Edited by: Janos G. Filep, Université de Montréal, Canada
Reviewed by: Mariya Hristova, University College London, United Kingdom; Daniel Ricklin, University of Basel, Switzerland
Specialty section: This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology
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Intravascular erythrocyte destruction, accompanied by the release of pro-oxidative and pro-inflammatory components hemoglobin and heme, is a common event in the pathogenesis of numerous diseases with heterogeneous etiology and clinical features. A frequent adverse effect related to massive hemolysis is the renal injury and inflammation. Nevertheless, it is still unclear whether heme––a danger-associated molecular pattern––and ligand for TLR4 or upstream hemolysis-derived products are responsible for these effects. Well-characterized animal models of hemolysis with kidney impairment are needed to investigate how hemolysis drives kidney injury and to test novel therapeutic strategies. Here, we characterized the pathological processes leading to acute kidney injury and inflammation during massive intravascular hemolysis, using a mouse model of phenylhydrazine (PHZ)-triggered erythrocyte destruction. We observed profound changes in mRNA levels for markers of tubular damage (Kim-1, NGAL) and regeneration (indirect marker of tubular injury, Ki-67), and tissue and vascular inflammation (IL-6, E-selectin, P-selectin, ICAM-1) in kidneys of PHZ-treated mice, associated with ultrastructural signs of tubular injury. Moreover, mass spectrometry revealed presence of markers of tubular damage in urine, including meprin-α, cytoskeletal keratins, α-1-antitrypsin, and α-1-microglobulin. Signs of renal injury and inflammation rapidly resolved and the renal function was preserved, despite major changes in metabolic parameters of PHZ-injected animals. Mechanistically, renal alterations were largely heme-independent, since injection of free heme could not reproduce them, and scavenging heme with hemopexin in PHZ-administered mice could not prevent them. Reduced overall health status of the mice suggested multiorgan involvement. We detected amylasemia and amylasuria, two markers of acute pancreatitis. We also provide detailed characterization of renal manifestations associated with acute intravascular hemolysis, which may be mediated by hemolysis-derived products upstream of heme release. This analysis provides a platform for further investigations of hemolytic diseases and associated renal injury and the evaluation of novel therapeutic strategies that target intravascular hemolysis.
Intravascular erythrocyte destruction, accompanied by the release of pro-oxidative and pro-inflammaotry components hemoglobin and heme, is a common event in the pathogenesis of numerous diseases with heterogeneous etiologic factors and clinical features, such as sickle-cell disease (SCD), microangiopathic hemolytic anemias, ABO mismatch transfusion reaction, paroxysmal nocturnal hemoglobinuria, autoimmune hemolytic anemia, malaria, cardiopulmonary bypass, mechanical heart valve-induced anemia and chemical-induced anemias, and many others (
Intravascular hemolysis is associated with acute kidney injury, most likely due to oxidative stress, cytotoxicity resulting in tubular necrosis, intratubular casts, and pro-inflammatory effects, such as production of IL-6 or MCP-1 (
The objective of this study is to characterize the pathological processes leading to kidney injury in acute drug-induced intravascular hemolysis. We aimed to characterize the inflammation and the renal phenotype of a mouse model of intravascular hemolysis triggered by PHZ and to find out to what extend it is dependent on the release of free heme.
Solution of PHZ of 25 mg/mL (Sigma-Aldrich) was prepared in PBS immediately before use. The Fe3+ form of heme [hemin (ferriprotoporphyrin IX), designated as heme (Frontier Scientific Inc. or Sigma-Aldrich)] was dissolved to 20 mM in 50-mM NaOH and 145-mM NaCl, and further diluted in PBS just before use. Plasma-purified Hx was provided by CSL Behring.
All experiments were conducted in accordance with the recommendations for the care and use of laboratory animals following the ARRIVE regulations and with the approval APAFIS#3764-201601121739330v3 of the French Ministry of Agriculture. C57Bl/6 mice were from Charles River Laboratories (L’Arbresle, France). Female C57Bl/6 mice were injected i.p. with 200 µL of PBS (Gibco), or freshly prepared heme [40 µmol/kg, corresponding to 28-µg/g body weight (
The follow-up of renal function was performed by placing 6-week-old C57Bl/6 female mice (Charles Rivers) in individual metabolic cages for acclimation for 5 days, with daily weight measurement and fixed, daily amounts of food. After stabilization of body weight (day −3) metabolic parameters were measured (diuresis, amount of excrements, water and food uptake, and body weight). Urine was collected at fixed times for 3 days at baseline, and 2 days after injections. PHZ, heme, or PBS were injected at day 0. Blood was taken from the retro-orbital sinus before and at day 2 after treatment. Blood gas was measured at day 2 after treatment (Alere system, self-calibrated epoc® BGEM Test Card). Organs were recovered at day 2. In alternative experiments in regular cages organs were recovered at day 4. Organs were snap-frozen in liquid nitrogen in Cryomatrix (Thermoscientific). Urinary protein and creatinin levels, as well as plasma urea concentrations were measured using Konelab equipment. For comparative purposes, similar experiments were performed in regular cages, with five animals per group.
