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Thrombin-Induced Podocyte Injury Is Protease-Activated 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 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 - 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 , 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 -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: .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.

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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 nM) induced a significant in- crease in terminal deoxynucleotidyl transfer- ase dUTP nick end labeling (TUNEL)-positive podocytes from 1.4%60.4% in media- only control to 49.2%67.3% (P,0.001; Figure 4C). Antibody blockade of either PAR3 (6.46%63.7%; P,0.001 versus thrombin) or PAR4 (8.9%63.7%; P,0.001 versus thrombin) resulted in significantly decreased injury. When the thrombin sig- nal was mimicked with APs, PAR4 AP sig- nificantly increased the TUNEL signal (40.1%68.2%; P,0.001 versus control and Figure 2. Thrombin colocalizes to podocytes during PAN-induced rat nephrosis. (A) P,0.001 versus scrambled peptide; Figure Representative immunofluorescence histology of representative glomeruli from nephrotic 4D), whereas PAR3 AP did not (7.01%6 rats 0–10 days after PAN (50 mg/kg) infusion (original magnification, 360). (B) Proteinuria 2.94%; P,0.96 versus control; P=0.91 versus values (solid line; ANOVA P,0.001) and relative proportion of synaptopodin-positive scrambled peptide). Further confirming the , pixels with colocalized thrombin (dashed line; ANOVA P 0.001) calculated utilizing the PAR specificity of 20 nM thrombin signaling colocalization coefficient feature of ZEN software (Zeiss USA). Values are mean6SEM a in human podocytes, thrombin-dependent from n=3 rats per time point; 20 random glomeruli per rat. #P,0.05; P,0.01; ^P,0.001 extracellular signal–related kinase 1/2 versus control. (ERK1/2) phosphorylation was inhibited by PAR3 and PAR4 antibody blockade (P,0.05; PAN-nephrotic rats (P,0.001); roughly in parallel to in- Figure 4F). Podocyte viability, by 3-(4,5-dimethylthiazol-2yl)- creasing proteinuria severity. 2,5-diphenyltetrazolium bromide (MTT) assay, was significantly reduced by thrombin (P,0.05), but not rescued by a pan-caspase In Vivo Podocyte Expression of PARs inhibitor (Z-VAD-FMK; Supplemental Figure 4A). Moreover, Podocyte expression of PAR2 and PAR3, but not PAR1, has podocytes demonstrated F-actin cytoskeletal rearrangement in re- previously been demonstrated in human kidney sections by sponse to thrombin exposure (P,0.001; Supplemental Figure 4B).

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Figure 3. In vivo rat podocyte expression of PARs. (A) Dot plot showing a single cell suspension of freshly isolated rat kidney glomerular cells stained with a FITC-conjugated antibody to synaptopodin. Gating strategy and the percentage of total cells expressing synaptopodin are shown. (B) Histograms showing the expression of PAR1–PAR4 on synaptopodin-positive podo- cytes. Unstained podocytes (gray), podocytes stained with APC-conjugated isotype control antibodies (dashed), and podocytes stained with APC-conjugated antibodies to PAR1–PAR4 (solid black) are shown. Each curve represents an analysis of 50,000 events. Data are representative of three separate experiments. APC, allophycocyanin; FSC-A, forward scatter-area; SSC-A, side scatter-area.

Thrombin Induces Rat Podocyte Injury in a PAR1- and studies were congruent with those observed in human po- PAR4-Dependent Manner docytes (P,0.05 and P,0.001, respectively; Supplemental Similarly, cultured rat podocytes expressed all four rat PARs Figure 5). by both RT-PCR (Figure 5A) and Western blot (Figure 5B). Because PAR signaling mechanisms are species-specific,34 Thrombin Induces PAR-PAR Interactions in Cultured the same techniques utilized to determine thrombin recep- Podocytes tor specificity in the human podocytes were performed. To assess for thrombin-dependent PAR3/PAR4 interactions in Thrombin (20 nM) significantly increased TUNEL positiv- human podocytes and PAR1/PAR4 interactions in rat podo- ity from 1.0%60% in the control to 33.6%610.77% cytes, we conducted proximity ligation assays (PLAs) in the (P=0.01; Figure 5C). TUNEL positivity was significantly re- presence and absence of thrombin. Because PARs are known to duced by preincubation with anti-PAR1 antibody (1.9%6 be rapidly endocytosed and degraded after cleavage-dependent 1.37%; P,0.02 versus thrombin) or anti-PAR4 antibody activation, we first performed a time-dependent thrombin ex- (3.5%66.1%; P=0.02 versus thrombin). Moreover, stimu- posure experiment with human podocytes to determine the lation with PAR1 AP induced TUNEL positivity to levels optimal exposure time for subsequent experiments (5 minutes; similar to thrombin exposure (37.5%68.27%; P,0.001 Supplemental Figure 6).39 We then performed PLA experi- versus control and P,0.05 versus scrambled peptide; Figure ments demonstrating the presence of PAR3/PAR4 interactions 5D), whereas PAR4 AP did not (19.01%66.35%; P=0.16 in human podocytes only in the presence of thrombin (4.156 versus control; P=0.94 versus scrambled peptide). Thrombin- 1.42 PLA signals/nucleus versus 060 PLA signals/nucleus in dependent ERK1/2 phosphorylation was inhibited by control; P,0.01; Figure 6, A and B). Similarly, PAR1/PAR4 preincubation with PAR1 and PAR4 antibodies (P,0.05; interactions were only observed after 5 minutes of thrombin Figure 5F). Podocyte viability and F-actin rearrangement exposure in rat podocytes (4.2760.46 PLA signals/nucleus

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Figure 4. Thrombin induces human podocyte injury in a PAR3- and PAR4-dependent manner. (A) PCR and (B) Western blot showing mRNA and protein expression for PARs in cultured human podocytes. (C) PAR-specific antibody blockade of either PAR3 or PAR4 c prevents thrombin-induced injury (^P,0.001 versus control; P,0.001 versus 20 nM thrombin; 2.5 mg/ml hirudin; n=3 per con- a dition). (D) Stimulation with PAR4-specific AP induces injury to a similar level as thrombin (^P,0.001 versus control; P,0.01 versus control and scrambled peptide; 20 nM thrombin; 20 mMAP;n=3 per condition); (E) Representative TUNEL images (original magnification, 320). (F) Thrombin-dependent ERK1/2 phosphorylation was inhibited by PAR3 and PAR4 antibody blockade (upper v panel: representative blots; lower panel: densitometric quantification; P,0.05 versus thrombin; n=10 per condition). Ab, antibody; AP, activation peptide.

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Figure 5. Thrombin induces rat podocyte injury in a PAR1- and PAR4-dependent manner. (A) PCR and (B) Western blot demonstrating PAR expression in cultured rat podocytes. (C) PAR-specific antibody blockade of either PAR1 or PAR4 prevents v thrombin-induced injury (#P,0.05 versus control; P,0.05 versus 20 nM thrombin; 2.5 mg/ml hirudin; n=3 per condition). (D) Stimulation with PAR1-specific AP induces injury to a similar level as thrombin (^P,0.001 versus control; *P,0.05 versus scrambled peptide; 20 nM thrombin; 20 mMAP;n=3 per condition). (E) Representative TUNEL images (original magnification, 320). (F) Thrombin-dependent ERK1/2 phosphorylation was inhibited by PAR1 and PAR4 antibody blockade (upper panel: v representative blots; lower panel: densitometric quantification; P,0.05 versus thrombin; n=6 per condition). Ab, antibody; AP, activation peptide.

