Direct Oral Anticoagulants Reduce Hypercoagulopathy and Preserve Podocyte Function in an Experimental Model of Glomerular Disease

Proteinuric glomerular diseases are a leading cause of chronic kidney disease (CKD). Both pre-CKD glomerular disease and established CKD are major risk factors for thrombosis. Glomerular capillary podocyte injury is a key determinant of CKD progression and results in massive proteinuria accompanied by an acquired hypercoagulopathy that drives thrombotic risk. Unfortunately, the routine use of anticoagulant prophylaxis during glomerular proteinuria (GP) remains controversial due to both a lack of agreement regarding indications and no randomized controlled trial data demonstrating both safety and efficacy. We have recently used rat glomerular disease models to reveal that: (1) Proteinuria is directly correlated with hypercoagulopathy and in vivo thrombosis and (2) Thrombin, the key effector enzyme of the coagulation system, directly injures podocytes during proteinuria. What is not yet known is the ability of direct oral anticoagulant (DOAC) therapy to improve these important CKD and thrombosis outcomes. Thus, the aim of the present study is to determine if DOACs simultaneously reduce podocytopathy and enable effective thromboprophylaxis during GP. We hypothesized that DOACs would simultaneously preserve podocyte function and reduce hypercoagulopathy, in a podocyte-specific rodent model of glomerular disease. We utilized the podocin promotor-human diphtheria toxin receptor (pDTR) transgenic rat model to induce highly specific podocyte injury following a single I.P. injection of 50 ng/kg diphtheria toxin (DT). DT-induced proteinuria was subsequently treated daily by oral gavage with 1) Dabigatran (20 mg/kg; Dabi), 2) Rivaroxaban (3 mg/kg; Riva), or 3) Sham (saline) and compared to healthy controls (n=3-6/group). Morning spot urine and citrated plasma samples were collected from each group at day 10 post-DT. Endogenous Thrombin Potential (ETP) was measured by Technothrombin TGA assay, without and with DOAC-Stop reagent. Glomeruli were isolated from the kidney, dissociated into single-cell suspensions and analyzed by flow cytometry following immunofluorescent antibody and TUNEL staining. Both Dabi and Riva significantly reduced proteinuria (Fig A) and podocytopathy (TUNEL positive podocyte fraction; Fig B), while concomitantly correcting elevated ETP levels (Fig C open symbols). Addition of DOAC-Stop (Fig C closed symbols) revealed an insignificant (P=0.18) trend toward partial ETP reduction, consistent with DOAC-mediated reduction of the underlying GP-mediated hypercoagulopathy (via indirect, antiproteinuric effects). In conclusion, dabigatran and rivaroxaban reduce proteinuria and enhance podocyte health in concert with alleviation of the acquired hypercoagulopathy in a podocyte-specific rodent model of glomerular disease. Overall these data suggest DOAC treatment as a novel approach to simultaneously reduce both podocytopathy and thrombotic co-morbidities during glomerular disease. Additional experiments using this model to determine DOAC efficacy on in vivo thrombosis are in progress. Results from these preclinical studies should inform subsequent randomized controlled DOAC trials that may transform care for patients with glomerular disease by mitigating their risk of both CKD progression and thrombosis. Disclosures No relevant conflicts of interest to declare.

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

AJP Renal Physiology ◽

10.1152/ajprenal.00162.2016 ◽

2016 ◽

Vol 311 (6) ◽

pp. F1308-F1317 ◽

Cited By ~ 28

Author(s):

Leopoldo Raij ◽

Runxia Tian ◽

Jenny S. Wong ◽

John C. He ◽

Kirk N. Campbell

Keyword(s):

Oxidative Stress ◽

Glomerular Filtration ◽

Biologically Active ◽

Current Evidence ◽

Glomerular Sclerosis ◽

Glomerular Diseases ◽

Podocyte Injury ◽

Filtration Barrier ◽

Focal Segmental Glomerular Sclerosis

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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Organoid single-cell profiling identifies a transcriptional signature of glomerular disease

10.1101/468850 ◽

2018 ◽

Author(s):

Jennifer L. Harder ◽

Rajasree Menon ◽

Edgar A. Otto ◽

Jian Zhou ◽

Sean Eddy ◽

...

