Concurrent IgA Nephropathy and Membranous Nephropathy, Is It an Overlap Syndrome?

IgA nephropathy (IgAN) and membranous nephropathy (MN) are common glomerulonephritis, the presence of which in the same patient– concurrent of IgAN and MN (cIgAN/MN) has been described occasionally. This study aims to show clinical-pathological features of cIgAN/MN and attempts to suggest underlying pathogenesis using disease-specific biomarkers and a genomics approach. This retrospective cohort study described the clinical and pathological data from 137 patients with cIgAN/MN diagnosed in Peking University First Hospital from 2005 to 2019. One hundred primary IgAN and 100 MN cases were randomly selected as disease controls between the same time interval. Moreover, disease-specific biomarkers and polygenic risk score models were conducted to reveal the underlying pathogenesis. The median age of the cIgAN/MN cases was 45-year-old, and 46% were women. Compared to IgAN, patients with cIgAN/MN had a higher level of 24-hour proteinuria excretion but lower microscopic hematuria. They had a lower median level of galactose-deficient IgA1 (Gd-IgA1, 4.00 versus 5.45 μg/ml, P=0.002) as well as the standardized genetic risk scores of developing IgAN (GRSs: 0.05 versus 0.68, P<0.001). Compared to MN, patients with cIgAN/MN had a lower proportion of nephrotic syndrome and a lower level of albumin-to-creatinine ratio. However, the 24-hour proteinuria levels, serum lipid profiles, proportion of hypertension, and pathology classification were similar. Patients with cIgAN/MN had lower levels of plasma autoantibodies against the M-type transmembrane phospholipase A2 receptor (PLA2R) (11.23 versus 36.59 U/ml, P=0.005). Intriguingly, there were no statistical differences in standardized GRSs of developing MN between them (2.77 versus 3.02, P=0.326). Compared to IgAN, cIgAN/MN may lean towards MN more according to clinical-pathological features, disease-specific biomarker levels, and disease-specific genetic risk scores.


INTRODUCTION
Immunoglobulin A nephropathy (IgAN) and membranous nephropathy (MN) are two types of common glomerulonephritis (GN) worldwide. It was reported that IgAN is the most common GN in patients less than 59-year-old, and MN is the most frequently observed GN in patients at age ≥60 years (1). These two diseases differ in clinical features, pathology, treatment, and prognosis. IgAN is more unlikely to develop hypo-albuminemia but more likely to present episodes of gross hematuria. IgAN can only be diagnosed with a kidney biopsy (2). However, a kidney biopsy may not be required to confirm the diagnosis of MN in patients with a compatible clinical and serological presentation like antibodies against PLA2R according to the 2021 KDIGO guideline (3,4). Proteinuria reduction to <1g/d is a surrogate marker of improved kidney outcome in IgAN, and immunosuppressive drugs should only be considered in patients with IgAN who remain proteinuria >1g/24h despite at least 90 days of optimized supportive care. Differently, immunosuppressive therapy may not be required in patients with MN when proteinuria <3.5 g/d and eGFR >60 ml/min/1.73 m 2 . Thus, elucidating the underlying disease is pivotal in guiding management and treatment decisions, which seems to be difficult when two diseases coexist in the same patient.
It is suggested that concurrent IgAN and MN (cIgAN/MN) in the same patient is rare, but more and more cases have been reported since 1983 (5)(6)(7)(8)(9)(10)(11). From the literature, all the patients had hematuria and nephrotic range proteinuria. In our previous reports on clinical-pathological features of 26 patients with cIgAN/MN, we observed that these patients displayed similar clinical features with MN patients and milder pathological lesions than IgAN patients (12), and they had comparable serum levels of Gd-IgA1 with IgAN, but lower detectable serum levels of anti-PLA2R compared with MN. These patients showed characteristics of both diseases. We thus suggested that cIgAN/MN might result from superimposed MN on a background of preexisting mild IgAN. However, the limitations of the previous studies are the small sample size and lack of pathogenesis investigation.
Thus, the aim of this study is to show clinical-pathological features with a larger sample size in 137 patients of cIgAN/MN and attempt to suggest underlying disease pathogenesis using disease-specific biomarkers and a genomics approach. Any attempts to address the issue should be of clinical relevance as different treatment strategies may lead to different prognoses.

ARTICLE TYPES
Original Research.

