Podocyte protease activated receptor 1 stimulation in mice produces focal segmental glomerulosclerosis mirroring human disease signaling events

Abstract

About 30% of patients who have a kidney transplant with underlying nephrotic syndrome (NS) experience rapid relapse of disease in their new graft. This is speculated to be due to a host-derived circulating factor acting on podocytes, the target cells in the kidney, leading to focal segmental glomerulosclerosis (FSGS). Our previous work suggests that podocyte membrane protease receptor 1 (PAR-1) is activated by a circulating factor in relapsing FSGS. Here, the role of PAR-1 was studied in human podocytes in vitro, and using a mouse model with developmental or inducible expression of podocyte-specific constitutively active PAR-1, and using biopsies from patients with nephrotic syndrome. In vitro podocyte PAR-1 activation caused a pro-migratory phenotype with phosphorylation of the kinase JNK, VASP protein and docking protein Paxillin. This signaling was mirrored in podocytes exposed to patient relapse-derived NS plasma and in patient disease biopsies. Both developmental and inducible activation of transgenic PAR-1 (NPHS2 Cre PAR-1Active+/−) caused early severe nephrotic syndrome, FSGS, kidney failure and, in the developmental model, premature death. We found that the non-selective cation channel protein TRPC6 could be a key modulator of PAR-1 signaling and TRPC6 knockout in our mouse model significantly improved proteinuria and extended lifespan. Thus, our work implicates podocyte PAR-1 activation as a key initiator of human NS circulating factor and that the PAR-1 signaling effects were partly modulated through TRPC6. About 30% of patients who have a kidney transplant with underlying nephrotic syndrome (NS) experience rapid relapse of disease in their new graft. This is speculated to be due to a host-derived circulating factor acting on podocytes, the target cells in the kidney, leading to focal segmental glomerulosclerosis (FSGS). Our previous work suggests that podocyte membrane protease receptor 1 (PAR-1) is activated by a circulating factor in relapsing FSGS. Here, the role of PAR-1 was studied in human podocytes in vitro, and using a mouse model with developmental or inducible expression of podocyte-specific constitutively active PAR-1, and using biopsies from patients with nephrotic syndrome. In vitro podocyte PAR-1 activation caused a pro-migratory phenotype with phosphorylation of the kinase JNK, VASP protein and docking protein Paxillin. This signaling was mirrored in podocytes exposed to patient relapse-derived NS plasma and in patient disease biopsies. Both developmental and inducible activation of transgenic PAR-1 (NPHS2 Cre PAR-1Active+/−) caused early severe nephrotic syndrome, FSGS, kidney failure and, in the developmental model, premature death. We found that the non-selective cation channel protein TRPC6 could be a key modulator of PAR-1 signaling and TRPC6 knockout in our mouse model significantly improved proteinuria and extended lifespan. Thus, our work implicates podocyte PAR-1 activation as a key initiator of human NS circulating factor and that the PAR-1 signaling effects were partly modulated through TRPC6. Translational StatementThere is compelling evidence implicating the role of a circulating factor in nephrotic syndrome, and we have previously shown that this factor signals via protease-activated receptor 1 (PAR-1). This work further extends our previous data showing that the signaling responses to PAR-1 agonists were conserved between 3 systems: podocytes in vitro, the mouse model expressing a transgenic overactive form of the PAR-1 receptor, and human circulating factor disease, demonstrating the significance. Looking downstream, TRPC6 (transient receptor potential cation channel subfamily c member 6) knockout podocytes demonstrated altered PAR-1 agonist signaling responses. Knockout of TRPC6 in the PAR-1–active mice significantly reduced their proteinuria and extended their lifespan. This work suggests that PAR-1 could be a key target in the treatment of nephrotic syndrome. There is compelling evidence implicating the role of a circulating factor in nephrotic syndrome, and we have previously shown that this factor signals via protease-activated receptor 1 (PAR-1). This work further extends our previous data showing that the signaling responses to PAR-1 