (48).

Whereas in many studies of genetic glomerulopathies the zebrafish has been used to model human disease-associated phenotypes, disruption of the glomerular filtration barrier can only be visualized by ultrastructural techniques like superresolution or electron microscopy that are not compatible with large-scale chemical screens. Edema formation in zebrafish embryos can indirectly report glomerular barrier impairments; however, despite being easily observed in brightfield microscopy it is not exclusively linked to renal impairment (49). This restricts its value as a phenotypic readout parameter in chemical kidney screens. Functional assessment of glomerular filtration and barrier integrity can be achieved by monitoring the temporal reporter activity after microinjection of fluorescently labeled inulin (50) or dextrans of different molecular weight (51–53) into the vascular system; however, this method is laborious and incompatible with extensive screening experiments. Additionally, a transgenic zebrafish that expresses GFP (green fluorescent protein)-tagged vitamin D-binding protein (VDBP), which acts as a tracer for proteinuria, has been reported (54) and may serve as an attractive alternative for high-content and high-throughput screening (53).

### ZEBRAFISH AS A MODEL FOR HUMAN CYSTIC KIDNEY DISEASES

Cystic diseases of the kidney are frequent monogenic disorders in humans (55, 56), with primary cilia dysfunction being the unifying cellular mechanism leading to most if not all cystic kidney diseases (56–58). Mutations in a variety of genes encoding the primary cilia/centrosome complex cause ciliopathies often associated with the development of renal cysts, in both human and zebrafish (59–64). However, it must be noted that cilia in the zebrafish pronephros are motile, whereas human renal cilia are thought to be non-motile (65, 66), suggesting a potential contribution of lack of fluid dynamics to cyst formation in the zebrafish model.

Mutations in polycystin-1 and polycystin-2 are responsible for autosomal dominant polycystic kidney disease (ADPKD), the most common human congenital renal disorder (67). In zebrafish, polycystin-2 morpholino knockdown or mutation of orthologous pkd genes induces kidney cysts, hydrocephalus, left/right asymmetry defects, and strong dorsal axis curvature (63, 68, 69). Autosomal recessive polycystic kidney disease (ARPKD) usually manifests perinatally or in childhood. In addition to PKHD1 (polycystic kidney and hepatic disease 1), mutations in DZIP1L (DAZ interacting zinc finger protein 1 like) have recently been associated with ARPKD (70, 71). Other than dzip1l morphants and dzip1l CRISPR mutants, Lu et al. injected the dzip1l translation-blocking morpholino into Tg(wt1b:egfp) transgenic embryos expressing GFP under Wilms' tumor suppressor (wt1b) promoter in the pronephros (72), allowing for in vivo fluorescence imaging of the cystic pronephros.

Nephronophthisis (NPHP), an autosomal recessive cystic kidney disease, represents the most frequent genetic cause of ESRD in the first three decades of life (55). Nephronophthisis can be accompanied by anomalies in other organs, such as cerebellar vermis hypoplasia, laterality defects, intellectual disability, shortening of bones, retinal degeneration, and hepatobiliary disease (56). These features are represented in a variety ofsyndromes, including Senior–Løken syndrome, Joubert syndrome, Bardet–Biedl syndrome, and Jeune asphyxiating thoracic dystrophy (73, 74). To date, NPHP-causing mutations have been identified in more than 20 genes (56). Morpholino knockdown in zebrafish has been performed for nphp2 to 6 (75–83) and nphp11 (84). Zebrafish mutants for arl13b/scohi<sup>459</sup> (85), ahi1lri<sup>46</sup> (86), and cc2d2a (87) develop pronephric cysts to varying degrees and serve as models for Joubert syndromerelated disease.

Intraflagellar transport (IFT) proteins are essential for the development and maintenance of motile and sensory cilia and localize to the cilium, basal body, and/or centrosome (88). Several zebrafish ift mutants demonstrating renal cysts were identified by forward genetic screens (63, 89). IFT80 mutations underlie a subset of Jeune asphyxiating thoracic dystrophy cases, of which 20% are associated with kidney abnormalities including renal cysts (90). Ift80 morphants show a dose-dependent phenotype with strong body curvature, large kidney cyst, and pericardial edema. IFT172 mutations were initially reported in Jeune and Mainzer-Saldino syndromes, but have also been observed in patients with Bardet-Biedl syndrome (91, 92). Zebrafish mutants and morphants for ift172 resemble these phenotypes, with renal cysts readily detectable in brightfield images (91). In our work, we have shown that morpholino knockdown of ift80 and ift172 in Tg(wt1b:egfp) with fluorescently labeled kidney structures allow for visualization of pronephric cysts, providing a model system for large-scale chemical screening studies to identify chemical modifiers of cyst formation (93).

### CHEMICAL SCREENING IN ZEBRAFISH—TECHNICAL ASPECTS AND CONSIDERATIONS

The zebrafish can cost-effectively bridge the gap between high-throughput experimentation in cellular models lacking physiological context and low-throughput models such as rodents that are closer to human biology (12, 13, 23, 94). The optically transparent embryos and larvae fit into wells of microtiter plates rendering them amenable for automated microscopy applications using existing screening technologies (12, 13, 17, 23). Due to accessibility to genetic manipulation (37, 95), a plethora of zebrafish transgenic and mutant lines has been generated (www.zfin.org, www.ezrc.kit.edu, www.zebrafish.org) (96), complemented by transient labeling, knockdown and genome-editing techniques (97). This provides an extraordinary rich toolkit to model and visualize the biological processes underlying development and disease. Several transgenic lines labeling pronephric structures such as podocytes (98, 99), tubules, and ducts (98, 100–106) or both (72) have been established. In combination with genetic alteration, either by transient gene knockdown using antisense morpholino oligos or by genome-editing technologies, these lines can mimic e.g., ARPKD (71), ADPKD (107), and other cystic kidney diseases (93, 108) and enable in vivo fluorescence microscopy of the diseased pronephros. Although controversies exist regarding the use of morphants generated by morpholino knockdown (109–113), they remain a valuable tool in altering target gene expression, provided that all mandatory control experiments to validate the observed disease-associated phenotypes have been conducted (110, 111).

To our knowledge, large-scale screening experiments evaluating fluorescent pronephric phenotypes in models of genetic kidney diseases have not been demonstrated. In a chemical modifier screen using a custom library of 115 compounds in pkd2hi<sup>4166</sup> and ift172hi<sup>2211</sup> mutants displaying renal cysts (114), morphological parameters such as body axis curvature and/or laterality defects were scored. Histone deacetylase inhibitors Tricostatin A and valproic acid attenuated these phenotypes, and cyst size-reducing effects were confirmed in secondary assays. Additionally, a chemical screen of ∼2,000 compounds identified histone deacetylase inhibitors to expand the pool of embryonic renal progenitor cells (115), a mechanism presumably involved in regeneration following acute kidney injury.

In combination with automated microscopy (116), zebrafish disease model systems allow performing large-scale phenotypic whole organism screening assays (117, 118). Phenotypic readouts encompass survival rates, overall morphology, physiological parameters, cell- and tissuespecific phenotypes, reporter gene expression patterns, and behavioral phenotypes (25, 27, 93, 119–123). Phenotypic screens within the context of a live vertebrate provide significant advantage over classical target-based in vitro assays as they do not require a priori knowledge about biochemical pathways affected, thus allowing unbiased identification of drug candidates or toxicological effects of substances. Furthermore, they intrinsically involve potential contributions of biodistribution, metabolism, and pharmacokinetics (1, 12, 13, 17, 95, 124).

Prior to large-scale zebrafish experiments careful planning and preparatory experiments are required to generate robust protocols and tailor conditions toward desired readouts in screening compatible disease models. In brief, handling of thousands of embryos causes logistical challenges, thus protocols for animal husbandry, micromanipulation, embryo culture, and treatment (e.g., anesthesia), and sample handling must be established. When image-based assays are carried out the usage of pigmentation mutant strains (e.g., casper line) or chemical treatment (e.g., 1-phenyl-2-thiourea, PTU) to block pigment formation is often necessary to adequately visualize internal structures. Moreover, to minimize false positive and negative results, non-specific developmental toxicity or off-target effects, control experiments must be carried out to titrate required compound concentration ranges as well as the treatment period during embryonic development. Importantly, controls should also be continuously carried out as a reference readout to benchmark observed phenotypic effects and normalize experimental variation. Finally, image acquisition routines must be balanced with analysis

(left panel) or 150µm (right panel). (E) Detailed visualization of kidney regions enabling scoring of kidney phenotypes. Shown are wildtype (first row) or cystic (other rows) kidneys of 72 hpf Tg(wt1b:egfp) embryos. (F) Automated quantitative analysis and phenotypic scoring using image processing techniques. Heatmap shows quantitative measurements of cystic areas as shown in (E). Figure panels are taken or modified from Westhoff et al. (93), Wittbrodt et al. (125), Pandey et al. (unpublished), and www.acquifer.de.

needs to ensure effective and robust scoring of phenotypic alterations.

Despite its amenability to large-scale experimentation, the full exploitation of the zebrafish model in screening assays is often hampered. While sample manipulation can be scaled efficiently, large-scale imaging, and phenotypic scoring remains challenging as available screening methodologies are usually optimized for in vitro assays (**Figure 2A**) (126). In comparison to cellular models, zebrafish embryos are large three-dimensional objects of complex morphology leading to random orientation of embryos within wells of microtiter plates (126, 127). This can obscure the view on target structures and leads to the generation of non-standardized image data. Therefore, novel sample preparations or automation strategies are needed, as it is unfeasible to upscale classical zebrafish mounting techniques. In our work, we developed orientation tools allowing the generation of agarose cavities within wells of microtiter plates for consistent positioning and orientation of zebrafish embryos (**Figure 2B**) (25, 93, 125). This enables the automated acquisition of consistent views of 48–96 hpf zebrafish larvae in large-scale screening scenarios (**Figure 2C**). For instance, we employed that methodology for imaging of embryonic kidneys in automated large-scale microscopy assays to score for morphological alterations of the pronephros upon compound exposure (93, 125, 128) (Westhoff et al. unpublished data) or capture phenotypic changes in cystic kidney disease models (129) (Pandey et al., unpublished data). Other more complex technical solutions employ microfluidic systems that combine automated detection and rotational orientation within glass capillaries followed by microscopic imaging (127, 130).

To date, the vast majority of zebrafish screens employ low magnification to capture the entire zebrafish embryo body, followed by subsequent analysis of phenotypic changes (13, 131). However, this significantly attenuates the power of using a fully developed live vertebrate embryo as in vivo visualization of morphological, physiological or genetic events on the cell- or tissue-specific level is hampered. The widespread usage of low resolution assays is largely due to the impracticality of positioning the regions of interest (ROI) within the small field of views of higher magnification objectives in combination with fixed scan-field coordinates of automated microscopes. Additionally, the spatiotemporal location of ROIs within the embryo body might be variable or unpredictable. To overcome that limitation, technologies have been developed that allow the automatic centering of ROIs in front of objective lenses of microscopes. Microfluidic systems (127, 130) can fulfill that requirement but usually require rather complex setups and are potentially challenged when overall morphological changes occur, or developmental and disease-associated processes are observed in time-lapse experiments. Several automated microscopy solutions for microplate-based screening have been reported that allow to teach or detect the position of ROIs followed by automatic centering and multidimensional image acquisition (126, 132– 134). This can be an expert operator manually selecting target structures for subsequent automated imaging or more advanced methods employing automatic detection by image processing. These automated smart imaging approaches are based on interfacing the imaging device with external software tools that automatically detect coordinates of features of interest and send back machine commands containing instructions for re-centering, higher resolution imaging, or tracking of target structures. While several solutions have been reported, their application is usually restricted to cellular models and they are often characterized by a high level of complexity requiring expert knowledge in image processing, general programming and hardand software interfacing. Therefore, to enable a widespread usage of such toolsets novel developments are needed that provide a simplified access to biomedical researchers. To this extent, we have developed a robotic microscopy platform (www.acquifer. de) with a smart imaging interface that allows to manually select ROIs, or to use any image processing software to send back human-readable script commands to the imaging device. We utilize this technique to e.g., acquire high-resolution datasets of cystic kidneys in a genetic zebrafish disease model to screen for modifiers of cystogenesis (**Figures 2D–F**) (129) (Pandey et al., unpublished data).

Due to the wide variety of potential zebrafish screening assays and thus phenotypic readouts there are myriad of potential quantitative descriptors that can be extracted from image-based datasets. This can include fluorescence intensity, morphological descriptors, or dynamic parameters and ranges from simple whole embryo signal intensity to spatiotemporal activity of fluorescent reporters or tissue dynamics and beyond. For instance, advanced segmentation techniques were used in the Tg(cdh17:egfp)pt<sup>305</sup> zebrafish line to detect the fluorescently labeled tubular cells of the kidney (105). A full discussion of analysis strategies and development of automated image processing pipelines is beyond the scope of this manuscript (135). However, as post-acquisition analysis strategies are of vital importance for the success of any screening assay, the design of scoring pipelines needs careful consideration at early stages of experimental planning.

### CONCLUSIONS

Phenotype-based, cost-effective whole-organism chemical screening in zebrafish offers a variety of advantages including the identification of disease-modifying drugs without knowledge of a validated target, the potential to identify compounds with polypharmacological efficacy, and the simultaneous assessment of compound efficacy, toxicity, biodistribution, and pharmacokinetics within a vertebrate model system. While a growing number of genes are being identified to cause human kidney diseases, therapeutic options to combat these diseases are often absent. Ideally, the use of genetically modified zebrafish mimicking human genetic disorders in conjunction with kidney-specific transgenic reporter lines or in conjunction with fluorescently-labeled functional reporter lines (or other secondary readouts), permit the implementation of chemical screening for disease-modifying substances in the field of genetic kidney diseases.

## AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.

## ACKNOWLEDGMENTS

We thank Franz Schaefer (University Children's Hospital Heidelberg), Felix Loosli (Karlsruhe Institute of Technology) and Jochen Wittbrodt (Centre for Organismal Studies (COS) Heidelberg) for general support. This work was partially funded by the RENALTRACT ITN project, that has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement N◦ 642937. JHW has received funding from Peter-Stiftung für die Nierenwissenschaft and Doktor Robert Pfleger-Stiftung. We acknowledge financial support by Deutsche Forschungsgemeinschaft within the funding programme Open Access Publishing, by the Baden-Württemberg Ministry of Science, Research and the Arts and by Ruprecht-Karls-Universität Heidelberg.

### REFERENCES


mechanosensation and is activated in polycystic kidney disease. Dev Cell (2006) 10:57–69. doi: 10.1016/j.devcel.2005.12.005


#### **Conflict of Interest Statement:** GP and JG are employees of DITABIS AG, Pforzheim, Germany.

The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Gehrig, Pandey and Westhoff. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## The Genetic and Cellular Basis of Autosomal Dominant Polycystic Kidney Disease—A Primer for Clinicians

#### *Adrián Cordido† , Lara Besada-Cerecedo† and Miguel A. García-González\**

*Grupo de Genética y Biología del Desarrollo de las Enfermedades Renales, Laboratorio de Nefrología (n.° 11), Instituto de Investigación Sanitaria (IDIS), Complexo Hospitalario de Santiago de Compostela (CHUS), Santiago de Compostela, Spain*

#### *Edited by:*

*Max Christoph Liebau, Universitätsklinikum Köln, Germany*

#### *Reviewed by:*

*Jan Halbritter, Leipzig University, Germany Erum Aftab Hartung, Children's Hospital of Philadelphia, United States Efthimia K. Basdra, National and Kapodistrian University of Athens, Greece*

#### *\*Correspondence:*

*Miguel A. García-González miguel.garcia.gonzalez@sergas.es*

*† These authors have contributed equally to this work.*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 30 September 2017 Accepted: 07 December 2017 Published: 18 December 2017*

#### *Citation:*

*Cordido A, Besada-Cerecedo L and García-González MA (2017) The Genetic and Cellular Basis of Autosomal Dominant Polycystic Kidney Disease—A Primer for Clinicians. Front. Pediatr. 5:279. doi: 10.3389/fped.2017.00279*

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common genetic disorders worldwide. In recent decades, the field has undergone a revolution, starting with the identification of causal ADPKD genes, including *PKD1*, *PKD2*, and the recently identified *GANAB*. In addition, advances defining the genetic mechanisms, protein localization and function, and the identification of numerous pathways involved in the disease process, have contributed to a better understanding of this illness. Together, this has led to a better prognosis, diagnosis, and treatment in clinical practice. In this mini review, we summarize and discuss new insights about the molecular mechanisms underlying ADPKD, including its genetics, protein function, and cellular pathways.

Keywords: autosomal dominant polycystic kidney disease, genetics, molecular biology, diagnosis, therapy

### INTRODUCTION

Polycystic kidney disease (PKD) is a heterogeneous group of monogenic disorders characterized by the bilateral formation and progressive expansion of renal cyst that lead to end stage renal disease (ESRD) (1). Several Mendelian diseases including autosomal dominant polycystic kidney disease (ADPKD), autosomal recessive polycystic kidney disease (ARPKD), and atypical forms of PKD can be grouped under this pathological entity.

Autosomal dominant polycystic kidney disease is the most common inherited kidney disease affecting ~1/400–1/1,000 individuals (2). The hallmark characteristic of ADPKD is the progressive development and expansion of cysts in the kidney leading to ESRD. It can be associated with several extrarenal manifestations including hypertension, symptomatic extrarenal cysts, and subarachnoid hemorrhage from intracranial aneurysms (3–5). The vast majority of the patients develop the disease between the ages of 20–40 s, but there have been sporadic cases that range in onset from late to childhood ("early onset," before 15 years old) or even *in utero* ("very early onset") (6).

### GENETICS OF THE ADPKD

Autosomal dominant polycystic kidney disease is genetically heterogeneous and associated with mutations in *PKD1* (responsible of ADPKD-Type I), *PKD2* (-Type II), and *GANAB*. *PKD1* is a complex gene mapping to chromosome 16 (16p13.3) (**Figure 1A**). Its genomic structure has a number of features that complicate its evaluation (7, 8): (a) it is highly GC-rich with a large number of

CpG dinucleotides, (b) 70% of *PKD1* is duplicated multiple times throughout chromosome 16 with high sequence fidelity (95% identity) (9), and (c) it contains a 2.5 kb polypyrimidine tract in intron 21 (the largest in the human genome) (10). In contrast to *PKD1*, the *PKD2* gene is located on chromosome 4 (4q21) and has simpler features and structure (11) (**Figure 1A**). Approximately, 80–85% of ADPKD families were associated with *PKD1* mutations, and 15–20% to *PKD2* mutations in the literature (12). Recently, Porath and colleagues identified causal mutations in *GANAB*, a gene on chromosome 11q12.3 (**Figure 1A**), in ADPKD patients that are negative for *PKD1* and *PKD2* mutations. They report that *GANAB* accounts for ~0.3% of total ADPKD and it is associated with a milder manifestation of PKD and autosomal dominant polycystic liver disease (ADPLD) (5).

### DIAGNOSIS OF ADPKD

The diagnosis of ADPKD is dependent on the stage of the disease. When the disease is fully established, the diagnosis is clinically based on patient's history and physical examination (13, 14). However, definitive diagnosis can be difficult due to other disorders having overlapping symptoms. Therefore, complementary approaches such as diagnostic imaging or genetic tests are necessary to confirm the diagnosis. Imaging techniques, including ultrasound, computed axial tomography, or nuclear magnetic resonance, allow for the detection of cysts in the kidney, liver, or pancreas (15). The magnetic resonance technique has proven to be more sensitive than ultrasound, allowing measurements of height-adjusted total kidney volume (htTKV) and better definition of the cysts without the use of contrast agents. However, these imaging tests are expensive (16) and are often not performed on a subset of the ADPKD population including those who are young individuals at risk or patients with atypical or *de novo* renal cystic disease (13) for whom complementary genetic tests is the method for definitive diagnosis. Direct DNA sequencing (DS) could offer a molecular diagnosis; however, the genetic analysis of the *PKD1* (responsible for most ADPKD cases) is complicated. The 5′-region of the gene (exon 1–34) is replicated in at least six highly homologous copies on chromosome 16 (7, 9, 17). To date, direct sequencing based on a Long-Rage PCR strategy with specific primers has been the accepted strategy by the ADPKD community (18). Isolated gene by gene sequencing is laborious and expensive, and provides limited amount of information to provide a better diagnosis and prognosis for the patients. Moreover, it has been described that the main mutation responsible of the disease may interact with other PKD or ciliopathy loci modifying the phenotype and extending the genetic complexity of the disease (18–20). For this reason, ADPKD experts are highlighting the necessity to screen all cystic genes in a common strategy to allow for a more accurate diagnosis, including those genes responsible of atypical forms of PKD. Under this context, next-generation sequencing strategies followed by the validation of variants by DS have become the recommended methodology allowing for faster, more cost-effective, and more reliable genetic diagnosis of large ADPKD cohorts (21).

### GENOTYPE–PHENOTYPE CORRELATIONS

It has been described that patients with mutations in *PKD1* gene have larger kidneys and earlier onset (mean age at ESRD, 53.4 versus 72.7 years old, respectively) with lower eGFR and higher htTKV than *PKD2* patients (22, 23). In addition, *GANAB* mutations seem to be associated with a mild renal phenotype, closer to a *PKD2* than a *PKD1* phenotype, revealing the importance of molecular diagnosis (5). Moreover, a strong correlation between the type of the mutation and the severity of the disease was observed, illustrating the importance of quantifying genetic heterogeneity in ADPKD. Truncating *PKD1* mutations (frameshift, splicing, and nonsense) have a more severe disease prognosis with lower eGFR; however. the type of mutation does not correlate with htTKV (23, 24). Non-truncating *PKD1* mutations (missense, inframe deletion/insertion) or mutations in *PKD2* are associated with a milder form of the disease. In addition, males with truncating *PKD1* are associated with larger kidneys and increased risk for ESRD, while women with truncating *PKD1* have a more severe liver phenotype (23, 24). In addition, disease manifestation in ADPKD patients from the same family, or patients with the same mutation, can have varying severity and differential disease progression, which may be due to the presence of variation in a modifying gene. This phenomenon is known as genetic interaction and epistasis, and usually aggravates or attenuates the phenotype cause by the primary mutation (17, 25).

Based on genetic and clinical data, Cornec-Le Gall and colleagues (26) developed a robust prognostic model, the PROPKD score (with a range from 0 to 9), to predict survival in ADPKD patients. They described critical variables associated with ESRD including age of onset (median age reported to be 70.6 years for low risk, 56.9 years for intermediate risk, and 49 years for high risk) and a scoring system to predict disease progression: sex (being male 1 point), need for antihypertensive therapy before 35 years old (2 points), occurrence of the first urologic event before 35 years old (2 points), and genetic status (having PKD2 mutations: 0 points, non-truncating PKD1 mutation: 2 points and truncating PKD1 mutations: 4 points). Three risk categories were then defined to describe the putative risk for progression to ESRD: low risk (0–3 points), intermediate risk (4–6 points), and high risk (7–9 points) (26).

### PKD PROTEINS: STRUCTURE AND FUNCTION

*PKD1* and *PKD2* encode the proteins PC1/Polycystin-1 and PC2/ Polycystin-2 or TRPP2, respectively. PC1 is a putative receptor for an unidentified ligand which contains a long extracellular N-terminal domain, 11 transmembrane domains and a short intracellular C-terminal domain (27). PC2/TRPP2 has similar characteristics to TRP channel, having six transmembrane segments, a pore loop domain (separating the first two transmembrane segments), and an N- and C-terminal domains (28) (**Figure 1B**). PC2 is a Ca2<sup>+</sup>-permeable non-selective ion cation channel and together with PC1 forms a receptor–channel complex implicated in the Ca2<sup>+</sup> pathway called PC complex (29).

In contrast to *PKD1* and *PKD2*, *GANAB* encodes the alpha subunit of glucosidase II (GIIα) which is the catalytic subunit of GII. GIIα together with the regulatory subunit of GII, GIIβ (also called hepatocystin) (5) form a functional holoenzyme in the endoplasmic reticulum (ER). This holoenzyme is implicated in the proper folding and translocation of glycoproteins into the ER, and its dysfunction has been reported to be associated with maturation and localization defects of PC1 (30).

### DISEASE MECHANISM

### Two-Hit Model for ADPKD

The human kidney has approximately one million nephrons, and an ADPKD patient will develop around a 1,000 cysts (31). ADPKD disease progression is highly variable and depends directly from the nature of the mutated gene. The "Two-Hit Model," in which two different mutations affect proper genetic/cellular interactions, has been the proposed theory to explain the kidney phenotype observed in ADPKD patients. While an individual has inherited a germ line mutation ("first hit"), the development of cysts does not occur until another mutation (somatic mutations) in either *PKD1* or *PKD2* occurs ("second hit") (32, 33).

### Localization of PKD Proteins: The Role of Primary Cilia

There have been a number of different localizations proposed for the PC1/PC2, including the ER, apical and basolateral cell membranes, or secreted exosomes (31). However, there is evidence supporting their presence in primary cilia based on their central role in ADPKD pathogenesis. Cilia are microtubule-based, nonmotile organelles on the apical surface of the cells and play an essential role in cellular detection and regulation of external signals. Dysfunction of this organelle result in a group of disorders called the ciliopathies (34). Data from animal models (*C. elegans*, *Drosophila*, and *Mus musculus*) support the idea that defects in function or structure of primary cilia contribute to the pathomechanisms of PKD (35). PKD proteins such as PC1, PC2, and polyductin/FPC (encoded from the ARPKD gene, *PKHD1*) localized to the primary cilium (36–38). These PKD proteins interact with each other (17, 29, 36, 39, 40) and form a functional complex with common downstream signaling pathways (41). In addition, deleted in azoospermia interacting protein 1-like, the protein encoded from the recently identified ARPKD gene (*DZIP1L*), was reported to localize to the centrioles and basal bodies of cilia and are also associated with ciliary trafficking defects (19).

There has also been additional evidence to support the functional role of PKD proteins within the cilium. Urine flow has been linked to an increase in intracellular calcium (42), likely driven by the mechanical response of the primary cilium (43). The large extracellular domain of PC1 has been proposed to be the flow mechanosensor that opens the PC2-channel, allowing calcium entry leading to mechanotransduction activation (44). A different model proposes that the primary cilia's role in flow sensing is required for proper centrosomal localization that results in oriented cell division (OCD). In addition, defects in cilia drive the loss of planar cell polarity and consequently abnormal OCD (45); however, this model is controversial and remains unclear (46, 47). Several observations support the idea of the mechanosensory role of polycystins in the primary cilium (48, 49). PC2 directly interacts with KIF3A and KIF3B, two essential proteins for ciliary assembly and function (37, 50). In addition, PC2 is required for the flow-mediated increase of cytosolic Ca2<sup>+</sup> (51, 52), and mechanical stimuli can induce proteolytic cleavage of the intracellular C-terminal domain of PC1 (53). Interestingly, there have been controversial results reporting that mechanosensation does not occur *via* Ca2<sup>+</sup> signaling within cilia (54). In spite of this, there are some unanswered questions as while Delling and colleagues do not exclude the presence of others mechanosensitive elements in primary cilia (55) and the cilia seems to increase cytoplasmic Ca2<sup>+</sup> concentration by diffusion (56).

A very interesting and unexpected finding by Ma and colleagues showed that loss of cilia results in a significant reduction of PKD severity (57). Authors reported that a simultaneous inactivation of polycystins and cilia assembly resulted in the reduction of the cystic phenotype associated with polycystins inactivation. These findings suggest that the polycystins modulate a pathway involved in the cilia signaling, but require intact cilia function (58).

### Threshold or Dosage Model

Genetic background influences the phenotypic variability of ADPKD. As we previously mentioned, patients with mutations in *PKD1* have worse prognosis than those with mutations in *PKD2* (59), and those with truncating *PKD1* mutations were associated with more severe polycystic renal pathology than those with non-truncating mutations (60). In addition, unaffected patients who carry a missense variant in *PKD1* indicate that some alleles are incompletely dominant in the disease (61). Similarly, other studies suggest that incomplete, penetrant alleles can influences disease severity in ADPKD (62).

These data support that a threshold or dosage model could explain cystogenesis in ADPKD (63). According to this model, cyst initiation and cystic expansion depends on PKD gene dosage, starting when the level of functional PC falls below the cystogenic threshold (58, 63). Defects in that threshold may occur by a combination of one or more factors: the nature of germline mutation ("first-hit"), somatic mutations ("second hit"), modifier genes or environmental factors such as renal injury or inflammation (58, 64). Several studies support this: (1) García-González and colleagues reported genetic interaction between ADPKD and ARPKD genes in a common pathway (17), (2) it has been reported that ADPLD genes (*Prkcsh* and *Sec63*), ARPKD gene (*Pkhd1*) and ADPKD gene (*Pkd1*) interact with each other suggesting a central role of PC1 in cystogenesis (65), and (3) a developmental window for cystogenesis has been identified, suggesting that timing of secondary events may influence the severity of ADPKD (66).

A crucial step in the protein maturation of functional PCs is also related to the *dosage model*. Autoproteolytic cleavage of PC1 at the GPS domain, mediated by larger GAIN [G protein-coupled receptor (GPCR)-autoproteolysis inducing] domain which includes a GPCR proteolysis site (GPF) motif, is crucial for PC1 maturation (67, 68). Besse and colleagues have described that specific isolated-PLD proteins (encoded by SEC61β, ALG8, and GANAβ), from the ER protein biogenesis pathway, are directly related to PC1 biogenesis (30). Furthermore, PC1 maturation requires PC2 in a dose-dependent manner (69). It is also known that mature PC assembles at the PC complex-bearing vesicles in the Golgi before trafficking to the ciliary/plasmatic membrane (70) (**Figure 2)**. In addition, Cai and colleagues described the effect of several mutations in *Pkd1* and *Pkd2* in the importance of PCs trafficking to cilia using *in vitro* and *in vivo* models, concluding that altered trafficking and dysfunctional maturation of PC complex underlie PKD pathology (71) These facts suggest a central role for PC1 in the cystogenesis process and in regulating the severity of ADPKD, ARPKD, and ADPLD (72).

### Signaling Pathways and Targeted Therapies in ADPKD

Several signaling pathways and transcription factors control the progression and development of cystogenesis (64, 73). Calcium signaling is one of the most studied pathways in the PKD field.

and is implicated in the Ca2+ pathway. PC2 also regulates intracellular calcium in the ER. PC mutations result in deregulation of Ca2+ leading an increase in cAMP and upregulation of the PKA and MAPK pathways. Abbreviations: RyR, ryanodine receptor; IP3R, IP3 receptor; PDE, phosphodiesterase; AC-VI, adenylyl cyclase 6; Gs and Gi, guanosine nucleotide-binding proteins; V2R, V2 receptor; cAMP, cyclic AMP; PKA, protein kinase A; MAPK, MAP kinases; SIRT1, sirtuin 1.

PC2 is a calcium permeable non-selective cation channel that is abundantly expressed in the ER and interacts with others calcium channel proteins (51). It binds to the inositol 1,4,5-trisphosphate receptor (IP3R) regulating Ca2<sup>+</sup> homeostasis and the activity of ryanodine receptors (74, 75). In contrast to PC2, PC1 accelerates the decay of the intracellular calcium response to ATP by increasing ER calcium uptake. All of this suggests a major role of polycystins in intracellular calcium hometostasis (76, 77). Cystic epithelial cells have an aberrant cross talk between intracellular calcium and cAMP signaling as elevated levels of cAMP stimulate cyst fluid secretion, enhancing protein kinase A activity (78–80). Furthermore, V2 receptor antagonists (Tolvaptan) ameliorate the progression of PKD by the inhibition of cAMP signaling pathway in both animal models (81, 82) and in clinical trials (83) (**Figure 2**). Importantly, adverse secondary effects could appear with Tolvaptan treatment such as polyuria, nocturia, and elevation of liver enzymes (84). Taking this into account, Tolvaptan is the first therapy approved for indication of ADPKD in several countries.

Alterations to other pathways have been reported to affect cystic volume or cystic progression, but to date; attempts to completely inhibit cystogenesis have been unsuccessful. The mTOR pathway is highly activated in cystic tissues independent of the PKD gene mutation (85). Preclinical trials with sirolimus and everolimus blocked cystic progression in a rodent model of ADPKD (86, 87). In addition, somatostatin analogs, as octreotide and lanreotide reduced hepatic and renal volume expansion in ADPKD (88–90). Treatment of PKD animal models, which have defective glucose metabolism associated with cystic expansion, with 2-deoxyglucose, an analog of glucose, also result in reduced cystic progression (91, 92) (**Figure 2**). Adding to the hunt for therapeutics, other alternate mechanisms also exist, such as sirtuin 1, microRNAs, and MCP1 which have been postulated as possible therapies for PKD (93–95) and recently, ongoing Phase-II and Phase-I clinical trials of a multi-kinase inhibitor, tesevatinib, are ongoing for ADPKD and children with ARPKD (96, 97).

### CONCLUSION

In the last two decades, there have been significant contributions toward our understanding of the genetic, cellular, and functional role of PKD genes and proteins, as well as, the identification of a number of pathways implicated in the pathogenesis of the disease. Nevertheless, several questions remain unresolved and controversial in the PKD community. The complexity of this disease is reflected along all scientific levels, starting at the

### REFERENCES


genetic level with critical refinement of the mutations, followed by the study of protein function and dosage to understand the spectrum of clinical manifestations in PKD, and finally the study of related pathways and modifier mechanisms that all should be taken into account for future clinical trials and personalized medicine.

### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

### ACKNOWLEDGMENTS

The authors would like to thank María Pardo and Perciliz Tan for their comments.

### FUNDING

This work was supported by grants from the Instituto de Salud Carlos III FEDER funds ISCIII RETIC REDINREN RD12/0016, PI11/00690, PI15/001467, Sociedad Española de Nefrología, and Xunta de Galicia.


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Cordido, Besada-Cerecedo and García-González. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

#### *Stéphanie De Rechter1,2\*, Luc Breysem3 and Djalila Mekahli1,2*

*1PKD Lab, Department of Development and Regeneration, KU Leuven, Leuven, Belgium, 2Department of Pediatric Nephrology, University Hospitals Leuven, Leuven, Belgium, 3Department of Radiology, University Hospitals Leuven, Leuven, Belgium*

Autosomal dominant polycystic kidney disease (ADPKD) affects 1 in 400 to 1,000 live births, making it the most common monogenic cause of renal failure. Although no definite cure is available yet, it is important to affect disease progression by influencing modifiable factors such as hypertension and proteinuria. Besides this symptomatic management, the only drug currently recommended in Europe for selected adult patients with rapid disease progression, is the vasopressin receptor antagonist tolvaptan. However, the question remains whether these preventive interventions should be initiated before extensive renal damage has occurred. As renal cyst formation and expansion begins early in life, frequently *in utero*, ADPKD should no longer be considered an adult-onset disease. Moreover, the presence of hypertension and proteinuria in affected children has been reported to correlate well with disease severity. Until now, it is controversial whether children at-risk for ADPKD should be tested for the presence of the disease, and if so, how this should be done. Herein, we review the spectrum of pediatric ADPKD and discuss the pro and contra of testing at-risk children and the challenges and unmet needs in pediatric ADPKD care.

#### *Edited by:*

*Miriam Schmidts, Radboud University Nijmegen, Netherlands*

#### *Reviewed by:*

*Consolato Sergi, University of Alberta Hospital, Canada Julia Hoefele, Technische Universität München, Germany*

*\*Correspondence: Stéphanie De Rechter stephanie.derechter@uzleuven.be*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 30 October 2017 Accepted: 04 December 2017 Published: 20 December 2017*

#### *Citation:*

*De Rechter S, Breysem L and Mekahli D (2017) Is Autosomal Dominant Polycystic Kidney Disease Becoming a Pediatric Disorder? Front. Pediatr. 5:272. doi: 10.3389/fped.2017.00272*

Keywords: autosomal dominant polycystic kidney disease, children, testing, prevention, treatment

### AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE (ADPKD) AND ITS SPECTRUM IN PEDIATRIC PATIENTS

Autosomal dominant polycystic kidney disease affects 1 in 400 to 1,000 live births, making it the most common monogenic cause of renal failure and representing a major socio-economic medical problem in the world (1). The disorder arises as a consequence of *PKD1* or *PKD2* gene mutations, encoding polycystin-1 (PC1) and -2 (PC2), and accounting for 85 and 15% of patients with a *PKD* gene mutation, respectively. Recently, a third gene has been identified to cause ADPKD when mutated, namely *GANAB* (Glucosidase, Alpha, Neutral AB form)*,* encoding the glucosidase IIa subunit (2). Although most cases are familial, in 10–25% of patients, a positive family history is absent, posing a diagnostic challenge. These cases are explained by *de novo* disease in up to 10–15% (3), missing parental medical records, germline or somatic mosaicism, or mild disease from hypomorphic *PKD1* and *PKD2* mutations (4).

**Abbreviations:** ACEi, angiotensin converting enzyme inhibitor; ADPKD, autosomal dominant polycystic kidney disease; ARB, angiotensin receptor blocker; CKD, chronic kidney disease; (e)GFR, (estimated) glomerular filtration rate; LVH, left ventricular hypertrophy; MRI, magnetic resonance imaging; RAAS, renin–angiotensin–aldosterone system; TKV, total kidney volume; VEO, very early onset; US, ultrasound.

Autosomal dominant polycystic kidney disease is characterized by a progressive growth of cysts in all nephron segments, ultimately leading to end-stage renal disease (ESRD) in 50% of patients in the sixth decade. ADPKD patients with a *PKD2* mutation have a milder phenotype and reach ESRD approximately 20 years later than *PKD1* patients. Within the *PKD1* population, patients with a truncating mutation are more severely affected than those with a non-truncating. Therefore, *PKD* gene mutation analysis is one of the elements forming the PROPKD scoring system, an algorithm to predict renal survival in adult ADPKD patients (5). Moreover, ADPKD as a bona fide ciliopathy is a systemic disorder, comprising extrarenal symptoms such as cyst formation in other organs, mostly in the liver, hypertension, intracranial arterial aneurysms, cardiac valvular defects, inguinal and abdominal herniation, etc. (6). Recently, there is also evidence of bone and mineral metabolism anomalies, from both studies in different animal ADPKD models (7–9) as from clinical studies in adult and pediatric patients (7, 10–14).

As the majority of patients remain *a-* or *pauci-*symptomatic until adulthood, ADPKD is still often considered as a late-onset disease (15). Historically, ADPKD was termed "adult" polycystic kidney disease due to this classic presentation (16). However, comorbidities might be underdiagnosed in childhood if not actively looked for by caregivers as not all cause complaints. Moreover, the glomerular filtration rate (GFR) masks the underlying renal damage for several decades, due to hyperfiltration and hypertrophy of unaffected residual nephrons (17). The parameter proven to be a strong and independent predictor for ESRD development in ADPDK patients was the factual anatomic damage, indirectly assessed as the height-adjusted total kidney volume (TKV) on magnetic resonance imaging (MRI) (18, 19). Although evidence is growing that renal injury starts with the formation of the first renal cysts *in utero* (20), a consensus for the management of children at-risk for or diagnosed with ADPKD is lacking. Indeed, of all ADPKD patients, 2–5% present in childhood with a broad phenotypic spectrum, ranging from severe neonatal presentations mimicking autosomal recessive polycystic kidney disease (21) to the incidental sonographic finding of renal cysts (22). Here, we aimed to summarize the most important manifestations of ADPKD in childhood.

### Renal Manifestations in Childhood

Possible presenting symptoms of renal disease in children with ADPKD are frequency, nocturia and/or, hematuria, urinary tract infection(s) and back, flank or abdominal pain (23). Often, the earliest biological renal manifestation of childhood ADPKD is a decreased urinary concentrating ability, resulting in polyuria and polydipsia (15). 58% of children (*N*= 53) showed a decreased renal concentrating capacity when performing a standardized renal concentrating capacity test after nasal drop application of desmopressin (24). In several study cohorts, children were included having a decreased estimated GFR (eGFR): 12 (25) to 39% (26) of the ADPKD children had eGFR levels < 90 mL/min/1.73 m2 , and ESRD was seldom observed. Indeed, cases of ESRD in childhood due to ADPKD are rare and in these cases, coexisting reasons for renal function decline should be ruled out. Other reasons might be the coinheritance of a hypomorphic *PKD1* allele in trans with an inactivating *PKD1* allele or the inheritance of two incompletely penetrant *PKD1* alleles—most likely leading to very early onset (VEO) ADPKD (27, 28). Children with ADPKD display glomerular hyperfiltration in 18 (29) to 21% (25). Importantly, the occurrence of hyperfiltration was associated with a significantly faster renal function impairment and a higher renal growth rate over time (29). Also, proteinuria and microalbuminuria are seen in ADPKD, and more frequently in children and adolescents than in adults. In different pediatric cohorts, the prevalence of micro-albuminuria and proteinuria ranges from 30 to 48% and 10 to 23%, respectively (25, 26, 30). From studies performed in both pediatric (30) and adult (31) patient cohorts, it was shown that patients with established proteinuria have a more aggressive course of renal disease. Indeed, adult proteinuric patients had higher mean arterial pressures, larger renal volumes and lower creatinine clearances compared to the non-proteinuric subjects (31). Similarly, proteinuric children had a more severe renal disease -defined as having more than 10 renal cysts—then those without proteinuria. However, unlike in adults, no correlation between proteinuria and hypertension, one of the most prevalent extrarenal manifestations, was seen in children (30). Last, hematuria, both possible after an antecedent of abdominal trauma, for instance in patients playing contact sports, as spontaneously (32), and nephrolithiasis are rather uncommon in children (15). A prevalence of the latter is lacking, as information comes from case reports (33). Moreover, nephrolithiasis is seen in up to 28% of ADPKD patients, which is 5–10 times the rate in the general population, due to both anatomic and metabolic abnormalities; but none in the patients below 20 years of age (34). Different from the general population is that nephrolithiasis in ADPKD patients should only be made after ruling out a superimposed urinary tract infection, cyst infection or hemorrhage and that screening for underlying urinary metabolic abnormalities should be performed from the first presentation (35).

The anatomic renal damage can often already be seen from a young age as renal cysts and/or uni- or bilateral nephromegaly. In a cohort of 47 ADPKD children, all had accelerated renal growth relative to the normal population, and half had nephromegaly (26). Affected individuals who do not yet have cysts detectable by ultrasound (US) were shown to have larger age-adjusted kidneys, suggesting ongoing microscopic cyst development and growth (36). Importantly, mathematical models computing volume changes of solitary and multiple cysts in function of time show that cysts formed early in life, are the main contributors to the final total cyst volume (37).

Another major symptom of childhood ADPKD, explained by anomalies in both the renal and cardiovascular system, namely hypertension, will be discussed in Section "Extrarenal Manifestations in Childhood: Cardiovascular Symptoms."

### Extrarenal Manifestations in Childhood: Cardiovascular Symptoms

Cardiovascular manifestations are the most prominent extrarenal manifestation in ADPKD, also in childhood, of which hypertension occurs in a majority of patients, prior to renal function decline. The reported incidence in childhood varies widely in different cohorts, namely from 5 to 44% (15): 6% (25), 15% (26), 21% (30), 33% (38), and 37% (29). The most reliable assessment probably is a recently published meta-analysis covering 14 studies, estimating 20% of ADPKD children having hypertension (39).

The pathogenesis of hypertension in ADPKD includes a complex and multifactorial process, involving the renin–angiotensin–aldosterone system (RAAS), the sympathetic nervous system, sodium retention, and endothelial dysfunction. First, the presence of growing renal cysts was shown to attenuate intrarenal blood vessels, suggesting the presence of local renal ischemia and hypoxia. This in turn suggests stimulation of the RAAS in ADPKD. Both increased circulating (40) as intrarenal (41) RAAS activity was demonstrated in patients with ADPKD. Second, it is known that RAAS activation stimulates the sympathetic nervous system and *vice versa*. Indeed, adult hypertensive ADPKD patients have significantly higher plasma catecholamine concentrations compared to patients with essential hypertension (42). Third, sodium retention plays a role. Interestingly, adult patients with normotension were shown to exhibit an increased total body sodium as compared with their unaffected siblings, suggesting an early role of RAAS (43). Last, expression of Pkd1 and Pkd2 was shown in the major vessels and in the cilia of endothelial and vascular smooth muscle cells. Decreased or absent PC1 or -2 levels are associated with abnormal vascular structure and function, *via* reduced nitric oxide (NO) levels due to diminished Ca2<sup>+</sup>-dependent endothelial NO syntheses activity, resulting in an altered endothelial response to shear stress with attenuation in vascular relaxation. Moreover, decreased NO production leads to the activation of the RAAS, next to its triggering by cyst enlargement and intra-renal ischemia (44). However, it is still unclear which factor hypertension in ADPKD is mainly due to. The early onset of hypertension in ADPKD, probably still underdetected and therefore left untreated, is associated with left ventricular hypertrophy (LVH) in half of the patients in their 40s (45). Importantly, cardiovascular complications appear to be the leading cause of death in patients with ADPKD. Therefore, LVH should be prevented in patients with ADPKD, as it is known to be associated with coronary heart disease, cardiac failure, arrhythmias, and sudden death (46).

A correlation between TKV and cyst volume, and hypertension was observed in a longitudinal MRI study in ADPKD children: the hypertensive subgroup had a larger increase in both cyst volume and number than the normotensive (47). Also, the prevalence of hypertension was found to be significantly higher in children with a decreased urinary concentrating capacity compared to those with a normal concentrating capacity. Significant negative correlations were shown between urinary concentrating capacity, blood pressure and the number of renal cysts (24).

Vascular dysfunction begins early in the course of ADPKD. In normotensive patients with preserved renal function, the endothelium-dependent dilatation and arterial stiffness were evaluated and compared to age and sex matched healthy controls, by measuring the brachial artery flow-mediated dilation (FMDBA) and the carotid-femoral pulse wave velocity (CFPWV), respectively. FMDBA was lower in 25% of patients, indicating an impaired endothelium-dependent dilatation, while CFPWV was higher in 14% of patients, designating increased arterial stiffness. Both are well-known independent predictors of future cardiovascular comorbidity and mortality (48).

Another possible cardiovascular feature in ADPKD, is mitral valve prolapse, occurring in 12% of children (15). Next, the association of ADPKD with intracranial aneurysm formation is well known (49). Almost all cases of ruptured intracranial aneurysms occur in adult patients with a median age at rupture of 40 years. In a study screening ADPKD patients, aged 7–87 years, the prevalence of intracranial aneurysm was 12.4%, reaching a peak in the 60–69 years age group; and higher in patients with a positive family history of hemorrhagic stroke or intracranial aneurysm. Only 1 patient had an aneurysm before the age of 29 years, but the authors do not state the exact age of this patient (49). However, case reports describe rupture at very young ages, even neonatal (50, 51). Although rare, this complication might be lethal in young patients.

### Extrarenal Manifestations in Childhood: Others

Recently, evidence has emerged showing bone involvement in ADPKD (7–14). Indeed, PC1 is highly expressed in both osteoblasts and osteocytes and a homozygous *PKD1* null mutation in mice was shown to cause polyhydramnios, hydrops fetalis, spina bifida, and osteochondrodysplasia (52, 53). Moreover, the PC1 C terminal tail regulates TAZ expression, an enhancer of RUNX2-mediated osteoblast differentiation and suppressor of PPARγ-stimulated adipocyte differentiation. A causal relationship between mutant PC1, decreased Runx2 levels, and impaired osteoblast differentiation leading to a defect skeletogenesis was described (9, 54).

It has been demonstrated that fibroblast growth factor 23 (FGF23), a phosphaturic hormone secreted by osteocytes, was studied in different ADPKD rodent models. FGF23 levels were raised, but a peripheral biologic resistance to FGF23 was hypothesized to account for the absence of a renal phosphate leak or hypophosphatemia. Importantly, FGF23 was detected in cells lining renal cysts, from a rodent model (7). In ADPKD adults in chronic kidney disease (CKD) stages 1–2, FGF23 levels were highly increased compared to both healthy controls and CKD matched controls. However, the majority of the ADPKD population displayed FGF23 resistance, given the absence of a phosphaturic phenotype, confirming the observations from the animal models. This was hypothesized to be due to the deficiency of the FGF coreceptor, namely serum Klotho (sKlotho) (13). FGF23 has also been studied in ADPKD children with preserved renal function. Compared to healthy controls, patients had significantly lower serum phosphate SDS. In a quarter of the patient population, this was accompanied by low TmP/GFR levels for age, based on the reference values (55). Although FGF23 levels were similar in both groups, it is inappropriately normal for the ADPKD cohort, given the hypophosphatemia in the latter. Also sKlotho levels were similar between both groups. Apart from the phenotypic differences in adulthood vs. childhood concerning FGF23 and phosphate metabolism, these anomalies seem to be ADPDK specific, as markedly FGF23 elevation is only found in non-ADPKD patients with advanced CKD stage, where it is thought to counteract phosphate retention and is classically accompanied by an increased parathyroid hormone and decreased 1,25-dihydroxyvitamin D levels (14). Moreover, given the observations in the pediatric cohort, it is an early disease phenomenon.

In the same pediatric ADPKD cohort, significantly lower values of bone alkaline phosphatase were seen compared to healthy controls (10). Based on these data and the findings from a bone biopsy study in which a low bone turnover was observed in young adult ADPKD patients with preserved kidney function (56), bone formation suppression is hypothesized in ADPKD.

Also sclerostin, known to inhibit the osteo-anabolic Wnt/βcatenin pathway, is an emerging player in the mineral metabolism phenotype of ADPKD. Higher osseous sclerostin expression has been reported in *jck* mouse model (57) and in a small cohort of adult ADPKD patients in ESRD (11). However, it could not be confirmed in a pediatric ADPKD cohort (10). This discrepancy remains to be elucidated, next to the clinical relevance of sclerostin overactivity in ADPKD, if confirmed in future studies.

In general, one of the main extrarenal manifestations of ADPKD is polycystic liver disease (PLD). In contrast to renal cyst formation, liver cyst formation does not affect liver function, although it can cause symptoms related to its mass effect when significant liver enlargement has occurred, or due to cyst bleeding or infection (58). In children, liver cyst formation is very rare (59). PLD is the result of ductal plate—the primitive biliary structure—malformations. Normally, ductal plate remodeling takes place from 12 weeks gestational age, up to term, but with a slowing down between the 20th and the 32nd week of gestation (60). Ductal plate malformation leads to a partial to complete arrest of ductal plate remodeling, and therefore a persistent excess of embryonic biliary structures (61). Typically, these structures are present from a very early disease stage, but remain asymptomatic until cyst growth initiates in adulthood (62).

### DIAGNOSING ADPKD IN CHILDREN

The diagnosis of ADPKD in children can be made either unintentionally or actively, *via* screening. First, children at-risk for ADPKD might present with symptoms such as urinary tract infection(s), hematuria, enuresis etcetera, leading to their diagnosis when further investigated. Children might be diagnosed unintentionally as well, starting from the routinely performed prenatal US during pregnancy. Prenatally, the typical—although not specific for ADPKD—sonographic characteristics are moderately enlarged hyperechogenic kidneys with an increased corticomedullary differentiation (CMD). Other prenatal sonographic features such as an absent or decreased CMD and cortical cysts are less frequently seen (21). Although a prenatal presentation of ADPKD is rare, this form is recurrent within families, suggesting a common genetic modifying background with low levels of PC1 function (63) or the association with other modifying genes such as *HNF1B* or *PKHD1* (64). In other cases, children might be diagnosed unintentionally postnatally, after the coincidental detection of renal cysts on an abdominal US performed for other reasons, most frequently abdominal pain. Second, active presymptomatic testing might be requested by the parents and/or the adolescents themselves. However, the diagnosis of ADPKD, especially its timing, represents controversies that we will discuss later in this review.

We first aim to demonstrate the challenges and limitations in the methods for the diagnosis of ADPKD in children and the lack of definite criteria for individuals under the age of 15 years. Indeed, presymptomatic testing in children at-risk for ADPKD, due to their positive familial history, can be executed by clinical evaluation and/or imaging. However, genetic evaluation is needed to resolve some cases. Most diagnoses of ADPKD are established on a positive family history combined with imaging [sonographic (65, 66) or MRI (67)] findings. However, for individuals under the age of 15 years, there are no validated diagnostic imaging criteria available (36, 68). As simple renal cysts occur rarely in childhood (69), a child with a positive family history for ADPKD, having a single cyst, preferably > 1 cm in diameter, is generally assumed to have the diagnosis of ADPKD, although this was never fully evaluated and validated. Moreover, based on renal US findings, one can only exclude disease presence in at-risk individuals aged 30–40 years or older (65–67). Based on MRI, this will probably be possible at an earlier age, but a cutoff age for a definite exclusion is not set yet. In case of a negative US and the patient and their family are willing to have a more definitive evaluation, gene sequencing of the *PKD* genes can be performed. However, this is not yet routinely used in clinical practice yet due to its high cost and challenging analysis. These are explained by the presence of six *PKD1* pseudogenes and the remarkable allelic heterogeneity. While the diagnostic accuracy of *PKD1* and *PKD2* gene mutation testing was found to be lower than the accuracy of US examination in adults above the age of 30 years, the relative accuracy of genetic vs. sonographic testing was comparable for children younger than 15 years in *PKD1* and superior in *PKD2* individuals (65). Also, in case the familial mutation has been identified, it is more cost effective to sequence the exon with the known mutation in at-risk individuals of this specific family. However, in that case, there is still a chance of a non-familial *de novo* mutation, not completely excluding the disease. Also, in case of a non *PKD1* non *PKD2* family, in which there is a clear ADPKD family history, but the *PKD* gene mutational analysis remains negative, exclusion of the disease in at-risk individuals will be possible only by means of a negative renal US at the age of 30–40 years or older (70).

Importantly, children who were diagnosed *in utero* or within their first 18 months of life, the so-called VEO group, represent a particularly high-risk group of ADPKD patients and should be managed accordingly. Indeed, this group was shown to have more hypertension and progression of eGFR < 90 mL/min/1.73 m2 by the last follow-up at the median age of 16 years; although they had similar rates of glomerular hyperfiltration compared to a non-VEO ADPKD control group (27).

### THE CONTROVERSY OF TESTING AT-RISK CHILDREN FOR THE PRESENCE OF ADPKD

Currently, apart from the Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference Report (71) and the European ADPKD Forum (EAF) Report (72), no guidelines are available for the clinical care of ADPKD families.

In the KDIGO consensus, it is recommended to have atrisk children screened for hypertension, starting at the age of 5 years, and repeated every 3 years in case of normotension. Presymptomatic screening of ADPKD by means of US or genetic analysis is not currently recommended for at-risk children, while it is accepted for at-risk adults, in whom the potential benefits of presymptomatic diagnosis usually outweigh the risks. However, on the other hand, this consensus suggests the following three options for at-risk children to be discussed thoroughly with their parents, so the parents can decide which approach will be taken. First, to screen the children and to disclose the results to the whole family; second to screen the children and disclose the results only to their parents, or last, not to screen the children. Moreover, according to the consensus, molecular genetic testing should be considered in cases of atypical renal imaging, discordant disease within a family; very mild PKD; in a patient without a family history for PKD; early and severe forms of PKD or PKD associated with syndromic features; and reproductive counseling (71).

In the EAF report, it is not discussed whether at risk children should be tested, although it is stated that genetic testing is vital for diagnosing ADPKD in children and that genetic counseling including informing the patients on the possibility of preimplantation genetic diagnosis for ADPKD should be offered at initial diagnosis (72).

Taken together, it is still a matter of controversy whether asymptomatic individuals at-risk for ADPKD, especially minors, should be tested for the presence of the disease or not (73). However, they and their family should be informed about the possibility of testing, and reasons why (not) to test. An important contra to testing, is the current absence of a cure (6). Although over the last decade, several drug targets based on the cellular mechanisms were tested in *in vitro* and *in vivo* models to slow down cystogenesis, only one drug demonstrated a moderate effect on disease progression in adult ADPKD patients, namely the vasopressin V2 receptor antagonist tolvaptan (74), as also concluded by a recent Cochrane review on interventional studies in adult patients (75). Despite its adverse effects of polydipsia and polyuria, it is currently the only drug available in Europe, indicated in selected adult patients with CKD stages 1–3, having evidence of rapidly progressing disease (76). Important to take into account are the methodological issues, limiting the interpretation of results of the published trials up till now. Though these are often encouraging, they are frequently restricted because of inadequate power, short study duration, patients' heterogeneity (large differences in TKV and/or underlying mutation class for instance), doses with inadequate pharmacological effects and uncertain target organ concentration (77). Moreover, the question remains whether it would be more efficacious to start treatment before extensive renal damage has occurred. A second contra is the potential induction of psychological stress in an at-risk individual and his/ her family, possibly causing feelings of (survival) guilt; although not only the certainty of having a diagnosis might be experienced as unbearable, but also the uncertainty when not testing (78, 79). A recently published mono-center survey performed in adults diagnosed with ADPKD, prior to the start of renal replacement therapy, showed that 22% had a clinically significant depression and 62% felt guilty about passing the disease on to their children. The latter represented one of the major psychological challenges confronting patients with ADPKD (80). A third argument against presymptomatic testing is the negative financial consequences a diagnosis with ADPKD might cause, such as difficulties obtaining insurances (81).

On the other hand however, presymptomatic testing not only has a prognostic value, by means of TKV measurement and *PKD* genotyping; it may also give caregivers the opportunity to target modifiable risk factors for disease progression from an early disease stage (82), thereby improving long-term renal outcome (71). This is supported by the observation of similar outcomes in a study comparing the presence of nephromegaly, hypertension, microalbuminuria and decreased eGFR between a cohort in which diagnosis was based on postnatal US screening vs. after presenting symptoms (26). This emphasizes the importance of regular screening for hypertension and proteinuria in at-risk children, but could also be seen as supporting argument for presymptomatic testing. Indeed, the significance of early treatment of disease symptoms should not be neglected. In children, this was demonstrated by slower cyst growth in ADPKD children with normotension compared to those with hypertension (47). Moreover, effective blood pressure control from childhood on may ameliorate the cardiovascular outcomes in these patients, known to be at high risk for early cardiovascular events (83, 84). Although glomerular hyperfiltration was shown to correlate with a more severe renal phenotype (29), there is no evidence (yet) for regular screening and/or treatment of it. Also, for some identified disease progression markers in adults, it remains to be determined to what extent their modification may influence the clinical course of ADPKD, such as for higher urinary sodium excretion and lower serum HDL cholesterol (85). A benefit accruing to all tested individuals, is their empowerment, namely the process through which people gain greater control over decisions and actions affecting their health and life, an important aspect is informed reproductive decision making (79).

A survey performed among European pediatric and adult nephrologist and geneticists before the KDIGO publication and the EMA approval of tolvaptan, demonstrated that their clinical practice and beliefs are very heterogeneous regarding testing of asymptomatic offspring and other ethical issues in the management of ADPKD families (86). In this study, agreement between respondents was observed regarding clinical testing of at-risk minors, although pediatric nephrologists supported this significantly stronger compared to geneticists. Also, the three groups of caregivers showed a moderate disagreement on the performance of genetic testing for ADPKD in at-risk minors. However, the differences observed in their counseling attitude were only attributable to their professional discipline. Therefore, ADPKD patients and their family tend to receive conflicting and/ or incomplete information from their caregivers. As suggested in the article, a reevaluation in a few years from now, to evaluate how the KDIGO consensus statement and the availability of tolvaptan and probably other upcoming treatment options influence both professionals and patients beliefs on these issues, would be interesting to perform (86).

### CHILDHOOD AS A CRITICAL THERAPEUTIC WINDOW FOR (FUTURE) THERAPIES

As suggested earlier, we should ask ourselves whether the best chance for preserving long term renal function in ADPKD is to take an early start. Starting prevention before extensive renal damage has occurred, thus at a young(er) age, might indeed improve the long term renal survival. Moreover, as suggested by Grantham et al., based on the assumption that decline in GFR is linked to the progressive increase in TKV, early treatment has the potential to delay renal dysfunction progression by more than a decade. When therapy is started at 18 years of age, reduction in the rate of TKV growth is predicted to shift from 5 to 2.5%, or 1.25% per year—a change that yields a vastly different effect on GFR decline than that induced by starting therapy later in life (87).