MATERIALS AND METHODS
All the study protocols complied with the principles of the Declaration of Helsinki and were approved by the Ethics Secondly, we screened those patients with concurrent MN (n=180). The diagnosis of cIgAN/MN was additionally confirmed by kidney biopsy with membranous thickening of the glomerular capillary wall; dominant staining for IgG, C3 in glomerular capillary walls on immunofluorescence microscopy; the existence of subepithelial electron-dense on electron microscopy ( Figure 1). Patients who were secondary MN, like autoimmune disease (lupus erythematosus), infection with hepatitis B, hepatitis C, or syphilis, certain medications (gold/ mercury salts and nonsteroidal anti-inflammatory drugs) or solid cancerous tumors or blood cancers were excluded from our study (n=43). The flowchart of the recruitment process was displayed in Figure 2.

The Selection of the Disease-Control Groups
Meanwhile, we searched our database for disease-control groups from the same time period. One hundred patients with primary MN and 100 with primary IgAN were retrieved using the stratified random sampling method as disease controls based on the year of hospital admission.

General and Clinical Information of All the Enrolled Patients
Demographics and clinical features at the time of renal biopsy performance included age, gender, blood pressure, urinary sediment microscopy, 24-hour urine protein excretion, serum levels of IgA and IgG, uric acid, lipid profiles, and creatinine. Hyperlipidemia was defined as the total cholesterol above 6.2 mmol/L or triglycerides above 2.3 mmol/L. The estimated glomerular filtration rate (eGFR) was calculated by the Chronic Kidney Disease Epidemiology Collaboration equation (13).

Pathological Features
All the kidney sections were processed for immunofluorescence examination, light microscopy, and electron microscopy. Pathological data were also recorded for each patient, including the degree of mesangial cell proliferation, the proportion of glomeruli with cellular/fibro cellular/fibrous crescents, the intensity of immunofluorescence staining (IgG/ C3/IgA), and electron-dense deposits on the mesangial area or glomerular basement membrane (GBM). Because in the original development of Oxford classification, those with secondary causes of mesangial IgA deposits or those with comorbid conditions were excluded. The Oxford MEST scores may be not proper in evaluating pathology lesions in patients with cIgAN/MN, which were not reported here. The pathological stages of MN were based on the electron microscopic findings, referring to the immune deposits in the GBM, GBM reaction to the deposits, and resolution of glomerular injury with resorption of the deposits (14).

Assay of Gd-IgA1 and IgA1
Levels of plasma Gd-IgA1 (1:200 dilution) were detected using the KM55 ELISA kit (27600, Immuno-Biological Laboratories, USA). All the experiments were done according to the manufacturer's instructions. Moreover, plasma IgA1 levels were determined using capture ELISA. High-absorption polystyrene plates (Thermo, USA) were coated with 2.5 mg/ml F(ab')2 fragment of goat anti-human IgA (Jackson ImmunoResearch, USA) overnight at 4°C. After washing and blocking with 1% bovine serum albumin in PBS with 0.1% Tween, the diluted plasma (1:80000) was added for incubation. The diluted plasma was then detected by the anti-human IgA1 horseradish peroxidase-conjugated antibody (25-783-72807, Gentaur, Belgium). The optical density at 450 nm was measured after the tetramethylbenzidine liquid substrate system was applied.

Single Nucleotide Polymorphisms (SNPs) Selection and Genotyping
Disease-specific genetic risk scores were calculated to deepen our understanding of the pathogenesis of cIgAN/MN. As previously reported, the IgAN risk score equation was based on the 15 SNPs associated with IgA nephropathy in the analysis of 20,612 individuals from 14 international case-control cohorts of European and Asian ancestry (16). The risk score was standardized using genotypes of 1,050 individuals from 52 worldwide populations included in the Human Genome Diversity Project (17). The MN risk score equation was derived from GWAS analysis of 12,820 individuals (3,782 primary MN and 9,038 controls), including 4,841 individuals of East Asian ancestry (1,632 cases and 3,209 controls) and 7,979 individuals of European ancestry (2,150 cases and 5,829 controls). We adopted the East Asians risk score, which is calculated separately for East Asians and standard-normalized using genotypes of healthy ancestry-matched controls (18).
Peripheral blood samples were collected from participants using anticoagulant EDTA. We obtained the genomic DNA from peripheral blood leukocytes using the salting-out method. Genotyping was performed using the Kompetitive Allele Specific PCR (KASP) assays (Compass Biotech, Tianjin, China) among 132 patients with cIgAN/MN, 96 with MN, and 98 with IgAN.