Keyword(s):

Gene Expression ◽

Kidney Disease ◽

Single Cells ◽

Gene Expression Signature ◽

Human Kidney ◽

Glomerular Disease ◽

Podocyte Injury ◽

Compensation Mechanisms ◽

Kidney Organoids

ABSTRACTPodocyte injury is central to many forms of kidney disease, but transcriptional signatures reflecting podocyte injury and compensation mechanisms are challenging to analyze in vivo. Human kidney organoids derived from pluripotent stem cells (PSCs), a new model for disease and regeneration, present an opportunity to explore the transcriptional plasticity of podocytes. Here, transcriptional profiling of over 12,000 single cells from human PSC-derived kidney organoid cultures was used to identify robust and reproducible cell-lineage gene expression signatures shared with developing human kidneys based on trajectory analysis. Surprisingly, the gene expression signature characteristic of developing glomerular epithelial cells was also observed in glomerular tissue from a kidney disease cohort. This signature correlated with proteinuria and inverse eGFR, and was confirmed in an independent podocytopathy cohort. Three genes in particular were further identified as critical components of the glomerular disease signature. We conclude that cells in human PSC-derived kidney organoids reliably recapitulate the developmental transcriptional program of podocytes and other cell lineages in the human kidney, and that the early transcriptional profile seen in developing podocytes is reactivated in glomerular disease. Our findings demonstrate an innovative approach to identifying novel molecular programs involved in the pathogenesis of glomerulopathies.

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Translating genetic findings in hereditary nephrotic syndrome: the missing loops

AJP Renal Physiology ◽

10.1152/ajprenal.00683.2014 ◽

2015 ◽

Vol 309 (1) ◽

pp. F24-F28 ◽

Cited By ~ 16

Author(s):

Gentzon Hall ◽

Rasheed A. Gbadegesin

Keyword(s):

Nephrotic Syndrome ◽

Single Gene ◽

Glomerular Disease ◽

Podocyte Injury ◽

In Vivo Models ◽

Common Cause ◽

Speed Up ◽

Generation Sequencing

Nephrotic syndrome (NS) is a clinicopathological entity characterized by proteinuria, hypoalbuminemia, peripheral edema, and hyperlipidemia. It is the most common cause of glomerular disease in children and adults. Although the molecular pathogenesis of NS is not completely understood, data from the study of familial NS suggest that it is a “podocytopathy.” Virtually all of the genes mutated in hereditary NS localize to the podocyte or its secreted products and the slit diaphragm. Since the completion of human genome sequence and the advent of next generation sequencing, at least 29 causes of single-gene NS have been identified. However, these findings have not been matched by therapeutic advances owing to suboptimal in vitro and in vivo models for the study of human glomerular disease and podocyte injury phenotypes. Multidisciplinary collaboration between clinicians, geneticists, cell biologists, and molecular physiologists has the potential to overcome this barrier and thereby speed up the translation of genetic findings into improved patient care.

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P0407THE INVESTIGATION OF SYNAPTOPODIN EXPRESSION OF PODOCYTES IN GLOMERONEPHRITIS

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p0407 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Zhigang Zhang

Keyword(s):

Molecular Mechanisms ◽

Cultured Cells ◽

Glomerular Diseases ◽

Podocyte Injury ◽

Adriamycin Nephropathy ◽

Assembly Result ◽

And Function ◽

Rhoa Signaling ◽

New Strategies

Abstract Background and Aims Synaptopodin, a proline-rich actin-associated protein, plays an important role in the regulation of podocytes processes structures and dynamics. The mutation or lack of synaptopodin may lead to the changes of podocytes structures and functions and cause the occurrence of proteinuria. But the underlying molecular mechanisms remain primarily elusive. Method we used cellular and pathological experiments to observe the expression changes synaptopodin in vivo and vitrio. Results The results showed that the reduction expression of synaptopodin and RhoA were found in the podocytes in different nephriris of human renal biopsy as well as in rat adriamycin nephropathy. The cultured cells treated with inflammatory cytokins such as TNF, IL-1 also showed decreased synaptopodin level in podocyte, which led to low RhoA level and disarrange the actin cytoskeleton assembly, result in the abnormal changes of podocyte morphology. Conclusion These data preliminarily proved that synaptopodin loss in podocyte injury plays an important role in the regulation of podocyte morphology and function through RhoA signaling pathway, and further researches are required to clarify the more mechanism, which may provide new strategies and methods for the prevention and treatment of glomerular diseases.