However, before heading toward cystogenesis inhibiting treatment options in children, disease manifestations requiring symptomatic treatment should be identified and managed adequately (16).

In both children and adults with ADPKD, hypertension is a predictor of worse renal outcome. Moreover, it is associated with increased cardiovascular morbidity and mortality (15).

In adults with ADPKD, LVH, a major cardiovascular risk factor, was significantly further decreased by rigorous (<120/80 mmHg) than standard blood pressure control (<140/90 mmHg), although this was not associated with a statistically significant difference in renal function between the two treatment arms (88). Moreover, from the HALT/PKD study, it was shown in an early ADPKD cohort that as compared with standard blood pressure control (120/70 to 130/80 mmHg), rigorous blood-pressure control (95/60 to 110/75 mmHg) was associated with a slower increase in TKV, no overall change in the eGFR, a greater decline in left ventricular mass index, and a greater reduction in albuminuria levels (89). These data may be particularly applicable to children as the study cohort represented early stages of ADPKD. However, a study using angiotensin converting enzyme inhibitors (ACEi) in children and young adolescents with ADPKD, over a period of 5 years, failed to demonstrate a significant effect on renal growth in the total cohort. Importantly, ACEi treatment was associated with stable renal function and left ventricular mass index in the (borderline) hypertensive children, who are at particular risk for increases in renal volume and left ventricular mass index and decreased renal function as compared with the other study groups (90).

KDIGO states that treatment of hypertension in pediatric ADPKD patients should follow prevailing pediatric guidelines; meaning a goal blood pressure below the 90th percentile for age, sex, and height; with the only exception that RAAS blockade is preferred as first-line treatment (71). However, given these findings, it was recommended recently to use an ACEi with a target blood pressure of <110/75 in young adults older than 18 years; and in adolescents and children with borderline hypertension (75–95th percentile) or hypertension (≥95th percentile), to use an ACEi to achieve a goal blood pressure ≤50th percentile. Angiotensin receptor blockers (ARBs) can be used in patients who do not tolerate ACEi (15). Combination of both RAAS blockers should be avoided as no benefit was demonstrated by adding an ARB (telmisartin) to an ACEi (lisinopril) on ADPKD progression in adult patients with early or moderately advanced kidney disease (89). Moreover, girls of childbearing potential and their families should be informed on fetal toxicity of ACEi and ARBs (15).

Next, in a 3–year, phase III clinical trial of pravastatin on TKV and left ventricular mass index, pravastatin was effective in slowing the progression of structural kidney disease in older children and young adults with ADPKD. There was no beneficial effect on the left ventricular mass index, nor on their urinary micro albumin excretion (91). However, a recently published *post hoc* analysis of the HALT PKD trials, comparing the outcomes of participants who never used statins with those who used statin for at least 3 years, failed to demonstrate a benefit of statin therapy in both subjects with a preserved renal function (Study A) as a reduced renal function (Study B) (92). Conclusions remain preliminary, as the first study consisted of a relatively small cohort, and the *post hoc* analysis of the HALT PKD is limited given the small number of statin users in Study A, different statin drugs and doses used, non-randomized allocation and advanced disease stage in Study B. Also, as with ACEi, potential risks to the fetus with statin exposure during pregnancy should be reviewed prior to treatment to females of appropriate pubertal development.

As mentioned before, currently, the only drug available for selected (<50 years, CKD stages 1-3a, rapidly progressing disease) adult patients in Europe (76) is the vasopressin V2 receptor antagonist tolvaptan. Tested in the TEMPO clinical trials, including adult patients with a relatively advanced disease stage (TKV > 750 mL), tolvaptan use was demonstrated to induce both a slower annual increase in TKV and a slower annual decline in kidney function in treated patients vs. controls (74). Despite its possible adverse effects—polydipsia, polyuria and significant liver enzyme elevation—tolvaptan is currently studied in a phase III double-blind placebo controlled clinical trial in a pediatric ADPKD cohort within Europe (NCT02442674) (93). Apart from this, there is one case report, describing the off-label use of tolvaptan in a severely affected neonate with ADPKD (94). Other coming exciting therapies, such as the AMPK activator metformin, are currently tested in adult patients, and will be tested in pediatric ADPKD depending on the results in adults (95). A general remark on (future) studies including children with ADPKD is that the subjects often are performed in tertiary and specialist centers. This might reflect highly selected populations, likely to contain a higher proportion of symptomatic children and "fast-progressors," influencing the results.

### CHALLENGES AND UNMET NEEDS IN PEDIATRIC ADPKD CARE

In the care of pediatric ADPKD and their families, there is a clear need of a more standardized management. Few initiatives have been taken so far, namely the KDIGO consensus and EAF report, however, conflicting recommendations still need standardization and completion such as testing of at-risk minors and the target blood pressure when treating hypertension (50th vs. 90th percentile) as discussed earlier. Moreover, there are no details provided on how blood pressure should be monitored, namely *via* an office blood pressure measurement or a 24-h ambulatory blood pressure monitoring. There is evidence that the latter should be performed routinely from a large pediatric cohort, in which 51.9% of patients lacked a physiological nocturnal blood pressure dipping and 18.2% had isolated nocturnal hypertension (Massella L et al. High prevalence of hypertension in a European cohort of children with ADPKD: results of the ADPKiDs study. IPNA; 2016; Iguacu, Brazil) (96).

Others recommendations are absent, such as on screening for intracranial aneurysms, both in adults and in children, a topic in which many areas of uncertainty remain, as observed in a recent survey among European French speaking nephrologists (97). However, there is recent evidence that systematic screening in all ADPKD patients, regardless of the family history for intracranial aneurysms, is cost-effective (98). Moreover, despite the recommendations, it will take time to implement these. Till now, many at risk children with ADPKD are not under regular follow-up and remain undiagnosed (39). Thus, leaving the important issue who should be responsible for informing them in adulthood: only the family, both the family and the caregivers or only the caregivers? From the caregivers' point of view, this is a shared responsibility for professionals with the family (86), although it would be interesting to know the opinion of ADPKD patients on this.

Next, there is a need of diagnostic imaging criteria under the age of 15 years apart from the currently used eminence-based guidelines. The most important is the need for clear prognostic criteria from childhood, as a validated method to identify slow vs. rapid progressors does not exist in the pediatric population, except in case of VEO ADPKD (27). However, this is necessary for the stratification of young patients in clinical trials, and possibly as new and better outcome measures. Indeed, TKV as a prognostic marker is well-assessed and accepted, but only in studies starting to include patients from the age of 15 years (87). Moreover, TKV measurements *via* MRI were shown to be more accurate and

### REFERENCES


reproducible compared to US in adults (99), however, unfeasible in many young children due to the need of sedation or general anesthesia. Therefore, new modalities should be tested for TKV measurement in ADPKD children. One promising option is a three dimensional US technique, which was shown a valuable alternative to MRI to measure, although underestimating MRI TKV (96, 100).

Untill now, most studies published were performed on small pediatric cohorts, while we need to build large cohorts before we will be able to define evidence-based recommendation for children with ADPKD. Therefore, we recently have launched our initiative of the "ADPedKD" international registry to collect retrospective and prospective longitudinal clinical, imaging, and genetic data of children until the age of 19 years.

### CONCLUSION

In conclusion, children and young adults with ADPKD may represent a critical therapeutic window to reduce future renal and cardiovascular risk. However, we are still far from standardized care for this population. Moreover, the ultimate goal should be to empower patients to make their own informed decisions.

According to the KDIGO consensus, the advised follow-up of at-risk children should encompass regular monitoring for disease manifestations such as hypertension and proteinuria, so treatment of these disease modifying factors is initiated as early as possible.

### AUTHOR CONTRIBUTIONS

The authors confirm being the sole contributors of this work and approved it for publication.

### FUNDING

DM is supported by the Clinical Research Fund of UZ Leuven, by the Fund for Scientific Research, Flanders G0B1313N and by a research grant from the European Society for Pediatric Nephrology. SR is supported by the Fund for Scientific Research, Flanders 11M5214N.

survival in autosomal dominant polycystic kidney disease. *J Am Soc Nephrol* (2015) 27:942–51. doi:10.1681/ASN.2015010016


autosomal dominant polycystic kidney disease. *J Clin Endocrinol Metab* (2017) 102:4210–7. doi:10.1210/jc.2017-01157


polycystic kidney disease is cost-effective. *Kidney Int* (2017). doi:10.1016/j.kint. 2017.08.016


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 De Rechter, Breysem and Mekahli. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Is It Ethical to Test Apparently "Healthy" Children for Autosomal Dominant Polycystic Kidney Disease and Risk Medicalizing Thousands?

#### *Tess Harris\**

*PKD International, Geneva, Switzerland*

Keywords: autosomal dominant polycystic kidney disease, genetic test, presymptomatic testing, genetic counseling, medicalization, next-generation sequencing

### INTRODUCTION

#### *Edited by:*

*Max Christoph Liebau, Klinik und Poliklinik für Kinder- und Jugendmedizin, Universitätsklinikum Köln, Germany*

#### *Reviewed by:*

*Julia Hoefele, Technische Universität München, Germany Djalila Mekahli, University Hospitals Leuven, Belgium Marva Moxey-Mims, Children's National Health System, United States*

#### *\*Correspondence:*

*Tess Harris tess.harris@pkdinternational.org*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 31 October 2017 Accepted: 20 December 2017 Published: 19 January 2018*

#### *Citation:*

*Harris T (2018) Is It Ethical to Test Apparently "Healthy" Children for Autosomal Dominant Polycystic Kidney Disease and Risk Medicalizing Thousands? Front. Pediatr. 5:291. doi: 10.3389/fped.2017.00291*

A frequent question asked by parents of children at risk of inheriting autosomal dominant polycystic kidney disease (ADPKD) is: "should my child be tested?" Until recently, most doctors answered "no." They regarded ADPKD as an "adult" condition and advised families to wait until their children were grown up and not to worry them during childhood. But is this an ethical response today given growing evidence of manifestation in children already diagnosed with ADPKD and the prospect of a treatment (1).

### BACKGROUND

Autosomal dominant polycystic kidney disease is the most common inherited renal disease worldwide, affecting 1–2 in 1,000 people (2), an estimated 12.5 million individuals across all ethnic groups. Mutations in three genes (*PKD1*, *PKD2*, and *GANAB*) cause multiple fluid-filled cysts to develop in both kidneys and other organs. The kidney cysts grow and expand throughout life causing complications, such as chronic and acute pain, infections, hematuria, stones, hernia, and disfiguring abdominal swelling. Over time, the cysts overwhelm and destroy healthy tissue resulting in kidney failure. ADPKD also causes cysts to form in the liver (polycystic liver disease or PLD), with some patients experiencing massive liver growth, infection, and pain. Brain aneurysms are four times as common in ADPKD as the general population and patients are at risk of cardiovascular disease from high blood pressure. Other, rarer manifestations can occur, such as cysts in the pancreas, brain, and seminal vesicles (**Figure 1**).

Autosomal dominant polycystic kidney disease accounts for one in 10 patients in end-stage renal disease (ESRD) requiring renal replacement therapy (dialysis or kidney transplant) and is a major health-care burden. Across Europe, ESRD in ADPKD is estimated to cost €1.5 billion/year (3). Those with severe PLD may require life-saving liver transplantation due to comorbidities from liver enlargement. Perversely, cystic livers remain functional and patients with PLD can spend years on transplant waiting lists owing to low Model for End-Stage Liver Disease score.

Pre-ESRD, ADPKD patients often experience significant diminution in quality of life. This is due not only to the clinical burden, but also to the impact that this chronic, progressive incurable, genetic disease has on personal and family life. Anxiety, depression, shock, fear of the future, and genetic guilt have been observed in patients (4). Average ESRD age in ADPKD is mid to late 50s (5). Kidney failure at an age when many people are economically productive can result in lost jobs and income—with consequent socio-economic burden.

There are no proven treatments that will stop cyst development. Management is focused on treating the symptoms: anti-hypertensive medication, antibiotics, pain relief, and then dialysis or transplantation. In 2015, the first-ever drug to treat ADPKD in adults was approved by regulators

in Japan, Canada, and the EU. In a trial, tolvaptan (a vasopressin V2 receptor antagonist) was shown to help slow ADPKD progression by reducing the rate at which kidneys become enlarged by cysts and helping slow the speed of kidney function decline (6). Tolvaptan does not alter liver cyst growth. In the EU, only adult ADPKD patients with CKD stage 1–3 and evidence of rapidly progressing disease are eligible for this therapy.

## ADPKD IN CHILDREN

Autosomal dominant polycystic kidney disease is present from conception but symptoms are primarily adult-onset and rare during childhood. Being an autosomal dominant condition, ADPKD carries an inheritance risk of 1 in 2 (50%) from an affected parent. However, *de novo* mutations have been found in 1 in 10–13 people (7). Despite continuous, silent cyst growth, kidney function is typically normal during childhood owing to compensatory hyperfiltration in the nephrons. Many people are not diagnosed until adulthood (even with family histories) when symptoms, such as hypertension, chronic back pain or blood in the urine trigger an investigation for ADPKD. Some patients are diagnosed incidentally, e.g., during scans for kidney stones or pregnancy (unpublished data from PKD Charity UK survey 2015).

Diagnosis is usually by ultrasound. However, cysts in children are not always detected and there is a chance of cyst development in later life. Genetic testing is uncommon owing to cost and genetic complexity. Rarely, genetic testing is used where diagnosis by imaging is inconclusive or to distinguish ADPKD from other cystic kidney diseases such as autosomal recessive polycystic kidney disease and ciliopathies which can "mimic" PKD (8).

There is wide variability in ADPKD progression, even in families with the same mutation. Individuals with ADPKD caused by *PKD1* mutations typically have a more severe progression than those with *PKD2* mutations. ESRD, for instance, can be up to 20 years earlier in patients with *PKD1* mutations (9). Clinical features do overlap, however, and clinicians do not differentiate in practice. Monitoring and treatment are indicated by kidney function progression and comorbidities.

Molecular mechanisms that may explain the variability are starting to be understood. It is suggested that stochastic, epistatic and environmental events may influence ADPKD expression in individuals (10). Advances in next-generation sequencing should provide more knowledge and insights but this technique is not widely available or affordable.

Unless a child has a diagnosis of ADPKD, they probably will not need treatment until later in life. Symptom rarity, perception of ADPKD being an "adult" condition, and lack of licensed treatments for children to slow disease progression, mean that historically doctors and parents felt safe to wait until the child was old enough to decide about testing for themselves.

Presymptomatic testing was discussed at the ADPKD Kidney Disease: Improving Global Outcomes (KDIGO) Consensus Conference (2014). The conference included patients and parents, and at that time there was agreement that minors should not be offered presymptomatic testing (11). Three approaches were proposed for parents considering screening or testing at-risk but undiagnosed children: "screen children as young as possible and disclose the results to the entire family; screen and disclose results only to the parents; do not screen." Parents would in all cases have the final decision regarding screening.

The KDIGO report's authors felt that negative outcomes of a positive diagnosis in a child outweighed positives (11). When anyone is diagnosed with ADPKD (or any other potentially life-changing, incurable genetic condition), there are long-term consequences. Career choices may be limited, e.g., patients may be unable to join the armed forces or pursue vigorous contact sports such as rugby or martial arts. Insurance policies for life, critical illness, and private health may be impossible to obtain; other types of insurance will incur higher premiums. A diagnosis has implications for family planning and may negatively influence a patient's perspective of their future (unpublished data from PKD Charity UK survey 2015).

Undiagnosed at-risk children may appear "healthy" on the outside. However, there is evidence of early symptoms that may be overlooked in the undiagnosed ADPKD population. In a meta-analysis, Marlais et al. estimated that at least one in five children with ADPKD have hypertension (12); in some centers one in three children are affected. Left ventricular hypertrophy is seen. Cyst and kidney growth is evident, sometimes accompanied by pain. Some children have hematuria and urinary tract infections. Early interventions such as anti-hypertensive medication are inexpensive to prescribe and provide long-term positive outcomes with minimal side-effects. Children can be advised about kidney protection during sports.

There is evidence of changing attitudes among clinicians. In a survey, European geneticists, pediatric and adult nephrologists generally agreed that at-risk minors be clinically tested for ADPKD but did not agree about genetic testing (13). The authors called for a consensus, in particular to clarify the sometimes "conflicting information given to families."

Autosomal dominant polycystic kidney disease was historically called an "adult" condition. The term "adult PKD" is still in use in the SNOMED CT International Codes (14). But it is clearly not just a disease of adulthood, so why are doctors—and parents—reluctant to test children for ADPKD?

### ETHICS OF PRESYMPTOMATIC TESTING IN CHILDREN

Although genetic testing is rarely used to diagnose ADPKD, declining costs mean this may become cost-effective and more available. Moreover, the author anticipates there will a rise in parents asking about screening with the potential of access to a licensed therapy (tolvaptan) at 18, as well as growth in clinicians' awareness of manifestations in children. In the author's opinion, international recommendations on presymptomatic testing—to encompass both imaging and genetic testing—should be developed as a priority to guide ADPKD screening/testing in children.

It is instructive to examine current recommendations for presymptomatic genetic testing in children and consider customizing them for ADPKD to include imaging. The prevailing view is that children should not be offered genetic tests for inherited conditions that develop in adult life and are currently untreatable. If a test is being considered, parents and doctors need to ask if the test is in the child's "best interests," i.e., will it do more harm than good to have a diagnosis or should it wait until the child is sufficiently mature and emotionally competent to decide for themselves?

The European Society of Human Genetics (ESHG) recommends (15) that children (minors) can decide for themselves about genetic testing when they are "well informed, have an adequate understanding of the test and its potential consequences, have the capacity to make this decision, are not exposed to external pressure, and have had appropriate counseling."

These are laudable recommendations but the author knows from personal family history of ADPKD and 10-year experience moderating a large ADPKD support group (PKD Charity UK) that information and counseling provided to families and individuals undergoing genetic tests are not offered to those considering an ultrasound test. Indeed, many families are given referrals for ultrasound tests without the opportunity to discuss pros and cons. This standard of care is unlikely to change in the short-term as the routine commissioning of genetic services for ADPKD is not expected for many years.

The ESHG recommendations state that presymptomatic testing can be done if preventive interventions can start in childhood. At present, unfortunately, there are no guidelines for the care of children with or at risk of ADPKD and thus no motivation or incentive for physicians to suggest presymptomatic testing (genetic or imaging).

### WHAT ARE THE MEDICALIZATION IMPLICATIONS OF PRESYMPTOMATIC TESTING OF CHILDREN AT RISK OF ADPKD?

It is estimated that 12,000 children (under 18 years) in the UK have ADPKD, of whom about 500 are seen in specialist clinics (unpublished survey of UK nephrologists 2015). The actual undiagnosed population is unknown but presumed to be many thousands. Most children will not have symptoms until adulthood. Many doctors and parents feel that medicalizing children, labeling them with a "disease" while asymptomatic, is inappropriate. Over 90% of parents of these children will also be dealing with the impact of ADPKD on one of them (and possibly other family members). Burdening them with caring for a diagnosed child could add unnecessary anxiety and stress. Moreover, medicalization incurs intervention costs, even if these are limited to hypertensive monitoring and low cost drug prescribing.

Medicalization can have benefits, however (16). Getting a diagnosis can be enabling and empowering. Early antihypertensive intervention may reduce cardiovascular risk (and consequent costs) in later life and can help families prepare their children for adulthood. These benefits need to be weighed against the costs to society; there is no immediate solution to this dilemma.

### OPINION

In the author' opinion, it is ethical to test apparently "healthy" children at risk of ADPKD, but only when there is a specific framework in place for presymptomatic ADPKD testing accompanied by approved international recommendations for follow-up management and long-term care. The framework should set quality standards which ensure: (i) that parents have the opportunity to discuss the implications in advance of referrals for imaging and/or genetic testing; (ii) that testing by ultrasound for ADPKD is considered equivalent to genetic testing and accompanied by access to appropriate counseling; (iii) that the child is involved in the decision-making if they are sufficiently mature and well informed; (iv) that parents are enabled to discuss the diagnostic implications of ADPKD with children and supported by appropriate services and information; and (v) that genetic testing is considered and provided where feasible and cost effective.

### REFERENCES


### AUTHOR CONTRIBUTIONS

TH contributed the content of the article, which expresses the personal opinion of TH and not necessarily the official views of the PKD Charity UK and PKD International.

### ACKNOWLEDGMENTS

The author acknowledges the inputs from families and individuals affected by ADPKD. TH is also a patient with ADPKD with several affected siblings and relatives.

severe polycystic kidney disease. *J Am Soc Nephrol* (2011) 22(11):2047–56. doi:10.1681/ASN.2010101080


**Conflict of Interest Statement:** The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Harris. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Gastrostomy Tube Insertion in Pediatric Patients With Autosomal Recessive Polycystic Kidney Disease (ARPKD): Current Practice

Kathrin Burgmaier <sup>1</sup> , Joy Brandt <sup>1</sup> , Rukshana Shroff <sup>2</sup> , Peter Witters <sup>3</sup> , Lutz T. Weber <sup>1</sup> , Jörg Dötsch<sup>1</sup> , Franz Schaefer <sup>4</sup> , Djalila Mekahli 5,6 and Max C. Liebau1,7 \*

<sup>1</sup> Department of Pediatrics, University Hospital of Cologne, Cologne, Germany, <sup>2</sup> Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom, <sup>3</sup> Department of Pediatric Gastroenterology and Hepatology, University Hospitals Leuven, Leuven, Belgium, <sup>4</sup> Division of Pediatric Nephrology, Center for Pediatric and Adolescent Medicine, University of Heidelberg, Heidelberg, Germany, <sup>5</sup> Department of Pediatric Nephrology, University Hospitals Leuven, Leuven, Belgium, <sup>6</sup> PKD Research Group, Department of Development and Regeneration, KU Leuven, University of Leuven, Leuven, Belgium, <sup>7</sup> Center for Molecular Medicine, University Hospital of Cologne, Cologne, Germany

#### Edited by:

Katherine MacRae Dell, Case Western Reserve University, United States

#### Reviewed by:

Praveen Kumar Conjeevaram Selvakumar, Cleveland Clinic, United States Vera Hermina Koch, Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, Brazil

> \*Correspondence: Max C. Liebau max.liebau@uk-koeln.de

#### Specialty section:

This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics

Received: 31 January 2018 Accepted: 15 May 2018 Published: 04 June 2018

#### Citation:

Burgmaier K, Brandt J, Shroff R, Witters P, Weber LT, Dötsch J, Schaefer F, Mekahli D and Liebau MC (2018) Gastrostomy Tube Insertion in Pediatric Patients With Autosomal Recessive Polycystic Kidney Disease (ARPKD): Current Practice. Front. Pediatr. 6:164. doi: 10.3389/fped.2018.00164 Introduction: Autosomal recessive polycystic kidney disease (ARPKD) is a severe hepatorenal disorder of childhood. Early renal disease in ARPKD may require renal replacement therapy and is associated with failure to thrive resulting in a need for nasogastric tube feeding or gastrostomy. In ARPKD patients, the benefit of a gastrostomy in nutrition and growth needs to be weighed against the potential risk of complications of congenital hepatic fibrosis (CHF) and portal hypertension like variceal bleeding. CHF in ARPKD has thus been considered as a relative contraindication for gastrostomy insertion. Yet, data on gastrostomies in pediatric patients with ARPKD is lacking.

Methods: We conducted a web-based survey study among pediatric nephrologists, pediatric hepatologists and pediatric gastroenterologists on their opinions on and experiences with gastrostomy insertion in ARPKD patients.

Results: 196 participants from 39 countries shared their opinion. 45% of participants support gastrostomy insertion in all ARPKD patients, but portal hypertension is considered to be a contraindication by a subgroup of participants. Patient-specific data was provided for 38 patients indicating complications of gastrostomy that were in principal comparable to non-ARPKD patients. Bleeding episodes were reported in 3/38 patients (7.9%). Two patients developed additional severe complications. Gastrostomy was retrospectively considered as the right decision for the patient in 35/38 (92.1%) of the cases.

Conclusions: This report on the results of an online survey gives first insights into the clinical practice of gastrostomy insertion in ARPKD patients. For the majority of participating physicians benefits of gastrostomy insertion retrospectively outweigh complications and risks. More data will be required to lay the foundation for clinical recommendations.

Keywords: ARPKD, congenital hepatic fibrosis, portal hypertension, peritoneal dialysis, PKHD1, pediatric polycystic kidney disease

**106**

### INTRODUCTION

Autosomal recessive polycystic kidney disease (ARPKD) is a rare but severe disorder mainly affecting the liver and the kidneys. The disorder represents one of the leading reasons for pediatric dialysis and kidney-, liver- or combined liver and kidney transplantation. Renal involvement may present very early in life with massively enlarged polycystic kidneys and end stage kidney disease (ESKD), requiring renal replacement therapy (1, 2). Liver involvement due to congenital hepatic fibrosis (CHF) tends to present later in life and is associated with portal hypertension in up to 60% of patients with subsequent development of splenomegaly and collateral circulation (1–3). Esophageal varices have been reported in up to 56% of ARPKD patients with the risk of variceal bleeding (1–3).

Chronic kidney disease (CKD) is associated with growth failure and it has been suggested that ARPKD patients are at a particularly high risk (3, 4). Immaturity and early uremia may affect enteral feeding. Severe ARPKD is also accompanied by massive kidney enlargement and pulmonary hypoplasia. Abdominal distension and ventilation may complicate nutrition resulting in a need for persistent nasogastric feeding.

Concerns have been raised concerning gastrostomy in ARPKD patients. Infection risk is an issue in patients on peritoneal dialysis (PD) (5, 6). If possible, gastrostomy insertion in PD patients should take place prior to or at the same time of PD catheter placement. If gastrostomy insertion becomes necessary after onset of PD, an open surgical procedure with protective sutures is recommended as opposed to the endoscopic technique (7). An increased risk of variceal bleeding in patients with portal hypertension and an increased risk of spleen injury in case of splenomegaly have been suggested for endoscopic gastrostomy insertion in patients with cystic fibrosis associated liver disease and portal hypertension (8) and patients with liver cirrhosis (9). Furthermore, the development of stomal varices after gastrostomy has been discussed (8, 10). ARPKD with accompanying portal hypertension and possible future liver transplantation has been classified as a relative contraindication for gastrostomy insertion in children by the French society of gastrointestinal endoscopy (11) and within the position paper of the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) on management of percutaneous endoscopic gastrostomy in children and adolescents (12).

To the best of our knowledge there are no previous studies addressing clinical approaches towards gastrostomy insertion and complications of gastrostomy in pediatric ARPKD patients. In order to gain insight into current practice, we conducted an anonymous web-based survey among pediatric nephrologists, pediatric hepatologists and pediatric gastroenterologists on their opinion and experiences concerning benefits, risks, and methods of gastrostomy insertion in ARPKD patients.

### METHODS

### Study Design, Survey Development, Survey Content, and Administration

Questionnaires on the current clinical practice in gastrostomy insertion in patients with ARPKD (Supplementary data 1) were designed and validated by an expert group of pediatric nephrologists, hepatologists, and gastroenterologists. The survey was conducted in two steps: the first part was designed to assess general data on the background of participants as well as opinions regarding gastrostomy in patients suffering from CKD in general and ARPKD patients in particular. An additional aim was to identify specific conditions regarded as contraindications. The second part of the survey was designed to address gastrostomy insertion in ARPKD more specifically with respect to age at insertion, technique of insertion, periinterventional antibiotic and antifungal prophylaxis, signs of hepatic ARPKD involvement and conduction of dialysis at time of insertion. We specifically asked for observed complications after gastrostomy insertion in patients and for management of gastrostomy in case of subsequent transplantation. The survey ended with a personal evaluation of the risk-benefit analysis and the management of gastrostomy in non-ARPKD patients prior to transplantation.

The survey was an anonymous, web-based, cross-sectional study on a voluntary basis. Invitation to participate in the survey was sent to members of the European Society of Pediatric Nephrology (ESPN) study group, the International Pediatric Peritoneal Dialysis Network (IPDN) study group, the ARegPKD Consortium, the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) Hepatology Interest Group, and the Pediatric Gastroenterology Internet Bulletin Board (PEDGI). The participants were asked to avoid duplicate entries in case of repeated invitation for the first part of the survey and to coordinate patient-specific entries within the own center in order to avoid multiple replies from a single center for the second part of the survey. The study was approved by the Ethics Committee of the Medical Faculty of the University of Cologne, Germany.

We received responses from 196 participants out of 39 countries with the largest groups of participants deriving from Germany (26), Poland (19), the United States of America (18), Belgium (17), the United Kingdom (14), France and Spain (each 11). Five or less participants each came from Argentinia, Australia, Austria, Brazil, Chile, China, Croatia, Denmark, Estonia, Georgia, Greece, Hungary, India, Iran, Kuwait, Lithuania, Macedonia, Netherlands, New Zealand, Oman, Pakistan, Peru, Portugal, Russia, Saudi Arabia, South Africa, South Korea, Sweden, Switzerland, and the United Arab Emirates. The profession of the participants was indicated as pediatric nephrologists (n = 141, 74.2%), pediatric hepatologists and/or gastroenterologists (n = 39, 20.5%), pediatrician (n = 3, 1.6%), pedatric nephrology/dialysis nurse (educator) (n = 2, 1.0%), dietitian (n = 2, 1.0%), and pediatric intensivist, adult nephrologist and trainee pediatric nephrology (each 1, each 0.5%). There were no replies by pediatric surgeons. 161/166 (97.0%) participants indicated that they took care of ARPKD patients in follow-up with a

**Abbreviations:** ARPKD, autosomal recessive polycystic kidney disease; CHF, congenital hepatic fibrosis; CKD, chronic kidney disease; ESKD, end-stage kidney disease; GI, gastrointestinal; HD, hemodialysis; HTN, hypertension; n.s., not significant; PD, peritoneal dialysis; PEG, percutaneous endoscopic gastrostomy.

median number of 6 ARPKD patients (minimum one patient, maximum 60 patients). 152/164 (92.7%) participants performed pediatric PD and 145/164 (88.4%) performed hemodialysis (HD) at their institution. 118/162 (72.8%) participants performed kidney transplantation at their center, 65/159 (40.9%) performed liver transplantation, and 53/158 (33.5%) performed combined liver and kidney transplantation. 99/158 (60.7%) participants indicated that they had experience with management of variceal/portal hypertensive bleeding at their institution.

Patient-specific data was entered by 21 participants for a total of 38 patients. Most patients were from Germany, the United States of America, Denmark, and Iran. All patients showed specific characteristics in terms of country of origin, age, gender, and treatment modalities making duplicate entries of a single patient unlikely. Information regarding 32/38 (84.2%) patients was entered by pediatric nephrologists, for 3 (7.9%) patients information was given by pediatric hepatologists, information regarding one patient (2.6%) was entered by a pediatric gastroenterologist, pediatrician in training, and a nurse, respectively.

### Statistical Analysis

Age at insertion of gastrostomy is given as median with interquartile range. All other variables were categorical and evaluated using descriptive statistics. Differences between nominal variables were calculated using Chi-Square test, significance was considered for p < 0.05. Data analysis was performed using IBM SPSS Statistics 22 for Windows.

### RESULTS

### General Attitude Toward Gastrostomy Insertion in CKD and ARPKD

In principle, 121/166 (72.9%) participants supported gastrostomy insertion in patients with CKD and ESKD (**Figure 1**). Regarding gastrostomy insertion in ARPKD patients, participants showed more detailed answers: while 71/158 (44.9%) supported insertion in all ARPKD patients, 15/158 (9.5%) did not support gastrostomy insertion in ARPKD patients. 23/158 (14.6%) participants supported gastrostomy insertion only in ARPKD patients without signs of portal hypertension, 10/158 (6.3%) only in patients without PD and 9/158 (5.7%) only in patients without signs of portal hypertension and/or without PD (**Figure 1**). There were significantly more nephrologists [61/117 (52.1%)] supporting gastrostomy in all ARPKD patients compared to gastroenterologists/hepatologists [7/28 (25.0%), p = 0.01]. There was a trend to a more restrictive support of gastrostomy insertion in patients with signs of portal hypertension and/or PD in pediatric gastroenterologists/hepatologists compared to pediatric nephrologists (n.s., **Figure 1**). A substantial number of participants (28/158, 17.7%) raised other or additional concerns, that encompassed general factors ("precise case-by-case-decision," "only in failure of all other techniques of feeding," "fear of increased risk of severe local infections after transplantation," "reluctance of surgeons or gastroenterologists to perform procedure"), PDassociated factors ("more strict indication in PD patients," "only surgical procedure in case of PD") and factors associated with portal hypertension ("more strict indication in case of portal hypertension," "future liver transplantation as contraindication") (**Figure 1**).

### Indications for Gastrostomy Insertion and Hepatic Involvement Prior to Insertion in ARPKD Patients

Indications for gastrostomy insertion are shown in **Figure 2A**. Median age at insertion was 1.41 years (interquartile range 0.50– 2.00 years). The youngest patient was 0.08 years, the oldest patient was 11.00 years old at gastrostomy insertion.

Prior to gastrostomy insertion more than half of the centers screened for varices in ARPKD patients (12/22, 54.5%). Most centers used esophagogastroduodenoscopy and/or ultrasound. Evaluation of the hepatic phenotype in specific patients indicated splenomegaly in 19/38 (50%) patients at time of gastrostomy insertion and evidence of collateral circulation (e.g., oesophageal varices) in 7/38 (18.4%) of patients (**Figure 2B**).

### Method of Gastrostomy Insertion in ARPKD Patients

In the identified 38 patients, gastrostomy was inserted via endoscopy in 21/38 (55.3%), laparoscopically in 4/38 (10.5%), and in an open surgical approach in 9/38 (23.7%) patients. This differs from the opinion indicated in the first part of the survey, in which participants reported to favor insertion via endoscopy (60/130, 46.2%) or laparoscopic (37/130, 28.5%), while open insertion was preferred by only 4/130 (3.1%) participants with 29/130 (22.3%) participants being unsure about their preference in ARPKD patients (**Figure 3A**). The preference of different methods of insertion did not differ between pediatric gastroenterologists/hepatologists and nephrologists. In general CKD/ESKD patients, 72/117 (61.5%) participants favored endoscopic insertion, 25/117 (21.4%) laparoscopic, and 2/117 (1.7%) open insertion, while 18/117 (15.4%) were unsure about their preferred method.

### Complications of Gastrostomy in ARPKD Patients

Reported complications of gastrostomy insertion encompassed excessive granulation tissue (7/38, 18.4%) and wound infection (5/38, 13.2%). In 3/38 (7.9%) patients each leakage of PD fluid or stomach fluid through gastrostomy was reported. One patient each (2.6% each) developed stomal varices, bleeding episodes due to variceal bleeding and excessive bleeding from gastrostomy exit site 9 months after insertion (**Figure 3B**). Other complications encompassed buried bumper, hernia, requirement of surgical closure after gastrostomy removal and a suspected association with a recurrent Clostridium difficile infection. One patient was reported to have suffered from preinterventional hypotension during anesthetic induction with subsequent cerebral infarction. 5/38 (13.2%) patients required

surgical revision (change to button, herniotomy, surgical closure, due to infection), including one patient who was reported to have developed fulminant sepsis with subsequent death. Detailed information regarding causality and temporal connection of gastrostomy with fulminant sepsis was not available due to the anonymous setting of the survey. 7/52 (13.5%) colleagues

who responded to the general questionnaire indicated the requirement for gastrostomy removal in ARPKD patients for various reasons (gastrocutaneous fistula, infection, discomfort of patient resp. parents'refusal of continuation, pretransplant removal due to sufficient weight gain).

### Antiinfectious Prophylaxis

Prophylactic antibiotics were applied to all patients undergoing gastrostomy insertion in 8/22 (36.4%) centers, while 3/22 (13.6%) centers indicated prophylactic antibiotics only in ARPKD patients. No prophylactic antibiotics were given in 7/22 (31.8%) centers. Cephalosporines were most frequently used [8/10 (80.0%) centers]. 2/22 (9.1%) centers used prophylactic antifungals (fluconazole) in both ARPKD and non-ARPKD patients.

### Dialysis and Transplantation

10/38 (26.3%) patients with gastrostomy already performed PD at time of gastrostomy insertion. 4/38 (10.5%) patients performed HD at time of gastrostomy insertion. 13/38 patients (34.2%) started PD after gastrostomy insertion, 2/38 (5.3%) patients started HD after gastrostomy insertion.

18/38 (47.4%) patients underwent transplantation with inserted gastrostomy (10 kidney transplantations, 8 combined liver and kidney transplantations). Gastrostomy was removed at timepoint of transplantation in 3 cases (one patient with kidney transplantation from one center in Iran, two patients with combined liver and kidney transplantation from one German center) and was kept in all other patients. In non-ARPKD patients gastrostomy was removed in 6/64 (9.4%) centers prior to transplantation. One colleague reported that gastrostomy is removed in non-ARPKD patients in case of liver, but not in case of kidney transplantation.

### General Evaluation of Gastrostomy in ARPKD Patients

63/158 (39.9%) participants indicated at least one ARPKD patient who had undergone gastrostomy insertion at their center with 7/65 (10.8%) colleagues recalling significant complications. Yet, in summary, 58/64 (90.6%) participants retrospectively summarized that gastrostomy insertion was the right decision for their patients, including 12/15 (80.0%) pediatric gastroenterologists/hepatologists, and 42/45 (93.3%) pediatric nephrologists (p = 0.14).

In summary 34/38 (89.5%) patients were considered to benefit from gastrostomy with respect to development and growth. Gastrostomy insertion was evaluated as right decision for the patient in 35/38 (92.1%) patients. In two cases gastrostomy insertion was interpreted as not beneficial for the patient, with one patient developing preinterventional hypotension with neurological sequelae and one patient dying from fulminant sepsis with unclear causality or temporal connection to gastrostomy insertion.

## DISCUSSION

Based on an online survey we report on the first data on current practice of gastrostomy insertion in patients with ARPKD. To the best of our knowledge, this pediatric ARPKD cohort is the first to be reported with details on method of insertion and associated complications.

When evaluating the opinion in a first survey with 196 participants, ARPKD was considered to be a special condition in comparison to other CKD/ESKD causes. Both portal hypertension and PD were mentioned as (relative) contraindications for gastrostomy in ARPKD patients. This reflects the presumption of these two conditions as precautions for gastrostomy insertion in ARPKD patients (5, 7, 11, 12). Interestingly, pediatric gastroenterologists/hepatologists seemed to be more cautious in inserting gastrostomy in comparison to pediatric nephrologists. These differences in opinions may point out the need of a multidisciplinary discussion of both indications and contraindications between pediatric nephrologists and gastroenterologists/hepatologists to offer a uniform concept to affected patients and their families.

The common indications of insertion in 38 ARPKD patients encompassed malnutrition, failure to thrive and safe medication administration. Weight gain can be a major challenge in CKD and ARPKD (6, 13), but is substantial for achievement of a sufficient body weight for transplantation (about 10 kg in many centers). After kidney transplantation, a safe way of medication and fluid application via gastrostomy can facilitate management. Importantly, the indication for gastrostomy tube insertion in a specific ARPKD patient needs to be assessed on a case-bycase basis implementing the aspects of malnutrition as well as the renal, hepatic and neurological phenotype of an individual patient.

Our series does not report greatly increased proportions of complications in ARPKD patients compared to other pediatric patients. The most frequent reported complications were excessive granulation tissue and wound infection not exceeding complication rates in larger series of children with percutaneous endoscopic or laparoscopic gastrostomy (12, 14–16). Two cases reported in our study deserve a closer look: in one patient who had undergone bilateral nephrectomy and who suffered from severe blood pressure variations, arterial hypotension developed during anesthetic induction for gastrostomy insertion. This lead to sequelae of neurological impairment and the attending physician's assessment that the gastrostomy insertion was the wrong decision for this specific patient. However, the reported complication may appear to be related rather to the general risks of anesthesia after bilateral nephrectomy than to the specific intervention of gastrostomy insertion. In another case, a colleague reported fulminant sepsis with death in an APRKD patient. From our data, we cannot specify whether this event was directly linked to gastrostomy insertion, as detailed information on this case was not available in our anonymous study.

Regarding the hepatic phenotype of patients requiring gastrostomy, conditions of portal hypertension and/or possible future liver transplantation are considered as relative contraindications for percutaneous endoscopic gastrostomy (PEG) insertion by the ESPGHAN (12). The position paper mentions the risk of de novo portosystemic shunts and peristomal varices which could cause severe bleeding and pose major challenges for future liver transplantation. Very limited evidence of similar scenarios dealing with portal hypertension in patients with PEG are cited from the literature (12) in form of only two studies with 2 respectively 5 patients suffering from portal hypertension receiving PEG (17, 18). The authors underline that careful preparation and adequate expertise are mandatory requirements for PEG insertions in these specific patients. In our survey, surprisingly, half of the 38 ARPKD patients showed some form of hepatic involvement/phenotype at the time of gastrostomy insertion. Apparently, the benefits of gastrostomy insertion were considered to outweigh potential complications in these patients. Bleeding-related complications were reported in 3 of 38 patients (7.9%): one patient each was reported to suffer from stomal varices respectively bleeding episodes due to variceal bleeding summing up to two patients (5.3%) with portal hypertension related bleeding complications. The third patient (1 of 38, 2.6%) was reported to suffer from excessive bleeding from gastrostomy exit site 9 months after insertion. It remained unclear whether this bleeding episode was related to portal hypertension or rather to gastrostomy tube induced mucosal irritation. In the literature, data are very limited regarding bleeding complications in pediatric patients with portal hypertension and gastrostomy. First data exist for children with cystic fibrosis associated liver disease: two case series with 7 and 37 patients report no bleeding episodes attributable to varices or development of stomal varices, minor complications in common frequency and no procedure-related mortality (8, 10). Compared to this, the rate of complications related to portal hypertension in our series may seem to be relevant and needs to be taken into consideration in the decision process prior to gastrostomy insertion. Assuming that the patient with excessive bleeding from gastrostomy exit site 9 months after insertion suffered from tube induced mucosal irritation, this rate seems to be within the same range of a single center experience from South Korea (19). In this study on 236 pediatric non-ARPKD patients undergoing gastrostomy insertion due to poor nutrition, swallowing difficulty, and upper gastrointestinal obstruction gastrointestinal (GI) bleeding due to gastrostomy irritation was reported in up to 5.4% of their cohort (19).

The ESPGHAN position paper on management of PEG insertion in children and adolescents does not indicate any evidence regarding the management of gastrostomy tubes in patients with potential upcoming liver transplantations (12). Almost half of the patients (18/38) with inserted gastrostomy in our dataset underwent transplantation with a gastrostomy in situ. Ten patients received isolated kidney transplantation and 8 patients received combined liver and kidney transplantation. In three patients (two with combined liver and kidney transplantation, one with isolated kidney transplantation), gastrostomy was removed at the timepoint of transplantation. No adverse events or severe complications were reported in the courses after transplantation. As there is no recommendation regarding timepoint of gastrostomy removal in children after transplantation, risks and benefits need to be outweighed in every single patient (20, 21). Our data without severe complications in the post-transplant courses in 15 ARPKD patients with gastrostomy in situ (9 patients with isolated kidney transplantation, 6 patients with combined liver and kidney transplantation) may set a basis for discussing the timepoint of gastrostomy removal.

Limitations of our survey include the anonymous questionnaire which does not allow queries for both participants and survey organizers in case of uncertainties. The set-up of the survey may result in a selection bias of centers. We can neither exclude a bias in participation due to personal experiences nor a bias in reporting in both positive and negative aspects. Due to pre-determined answer possibilities, specifications were not possible in all questions. Answers of questions might be biased to a nephrologic point of view, since three quarter of all participants indicated to be pediatric nephrologists and only 20% indicated to be pediatric gastroenterologists/hepatologists. We did not receive replies from pediatric surgeons potentially precluding addition of information from a surgical point of view. On the other hand, regular patient care and follow-up is provided by pediatric nephrologists and gastroenterologists/hepatologists in most pediatric ARPKD patients. Furthermore, pediatric gastroenterologists/hepatologists take care of gastrostomy insertion and follow-up in the first place in many centers consulting pediatric surgeons only in case of uncertainties or contraindications for non-surgical insertion. As further limitation, patient-specific data did not encompass a longitudinal follow-up of data on growth and development.

The choice of insertion method in complex patients—such as children with ARPKD—is subject to local habits and experiences at specific centers. Since the cohort of pediatric ARPKD patients displays major phenotypic variability with respect to growth and development as well as renal and hepatic phenotype, the results of our survey can neither be extrapolated to daily clinical practice without critical case-by-case discussion in a multidisciplinary team nor replace future clinical practice recommendations or guidelines.

Despite these limitations, this study sets a first basis of reporting encouraging international experiences with gastrostomy insertion in pediatric ARPKD patients. In summary, gastrostomy insertion was evaluated as a correct decision for the patient and outweighing developing complications in most

### REFERENCES


cases. Due to the concomitant hepatorenal affection, children with ARPKD rely on a multidisciplinary collaboration of both pediatric nephrologists and gastroenterologists/hepatologists (22). In order to set a basis for development of management recommendations in a multidisciplinary approach, international initiatives like the recently established ARPKD registry Study ARegPKD will further help to define indications and contraindications of gastrostomy insertion in ARPKD patients (23, 24).

### AUTHOR CONTRIBUTIONS

KB, JB, RS, PW, LW, JD, FS, DM, and ML drafted the manuscript. KB, JB, RS, PW, FS, DM, and ML designed the questionnaire. All authors reviewed and approved the final manuscript.

### DATA AVAILABILITY

The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.

### ACKNOWLEDGMENTS

We thank all participating members of the mentioned societies for their input and support. ML was supported by grants of the GPN, the European Society for Paediatric Nephrology, the German PKD foundation, the Koeln Fortune program, the GEROK program of the Medical Faculty of University of Cologne, and the Marga and Walter Boll-Foundation. ML and FS are supported by the the German Federal Ministry of Research and Education (BMBF grant 01GM1515). The Pediatric Study Center Cologne was supported by the German Federal Ministry of Research and Education (BMBF grant 01KN1106). KB was supported by the Koeln Fortune program of the Medical Faculty of University of Cologne.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fped. 2018.00164/full#supplementary-material


2012 update. Perit Dial Int J Int Soc Perit Dial. (2012) **32**(Suppl. 2):S32–86. doi: 10.3747/pdi.2011.00091


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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Burgmaier, Brandt, Shroff, Witters, Weber, Dötsch, Schaefer, Mekahli and Liebau. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Managing Bardet–Biedl Syndrome—now and in the Future

*Elizabeth Forsythe\*, Joanna Kenny, Chiara Bacchelli and Philip L. Beales*

*Genetics and Genomic Medicine Programme, UCL Great Ormond Street Institute of Child Health, University College London, London, United Kingdom*

Bardet–Biedl syndrome is a rare autosomal recessive multisystem disorder caused by defects in genes encoding for proteins that localize to the primary cilium/basal body complex. Twenty-one disease-causing genes have been identified to date. It is one of the most well-studied conditions in the family of diseases caused by defective cilia collectively known as ciliopathies. In this review, we provide an update on diagnostic developments, clinical features, and progress in the management of Bardet–Biedl syndrome. Advances in diagnostic technologies including exome and whole genome sequencing are expanding the spectrum of patients who are diagnosed with Bardet– Biedl syndrome and increasing the number of cases with diagnostic uncertainty. As a result of the diagnostic developments, a small number of patients with only one or two clinical features of Bardet–Biedl syndrome are being diagnosed. Our understanding of the syndrome-associated renal disease has evolved and is reviewed here. Novel interventions are developing at a rapid pace and are explored in this review including genetic therapeutics such as gene therapy, exon skipping therapy, nonsense suppression therapy, and gene editing. Other non-genetic therapies such as gene repurposing, targeted therapies, and non-pharmacological interventions are also discussed.

Keywords: Bardet–Biedl syndrome, genetic therapies, pharmacogenomics, genome editing, targeted therapies, drug repurposing

### INTRODUCTION

Bardet–Biedl syndrome (BBS), sometimes known as Laurence–Moon–Bardet-Biedl syndrome, is a rare autosomal recessive ciliopathy characterized by rod-cone dystrophy, learning difficulties, polydactyly, obesity, genital malformations, and renal abnormalities.

In the 1880s, a family with retinitis pigmentosa, obesity, and intellectual impairment was described by doctors Laurence and Moon. The affected family members later went on to develop a spastic paraparesis. In 1920 and 1922, respectively, doctors Bardet and Biedl independently described two families with obesity, retinitis pigmentosa, and polydactyly. From 1925, the syndrome was known as Laurence–Moon–Bardet–Biedl syndrome, but there was disagreement as to whether they were the same entity. Later, it was considered as two entities, Laurence–Moon and Bardet–Biedl syndromes, but mutations in known BBS genes have been seen in families with both syndromes (1, 2). Today, it is most usually known as BBS.

It is a pleiotropic disorder and has a prevalence of around 1:100,000 in North America and Europe, but it is significantly more common in certain isolated communities including Newfoundland (1:18,000) (2) and Kuwaiti Bedouins (1: 13,500) (3, 4). In the last 2 decades, 21 BBS genes (BBS1– BBS21) (5–7) have been identified, mutations in which account for 80% of cases with a clinical diagnosis of BBS (1). **Table 1** outlines the 21 BBS genes.

Mutations in *BBS1* and *BBS10* account for the majority of genotypes (~51 and ~20%, respectively) in Northern Europe and North America (4).

#### *Edited by:*

*Miriam Schmidts, Radboud University Nijmegen, Netherlands*

#### *Reviewed by:*

*Diana Valverde, University of Vigo, Spain John Andrew Sayer, Newcastle University, United Kingdom*

*\*Correspondence: Elizabeth Forsythe* 

*elizabeth.forsythe@ucl.ac.uk*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 14 December 2017 Accepted: 25 January 2018 Published: 13 February 2018*

#### *Citation:*

*Forsythe E, Kenny J, Bacchelli C and Beales PL (2018) Managing Bardet– Biedl Syndrome—Now and in the Future. Front. Pediatr. 6:23. doi: 10.3389/fped.2018.00023*

Bardet–Biedl syndrome proteins localize to the primary cilium/basal body complex, a ubiquitously expressed highly evolutionarily conserved organelle functioning primarily for cell-to-cell signaling. The genes that cause BBS can also cause other ciliopathies, with the classic example being *CEP290*, which can cause Joubert syndrome, Leber congenital amaurosis, Meckel syndrome, and Senior-Loken syndrome in addition to BBS (8).

### DIAGNOSING BBS

Bardet–Biedl syndrome is a pleiotropic disorder and diagnosis is based on the presence of at least four major features or three major features and at least two minor features in accordance with


the diagnostic criteria published by Beales et al. (9). **Figure 1** demonstrates the clinical features associated with BBS and highlights the relative frequencies at which these features are observed.

Molecular confirmation of BBS has evolved over the last decade from targeted sequencing of common genetic variants, including the common *BBS1* p.M390R, *BBS2* p.Y24X, *BBS2* p.R275X, and *BBS10* c.91fsX5 mutations to next-generation sequencing gene panels containing all known BBS genes. The frequency at which molecular confirmation is achieved has increased accordingly from approximately 40–80% (1).

The age at which patients are diagnosed is extremely variable and is driven by the age of onset of symptomatic rod-cone dystrophy. While this may manifest in infancy, it is more usually seen between the ages of 5 and 10 years of age and typically presents with night blindness (9). Isolated polydactyly at birth or obesity, generally seen from infancy, do not usually prompt referral. Siblings of affected children are generally diagnosed earlier. Antenatal diagnosis is extremely rare in the absence of a family history, but BBS may be suspected from the identification of echogenic kidneys and polydactyly on ultrasound scanning. Children presenting with renal anomalies or renal failure may be diagnosed earlier than those without, but there are insufficient data to confirm this. A subset of individuals present with isolated rod-cone dystrophy with notable absence of other BBS-related features and are often diagnosed in adulthood. These individuals are now being picked up because of the introduction of panelbased genetic testing and major diagnostic studies such as the UK 100,000 genomes project (10) and the Deciphering Developmental Disorders (exome) study (11). They were previously overlooked as there are many causes of rod-cone dystrophy, and it was not understood that BBS genes could cause this feature in isolation.

Currently, diagnostic gene panels are the diagnostic tool of choice. The use of whole exome sequencing (WES) and whole genome sequencing (WGS) may increase coverage, aid in the discovery of novel genes, and allow for the identification of


Figure 1 | Clinical and diagnostic features of Bardet–Biedl syndrome. (i) Clinical features associated with Bardet–Biedl syndrome. (A–D) Typical facial features are often subtle and not always present. Typical facial features include malar hypoplasia, a depressed nasal bridge, deep set eyes, and retrognathia. (E) Brachydactyly. (F) Dental crowding. (G) High palate. (H) Rod-cone dystrophy. (ii) Diagnostic features of Bardet–Biedl syndrome. At least four major features or three major and two minor features are required to make a clinical diagnosis. Informed consent was obtained and republished with permission (4).

non-coding variants. However, along with increased expense, disadvantage of the more advanced diagnostic sequencing techniques is the identification of pathogenic variants in non-BBS genes and of variants of unknown significance (VUS) in BBS genes (12). This can result in a diagnostic conundrum in particular where patients manifest only one or two non-specific major diagnostic criteria such as obesity and/or learning difficulties. The use of WES and WGS requires careful consenting of patients and having a plan in place to deal with VUS and unlooked for results.

Variable expressivity is the hallmark of BBS (8); patients with the same genotype and even siblings frequently manifest symptoms differently. As a result, although genotype–phenotype correlations exist on a population basis, it is not possible to make individual predictions about symptomatic manifestations (13). As a group, patients with mutations in *BBS1* are usually less severely affected than patients with mutations in other BBS genes. On average, they develop visual deterioration later in life (14), are less likely to develop renal disease (13), and more likely to have a better endocrine biochemical profile (15) with a lower prevalence of metabolic syndrome (16, 17). It is not possible to delineate if this is a consequence of the common missense mutation *BBS1* p.M390R identified in 80% of Northern European patients (4) or if the milder phenotype is representative of an overall less severe phenotype associated with *BBS1* mutations (17).

Suggestion that BBS would be a candidate for triallelic inheritance, whereby a third mutation is required to either manifest the condition or adding mutational load, has gathered limited evidence (18–20). In practice, the phenotypic variability observed in patients with the same genotype and within families is likely to reflect a complex interplay between multiple genetic factors and environmental influences.

In the future, it may be possible to identify phenotypic modifiers and further elucidate the cause for variability through analysis of the "Omics" (genomics, epigenomics, transcriptomics, proteomics, and metabolomics) whereby the complex interplay of genes, transcription, protein expression, and metabolism is considered as part of the phenotypic analysis (21, 22).

### RENAL DISEASE IN BBS

Bardet–Biedl syndrome has classically been associated with polycystic kidney disease, a typical feature of ciliopathies with renal manifestations (13, 16, 23–29). The prevalence of renal disease in BBS has been estimated at 53–82% (13, 16, 25). A recent study of 350 patients from the United Kingdom estimated that 50% of patients will develop functional renal disease and demonstrated that cystic or dysplastic disease only accounts for 30% of patients with renal disease, where the remainder have hydronephrosis, scarred or atrophic kidneys, loss of corticomedullary function, or developmental abnormalities (13). Around 8% of patients go on to develop end-stage renal disease requiring dialysis or transplantation (13). The majority of patients who develop end-stage renal disease do so in early childhood (before the age of 5), and in most cases, deterioration is rapid with frequent requirement for dialysis within the first year of life (13). Some patients develop sudden renal failure in adulthood for unknown reasons, and a further group of patients develop end-stage kidney disease as a result of comorbidities including type 2 diabetes and hypertension (13). The prevalence of these comorbidities is thought to be higher in BBS patients than the normal population and in 1 study of 69 patients were found in 22 and 35%, respectively (17). The risk of type 2 diabetes relates to obesity and is treated using standard protocols. Many patients with structural renal abnormalities do not go on to develop functional renal disease (17).

The molecular mechanistic pathways leading to renal disease in BBS remain unelucidated (29). It has been suggested that aberrant mTOR signaling may contribute to the development of cystic kidney disease (28). Another theory proposes that ciliary dysfunction leads to aberrant non-canonical Wnt signaling and planar cell polarity, which may contribute to the development of cysts (30). Molecular evidence supporting these theories remains limited. Furthermore, they do not explain why only some patients develop renal dysplasia or indeed the heterogeneous types of renal disease developed by patients.

### CURRENT MANAGEMENT OF BBS

Bardet–Biedl syndrome is currently treated symptomatically focusing in particular on aggressive management of diabetes, hypertension, and metabolic syndrome to minimize the secondary impact that these conditions have on vulnerable organ systems already affected by BBS, in particular the eyes and kidneys (17). Weight management is a continual struggle for the majority of patients (31). Some elect to have bariatric surgery (32) while others take antiobesity medication, but for the majority of patients, dietetic input provides the safest and most effective weight loss strategy (33).

In the UK, patients who attend the national BBS clinics are invited to attend a multidisciplinary clinic for annual review by a geneticist, ophthalmologist, nephrologist, endocrinologist, psychologist, dietitian, speech and language therapist, nurse, and a patient support group representative. This provides a platform for regular review and individualized risk assessment in particular with reference to renal and endocrine deterioration. All patients are genotyped using a service-developed diagnostic gene panel. The clinic also provides an opportunity for research into the natural progression of BBS, and it is expected that patients will eventually be stratified according to genotype and their need for clinical follow-up.

### FUTURE THERAPIES FOR BBS

The last decade has seen significant advances in the development of therapeutic modalities, which could potentially be applicable to patients with BBS and related ciliopathies. However, the large number of disease-causing genes and private mutations, which are those seen in only a single family, present a unique challenge in developing genetic therapies for BBS (34). **Figure 2** outlines potential future therapies, which are likely to benefit patients with BBS in the future.

### Genetic Therapies

A particular focus for therapeutic intervention in the ciliopathies has been the development of therapy for rod-cone dystrophy

are indicated in light blue. The last column indicates the percentage of BBS patients who could benefit from this type of intervention. BBS, Bardet–Biedl syndrome; LCA, leber congenital amaurosis; PCD, primary ciliary dyskinesia; RP, retinitis pigmentosa; US, Usher syndrome.

(34–36). The eye offers an attractive organ for therapeutic intervention in BBS due to the ease of access, the presence of a control (other) eye, the small amount of tissue that needs to be infiltrated, and a window of opportunity to develop treatment as patients typically do not develop symptoms before mid to late childhood (37).

Novel disease models are being generated with the potential to develop *in vitro* organ systems for the assessment of new therapies. A promising development is the generation of induced pluripotent stem cells (38–43). These cells are generated when adult cells are reprogrammed and subsequently differentiated into another cell type through the addition of growth factors (41). A number of cell types have been used for reprogramming including dermal fibroblasts (44), renal epithelial cells (45), keratinocytes (46), and peripheral blood cells (47).

Urine-derived renal epithelial cells have been used to model ciliopathies, such as Joubert syndrome to assess the effect of potential therapeutics (48, 49) and BBS to derive mechanistic insights into disease pathogenesis (unpublished data). This is a particularly attractive model for many ciliopathies including BBS as it is non-invasive and offers an organ-specific relevant disease model.

To our knowledge, currently, there are no efforts in progress to develop genetic therapies targeted to the renal manifestations of BBS. This is likely to be a function of a number of issues including that the onset of renal manifestations is often antenatal precluding a therapeutic window of opportunity. Furthermore, the natural history of renal disease in BBS is not well understood, and the cause of renal disease can be both primary (e.g., cystic renal disease) and secondary to metabolic syndrome, hypertension, or diabetes all of which occur more frequently in BBS than the general population (13). In addition, the kidney is more difficult to target with genetic therapies but may prove to be a more amenable target for pharmacological therapies.

