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Podocyte Integrin-b3 and Activated Protein C Coordinately Restrict RhoA Signaling and Ameliorate Diabetic Nephropathy

Thati Madhusudhan,1,2 Sanchita Ghosh,1,3 Hongjie Wang,1,4 Wei Dong,1 Dheerendra Gupta,1,3 Ahmed Elwakiel,1,3 Stoyan Stoyanov,5 Moh’d Mohanad Al-Dabet,1,3,6 Shruthi Krishnan,1,3 Ronald Biemann,1,3 Sumra Nazir,1 Silke Zimmermann,1,3 Akash Mathew ,1,3 Ihsan Gadi,1,3 Rajiv Rana,1,3 Jinyang Zeng-Brouwers,7 Marcus J. Moeller,8 Liliana Schaefer,7 Charles T. Esmon,9 Shrey Kohli,1,3 Jochen Reiser,10 Alireza R. Rezaie,11 Wolfram Ruf,2,12 and Berend Isermann1,3

Due to the number of contributing authors, the affiliations are listed at the end of this article.

ABSTRACT Background Diabetic nephropathy (dNP), now the leading cause of ESKD, lacks efficient therapies. Coagulation protease–dependent signaling modulates dNP, in part via the G protein–coupled, protease-activated receptors (PARs). Specifically, the cytoprotective protease-activated protein C (aPC) protects from dNP, but the mechanisms are not clear. Methods A combination of in vitro approaches and mouse models evaluated the role of aPC-integrin interaction and related signaling in dNP.

Results The zymogen protein C and aPC bind to podocyte integrin-b3, a subunit of integrin-avb3.Deficiency of this integrin impairs thrombin-mediated generation of aPC on podocytes. The interaction of aPC with integrin- avb3 induces transient binding of integrin-b3 with Ga13 and controls PAR-dependent RhoA signaling in podo- cytes. Binding of aPC to integrin-b3 via its RGD sequence is required for the temporal restriction of RhoA signaling in podocytes. In podocytes lacking integrin-b3, aPC induces sustained RhoA activation, mimicking the effect of thrombin. In vivo, overexpression of wild-type aPC suppresses pathologic renal RhoA activation and protects against dNP. Disrupting the aPC–integrin-b3 interaction by specifically deleting podocyte integrin-b3 or by abolishing aPC’s integrin-binding RGD sequence enhances RhoA signaling in mice with high aPC levels and abolishes aPC’s nephroprotective effect. Pharmacologic inhibition of PAR1, the pivotal thrombin receptor, restricts RhoA activation and nephroprotects RGE-aPChigh and wild-type mice.

Conclusions aPC–integrin-avb3 acts as a rheostat, controlling PAR1-dependent RhoA activation in podo- cytes in diabetic nephropathy. These results identify integrin-avb3 as an essential coreceptor for aPC that is required for nephroprotective aPC-PAR signaling in dNP.

JASN 31: ccc–ccc, 2020. doi: https://doi.org/10.1681/ASN.2019111163

Diabetic nephropathy (dNP) is now the leading cause Received November 11, 2019. Accepted April 30, 2020. 1 of ESKD in industrialized countries. Early disease M.T., S.G., and H.W. contributed equally to this work. stages of dNP are in principle reversible,2 yet specific Published online ahead of print. Publication date available at medical approaches exploiting this therapeutic win- www.jasn.org. dow are scarce. Recent findings have established that coagulation proteases and their receptors modulate Correspondence: Dr. Madhusudhan Thati or Prof. Berend Iser- mann, Institute of Laboratory Medicine, Clinical Chemistry and dNP. Deciphering the underlying mechanism may Molecular Diagnostics, University Hospital Leipzig, Liebigstr. offer new therapeutic strategies for this condition. 27A, 04103 Leipzig, Germany E-mail: [email protected] or Beyond controlling hemostasis, coagulation [email protected] proteases modulate cellular homeostasis via Copyright © 2020 by the American Society of Nephrology

JASN 31: ccc–ccc, 2020 ISSN : 1046-6673/3108-ccc 1 BASIC RESEARCH www.jasn.org receptor-dependent mechanisms in a highly context-specific Significance Statement fashion.3–5 In particular, the cytoprotective effects of the anticoagulant serine protease-activated protein C (aPC) Signaling to integrins is complex and depends on ligands and seem appealing, because these effects and the anticoagulant their binding sites. Signaling-competent integrin ligands that effects of aPC depend largely on disjunct molecular structures protect podocyte function remain unknown. This study demon- strates that the coagulation protease-activated protein C (aPC) fi 4,6 and can hence be speci cally exploited. Accordingly, aPC binds via its RGD sequence to podocyte integrin-b . Disruption of fi 3 variants with reduced anticoagulant ef cacy are being inves- the aPC–integrin-b3 interaction results in excess RhoA activation and tigated in clinical stroke trials, and small compounds called podocyte dysfunction. These findings identify the RGD-mediated – b parmodulins that mimic biased aPC signaling via the pivotal G aPC integrin- 3 interaction as a rheostat of RhoA signaling, which protein–coupled aPC receptor, protease-activated receptor 1 is disrupted in diabetic nephropathy. Protease-activated receptor 1 (PAR1) antagonism could ameliorate excess RhoA signaling in (PAR1), have been developed.6–8 theabsenceofaPC–integrin-b3 interaction. These data identify a Activation of the zymogen protein C (PC) by the new function of podocyte integrin-b3 and provide a mechanistic thrombomodulin-thrombin complex is enhanced by the cor- rationale for PAR antagonism as a therapeutic approach for eceptor endothelial PC receptor (EPCR) on endothelial cells. diabetic nephropathy. In addition, EPCR promotes biased signaling of aPC via PAR1 9–13 on endothelial cells. However, aPC also conveys cytopro- The following inhibitors, agonists, and antagonists were — tective effects in extravascular compartments e.g.,theskin, used in this study: RGDS, Cyclo-RGDfv, human thrombin, brain, and kidneys, targeting keratinocytes, neurons, and renal and SCH79797 (Sigma). Other reagents included DMEM, 14–16— epithelial cells (podocytes, tubular cells), respectively in trypsin-EDTA,penicillin,streptomycin,FCS,FBS,and 15,17–19 a manner partially independent of EPCR. aPC HEPES from Thermo Fisher Scientific (Dreieich, Germany); fi signaling on podocytes depends on species-speci cPARhet- BCA reagent from Perbio Science (Bonn, Germany); VECTA- erodimers (PAR2/PAR3 on human podocytes and PAR1/PAR3 SHIELD mounting medium with 49,6-diamidino-2-phenylin- on mouse podocytes), but appears to be independent of dole (DAPI), antigen-unmasking solution (Tris-based), and 15 EPCR. It remains unknown whether receptors other than an M.O.M. kit from Vector Laboratories; RhoA and Rac1 pull- PARs are required for aPC-mediated signaling in podocytes. down assays from Cytoskeleton, Inc.; polyvinylidene difluoride In addition to PARs and EPCR, various other receptors membranes and Immobilon Western Chemiluminescent HRP fi 20,21 interacting with aPC have been identi ed. aPC binds Substrate from Merck Millipore; streptozotocin (STZ) from b -integrins via its RGD sequence, regulating the adhesion of Enzo Life Sciences (Lörrach, Germany); Accu-Chek test strips, 19,22 neutrophils and macrophages. Apotentialroleofan an Accu-Check glucometer, and protease inhibitor cocktail from integrin-aPC interaction on podocytes appears plausible given Roche Diagnostics (Mannheim, Germany); albumin fraction V, ’ 15,23,24 aPC s nephroprotective effects and the pivotal func- Gill II hematoxylin, acrylamide, agarose, Periodic acid–Schiff 25,26 tions of integrins in podocytes. The functions of podo- (PAS) reagent, and ROTIHistokitt synthetic mounting medium cyte integrins appear to be twofold, because both integrin from Carl Roth (Karlsruhe, Germany); and aqueous mounting absence/integrin defects and excess integrin activation promote medium from Zytomed (Berlin, Germany). Wild-type aPC 27,28 b renal pathologies. Because podocytes express integrin- 1 and RGE-aPC were expressed and purified as described b 25 and integrin- 3, we hypothesized that aPC interacts via its previously.22,29 RGD sequence with podocyte integrin-b1 and/or integrin-b3 to modulate podocyte function. Animals d d APChigh and EPCR / mice have been previously de- 10,30 LoxP/LoxP scribed. Integrin-b3 mice (provided by Katherine METHODS Weilbaecher) were crossed with PodCre mice to generate mice 31,32 with podocyte-specific deletion of integrin-b3. All mice Reagents were backcrossed onto the C57BL/6J background for at least The following antibodies were used in this study: antibodies eight generations and were routinely maintained on the against integrin-b1 (sc-59827) and Ga13 (sc-410) from Santa C57BL/6J background. Only littermates were used as controls Cruz Biotechnology; antibodies against mouse nephrin in this study, and the mice were randomly assigned to the (AF3159) from R&D Systems; antibodies against integrin-b3 experimental groups. The presence of targeted genes and (13166), RhoA (2117), Rac1 (4651), and anti-rabbit horserad- transgenes was routinely determined by PCR analyses of tail ish peroxidase (HRP)–conjugated secondary antibody from DNA. Wild-type C57BL/6, db/dm, and db/db (C57BL/KSJRj-db) Cell Signaling Technology; antibodies against active- mice were obtained from Janvier (Le Genest-Saint-Isle, France). conformation integrin-b3 AP5 (EBW107) from Kerafast, In some mice, we intraperitoneally injected aPC (1 mg/kg body Inc.; antibodies against PC (ab36407) and WT-1 (ab89901) wt, every alternate day) or an equal volume of PBS starting at from Abcam; and an antibody against human PC (PA5-28321; 18 weeks of age until 1 day before the analyses.24,33 Mice were Thermo Fisher Scientific). The mAb HAPC1573 has been de- analyzed at age 24 weeks. The animal experiments were con- scribed previously.8 ducted following standards and procedures approved by the