Moreover, 3-μm-thick sections of fixed (PFA 4%) frozen kidneys were cut with Cryostat Leica AS-LMD. Heme oxygenase 1 (HO-1) expression was studied using rabbit anti-mouse HO-1 (Abcam, Ab13243) followed by a polymer anti-rabbit IgG-HRP (DAKO, K4003). Staining was revealed with DAB solution. Slides were scanned by Nanozoomer (Hamamatsu). Hematoxylin–Eosin, Perl’s Prussian blue, and PAS coloration were performed by routine procedures using sections of paraffin-embedded kidneys at days 1, 2, and 4. Coloration of slides was scanned by Slide Scanner Axio Scan (Zeiss).
Snap-frozen kidney sections were recovered in RLT buffer (Qiagen) + 1% β-mercaptoethanol (Gibco) and used for mRNA extraction with Qiagen RNeasy miniKit. The quality and quantity of mRNA were evaluated with bioanalyser Agilent 2100 using Agilent TNA 6000 NanoKit and if the RNA integrity number was >7 the mRNA was retrotranscribed to cDNA. Gene markers of early tubular and endothelium activation/injury relevant for hemolysis and SCD were analyzed by low-density array (LDA, ThermoFisher) including NGAL, Kim-1, HO-1, Il-6, Ki67, ICAM-1, E-selectin and P-selectin, Caspase-3, and CD31 (
Urine of the mice recovered from the metabolic cages was centrifuged and diluted 1/2 with a sample buffer containing glycerol and bromophenol blue (but not SDS) and 20 µL are deposited on 10 wells 12% gels and migrated for 40 min. Staining the gel-resolved proteins was performed with Coomassie blue.
After discoloration, bands of interest were excised from the Coomassie blue-stained SDS page gel at the same level for the PBS, heme and PHZ-treated animals. Samples were then reduced with the addition of DTT (4.2 mM, final concentration) for 45 min at 37°C, then alkylated with iodoacetamide (7.6 mM, final concentration) and subjected to in-gel tryptic digestion using porcine trypsin (Promega, France) at 12.5 ng/µL. The dried peptides extracts were dissolved in 12 µL of solvent A (2% acetonitrile, 0.1% formic acid) and analyzed by online nanoLC using an Ultimate 3000 System (Dionex) coupled to a LTQ-XL Orbitrap mass spectrometer (Thermo Fisher Scientific). Each peptide extract (5 µL) was loaded on a C18 precolumn (Acclaim PepMap C18, 5-mm length × 300-µm I.D., 5-µm particle size, 100-Å porosity, Dionex) at 20 µL/min in solvent A. After 5-min desalting, the precolumn was switched online with a C18 capillary column (Acclaim PepMap C18, 15-cm length × 75-µm ID × 3-µm particle size, 100-Å porosity, Dionex) equilibrated in solvent A. Peptides were eluted using a 0–70% gradient of solvent B (80% acetonitrile, 0.1% formic acid) during 50 min at a flow rate of 300 nL/min. The LTQ-XL Orbitrap was operated in data-dependent acquisition mode with the XCalibur software. Survey scan MS were acquired in the Orbitrap in the 400–1,600
Peak lists extraction from XCalibur raw files were automatically performed using Proteome Discover software (version 1.4, Thermo Fisher scientific). Database searches were performed using the Mascot server v2.2.07 with the following parameters: database
The gels with urine proteins, resolved by electrophoresis as above were transferred to nitrocellulose membranes using iBlot equipment (Invitrogen) and stained with Rabbit polyclonal anti-pancreatic alpha amylase antibody (Abcam, ab199132) using SNAP technology (Merk Millipore), followed by immunodetection with Goat anti Rabbit IgG-HRP (Santa Cruz, 1/5,000). The signal was revealed by chemiluminescence.
The levels of α-amylase were detected in mouse plasma, using Colorimetric Amylase Assay Kit (Abcam, ab102523) using the protocol provided by the manufacturer.