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Figure 6. Thrombin induces PAR3/PAR4 interactions in human podocytes. (A) Representative PLA images demonstrating the presence of PAR3/PAR4 interactions in human podocytes only in the presence of thrombin (original magnification, 320). (B) Proximity signals per a nucleus ( P,0.01 versus control; n=4 per condition). versus 060 PLA signals/nucleus; P,0.001; Figure 7, A and B). in podocytes after thrombin exposure in this study.36 In rat Importantly, no significant PLA signal was detected with podocytes, thrombin-mediated podocyte injury occurs via isotype-matched, nonspecific control antibodies in either PAR1 in a PAR4-dependent manner and thrombin induced species of podocyte (Figures 6 and 7). Coimmunoprecipitation PAR1/PAR4 interactions. Thus, interrupting thrombin sig- (co-IP) assays were utilized to confirm these PLA findings and naling may offer a novel approach to reduce podocyte injury to screen for other PAR-PAR interactions not predicted by during NS. theantibodyblockadeandAPstimulation experiments (Fig- Thrombin generation is enhanced during NS.12,14 High ure 8). Thus, in human podocytes, thrombin (20 nM) signif- concentrations of thrombin may contribute to podocyte in- icantly increased PAR3-PAR4 co-IP (P,0.05; Figure 8, A–C), jury and nonspecific thrombin inhibition reduces proteinuria and PAR1-PAR4 interactions were enhanced in rat podocytes in experimental NS.15–18,20 Prothrombin (69 kDa) is slightly (P,0.05; Figure 8, D–F). Co-IP was negligible for other com- larger than albumin (66 kDa), the major plasma protein ex- binations tested in both species. creted during NS, but has been observed in the urine of ne- phrotic children and PAN-nephrotic rats.3,40,41 In this study, urinary F1.2 and TAT increased in proportion to proteinuria DISCUSSION severity but remained stable in the plasma compartment, sug- gestive of urinary thrombin activity. Importantly, thrombin In this study, highly-specific thrombin inhibition partially increasingly colocalized to podocytes during PAN NS in par- ameliorated proteinuria in experimental PAN-induced NS allel to proteinuria severity. Podocytes have been shown to to a similar degree as nonspecific thrombin inhibition. A sim- express tissue factor, perhaps inaninduciblefashionduring ilar benefitwasobservedinthepodocyte-specifichDTR disease and/or after stress.29–31,42 It is thus reasonable to hy- NS model. We demonstrated that thrombin interacts in vivo pothesize that thrombin is generated in the ultrafiltrate, per- with podocytes during PAN NS. We established that podocytes haps via prothrombinase formation on the podocyte surface. express the PAR family of coagulation receptors in vivo and Moreover, the antiproteinuric effect of AT, which has been that thrombin induces in vitro podocyte injury in a PAR- confirmed in this study, appears to be reproduced by dependent manner. PAR3/PAR4 interactions have not been highly-specific thrombin inhibition with hirudin, a renally previously observed in human cell biology, but were observed excreted direct thrombin inhibitor,43 suggesting that

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Figure 7. Thrombin induces PAR1/PAR4 interactions in rat podocytes. (A) Representative PLA images demonstrating the presence of PAR1/PAR4 interactions in rat podocytes only in the presence of thrombin (original magnification, 320). (B) Proximity signals per nucleus (^P,0.001 versus control; n=4 per condition). thrombin inhibition may reduce in vivo podocyte injury. This subtle changes in local thrombin activity may be biologically hypothesis is further supported by the beneficial effect of relevant to podocytes. In pilot experiments, we exposed cultured hirudin on proteinuria in the podocyte-specifichDTRNS podocytes to a range of thrombin concentrations around 20 nM, model.38 with maximal injury observed at 20 nM. Thrombin-induced Using flow cytometry techniques to identify podocytes podocyte injury appears to be mediated by PAR1 in a PAR4- within a suspension of freshly isolated rat glomerular cells, dependent manner in rat podocytes, whereas it is mediated by we were able to demonstrate that these cells also express all PAR4 in a PAR3-dependent manner in human podocytes. The four PARs. These data are consistent with the observations that ERK1/2 phosphorylation data suggest that thrombin-dependent cultured mouse and human podocytes express PARs.34,35 podocyte injury may be mediated via the MAPK signaling path- In vitro, both human and rat differentiated podocytes ex- way, which has been implicated in other cell types after thrombin pressed all four members of the PAR family, at both the mRNA exposure.47,48 Importantly, podocyte viability is reduced upon and protein level.34,35 Interestingly 20 nM thrombin, previ- thrombin exposure, an effect not reversed by caspase inhibition, ously demonstrated to exacerbate glucose-induced podocyte suggesting that thrombin-induced podocyte injury is caspase injury, injured podocytes independently of both glucose and independent. The applicability of these PAR data to in vivo ne- PAN.20 In addition to providing important confirmation of phrosis awaits additional studies with appropriate PAR-specific our previous observation, these data provide further evidence inhibitor treatments. that high-concentration thrombin directly injures podocytes. PAR-PAR interactions have previously been published for Importantly, on the basis of peak thrombin generation in most PAR combinations, including both homodimer and het- PAN-nephrotic plasma (approximately 300 nM),12 20 nM erodimer interactions.36 These interactions may be cofactor concentrations are speculated to be possible within Bowman’s phenomena, wherein a protease binding to the first PAR facil- space during NS. However, the precise concentrations of itates cleavage of a neighboring PAR.36 This mechanism was thrombin achieved within the ultrafiltrate or on the podocyte originally described for PAR3-dependent activation of PAR4 surface have not been established. Nonetheless, 20 nM is ap- in thrombin-dependent mouse platelet activation.49,50 Alter- proximately three orders of magnitude below the published natively, transactivation involves activation of a PAR by the Michaelis constant for thrombin cleavage of PAR1 and PAR4 tethered ligand of a nearby PAR, as has been described for (26–28 mM and 56–61 mM, respectively),44–46 suggesting that PAR1 transactivation of PAR2.36,51 Additionally, substantial

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to speculate, on the basis of similarities in sequence and molecular mass that the di- mensions of the remaining PARs are similar. Therefore, it is possible that the PLA signals observed in these studies represent close proximity (#400 Å), but not direct molecu- lar interactions. However, these interactions were confirmed by co-IP assays and are con- sistent with our functional assay data (PAR antibody blockade and AP stimulation), strongly supporting the notion that these PARs interact with one another in a bio- logically meaningful way. Importantly, PAR dimers are known to be dependent on interactions between hydrophobic trans- membrane domains,60,61 thus the co-IP ex- periments included bis(sulfosuccinimidyl) suberate, an 11.4-Å-long crosslinking re- agent,62 to stabilize PARdimers during aque- ous phases of the protocol, suggesting that the PARs are in direct contact with one an- other. This hypothesis is further supported by studies demonstrating that PAR-PAR heterodimers are involved in activated protein C signaling events in both mouse and human podocytes.20,33,34 Nonetheless, confirmation of these interactions await Figure 8. PAR-PAR interactions in human and rat podocytes confirmed by co-IP. (A–C) molecular biology studies designed to de- fi Human podocyte PAR co-IP experiments: (A) Representative blots after 5 minutes of ne which PAR domains are involved in exposure to 20 nM thrombin. (B) Relative PAR3 protein after PAR4 immunoprecipi- the formation of these complexes. tation. (C) Relative PAR4 protein after PAR3 immunoprecipitation. (D–F) Rat podocyte Previous investigations have demon- PAR co-IP experiments: (D) representative blots after 5 minutes of exposure to 20 nM strated that anticoagulants improve protein- thrombin; (E) relative PAR1 protein after PAR4 immunoprecipitation; (F) relative PAR4 uriainanimalmodelsofNS.15–18 Conversely, # protein after PAR1 immunoprecipitation. P,0.05, n=3–6 per condition. Con, Control; thrombin may exacerbate podocyte injury.20 Thr, Thrombin. However, these studies stopped short of determining (1) the thrombin specificity evidence suggests that the PARs may homo- or heterodimer- of anticoagulant effects on proteinuria and (2) the molecular ize after activation-dependent allosteric changes in PAR receptors involved in thrombin-mediated podocyte injury. conformation.39,52–54 This mechanism, which may alter Thus, in this study, we have demonstrated that (1)highly- the downstream G-protein and/or b-arrestin pathways involved specific thrombin inhibition reduces proteinuria in experi- in intracellular signaling, has been recently demonstrated for mental rat nephrosis; (2) thrombin-induced podocyte injury PAR1/PAR4 heterodimer-dependent thrombin-mediated acti- is mediated by PARs in a species-specific manner, consistent vation in human platelets.52 However, interactions between hu- with species-specific PAR signaling in other cell types54;and man PAR3/PAR4 have not, to our knowledge, been described (3) that these receptors are expressed by podocytes in vivo. previously.36 In this study, proximity ligation was chosen to con- Further, our data suggest that this injury may be dependent on firm PAR-PAR interactions because this technique allows for specific PAR-PAR interactions. Taken together, these data, light microscopy detection of molecular interactions without along with the previously published observations, suggest genetic manipulation of the cultured cells, such as may be re- that ultrafiltered prothrombin during nephrotic-range pro- quired for other techniques.36 This technique has recently been teinuria may be a key driver of ongoing and potentially pro- utilized to detect protein-protein interactions involved in both gressive podocyte injury. Thus, strategies to interrupt this integrin signaling and non-PAR GPCR interactions.55–57 Prox- pathway may lead to novel therapeutic approaches benefitting imity ligation detects target epitopes that are #400 Å apart.55–58 patients who are resistant to conventional therapy, or as an The crystal structure of PAR1 reveals that its dimensions are adjuvant therapy to accelerate remission and slow progression approximately 88.9388.9388.7 Å.59 Although the crystal struc- toward ESRD.8 PAR3 is not involved in human platelet biology ture of the other PARs have not been published, it is reasonable but is involved in the activated protein C podocytoprotective