2 JASN JASN 31: ccc–ccc,2020 www.jasn.org BASIC RESEARCH local Animal Care and Use Committee (Landesverwaltungsamt which to study therapeutic approaches in early dNP. Sub- Halle, Germany). groups of mice received various interventions. We intraper- itoneally injected the mice in the different subgroups with Generation of RGE-APChigh Mutant Mice the PAR1 antagonist SCH79797 (15 mg/kg), Cyclo-RGDfv To generate mice expressing a human hyperactivatable PC (0.5 or 2 mg/kg body wt, dissolved in PBS), or PBS once daily (PROC) mutant with an additional mutation in the RGD se- starting 10 weeks after the last STZ injection until 1 day before quence (RGE-APChigh mice), the same construct previously the analyses (week 24).35–39 used to generate APChigh mice was used. A point mutation (D222E) was generated by site-directed mutagenesis using Determination of Albuminuria the QuikChange mutagenesis kit (Agilent, Santa Clara, CA; Mouse urine albumin and creatinine were measured as pre- Supplemental Figure 1A). Correct mutagenesis was confirmed viously described.10,40,41 by the presence of a newly generated BanII site resulting in a 521-bp and a 203-bp fragment upon BanII digestion of a PCR Histology and PAS Staining amplimer (using the primers P1, 59-CAA GCC GGT TTA CTC Tissues were processed as previously described.10,15 Sections TGA CCC-39,andP2,59-CCT CTATGC ACT CCC GCT CCA (5 mm thick) were used for histologic analyses. At least 50 dif- GGC-39). Before pronucleus injection, correct mutagenesis ferent superficial glomeruli per mouse were randomly chosen was additionally confirmed by sequencing. Transgenic mice for analysis. Fractional mesangial area (FMA) was calculated were generated in the transgenic core facility at the medical as the percentage of PAS-positive area in the glomerular tuft faculty of Heidelberg University. The transgenic RGE-APChigh area.33 All histologic analyses were conducted by a blinded mice were identified by PCR using the abovementioned pri- investigator. mers, P1 and P2, followed by BanII digestion of the amplimer (Supplemental Figure 1B). Transgenic RGE-APChigh mice ap- Immunohistochemistry peared grossly normal, reproduced normally, and did not show Animals were perfused with ice-cold PBS and then with 4% any signs of spontaneous bleeding. buffered paraformaldehyde (PFA). Tissues were fixed in 4% Based on expression analyses using semiquantitative RT-PCR, buffered PFA for 2 days, embedded in paraffin, and processed the transgene is expressed predominantly in the liver but also in for sectioning. Double immunofluorescence of active the brain and at very low levels in the kidney (Supplemental integrin-b3 (AP5) and nephrin was performed on a subset Figure 1C), which is consistent with the previously reported ex- of mouse renal tissue sections as previously described.26,42 pression pattern of a hyperactivatable D167F/D172K PC mutant WT-1 immunohistochemistry was performed on mouse renal using the same promoter.10 tissue sections to estimate podocyte loss. In both cases, sec- The levels of APC in mice were determined using a capture tions were fixed in ice-cold acetone for 1 minute after antigen assay with the antibody HAPC1555, which is highly specificfor retrieval in Tris-based antigen-unmasking solution, incubated activated human PC and does not detect mouse (activated) PC.34 in PBS (containing 0.025% Tween 20) for 5 minutes, and The mean plasma level of aPC was 5.3 ng/ml in RGE-APChigh blocked in M.O.M. blocking solution (as applicable) accord- mice, quite comparable to the values determined in parallel in ing to the manufacturer’s instructions for 1 hour. The slides APChigh mice (5.8 ng/ml; Supplemental Figure 1D). The total were then incubated with primary antibodies against active blood volume loss after a standardized tail injury was markedly integrin-b3 (AP5; 1:100) and nephrin (1:250) or WT-1 greater in RGE-APChigh mice (102611 ml) than in wild-type (1:200) for 48 hours at 4°C. The slides were rinsed three times littermates (4.561.2 ml), but did not differ significantly from in PBS (containing 0.025% Tween 20). The sections were then that observed in APChigh mice (98631 ml; Supplemental incubated with corresponding fluorescently labeled secondary Figure 1E), establishing that the mutant hyperactivatable antibodies (AP5 and nephrin) or anti-rabbit HRP-conjugated RGE-PC mutant has an anticoagulant effect comparable to secondary antibody (WT-1) for 120 minutes before again be- that of hyperactivatable RGD-PC. ing rinsed three times in PBS (containing 0.025% Tween 20). The slides were covered with VECTASHIELD mounting STZ Model and In Vivo Interventions in Mice medium containing the nuclear stain DAPI or hematoxylin Insulinopenia and persistent hyperglycemia were induced in counterstaining, and the specimens were analyzed on a Leica experimental mice using low-dose STZ as described previ- SP5 confocal microscope. WT-1–positive cells were counted ously.10 Blood glucose levels were determined at least weekly to estimate podocyte loss in each experimental group. For in mice to ensure persistent hyperglycemia. Mice displaying immunofluorescence, DAPI-stained and fluorescently labeled blood glucose levels .500 mg/dl received individual injections images were acquired separately. The exposure settings and of 1–2 U insulin glargine. Blood glucose levels shown within laser gain were kept the same for each condition. A total of the manuscript reflect the last blood glucose measurement 30 fields were acquired per condition, with a single focal plane before euthanizing the mice. The STZ model results in mild per field. The images were analyzed with ImageJ/Fiji. For albuminuria and thus reflects early stages of dNP,which are in quantification, each individual glomerulus was selected using principle reversible,2 making this an attractive model with the drawing tool (freeform). The images were separated into

JASN 31: ccc–ccc, 2020 Integrin-b3 and aPC Inhibit RhoA 3 BASIC RESEARCH www.jasn.org their individual channels, and a threshold was determined to HAPC1573 (1:1 ratio for 10 minutes under gentle agitation; select only the active integrin-b3 fluorescence that overlapped HAPC1573 specifically inhibits aPC’s anticoagulant function), with nephrin. The area, integrated intensity, and mean gray or RGE-aPC (20 nM).22,24 A standardized scratch injury was value were measured for each selected region of the glomerulus. generated using a sterile pipette tip and images of the scratch This was repeated for a selected region within the glomeruli with injury were obtained after 24 hours, as previously described.36 no active integrin staining and therefore with no fluorescence. The number of podocytes that migrated into the scratch were The fluorescence quantified for this region was considered to be counted. the background. The mean fluorescence intensity for active integrin-b3 was calculated by subtracting the background In Vitro aPC Generation Assay fluorescence from the integrated density of active integrin-b3 Cell-dependent PC activation was determined as described fluorescence. These steps were repeated for at least 30 glomeruli previously with modifications.10,46 Here, 250,000 differenti- per kidney section from three mice per group. ated mouse podocytes (control and integrin-b3 knockdown KD [integrin-b3 ]) or mouse trophoblast cells were resuspended Transmission Electron Microscopy in 20 mM HEPES-hydrochloride (pH 7.5) containing 0.1% Ultrastructural images of the glomerular filtration barrier BSA, 0.15 M sodium chloride (NaCl), 5 mM calcium chloride, were obtained by transmission electron microscopy as previ- and20mMbenzamidineandmixedat37°Cwitha25ml ously described.40 The thickness of the glomerular basement volume of the same buffer containing 1 mg of PC and 10 nM membrane (GBM) was analyzed using ImageJ software. For human bovine thrombin to initiate activation. The reactions each image, the basement membrane thickness was deter- were stopped at each defined time interval by taking a 10-ml mined at 15 adjacent and evenly distributed locations. Photo- aliquot of the cell suspension and mixing it with 60 U/ml micrographs of the GBM were analyzed for the density of tight hirudin. aPC concentrations were determined based on the slit pores between the podocyte foot processes using randomly amidolytic activity toward 1 mM Spectrozyme PCa substrate chosen electron micrographs of ten glomeruli per kidney for in 0.15 M NaCl, 5 mM calcium chloride, and 20 mM HEPES- each mouse. Tight slit pores were identified by the obliteration hydrochloride, pH 7.5. Substrate cleavage was measured in a of spaces between adjacent foot processes.24,43 The numbers BioTek microplate reader at 405 nm. of tight slit pores were counted and divided by the GBM length (mm) to determine the density of tight slit pores. A RhoA and Rac1 Pull-Down Assay total of 500 foot processes from each group were evaluated for RhoA and Rac1 activation assays using Rhotekin-RBD and the analysis of slit pore density. All histologic analyses were PAK-PBD affinity beads, respectively, were performed accord- conducted by a blinded investigator. ing to the manufacturer’s instructions (Cytoskeleton, Inc.). Conditionally immortalized human podocyte cultures were Cell Culture exposed to aPC (20 nM), thrombin (10 nM), or specific Conditionally immortalized mouse podocytes and human po- antagonists (RGDS, 50 mg/ml) for the desired period. The docytes were obtained from Prof. Jochen Reiser and cultured culture medium was aspirated, and the cells were rinsed twice as described elsewhere.44 The cell lines tested negative for with ice-cold PBS before ice-cold cell lysis buffer (50 mM Tris mycoplasma. Experiments were performed after 14 days of pH 7.5, 10 mM magnesium chloride, 0.5 M NaCl, and 2% differentiation. For immunoblotting analysis, cells were se- Igepal) was added. The cells were harvested using a cell rum starved for 8 hours before adding glucose for further scraper, and the cell lysates were immediately clarified by cen- 24 hours. Lysates were prepared at the desired time points trifugation at 10,000 3 g at 4°C for 1 minute. The protein after aPC or thrombin treatment. Murine trophoblast stem concentration in each sample was determined, and equal con- cells were cultured and differentiated as described centrations of protein (200 mg) were incubated for 1 hour at previously.45 4°C with Rhotekin-RBD beads. The lysates were pelleted by For in vitro AP5 immunocytochemical analyses, confluent centrifugation at 4000 3 g, and the supernatant was aspirated. differentiated podocytes were serum starved for 30 minutes The pellet with Rhotekin-RBD beads was resuspended in 20 ml and then stimulated with glucose (25 mM, 48 hours before the of 23 Laemmli buffer and boiled for 2 minutes. The active experiment; control, 5 mM glucose; osmotic control, 25 mM RhoA-GTP or Rac1-GTP and total RhoA or Rac1 in each sam- mannitol). Subsets of cells were treated with 20 nM aPC (for ple were quantified by Western blot analysis using anti-RhoA the last 3 hours).24 For cell migration assay (scratch assay), or anti-Rac1 monoclonal antibodies, respectively (Cell Signaling confluent differentiated podocytes were stimulated with pu- Technology). romycin aminonucleoside (PAN; 30 mg/ml, 30 minutes before the experiment; control, PBS) or with glucose (25 mM, Immunocytochemistry (Active Integrin-b3 Staining) 24 hours before the experiment; control, 5 mM glucose; Human podocytes were plated on collagen-coated glass cov- osmotic control, 25 mM mannitol). Subsets of cells were erslips and exposed to high glucose (25 mM) without or with pretreated (3 hours, then freshly added every 12 hours) with additional aPC (20 nM) on the day of differentiation. The aPC alone (20 nM), aPC preincubated with the antibody coverslips were washed with ice-cold 13 PBS and fixed in