#### Gene Therapy

Traditional gene replacement therapy has achieved significant success in the treatment of ciliopathy-related eye diseases including Usher syndrome and Leber congenital amaurosis in recent years (50–52). The premise involves the generation of viral or non-viral vectors carrying a wild-type gene of choice with the aim of integrating the gene into the host genome. The highest chance of success is achieved in diseases where only a small amount of healthy gene expression is required to generate a phenotypic effect (53).

A major challenge in developing genetic therapies for BBS is the generation of a long lasting therapy. A successful example of this is the retinal gene therapy (Luxturna), which has been successfully developed for RPE65-associated Leber congenital amaurosis. It is likely to obtain approval from the Food and Drug Administration (FDA) in 2018 as the first gene therapy for ocular disease. Extensive optimization was required before RPE65 gene therapy could be launched for FDA approval due to concerns about the sustainability and long-term maintenance of visual function (36, 37, 54).

Work on retinal gene replacement therapy for BBS is ongoing in animal models, and recent efforts were published demonstrating encouraging results in knock-in mouse models with the most common genotype in humans *BBS1* p.M390R (55). Viral AAV vectors were generated containing the wild-type *Bbs1* construct and injected subretinally rescuing BBSome formation and rhodopsin localization and showing trends toward improved electroretinogram function in mice (55).

Challenges of developing safe and effective gene therapy include ensuring that the gene expression is proportionate to the requirements of the host organism avoiding overexpression and thus cell toxicity (36, 51, 55), identifying an appropriate time to administer therapy (36) (in the case of rod-cone dystrophy ideally before photoreceptor death), avoiding generation of an immune response, and developing safe and effective vectors (53).

#### Readthrough Therapy

Readthrough therapy exploits the natural inconsistency of the genetic proofreading mechanism in the process of RNA translation into protein (56). Nonsense mutations account for an estimated 11% of the total mutational load in BBS (own unpublished data) and lead to premature termination of protein synthesis and subsequent degradation via nonsense mediated decay (57). Readthrough therapy acts by destabilizing the translational ribosome's response to a nonsense mutation, hence allowing the insertion of a near cognate amino-acyl-tRNA. This allows translation to continue and a full-length protein to be produced (58). The effect mimics that of a missense mutation in the same locus, which may result in a less severe clinical phenotype. Readthrough therapy has been applied to a number of different conditions and been tested in clinical trials for both cystic fibrosis (59–61) and Duchenne muscular dystrophy (62, 63). The effect of readthrough therapy has been assessed on some ciliopathies at the preclinical stage including primary ciliary dyskinesia (64), Usher syndrome (65), and retinitis pigmentosa (RP2) (66).

#### Exon Skipping Therapy

Exon skipping therapy operates at the level of RNA transcription allowing the transcriptional machinery to "skip" exons containing undesirable genetic sequences (67, 68). Antisense oligonucleotides are designed to target an exon/intron of interest. This allows for a novel splicing product that may retain much of its wild-type function depending on the quantity and importance of functional motifs coded for by the exon. This form of genetic therapy is often used to target mutations, which disrupt the genetic reading frame and may otherwise result in a complete lack of functional protein. Successful application of exon skipping therapy has been developed to the level of clinical trials for Duchenne muscular dystrophy and spinal muscular atrophy (69, 70). Exon skipping therapy for ciliopathies has been developed to the stage of preclinical trials for Leber Congenital Amaurosis and Usher syndrome (67, 71). In BBS, this form of therapy could benefit up to 9% of patients. The primary challenge is that many mutations suitable for exon skipping therapy in BBS are private and thus require truly individualized therapy. The common frameshift mutation in BBS (*BBS10* c.91fsX5) is not a suitable candidate as the gene contains only two exons.

#### Genome Editing

Genome engineering offers an attractive future prospect in the management of genetic diseases, allowing DNA to be deleted, replaced, or corrected (72). Targeted endonucleases create double-stranded breaks at specific points in the genome allowing for DNA repair, which can restore the wild-type genotype (43). Promising results have been achieved in cells from patients with the motile ciliopathy primary ciliary dyskinesia where ciliation was restored on replacement of the wild-type *DNAH11* sequence (73). Preclinical work and clinical trials for other diseases using genome editing techniques are progressing with significant advances in animal models of epidermolysis bullosa (74). Initial reports of successful gene editing in humans include gene editing on embryos at risk for hypertrophic cardiomyopathy caused by *MYBPC3* mutation (75) and treatment-resistant leukemia (75, 76). The potential applications are promising, but this technique requires significant refinement in specificity, efficacy, and safety before this can be applied in a clinical setting. Off target effects, whereby genome engineering may erroneously occur on an unintended gene causing DNA damage are a major challenge in bringing this technique forward (72).

While genome editing can be used to target specific organ systems, it can also be applied *ex vivo* to correct or insert mutations to directly comparable cell types which apart from the mutation of interest are genetically identical, thus eliminating genetic background noise and providing an ideal model system (77).

### Non-Genetic Personalized Therapies Targeted Therapies

A novel development in the therapeutic landscape for BBS is the application of targeted drug therapies. An example of this is the ongoing work on the melanocortin receptor agonists as potential therapeutic intervention against obesity in BBS (78). Our understanding of how aberrant BBS proteins cause signal disruption is still evolving; however, there is emerging evidence suggesting that BBS results in defects in the hypothalamic leptin–melanocortin axis (79). This in turn causes leptin resistance culminating in obesity (78).

Seo et al. demonstrated that intravenous administration of a melanocortin receptor agonist decreased both body weight and food intake in wild-type and *Bbs* knockout mice (78). A phase 2/3 clinical trial is in process assessing the effect of novel melanocortin receptor agonist setmelanotide on obesity in BBS and other forms of syndromic obesity (80).

#### Drug Repurposing

Future therapies may include repurposing of drugs, which are already FDA approved. Early work on *Bbs* zebrafish indicated that rapamycin may be a candidate for rectifying the renal cystic phenotype (81). Further work on higher animals has not been published, but drug repurposing offers an attractive and economical option for management in a clinical setting since the cost and failure rate of developing novel therapeutics remain high.

### NON-PHARMACOLOGICAL FUTURE INTERVENTIONS

A major challenge in any clinical service is harnessing the advances in technology and managing increasing clinical pressures. To this end, the UK National BBS clinic is implementing several new strategies to meet the increasing need for transparency and access to clinical services.

There is an increasing move to offer patients easy access to their medical notes and to data sharing within the UK National Health Service to optimize patient management (82). These issues are being addressed in the UK National BBS clinic through the development of a cloud-based medical notes system, which is accessible to all clinicians in the service. In the future, patients will be able to access their personal medical records through smart phones or other devices, so that medical information can be accessed wherever they are.

Telemedicine and virtual clinics, whereby patient consultations can take place *via* a screen in the patient's own home, are an attractive alternative for BBS patients who are stable and in particular for those with visual impairment where travel can present a considerable obstacle to attending hospital appointments. It also offers an advantage to clinicians where hospital resources and clinic space are at a premium.

### PHARMACOGENOMICS

Pharmacogenomic profiling, whereby the effect of the genome on drug response is determined, could offer significant advantages to those patients with complex medical needs who are subject

### REFERENCES


to polypharmacy (83) including BBS patients. Although several private companies offer pharmacogenomics gene panels, this is not currently available in the UK National Health Service, and the evidence base is unclear for many of these private initiatives. However, there is a growing evidence base supporting pharmacogenomic profiling, and this is likely to become available in the National Health Service in the coming years.

### CONCLUSION

Bardet–Biedl syndrome provides a robust model disease for future opportunities in genetic and non-genetic therapeutic management of rare diseases. A significant advantage in the United Kingdom is the presence of the national BBS clinics, which offer a centralized hub for clinical and scientific expertise. The multiorgan effects and wide range of genes and mutation types mean that a number of different genetic therapeutic modalities must be considered, as well as non-genetic pharmacological interventions and non-pharmacological approaches to optimize management of this rare disease in the future.

### AUTHOR CONTRIBUTIONS

EF, JK, CB, and PB made substantial contributions to the writing, drafting, and revision of this manuscript. All authors approved the final published version of the manuscript.

### FUNDING

EF is funded by the Medical Research Council. JK is funded in part by Innovate UK. PB is an NIHR Senior Investigator. This work was supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre (GOSH BRC). The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health.

in Bardet-Biedl syndrome. *J Hum Genet* (2016) 61(5):447–50. doi:10.1038/ jhg.2015.162


*Arch Ophthalmol* (2012) 130(7):901–7. doi:10.1001/archophthalmol. 2012.89


research and clinical perspectives. *Prog Retin Eye Res* (2017). doi:10.1016/j. preteyeres.2017.10.004


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Forsythe, Kenny, Bacchelli and Beales. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Many Genes—One Disease? Genetics of Nephronophthisis (NPHP) and NPHP-Associated Disorders

*Shalabh Srivastava1,2, Elisa Molinari1 , Shreya Raman3 and John A. Sayer1,4\**

*<sup>1</sup> Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom, 2Renal Unit, City Hospitals Sunderland and South Tyneside NHS Foundation Trust, Sunderland, United Kingdom, 3Department of Histopathology, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, United Kingdom, 4Renal Services, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, United Kingdom*

Nephronophthisis (NPHP) is a renal ciliopathy and an autosomal recessive cause of cystic kidney disease, renal fibrosis, and end-stage renal failure, affecting children and young adults. Molecular genetic studies have identified more than 20 genes underlying this disorder, whose protein products are all related to cilia, centrosome, or mitotic spindle function. In around 15% of cases, there are additional features of a ciliopathy syndrome, including retinal defects, liver fibrosis, skeletal abnormalities, and brain developmental disorders. Alongside, gene identification has arisen molecular mechanistic insights into the disease pathogenesis. The genetic causes of NPHP are discussed in terms of how they help us to define treatable disease pathways including the cyclic adenosine monophosphate pathway, the mTOR pathway, Hedgehog signaling pathways, and DNA damage response pathways. While the underlying pathology of the many types of NPHP remains similar, the defined disease mechanisms are diverse, and a personalized medicine approach for therapy in NPHP patients is likely to be required.

#### *Edited by:*

*Max Christoph Liebau, Universitätsklinikum Köln, Germany*

#### *Reviewed by:*

*Ruxandra Bachmann-Gagescu, University of Zurich, Switzerland Katja Höpker, Universitätsklinikum Köln, Germany*

#### *\*Correspondence:*

*John A. Sayer john.sayer@ncl.ac.uk*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 16 October 2017 Accepted: 14 December 2017 Published: 05 January 2018*

#### *Citation:*

*Srivastava S, Molinari E, Raman S and Sayer JA (2018) Many Genes— One Disease? Genetics of Nephronophthisis (NPHP) and NPHP-Associated Disorders. Front. Pediatr. 5:287. doi: 10.3389/fped.2017.00287*

Keywords: ciliopathy, molecular genetics, nephronophthisis, cilia, centrosome, DNA damage, cyclic adenosine monophosphate, Joubert syndrome

### INTRODUCTION

Nephronophthisis (NPHP) is an autosomal recessive inherited kidney disease, which leads to endstage renal disease (ESRD) typically within the first three decades of life (1). Traditionally, this disease was diagnosed using clinical and histological features. However, over recent years, many of the genetic causes underlying NPHP have been identified allowing both a precise molecular diagnosis to be made and some mechanistic insights into the underlying disease process. The known NPHP genes encode proteins that are almost all expressed in centrosomes and primary cilia. NPHP is therefore considered to be a ciliopathy disease (2), consistent with the fact that extrarenal manifestations, consistent will a ciliopathy syndrome, occur in around 20% of cases. Here we will review the clinical and histological features of the disease and its conventional classification before reviewing the underlying genetic causes and the ciliopathy syndromes associated with NPHP.

Based on the original histological descriptions, which included corticomedullary cysts, atrophy, and interstitial fibrosis, NPHP literally means disappearance or disintegration of nephrons (3). The clinical symptoms of NPHP, which reflect reduction in GFR and loss of distal tubular function (4), include polyuria, polydipsia, secondary enuresis, and growth retardation. Unfortunately, NPHP is associated with a progressive loss of kidney function and ESRD typically

**122**

H&E stain shows tubular atrophy with hyaline casts, moderate interstitial fibrosis, and patchy mononuclear inflammation. (C) Electron microscopy image. Tubular basement membrane demonstrates thickening and multilayering. Scale bar 2 µm.

occurs before 30 years of age. Cases have historically been classified based on the age of onset of ESRD as infantile, juvenile, adolescent, and late onset. These are worth reviewing, although it is worth noting that a single genotype may present at a wide range of ages.

Juvenile NPHP is the classical form of NPHP and is characterized by polyuria and polydipsia symptoms and often anemia in patients within the first decade of life. Progressive loss of kidney function leads to ESRD at a median age of 13 years (5).

Kidneys affected by NPHP are grossly normal or have a shrunken appearance, typical of ESRD. There may be corticomedullary cysts that are up to 1.5 cm in size. Cysts often develop in later stages of the disease. The renal ultrasound scan appearances may often display a loss of corticomedullary differentiation.

Where renal biopsies have been performed in NPHP patients, distinct histological features have been reported. The histological changes can be divided into early or late stages of disease. In the early stages of the NPHP, there is interstitial fibrosis with sparse inflammation and lack of infiltration with neutrophils or monocytes. The tubules are tortuous and atrophic with segmented tubular basement membrane thickening (6). The distal tubules have focal diverticulum like protrusions. The glomeruli are usually normal but there may be periglomerular fibrosis that can extend into the glomerular tuft leading to focal or global collapse of the tuft and obsolescence of the glomeruli (7, 8). In later stages of the disease, the tubules may demonstrate basement membrane abnormalities with both atrophy and thickening. There often is cystic dilatation of the distal tubules, and the glomeruli may show collapse and severe periglomerular fibrosis (8, 9) (**Figure 1**). NPHP is not an immune mediated disease, and consequently there is no immune or complement deposition (6, 8). Electron microscopy may reveal tubular basement membrane duplication, thickening, and folding (6, 8). When examining clinical, pathological, and histological features of NPHP, it must be remembered that a separate disorder, known as medullary cystic kidney disease may share similar features. Medullary cystic kidney disease is an autosomal dominant condition, which is now classified under the term autosomal dominant tubulointerstitial kidney disease (ADTKD). Typical extrarenal manifestations include gout and anemia. A comparison of NPHP and ADTKD, alongside is given in **Table 1** and has been discussed elsewhere (10).

Table 1 | Comparison of nephronophthisis (NPHP) with autosomal dominant tubulointerstitial kidney disease (ADTKD).


Infantile NPHP is rare, but is noteworthy, due to its severe phenotype with ESRD typically occurring during the first year of life (7). There may be antenatal presentations with oligohydramnios and bilateral enlarged cystic kidneys. Infantile NPHP is usually caused by mutations in *INVS* (11) and *NPHP3* (12) but has been reported for other genetic forms of NPHP such as *NEK8* (13) and *CEP83* (14). The macroscopic and histological kidney phenotype is markedly different from other varieties of NPHP, with enlarged cystic kidneys, as opposed to micro and small corticomedullary cysts. Histologically, infantile NPHP lacks the tubular basement membrane changes seen in other NPHP phenotypes and may resemble autosomal recessive polycystic kidney disease. There may also be severe cardiac anomalies including situs inversus and ventricular septal defects (15).

The adolescent form of NPHP was originally described in a large Venezuelan pedigree (16). Biallelic mutations in *NPHP3* were found in this family, resulting in ESRD at a median age of 19 years (16). It is now known that *NPHP3* mutations may lead to a broad range of phenotypes including perinatal lethal Meckel–Gruber syndrome and infantile presentations. The term "adolescent NPHP" is thus somewhat arbitrary and merely extends the phenotypic spectrum from juvenile NPHP.

A number of case reports have highlighted the fact that NPHP may first present later in life. Georges et al. reported three (genetically unsolved) families with retinal dystrophy, NPHP on renal biopsy and slowly progressive renal failure and ESRD between the ages of 42 and 56 years (17). In another family with a homozygous *NPHP1* deletion (18) ESRD was reported in three patients between 27 and 43 years of age. These cases of NPHP extend the age of ESRD from birth to up to the sixth decade of life.

### AN APPROACH TO THE CLINICAL DIAGNOSIS OF NPHP

Clinical recognition of NPHP is important, and the renal and extrarenal features of a ciliopathy syndrome (discussed below) may allow a clinical diagnosis to be made. NPHP occurs in isolation in around 80% of cases and is associated with a variety of other ciliopathy phenotypes in 20% of cases. A detailed review with specific emphasis on the family history and extrarenal features known to be associated with NPHP is therefore an essential prerequisite to an exact diagnosis. NPHP is characterized by a urinary concentrating defect early on in life that leads to polyuria and polydipsia. The onset of the disease may be easily missed, as there is typically no severe hypertension, minimal or no proteinuria, and a bland urine sediment. Clinical spectrums of disease are wide and widening. Besides extensive investigations of renal function, clinical phenotyping should also encompass a full neurological screening to assess for cerebellar signs and fundoscopy to assess for retinal degeneration. A formal ophthalmological examination is advised. The role of renal biopsy in diagnosing NPHP is contentious and should be limited to cases where a tissue diagnosis will serve to distinguish it from other differential diagnoses. In most cases, a histopathological diagnosis should be superseded by a molecular genetic diagnostic approach, because genetic screening allows for early diagnosis and prevents complications of renal biopsy. *NPHP1* mutations and deletions are the most frequent genetic cause of NPHP and may be screened for using standard PCR assays (19). Given the large numbers of other NPHP genes involved multiplex PCR (20), targeted exon capture or whole-exome sequencing approaches are recommended (21).

### EXTRARENAL MANIFESTATIONS OF NPHP

There are several important additional phenotypes that may be associated with NPHP (**Table 2**). These multisystem features are consistent with the fact that NPHP is a ciliopathy and may affect retina, brain, liver, and other tissues either by prenatalonset dysplasia or by postnatal organ degeneration and fibrosis. Extrarenal manifestations are seen in ~20% of cases (22). In a recent study where 89 patients with NPHP mutations were analyzed, *NPHP1* mutations were the most common genetic cause and gave rise to typical renal presentations. These included increased echogenicity of the kidney and loss of corticomedullary differentiation, with cystic kidney disease presenting later Table 2 | Extrarenal manifestations of nephronophthisis (NPHP) and their associated syndromes.


in the disease course (median age 12.3 years) and ESRD at a median age of 12.8 years (23). Extrarenal manifestations of *NPHP1* mutations were seen more frequently than expected, with 8% presenting with liver symptoms, 19% having developmental delay and 7% epilepsy and seizures (23). The important syndromes associated with NPHP are briefly described below.

### NPHP with Retinitis Pigmentosa (Senior–Løken Syndrome)

Retinal dysplasia and degeneration is seen in 10–15% of patients with NPHP and may lead to an early and severe visual loss resembling Leber congenital amaurosis (LCA) (24, 25). Later onset forms present initially with night blindness, which then progresses to visual loss.

### Cerebellar Vermis Aplasia/Hypoplasia with NPHP (Joubert Syndrome)

Joubert syndrome is a developmental disorder characterized by cerebellar vermis hypoplasia (26). Brain imaging (MRI) reveals the typical sign known as the "molar tooth sign." Clinical features include hypotonia, cerebellar ataxia, neonatal tachypnea, and developmental delay. There may also be ocular coloboma, polydactyly, and hepatic fibrosis. NPHP is found in up to 30% of Joubert syndrome patients (27–29). Large cohorts of Joubert syndrome patients have been described, allowing some genotype/phenotype correlations to be made in the more frequent genetic causes. Mutations in *TMEM67* in Joubert syndrome are the most frequently associated with kidney disease, whereas mutations in *CEP290* were most likely to give retinal, renal, and brain phenotypes (28). In a recent cohort analysis of 97 patients with Joubert syndrome, renal phenotypes were detect in 30% of cases and was commonly associated with NPHP genes including *CEP290*, *TMEM67*, and *AHI1* (29). In this study, renal phenotypes in Joubert syndrome extended beyond classical NPHP and included an overlapping phenotype resembling autosomal recessive polycystic kidney disease and NPHP (mimicking infantile NPHP), unilateral multicystic dysplastic kidney and indeterminate cystic kidney disease phenotypes (29).

### Oculomotor Apraxia (OMA) Type Cogan

Oculomotor apraxia type Cogan is an eye movement disorder. It is characterized by abnormal horizontal eye movements that include nystagmus and difficulty with saccades (smooth visual pursuits) and has been associated with NPHP (2, 30). OMA may be a mild form of Joubert syndrome, as cerebellar vermis aplasia has been described in this condition (31).

### Perinatal Lethality (Meckel–Gruber Syndrome)

Meckel–Gruber syndrome is characterized by occipital encephalocele, polydactyly, bile ductal proliferation, and cystic kidney dysplasia. Typically, the condition is perinatally lethal. The syndrome is associated with severe biallelic mutations in NPHP genes that include *NPHP3*, *CEP290*, and *RPGRIP1L* (1, 32–35).

### Skeletal Defects [Jeune Syndrome (JS), Sensenbrenner Syndrome, and Saldino-Mainzer Syndrome]

Various skeletal defects have been reported in association with NPHP. These include cone-shaped epiphyses (16, 36), shortening of limbs, and ribs, scoliosis, polydactyly, brachydactyly, and craniosynostosis. Mutations are in genes encoding intraflagellar transport (IFT) proteins including TTC21B and WDR19 (37–41).

### Episodic Hyperpnea (Joubert Syndrome)

The original report of Joubert syndrome (42) described episodes of fast breathing followed by a period of apnea. This feature is demonstrable only when the patient is awake. Abnormal respiratory pattern is not a consistent feature of Joubert syndrome and the reported incidence varies (44–71%) (43).

### Anosmia As an Extrarenal Manifestation of Renal Ciliopathies

Several renal ciliopathy syndromes have been associated with anosmia, secondary to olfactory cilia defects. This has been studied in most detail in Bardet–Biedl syndrome (BBS) (44, 45) but has been reported in patients with LCA secondary to mutations in *CEP290* and in a murine model of *Cep290* (46). Such data also suggest a link between ciliary defects in the olfactory neurons and kidney disease. Indeed, proteins that mediate olfactory-like chemosensory signaling pathways were found expressed in the renal tissue (47), including adenylate cyclase III, which is localized to the primary cilium (48). These pathways may be vital in tubuloglomerular feedback and blood pressure control. There is a real need now to assess patients with renal ciliopathies/NPHP (and indeed corresponding murine models) for defects in smell and to determine a role for olfactory-like signaling within the kidney.

## KNOWN GENETIC CAUSES OF NPHP

There are now more than 20 genes that if mutated may lead to NPHP (**Table 3**). It is worth reviewing these genetic causes as they all point toward some mechanistic insights into the pathogenesis of NPHP. Finding commonality among the genetic causes relies on a connection to the centrosome/basal body/ primary cilium, although this may not be true for every genetic cause.

The most common genetic cause of NPHP is mutations in *NPHP1*, which account for around 20% of cases. The most common *NPHP1* gene defect is a large homozygous deletion affecting the whole gene (49, 79). Each of the remaining NPHP genes probably accounts for 1% or less of all cases of NPHP, meaning that around two-thirds of cases remain genetically unsolved (2). It is noteworthy that mutations in a single NPHP gene may give an extremely wide spectrum of clinical phenotypes that may include isolated NPHP, NPHP with additional features, such as Senior– Løken syndrome and Joubert syndrome and severe neonatal lethal forms, such as of Meckel–Gruber syndrome. Linkage studies and painstaking mapping approaches led to the identification of *NPHP1* in 1997 (49). Similar approaches for the next decade (sometimes combined with candidate gene screens) allowed the discovery of eight genes (at a rate of around one new gene per year). Since 2010, next-generation sequencing approaches have been utilized (80) allowing the detection of NPHP genes at a much faster rate.

### *NPHP1*

*NPHP1* encodes nephrocystin-1 (alias nephrocystin). It was shown to interact with p130cas, tensin, filamin, and focal adhesion kinase 2, all molecules involved in cell–cell adhesion and cell signaling (81–83). In the primary cilium, nephrocystin-1 interacts with nephrocystin-4 and RPGRIP1L at the transition zone and links it to inversin (77).

### *INVS*

INVS causes ESRD in the first 2 years of life and presents typically as an infantile form of NPHP as described earlier. The frequency of *INVS* mutations has been reported to be as high as 78% in the group of patients reaching ESRD before 2 years of age (12). The kidney size in INVS is often enlarged unlike most other forms of NPHP in which the kidneys are normal in size or shrunken (12). The distribution of cysts is corticomedullary and is more reminiscent of autosomal recessive polycystic kidney disease, given the kidneys can be massively enlarged.

Inversin, the gene product of *INVS* interacts with nephrocystin-1 and nephrocystin-3 and plays a vital role in intercellular adhesion (84). It localizes to the cilium and serves as a switch between the canonical and non-canonical Wnt pathway (85). Otto et al. established a link between cystogenesis and the primary cilia in humans disease during the study of this disease in 2003 (11) establishing this as a landmark paper in the study of NPHP and ciliopathies. Inversin is also plays a role in planar cell polarity (PCP) processes, discussed below. Loss of inversin leads to abnormal mitotic spindle orientation (86), which may drive cystogenesis.


*BBS, Bardet–Biedl syndrome; COACH, cerebellar vermis hypo/aplasia, oligophrenia (mental retardation), ataxia, ocular coloboma, and hepatic fibrosis; JBTS, Joubert syndrome; LCA, Leber congenital amaurosis; MKS, Meckel syndrome; NPHP, nephronophthisis; SLNS, Senior–Løken syndrome.*

### *NPHP3*

Omran et al. first described mutations in *NPHP3* in a large Venezuelan family in 2000 (87). It is characterized by NPHP, situs inversus, and structural heart defects. Hoff et al. uncovered a link between nephrocystin-3, inversin, and NEK8 (65) in a report on the role of *ANKS6*, linking the above proteins at the proximal part of the primary cilium known as the inversin compartment. This may explain the overlap seen in the phenotype of patients with mutation in *INVS*, *NPHP3*, and *NEK8*.

### *NPHP4*

*NPHP4* was identified by homozygosity mapping and genome wide linkage analysis by Mollet et al. in patients with NPHP who did not have mutations in the *NPHP1*, *2*, and *3* genes. Nephrocystin-4 localizes to the primary cilia and cortical actin cytoskeleton in the polarized cells. In dividing cells, it localizes to the centrosomes. It has been shown to interact with p130 (Cas), tensin, and filamin (88).

### *IQCB1*

Patients with *IQCB1* mutations are characterized by the presence of retinitis pigmentosa with NPHP (renal–retinal or Senior–Løken syndrome). In a study investigating the association of retinitis pigmentosa with NPHP, Otto et al. found a novel gene *IQCB1* that associates with retinitis pigmentosa GTPase regulator (RPGR) and calmodulin in the retinal connecting cilia, an analogous structure of the ciliary transition zone (53).

### *CEP290*

Mutations in the *CEP290* gene underlie NPHP6 and are the leading cause of Joubert syndrome and related diseases, a cerebello–retinal–renal syndrome. The association of *CEP290* with NPHP was established in 2006 in a cohort of families with Joubert syndrome, Senior–Løken syndrome, and NPHP (54, 55). CEP290 was found to interact with the transcription factor ATF4, which is involved in cyclic adenosine monophosphate (cAMP) mediated cyst formation (54). *CEP290* mutations are the most common inherited cause of retinal degeneration (LCA). Mutations in *CEP290* may also cause BBS phenotypes (89).

TMEM67 (33, 90) and CC2D2A (91, 92) are both interacting partners of CEP290 and can cause severe ciliopathy phenotypes including Meckel–Gruber syndrome and Joubert syndrome. There is emerging evidence of the role of *CEP290* in ciliogenesis (93), cell signaling (94, 95), DNA damage response (DDR) (96), and consequently renal cystogenesis (97).

### *GLIS2*

In 2007, Attanasio et al. reported a mutation in *GLIS2* as a novel cause for NPHP. The loss of this transcription factor leads to increased fibrosis and apoptosis (56). In a recent paper, *GLIS2* loss has been found to increase cell senescence. *Kif3a* null mice show increased cyst formation due to unrestrained proliferation, destabilization of p53 and increased DNA damage. This is partially rescued by ablation of *GLIS2* and pharmacological stabilization of p53 (98).

### *RPGRIP1L*

Arts et al. identified mutations in *RPGRIP1L* as causative for Joubert syndrome in three families in 2007. This protein localizes to the basal body and interacts with NPHP4 (57).

### *NEK8*

Otto et al. identified *NEK8* as the causative gene for Joubert syndrome after observing that the *jck* mouse harbors a mutation in the highly conserved RCC1 domain of Nek8. They performed a mutational analysis of a worldwide cohort of patients and established the pathogenic role of *NEK8* mutations in humans (13). More recently, *NEK8* loss was implicated in increased DNA damage in the pathogenesis of NPHP (99). This established one of the first associations between the role DDR and cystic kidney disease. Grampa et al. have recently described the association of *NEK8* with deregulation of the Hippo pathway and its role in severe syndromic renal cystic dysplasia (100). Al-Hamed et al. described a stillborn fetus with cystic kidneys, oligohydramnios, CVA and bilateral bowing of the femur secondary to *NEK8* mutation (101).

### *SDCCAG8*

*SDCCAG8* was the first NPHP gene to be identified using nextgeneration sequencing approaches (58). Patients with mutations in this gene were diagnosed with Senior–Løken syndrome, but may also have features suggestive of BBS (102). The encoded protein SDCCAG8 localizes to centrioles and directly interacts with the ciliopathy-associated protein OFD1. A recently described murine model of SDCCAG8 has implicated elevated levels of DDR signaling as a potential mechanism of kidney disease (59).

### *TMEM67*

Otto et al. screened a cohort of 62 patients with NPHP and liver fibrosis and found hypomorphic mutations in *TMEM67* in 8% of the patients (61). *TMEM67* has been implicated in the pathogenesis of Meckel–Gruber syndrome, Joubert syndrome, and COACH syndrome (cerebellar vermis hypo/aplasia, oligophrenia, congenital ataxia, coloboma and congenital hepatic fibrosis) (33, 34). Liver fibrosis is a frequent feature of *TMEM67* mutations, and any patient with NPHP along with liver involvement should have tests for mutations in *TMEM67*. In a cohort of 100 patients with Joubert syndrome, mutations in *TMEM67* were most frequently associated with kidney disease (28).

### *TTC21B*

Davis et al. reported the association of *TTC21B* mutations with both isolated NPHP and JS (37). *TTC21B* encodes the retrograde IFT protein IFT139, which has been shown to regulate Hedgehog signaling (103).

### *WDR19*

*WDR19* mutations have been reported in patients with ciliopathy syndromes including Sensenbrenner syndrome, Joubert syndrome, Senior–Løken syndrome, and isolated NPHP (38, 41, 104). *WDR19* encodes for IFT144, a protein that participates in retrograde IFT and is important for ciliogenesis.

### *ZNF423*

*ZNF423* mutations have shown to cause Joubert syndrome with NPHP (63). The encoded protein ZNF423 is a nuclear protein which functions as a DNA-binding transcription factor and interacts with DDR protein PARP1 [poly (ADP-ribose) polymerase 1] and also CEP290 (63).

### *CEP164*

Mutations in *CEP164* may cause NPHP and related ciliopathy syndromes including Senior–Løken syndrome (63). The CEP164 protein is a regulator of ciliogenesis and is essential for the formation of the distal appendage of the centriole (105). Loss of *CEP164* induces DNA damage (63).

### *ANKS6*

*ANKS6* mutations lead to NPHP. ANKS6 localizes to the inversin compartment and links the NPHP proteins NPHP2, NPHP3, and NPHP9 to NEK8. This functional role of ANKS6 in an NPHP module may explain the phenotypic overlap that includes abnormalities in heart and liver, seen in the patients carrying individual mutations in these genes (65, 66).

### *IFT172*

Intraflagellar transport is vital in maintaining the cilium and in executing its functions. IFT-A module has six components, and mutations in genes encoding these proteins have all been associated with ciliopathy diseases. IFT-B has 14 components. Halbritter et al. established the first link between IFT-B component IFT172 and skeletal ciliopathies. Some patients in this cohort had NPHP (67). Mutations in IFT172 may also cause BBS syndrome (106).

### *CEP83*

*CEP83* mutations have recently been described to cause infantile NPHP (14). *CEP83* encodes a centriolar distal appendage protein, CEP83. In the seven families so far described, the NPHP phenotype was early-onset (juvenile), and in some was also associated with hydrocephalus and learning difficulties (14).

### *DCDC2*

Schueler et al. reported a novel mutation in the gene *DCDC2*, in patients presenting with an NPHP and hepatic fibrosis phenotype (69). DCDC2 localizes to the ciliary axoneme and the mitotic spindles. *DCDC2* knockdown inhibits ciliogenesis. It interacts with DVL, and *DCDC2* knockdown leads to defects in Wnt signaling and may contribute to the liver fibrosis (69).

### *MAPKBP1*

Macia et al. have recently described a novel gene, *MAPKBP1*, in five families with eight individuals presenting with juvenile or late-onset NPHP with massive fibrosis. This gene encodes MAPKBP1, a scaffolding protein for JNK signaling. Interestingly, this protein does not localize to the primary cilium instead it localizes to the mitotic spindle pole. The authors also report increased DDR signaling in murine fibroblasts upon knockdown of *Mapkbp1* (70).

### *AHI1*

Mutations in AHI1 were initially described in patients with Joubert syndrome and no kidney involvement (71, 72). However, AHI1 mutations may cause NPHP phenotypes (73) and may cause multicystic dysplastic kidneys also (29). AHI1 is localized to the basal body and cell–cell junctions (74).

### *CC2D2A*

Mutations in *CC2D2A* have been reported to cause Joubert syndrome with and without cystic kidney disease (92). CC2D2A is localized to the basal body and colocalizes with CEP290 (92). Mutations in *CC2D2A* may also cause antenatal cystic kidney disease phenotypes and severe brain phenotypes (typical of Meckel–Gruber syndrome) leading to fetal death (101).

### EVIDENCE OF OLIGOGENICITY AND TRIALLELISM IN NPHP

Alongside the novel findings relating to gene discovery in NPHP has been the continued theme of wide phenotypic variability, especially in extrarenal manifestations. The type of mutation may influence the phenotype in certain circumstances. Examples include *CC2D2A* (107, 108) and *TMEM67* (109) where two truncating mutations tend to lead to more severe phenotypes than missense mutations. With the now frequent sequencing of NPHP cohorts (110, 111) and the use of high-throughput genetic sequencing platforms (112), a few findings of oligogenicity and triallelism within NPHP have been reported; however, these are controversial as these findings are anecdotal. As an example, a heterozygous *AHI1* mutation when inherited with biallelic *NPHP1* mutations seems to lead to a more severe brain phenotype (111). Thus, a concept of mutation burden seems relevant to NPHP, and like BBS (113) it will be important that these variants are reported and that they are assessed in terms of their pathogenicity. Interestingly, *NPHP1* mutations and copy number variants, as well as causing NPHP and Joubert syndrome, may also contribute to the mutational burden of BBS (114, 115). More recent nextgeneration sequencing data studying Joubert syndrome suggest that rare disease variants are frequently found in addition to the causal biallelic variants. Typically, over one-third of affected individuals carry rare disease variants in addition to the causal mutations but importantly they did not correlate with disease severity (116). This study also found no evidence or support for triallelism. Discordant phenotypes between affected siblings were observed in 60% of subjects who shared causal mutations, suggesting that modifier alleles are important but elusive (116). Therefore, using third alleles and other NPHP gene variants of uncertain significance to determine additional phenotypes and disease severity and inform genetic counseling is not presently advised, or should only be done with the utmost care.

### PATHOGENESIS OF NPHP

There are various theories behind the pathogenesis of the NPHP disease process. The very early hypotheses were based entirely on the histopathological description of the disease and led to the widespread belief that this disease was caused by some unknown nephrotoxic agent or an enzyme defect (8). The frequent finding of tubular basement membrane thickening led to a basement membrane hypothesis for the pathogenesis of NPHP. It was observed that nephrocystin-1, the protein product of *NPHP1* had a high degree of sequence conservation with CRK (a focal adhesion protein) (117), contained an SH3 domain and interacted with other proteins including p130Cas and ACK1 (49, 118). Nephrocystin-1 was shown to localize to adherens junctions and focal adhesions. This supported a hypothesis that nephrocystin-1 has an important role in the maintenance of the tubular epithelium and that abnormal cell–cell and cell–matrix interactions were the underlying defect in NPHP. Many years later, the debate of the initial pathogenic defect in NPHP continues, with the focus on NPHP as a ciliopathy (119). This hypothesis is strongly supported by multiple gene discoveries in NPHP with nearly all the affected genes coding for the components of the cilia, basal body or centrosome. Defects in primary cilia associated with cystic kidney disease were initially noted in *Ift88* mutant mice (120). This link between NPHP and cilia was confirmed in human disease established after the discovery that *INVS* mutations cause infantile NPHP and that the encoded protein inversin interacts with nephrocystin-1 and β-tubulin, colocalizing with them to the primary cilia of renal tubular cells (11). There is now almost universal agreement that the primary cilia are at the center of the disease process especially in terms of cystogenesis although it should not be forgotten that nephrocystins may have multiple subcellular localizations (54) and may play different roles in different tissues (1). There is an interesting overlap with the localization and function of NPHP genes and other "cystogenes" such as *PKD1*, *PKD2*, and the many other inherited causes of cystic kidney disease. The functional role of primary cilia in the human nephron is not fully understood. It was initially thought that the encoded proteins from *PKD1* and *PKD2*, namely, polycystin-1 and polycystin-2, were able to sense luminal flow of urine, and ciliary deflection stimulated calcium entry into the cell *via* the polycystin proteins leading to downstream signaling cascades (121). However, more recent studies have challenged this hypothesis (122, 123).

### A COMPARISON OF NPHP AND ADPKD PATHOPHYSIOLOGY

A detailed discussion of the underlying pathophysiology of ADPKD has been recently published and is beyond the scope of this review (124). It is worth highlighting, however, that disease pathways in ADPKD involve cAMP, ciliary dysfunction, PCP and centrosome number as well as many others (124). Thus, these mechanisms of disease are shared with those of NPHP. Indeed, the development of tolvaptan, a vasopressin V2 receptor (V2R) antagonist for the use in patients with ADPKD was pioneered in murine models of NPHP (51). However, fluid secretion and proliferation seems less prominent in NPHP, while fibrosis and scarring are more prominent pathological features. The pairing of potential disease mechanisms in NPHP with targeted therapeutics will hopefully allow better treatments for NPHP in the near future (125).

### OTHER KEY MOLECULAR PATHWAYS IMPLICATED IN NPHP

### Planar Cell Polarity

Planar cell polarity is an evolutionary conserved mechanism by which cells maintain their orientation in a plane perpendicular to the apical-basal polarity of a cell layer. This is achieved by the correct alignment and orientation of cell division, orchestrated by the mitotic spindle and centrosomes. The maintenance of tubular diameter is dependent upon PCP signaling, and when this is defective, tubular dilatation rather than elongation is thought to contribute to cystogenesis (126, 127). Non-canonical Wnt signaling is vital for these signaling events and mutations in *INVS* are thought to lead to defective regulation of this pathway (84, 85). Mutations in *DCDC2*, leading to NPHP type 19 have also been implicated in this pathway, lending weight to this mechanism of cytogenesis and NPHP. Loss of Dcdc2 in IMCD3 cells led to an activation of Wnt signaling, leading to a loss of cilia, which was amenable to treatment with Wnt inhibitor treatment (69). Other connections to the Wnt pathway in NPHP includes CEP164 which interacts with disheveled protein 3 (DVL3) (63). The disheveled protein is a key component of the Wnt pathway, and part of the switch between canonical and non-canonical Wnt signaling. Defective Wnt signaling has also been demonstrated in murine models of Joubert syndrome. *Ahi1* mutant mice showed defect in cerebellar midline fusion in sites of reduced Wnt activity (128) while renal tissues from the same mice demonstrated abnormal Wnt signaling in late stages of NPHP (129). A murine gene trap model of *Cep290* similarly showed Wnt pathway changes (reduced Tcf1 protein) only at later stages of the disease (murine kidney tissue aged 1 year) implicating this pathway in renal fibrosis (95). The relationship between Wnt signaling and cystic kidney disease has been recently reviewed (130).

### cAMP Signaling

A huge amount of data have demonstrated the key role of elevated cAMP in mural epithelial cell proliferation and fluid secretion, which are the main drivers of cyst formation in polycystic kidney disease (131). However, some lines of evidence suggest that high cAMP levels are also implicated in junctional and polarity defects in NPHP. Levels of cAMP were found to be elevated in *Nphp3-*, *Nphp6-*, and *Nphp8-*stable knockdown mIMCD3 lines. When these cells were examined in a 3D spheroid culture system, they formed abnormal spheroids with no lumen and/or misaligned nuclei. Treatment with octreotide, an inhibitor of cAMP production, could rescue these structural abnormalities, linking high cAMP levels to cell polarity defects (132). Furthermore, it was shown that treatment with the cAMP analog 8-bromo-cAMP resulted in a dose-dependent loss of SDCCAG8 (NPHP10) protein in cell–cell junctions of the renal epithelial cell line MDCK-II, highlighting the potential role of high cAMP in perturbation of tissue architecture (58). Importantly, elevated cAMP levels and expression levels of the cAMP-dependent gene *Aquaporin-2* were found in the renal tissue of *Pcy* mice that carry a missense mutation in *Nphp3* (51).

### mTOR Pathway

Increased mTOR (mechanistic target of rapamycin) activity was found in cystic kidney (133, 134) and in particular in cyst-lining epithelium (135) of several NPHP mouse models. mTOR is an atypical serine/threonine kinase that, by integrating a variety of signals from nutrients and growth factors, regulates cell growth and proliferation.

The detailed subcellular localization of mTOR pathway components is not clear but it has been showed that primary cilium is important for the regulation of mTOR pathway (136, 137). It has been proposed that the flow-dependent bending of primary cilium represents a mechanosensory signal that controls cell size through the regulation of mTOR activity (137). Consequently, cilia abnormalities may ultimately result in cell growth deregulation, which could be potentially critical for the tubular geometry of the kidney.

### THE ROLE OF CILIA IN SONIC HEDGEHOG SIGNALING AND CELL CYCLE

The Hedgehog (Hh) signaling pathway is a key developmental pathway and was first discovered in *Drosophila* (138). There are three mammalian Hh homologs, Desert, Indian, and Sonic. The sonic Hh (Shh) pathway is essential for development (139), patterning, organogenesis, and cell signaling (140). It acts as a morphogen and a mitogen and dysregulation of the pathway can lead to severe developmental defects and can give rise to various cancers (141). Shh signaling is intimately related to the primary cilium (141). The receptor Patched (Ptch1) is 12-pass transmembrane protein localized to the primary cilium. It has an inhibitory effect on the translocation of Smoothened (Smo, a G-protein-coupled-like receptor). The secreted ligand Shh binds to Ptch1 and triggers internalization of Ptch1 into endocytic vesicles. This allows the translocation of Smo into the primary cilium and its stepwise activation (142). Downstream effector Glioma proteins (Gli2,3) remain in a neutral state under the effect of suppressor of fused and by sequential phosphorylation by protein kinase A, glycogen synthase kinase 3β, and casein kinase 1, they undergo proteolytic conversion to their repressor form. Smo, when enriched in the primary cilium and activated, promote the conversion of Gli repressor (Gli3r) forms into full-length activator forms. The Gli activators (Gli3a) induce the expression of Hh target genes *cyclin D1*, *Gli1*, *Gli2*, *N-myc*, and *Ptch1.* An intact Hh pathway is important for ciliogenesis. The evidence implicating the defects of the Hh signaling in NPHP, renal development and cystogenesis is evolving (77, 103). Loss of the transcription factor Glis2 (Gli-similar zinc finger protein) causes NPHP type 7 (56), Shh knockout mouse embryos showed either renal agenesis or cystic dysplasia (143) and upregulated Indian Hh has been implicated in cystogenesis (144). A subset of BBS proteins has been shown to modulate Shh signaling and interact with IFT proteins (145). More recently, Hh signaling has been shown to be dysregulated in models of cystic kidney disease including *Thm1*, *Pkd1*, *jck* (103) and *Cep290* (95).

### THE CILIARY TRANSITION ZONE AND LINKS TO NPHP

Between the basal body and the ciliary axoneme lies the transition zone, a physical barrier between the ciliary membrane and the apical plasma membrane of the cell. Several genes causing NPHP encode transition zone components, including *NPHP1*, *RPGRIP1L*, *NPHP4*, and *CEP290*. The transition zone controls the protein entry into and exit from the primary cilium and the composition of the ciliary membrane, which directly impacts ciliary signaling pathways such as Hedgehog signaling. The hedgehog signaling molecule Smoothened has been shown to accumulate in discrete clusters in the transition zone, and RPGRIP1L mutations disrupted this localization, leading to disrupted signaling (146). These data support the hypothesis of the transition zone as a gatekeeper of the cilium and that defects can account for phenotypes such as NPHP.

### DDR PATHWAYS AND NPHP

The DDR signaling pathway allows the cell to detect DNA damage, apply an arrest in cell cycle and promote repair of the DNA. Repair of double stranded DNA breaks is particularly important for the maintenance of chromosome integrity. The DNA damage pathway ensures that damaged cells do not progress through S phase and into mitosis before repair is complete. Recently, several of the proteins implicated in NPHP including NEK8 (99), CEP164 (147), ZNF423 (63), SDCCAG8 (58, 59), and CEP290 (96) have been implicated in this pathway, suggesting a nuclear (non-ciliary) role. Following DNA damage, ZNF423, CEP164, and SDCCAG8 proteins have been shown to colocalize to nuclear foci positive for TIP60, a marker of sites of DNA damage and knockdown of CEP164 or ZNF423 causes increased sensitivity to DNA damaging agents (63). These observations provided a hypothesis that may explain why some NPHP genes with null mutations (such as *NPHP3*, *CEP290*, and *RPGRIP1L*) present as severe congenital-onset dysplasia and malformation in multiple organs including the kidney, brain and eye while hypomorphic mutations in the same genes produce milder phenotypes, which include late-onset degeneration and fibrosis leading to NPHP in the kidney and retinal degeneration in the eye. During periods of high proliferation and replication stress such as morphogenesis, DDR signaling is essential, and defects may lead to tissue dysplasia. By contrast, during maintenance of tissues in postnatal life, low replication stress would be expected, and defects would produce a degenerative phenotype. This hypothesis may go some way to explain the organ specific phenotypes seen in Joubert syndrome and other syndromes associated with NPHP (63). DDR defects and replication stress may also be an explanation for the fibrosis seen in association with NPHP and represents drugable target for the disease, which may be independent from and more reversible than cystogenesis (96, 148).

### AN INTEGRATION OF SIGNALING PATHWAYS IN THE DEVELOPMENT OF NPHP

It remains clear that given the genetic heterogeneity of NPHP and the numerous mechanistic pathways discussed that there is not one unifying pathology leading toward NPHP. The renal histology of NPHP points to a common endpoint of tubular damage and fibrosis, which may have multiple triggers. With each new gene discovery paper, there seems to be better clarity toward molecular diagnosis but more confusion regarding the signaling pathways underlying disease.

The clear themes concerning NPHP are that this disease is a manifestation of a renal ciliopathy with almost all NPHPassociated genes encoding gene products known to localize to primary cilia and regulate ciliary function and structure (149), with both the Hedgehog and Wnt signaling pathways implicated downstream from abnormal ciliary signaling. The function of the ciliary transition zone as a gatekeeper for ciliary protein entry and exit is clearly fundamental to ciliary signaling processes. Protein interaction studies of NPHP proteins now allow the proteins to be grouped into four distinct modules. These are the NPHP1–4–8 (NPHP1, NPHP4, and RPGRIP1L) module, the NPHP2–3–9-ANKS6 (INVS, NPHP3, NEK8, and ANKS6) module, the NPHP5–6 (IQCB1 and CEP290) module and the MKS module (MKS1, CC2D2A, and TCTN2). This points to the fact that each NPHP protein has a distinct localization and function within the centrosome/ transition zone/cilium.

However, there is also growing evidence for a nuclear/DDR function of some NPHP-associated proteins, which may be important in disease initiation and progression. Whether this is independent of roles in the primary cilium is not known. It is possible that loss of ciliary function may be a downstream effect of nuclear events affecting cell cycle progression as a result of replication stress (148). The intimate relationship between ciliogenesis and DDR has recently been discussed (150). Centrosomal proteins including NEK8 and CEP290 that are mutated in ciliopathy disorders and are known to have functional roles in DDR are discussed in detail. Overall, it seems likely, given the body of evidence concerning cilia and cystic kidney disease that ciliary dysfunction is a relatively specific subcellular phenotype and final common pathway leading to NPHP, but other pathways may feed into this and be interrelated.

### TREATMENT OF NPHP

Nephronophthisis is incurable at present, but a range of potential therapeutic interventions has arisen from several lines of investigation into the pathogenesis of NPHP. Elevated renal cAMP levels were found associated with the cystic phenotype of NPHP and the modulation of cAMP production has been extensively explored as a potential strategy in the treatment of cystic kidney disease (51, 151–154). V2R antagonists are able to slow the rate of cAMP production by inhibiting V2R that, by coupling with G proteins, regulates the activity of adenylate cyclase and mediates urine concentration. Indeed, V2R antagonists OPC31260 and tolvaptan were shown to be effective in reducing renal accumulation of cAMP and rescuing the cystic kidney phenotype in *Pcy* mice (a model of NPHP3) (16, 51, 153). The use of tolvaptan has now moved successfully from preclinical models, through clinical trials (155) and into clinical practice (156). Its use in childhood ADPKD is currently being investigated in clinical trials.

As discussed, several lines of evidence support a direct link between DNA damage and the loss of NPHP proteins. In particular, both the NPHP type nine associated protein NEK8 and Cep290 were shown to be important regulators of DNA damage as their loss leads to increased sensitivity to replication stress and increased levels of CDKs (96, 99). Interestingly, CDK inhibition is able to suppress the DNA damage caused by loss of NEK8 or Cep290, therefore providing a rationale for CDK inhibition as a potential strategy in the treatment of NPHP (96, 99). Indeed, the CDK inhibitor roscovitine and its analog S-CR8 significantly halted the progression of cystic phenotype and attenuated loss of kidney function in *jck* mice (carrying a mutation in *Nek8*) (99, 157). Furthermore, roscovitine was able to ameliorate the ciliary phenotype of renal epithelial cells derived from a patient with NPHP secondary to a mutation in *CEP290*. Interestingly, it was shown that the treatment with purmorphamine, an agonist of the Shh pathway, is not only as effective as roscovitine in rescuing the ciliary defect, but is also able to decrease CDK5 protein levels in patient cells, suggesting a possible convergence of these signaling pathways (158).

Given the pivotal role played by the primary cilium in the context of Hh pathway, a manipulation of Hh signaling appears as an appealing strategy in the treatment of ciliopathies such as NPHP. It has been shown that genetic deletion of *Gli2* can ameliorate the cystic kidney phenotype in an orthologous mouse model of TTC21B (56), while Hh agonism mediated by purmorphamine treatment is able to rescue the architectural defect displayed by 3D cultures of CEP290 renal epithelial cells (95).

Hyperactivation of mTOR (mechanistic target of rapamycin) pathway was found to be associated with cystic kidney disease and rapamycin has proven to be effective in several rodent [Han:SPRD rat (134, 159), LPK rat (135), *Pcy* mouse (133), and zebrafish models (*invs*, *iqcb1*, and *cep290* morphant) of NPHP (160)].

The zebrafish models of NPHP are proving also to be extremely useful for high-throughput drug screens to determine their effect on kidney development (161).

There is hope therefore that these and other animal models of NPHP will provide valuable insights for future personalized medicine treatments of NPHP in affected patients (162). However, despite the great number of promising interventions that has arisen from preclinical studies, no clinical trials have yet been conducted to test their therapeutic potential in NPHP patients, most of whom eligible for treatment would be less than 18 years of age (125).

To date, options for the treatment of NPHP remain supportive. Control of blood pressure is a priority in children and young adults affected. Management of complications arising from progressive renal failure such as anemia, symptoms of uremia and fluid overload are important alongside preparation for future renal replacement therapy. This disease does not recur in a transplant and renal transplantation remains the ideal mode of renal replacement therapy.

## CONCLUSION

The clinical and pathological diagnosis of NPHP is important, given its progression to ESRD and its associated extrarenal manifestations. Molecular genetic investigations allows a diagnosis in around one-third of cases and can give insights into the associated disease features, the underlying mechanisms and hopefully pave the way for individualized treatments for the underlying kidney disease. As this review demonstrates, it is true that many genes cause NPHP and while most of the identified molecular causes implicate the primary cilium in the pathogenesis of NPHP, it has also become apparent that there are important differences in the underlying pathophysiology. The traditional descriptions of NPHP of infantile, juvenile, and adolescent may now seem dated; however, they highlight the fact that different genetic forms of the disease disrupt the kidney by different mechanisms, demanding a precision medicine approach to the diagnosis, understanding and treatment of NPHP and its associated syndromes.

### REFERENCES


### AUTHOR CONTRIBUTIONS

JS conceived, drafted, and wrote the manuscript. SS and EM drafted and revised the manuscript. SR drafted the manuscript and provided figures.

### ACKNOWLEDGMENTS

SS is a Kidney Research Clinical Training Fellow. EM is funded by Kids Kidney Research. JAS is funded by the MRC MR/M012212/1, the Newcastle upon Tyne Hospitals NHS Charity and Northern Counties Kidney Research Fund.

### FUNDING

The authors gratefully acknowledge funding from Kidney Research UK, Kids Kidney Research, and Northern Counties Kidney Research Fund.


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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer KH and handling Editor declared their shared affiliation.

*Copyright © 2018 Srivastava, Molinari, Raman and Sayer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Meckel–Gruber Syndrome: An Update on Diagnosis, Clinical Management, and Research Advances

*Verity Hartill1,2, Katarzyna Szymanska2 , Saghira Malik Sharif1 , Gabrielle Wheway3 and Colin A. Johnson2 \**

*1Department of Clinical Genetics, Yorkshire Regional Genetics Service, Leeds Teaching Hospitals NHS Trust, Leeds, United Kingdom, 2 Leeds Institute of Biomedical and Clinical Sciences, University of Leeds, Leeds, United Kingdom, <sup>3</sup> Faculty of Health and Applied Sciences, Department of Applied Sciences, UWE Bristol, Bristol, United Kingdom*

#### *Edited by:*

*Miriam Schmidts, Radboud University Nijmegen, Netherlands*

#### *Reviewed by:*

*Julia Hoefele, Technische Universität München, Germany Jan Halbritter, Leipzig University, Germany*

> *\*Correspondence: Colin A. Johnson c.johnson@leeds.ac.uk*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 26 September 2017 Accepted: 02 November 2017 Published: 20 November 2017*

#### *Citation:*

*Hartill V, Szymanska K, Sharif SM, Wheway G and Johnson CA (2017) Meckel–Gruber Syndrome: An Update on Diagnosis, Clinical Management, and Research Advances. Front. Pediatr. 5:244. doi: 10.3389/fped.2017.00244*

Meckel–Gruber syndrome (MKS) is a lethal autosomal recessive congenital anomaly syndrome caused by mutations in genes encoding proteins that are structural or functional components of the primary cilium. Conditions that are caused by mutations in ciliary genes are collectively termed the ciliopathies, and MKS represents the most severe condition in this group of disorders. The primary cilium is a microtubule-based organelle, projecting from the apical surface of vertebrate cells. It acts as an "antenna" that receives and transduces chemosensory and mechanosensory signals, but also regulates diverse signaling pathways, such as Wnt and Shh, that have important roles during embryonic development. Most MKS proteins localize to a distinct ciliary compartment called the transition zone (TZ) that regulates the trafficking of cargo proteins or lipids. In this review, we provide an up-to-date summary of MKS clinical features, molecular genetics, and clinical diagnosis. MKS has a highly variable phenotype, extreme genetic heterogeneity, and displays allelism with other related ciliopathies such as Joubert syndrome, presenting significant challenges to diagnosis. Recent advances in genetic technology, with the widespread use of multi-gene panels for molecular testing, have significantly improved diagnosis, genetic counseling, and the clinical management of MKS families. These include the description of some limited genotype–phenotype correlations. We discuss recent insights into the molecular basis of disease in MKS, since the functions of some of the relevant ciliary proteins have now been determined. A common molecular etiology appears to be disruption of ciliary TZ structure and function, affecting essential developmental signaling and the regulation of secondary messengers.

Keywords: Meckel–Gruber syndrome, renal cystic dysplasia, oligohydramnios, primary cilium, transition zone, Shh signaling

### CLINICAL FEATURES

Meckel–Gruber syndrome (MKS, OMIM number #249000), sometimes simply termed Meckel syndrome, was first described by Johann Friedrich Meckel in 1822. Meckel noted two siblings who died soon after birth with typical features that included occipital encephalocele, polycystic kidneys, and polydactyly (1). Gruber described the same condition later, naming it dysencephalia splanchnocystica (2). In the 1960s, the condition was further delineated by Opitz and Howe (3) with subsequent refinement of the diagnostic criteria (4–7).

Meckel–Gruber syndrome is a lethal developmental syndrome characterized by posterior fossa abnormalities (most frequently occipital encephalocele) (**Figures 1A,B**), bilateral enlarged cystic kidneys (**Figures 1C–E**), and hepatic developmental defects that include ductal plate malformation associated with hepatic fibrosis and cysts (**Figure 1F**) (8). A common additional feature is postaxial polydactyly, usually affecting both hands and feet (**Figure 1A**), which is seen in 70–80% of cases (5–7, 9). Other occasional features, seen in 25–40% of fetuses include bowing and shortening of the long bones, abnormalities of the male genitalia, microcephaly or anencephaly, cleft lip and palate, and other craniofacial abnormalities, congenital heart defects, and pulmonary hypoplasia (5, 6, 8, 9). Rare features (seen in less than 20% of cases) include cystic dysplasia of the lungs or thyroid, retinal colobomata, and *situs* defects (8–14). Central nervous system (CNS) defects are considered to be obligate features of MKS but appear to have a variable presentation, varying between total craniorachischisis, partial defects of the corpus callosum, Dandy–Walker malformation, and most frequently occipital encephalocele (15).

Renal cystic dysplasia is the most characteristic feature of MKS, but differs from those typical of polycystic kidney disease. The degree of cyst formation varies between individuals with MKS, but the kidneys will often be grossly enlarged, causing massive swelling of the abdomen (**Figures 1A,D**). Large, fluid-filled cysts are visible by eye in most affected individuals. However, in other small cysts, cystic changes of the proximal tubules and the absence of normal renal parenchyma are visible by microscopic investigation (**Figure 1E**). Cysts develop first in the glomeruli in the cortex, and cystogenesis progresses along the tubules and collecting ducts in the medulla. Abnormal fetal renal function is a frequent cause of oligohydramnios or anhydramnios, a common complication of an MKS pregnancy (7, 9). Potter's sequence is frequent (pulmonary hypoplasia, growth restriction, club feet, and contractures) with a distinctive facies (comprising slanting forehead, flattened nose, and low-set ears) (**Figure 1A**), and is secondary to oligohydramnios or anhydramnios during pregnancy. In addition to cystic kidney dysplasia, hepatic involvement is an obligate feature of MKS (6). The typical histology shows bile duct proliferation and dilation (**Figure 1F**), associated with excess collagenous connective tissues (8), which are thought to arise from the incomplete development of the hepatic biliary system (16, 17).

### GENETICS AND ALLELISM

Autosomal recessive (AR) inheritance for MKS is confirmed by equal occurrence in males and females, concordance in monozygotic twins, examples of affected siblings, and, in some cases, parental consanguinity. If the condition has occurred in previous pregnancies then the recurrence risk is 25%. The worldwide incidence of MKS has been estimated at 1 in 135,000 live births (18), but higher incidences of MKS are observed in endogamous populations, such as Gujarati Indians (19), Tatars (20), Hutterites (21), and in Finland (22) where 1 in every 9,000 live births is affected. MKS incidence is also higher in consanguineous populations, such as Kuwaiti Bedouin tribes (1 in 3,530 births) (23) and populations in Saudi Arabia (1 in 3,500 births) (24).