Genetic Risk Score Assessment
Individuals with 100% non-missing genotypes across all the scored loci were analyzed. The weighted genetic risk score (GRS) assessment was adopted to evaluate individual disease risk. The weighted-GRS utilized the allelic odds ratios to account for the strength of the genetic association within each allele because alleles might have different odds ratios. The weighted GRS was the weighted sum of risk allele counts, where the weight for each SNP was the natural log of the odds ratio. Details about the calculation formula were derived from http://www. columbiamedicine.org/divisions/kiryluk/resources.php.

Follow-Up Data
Follow-up was defined as the interval between renal biopsy and the last outpatient visit. Full details about the follow-up data, such as general clinical course, medication history, renal function, and urinalyses, were recorded. Time-averaged proteinuria (TA-proteinuria) was calculated as the weighted mean of all the 24-hour urine protein excretion measurements during follow-up, with the weight representing the time elapsed since the previous measurement (19). Patients were also classified according to the magnitude of TA-proteinuria (>1.0 or ≤1.0 g/d). Complete remission was defined as urinary protein excretion <0.5 g/24 h confirmed by two values at least one month apart, accompanied by a normal serum albumin concentration and normal serum creatinine level. Partial remission was defined as urinary protein excretion <3.5 g/24 h and reduced by at least 50% from peak values, accompanied by an improvement or normalization of the serum albumin concentration as well as stable serum creatinine.

Statistical Analysis
Continuous variables in this study were compared using an unpaired t-test or analysis of variance (ANOVA) between groups if the variables were normally distributed; otherwise, a Mann-Whitney U test or Kruskal-Wallis test was performed. Categorical variables were compared using the chi-square test or Fisher's exact test. Cumulative proteinuria remission rates were calculated according to the Kaplan-Meier method (log-rank test).
The statistical analysis was performed with the STATA 15.0 (Texas, USA). A two-tailed P-value <0.05 was considered statistically significant.

Demographics
From

Gd-IgA1
Compared to IgAN, the level of plasma IgA in patients with cIgAN/MN was lower (2.37 versus 3.00 g/L, P<0.001). Although the level of plasma IgA1 was similar, they had a lower median level of Gd-IgA1 (4.00 versus 5.45 mg/ml, P=0.002). In contrast, patients with cIgAN/MN had a comparable level of Gd-IgA1 compared to that of MN (4.00 versus 3.64 mg/ml, P=0.100).

Anti-PLA2R
Compared to MN, patients with cIgAN/MN had a lower frequency of plasma anti-PLA2R antibody positivity (40.43% versus 59.14%, P=0.036; 20U/ml as the cut-off value) and lower titers of antibody (11.23 versus 36.59 U/ml, P=0.005). Of certain, patients with IgAN did not have detectable anti-PLA2R antibodies in sera ( Table 3 and Figure 3).

Disease-Specific Genetic Risk Scores (GRSs)
To determine an individual's risk of developing IgAN and MN based on specific genetic markers, we calculated polygenic risk scores, which bridge the gap between initial discovery efforts and clinical applications for the estimation of disease risk using genetics. With the most updated genetic risk models suggested by Kiryluk (Table 4 and Figure 4).

IgAN-GRSs
Compared to IgAN, patients with cIgAN/MN had lower standardized IgAN-GRSs (0.05 versus 0.68, P<0.001). By stratifying the IgAN-GRSs into three risk groups as reported (high >1; average -1~1; low<-1), patients with cIgAN/MN showed a significantly lower frequency of high-risk group     Comparisons across patients with or without complete follow-up data were performed, confirming no selection bias   Table 1). We updated all the details among the patients who were regularly followed up until February 09 th , 2022. The median follow-up period was 53 months. Twenty-two (73.33%) patients with cIgAN/MN achieved complete or partial remission, which was similar to MN (68.42%, P=0.66). 26.67% and 31.58% of patients with cIgAN/MN and MN showed no proteinuria remission. However, the percentages of glucocorticoids and other immunosuppressive agents in patients with cIgAN/MN were lower than those in MN. We assume milder pathology lesions and low anti-PLA2R titers in patients with cIgAN/MN might explain this difference. Kaplan-Meier analysis ( Figure 5) showed that the cumulative incidence of complete or partial remission was similar between patients with cIgAN/MN and MN or IgAN. Compared to IgAN, the cumulative incidence of persistent proteinuria after therapy in patients with cIgAN/MN was higher than in IgAN (P=0.001).