4 JASN JASN 31: ccc–ccc,2020 www.jasn.org BASIC RESEARCH

3.7% PFA at room temperature for 15 minutes. The coverslips DMEM (Gibco), 10% FBS, and penicillin-streptomycin were washed (33 PBS, 0.3% Triton X-100, and 0.25% Tween (50 mg/ml and 50 mg/ml). HEK293T cells were seeded at a 20 for 10 minutes per wash) and blocked (5% donkey serum in density of 113106 cells per 15-cm2 dish 24 hours before trans- 13 PBS, 0.3% Triton X-100, and 0.25% Tween 20) for 1 hour. fection. Lentiviruses were produced by transfecting HEK293T The sections were then incubated (overnight at 4°C) with the cells with short hairpin RNA (targeting integrin-b3; the plasmids primary antibody AP5, which specifically recognizes the active were obtained from Dharmacon) together with two helper plas- conformation of integrin-b3.Afterwashing(33 PBS for mids (the packaging plasmid psPAX2 and the VSV-G–expressing 10 minutes per wash), the sections were incubated with sec- plasmid pMD2.g; both from Addgene). The transfections were ondary anti-mouse tetramethylrhodamine B isothiocyanate– carried out using the calcium-phosphate method (2 M calcium conjugated antibodies and FITC-conjugated phalloidin for chloride and 23 HEPES buffered saline, pH 7.4) with the 1 hour in the dark at 37°C. After washing with 13 PBS (33 plasmids in a 3:2:1 ratio (short hairpin RNA plasmid, for 10 minutes per wash in the dark), the coverslips were psPAX2:pMDg.2). The virus-containing medium was harves- mounted on glass slides and prepared for imaging using an ted 48 hours and 72 hours after transfection and subsequently LSM 700 Imager.M2 (Carl Zeiss AG, Jena, Germany). The precleaned with a 0.45-mm filter (Millipore) as described.47 exposure settings and laser gain were kept the same for each The virus-containing medium was mixed with 50% PEG condition. A total of 50 fields with z-stacks were acquired per 6000, 4 M NaCl, and 13 PBS (without calcium and magne- condition per coverslip. The images were analyzed with Im- sium) at volume percentages of 26%, 11%, and 12%, respec- ageJ/Fiji. For quantification, the images were separated into tively, and incubated on a shaker at 4°C for 4 hours. After their individual channels, and a threshold was determined to incubation, the solution was centrifuged at 1600 3 g at 4°C select only the active integrin-b3 fluorescence in the periphery for 60 minutes. After centrifugation, the supernatant was care- of the cells per z-stack. The nuclei (reflecting the number of fully removed without disturbing the viral pellet. Cell culture cells) were counted using the Point Picker plugin. The average medium was added to the pellet for resuspension, and the 50 ml integrated density was determined for every field. Falcon tubes were kept at 4°C in a shaker for recovery over- night. The viral particles were resuspended in 1/20th of In Situ Proximity Ligation Assay the original volume. The protocol for concentrating the viral The Duolink in situ proximity ligation assay (PLA) kit was particles was adapted from that of Kutner et al.48 The cells used according to the manufacturer’s instructions (Olink were treated with two rounds of the enriched lentivirus, and Biosciences, Sigma Aldrich). Briefly, paraffinmousekidney knockdown efficiency was ascertained after 4 days by sections were dewaxed and rehydrated followed by antigen immunoblotting. retrieval in Tris-based antigen-unmasking solution and then incubated in PBS (containing 0.025% Tween 20) for Microscale Thermophoresis Binding Assay 30 minutes. Blocking was performed in M.O.M. blocking so- Protein-protein interactions between human integrin-aVb3 lution, according to the manufacturer’s instructions, for and human aPC or human PC were determined by means of 30 minutes followed by PLA blocking solution for another a microscale thermophoresis binding assay (NanoTemper 30 minutes. Then slides were incubated with three primary anti- Technologies, Munich, Germany). Integrin-aVb3 was labeled bodies(overnightat4°C)raisedindifferent species and recogniz- with the fluorophore NT-647(Monolith NT Protein Labeling 49 ing the target antigens of interest: rabbit anti-integrin-b3 (1:100), Kit; NanoTemper Technologies, Munich, Germany). A mouse anti-PC (1:100), and goat anti-nephrin (1:250). After 14-fold titration series of aPC or PC (1 mM–0.122 mM) diluted washing (23 with buffers A and B provided in the PLA kit, each 1:1 with PBS containing 0.05% Tween 20 was performed. The 10 minutes), sections were incubated with species-specific sec- concentration of NT-647–labeled integrin-aVb3 was kept ondary antibodies (against integrin-b3 and PC) with a unique constant (15 nM). The binding partners were incubated for short DNA strand attached (PLA probes). Antibody-attached 30 minutes in the dark to enable binding. The reaction was oligonucleotides were linked by enzymatic ligation, amplified then aspirated into glass capillaries that were sealed with wax, via rolling circle amplification using a polymerase, and ampli- and the thermophoretic movement of the labeled proteins fied DNA was detected by fluorescent-labeled complementary was monitored with a laser (on for 30 seconds and off for oligonucleotide probes. Additionally, slides were incubated 5 seconds) at a laser power of 80%. Fluorescence was mea- with fluorescently labeled secondary antibody against nephrin sured before laser heating (FInitial)andafter30secondsof for 120 minutes before being rinsed and mounted with laser-on time (FHot). The Monolith NT.115 device and the NT VECTASHIELD mounting medium containing DAPI for Analysis software version 1.427 (NanoTemper Technologies) nuclear staining. were used for analysis. The normalized fluorescence FNorm5FHot/FInitial reflected the concentration ratio of the Production of Lentiviral Particles labeled molecules. FNorm was plotted directly and multiplied by HEK293T cells (CRL-11268; ATCC) were grown at 37°C in a a factor of 10, yielding a relative change in fluorescence per humidified atmosphere with 5% carbon dioxide in an incuba- mill. Thus, the fraction of bound ligand molecules can be de- tor (Thermo Fisher Scientific). The culture medium contained rived from the measured change in normalized FNorm.Kd was

JASN 31: ccc–ccc, 2020 Integrin-b3 and aPC Inhibit RhoA 5 BASIC RESEARCH www.jasn.org calculated from three independent thermophoresis measure- role of EPCR in vivo, we induced persistent hyperglycemia ments with NanoTemper Software (NanoTemper Technologies). in mice expressing low levels of EPCR (below 10% compared d d with wild-type mice, EPCR / mice).30 Albuminuria and glo- Statistical Analyses merular damage, as reflected by the FMA, were comparable d d The data are summarized as the mean6SEM. The in diabetic EPCR / and diabetic wild-type mice (Figure 1). Kolmogorov–Smirnov test or D’Agostino–Pearson normality This finding contrasts earlier results in mice with markedly test was used to determine whether the data followed a Gaussian impaired ability to activate PC (TMPro/Pro mice), which are distribution. Statistical analyses were performed with t test or also characterized by a limited capability to restrict thrombin ANOVA as appropriate. StatistiXL software (www.statistixl. activity and enhanced PAR1-signaling.10,50 These data support com) and Prism 5 (www.graphpad.com) software were used the conclusion that EPCR is not required for aPC’s cytopro- for the statistical analyses. Statistical significance was accepted tective effects in the context of dNP and raise the question at values of P,0.05. whether other coreceptors are required for PC activation and aPC signaling via PARs on podocytes. 25 Given the relevance of b1 and b3 integrins on podocytes RESULTS and the known binding of aPC to integrins,19,22 we hypothe- sized that aPC’s cytoprotective effect requires aPC binding to

Integrin-avb3 Binds To PC and aPC and Enhances PC podocyte integrins. Indeed, immunoprecipitation studies re- Activation on Podocytes vealed that human aPC readily binds to integrin-b3 on human We previously proposed that EPCR is not required for the podocytes in a concentration-dependent manner, whereas 15 cytoprotective effect of aPC in podocytes. To address the very weak binding to integrin-b1 was observed (Figure 2A).