Meckel–Gruber syndrome has extreme genetic heterogeneity and displays allelism with other ciliopathies such as Joubert syndrome (JBTS), COACH syndrome (cerebellar vermis hypo/ aplasia, oligophrenia, congenital ataxia, ocular coloboma, and hepatic fibrosis), oro-facio-digital syndrome (OFD), nephronophthisis (NPHP), and Bardet–Biedl syndrome (BBS). To date, mutations in 14 genes (including *TXNDC15*) are identified as causative for MKS (**Table 1**). Mutations in a further three genes, specifically *C5orf42* (25), *CSPP1* (26), and *CEP55* (27), are implicated on the basis of pathogenic private mutations in individual families with either MKS or MKS-like phenotypes (**Table 1**). Two other genes, comprising *SEC8* (also known as *EXOC4*) (25) and *EXOC3L2* (28), have private mutations as a cause of probable MKS phenotypes. In total, mutations in these genes appear to explain only 50–60% of MKS cases (28, 29).

Mutations in *TMEM67*, *RPGRIP1L*, and *TMEM216* can cause both JBTS and MKS, demonstrating allelism between these conditions (31, 35, 36, 46) (**Table 1**). Biallelic mutations in the known MKS genes can also cause other related and allelic ciliopathies. Most notably, mutations in *CEP290* (*MKS4*) can also occur in patients with NPHP (47), Senior-Løken syndrome (SLSN), JBTS (34), BBS (48), COACH syndrome (49), and Leber congenital amaurosis (50). The variable expressivity of the MKS phenotype can complicate the diagnosis of the condition, since this can extend to intra-familial variation for individuals that carry the same pathogenic mutation within families (31), even between monozygotic twins (7). The effect of non-Mendelian inheritance modes or the influence of genetic modifier alleles has frequently been proposed to explain some of this variation. However, in a cohort of JBTS patients who shared causal alleles but had discordant phenotypic features, digenic, or oligogenic inheritance modes were largely excluded as disease mechanisms (51). A second recent study suggests that ciliopathy phenotypes, including those for MKS, are allele-specific and that stochastic effects have a more important role on phenotypic variability than modifier alleles (28).

### GENOTYPE–PHENOTYPE CORRELATIONS

In recent years, the identification of pathogenic alleles specific to MKS, limited genotype–phenotype correlations and founder mutations for particular ethnic groups (**Table 1**) have facilitated the rapid and accurate genetic diagnosis of this condition. Mutations in *MKS1* cause ca. 7% of all MKS cases and ca. 70% cases in Finland. Most Finnish MKS patients share the common "Finn major" mutation (*MKS1* IVS15-7\_35del; **Table 1**), which is thought to have arisen in a founder of the Finnish population (52). Skeletal involvement including shortening and bowing of the long bones, polydactyly, and occipital encephalocele are frequently seen in patients with the Finn major mutation (18). More generally, *MKS1* mutations are associated with higher rates of polydactyly and occipital encephalocele, bone dysplasia, cleft

palate, and *situs* defects (52). All *MKS1* mutations reported in MKS patients are null mutations, but a hypomorphic mutation has been reported in an individual with BBS (48), and recently in individuals with JBTS (53, 54).

*TMEM67* mutations are estimated to cause 16% of MKS cases (55), making them the most frequent cause of MKS. Both CNS malformations and polydactyly are less frequent in *TMEM67* mutated individuals than those with *MKS1* mutations (56).

#### Figure 1 | Continued

Clinical features of Meckel–Gruber syndrome (MKS) and schematic of primary cilia structure. (A) Typical external features for a fetus with MKS at gestation age 16/40, showing typical clinical features comprising occipital encephalocele, massive flank masses due to bilateral renal cystic dysplasia, postaxial hexadactyly of all limbs, and a typical Potter's sequence facies with a slanting forehead and flattened nose. (B,C) Ultrasound findings at 14+/40 weeks of gestation for MKS showing (B) encephalocele (arrowheads), and (C) large cystic kidneys (arrowhead). (D) Massive swelling of the abdomen of a fetus at gestation age 18+/40 with MKS due to grossly enlarged, cystic kidneys. (E) Hematoxylin-eosin staining of MKS fetal kidney at gestation age 18+/40 showing cystic dysplasia, comprising large, fluid-filled cysts, small cysts, and cystic swelling of the proximal tubules and glomeruli, with the absence of normal renal parenchyma. (F) Immunohistochemical staining of MKS fetal liver at gestation age 18+/40 for cytokeratin-19, showing the retention of embryonic bile duct structures (the ductal plate malformation) without the formation of patent bile ducts (arrowhead). PV, hepatic portal vein. (G) Left: simplified schematic of cilium ultrastructure (individual components are not to scale). For the purposes of clarity, the ciliary axoneme is represented by two doublets of microtubules (the A- and B-tubules; gray rods), and the nine-fold symmetry in the structure is indicated by dark gray ovals in the mother centriole. The axoneme is bound by ciliary membrane (red/brown line and shading). The ciliary transition zone (TZ) is characterized by Y-shaped links (pink) that mediate interactions with the ciliary membrane. The permeability barrier called the "ciliary gate" is indicated by the dashed pink ovals and pink shading, and is thought to consist of transition fibers and TZ proteins. Ciliary cargo proteins (purple) are trafficking by vesicular transport mediated by coordinated interactions between the Rab family of small GTPases (RAB11 and RAB8; R8), the RAB8 activator protein Rabin8 (R), and the exocyst protein complex (Ex) at the ciliary base. The exocyst tethers vesicles to the periciliary membrane, facilitating transport through the ciliary gate (green arrow), followed by transport within the cilium mediated by intraflagellar transport (IFT; dashed black arrows). Right: schematic of a TZ model, showing the possible location of TZ proteins mutated in MKS and related ciliopathies (pink ovals) forming the "MKS-JBTS module," RPGRIP1L (blue oval) which is thought to be a component of a ciliary assembly module, and a third functional module (brown) that is associated with nephronophthisis (NPHP) proteins. The selective transport of cargo transmembrane proteins (lilac or purple) is indicated by arrows. The proteins include G-protein-coupled receptors (GPCRs), and effectors of the Shh signaling pathway (Smoothened and Patched). Images (A–C) are used by kind permission of Dr. Riitta Salonen (Rinnekoti Foundation, Helsinki, Finland) from the Robert J. Gorlin Slide Collection. JBTS, Joubert syndrome.



*Assigned locus names are listed, with unassigned ones indicated by NA. Alias names of genes or loci summarize the alternative gene symbols, names, and any reported allelism with other ciliopathies (indicated by the gene symbol or locus name). Any reported founder mutations in specific populations are also listed, in addition to the original gene discovery paper or papers.*

*CORS, cerebellar–ocular–renal syndrome; JBTS, Joubert syndrome; MKS, Meckel–Gruber syndrome; NPHP, nephronophthisis; OFD, oro-facio-digital syndrome; SLSN, Senior-Løken syndrome.*

*TMEM67* mutations also account for 57–83% (49, 55, 57) of the JBTS variant phenotype of COACH syndrome, demonstrating a strong genotype–phenotype correlation of *TMEM67* mutations with hepatic developmental defects or coloboma (49, 58). This correlation indicates that JBTS patients with hepatic involvement are prioritized for *TMEM67* mutation testing (57). Other genotype–phenotype correlations for MKS or JBTS have been identified by mutation type, or by the location of missense mutations within protein domains. For example, *CC2D2A* missense mutations are associated with JBTS whereas MKS is caused by null alleles (59). Missense mutations in exons 8–15 of *TMEM67*, particularly in combination with a second truncating mutation, are also associated with MKS (29, 55). Homozygous truncating mutations in *RPGRIP1L* are associated with MKS, whereas missense mutations (either in the homozygous state or compound heterozygous with a truncating mutation) are prevalent for JBTS patients. MKS phenotypes associated with *RPGRIP1L* mutations tend to be severe, including anencephaly and shortening and bowing of the long bones, in addition to the classic triad (36).

### DIAGNOSIS, CLINICAL MANAGEMENT, AND GENETIC COUNSELING

Meckel–Gruber syndrome is lethal *in utero* or immediately after birth, often due to pulmonary hypoplasia, although an unusual survivor has been described aged 28 months (60). MKS has a broad, multi-organ phenotype with considerable variation, but it is generally diagnosed by the presence of cystic kidney dysplasia (**Figure 1C**), in addition to at least one other hallmark feature of the disease. These comprise occipital encephalocele (**Figure 1B**), polydactyly (**Figure 1A**), or hepatic developmental abnormalities such as the ductal plate malformation (**Figure 1F**) (6, 7). The presence of abnormal intrahepatic bile ducts and cystic kidneys has been proposed to be invariant features of MKS and appears to be pathognomonic for the condition (6, 17). Salonen (6) has proposed that the minimal diagnostic criteria for MKS are CNS malformation, bilateral multicystic kidneys, and hepatic fibrotic changes.

Transabdominal ultrasonography, performed at 10–14 weeks gestation, has been shown to successfully detect several of the fetal anomalies associated with MKS, including polycystic kidneys (from 9 weeks gestation), occipital encephalocele (from 13 weeks), and polydactyly (from 11 weeks), in both high-risk and low-risk pregnancies (**Figure 1B**) (60–64). Visualization can be compromised by oligohydramnios (65), but this is less problematic when performed in the first trimester of pregnancy (62). Further investigation of anomalies is possible by transvaginal scanning. Enlargement of the fetal trunk can give an early indication of multicystic renal dysplasia (66), in addition to unusually heterogeneous corticomedullary differentiation, reduced echogenicity of the medulla, increased echogenicity of the cortex, and the visualization of small medullary cysts (**Figure 1C**) (67). The fetal bladder can also be visualized by ultrasonography from 11 weeks, and the absence of a visible fetal bladder is often indicative of renal dysfunction.

Fetal MRI is an alternative if ultrasonography findings are inconclusive or lack of amniotic fluid prevents clear imaging. MRI offers better soft-tissue resolution than ultrasonography, and can provide clearer images of intracranial structures to enable an accurate diagnosis of CNS malformations, but is rarely performed before 18 weeks gestation. Fetal movement and maternal aortic pulsation do not preclude a successful diagnosis of MKS using MRI (68), since imaging artifacts caused by fetal movement can be minimized by a fetal neuromuscular blockade (69) or general anesthesia of the mother. Transabdominal or vaginal endoscopy in the first trimester of pregnancy allows diagnosis of MKS by visualization of the surface anatomy of the embryo. Fetoscopy enables the direct observation of polydactyly and occipital encephalocele from 11 weeks gestational age (70). Prenatal diagnosis is also possible by using a combination of these imaging techniques, α-fetoprotein testing of amniotic fluid, and DNA testing of fetus and parents. For example, elevated levels of maternal α-fetoprotein during antenatal screening may be associated with MKS (61).

Definitive diagnosis is often possible by using DNA testing to screen for mutations in the known MKS genes. Molecular diagnostic strategies include mutation screening of individual genes or targeted clonal sequencing of multi-gene panels. Single gene testing has low diagnostic sensitivity because of the absence of clear genotype–phenotype correlations for MKS. However, once a pathogenic variant in a family has been identified, then prenatal genetic diagnosis by chorionic villus sampling can be prioritized for at-risk pregnancies. Multi-gene panels can include MKS genes, and other ciliopathy genes of interest, that can be tested by sequence analysis and deletion/duplication analysis (71). However, their diagnostic sensitivity can vary between laboratories because panels can include genes that are unrelated to MKS. Referring clinicians should therefore decide which multi-gene panel has the best balance of sensitivity and affordability. If testing by a multi-gene panel does not confirm a clinical diagnosis of MKS, then more comprehensive testing by either whole-exome sequencing or whole-genome sequencing (WES or WGS) should be considered for obligate carriers. However, these tests may support or suggest unexpected diagnoses, in view of the broad range of possible differential diagnoses for MKS. These are extensive and include Smith–Lemli–Opitz syndrome, trisomy 13, hydrolethalus syndrome, and other related ciliopathies that include AR polycystic kidney disease, BBS, JBTS, and OFD. In particular, the prenatal features of BBS (polydactyly, renal defects, hepatic anomalies, genital hypoplasia, and heart malformations) can lead to a misdiagnosis of MKS (72). The extensive range of differential diagnoses and the broad allelism between different ciliopathies highlights the clinical need for accurate molecular diagnosis and the refinement of genotype–phenotype correlations (71, 73).

Since MKS has an AR inheritance pattern, couples with a previously affected child should have the opportunity for genetic counseling with a medical professional in order to discuss the nature, inheritance, and implications of an MKS diagnosis. The possibility of prenatal testing needs appropriate counseling of parents if it is being considered for the purpose of pregnancy termination in addition to early diagnosis. Individual opinions regarding prenatal testing and pregnancy termination may depend on a number of factors such as religious and cultural beliefs, and previous experiences of the medical condition (74, 75). An accurate perception of their genetic risk, a clear impact of genetic disorder and previous experience of having an affected child can help parents to make an informed decision (76). It is therefore appropriate to discuss these issues at an early stage to ensure that families can make informed medical and personal decisions.

With consanguineous MKS families, geneticists will encourage disclosure to at-risk relatives. The fact that these individuals often do not seek carrier testing suggests that many families are not sharing this information. In families with an AR disorder who practice consanguinity, the concern that their children's carrier status would affect their marriage prospects can contribute to non-disclosure of this important information to their wider family (76–78). Furthermore, there is a widespread assumption among healthcare professionals that Muslim patients will not accept termination of pregnancy (79). However, under a *fatwā* (an Islamic legal pronouncement), termination of pregnancy is permitted before "ensoulment" at 120 days in cases of severe disability such as MKS, or where there is a risk to the mother's life (75, 80), but knowledge of the *fatwā* is limited among Muslim populations (75, 81).

### INSIGHTS INTO MOLECULAR PATHOMECHANISMS

The primary cilium is a microtubule-based organelle, projecting from the apical surface of vertebrate cells. It acts as an "antenna" that receives and transduces chemosensory and mechanosensory signals (**Figure 1G**). Primary cilia are thought to be hubs of developmental signaling that regulate diverse pathways such as Wnt and Shh that have essential roles during embryonic development. In particular, mutations MKS genes cause defective Shh signaling in several mouse models of ciliopathies (82–85). Primary cilia have a complex architecture that defines the compartmentalization of ciliary proteins, and many genes that are mutated in MKS encode proteins that localize to the ciliary TZ. This is a distinct molecular compartment located at proximal regions of cilia (86, 87) that has been defined by genetic and biochemical approaches to consist of protein complexes forming the so-called "MKS-JBTS module" (**Figure 1G**). The TZ connects the microtubules of the ciliary axoneme to the plasma and ciliary membranes, and it has been suggested to both limit and assist the regulated trafficking of cargo proteins and lipids into and out of the cilium (88). The TZ is therefore thought to modulate the composition of essential ciliary compartments such as the ciliary membrane, axoneme, and associated proteins (88). A recent study demonstrated that the disruption of ciliary TZ architecture causes JBTS (89), but how this affects ciliary trafficking and causes the MKS or other ciliopathy phenotypes remains a research question of paramount importance.

Genetic, biochemical, and proteomic approaches have delineated the networks of protein-protein interactions that underlie the functional "MKS-JBTS module" at the TZ, as well providing insights into the structural basis of selective permeability at this ciliary compartment (90, 91). For example, several small tetraspanin-like transmembrane proteins (TMEMs) that are mutated as a cause of MKS and other ciliopathies (TMEM216, TMEM231, and TMEM107) appear to localize to the specialized ciliary membrane at the TZ. Other transmembrane proteins at this compartment include the Tectonic proteins (TCTN1, TCTN2, and TCTN3) and the Frizzled-like receptor TMEM67. In turn, the TMEM and Tectonic transmembrane proteins are likely to mediate interactions with and modulate the function of membrane-targeting proteins such as RPGRIP1L and CC2D2A that contain C2 domains (90, 91). TCTN1, for example, forms a biochemical complex at the TZ with CC2D2A, other ciliary transmembrane proteins (TCTN2, TMEM216, and TMEM67), and other known ciliopathy proteins (MKS1, CEP290, and B9D1) (92). The role of CEP290 and the B9 domain-containing proteins B9D1 (MKS9) and B9D2 (MKS10) at the TZ is undefined, but by analogy with the C2 domain-containing proteins, they are likely to mediate protein–protein interactions through their coiled-coil or B9 domains. One plausible model suggests that these proteins link TMEMs with either vesicular cargo at the TZ during the process of ciliogenesis, or then subsequently during the ciliary trafficking of cargo proteins through the TZ (**Figure 1G**). However, functional work on AHI1/jouberin, a ciliary TZ protein that contains WD40 and Src Homology 3 protein interaction domains, tends to support a general role for TZ proteins during vesicular trafficking because AHI1/jouberin interacts with RAB8A (93). RAB8A is an important small GTP/ GDP-binding protein that is essential for vesicular protein trafficking from the endoplasmic reticulum to the Golgi apparatus and plasma membrane (93). Interestingly, AHI/jouberin also directly interacts and sequestrates β-catenin (a downstream effector of canonical Wnt signaling) at the cilium (94, 95) and it therefore has functions that are additional and distinct to those of CEP290.

Several recent studies have provided mechanistic insights into how the disruption of selective permeability at the TZ results in the incorrect trafficking of proteins at the cilium, with a focus on the trafficking of enzymes, receptors, and other transmembrane proteins involved in intracellular signaling. Although two TMEMs, TMEM17 and TMEM231, localize to the ciliary TZ, other TZ proteins (CC2D2A and B9D1) are required for TMEM231 localization to the TZ (82). In turn, these functional interactions in the TZ regulated the trafficking of ciliary G-protein-coupled receptors (GPCRs; somatostatin and serotonin receptors SSTR3 and HTR6) into the ciliary membrane, and both B9D2 and TMEM231 mutations caused defective ciliogenesis and Shh signaling in mice (82). In a separate study, loss of TCTN1 did not affect ciliogenesis, but nevertheless caused defective ciliary localizations of the transmembrane signaling proteins Smoothened and PKD2/ polycystin-2, adenylyl cyclase 3 (ACIII; forming the secondary messenger cAMP), and ARL13B (a small Arf-family GTPase that localizes to the ciliary membrane) (92). Furthermore, the normal trafficking and localization of Smoothened at both the TZ and cilium is lost as a consequence of JBTS-associated mutations in RPGRIP1L that disrupt ciliary TZ architecture (89). In turn, the disruption of ciliary structure and function prevent Smoothened from mediating correct developmental Shh signaling (89). Disrupted compartmentalization could also prevent the correct ciliary transport of KIF7, a kinesin motor-protein that controls Shh signaling by regulating the amounts of activator and repressor isoforms of GLI transcription factors (96, 97). GLI proteins mediate gene expression changes as a consequence of Shh signaling, and the regulatory roles of KIF7 appear to be both negative, by preventing the incorrect activation of GLI2 in the absence of Shh ligand, and positive, by preventing the processing of GLI3 into the repressor isoform.

### FUTURE PERSPECTIVES

At the present time, the molecular cause for only 60% of cases can be explained by mutations in the known MKS genes (28, 29, 71). The entire series of causative genes for MKS, MKS-like, and related ciliopathy phenotypes are therefore incomplete, but the remaining genes will be either uncommon or carry private mutations that are likely to be represented by single families. However, the widespread use of targeted clonal sequencing techniques such as multi-gene panels and WGS now allows the affordable re-investigation of molecular causes in the known MKS genes for individuals that have previously been mutation negative. These studies are likely to identify potential pathogenic causes that include intronic mutations, copy number variations, and other genic variants in promoter sequences or *cis*-regulatory elements. Although this will improve diagnostic rates for MKS families, these efforts are complicated by both allelism and extreme phenotypic variability for MKS and related ciliopathies. The mechanistic basis of phenotypic variability in MKS still remains largely unclear, and it is undefined what the relative contributions are made by modifier alleles in other genes, in contrast to the general stochastic effects of variability in developmental signaling during embryogenesis. A future research challenge is to delineate the complex relationships between ciliary architecture and organization, and how these relate to and regulate ciliary function.

### AUTHOR CONTRIBUTIONS

VH, KS, SS, GW, and CJ made substantial contributions to the writing, drafting, and revision of this manuscript. All authors approved the final published version of the manuscript.

### REFERENCES


### ACKNOWLEDGMENTS

We thank Dr. Riitta Salonen (Rinnekoti Foundation, Helsinki, Finland) for permission to use clinical images.

### FUNDING

This work was supported by funding from the Medical Research Council (grant numbers MR/K011154/1 and MR/M000532/1) and the European Community's Seventh Framework Programme FP7/2009 under grant agreement no. 241955 SYSCILIA (to CJ). VH was supported by a British Heart Foundation clinical training fellowship (grant number FS/13/32/30069). GW was supported by National Eye Research Centre Small Grant SAC019 and Wellcome Trust Seed Award in Science 204378/Z/16/Z.


by complex de-regulated ciliogenesis, Shh and Wnt signalling defects. *Hum Mol Genet* (2013) 22:1358–72. doi:10.1093/hmg/dds546


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Hartill, Szymanska, Sharif, Wheway and Johnson. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Network for early Onset cystic Kidney Diseases—A comprehensive Multidisciplinary Approach to Hereditary cystic Kidney Diseases in childhood

*Jens Christian König\*, Andrea Titieni, Martin Konrad and The NEOCYST Consortium*

*Department of General Pediatrics, University Children's Hospital Münster, Münster, Germany*

#### *Edited by:*

*Miriam Schmidts, Radboud University Nijmegen, Netherlands*

#### *Reviewed by:*

*Laura Malaga-Dieguez, New York University School of Medicine, United States Julia Hoefele, Technische Universität München, Germany*

> *\*Correspondence: Jens Christian König jens.koenig@ukmuenster.de*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 18 October 2017 Accepted: 25 January 2018 Published: 13 February 2018*

#### *Citation:*

*König JC, Titieni A, Konrad M and the NEOCYST Consortium (2018) Network for Early Onset Cystic Kidney Diseases—A Comprehensive Multidisciplinary Approach to Hereditary Cystic Kidney Diseases in Childhood. Front. Pediatr. 6:24. doi: 10.3389/fped.2018.00024*

Hereditary cystic kidney diseases comprise a complex group of genetic disorders representing one of the most common causes of end-stage renal failure in childhood. The main representatives are autosomal recessive polycystic kidney disease, nephronophthisis, Bardet–Biedl syndrome, and hepatocyte nuclear factor-1beta nephropathy. Within the last years, genetic efforts have brought tremendous progress for the molecular understanding of hereditary cystic kidney diseases identifying more than 70 genes. Yet, genetic heterogeneity, phenotypic variability, a lack of reliable genotype– phenotype correlations and the absence of disease-specific biomarkers remain major challenges for physicians treating children with cystic kidney diseases. To tackle these challenges comprehensive scientific approaches are urgently needed that match the ongoing "revolution" in genetics and molecular biology with an improved efficacy of clinical data collection. Network for early onset cystic kidney diseases (NEOCYST) is a multidisciplinary, multicenter collaborative combining a detailed collection of clinical data with translational scientific approaches addressing the genetic, molecular, and functional background of hereditary cystic kidney diseases. Consisting of seven work packages, including an international registry as well as a biobank, NEOCYST is not only dedicated to current scientific questions, but also provides a platform for longitudinal clinical surveillance and provides precious sources for high-quality research projects and future clinical trials. Funded by the German Federal Government, the NEOCYST collaborative started in February 2016. Here, we would like to introduce the rationale, design, and objectives of the network followed by a short overview on the current state of progress.

Keywords: hereditary cystic kidney diseases, ciliopathy, nephronophthisis, autosomal recessive polycystic kidney disease, Bardet–Biedl syndrome, hepatocyte nuclear factor-1beta nephropathy

## BACKGROUND

Hereditary cystic kidney diseases comprise a group of slowly progressive, chronically debilitating disorders with a high level of complexity. Despite rare individually, as a group they represent one of the most common causes of end-stage renal failure in childhood and, therefore, have an enormous socioeconomic impact. The main representatives of this group of diseases are autosomal recessive polycystic kidney disease (ARPKD), nephronophthisis and nephronophthisis-related ciliopathies (NPH/NPH-RC), Bardet–Biedl syndrome (BBS), and hepatocyte nuclear factor-1beta (HNF1B) nephropathy (1). The incidence ranges from 1:5,000 to 1:100,000 and the estimated overall prevalence is about 300–450 children in Germany (2–7).

Most early onset cystic kidney diseases are inherited as an autosomal recessive trait. One important exception is the HNF1B nephropathy, which follows autosomal dominant inheritance (6). Within the past 20 years scientific efforts have brought tremendous progress for the molecular understanding of hereditary cystic kidney diseases. To date, mutations in more than 70 individual genes have been identified (**Table 1**)


TABLE 1 | Continued


(8). Yet, genetic heterogeneity is a major problem since, there is a significant amount of genetic as well as phenotypic overlap and reliable data on genotype–phenotype correlations are still lacking (9). Moreover, the phenotypic spectrum covered by cystic kidney diseases is highly complex and extremely variable. Although all disease entities are characterized by the development of renal cysts as a common characteristic, clinical courses as well as the onset of chronic renal failure differ significantly. Furthermore, most phenotypes are not limited to the kidneys, but comprise extrarenal organ manifestations (10, 11).


obesity, developmental delay, postaxial hexadactyly, hypogonadism, and slowly progressive CKD. The clinical presentation is highly variable and can be accompanied by further organ involvement (5). The renal phenotype usually resembles NPH, including polyuria and small- to normal-sized kidneys showing cystic lesions at the corticomedullary junction. However, renal involvement is not mandatory and reported in only 31–42% of BBS (18).

• HNF1B nephropathy: HNF1B is a transcription factor playing a central role in the tissue-specific regulation of gene expression in various organs, such as kidney, liver, biliary duct, pancreas, and genital organs. Initially, mutations in the *HNF1B* gene were described in association with maturity onset diabetes of the young (MODY type 5). Subsequently, it was shown that in patients with congenital cystic renal dysplasia *HNF1B* mutations could frequently be identified (renal cysts and diabetes syndrome, RCAD). In contrast to the NPH spectrum, renal cysts may already be detected prenatally, but renal ultrasound presentation is variable including unilateral and bilateral renal dysplasia with different numbers and sizes of cysts. Depending on the extent of renal dysplasia some patients develop renal failure in early childhood while others preserve normal renal function for their whole life (6). Beyond the renal involvement, the clinical spectrum comprises elevated liver enzymes, hyperuricemia, genital tract malformations, and electrolyte disturbances, such as hypomagnesemia and hypokalemia (11).

### GENETIC AND PHENOTYPIC HETEROGENEITY

Although the clinical characteristics of the different hereditary cystic kidney diseases appear quite discriminative, there is significantly genetic as well as phenotypic overlap that hampers an early diagnosis and an individual clinical management (**Figure 1**) (1). The tremendous progress that has been achieved within the genetic field had major impact on the classification of cystic kidney diseases. However, the newly generated insights seem to further complicate the clinical situation for physicians dealing with affected individuals: it has become increasingly evident that so far well-defined clinical entities can be caused by mutations in multiple genes. Even in ARPKD, which for a long time has been assumed to be a single gene disease, modern NGS-based sequencing techniques just recently were able to identify a new genetic cause encoding a ciliary transition zone protein (26). Also, mutations in the same gene can cause very different phenotypes that range from lethal early embryonic multivisceral manifestations to single organ involvement starting in adolescence (**Figure 1**) (1, 27). Thus, recent advances in genomics challenged the classical Mendelian conditions and highlighted the genetic complexity of hereditary cystic kidney diseases and related ciliopathies (28). This complexity has been attributed to allelic heterogeneity, locus heterogeneity, reduced penetrance, variable expressivity, modifier genes, and/or environmental factors (29).

Based on these newly generated insights it has become clear that the "diagnostic odyssey" experienced by patients does not end with the identification of a disease-causing genotype (30). Neither does the traditional approach using textbook signs nor symptoms to guide diagnosis and management seem to be sufficient any longer (31). Rather a systematic and deep phenotyping approach supplementary to careful genotyping is critical in order to discover nonobvious phenotypes and to determine a precise diagnosis. Thus, physicians addressing hereditary cystic kidney diseases should combine both—detailed, multisystemic phenotyping and careful assessment of genotype pathogenicity—in order to capture all facets of the underlying disease (30). Against this background, it will be important to overcome the financial restraints associated with modern NGS-based sequencing currently hampering a comprehensive genetic characterization.

### MOLECULAR UNDERSTANDING

The genetic discoveries revolutionized our molecular understanding of cystic kidney diseases. The observation that most genes that have been identified so far encode proteins that co-localize in primary cilia and even interact as functional ciliary clusters suggested the existence of a common pathophysiological pathway and lead to the so-called ciliary hypothesis (**Figure 2**) (32, 33). However, different molecular mechanisms have been described to be altered in renal cyst formation, including cell-proliferation, apoptosis, DNA repair, fluid secretion into the cyst lumen, altered

kidney disease; ADPKD, autosomal dominant polycystic kidney disease.

ciliary function (e.g., disrupting intraflagellar transport), for most others the exact pathophysiological mechanisms still remain to be unraveled. Abbreviations: NPH, nephronophthisis; JBTS, Joubert syndrome; BBS, Bardet–Biedl syndrome; MKS, Meckel–Gruber syndrome; ARPKD, autosomal recessive polycystic

apico-basal cell polarity, directional cell migration, cell–cell adhesion, interaction with the extracellular matrix and ciliary function. Additionally, various intracellular signaling pathways were identified to be activated in epithelia developing cystic lesion, such as intracellular calcium signaling, cAMP triggered fluid secretion, the wnt-, hedgehog, mTOR-, notch-, YAP-hippo-, and other pathways. Whether or not these observations are related to each other and how the mentioned mechanisms could be connected with an altered ciliary function remains poorly understood and is the topic of ongoing research. Details on these topics are beyond the scope of this perspective. However, this has been excellently reviewed by Ong et al. recently (34–36).

### NETWORK FOR EARLY ONSET CYSTIC KIDNEY DISEASES (NEOCYST)—A COMPREHENSIVE APPROACH ON HEREDITARY CYSTIC KIDNEY DISEASES

Despite all the achievements mentioned above, enormous challenges remain to be solved: the complex phenotypic spectrum, genetic heterogeneity, a lack of reliable genotype–phenotype correlations, a limited molecular understanding and the absence of disease-specific biomarkers still hamper an early diagnosis and individual counseling. Data from international registry studies like the NEPHREG or the ARegPKD registry will certainly help to define robust genotype–phenotype associations by increasing sample sizes and following individual disease courses in a longitudinal fashion (10, 37). However, in order to achieve a deeper clinical as well as molecular understanding, international collaborative efforts comprising various subspecialists will be necessary to move knowledge forward especially in the context of rare diseases. A detailed molecular understanding will be fundamental in order to identify new therapeutic targets and develop disease specific treatment approaches. Therefore, future research activities should go one step further and try to verify the hypotheses gained from cell culture experiments in specimen harvested from actual patients.

To address all these questions, the NEOCYST was initiated in 2016 in order to match the ongoing "revolution" in genetics and molecular biology with an improved efficacy of clinical data collection. Here, we would like to introduce the structure, the goals, and the individual work-packages of the NEOCYST collaborative accompanied by a short overview on the current state of progress.

### DESIGN

Network for early onset cystic kidney diseases is a multidisciplinary, multicenter observational study of hereditary cystic kidney diseases in childhood. It combines a detailed and comprehensive clinical data collection with translational scientific approaches covering the genetic, molecular, and functional background.

### AIMS

The primary goal of the NEOCYST consortium is to improve the clinical situation and the management of children with hereditary cystic kidney diseases. In order to reach this goal a multimodal approach was chosen including:


### STUDY COHORT

Eligible are patients with the diagnosis of ARPKD, isolated NPH or NPH-related ciliopathies, BBS, and HNF1B-nephropathy. While for the first four study, inclusion is justified either by clinical or genetic criteria, HNF1B-nephropathy has to be confirmed genetically in order to avoid phenotypic overlap with other urinary tract malformations. For the clinical diagnosis of NPH at least two of the following criteria have to be met: (i) characteristic clinical course with polyuria/polydipsia; (ii) chronic kidney disease; (iii) kidney ultrasound or biopsy suggestive of NPH (24); (iv) pedigree compatible with autosomal recessive inheritance. The clinical diagnosis of BBS is based on the diagnostic criteria according to Beales et al. (38). Phenotypes reassembling a NPHrelated ciliopathy are classified by the clinical criteria published by Bergmann (1).

Exclusion criteria encompass the definite genetic or clinical diagnosis of other cystic kidney diseases, in particular autosomal dominant polycystic kidney disease (ADPKD) which is addressed by other clinical registry studies.

Patient inclusion is preceded by a written informed consent and an approval by the local ethics committee of each contributing center, guaranteeing accordance with the principles of the Declaration of Helsinki and Good Clinical Practice guidelines.

### WORK PACKAGES

To address the goals mentioned above the NEOCYST consortium comprises seven work packages (**Figure 3**). Subsequently, we will outline the main topics covered by these work-packages:

• Clinical registry study: the web-based NEOCYST registry is a newly created retro- and prospective clinical registry providing a detailed genetic and phenotypic characterization of all hereditary cystic kidney diseases in one common database collecting cross-sectional as well as longitudinal data. Three pre-existing registries on NPH-RC,1 BBS, and HNF1B were merged to build the fundaments of this new database. Additionally, technical bridgehead components

<sup>1</sup>www.nephreg.de.

have been implemented that allow a direct comparative data analysis with the international ARPKD registry2 (37). Thereby, NEOCYST is the first registry study providing a comprehensive approach to early onset hereditary cystic kidney diseases and allowing back-to-back analyses on similarities and differences of the individual disease entities. At the same time, the database supplies technical features that enable external international cooperation and provide a platform for further research projects and future clinical trials throughout Europe.


techniques, such as proteomics, 3D spheroid models, cell (re)programming, and genomic engineering are applied to analyze different signaling pathways and their impact on cystogenesis *in vivo* and *in vitro*. Additionally, in order to characterize altered ciliary processes in hereditary cystic kidney diseases, nasal brushes from affected patients are generated and techniques like high-frequency video microscopy, freeze-fracture analysis, and immunohistological staining are applied to motile as well as immotile cilia. Finally, it is a goal to identify disease-specific proteome marker profiles from patients' spot urine using a blinded prospective approach based on capillary electrophoresis coupled with mass spectrometry.


<sup>2</sup>www.aregpkd.org.

management of hereditary cystic kidney diseases are scarcely available. Thus, NEOCYST has set itself the task to develop and elaborate recommendations on different topics concerning hereditary cystic kidney diseases based on the given evidence in the literature as well as experts' opinions.

### CURRENT STATUS

The NEOCYST project started in February 2016 after initiation and approval of funding by the Federal Ministry of Education and Research (BMBF). After 24 months crucial milestones have been reached.

First of all, a homepage3 has been installed addressing patients, clinicians, and researchers to the same extent. Valuable information on the NEOCYST collaborative, the study goals as well as the medical background is provided in German and English language. A download domain contains all necessary study documents.

Furthermore, in September 2017 the technical implementation of the new online NEOCYST database has been completed merging three pre-existing clinical registries. Up-to-date 344 patients have been included comprising 194 patients with NPH and related ciliopathies, 88 patients with BBS, and 62 patients with HNF1B nephropathy. Especially, regarding HNF1B a rapid increase of participants has been observed ever since HNF1B became a part of the regular genetic screening in patients with hereditary cystic kidney diseases. However, we still assume a high number of unreported cases anticipating a further expansion of the study cohort within the next few years. The database is accessible *via* the NEOCYST-homepage (see text footnote 3).

Major success has been achieved by the genetic workgroup in identifying *DZIP1L* as a new cause for early onset cystic kidney diseases. *DZIP1L* encodes a protein located at the ciliarytransition-zone leading to a phenotype mimicking ARPKD when mutated (26). Additionally, mutations in *MKS1* so far only associated with lethal Meckel–Gruber syndrome were found also to be responsible for a milder phenotype resembling Joubert syndrome accompanied by agenesis of the corpus callosum (39).

The number of biological samples stored in the biobank is continuously growing and substantial progress has been achieved by the projects attended to the molecular biology of cystic kidney diseases. Methods that have been established in another context were successfully transferred to the scientific issues addressed by the NEOCYST project. Detailed imaging and structural analyses of primary and motile cilia have been elaborated and brought to a new scale. First results suggest that structural abnormalities and differences in ciliary protein composition specific for the underlying genetic defect. Furthermore, urine-derived renal epithelial cells, URECs have been established as a valid model on studying cell polarity and epithelial morphogenesis in cystic kidney diseases producing distinguishable patterns of cell clusters. Most results are still preliminary and part of an ongoing research process that will be the subject of future publications.

So far, 19 manuscripts from the NEOCYST cohort have been published or accepted for publication—including a positional paper on the "perinatal management of early onset cystic kidney diseases" (40). Two further positional papers on "imaging of early onset cystic kidney diseases" and "management of early onset ADPKD" have been finalized and are about to be published.

### CONCLUSION

Since initiation in February 2016, the NEOCYST collaborative has made substantial progress in addressing clinical, genetic, and molecular questions related to hereditary cystic kidney diseases. However, the mentioned achievements just represent the first steps of an ongoing process and further scientific initiatives and funding as well as international cooperation will be needed in order to answer these questions. Thus, any participation by international centers is warmly welcome. By setting up the infrastructure of an international clinical registry accompanied by several biological projects and the longtime storage of biomaterial, NEOCYST provides a platform that guarantees a detailed collection of precious longitudinal clinical data going along with further high-quality research approaches and enabling future clinical trials.

### NEOCYST CONSORTIUM

**C. Bergmann**, Ingelheim, Germany; **M. Cetiner**, Essen, Germany; **J. Drube**, Hannover, Germany; **C. Gimpel**, Freiburg, Germany; **J. Göbel**, Frankfurt, Germany; **D. Haffner**, Hannover, Germany; **T. Illig**, Hannover, Germany; **N. Klopp**, Hannover, Germany; **J. König**, Münster, Germany; **M. Konrad**, Münster, Germany; **M. Lablans**, Heidelberg, Germany; **M. C. Liebau**, Cologne, Germany; **S. Lienkamp**, Freiburg, Germany; **C. Okorn**, Essen, Germany; **H. Omran**, Münster, Germany; **L. Pape**, Hannover, Germany; **P. Pennekamp**, Münster, Germany; **F. Schaefer**, Heidelberg, Germany; **B. Schermer**, Cologne, Germany; **H. Storf**, Frankfurt, Germany; **A. Titieni**, Münster, Germany; **F. Ückert**, Heidelberg, Germany; **S. Weber**, Marburg, Germany; **W. Ziegler**, Hannover, Germany.

### AUTHOR CONTRIBUTIONS

JK, AT, and MK drafted the manuscript. MK is the principle investigator and JK the central coordinator of the described consortium.

### FUNDING

The authors thank GPN for their support. The NEOCYST consortium is funded by the German Federal Ministry of Research and Education (BMBF, grant 01GM1515).

<sup>3</sup>www.neocyst.de.

### REFERENCES


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 König, Titieni, Konrad and the NEOCYST Consortium. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## The KOUNCIL Consortium: From Genetic Defects to Therapeutic Development for Nephronophthisis

Kirsten Y. Renkema<sup>1</sup> \*, Rachel H. Giles <sup>2</sup> , Marc R. Lilien<sup>3</sup> , Philip L. Beales <sup>4</sup> , Ronald Roepman<sup>5</sup> , Machteld M. Oud<sup>5</sup> , Heleen H. Arts <sup>6</sup> and Nine V. A. M. Knoers <sup>1</sup>

<sup>1</sup> Department of Genetics, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands, <sup>2</sup> Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands, <sup>3</sup> Department of Pediatric Nephrology, Wilhelmina Children's Hospital, University Medical Center Utrecht, Utrecht University, Utrecht, Netherlands, <sup>4</sup> UCL Great Ormond Street Institute of Child Health, London, United Kingdom, <sup>5</sup> Department of Genetics, Radboud University Medical Center, Nijmegen, Netherlands, <sup>6</sup> Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada

#### Edited by:

Miriam Schmidts, Radboud University Nijmegen, Netherlands

#### Reviewed by:

Jens Christian König, Department of Pediatrics, University Hospital Würzburg, Germany Andrew Mallett, Royal Brisbane and Women's Hospital, Australia

> \*Correspondence: Kirsten Y. Renkema k.renkema@umcutrecht.nl

#### Specialty section:

This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics

Received: 09 February 2018 Accepted: 18 April 2018 Published: 07 May 2018

#### Citation:

Renkema KY, Giles RH, Lilien MR, Beales PL, Roepman R, Oud MM, Arts HH and Knoers NVAM (2018) The KOUNCIL Consortium: From Genetic Defects to Therapeutic Development for Nephronophthisis. Front. Pediatr. 6:131. doi: 10.3389/fped.2018.00131 Nephronophthisis (NPH) is the most common monogenic cause of renal failure in children. Treatment options are limited to dialysis and transplantation. Therapeutics to significantly delay or prevent end-stage renal disease (ESRD) in children are currently not available. In the Dutch-Anglo KOUNCIL (Kidney-Oriented UNderstanding of correcting CILiopathies) consortium, several groups and specialties united to perform scientific groundwork with the aim to develop genetic and therapeutic personalized care for NPH patients. At the start of this consortium, a genetic diagnosis for NPH was available for only 30–40% of patients, which improved to 50–60% during the course of the 4-year KOUNCIL project. Other major accomplishments of the consortium were (1) the establishment of a Dutch renal ciliopathy patient database with genotype and phenotype data; (2) composition of a proteomics-based integrated network of protein modules disrupted in NPH; (3) the development of non-invasive, urine-based assays that allow functional assessment of genomic variants in NPH and of therapeutic efficiency of drugs; and (4) chemical screening toward the identification of compounds that delay or prevent disease progression in NPH, which resulted in four potential medical interventions for NPH. In conclusion, the KOUNCIL consortium effectively channeled complementary approaches to broaden our understanding of NPH pathogenesis, resulted in 54 publications, improvement of genome diagnostics for NPH patients, awareness in the nephrology and clinical genetics communities for NPH, and new avenues for patient management.

Keywords: cilia, pediatric kidney disease, nephronophthisis, renal ciliopathy, genetics

### INTRODUCTION

Nephronophthisis (NPH; OMIM Phenotypic Series PS256100) is an autosomal recessive, genetically heterogeneous disorder that results in chronic renal disease in children and young adults (1). NPH is initially characterized by polydipsia and polyuria. Progression of the disease is characterized by renal interstitial fibrosis, tubular basement membrane disruption, and—in a subset of cases—renal cyst formation, eventually leading to renal failure. NPH can occur as an isolated disorder but is also often accompanied by a variety of extrarenal manifestations such as in Senior-Løken-, Joubert-, Bardet-Biedl-, Meckel-Gruber-, Jeune-, Short-Rib-Polydactyly-, and Sensenbrenner syndromes. These disorders overlap phenotypically, as well as genetically and functionally. All are thought to result from defective ciliary signaling, and are classified as renal ciliopathies or nephronophthisis-related ciliopathies (NPH-RCs). Although NPH patients are usually diagnosed when renal failure has developed, NPH patients typically experience a 5–10-year period between diagnosis and renal replacement therapy, which offers a therapeutic window of opportunity. Currently, no medical interventions are available for NPH patients. We anticipate that the identified molecular defect in each patient will ultimately steer the development and use of personalized therapies (2).

Gene identification is essential for development of personalized therapies. However, genetic causes of NPH are unknown in ∼50% of cases (3, 4). Twenty genes are known to date to be mutated in NPH. The vast majority of gene mutations results in loss-of-function of proteins that are important for ciliary architecture or regulation of signaling cascades. Cilia are the tail-like microtubule-based protrusions from the plasma membrane that reside on the apical surface of virtually every vertebrate cell, including renal tubular cells. Cilia act as the cell's antenna by sensing the extracellular environment through an array of specific receptors and transmitting a signal response to the cell that reacts upon the signal. Ciliary gene disruption can disrupt cilia-dependent signaling cascades, which may lead to apoptosis and misaligned cell division (5). This results in heterogeneous phenotypes that affect multiple organs, including kidneys (6).

Despite steady progress in gene discovery over the past decade, a significant number of genes mutated in NPH still await identification because a significant proportion of NPH cases remains unexplained. To address this issue and advance NPH research and diagnostics collectively, our research groups united in a Dutch-Anglo consortium, funded by the Dutch Kidney Foundation. In this review, we aim to demonstrate the value of synergistic consortia in obtaining the critical mass required for patient cohort recruitment, while combining complementary expertise to work toward gene identification and targeted therapies for NPH.

### THE KOUNCIL CONSORTIUM

The KOUNCIL (**K**idney-**O**riented **UN**derstanding of correcting **CIL**iopathies) consortium was founded to perform scientific groundwork for the improvement of personalized care for NPH patients. Our consortium augmented the individual expertise of the collaborating workgroups toward reaching the challenging goals. The consortium included research groups with established expertise in renal ciliopathy research and diagnostics, including clinical geneticists, pediatric nephrologists, molecular biologists, cell biologists, and bioinformaticians (**Figure 1**). The international scientific advisory board of the Dutch Kidney Foundation reviewed and supported the consortium, and a tailored internal scientific advisory board reviewed our progress and suggested solutions to problems encountered.

We designed and conducted an integrated work plan aimed to (1) identify novel genes associated with NPH and related disorders by using next generation sequencing (NGS) approaches, (2) gain insights in genotype-phenotype relations through the setup and analysis of a renal ciliopathy database, (3) increase our understanding of the composition and function NPH-related protein modules in the ciliary proteome and explore mutational effects by state-of-the-art proteomics, and (4) initiate the development of targeted therapies to delay or prevent renal failure in NPH patients by drug screening in zebrafish and 3D culturing of cells derived from patients' urine samples.

By bringing together the three university medical centers in our project we maximized patient inclusion. This was an important aspect of our study as NPH and -related disorders are individually rare. Combining our efforts has led to significant conclusions and high impact findings. The interactions within and between the different groups and work packages have been very open and effective, and by connecting different disciplines in the KOUNCIL project, a robust workflow could be implemented in the clinic that results from this synergy. We believe that without the close and successful collaboration within the consortium, the results would have been less optimal.

### RESULTS OF KOUNCIL

### Patient Recruitment, Database Generation, and Diagnostics

The success of research consortia relies heavily on patient numbers, diagnostic accuracy, and deep-phenotyping of patients. In KOUNCIL, we used the AGORA data- and biobank protocols to systematically recruit and include patients (http://www. agoraproject.nl) (7). Furthermore, we involved the professional Society for Dutch Pediatric Nephrologists and requested participation of pediatric nephrologists in recruiting patients for the renal ciliopathy database. Patients were also directly informed via the Dutch Kidney Patient Association and the KOUNCIL website (http://www.kouncil.nl). Clinical data including renal and extrarenal phenotypic information was obtained and manually curated for inclusion in the renal ciliopathy database (**Figure 2**). So far, 88 patients with NPH and -related disorders have been included in our coded database. Inclusion is ongoing and the database will be maintained as a resource during the following years. The database is a valuable source of clinical and genetic information, and is currently being assessed for investigations on genotype-phenotype correlations to improve early diagnosis, prognosis, and genetic counseling. Furthermore, the database offers opportunities to match with other renal ciliopathy databases such as the German language cohort Nephreg (http://www.nephreg.de), and we hope to follow up on that initiative.

We applied NGS approaches to identify causative genetic defects in 50 patients with unexplained renal ciliopathies. These methods include targeted gene panel sequencing and whole exome sequencing that facilitate the parallel analysis of multiple

genes in one sequencing run in a cost- and time-efficient manner. This has led to the identification and confirmation of multiple genes for renal ciliopathy-related diseases: WDR60 (8), WDR34 (9), IFT172 (10), DCDC2 (11), CEP120 (12), ICK (13), EXTL3 (14), and TCTEX1D2 (15). Causal variant identification in new and known genes resulted in a genetic diagnosis for 31 patients from our cohort, who had not received a prior molecular diagnosis. We have implemented renal ciliopathy-specific gene screening in our genome diagnostic divisions and ensure high sensitivity and optimal diagnostic yield by periodically updating available tests.

### Understanding Functional Defects Underlying Renal Ciliopathies

Cilia are regulated by defined protein complexes, some of which act as molecular machines (16). It is estimated that 1,000–2,000 proteins contribute to ciliary architecture or signaling. Through large-scale interaction assays, many of these proteins have been delineated in functional modules that are interconnected. Disrupting ciliary protein-protein interactions interferes with modules regulating cilia function. Because different ciliopathies are characterized by multiple and overlapping phenotypes, we hypothesized that there is also overlap in the molecular defects and protein modules that are disrupted in the different ciliopathies, including eye, brain, and kidney abnormalities (**Figure 3**) (17). Work that was performed prior to KOUNCIL suggested that proteomics-based dissection of molecular modules can relate biological mechanisms to specific genotypes in ciliopathy patients (18). Proof of principle ignited our interest to expand this approach to renal ciliopathies. Although our approach involving state-of-the art quantitative proteomics such as Stable Isotope Labeling with Amino acids in Cell culture (SILAC) combined with mass spectrometry proved technically challenging and involved multiple rounds of troubleshooting and adaptation, we were eventually successful in increasing our understanding of the ciliary protein complexes that are disturbed in NPH and allied ciliopathies (**Figure 4** and unpublished data), which is indispensable for the development of targeted therapies (17, 18). Another lesson learned was that patients remain their own best model of disease, since patient cells such as urine-derived renal epithelial cells (URECs) and fibroblasts delivered the most relevant information about possible pathogenic effects of detected genomic variation and more generally, mechanisms of disease (**Figure 4**) (20). Thus, implementation of the developed non-invasive methods for patient-derived cell investigations has proven to be tremendously useful in diagnostics for renal diseases and therapy development.

### Advancing Therapeutics for NPH

Since there is no targeted cure for NPH, research toward the development of therapies that slow renal disease progression in NPH patients is warranted. We aimed to identify drugs that delay or prevent renal cyst formation, and used zebrafish embryos with pronephric cysts for a high-throughput drug screen to achieve this goal. We first developed a robust zebrafish model for NPH, using both morpholinos and CRISPR/Cas9 gene editing

technologies. A popular drug screening strategy is repurposing, whereby already FDA-approved drugs are investigated for new uses. We hypothesized that such a strategy is likely effective toward the identification of drugs that slow renal failure in NPH as it would allow a fast conversion from drug identification to actual clinical use. As such, we tested a library of FDA-approved compounds for inhibitory effects on renal cystogenesis. Two approaches were taken to screen for effective compounds. First, we performed a screen of 640 drugs in zebrafish embryos with NPH, which was a non-hypothesis driven approach. Secondly,

FIGURE 4 | Defective retrograde transport in urine-derived cells from a Mainzer-Saldino syndrome patient. URECs derived from a patient (referred to as "patient" or "II-3") with compound heterozygous variants in IFT140 (one pathogenic frameshift and one missense variant of uncertain significance), were compared to URECs from three control individuals (C1-3). (A) URECs stained for ciliary marker acetylated-α-tubulin (red) and RPGRIP1L (green). (B) Analysis of retrograde transport in controls, healthy family members (I-1, I-2, and II-2) and patient (II-3) URECs. Cilia were visualized with acetylated-α-tubulin (red) and RPGRIP1L (pink). The cells were analyzed for the presence of IFT88 (green) accumulation at the ciliary tip. Patient II-3 showed a significant tip accumulation of IFT88 in 41% of the cells (Fisher's exact two-tailed test showed a p < 0.0001 when comparing the cells of the patient to those of the controls). Scale bar represents 5µm. (C) A schematic overview of the cilium in controls and in the patient with pathogenic variants in IFT140. The control shows normal intraflagellar transport with the IFT-B complex transporting proteins from the base of the cilium to the tip (axoneme is shown in red and the transition zone marks the base of the cilium in pink) and the IFT-A complex transporting proteins back down to the base. The image shows defective retrograde transport leading to an abnormal bulge at the ciliary tip, which is filled with IFT-B proteins. This image was adapted from Oud et al. (19). https://ciliajournal.biomedcentral.com/articles/10.1186/s13630-018-0055-2.

we extensively examined 14 therapies based on known beneficial effects in polycystic kidney disease (PKD), as a candidate-selected approach. From the second approach, four individual drugs and one drug combination significantly rescued the NPH phenotype in zebrafish embryos with acceptable toxicity profiles. These drugs warrant further in vivo investigations for their potential in modulating NPH-progression.

To further advance therapy development, a major effort in KOUNCIL was the standardization of the use of URECs in molecular and cellular diagnostics (20). The 3D culturing of URECs resulted in clear characteristic growth patterns in NPH patients. This allowed us to adapt a non-invasive drug screening methodology in patient-derived 3D spheroids from urine. Although expansion and validation of the URECs and 3D models is ongoing, we are excited by the potential this patientcentered approach offers.

### ADVANCES FOR NPH-RC PATIENTS AND THEIR FAMILIES

Our project advanced medical care for kidney patients and their families. First, NGS-based methods that were implemented in DNA diagnostics at the start of our project almost doubled the diagnostic yield for NPH-RC. Secondly, NGS-based DNA diagnostics for renal ciliopathies allow clinicians to make the NPH diagnosis at a much earlier stage, which resolves the need for invasive renal biopsies for diagnostic purposes. Third, a solidified work flow has been implemented for renal ciliopathy patients in our dedicated multidisciplinary outpatient clinics for kidney diseases in the Radboud university medical center, Nijmegen, The Netherlands and the University Medical Center Utrecht, Utrecht, The Netherlands. In London, UK, multidisciplinary clinics for Bardet-Biedl Syndrome (BBS) patients are provided. NPH-RC patients are seen by a nephrologist, ophthalmologist, geneticist, dietician, general pediatricians, endocrinologist (diabetes type II), and a clinical psychologist. The aim is to provide a "one stop" visit to ensure that patients receive specialized and expert attention and management. This should result in a major change in how NPH and BBS is managed, with a focus on diagnosis, early intervention and appropriate health management. These multidisciplinary clinics form a role model for other hospitals worldwide and will hopefully be more widely implemented in the near future.

The fourth improvement in patient care results from the setup of a renal ciliopathy database, which increased our insight into the relation between genotypes and phenotypes of NPH-RC patients. In the future, the database can be connected to other databases and converted to an interactive, international online registry. This will in turn allow for more refined insights in genotype-phenotype correlations and prognosis (6). Finally, we have developed a non-invasive protocol for research on renal cells that does not require patients (and their parents) to visit the hospital. In conclusion, KOUNCIL improved opportunities for patient management, led to advancements in the diagnostic trajectory and resulted in optimal genetic counseling for patients and their families.

### SCIENTIFIC IMPACT

KOUNCIL and KOUNCIL collaborations revealed several new renal ciliopathy genes and molecular mechanisms of disease, which significantly improved the diagnostic yield for NPH and related disorders as well as advanced knowledge of ciliopathy pathophysiology. In combination with the phenotypic characteristics sampled in our coded renal ciliopathy database, the power of genotype-phenotype correlations for NPH is just emerging and will require further prospective validation. Continued genetic screening and ongoing deep phenotyping of NPH-RC patients will further dissect these correlations, ultimately improving patient counseling. Understanding the impact of allelic variants from patients on disease manifestations demands understanding of the extensive molecular connectivity of cilium-directed pathways; to this end, we have developed a proteomics-based approach to evaluate the assembly defects of the ciliary transport particles (IFT) in a subset of ciliopathy patients. Although ciliopathies represent a class of rare pediatric diseases, through the work of KOUNCIL, we have found novel insights into the molecular mechanisms of chronic renal disease progression widespread in the general population. Preliminary work suggesting that cilia are key repressors of cellular fibrosis in the kidney, will direct and further streamline future efforts to develop therapeutic intervention with a focus on fibrosis treatment.

It should be noted that development of methodologies independent of animal experimentation are a priority for most scientific bodies today. We have generated a robust protocol growing renal cells from urine that is non-invasive to patients, yet is patient-specific, and is amenable to screening (20). We showed that functional tests in URECs and CRISPR/Cas9-derived knockdown cells can more widely clarify the pathogenicity of genetic variants of unknown significance identified in NPH-RC patients. Growing 3D spheroids from urine ("urinoids") may also be appropriate for variant interpretation and with appropriate quality assurance measures, are potentially adaptable for diagnostic support (21).

### CONCLUSIONS

The KOUNCIL consortium, consisting of pediatric nephrologists, clinical geneticists, molecular biologists and

### REFERENCES


bioinformaticians, provided a unique, coherent inter- and transdisciplinary team to combat juvenile kidney failure. The resulting five PhD theses from three centers in two countries implicated the relevance of our consortium for the next generation of renal clinicians and scientists, and will continue to have a high impact on the renal field.

This consortium aimed to improve the understanding of genetics and biology of renal ciliopathies with innovative NGS and biochemical techniques, and set the first important steps toward therapeutic approaches to delay the progressive degenerative defects in the kidney. The results of this consortium project have already significantly impacted the clinical management of NPH-RC by improvements in diagnosis, prognosis, and accuracy of genetic risk assessment. Our project has provided us with optimal preparations for the development and refinement of future personalized therapies, and is a message of hope for NPH patients for whom there is currently no satisfactory treatment.

### AUTHOR CONTRIBUTIONS

KR, RG, and HA performed the writing of the manuscript; ML, MO, and RR provided figures; RG, PB, RR, HA, and NK were work package leaders in the KOUNCIL consortium. All authors provided feedback on the revised manuscript.

### ACKNOWLEDGMENTS

We thank the patients and parents who participated in the research performed within the KOUNCIL consortium. We also thank the scientific advisory board of the KOUNCIL consortium: Dr. Richard Sandford, Prof. Dr. Christine Mummery, and Prof. Dr. Irene van Langen; and the international scientific advisory board of Dutch Kidney Foundation chaired by Prof. Dr. Jo Berden. The KOUNCIL consortium was financially supported by the Dutch Kidney Foundation under grant agreement number CP11.18. The authors thank Simone Dusseljee for project management and Dr. Arjen Rienks and Dr. Eveline Martens of the Dutch Kidney Foundation for program management. PB is an NIHR Senior Investigator.

diagnosis of nephronophthisis-related ciliopathy. Exp Mol Med. (2016) **48**:e251. doi: 10.1038/emm.2016.63


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The handling Editor declared a shared affiliation, though no other collaboration, with two of the authors: RR and MO.

Copyright © 2018 Renkema, Giles, Lilien, Beales, Roepman, Oud, Arts and Knoers. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Congenital Heart Defects and Ciliopathies Associated With Renal Phenotypes

#### George C. Gabriel <sup>1</sup> , Gregory J. Pazour <sup>2</sup> and Cecilia W. Lo<sup>1</sup> \*

<sup>1</sup> Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA, United States, <sup>2</sup> Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, United States

#### Edited by:

Miriam Schmidts, Radboud University Nijmegen, Netherlands

#### Reviewed by:

Kathryn Hentges, University of Manchester, United Kingdom Claudio Cortes, UMR7288 Institut de Biologie du Développement de Marseille (IBDM), France

> \*Correspondence: Cecilia W. Lo cel36@pitt.edu

#### Specialty section:

This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics

Received: 29 December 2017 Accepted: 29 May 2018 Published: 15 June 2018

#### Citation:

Gabriel GC, Pazour GJ and Lo CW (2018) Congenital Heart Defects and Ciliopathies Associated With Renal Phenotypes. Front. Pediatr. 6:175. doi: 10.3389/fped.2018.00175 Congenital heart disease (CHD) is one of the most common birth defects, and recent studies indicate cilia-related mutations play a central role in the genetic etiology of CHD. As cilia are also known to have important roles in kidney development and disease, it is not surprising that renal anomalies were found to be enriched among CHD mutant mice recovered in a large-scale mouse forward genetic screen. Indeed 42% of mutations identified to cause both CHD and renal anomalies were cilia-related. Many of these cilia mutations comprise cilia transition zone or inversin compartment components, consistent with the known role of these cilia proteins in a wide variety of ciliopathies. The high prevalence of CHD with congenital anomalies of the kidney and urinary tract (CAKUT) observed in mice was also corroborated with clinical studies that showed 20–30% of CHD patients have renal anomalies. Together these findings suggest CHD patients may benefit from early screening for renal anomalies to allow early diagnosis and intervention to improve outcome for this vulnerable patient population.

