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Thrombin-Induced Podocyte Injury Is Protease-Activated Receptor Dependent

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Thrombin-Induced Podocyte Injury Is Protease-Activated Receptor Dependent BASIC RESEARCH www.jasn.org Thrombin-Induced Podocyte Injury Is Protease-Activated Receptor Dependent † ‡ Ruchika Sharma,* Amanda P. Waller,* Shipra Agrawal,* Katelyn J. Wolfgang,* Hiep Luu,* | †† Khurrum Shahzad,§ Berend Isermann,§ William E. Smoyer,*¶** Marvin T. Nieman, and † Bryce A. Kerlin* ** *Center for Clinical and Translational Research, The Research Institute at Nationwide Children’s Hospital, †Division of Hematology, Oncology, and BMT, and ¶Division of Nephrology, Nationwide Children’s Hospital, Columbus, Ohio; ‡Heritage College of Osteopathic Medicine, Ohio University, Athens, Ohio; §Institute of Clinical Chemistry and Pathobiochemistry, Otto-von-Guericke University, Magdeburg, Germany; **Department of Pediatrics, College of Medicine, The Ohio State University, Columbus, Ohio; |Department of Biotechnology, University of Sargodha, Sargodha, Pakistan; and ††Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio ABSTRACT Nephrotic syndrome is characterized by massive proteinuria and injury of specialized glomerular epithelial cells called podocytes. Studies have shown that, whereas low-concentration thrombin may be cytoprotective, higher thrombin concentrations may contribute to podocyte injury. We and others have demonstrated that ex vivo plasma thrombin generation is enhanced during nephrosis, suggesting that thrombin may contribute to nephrotic pro- gression. Moreover, nonspecific thrombin inhibition has been shown to decrease proteinuria in nephrotic animal models. We thus hypothesized that thrombin contributes to podocyte injury in a protease-activated receptor- specific manner during nephrosis. Here, we show that specific inhibition of thrombin with hirudin reduced pro- teinuria in two rat nephrosis models, and thrombin colocalized with a podocyte-specific marker in rat glomeruli. Furthermore, flow cytometry immunophenotyping revealed that rat podocytes express the protease-activated receptor family of coagulation receptors in vivo. High-concentration thrombin directly injured conditionally immor- talized human and rat podocytes. Using receptor-blocking antibodies and activation peptides, we determined that thrombin-mediated injury depended upon interactions between protease-activated receptor 3 and protease- activated receptor 4 in human podocytes, and between protease-activated receptor 1 and protease-activated receptor 4 in rat podocytes. Proximity ligation and coimmunoprecipitation assays confirmed thrombin- dependent interactions between human protease-activated receptor 3 and protease-activated receptor 4, and between rat protease-activated receptor 1 and protease-activated receptor 4 in cultured podocytes. Collectively, these data implicate thrombinuria as a contributor to podocyte injury during nephrosis, and suggest that thrombin and/or podocyte-expressed thrombin receptors may be novel therapeutic targets for nephrotic syndrome. J Am Soc Nephrol 28: 2618–2630, 2017. doi: https://doi.org/10.1681/ASN.2016070789 Nephrotic syndrome (NS) is the third leading cause Received July 22, 2016. Accepted March 16, 2017. of ESRD, which is the eighth leading cause of death R.S. and A.P.W. contributed equally to this work. in the United States.1,2 NS is characterized by mas- Published online ahead of print. Publication date available at sive proteinuria and persistent proteinuria is asso- www.jasn.org. ciated with progression to ESRD.3–7 Currently, NS therapies are limited to immunosuppressive agents, Correspondence: Dr. Bryce A. Kerlin, Center for Clinical fi and Translational Research, The Research Institute at Nation- which may be associated with clinically signi cant wide Children’s Hospital, 700 Children’sDr.,W325,Colum- side effects and/or treatment resistance.8,9 Protein- bus, OH 43205-2696. Email: Bryce.Kerlin@NationwideChildrens. uria results primarily from podocyte damage and org loss, although other glomerular defects may Copyright © 2017 by the American Society of Nephrology 2618 ISSN : 1046-6673/2809-2618 JAmSocNephrol28: 2618–2630, 2017 www.jasn.org BASIC RESEARCH contribute.10,11 NS-associated proteinuria induces coagulation thrombin interacts with podocytes in situ;and(3)which protein imbalance, resulting in a complex coagulopathy notable PARs are involved in thrombin-mediated podocyte injury. for enhanced thrombin generation.3,12–14 Nonspecific throm- bin inhibition, with heparin or antithrombin (AT), is known to attenuate proteinuria in the doxorubicin and puromycin RESULTS aminonucleoside (PAN) models of NS.15–18 Thrombin demonstrates opposing, concentration-dependent effects Thrombin Inhibition Reduces Proteinuria in the on endothelial cells, wherein low concentrations mediate PAN-Induced