A B ns ns 800 1000 *** *** *** *** 800 600

600 400 400 200 200 Blood glucose (mg/dL) 0 0 Albuminuria (alb/crea, μ g/mg) C DM C DM CDMC DM δ/δ WT EPCRδ/δ WT EPCR

C *** ns 40 Wild type EPCR δ/δ *** Control DM Control DM 30

20 FMA

10 (% glomerular area)

0 CDMC DM WT EPCRδ/δ

Figure 1. EPCR deficiency has no effect on dNP. (A and B) Dot plot summarizing (A) urine albumin levels (albumin-creatinine ratio) or d d (B) blood glucose levels in control and diabetic wild-type (WT) and EPCR-deficient (EPCR / ) mice. Albuminuria and blood glucose d d levels are not different between control or diabetic EPCR / mice as compared with control or diabetic wild-type mice, respectively. (C) Representative images of glomeruli (left; PAS staining of paraffin-fixed sections; scale bar, 5 mm) and dot plot summarizing data for the FMA (right). Data shown as dot plots represent mean6SEM of at least six mice per group. ***P,0.005. (A and B) ANOVA with Tukey-adjusted d d post hoc comparison; diabetic mice (DM) were compared with corresponding (WT or EPCR / ) nondiabetic control mice and diabetic d d WT mice were compared with diabetic EPCR / mice. C, control; DM, mice with persistent hyperglycemia after STZ injection.

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A D β IP 1 Zymogen PC: K = 12.4 ± 3.4 1,35 D 55 kDa IB: aPC aPC: KD = 61.5 ± 10.3 1,15 *** β ** 138 kDa 1 0,95 * 0 20 50 100 aPC (nM) *** 0,75 *** β 3 IP 0,55 ***

55 kDa IB: aPC Fraction Bound 0,35

β 100 kDa 3 0,15 0 20 50 100 aPC (nM) -0,05

B -0,25 human mouse 0,01 0,1 1 10 100 1000 10000 Concentration (PC or aPC, nM) E * 0.8 * 24 24 0.7 *** 18 18 0.6 12 12 *** 0.5 6 6 0.4 0 0 ** Protein Clevels (AU) Protein Clevels (AU) Control DM Control DM 0.3

OD (at 405 nm) 0.2 C * * 0.1 0.0 0.0 2.5 5.0 7.5 10.0 Time (min) negative control control podocytes

β KD integrin 3 podocytes trophoblast cells

β PLA (protein C & integrin 3), nephrin, DAPI

Figure 2. The PC–integrin-avb3 interaction enhances aPC generation in podocytes. (A) Representative immunoblot images of aPC (55 kDa, top) after immunoprecipitation of integrin-b1 (b1 IP, left, 138 kDa) or integrin-b3 (b3 IP, right, 100 kDa) in human podocytes. Human aPC binds in particular to integrin-b3 on human podocytes; the antibody used to detect aPC recognizes both the zymogen and the activated form. The lower gel shows the loading control (integrin-b1 or integrin-b3). The concentration of aPC is shown at the bottom in nanomolar, and the incubation time was 10 minutes. (B and C) Experimental evidence that PC/aPC is glomerularily filtered and interacts with b3 integrin on podocytes. (B) PC is detectable in the urine of nondiabetic humans and mice (control) and markedly increases in patients with diabetes (DM) and diabetic mice (db/db mice, DM). Representative immunoblot images and bar graph summarizing results of at least five humans or mice per group. *P,0.05, t test comparing DM versus control. (C) Representative image, showing colocalization (yellow, white arrows) of the PC–integrin-b3 complex (red, detected by PLA) with nephrin (green, podocyte marker, immunofluorescence staining) in mouse kidney sections. Scale bar, 20 mm. (D) Binding of the serine protease aPC (green) or the zymogen PC (red) to integrin-avb3 as analyzed by microscale thermophoresis. The data are presented as the mean6SEM for three independent repeat experiments. *P,0.05, **P,0.01, ***P,0.005 comparing PC versus aPC; t test with Bonferroni correction. (E) Cell- based in vitro aPC generation assay. Thrombin (10 nM)-mediated PC activation was determined in the presence of trophoblast cells KD (positive control, purple), control mouse podocytes (red), or integrin-b3–deficient (integrin-b3 , blue) podocytes. aPC generation in the absence of cells served as a negative control (black). The data are presented as the mean6SEM for three independent repeat KD experiments. Comparative results were obtained with an independent integrin-b3 cell line. *P,0.05, **P,0.01, ***P,0.005 versus KD control podocytes; one-way ANOVA with Bonferroni-adjusted post hoc comparison of trophoblast cells or integrin-b3 podocytes with control podocytes.

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A B *** *** *** 15 4 *** *** ***

NG (5 mM) 10 (AU) 3 3 ns ns ns ns 5 2 0 1 *** RhoA-GTP Active integrin β 0 RhoA HG (25 mM) NG NG + aPC PBS + +aPC +PBS + aPC + PBS IgG Control aPC(NG) Mannitol Mannitol only aPC only β HG Mannitol Active integrin 3 (AP5) HG Phalloidin DAPI

C D RhoA-GTP aPC (20 nM) RGDS (50 µg/ml) + RhoA

Gα13IP aPC (20 nM) aPC (20 nM) 100 kDa RGDS β RhoA-GTP IB: 3 RhoA + aPC (20 nM) IB: Gα13 44 kDa 0 10 15 20 30 0 10 15 20 30 Time (min) RhoA-GTP RGDS 2.0 RhoA *** aPC (AU) RGDS + aPC 3 1.5 *** β KD podocytes RhoA-GTP 3 ns ns RhoA + aPC (20 nM) 1.0 ns ns 0.5 * * RhoA-GTP Thrombin IP of integrin β

13 RhoA (10 nM) α

G 0.0 0 10152030 Time (min) 010152030 Time (min) E 4 ** ### *** ** ### 100 *** 100 3 *** ### ### × 80 80 × 2 60 60 +++ +++ +++ 40 40 ++ × 1 20 20 fold change from 0 min 0 0 RhoA GTP / total 0 No. of migrated podocytes PBS PBS 0 10152030 Time (min) Control Control RGE-aPC Mannitol RGE-aPC aPC (20 nM) aPC (20 nM) aPC (20 nM) RGDS (50 µg/ml) + aPC (20 nM) aPC + HAPC1573 + aPC aPC + HAPC1573 + aPC RGDS (50 µg/ml) only Integrin β KD + aPC (20 nM) PAN (30 µg/ml) HG (25 mM) 3 × thrombin (10 nM)

Figure 3. aPC–integrin-avb3 temporally regulates RhoA activation in podocytes. (A) Representative immunofluorescence images (left) of active integrin-b3, as determined by the conformation-specific antibody AP5, in human podocytes without (normal glucose con- centration, 5 mM glucose, NG) or with high glucose (HG, 25 mM) stimulation in the absence (PBS) or presence of aPC (20 nM). Mannitol is used as an osmotic control. Cells stained with nonspecific IgG served as staining controls. The dot plot at the right summarizes the results. All groups were compared with control and HG1PBS to HG1aPC. Scale bar, 20 mm. (B) Representative immunoblot images