2626 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 2618–2630, 2017 www.jasn.org BASIC RESEARCH phenomenon.34,36 Thus, an intriguing approach may be to dis- (Thermo Fisher Scientific, Grand Island, NY). Images were captured rupt PAR3/PAR4 interactions if they prove to be necessary to in with a BZ900 microscope (Keyence, Osaka, Japan) and analyzed with vivo thrombin-mediated podocyte injury. Such an approach ZEN software (Zeiss USA, Thornwood, NY). Colocalization analysis could avoid the bleeding risks associated with currently available was performed on a pixel-by-pixel basis, wherein every pixel within anticoagulants and also leave activated protein C–mediated the defined region of interest (glomerulus, see red demarcation line in PAR2/PAR3 heterodimer-dependent cytoprotection intact. “false color” images of Figure 2A) is plotted in a scattergram on the basis of fluorescence intensity of each channel. The software calcu- lates the colocalization coefficient as the proportion of colocalized CONCISE METHODS pixels (positive in both channels) within the total population of a single channel, green (SYNPO-positive) for the purposes of this For full details on materials and methods, please see Supplemental study.67 For normal human kidney PAR4 expression, immunofluo- Material. rescence staining was performed on previously reported paraffin- embedded sections.34 Briefly, after rehydration, sections were boiled Animals for 30 minutes in water for antigen retrieval, blocked in 10% horse All procedures were approved by The Research Institute at Nationwide serum for 1 hour, incubated with primary antibodies (1:50 or block- Children’s Hospital Institutional Animal Care and Use Committee ing solution only for negative controls) in a humidified chamber for and performed in accordance with the National Institutes of Health 3 days at 4°C, and washed in PBS. Secondary antibodies (1:200) were Guide for the Care and Use of Laboratory Animals. For the AT and added in humidified chambers overnight at 4°C, washed in PBS, and hirudin experiments, AT (500 IU/kg daily via tail vein) or hirudin mounted under coverslips. Images were obtained with a Leica SP5 (1 mg/kg subcutaneous twice daily) was administered beginning im- confocal microscope. mediately after PAN injection (Wistar rats).18,63 Control animals re- ceived vehicle injections by the respective route/frequency and all Podocyte Flow Cytometry animals were euthanized on day 10. The hDTR transgenic rat model Glomeruli were isolated using sequential sieving, as described was kindly provided by Dr. Roger C. Wiggins (University of Michigan, previously.66,68,69 Glomerular cells were dissociated as previously Ann Arbor). The hDTR rats received a single dose of intraperitoneal described, with minor modifications.70 Briefly, glomeruli were sus- diphtheria toxin (DT; 25 ng/kg) on day 0, after which subcutaneous pended in 2 ml digestion buffer (300 U/ml type 4 collagenase and hirudin or vehicle was administered per the above dose and frequency.38 50 U/ml DNase I [both from Worthington Biochemicals, Lakewood, Urine was collected for proteinuria analysis on days 0 (before PAN or NJ] with 2.8 U/ml Dispase II [Sigma-Aldrich, St. Louis, MO] in DT injection) and 10. In experiments to evaluate urinary markers of HBSS [Gibco; ThermoFisher, Grand Island, NY]) and incubated at thrombin activity, varying doses of PAN (0–150 mg/kg) were utilized to 37°C on a thermomixer shaking at 1150 rpm for 32 minutes, inter- induce dose-dependent proteinuria, as previously described.12 After mittently shearing the cell suspension with a pipet tip. Tissue debris urine collection on day 9, the rats were anesthetized with 3% isoflurane was removed by filtration (40 mmpores)andwashedwithHBSS. and blood was collected from the inferior vena cava using a 23-G needle Cells were concentrated by centrifugation (1500 rpm for 5 minutes at into 0.32% sodium citrate/1.45 mM corn inhibitor, final con- 4°C), resuspended in 2 ml HBSS with 0.1% BSA and DAPI (1 mg/ml), centrations (Haematologic Technologies Inc., Essex Junction, VT).12,64 and cell concentration measured. Aliquots of approximately 33106 For the immunofluorescence colocalization experiments, Wistar rats cells/ml were washed with BD BSA stain buffer (Fisher Scientific, received a single dose of intravenous PAN (50 mg/kg) on day 0 to induce Pittsburgh, PA), blocked with mouse anti-rat CD32 (10 mg/ml; BD nephrosis, as previously described.12,18,65,66 After urine was collected at Biosciences, San Jose, CA), and stained with FITC-labeled anti-rat the time points indicated in Figure 2, the animals were euthanized using synaptopodin antibody (1:5) and allophycocyanin-labeled anti-rat the above protocol and kidneys collected for immunofluorescence his- PAR antibodies (PAR1 and PAR2 at 4.4 mg/ml, PAR3 and PAR4 at tology. Kidneys were harvested from healthy Wistar rats for the flow 3 mg/ml). Fluorescence-labeled, isotype-matched, nonspecificanti- cytometry immunophenotyping experiments. bodies were used for controls. Cells were analyzed on a LSR II flow cytometer (BD Biosciences) and PAR expression was analyzed only Immunofluorescence Histology on cells within the synaptopodin-positive gate. Isotype controls, un- Paraffin-embedded rat kidney sections were deparaffinized and anti- stained cells, and an open channel were used to identify and calibrate gen retrieval was performed at 100°C. Super Block (Scytek Labora- for autofluoresence. Each curve in Figure 3 represents an analysis of tories, Inc., West Logan, UT) was applied for two consecutive 50,000 events. Results were confirmed using three rats in three sep- 30-minute intervals at 37°C (250 ml/slide) to reduce nonspecific arate experiments. staining. Primary antibodies for SYNPO (1:5 dilution) and thrombin (1:100 dilution) were incubated for 48 hours at 4°C in Super Block. Proximity Ligation Assay Slides were washed for 10 minutes at room temperature with 0.5% PLA experiments were performed on human podocytes (grown in Tween-PBS three times. Secondary antibodies (Alexa Fluor 594 anti- an eight-well chamber slide system) and rat podocytes (grown in rabbit IgG and Alexa Fluor 488 anti-mouse IgG; 2 mg/ml each) were six-well plates with coverslips) and analyzed using the DUOLink applied overnight at 4°C. Sections were mounted with ProLong Gold kit (Sigma-Aldrich) according to the manufacturer’s instructions Antifade Mountant with 4’,6-diamidino-2-phenylindole (DAPI) stain and as described previously.71,72 After blocking, anti-PAR