Primary human umbilical vein endothelial cells (HUVEC) were cultured as described previously (
Results were analyzed using a statistical software package (GraphPad Prism 5) as indicated in figures legends. Briefly,
To find out to what extend the acute intravascular hemolysis and heme induce renal injury, we evaluated the parameters of the kidney function, placing the mice in metabolic cages. Blood gas analyses revealed that heme- and PHZ-treated mice had decreased blood hemoglobin at day 2 compared with PBS controls of about 8% (not reaching significance) and significant 46%, respectively (Figure
Evaluation of the renal function parameters in the phenylhydrazine (PHZ) and heme-injected mice in metabolic cages.
Kidneys (Figure
Renal histology of phenylhydrazine (PHZ)-treated mice.
Furthermore, we analyzed at gene level a number of more sensitive markers for inflammation and tissue injury in the acute hemolysis model. Sustained expression of markers of tubular injury NGAL and Kim-1, till days 2 and 4, respectively (Figures
Hemolysis inducing renal injury. Kinetics of mRNA levels in renal tissue of
To find out whether the endothelial activation was due to intravascular hemolysis or related to direct effects of PHZ, endothelial cells (HUVEC) were exposed to PHZ
To find out whether these changes are heme-dependent or occur due to upstream products (as hemoglobin), we investigated expression of NGAL, Kim-1, and IL-6 in heme-treated mice at day 2. We detected a significant increase of NGAL expression (Figure
The renal injury which is largely heme-independent. mRNA levels of NGAL
We analyzed the expression of the same gene panel in liver and spleen and observed similar alterations of a set of genes in the PHZ-treated mice, indicating that this model is associated with multiorgan lesions (data not shown).
Strong upregulation of the cytoprotective enzyme HO-1 till day 4 at both gene (Figures
Heme oxygenase 1 (HO-1) expression induced in tubular kidneys in response to hemolysis.
To find out if the tubular necrosis and the oxidative and inflammatory stress in the kidney result in alteration of proteinuria, the profile of the urinary protein content was examined by electrophoresis (Figure
Phenylhydrazine (PHZ) triggered pancreatic α-amylase activity.
Nevertheless, when samples were deposited at equal volume (for qualitative analyses) several proteins that were not present in the PBS controls were specifically identified in the PHZ and heme samples. To identify these proteins and find out whether they reflect alteration of the tubular or glomerular function, a mass spectrometry analyses were performed by in-gel trypsin digestion of the proteins. Urine of PHZ-treated animals was positive for meprin-α, cytoskeletal keratins, α-1-antitrypsine and α-1-microglobulin (protein AMBP), while heme-treated mice urine was positive for meprin-α only (Table
Proteins, identified by mass spectrometry in each of the bands, excised from the gel of urinary electrophoresis.
MW kDa | Heme | PHZ | PBS | BSA |
---|---|---|---|---|
Band 200 | Meprin A subunit alpha P28825 | Meprin A subunit alpha P28825 | Uromodulin Q91 × 17 | |
Band 75 | Serum albumin P07724 |
|||
Band 70 | Uromodulin Q91 × 17 | Serotransferrin Q0921I1 |
Serotransferrin Q0921I1 | |
Band 65 | Serotransferrin Q0921I1 |
|||
Band 60 | Serum albumin P07724 | Serum albumin P07724 | Serum albumin P07724 | Serum albumin P02769 |
Band 50–55 | Pancreatic alpha-amylase P00688 | Serum albumin P07724 |
||
Band 40 | Pancreatic alpha-amylase P00688 | Serum albumin P07724 | ||
Band 38 | Keratin, type-II cytoskeletal 79 Q8VED5 |
|||
Band 35 | Keratin, type-I cytoskeletal 42 Q6IFX2 |
|||
Band 25 | Kallikrein P15947 | Kallikrein P15947 |
Kallikrein P15947 |
The gels revealed also a presence of a thick ~55-kD band, characteristic for the heme and to less extend to PHZ-injected mice, which was absent in the PBS controls (Figure
Presence of pancreatic α-amylase in the urine could suggest an acute pancreatitis, caused by heme toxicity and hemolysis. To test this hypothesis, measurement of plasmatic pancreatic α-amylase was performed and these were found indeed increased in PHZ-treated animals as compared with controls (Figure
Here, we provide a detailed description of the renal phenotype of a mouse model of intravascular hemolysis, triggered by injection of PHZ. Hemolysis induced rapidly renal inflammation and mild tubular injury, which were largely heme-independent. In addition, we found signs of multiorgan injury, as revealed by major alterations of multiple metabolic parameters in mice, including acute pancreatitis in response to heme and PHZ.