DISCUSSION
The present study mainly shows the clinical and pathological data about 137 patients with cIgAN/MN. Additionally, diseasespecific biomarkers and genomics analysis were checked for a better understanding of the pathogenesis of cIgAN/MN. Subsequently, more cases had been noticed in the clinic, and the reported patients had hematuria and nephrotic range proteinuria (6,(8)(9)(10). Magil et al. also showed an isolated case, suggesting that cIgAN/MN might be associated with hepatitis B surface antigenemia (7). Other related types of research showed that IgAN and MN occurred separately in one patient after some Complete remission is defined as urinary protein excretion <0.5 g/24 h confirmed by two values at least one month apart, accompanied by a normal serum albumin concentration and normal serum creatinine level. Partial remission was defined as urinary protein excretion <3.5 g/24 h and reduced by at least 50% from peak values, accompanied by an improvement or normalization of the serum albumin concentration as well as stable serum creatinine. c Categorical variables were compared using the chi-square test or Fisher's exact test.
ACEI, angiotensin-converting enzyme inhibitors; ARB, angiotensin II receptor blockers. years' interval (20,21 Moreover, we used disease-specific biomarkers and a genomics approach to shed some light on its pathogenesis. The plasma level of Gd-IgA1 in patients with cIgAN/MN is much lower than IgAN but had no significant difference with MN. Thus, Gd-IgA1 seems not to be a significant pathogenic factor in cIgAN/MN. On the other hand, nearly 40.43% of patients with cIgAN/MN had detectable plasma levels of anti-PLA2R antibodies. Although the plasma level of anti-PLA2R antibodies in cIgAN/MN is much lower than that in MN, anti-PLA2R antibodies may at least in part contribute to the pathogenesis of cIgAN/MN. To gain more insights into the pathogenesis of cIgAN/MN from a genetic perspective, we genotyped the majority of the subjects examined. The genetic risk score of developing IgAN in patients with cIgAN/MN is much lower than IgAN. Stratified analyses also showed that the high-risk group of developing IgAN in patients with cIgAN/MN is much lower than IgAN. Intriguingly, after calculating the genetic risk scores of developing MN, we did not observe a significant difference between patients with cIgAN/MN and MN. Thus, the genetic background of cIgAN/MN resembles that of MN. Therefore, the present study may demonstrate that cIgAN/MN seems to be just concurrent, not the overlap syndrome of IgAN and MN. cIgAN/ MN may result from superimposed MN on a background of mild IgAN or IgA deposition (summarized in Figure 6).
Despite a relatively large-scale study on cIgAN/MN, we should note some limitations. First, the pathogenesis of cIgAN/MN is still not clear, but disease-specific markers in the circulatory system may be highlighted in disease differentiation. Second, patients with cIgAN/MN enrolled in the present study are diagnosed simultaneously by renal biopsy. We cannot rule out the possibility that IgAN or MN may superimpose on the basis of preexisting one of the other glomerular diseases. Third, our follow-up data are not complete. Only one-third of the patients enrolled from pathology dataset had been regularly followed-up. The composite endpoint, such as the decline rate in eGFR and the receipt of renal replacement therapy, should be further recorded in long-term follow-up. Fourth, as a growing number of cases will be discovered in the clinic, additional research with larger sample sizes is still warranted. More studies focusing on other biomarkers related to underlying mechanisms, such as T cells and activation of the complement system, will also be conducted (22)(23)(24)(25)(26). Fifth, we mainly recruited participants of Chinese Han ethnicity. IgA deposition was different among various ethnicities (27)(28)(29)(30). Replication from different ethnicities or different populations was needed for similar studies.
In conclusion, although MN is concurrent with IgA deposition and mesangial proliferation in pathology, from the clinical, diagnostic, and prognostic point of view, cIgAN/MN is more likely to be MN accompanied by IgA deposition and may not be an independent disease. FIGURE 6 | The symbol "<" or ">" indicates that the specific item is lower or higher than the other group, respectively. Moreover, the symbol "⊙" means no statistical difference between the two groups.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

ETHICS STATEMENT
All the study protocols complied with the principles of the Declaration of Helsinki and were approved by the Ethics Committee of Peking University First Hospital (Institutional Review Board number: 2021[Y148]). The patients/participants provided their written informed consent to participate in this study.