Keywords: cilia, CAKUT, congenital heart disease, congenital abnormalities, ciliopathies, genetic syndromes

Congenital heart disease (CHD) is the most common birth defect, occurring in up to 1% of live births (1). Interestingly, CHD often presents in combination with extra cardiac defects including congenital anomalies of the kidney and urinary tract (CAKUT) (2–4). In fact, renal or urinary system defects are associated with 23.1% of congenital heart defects (5). These findings suggest an overlapping genetic etiology for CHD and CAKUT. Consistent with this is the fact that several previously reported mouse models (not individually discussed in this review) have found genetic disruption of a single gene can affect both heart and kidney development, and that many genetic syndromes can present with both cardiac and renal anomalies (6–9). Hence, insights into the genetic etiology of CHD may help elucidate the etiology of CAKUT. Relevant to this is the recent unexpected finding from a large-scale forward genetic screen in mice indicating a significant role for cilia-related mutations in the pathogenesis of CHD (10). As cilia mutations are well-described in the context of CAKUT, this would suggest cilia defects may have a central role in mediating both CHD and CAKUT phenotypes. Below, we briefly discuss CAKUT and its association with CHD, and its shared genetic etiology involving cilia-related mutations.

### ASSOCIATION OF HEART AND KIDNEY PHENOTYPES IN GENETIC SYNDROMES

CAKUT represent a broad range of kidney and urinary tract defects. This can include abnormalities in the shape, size, or structure of the kidney including kidney agenesis, kidney hypoplasia or dysplasia, horseshoe/fused kidney, cystic kidneys, or duplex/multiplex kidney, and multiple collecting ducts or ureters (11). These defects can occur in combination such as with the finding of cystic dysplastic kidneys. These structural abnormalities can result in vesicoureteral reflux, ureteropelvic junction obstruction, hydroureter, and hydronephrosis (10, 12). In humans, monogenic mutations in several genes have been implicated in CAKUT development; however, these mutations explain only 5–20% of patient disease (13). Additional causes of CAKUT phenotypes include genetic syndromes, which can present with a spectrum of phenotypes often involving defects in many organs including both the heart and the kidney.

In DiGeorge syndrome, heterozygous deletion of chromosome 22q11.2 is associated with multiple organ defects, including the heart, the kidney, the thymus, and the nervous system. It has an estimated prevalence of 1:3,000 to 1:4,000 live births, though prenatal chromosomal analysis suggests this may be an underestimate, and affected patients characteristically present with CHD, thymus hypoplasia or agenesis, and craniofacial defects (14–16). Approximately 20–30% of DiGeorge syndrome patients also exhibit CAKUT (17). Haploinsufficiency of TBX1, a transcription factor within the T-box family, has been shown to contribute to the cardiac phenotypes associated with this disorder (18, 19). More recently, another gene within the 22q11.2 locus, CRKL, an adaptor protein that regulates tyrosine kinase signaling, was found to contribute to developmental kidney defects in the context of DiGeorge syndrome (20, 21).

Another syndrome known as VACTERL association also presents with both congenital heart defects and renal abnormalities. Occurring in 1:10,000 to 1:40,000 live births, VACTERL association is a birth defect associated with at least 3 of the following phenotypes: vertebral defects, anal atresia, cardiac defects, trachea-esophageal fistula, renal defects, and limb defects (22). In a large study of patients diagnosed with VACTERL association, 80% exhibited kidney defects and 48% exhibited cardiac defects (23). In humans, several genes have been related to VACTERL association including FGF8, FOXF1, HOXD13, LPP, TRAP1, and ZIC3, all of which have been implicated in kidney developmental anomalies, with ZIC3 being an X-linked gene also shown to cause heterotaxy (24).

A rarer genetic disorder known as Fraser syndrome can also cause both CHD and genitourinary abnormalities. It is a recessive disorder with a prevalence of only 1 in 200,000 (25). Patients with Fraser syndrome can present with eye defects, syndactyly, kidney defects, most commonly kidney agenesis, and heart defects (25). Fraser syndrome is associated with mutations in FRAS1, FREM2, or GRIP1, all of which are known to interact to form a FRAS/FREM complex that is localized to the basement membrane (26, 27). Mutations in these genes cause loss of the FRAS/FREM complex that likely drives the disease phenotypes observed in Fraser syndrome, although the precise disease mechanism remains unclear (27, 28).

### CILIA AND THE GENETIC LANDSCAPE OF CHD

The close association of renal anomalies with CHD suggests insights into the genetics of CHD can yield significant insights into the mechanism of disease pathogenesis for CAKUT. Interestingly, the pursuit of a large-scale forward genetic screen in mice for mutations causing CHD yielded a large preponderance of mutations in cilia-related genes (29). From screening more than 100,000 ethylnitrosourea (ENU) mutagenized mice using in utero fetal ultrasound phenotyping, we recovered over 200 mouse lines with a wide spectrum of CHD. Using whole exome sequencing nearly 100 CHDcausing mutations were recovered in 61 genes, 34 (55.7%) of which were cilia-related (29). As this is a phenotype driven mutagenesis screen in which fetal echocardiography was used to identify mutants with CHD, these findings point to cilia as playing a central role in the pathogenesis of CHD. This is corroborated with the completion of the screen, showing over 50 genes are cilia-related among 100 genes recovered causing CHD. Moreover, analysis of de novo mutations identified in whole exome sequencing analysis from CHD patients also identified similar mutations contributing to CHD pathogenesis (30).

### CILIA AND CILIOPATHIES IN CHD, RENAL ANOMALIES, AND OTHER HUMAN DISEASES

Cilia are microtubule-based organelles that have well-described compartments including an axoneme that projects from the apical cell surface, and a basal body base with an overlying transition zone and inversin compartment that regulate protein trafficking into and out of the cilium (**Figure 1**) (32). Cilia can


<sup>a</sup> Gene with dark highlighting are ciliary components based on proteomic and other studies.

Gene names followed by \* indicate mutations that cause situs defects.

Parenthesis indicate the number of mutants analyzed.

Adapted from San Agustin et al. (10).

be motile with the expression of motor dyneins that drive ciliary motility, or nonmotile, termed primary cilia, which extend from almost all cell types in the body, including cells of the developing heart, and are known to serve important cell signaling functions by mediating various signal transduction processes (33–35). Motile cilia are required for left-right patterning as well as in mediating sperm motility, mucociliary clearance in the airway, and also cerebral spinal fluid flow in the brain. Importantly, it is now appreciated that cilia mutations can cause a wide spectrum of human diseases collectively known as ciliopathies (31). Many of these ciliopathies can present with renal anomalies, such as in nephronepthesis, Meckel-Gruber syndrome (MKS), Bardet-Biedl syndrome (BBS), Joubert syndrome, Alstrom syndrome and polycystic kidney disease among others (36). The renal defects observed may include CAKUT, polycystic kidney disease or progressive renal dysfunction. Other structural birth defects observed in the ciliopathies include CHD, cardiomyopathy, skeletal malformations, brain abnormalities, blindness, and obesity (31).

Many of the mutations known to cause renal anomalies within the ciliopathy spectrum are associated with proteins localized in the cilia transition zone or inversin compartment. Thus, among the 61 genes recovered causing CHD, half (N = 34) were cilia-related, including 11 affecting transition zone or inversin compartment components (29). While these are mainly thought to disrupt primary cilia function, recent studies showed ciliopathies generally considered to involve primary cilia defects may also impact motile cilia function. This is indicated by the observation of airway clearance defects in patients diagnosed with ciliopathies such as Leber congenital amaurosis or Sensenbrenner syndrome (37, 38). This is not unexpected given over 70% of proteins required for primary cilia function are also expressed in motile cilia (39). Indeed, airway clearance defects are the hallmark of a ciliopathy known as primary ciliary dyskinesia (PCD).

PCD patients have severe sinopulmonary disease and typically this is associated with mutations in genes required for motile cilia function, such as motor dyneins (40–42). It is interesting to note some isolated reports of bronchiectasis associated with polycystic kidney disease, although the connection with airway clearance defects is unknown (43). PCD patients also can exhibit laterality defects, a reflection of the requirement for

FIGURE 3 | Mutations in interacting proteins cause a similar spectrum of both heart and kidney phenotypes. Mutations in four interacting proteins, Anks6, Bicc1, Nek8, and Wwtr1 cause similar phenotypes including both congenital heart disease and cystic kidney disease (Unpublished data).

#### TABLE 2 | Clinical characteristics of congenital heart disease patients.


\*D-TGA, D-transposition of the great arteries; DORV, double outlet right ventricle; ASD, atrial septal defect; VSD, ventricular septal defect; HLHS, hypoplastic left heart syndrome; TOF, Tetralogy of Fallot; AVSD, atrioventricular septal defect; PA, pulmonary atresia; TAPVR, total anomalous pulmonary venous return. Adapted from San Agustin et al. (10).

motile cilia in the specification of the left-right body axis (44, 45). Consistent with this, PCD patients can display a spectrum of phenotypes including situs solitus (normal visceral organ situs), situs inversus totalis also known as Kartagener's syndrome (reversed visceral organ situs), and heterotaxy, or the randomization of visceral organ situs. In this context, CHD is often associated with heterotaxy as a result of defects in motile cilia function.

Interestingly, some ciliopathies thought to affect only primary cilia function also can be associated with laterality defects, one prominent example being MKS, a ciliopathy that causes severe birth defects and is usually prenatal or neonatal lethal. Studies of Mks1 mutant mice showed defects involving not only the primary cilia, but also laterality defects associated with the loss of motile cilia function in the embryonic node (46). Together, these findings suggest that the distinction of ciliopathies as either affecting motile or primary cilia can be problematic, given the promiscuity of cilia genes in having functional roles in both primary and motile cilia.

Intriguingly, primary cilia proteins can be found in other cellular locations, suggesting that they may have other nonciliary functions (47). In fact, ciliary proteins are also involved in a wide range of cellular processes including cell cycle progression, cytoskeletal organization, and vesicular trafficking (48–50). Further, cilia themselves are involved in several cell signaling pathways including the sonic hedgehog, TGFβ, wnt, and notch pathways, and defects can arise in these pathways through defective cilia or pathway components outside of the cilium (51). Thus, future research will be important to determine the precise roles cilia and cilia associated proteins play in CHD and CAKUT development.

### MUTATIONS CAUSING CHD AND CAKUT PHENOTYPES

Extra-cardiac phenotypes, including renal anomalies, were recovered in many CHD mutant mice even though the screen was entirely focused on CHD phenotyping. This included a spectrum of kidney phenotypes including duplex kidney, multiplex kidney, hydronephrosis, kidney agenesis, and cystic kidney (**Figure 2**), with the most commonly observed renal phenotype being duplex kidneys (10). Among the 39 mutant mouse lines recovered with congenital kidney abnormalities, the most common CHD phenotype is double outlet right ventricle (DORV), occurring in 19/39 lines (48.7%). This is followed by atrioventricular septal defect, which occurred in 15/39 lines (38.5%; **Table 1**). Other CHD phenotypes associated with kidney defects included transposition of the great arteries (TGA), persistent truncus arteriosus (PTA), ventricular septal defect (VSD), aortic arch anomalies, and biventricular hypertrophy (**Table 1**).

Previous attempts to recover CAKUT genes have been confounded by incomplete penetrance and the probable involvement of more complex genetics. The few CAKUT genes previously identified are largely those that mediate the early steps in kidney development and patterning. Other than Pax2, which causes renal coloboma syndrome, and Hnf1b, which causes renal cystic disease and diabetes, most genes identified to cause CAKUT are associated with disease in only a few patients and hence, the evidence supporting pathogenesis is limited (52). Among the 135 CHD mouse lines recovered, 39 (28.8%) had kidney defects caused by mutations in 26 genes, 11 (42%) being known cilia genes (**Table 1**, **Figure 1**) (10). Remarkably, this included four cilia genes that encoded known direct proteinprotein interactors: Anks6, Nek8, Bicc1, and Wwtr1 (53–56). These mutations were found in four independent CHD mouse lines, with mutation in each gene causing a similar spectrum of kidney and heart defect phenotypes comprising double outlet right ventricle and cystic kidney disease (**Figure 3**).

### CLINICAL STUDY SHOWING ASSOCIATION OF CHD WITH CAKUT PHENOTYPES

The relevance of CAKUT abnormalities in CHD was confirmed with a clinical study of 77 CHD patients recruited from the Children's Hospital of Pittsburgh. Retrospective review of the medical records of these 77 patients revealed 23 (30%) also had some form of renal defect such as renal cysts, kidney agenesis, cystic dysplastic kidneys, and horseshoe kidney (**Figure 4**, **Table 2**) (10). These findings are in agreement with a previous epidemiological study in the Atlanta metro area, which showed 23% of 8,000 subjects with CHD also had renal abnormalities (1). Thus, both the mouse and human studies point to a significant overlap in the genetic etiology of CHD and kidney abnormalities. This probably reflects the conservation of developmental pathways and cell signaling mechanisms that regulate cardiovascular and renal development, including a central role for cilia in the pathogenesis of CHD and renal birth defects. Together these findings suggest the routine evaluation of CHD patients for renal anomalies with simple non-invasive renal ultrasound may be warranted. This may allow early diagnosis and early therapeutic intervention to treat and manage any renal dysfunction that could negatively impact the long-term outcome of this high-risk patient population.

### REFERENCES


### CONCLUSION

Studies in both CHD mutant mice and CHD patients showed CAKUT is highly associated with CHD. This is supported by the finding of CAKUT and CHD in various genetic syndromes, and the finding that many mutations identified to cause CHD can also cause CAKUT phenotypes. Among genes identified to cause both CHD and CAKUT there is a significant enrichment of cilia genes, indicating a central role for cilia in the pathogenesis of CHD and CAKUT phenotypes. However, as half of the mutations identified to cause CHD and renal anomalies are not known to be cilia-related, the association of CHD with renal anomalies likely extend beyond the role of cilia in heart and kidney development. The clinical relevance of these findings in mice were shown with the observation that 23–30% of CHD patients also exhibited renal anomalies. This would suggest CHD patients may benefit from routine evaluation for renal anomalies. This might improve the long-term outcome of this high-risk patient population by reducing potential renal complications with early diagnosis and therapeutic intervention.

### AUTHOR CONTRIBUTIONS

All authors listed contributed intellectually to this work and approved it for publication.

### ACKNOWLEDGMENTS

This work was supported by NIH grants HL098180, 1S10 OD010340, HL132024, and GM104412 to CL and GM060992 and DK103632 to GP.


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Gabriel, Pazour and Lo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Cystic Kidney Diseases From the Adult Nephrologist's Point of View

#### *Roman-Ulrich Müller\* and Thomas Benzing*

*Department II of Internal Medicine and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany*

Cystic kidney diseases affect patients of all age groups with the onset spanning from prenatal disease to late adulthood. Autosomal-dominant polycystic kidney disease (ADPKD) is by far the most common renal cystic disease. However, there are various cystic kidney diseases, the onset of which occurs at different times in life and depends on the type of the disease and the causative genes involved. When genetic kidney diseases are discussed in the adult setting this view is usually limited on autosomal-dominant kidney disease, the most frequent genetic disorder causing adult onset ESRD. Other diseases—such as autosomal-recessive polycystic kidney disease—are often being viewed as a disorder only important in pediatric nephrology. However, more recent data has revealed that, despite clear age peaks of onset for each disorder, all of them can also show highly variable phenotypes with classical adult onset genetic diseases being of importance in pediatrics and *vice versa*. Furthermore, the affected children need to be seen by adult nephrologists in the long term after transition, requiring knowledge on the underlying pediatric disease, potential extrarenal manifestations, and genetic counseling. Consequently, the view on these diseases should be widened on both ends. Close interaction between pediatric and adult nephrology is key to appropriate care of patients

suffering from genetic kidney disease to profit from each other's experience. Keywords: polycystic kidney diseases, autosomal dominant polycystic kidney disease, autosomal-recessive polycystic kidney disease, tuberous sclerosis complex, von Hippel–Lindau disease, nephronophthisis, genetic

## INTRODUCTION

kidney disease, Birt–Hogg–Dubé syndrome

While pediatric nephrologists are rather experienced in managing patients with genetic kidney diseases, most adult nephrologists are faced with congenital disease less frequently (1, 2). This is mainly due to the reason that genetic disorders are the minority of causes of end-stage renal disease in the adult population whereas in children they are common. Autosomal-dominant polycystic kidney disease (ADPKD) reveals most of its burden in adulthood and has long been considered to be the only cystic kidney disease in adults. Other cystic kidney diseases—primarily nephronophthisis (NPH) and autosomal-recessive polycystic kidney disease (ARPKD)—start much earlier and are entities that have primarily been studied and treated by pediatric nephrologists. However, with the advent of the first targeted treatment strategies—such as tolvaptan in ADPKD patients—the question arises whether early detection of disease and an early commencement of a potential therapy may be beneficial.

Genetic causes of kidney disease are on the brink to become much more of a visible problem in adult nephrology. This is not limited to cystic kidney diseases, but also true for other disorders, such as the MCD-FSGS spectrum. On the one hand, an increasing number of children suffering from kidney disease reach adulthood raising the question of transition and continued care in the adult nephrology setting. On the other hand, the advent of novel sequencing technologies has paved the way for elucidating more complex genetic factors in kidney disease and will allow for novel associations

#### *Edited by:*

*Miriam Schmidts, Radboud University Nijmegen, Netherlands*

#### *Reviewed by:*

*Andrew Mallett, Royal Brisbane and Women's Hospital, Australia Cynthia J. Willey, University of Rhode Island, United States Francois Jouret, University of Liège, Belgium*

#### *\*Correspondence:*

*Roman-Ulrich Müller roman-ulrich.mueller@uk-koeln.de*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 03 December 2017 Accepted: 05 March 2018 Published: 22 March 2018*

#### *Citation:*

*Müller R-U and Benzing T (2018) Cystic Kidney Diseases From the Adult Nephrologist's Point of View. Front. Pediatr. 6:65. doi: 10.3389/fped.2018.00065*

**171**

between the genetic background and pathogenic aspects. Clearly, chronic kidney disease in general in adulthood has an important genetic component. However, many of the genetic alterations are not simply monogenetic causes, but must rather be considered as complex genetic diseases. Consequently, knowledge on genetic diseases of the kidney will play an increasingly important role in adult nephrology and profit from a close interaction with colleagues from pediatric nephrology. Here, we will use the example of cystic kidney diseases—and ADPKD specifically—to point out the most important aspects and advances in the care of adult patients.

### CYSTIC KIDNEY DISEASE—A CILIOPATHY

The introduction of next-generation sequencing techniques in the last decade—starting in basic research and being currently introduced in clinical diagnostics—made the large-scale analysis of potential disease-causing mutations in patients with cystic kidney diseases feasible (3–6). This has led to the identification of numerous novel disease-causing genes enabling the discovery of more than 100 genes to be involved in cystic kidney diseases (7). Nonetheless, a significant proportion of cases remains unsolved which will require further research in well-characterized families and cohorts (3, 5). Despite this enormous genetic complexity of cystic kidney diseases, they are unified by a single pathophysiological concept. Nearly all protein products of disease-causing genes identified so far have been linked to the generation or function of the primary cilium—a membranous protrusion of the apical cell membrane of close to all cell types that is supported by a microtubular skeleton (7). Primary cilia may function as chemo- and mechanoreceptors and ciliary dysfunction which initiates numerous signaling aberrations in epithelial kidney cells—like increased cAMP generation or mTOR-signaling—that are crucial to cyst formation (8). This has led to the definition of the cystic disease complex as a ciliopathy. The fact that pretty much all tissues in the human body contain ciliated cells explains why ciliary defects can be the basis to complex syndromes affecting different organs—e.g., the combination of cystic kidneys with *situs inversus* and retinitis pigmentosa observed in several NPH-associated syndromes (9).

### ADPKD—EPIDEMIOLOGY

Autosomal-dominant polycystic kidney disease is the most common entity among cystic kidney diseases (10). Its lifetime morbid risk is estimated to be around 1:1,000 while point prevalence which rather reflects clinically relevant disease—has recently been shown to be approximately 1:2,500 (11–14). The frequency of ADPKD is thus comparable to much more commonly known disorders, such as cystic fibrosis or multiple sclerosis. Consequently, it is also the most frequent genetic cause of end-stage renal disease in adults with a prevalence of 5–10% in the dialysis population (15). However, a decline in renal function is rarely observed in childhood leading to ADPKD being primarily viewed as a disease of adult patients. Nonetheless, with the advent of novel targeted therapeutic strategies—e.g., tolvaptan and somatostatin-analogs—and seeing that a subpopulation of children with ADPKD suffer from clinically meaningful hypertension (16), ADPKD has gained increasing attention among pediatric nephrologists—a fact that is reflected by the initiation of a pediatric ADPKD registry in 2017 (www.adpedkd.org) that has successfully started recruiting patients. Nonetheless, the predominant time at which ADPKD gets manifest is above the age of 30 years and ESRD is reached at an average age of 50–60 years (17). Consequently, while there is no clear consensus of how to care for children of affected parents, the period in life during which interventions are necessary is primarily adulthood for now. Obviously, this may change in the future, e.g., with upcoming clinical trials on drugs such as the V2 receptor antagonist tolvaptan in pediatric cohorts (18).

## DIFFERENTIAL DIAGNOSIS OF ADPKD

Due to its typical clinical manifestation, the diagnosis of ADPKD can be made solely on clinical grounds in the vast majority of affected patients. This is primarily based on three considerations: (1) morphology of the kidneys, (2) extrarenal manifestations, and (3) mode of inheritance.

Using these criteria ADPKD can—in most cases—clearly be separated from the rarer causes of cystic kidney diseases in adulthood. This is very important, since—even though ADPKD is by far the most common diagnosis—distinguishing other disorders, including autosomal-dominant tubulointerstitial disease (ADTKD), NPH, and ARPKD and a clear separation from simple renal cysts is crucial to make the correct diagnosis. This has become even more important with targeted therapies getting available for particular disorders.

### Kidney Morphology

With the morphology of the kidneys being the key to diagnosing or excluding ADPKD in individuals at risk, imaging modalities are highly important screening tools. As to this point, several different key factors have to be taken into consideration: (1) kidney size, (2) number of cysts, (3) unilateral/bilateral disease, and (4) distribution of the cysts.

(1) Autosomal-dominant polycystic kidney disease generally goes along with enlarged kidneys. Since the disease is gradually progressing during lifetime, size has to be considered in an agedependent fashion. The correlation of age-adjusted kidney size with disease progression allows for using the information obtained by imaging to—beyond making a diagnosis—help guiding treatment decisions using, e.g., MRI volumetry (19) (**Figure 1**). Large polycystic kidneys are also found in ARPKD as well as the much less common tumor-associated syndromes (tuberous sclerosis, Von Hippel–Lindau disease, Birt–Hogg–Dubé syndrome) and *HNF1b*associated kidney disease (20–22). In contrast to this, diseases of the NPH complex are characterized by small (to normal) sized kidneys. (2) Ravine et al. were the first to come up with a classification based on cyst numbers that allowed for diagnosing ADPKD in individuals with a positive family history using ultrasonography more than 20 years ago (23). Since then this approach has been revised twice in order to account for the advances in imaging technologies that strongly increased sensitivity of cyst detection accompanied with decreasing positive predictive values (24, 25). York Pei and colleagues validated novel criteria in a well-designed

polycystic kidney disease (ADPKD). These images reveal the classical features of ADPKD: strongly enlarged kidneys showing a ubiquitous distribution of cysts throughout the parenchyma. Kidney volume—an important prognostic feature now used in algorithm for making treatment decisions—can be obtained by planimetry (A,B) as done in the clinical trials. However, for everyday clinical decisions volumetry based on measuring the axes and using the ellipsoid formula has been shown to be sufficient (19) (C,D). From Müller et al. (27) (images kindly provided by Dr. Thorsten Persigehl, Institute of Radiology, University of Cologne).

study in 2015 that now allows for clear detection of ADPKD by ultrasound from the age of 30 onwards. Interestingly, all affected individuals showed more than ten cysts when measured by MRI clearly distinguishing them from unaffected individuals. This held true already from the age of 16 years providing data that imagingbased exclusion of disease may be possible starting in adolescence (25). One caveat regarding the diagnosis of ADPKD based on cyst number is the fact that these approaches have only been validated in patients showing a positive family history. (3) However, there is still more conclusions that can be drawn from kidney morphology in cystic kidney diseases. First, based on the fact that these are genetic diseases that go along with the same genetic alteration in all kidney cells, bilateral disease is to be expected. There is few and very rare exceptions to this rule due to potential genetic somatic mosaicism (26). (4) In ADPKD, the bilateral cysts are generally distributed ubiquitously throughout the parenchyma, since cyst formation occurs along the entire nephron (**Figure 1**). This leads to an entirely different pattern compared to NPH, where the cysts are strictly localized at the corticomedullary junction. In ARPKD, cyst formation occurs primarily in the collecting duct, however, the imaging findings can be similar to ADPKD. The same holds true for the tumor syndromes mentioned above and *HNF1b*-associated disease that can all phenocopy ADPKD as to kidney morphology. Here, apart from the fact that—due to its frequency—ADPKD is clearly the most likely diagnosis, extrarenal findings help to distinguish the different entities.

### Renal Manifestations Beyond Cysts

As to the kidneys themselves, ADPKD is not only characterized by renal cysts but also includes kidney stones, flank pain, cyst infections, and macrohematuria. While these manifestations are common in adult patients and may lead to the diagnosis especially in cases with a negative family history—they are rare in childhood. Importantly, early onset of these symptoms may indicate rapid progression of disease as pointed out by the data from the French GENKYST cohort (PROPKD score, see section on "Targeted therapies") (28).

### Extrarenal Manifestations

As explained above cystic kidney diseases are one of the best characterized ciliopathies. Knowing that cilia are present on nearly all cells of the human body and are expected to play important roles in cellular polarity signaling and cell proliferation it is not surprising that the clinical manifestation of a ciliopathy is normally not limited to one organ, but causes a multitude of syndromic clinical pictures. Knowledge of the extrarenal manifestations is crucial to patient care, since they do not only explain symptoms but may also require specific interventions/diagnostic strategies—e.g., in the case of intracranial aneurysms in ADPKD. As to diagnosing a specific cystic kidney disease these manifestations are very helpful, especially in cases where the decision is not clear based on kidney morphology. Hence, a complete workup of these aspects by patient history and clinical examination is essential when seeing PKD patients.

### Extrarenal Manifestations in ADPKD

The most common extrarenal finding in ADPKD patients is extrarenal cysts with the majority of patients also showing liver cysts (and less frequently in other organs, such as the seminal vesicles, pancreas, or spleen). Furthermore, diverticulosis is a common manifestation. As to cardiac manifestations cardiac valve abnormalities primarily mitral valve prolaps and insufficiency—are the most common findings, a fact that makes echocardiography a useful tool in the workup of these patients. Abdominal and inguinal hernias are also associated with ADPKD and are probably a consequence of both an altered strength of the abdominal wall and the increased intra-abdominal pressure due to enlarged kidneys and liver. An aspect that may be especially worrisome for ADPKD patients is the increased occurrence of intracranial aneurysms. Here, a tailored screening strategy in high-risk patients (e.g., those with a positive family history) as well as an interdisciplinary workup (neurosurgeons, neuroradiologists, nephrologists) are crucial—especially when taking into account that interventions to close these aneurysms can also lead to significant morbidity and mortality (29, 30). A more complete workup of potential extrarenal associations with ADPKD and their management can be found in the recent literature (10, 31, 32).

### Extrarenal Manifestations of Tumor Syndromes Associated With Polycystic Kidneys

In contrast to ADPKD, the tumor syndromes—associated with polycystic kidneys that often resemble ADPKD—tuberous sclerosis, BHD syndrome, and VHL syndrome—all go along with a significantly increased risk of kidney cancer (clear-cell renal cell carcinoma in VHL and TSC; chromophobe renal cell carcinoma in BHD). Furthermore, all of these syndromes have characteristic extrarenal manifestations—TSC: e.g., giant-cell astrocytomas and other CNS tumors, renal angiomyolipomas, pulmonary lymphangioleiomyomatosis, cardiac rhabdomyomas, and multiple dermatological signs (facial angiofibromas, periungual fibromas, ash leaf spots, Shagreen patches, etc.); BHD: recurrent pneumothoraces and fibrofolliculomas; VHL: e.g., CNS/retinal hemangioblastomas, pancreatic neuroendocrine tumors, and pheochromocytoma (33). All of these can and should be used for differential diagnosis. While VHL and BHD patients often do not show any symptoms before late adolescence or adulthood, TSC—especially due to the CNS and cardiac manifestations—is frequently already highly symptomatic in early childhood. Nonetheless, there are a considerable proportion of TSC patients that are not diagnosed before adulthood and present with an oligosymptomatic course that, e.g., is mainly characterized by multiple renal angiomyolipomas.

### Extrarenal Manifestations in ARPKD and HNF1B-Associated Kidney Disease

Another disease that can phenocopy the renal phenotype of ADPKD patients is ARPKD. However, the spectrum of extrarenal manifestations differs between these two entities. While in childhood pulmonary hypoplasia is one of the most dramatic findings, patients reaching adulthood without having been diagnosed do generally not show pulmonary symptoms. However, ARPKD has an obligatory association with liver fibrosis (in combination with dilated bile ducts—Caroli syndrome). Consequently, these patients need a workup, including liver imaging (sonography, fibroscan) and gastroscopy (screening for varices). Combined kidney-liver transplantation always needs to be considered when reaching end-stage renal disease (34).

HNF1b is a key transcription factor influencing numerous renal and extrarenal disease genes. Consequently, its mutation can lead to a range of symptoms. As to extrarenal manifestationassociated disorders, which include elevated liver enzymes, MODY diabetes mellitus, pancreatic insufficiency, hypomagnesemia, hyperparathyroidism, gout, and mental retardation (22).

### Extrarenal Manifestations in Diseases of the NPH Complex

Even though diseases of the NPH complex are much less common in the adult population knowledge about their characteristics is important for identifying individuals affected by non-ADPKD cystic kidney diseases. Due to the nature of these disorders as a ciliopathy a wide range of affected tissues and symptoms are possible. In the following, we will summarize the most common examples that may sometimes not be diagnosed before reaching adulthood (9). Importantly, the attribution of a disorder to the NPH complex is—due to genetic complexity—not primarily guided by genetic diagnostics, but by the clinical characterization of the syndromic pattern of manifestations. The most frequent extrarenal finding in NPH patients is retinitis pigmentosa, which is characterized by night blindness followed by tunnel vision and eventually blindness. The combination of NPH and retinitis pigmentosa is classified as Senior–Løken syndrome. Furthermore—based on the role of cilia at the primary node during embryonal determination of laterality—many patients with NPH-associated syndromes show *situs inversus* as a characteristic finding. Another entity to be specifically named here is Bardet– Biedl syndrome which is characterized by polydactyly, juvenile obesity (often accompanied by diabetes mellitus), mental retardation of different degrees, retinitis pigmentosa, anosmia, and hypogonadism. Interestingly, Bardet–Biedl patients often show large kidneys at birth. While kidney size in the majority returns to normal by the age of 1–2 years, renal phenotypes that resemble ADPKD have been described in adult patients. Most other diseases of the NPH complex are nearly exclusively diagnosed in early childhood, such as Joubert syndrome (characterized by cerebellar vermis hypoplasia) or result in perinatal lethality as for Meckel–Gruber syndrome (20).

### The Role of Genetic Diagnostics

Using the clinical criteria mentioned above, the diagnosis can be made correctly in the vast majority of patients without requiring a molecular genetics workup (20, 25, 35). In NPH, there is only very limited phenotype–genotype correlation which makes targeted genetics difficult. However, since *NPHP1* is the most commonly affected gene, testing for alterations in *NPHP1* may be of diagnostic utility (36, 37). This may change in the future with always cheaper and easier access to next-generation sequencing panels. ADPKD is—in patients with a positive family history—diagnosed almost exclusively on clinical grounds. However, molecular genetics may play a major important role in these patients for predicting disease progression (which may be a piece of the puzzle for making therapeutic decisions) in the future (28). The tumor syndromes on the other hand require different considerations when discussing molecular genetics. Here, a clear diagnosis based on the mutation is essential to distinguish these entities from ADPKD which then allows for the design of screening strategies for individual patients and prognostic testing in family members. An overview of a diagnostic strategy in cystic kidney diseases including clinical and genetic considerations is illustrated in (**Figure 2**). Importantly, current clinical diagnostic algorithms may require adaptation in the future due to novel culprit genes and associated disease entities identified—e.g., *GANAB* recently described as a new gene in ADPKD (5). Obviously, diseases not expected in adulthood—e.g., ARPKD and rare disorders that go along with variable clinical manifestations like HNF1b-associated kidney disease—may also require a molecular genetic diagnosis.

### CLINICAL TRIALS TO PREVENT DISEASE PROGRESSION OF CYSTIC KIDNEY DISEASE

### Supportive Measures

Knowledge of the various manifestations is key to the therapy of cystic kidney diseases in general. ESRD can obviously be addressed by renal transplantation in all entities (with a combined liver transplantation to be considered in ARPKD patients). Yet, data from randomized trials as well as a targeted therapy are only available for ADPKD. Until the year 2015 merely

supportive measures were available. The most important recommendations to all of the affected individuals are summarized in **Table 1**, however, the degree of evidence is limited for some of these recommendations. As an example, sufficient fluid intake is mainly based on pathophysiological considerations regarding ADH-suppression (38–40). Here, a recently initiated randomized trial will hopefully clarify the impact of fluid intake (41). On the contrary, there is solid data regarding blood pressure control which was recently emphasized again by the randomized HALT-PKD trials (42). Study arm A showed that lowering blood pressure to 95/60–110/70 mmHg compared to 120/70–130/80 mmHg

BBS, Bardet–Biedl syndrome; JBTS, Joubert syndrome; SLSN, Senior–Loken syndrome; MKS, Meckel–Gruber syndrome.

using an ACE-inhibitor or an AT1-blocker leads to a significant decrease in the kidney growth rate (42) in young patients with preserved kidney function (15–49 years of age, CKD stages G1 and 2). Dual RAAS-blockade once again did not show any additional benefit (43). Interestingly, a *post hoc* analysis of the same trial confirmed that salt restriction should be part of the general management of ADPKD patients (44).

### Targeted Therapies—Tolvaptan

The TEMPO 3:4 trial which was published in 2012 entirely changed the approach to ADPKD, since it led to the approval of Table 1 | Supportive measures in autosomal-dominant polycystic kidney disease (ADPKD).

#### Supportive measures in ADPKD

Blood pressure control (42) Limiting NaCl intake to <5–7 g/day (44) Sufficient fluid intake (>3 L/day) (38–41) Avoid estrogen intake (which stimulates liver growth) (45–47) Healthy diet (e.g., Mediterranean diet) (48, 49)

*While strict blood pressure control is based upon the results from an RCT (HALT-PKD study arm A), the strongest data regarding limiting salt intake specifically in ADPKD relies on a post hoc analysis of HALT-PKD. The effect of estrogens was observed both in pregnant women and more specifically in a small cohort regarding postmenopausal substitution. As to healthy diets, trial results regarding cardiovascular endpoints—the risk of which is increased in ADPKD—can be extrapolated to this cohort. Sufficient fluid intake inhibits ADH secretion, however, its impact on disease progression still needs to be confirmed.*

the very first targeted therapy in Europe. TEMPO 3:4 showed in a double-blind randomized design—that vasopressin receptor (V2R) inhibition could significantly slow down the rate of kidney growth by close to 50% (50). Even more importantly, eGFR loss was decreased by about 26%, an effect size that is comparable to the large trials on RAAS-blockade in diabetic nephropathy. This effect has recently been confirmed by the REPRISE trial that showed tolvaptan to be effective not only in early-stage ADPKD but also in patients having reached early CKD stage G4 (51). As expected V2R-blockade goes along with significant polyuria. Nonetheless, close to 80% of patients enrolled did continue through 3 years of the trial; a finding that is confirmed by recent real-world experiences (52). Furthermore, even though rare and reversible, liver toxicity is possible and requires regular screening of liver enzymes. In order for ADPKD patients to benefit from this therapy, careful selection of patients in whom the treatment is started is of utmost importance. Individuals that will not reach ESRD during lifetime are not suitable candidates for such an approach making only rapid progressors candidates for this therapy. Consequently, several publications have been published in the past 2 years trying to give guidance to nephrologists as to patient selection (27, 53). Past data on eGFR loss is the best indicator of rapid disease progression, yet, these data are not always available and do not help in CKD stage G1. Consequently, a row of predictors of disease progression in ADPKD—including models using total kidney volume ("Mayo classification") (19), mutation status, and clinical symptoms ("PROPKD Score") (28)—have been validated that can help in treatment decisions and patient counseling. While ultrasonography is usually sufficient to make a diagnosis of ADPKD, MRI volumetry has proven very helpful to obtain more precise kidney volumes as the basis to treatment decisions (**Figure 1**). A more detailed summary of these criteria can be found in the recent literature (27, 53).

### Targeted Therapies Beyond Tolvaptan

Even though tolvaptan is a major step forward in the care of ADPKD patients it only slows down disease progression and will not prevent reaching ESRD in the vast majority of patients. Consequently, additional strategies targeting others of the multiple dysregulated cellular signal transduction pathways are necessary (54). One promising target was the mTOR-signaling pathway based on data from mouse models (55, 56). However, two clinical trials did unfortunately not show any benefit in the patient setting (57, 58). The use of somatostatin-analogs targets the same intracellular secondary messenger—cAMP—as tolvaptan. A small trial has shown a potential benefit in ADPKD patients (59). Currently, two phase 3 trials—the LIPS trial in France and the DIPAK1 trial in the Netherlands—are examining this approach in larger cohorts. Somatostatin-analogs would have the advantage to also influence liver disease in ADPKD, while an impact on the hepatic involvement has by now not been shown in the case of tolvaptan. Interestingly, with the use of pravastatin in ADPKD, a double-blind randomized trial in children and young adults has added another potential target (60). Even though a recent *post hoc* analysis of the HALT-PKD trials could not confirm a disease-modifying effect of statin therapy in ADPKD, final clarification of the potential of these drugs will need a larger prospective clinical trial (61). With the overactivation of several kinases in ADPKD, re-purposing of kinase inhibitors has gained interest in the past years. In this line, a phase 2 trial examining the potential of bosutinib—a src/bcr-abl tyrosine kinase inhibitor—has recently been published. In this 2-year trial, bosutinib was able to slow down kidney growth. However, there was no effect on eGFR loss and close to 50% of the patients discontinued the initial treatment period, primarily due to treatment-associated adverse events (62). Consequently, more efforts are necessary to continue translating basic research in ADPKD to clinically meaningful therapies. Apart from the identification of novel treatment targets and their translation into clinical trials—e.g., as for CDC25 inhibition using vitamin K3, treatment with niacinamide, re-purposing of metformin, dietary interventions, or reversal of the Warburg effect (63–67)—combination therapies could also help in improving the effects and reducing side effects of pharmacological therapies in ADPKD in the future (68, 69). Furthermore, as to tolvaptan, a trial that started recruitment in the past year is currently examining V2R-inhibition as a therapeutic principle in a pediatric cohort. Even though clinical disease onset in ADPKD does primarily occur in adulthood, patients with clear indicators of very rapid disease progression could profit from an early start of therapy making this trial an interesting endeavor.

### CONCLUSION

There has been an enormous breakthrough in the understanding of polycystic kidney diseases both in the adult as well as in the pediatric population. A large number of underlying gene defects have been identified and pathogenic pathways characterized. In ADPKD this resulted in the development of the first available treatment strategies and the approval of tolvaptan to prevent progression of the disorder. Even though ADPKD is primarily a disease of adulthood and ADPKD patients are thus mainly seen by adult nephrologists, a close interaction between pediatric and adult nephrology will be of benefit to patients and their families. Not only do genetic diseases always involve more than one individual. Unusual courses can also lead to onset of classical diseases of adulthood (e.g., ADPKD) in children and of classical pediatric diseases (e.g., ARPKD and NPH complex) in adult patients. Adult nephrologists can benefit from the wide experience of pediatric disciplines regarding genetic diseases on the one hand. On the other hand, this interaction helps in combining the knowledge on childhood interventions with outcome in adulthood and allows to learn from each other's experiences in the treatment of polycystic kidney diseases.

### REFERENCES


### AUTHOR CONTRIBUTIONS

R-UM and TB wrote the manuscript. R-UM designed the figures.

### FUNDING

Ministerium für Kultur und Wissenschaft des Landes NRW (Nachwuchsgruppen.NRW 2015-2021) Deutsche Nierenstiftung Marga und Walter Boll Stiftung.

ClinicalTrials.gov (2017). Available from: https://clinicaltrials.gov/ct2/show/ NCT02964273. (accessed October 23, 2017).


**Conflict of Interest Statement:** R-UM and TB have received personal fees for participation in advisory boards and as an expert speaker from Otsuka Pharmaceutical. The Department II of Internal Medicine has received research funding from Otsuka Pharmaceutical.

*Copyright © 2018 Müller and Benzing. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Bartter Syndrome Type 3: Phenotype-Genotype Correlation and Favorable Response to Ibuprofen

Xuejun Yang, Gaofu Zhang, Mo Wang, Haiping Yang and Qiu Li\*

Department of Nephrology, Children's Hospital of Chongqing Medical University, Chongqing, China

Objective: To investigate the phenotype-genotype correlation in different genetic kinds of Bartter syndrome type 3 in children.

Methods: Clinical and genetic data of 2 patients with different mutations in Bartter syndrome type 3 was analyzed while the prognosis was compared after a 6-year follow-up or 2-year follow-up, respectively.

Results: Bartter syndrome is a kind of autosomal recessive inherited renal disorder. The manifestation and prognosis of Bartter syndrome change with mutation types, and severe mutation were often accompanied with unfavorable prognosis. Comprehensive therapy with ibuprofen, antisterone, captopril, and potassium have remarkable effect, while ibuprofen may improve growth retardation partly.

#### Edited by:

Miriam Schmidts, Radboud University Nijmegen, Netherlands

#### Reviewed by:

David J. Sas, Mayo Clinic, United States Verdiana Ravarotto, Università degli Studi di Padova, Italy

> \*Correspondence: Qiu Li liqiu809@126.com

#### Specialty section:

This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics

Received: 15 December 2017 Accepted: 08 May 2018 Published: 30 May 2018

#### Citation:

Yang X, Zhang G, Wang M, Yang H and Li Q (2018) Bartter Syndrome Type 3: Phenotype-Genotype Correlation and Favorable Response to Ibuprofen. Front. Pediatr. 6:153. doi: 10.3389/fped.2018.00153 Conclusion: Bartter syndrome should be considered when children have unreasonable continuous electrolyte disturbance, metabolic alkalosis and growth retardation.As a genetic disease, its clinical features depend on the mutation type. It can be ameliorated by electrolyte supplementation, prostaglandin synthetase inhibitors, angiotensin-converting enzyme inhibitors and potassium-sparing diuretic. Considering the following electrolyte disturbances, infections, growth retardation, kidney failure and even death, Bartter syndrome need lifelong treatment, early diagnosis and treatment is the most important.

#### Keywords: Bartter syndrome, hypokalemia, alkalosis, children, CLCNKB

### INTRODUCTION

In this study, we reported two cases of Chinese patients with Bartter syndrome type 3 caused by different mutations of CLCNKB, which lead to different symptoms and different response to therapeutic measures (the written informed consent was obtained from the parents to publish the case report).

## BACKGROUND

Bartter syndrome is a rare diseases, which results from congenital defects in the renal tubular system regulating the reabsorption of sodium, potassium and chloride (1). Based on the different pathogenic genes, Bartter syndrome was classified into five types (Type 1–5) (2). Meanwhile, classical Bartter syndrome, neonatal Bartter syndrome and variant Bartter syndrome (Gitelman syndrome) are the three clinical types of Bartter syndrome (3). Hypokalemia, hypochloremia, metabolic alkalosis, and growth retardation are the most common manifestations of Bartter syndrome (4), while the manifestation and prognosis change with mutation types, and patients with severe mutation often have a unfavorable prognosis. Moreover, comprehensive therapy with electrolyte supplementation, adequate fluid intake, prostaglandin synthetase inhibitors, angiotensin-converting enzyme inhibitors, and potassium-sparing diuretic have remarkable effects (4, 5).

### CASE PRESENTATION

### Case 1

### Clinical Features

A 4-month-old female baby was sent to a children's hospital because of repeated vomiting and growth retardation. Her vomiting, which was not bilious or projectile, had lasted for 2 months and became more and more forceful recently. The baby looked thin and week, with a weight of 3.5 kg (≤3SD), a height of 54 cm (≤3SD), and a head circumference of 37 cm (≤3SD) (6).

The infant was born to a healthy 25-year-old G1P1 mother via spontaneous vaginal delivery at 38 weeks gestational age without antenatal polyhydramnios, with a birth weight of 2.9 kg and height 49 cm, and the Apgar scores was 9, 8, 10, at 1, 5, and 10 min, respectively. The patient's parents and relatives did not have any apparent clinical symptoms such as vomiting. There's no family history of consanguineous marriage and hereditary disease.

After admission, physical examination on the baby was performed. This revealed dehydration and delayed development, which manifested as disability of rising her head. Her blood pressure was 80/60 mmHg, pulse was 139 beats/min, and respiratory rate was 46/min. No rash, edema or hepatosplenomegaly was found. Circulatory, respiratory and neurologic examination did not reveal other specific deficit. Ultrasound of the gastrointestinal tract was normal, while ultrasound of the kidneys showed echo enhancement in both kidney, compatoible with nephrocalcinosis. Electrocadiography showed a low and flat T wave, accompanied with U wave. Serum electrolytes revealed hyponatremia, hypokalemia, and hypochloremia as follows: Na<sup>+</sup> 122.5 mmol/l (normal range 135–145 mmol/l), K<sup>+</sup> 1.8 mmol/l (normal range 3.5–5.5 mmol/l), Cl<sup>−</sup> 56.6 mmol/l (normal range 95–110 mmol/l), Mg2<sup>+</sup> 1.22 mmol/l (normal range 0.8–1.6 mmol/l), Ca2<sup>+</sup> 2.57 mmol/l (normal range 2.15–2.75 mmol/l). Blood gas analysis showed metabolic alkalosis (pH 7.8, HCO<sup>−</sup> 3 35.7 mmol/L, pCO<sup>2</sup> 5.6 Kpa). The serum aldosterone level was high (366 pg/ml, normal range 65–296 pg/ml), as well as the rennin activity (8.57 ng/ml/h, normal range 0.05–0.79 ng/ml/h), and the angiotensin II activity (1,084 pg/ml, normal range 28.2–52.5 pg/ml).

In consideration of vomiting, growth retardation, hypokalemia, hypochloremia, and metabolic alkalosis, the infant was treated as a suspect case of Bartter syndrome on the second day. Spironolactone (1 mg/kg/d), catopril (1 mg/kg/d) for oral and adequate intravenous fluid therapy were given. Since the parents refused, prostaglandin synthetase inhibitors such as ibuprofen or indomethacin were not given at that time. On day 6, on account of the discontinued vomiting, normal serum electrolytes and blood gas analysis, the intravenous therapy was replaced of oral KCl solution (10 mmol/kg/d). On day 11, the baby was dismissed from hospital in-patient care with the therapy of KCl and increased fluid intake with age, then started a regular follow-up from then on. During the first 2 years, the baby did not vomit again. Serum electrolytes and blood gas analysis checked every month were normal. In the third year of follow up, when the girl was 4 years old, obvious growth retardation [weight 8.5 kg (≤3SD), height 75 cm (≤3SD)] was still observed (6). After a conversation with her parents, they agreed to start treatment with ibuprofen (30 mg/kg/d, 3 times a day). This led to improved length and wait gain in the following period. However, at the age of 6 years, the girl's weight was 14.9 kg (−3SD∼−2SD) (**Figure 1A**), while the height was 105.4 cm (−2SD∼−SD) (6) (**Figure 1B**).

### Mutation Analysis

Informed consent was obtained from the parents for mutational analysis of known Bartter syndrome genes. Genomic DNAs of the patients and their parents were extracted from peripheral blood, while DNA samples from 50 healthy unrelated Chinese people were severed as normal controls. Targeted sequencing using nextgeneration sequencing was conducted for genes responsible for Bartter syndrome (The detailed methods were in supplemental file 1).

As a result, two mutations of CLCNKB were identified. One is a homozygous transition (A–G) at the −2 position of the splicing acceptor site of intron 12 (NM\_000085.4:C.1228-2A>G) from her mother (**Figure 2A**), which may resulted in the abnormal splice of exon 12. Another one is a heterozygous loss of exons 1–18(NM\_000085.4: Ex1\_18 del) from her father (**Figure 2B**). However, neither of these two mutations were detected in the control samples. Given the predicted devastating effect on protein structure of the 2 alleles, segregation within the family and no other mutations detected in known Bartter genes, we regarded the mutations as causative of Bartter syndrome type 3 (OMIM: 607364) in the baby.

### Case 2

### Clinical Features

A 4<sup>2</sup> /12-year-old boy was brought to hospital because of persistent hypokalemia and growth retardation. His serum potassium was 2.1 mmol/L the day before in a local hospital. He was born to a healthy 20-year-old G1P1 mother via spontaneous vaginal delivery at 39+<sup>2</sup> weeks gestational age without antenatal polyhydramnios, with a birth weight of 3.4 kg and height 50 cm, and the Apgar scorea were normal. However, the patient's parents were first cousins without family history of hereditary disease.

On admisssion, his weight was 9.9 kg (≤3SD) and height was 83.2 cm (≤3SD) (6). His blood pressure was 92/58 mmHg, pulse was 101 beats/min, and respiratory rate was 31/min. Besides dental enamel dysplasia, no rash, edema or hepatosplenomegaly was found. No disorder showed in circulatory, respiratory, or neurologic examination. Ultrasound of the gastrointestinal tract and electrocadiography were normal while renal ultrasound examination showed echo enhancement in both kidney similar

to what was observed in case 1 above. Serum electrolytes revealed hyponatremia, hypokalemia, and hypochloremia as follows: Na<sup>+</sup> 111.9, K<sup>+</sup> 2.3, Cl<sup>−</sup> 70.3, Mg2<sup>+</sup> 0.97, Ca2<sup>+</sup> 2.52 mmol/L. Blood gas analysis showed metabolic alkalosis (pH 7.57, HCO<sup>−</sup> 3 39.4 mmol/L, pCO<sup>2</sup> 5.73 Kpa). The serum aldosterone level was high (422 pg/ml), as well as the rennin activity (11.15 ng/ml/h), and the angiotensin II activity (1,459 pg/ml).

With the presentations of growth retardation, hypokalemia and metabolic alkalosis, the boy was clinically diagnosed as Bartter syndrome type 3. Spironolactone (1 mg/kg/d), catopril (1 mg/kg/d), ibuprofen (30 mg/kg/d) for oral and intravenous fluid therapy were given. On day 3, on account of the corrective serum electrolytes and blood gas analysis, the intravenous therapy was replaced by oral KCl solution (10 mmol/kg/d). On day 7, the boy left the hospital with the therapy of KCl and increased fluid intake with age, then started a regular follow-up by telephone from then on (Since he lived in a small village far from our hospital, his parents arranged him to attend a local clinic and informed us about the results via telephone). During these 2 years, serum electrolytes and blood gas analysis checked every month showed that hypokalemia, hypochloremia (K<sup>+</sup> 2.8–4.0 mmol/L, Cl<sup>−</sup> 84.1–100.5 mmol/L) and metabolic alkalosis (pH 7.37–7.58, HCO<sup>−</sup> 3 28.6–35.7 mmol/L, pCO<sup>2</sup> 4.0–6.0 Kpa) still existed. At the age of six, the boy came back to us, still suffering from a severe growth retardation [weight 11.2 kg (≤3SD), height 89.4 cm (≤3SD)] **(6)** (**Figures 1C,D**), hypokalemia, hypochloremia (K<sup>+</sup> 2.38 mmol/L, Cl<sup>−</sup> 92.6 mmol/L) and metabolic alkalosis (pH 7.50, HCO<sup>−</sup> 3 32.3 mmol/L, pCO<sup>2</sup> 5.9 Kpa).

### Mutation Analysis

In view of consanguineous marriage, NGS targeted sequencing of known Bartter syndrome genes and whole exome sequencing were both performed and revealed a mutation of CLCNKB. Unfortunately for the patient, since he descended from a consanguineous marriage, he inherited a large homozygous loss of exons 1–18 (NM\_000085.4: Ex1\_18 del) from his parents (**Figures 3A,B**), which was showed better by PCR electrophoresis of CLCNKB (**Figure 3C**). Meanwhile, another mutation of FAM83H was also identified, which was a homozygous transition

(G to A) in exon 2 (NM\_198488.2:C.154G>A), leading to amelogenesis imperfecta type 3 (OMIM: 130900).

### DISCUSSION

Bartter syndrome autosomal-recessively inherited and characterized by the association of hypokalemia, hypochloremia, metabolic alkalosis, growth retardation and the activation of the renin-aldosterone axis (4). Since first reported by Bartter et al. (4), more and more patients have been diagnosed. However, a report in 2008 showed its incidence was about 1/1,00,000 (7). Based on the different underlying disease causing genes, Bartter syndrome was classified into five types with mutations in SLC12A1, KCNJ1, CLCNKB, BSND, and CASR identified to date (8) (**Table 1**).

Bartter syndrome type 3, caused by the mutation in CLCNKB, which encodes a protein called ClC-Kb. ClC-Kb is a member of the voltage dependent chloride channel family (ClC), which

TABLE 1 | Genetic classification of Bartter syndrome.


\*AR, autosomal recessive inheritance; AD, autosomal dominant inheritance.

is expressed in the thick ascending limb of Henle's loop, distal tubule and cortical collecting tubule and regulates the tubular reabsorption of chloride in the kidney (9). As a result, mutations inactivate ClC-Kb, reducing chloride as well as sodium reabsorption in the renal tubules. Moreover, the loss of sodium chloride and water activates the renin-angiotensin-aldosterone

system (RAAS), which contributes to the loss of potassium and renal fibrosis (10, 11). Up to now, more than 50 mutations of CLCNKB have been identified, most of which have unclear functional effect (12). In the present study, we identified two different CLNCKB mutations with one individual being compound heterozygous for a splice site mutations and deletion of the entrie gene and the seocnd case being homozygous for the deletion. None of varaints has been found in control samples. Brochard et al. (13) reported a similar CLCNKB gene splice mutation at the +1 position of the splicing acceptor site of intron 11 (NM\_000085.4:C.1107+1G>T), which may lead to the loss of splice donor site. For another, according to Simon et al. (10), many patients have homozygous CLCNKB deletions which result in loss of normal gene function. In view of the foregoing and the patients' clinical features, we believe these mutations are pathogenetic.

As classical Bartter syndrome, Bartter syndrome type 3 is always accompanied with the mildest presentation, begins in infancy or later and often manifests with dehydration, electrolyte imbalance, polyuria, polydipsia, vomiting, and growth retardation (14). However, in both cases, we found echo enhancement in kidney, which may suggest nephrocalcinosis. Nephrocalcinosis and nephrolithiasis are usually found in antenatal and neonatal Bartter syndrome. Nevertheless, recently some studies have mentioned nephrocalcinosis in classical Bartter syndrome (15, 16). It is considered that CLCNKB mutations most commonly cause the classic Bartter phenotype, but in a minority of patients, they can also cause phenotypes that overlap with either antenatal Bartter syndrome/neonatal Bartter syndrome or Gitelman sydrome, such as nephrocalcinosis and nephrolithiasis. Exactly what causes nephrocalcinosis is unknown, but several factors may contribute to the condition, such as hypercalciuria, marginal hyperuricosuria, and hyperoxaluria (15).

For all types of Bartter syndrome, phenotypes depend on genotypes (17). Serious mutations always bring severe damage to patients, as in our present cases. Comprehensive therapy with electrolyte supplementation, adequate fluid intake, prostaglandin synthetase inhibitors, angiotensin-converting enzyme inhibitors and potassium-sparing diuretic have remarkable effect (5). Futhermore, for the past few years, more and more studies have raised that, on the basis of salt substitution, prostaglandin synthetase inhibitors may play an important part in improving growth retardation (18–20), which was also suggested in case 1. According to long-term follow-up studies in Bartter Syndrome, with appropriate treatment, patients with Bartter syndrome can achieve normal electrolyte values and growth parameters (21, 22), as we also observed in case 1 regarding the electrolytes while growth retardation remained. Case 2 however still showed electrolyte imbalance, alkalosis and growth retardation even after treatment with salt substitution and prostaglandin synthetase inhibitors. The large homozygous deletion of 18 exons of CLCNKB in case 2 may cause more severe loss of function than the compound heterozygous mutations in case 1. Secondly, considering the parental consangouinous marriage, case 2 may carry additional modifiying alleles such as the mentioned mutation of FAM83H, which may exacerbate his condition. Flyybjerg et al. (23) suggested that hypokalemia is a causative factor of growth retardation, while Masanori et

### REFERENCES


al. (24) stated that sometimes classical Bartter syndrome may be complicated with growth hormone deficiency which could also be the case in case 2. Thus, more laboratory examinations such as growth hormone, whole genome sequencing and longer follow up will be necessary to establish the exact cause for reduced treatment response in Bartter Syndrome patients such as case 2.

In summary, we reported different prognosis of two Chinese patients with different CLCNKB gene mutations leading to Bartter syndrome type 3, as well as the effect of prostaglandin synthetase inhibitors in improving growth retardation. Since only two cases are limited in estimating the correlation between phenotype and genotype, more cases should be collected and analyzed in the future. However, considering the patients like which in case 2 still suffer from unsatisfied prognosis, some new treatment such as renin inhibitor (25), as well as more functional study of the mutations, examinations and long follow-up should be taken into account.

### ETHICS STATEMENT

This research was approved by the Ethics review committee of Chlidren's Hospital of Chongqing Medical University. Since this is a case report, no protocol or ethics committee was utilized for this report. Any identifiable information has been removed from the manuscript.

## AUTHOR CONTRIBUTIONS

XY and GZ assumed clinical duties of this patient while he was hospitalized and drafted the initial manuscript. XY, HY, MW, and QL all reviewed and revised the manuscript. All authors approved the final case report as submitted and agree to be accountable for all aspects of the work.

### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fped. 2018.00153/full#supplementary-material


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Yang, Zhang, Wang, Yang and Li. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Nephropathic Cystinosis: Symptoms, Treatment, and Perspectives of a Systemic Disease

#### *Sören Bäumner\* and Lutz T. Weber*

*Pediatric Nephrology, Children's and Adolescents' Hospital, University Hospital Cologne, Cologne, Germany*

Cystinosis is a rare autosomal recessive lysosomal storage disorder caused by mutations in the *CTNS* gene. Main dysfunction is a defective clearance of cystine from lysosomes that leads to accumulation of cystine crystals in every tissue of the body. There are three different forms: infantile nephropathic cystinosis, which is the most common form, juvenile nephropatic, and non-nephropathic cystinosis. Mostly, first symptom in infantile nephropathic cystinosis is renal Fanconi syndrome that occurs within the first year of life. Another prominent symptom is photophobia due to corneal crystal deposition. Cystine depletion therapy with cysteamine delays end-stage renal failure but does not stop progression of the disease. A new cysteamine formulation with delayed-release simplifies the administration schedule but still does not cure cystinosis. Even long-term depletion treatment resulting in bypassing the defective lysosomal transporter cannot reverse Fanconi syndrome. A future perspective offering a curative therapy may be transplantation of *CTNS*-carrying stem cells that has successfully been performed in mice.