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54 The presence of PC and aPC in the urine of humans and AP5, which detects an active conformation of integrin-b3. mice and the binding of aPC to integrin-b3 on podocytes, as Interestingly, compared with control medium (5 mM glucose), determined by PLA, indicate that PC/aPC can cross the glo- exposure of podocytes to high glucose medium (25 mM), but merular filtration barrier and bind to podocyte integrin-b3 not increased mannitol concentrations (25 mM), reduced the (Figure 2, B and C, Supplemental Figure 2). To determine active integrin-b3 conformation. Concomitant exposure to the specific interaction of PC and aPC with the functionally aPC abolished the glucose-dependent reduction in active important podocyte integrin heterodimer avb3, we analyzed integrin-b3, whereas aPC (control medium, 5 mM glucose) protein-protein interactions in a cellfree system using micro- had no effect (Figure 3A). Hence, aPC maintains an active scale thermophoresis, which demonstrated a strong interac- integrin-b3 conformation congruent with signaling of aPC tion of both PC and aPC with integrin-avb3. Intriguingly, the via integrin-b3. Integrins modulate the cytoskeleton in part 55 zymogen PC bound to integrin-avb3 with slightly higher af- via RhoA signaling. In agreement with the cytoskeletal finity (Kd512.463.4 nM) than aPC (Kd561.5610.3 nM; changes observed, high glucose induced RhoA activation, Figure 2D). which was not observed in the presence of aPC (Figure 3B). The higher affinity of PC than aPC for integrin-avb3 Binding of the RGD-containing coagulation factor raised the question of whether PC binding to integrin-avb3 fibrin/fibrinogen to integrin-b3 induces interactions of the promotes aPC generation. To answer this question, we assessed integrin-b3 cytoplasmic tail with components of canonical thrombin-mediated PC activation by control or integrin- G protein-coupled receptor (GPCR) signaling, e.g.,Ga13 and KD 42,56 b3–deficient (integrin-b3 ; Supplemental Figure 3 Ga12. To determine whether binding of aPC to integrin-b3 Figure 3Supplemental Figure 3) podocytes using trophoblast on podocytes induces G-protein recruitment, we analyzed the 51,52 cellsaspositivecontrols(Figure2E). PC activation was integrin-b3 interaction with Ga13 after stimulation with aPC. reduced to approximately 50% of that by control podocytes aPC transiently induced recruitment of Ga13 to integrin-b3, KD in two independent podocyte integrin-b3 cell lines which peaked after 10–15 minutes (Figure 3C), and this effect (Figure 2E). These results support a model in which was completely abolished by the antagonistic peptide RGDS. integrin-b3 enhances aPC generation, potentially enhancing Fibrin/fibrinogen outside-in signaling via integrin-b3 recruits cytoprotective aPC-PAR signaling on podocytes while sup- Ga13 in platelets, which negatively regulates thrombin–PAR–Src- pressing local thrombin generation and cell-disruptive dependent RhoA activation.42,56 Likewise, aPC inhibited Src ac- thrombin-PAR signaling.15,53 tivity, as deduced from an increased ratio of inactivating Y527 phosphorylation to activating Y416 phosphorylation in podo- Activated PC Induces a Temporal Interaction of cytes (Supplemental Figure 4A). The aPC-induced inhibition Integrin-b3 with Ga13 of Src activity was abolished or even reversed in the presence Ligand binding to integrins may modify their conformation. of an avb3 antagonist (Supplemental Figure 4A). These data Therefore, we determined the levels of active integrin-b3 on suggest that aPC modulates Ga13 signaling upon binding to human podocytes using the conformation-specificmAb integrin-b3. showing levels of RhoA-GTP (21 kDa) and total RhoA (21 kDa) obtained from the RhoA pull-down assayanddotblotsummarizing the data. Compared with control (5 mM glucose, NG) and the osmotic control mannitol (25 mM), high glucose concentrations (25 mM, HG, 3 hours) induce RhoA activation in human podocytes, which is prevented by concomitant exposure to aPC (aPC, 3 20 nM). (C) Representative integrin-b immunoblot images (top; IB: b3)ofGa13 immunoprecipitate showing time-dependent in- teraction of Ga13 with integrin-b3 upon stimulation of human podocytes with aPC. Ga13 (44 kDa) immunoblots were used as loading controls (top; IB: Ga13). Preincubation of cells with RGDS abolished the aPC-induced time-dependent interaction of Ga13 with integrin-b3. The line graph summarizes the results from three repeat experiments (bottom), with each dot representing an indi- vidual measurement. All groups were compared with control. (D) Representative immunoblot images (top) showing the levels of RhoA-GTP (21 kDa) and total RhoA (21 kDa) obtained from the RhoA pull-down assay and line graphs summarizing the kinetic data (bottom). RhoA activation was transient (peaking at 10 minutes) in human podocytes stimulated with aPC only (aPC), whereas sustained RhoA activation over 30 minutes was observed in podocytes stimulated with aPC and RGDS (aPC1RGDS), in integrin- KD b3–deficient podocytes stimulated with aPC (after lentiviral short hairpin RNA–mediated knockdown of integrin-b3,aPC-b3 ), or in thrombin-stimulated podocytes. RGDS alone had no effect (RGDS). All groups were compared with control. (E) Dot plot summarizing how PAN or high glucose(HG,25mM)inducedpodocytemigration(as determined by scratch assay) after treatment with PBS (PBS, control), aPC, aPC preincubated with the antibody HAPC1573 (a mouse mAb that blocks aPC’s anticoagulant effect), or RGE-aPC. In addition, mannitol as an osmotic control is shown for the glucose stimulation experiment. All groups were compared with control. The data are shown as dots of at least three (A–D) or five (E) independent repeat experiments in the dot plot,includingmeanandSEMin(A,B,andE),orasdotsrepresenting individual data points from three independent repeat experiments in the line graphs in (C and D). *P,0.05, **P,0.01, ***P,0.005 (A, B, and E). ***P,0.005 (aPC versus time point

0 minute), ### P,0.005 (RGDS 1 aPC versus time point 0 minute), 11P,0.01, 111P,0.005 (integrin b3KD 1 aPC versus time point 0 minute) in D. (A, B, D, and E) one-way ANOVA with Tukey-adjusted post hoc comparison of treated cells with untreated cells (time point, 0 minute).

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A 40 ns 40 db/mdb/db db/db + aPC IgGControl * 30 **** **** 30 **** ****

20 20

CDM DM + aPC IgGControl MFI (AP5) 10 10

0 0 C DM db/m db/db DM+aPC β3

Active integrin (AP5) Nephrin DAPI db/db+aPC

B C Wild type β3ΔPod β3ΔPod APChigh 1000 ** Control DM Control DM Control DM 800 ** 600 * 400 μ g/mg) 200

Albuminuria (aIb/crea, 0 CDMCC DM DM WT β ΔPod β ΔPod 3 3 APChigh

D E *** F *** 60 *** 1000 *** *** *** *** ** *** 3 *** ** 800 *** ** 40 2 *** 600

FMA *** 400 20 1 200 GBM width (nm) (per μ m of CBM) (% glomerular area)

0 0 Tight-slit pore density 0 CDMCC DM DM CDMCC DM DM CDMCC DM DM

Δ Δ Δ Δ β Δ WT β Pod β Pod WT β Pod β Pod WT 3Pod β Pod 3 3 3 3 3 APChigh APChigh APChigh G H β Δ ** 3 Pod 20 ** *** aPC 15 RhoA-GTP ****** 10 RhoA Sustained β PAR-1 -actin RhoA (AU) 5 PAR1 Rho signaling CDMDMCDMC / total GTP RhoA RhoA WT ΔPod ΔPod high 0 β3 β3 APC CDMCC DM DM Podocyte

WT β ΔPod β ΔPod 3 3 APChigh

Figure 4. Podocyte-specific deletion of integrin-b3 abrogates the cytoprotective effect of aPC in dNP. (A) Representative immuno- fluorescence images (left) of glomeruli of nondiabetic mice (db/m or control [C]), diabetic mice (db/db or STZ-induced diabetes [DM]), or diabetic mice treated with aPC (db/db1aPC or DM1aPC). The conformation-specific antibody AP5 was used to detect active integrin-b3 (red). Podocytes were identified by nephrin staining (green); yellow reflects the colocalization of AP5 and nephrin.

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Integrin-b3–aPC Interaction Is Required for Transient Podocyte-Specific Genetic Ablation of Integrin-b3 RhoA Activation in Podocytes Abrogates the Cytoprotective Effect of aPC Because outside-in signaling of integrin-b3 via Ga13 negatively We and others have previously shown that both genetically regulates thrombin-PAR–dependent RhoA activation in plate- induced as well as injections of aPC ameliorate experimental 42,56 10,23,24,33 lets, we next exposed podocytes to aPC and determined dNP. To evaluate the relevance of aPC–integrin-b3 RhoA activity. aPC transiently activated RhoA, with a peak signaling for aPC-mediated nephroprotection in vivo,wefirst after 10 minutes (Figure 3D). In contrast to the aPC-dependent used the conformation-specific antibody AP5 to determine transient RhoA activation, aPC induced a sustained suppression integrin-b3 regulation by aPC in vivo. In models, FSGS integ- of Rac1 in podocytes (Supplemental Figure 4B). Blocking the rin activation, as reflected by AP5 staining, is induced via 36,57 aPC-integrin interaction with the RGDS peptide caused sus- RGD-independent ligand binding to integrin-b3. In con- tained aPC-dependent RhoA activation, whereas the RGDS trast, AP5 staining is reduced in models of dNP.54 Our above peptide alone had no effect. Similarly, aPC triggered sustained results (Figure 3) suggest that aPC, which is reduced in dia- KD 3 RhoA activation in integrin-b3 podocytes (Figure 3D). In- betes mellitus, induces transient integrin-b3 activation via its triguingly, stimulation of podocytes with thrombin (10 nM) RGD sequence. Taken together, these observations suggest a also resulted in sustained RhoA activation, which was not al- concept of bimodal integrin activation through various bind- tered in the presence of RGDS peptide (Figure 3D, Supplemen- ing sites. Congruently, we observed less integrin-b3 activation tal Figure 4C). These data indicate that both aPC and thrombin (as demonstrated by AP5 levels) in podocytes in 24-week-old induce RhoA activation but that aPC specifically restricts RhoA db/db mice (model of type 2 diabetes mellitus) compared with activation by binding via its RGD sequence to integrin-b3. age-matched nondiabetic db/m mice (Figure 4A, Supplemen- To ascertain the mechanistic relevance of aPC–integrin-b3 tal Figure 5). In vivo treatment of db/db mice with aPC fol- binding on podocytes, we used the well established PAN- lowing an established protocol known to be nephroprotective induced podocyte injury model. Treatment with PAN induces (a dose of 1 mg/kg body wt for 6 weeks)24 restored AP5 stain- loss of actin fibers and promotes pathologic podocyte migra- ing to levels observed in db/m mice (Figure 4A). Similar tion, which is an in vitro correlate of podocyte foot process resultswereobtainedinanSTZ-inducedmodeloftype1 effacement in vivo.36 Wild-type aPC, but not an integrin diabetes mellitus (DM, a murine model reflecting early and binding–deficient aPC mutant (RGE-aPC), inhibited theoretically reversible dNP).2 Following sustained hypergly- PAN-induced podocyte migration, demonstrating the rele- cemia for 16 weeks, colocalization of AP5 with nephrin was vance of aPC’s RGD sequence for its cytoprotective effect in reduced, but treatment of mice with aPC restored AP5 staining podocytes (Figure 3E). The effect of aPC was independent (Figure 4A, Supplemental Figure 5). The maintained presence of its anticoagulation property, because preincubation with of podocyte-active integrin-b3 supports a model in which the mAb HAPC1573, which specifically blocks aPC’santi- aPC conveys its cytoprotective effect by interacting with and coagulant activity, did not interfere with the aPC-mediated signaling through integrin-b3 via its RGD sequence. 15 inhibition of podocyte migration (Figure 3E). Similar re- To scrutinize the in vivo relevance of the aPC–integrin-b3 sults were obtained when using high glucose (25 mM) in- interaction, we first generated mice with podocyte-specific LoxP/LoxP stead of PAN as a stimulus, demonstrating the relevance of deletion of integrin-b3 by crossing integrin-b3 mice Cre DPod these observations for hyperglycemia-induced podocyte with Pod mice (b3 mice). These mice were then crossed dysfunction (Figure 3E). Taken together, these results with mice expressing a hyperactivatable human PC mutant, high 10 show that aPC–integrin-b3 interaction is required for sup- resulting in high plasma levels of aPC (APC mice). Stable pressing of podocyte injury– or hyperglycemia-induced hyperglycemia, resembling diabetes mellitus, was induced RhoA activation. in these mice using STZ (DM; Supplemental Figure 6A).