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Mitochondrial dysfunction is an early event in aldosterone-induced podocyte injury

AJP Renal Physiology ◽

10.1152/ajprenal.00570.2012 ◽

2013 ◽

Vol 305 (4) ◽

pp. F520-F531 ◽

Cited By ~ 42

Author(s):

Min Su ◽

Asish-Roopchand Dhoopun ◽

Yanggang Yuan ◽

Songming Huang ◽

Chunhua Zhu ◽

...

Keyword(s):

Mitochondrial Dysfunction ◽

Early Event ◽

Mtdna Copy Number ◽

Glomerular Diseases ◽

Podocyte Injury ◽

Atp Production ◽

Podocyte Damage ◽

Mitochondrial Transcription Factor

We previously showed that mitochondrial dysfunction (MtD) is involved in an aldosterone (Aldo)-induced podocyte injury. Here, the potential role of MtD in the initiation of podocyte damage was investigated. We detected the dynamic changes of urinary protein, urinary F2-isoprostane and renal malondialdehyde levels, kidney ultrastructure morphology, mitochondrial DNA (mtDNA) copy number, mitochondrial membrane potential (ΔΨm), and nephrin and podocin expressions in Aldo-infused mice. Aldo infusion first induced renal oxidative stress, as evidenced by increased levels of urinary F2-isoprostane and renal malondialdehyde, and MtD, as demonstrated by reduced mtDNA, ΔΨm, and ATP production. Later, at 5 days after Aldo infusion, proteinuria and podocyte injury began to appear. In cultured podocytes, Aldo or hydrogen peroxide (H2O2) induced MtD after 2–8 h of treatment, whereas the podocyte damage, as shown by decreased nephrin and podocin expressions, occurred later after 12 h of treatment. Thus Aldo treatment both in vitro and in vivo indicated that MtD occurred before podocyte damage. Additionally, MtDNA depletion by ethidium bromide or mitochondrial transcription factor A (TFAM) RNAi induced MtD, further promoting podocyte damage. TFAM expression was found to be reduced in Aldo-infused mice and Aldo-treated podocytes. Adenoviral vector-mediated overexpression of TFAM prevented Aldo-induced MtD and protected against podocyte injury. Together, these findings support MtD as an early event in podocyte injury, and manipulation of TFAM may be a novel strategy for treatment of glomerular diseases such as podocytopathy.

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Autophagy is activated to protect against podocyte injury in adriamycin-induced nephropathy

AJP Renal Physiology ◽

10.1152/ajprenal.00114.2017 ◽

2017 ◽

Vol 313 (1) ◽

pp. F74-F84 ◽

Cited By ~ 22

Author(s):

Mixuan Yi ◽

Lei Zhang ◽

Yu Liu ◽

Man J. Livingston ◽

Jian-Kang Chen ◽

...

Keyword(s):