agonists were conserved between 3 systems: podocytes in vitro, the mouse model expressing a transgenic overactive form of the PAR-1 receptor, and human circulating factor disease, demonstrating the significance. Looking downstream, TRPC6 (transient receptor potential cation channel subfamily c member 6) knockout podocytes demonstrated altered PAR-1 agonist signaling responses. Knockout of TRPC6 in the PAR-1–active mice significantly reduced their proteinuria and extended their lifespan. This work suggests that PAR-1 could be a key target in the treatment of nephrotic syndrome. Idiopathic nephrotic syndrome (INS) is one of the most difficult clinical conditions to manage in nephrology. Breakdown of the glomerular filtration barrier results in massive proteinuria, with the underlying trigger not well understood. A proportion of patients will not respond to any therapy, leading inevitably to renal failure and transplantation. In patients without a detectable genetic mutation, up to 50% suffer recurrence of disease.1Bierzynska A. Saleem M.A. Deriving and understanding the risk of post-transplant recurrence of nephrotic syndrome in the light of current molecular and genetic advances.Pediatr Nephrol. 2018; 33: 2027-2035Crossref PubMed Scopus (27) Google Scholar This leads to the reduced lifespan of the graft, and often after graft loss, the patient is deemed untransplantable given the very high risk of further recurrences. There is ample evidence that recurrent INS is caused by an as yet unknown circulating factor, likely produced by the immune system. Despite decades of study, the identity of such circulating factor(s) remains unknown.1Bierzynska A. Saleem M.A. Deriving and understanding the risk of post-transplant recurrence of nephrotic syndrome in the light of current molecular and genetic advances.Pediatr Nephrol. 2018; 33: 2027-2035Crossref PubMed Scopus (27) Google Scholar Here we have tested the hypothesis that the circulating factor works through podocyte protease-activated receptor 1 (PAR-1). PARs 1 to 4 are part of the well-known family of G protein–coupled receptors and are activated by serine proteases. PAR-1 and PAR-2 have been demonstrated to be expressed in the glomerulus, and immunohistochemical staining shows high PAR-1 expression in the podocytes.2Palygin O. Ilatovskaya D.V. Staruschenko A. Protease-activated receptors in kidney disease progression.Am J Physiol Renal Physiol. 2016; 311: F1140-F1144Crossref PubMed Scopus (35) Google Scholar Circulating plasma carries many proteases with diverse biological roles, which are tightly regulated by cognate antiproteases. We previously showed data suggesting that there is excess circulating protease activity in the plasma of post-transplant INS patients with active post-transplant disease (referred to as relapse plasma) that can signal to podocytes resulting in vasodilator-stimulated phosphoprotein (VASP) phosphorylation and increased podocyte motility, with the signaling being podocyte specific.3Harris J.J. McCarthy H.J. Ni L. et al.Active proteases in nephrotic plasma lead to a podocin-dependent phosphorylation of VASP in podocytes via protease activated receptor-1.J Pathol. 2013; 229: 660-671Crossref PubMed Scopus (64) Google Scholar This effect is not present in response to remission plasma from the same patients or control plasma. Small, interfering RNA knockdown of PARs in podocytes demonstrated a role for the PAR-1 protease receptor in this response to relapse plasma. We hypothesized that there is a protease(s) that is present in the circulation that becomes dysregulated early in the pathogenesis of INS (either by upregulation or loss of a cognate antiprotease). Both T cells and memory B cells have been postulated as sources of the circulating factor,4Bierzynska A. McCarthy H.J. Soderquest K. et al.Genomic and clinical profiling of a national nephrotic syndrome cohort advocates a precision medicine approach to disease management.Kidney Int. 2017; 91: 937-947Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar,5Oleinika K. Mauri C. Salama A.D. Effector and regulatory B cells in immune-mediated kidney disease.Nat Rev Nephrol. 2019; 15: 11-26Crossref PubMed Scopus (76) Google Scholar and our subsequent work suggests that the T helper cell 17 (Th17) subset could be a source of circulating proteases.6May C.J. Welsh G.I. Chesor M. et al.Human Th17 cells produce a soluble mediator that increases podocyte motility via signalling pathways which mimic PAR-1 activation.Am J Physiol Renal Physiol. 