The nuclei were stained with DAPI (blue). Dot plots summarizing the results are shown at the right. Diabetic mice without or with aPC treatment were compared with nondiabetic control mice (db/m or C) and among each other (db/db versus db/db1aPC and DM versus DM1aPC). Scale bar, 10 mm. (B) Dot plot summarizing urine albumin levels (the albumin-creatinine ratio) in control (C) and diabetic high high (DM) wild-type (WT) mice, b3DPod mice and b3DPod mice crossed with APC mice (b3DPod APC ). (C) Representative images of glomeruli (top; PAS staining of paraffin-fixed sections; scale bar, 5 mm) and the glomerular filtration barrier (bottom, transmission electron microscopy; scale bar, 0.2 mm) and dot plots summarizing (D) the data for the FMA, (E) the width of the GBM (representative of arrows in the far-left image of [C] only), and (F) tight slit pore density, reflecting foot process effacement. (G) Representative immunoblots (left) of RhoA-GTP (21 kDa), total RhoA (21 kDa), and b-actin (42 kDa) from renal tissue lysates from experimental mice and dot plots summarizing the data from the experimental groups (right). (H) Schematic representation of the working model: aPC cannot interact with integrin-avb3 in b3DPod mice, resulting in unopposed PAR1-RhoA signaling, aggravating podocyte dysfunction and hence promoting dNP in mice with increased aPC levels. The data shown in dot plots represent the mean6SEM of at least ten mice (A), five mice (B and D–F), or four mice (G) per group. *P,0.05, **P,0.01, ***P,0.005. (A and C–E) ANOVA with Tukey-adjusted post hoc comparison; for each genotype diabetic mice were compared do nondiabetic mice, and diabetic mutant mice were compared with diabetic wild-type mice. C, nondiabetic control mice; DM, mice with persistent hyperglycemia after STZ injection; MFI, mean fluorescent intensity.

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A B 1000 *** *** Cyclo-RGDfv 800 0.5 or 2 mg/kg 600

400 * 8w 18w 24w 18w 200 Streptozotocin Ex vivo analysis 0 Albuminuria (aIb/crea, μ g/mg) CDMDMDM + 0.5 + 2.0 DM + CycloRGDfv CycloRGDfv (mg/kg) C D Control DM 0.5 mg/kg 2 mg/kg 60 *** *** ***

40 *** FMA 20 (% glomerular area) 0 C DM DM DM + 0.5 + 2.0 E G CycloRGDfv (mg/kg) 1500 *** *** *** 15 *** ****** 1000 *** 10 ***

500 5 GBM width (nm)

0 Rhoa GTP / total RhoA (AU) 0 C DM DM DM + 0.5 + 2.0 RhoA-GTP CycloRGDfv (mg/kg) RhoA F *** β-actin 3 ** *** *** *** C DM DM DM + 0.5 + 2.0 2 +CycloRGOfv (mg/kg)

1 (per mm of GBM) Tight-slit pore density

0 C DM DM DM + 0.5 + 2.0 CycloRGDfv (mg/kg)

Figure 5. Integrin-avb3 antagonism dose-dependently modulates dNP. (A) Scheme showing the experimental approach and dosing of the integrin-avb3 antagonist Cyclo-RGDfv in wild-type mice with STZ-induced persistent hyperglycemia (DM). (B) Dot plot summarizing urine albumin levels (albumin-creatinine ratio) in control (C) and diabetic (DM) mice; diabetic control mice received PBS. (C) Repre- sentative images of glomeruli (top; PAS staining of paraffin-fixed sections; scale bar, 5 mm) and the glomerular filtration barrier (bottom, transmission electron microscopy; scale bar, 0.2 mm) and dot plots summarizing the data for (D) the FMA, (E) the width of the GBM

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DPod Compared with diabetic wild-type mice, diabetic b3 mice matrix accumulation (FMA), GBM widths, tight slit pore den- were partially protected against dNP based on improved mor- sity, podocyte loss (Figure 5, B–F, Supplemental Figure 7, B phologic indices of dNP (FMA, GBM width; Figure 4, C–E). and C), and RhoA activation (Figure 5G). These data are high Strikingly, podocyte-specific deletion of integrin-b3 in APC consistent with cytoprotective aPC signaling via podocyte DPod high 26,36,58 mice (b3 -APC mice) not only abolished aPC’s protec- integrin-b3 and suggest that low doses of an integrin- tive effect but actually exacerbated albuminuria and morpho- avb3 inhibitor abolish the nephroprotective effect of endoge- logic features of dNP (FMA, podocyte loss, GBM width, and nous aPC in the context of dNP. tight slit pore density; Figure 4, B–F, Supplemental Figure 6). We next used a higher dose of Cyclo-RGDfv (2 mg/kg) Furthermore, integrin-b3 deficiency in podocytes of diabetic with the same experimental design (Figure 5A). In contrast high DPod high APC mice (b3 -APC mice) resulted in increased to low-dose Cyclo-RGDfv–mediated inhibition, high-dose RhoA activity compared with that of diabetic wild-type or Cyclo-RGDfv–mediated integrin-avb3 inhibition partially re- DPod diabetic b3 mice (Figure 4G). These results are congru- versed albuminuria, dNP morphologic features (FMA, GBM ent with the above in vitro data, demonstrating that the width, tight slit pore density, and podocyte loss; Figure 5, B–F, aPC–integrin-b3 interaction via the RGD sequence is re- Supplemental Figure 7, B and C), and RhoA activation quired to limit RhoA activation. Increased aPC levels and (Figure 5G). concomitantly reduced podocyte integrin-b3 expression in These data illustrate that integrin-avb3 inhibition is a DPod high b3 -APC mice results in unbalanced PAR1 activation by double-edged sword in dNP. The aggravation of dNP upon aPC (Figure 4H). This triggers uncontrolled RhoA signaling low-dose integrin-avb3 inhibition is consistent with the pro- and podocyte dysfunction, mimicking the effect of thrombin posed cytoprotective signaling of aPC via integrin-avb3 in through PAR1 signaling. stressed podocytes. The partial improvement of dNP in mice with isolated integrin-b3 deficiency in podocytes (the reduced FMA and Targeted Disruption of the aPC–Integrin Interaction In DPod GBM width in b3 mice) is consistent with a pathologic Vivo Promotes Excess PAR1 and RhoA Signaling and 26,36,58 role of activated integrin-b3 in podocytes, whereas the Exacerbates dNP aggravation of albuminuria, histologic indices, and RhoA ac- To directly demonstrate the relevance of the proposed neph- tivation in mice with combined podocyte integrin-b3 defi- roprotective aPC–integrin-b3 interaction via aPC’sRGDse- DPod high ciency and increased aPC plasma levels (b3 -APC quence in vivo, we generated transgenic mice expressing a mice) elucidates a previously unknown protective function human hyperactivatable aPC mutant lacking the endogenous high of integrin-b3 in dNP. RGD integrin-binding site (RGE-APC mice; Supplemental Figure 1). These model mice complemented the previously Integrin-avb3 Antagonism Dose-Dependently generated model mice expressing a hyperactivatable human Modulates dNP aPC mutant (APChigh mice, containing the endogenous RGD DPod The partial protection from dNP in b3 mice and the sequence), enabling us to directly determine the role of aPC’s DPod high aggravation of dNP in b3 -APC mice indicate a dual RGD sequence in vivo. Blood loss upon standardized tail in- high function of podocyte integrin-b3 that is reminiscent of the jury and aPC plasma levels were comparable in APC mice dose-dependent effects of integrin inhibitors on tumor an- and RGE-APChigh mice (Supplemental Figure 1, D and E), giogenesis.37 To address whether integrin inhibition has confirming similar expression and anticoagulant function. dose-dependent effects in the context of dNP, we tested Persistent hyperglycemia was induced in wild-type, high high two different concentrations of the integrin-avb3 inhibitor APC ,andRGE-APC mice (Figure 6A, Supplemental Cyclo-RGDfv (0.5 mg/kg and 2.0 mg/kg body wt). Interven- Figure 8A). Compared with wild-type mice, APChigh mice tions with Cyclo-RGDfv were initiated in mice with stable were protected against dNP, as reflected by reduced albumin- hyperglycemia for 10 weeks and hence after establishment of uria and markedly reduced morphologic markers of dNP albuminuria (Figure 5A, Supplemental Figure 7). (FMA, GBM width, tight slit pore density, and podocyte Compared with vehicle-treated diabetic wild-type (DM) numbers; Figure 6, B–F, Supplemental Figure 8, B and C), mice,diabeticmicetreatedwithlow-doseCyclo-RGDfv in agreement with the findings of previous studies.10 In con- (0.5 mg/kg) exhibited enhanced albuminuria, extracellular trast, disruption of aPC-integrin binding in RGE-APChigh mice

(representative of arrows in the far-left image of [C] only), and (F) tight slit pore density, reflecting foot process effacement. (G) Representative immunoblots of RhoA-GTP (21 kDa), total RhoA (21 kDa), and b-actin (42 kDa) from renal tissue lysates from experimental mice and a dot plot summarizing the data. The data shown in the dot plots represent the mean6SEM of at least eight mice (B–F) or five mice (G) per group; each dot represents data from one mouse. *P,0.05, **P,0.01, ***P,0.001. (B and D–F) ANOVA with Tukey-adjusted post hoc comparison; diabetic mice were compared do nondiabetic mice, and diabetic mice treated with Cyclo-RGDfv were compared with diabetic control mice. C, nondiabetic control mice; DM, mice with persistent hyperglycemia after STZ injection.