Therapeutic Potential ◽

Glomerular Disease ◽

Podocyte Injury ◽

Knockout Mouse Model ◽

Filtration Barrier ◽

Induced Apoptosis ◽

And Function

Podocytes are highly differentiated epithelial cells wrapping glomerular capillaries to form the filtration barrier in kidneys. As such, podocyte injury or dysfunction is a critical pathogenic event in glomerular disease. Autophagy plays an important role in the maintenance of the homeostasis and function of podocytes. However, it is less clear whether and how autophagy contributes to podocyte injury in glomerular disease. Here, we have examined the role of autophagy in adriamycin-induced nephropathy, a classic model of glomerular disease. We show that autophagy was induced by adriamycin in cultured podocytes in vitro and in podocytes in mice. In cultured podocytes, activation of autophagy with rapamycin led to the suppression of adriamycin-induced apoptosis, whereas inhibition of autophagy with chloroquine enhanced podocyte apoptosis during adriamycin treatment. To determine the role of autophagy in vivo, we established an inducible podocyte-specific autophagy-related gene 7 knockout mouse model (Podo-Atg7-KO). Compared with wild-type littermates, Podo-Atg7-KO mice showed higher levels of podocyte injury, glomerulopathy, and proteinuria during adriamycin treatment. Together, these observations support an important role of autophagy in protecting podocytes under the pathological conditions of glomerular disease, suggesting the therapeutic potential of autophagy induction.

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mPGES-1-derived PGE2 contributes to adriamycin-induced podocyte injury

AJP Renal Physiology ◽

10.1152/ajprenal.00499.2015 ◽

2016 ◽

Vol 310 (6) ◽

pp. F492-F498 ◽

Cited By ~ 6

Author(s):

Jing Yu ◽

Wei Gong ◽

Yimei Wu ◽

Shuzhen Li ◽

Yiyun Cui ◽

...

Keyword(s):

Underlying Mechanism ◽

Pathological Feature ◽

Glomerular Diseases ◽

Podocyte Injury ◽

Phenotypic Alteration ◽

Tnf Α ◽

Progression Of Kidney Disease

Podocyte damage is a common pathological feature in many types of glomerular diseases and is involved in the occurrence and progression of kidney disease. However, the pathogenic mechanisms leading to podocyte injury are still uncertain. The present study was undertaken to investigate the role of microsomal PGE synthase (mPGES)-1 in adriamycin (ADR)-induced podocyte injury as well as the underlying mechanism. In both mouse kidneys and in vitro podocytes, application of ADR remarkably enhanced mPGES-1 expression in line with a stimulation of cyclooxygenase-2. Interestingly, inhibition of mPGES-1 with a small interfering RNA approach significantly attenuated ADR-induced downregualtion of podocin and nephrin. Moreover, ADR-induced podocyte apoptosis was also markedly blocked in parallel with blunted caspase-3 induction. In agreement with the improvement of cell phenotypic alteration and apoptosis, the enhanced inflammatory markers of IL-1β and TNF-α were also significantly suppressed by mPGES-1 silencing. More importantly, in mPGES-1-deficient mice, albuminuria induced by ADR showed a remarkable attenuation in line with decreased urinary output of PGE2 and TNF-α, highly suggesting an in vivo role of mPGES-1 in mediating podocyte injury. In summary, findings from the present study offered the first evidence demonstrating a pathogenic role of mPGES-1 in mediating ADR-induced podocyte injury possibly via triggering an inflammatory response.

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Proteinuria Severity Is Directly Correlated with Markers of Hypofibrinolysis in a Podocyte Specific Experimental Model of Nephrotic Syndrome

Blood ◽

10.1182/blood.v126.23.1106.1106 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1106-1106 ◽

Cited By ~ 1

Author(s):

Amanda P Waller ◽

Ruchika Sharma ◽

Shipra Agrawal ◽

Roger C Wiggins ◽

William E Smoyer ◽

...

Keyword(s):

Nephrotic Syndrome ◽

Animal Models ◽

Experimental Model ◽

Diphtheria Toxin ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Clot Lysis ◽

