Podocyte injury: the role of proteinuria, urinary plasminogen, and oxidative stress

Podocytes are the key target for injury in proteinuric glomerular diseases that result in podocyte loss, progressive focal segmental glomerular sclerosis (FSGS), and renal failure. Current evidence suggests that the initiation of podocyte injury and associated proteinuria can be separated from factors that drive and maintain these pathogenic processes leading to FSGS. In nephrotic urine aberrant glomerular filtration of plasminogen (Plg) is activated to the biologically active serine protease plasmin by urokinase-type plasminogen activator (uPA). In vivo inhibition of uPA mitigates Plg activation and development of FSGS in several proteinuric models of renal disease including 5/6 nephrectomy. Here, we show that Plg is markedly increased in the urine in two murine models of proteinuric kidney disease associated with podocyte injury: Tg26 HIV-associated nephropathy and the Cd2ap −/− model of FSGS. We show that human podocytes express uPA and three Plg receptors: uPAR, tPA, and Plg-RKT. We demonstrate that Plg treatment of podocytes specifically upregulates NADPH oxidase isoforms NOX2/NOX4 and increases production of mitochondrial-dependent superoxide anion (O2−) that promotes endothelin-1 synthesis. Plg via O2− also promotes expression of the B scavenger receptor CD36 and subsequent increased intracellular cholesterol uptake resulting in podocyte apoptosis. Taken together, our findings suggest that following disruption of the glomerular filtration barrier at the onset of proteinuric disease, podocytes are exposed to Plg resulting in further injury mediated by oxidative stress. We suggest that chronic exposure to Plg could serve as a “second hit” in glomerular disease and that Plg is potentially an attractive target for therapeutic intervention.

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The podocyte as a direct target for treatment of glomerular disease?

AJP Renal Physiology ◽

10.1152/ajprenal.00184.2016 ◽

2016 ◽

Vol 311 (1) ◽

pp. F46-F51 ◽

Cited By ~ 32

Author(s):

Sandeep K. Mallipattu ◽

John C. He

Keyword(s):

The United States ◽

Glomerular Disease ◽

Glomerular Sclerosis ◽

Systemic Effects ◽

Glomerular Diseases ◽

Podocyte Injury ◽

Filtration Barrier ◽

Focal Segmental Glomerular Sclerosis ◽

Control And Prevention ◽

Segmental Glomerular Sclerosis

The Centers for Disease Control and Prevention estimates more than 10% of adults in the United States, over 20 million Americans, have chronic kidney disease (CKD). A failure to maintain the glomerular filtration barrier directly contributes to the onset of CKD. The visceral epithelial cells, podocytes, are integral to the maintenance of this renal filtration barrier. Direct podocyte injury contributes to the onset and progression of glomerular diseases such as minimal change disease (MCD), focal segmental glomerular sclerosis (FSGS), diabetic nephropathy, and HIV-associated nephropathy (HIVAN). Since podocytes are terminally differentiated with minimal capacity to self-replicate, they are extremely sensitive to cellular injury. In the past two decades, our understanding of the mechanism(s) by which podocyte injury occurs has greatly expanded. With this newfound knowledge, therapeutic strategies have shifted to identifying targets directed specifically at the podocyte. Although the systemic effects of these agents are important, their direct effect on the podocyte proves to be essential in ameliorating glomerular disease. In this review, we highlight the mechanisms by which these agents directly target the podocyte independent of its systemic effects.

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P0053A METHOD FOR THE ISOLATION OF FLUORESCENT GLOMERULI FROM ZEBRAFISH LARVAE

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p0053 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Anna Iervolino ◽

Tim Lange ◽

Florian Siegerist ◽

Maximilian Schindler ◽

Giovambattista Capasso ◽

...

Keyword(s):