Diverse experimental models of intravascular hemolysis have been described, but the most common one is drug-induced hemolysis with PHZ. Using this model, the protective role of hemoglobin- and heme-scavenging proteins, such as haptoglobin and Hx, as well as heme-degrading enzyme HO-1 function have been demonstrated (
A main concern in this model is the specificity of the used drug. Indeed, PHZ is classically applied to trigger experimental erythrocytes lysis, but its effects on other cell types are poorly documented. Our results indicate that even high doses of PHZ do not cause endothelial cell activation and the observed expression of endothelial activation markers is, therefore, related to the hemolysis-derived products, rather than to direct effects of PHZ itself.
Kidneys are the primary route for hemoglobin clearance after saturation of the natural scavenging systems, and they are therefore highly susceptible to organ dysfunction during hemolysis. Renal lesions are described as major complications of hemolysis (
Renal inflammation has rarely been studied in detail in hemolytic models. In PHZ-injected mice, we detected IL-6 as a marker of tissue inflammation, as previously reported in other hemolysis models (
Taken together, our results indicate that kidneys respond to hemolysis through increased endothelial activation, since we found pro-inflammatory changes in renal endothelial cells, with upregulated expression of P-selectin, E-selectin, and ICAM-1. These adhesion molecules are thought to contribute to renal inflammation (
Detailed examination of metabolic parameters and renal function in PHZ-treated mice revealed extensive anemia, but only minor alterations of uremia, and no difference in parameters related to glomerular filtration at 48 h. We detected increased proteinuria and severe, transient oliguria (at 24 h). These may be explained by reduced water uptake. We also detected reduced food uptake, hence reduced excrements, with subsequently reduced body weight. Altogether, obtained results indicate that murine kidneys are relatively resistant to hemolysis-induced acute kidney injury. Local inflammation and tubular and vascular stress appeared on the first day after hemolysis but resolved rapidly. Therefore, extensive analyses of metabolic parameters must be performed in murine models of intravascular hemolysis, in order to determine to what extend renal injury is related to the alteration of kidney function, or merely to the altered overall health status of the anemic animals.
The poor overall health status of the PHZ- and heme-injected animals suggests that other organs may be more affected than kidneys. Indeed, we detected alteration of the mRNA level of these markers also in the liver and spleen, as expected from alternative hemolysis models (
We sought to determine the contribution of hemolysis-derived products other than heme, to the adverse effects of intravascular hemolysis. Heme is a well-known TLR-4 ligand (
One may argue that the dose of heme injected was insufficient to trigger more significant kidney injury, or that part of it did not reach the circulation after intraperitoneal injection. Indeed, crystalline heme is poorly soluble in aqueous solutions and prone to aggregation upon injection
In multiple studies, heme injection was used for tissue precon-ditioning, as a mean to enhance HO-1 activity in order to protect organs from subsequent challenges. This was studied in animal models (
Our results, added to the literature, converge to suggest that the injection of heme remains a method of choice for targeted, mechanistic studies, or as a tool to provoke vaso-occlusions in murine models of SCDs. However, PHZ-induced intravascular hemolysis bears several advantages over heme injection, as it emulates the pathological process of erythrocyte destruction, combined with hemoglobin and heme release. Also, it gives reproducible results and is easy to handle. PHZ administration, studied herein, may thus be preferred to study erythrocyte degradation in physiological processes and hemolytic diseases, to establish the benefits of HO-1-mediated therapy, or to investigate the effects of hemoglobin and heme scavengers.
All experiments were conducted in accordance with the recommendations for the care and use of laboratory animals and with the approval APAFIS#3764-201601121739330v3 of the French Ministry of Agriculture. C57Bl/6 mice were from Charles River Laboratories (L’Arbresle, France).
Study design: LTR, NM, JDD. Perform research: NM, AG, MLF, SC, MD, TRR, RN, MR, SK, NB, TG, VL, FG, RD. Discussed the data: LTR, JDD, OBB, RD, NM, AG, SC, MLF, PH, MR, SM, VL, FG, OM, MF, MD, TRR. LR, NM and JDD wrote the manuscript. All authors approved the submission.
NB, TG, and SM are employees of CSL Behring. LR receives research funding from CSL Behring. The remaining authors declare no conflict of interest.
The cytometry and microscopy analysis were performed at the Centre d’Imagerie Cellulaire et de Cytométrie (CICC) and Centre de Recherche des Cordeliers UMRS1138 (Paris, France). CICC is a member of the UPMC flow-cytometry network (RECYF). We are grateful for excellent technical assistance of the CEF team of the Centre de Recherche des Cordeliers for their support with the animal experimentation. We also thank Dr. Gaëlle Brideau and the members of the Plateforme d’Exploration Fonctionnelle Rénale des Cordeliers for the help with renal function analyses.