#### *Edited by:*

*Miriam Schmidts, Radboud University Nijmegen, Netherlands*

#### *Reviewed by:*

*Frederick Jeffrey Kaskel, Children's Hospital at Montefiore, United States Vera Hermina Koch, Instituto da Criança do Hospital Das Clinicas FMUSP, Brazil*

*\*Correspondence:*

*Sören Bäumner soeren.baeumner@uk-koeln.de*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 19 December 2017 Accepted: 27 February 2018 Published: 14 March 2018*

#### *Citation:*

*Bäumner S and Weber LT (2018) Nephropathic Cystinosis: Symptoms, Treatment, and Perspectives of a Systemic Disease. Front. Pediatr. 6:58. doi: 10.3389/fped.2018.00058*

Keywords: nephropathic cystinosis, cysteamine, renal Fanconi syndrome, *CTNS* gene, hematopoietic stem cell therapy

### INTRODUCTION

Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disorder leading to endstage renal disease and many extra-renal complications with crystal deposition in the conjunctiva and cornea being the most prominent. It is caused by mutations in the *CTNS* gene on chromosome 17p13, which encodes the lysosomal cystin transporter cystinosin (1). First descriptions date to the beginning of the twentieth century, when cystine crystals were found in liver and spleen of a toddler, who died from dehydration and failure to thrive (2). More insight gave studies in the 1980s, where a defective clearance of cystine from lysosomes could be demonstrated (3, 4). The encoding *CTNS* gene was found in 1998 (5). Since then more than 100 pathogenic mutations in the *CTNS* gene have been described (6).

Central column of treatment is a depletion therapy with cysteamine that has proven to slow down progression of renal failure and to prevent or slow down extra-renal manifestations, even though it is not a curative therapy.

There are three different forms of cystinosis, which differ in age at manifestation and severity of the symptoms: (i) infantile nephropathic cystinosis, which is the most common and severe form; (ii) juvenile nephropathic cystinosis, which is characterized by later onset of symptoms and slower progression; and (iii) non-nephropatic cystinosis, with a mainly ocular manifestation, also known as adult form. Each form shows different mutations in the *CTNS* gene (7). Cystinosis occurs approximately in 1–2 of 100,000 live births (6).

### INFANTILE NEPHROPATHIC CYSTINOSIS

Neonates are clinically asymptomatic at birth with normal birthweight and normal length, even though cystine accumulation already starts *in utero*. First symptoms occur within the first year of life, usually presenting as renal Fanconi syndrome, a dysfunction of the proximal tubule that leads to polydipsia, polyuria, dehydration, proximal renal tubular acidosis, urinary loss of electrolytes, and growth retardation. In the urine, glucosuria and aminoaciduria can be found. In the case of glucosuria and normal serum glucose levels, one should always think of renal glucosuria or Fanconi syndrome. Glucosuria is the only parameter to be detected by urine dipstick in the Fanconi tubulopathy. The high protein turnover in the proximal tubule may explain why Fanconi syndrome is the first symptom of cystinosis.

Without treatment end-stage, renal failure occurs at a median age of 10 years. About 95% of cystinosis patients suffer from this type (6, 8). Historically cystinosis accounts for 5% of childhood renal failure (9).

### JUVENILE NEPHROPATHIC CYSTINOSIS

Patients with the juvenile type of nephropathic cystinosis develop symptoms at an older age, often presenting with more unspecific symptoms than patients with infantile cystinosis like nephrotic syndrome or mild proximal tubulopathy, but not necessarily the complete picture of Fanconi syndrome. Endstage renal disease may occur. Most patients are diagnosed in the second decade of life, when onset of photophobia leads to ocular examination and cystine crystals in the cornea can be found. This form accounts for approximately 5% of all cases of cystinosis (7, 10).

### NON-NEPHROPATHIC CYSTINOSIS

This type presents only with ocular symptoms, as deposits are limited to cornea and conjunctiva and is also known as the adult form of cystinosis. Of note, there might be a continuum between milder forms of cystinosis, since non-nephropathic and juvenile forms have been described within one family. Therefore, renal function of every patient with non-nephropathic cystinosis should be monitored closely (10, 11).

### DIAGNOSIS

First clinical signs in patients with infantile nephropathic cystinosis are polyuria, polydipsia, and failure to thrive. These symptoms reflect renal Fanconi syndrome in combination with metabolic acidosis and loss of electrolytes, especially phosphate (1). Since cystinosis is the most common reason for renal Fanconi syndrome at this age, this differential diagnosis should always be considered. Less common reasons for secondary Fanconi syndrome may be Dent's disease, Lowe's syndrome, inherited fructose intolerance, galactosemia, or tyrosinemia (12). Corneal cystine deposits, which are pathognomonic in untreated cystinosis, can rarely be found in the first year of life but are visible in almost every untreated patient at the age of 16 months (13). Confirmation of the diagnosis is made by measurement of elevated cystine levels in white blood cells followed by genetic testing for mutations in the *CTNS* gene (14). For cystine levels see **Table 1**.

### GENETICS

Confirmation of the diagnosis can be made by genetic testing. The *CTNS* gene, which encodes for the lysosomal carrier cystinosin, is located on the short arm of chromosome 17 (p13) (5). The most frequent mutation in Northern Europe is a 57-kb deletion that accounts for approximately 75% of all cases of nephropathic cystinosis (7, 15). Up to now, more than 100 mutations are known in the *CTNS* gene (6, 14). Most mutations in the *CTNS* gene result in a total loss of transport activity of cystinosin. The phenotype is the infantile nephropathic cystinosis. Patients with the milder juvenile or non-nephropathic form show different mutations suggesting that there is a genotype–phenotype correlation (11, 16–18). Still the pathogenesis is not fully understood because no disease model explains the link between lysosomal cystine accumulation and renal Fanconi syndrome completely (19).

### PATHOPHYSIOLOGY AND CLINICAL PRESENTATION

Cystine is a disulfide of the amino acid cysteine. It is generated by lysosomal protein hydrolysis. Because no enzymatic dysfunction was found in cystinotic cells, research focused on defect transporter proteins leading to the identification of the sevendomain transmembrane protein cystinosin, which provides the transport of cystine from lysosomes to cytoplasm (20). Defective transport leads to accumulation and crystallization of cystine in the lysosomal compartment. Affected cells suffer from mitochondrial dysfunction, oxidative stress, and inflammation and, in the end, undergo apoptosis. Since lysosomes are part of every cell type cystine accumulation occurs throughout the whole body, making cystinosis a systemic disease (2). Over time, further symptoms can be seen in virtually every organ. The following sections represent just a selection of cystinosis manifestations. See also **Figure 1**.

### KIDNEY

Nonetheless, there is a different susceptibility of cell types to cystine accumulation with renal cells being especially susceptible. This is the reason why cystinosis presents primarily with renal symptoms (see above). Firstly impaired tissue is the proximal

Table 1 | Diagnosis of cystinosis by measuring cystine levels.


tubule leading to renal Fanconi syndrome, which is the major symptom of cystinosis. A characteristic histopathological sign is the so-called swan neck deformity, which describes the loss of proximal tubular cells. Electron microscopy shows cystine crystals in the tubular cells (10, 21, 22). Simultaneously, glomerular lesions occur due to involvement of podocytes that present histologically with the picture of focal and segmental sclerosis and lead clinically to glomerular proteinuria and progressive deterioration of renal function (2). Using electron microscopy, podocytes appear hypertrophic, multinucleate and have foot process effacement which is pathognomonic for cystinosis (23). Cystinosis does not recur in the kidney graft after transplantation has been performed.

### EYE

Probably, the subjectively most impairing early problem is photophobia due to corneal deposition of cystine crystals (see **Figure 2**) which begins at a median age of 3–4 years, when no treatment is offered. The natural course of cystinosis in the eyes leads to blepharospams, corneal erosions, superficial punctuate keratopathy, and band keratopathy. Involvement of the retina, which can be seen constantly without treatment, causes loss of vision later in life (24, 25). Oral depletion treatment with cysteamine has no influence on the ocular manifestation since there is no vascularization in the cornea. So a topical treatment has been established, bearing new challenges for the patients because cysteamine eye drops have to be applied 6–12 times per day (26).

### BONE AND MUSCLE

Increased urinary loss of phosphate, calcium, and disturbances in vitamin D metabolism cause hypophosphatemic rickets in cystinosis patients. Clinical signs are genua vara, frontal bossing, rachitic rosary, and metaphyseal widening on skeletal X-rays (12). However, there are patients who appear clinically similar without the laboratory findings of disturbed vitamin D metabolism mentioned above despite being treated with cysteamine. This finding might be explained by copper deficiency due to cysteamine toxicity, which may interfere with collagen cross-linking (28). Growth retardation is a frequent finding in cystinosis patients and is usually treated with growth hormone.

Even though X-ray absorptiometry shows bone densities within the normal range, bone fractures can be seen more often

in cystinosis. Possibly, it might be that intra-osseous cystine crystals lead to falsely elevated X-ray absorptiometry levels making X-ray absorptiometry an ineffective tool to assess fracture risk in cystinosis patients (29). Three-dimensional peripheral computed tomography should be the preferred method to assess fracture risk in growth-retarded children with (chronic kidney) disease (30).

### NEUROLOGY

Accumulation of cystine crystals in the brain leads to neurocognitive impairment. Early signs for brain involvement are nonverbal learning difficulties resulting in poor executive functions, whereas verbal and general intelligence are normal (6, 31). Cysteamine can cross the blood–brain barrier. Depletion treatment with cysteamine can improve neurological outcome even when patients are already symptomatic, but it has also been shown that impairment in visual-motor function occurs even if cysteamine treatment was started before neurologic impairment became noticeable. This suggests that it is not only accumulation, which causes learning difficulties in cystinosis patients (32, 33). Long-term neurological complications are cystine encephalopathy, presenting with cerebellar and pyramidal signs, mental deterioration and pseudo-bulbar palsy, as well as distal myopathy, which begins with weakness of the extremities and results in dysphagia and pulmonary insufficiency. Treatment with cysteamine has been shown to have a beneficial and potential reversible effect on both cystine encephalopathy and distal myopathy (34, 35).

## ENDOCRINOLOGY

The most frequent endocrine finding is hypothyroidism, which appears approximately in half of all cystinosis patients and can easily be treated with substitution of thyroid hormones (6). Another frequent finding is hypogonadism. Male cystinosis patients are prone to be infertile due to azoospermia even if treated with cysteamine since early age. It has been shown that spermatogenesis at testicular level can be intact. The underlying mechanisms remain unclear (36). A successful conception after percutaneous epididymal sperm aspiration followed by intracytoplasmic sperm injection has recently been reported (37). Female cystinosis patients are fertile and several successful pregnancies have been described (38). Other affected endocrine organs are exocrine and endocrine pancreas resulting in diabetes mellitus in 5% of all patients (6, 12).

### SYSTEMIC DEPLETION THERAPY

The first trials to deplete cystine from cells used 1,4-dithiothreitol (DTT) or ascorbic acid with moderate success (19). The cystine depleting therapy with cysteamine was first described in 1976 and is still the golden standard in cystinosis therapy (6, 39). Cysteamine induces a thiol-disulfide interchange reaction that generates equimolar amounts of cysteine and cysteinecysteamine molecules from cystine (40). These molecules can exit the lysosomes using alternative cationic transporters and bypass the defective cystinosin transporter, as shown in **Figure 3** (41). Cysteamine treatment can be monitored by

Cysteamine induces a chemical reaction resulting in cysteamine–cysteine and cysteine. Both molecules exit the lysosome bypassing the cystinosin transporter protein (green arrows). Adapted from Ref. (6).

measuring intracellular cystine levels in white blood cells, which is considered to reflect the cystine concentration of the body's other tissues. Target levels are usually <1.0 nmol hemicystine/mg protein. Since cystine levels in healthy people are <0.2 nmol hemicystine/mg protein the optimal range for cystinosis patients is considered to be <0.5 nmol hemicystine/ mg protein (12, 42, 43).

Cysteamine has been available as a commercial drug since 1997 in an immediate-release formulation that demands a strict 6-h administration schedule to maintain effective plasma levels. A new delayed-release formulation has been available since 2013 in the USA and since 2014 in Europe which must only be administered twice daily. Both formulas use cysteamine bitartrate since the bitartrate formulation needs lower doses to maintain plasma levels than cysteamine hydrochloride or phosphocysteamine (44). The new delayed-release formulation is composed of enteric-coated, microspheronised beads encapsulated in hard gelatin that allow to extend the intake up to 12 h (45). The maximum plasma levels of delayed-release cysteamine are reached about 3 h after administration, whereas maximum levels of the immediate formulation can be found after about 1 h already (46). Both extended- and immediate-release cysteamine have been proven to reach cystine target levels, which are thought necessary to slow down progress of cystinosis-related symptoms. The 12-h administration of the extended-release formulation supports therapy adherence by simplifying the administration schedule whereas experiences of side effects differ from center to center (43, 45, 47). Since new therapeutic strategies in many fields of pediatric diseases enabled patients to reach adolescence and adulthood, e.g., patients with cystic fibrosis or diabetes mellitus, new problems with therapy adherence at this age arose leading to the concept of a controlled transition from pediatric to adult health-care services. This can also be seen in cystinosis patients for whom special transition protocols are recommended (48, 49).

Most reported side effects of cysteamine therapy are halitosis, disagreeable sweat odor, and gastrointestinal side effects like nausea and abdominal pain. Proper dosing up to a maximum of 1.95 g/m2 /day with a gradually increasing application schedule avoids side effects as lethargy, hyperthermia, and rash (50–52). Recent reports of new adverse events like bruise-like skin lesions, bone abnormalities, and muscle weakness in cystinosis patients with Fanconi syndrome showed that they have an increased urinary copper excretion under cysteamine therapy. This led to the hypothesis that cysteamine toxicity causes copper deficiency because of the structural similarity of cysteamine to D-penicillamine resulting in a reduced formation of aldehydes required for collagen cross-linking (28).

### EYE DROPS

Systemic depletion therapy with cysteamine reduces posterior segment complications like pigmentary changes that can lead to retinopathy and loss of vision, but does not prevent deposition of cystine crystals in cornea and conjunctiva. It has been shown that photophobia is associated with crystal density, infiltration of inflammatory cells, and nerve damage within the cornea. Therefore, a topical treatment is necessary and has proven to be effective (24, 53, 54). Cysteamine eye drop formulations are aqueous solutions that have to be administered 6–12 times per day. Because cysteamine is unstable at room temperature and to light exposure, storage and transport of the eye drops are challenging making therapy adherence difficult. A new, gel-like viscous formulation with a fivefold higher concentration of cysteamine has been developed, which has to be administered only four times daily. The gel-like formulation increases the contact time to the cornea allowing the cysteamine to penetrate more deeply into the corneal layers. On the other hand, using the gel-like drops produce side effects like stinging, burning, and vision blurring that were more common compared to the aqueous formulation. The reason may be the higher cysteamine concentration and the viscous consistency but it did not lead to lower therapy adherence (24). Patients untreated with eye drops sometimes develop severe corneal lesions that require a corneal transplant. A topical treatment is necessary even after corneal transplantation because cystinosin-deficient host cells can reinvade into the transplanted cornea (14).

### FUTURE PERSPECTIVES

Even though the depleting therapy with cysteamine—oral and topical—has improved the prognosis of cystinosis patients dramatically, it is still not a curative therapy because the defective lysosomal transport protein cystinosin is only bypassed. New formulations of oral and topic cysteamine can alleviate the medical schedule and through this support therapy adherence. But cysteamine has only a delaying effect on complications like end-stage renal failure. Evidence grows that dysfunction of the lysosomal transport protein cystinosin leads to several disturbed intracellular interactions that cannot be corrected by depleting cystine from the lysosomal compartment: for example, cystinotic cells show (i) impaired chaperone-mediated autophagy (55); (ii) reduced levels of transcription factor EB, a key factor in regulating lysosomal biogenesis and clearance (56); and (iii) downregulation of the mammalian target of rapamycin pathway in proximal tubular cells (57). All these mechanisms are not influenced by cysteamine treatment and are associated with the persistence of renal Fanconi syndrome (8). Also altered cellular energy homeostasis or increased oxidative stress found in cystinotic fibroblasts offer new potential targets for the treatment of cystinosis, but still do not offer a causal therapy (58, 59).

The most promising approach to cure cystinosis is the transplantation of hematopoietic *CTNS*-carrying stem cells. Stem cells from wild-type donors were transplanted into irradiated *CTNS*-knockout mice (60). Whereas transplantation of mesenchymal stem cells did not integrate efficiently, the transplantation of hematopoietic stem cells led to stable engraftment in mice. This resulted in a long-term improvement of renal function including Fanconi syndrome even though the stem cells did not reprogram proximal tubular cells. Confocal microscopy showed that most transplanted stem cells differentiated into interstitial lymphoid, dendritic, or fibroblastic cells and did not replace renal epithelium cells (61). The reduction of cystine content in different tissues reached up to 94% (60).

Transplantation of hematopoietic stem cells was still effective in older mice, suggesting that stem cell therapy might still be an option even if tissue injury is already manifested. The exact mechanism of stem cell therapy remains unclear. It is possible that stem cell therapy protects healthy tissue from being harmed by cystine crystals or that damaged tissue is being reversed (8). Further problems of allogeneic stem cell transplantation like graft-versus-host-disease, which cause high morbidity and mortality in transplanted patients, still need to be considered. A new approach uses genetically modified autologous hematopoietic stem cells. For transfer a self-inactivating-lentivirus vector is used in an attempt to lower the risks of allogeneic stem cell transplantation (62).

### TRANSITION AND TRANSFER

The above-mentioned recommendations of structured transition programs (48, 49) must by no means attach little value but transition has paramount importance in rare diseases that require interdisciplinary lifelong care. Pediatric cystinosis patients in general experience comprehensive, interdisciplinary, and structured care led and coordinated by pediatric nephrologists with the Fanconi tubulopathy and chronic kidney disease being the leading symptoms. This coordinated guardian care may change when the patient is transferred to adult care. Lack of knowledge about rare diseases might be a problem just as appointments with separate professionals focusing on their own specialties resulting in "fragmented" care (48). Therefore, in Germany a current initiative aims to transfer pediatric cystinosis patients to adult Morbus Fabry centers. By this, young adult patients encounter a patientcentered care with high awareness of rare diseases that is led by an adult nephrologist and provides interdisciplinary resources and institutional support perfectly applicable to the needs of cystinosis patients.

## CONCLUSION

Over the last decades, our knowledge and understanding of cystinosis has improved continuously. This led to new therapy options and simplified medicine formulations, which dramatically improved life expectancy and life quality of cystinosis patients. But still there is no curative therapy. The new approach of stem cell transplantation gives hope to become a curative treatment that would mean another big step for cystinosis patients to further improve life expectancy and quality.

### AUTHOR CONTRIBUTIONS

SB and LW were responsible for concept and creation of this manuscript. Both authors revised the manuscript and approved the final version to be published.

### ACKNOWLEDGMENTS

The authors thank Petra Kleinwächter for preparing the figures.

### REFERENCES


in vivo confocal microscopy and anterior-segment optical coherence tomography study. *Invest Ophthalmol Vis Sci* (2015) 56(5):3218–25. doi:10.1167/ iovs.15-16499


**Conflict of Interest Statement:** LW has received travel grants and speaker's honoraria of Raptor Pharmaceuticals, Horizon Pharma, and Chiesi GmbH. SB has received travel grants and speaker's honoraria of Orphan Europe declares no conflict of interest.

*Copyright © 2018 Bäumner and Weber. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Novel Aspects of Renal Magnesium Homeostasis

*Paula Giménez-Mascarell1 , Carlotta Else Schirrmacher <sup>2</sup> , Luis Alfonso Martínez-Cruz1 \* and Dominik Müller2 \**

*1CIC bioGUNE, Bizkaia Science and Technology Park, Derio, Spain, 2Department of Pediatric Gastroenterology, Nephrology and Metabolism, Charité – Universitätsmedizin Berlin, Berlin, Germany*

Magnesium (Mg2+) is indispensable for several vital functions, such as neurotransmission, cardiac conductance, blood glucose, blood pressure regulation, and proper function of more than 300 enzymes. Thus, Mg2+ homeostasis is subject to tight regulation. Besides the fast and immediate regulation of plasma Mg2+, a major part of Mg2+ homeostasis is realized by a concerted action of epithelial molecular structures that tightly control intestinal uptake and renal absorption. This mechanism is provided by a combination of para- and transcellular pathways. Whereas the first pathway provides the organism with a maximal amount of vital substances by a minimal energy expenditure, the latter enables controlling and fine-tuning by means of local and regional regulatory systems and also, hormonal control. The paracellular pathway is driven by an electrochemical gradient and realized in principal by the tight junction (TJ), a supramolecular organization of membrane-bound proteins and their adaptor and scaffolding proteins. TJ determinants are claudins (CLDN), a family of membrane spanning proteins that generate a barrier or a pore between two adjacent epithelial cells. Many insights into molecular mechanisms of Mg2+ handling have been achieved by the identification of alterations and mutations in human genes which cause disorders of paracellular Mg2+ pathways (CLDN10, CLDN14, CLDN16, CLDN19). Also, in the distal convoluted tubule, a basolateral protein, CNNM2, causes if mutated, familial dominant and also recessive renal Mg2+ wasting, albeit its true function has not been clarified yet, but is assumed to play a key role in the transcellular pathway. Moreover, mutations in human genes that are involved in regulating these proteins directly or indirectly cause, if mutated human diseases, mostly in combination with comorbidities as diabetes, cystic renal disease, or metabolic abnormalities. Generation and characterization of animal models harboring the corresponding mutations have further contributed to the elucidation of physiology and pathophysiology of Mg2+ disorders. Finally, high-end crystallization techniques allow understanding of Mg2+ handling in more detail. As this field is rapidly growing, we describe here the principles of physiology and pathophysiology of epithelial transport of renal Mg2+ homeostasis with emphasis on recently identified mechanisms involved.

Keywords: magnesium, crystallography, CNNM2, kidney, genetics

### INTRODUCTION

Magnesium (in its ionized and biologically active form: Mg2<sup>+</sup>) belongs to the group of alkaline earth metals and is the second most abundant intracellular divalent cation. It is the eleventh most abundant element by mass in the human body. Mg2<sup>+</sup> is indispensable for several vital functions, such as neurotransmission, cardiac conductance, blood glucose control, and blood pressure regulation.

#### *Edited by:*

*Max Christoph Liebau, Universitätsklinikum Köln, Germany*

#### *Reviewed by:*

*Tom Nijenhuis, Radboud University Nijmegen, Netherlands Jakub Zieg, University Hospital in Motol, Czechia*

#### *\*Correspondence:*

*Luis Alfonso Martínez-Cruz amartinez@cicbiogune.es; Dominik Müller dominik.mueller@charite.de*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 31 October 2017 Accepted: 13 March 2018 Published: 09 April 2018*

#### *Citation:*

*Giménez-Mascarell P, Schirrmacher CE, Martínez-Cruz LA and Müller D (2018) Novel Aspects of Renal Magnesium Homeostasis. Front. Pediatr. 6:77. doi: 10.3389/fped.2018.00077*

**194**

In muscle, Mg2<sup>+</sup> regulates contraction by antagonizing calcium (1–4). Mg2<sup>+</sup> has also been identified as a second messenger, e.g., in T-Cells, where mutations in the gene MAGT1 have been linked to human immunodeficiency. There, mutations disable transient Mg2<sup>+</sup> influx induced by the activation of the T-cell receptor (5).

Adenosine triphosphosphate (ATP) is the major source of cell energy, and must bind Mg2<sup>+</sup> in order to be biologically active. The resulting complex, Mg2<sup>+</sup>-ATP is vital for the stability of all polyphosphate compounds in cells, including those associated with synthesis of DNA and RNA. More than 300 enzymes are dependent on Mg2<sup>+</sup> for their biocatalytic function, including those that utilize or synthesize ATP, or those that use other nucleotides to synthesize DNA and RNA (6). In plants, Mg2<sup>+</sup> is the central ion of chlorophyll and, therefore, vital for photosynthesis. In higher organisms, hemoglobin, the essential O2 carrier, has high structural similarities with chlorophyll but here, Fe2+ replaced Mg2<sup>+</sup> as the central ion. Magnesium is an essential mineral nutrient (i.e., element) and is present in every cell type and in every organism. In the blood and serum, Mg2<sup>+</sup> is mostly bound to serum albumin (like the most abundant divalent cation, Ca2+) and stored in muscle fibers and in bone. The biologically active form is the ionized form and dietary sources rich of magnesium are plants [Almonds, Cashews, Cocoa, Pumpkin Seeds, Spinach, and Fish (Halibut, Mackeral)]. Clinically, deficiency of Mg2<sup>+</sup> causes nausea, appetite loss, fatigue, and general weakness. At a later stage, patients present with numbness, tingling, muscle cramps, cerebral seizures, and cardiac arrhythmias. Moreover, Mg2<sup>+</sup> deficiency can lead to hypocalcemia and hypokalemia (7). On the other hand, little is known about the consequences of hypermagnesemia as can be encountered, e.g., in patients with end stage renal disease (8). However, Mg2<sup>+</sup> complexes are involved in the development of vascular calcifications, a major cause of morbidity and mortality of patients with chronic kidney disease (9).

Compared to Ca2<sup>+</sup> storage (around 1000 g in adults), the whole body content of Mg2<sup>+</sup> only sums up to 20 g. In addition, Mg2+ stores are not as readily accessible as Ca2<sup>+</sup> stores by, e.g., PTH action. Therefore, the organisms' intra- and extracellular Mg2<sup>+</sup> levels are kept tightly within narrow limits. Likewise, the available, ionized, biologically active and relevant form (Mg2<sup>+</sup>) is dependent on a more or less a continuous nutritional supply. Mg2<sup>+</sup> serum concentrations in adults range from 0.7 to 1.1 mmol/l, while newborns and toddlers can have lower limit serum concentrations (e.g., from 0.45 mmol/l on). The daily need of Mg2<sup>+</sup> in adults is around 300–350 mg/day and fractional intestinal uptake varies depending on the amount of intake. Fine et al. found that intestinal Mg2<sup>+</sup> absorption increases with intake but also that fractional Mg2<sup>+</sup> absorption fells progressively (from 65% at low to 11% at high intake) (10). The authors described the intestinal absorption by an equation containing a hyperbolic function plus a linear function. They concluded that Mg2<sup>+</sup> absorption must, therefore, be realized by a twin-mechanism that simultaneously reaches an absorptive maximum, and in addition a mechanism that endlessly absorbs a defined fraction (7%, as the authors concluded) (10). Although the molecular mechanisms have not been identified at that time, their and the findings of other groups later, predicted the coexistence of two distinct mechanisms of Mg2<sup>+</sup> absorption, the trans- and paracellular transport in the intestine. Moreover, as we know nowadays, the same holds true for the kidney.

In both organs, the intestine and the kidney, Mg2<sup>+</sup> is absorbed *via* paracellular as well as by transcellular routes. Whereas the first pathway is in principal driven by an existing electrochemical gradient, the latter requires energy-consuming mechanisms in order to conduct apical uptake, buffering, transport, and basolateral extrusion (**Figure 1**). The paracellular route guarantees the organism to retrieve a considerable amount of vital substances with a minimal energetic expenditure. On the other side, the

energy-consuming transcellular route provides a fine-tuning at the several steps of transport and is, therefore, subject to precise local, regional, or global regulation. This can be realized by hormones such as 1,25(OH)D2 or parathyroid hormone according to the basic but also the actual needs of the organism (e.g., growth).

In the kidney, the driving force of transepithelial Mg2+ absorption is provided by a continuously maintained electrochemical gradient between the apical (tubular fluid) and the basolateral (blood) compartment. This process is realized by a battery of transcellular (and apical to basolateral) and paracellular transport mechanisms. As a principle, paracellular transport mainly takes place where the transepithelial concentration gradient is high, i.e., the proximal jejunum in the gut and in the proximal tubule (PT) as well the thick ascending loop of Henle in the kidney. By contrast, transcellular transport takes place in the late ileum, colon, and the distal part of the renal tubule (11, 12).

### MAGNESIUM HANDLING ALONG THE NEPHRON

After glomerular filtration, the non-protein-bound fraction, Mg2<sup>+</sup> is absorbed along the nephron before it is finally lost irretrievable for the organism within the urine. Under normal conditions (Glomerular filtration rate >90 ml/min/1.73 m2 ), more than 95% of filtered Mg2<sup>+</sup> in the pro-urine is reabsorbed along the tubular system by several coordinated transport processes (13). Besides their anatomical localization, regulatory mechanisms of renal Mg2<sup>+</sup> homeostasis can also be classified by their involvement in the hierarchy of transport. Therefore, we classify here the mechanisms of Mg2<sup>+</sup> transport and homeostasis according to their involvement in the network of Mg2<sup>+</sup> handling (**Table 1**). The first category comprises proteins or elements that transport Mg2+ by itself, e.g., a direct Mg2<sup>+</sup> transport mechanism. The second category includes proteins and mechanisms that regulate elements of category 1. The third category comprises members that influence Mg2<sup>+</sup> handling in a more remote way, e.g., by regulating ions, other than Mg2<sup>+</sup> but thereby influence Mg2<sup>+</sup> handling. To the latter category belong also proteins that influence Mg2<sup>+</sup> homeostasis, e.g., by causing polyuria and thereby a "wash out" of Mg2<sup>+</sup> by reducing the time of contact of absorbing mechanisms with the corresponding substance (as can be seen

Table 1 | Protein classification based on the role played by each molecule in renal magnesium handling.


*bProteins expressed in the DCT. c Proteins which role is under debate.* by the treatment with furosemide). With this newly proposed classification, we aim to focus on the current knowledge on Mg2<sup>+</sup> handling toward a more interactive model, the magnesiome, and the Mg2<sup>+</sup> interactome.

Anatomically, the major sites of renal Mg2<sup>+</sup> reabsorption are, besides the PT (10–20%), the thick ascending loop of Henle (TAL, 65–70%) and the distal convoluted tubule (DCT, 10%) (**Figure 2**). Beyond the DCT, no significant mechanisms of Mg2<sup>+</sup> absorption have been described so far (11, 12).

### PT and Thick Ascending Part of Henle's Loop

Mg2<sup>+</sup> absorption in the PT and TAL occurs mainly *via* paracellular route and in the DCT the transcellular route (13). The paracellular route is mainly determined by the Tight Junction (TJ), a supramolecular organization of membrane-bound proteins and their intracellular adaptor- and scaffolding proteins. The major proteins of the TJ comprise Claudins (from lat. *claudere*: to seal), a protein family consisting of at least 24 members in Eukaryotes enabling the TJ to function as either (a) barrier, (b) fence, or (c) channel (**Figure 3**). The *Barrier* function of the TJ enables the organism to increase transepithelial resistance (TER; Ω × cm2 ) where needed. As such, the PT has a low resistance (6–10 Ω × cm2 ) whereas downstream, toward TAL (11–34 Ω × cm2 ) and the Collecting Duct (60 Ω × cm2 ; MDCK cells) TER is constantly increasing (14, 15). The bladder, in order to fulfill its function of a tight reservoir, has the highest epithelial resistance [>300 kΩ × cm2 (16); **Figure 3A**]. The *Fence* function of the TJ is a key element that contributes to the apical-to-basolateral orientation of epithelial cells. In order to guarantee a coordinated, regulated transcellular transport, membrane-bound proteins must be oriented stable at either apical or the basolateral side. This principal requirement of all epithelial tissues is mainly realized by the TJ (17). Likewise, the loss of the apical-to-basolateral orientation, i.e., the loss of polarity and even cell-to-cell contact is an early event in tumor development. Several claudins have been shown to be potential markers of gastrointestinal tumors and their progression (18). Moreover, other Claudins (e.g., Claudin-3) have been demonstrated to be involved, in men and animals in tumor invasiveness and in autoimmune disorders (18, 19). Also, several Claudins are vital for teeth development in mice and humans (20). A great step toward the determination of the role of Claudins has been achieved by the resolution of the crystal structure of the Claudin protein Claudin-15 (21, 22) (**Figure 3B**). Although their contribution to human disease has been demonstrated, proteins that directly interfere with Claudins have only been preliminarily characterized (23, 24). The *Channel* function of the TJ is crucial for ions (like Mg2+ and Ca2+) and H20 absorption and is dependent on an existing electro-chemical gradient (17). The *Channel* function is realized by a surplus of one charge on one side of the epithelial layer than on the other side (e.g., apical vs. basolateral or vice versa) or on the surplus of a molecule or substance over the epithelial layer. Although the TJ does not possess pumps or antiporters, transport can nevertheless be selective by the variation and regulation of TJ composition and protein expression (25). Thus, the expression of different Claudins along epithelial

tissues and their regulation and expression can determine Mg2<sup>+</sup> spatial and temporal reabsorption (26) (**Figure 4**).

Although our knowledge on Mg2<sup>+</sup> handling in the more distal nephron has increased significantly, little is known about Mg2<sup>+</sup> transport in the PT, where it is believed to be transported by the paracellular way. There, terminus "bulk reabsorption" is used widely although this phenomenon has not been elucidated in detailed molecular or physiological context.

Bartter's Syndrome, first described by the endocrinologist Frederick Bartter, is characterized by renal wasting of Na<sup>+</sup>, K<sup>+</sup>, and polyuria. Clinically, hyperaldosteronism in preterm borns, polyhydramnion and prematurity became a hallmark. The seminal work of the Lifton and Hildebrandt groups showed that mutations in the genes that code for the apical transport in the TAL of Na+, K+ and Cl− (NKCC2, ROMK2) as well as the basolateral extrusion mechanisms (ClC-Ka, Barttin) are essential for Na<sup>+</sup> K<sup>+</sup> and Cl<sup>−</sup> handling in the TAL (Bartter's Syndrome types 1–4)

(28–33) Here, Mg2<sup>+</sup> wasting is not caused by a direct mechanisms, and likewise Mg2<sup>+</sup> wasting is a clinical hint, but not a mandatory feature of Bartter's syndrome. Mg2<sup>+</sup> wasting in Bartter's Syndrome is also believed to be secondary to polyurias (and polydipsia) present in these patients. Thus, all the proteins are classified into Category 3 (**Table 1**) supported by the fact that also mice with a targeted deletion of NKCC2 show a Bartter-like phenotype but no hypomagnesemia (34). Interestingly, a transient form of Bartter's syndrome has been described in several patients recently. Patients displayed the prenatal (Polyhydramnion, Prematurity) and postnatal (Hyponatrimia, Hypokalemia) clinical hallmarks of Bartter's syndrome. The authors have shown that these disorders are caused by mutations in the gene encoding melanoma-associated antigen D2, explaining that fact that only males were affected (35, 36). Although the authors have not reported on Mg2<sup>+</sup> levels, it is intriguing that this disorder is a more common cause of neonatal hypomagnesemia than anticipated that far.

The TAL is separated anatomically but also by its functional elements in the medullary and the cortical part (mTAL and cTAL). An important driving force in epithelial tissues and, therefore, also in the TAL, in order to maintain a transepithelial gradient is generated by the basolateral Na<sup>+</sup> K<sup>+</sup> ATPase. Mutations in the gene (FYXD2) coding for the γ-subunit of this protein have been shown to cause dominant familial Hypomagnesemia. Thus, it has been shown that this protein is a "conditio-sine-qua-non" for generating the necessary transepithelial gradient for transport systems and, thus, also for renal Mg2<sup>+</sup> handling (11, 12, 37), this protein can be classified into the Category 2.

During the last years, several proteins have been shown to be involved in renal paracellular ion transport. However, a clear phenotype–genotype correlation has established in some but not in all of the genes and proteins involved. An example is provided by CLDN14. Mutations in CLDN14 cause non-syndromic deafness in men and mice (38, 39). Affected human individuals do not display overt renal abnormalities and the same holds true for mice with targeted deletions in CLDN14 (38). On the other hand, genome-wide association studies identified CLDN14 variants as a major risk gene associated with hypercalciuric stone disease (4, 40, 41). Furthermore, in the TAL, mutations in human genes (CLDN10, CLDN14, CLDN16, and CLDN19), that define the paracellular pathway (**Figure 4**) of Mg2<sup>+</sup> absorption have been shown to cause rare human disorders. In the TAL, where significant Mg2+ and Ca2<sup>+</sup> transcellular transport is absent, paracellular transport is of vital importance and is driven by a lumen-positive potential. Mutations in CLDN16 cause an autosomal-recessive disorder called "Familial Hypomagnesemia with Hypercalciuria and Nephrocalcinosis" (FHHNC) (42). Patients affected display renal Mg2+ and Ca2<sup>+</sup> wasting accompanied by nephrocalcinosis. This disorder causes in most of the cases end stage renal disease, leading in many, but not all cases to the need for renal transplantation (43). A clinical significant problem is given by the fact that there currently is no general screening procedure at the neonatal stage or later for toddles and schoolchildren on hypercalciuria or hypermagnesemia. Thus, if such patients are referred to a secondary or tertiary center, respectively, chronic renal insufficiency and calcifications have often already progressed to a severe and often, irreversible state. In contrast to the human situation, mice with targeted deletion of CLDN16 recapitulate human renal Mg2+ and Ca2<sup>+</sup> wasting, but do not show any signs of overt renal calcifications and, moreover, no signs of apparent or progressing renal insufficiency (44). There is currently no explanation for this significant difference; however, it is intriguing that solving this striking difference, a possible route for treatment of patients may be opened. A novel recent finding was that the absence of CLDN16 in ameloblasts explains the clinical finding of amelogenesis imperfecta in FHHNC patients and mice with CLDN16 deficiency (45). A similar finding was reported for patients with mutations in the genes coding for CLDN19 and similar for mice (CLDN3) (20, 46).

In 2006, Konrad et al. reported that mutations in human CLDN19 also lead to renal Mg2+ and Ca2<sup>+</sup> wasting, clinically an almost phenocopy of patients with CLDN16 mutations (47). Although still a matter of debate, it is believed that CLDN16 and CLDN19 interact and form a heteromeric paracellular channel, with a cation selectivity including Ca2+ and Mg2<sup>+</sup> (48) (**Figure 4**). However, they could also show that patients harboring CLDN19 mutations suffer from severe ocular involvement, as Claudin-19, but not Claudin-16 is expressed in the retinal pigment epithelium TJs, leading besides the renal Mg2+ and Ca2<sup>+</sup> wasting to major vision problems (47, 49).

Another Claudin (Claudin-10) that contributes to Mg2<sup>+</sup> handling exists at least in two principal forms. Claudin-10a and -10b, both confer different electrophysiological properties (anion-selective channel vs. cation-selective channel with a high preference for Na<sup>+</sup>) and their tissue distribution (Kidney and Uterus for Claudin-10a and ubiquitously for Claudin-10b) (50–52) (**Figure 4**). The expression of the Claudin-10b in almost every epithelial tissue might explain that mice with a targeted deletion die soon after birth. In sharp contrast, the generation of a kidney specific CLDN10-KO mouse (by the use of a ksp-Cre deleter strain) led to a vital mouse model. However, these mice displayed hypermagnesemia, hypocalciuria, nephrocalcinosis, and polyuria (53). Isolated mouse tubules of the TAL demonstrated a decreased paracellular Na<sup>+</sup> permeability as well as higher expression of Claudin-16. Interestingly, recently four independent groups have reported mutations in human CLDN10 (54–57). Bongers et al. reported on two non-related patients presenting with alkalosis, hypokalemia, hypocalciuria, and hypercalcemia and a serum Mg2<sup>+</sup> in the upper range of normal. They identified heterozygous mutations (P149R, Glu157\_Tyr192del, and D73N) in two unrelated families. Hadj-Rabia et al. reported mutations in six patients from two unrelated families (S131L, M1T), resulting in an absence of CLDN10 at the plasma membrane. Affected members had high serum Mg2<sup>+</sup> levels and renal loss of K<sup>+</sup>, Na<sup>+</sup> and Cl<sup>−</sup>. Of interest is that patients also suffered from a variety of skin and teeth disorders (hypolacrymia, ichthyosis, xerostomia, and severe enamel wear). A similar renal phenotype was reported by Klar and colleagues (55). They identified a CLDN10 Mutation (N48K) in two distantly related families with 13 affected individuals presenting with anhidrosis and the inability to produce tears. Although serum levels of Na<sup>+</sup> and K<sup>+</sup> were in the normal range, all patients present with high Mg2<sup>+</sup> serum levels. All groups identified homozygous or compound heterozygous mutations in CLDN10 and demonstrated thereby unequivocally the importance of Claudin-10 for human Mg2<sup>+</sup> homeostasis. However, the recently described homozygote mutation (G163A) in a patient by Terliesner was reported to have normomagnesemia (57). So far, several different mutations in human CLDN10 have been shown to cause a renal tubular disorder that is characterized by hypokalemia, alkalosis, and hypermagnesemia. Moreover, as Claudin-10 is expressed in skin tissues, and several different symptoms of disordered dermal Na+ homeostasis could, therefore, be attributed to this defect.

Interestingly, the full CLDN10 knock-out mouse dies a few hours after birth, indicating that one or more organs different than the kidney and skin must be, if deficient for Claudin-10, vital for survival. Thus, the fact that human mutation does not lead to a lethal phenotype are intriguing in terms of compensatory mechanisms. However, the fact that Claudin-10 is expressed in the lung leads to spectate that the primary cause of postnatal death is caused by the absence of Claudin-10 in the lung (58, 59).

A corresponding mouse model has been generated by Breiderhoff and colleagues, by crossbreeding Claudin-16 deficient mice with a kidney-specific Claudin-10-deficient mouse strain (44, 53). Combining a hypomagnesemic model (CLDN16<sup>−</sup>/ <sup>−</sup>) with a hypermagnesemic model (kidney specific CLDN10<sup>−</sup>/<sup>−</sup>) resulted in a normomagnesemic mouse, thus a "restored" normal phenotype (60). These findings point at the high compensatory, and more than so far anticipated capacity of the DCT. Put in perspective, the development of a selective renal Claudin-10b blocking agent could be a therapeutic option for Claudin-16 patients, since obviously the block of a Na<sup>+</sup> pore restores the capacity of Mg2+ and Ca2<sup>+</sup> recovery more distantly, i.e., the DCT.

### Distal Convoluted Tubule

In the DCT, Mg2+ reabsorption takes place mainly by transcellular route. Here, apical uptake, intracellular buffering, transport, and the extrusion at the basolateral site is concerted by a highly defined and regulated (e.g., hormonal) molecular machinery that has been recently reviewed in Ref. (5, 27). Among the eight different types of Mg2<sup>+</sup> transport factors identified in eukaryotes (TRPM6/M7, Mrs2, MMgT, MagT1, SLC41 family, NIPA, HIP14, and CNNMs) (61–67), only three are expressed at the DCT. The selected list includes (1) the transient receptor potential channel melastatin member 6 (TRPM6) (68), (2) the third member of the solute carrier SLC41A family (SLC41A3) (69), and (3) the "Cyclin and CBS Domain Divalent Metal Cation Transport Mediator-2" (CNNM2), also referred to as ACDP2 (ancient conserved domain protein-2) (63, 65) (**Figure 4**).

These three proteins are classified in the first category of **Table 1** as they are directly involved in Mg2<sup>+</sup> transport or have been related to direct Mg2<sup>+</sup> handling in the DCT.

TRPM6 was the first molecularly identified protein involved in active Mg2<sup>+</sup> reabsorption (68). This channel, which associates in homotetramers, and may also interact with its closest homolog TRPM7 to form heterotetrameric species (70), is five times more permeable to Mg2+ than to Ca2<sup>+</sup>, and permits the reabsorption of these cations through the apical membrane of the epithelial cells (68). The three-dimensional structure of TRPM6 still remains unsolved, but its domain distribution is known and includes a cytosolic N-terminus followed by a transmembrane region of six α-helices and a long intracellular C-terminus that contains a serine-threonine active kinase domain similar to that present in α-kinases. The ion pore is putatively located between the fifth and sixth α-helices of the transmembrane section (71). Interestingly, mutations that impair the phosphorylation of threonine at position 1851 (72) decrease the protein transport activity. These findings have led to propose that autophosphorylation is a key step in the regulatory mechanism of Mg2<sup>+</sup> transport through this channel (73). Clinical or genetical disturbances in TRPM6 are linked to different diseases. For example, mutations in its amino acid sequence cause the rare autosomal-recessive familial hypomagnesemia with secondary hypocalcemia (74–76). Other variants have been linked to hypoparathyroidism (77) and breast cancer (78). Of note, genetic ablation of the TRPM6 gene in mice results in early embryonic lethality (79). Interestingly, TRPM6<sup>±</sup> mice showed reduced expression of the channel in kidney and colon, resulting in mild hypomagnesemia with no hypocalcemia (80).

A set of other mutations in genes that are not primarily associated with Mg2<sup>+</sup>, but relevant for the establishment of the apical membrane potential to drive Mg2<sup>+</sup> entry through TRPM6 (and, therefore, classified in categories 2 and 3 in **Table 1**), have been identified causing secondary Mg2<sup>+</sup> wasting. The corresponding genes code for transcriptional factors (HNF1B and PCBD1), growth factors (EGF), (co)-transporters (NCC, encoded by gene SLC12A3), or even ion-channels (Kir4.1 encoded by KCNJ10) (66, 81–84) (**Figure 4**). According to the gene mutated, the resulting phenotype comprises cystic kidney disease, diabetes, electrolyte disturbances other than Mg2<sup>+</sup>, or seizures. Clinically, hypomagnesemia is, compared to coexisting diabetes (MODY5), the chronic kidney disease and the hyperphenylinaemia a subordinated problem to the patient. However, identifying hypomagnesemia might be of value to identify the comorbidities at an early stage (85). Furthermore, elucidating the mechanisms that contribute to the disordered handling of Mg2<sup>+</sup> in these patients may also enable a better understanding and treatment of diabetes, cyctic kidney disease, and hyperphenylinemia.

Inactivating mutations in SLC12A3 cause Gitelman syndrome, the most frequent cause of hereditary hypomagnesemia and characterized by hypokalemic metabolic alkalosis with hypomagnesemia and hypocalciuria. It has been proposed that a decrease activity of the NCC protein affects the membrane potential necessary for Mg2<sup>+</sup> reabsorption in the apical membrane of DCT by TRPM6 (86–89).

SLC41A3 was originally described by Quamme as part of the solute carrier family 41 (65), which encompasses three integral cytoplasmic membrane putative Mg2<sup>+</sup> transporters (SLC41A1, -A2, and -A3) (90). Mutations in this family are linked to Parkinson's disease (91), diabetes (92), and nephrolithiasis (93). As in the case of TRPM6, the three-dimensional structure of these transporters remains unsolved, but is known to be built up of 10 or 11 transmembrane α-helices (94). SLC41A3, whose molecular function and interaction partners remain also elusive, is the highest enriched member in the DCT. Recently, de Baaij et al. found that, a Slc41a3−/− knockout mice suffer from hypomagnesemia and normomagnesiuria, accompanied by upregulation of TRPM6 and SLC41A (69). These results underlined the relevant role played by SLC41A3 in Mg2<sup>+</sup> reabsorption.

The third identified Mg2<sup>+</sup> transport mediator expressed in the DCT is CNNM2, which belongs to the Cyclin M family. This family encompasses four different members (CNNM1-4). Mutations in CNNM2 cause dominant familial hypomagnesemia (67), and have been linked to brain development anomalies (95), hypertension, diabetes, and obesity (96, 97). Moreover, the CNNM2 locus has been linked by GWAS to neuro-psychiatric disorders (e.g., Schizophrenia) (98, 99). In 2014, Arjona et al. found that knockdown of CNNM2 orthologs in zebrafish results in brain abnormalities, increase of spontaneous contractions, and Mg2<sup>+</sup> waste (95). These authors also identified five new families with mutations in CNNM2 that suffered hypomagnesemia with mental retardation and seizures. These findings suggested an essential role of CNNM2 in Mg2<sup>+</sup> homeostasis and brain development. The relevant role of CNNM2 is underlined by the fact that mice lacking CNNM2 are embryonic lethal (100). Heterozygous (Cnnm2<sup>+</sup>/<sup>−</sup>) mice show lower Mg2<sup>+</sup> levels in serum, thus suggesting defects in Mg2<sup>+</sup> reabsorption in kidney. In addition, these animals showed lower blood pressure than compared to control mice. These results highlighted the importance of Mg2<sup>+</sup> and its reabsorption in the kidney to maintain blood pressure (100).

The CNNMs represent the least-studied members across the mammalian transporters and share with MgtE and with the CLC family of chloride channels the presence of a cystathionine β-synthase (CBS) domain pair in their amino acid sequence (101–103). The four CNNM family members were first identified in 2003 by Wang et al. (61, 104) and show very strong homology to the bacterial CorC protein [which is involved in Mg2<sup>+</sup> and cobalt (Co2<sup>+</sup>) efflux (63)], and with the Mam3p proteins (67, 105). It was initially suggested that CNNMs might be involved in cell-cycle regulation (61), as they contain a cyclin box-like motif and are located in the plasma membrane. However, the cyclin M function has that far not been proved *in vivo*.

The second member of the Cnnm family, CNNM2, is abundant in brain and kidney (64, 65), and shows a complex modular architecture composed by four structural domains (**Figure 5**) (106). The N-terminal section (likely to be an extracellular compoment) consists of a β-stranded enriched region (residues 1–250) and precedes a DUF21 domain (residues 251–400, Pfam code PF01595) with three or four transmembrane α-helices (107).

The following intracellular region includes a CBS domain pair (so called "Bateman" module; Pfam code PF00571) (106) and a C-terminal cyclic nucleotide monophosphate (cNMP) like binding domain (Pfam code PF00027) (106) (**Figure 5**). Although the concrete function of each domain remains unknown, recent biophysical and structural data supports a regulatory role for the Bateman module.

### Mechanisms of Mg2**+** Transport in the DCT and CNNM2

Despite the universally recognized relevance of magnesium in maintaining key life processes as mentioned above, current knowledge about the CNNMs and their role in the DCT, as well as the molecular mechanisms involved in its transport across the cellular membranes remain still to be explored. This is largely due to the scarce structural information available on Mg2<sup>+</sup> transporters and channels, that so far was limited to the crystal structures of two prokaryotic proteins: (i) CorA from *Thermotoga maritima* (108–111) and (ii) MgtE from *Thermus thermophilus* (112–115). These proteins are homologs of two eukaryotic Mg2+ transport mediators: the mitochondrial Mg2<sup>+</sup> channel Mrs2 and the solute carrier (SLC) family 41 members (homologs of CorA and MgtE, respectively). CorA represents the major transport machinery responsible for Mg2<sup>+</sup> uptake in bacteria and it translocates Mg2<sup>+</sup> by using an inwardly biased electrochemical gradient that serves as the driving force for Mg2<sup>+</sup> permeation (108, 109, 111, 116).

MgtE is a dimeric Mg2<sup>+</sup> selective channel (117) that permeates Mg2+ ions and maintains the intracellular Mg2<sup>+</sup> homeostasis in bacteria. MgtE shares with CNNM2 (but not with SLC41) the presence of an intracellular CBS domain pair (101, 102). An ATP/Mg-dependent open-to-close gating process that involves binding of the nucleotide at this region defines the threshold of intracellular Mg2<sup>+</sup> for the channel inactivation and provides sensory capacity to this protein (113, 114, 118).

The recent elucidation of the crystal structure of the Bateman module of CNNM2 (119, 120) has shed new light on the molecular mechanisms underlying Mg2+ transport through the basolateral membrane of the DCT (**Figure 6**). We and others recently confirmed that this region may host ATP in a Mg2<sup>+</sup>-dependent manner, as well as independent Mg2<sup>+</sup> atoms that interact with some acidic clusters located in the CBS1 motifs (106, 119). The fact that the Mg2<sup>+</sup> independent sites are far away from the nucleotide suggest that, as observed in MgtE (114), binding of

Figure 6 | Crystal structure of the Bateman module of CNNM2. (A) The Bateman module of CNNM2 consists of two consecutive cystathionine β-synthase (CBS) motifs (CBS1, residues 445–508; CBS2, residues 509–578). A long extended loop links strands β5 and β6 in the CBS2 motif. The H0 helix connects CBS1 with the DUF21 transmembrane domain in the full-length protein. The H4 helix connects CBS2 with the cyclic nucleotide monophosphate domain. Nucleotides, ca. AMP (blue), ADP (green), or adenosine triphosphosphate (ATP) (orange) bind independently at the S2 site, thus disrupting the interactions formerly existing in the cavity between residues of the CBS1 and CBS2 motifs. This induces a displacement of helices H0, H1, and H4 in each Bateman subunit. The apo- and the nucleotide-bound Bateman module are represented in green and marine, respectively. The crystal structure of the T568I protein variant is in red. As shown, the T568I mutation mimics the structural effect of ATP binding; although in the first case, the structural change is irreversible, thus locking the protein in the nucleotide-bound like conformation. (B,C) *Conformational changes induced by ATP*. The Bateman module of CNNM2 associates in disk-like dimers known as "CBS modules," which adopt a *twisted* (B) or a *flat* (C) state depending on whether the site S2 of each subunit is empty or hosts an ATP (or MgATP) molecule, respectively. Note that the H0 helices connecting the CBS2 motif with the DUF21 domain are differently oriented in each case, thus likely transmitting the transformation suffered by the Bateman module to the transmembrane region.

Mg2<sup>+</sup> at concrete positions may not be directly coupled with ATP binding, although it may complement its effect in conformational transformations suffered by the whole module (106, 121).

The Bateman module of CNNM2 features two major cavities (named as S1 and S2) that are located at opposite ends of the central β-sheets of the CBS motifs. In contrast with S1, which is partially occluded and full of bulky residues, the site S2 is fully accessible and can accommodate phospho-nucleotides, such as AMP, ADP, or ATP (106, 121) (**Figure 6**). Site S2 is built by three different structural blocks: (i) the central residues from the linker preceding the first β-strand (β1) of the CBS1 domain, (ii) the C-terminal residues from the last β-strand (β6) of the CBS2 motif, and (iii) the first two turns of helix H4 of CBS2 (**Figure 6**). The upper and right walls of the cavity are mainly hydrophobic and help accommodating the bulky adenine ring of ATP (106, 121). By contrast, the left wall of the cleft is hydrophilic and is built from the last β-strand and the following α-helix (H4) of the CBS2 motif. A conserved threonine (T568) and an aspartate residue (D571) from this helix are key in help orienting the ribose ring of the nucleosides inside the cavity, and if mutated, impede the allocation of ATP inside (106). Interestingly, the repulsive effect otherwise exerted by the acidic cluster formed by residues E570, D571, and E574 (at the first turn of α-helix H4 of CBS2) over the polyphosphate chain of ATP (106) is neutralized by the Mg atom that accompanies the ATP molecule. The positive dipole end of helix H4 and the nearby arginine residue, R480, complement the neutralizing effect (106). The Bateman module of CNNMs associates in head-to-head oriented disk-like dimers known as *CBS modules* (**Figures 6B,C**) (106, 121).

At low concentrations of Mg2<sup>+</sup> and in the absence of MgATP, the CBS module adopts a "twisted" shape (**Figure 6B**), in which the CBS2 domains from complementary subunits remain in contact while the CBS1 motifs are separated and retain only scarce hydrophobic interactions. Binding of MgATP at site S2 disrupts a network of H-bonds centered on the conserved threonine at

Figure 7 | Mechanism of Mg2+ transport at the distal convoluted tubule (DCT). (A) Mg2+ enters into the DCT epithelial cells through the apical membrane with the help of TRPM6/7 channels. At low Mg2+ concentrations, the cystathionine β-synthase (CBS) module of CNNM2, located at the basolateral membrane, remains in its twisted conformation. (B,C) Upon increasing the intracellular concentration of Mg2+, binding of these cations and of MgATP to CNNM2, triggers the progression of the CBS module toward its flat state, and the transport of Mg2+ through the basolateral membrane toward the blood torrent. In addition, an increased intracellular Mg2+ concentration inhibits apical transport by TRPM6.

position 568 (T568) as well as a salt bridge between R480 and E570 and causes the displacement the C-terminal helix of the CBS2 domain (helix H4) as well as of the long α-helix (helix H0) that connects the Bateman module with the DUF21 domain (**Figure 5**). These structural changes, which occur concomitantly in the two complementary subunits of the dimer, trigger an overall rearrangement of the CBS module that makes it to evolve from a "twisted" (**Figure 6B**) toward a "flat" disk structure (**Figure 6C**). The conformational effect of ATP is likely transferred to the DUF21 transmembrane domain through helix (H0) that connects it with the CBS2 motif. This sequence of events has been postulated as the mechanism by which CNNM2 might regulate the gating of Mg2<sup>+</sup> ions through the basolateral cell membrane (106, 121) (**Figure 7**).

### CNNM2 and MgtE Differ in Their CBS-Domain-Mediated Gating Mechanism

Interestingly, an ATP/Mg-mediated gating process ruled out by CBS domains has also been postulated as the mechanism by which the MgtE transporter senses and regulate the Mg2<sup>+</sup> homeostasis in bacteria (114). In contrast with the twisted-to-flat transformation observed in CNNM, the rearrangement of the Bateman modules of MgtE responds to an open-to-close mechanism (113–115). In the absence of Mg2<sup>+</sup> ions, the complementary CBS2 motifs of MgtE subunits remain apart in the dimer due to the repulsion exerted by acidic clusters located at the interfacial helices of the CBS2 domains. In this state, the CBS module impairs the transport of Mg through the membrane and maintains the protein in an "open" state. While the intracellular concentration of Mg2<sup>+</sup> is low, the CBS module remains open and allows the influx of Mg2<sup>+</sup> ions toward the interior of the cell. Upon increasing the intracellular concentration of Mg2<sup>+</sup>, the pre-existing repulsive acidic clusters become sequentially neutralized by newly bound Mg2<sup>+</sup> ions, thus allowing the approximation of the CBS2 motifs. The sequential binding of Mg atoms progressively causes a closure of the CBS module that, when completed, adopts a "flat" disk-like arrangement as that observed in the MgATP/Mg2<sup>+</sup> bound form of CNNM2 (106). The new state is transferred to the transmembrane region and results in the closure of the membrane pore (114).

### CONCLUSION

The identification of mutations in human genes has led to a deeper understanding of Mg2<sup>+</sup> handling in health and disease.

### REFERENCES


On that basis, technologies, such as mouse genetic engineering as well as crystallography, have contributed in this field of physiology and pathophysiology. However, our current knowledge of the molecular mechanisms underlying magnesium transport through the cell membranes is very scarce, and represents an incipient field of research that will mature, as we are able to identify new molecular partners involved in this process. Proteins involved in trans- and paracellular pathways have the potential of being key players in Magnesium Homeostasis and also other disorders, such as diabetes, hypertension, and schizophrenia, thus being a potential target for pharmaceutical interventions.