Glomerular Filtration ◽

Fluorescent Protein ◽

Transgenic Zebrafish ◽

Renal Tubules ◽

Glomerular Filtration Barrier ◽

Podocyte Injury ◽

Zebrafish Larvae ◽

Filtration Barrier ◽

Glomerular Damage

Abstract Background and Aims The zebrafish is a powerful animal model to study the glomerular morphology and the function of the permselectivity of the glomerular filtration barrier. Since zebrafish larvae develop quickly and can be bred to transparency, in vivo observation of these animals is possible. At 48 hours post fertilization (dpf), zebrafish develop a single filtering glomerulus which is attached to a pair of renal tubules. Like in mammals, the glomerular filtration barrier consists of a fenestrated endothelium, the glomerular basement membrane (GBM) and interdigitating podocyte foot processes bridged by a molecularly conserved slit diaphragm. By the use of genetically modified zebrafish strains with fluorescently labeled podocytes, it is possible to study alterations of the glomerulus during the development of renal disease directly in vivo and in vitro. As an injury model we used the nitroreductase/metronidazole (NTR/MTZ) zebrafish line to induce podocyte apoptosis and detachment from the GBM. Moreover, treatment of these larvae with MTZ induces glomerular injury that mimics focal segmental glomerulosclerosis (FSGS). The aim of our study was to establish a glomeruli isolation method which allows us to identify deregulation of miRNAs and mRNAs in the injured glomeruli by sequencing. Method The transgenic zebrafish strain Cherry (Tg(nphs2:Eco.nfsB-mCherry); mitfaw2/w2; mpv17a9/a9) which expresses the prokaryotic enzyme nitroreductase (NTR) fused to mCherry, a red fluorescent protein, under the control of the podocyte-specific podocin (nphs2) promoter in a transparent zebrafish strain, was used. The NTR/MTZ is a model of cell ablation to mimic podocyte injury. The prodrug MTZ (80 µM) is converted into a cytotoxin by NTR leading to a dose-dependent apoptosis exclusively in NTR-expressing podocytes. To induce podocyte injury, we treated Cherry larvae at 4 days post fertilization with MTZ (80 µM) freshly dissolved in 0.1% DMSO-E3 medium for 48 hours. Control larvae were treated with 0.1% DMSO-E3 medium. The treatment was stopped by a MTZ washout at 6 dpf. In order to perform the miRNA and mRNA sequencing on glomeruli isolated from MTZ-treated and control larvae we tried to establish a method to obtain total RNA samples of good quality. For this purpose, three different approaches were tested and validated: 1) Sieving method, 2) Fluorescence-Activated Cell Sorting method (FACS), and 3) manual isolation of glomeruli by using a micropipette. Results Zebrafish larvae developed a glomerular damage similar to FSGS after MTZ-treatment. MTZ-treated larvae showed severe pericardial edema, a reduction of the nephrin and podocin expression, proteinuria and an increased mortality rate at 8 dpf. After many tests we showed that glomeruli isolation using the sieving method and FACS were not efficient due to contaminations with other organs (sieving) and a loss of a large amount of cells per sample (FACS), respectively. Samples of the required quality for sequencing resulted only from the manual glomeruli isolation. Conclusion Here we describe methods to isolate fluorescent glomeruli from transgenic zebrafish larvae. For our studies, we used the NTZ/MTR kidney disease model in order to identify mRNAs and miRNAs regulated in response to glomerular damage. This technique will further allow to screen for healing drugs in high-throughput experiments.

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Podocyte Autophagy in Homeostasis and Disease

Journal of Clinical Medicine ◽

10.3390/jcm10061184 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1184

Author(s):

Qisheng Lin ◽

Khadija Banu ◽

Zhaohui Ni ◽

Jeremy S. Leventhal ◽

Madhav C. Menon

Keyword(s):

Glomerular Filtration ◽

Functional Role ◽

Specific Reference ◽

Protective Mechanism ◽

Glomerular Filtration Barrier ◽

Glomerular Diseases ◽

Podocyte Injury ◽

Filtration Barrier ◽

Cellular Autophagy ◽

Survival Mechanisms

Autophagy is a protective mechanism that removes dysfunctional components and provides nutrition for cells. Podocytes are terminally differentiated specialized epithelial cells that wrap around the capillaries of the glomerular filtration barrier and show high autophagy level at the baseline. Here, we provide an overview of cellular autophagy and its regulation in homeostasis with specific reference to podocytes. We discuss recent data that have focused on the functional role and regulation of autophagy during podocyte injury in experimental and clinical glomerular diseases. A thorough understanding of podocyte autophagy could shed novel insights into podocyte survival mechanisms with injury and offer potential targets for novel therapeutics for glomerular disease.

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Plasminogenuria is associated with podocyte injury, edema, and kidney dysfunction in incident glomerular disease

10.1101/19006809 ◽

2019 ◽

Author(s):

Marc A. Egerman ◽

Jenny S. Wong ◽

Tian Runxia ◽

Gohar Mosoyan ◽

Kinsuk Chauhan ◽

...