and Human Diphtheria Toxin Receptor cytoprotection and higher concentrations activate a proinflam- Rat Models of NS matory phenotype.19 A similar effect has recently been noted We confirmed that intravenous AT (500 IU/kg daily) signifi- in glucose-challenged podocytes, wherein 50 pM thrombin cantly reduced day 10 PAN-induced proteinuria to 4.261.2 is cytoprotective and 20 nM thrombin exacerbates glucose- mg/mg (versus 10.462.1 mg/mg in sham controls; P=0.04; mediated cytotoxicity.20 However, the receptors involved in Figure 1A).18 Moreover, twice daily subcutaneous hirudin thrombin-dependent podocyte injury, which may represent (1 mg/kg), a highly-specific thrombin inhibitor, also signifi- novel therapeutic targets for NS, are not yet known. cantly reduced proteinuria (3.560.8 mg/mg; P=0.02 versus During NS plasma proteins, predominantly albumin (66 sham). The antiproteinuric benefit of hirudin was not signif- kDa) but also coagulation proteins of similar molecular mass, icantly different than that of AT (P=0.71). Similarly, using the are inappropriatelyexcreted in the plasma ultrafiltrate.3,5,6,13,21 podocyte-specific human diphtheria toxin receptor (hDTR) Coagulant enzymatic activity may proceed in the ultrafiltrate, transgenic rat model, hirudin reduced proteinuria to 5.460.5 which has a pH and calcium concentration similar to mg/mg (versus 9.061.2 mg/mg in sham controls; P,0.05; plasma.22–28 Moreover, podocytes express tissue factor, a Figure 1B).38 potent coagulation initiator, perhaps in a stress-induced manner.29–31 Thus, during NS podocytes may be exposed Thrombin Colocalizes to Podocytes during PAN-Induced to coagulation factors, including prothrombin (69 kDa), Rat Nephrosis which may be converted to thrombin (37.4 kDa).3,32 Recent Plasma levels of prothrombin fragment 1.2 (F1.2), an in vivo studies have demonstrated that the protease-activated recep- marker of thrombin generation, and thrombin-antithrombin tor (PAR) family of G protein-coupled receptors (GPCRs) is (TAT) complex, a product of thrombin regulation, remain expressed by podocytes.20,33–35 PARs are specialized GPCRs stable in the presence of increasing proteinuria severity that become activated after N-terminal cleavage by coagula- whereas urinary levels increase significantly, consistent with tion enzymes and other proteases, which exposes a tethered urinary thrombin activity (F1.2: R2=0.62, P,0.001 and TAT: ligand.36,37 Importantly, these receptors appear to be relevant R2=0.77, P,0.001; Supplemental Figure 1). Thus, immuno- to glomerular diseases.20,33–35 We hypothesized that throm- fluorescence colocalization was utilized to determine the pat- bin contributes to podocyte injury in a PAR-specific manner tern of thrombin-podocyte interactions during the course of during NS. Totest this hypothesis, we performed experiments proteinuria development (Figure 2). These experiments to determine (1) if hirudin, a highly specificthrombininhib- demonstrate that thrombin colocalization to podocytes in- itor, recapitulates the antiproteinuric effects of AT; (2)if creases during the course of proteinuria development in Figure 1. Thrombin inhibition reduces proteinuria in the PAN-induced and hDTR rat models of NS. (A) Rats were given 50 mg/kg PAN (intravenous) on day 0, both twice daily subcutaneous hirudin (1 mg/kg; n=8) and daily intravenous AT (500 IU/kg; n=7) reduced day 10 mean6SEM urine protein-to-creatinine ratio versus control animals (n=8), which received sham treatment with the carrier solution (normal saline) by both routes at the respective frequencies. (B) Rats were given 25 ng/kg DT (intraperitoneal) on day 0. Twice daily hirudin (1 mg/kg; n=5) reduced day 10 mean6SEM urine protein-to-creatinine ratio versus control animals (n=4) receiving sham treatment. *P,0.05 versus day 0; #P,0.05 versus control. J Am Soc Nephrol 28: 2618–2630, 2017 Thrombin-Induced Podocyte Injury 2619 BASIC RESEARCH www.jasn.org immunofluorescence.34 Immunofluores- cence histology demonstrated PAR4 hu- man podocyte colocalization in a cell body predominant pattern, similar to PAR3 (Supplemental Figure 2). Moreover, flow cytometry immunophenotyping re- vealed that podocytes, freshly isolated from healthy rats, express all four PARs (Figure 3). Thrombin Induces Human Podocyte Injury in a PAR3- and PAR4- Dependent Manner As previously reported, all four human PARs were detected by RT-PCR (Figure 4A) and Western blot (Figure 4B) in cul- tured human podocytes.34,35 Thrombin exacerbated PAN-induced podocyte injury with maximal effects at 20 nM (Supple- mental Figure 3A). Moreover, 20 nM thrombin was found to induce maximal podocyte injury independently of PAN (Supplemental Figure 3B). To determine which PARs may be involved in thrombin- mediated podocyte injury, a series of PAR antibody blocking and PAR activation pep- tide (AP) experiments were conducted. Thrombin (20
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