Plasma Clot ◽

Podocyte Injury ◽

Thrombotic Risk

Abstract Introduction Nephrotic syndrome (NS) is characterized by massive proteinuria (secondary to podocyte injury or dysfunction), hypoalbuminemia, and edema, and is associated with a complex acquired hypercoagulopathy and a high prevalence (~25%) of life-threatening thrombotic complications. However, anticoagulation is associated with a substantial risk for adverse bleeding events. Recently published epidemiology studies suggest that proteinuria severity is directly correlated with thrombotic risk. However, further validation of this candidate biomarker for thrombotic risk requires appropriate validation and adequate pathophysiologic explanation. We have recently demonstrated that proteinuria severity is directly proportional to hypercoagulability (as assessed by ex vivo and in vivo markers of thrombotic capacity),in two well-established animal models of NS (puromycin aminonucleoside (PAN) and Adriamycin (ADR) rats). Thromboelastometry studies also suggested a resistance to fibrinolysis during rat NS. Thus, the aim of the present study was to further delineate the relationship between proteinuria severity and hypofibrinolysis using a podocyte-specific rat model of NS. We hypothesized that hypofibrinolysis is directly proportional to severity of proteinuria. Methods Using a transgenic rat which expresses the human diphtheria toxin receptor (hDTR) on a podocyte specific promoter (podocin), we compared markers of global hemostasis (ROTEM) and fibrinolysis to proteinuria severity. A range of proteinuria severity was induced by a single I.P. injection of diphtheria toxin (0, 25, 50 & 75 ng/kg; n= 7-8/group). On Day 10 post-injection, morning spot urines were collected and analyzed for protein:creatinine ratio. Rats were then anesthetized and venous blood (IVC) was collected into 0.32% NaCitrate/1.45 µM Corn Trypsin Inhibitor [final concentrations] and immediately analyzed with ROTEM (whole blood; intem) before being spun down to platelet poor plasma (PPP). Plasma clot lysis assay (CLA) was performed using urokinase (50 IU). Thrombin and plasmin generation assays are currently being performed. Results There were significant differences (P<0.005) between the highest proteinuria hDTR rat group and controls, in both hypercoagulopathic (clot formation time, maximum clot firmness, and clot size (amplitude at 10 & 20 min)), and hypofibrinolytic (amount of lysis at 60 min; LI60) ROTEM parameters. Importantly, there was a significant negative correlation between proteinuria severity and LI60 (R2 =0.362, P=0.02), suggesting that hypofibrinolysis is directly proportional to podocyte injury and therefore disease severity during NS. Preliminary results from the CLA (n=2 control & 2 high proteinuria) also suggest a marked impairment (~50% difference) in plasma clot lysis time in proteinuria rats. Conclusions These results demonstrate that proteinuria severity is directly proportional to both hypercoagulability and hypofibrinolytic capacity in a podocyte-specific rodent model of NS, thus confirming our recent findings in two other well-established animal models of NS in a third, more specific, experimental model of glomerular disease. Importantly, these data also strongly suggest a marked impairment in fibrinolysis during NS, which is directly correlated with proteinuria severity. Therefore it appears that severe proteinuria is associated with both hypercoagulopathic and hypofibrinolytic defects in the coagulation system. Future studies will delve into the molecular mechanisms involved in these defects. Disclosures No relevant conflicts of interest to declare.

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Podocytes and Proteinuric Kidney Disease

BANTAO Journal ◽

10.2478/bj-2014-0004 ◽

2015 ◽

Vol 12 (1) ◽

pp. 16-19

Author(s):

Alketa Koroshi ◽

Alma Idrizi

Keyword(s):

Treatment Strategies ◽

Glomerular Disease ◽

Glomerular Sclerosis ◽

Endstage Renal Disease ◽

Glomerular Diseases ◽

Podocyte Injury ◽

Glomerular Function ◽

Podocyte Loss ◽

Insight Into

Abstract Glomerular disease is the most common cause of endstage renal disease (ESRD), accounting for almost two thirds of cases. In glomerular disease, alterations of po-docytes are of particular importance. Podocyte loss represents a central mediator of glomerular sclerosis. Toxic, genetic, immune, infectious, oxidant, metabolic, hemody-namic, and other mechanisms can all target the podo-cytes. These mechanisms provide new insight into the unique dynamic microenvironment that each individual podocyte inhabits and how it can turn hostile to survival. At the same time, they raise new therapeutic challenges to preserve glomerular function by containing podocyte injury and limiting its spread, both in podo-cytopathies and in other progressive glomerular diseases. Treatment strategies should aim at enhancing podocyte survival. The renin-angiotensin axis blockade, apart from its antifibrotic and intraglomerular hemodynamic effects, has an important role in preventing podocyte loss. However, only long-term observational studies can clarify if many patients will benefit from podocyte-targeted treatment such as abatacept or similar agents.

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