J Am Soc Nephrol 28: 2618–2630, 2017 Thrombin-Induced Podocyte Injury 2627 BASIC RESEARCH www.jasn.org antibodies were applied at 10 mg/ml; nonspecific rabbit IgG (Santa DISCLOSURES Cruz Biotechnology, Dallas, TX) and mouse IgG2b (Bio-Rad, None. Hercules, CA) were used as controls. Oligonucleotide-conjugated secondary antibodies against mouse and rabbit primary anti- bodies were utilized to detect protein-protein associations, visu- REFERENCES alized as bright red dots by microscopy. Each slide was mounted with PLA mounting reagent containing DAPI and counterstained 1. Murphy SL, Xu J, Kochanek KD: Deaths: Final data for 2010. National for actin using Alexa Fluor 488 phalloidin staining solution, vital statistics reports: From the centers for disease control and pre- – which was added at 2 U/coverslip for 20 minutes before mount- vention, national center for health statistics. Nat Vital Stat Report 61: 1 117, 2013 ing the slides. The PLA signal was quantified with a BZ900 2. U.S. Renal Data System: USRDS 2013 Annual Data Report: Atlas of microscope and software (Keyence) by manually counting the Chronic Kidney Disease and End-Stage Renal Disease in the United number of red dots per nucleus in at least five random fields per States, Bethesda, MD, National Institutes of Health, National Institute condition. of Diabetes and Digestive and Kidney Diseases, 2013 3. Kerlin BA, Ayoob R, Smoyer WE: Epidemiology and pathophysiology of Statistical Analyses nephrotic syndrome-associated thromboembolic disease. Clin J Am – Statistical significance was determined by the unpaired paired t test for Soc Nephrol 7: 513 520, 2012 4. Hanko JB, Mullan RN, O’Rourke DM, McNamee PT, Maxwell AP, single comparisons and one- or two- way ANOVA for multiple group Courtney AE: The changing pattern of adult primary glomerular dis- comparisons, using SigmaStat software (Systat, San Jose, CA). Statis- ease. Nephrol Dial Transplant 24: 3050–3054, 2009 tical significance was defined as P,0.05. Data are presented as 5. Eddy AA, Symons JM: Nephrotic syndrome in childhood. Lancet 362: mean6SEM. 629–639, 2003 6. Orth SR, Ritz E: The nephrotic syndrome. N Engl J Med 338: 1202– 1211, 1998 7. Korbet SM, Schwartz MM, Lewis EJ: Primary focal segmental glomer- ACKNOWLEDGMENTS ulosclerosis: Clinical course and response to therapy. Am J Kidney Dis 23: 773–783, 1994 The authors thank Dr. Charles J. Lockwood and Dr. Matthias Kretzler 8. Greenbaum LA, Benndorf R, Smoyer WE: Childhood nephrotic – – for meaningful critique of experimental designs, Melinda A. Chanley, syndrome Current and future therapies. Nat Rev Nephrol 8: 445 458, 2012 Adam Guess, Xiaojing Nie, Victoria Best, and Cynthia McAllister for 9. Ding WY, Saleem MA: Current concepts of the podocyte in nephrotic contributing to the refinement of analytic techniques, and Dr. Roger syndrome. Kidney Res Clin Pract 31: 87–93, 2012 C. Wiggins for providing the human diphtheria toxin receptor rat 10. Schönenberger E, Ehrich JH, Haller H, Schiffer M: The podocyte as a model. R.S., A.P.W., S.A., K.J.W., H.L., and K.S. conducted the ex- direct target of immunosuppressive agents. Nephrol Dial Transplant – periments, analyzed the data, prepared the figures, and wrote the 26: 18 24, 2011 11. Chiang CK, Inagi R: Glomerular diseases: Genetic causes and future manuscript. M.T.N. prepared and provided critical reagents and wrote therapeutics. Nat Rev Nephrol 6: 539–554, 2010 the paper. W.E.S. provided cell cultures and animal models and edited 12. Kerlin BA, Waller AP, Sharma R, Chanley MA, Nieman MT, Smoyer WE: the paper. B.I. advised on study design and reagent choices and Disease severity correlates with thrombotic capacity in experimental wrote the paper. B.A.K. conducted the experiments, analyzed the data, nephrotic syndrome. JAmSocNephrol26: 3009–3019, 2015 wrote the manuscript, and was responsible for overseeing and co- 13. Loscalzo J: Venous thrombosis in the nephrotic syndrome. NEngl – ordinating the study. JMed368: 956 958, 2013 14. Mahmoodi BK, Mulder AB, Waanders F, Spronk HM, Mulder R, This work was supported by National Institutes of Health, Na- Slagman MC, Vogt L, Navis G, Ten Cate H, Kluin-Nelemans HC, tionalInstitute of DiabetesandDigestiveandKidneyDiseases grants Laverman GD: The impact of antiproteinuric therapy on the pro- L40DK103299, U54DK083912-05S1, U54DK083912-07S1, and thrombotic state in patients with overt proteinuria. JThrombHaemost K08DK103982 (to B.A.K.), andR01DK095959 (toW.E.S.);National 9: 2416–2423, 2011 Heart, Lung, and Blood Institute grant R01HL098217 (to M.T.N.); 15. Baroni EA, Costa RS, da Silva CG, Coimbra TM: Heparin treatment reduces glomerular injury in rats with adriamycin-induced nephropathy Deutsche Forschungsgemeinschaft DFG IS67/5-3 and DFG IS67/ but does not modify tubulointerstitial damage or the renal production 8-1 (to B.I.); the CSL Behring Foundation Professor Heimburger of transforming growth factor-beta. Nephron 84: 248–257, 2000 Award (to B.A.K.); the George and Elizabeth Kelly Foundation 16. Benchetrit S, Golan E, Podjarny E, Green J, Rashid G, Bernheim J, (Lewis Center, OH; to B.A.K.); the Nationwide Children’sHospital Hershkovitz R, Bernheim J: Low molecular weight heparin reduces Foundation Hitchcock-Wilson Fellowship in Pediatric Hematol- proteinuria and modulates glomerular TNF-alpha production in the – ’ early phase of adriamycin nephropathy. Nephron 87: 155 160, 2001 ogy, Oncology, and BMT (to R.S.); and the Nationwide Children s 17. Diamond JR, Karnovsky MJ: Nonanticoagulant protective effect of Hospital Foundation Joan Fellowship in Pediatric Hemostasis- heparin in chronic aminonucleoside nephrosis. Ren Physiol 9: 366–374, Thrombosis (to R.S.). 1986 Portions of this work were previously presented at meetings of the 18. Yamashita J, Nakajima K, Ohno Y, Kaneshiro Y, Matsuo T, Tanaka H, American Society of Hematology, Orlando, FL December 5–8, 2015, Kaneko K: Protective effects of antithrombin on puromycin amino- – – nucleoside nephrosis in rats. Eur J Pharmacol 589: 239 244, 2008 American Society of Nephrology, San Diego, CA, November 3 8, 19. Bae JS, Kim YU, Park MK, Rezaie AR: Concentration dependent dual 2015, and International Society on Thrombosis and Haemostasis, effect of thrombin in endothelial cells via Par-1 and Pi3 Kinase. JCell Milwaukee, WI, June 23–26, 2014. Physiol 219: 744–751, 2009

2628 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 2618–2630, 2017 www.jasn.org BASIC RESEARCH