Keyword(s):

Oxidative Stress ◽

Kidney Disease ◽

Glomerular Disease ◽

Causative Role ◽

Glomerular Injury ◽

Puromycin Aminonucleoside ◽

Glomerular Diseases ◽

Podocyte Injury ◽

Cardiovascular Morbidity And Mortality

ABSTRACTUrinary plasminogen/plasmin, or plasmin(ogen)uria, has been demonstrated in proteinuric patients and exposure of cultured podocytes to plasminogen results in injury via oxidative stress pathways. A causative role for plasmin(ogen) as a “second hit” in kidney disease progression has yet to be demonstrated in vivo, and the association between plasmin(ogen)uria and kidney function in glomerular diseases remains unclear. We performed comparative studies in a puromycin aminonucleoside (PAN) nephropathy rat model treated with amiloride, an inhibitor of plasminogen activation, and measured changes in plasmin(ogen)uria and urinary endothelin-1 (ET1). In a glomerular disease biorepository cohort (n=128), we measured time-of-biopsy albuminuria, proteinuria, and plasmin(ogen)uria for correlations with renal outcomes. Increased glomerular plasmin(ogen) was found in PAN rats and FSGS patients. PAN nephropathy was associated with increases in plasmin(ogen)uria, proteinuria, and urinary ET1. Amiloride was protective against PAN-induced glomerular injury, reducing urinary ET1 and oxidative stress. In patients, we found associations between plasmin(ogen)uria and edema status as well as eGFR. Our study demonstrates a role for plasmin(ogen)-induced podocyte injury in the PAN nephropathy model, with amiloride having podocyte-protective properties. In one of largest glomerular disease cohorts to study plasminogen, we validated previous findings while suggesting a potentially novel relationship between plasmin(ogen)uria and eGFR. Together, these findings suggest a role for plasmin(ogen) in mediating glomerular injury and as a viable targetable biomarker for podocyte-sparing treatments.TRANSLATIONAL STATEMENTProteinuria is associated with CKD progression, and increased cardiovascular morbidity and mortality. The underlying mechanisms of podocyte injury, the hallmark of proteinuric kidney disease, are poorly understood with limited, non-specific therapeutic options. This study adds to the evidence that plasmin(ogen) in the urine of proteinuric patients is associated with podocyte injury, edema, and impaired renal function. Previously published results from us and others, taken together with our current rodent model and human data, suggest that urinary plasmin(ogen) is a potential targetable biomarker. Efforts to decrease plasmin(ogen)-mediated podocyte injury could be part of a novel therapeutic strategy for glomerular disease.

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Adriamycin does not damage podocytes of zebrafish larvae

PLoS ONE ◽

10.1371/journal.pone.0242436 ◽

2020 ◽

Vol 15 (11) ◽

pp. e0242436

Author(s):

Maximilian Schindler ◽

Antje Blumenthal ◽

Marcus Johannes Moeller ◽

Karlhans Endlich ◽

Nicole Endlich

Keyword(s):

Glomerular Filtration ◽

Rapid Development ◽

Glomerular Injury ◽

Glomerular Filtration Barrier ◽

Glomerular Diseases ◽

Zebrafish Larvae ◽

Filtration Barrier ◽

Suitable Animal Model ◽

Similar Phenotype

Podocytes are highly specialized epithelial cells that are essential for an intact glomerular filtration barrier in the kidney. Several glomerular diseases like focal segmental glomerulosclerosis (FSGS) are initially due to podocyte injury and loss. Since causative treatments for FSGS are not available until today, drug screening is of great relevance. In order to test a high number of drugs, FSGS needs to be reliably induced in a suitable animal model. The zebrafish larva is an ideal model for kidney research due to the vast amount of offsprings, the rapid development of a simple kidney and a remarkable homology to the mammalian glomerulus. Zebrafish larvae possess a size-selective glomerular filtration barrier at 4 days post fertilization including podocytes with interdigitating foot processes that are connected by a slit membrane. Adriamycin is an anthracycline which is often used in mice and rats to induce a FSGS-like phenotype. In this study, we aimed to induce a similar phenotype to zebrafish larvae by adding adriamycin to the tank water in different concentrations. Surprisingly, zebrafish larvae did not develop glomerular injury and displayed an intact filtration barrier after treatment with adriamycin. This was shown by (immuno-) histology, our filtration assay, in vivo imaging by 2-photon microcopy, RT-(q)PCR as well as transmission electron microscopy. To summarize, adriamycin is unable to induce a podocyte-related damage in zebrafish larvae and therefore major effort must be made to establish FSGS in zebrafish larvae to identify effective drugs by screenings.