2019; 317: F913-F921Crossref PubMed Scopus (12) Google Scholar This increased activity of the circulating protease stimulates PAR-1 and induces specific signaling cascades. To test this at the receptor level, we now activated PAR-1 in vitro in conditionally immortalized human podocytes. We then used this signaling data to assess pathologic signaling in human nephrotic patient kidney biopsies and NPHS2 Cre PAR-1Active+/− mice. Furthermore, we hypothesized that podocyte TRPC6 (transient receptor potential cation channel subfamily c member 6) mediates PAR-1 signaling and is activated in response to human plasma or PAR-1 agonism. TRPC6 is an ion channel that binds to the podocyte-specific membrane protein podocin,7Dryer S.E. Reiser J. TRPC6 channels and their binding partners in podocytes: role in glomerular filtration and pathophysiology.Am J Physiol Renal Physiol. 2010; 299: F689-F701Crossref PubMed Scopus (122) Google Scholar and activating mutations in this channel in patients are able to cause focal segmental glomerular sclerosis (FSGS).8Hall G. Wang L. Spurney R.F. TRPC channels in proteinuric kidney diseases.Cells. 2019; 9: 44Crossref PubMed Google Scholar Both gain-of-function and loss-of-function mutations are known to cause FSGS.9Riehle M. Büscher A.K. Gohlke B.O. et al.TRPC6 G757D loss-of-function mutation associates with FSGS.J Am Soc Nephrol. 2016; 27: 2771-2783Crossref PubMed Scopus (79) Google Scholar In addition, it has been shown in podocytes that thrombin treatment can increase intracellular calcium concentrations. This increase is blocked by treatment with a TRPC3/6 inhibitor.10Guan Y. Nakano D. Zhang Y. et al.A protease-activated receptor-1 antagonist protects against podocyte injury in a mouse model of nephropathy.J Pharmacol Sci. 2017; 135: 81-88Crossref Scopus (19) Google Scholar The PAR-1 active construct was a kind gift of Dr. Shaun Coughlin, University of California San Francisco.11Hammes S.R. Shapiro M.J. Coughlin S.R. Shutoff and agonist-triggered internalization of protease-activated receptor 1 can be separated by mutation of putative phosphorylation sites in the cytoplasmic tail.Biochemistry. 1999; 38: 9308-9316Crossref PubMed Scopus (39) Google Scholar Briefly, all serine and threonine residues in the C-terminal tails have been substituted for alanines. This renders the PAR-1 receptor phosphor-null at the C-terminal tail. This form of the receptor is defective in both shutoff and agonist-triggered internalization. All animal experiments and procedures were approved by the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986, and the Guide for the Care and Use of Laboratory Animals was followed during experiments. SV129 transgenic mice were generated by Genoway. These mice were bred with NPHS2 Cre hemizygous mice or NPHS2 rtTA Tet O Cre mice to generate developmental and inducible animals, respectively. The Pod Cre PAR-1 mice were crossed with whole-body TRPC6 knockout (KO) mice on a C57/Bl6 background. These mice were back-crossed to enrich for SV129. Inducible transgenic mice were fed in in their to the inducible between 4 and of and for 3 The of treatment has been The were at and and their were There is in this to with the of In for the of animal podocytes were conditionally immortalized using the system. This podocytes to at the of and at L. J. A. et of recurrent focal segmental glomerulosclerosis after J PubMed Scopus Google Scholar were as previously L. Saleem M. of the PubMed Scopus Google Scholar podocyte cell were generated from a TRPC6 mouse on a as the in a previous L. Saleem M. of the PubMed Scopus Google Scholar glomerular cells were generated and were conditionally immortalized using the same as used in the podocytes. cell were generated by as A. an source for the and of of human Scopus Google Scholar podocytes were with PAR-1 agonist at a of for the were with thrombin as a PAR-1 agonist at a of for the were with nephrotic patient plasma as previously J.J. McCarthy H.J. Ni L. et al.Active proteases in nephrotic plasma lead to a podocin-dependent phosphorylation of VASP in podocytes via protease activated receptor-1.J Pathol. 