20. Wang H, Madhusudhan T, He T, Hummel B, Schmidt S, Vinnikov IA, 37. Madhusudhan T, Kerlin BA, Isermann B: The emerging role of co- Shahzad K, Kashif M, Muller-Krebs S, Schwenger V, Bierhaus A, agulation proteases in kidney disease. Nat Rev Nephrol 12: 94–109, Rudofsky G, Nawroth PP, Isermann B: Low but sustained coagulation 2016 activation ameliorates glucose-induced podocyte apoptosis: Pro- 38. Wharram BL, Goyal M, Wiggins JE, Sanden SK, Hussain S, Filipiak WE, tective effect of factor V Leiden in diabetic nephropathy. Blood 117: Saunders TL, Dysko RC, Kohno K, Holzman LB, Wiggins RC: Podocyte 5231–5242, 2011 depletion causes glomerulosclerosis: Diphtheria toxin-induced podo- 21. Schlegel N: Thromboembolic risks and complications in nephrotic cyte depletion in rats expressing human diphtheria toxin receptor children. Semin Thromb Hemost 23: 271–280, 1997 transgene. JAmSocNephrol16: 2941–2952, 2005 22. Cunningham MA, Kitching AR, Tipping PG, Holdsworth SR: Fibrin in- 39. Soh UJ, Dores MR, Chen B, Trejo J: Signal transduction by protease- dependent proinflammatory effects of tissue factor in experimental activated receptors. Br J Pharmacol 160: 191–203, 2010 crescentic glomerulonephritis. Kidney Int 66: 647–654, 2004 40. Andersen RF, Palmfeldt J, Jespersen B, Gregersen N, Rittig S: Plasma 23. Erlich JH, Holdsworth SR, Tipping PG: Tissue factor initiates glomerular and urine proteomic profiles in childhood idiopathic nephrotic syn- fibrin deposition and promotes major histocompatibility complex class drome. Proteomics Clin Appl 6: 382–393, 2012 II expression in crescentic glomerulonephritis. Am J Pathol 150: 873– 41. Cruz C, Juárez-Nicolás F, Tapia E, Correa-Rotter R, Pedraza-Chaverrí J: 880, 1997 Abnormalities of coagulation in experimental nephrotic syndrome. 24. Haraguchi M, Border WA, Huang Y, Noble NA: t-PA promotes glo- Nephron 68: 489–496, 1994 merular plasmin generation and matrix degradation in experimental 42. Quaggin SE: Transcriptional regulation of podocyte specification and glomerulonephritis. Kidney Int 59: 2146–2155, 2001 differentiation. Microsc Res Tech 57: 208–211, 2002 25. Hertig A, Rondeau E: Role of the coagulation/fibrinolysis system in 43. Richter M, Cyranka U, Nowak G, Walsmann P: Pharmacokinetics of fibrin-associated glomerular injury. JAmSocNephrol15: 844–853, 125I-hirudin in rats and dogs. Folia Haematol Int Mag Klin Morphol 2004 Blutforsch 115: 64–69, 1988 26. Lelongt B, Bengatta S, Delauche M, Lund LR, Werb Z, Ronco PM: Matrix 44. Jacques SL, LeMasurier M, Sheridan PJ, Seeley SK, Kuliopulos A: metalloproteinase 9 protects mice from anti-glomerular basement Substrate-assisted catalysis of the PAR1 . Enhance- membrane nephritis through its fibrinolytic activity. JExpMed193: ment of macromolecular association and cleavage. JBiolChem275: 793–802, 2001 40671–40678, 2000 27. Malliaros J, Holdsworth SR, Wojta J, Erlich J, Tipping PG: Glomerular 45. Jacques SL, Kuliopulos A: Protease-activated receptor-4 uses dual fibrinolytic activity in anti-GBM glomerulonephritis in rabbits. Kidney prolines and an anionic retention motif for thrombin recognition and Int 44: 557–564, 1993 cleavage. Biochem J 376: 733–740, 2003 28. Motojima M, Matsusaka T, Kon V, Ichikawa I: Fibrinogen that appears in 46. Nieman MT, Schmaier AH: Interaction of thrombin with PAR1 and PAR4 Bowman’s space of proteinuric kidneys in vivo activates podocyte Toll- at the thrombin cleavage site. Biochemistry 46: 8603–8610, 2007 like receptors 2 and 4 in vitro. Nephron Exp Nephrol 114: e39–e47, 47. Lockwood CJ, Kayisli UA, Stocco C, Murk W, Vatandaslar E, Buchwalder 2010 LF, Schatz F: Abruption-induced preterm delivery is associated with 29. Apostolopoulos J, Moussa L, Tipping PG: The cytoplasmic domain of thrombin-mediated functional progesterone withdrawal in decidual tissue factor restricts physiological albuminuria and pathological pro- cells. Am J Pathol 181: 2138–2148, 2012 teinuria associated with glomerulonephritis in mice. Nephron Exp 48. Bretschneider E, Kaufmann R, Braun M, Nowak G, Glusa E, Schrör K: Nephrol 116: e72–e83, 2010 Evidence for functionally active protease-activated receptor-4 (PAR-4) 30. Yamabe H, Yoshikawa S, Ohsawa H, Inuma H, Miyata M, Sasaki T, in human vascular smooth muscle cells. Br J Pharmacol 132: 1441– Kaizuka M, Tamura N, Onodera K: Tissue factor production by cultured 1446, 2001 rat glomerular epithelial cells. Nephrol Dial Transplant 8: 519–523, 49. Kahn ML, Zheng YW, Huang W, Bigornia V, Zeng D, Moff S, Farese RV Jr., 1993 Tam C, Coughlin SR: A dual thrombin receptor system for platelet acti- 31. Narita I, Shimada M, Yamabe H, Kinjo T, Tanno T, Nishizaki K, Kawai M, vation. Nature 394: 690–694, 1998 Nakamura M, Murakami R, Nakamura N, Tomita H, Saleem MA, 50. Nakanishi-Matsui M, Zheng YW, Sulciner DJ, Weiss EJ, Ludeman MJ, Mathieson PW, Okumura K: NF-kappaB-dependent increase in tissue Coughlin SR: PAR3 is a cofactor for PAR4 activation by thrombin. Na- factor expression is responsible for hypoxic podocyte injury. Clin Exp ture 404: 609–613, 2000 Nephrol 20: 679–688, 2015 51. O’Brien PJ, Prevost N, Molino M, Hollinger MK, Woolkalis MJ, Woulfe 32. Krishnaswamy S, Mann KG, Nesheim ME: The prothrombinase-cata- DS, Brass LF: Thrombin responses in human endothelial cells. Contri- lyzed activation of prothrombin proceeds through the intermediate butions from receptors other than PAR1 include the transactivation of meizothrombin in an ordered, sequential reaction. JBiolChem261: PAR2 by thrombin-cleaved PAR1. J Biol Chem 275: 13502–13509, 8977–8984, 1986 2000 33. Isermann B, Vinnikov IA, Madhusudhan T, Herzog S, Kashif M, 52. Nieman MT: Protease-activated receptor 4 uses anionic residues to Blautzik J, Corat MA, Zeier M, Blessing E, Oh J, Gerlitz B, Berg DT, interact with alpha-thrombin in the absence or presence of protease- GrinnellBW,ChavakisT,EsmonCT,WeilerH,BierhausA,Nawroth activated receptor 1. Biochemistry 47: 13279–13286, 2008 PP: Activated protein C protects against diabetic nephropathy by 53. Coughlin SR: Protease-activated receptors in hemostasis, thrombosis inhibiting endothelial and podocyte apoptosis. Nat Med 13: 1349– and vascular biology. J Thromb Haemost 3: 1800–1814, 2005 1358, 2007 54. Canto I, Soh UJ, Trejo J: Allosteric modulation of protease-activated 34. Madhusudhan T, Wang H, Straub BK, Gröne E, Zhou Q, Shahzad K, receptor signaling. Mini Rev Med Chem 12: 804–811, 2012 Müller-Krebs S, Schwenger V, Gerlitz B, Grinnell BW, GriffinJH,Reiser 55. Borroto-Escuela DO, Romero-Fernandez W, Garriga P, Ciruela F, J, Gröne HJ, Esmon CT, Nawroth PP, Isermann B: Cytoprotective sig- Narvaez M, Tarakanov AO, Palkovits M, Agnati LF, Fuxe K: G protein- naling by activated protein C requires protease-activated receptor-3 in coupled receptor heterodimerization in the brain. Methods Enzymol podocytes. Blood 119: 874–883, 2012 521: 281–294, 2013 35. Harris JJ, McCarthy HJ, Ni L, Wherlock M, Kang H, Wetzels JF, Welsh 56. Kasirer-Friede A, Kang J, Kahner B, Ye F, Ginsberg MH, Shattil SJ: GI, Saleem MA: Active proteases in nephrotic plasma lead to a podocin- ADAP interactions with talin and kindlin promote platelet integrin dependent phosphorylation of VASP in podocytes via protease activated aIIbb3 activation and stable fibrinogen binding. Blood 123: 3156– receptor-1. JPathol229: 660–671, 2013 3165, 2014 36. Lin H, Liu AP, Smith TH, Trejo J: Cofactoring and dimerization of 57. Trifilieff P, Rives ML, Urizar E, Piskorowski RA, Vishwasrao HD, Castrillon proteinase-activated receptors. Pharmacol Rev 65: 1198–1213, 2013 J, Schmauss C, Slättman M, Gullberg M, Javitch JA: Detection of