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Podocyte Lysosome Dysfunction in Chronic Glomerular Diseases

International Journal of Molecular Sciences ◽

10.3390/ijms21051559 ◽

2020 ◽

Vol 21 (5) ◽

pp. 1559 ◽

Cited By ~ 1

Author(s):

Guangbi Li ◽

Jason Kidd ◽

Pin-Lan Li

Keyword(s):

Structural Integrity ◽

Hydrolytic Enzymes ◽

Therapeutic Strategies ◽

Normal Function ◽

Current Evidence ◽

Autophagic Flux ◽

Glomerular Diseases ◽

Podocyte Injury ◽

Membrane Bound

Podocytes are visceral epithelial cells covering the outer surface of glomerular capillaries in the kidney. Blood is filtered through the slit diaphragm of podocytes to form urine. The functional and structural integrity of podocytes is essential for the normal function of the kidney. As a membrane-bound organelle, lysosomes are responsible for the degradation of molecules via hydrolytic enzymes. In addition to its degradative properties, recent studies have revealed that lysosomes may serve as a platform mediating cellular signaling in different types of cells. In the last decade, increasing evidence has revealed that the normal function of the lysosome is important for the maintenance of podocyte homeostasis. Podocytes have no ability to proliferate under most pathological conditions; therefore, lysosome-dependent autophagic flux is critical for podocyte survival. In addition, new insights into the pathogenic role of lysosome and associated signaling in podocyte injury and chronic kidney disease have recently emerged. Targeting lysosomal functions or signaling pathways are considered potential therapeutic strategies for some chronic glomerular diseases. This review briefly summarizes current evidence demonstrating the regulation of lysosomal function and signaling mechanisms as well as the canonical and noncanonical roles of podocyte lysosome dysfunction in the development of chronic glomerular diseases and associated therapeutic strategies.

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Early Glomerular Filtration Defect and Severe Renal Disease in Podocin-Deficient Mice

Molecular and Cellular Biology ◽

10.1128/mcb.24.2.550-560.2004 ◽

2004 ◽

Vol 24 (2) ◽

pp. 550-560 ◽

Cited By ~ 180

Author(s):

Séverine Roselli ◽

Laurence Heidet ◽

Mireille Sich ◽

Anna Henger ◽

Matthias Kretzler ◽

...

Keyword(s):

Renal Disease ◽

Glomerular Filtration ◽

Molecular Mechanisms ◽

Slit Diaphragm ◽

Vascular Lesions ◽

Suitable Model ◽

Glomerular Diseases ◽

Filtration Barrier ◽

Severe Renal Disease

ABSTRACT Podocytes are specialized epithelial cells covering the basement membrane of the glomerulus in the kidney. The molecular mechanisms underlying the role of podocytes in glomerular filtration are still largely unknown. We generated podocin-deficient (Nphs2 −/−) mice to investigate the function of podocin, a protein expressed at the insertion of the slit diaphragm in podocytes and defective in a subset of patients with steroid-resistant nephrotic syndrome and focal and segmental glomerulosclerosis. Nphs2 −/− mice developed proteinuria during the antenatal period and died a few days after birth from renal failure caused by massive mesangial sclerosis. Electron microscopy revealed the extensive fusion of podocyte foot processes and the lack of a slit diaphragm in the remaining foot process junctions. Using real-time PCR and immunolabeling, we showed that the expression of other slit diaphragm components was modified in Nphs2 −/− kidneys: the expression of the nephrin gene was downregulated, whereas that of the ZO1 and CD2AP genes appeared to be upregulated. Interestingly, the progression of the renal disease, as well as the presence or absence of renal vascular lesions, depends on the genetic background. Our data demonstrate the crucial role of podocin in the establishment of the glomerular filtration barrier and provide a suitable model for mapping and identifying modifier genes involved in glomerular diseases caused by podocyte injuries.

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Insulin Resistance, Oxidative Stress, and Podocyte Injury: Role of Rosuvastatin Modulation of Filtration Barrier Injury

American Journal of Nephrology ◽

10.1159/000109394 ◽

2007 ◽

Vol 28 (1) ◽

pp. 67-75 ◽

Cited By ~ 31

Author(s):

Adam Whaley-Connell ◽

Vincent G. DeMarco ◽

Guido Lastra ◽

Camila Manrique ◽

Ravi Nistala ◽

...

Keyword(s):

Oxidative Stress ◽

Insulin Resistance ◽

Podocyte Injury ◽

Filtration Barrier

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WT1-interacting protein and ZO-1 translocate into podocyte nuclei after puromycin aminonucleoside treatment

AJP Renal Physiology ◽

10.1152/ajprenal.00389.2004 ◽

2005 ◽

Vol 289 (2) ◽

pp. F431-F441 ◽

Cited By ~ 53

Author(s):

Maribel Rico ◽

Amitava Mukherjee ◽

Martha Konieczkowski ◽

Leslie A. Bruggeman ◽

R. Tyler Miller ◽

...