2013; 229: 660-671Crossref PubMed Scopus (64) Google Scholar plasma was from the same protein were using a with and 3 and were with kinase protein kinase All were used a in and with the at 4 were using a or mouse were used a in and with the for 1 at The were with a of and with a signal which are in a were using the from The in this has been using All the of the This is to both a control and the of the control of the in were in were the podocytes at the of In the were and a was by the with a were then in to any and The were then and the was after and using a and a The of the was and podocyte was by in the PAR-1Active+/− mice were generated by The NPHS2 Cre and NPHS2 Cre rtTA mice were a gift from TRPC6 mice were from the were and in being were by our was using a and was using a The to during at the were The were from the of the The was using an for the the the glomerular signal The is in J. et a potential of glomerular increases glomerular cell and intracellular J Pathol. Full Text Full Text PDF PubMed Scopus (35) Google Scholar A was of the from the developmental from of the control and PAR-1Active+/− mice. was to the with were for A of this can be found M. et and in the of in renal J 2017; Scholar Briefly, the was against and the to of were as a of the effect on and were and using The have been used to was via the UK the UK Renal The PAR-1 receptor is expressed by the as shown in the protein et effects of in a model of kidney 2013; PubMed Scopus Google Scholar in RNA expression Scholar and in RNA a in cell 2017; PubMed Scopus Google Scholar immortalized human podocytes were with PAR-1 agonist This agonist is a and is specific for the PAR-1 This treatment a increase in the phosphorylation of VASP at serine work by the has shown a response to NS patient which was reduced after knockdown of the PAR-1 J.J. McCarthy H.J. Ni L. et al.Active proteases in nephrotic plasma lead to a podocin-dependent phosphorylation of VASP in podocytes via protease activated receptor-1.J Pathol. 2013; 229: 660-671Crossref PubMed Scopus (64) Google Scholar In addition, we now that JNK, and all increases in phosphorylation in response to PAR-1 agonist treatment The JNK, and phosphorylation are podocyte such phosphorylation was in PAR-1 glomerular cells phosphorylation of was showing that the glomerular cells are PAR-1 of at serine is with an increase in cell G.I. Saleem M.A. The podocyte to a in and disease.Nat Rev Nephrol. PubMed Scopus Google Scholar signaling pathways were in response to PAR-1 agonist that PAR-1 agonist treatment significantly increased podocyte motility which is a of increased podocyte in J. M. G. and genetic of J PubMed Scopus Google Scholar PAR-1 activating were with a TRPC6 M.A. Welsh G.I. et is a for calcium signalling in human conditionally podocytes and PubMed Scopus Google Scholar with PAR-1 agonist significantly the and VASP The significantly reduced the signaling response to PAR-1 agonist treatment for JNK, and significantly the JNK, and VASP signaling pathways with treatment significantly reduced JNK, and VASP relapse plasma significantly the JNK, and VASP pathways with remission plasma treatment This signaling response could be significantly reduced using the TRPC6 from patients with NS were via the UK Renal The biopsies were for and increases in the glomerular phosphorylation of VASP were in patients with disease and with with a factor disease, has syndrome, which is a genetic caused by a mutation that FSGS. there was signal in biopsies. A increase in was in patients with patients with patient signal of PAR-1 in in the caused glomerular with that human A construct a C-terminal form of the PAR-1 receptor was the of SV129 mice. This form of the PAR-1 receptor is constitutively active it remains at the membrane activated being to the for We the specific of PAR-1 in the podocyte by the protein in cells The NPHS2 Cre PAR-1Active+/− mice expressed transgenic PAR-1 by Cre were at and were from their control the NPHS2 Cre PAR-1Active+/− mice of were NPHS2 Cre PAR-1Active+/− mice were in renal failure as by their significantly of and with and Furthermore, NPHS2 Cre PAR-1Active+/− mice between the of and were with were at and The NPHS2 Cre PAR-1Active+/− mice glomerular with on both the and there was an of the glomerulus, and this is of the of were of from kidney failure in the is there were of This of is of human FSGS. demonstrated that at 1 of the in the mice as early as the of the glomerular membrane was with of podocyte was found that the PAR-1Active+/− mice showed evidence