J Am Soc Nephrol 28: 2618–2630, 2017 Thrombin-Induced Podocyte Injury 2629 BASIC RESEARCH www.jasn.org

antigen interactions ex vivo by proximity ligation assay: Endogenous 66. Agrawal S, Chanley MA, Westbrook D, Nie X, Kitao T, Guess AJ, dopamine D2- complexes in the striatum. Bi- Benndorf R, Hidalgo G, Smoyer WE: Pioglitazone enhances the bene- otechniques 51: 111–118, 2011 ficial effects of glucocorticoids in experimental nephrotic syndrome. Sci 58. Lowder MA, Appelbaum JS, Hobert EM, Schepartz A: Visualizing pro- Rep 6: 24392, 2016 tein partnerships in living cells and organisms. Curr Opin Chem Biol 15: 67. Manders EMM, Verbeek FJ, Aten JA: Measurement of co-localization 781–788, 2011 of objects in dual-colour confocal images. JMicrosc169: 375–382, 59. Zhang C, Srinivasan Y, Arlow DH, Fung JJ, Palmer D, Zheng Y, Green 1993 HF, Pandey A, Dror RO, Shaw DE, Weis WI, Coughlin SR, Kobilka BK: 68. Agrawal S, Guess AJ, Chanley MA, Smoyer WE: Albumin-induced po- High-resolution crystal structure of human protease-activated receptor docyte injury and protection are associated with regulation of COX-2. 1. Nature 492: 387–392, 2012 Kidney Int 86: 1150–1160, 2014 60. Arachiche A, Mumaw MM, de la Fuente M, Nieman MT: Protease- 69. Smoyer WE, Gupta A, Mundel P, Ballew JD, Welsh MJ: Altered ex- activated receptor 1 (PAR1) and PAR4 heterodimers are required for pression of glomerular heat shock protein 27 in experimental nephrotic PAR1-enhanced cleavage of PAR4 by a-thrombin. J Biol Chem 288: syndrome. JClinInvest97: 2697–2704, 1996 32553–32562, 2013 70. Boerries M, Grahammer F, Eiselein S, Buck M, Meyer C, Goedel M, 61. de la Fuente M, Noble DN, Verma S, Nieman MT: Mapping human Bechtel W, Zschiedrich S, Pfeifer D, Laloë D, Arrondel C, Gonçalves S, protease-activated receptor 4 (PAR4) homodimer interface to trans- Krüger M, Harvey SJ, Busch H, Dengjel J, Huber TB: Molecular fin- membrane helix 4. JBiolChem287: 10414–10423, 2012 gerprinting of the podocyte reveals novel and protein regulatory 62. Kang S, Hawkridge AM, Johnson KL, Muddiman DC, Prevelige PE Jr.: networks. Kidney Int 83: 1052–1064, 2013 Identification of subunit-subunit interactions in bacteriophage P22 71. Spears M, Cunningham CA, Taylor KJ, Mallon EA, Thomas JS, Kerr GR, procapsids by chemical cross-linking and mass spectrometry. J Pro- Jack WJ, Kunkler IH, Cameron DA, Chetty U, Bartlett JM: Proximity li- teome Res 5: 370–377, 2006 gation assays for isoform-specific Akt activation in breast cancer iden- 63. Hölschermann H, Bohle RM, Schmidt H, Zeller H, Fink L, Stahl U, Grimm tify activated Akt1 as a driver of progression. JPathol227: 481–489, H, Tillmanns H, Haberbosch W: Hirudin reduces tissue factor expres- 2012 sion and attenuates graft arteriosclerosis in rat cardiac allografts. Cir- 72. Spears M, Taylor KJ, Munro AF, Cunningham CA, Mallon EA, Twelves culation 102: 357–363, 2000 CJ, Cameron DA, Thomas J, Bartlett JM: In situ detection of HER2: 64. Dargaud Y, Luddington R, Gray E, Lecompte T, Siegemund T, Baglin T, HER2 and HER2:HER3 protein-protein interactions demonstrates Hogwood J, Regnault V, Siegemund A, Negrier C: Standardisation of prognostic significance in early breast cancer. Breast Cancer Res Treat thrombin generation test–Which reference plasma for TGT? An in- 132: 463–470, 2012 ternational multicentre study. Thromb Res 125: 353–356, 2010 65. Pippin JW, Brinkkoetter PT, Cormack-Aboud FC, Durvasula RV, Hauser PV, Kowalewska J, Krofft RD, Logar CM, Marshall CB, Ohse T, Shankland SJ: Inducible rodent models of acquired podocyte diseases. This article contains supplemental material online at http://jasn.asnjournals. Am J Physiol Renal Physiol 296: F213–F229, 2009 org/lookup/suppl/doi:10.1681/ASN.2016070789/-/DCSupplemental.

2630 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 2618–2630, 2017 Online Supplement

Thrombin-Induced Podocyte Injury is Protease Activated Receptor (PAR)-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

SUPPLEMENTAL METHODS

Antibodies, Peptides, and Reagents

Antibodies against PARs were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), as follows:

anti–PAR1 (human and rat: ATAP-2), anti–PAR2 (human and rat: SAM-11), anti–PAR3 (human: 8E8; rat: 8E8 or M-20), and anti–PAR4 (human and rat: H-120). For the human kidney section immunofluorescence, primary antibodies were goat anti-synaptopodin (P-19) sc-21537 (also from Santa Cruz); secondary antibodies were donkey anti-goat FITC (A16006) and donkey anti-rabbit TRITC (A25466), both from Novex (Thermo Fisher

Scientific Inc., Waltham, MA). Human PAR-activation peptides (AP) were purchased from Peptides

International (Louisville, KY, USA); Rat PAR-APs were purchased from Ohio Peptide, LLC (Columbus, OH,

USA). Specifically, human PAR-APs included PAR1 AP (TFFLRNPNDK), PAR2 AP (SLIGRL); PAR3 AP

(TFRGAP); PAR4 AP (AYPGKF) and a scrambled control peptide (FSLLRN).1, 2 Rat PAR-APs included PAR1

AP (SFFLRN), PAR3 AP (SFNGNE), PAR4 AP (GFPGKP).3 Rat PAR2 AP was the same as that for human

PAR2 AP. Rabbit polyclonal thrombin antibody (bs-1914R; raised against the thrombin heavy chain) was

purchased from Antibodies-Online, Inc. (Atlanta, GA, USA). Mouse monoclonal synaptopodin (SYNPO)

antibody (10R-S125A) was purchased from Fitzgerald Industries International (Acton, MA, USA). Anti-rabbit

IgG conjugated to Alexa Flour 594 and anti-mouse IgG Alexa Flour 488) were purchased from Invitrogen

(Carlsbad, CA, USA). Human α-thrombin was purchased from MP Biomedicals LLC (Santa Ana, CA, USA).

Human plasma-derived AT (Kybernin® P) was a kind gift from CSL Behring (Marburg, Germany). Hirudin was

purchased from Bachem Americas, Inc. (Torrance, CA). Alexa Fluor 488 Phalloidin was purchased from

Molecular Probes, Inc. (Eugene, OR, USA). FITC and APC antibody conjugation kits were purchased from

Abcam (Cambridge, MA, USA). Matched, isotype controls for flow cytometry were as follows: mouse IgG1-

FITC, mouse IgG1-APC, mouse IgG2a-APC, mouse IgG2b-APC, and rabbit IgG-APC; matched to the SYNPO and PAR1 – PAR4 antibodies, respectively (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

Urine Analysis

Urine protein-to-creatinine ratios were determined by Antech GLP (Morrisville, NC) using standard techniques

that are fully compliant with the Good Laboratory Practice regulations and other regulatory requirements, as

previously described and are reported as mg protein/mg creatinine (mg/mg).4

Enzyme-Linked Immunosorbent Assays

Plasma and urinary concentrations of thrombin-antithrombin (TAT) complex (Enzygnost; Siemens, Tarrytown,

NY, USA) and prothrombin fragment 1.2 (F1.2; My Biosource, San Diego, CA, USA) were measured by ELISA

assay kits, validated for use in the rat, as previously described.4

Cell Culture

Conditionally immortalized human and rat podocyte cell lines, which have recently been comprehensively

characterized and verified to be pathogen free, were cultured as previously described.5-8 In brief, human

podocytes were grown on plates coated with collagen type 1 at 33°C. Under these conditions, podocytes

proliferate and remain undifferentiated; differentiation was subsequently induced by incubation at 37°C.

Experiments were performed after 14 days of differentiation, which was confirmed by determining expression

of SYNPO and Nephrin (NPHS1) mRNA.6 Rat podocytes were grown in Dulbecco’s modified Eagle’s/F-12

media containing 10% fetal bovine serum (Fisher, USA), penicillin (100 U/ml), streptomycin (100 mg/ml), and

0.1% Insulin-Trans-Cel-X (Gibco BioChemical, Grand Island, NY, USA) at 33 °C. They were differentiated

under ‘restrictive conditions’ at 37°C in 5% CO2 for ≥4 days. Differentiation was confirmed by demonstrating presence of SYNPO mRNA.6 All experiments were performed on differentiated human and rat podocytes.