### AUTHOR CONTRIBUTIONS

All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

### FUNDING

This work was supported in part by Departamento de Educación, Universidades e Investigación del Gobierno Vasco Grant PI2010-17, Departamento de Industria, Innovación, Comercio y Turismo del Gobierno Vasco Grants ETORTEK IE05-14 and IE07-202, Diputación Foral de Bizkaia Grants 7/13/08/2006/11 and 7/13/08/2005/14, Spanish Ministerio de Ciencia e Innovación (MICINN), Grant BFU2010-17857, Spanish Ministry of Economy and Competitiveness Grant BFU2013-47531-R, BFU2016-77408-R from Spanish Ministry of Economy and Competitiveness (MINECO) and Ministerio de Ciencia e Innovación CONSOLIDER-INGENIO 2010 Program Grant CSD2008-00005 (to LM-C). We also thank MINECO for the Severo Ochoa Excellence Accreditation (SEV-2016-0644) and by a PhD fellowship from MINECO (REF BES-2014-068464) awarded to PG-M. DM was supported by the German Research Foundation (DFG, Graduate School 2318) and by The Berlin Institute of Health (BIH) G(CRG 12.01.134).


paracellular sodium permeability and leads to hypermagnesemia and nephrocalcinosis. *Proc Natl Acad Sci U S A* (2012) 109(35):14241–6. doi:10.1073/ pnas.1203834109


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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Giménez-Mascarell, Schirrmacher, Martínez-Cruz and Müller. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Update on Hereditary Kidney Stone Disease and Introduction of a New Clinical Patient Registry in Germany

#### *Jan Halbritter1 \*, Anna Seidel1 , Luise Müller1 , Ria Schönauer1 and Bernd Hoppe2*

*1Division of Nephrology, Department of Internal Medicine, University of Leipzig, Leipzig, Germany, 2Division of Pediatric Nephrology, University Children's Hospital, Bonn, Germany*

Kidney stone disease is an increasingly prevalent condition with remarkable clinical heterogeneity, with regards to stone composition, age of manifestation, rate of recurrence, and impairment of kidney function. Calcium-based kidney stones account for the vast majority of cases, but their etiology is poorly understood, notably their genetic drivers. As recent studies indicate, hereditary conditions are most likely underestimated in prevalence, and new disease genes are constantly being identified. As a consequence, there is an urgent need of a more efficient documentation and collection of cases with underlying hereditary conditions, to better understand shared phenotypic presentation and common molecular mechanisms. By implementation of a centralized patient registry on hereditary kidney stone disease in Germany, we aim to help closing the vast knowledge gap on genetics of kidney stone disease. In this context, clinical registries are indispensable for several reasons: first, delineating better phenotype–genotype associations will allow more precise patient stratification in future clinical research studies. Second, identifying new disease genes and new mechanisms will further reduce the rate of unknown nephrolithiasis/nephrocalcinosis etiology; and third, deciphering new molecular targets will pave the way to develop drugs for recurrence prevention in severely affected families.

#### *Edited by:*

*Max Christoph Liebau, Universitätsklinikum Köln, Germany*

#### *Reviewed by:*

*Fatos Yalcinkaya, Ankara University, Turkey Gianpaolo De Filippo, Bicêtre Hospital, France David J. Sas, Mayo Clinic, United States*

*\*Correspondence:*

*Jan Halbritter jan.halbritter@medizin. uni-leipzig.de*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 04 January 2018 Accepted: 19 February 2018 Published: 07 March 2018*

#### *Citation:*

*Halbritter J, Seidel A, Müller L, Schönauer R and Hoppe B (2018) Update on Hereditary Kidney Stone Disease and Introduction of a New Clinical Patient Registry in Germany. Front. Pediatr. 6:47. doi: 10.3389/fped.2018.00047*

#### Keywords: nephrolithiasis, hereditary, nephrocalcinosis, kidney stone disease, monogenic, registry

Incidence and prevalence of kidney stone disease continues to rise in the general population. With a lifetime prevalence of up to 10%, nephrolithiasis (NL) and nephrocalcinosis (NC) are therefore major health burdens, especially in the Western World (1). NL and NC are associated with significant morbidity and progression to chronic kidney disease due to recurrence, repetitive surgical/endoscopic intervention, and concomitant inflammation. On a simplified level, kidney stone formation results from an imbalance of urinary inhibitors (e.g., citrate, magnesium, uromoduline, and pyrophosphate) and promoters (e.g., oxalate, calcium, phosphate, urate, and cystine) of crystallization, exceeding supersaturation with consecutive aggregation, nucleation, and stone growth at Randall's plaque (**Figure 1**). This imbalance can be due to altered enteral and/or renal handling of either promotors or inhibitors, such as enteral malsecretion of oxalate or renal malreabsorption of calcium (**Figure 1**).

The underlying etiology of NL is thought to be multifactorial with an environmental, notable dietary, hormonal, and genetic component. In twin studies, the heritability of kidney stones has been estimated at 56% (3), and up to two-thirds of hypercalciuric stone formers have relatives with NL (4). Although calcium-containing kidney stones account for more than 80% of all, the genetic basis of such stones remains largely unknown (5). Except for variants in *CLDN14*, *TRPV5*, *SLC34A1*, *ALPL*, *CASR*, and *UMOD*, genome-wide association studies have yet to yield substantial genetic factors (6–8). However, risk alleles have been identified within genes that were also found to transmit

**207**

the disease on a Mendelian basis, such as *CASR*, *SLC34A1*, and *SLC2A9* (9, 10). To date, more than 30 single genes with an Online Mendelian Inheritance in Man-defined phenotype have been identified to be implicated in NL/NC, if mutated (**Table 1**).

Modes of inheritance in monogenic forms include autosomaldominant, autosomal-recessive, and X-linked transmission. Interestingly, in several of these genes, both recessive and dominant modes of inheritance have been reported: *SLC7A9*, *SLC34A1*, *SLC34A3*, *SLC2A9*, *SLC22A12*, and *SLC4A1*. While most of the syndromic and severe congenital disorders exhibit a recessive inheritance pattern (Bartter, Lowe, Dent, FHHNC, and distal renal tubular acidosis with sensorineural deafness), milder conditions are rather associated with mutations in dominant genes. The majority of encoded proteins constitute renal solute transporters (e.g., SLC34A1, SLC34A3, and SLC9A3R1), but also chloride channels (CLCN5), tight-junction proteins (e.g., CLDN16/CLDN19), and metabolizing enzymes (e.g., AGXT, APRT, and CYP24A1) have been found defective in patients with NL/NC. Hence, the underlying defect is mostly located in the tubular system of the kidney itself and can therefore be attributed as tubulopathy. Conversely, *a priori* extrarenal conditions, as in primary hyperoxaluria (PH) where dysfunction of liver enzymes (AGXT, GRHPR, and HOGA1) cause oxalate accumulation with secondary renal affection, are conceivable causes of NL/NC. Although each disease phenotype is thought to represent a relatively rare entity, single-gene causes may account for a significant number of patients by their broad genetic heterogeneity (42). Apart from genetic heterogeneity, there is also an allelic variation, where truncating variants rather result in a loss of function and missense variants (hypomorphs) may cause rather subtle defects, which can be clinically overseen, especially in adult stone formers. Another recently appreciated phenomenon is about gene dosage effects in several of the aforementioned kidney stone genes. In *SLC34A3* for instance, encoding one of the main phosphate transporters in the proximal tubule (NaPiIIc), it was shown that heterozygous individuals can no longer be merely regarded as healthy carriers, as they display renal calcifications and/or bone manifestation significantly more frequent than wild-type individuals; but still to a lesser degree than biallelic (homozygous and compound heterozygous) individuals (43). Similar observations were reported for families with mutations in *CYP24A1* (44). The contribution of monogenic disorders to the overall prevalence of kidney stone disease has not been studied comprehensively in the past. Especially, genetic evidence based on broad screenings of a multitude of causative genes in large patient cohorts is lacking. Comprehensive genetic testing has been too costly and inefficient in the past. For most individuals with NL/NC, mutation analysis for a causative genetic defect has therefore not been accessible, despite the fact that knowledge of the molecular cause of NL/NC may have important consequences for prognosis, prophylaxis and/or treatment. Only rough estimates have been derived from clinical observation studies: based on a huge data collection of stone composition analysis, it was concluded that monogenic causes do not exceed 9.6% in children and 1.6% in adults (45). In the last decade, however, this situation has begun to change, with the advent of high-throughput sequencing techniques.


Table 1 | Genes, known to cause monogenic forms of NL/NC.

### HIGH-THROUGHPUT MUTATION ANALYSIS IN PATIENTS WITH NL/NC

To investigate patients with kidney stone disease for the presence of pathogenic mutations in known disease genes, we established a gene panel based on microfluidic multiplex-PCR and consecutive NextGen sequencing (Fluidigm™/NGS) (46, 47).

In a "pilot-study," we consecutively recruited 268 genetically unresolved individuals from typical kidney stone clinics; 102 pediatric and 166 adult probands. As a result, we identified 50 deleterious variants in 14 out of 30 analyzed genes, leading to a molecular diagnosis in 15% of all cases. In the pediatric subgroup, we detected a causative mutation in 21%, while among adults, deleterious variants were present in 11% (**Figure 2A**) (48). Mutations in the cystinuria-gene *SLC7A9* were found most frequently in the adult cohort (**Figure 2B**). Two follow-up studies were able to confirm these results. First, in an exclusively pediatric cohort of 143 NL/NC patients, 17% of cases were explained by mutations in 14 different genes (49). Second, in a cohort of 51 families with age of NL/NC manifestation before 25 years, targeted WES was used to detect a genetic cause in almost 30% (50). Not surprisingly, recessive mutations were more frequently found among neonates and in cases of congenital disease, whereas dominant conditions usually manifested later in life. These data indicate that genetic kidney stone disease is an underdiagnosed condition, despite the fact that the molecular diagnosis will potentially influence prognosis, prophylaxis, and/or treatment. A limitation worth mentioning, however, is a potential selection bias due to recruitment from specialist kidney stone clinics in all of the three aforementioned studies.

### IDENTIFICATION OF NOVEL HUMAN DISEASE GENES BY CANDIDATE-GENE APPROACH

High-throughput mutation analysis is also used to screen for pathogenic variants in various candidate genes. One of the most interesting recent findings was the discovery of human mutations in *SLC26A1* (32). Since the first description of Ca-oxalate (CaOx) kidney stone formation and NC in Slc26a1 (Sat1)-knockout mice by Dawson et al. in 2010, *SLC26A1* has been a bona fide NL-candidate gene (51). SLC26A1 encodes an anion exchanger expressed at the basolateral membrane of proximal renal tubules, ileum, and jejunum. Consequently, by using a candidate-gene approach, pathogenic variants were identified in humans with a history of early onset CaOx-NL, namely, two unrelated individuals with biallelic missense variants (32). Functionally, pathogenicity of the identified variants was demonstrated *in vitro*, leading to intracellular mis-trafficking and impaired transport activity (32). Defective SLC26A1 therefore constitutes a new cause of CaOx-NL and should be considered when testing individuals for causes of recurrent CaOx-stone formation.

### NEW CLINICAL PATIENT REGISTRY FOR HEREDITARY KIDNEY STONE DISEASE

Most epidemiological data on increasing prevalence in Western countries are derived from US databases. Although urgently needed, centralized European databases are not available at the time. As aforementioned genetic studies on prevalence of hereditary kidney stone disease were executed with small cohorts from specialized centers in both Europe and the US, a translation to the general situation in Europe is not valid. While in the US, the *Rare Kidney Stone Consortium* constitutes a platform that integrates and coordinates registry, basic science, and clinical research activities for rare conditions such as cystinuria, PH, APRT deficiency, Dent and Lowe disease, no comparable data collection on patients with hereditary kidney stone disease has been implemented neither in Europe nor in Germany today. In collaboration with the existing European PH registry, *OxalEurope* (Prof. Bernd Hoppe, University of Bonn), and through funding by *Deutsche Forschungsgemeinschaft* and *Else Kröner-Fresenius Stiftung*, we recently established a clinical

Table 2 | Inclusion criteria for mutation analysis in clinical patient registry.

#### Clinical criteria

Pediatric age of onset or onset during early adulthood (<40 years) plus Positive family history or Recurrence (>3×) or Indicative phenotype (e.g., RTA, cystinuria, and NC) or Established molecular genetic diagnosis

patient "Registry for hereditary kidney stone disease" at the University of Leipzig. The registry is nationally supported by the German Societies of Adult Nephrology (DG*f*N) and Pediatric Nephrology (GPN). It is further enrolled at the German Clinical Trials Register (DRKS-ID: DRKS00012891). As a fundamental part of study recruitment, high-throughput mutation analysis for known and novel kidney stone genes is offered on a research basis for patients without an established molecular diagnosis but with a clinical picture that points to an underlying genetic susceptibility: e.g., early age of onset (<40 years), positive family history, indicative phenotypes such as NC, cystinuria, or RTA, and severely recurrent NL (>3×) (**Table 2**). While patients with an already established genetic diagnosis are generally enrolled, cases with secondary NL/NC causes, such as malignancy, sarcoidosis, and primary hyperparathyroidism, do not get included in genetic analysis. To actively enroll patients, a clinical center will usually need approval by the local Institutional Review Board; a process for which we offer our help and assistance by providing respective templates. Upon ethics approval, consent form and clinical data sheets (e.g., clinical questionnaire) can be downloaded from our registry website (http://www.mksregistry.net). To ensure thorough clinical phenotyping, we will be asking for substantial patient information such as ethnicity, consanguinity, family history, age of onset, recurrence (defined as every putatively new kidney stone event), daily fluid intake, surgical interventions, and extrarenal involvement among others. The documents can be filled in by the patient with the help of the enrolling physician. In addition, biochemical serum parameters, including creatinine, eGFR, PTH, vitamin D, electrolytes, uric acid, and urinalysis (pH, calcium, phosphate, magnesium, uric acid, citrate, oxalate, and cystine, preferably from 24-h urine, if not spot urine), as well as data on stone composition analysis will be requested upon enrollment. Taking into account that 24-h urine collection and stone composition analysis is not routinely performed at all institutions, we include these parameters upon availability. 2-yearly clinical follow-up visits of enrolled patients are desirable but not mandatory. After registration, recruiting clinical centers will be provided with a personalized login to enter patient data *via* our registry website

### REFERENCES


(http://www.mks-registry.net). Alternatively, we offer entering the data electronically when sent to us on paper. Entered data will be stored on a secured server and can be accessed by participating clinical centers to view their own patient data. The following websites provide further information:

https://www.dgfn.eu/hereditaere-nierensteinleiden.html https://www.drks.de/drks\_web/navigate.do?navigationId=trial. HTML&TRIAL\_ID=DRKS00012891

In summary, kidney stone disease is an increasingly prevalent condition which is clinically heterogeneous and poorly understood, notably its genetic drivers. As a series of recent studies indicated, monogenic conditions are most likely underestimated in prevalence. By implementation of a centralized patient registry on hereditary kidney stone disease, we will contribute to overcome, at least in part, the vast knowledge gap on genetics of kidney stone disease. In this context, clinical registries are valuable sources for several reasons: first, delineating better phenotype–genotype associations will be crucial for more precise patient stratification in future clinical research studies. Second, identifying new disease genes with new disease mechanisms will diminish the gap of unknown NL/NC etiology; and third, deciphering new molecular targets helps to pave the way for developing drugs of recurrence prevention in severely affected families.

### ETHICS STATEMENT

This study was carried out in accordance with the recommendations of "Ethikkommission an der Medizinischen Fakultät der Universität Leipzig" with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the "Ethikkommission an der Medizinischen Fakultät der Universität Leipzig."

### AUTHOR CONTRIBUTIONS

JH conceived and wrote the manuscript. BH, AS, LM, and RS edited the manuscript and built up the registry's infrastructure that is introduced to the reader.

### FUNDING

The research is funded by project grants from DFG (HA 6908/2-1) and EKFS (2016\_A52) to JH. This work was further supported by the Federal Ministry of Education and Research (BMBF), Germany, FKZ: 01EO1501 to JH.

(VET) Registry. *Kidney Int* (2005) 67:1053–61. doi:10.1111/j.1523-1755.2005. 00170.x


with kidney stones and bone mineral density. *Nat Genet* (2009) 41:926–30. doi:10.1038/ng.404


diagnostiques et thérapeutiques. *Nephrol Ther* (2008) 4:231–55. doi:10.1016/j. nephro.2007.12.005


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Halbritter, Seidel, Müller, Schönauer and Hoppe. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Renal Cell Carcinoma in von Hippel–Lindau Disease—From Tumor Genetics to Novel Therapeutic Strategies

*Emily Kim1,2 and Stefan Zschiedrich3 \**

*1Department of Radiation Oncology, Faculty of Medicine, Albert Ludwigs University of Freiburg, Freiburg, Germany, 2German Cancer Consortium (DKTK), Partner Site Freiburg, Freiburg, Germany, 3Renal Division, Department of Medicine IV, Faculty of Medicine, Albert Ludwigs University of Freiburg, Freiburg, Germany*

von Hippel–Lindau (VHL) disease is an autosomal dominant syndrome caused by mutations in the VHL tumor-suppressor gene, leading to the dysregulation of many hypoxia-induced genes. Affected individuals are at increased risk of developing recurrent and bilateral kidney cysts and dysplastic lesions which may progress to clear cell renal cell carcinoma (ccRCC). Following the eponymous *VHL* gene inactivation, ccRCCs evolve through additional genetic alterations, resulting in both intratumor and intertumor heterogeneity. Genomic studies have identified frequent mutations in genes involved in epigenetic regulation and phosphoinositide 3-kinase–AKT–mechanistic target of rapamycin (mTOR) pathway activation. Currently, local therapeutic options include nephron-sparing surgery and alternative ablative procedures. For advanced metastatic disease, systemic treatment, including inhibition of vascular endothelial growth factor pathways and mTOR pathways, as well as immunotherapy are available. Multimodal therapy, targeting multiple signaling pathways and/or enhancing the immune response, is currently being investigated. A deeper understanding of the fundamental biology of ccRCC development and progression, as well as the development of novel and targeted therapies will be accelerated by new preclinical models, which will greatly inform the search for clinical biomarkers for diagnosis, prognosis, and response to treatment.

Keywords: von Hippel–Lindau disease, renal cell carcinoma, cancer genetics, predictive biomarkers, preclinical models, new therapeutic targets

### INTRODUCTION

von Hippel–Lindau (VHL) disease is an autosomal dominant, multiorgan visceral cysts and tumor syndrome. The disease name derives from the German ophthalmologist Eugen von Hippel who studied two cases of striking retinal angiomas and the Swedish pathologist Arvid Lindau who detected a connection between cerebellar hemangioblastomas, retinal angiomas, and other visceral tumors (1, 2). The first report of VHL dates from 1894, when Collins described vascular intraocular tumors in two siblings (3). VHL manifestations can be found in retinal hemangioblastoma, cerebellar and spinal hemangioblastoma, renal cysts and clear cell renal cell carcinoma (ccRCC), liver hemangioma, pancreatic cysts, pancreatic microcystic serous adenoma and pancreatic neuroendocrine tumors, pheochromocytoma (PCC), epididymal and broad ligament cystadenoma, and endolymphatic sac

#### *Edited by:*

*Max Christoph Liebau, Universitätsklinikum Köln, Germany*

#### *Reviewed by:*

*Michal Malina, Charles University, Czechia Elena Ranieri, University of Foggia, Italy*

*\*Correspondence: Stefan Zschiedrich stefan.zschiedrich@uniklinikfreiburg.de*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 20 October 2017 Accepted: 16 January 2018 Published: 09 February 2018*

#### *Citation:*

*Kim E and Zschiedrich S (2018) Renal Cell Carcinoma in von Hippel–Lindau Disease—From Tumor Genetics to Novel Therapeutic Strategies. Front. Pediatr. 6:16. doi: 10.3389/fped.2018.00016*

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tumors. In 1964, Melmon and Rosen suggested clinical diagnostic criteria that are still valid today. VHL disease has been classified depending on the presence of PCC in VHL type 1 (without PCC) and type 2 (with PCC). VHL type 2 is further subclassified in type 2A (with PCC but without ccRCC), type 2B (with PCC and ccRCC), and type 2C (PCC only) (4–6). Practically, however, practitioners do not rely on this classification because families can move between clinical subtypes.

von Hippel–Lindau disease is caused by mutations in the *VHL* tumor-suppressor gene, located on chromosome 3p25- 26. *VHL* was mapped to chromosome 3 in 1988 and cloned in 1993 (7, 8). The incidence of VHL disease is approximately 1:36,000 (9). Although VHL disease typically presents in early adulthood, manifestation of retinal angiomas, PCCs, and ccRCCs has been reported earlier; therefore, guidelines recommend starting surveillance programs for eye examination at 2–5 years and abdominal imaging at 6–10 years (10–12).

Most patients develop ccRCCs that arise from microscopic precursor lesions present in both kidneys. The number of nonmalignant cysts lined with clear cells in an average VHL kidney was estimated to be 1,100, and the number of clear cell renal neoplasms (solid and cystic) to be 600 (13). Optimally, early RCC can be detected, observed, and surgically removed before progression to metastatic disease (14). However, repeated surgery for multifocal bilateral disease is followed by increased risk of end-stage renal disease (ESRD) requiring renal transplantation or dialysis.

### THE ROLE OF VHL

Whether hereditary or sporadic, ccRCC is characterized by mutations in the VHL tumor-suppressor gene on chromosome 3 (3p25-26) and a subsequent loss of heterozygosity. VHL, Elongin B (encoded by *TCEB1*), and Elongin C form a stable complex that targets hypoxia-inducible factor α (HIFα) subunits for proteolytic degradation under normoxic conditions (15). In the presence of hypoxia or in the absence of functional VHL tumor-suppressor protein, HIFα subunits HIF1α and HIF2α are stabilized, binding together with ARNT (HIF1β) to hypoxia-response elements to activate genes involved in angiogenesis, cell cycle, cell proliferation, glucose, and lipid metabolism, among others. Mutations of *TCEB1* that abrogate binding of Elongin B to VHL can also increase HIFα expression in ccRCCs with intact *VHL* (16). Mutations in *VHL* and *TCEB1* were mutually exclusive, supporting a permissive role for VHL complex degradation and HIFα stabilization in tumorigenesis.

### ADDITIONAL GENE MUTATIONS

Approximately 15 years after *VHL* was identified as the genetic basis for VHL, further driver mutations for ccRCC were identified, summarized in **Table 1**. Exome sequencing of tumors identified additional inactivating mutations on chromosome 3 in tumor suppressors polybromo-1 (*PBRM1*), SET domaincontaining 2 (*SETD2*), and BRCA1-associated protein-1 (*BAP1*), which are chromatin and histone regulators located at 3p21 (17–19). *PBRM1* and *BAP1* mutations are mutually exclusive, with *BAP1* mutations correlating with higher grade disease (19, 20). Large-scale genomic sequencing showed that these three tumor-suppressor genes, located near *VHL* at 3p21, are the most frequently mutated genes in ccRCC after *VHL* (16, 21). Other significantly mutated genes included histone-modifiers *KDM5C* and *KDM6A*, previously implicated in ccRCC (17–19, 22), genes in the phosphoinositide 3-kinase (PI3K)/AKT pathway, such as mechanistic target of rapamycin (*mTOR*), *PIK3CA*, and *PTEN*, as well as *TCEB1* (Elongin C) (16, 21). The tumor-suppressor gene *TP53* is infrequently mutated, playing a lesser role than in many other solid tumors.

### TUMOR HETEROGENEITY

Clear cell renal cell carcinoma has a moderate somatic mutation frequency compared with other solid tumors and these mutations progress in a branched evolutionary manner (24, 25). The evolutionary history of sporadic ccRCC in 10 individuals was investigated by exome sequencing of multiregion samples from primary ccRCCs and metastases (26). Intratumor heterogeneity was present in all tumors, indicating that a single biopsy underestimates the genomic complexity of a tumor. Tumor phylogeny, similar to an evolutionary tree, showed that inactivation of *VHL* and loss of chromosome 3p were ubiquitous early truncal events. *PBRM1* inactivation was a frequent mutation, occurring early as a truncal mutation in three tumors and as a later event in three


*Loss of heterozygosity at 3p was reported in over 90% of cases, and mutations in components of the PI3K–AKT–mTOR pathway in 28–76%.*

others. Distinct subclones, spatially separated within a single tumor, contained mutations that appeared in a branched rather than linear manner. Subclonal driver mutations were similar to those identified by earlier studies, such as chromatin modifiers, regulators of mTORC1 pathway, and tumor-suppressor *TP53*. Parallel evolution was observed in certain genes, in which different evolutionary paths or branches in a tumor resulted in inactivation of the same gene by separate mechanisms. Recurrent mutations in *PBRM1*, *SETD2*, *BAP1*, and *KDM5C* suggest an evolutionary selection for epigenetic dysregulation in tumorigenesis. Branched subclonal mutations were highly variable and contained more C>T transitions than truncal mutations, potentially useful as prognostic and predictive biomarkers.

Genomic analysis of ccRCC has established the fundamental role for VHL inactivation and HIF dysregulation, the importance of chromatin regulation and histone modification, and the involvement of the mTORC1 pathway. The central role of chromosome remodeling in the development and the progression of ccRCC implicates epigenetic dysregulation as a permissive factor in tumorigenesis and a novel target for therapeutic agents and candidate biomarkers. However, the molecular mechanisms whereby epigenetic alterations result in transcriptional dysregulation is currently unclear. Mutations and copy number alterations were detected in *mTOR* (mammalian target of rapamycin), *PIK3CA* (PI3K catalytic subunit-α), TSC1, and *PTEN* (16, 21). Mutually exclusive gene alterations of the PI3K/Akt/mTOR pathway were detected in approximately 28% of tumors (21). Activation of the PI3K/Akt/mTOR pathway may underlie the efficacy of mTOR inhibitors, such as everolimus and temsirolimus.

In kidneys of individuals with VHL disease, VHL-deficient lesions with constitutive HIF activation were detectable by carbonic anhydrase IX staining, allowing the progression from single cells to ccRCC to be easily observed (27). HIF activation occurs extremely early in the disease. Most lesions are single cells, with very few multicellular dysplastic lesions and cystic lesions, showing that loss of VHL function alone is insufficient for ccRCC formation (27). Renal cysts are classified as benign, atypical, and malignant. HIF-1α is expressed in all VHL-deficient renal cells, whereas HIF-2α is highly expressed in renal tubular cysts and ccRCC (27).

Multiregion whole exome sequencing of four tumors from one individual with VHL disease delineated multifocal tumors of independent clonal origins (28). Each tumor exhibited a loss of chromosome 3p, each with a distinctly different breakpoint. Tumor evolution was more linear than branching compared with sporadic ccRCC, with markedly less intratumor heterogeneity. Convergent mTOR pathway activation was observed in all tumors through distinct gene mutations. The evolutionary history of 40 tumors from 6 individuals with VHL was examined by whole-genome sequencing (29). Tumors showed more genetic homogeneity than sporadically occurring tumors, which are generally removed at a later stage. However, the lack of overlapping sets of single-nucleotide variants as well as copy number variants between tumors indicated that ccRCCs evolved independently. A similar approach with different *VHL* subtypes could elucidate the effect of genetic background on the disease. For example, type 1 VHL disease (without PCC) is associated with truncation or exon deletion of germline VHL, whereas missense mutation is associated with type 2 disease (with PCC) (30). ccRCC occurs in Types 1 and 2B, which poorly downregulate HIF-1α but not 2A and 2C (6, 31).

### PRECLINICAL MODELS

Tumor xenografts using human ccRCC cell lines or tissue have been extensively used in mice to evaluate potential therapies. Likewise, injecting zebrafish with patient-derived xenografts and human cell lines is a rapid, low-cost preclinical model system of cancer (32, 33). Genetically engineered animal models of biallelic mutation of *VHL* alone in both mouse and zebrafish recapitulate features of early human disease, but not the formation of ccRCC. HIF activation appears to be necessary, but not sufficient for tumor formation in animal models of ccRCC.

Initial animal models were developed by genetically modifying levels of VHL and HIFα. Homozygous deletion of *Vhl* is embryonic lethal at 10.5–12.5 days in mice due to defective placental vasculogenesis; heterozygous mice fail to develop kidney tumors (34). Kidney-specific inactivation of *Vhl* is insufficient for ccRCC development, but results in multiple cysts with constitutive HIF-α expression and metabolic alterations marked by lipid and glycogen accumulation similar to early human disease (35). Transgenic overexpression of HIF-1α or HIF-2α resulted in simple cysts (36, 37). Likewise in zebrafish embryos, homozygous inactivation of *VHL* (*vhl<sup>−</sup>/<sup>−</sup>*) results in a kidney with enlarged proximal pronephric tubules, disorganized cilia, accumulated lipid and glycogen, cell proliferation, and apoptosis. This phenotype was rescued by a specific HIF2α inhibitor, showing that the zebrafish model system could be used to facilitate rapid screening of candidate drugs (38).

The identification of additional mutations underlying ccRCC has informed the development of genetically engineered mouse models that are more analogous to human disease. After *VHL*, the most frequently mutated genes in ccRCC were chromosome and histone regulators *PBRM1*, *SETD2*, and *BAP1* (16, 21). Epigenetic genes were targeted in recent studies. Kidneyspecific dual inactivation of *Vhl* and *Bap1* or *Pbrm1* using *Pax8-Cre* in mice recapitulated human ccRCC with cytoplasmic accumulation of glycogen and lipids (39). Bap1-deficient cystic tumors were high grade, whereas Pbrm1-deficient solid tumors showed a longer latency. Pbrm1-deficient tumors were converted from low to high grade by disruption of one *Tsc1* allele, resulting in mTORC1 activation. Intriguingly, ccRCC appeared to arise from Bowman capsule cells rather than the proximal tubule based on gene inactivation using more specific *Pax8-Cre* drivers.

Kidney-specific inactivation of *Vhl* and *Pbrm1* using *Ksp-Cre* model the histopathological and molecular features, and gradual onset of human ccRCC (40). *Ksp-Cre* is expressed both in renal tubular cells and the Bowman capsule. Bilateral, multifocal tumors were marked by the clear cytoplasm, high glycogen, and carbonic anhydrase IX staining similar to human ccRCC. A stepwise progression was observed from normal to cystic lesions over 6 months, developing into multifocal ccRCC at ~10 months. Loss of PBRM1 further amplified the activation of HIF1 (hetereodimer HIF-1α and HIF-1β) and STAT3 pathways caused by loss of *Vhl*. Activation of mTORC1 was implicated as the third event leading to ccRCC.

Inactivation of *Vhl*, *Rb1*, and *Trp53* in mice induced precursor cysts and gradually developing ccRCC tumors (41). Mutations were observed in genes involved in the primary cilium, *Kif3a* and *Kif3b*. Transcriptional analysis showed a gene expression profile similar to that observed in human ccRCC, with upregulation of HIF-1α and HIF-2α, mTOR activity. Mouse tumors showed variable response to anti-angiogenic therapy, and partial response to acriflavine, which interferes with the dimerization of HIF-1α and HIF-2α.

Zebrafish with multiple mutations of VHL disease-related genes are another potential preclinical model for exploring the progression and metastasis of ccRCC with the advantage of live imaging.

### CURRENT TREATMENT STRATEGIES

### Active Surveillance

Clear cell renal cell carcinomas grow slowly and small tumors <3 cm are at low risk to metastasize in VHL (42, 43). Currently, active surveillance until a threshold size of 3–4 cm is recommended for surgical intervention (43–46), resulting in a recurrence-free survival rate of 76% at 5 years and 20% at 8 years (47).

Regular screening is advised to detect RCC at an early stage and small tumors are followed with serial imaging. To improve the quality of life and survival of these patients, a balance between two goals is paramount: preventing metastases and preserving renal function. The goal is to treat before the tumor metastasizes, but to minimize consequences of the treatment such as compromised renal function. Through better surveillance by regularly scheduled imaging, individuals are living longer; however, a longer lifespan increases the probability of developing multiple RCCs and other sequelae of the disease.

### Treatment of Localized Disease Surgery

Early RCC can be detected, observed, and surgically removed before progression to metastatic disease (14, 48). However, repeated surgery for multicentric bilateral disease is followed by increased risk of ESRD requiring renal replacement therapy. Nephron-sparing surgery at a tumor size of 3–4 cm is the current treatment standard, replacing radical nephrectomy which compromised renal function and resulted in early dialysis. However, repeated partial nephrectomy reduces renal function, eventually causing ESRD. Delaying the interval to kidney surgery without increasing the risk of metastases prolongs sufficient renal function and delays dialysis. Independently of VHL disease, chronic kidney disease is associated with increased risk of death, cardiovascular events, and hospitalization (49). Progression to ESRD not only impacts quality of life but further increases morbidity—the yearly mortality rate of patients receiving long-term dialysis is 15–20% (50).

Locally recurrent disease is not uncommon after both partial nephrectomy and ablative therapy. Therapy options include observation, initial or repeat ablation, initial or repeat partial nephrectomy, radical nephrectomy, or systemic therapy (51). Salvage operation has a high major complication rate approaching 20% (52).

As renal surveillance of VHL patients has shifted surgical treatment from the resection of large tumors to the management of multiple small asymptomatic tumors, nephron-sparing therapeutic options are increasingly used. These minimally invasive procedures include percutaneous radiofrequency ablation (RFA), microwave ablation (MWA), cryoablation, and stereotactic body radiotherapy (SBRT) which are compared with surgery in **Table 2**.

#### Radiofrequency Ablation

Initially a treatment for non-surgical candidates, RFA has been used as a first-line procedure used to treat smaller and less numerous stage T1a lesions, and for renal salvage (46). Tumor necrosis is achieved with the heat of 50–100°C of radiofrequency energy transmitted by one or multiple needles in the tumor tissue. Placement of these needles can be achieved both laparoscopically or percutaneous (51).

Short-term local recurrence rates of RFA compare favorably with partial nephrectomy. A study of RFA treated T1 RCC reported a 13% retreatment rate for residual disease, a recurrencefree survival of 94%, and disease-free survival of 88% at 5 years (53). A study of T1a tumors treated with RFA versus partial nephrectomy reported a recurrence-free survival of 91.7 versus 94.6%, and an identical disease-free survival of 89% at 5 years (54). A recent study of 20 RCCs of 1–4 cm diameter in 9 VHL patients treated with RFA showed no recurrence and preserved



kidney function with a median follow-up of 102 months (55). Zagoria and colleagues concluded that long-term tumor control could be achieved in lesions treated smaller than 4 cm diameter (56). Another group reported increased risk of residual tumor for lesions >3.5 cm diameter (57). Clearly, there is a decrease of disease-free survival with every centimeter increase of the lesion (56). Although RFA is performed in many centers, conclusions about long-term efficacy are limited by the relatively short followup interval and small study sizes.

#### Microwave Ablation

Electromagnetic microwaves, transmitted from one or multiple antennas placed in the tumor can create a thermal field that causes tumor tissue necrosis (58). The antennas can be placed percutaneously, laparoscopically, or less often in an open approach. The potential benefit of Microwave Ablation (MWA) compared with RFA is that intratumoral temperatures with MWA are less affected by the heat sink effect, since MWA is less dependent on the electrical conductivity of the tissue. Larger tumors can be ablated with MWA compared to RFA; therefore, a maximum diameter of 4 cm was recommended in most studies (59–62). Several authors suggest MWA can be performed in tumors close to renal sinus or collecting system—a clear advantage over the other ablation techniques (62, 63).

#### Cryoablation

Cryoablation either laparoscopic or percutaneous, is a minimally invasive procedure that freezes and destroys small tumors (64–67). The cryoprobe is cooled down to −185 to −195°C by a nitrogenbased liquid guided through the tip of the probe. Cryoablation is less precise than RFA as it requires three applicators and a tumor margin of 10 mm. Thus, RFA of a 2-cm mass ablates approximately 10 cm3 of normal tissue, whereas cryoablation treatment ablates 30 cm3 of normal tissue (68). The location of the tumor within the kidney plays a critical role in treatment success, as centrally located tumors more frequently fail effective tumor cell destruction (69–71). Treatment failure is also significantly associated with tumor size >4 cm (72).

#### Stereotactic Body Radiotherapy

Stereotactic body radiotherapy (SBRT) is a method of external beam radiotherapy that precisely targets tumors with high individual doses. Multiple 3D conformal beams or intensity-modulated RT ensure the delivery of highly conformal dose distribution with a steep gradient falloff at the tumor margin to minimize injury of surrounding normal tissue (73, 74). SBRT provides superior renal tumor control compared with conventional radiation therapy, both *in vitro* and *in vivo* (75–77). Despite concerns that radiation may accelerate mutational events, stereotactic treatment of VHL tumors (primary and metastatic RCC, as well as hemangioblastomas) showed no increase in tumor formation of surrounding tissue after doses of 30–40 Gy (78–81). Two reviews reported a local control rate of over 90% for large primary RCCs and up to 98% for small tumors (82, 83). The treatment is non-invasive, with a low toxicity and mild deterioration of renal function (84). Lesions close to collecting vessels are also amenable to therapy (85). Mild side effects included nausea, fatigue, skin rash, and local pain. Similar to other ablative therapies, assessment of the long-term efficacy is limited by the short follow-up interval and small study size.

In summary, because of the lack of comparing long-term studies, partial nephrectomy for tumors of 3–4 cm diameter is still the standard of care. However, minimal-invasive physically treatment with RFA, MWA, cryoablation, and SBRT carries certain treatment advantages. The choice of resection versus ablative treatment is dependent upon the tumor localization, treatment availability, and experience of the VHL center.

### Treatment of Advanced ccRCC

Advances in imaging and localized therapy have greatly improved detection and survival rates for VHL patients with early ccRCC. Currently, metastatic ccRCC is difficult to cure despite the availability of multiple systemic therapies. Before 2005, systemic therapy consisted of cytokine therapy with interleukin-2 and interferon-alpha, which was marked by severe toxicity and low response rates. Drugs that target the VHL–HIF–vascular endothelial growth factor (VEGF) pathway such as VEGF receptor inhibitors (sunitinib, sorafenib, pazopanib, and axitinib) significantly improved outcome (86–89). Bevacizumab, an anti-VEGF monoclonal antibody, was the first recombinant human monoclonal antibody that showed clinical efficacy in advanced disease (90). Today, sunitinib and pazopanib are approved firstline tyrosine kinase inhibitors for metastatic ccRCC.

Mechanistic target of rapamycin inhibitors like everolimus and temsirolimus aim at the "mechanistic target of rapamycin" complex mTORC1 which controls fundamental cellular functions such as growth, proliferation, and apoptosis. Intravenous temsirolimus was approved in 2007 following a study showing improved progressive free survival (PFS) compared with interferon alone or combination therapy with interferon and temsirolimus (91). For oral everolimus, PFS was longer in the treated group versus placebo group (92).

In 2016, cabozantinib was approved as a second-line inhibitor of VEGF receptor and a broad range of type III receptor tyrosine kinases. Both PFS and median overal survival were longer for cabozantinib treated patients versus everolimus treatment (93, 94). Levantinib, an oral multityrosine kinase, showed a synergistic effect with everolimus in patients previously pretreated for advanced ccRCC (95, 96).

Checkpoint inhibitors target "programmed cell death 1" (PD1), PD1-ligand, and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) to activate T cell function. Nivolumab is a fully humanized IgG4 antibody against PD1, a negative regulator of T cell function, which improved survival in a subset of patients (97). Regardless of the targeted pathway, single agent treatment shows limited efficacy, with eventual treatment failure. Building on clinical experience favoring multiagent therapy (98), ongoing clinical trials are investigating combinations of kinase inhibitor treatments with immunotherapy, as well as combinations of immune modulators nivolumab and ipilimumab, an inhibitor of CTLA-4. A regression of metastases was observed after stereotactic radiotherapy in 4 of 28 renal cell carcinoma patients (99), suggesting that radiotherapy could enhance an immune response (98). Clinical trials testing the combination of radiotherapy with immunotherapy or targeted therapy are in progress (NCT02781506, NCT02019576, and NCT02978404).

Therapeutic options of advanced ccRCC have rapidly advanced over the past decade. Effective agents in advanced ccRCC have now been tested in earlier ccRCC stages and become appropriate first-line therapy drugs (100). Ideally, these drugs could serve as candidate perioperative agents with the potential to optimize postoperative outcome (101). In terms of precision medicine, the challenge now is to match a given patient with the optimal therapeutic agents with the help of robust molecular biomarkers.

### THERAPEUTIC TARGETS AND FUTURE TRIALS

Traditional chemotherapy and conventional radiotherapy, unspecifically directed against highly proliferative cells, is poorly effective in ccRCC. Multiple dysregulated signaling pathways have recently been identified and therapies that target these pathways have shown clinical efficacy in a subset of patients. ccRCCs are highly vascular and treated by currently approved anti-angiogenic agents. Downstream components of the VHL–HIF–VEGF pathway are modulated by receptor tyrosine kinase inhibitors (axitinib, cabozantinib, levantinib, pazopanib, sorafenib, and sunitinib), as well as anti-VEGF monoclonal antibodies (bevacizumab). The PI3K/Akt/mTOR pathway is targetable by mTOR inhibitors (everolimus, temsirolimus), which reduces the accumulation of HIF protein.

Genomic profiling is a potential guide for treatment and prognosis. No predictive biomarkers have been validated for selecting treatment, but several candidate biomarkers for treatment response have been identified through retrospective patient studies using tumor DNA. For example, mutations in *MTOR*, *TSC1*, or *TSC2* were associated with response to mTOR inhibitors, 21% in responders versus 11% in non-responders (102). But many responders (56%) had no mTOR pathway mutation. A positive response to first-line everolimus was associated with *PBRM1* mutations, and a negative response with *BAP1* mutations. In addition, *KDM5* mutations were associated with better response with first-line sunitinib than with everolimus (103). These genomic biomarkers are currently being evaluated in prospective studies. Other potential biomarkers include RNA sequencing to detect structural rearrangement and transcription levels, microRNA sequencing, DNA methylation profiling, and metabolomics.

Given the inherent intratumor heterogeneity of ccRCC, therapy that targets truncal events involving *VHL* or chromosome 3p may be more effective than targeting subclonal pathways. *VHL* and *TCEB1* are mutually exclusive mutations in ccRCC, resulting in VHL complex degradation and HIFα stabilization. HIF-2α, constitutively activated in ccRCC, is mainly expressed in renal, lung, hepatic, and endothelial cells. Although transcription factors are difficult to target, a novel HIF-2α inhibitor PT2399 was recently developed which prevents binding of HIF-2α to ARNT/ HIF-1β to activate a HIF-responsive promoter. Treatment with a HIF-2α inhibitor was investigated by grafting human ccRCC cell lines or patient tumor cells into nude mice, and by inactivating *vhl* in a zebrafish model.

Specific HIF-2α inhibition resulted in tumor regression in a subset of ccRCC cell line xenografts in mouse models of primary and metastatic ccRCC, in 10 of 18 patient-derived RCC xenografts in mice, and a patient with extensively pretreated metastatic ccRCC, who remained progression free for 11 months. Sensitivity to PT2399 correlated with a higher level of HIF-2α expression, and the presence of p53 (104, 105). Clinical trials with the HIF-2α inhibitor PT2385 are currently in progress (NCT02293980 and NCT03108066). Given the diversity of response, predictive biomarkers, such as HIF-2α and p53, may be useful for effective, targeted treatment. In zebrafish, treatment of *vhl<sup>−</sup>/<sup>−</sup>* embryos with a specific HIF2α inhibitor rescued pronephric abnormalities similar to human precancerous disease (38).

### CONCLUSION

Genomic sequencing has revolutionized the understanding of ccRCC by identifying multiple driver genes beyond *VHL*. Genetically engineered animal models to investigate combinations of *VHL*, epigenetic, and other genes provide a powerful preclinical model for elucidating the biology of ccRCC, developing novel combinatorial therapies, and identifying candidate biomarkers for clinical validation. In particular, uncovering the molecular basis driving tumor heterogeneity and the role of epigenetic genes will identify new pathways for intervention. Insights from these model systems will be clinically applicable to both hereditary and sporadic ccRCCs.

Surveillance and surgery remain standard of care in early ccRCC, while ablative therapies provide options for alternative treatment. For early ccRCC, current recommendations for intervention based solely on size could be better informed by prognostic tumor markers indicating a potential for aggressive growth, progression, and metastasis. Non-invasive biomarkers in blood or urine would be ideal for surveillance to avoid tissue biopsy. For advanced disease, a multiagent approach is supported by both clinical and preclinical observations, and ongoing clinical trials are currently in progress to evaluate treatment regimens and prognostic genomic biomarkers. In the future, genomic profiling is likely to be augmented by transcriptional and metabolomics analysis, as well as DNA methylation status. The efficacy of targeted therapy informed by tumor profiling may be limited by intratumor and intertumor heterogeneity. Immunotherapy has the potential to circumvent the high mutational load; clinical trials are in progress with single agent and multimodal therapy, targeting multiple signaling pathways or enhancing the immune response with stereotactic radiation.

### AUTHOR CONTRIBUTIONS

Both authors made substantial contribution to the work and approved the final version for publication.

### FUNDING

The article processing charge was funded by the German Research Foundation (DFG) and the University of Freiburg in the funding programme Open Access Publishing.

### REFERENCES


suppressors in clear cell renal cell carcinoma. *Eur Urol* (2013) 63:848–54. doi:10.1016/j.eururo.2012.09.005


with von Hippel-Lindau disease. *J Urol* (2004) 172:63–5. doi:10.1097/01. ju.0000132127.79974.3f


**Conflict of Interest Statement:** The authors declare the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Kim and Zschiedrich. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Identification of a Novel Heterozygous *De Novo* 7-bp Frameshift Deletion in *PBX1* by Whole-Exome Sequencing Causing a Multi-Organ Syndrome Including Bilateral Dysplastic Kidneys and Hypoplastic Clavicles

*Korbinian Maria Riedhammer 1,2, Corinna Siegel <sup>2</sup> , Bader Alhaddad2 , Carmen Montoya3 , Reka Kovacs-Nagy <sup>2</sup> , Matias Wagner 2,4,5, Thomas Meitinger 2,4 and Julia Hoefele <sup>2</sup> \**

#### *Edited by:*

*Miriam Schmidts, Radboud University Nijmegen, Netherlands*

#### *Reviewed by:*

*Kirsten Renkema, University Medical Center Utrecht, Netherlands Jan Halbritter, Leipzig University, Germany*

> *\*Correspondence: Julia Hoefele julia.hoefele@tum.de*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 18 August 2017 Accepted: 07 November 2017 Published: 24 November 2017*

#### *Citation:*

*Riedhammer KM, Siegel C, Alhaddad B, Montoya C, Kovacs-Nagy R, Wagner M, Meitinger T and Hoefele J (2017) Identification of a Novel Heterozygous De Novo 7-bp Frameshift Deletion in PBX1 by Whole-Exome Sequencing Causing a Multi-Organ Syndrome Including Bilateral Dysplastic Kidneys and Hypoplastic Clavicles. Front. Pediatr. 5:251. doi: 10.3389/fped.2017.00251*

*Genetics, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany, 3KfH Center of Pediatric Nephrology, Children's Hospital Munich Schwabing, Munich, Germany, 4 Institute of Human Genetics, Helmholtz Zentrum Munich, Neuherberg, Germany, 5 Institute of Neurogenomics, Helmholtz Zentrum Munich, Neuherberg, Germany*

*1Department of Nephrology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany, 2 Institute of Human* 

Introduction: Congenital anomalies of the kidney and urinary tract (CAKUT) represent the primary cause of chronic kidney disease in children. Many genes have been attributed to the genesis of this disorder. Recently, haploinsufficiency of *PBX1* caused by microdeletions has been shown to result in bilateral renal hypoplasia and other organ malformations.

Materials and methods: Here, we report on a 14-year-old male patient with congenital bilateral dysplastic kidneys, cryptorchidism, hypoplastic clavicles, developmental delay, impaired intelligence, and minor dysmorphic features. Presuming a syndromic origin, we performed SNP array analysis to scan for large copy number variations (CNVs) followed by whole-exome sequencing (WES). Sanger sequencing was done to confirm the variant's *de novo* status.

Results: SNP array analysis did not reveal any microdeletions or -duplications larger than 50 or 100 kb, respectively. WES identified a novel heterozygous 7-bp frameshift deletion in *PBX1* (c.413\_419del, p.Gly138Valfs\*40) resulting in a loss-of-function. The *de novo* status could be confirmed by Sanger sequencing.

Discussion: By WES, we identified a novel heterozygous *de novo* 7-bp frameshift deletion in *PBX1*. Our findings expand the spectrum of causative variants in *PBX1*-related CAKUT. In this case, WES proved to be the apt technique to detect the variant responsible for the patient's phenotype, as single gene testing is not feasible given the multitude of genes involved in CAKUT and SNP array analysis misses rare single-nucleotide variants and small Indels.

Keywords: CAKUT, *PBX1*, dysplastic kidneys, hypoplastic clavicles, developmental delay

## INTRODUCTION

Congenital anomalies of the kidney and urinary tract (CAKUT) represent the primary cause of chronic kidney disease in children. CAKUT is the collective term for many different renal and urinary tract malformations. In recent years, a multitude of monogenic disease-causing genes has been discovered (1). Disruption of the normal nephrogenesis by pathogenic variants in genes involved in kidney development is a basic principle of CAKUT (2).

When it comes to heterogeneous diseases like CAKUT, with many different genes involved, large-scale next-generation sequencing has become an extremely useful tool for the unbiased detection of pathogenic variants (3). In whole-exome sequencing (WES), the coding regions (the exome) of the human genome are enriched and sequenced. This has proven to be both an economic, as the exome only comprises 1% of the genome, and a pragmatic approach, as about 85% of disease-related variants can be found in the exome (4).

*PBX1* encodes a transcription factor that has already been linked to nephrogenesis as shown by *Pbx1*-deficient mice (5). Additionally, earlier mouse models revealed its role in bone formation (6). In 2017, microdeletions comprising *PBX1* as a minimal common region could be identified by microarray analysis in eight patients with syndromic CAKUT with predominantly renal hypoplasia. In the same publication, it was shown that *PBX1* was strongly expressed in the fetal kidney and brain (7). Here, we report on a 14-year-old male patient presenting to the pediatric nephrology department with the predominant clinical features of bilateral dysplastic kidneys, hypoplastic clavicles, and developmental delay.

### MATERIALS AND METHODS

This study was approved by the local Ethics Committee of the Technical University of Munich and performed according to the standard of the Helsinki Declaration of 1975. Written informed consent was obtained from the parents of the participant for publication of this case report. Blood samples were collected after written informed consent.

DNA was extracted from peripheral blood using the Gentra Puregene Blood Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The DNA sample of the patient was analyzed by using the SNP Array Affymetrix® CytoScanTM 750 K Array (Affymetrix® Inc., Santa Clara, CA, USA) with an average space between two oligonucleotides of 4 kb. Scanning was performed by the Affymetrix® GeneChip Scanner 3000 7G (resolution 0.51–2.5 µm). The data analysis was conducted using the Affymetrix® Chromosome Analysis Suite Software (ChAS), version 3.0, hg19.

Exome sequencing was performed using a Sure Select Human All Exon 60 Mb V6 Kit (Agilent) and a HiSeq4000 (Illumina) as previously described (8, 9). Reads were aligned to the UCSC human reference assembly (hg19) with BWA v.0.5.8. More than 98% of the exome was covered at least 20×. *PBX1* was covered >20× to 100%. Single-nucleotide variants and small insertions and deletions were detected with SAMtools v.0.1.7. Variant prioritization was performed based on an autosomal recessive pattern of inheritance (homozygous or putative compound heterozygous variants with a minor allele frequency <0.1%) as well as an autosomal dominant pattern of inheritance (heterozygous variants with a minor allele frequency <0.001%).

Sanger sequencing was used to confirm the identified variant and to test the patient's parents. Oligonucleotide primer sequences are available upon request.

## CASE REPORT AND RESULTS

The 14-year-old boy is the first child of healthy parents (**Figure 1**). He has one younger healthy brother and one younger healthy half-brother. The patient was born at a gestational age of 38 weeks [birth weight: 2,840 g (25th–50th percentile), birth length: 48 cm (10th–25th percentile)]. There were no obvious malformations noted at birth. During the neonatal period, a slender thorax and short clavicles were identified clinically. Body height was on the third percentile during infancy and early childhood (**Figure 2**) and a global developmental delay (including motor and speech delay) was diagnosed on regular pediatric screening examinations.

An ultrasound of the kidneys at 3 months of age revealed small kidneys with hyperechogenic parenchyma. The boy was then regularly seen by a pediatric nephrologist. By the age of six, the right kidney had a length of 6.2 cm (<1st percentile, see **Figure 3**), the left kidney had a length of 5.8 cm (<1st percentile, see **Figure 4**). Kidney function ranged between eGFR 59 mL/ min/1.73 m2 at 4 years of age, 90 mL/min/1.73 m2 at 8 years of age, and 69 mL/min/1.73 m2 at 13 years of age (Schwartz estimate, CKD II). Moreover, the patient had bilateral cryptorchidism for which he received hormonal therapy by the age of 2 years.

Psychological intelligence testing by the age of 13 revealed a below average speech comprehension (IQ 81), a reduced information processing speed (IQ 74) and an impaired auditory working memory (IQ 77).

On orthopedic examination the patient had a slim shoulder profile, impaired abduction of the arms, and a hunched back. Thoracic X-ray revealed hypoplastic clavicles (**Figure 5**, only left clavicle visible). He also had some dysmorphic features (wide nasal bridge, short neck, bilateral overfolding of the helix, and bilateral clinodactyly).

In this child, we presumed a syndromic origin and initially performed SNP array analysis. However, no microdeletions or

duplications (copy number variations, CNVs) larger than 50 or 100 kb, respectively, could be detected. As CAKUT have been associated with a large number of genes, we then performed WES. By WES, we could identify a novel heterozygous 7-bp deletion in *PBX1* leading to a frameshift (c.413\_419delGGGCAGG, p.Gly138Valfs\*40), resulting in either nonsense-mediated decay of the mRNA or a truncated protein lacking the DNA-binding domain. The variant is not listed in 60,000 control individuals of the Exome Aggregation Consortium (ExAC) browser. The ExAC browser does not list any high-confidence loss-of-function variants in *PBX1* indicating that *PBX1* is intolerant to loss-offunction variants. To verify the *de novo* status of the variant, we performed Sanger sequencing in the patient and his parents. The variant could not be detected in the blood DNA of both parents (**Figure 6**).

### DISCUSSION

CAKUT represent the primary cause of chronic kidney disease in children and many genes have been attributed to the genesis of this disorder with both dominant and recessive modes of inheritance.

Figure 3 | Ultrasound of the right kidney with a length of 6.2 cm (<1st percentile).

Figure 4 | Ultrasound of the left kidney with a length of 5.8 cm (<1st percentile).

Figure 5 | X-ray (detail of a babygram) of the patient at the age of 7 months showing a hook-shaped hypoplastic left clavicle.

Figure 6 | Partial nucleotide sequence of exon 3 of *PBX1* of the patient and his parents showing the heterozygous *de novo* variant c.413\_419delGGGCAGG, p.Gly138Valfs\*40. Shown reference sequence: TTCTGGAGGGGCAGG. Genomic position of the variant: chr1:164761876–164761882 (hg19, transcript NM\_002585.3).


(*Continued*)


*Patients K175, K179, K186, K181, K136 see Ref. (11); patients PT1-8 see Ref. (7). Reference genome for deletion coordinates: hg19; ASD, atrial septal defect; eGFR, estimated glomerular filtration rate; VSD, ventricular septal defects.*

CAKUT mainly occur as part of (multi-organ) syndromes but there are also isolated cases described in the literature (1, 2, 10). Just recently, haploinsufficiency of *PBX1* caused by microdeletions was shown to result in bilateral renal hypoplasia and other organ malformations (7). Furthermore, a targeted exome sequencing of 330 genes in 204 unrelated CAKUT patients could identify five novel *de novo* heterozygous loss-of-function variants/deletions in *PBX1* (11).

*PBX1* encodes a transcription factor which promotes protein–protein interaction and is important for organogenesis (12). *Pbx1<sup>−</sup>/<sup>−</sup>* mice die at an embryonic age and show extensive organ malformations including hypoplastic kidneys with unilateral agenesis (5). In a further publication, *Pbx1*-deficient mice exhibited besides multiple organ malformations—a pronounced skeletal phenotype with a slender thorax, hunched posture, and axial malformations. *PBX1* is highly expressed in proliferating chondrocytes (6). Additionally*,* there is strong *PBX1* expression in the fetal brain (7), and it regulates patterning of the cerebral cortex in progenitor neurons in mice (13). To date, two publications reported CAKUT phenotypes related to pathogenic *PBX1* variants/microdeletions; however, only two of the eight patients published by Le Tanno et al. had heterozygous microdeletions only encompassing *PBX1* (7). The phenotype of these patients involved, among other things, bilateral renal hypoplasia with hyperechogenic parenchyma, cryptorchidism, skeletal malformations, and developmental delay. The other patients in this publication had larger deletions involving a more extensive set of genes contributing to the phenotype. The five patients with novel loss-of-function variants/deletions in *PBX1* identified in a targeted exome sequencing study (11) lacked a detailed genotype–phenotype correlation, as only limited information on the extrarenal phenotype was provided. See **Table 1** for a detailed summary of the genetic changes in *PBX1* and clinical features described so far.

The patient in our report displayed a complex clinical picture with kidney and skeletal malformations and a neuronal phenotype with developmental delay and impairment of intelligence. In addition to the previously published data from *PBX1* mouse models, microdeletions and loss-of-function variants/ deletions mentioned above, we provide a detailed description

### REFERENCES


of the phenotype and make the case for an improved diagnostic approach in CAKUT: in this patient our diagnostic algorithm involved SNP array analysis which did not yield a positive result. We then performed WES and identified a novel heterozygous *de novo* 7-bp frameshift deletion in *PBX1* (c.413\_419del, p.Gly138Valfs\*40). For future CAKUT cases, we recommend directly employing whole-exome or whole-genome sequencing, as these are the apt techniques to identify new pathogenic variants/CNVs in this genetically heterogeneous syndrome. This is especially true for syndromal and familial CAKUT. In patients with isolated CAKUT, however, diagnostic yield is probably rather low, as less than 10% carry variants in 20 known CAKUT genes (2).

### ETHICS STATEMENT

This study was carried out in accordance with the recommendations of the Ethics Committee of the Technical University of Munich with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Ethics Committee of the Technical University of Munich.

### AUTHOR CONTRIBUTIONS

KR, MW, TM, and JH were responsible for writing and revision of the manuscript. CS and CM cared for the patient and provided the clinical data. KR, BA, RK-N, and MW did the exome analysis.

### ACKNOWLEDGMENTS

The authors would like to thank the family for participation and Dr. M. Steinborn from the Department of Diagnostic and Pediatric Radiology, Schwabing Hospital, Munich, Germany, for permission to use the X-ray. This work was supported by the German Research Foundation (DFG) and the Technical University of Munich (TUM) in the framework of the Open Access Publishing Program.


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Riedhammer, Siegel, Alhaddad, Montoya, Kovacs-Nagy, Wagner, Meitinger and Hoefele. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Update on Genetic Conditions Affecting the Skin and the Kidneys

*Antonia Reimer1,2, Yinghong He1 and Cristina Has1 \**

*1Department of Dermatology, Faculty of Medicine, Medical Center, University of Freiburg, Freiburg, Germany, 2Berta-Ottenstein-Programme, Faculty of Medicine, University of Freiburg, Freiburg, Germany*

Genetic conditions affecting the skin and kidney are clinically and genetically heterogeneous, and target molecular components present in both organs. The molecular pathology involves defects of cell–matrix adhesion, metabolic or signaling pathways, as well as tumor suppressor genes. This article gives a clinically oriented overview of this group of disorders, highlighting entities which have been recently described, as well as the progress made in understanding well-known entities. The genetic bases as well as molecular cell biological mechanisms are described, with therapeutic applications.

#### *Edited by:*

*Miriam Schmidts, Radboud University Nijmegen, Netherlands*

#### *Reviewed by:*

*Katja Höpker, University Hospital Cologne, Germany Roman-Ulrich Mueller, University Hospital Cologne, Germany*

#### *\*Correspondence:*

*Cristina Has cristina.has@uniklinik-freiburg.de*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 26 October 2017 Accepted: 14 February 2018 Published: 02 March 2018*

#### *Citation:*

*Reimer A, He Y and Has C (2018) Update on Genetic Conditions Affecting the Skin and the Kidneys. Front. Pediatr. 6:43. doi: 10.3389/fped.2018.00043*

Keywords: epidermolysis bullosa, mosaicism, genodermatosis, kidney, mutation, RASopathy, nevus, renal anomaly

### INTRODUCTION

Anomalies of both skin and kidney occur in a vast number of genetic conditions. There are two major reasons for this concomitant occurrence of clinical manifestations. First, skin and kidney share a common embryological background represented by mesoderm for dermal connective tissue and kidneys. Second, various molecules involved in adhesion (e.g., integrin α3, CD151), cholesterol biosynthesis (e.g., NSDHL), or signaling pathways [e.g., Wnt, hedgehog (Hh), Ras/ MAPK] are highly relevant for the development, structure, and function of both organs. In some syndromes, cutaneous and renal involvements are among the striking, pathognomonic features. Many of these disorders are recognizable at birth or early childhood.