of the control showed evidence of sclerosis and There was a increase in sclerosis in the PAR-1Active+/− mice at by mouse and a increase in sclerosis the mice were by A podocyte-specific inducible PAR-1Active+/− mouse was generated to to activation of podocyte PAR-1 in mice this The were at between and of with The inducible demonstrated of NS 3 significantly increased of and glomerular on and The of and were to the results in the developmental model, an followed by with and by a a increase in segmental sclerosis in the inducible and there was a increase in VASP phosphorylation in the of the NPHS2 Cre PAR-1Active+/− mice at There were increases in the staining of all in a podocyte signaling was significantly increased in the of the NPHS2 Cre PAR-1Active+/− mice at We that 1 of was a for and the for all the was in the same was on kidney from 3 with to This is the same signaling response as in the human podocytes in vitro in response to both PAR-1 agonist and relapse plasma. is a specific TRPC6 agonist that stimulates calcium in et al.TRPC6 binds to and of channel and podocyte cell and Am Soc Nephrol. 2019; PubMed Scopus Google Scholar This calcium was by treatment with relapse plasma from nephrotic patients with remission plasma To test TRPC6 mediates podocyte signaling pathways of PAR-1, a mouse podocyte cell was with PAR-1 agonist L. Spurney R.F. Knockout of TRPC6 and glomerular injury in Int. 2019; Full Text Full Text PDF PubMed Scopus Google Scholar phosphorylation of and VASP was in mouse podocytes with phosphorylation in TRPC6 podocytes and for there to be evidence of of the podocytes after PAR-1 agonist treatment with evidence of of in the TRPC6 mice is that the TRPC6 podocytes a response to PAR-1 agonist In we crossed the Pod Cre PAR-1Active+/− mouse with a mouse to generate the developmental mouse on a background. The whole-body of TRPC6 can glomerular disease is not to cause et induces activation and cell in response to Full Text Full Text PDF PubMed Scopus Google Scholar The TRPC6 mice crossed with significantly reduced proteinuria with mice TRPC6 in the Pod Cre PAR-1Active+/− significantly improved lifespan These are on a the which are known to be to the control mice on this are proteinuric their on a background. by a in glomerulosclerosis between the and of the PAR-1–active mice and There was a in glomerular signaling in the mice at in the Pod Cre PAR-1Active+/− mice the Pod Cre PAR-1Active+/− TRPC6 mice There was between the in of and signaling pathways and Despite the of the Pod Cre PAR-1Active+/− mice a filtration barrier at with a of the barrier by Pod Cre PAR-1Active+/− TRPC6 mice of the of the filtration barrier the of the podocyte in mice that TRPC6 in the mice caused a of significantly The PAR-1 receptor is in podocytes of the kidney The treatment of human podocytes in vitro with PAR-1 agonist and active human nephrotic plasma leads to activation of the same pathways JNK, and plasma a This suggests that there could be an in relapse plasma that stimulates a signaling response via the PAR-1 work by has that of PAR-1 using can the signaling response of the podocyte to nephrotic M. J. C. et nephrotic plasma signalling pathways of protease-activated receptor Scholar This signaling response is on TRPC6. The podocyte-specific VASP and signaling response in vitro was in human nephrotic patient biopsies. the and VASP signaling was significantly increased in biopsies of patients with likely circulating factor has syndrome, a genetic form of FSGS. to FSGS, it is not caused by a circulating there was evidence of VASP or signaling in this The developmental mouse model the signaling a that and was of human FSGS. The NPHS2 Cre PAR-1Active+/− mice of renal failure of We were able to significantly proteinuria and increase lifespan of PAR-1Active+/− mice by by TRPC6 This TRPC6 as an part of the PAR-1 signaling by a of and in the of These data that the signaling from the PAR-1 receptor to and is via TRPC6. of TRPC6 the phosphorylation of and PAR-1 is The TRPC6 PAR-1 mice the PAR-1 TRPC6 mice of renal This suggests that phosphorylation of both and VASP of PAR-1 activation is A role for TRPC6 in has been In TRPC6 mediates activity and cell of activation of protein kinase in response to et induces activation and cell in response to Full Text Full Text PDF PubMed Scopus Google et receptor 1 activation induces and increases A. PubMed