RNA Extraction and Polymerase Chain Reaction

Podocytes were lysed in RLT buffer (Qiagen,Valencia, CA) containing 1% β-Mercaptoethanol. Total RNA and

subsequently cDNA were isolated from podocytes using the RNeasy kit (Qiagen) and Protoscript First Strand

cDNA Synthesis kit (New England Biolabs; Ipswich, MA), respectively, according to the manufacturer’s

instructions. Purity and yield of RNA and cDNA were confirmed by measuring the absorbance at 260 and

280 nm. cDNA was amplified by polymerase chain reaction (PCR); 0.5 μL cDNA (diluted in a total volume of 11 μL RNase-free H2O), 0.5 uL forward (fwd) and reverse (rvs) primers (below), and 12.5 uL HotStarTaq Plus

Master Mix (Qiagen). Primers were obtained from Invitrogen (Carlsbad, CA). Primers were obtained from

Invitrogen (Carlsbad, CA). PCR was performed as follows: 1 cycle at 95 °C for 5 min, 40 cycles at 94 °C for

30 sec and 55 °C for 30 sec and 72 °C for 30 sec, with a final extension of 72 °C for 10 min. PCR products were electrophoresed on a 1.8% agarose gel, and visualized using a Universal Hood II GelDoc and accompanying

Quantity One 1-D analysis software (Bio-Rad; Hercules, CA).

Human Podocyte primer pairs (5’ → 3’):

β-actin (ACTB): (fwd) ACAGAGCCTCGCCTTTGCCG; (rvs) ACAGAGCCTCGCCTTTGCCG

Synaptopodin (SYNPO): (fwd) AGAGTGGCCCAGAAACCAG; (rvs) TGGCTCTCCAAGGTGAACTC

Nephrin (NPHS1): (fwd) CCCATGGAGGAGACAGTCAT; (rvs) ACGTTCAGGATGAGCGACTT

PAR1 (F2R): (fwd) CCTGGCTGACACTCTTTGTCC; (rvs) ACTGCCGGAAAAGTAATAGCTG

PAR2 (F2RL1): (fwd) TTCCCAGCC TTCCTCACAG; (rvs) TCTTTGAGGTGAGGGATAC

PAR3 (F2RL2): (fwd) TCCTGGTGTTTGTAGTTGGTGT; (rvs) CCAGTTGTTCCCATTGAGATGA

PAR4 (F2RL3): (fwd) CTGGGCAACCTCTATGGTG; (rvs) GCACCTTGTCCCTGAACTCG

Rat Podocyte primer pairs (5’ → 3’):

β-actin (Actb): (fwd) CGGTCCACACCCGCCACC; (rvs) CTTGCTCTGGGCCTCGTCGC

Synaptopodin (Synpo): (fwd) CAAACCCAACACTCCACGCG; (rvs) TGCATGCCAATGAGCAGAGA

PAR1 (F2r): (fwd) AATTGGCAAGGGAGGGGATG; (rvs) CGGTTTAGCTGATAGGCCGT

PAR2 (F2rl1): (fwd) GAACGAAGAAGAAGCACC; (rvs) GGAACAGAAAGACTCCAATG

PAR3 (F2rl2): (fwd) ACAGCTGCGTAGACCCTTTC; (rvs) TAATGAAGGTCGCGCCAAGT

PAR4 (F2rl3): (fwd) GCTGCGTAGACCCTTTCATC; (rvs) AGGGTTCAGGAGGGACAGTT

Western Blotting

Western Blot experiments were performed with human and rat podocyte protein lysates for the detection of

PAR protein expression. Adherent human and rat podocytes were washed with ice-cold phosphate buffered saline (PBS) and lysed with 450 µL MPER lysis buffer (Thermo Fisher Scientific Inc., Waltham, MA) containing protease and phosphatase inhibitor cocktails (Fisher). Protein concentration of lysates was determined using

the bicinchoninic acid (BCA) protein assay reagent (Fisher). Appropriate amounts of protein (30-60 µg) were

separated by 10% SDS-PAGE and transferred to PVDF membranes, blocked for 1 h with 5% milk in PBS-T

(PBS containing 0.1% Tween 20), and incubated with desired PAR primary antibodies (1:1000 in 2% PBS blotto) overnight at 4°C. Membranes were washed with TBST and incubated with anti-mouse IgG (1:2000), anti-rabbit IgG (1:10000), or anti-goat (1:5000) horseradish peroxidase conjugated antibodies, as appropriate.

Proteins were detected on x-ray film using the ECL chemiluminescence reagent (Fisher), and the density of each band was quantified using Image J software. Accuracy of protein loading was confirmed by GAPDH

(1:10000; Millipore) Western Blot.

For the co-immunoprecipitation assays, BS3 (bis(sulfosuccinimidyl)suberate; Thermo Fisher), a water soluble covalent protein linker, was utilized during podocyte thrombin exposure to enhance interaction stability during the aqueous phases of the protocol. Thrombin-dependent PAR homo- and hetero-dimerization is dependent upon interactions between hydrophobic PAR transmembrane domains.9, 10 Similarly, BS3 has thus been utilized to enhance oligomer detection of another G protein-coupled receptor (M2 Muscarinic Cholinergic

Receptor).11 Briefly, thrombin (20 nM) was added to differentiated podocyte cultures at time zero, 2.5 minutes later 2 mM BS3 (bis(sulfosuccinimidyl)suberate; Thermo Fisher) was added, followed by protein lysate preparation at 5 minutes. PAR antibodies were adsorbed to sepharose beads for the respective pull-down conditions and incubated overnight with 500 µg of total protein lysates. Beads were then washed 5 times in

RIPA buffer, resolved in sample buffer, and boiled for 5 minutes at 95 °C for elution of bound protein complexes, which were subjected to Western Blot for the respective target PARs using the conditions

described above. The membranes were subsequently stripped and re-blotted with the respective pull-down antibodies to confirm appropriate immunoprecipitation (data not shown).

Antibodies against total and phosphorylated ERK1/2 MAPK (137F5 and 20G11, respectively) were purchased

from Cell Signaling Technology (Danvers, MA). Western Blot experiments were performed as above on human

and rat podocyte protein lysates obtained following exposure to thrombin with or without PAR antibody

blockade. Total protein (30 µg) was loaded onto 12% gels. Total and phosphorylated ERK1/2 primary antibodies were used at 1:1000 in 2% PBS blotto overnight at 4°C. Membranes were washed with TBST and

incubated with anti-rabbit IgG (1:5000) horseradish peroxidase conjugated antibody. Relative levels of protein

were quantified by optical density analysis using ImageJ open source software (National Institutes of Health;

Bethesda, MD).

Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assays

Differentiated podocytes were serum-starved overnight in serum-free medium. After 36 hours of exposure to

thrombin or PAR activation peptides (AP), podocytes were fixed in 4% neutral buffered formalin, washed in

PBS, and injury was determined using TUNEL assay (Roche Diagnostics; Indianapolis, IN, USA), according to

the manufacturer’s directions and as previously described.1, 12 Briefly, podocytes were incubated with terminal deoxynucleotidyl transferase in the presence of fluorescein-labeled dUTP (60 minutes at 37 °C) and counterstained with Hoechst 33258 (3.5 µg/mL). Random images were captured with a Ziess 510 META confocal microscope (Zeiss; Thornwood, NY, USA) coupled to a Zeiss LSM 700, and the frequency (%) of

TUNEL positive podocytes was determined by a blinded investigator (RS). Varying concentrations of thrombin

(from 0 – 50 nM) were used to evaluate thrombin-mediated exacerbation of PAN-induced podocyte injury.

Thrombin (20 or 30 nM) without PAN was evaluated for PAN-independent podocyte injury. Subsequent experiments were conducted using 20 nM thrombin (final concentration), which was added every 12 hours.12

For anti-PAR experiments, podocytes were pretreated with anti–PAR antibodies (10 µg/mL) for 30 minutes before thrombin exposure.1 In separate experiments, podocytes were exposed to PAR APs and scrambled

(control) peptide at 20 µM (final concentration).1

Viability and F-actin Rearrangement Assays

Podocyte viability was assessed using the MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide;

Sigma) assay, as described previously.13 Briefly, podocytes were differentiated in 6-well plates, serum starved

overnight, then exposed to thrombin (20 nM) for 48 hours in the presence or absence of pan-caspase inhibitor

(Z-VAD-FMK, R&D Systems Inc., Minneapolis, MN, USA) pretreatment (40 µM). Afterward, the podocytes

were incubated with MTT (500 μg/ml) for 4 hours at 37 °C. MTT formazan crystals were extracted in dimethyl

sulfoxide containing 0.01 M glycine and absorbance was measured at 570 nm on a SpectraMax M2 plate reader (Molecular Devices, Sunnyvale, CA). Control cells (100% viable) were not exposed to Z-VAD-FMK or thrombin, whereas Triton X-100 treated cells were used as negative controls (0% viable). Each experiment consisted of 3 wells per treatment and experiments were repeated 5-11 times. In preliminary experiments, 40

µM Z-VAD-FMK was determined to be the minimal concentration necessary to maximally inhibit caspase-3

activity in differentiated human podocytes exposed to PAN (30 µg/mL; data not shown).