Renal anomalies include congenital abnormalities of the kidney and urinary tract (CAKUT) (e.g., renal hypoplasia or aplasia, horseshoe deformations, anomalies of the urine collection system), malfunctioning of glomerular filtration, and the predisposition for tumors. The spectrum of skin anomalies is wide including pigmentation anomalies, skin dryness and ichthyosis, vascular anomalies (e.g., nevi flammei and hemangiomas), benign and malign skin tumors, abnormal hair, and nail dystrophy.

In this overview, genetic conditions affecting the skin and the kidneys are divided into three main groups:


**Abbreviations:** CAKUT, congenital abnormalities of the kidney and urinary tract; EB, epidermolysis bullosa; Hh, hedgehog; HLRCC, hereditary leiomyomatosis and renal cell cancer; LEOPARD, Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormalities of genitalia, Retardation of growth and Deafness; NF1, neurofibromatosis type 1; NS, Noonan syndrome; TSC, tuberous sclerosis complex.

### MONOGENIC DISORDERS WITH SKIN AND RENAL INVOLVEMENT

In certain monogenic disorders, such as epidermolysis bullosa (EB), RASopathies or disorders with tumor predisposition, cutaneous, as well as primary or secondary renal involvement may occur. In this section, we update the clinical and molecular features of the most relevant disorders of this vast group. In a large number of other genodermatoses and genetic syndromes, renal involvement may occur, but is not a defining feature. The clinical and molecular characteristics of these rare disorders are updated in **Tables 1** and **2**.

### Epidermolysis Bullosa

Epidermolysis bullosa encompasses disorders defined by cutaneous and mucosal fragility. Classification into four major EB types, EB simplex, junctional EB, dystrophic EB, and the Kindler syndrome, is based on the ultrastructural level of skin cleavage (2, 3). Renal and urinary tract anomalies may occur in all EB types, in particular in junctional and dystrophic EB. In patients with severe dystrophic EB due to absence of collagen VII various renal pathologies may occur and lead to chronic renal failure. Hydronephrosis, poststreptococcal glomerulonephritis, IgA mesangial disease, or renal amyloidosis has been reported in dystrophic EB case series (4, 5). The mechanisms may include repetitive vesiculation within the lining epithelia of the urinary tract, and chronic systemic inflammation (6). Only EB types for which reno-urinary involvement is a primary feature will be described here.

### Interstitial Lung Disease, Nephrotic Syndrome, and EB (ILNEB; MIM 614748)

#### *Clinical Features*

ILNEB is a rare autosomal recessive multiorgan disorder affecting the skin, kidneys and lungs. So far, 11 cases have been identified [reviewed in Ref. (5), and own unreported data], but the disease may be under recognized.

The clinical manifestations of ILNEB encompass the triad of early onset interstitial lung disease with respiratory distress, variable renal anomalies, and skin fragility. Since integrin α3 is widely expressed, clinical manifestations may occur in other organs, but are not characterized yet, because of the small number of cases and the early lethality. Skin involvement may include blistering, erosions or nail dystrophies, or may remain clinically unrecognized. The following renal anomalies were reported: congenital nephrotic syndrome, focal–segmental glomerulosclerosis, bilateral renal cysts, and a spectrum of CAKUT, including renal hypodysplasia, unilateral kidney hypoplasia, and ectopic conjoint kidney (7–12). Recently, two siblings of 13 and 9 years with viable ILNEB phenotypes presenting with growth retardation, severe pulmonary fibrosis, skin atrophy and erythema, scarce eyelashes/ eyebrows, and nail anomalies (pachyonychia) but without renal features were described (13).

#### *Genetics and Molecular Pathology*

This disease is caused by mutations in the gene for integrin α3 (*ITGA3*) (7). Thus far, 10 *ITGA3* mutations have been reported: 2 frameshift, 2 splicing, and 6 missense mutations (5). Loss-offunction mutations were associated with lethality before the age of 2 years. The consequences of missense mutations cannot be easily predicted. Some of them were shown to disturb the posttranslational modifications of integrin α3, which proved to be critical for the heterodimerization with integrin β1 and localization to the cell membrane (8, 9, 14).

Integrin α3 is the main integrin linking podocyte foot processes to the glomerular basement membrane [reviewed in Ref. (15, 16)]. In keratinocytes, it is located at cell–matrix adhesions, promoting epidermal adhesion primarily by maintaining the integrity of the basement membrane (17). The integrin α3 subunit is a widely expressed type I transmembrane protein consisting of a large extracellular region, a single transmembrane domain, and a short cytoplasmic tail (18). It forms obligate heterodimers with β1 integrin serving as a receptor for laminins, the major components of epithelial basement membranes (19). Integrin α3 is reduced or lost in several acquired conditions with glomerular disease, in which it is associated with reduction in podocyte adhesion to the glomerular basement membrane. For example, in podocytes of early-stage diabetic nephropathy integrin α3 expression was upregulated (20), while expression was suppressed with progression of the disease (21). In patients with primary focal segmental glomerulosclerosis, podocyte depletion was accompanied by reduced podocyte expression of α3β1 integrins (22). Moreover, integrin α3 is involved in podocyte foot process effacement during nephrotic syndrome (23).

### Nephropathy with Pretibial EB and Deafness (MIM 609057)

#### *Clinical Features*

Two siblings with congenital nephrotic syndrome and pretibial EB were first described in 1988 (24). The disease-causing mutation in the gene for the tetraspanin CD151 was identified in 2004 (25), and very recently an additional case was reported (26). The first two cases had proteinuria in the nephrotic range and end-stage renal failure requiring hemodialysis or peritoneal dialysis from the age of 14 or 16 years on, respectively (24). The third case was a 33-year-old male with nephropathy manifesting with proteinuria below the nephrotic range, multiple episodes of pyelonephritis, and urinary incontinence, manifesting as a combination of overflow incontinence and intermittent urge incontinence (26). Additional manifestations included pretibial or extensive skin blistering, poikiloderma, nail dystrophy, hair loss, dystrophic teeth, involvement of the ocular, oral, gastrointestinal, and urogenital mucosal membranes (25, 26).

### *Genetics and Molecular Pathology*

A homozygous single-nucleotide duplication in the *CD151* gene leading to frameshift and a premature stop codon was identified in the first two cases (25). Flow cytometry analysis demonstrated absence of reactivity for CD151, suggesting that the predicted truncated polypeptide was not functional. In the third case, a homozygous *CD151* splice site mutation, affecting a canonical donor splice site junction was found (26). Immunofluorescence staining and western blot analysis confirmed that the splice site mutation led to absence of CD151 in the cells of the patient (26).

Genetic Disorders with Skin and Kidney Involvement

#### Table 1 | Genodermatoses with reno-urinary involvement.




(*Continued*) Genetic Disorders with Skin and Kidney Involvement

Reimer et al.

# Frontiers in Pediatrics | www.frontiersin.org

#### TABLE 2 | Continued


CD151 (syn. Raph blood group, TSPAN24) is a member of the tetraspanin family of cell surface proteins and acts as a stabilizer of integrins (27). CD151 forms complexes with integrin α3β1 in cell culture and *in vivo* (28, 29). These complexes are assembled early during the integrin biosynthesis and precede the interaction of CD151 with other tetraspanins (30). CD151 also regulates glycosylation of α3β1 (31). CD151 is widely expressed in epithelia, endothelia, muscle cells, renal glomerular podocytes, Schwann and dendritic cells, in platelets and megakaryocytes. CD151 is involved in the formation and/or maintenance of the glomerular basement membrane (32).

### Junctional EB with Pyloric Atresia (MIM 226730)

#### *Clinical Features*

Junctional EB with pyloric atresia manifests with aplasia cutis congenita (**Figure 1**), generalized skin blistering, and pyloric atresia. Several acquired complications of the reno-urinary system are reported, including pyelonephritis, hydronephrosis, urinary retention, development of bladder hypertrophy, and urethral meatal stenosis (4, 33, 34).

#### *Genetics and Molecular Pathology*

The disease is caused by mutations in the genes coding for the α6 or β4 integrin subunits, most mutations residing in *ITGB4*. Absence of α6β4 integrin is associated with a high rate of lethality in the first months of life, while missense and splicing mutations lead to moderate disease severity and reno-urinary manifestations.

Integrin α6β4 is a heterodimer composed of two type I transmembrane subunits localized in hemidesmosomes', which anchor keratin intermediate filaments to the cell membrane and extracellular matrix. The intracellular region of α6β4 consists of the short tail of α6 and a large β4 cytoplasmic domain, which interacts with plectin and collagen XVII in keratinocytes. The ligands of α6 integrin are CD151, collagen XVII and laminin 332. Integrin α6β4 has a major adhesive function and promotes polarization of the cells (35). α6β4 is expressed in the epithelial cells within the medulla of the kidney. In a mouse model, α6β4 was not required for morphogenesis of the urinary tract, but for maintaining the integrity of the kidney collecting system. Collecting duct anomalies appeared as the animals aged. α6-null collecting duct cells were not able to withstand mechanical stress and detached from the basement membrane (36, 37).

deficiency (right panel) [clinical pictures, courtesy of Dr. P. Häusermann (Department of Dermatology Basel)].

### Junctional EB Generalized Severe (Formerly: Herlitz EB; MIM 226700) *Clinical Features*

Junctional EB generalized severe is caused by complete lack of laminin 332, the major laminin expressed in the cutaneous basement membrane. Laminin 332 is a heterotrimeric glycoprotein consisting of three polypeptide chains: laminin α3, β3, and γ2, encoded by *LAMA3*, *LAMB3*, and *LAMC2*, respectively. The clinical picture is dominated by mucocutaneous blistering from birth onward. Extensive generalized blistering leads to loss of fluids and protein and failure to thrive. The most common complications are anemia, dyspnea, infections, and sepsis. Affected children show multiorgan involvement and commonly die before the age of 2 years (38). In an infant with *LAMB3* mutations, nephrotic syndrome with albuminuria due to failure of the glomerular filtration barrier, and high urinary *N*-acetylglucosaminidase levels, also indicating renal tubular involvement were reported (39).

#### *Genetics and Molecular Pathology*

Laminin 332 is the major laminin expressed by keratinocytes, but is also present in multiple epithelial basement membranes, including those of kidney. Like all laminins, it is a glycoprotein composed of three chains (α3, β3, and γ2) bound through disulfide bonds (5). In junctional EB generalized severe, mutations are found in one of the three genes encoding the laminin 332 chains. In the majority of cases, mutations are located in *LAMB3* and lead to premature termination codons, mRNA decay, and absence of laminin 332.

### RASopathies

RASopathies represent an expanding common group of neurodevelopmental disorders caused by germline mutations in genes encoding components of the Ras/MAPK pathway (40). Collectively, they affect >1 in 1,000 individuals (41). The Ras/MAPK pathway is a conserved omnipresent intracellular signaling pathway that is critical in regulating cell cycle, differentiation, growth, apoptosis, and senescence (40). The group of RASopathies includes neurofibromatosis type 1 (NF1), Noonan syndrome (NS), NS with multiple lentigines, Legius syndrome, Costello syndrome, cardio-facio-cutaneous syndrome, capillary malformation-arteriovenous malformation, and autosomal dominant intellectual disability type 5. Because of the common

molecular mechanisms, phenotypic features of these syndromes are overlapping.

### NF1 (von Recklinghausen Disease, MIM 162200) *Clinical Features*

With an incidence of 1:2,500–3,000 (42), NF1 is one of the most common disorders of this group. NF1 follows autosomal dominant inheritance, about half of all cases occur due to spontaneous mutations. Diagnosis of NF1 is established following a set of clinical diagnostic criteria established in 1988 [**Table 3**, diagnosis of NF1 is probable when more than two criteria are present (43)]. Most cases are diagnosed in childhood, but when the complete set of criteria is not yet evident, follow-up and reevaluation are necessary. Cutaneous features include café-au-lait macules, cutaneous and internal neurofibromas, or plexiform neurofibromas and axillary freckling. Renal involvement occurs sporadically, manifestations include hypertension due to renal artery stenosis, renal neurofibromas, and renal metastases of malignant schwannomas. The cooccurrence of NF1 and Wilms' tumor has been reported in some cohorts (44, 45).

Individuals with NF1 have a high risk of developing malignancies, especially malignant peripheral nerve sheath tumors (46). Life expectancy has been found to be approximately 8 years lower than in the normal population (47).

The cutaneous features progress with age. Neurofibromas as the main cutaneous finding in NF1 can be itchy, lead to disfigurement, and cause psychological strain. They can be treated with excisions or laser ablation (Er:YAG or CO2 laser) (48, 49), both with risk for hypertrophic scarring and recurrence (42).

In uncomplicated cases, clinical evaluation in childhood should be performed annually and include auxologic measurements, cardiovascular assessment, skin examination, and developmental progress (42). In childhood, visual assessment should be performed every 6–12 months for early detection of optical pathway glioma until the age of 7 years (42).

#### *Genetics and Molecular Pathology*

Monoallelic loss-of-function variants in *NF1* coding for neurofibromin 1 are disease-causing in NF1. Neurofibromin is a multifunctional tumor suppressive protein which functions as a GTPase-activating protein. Neurofibromin inhibits cell proliferation and growth by blocking RAS-mediated signal transduction and modulates cell motility and adhesion (50).

The mutational spectrum is highly heterogeneous including nonsense and missense mutations, splice site mutations (about 30% of cases), small insertion–deletions, whole-gene deletions

Table 3 | Diagnostic criteria for neurofibromatosis 1 (NF1) (43).

• 6 or more café-au-lait macules (>0.5 cm in children or >1.5 cm in adults)

• 2 or more cutaneous/subcutaneous neurofibromas or one plexiform


(4–5%), and structural rearrangements (51). Penetrance is complete after childhood, but NF1 is characterized by extreme clinical variability which is poorly understood, as are genotype– phenotype correlations. Intra- and interfamilial evaluation of the NF1 phenotype suggests that genetic modifiers which are not linked to the *NF1* locus contribute to the variable expressivity of the disease (52, 53). Differently skewed expression of the *NF1* alleles as well as somatic "second hit" variants or loss of heterozygosity may account in part for the phenotypic variability (54, 55). In addition to NF1, atypical manifestations, such as familial spinal neurofibromatosis, multiple spinal ganglioneuromas, optic gliomas, or Lentigines, Electrocardiographic abnormalities, Ocular hypertelorism, Pulmonary stenosis, Abnormalities of genitalia, Retardation of growth and Deafness (LEOPARD) syndrome, have been associated with *NF1* mutations. Finally, incidental occurrence of *NF1* mutations together with mutations in other genes may account for atypical phenotypic associations.

### NS with Multiple Lentigines (syn. LEOPARD Syndrome, Multiple Lentigines Syndrome, Lentiginosis profusa and Progressive Cardiomyopathic Lentiginosis; MIM 151100) *Clinical Features*

Noonan syndrome with multiple lentigines is a rare RASopathy that manifests in childhood. The incidence is unknown; so far, more than 200 cases were published. The characteristic cutaneous appearance is described well by the acronym LEOPARD: the skin appears spotted due to thousands of dark brown lentigines of 1–5 mm size which are distributed on the entire body (including sun-protected areas), cooccurring with café-au-lait macules (hence sometimes confused with NF1), hypomelanotic macules, and sometimes axillary freckling. Apart from LEOPARD are defining features (56). CAKUT, including horseshoe kidneys, occur in 11% of affected individuals (57). NS with multiple lentigines is sometimes difficult to distinguish from NF1 and the allelic NS (58), especially in early childhood when pigmentation anomalies are not yet pronounced (59). The prognosis is generally good, but can be limited by hypertrophic cardiomyopathy, arrhythmias, and sudden cardiac death. Annual cardiologic check-up should be performed life-long, and hearing assessment should be undertaken until adulthood. If auxologic follow-up indicates small statue, growth hormone therapy should be considered (56). Intense pulsed light has been used for cosmetic treatment of lentigines (60).

#### *Genetics and Molecular Pathology*

Noonan syndrome with multiple lentigines is allelic with NS and with the cardio-facio-cutaneous syndrome. The genetic basis of NS with multiple lentigines is heterogeneous including heterozygous pathogenic variants in one of four genes *PTPN11* (90% of cases), *RAF1* (less than 5% of cases), *BRAF* and *MAP2K1* (both in single cases) (61). One or more additional, as-yet undefined genes are probably associated with about 5% of cases in whom no pathogenic variant has been identified (61). Genotype–phenotype correlations are not well established (62).

All involved genes code for components of the Ras/MAPK pathway:


Somatic mutations in all these genes are present in various types of cancers. Indeed, individuals with NS have a threefold increased risk of malignancies, such as juvenile myelomonocytic leukemia, acute lymphoblastic leukemia, rhabdomyosarcoma, and neuroblastoma (63, 64).

### Genetic Tumor Predisposition Syndromes Affecting both Skin and Kidney

This is a large group of disorders characterized by both numerous hamartomas (benign tumors that can develop in basically all tissues) and premature development of malignant tumors during childhood. The molecular pathology of these conditions is highly heterogeneous. The most common conditions are described below or in **Table 4**. The tumors in these syndromes can occur in both cutaneous and extracutaneous locations, including the kidney (**Table 4**). Other tumor predisposition syndromes which usually manifest in adult age are only briefly mentioned.

#### Tuberous Sclerosis Complex (TSC, TSC1 and TSC2, syn. Bourneville Disease; MIM 191100) *Clinical Features*

Tuberous sclerosis complex occurs with an estimated incidence of 1:5,800–1:10,000 (65). It is mostly diagnosed in infancy when it manifests with skin findings and seizures due to cerebral hamartomas and giant cell astrocytomas. The diagnosis of TSC is made according to clinical criteria (66) (**Table 5**, either two major features are required or, alternatively, one major and two or more minor features). Typical cutaneous features are hypopigmented macules (best seen in Wood's light), angiofibromas (mostly facial), periungual fibromas, and connective tissue nevi (shagreen patches). The frequency of cutaneous findings increases with age, but polygonal hypomelanotic macules, known as "ash-leaf spots," are the earliest manifestation and are invariably present at birth. Renal involvement is also common, with angiomyolipomas and cysts as the most frequent renal manifestations found in 17% of children with TSC by age 2 years and 65% of 14 years old children with TSC (67). Renal cell carcinoma is more common in TSC than in the overall population (68).

There is large variability in the clinical course, neurological development, and life expectancy in TSC, mostly depending on the number and location of hamartomas. While cutaneous features are crucial for clinical diagnosis, central nervous system tumors are the main cause of morbidity and mortality, while renal disease is the second leading cause of early death (69).

As TSC involves multiple organ systems, interdisciplinary care is necessary. Skin examinations should be performed annually. Diagnostic work-up of the kidney should include annual assessment of renal function and blood pressure and imaging (preferably with MRI) every 1–3 years (70). Since 2005, mTOR inhibitors have been evaluated for the use in TSC. Everolimus (Votubia®) is approved as a system therapeutic for use in children of 3 years and older with subependymal giant cell astrocytomas and for adults with complicated renal angiomyolipomas (71). Cutaneous lesions can be treated surgically, using laser (CO2/ Erbium:YAG/Dye laser combination, or CO2, or Nd:YAG, or pulsed-dye laser) (72–74) and pharmacologically using topical mTOR inhibitors (75, 76). Surgical intervention can be considered as a therapeutic option for painful hemorrhagic renal angiomyolipomas and cerebral lesions.

Table 4 | Genetic tumor predisposition syndromes with cutaneous and reno-urinary involvement.


Table 5 | Diagnostic criteria of tuberous sclerosis complex [adapted from Ref. (66)].


#### *Genetics and Molecular Pathology*

Tuberous sclerosis complex is caused by monoallelic mutations in *TSC1* (about 20% of cases) or *TSC2* (about 70% of cases) (69) (Leiden open variation database). Two-thirds of TSC cases result from *de novo* pathogenic variants, and in about 10% no mutation can be detected (69). Large gene rearrangements, intronic pathogenic variants, and somatic or germ line mosaicism may explain the failure to detect mutations (77, 78). Specialized methods, such as targeted-deep sequencing of introns and exons and high-resolution SNP arrays improved the mutation detection rate to 94% (79). Genotype–phenotype correlations revealed that *TSC2* mutations lead to earlier onset and more severe phenotype, as compared with *TSC1* mutations (80). The occurrence of autosomal dominant polycystic kidney disease in TSC may be due to a contiguous deletion of *TSC2* and *PKD1* (81).

*TSC1* and *TSC2* code for hamartin and tuberin which form heterodimers within the TSC protein complex. Loss-of-function mutations in either *TSC1* or *TSC2* lead to constitutive activation of the mammalian target of rapamycin complex 1 (mTORC1) that is uncoupled from inhibitory mechanisms. Thus tumor cells in TSC have increased activation of mTORC1 signaling, resulting in increased protein synthesis and cell growth, and reduced autophagy (82). In fact, somatic inactivation of normal alleles is expected to drive mTOR activation, but second hit mutations are not always observed. The pathogenesis of angiofibromas involves UV-induced mutations suggesting that sun exposure is the initiating event (83). In angiomyolipomas, about 70% of the second-hit events are loss-of-heterozygosity mutations (84). A recent study showed that in TSC, somatic mutation rates were lower than most malignant tumors, while whole or arm-level chromosome gains and losses were the most remarkable finding in over 10% of patients (79).

### Basal Cell Nevus Syndrome (syn. Gorlin Syndrome, Gorlin–Goltz Syndrome, Nevoid Basal Cell Carcinoma Syndrome; MIM 109400) *Clinical Features*

The basal cell nevus syndrome is a rare autosomal dominant condition, occurring with an estimated incidence of 1:30,000 (85). It formally belongs to the group of hamartoses, but is mainly ranked among the tumor predisposition syndromes. Its characteristic feature is the occurrence of multiple basal cell carcinomas from young adulthood onward. Development of basal cell carcinoma in infancy has also been described (86). Other skin manifestations include palmar and plantar punctate dyskeratotic pits and facial milia. Skeletal abnormalities (e.g., polydactily), jaw cysts, and medulloblastoma occurring in 5% of patients are early features that can hint toward the diagnosis of basal cell nevus syndrome. Renal anomalies, such as renal agenesis (87) or Wilms tumors (88, 89), were reported in single cases. Diagnosis can be difficult in childhood due to few or unspecific findings. In suspected basal cell nevus syndrome, a systematic work-up including examinations by a dermatologist, a radiologist, a dentist, a gynecologist, a cardiologist, and a geneticist is recommended (90). After the occurrence of the first basal cell carcinoma, dermatologic examinations should be performed every 6–12 months. A baseline cerebral MRI with yearly controls until the age of 8 years is recommended. Echocardiography should be performed at baseline to rule out cardiac fibromas. X-ray of the jaw should be repeated yearly until a first jaw cyst is detected, after that every 6 months or according to symptoms. For scoliosis detection, an X-ray at the age of 1 year or at time of diagnosis is recommended. If normal, it is only repeated in case of symptoms. If scoliosis is present, regular follow-ups are appointed. Other baseline evaluations include pelvic ultrasound and ophthalmologic assessments. Psychological evaluation and support is advisable (90).

For many years, excision of basal carcinoma was the main treatment option for this condition. Understanding of the molecular pathology has recently led to development and approval of vismodegib (Erivedge®) as an effective therapy. Vismodegib targets the sonic Hh pathway and leads to regression of existing and inhibits the development of new tumors (91). Radiation should be avoided as it will trigger the eruption of multiple new tumors (92).

#### *Genetics and Molecular Pathology*

The genetic basis of the basal cell nevus syndrome is heterogeneous. The main cause is represented by monoallelic germline pathogenic variants in *PTCH1* responsible for approximately 85% of the cases. *SUFU* pathogenic variants reside in about 5% of the cases (93). Rare causes are *PTCH2* and *SMO* mutations: a missense mutation in *PTCH2* was disclosed in a Chinese family (94), and a *SMO* mutation in a single case with a segmental basal cell nevus syndrome (95). In about 15–27% of cases, the genetic basis remains unclear (93). Low level of postzygotic mosaicism may explain some of the genetically unsolved cases (96). *PTCH1* pathogenic missense variants have also been associated with holoprosencephaly (97).

All these genes encode key players in the Hh signaling pathway, which is essential for development of vertebrates and drives proliferation, migration, and differentiation of progenitor cells (98):


Activation of the Hh pathway is initiated by the Hh ligand binding and inhibition of the transmembrane receptor patched 1, allowing the signal transducer smoothened to activate Gli transcription factors and amplify the expression of Hh target genes (98). Somatic mutations that activate the Hh signaling pathway drive growth of various cancers including basal cell carcinomas, medulloblastomas, pancreatic, prostate, and small cell lung cancer, that account for up to 25% of all human cancer deaths (99).

## Birt–Hogg–Dubé Syndrome

#### *Clinical Features*

The Birt–Hogg–Dubé syndrome is an autosomal dominant disorder which manifests with cutaneous lesions, pulmonary cysts and/or history of pneumothorax, and various types of renal tumors (100). Skin involvement occurs during the second, third, or fourth decade of life and progresses with age. It includes various benign tumors such as fibrofolliculomas, trichodiscomas/angiofibromas, perifollicular fibromas, and acrochordons. Fibrofolliculomas are the most common phenotypic features of the Birt–Hogg–Dubé syndrome, occurring in more than 85% of the patients over the age of 25 years (101). They appear as multiple, small, skin-colored papules disseminated on the face, neck, and upper trunk. Treatment by laser ablation results in temporary improvement, but relapse usually occurs.

Individuals with Birt–Hogg–Dubé syndrome have a sevenfold increased risk to develop renal tumors, that are typically bilateral and multifocal (102, 103). They are usually diagnosed in adults (median of diagnosis is 48 years, but have been described as early as 20 years of age) and have a slow progression (103). The histologic types of renal tumors found in individuals with Birt–Hogg–Dubé syndrome are: by far predominant are chromophobe renal cell carcinomas, followed by hybrid oncocytic tumors and oncocytomas, while clear cell renal cell carcinomas are uncommon. Yearly screening by renal MRI is indicated in individuals with Birt–Hogg–Dubé syndrome age 18 years or older. In some families, renal tumors and/or spontaneous pneumothorax occur without cutaneous manifestations.

### *Genetics and Molecular Pathology*

The Birt–Hogg–Dubé syndrome is caused by monoallelic pathogenic variants in *FLCN,* encoding folliculin. Mutation analysis detects disease-causing variants in 88% of the affected families; the deletion or duplication of a cytosine at position c.1285 is a mutational hot spot. Partial- or whole-gene deletions account for 3–5% of the cases and must be identified with specific methods. About 7–9% of the cases remain genetically unsolved. The protein folliculin forms a complex with folliculin-interacting protein 1 or 2 and binds to the 5′ AMP-activated protein kinase suppressing tumorigenesis (104). Moreover, it plays a role in mTOR activation (105–107).

### Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) *Clinical Features*

Hereditary leiomyomatosis and renal cell cancer is characterized by the occurrence of cutaneous and uterine leiomyomata, and/or a single, unilateral, and aggressive renal tumor (108). Cutaneous leiomyomata may be multiple or single, appear in adults (mean age of 25 years), and increase in size and number with age. They manifest as skin-colored papules or nodules, disseminated on the trunk, extremities, and face. The treatment consists of surgical or laser excision, or cryoablation. Renal tumors occur in about 10–16% of individuals with HLRCC at a median age of 44 years and cause hematuria and lower back pain. Histologically they are classically classified as type 2 papillary (108).

### *Genetics and Molecular Pathology*

Hereditary leiomyomatosis and renal cell cancer is caused by monoallelic *FH* mutations that lead to reduced activity of the enzyme fumarate hydratase (109). In tumor tissue, somatic variants and loss of heterozygosity are found. No genotype–phenotype correlations are known, and there is significant intrafamilial variability. Biallelic mutations resulting in fumarase deficiency cause an inborn error of metabolism characterized by rapidly progressive neurologic impairment including hypotonia, seizures, and cerebral atrophy (110).

### DISORDERS DUE TO POSTZYGOTIC MOSAICISM

The disorders in this group are caused by mutations that are mostly lethal if occurring as a germline mutation affecting all cells. However, if these mutations occur postzygotic in early embryogenesis, disorders with unilateral or segmental manifestations result, as proposed by Happle (111, 112). His hypothesis is supported by the elucidation of the molecular basis of several segmental disorders since the implementation of next-generation sequencing technologies.

### Linear Sebaceous Nevus Sequence [Schimmelpenning–Feuerstein–Mims Syndrome, Nevus Sebaceous of Jadassohn; MIM 163200]

### Clinical Features

This syndrome belongs to the group of epidermal nevus syndromes. More than 100 sporadic cases have been described, the incidence is not known. While solitary sebaceous nevi are a reasonably common finding in infants, the sebaceous nevi in this syndrome are associated with seizures, mental retardation, skeletal and ophthalmic anomalies, and asymmetric growth. At birth, one (or multiple) sebaceous nevi is/are found mostly in the mid-face. Involvement of the head/neck area is possible, as are locations on trunk or extremities. The sebaceous nevus shows a linear configuration along the lines of Blaschko. While it is mostly flat and wax-like in infancy, verrucous changes, hyperpigmentation, hyperkeratosis, and hypertrophy are seen toward puberty. In adulthood, development of (mostly benign) tumors within the sebaceous nevus is noted. Skeletal, ophthalmic, and renal involvements occur occasionally, the latter comprising CAKUT, such as double urinary collecting system and horseshoe kidneys, and renal hamartoma and nephroblastoma.

Surgical treatment of sebaceous nevi can be offered for cosmetic or psychological reasons. Excision is generally not indicated because of cancer prophylaxis, as the risk for malignant tumors is very low (113). Therapeutic options include excision and laser ablation (114). Regarding the main complications, seizures, neurological retardation, and rickets, interdisciplinary care for children with linear sebaceous nevus sequence should be offered.

#### Genetics and Molecular Pathology

This syndrome can be considered a mosaic RASopathy because it is caused by postzygotic pathogenic variants in *HRAS* (HRas Proto-Oncogene, GTPase), *KRAS* (KRAS Proto-Oncogene, GTPase), or the *NRAS* (NRas Proto-Oncogene, GTPase) genes (115, 116). The recurrent activating mutations induce constitutive activation of the MAPK and PI3K/AKT signaling pathways. Somatic mutations in the *HRAS* and/or *KRAS* genes were also found in isolated sebaceous nevi. These mutations are only present in keratinocytes which give rise to the cutaneous lesions.

The proteins encoded by the Ras oncogene family have intrinsic GTPase activity and function in signal transduction pathways important for cell growth, proliferation, and survival. Defects in these genes are present in various cancers.

### Neurocutaneous Melanocytosis (syn. Neurocutaneous Melanosis Sequence, Neuromelanosis; MIM 249400) Clinical Features

The landmark lesion of this syndrome, a giant pigmented nevus, is seen at birth. It is usually located in the posterior head or trunk (117). Sometimes, three or more large congenital nevi are found rather than a single giant nevus. Numerous disseminated ("satellite") nevi can be present at birth and more will develop in the course of disease. The presence and proliferation of melaninproducing cells within cranium and spine leads to increased intracranial pressure, seizures, mental deterioration, and death in early childhood (117). Leptomeningeal and intracranial melanoma occur in a significant portion of patients. Occasional abnormalities found in neurocutaneous melanocytosis are cerebral malformations such as syringomyelia and Dandy–Walker malformation, CAKUT, and unilateral renal cysts. Other tumors occurring in the syndrome include rhabdomyosarcoma, liposarcoma, and malignant peripheral nerve sheath tumors.

There is a risk of development of cutaneous malignant melanoma within the congenital nevi. They develop in the depth of the lesion and can be felt earlier than they can be seen. This has been suggested to be as high as 15% (118) in giant congenital melanocytic nevi, although others have reported incidences of 0.7% (119). The amount of patients with the full picture of neurocutaneous melanocytosis developing malignant melanoma is not known, probably as most of these patients die before developing melanomas.

They clinical course is mostly determined by neurologic symptoms. If these occur, there is no effective therapeutic approach. If the child shows normal psychomotoric development, excision for the giant melanocytic nevi is recommended. Such surgical procedures may require several steps and the use of tissue expanders. Dermabrasio is not considered as a therapeutic option any more. To date, a causal therapy is not available, but a recent *in vitro* assessment of inhibitors of the NRAS-signaling pathway (drugs also successfully used in the therapy of malignant melanoma) showed promising results (120).

#### Genetics and Molecular Pathology

Somatic oncogenic missense mutations affecting codon 61 of the *NRAS* gene were identified in affected cutaneous (melanocytes) and nervous tissues from patients with congenital melanocytic nevus syndrome and/or neurocutaneous melanosis (118).

### CHILD Syndrome (Congenital Hemidysplasia with Ichthyosiform Erythroderma and Limb Defects; MIM 308050)

#### Clinical Features

This epidermal nevus syndrome was coined with the acronyme "CHILD" by Happle and colleagues in 1980 (121) to sum up the main findings in children with this condition: a characteristic, mostly unilateral epidermal nevus in combination with ipsilateral congenital hemidysplasia of bones (affecting any part of the body, mainly limbs). The epidermal nevus is usually present at birth but can also develop in the first weeks of life. Spontaneous involution is sometimes witnessed (113). The CHILD nevus is red and scaly. It can show strict lateralization (right side more frequently than left side, 3:2) and midline demarcation, but it can also follow lines of Blaschko, and both patterns may be present in an affected individual (113) (**Figure 2**). Next to cardiovascular anomalies, renal findings comprise CAKUT, such as renal agenesis (122) or hypoplasia (123), to unilateral hydronephrosis, but their frequency is unknown (124).

Addressing the molecular pathology of CHILD, a therapeutic approach for treating the epidermal nevus combining simvastatin and cholesterol for topical use proved to be effective (125, 126) (**Figure 2**).

Figure 2 | Unilateral epidermal nevus in a patient with CHILD syndrome, before (left panel) and after 5 years topical application of a simvastatin/ cholesterol cream (right panel).

#### Genetics, Molecular Pathology

CHILD syndrome is caused by monoallelic loss-of-function pathogenic variants in the *NSDHL* gene encoding the NAD(P) H steroid dehydrogenase-like protein, which is a C4 demethylase involved in postsqualene cholesterol biosynthesis (127). The enzyme is located within the membranes of the endoplasmic reticulum. Its deficiency leads to impaired cholesterol processing, causing abnormal sonic Hh signaling, which affects spatial patterning during embryogenesis (128). The cutaneous features may result from a dual mechanism: accumulation of cholesterol precursors and cholesterol deficiency (128).

This X-linked dominant disorder is lethal in male during gestation and thus predominantly affects females. The CK syndrome [initials of the original proband (129)] is an X-linked recessive disorder that affects males being also caused by pathogenic *NSDHL* variants (130). In CHILD syndrome, mosaicism results from inactivation of an X-chromosome in females. Interindividual differences in the pattern of X inactivation explain the phenotypic variations.

### Focal Dermal Hypoplasia (Goltz syndrome; MIM 305600) Clinical Features

Focal dermal hypoplasia is rare; more than 175 cases have been reported. The focal dermal hypoplasia is mostly encountered in females (90%), as its X-linked dominant inheritance leads to lethality in male fetuses. Affected males usually show a mosaic form of focal dermal hypoplasia. This syndrome is evident

at birth, when skin and skeletal symptoms are predominant.

Children with focal dermal hypoplasia show skin atrophy with Blaschko linear arrangement, appearing as depressed or slightly raised red macules. This finding explains the original name "focal dermal hypoplasia" (131). Over the course of disease, fatty tissue can herniate through gaps in the underdeveloped connective tissue forming lipomatous papules. Papillomas and angiofibroma occur on the face and in the urogenitoanal region (132). Additional findings are patchy alopecia and thin hair. Affected children show facial dysmorphies and asymmetric skeletal deformities (e.g., syndactyly, polydactyly, amelia, scoliosis). Renal anomalies occur occasionally and include horseshoes kidneys and hydronephrosis.

No specific therapy for focal dermal hypoplasia exists. Papillomas can be surgically removed, but may reoccur.

#### Genetics and Molecular Pathology

Focal dermal hypoplasia is an X-linked dominant disorder which reflects mosaicism resulting from inactivation of an X-chromosome in females. The pathogenic variants affect *PORCN* (133). *PORCN* is a gene of the porcupine family, which code for endoplasmic reticulum proteins with multiple transmembrane domains involved in the processing of Wnt (wingless and int homolog) proteins. Mutations in different players of the Wnt signaling pathway have been described before to cause CAKUT (134), explaining the pathogenesis of CAKUT in focal dermal hypoplasia. The disease is lethal in males; live-born affected males are rare and nearly always have somatic mosaicism for a *de novo* postzygotic pathogenic variant. Postzygotic mutations may also cause mild disease in females (135).


*AD, autosomal dominant; AR, autosomal recessive; NA, not available; XLD, X-linked dominant; XLR, X-linked recess.*

### CHROMOSOMAL ABERRATIONS

In case of chromosomal aberrations, e.g., deletions or trisomies, a large number of genes are affected by the defect. Therefore, the resulting clinical picture is broad and includes renal and cutaneous anomalies in some syndromes (**Table 6**). However, these are not defining for the clinical picture.

### Differential Diagnosis in Newborns

Several acquired conditions affect both skin and kidney either pre- or postnatally. For example, maternal intake of valproate leads to fetal valproate syndrome commonly showing hemangiomas, altered pigmentation, and occasional renal malformations. Intake of phenytoin during pregnancy causes fetal hydantoin syndrome, in which hirsutism and coarse hair are common and renal malformations can occur. The oligohydramnios sequence (Potter syndrome) arises from lack of amniotic fluid. This anhydramnion or oligohydramnion can either be caused by primary renal problems such as agenesis, severe polycystic kidney deformation or obstruction of the urinary tract, or by chronic leakage from the amniotic sac. Fetal development, especially of the lungs, and life expectancy are severely limited.

### CONCLUSION

Genetic disorders affecting the skin and the kidneys cover a broad range of phenotypes and molecular mechanisms, which have been largely uncovered in the last decades. Many of these conditions comprise involvement of multiple organs and systems. Although, in many cases, the cutaneous findings (e.g., café-au-lait spots, angiofibromas, nevi) have no significant impact on the prognosis, they represent precious signs for the

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clinical diagnosis and should alert pediatricians to carefully evaluate the patients.


### AUTHOR CONTRIBUTIONS

AR wrote most of the clinical part (clinical features) and the tables. YH prepared the figures and reviewed the manuscript. CH drafted the manuscript, wrote the genetics and molecular part, and reviewed the entire manuscript.

### FUNDING

CH was supported by the Deutsche Forschungsgemeinschaft (DFG) CRC/SFB 1140 and AR by the Berta-Ottenstein Programme of the Faculty of Medicine, University of Freiburg. YH was a fellow of the Else-Kröner-Fresenius Foundation.


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mechanism for phenotypic variability in neurofibromatosis type 1. *J Child Neurol* (2012) 27:695–702. doi:10.1177/0883073811423439


development of a basal cell nevus syndrome patient registry. *JAMA Dermatol* (2016). doi:10.1001/jamadermatol.2016.4347


cause CK syndrome. *Am J Hum Genet* (2010) 87:905–14. doi:10.1016/ j.ajhg.2010.11.004


**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2018 Reimer, He and Has. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

## Developmental Programming of Renal Function and Re-Programming Approaches

#### *Eva Nüsken, Jörg Dötsch, Lutz T. Weber and Kai-Dietrich Nüsken\**

*Pediatric Nephrology, Department of Pediatrics, Medical Faculty, University of Cologne, Cologne, Germany*

Chronic kidney disease affects more than 10% of the population. Programming studies have examined the interrelationship between environmental factors in early life and differences in morbidity and mortality between individuals. A number of important principles has been identified, namely permanent structural modifications of organs and cells, long-lasting adjustments of endocrine regulatory circuits, as well as altered gene transcription. Risk factors include intrauterine deficiencies by disturbed placental function or maternal malnutrition, prematurity, intrauterine and postnatal stress, intrauterine and postnatal overnutrition, as well as dietary dysbalances in postnatal life. This mini-review discusses critical developmental periods and long-term sequelae of renal programming in humans and presents studies examining the underlying mechanisms as well as interventional approaches to "re-program" renal susceptibility toward disease. Clinical manifestations of programmed kidney disease include arterial hypertension, proteinuria, aggravation of inflammatory glomerular disease, and loss of kidney function. Nephron number, regulation of the renin–angiotensin–aldosterone system, renal sodium transport, vasomotor and endothelial function, myogenic response, and tubuloglomerular feedback have been identified as being vulnerable to environmental factors. Oxidative stress levels, metabolic pathways, including insulin, leptin, steroids, and arachidonic acid, DNA methylation, and histone configuration may be significantly altered by adverse environmental conditions. Studies on re-programming interventions focused on dietary or anti-oxidative approaches so far. Further studies that broaden our understanding of renal programming mechanisms are needed to ultimately develop preventive strategies. Targeted re-programming interventions in animal models focusing on known mechanisms will contribute to new concepts which finally will have to be translated to human application. Early nutritional concepts with specific modifications in macro- or micronutrients are among the most promising approaches to improve future renal health.

Keywords: kidney development, nephron number, renin–angiotensin–aldosterone system, renal sodium transport, blood pressure, early nutrition, re-programming intervention

### INTRODUCTION

Prevention of chronic kidney disease is a major public health challenge (1). Although diabetes mellitus is the most common cause of chronic kidney disease worldwide (2), developmental programming processes that have been reviewed by us (3, 4) and others (5–7) before substantially contribute to differences in morbidity and mortality between individuals. The normal development of the kidney

#### *Edited by:*

*Miriam Schmidts, Radboud University Nijmegen, Netherlands*

#### *Reviewed by:*

*Jan Michael Williams, University of Mississippi Medical Center School of Dentistry, United States Larry T. Patterson, Children's National Health System, United States*

*\*Correspondence: Kai-Dietrich Nüsken kai-dietrich.nuesken@uk-koeln.de*

#### *Specialty section:*

*This article was submitted to Pediatric Nephrology, a section of the journal Frontiers in Pediatrics*

*Received: 20 October 2017 Accepted: 08 February 2018 Published: 27 February 2018*

#### *Citation:*

*Nüsken E, Dötsch J, Weber LT and Nüsken KD (2018) Developmental Programming of Renal Function and Re-Programming Approaches. Front. Pediatr. 6:36. doi: 10.3389/fped.2018.00036*

**247**

can be disturbed by multiple environmental factors, including intrauterine deficiencies by disturbed placental function or maternal malnutrition, prematurity, intrauterine and postnatal stress, intrauterine and postnatal overnutrition, as well as dietary dysbalances of macro- and micronutrients. Since developmental steps take place during unique developmental periods, timing, and duration of an adverse environment specifically impact on developmental programming. Adverse kidney programming increases the incidence of severe renal and cardiovascular sequels later in life. This includes arterial hypertension and associated end organ damage, the aggravation of inflammatory glomerular disease and the occurrence of end-stage renal disease. Specific "re-programming" interventions may mitigate or even prevent programmed disease. Consequently, our mini-review will address the following topics:


### CRITICAL DEVELOPMENTAL PERIODS OF RENAL PROGRAMMING IN HUMANS

In humans the pronephros begins to form around day 22, urine production starts after 10 weeks (8), and maximum renal growth occurs between 26 and 34 weeks of gestation (9). Around week 36, nephrogenesis is completed and the number of nephrons is determined (8, 10). In preterm infants, adaptation to extrauterine conditions impairs nephrogenesis, and the children end up with fewer nephrons and a higher percentage of morphologically abnormal glomeruli (5, 6, 11, 12). In small for gestational age (SGA) fetuses, intrauterine renal growth is reduced compared to appropriate for gestational age controls (9). In both term and preterm infants, glomerular and tubular functions undergo further maturational changes during the first months of life (8, 13). In these vulnerable periods, babies are often exposed to nephrotoxic medication, such as non-steroidal anti-inflammatory drugs (14), antibiotics, or diuretics, during neonatal intensive care unit treatment (15, 16).

### LONG-TERM SEQUELAE OF RENAL PROGRAMMING IN HUMANS

### Blood Pressure and Loss of Kidney Function

Hypertension is the most important risk factor for cardiovascular events and mortality worldwide (17). Elevated blood pressure contributes to progression of renal insufficiency (18) and is a strong independent risk factor for end-stage renal disease (19). *Vice versa*, decreased renal function is associated with increased blood pressure and cardiovascular morbidity (20). Early detection of blood pressure elevation plays a major role in the prevention of end organ damage (21). Many studies, including a systematic meta-analysis of studies tracking blood pressure during life course, demonstrated that childhood blood pressure predicts blood pressure (22–24) and vascular end organ damage in adulthood (25). Abnormal birth weight, either low or high, increases the risk for blood pressure elevation and loss of renal function in a U-shaped manner (26–31). In SGA individuals, some studies demonstrated elevated blood pressure in childhood (32) or adulthood (33, 34) especially when rapid postnatal catch-up growth and later adiposity were present (35). Further risk factors include high maternal BMI (36) or elevated protein/carbohydrate ratios in maternal diet during pregnancy (37, 38), rapid postnatal weight gain (39), or being born large for gestational age (36, 40, 41).

### Proteinuria and Loss of Kidney Function

Several risk factors during early life predispose toward proteinuria and related decline of renal function. Accordingly, the prevalence of microalbuminuria among adults whose mothers had been exposed to the Dutch Hunger Winter 1944/45 was elevated (42). Chinese women born in the famine years 1959–1961 had a higher risk to develop more severe stages of proteinuria in their forties (43). Low birthweight itself is associated with elevated risks for albuminuria (OR, 1.81; 95% CI, 1.19–2.77), end-stage renal disease (OR, 1.58; 95% CI, 1.33–1.88), or low estimated glomerular filtration rate (GFR) (29, 44, 45). A birthweight-dependent decline in GFR may already be seen in childhood (29, 46, 47).

### Glomerular Disease and Inflammation

Furthermore, a number of studies have evaluated the association between perinatal risk factors and later glomerular disease. Thus, SGA individuals have a higher risk to experience steroid resistance and a more severe course in nephrotic syndrome (48, 49). In IgA-nephropathy, they develop arterial hypertension and glomerulosclerosis more often (50).

### MECHANISMS OF RENAL PROGRAMMING

In human studies, it is difficult to establish mechanistic links in the field of developmental programming since there usually is a large delay between an adverse event and the related clinical phenotype. This makes it very challenging to distinguish the underlying causes from multiple modifying factors. Thus, animal models providing the possibility of equalized postnatal conditions and specific interventions are especially valuable. In rodents, kidney development during the early postnatal period corresponds to the third trimester in humans (10). For an overview of mechanisms see **Figure 1**.

### Nephron Number

Nephron number in humans ranges from ~200,000 to >2.5 million nephrons per kidney (51). The well-known hypothesis of Brenner et al. linked decreased glomerular number with increased glomerular size, hyperfiltration, hypertension, and progressive glomerular injury (7, 52). Nephron number positively correlates with birth weight (53, 54) and is reduced after low-protein (LP) diet throughout pregnancy (55–59), utero-placental insufficiency

(60–62), intrauterine glucocorticoid exposure (63), preterm birth (11, 64), and oxidative stress (65). In addition, a diet deficient in vitamin A (58, 66), zinc (67), or iron (68) is associated with low nephron count. Finally, nephrons get lost with age (69). Interestingly, low nephron number in young individuals is not necessarily associated with hypertension (70, 71). Thus, modulating factors such as early hyperalimentation and aging processes certainly have an impact on renal outcome in individuals with low nephron count (71, 72).

### Renin–Angiotensin–Aldosterone System (RAAS)

Dysregulation of all or single components of the RAAS system may severely impair renal development (73, 74). Both activating and deactivating effects on the RAAS can induce a vicious circle of persisting hormonal dysbalances which may finally contribute to the development of arterial hypertension and renal failure.

In the fetal and perinatal period, downregulation of the RAAS has been identified as a relevant mechanism. In neonatal rats after LP diet during gestation, renal renin and angiotensin II levels (75) as well as angiotensin II receptors type 1 (AT1R) and 2 (AT2R) protein expressions (76) were reduced. Similarly, renal AT2R gene and protein expressions were reduced in fetal rats after prenatal caffeine exposure (77). Fetal angiotensin II levels in plasma were decreased after maternal high-salt diet in sheep (78).

Later in life, most environmental influences during early childhood end up with a RAAS activation. Adult rat offspring from the LP diet model showed elevated blood pressure (59), increased AT1R expression (79, 80) and elevated plasma angiotensin-converting enzyme (ACE) activity going along with slightly elevated angiotensin II levels (81). When challenged with angiotensin II infusion, adult LP offspring reacted with a greater decline in GFR than controls (80). In another LP study, there were more angiotensin II-positive cells in the cortical tubulointerstitium of adult offspring (82). Offspring from diabetic mothers had marked upregulation of angiotensinogen (AGT) and AT1R gene expression as well as increased ACE:ACE2 mRNA ratio (83). Some environmental influences induce RAAS activation already in fetal life. Thus, ovine offspring exposed to high salt during gestation presented with increased gene expression of AGT, ACE, AT1R, and increased ACE:ACE2 and AT1R:AT2R mRNA ratio (78). In the human situation, plasma renin concentrations were elevated in umbilical veins of SGA infants, and birth weight was inversely associated with circulating aldosterone concentrations (84). Treatment of human proximal tubule epithelial cells with palmitic acid demonstrated susceptibility to nutritional factors, as it induced intracellular endoplasmic reticulum (ER) stress and increased angiotensin II concentrations in cell medium. Co-treatment with AT1R-blocker or renin-inhibitor prevented ER stress (85).

### Renal Sodium Transport

In rats, LP nutrition or dexamethasone treatment during gestation both resulted in an upregulation of the bumetanide-sensitive Na-K-2Cl cotransporter and of the thiazide-sensitive Na-Cl cotransporter in the offspring (86, 87). Adult offspring exposed to LP nutrition during gestation and lactation presented with a reduced diuretic response after a single dose of furosemide (88). After prenatal dexamethasone treatment, proximal tubule Na/H exchanger protein expression was increased, going along with an increase in proximal tubule sodium and volume reabsorption (86, 89). Sodium uptake in renal proximal tubule cells from adult male sheep was enhanced after prenatal betamethasone exposure (90). In rat offspring exposed to experimental utero-placental insufficiency (91) or maternal diabetes (92), sodium-dependent hypertension was observed.

## Vasomotor and Endothelial Function

Another interesting aspect is the maturation of vascular smooth muscle function and small artery resistance regulation. Sympathectomy suppresses maturation of the gene program involved in small artery resistance regulation (93). Intrauterine and perinatal stress could, therefore, have a major impact on vascular tone regulation. In addition, vasomotor function can be impaired by perinatal hyperoxia (65) and LP diet (94). Endothelial dysfunction has also been described after intrauterine deficiency and may add to hypertension and glomerular damage (95).

### Myogenic Response and Tubuloglomerular Feedback (TGF)

An impaired myogenic response as well as a disturbed TGF are important contributors to glomerular damage in diabetic and hypertensive nephropathy (96). An altered myogenic response has been described in intrauterine growth-restricted (IUGR) neonates, which may be beneficial postnatally, but harmful in the long run (97). The TGF mechanism matures during fetal life and could, therefore, be susceptible to programming *in utero* (98). However, no study has examined the specific consequences of disturbed intrauterine environment for TGF function.

### Epigenetic Mechanisms

The molecular details of kidney development have been extensively studied (99, 100). Altered DNA methylation, histone modification, and other mechanisms modifying the renal transcriptome may significantly impair renal organogenesis and predispose toward renal disease which has lately been reviewed in detail (101). In this context, it is important to separate epigenetic changes during kidney disease (102) from epigenetic changes during early life leading to "programmed" disease. So far, there is little evidence that single, kidney-specific epigenetic alterations during early life might actually cause renal disease later on. Candidate genes would be all genes which are activated during specific developmental windows. Pax-2, for example, is essential for kidney development, ontogenetically regulated and can be reactivated in repair processes after acute kidney injury (103, 104). Global alterations of methylation associated with hypertension were observed after significant periconceptional deficiency of B vitamins and methionine (105). Thus, nutritional modifications may induce temporary or permanent epigenetic alterations that certainly have the potential to modulate kidney disease.

### Oxidative Stress

Oxidative stress and inflammation are major contributors to vascular remodeling and hypertension (106). In LP (94, 107) and maternal smoking models (108), it was shown that oxidative stress during critical developmental steps may significantly contribute to renal susceptibility toward disease. In addition, both IUGR offspring after global undernutrition of the dam (109) and after high-fat died during gestation and lactation (110) showed increased oxidative stress and elevated blood pressure later in life. Reduction of oxidative stress during early life can prevent programmed hypertension and renal damage (94, 107, 108).

### Metabolism

Rapid postnatal weight gain and early life obesity have been associated with adverse renal outcome (111, 112). Interplay between adiposity, leptin, and insulin resistance with RAAS regulation and sympathetic activity has been described (113, 114). Early postnatal overfeeding in rats by litter size reduction induced increased early postnatal weight gain and was associated with increased blood pressure, glomerulosclerosis, and proteinuria in adulthood (71). In a similar study, postnatal overfeeding resulted in decreased GFR, increased proteinuria and increased deposition of collagens. On the molecular level, intrinsic renal leptin resistance could be demonstrated (115). Dysregulation of renal leptin and Akt/AMPKα signaling associated with increased renal matrix deposition could also be shown in overweight offspring from mothers fed a high-fat diet during gestation and lactation (116). Maternal LP nutrition during rat gestation persistently decreased the expression of renal 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) (117, 118) and increased the expression of the renal glucocorticoid receptor in the offspring (118). The same was shown for sheep offspring exposed to temporary maternal calorie restriction (119).

### Arachidonic Acid Metabolism Pathway

Finally, there is evidence that the arachidonic acid metabolism pathway could be involved in the development of programmed hypertension (120, 121). 20-hydroxyeicosatetraenoic acid (20- HETE), a metabolite of arachidonic acid, contributes to the normal myogenic pressure response. Physiologically, arachidonic acid is released from cell membranes by phospholipase A2, converted to 20-HETE, which then adds to vasoconstriction of the afferent arteriole (96, 122). However, 20-HETE has also been linked to systemic hypertension and endothelial dysfunction in rats (123). Further arachidonic acid metabolites like Cox-2 derived prostaglandins contribute to counter regulatory vasodilation of the afferent arteriole after TGF-mediated vasoconstriction (124) and oxidative stress in the kidney (125), and therefore modulate intraglomerular pressure and GFR as well as renal inflammation (82). Thus, nutritional intake of arachidonic acid may significantly affect blood pressure, kidney function, and kidney survival.

### POTENTIAL THERAPEUTIC "RE-PROGRAMMING" INTERVENTIONS

The ultimate goal of all research on programmed disease is to develop preventive strategies. So far, the number of studies on reprogramming interventions is still limited and mainly restricted to dietary or anti-oxidative approaches.

### Early Dietary Interventions

Data on nutritional interventions are available from both animal and human studies. A meta-analysis showed slightly, but significantly lower blood pressure in infants, children, and adolescents who were breast fed during infancy compared to those being formula fed (126). Micronutrient (127), calcium (128), vitamin A (129, 130), and iron (131) supplementation during pregnancy as well as long-chain polyunsaturated fatty acid (LCPUFA) supplementation in infant formula (132) may be beneficial to renal outcome.

In detail, children of women receiving a multiple micronutrient supplementation during the second and third trimesters of pregnancy were heavier and had lower systolic blood pressure during infancy (127). Calcium supplementation from the 20th gestational week until delivery lowered systolic blood pressure in children aged 7 years, with a stronger effect when children were overweight (128). Supplementation of iron and folate until the end of pregnancy in rural Bangladesh caused a slightly decreased diastolic blood pressure and a slightly increased GFR in infants at the age of 4.5 years when started at the ninth, but not when started at the 20th gestational week (131). Another dietary intervention with micronutrient supplementation in malnourished pregnant Nepalese women until 3 months postpartum showed that folic acid or the combination of folic acid, iron, and zinc reduced the risk of microalbuminuria, but not blood pressure in the children aged 6–8 years (133). The effects of retinoic acid have mainly been studied in animals. Decreased availability of retinoic acid induced by down-regulated vitamin A metabolism after previous overexposure to vitamin A strongly impairs metanephric kidney development, which can be restored by adequate retinoic acid supplementation (129). In rat offspring exposed to LP diet of the dam during pregnancy, a single injection of retinoic acid to the dam at midgestation increased postnatal nephron number at 4 weeks of age (130). Postnatal administration of retinoic acid in preterm baboons, however, did not alter kidney growth or nephron number, presumably because the timing of the intervention was chosen too late (134).

Long-chain polyunsaturated fatty acid supplementation with arachidonic acid and docosahexaenoic acid (ratio 2:1) in infant milk formula (IF) during the first 4 months of life lowered blood pressure at 6 years of age compared to IF without LCPUFAs. Blood pressure of children fed LCPUFA-IF was similar compared to breast fed children (132). A diet sufficient in ω-3 PUFAs reduced blood pressure levels compared to a diet almost free of ω-3 PUFAs in TGR(mRen-2)27 rats which have high angiotensin II levels (135). Finally, the importance of the amino acid composition was demonstrated. Addition of glycine to maternal LP diet throughout gestation normalized body weight and blood pressure at 4 weeks of age in rat offspring, whereas alanine or urea had no effect (136).

### Anti-Oxidative Substances

Re-programming interventions with anti-oxidative substances have only been performed in animals. Supplementation of maternal LP diet with anti-oxidative (ACH09)-derived polyphenols extracted from grape skins reduced signs of renal oxidative stress in the offspring on the first postnatal day and attenuated the adverse effects of maternal LP diet on glomerular number and maturity (107). Administration of a lipid peroxidation inhibitor along with LP diet in gestation reduced prenatal oxidative stress and prevented programming of elevated blood pressure, enhanced vasoconstriction after angiotensin II administration and reduced vasodilation after sodium nitroprusside administration in adult animals (94). Similarly, treatment of previously malnourished dams with α-tocopherol during lactation prevented the development of hypertension in the offspring. In addition, upregulated angiotensin II levels and down-regulated Cox-2 expression in

### REFERENCES


the tubulointersititum were brought back to control levels and oxidative stress as well as macrophage infiltration was prevented. However, treatment of control dams with α-tocopherol resulted in arterial hypertension of the offspring (82).

### CONCLUSION AND FUTURE DIRECTIONS

The concept of "developmental origins of health and disease" highlights the interrelationship between environmental factors throughout life and differences in morbidity and mortality between individuals. Chronic kidney disease affects more than 10% of the population (1). High blood pressure, childhood underweight, and suboptimal breastfeeding are among the top risk factors contributing to global burden of disease (137). Prematurity, IUGR, overweight in early life, and other conditions have been associated with the development of arterial hypertension, proteinuria, and decline of renal function. Around 11% of all live-born infants worldwide are born preterm (138). IUGR is seen in 3–7% of all pregnancies (139). During childhood, 5–6% of girls and 7–8% of boys become overweight (140). Thus, renal programming is not a rare phenomenon but affects large parts of the population. Further studies that broaden our understanding of renal programming mechanisms are needed to ultimately develop preventive strategies. Targeted re-programming interventions in animal models focusing on known mechanisms will contribute to new concepts which finally will have to be translated to human application. Early nutritional concepts with specific modifications in macro- or micronutrients are among the most promising approaches to improve future renal health.

## AUTHOR CONTRIBUTIONS

EN performed the majority of literature research, and designed and wrote the review. K-DN contributed to literature research, and designed and wrote the review. JD and LW contributed to literature research and writing. All authors revised and approved the review.


**Conflict of Interest Statement:** The authors declare that the article was written in absence of any commercial or financial relationships that could be a potential conflict of interest to the topic.

*Copyright © 2018 Nüsken, Dötsch, Weber and Nüsken. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*