Keyword(s):

Adherens Junctions ◽

Slit Diaphragm ◽

Cell Junctions ◽

Puromycin Aminonucleoside ◽

Glomerular Diseases ◽

Podocyte Injury ◽

Interacting Protein ◽

Filtration Barrier ◽

Cell Cell

Podocyte differentiation is required for normal glomerular filtration barrier function and is regulated by the transcription factor WT1. We identified WT1-interacting protein (WTIP) and hypothesized that it functions as both a scaffold for slit diaphragm proteins and a corepressor of WT1 transcriptional activity by shuttling from cell-cell junctions to the nucleus after injury. Endogenous WTIP colocalizes with zonula occludens-1 (ZO-1) in cultured mouse podocyte adherens junctions. To model podocyte injury in vitro, we incubated differentiated podocytes with puromycin aminonucleoside (PAN; 100 μg/ml) for 24 h, which disassembled cell-cell contacts, rearranged actin cytoskeleton, and caused process retraction. Podocyte synaptopodin expression diminished after PAN treatment, consistent with podocyte dedifferentiation in some human glomerular diseases. To assess podocyte function, we measured albumin flux across differentiated podocytes cultured on collagen-coated Transwell filters. Albumin transit across PAN-treated cells increased to levels observed with undifferentiated podocytes. Consistent with our hypothesis, WTIP, as well as ZO-1, translocated from podocyte adherens junctions to nuclei in PAN-treated cells. Because WTIP is a transcriptional corepressor for WT1, we examined the effect of PAN on expression of retinoblastoma binding protein Rbbp7 (also known as RbAp46), a WT1 target gene expressed in S-shaped bodies during nephrogenesis. Rbbp7 expression in PAN-treated podocytes was reduced compared with untreated cells. In conclusion, WTIP translocates from cell-cell junctions to the nucleus in PAN-treated podocytes. We suggest that WTIP monitors slit diaphragm protein assembly and shuttles into the nucleus after podocyte injury, translating changes in slit diaphragm structure into altered gene expression and a less differentiated phenotype.

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Overexpression of TGF-β Inducible microRNA-143 in Zebrafish Leads to Impairment of the Glomerular Filtration Barrier by Targeting Proteoglycans

Cellular Physiology and Biochemistry ◽

10.1159/000453142 ◽

2016 ◽

Vol 40 (5) ◽

pp. 819-830 ◽

Cited By ~ 10

Author(s):

Janina Müller-Deile ◽

Finn Gellrich ◽

Heiko Schenk ◽

Patricia Schroder ◽

Jenny Nyström ◽

...

Keyword(s):

Endothelial Cells ◽

Glomerular Filtration ◽

Cell Types ◽

Endothelial Damage ◽

Micro Rna ◽

Glomerular Filtration Barrier ◽

Glomerular Diseases ◽

Filtration Barrier ◽

Glomerular Endothelial Cells ◽

Apoptotic Pathways

Background: TGF-β is known as an important stress factor of podocytes in glomerular diseases. Apart from activation of direct pro-apoptotic pathways we wanted to analyze micro-RNA (miRs) driven regulation of components involved in the integrity of the glomerular filtration barrier induced by TGF-β. Since miR-143-3p (miR-143) is described as a TGF-β inducible miR in other cell types, we examined this specific miR and its ability to induce glomerular pathology. Methods: We analyzed miR-143 expression in cultured human podocytes after stimulation with TGF-β. We also microinjected zebrafish eggs with a miR-143 mimic or with morpholinos specific for its targets syndecan and versican and compared phenotype and proteinuria development. Results: We detected a time dependent, TGF-β inducible expression of miR-143 in human podocytes. Targets of miR-143 relevant in glomerular biology are syndecans and versican, which are known components of the glycocalyx. We found that syndecan 1 and 4 were predominantly expressed in podocytes while syndecan 3 was largely expressed in glomerular endothelial cells. Versican could be detected in both cell types. After injection of a miR-143 mimic in zebrafish larvae, syndecan 3, 4 and versican were significantly downregulated. Moreover, miR-143 overexpression or versican knockdown by morpholino caused loss of plasma proteins, edema, podocyte effacement and endothelial damage. In contrast, knockdown of syndecan 3 and syndecan 4 had no effects on glomerular filtration barrier. Conclusion: Expression of versican and syndecan isoforms is indispensable for proper barrier function. Podocyte-derived miR-143 is a mediator for paracrine and autocrine cross talk between podocytes and glomerular endothelial cells and can alter expression of glomerular glycocalyx proteins.

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