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A C Wild type APChigh RGE-APChigh DM + DM + Control DM SCH79797 Control DM Control DM SCH79797 SCH79797

8w 18w 24w

Streptozotocin Ex vivo analysis

B D 60 800 *** *** *** *** *** *** 600 * *** 40 ** * *** *** 400 20 200 FMA (% glomerular area) 0

Albuminuria (alb/crea, µg/mg) 0 CDMDM CDMCDMDM CDMDM CDMCDMDM +S +S +S +S high high WT APC RGE-APC WT APChigh RGE-APChigh E G * *** *** *** 1000 *** *** *** *** 25 *** *** 800 20

600 15 * ** * 400 10

GBM width (nm) 200 5

0 0 RhoA GTP / Total (AU) CDMDM CDMCDMDM RhoA GTP +S +S RhoA WT APChigh RGE-APChigh F β-actin ** *** 4 *** ** C DMDM C DM CDMDM +S +S *** 3 WT APChigh RGE-APChigh *** *** H 2 APChigh RGE-APChigh

aPC aPC 1 (per µm of GBM) Tight-slit pore density 0 SCH79797 CDMDM CDMCDMDM α β Transient α β Sustained PAR-1 v 3 PAR-1 v 3 +S +S PAR1 RhoA PAR1 RhoA RhoA signaling RhoA signaling WT APChigh RGE-APChigh Podocyte Podocyte

Figure 6. aPC protects against dNP via its RGD sequence. (A) Experimental design. SCH79797 was used as a PAR1 antagonist in a subgroup of mice to counteract excess PAR1 signaling. (B) Dot plot summarizing urine albumin levels (albumin-creatinine ratio) in

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exacerbated albuminuria, FMA, GBM width, tight slit pore RhoA signaling in the absence of the aPC–integrin-b3 density, and podocyte loss (Figure 6, B–F, Supplemental interaction. Figure 8, B and C), demonstrating a crucial role of aPC’s RGD site in its cytoprotective effect in dNP. These functional and morphologic changes were associated with corresponding DISCUSSION changes in RhoA activation (Figure 6G). Thus, compared with that in diabetic wild-type mice, RhoA activation remained The roles of integrins in podocyte function are well estab- low in diabetic APChigh mice but was markedly enhanced in lished. Likewise, accumulating evidence supports a role of RGE-APChigh mice. coagulation proteases in regulating podocyte function. GiventhesustainedRhoAactivationuponthrombin Within this study, we established a new mechanism through stimulation of podocytes in vitro (Figure 3D), we next hypoth- which integrins and coagulation proteases coordinately esized that loss of nephroprotection and enhanced RhoA control podocyte function. Integrin-b3 and the serine pro- activation in RGE-APChigh mice reflects unopposed PAR1 tease aPC coordinately regulate transient RhoA activation in signaling. To evaluate this hypothesis, we inhibited PAR1 us- podocytes. The transient activation of RhoA depends on 35 ing a selective nonpeptide PAR1 antagonist (SCH79797). aPC binding to integrin-b3 via its RGD sequence. In the Treatment with SCH79797 initiated 10 weeks after induction absence of aPC’s RGD sequence or of podocyte integrin- of hyperglycemia (Figure 6A) markedly reduced RhoA activa- b3, aPC induces sustained RhoA signaling and podocyte tion, reversed albuminuria, and attenuated morphologic fea- dysfunction, mimicking the effect of thrombin. In support tures of dNP (FMA, GBM width, tight slit pore density, and of the development of aberrant PAR1 signaling upon disrup- high podocyte loss) in diabetic RGE-APC mice (Figure 6, B–G, tion of the aPC–integrin-b3 interaction, PAR1 inhibition coun- Supplemental Figure 8, B and C). Thus, pharmacologic mit- teracted the enhanced RhoA activation and maintained renal igation of PAR1 signaling reverses the markedly aggravated function in mice expressing an aPC variant lacking the RGE renal phenotype in diabetic mice expressing RGE-APChigh. sequence. These data support a pathophysiologic role of in- Endogenous aPC ameliorates dNP, as indicated by the creased RhoA and PAR1 signaling in dNP60–62 and identify aggravated renal phenotype in diabetic mice with markedly aPC as a rheostat that controls RhoA activation by interacting impaired PC activation ability and exaggerated thrombin gen- with integrin-b3. eration (TMPro/Pro mice).10 Considering the impairment of The roles of integrins in podocytes are well established.27 aPC generation in diabetic mice and patients with diabe- However, integrin activation is a complex process that can be tes,10,59 we hypothesized that PAR1 inhibition may reduce mediated by RGD and non-RGD binding sites. Thus, suPAR RhoA activation and ameliorate dNP in wild-type mice. In- regulates integrin activation and glomerular disease in mice deed, PAR1 antagonism in diabetic wild-type mice reversed through a non-RGD binding site.58 By contrast, this study albuminuria, ameliorated morphologic features of dNP demonstrates that PC/aPC crosses the glomerular filtration (FMA, GBM width, tight slit pore density, and podocyte loss), barrier and identifies the serine protease aPC as a physiologic and reduced RhoA activation (Figure 6, B–G, Supplemental integrin-b3 ligand at the RGD binding site that conveys cyto- Figure 8, B and C). Thus, the aPC-integrin interaction is essential and nephroprotective effects. Inducible costimulator ligand for aPC’s nephroprotective effect, and disruption of the aPC- was recently identified as yet another RGD-binding integrin integrin interaction or reduced aPC generation (as observed in ligand that fine tunes integrin function in podocytes.63 To- diabetes mellitus) promotes PAR1-dependent excess RhoA gether, these studies demonstrate the need to fine tune integ- signaling and thereby dNP (Figure 6H). Importantly, pharma- rin function and to maintain RhoA signaling in a physiologic cologic inhibition of aberrant PAR signaling counteracts excess balance in the context of glomerular disease.

control (C) and diabetic (DM) mice. Some diabetic mice received additional SCH79797 treatment (DM1S). (C) Representative images of glomeruli (top; PAS staining of paraffin-fixed sections; scale bar, 5 mm) and the glomerular filtration barrier (bottom; transmission electron microscopy; scale bar, 0.2 mm) and dot plots summarizing the data for (D) the FMA, (E) the width of the GBM (representative of arrows in the far-left image only), and (F) tight-slit pore density, reflecting foot process effacement. (G) Representative immunoblots (bottom) of RhoA-GTP (21 kDa), total RhoA (21 kDa), and b-actin (42 kDa) from renal tissue lysates from experimental mice and a dot plot summarizing the data (top). (H) Schematic representation of the working model: in APChigh mice, aPC activates PAR1 via its proteolytic activity, thus promoting RhoA activation, but at the same time binds to integrin-b3, restricting RhoA activity and resulting in high transient RhoA signaling. In contrast, in RGE-APC mice, aPC cannot bind to integrin-b3, resulting in sustained RhoA signaling (bottom). SCH79797, a PAR1 antagonist, counteracts the enhanced PAR1 and RhoA signaling in RGE-APChigh mice. The data shown in dot plots represent the mean6SEM of at least six mice per group. *P,0.05, **P,0.01, ***P,0.001. (B and D–F) ANOVA with Tukey-adjusted post hoc comparison; for each genotype diabetic mice were compared do nondiabetic mice, diabetic mutant mice were compared with diabetic wild-type mice, and diabetic mice treated with SCH79797 were compared with untreated diabetic mice of the same genotype. C, nondiabetic control mice; DM, mice with persistent hyperglycemia after STZ injection; DM1S, diabetic mice treated with the PAR1 antagonist SCH79797.