Scopus Google Scholar have PAR-1 activation to have a role in for in the M.A. et receptor 1 mediates renal in PubMed Scopus Google Scholar role in glomerular via the podocyte with known and has previously been We have a receptor that we a circulating factor could work We evidence that the same signaling pathways in vitro in response to PAR-1 agonist and to patient plasma are present and in human nephrotic patient biopsies. the signaling was present in the NPHS2 Cre PAR-1Active+/− mice. that of the PAR-1 receptor can an NS that is to human and have a signaling response that could be used to patients and with circulating factor have shown that mice with PAR-1 agonists a severe phenotype in a M. J. Protease-activated receptor-1 protects against in 2016; PubMed Scopus Google Scholar The PAR-1 antagonist is known to against in Y. Nakano D. Zhang Y. et al.A protease-activated receptor-1 antagonist protects against podocyte injury in a mouse model of nephropathy.J Pharmacol Sci. 2017; 135: 81-88Crossref Scopus (19) Google Scholar the PAR-1 mice not glomerular et podocyte injury is protease-activated receptor Am Soc Nephrol. 2017; PubMed Scopus Google Scholar is not tested in the current is the of PAR-1 receptor Despite et et podocyte injury is protease-activated receptor Am Soc Nephrol. 2017; PubMed Scopus Google Scholar that it is and that are in human podocytes and disease, we evidence that the PAR-1 receptor can pathologic signaling and in human A.D. et disease of the Nephrol. 2010; PubMed Scopus Google Scholar This work not or that the circulating factor(s) is a protease the of the circulating factor disease, leading to the active disease and there is a in activated proteases in the circulation from a of potential biological cascades. These serine proteases and the PAR-1 receptor signaling in the podocytes that generate a This activation of PAR-1, as by our model, has a effect on podocyte and the of the glomerular filtration the barrier and the of are and sclerosis of the massive proteinuria with renal we hypothesized that the Th17 subset of T that are with could be proteases as putative circulating The active of disease has been to an increase in Th17 and an increase in Th17 cells and their has been during active Y. et in patients with nephrotic PubMed Scopus Google Scholar We have shown that human Th17 cell induces the same signaling pathways in podocytes that can be blocked by a PAR-1 JNK, and C.J. Welsh G.I. Chesor M. et al.Human Th17 cells produce a soluble mediator that increases podocyte motility via signalling pathways which mimic PAR-1 activation.Am J Physiol Renal Physiol. 2019; 317: F913-F921Crossref PubMed Scopus (12) Google Scholar In this we present a putative in model of circulating factor disease, which will be in further disease We have the of PAR-1 receptor signaling in the pathogenesis of In the we have a model of circulating factor disease that will be in treatment All the This work was by a UK was by and We for this work from the with cell protease-activated receptor 1 (PAR-1) signaling were with PAR-1 There was phosphorylation of vasodilator-stimulated phosphoprotein or kinase in response to PAR-1 of protein kinase was in response to PAR-1 agonist This shows that the can respond to PAR-1 agonist and that the signaling response is not podocyte suggesting that the studied pathways are for the control is human podocytes with PAR-1 agonist for clinical Protease-activated receptor 1 (PAR-1) active The PAR-1 active construct is of a at the the mouse of the the transgenic PAR-1 receptor is a a and the The and is by Cre the at sites and the Cre the the the and is and the PAR-1–active is In the developmental model, Cre is expressed the control of a in the inducible model, Cre is expressed after treatment with for Pod Cre protease-activated receptor 1 receptor potential cation channel subfamily c member and Pod Cre PAR-1Active+/− TRPC6 knockout (KO) The shown in the are from mice Pod Cre PAR-1Active+/− TRPC6 and 3 Pod Cre PAR-1Active+/− TRPC6 are of the in on a further in the understanding of focal and segmental of focal and segmental glomerular sclerosis are known and a in nephrology. The by et light on previous data the key role of the protease-activated receptor The new evidence is on in in animal and on patient biopsies and a in the understanding of this pathologic PDF

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