To assess F-actin cytoskelatal rearrangement, differentiated podocytes were grown on coverslips. Following

exposure to thrombin (20 nM) for 72 hours, the coverslips were stained with phalloidin and DAPI. Images of

random individual cells that were well spaced from their neighbors were collected with a BZ900 inverted

confocal microscope. Each cell was subsequently scored as follows: (a) Filament thickness: uniform thickness

(1 point); mixture of thick and thin filaments (2 points), predominantly thin filaments (3 points); (b) Filament pattern: organized (0 points), disarrayed (1 point); (c) Filament condensation: none (0 points), condensed

clumps or clouds (1 point). Thus, each cell was scored on a 5 point scale.

1. Madhusudhan, T, Wang, H, Straub, BK, Grone, E, Zhou, Q, Shahzad, K, Muller-Krebs, S, Schwenger, V, Gerlitz, B, Grinnell, BW, Griffin, JH, Reiser, J, Grone, HJ, Esmon, CT, Nawroth, PP, Isermann, B: Cytoprotective signaling by activated protein C requires protease-activated receptor-3 in podocytes. Blood, 119: 874-883, 2012. 2. Henriksen, RA, Hanks, VK: PAR-4 agonist AYPGKF stimulates thromboxane production by human platelets. Arteriosclerosis, thrombosis, and vascular biology, 22: 861-866, 2002. 3. Wang, H, Ubl, JJ, Reiser, G: Four subtypes of protease-activated receptors, co-expressed in rat astrocytes, evoke different physiological signaling. Glia, 37: 53-63, 2002. 4. Kerlin, BA, Waller, AP, Sharma, R, Chanley, MA, Nieman, MT, Smoyer, WE: Disease Severity Correlates with Thrombotic Capacity in Experimental Nephrotic Syndrome. Journal of the American Society of Nephrology : JASN, 26: 3009-3019, 2015. 5. Saleem, MA, O'Hare, MJ, Reiser, J, Coward, RJ, Inward, CD, Farren, T, Xing, CY, Ni, L, Mathieson, PW, Mundel, P: A conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression. Journal of the American Society of Nephrology : JASN, 13: 630-638, 2002. 6. Chittiprol, S, Chen, P, Petrovic-Djergovic, D, Eichler, T, Ransom, RF: Marker expression, behaviors, and responses vary in different lines of conditionally immortalized cultured podocytes. American journal of physiology Renal physiology, 301: F660-671, 2011. 7. Eto, N, Wada, T, Inagi, R, Takano, H, Shimizu, A, Kato, H, Kurihara, H, Kawachi, H, Shankland, SJ, Fujita, T, Nangaku, M: Podocyte protection by darbepoetin: preservation of the cytoskeleton and nephrin expression. Kidney international, 72: 455-463, 2007. 8. Yaoita, E, Kurihara, H, Sakai, T, Ohshiro, K, Yamamoto, T: Phenotypic modulation of parietal epithelial cells of Bowman's capsule in culture. Cell and tissue research, 304: 339-349, 2001. 9. Arachiche, A, Mumaw, MM, de la Fuente, M, Nieman, MT: Protease-activated receptor 1 (PAR1) and PAR4 heterodimers are required for PAR1-enhanced cleavage of PAR4 by alpha-thrombin. The Journal of biological chemistry, 288: 32553-32562, 2013. 10. de la Fuente, M, Noble, DN, Verma, S, Nieman, MT: Mapping human protease-activated receptor 4 (PAR4) homodimer interface to transmembrane helix 4. The Journal of biological chemistry, 287: 10414-10423, 2012. 11. Redka, DS, Pisterzi, LF, Wells, JW: Binding of orthosteric ligands to the allosteric site of the M(2) muscarinic cholinergic receptor. Molecular pharmacology, 74: 834-843, 2008. 12. Wang, H, Madhusudhan, T, He, T, Hummel, B, Schmidt, S, Vinnikov, IA, Shahzad, K, Kashif, M, Muller- Krebs, S, Schwenger, V, Bierhaus, A, Rudofsky, G, Nawroth, PP, Isermann, B: Low but sustained coagulation activation ameliorates glucose-induced podocyte apoptosis: protective effect of factor V Leiden in diabetic nephropathy. Blood, 117: 5231-5242, 2011. 13. Agrawal, S, Guess, AJ, Benndorf, R, Smoyer, WE: Comparison of direct action of thiazolidinediones and glucocorticoids on renal podocytes: protection from injury and molecular effects. Molecular pharmacology, 80: 389-399, 2011.

A B

Supplemental Figure 1: During PAN-Induced Rat Nephrosis Urinary Markers of Thrombin Activation and Regulation Increase. At day 9 of PAN-nephrosis, urinary byproducts of thrombin activation, F1.2 (A), and regulation, thrombin-antithrombin complex (TAT; B), increase directly with proteinuria severity while plasma levels remain stable (n=30 rats administered varying PAN doses 0-150 mg/kg). #P<0.05 vs. control; ^P<0.001 vs. control. SYNPO PAR4 Merge *

* *

*

Inset

Supplemental Figure 2: Human Podocytes Express PAR4 In Vivo. Immunofluorescent histology of representative human glomeruli (60x) demonstrating predominantly cell body (asterisks) colocalization (yellow) of synaptopodin (green; FITC) with PAR4 (orange; TRITC) on podocytes, whereas glomerular endothelial cells (arrow heads) express only PAR4 (orange;TRITC); representative of 4 tissue sections with 1-2 glomeruli per section from normal adult human kidney sections. A

B

Supplemental Figure 3: Thrombin Alters PAN-Induced Human Podocyte Injury in a Concentration-Dependent Manner with Maximal Effects at 20 nM. (A) Thrombin diminishes podocyte injury at low concentration (50 pM) and exacerbates injury at concentrations ≥20 nM. (B) Thrombin induces podocyte injury, independently of PAN at concentrations ≥20 nM (#P<0.05, αP<0.01, ^P<0.001 vs control; ωP<0.05, φP<0.01 vs PAN; n=3-4 per condition). C

Control

A B Thrombin

* *

*

Hirudin * Thrombin +

Supplemental Figure 4: Thrombin-Induced Human Podocyte Injury. (A) Cell viability (MTT assay) is reduced by thrombin (20 nM) and is not rescued by pancaspase-inhibition (40 µM Z-VAD-FMK; #P<0.05; n=5-9 per condition). (B) Phalloidin staining scores of individual cells (symbols) and mean ± SEM showing F-actin cytoskeletal rearrangement by thrombin (20 nM), partially rescued by hirudin (n=55-75 cells per condition; ^P<0.001). (C) Representative phalloidin stained cells: (Top) Control cell with well-organized, mixed fiber thickness; score=2, (Middle) Thrombin-exposed cell with thin, organized fibers with condensed clouds (arrows); score=4, (Bottom) Thrombin+Hirudin-exposed cell with organized, mixed thickness fibers and clumps (asterisks); score=3. C

* Control * *

A B

* Thrombin *

Hirudin Thrombin +

Supplemental Figure 5: Thrombin-Induced Rat Podocyte Injury. (A) Cell viability (MTT assay) is reduced by thrombin (20 nM) and is not rescued by pancaspase-inhibition (40 µM Z-VAD-FMK; #P<0.05; n=9-11 per condition). (B) Phalloidin staining scores of individual cells (symbols) and mean ± SEM showing F-actin cytoskeletal rearrangement by thrombin (20 nM), partially rescued by hirudin (n=64-91 cells per condition; ^P<0.001). (C) Representative phalloidin stained cells: (Top) Control cell with organized, normal fiber thickness and condensed clumps (asterisk); score=2, (Middle) Thrombin-exposed cell with a mixed thickness, disorganized fibers and condensed clumps (asterisk) & clouds (arrows); score=4, (Bottom) Thrombin+Hirudin-exposed cell with thin, organized fibers; score=3. Supplemental Figure 6: Thrombin Induces Maximal PAR3/PAR4 Interactions in Human Podocytes at 5 Minutes. Mean ± SEM number of proximity signals per nucleus over time with representative images in upper panels (#P<0.05, ^P<0.001 vs. control; n=3 per condition).