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This study identifies aPC as a RGD-dependent integrin-b3 been demonstrated to occur in several cell types and different ligand on podocytes, which promotes nephroprotection at contexts,15,17–19 but the coreceptors required in the absence of two levels. First, as indicated by the decreased thrombin- EPCR remain largely unknown. The current data and obser- 19 dependent PC activation in integrin-b3 knockdown cells, vations by Cao et al. show that RGD-binding integrins can binding of PC to podocyte integrin-b3 enhances thrombin- functionally substitute for EPCR in propagating aPC- mediated aPC generation, inhibiting potentially harmful dependent cytoprotective effects. Importantly, this study 53 thrombin generation. Second, the binding of aPC to demonstrates that integrin-b3 enhances local PC activation 60 integrin-b3 restricts excessive and harmful RhoA signaling. as well as conveys aPC signaling in podocytes. This identifies The aPC-mediated regulation of RhoA activation is linked a new function of the PC/aPC–integrin-b3 interaction in ad- with integrin-dependent signaling through Ga13,asaPCin- dition to the previously shown functions related to neutrophil 19,22 duced a transient integrin-b3–Ga13 interaction via its RGD and macrophage adhesion. The existence of two distinct sequence. coreceptors of aPC-PAR signaling on podocytes and endothe- The aPC-integrin–dependent inhibition of RhoA signaling lial cells, integrin-b3 and EPCR, respectively, provides new via G-protein signaling has similarities with the pathway insights into the cell specificity of aPC signaling and provides described by Gong et al.42 Fibrinogen/fibrin binding to integrin- a plausible explanation for the disjunct aPC-mediated effects b3 on platelets induces a functional interaction of the cytoplas- on RhoA and Rac1 signaling in podocytes and endothelial 64,65 mic integrin domain with Ga13 to restrict RhoA activation, cells, respectively (Figure 3D, Supplemental 4B ). counteracting agonist (thrombin)-induced GPCR signaling.42 The study identifies additional questions. For example, ex- 42 Although the data of Gong et al. suggest that two different pression of integrin-b3 is not restricted to podocytes and it ligands control GPCR-Ga13 (thrombin) and integrin-Ga13 remains to be evaluated whether binding of aPC to integrin-b3 (fibrinogen/fibrin) signaling, our data indicate that a single modulates the function of cells other than podocytes within protein, aPC, induces both G protein–dependent RhoA activa- the kidney. However, the results obtained in mice with targe- tion through PARs and RhoA inhibition through noncanonical ted deletion of integrin-b3 specifically in podocytes establish integrin-Ga13 signaling (Figure 6H, left). Depletion of integrin- a salutary function for aPC–integrin-b3 signaling in dNP b3 or mutation of aPC’s RGD sequence abolishes aPC’s rheo- in vivo. Furthermore, species-specific differences in PAR sig- stat function and results in unopposed PAR1-mediated RhoA naling in podocytes (e.g., signaling of aPC via PAR3/PAR2 in activation in mice that can be ameliorated by pharmacologic humans but via PAR3/PAR1 in mouse podocytes) preclude the inhibition of PAR1 (Figure 6H, right). These results thus direct translation of these preclinical results from murine provide new insights into the mechanisms of cytoprotective models into humans.15,53 However, in vitro work using human signaling mediated by podocyte integrins. podocytes suggests that modulation of PAR signaling is a Consistent with the existence of cytoprotective signaling promising approach for the treatment of human glomerular 15 through integrin-b3 in podocytes, inhibition of integrin- disease (e.g., and this study), which warrants further avb3 in dNP is a double-edged sword, as indicated by analyses. the dose-dependent effects of integrin-avb3 inhibition in ex- perimental dNP models. This finding is consistent with the dose-dependent pro- and antiangiogenic effects of integrin ACKNOWLEDGMENTS inhibition.37 Enhancement of angiogenesis by low-dose integ- rin inhibitors has been attributed to altered avb3 and vascular We thank Dr. Katherine Weilbaecher, Washington University endothelial growth factor receptor-2 trafficking.37 Integrins School of Medicine, for providing b3LoxP/LoxP mice and Elliot d d control endosomal and lysosomal trafficking of several dis- Rosen for providing EPCR / mice. We also thank Ibrahim Sögüt, tinct classes of transmembrane receptors, including receptors Kuheli Banerjee, Anubhuti Gupta, Satish Ranjan, Kathrin Deneser, known to regulate podocyte function (e.g., uPAR, EGF recep- Julia Judin, Juliane Friedrich, René Rudat, and Rumiya Makarova for tor, and GPCRs).26 Whether integrin inhibitors likewise mod- their excellent technical support. ulate PAR trafficking on podocytes remains unknown. Another Dr. Sanschita Ghosh, Dr. Madhusudhan Thati, and Dr. Hongjie potential explanation for the dose-dependent effect of avb3 Wang designed and conducted in vitro work, mouse experiments, and inhibition in dNP is the expression of integrins by different ex vivo analysis; Dr. Moh’d Mohanad Al-Dabet, Dr. Wei Dong, renal cell types (e.g., podocytes versus endothelial cells). Alter- Dr. Ahmed Elwakiel, Dr. Ihsan Gadi, Dr. Dheerendra Gupta, natively, different concentrations of integrin-avb3 inhibitors Dr. Sumra Nazir, Dr. Rajiv Rana, and Dr. Silke Zimmermann assisted may differentially regulate integrin inside-out versus outside- with animal experiments and ex vivo analysis; Dr. Shrey Kohli, in signaling. Dr. Shruthi Krishnan, and Dr. Akash Mathew conducted in vitro Intriguingly, podocyte integrin-b3 appears to substitute for experiments; Dr. Ronald Biemann and Dr. Stoyan Stoyanov assisted known functions of EPCR on endothelial cells, i.e., PC activa- with image acquisition and analyses; Dr. Jinyang Zeng-Brouwers and tion and aPC signaling. EPCR, the pivotal coreceptor for aPC Dr. Liliana Schaefer conducted the microscale thermophoresis bind- signaling in endothelial cells, is not required for aPC signaling ing assay; Dr. Charles T. Esmon, Dr. Marcus J. Moeller, Dr. Jochen in podocytes.15 Signaling of aPC independent of EPCR has Reiser, Dr. Alireza R. Rezaie, and Dr. Wolfram Ruf provided

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reagents and critically reviewed the manuscript; and Dr. Berend 2. Gaede P, Tarnow L, Vedel P, Parving HH, Pedersen O: Remission to Isermann and Dr. Madhusudhan Thati conceptually designed the normoalbuminuria during multifactorial treatment preserves kidney study and prepared the manuscript. function in patients with type 2 diabetes and microalbuminuria. Nephrol Dial Transplant 19: 2784–2788, 2004 3. Bock F, Shahzad K, Vergnolle N, Isermann B: Activated protein C DISCLOSURES based therapeutic strategies in chronic diseases. Thromb Haemost 111: 610–617, 2014 4. Isermann B: Homeostatic effects of coagulation protease-dependent J. Reiser is cofounder of Trisaq, a biotechnology company that develops new signaling and protease activated receptors. JThrombHaemost15: drugs for kidney diseases. All remaining authors have nothing to disclose. 1273–1284, 2017 5. Arakaki AKS, Pan WA, Trejo J: GPCRs in cancer: protease-activated receptors, endocytic adaptors and signaling. Int J Mol Sci 19: 1886, FUNDING 2018 6. Griffin JH, Zlokovic BV, Mosnier LO: Activated protein C: biased for This work was supported by DFG grants TH 1789/1-1 (to M. Thati); WA translation. Blood 125: 2898–2907, 2015 3663/2-1 (to H. Wang); IS-67/8-1, IS-67/11-1, SFB854/B26, RTG2408/P7, and 7. Aisiku O, Peters CG, De Ceunynck K, Ghosh CC, Dilks JR, Fustolo- RTG2408/P9 (to B. Isermann); RTG2408/P5 and KO 5736/1-1 (to S. Kohli); Gunnink SF, et al.: Parmodulins inhibit thrombus formation without in- MO 1082/7-1 (to M.J. Moeller); project number 236360313 – SFB 1118 (to ducing endothelial injury caused by vorapaxar. Blood 125: 1976–1985, B. Isermann); and project number 259130777 – SFB 1177 and project C2 2015 SCHA 1082/6-1 (to L. Schaefer). This work was also supported by the 8. Nazir S, Gadi I, Al-Dabet MM, Elwakiel A, Kohli S, Ghosh S, et al.: Cy- BMBF STOP-FSGS consortium grant 01GM1518A (to M.J. Moeller); toprotective activated protein C averts Nlrp3 inflammasome-induced National Heart, Lung, and Blood Institute grant HL 101917 (to A.R. Rezaie); ischemia-reperfusion injury via mTORC1 inhibition. Blood 130: the Stiftung Pathobiochemie und Molekulare Diagnostik (to M. Thati); Center 2664–2677, 2017 of Thrombosis and Hemostasis Mainz funded by BMBF grant 01EO1503 (to 9. Riewald M, Petrovan RJ, Donner A, Mueller BM, Ruf W: Activation of W. Ruf); Boehringer Ingelheim (to M. Thati and W. Ruf); and the Alexander endothelial cell protease activated receptor 1 by the protein C path- von Humboldt-Stiftung (to W. Ruf). The work was also supported by a way. Science 296: 1880–1882, 2002 Deutscher Akademischer Austauschdienst scholarship (to M.M. Al-Dabet). 10. Isermann B, Vinnikov IA, Madhusudhan T, Herzog S, Kashif M, Blautzik J, et al.: Activated protein C protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis. Nat Med 13: DATA SHARING STATEMENT 1349–1358, 2007 11. Mosnier LO, Zlokovic BV, Griffin JH: The cytoprotective protein C All data associated with this study are available in the main text pathway. Blood 109: 3161–3172, 2007 or the Supplemental Materials. 12. Rezaie AR: The occupancy of endothelial protein C receptor by its ligand modulates the par-1 dependent signaling specificity of coagulation proteases. IUBMB Life 63: 390–396, 2011 SUPPLEMENTAL MATERIAL 13. Mosnier LO, Sinha RK, Burnier L, Bouwens EA, GriffinJH:Biasedago- nism of protease-activated receptor 1 by activated protein C caused by This article contains the following supplemental material online at noncanonical cleavage at Arg46. Blood 120: 5237–5246, 2012 http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019111163/-/ 14. Guo H, Liu D, Gelbard H, Cheng T, Insalaco R, Fernández JA, et al.: DCSupplemental. Activated protein C prevents neuronal apoptosis via protease activated receptors 1 and 3. 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AFFILIATIONS

1Institute of Clinical Chemistry and Pathobiochemistry, Otto von Guericke University Magdeburg, Magdeburg, Germany 2Center for Thrombosis and Hemostasis, University Medical Center Mainz, Mainz, Germany 3Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany 4Department of Cardiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 5German Center for Neurodegenerative Diseases, Otto von Guericke University Magdeburg, Magdeburg, Germany 6Department of Medical Laboratories, Faculty of Health Sciences, American University of Madaba, Amman, Jordan 7Institute of Pharmacology, University Hospital and Goethe University, Frankfurt, Germany 8Division of Nephrology and Immunology, University Hospital of the Rheinisch-Westfälische Technische Hochschule, Aachen University of Technology, Aachen, Germany 9Coagulation Biology Laboratory, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 10Department of Medicine, Rush University Medical Center, Chicago, Illinois 11Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 12Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, California

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