Phosphorylation of slit diaphragm proteins NEPHRIN and NEPH1 upon binding of HGF promotes podocyte repair

Abstract

Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain–containing protein tyrosine phosphatase-2–dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity. Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain–containing protein tyrosine phosphatase-2–dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity. With increasing knowledge of podocyte biology, it has become clear that normal glomerular filtration relies heavily on properly functioning podocytes. Although many proteins are involved in podocyte function, NEPHRIN and NEPH1 are key proteins that constitute the building blocks of the slit diaphragm and are critical for podocyte stability and integrity (1Benzing T. Signaling at the slit diaphragm.J. Am. Soc. Nephrol. 2004; 15: 1382-1391Crossref PubMed Scopus (217) Google Scholar, 2Nihalani D. Solanki A.K. Arif E. Srivastava P. Rahman B. Zuo X. Dang Y. Fogelgren B. Fermin D. Gillies C.E. Sampson M.G. Lipschutz J.H. Disruption of the exocyst induces podocyte loss and dysfunction.J. Biol. Chem. 2019; 294: 10104-10119Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). Of importance, mutations or genetic deletions of NEPHRIN and NEPH1 lead to dysfunctional podocytes, which, in turn, result in loss of renal filtration function (2Nihalani D. Solanki A.K. Arif E. Srivastava P. Rahman B. Zuo X. Dang Y. Fogelgren B. Fermin D. Gillies C.E. Sampson M.G. Lipschutz J.H. Disruption of the exocyst induces podocyte loss and dysfunction.J. Biol. Chem. 2019; 294: 10104-10119Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 3Patrakka J. Tryggvason K. Nephrin--a unique structural and signaling protein of the kidney filter.Trends Mol. Med. 2007; 13: 396-403Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 4Donoviel D.B. Freed D.D. Vogel H. Potter D.G. Hawkins E. Barrish J.P. Mathur B.N. Turner C.A. Geske R. Montgomery C.A. Starbuck M. Brandt M. Gupta A. Ramirez-Solis R. Zambrowicz B.P. and in a novel protein with to Biol. PubMed Scopus Google Scholar, M. T. A. H. E. T. P. D. H. slit and show and Am. Soc. Nephrol. 13: PubMed Scopus Google Scholar). Although that the extracellular domains of NEPHRIN and NEPH1 a function their integrity to the slit their domains were to signaling to actin in podocytes (1Benzing T. Signaling at the slit diaphragm.J. Am. Soc. Nephrol. 2004; 15: 1382-1391Crossref PubMed Scopus (217) Google Scholar, E. B. D. diaphragm protein and novel for of podocytes glomerular Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, T. slit signaling to podocyte Nephrol. PubMed Scopus Google Scholar). This that NEPHRIN and NEPH1 that is by phosphorylation to of However, the phosphorylation of these proteins remain unknown. the knowledge of a specific that induces phosphorylation of these the function to their extracellular domains structural of the slit In this a receptor-based phosphorylation was in engagement of the extracellular domains of NEPHRIN and NEPH1 with the hepatocyte growth factor (HGF) induced their We SH2 domain–containing protein tyrosine as a novel for these proteins. Furthermore, using in vitro and ex vivo of injury demonstrate in to is in an phosphorylation of NEPHRIN and NEPH1 to actin and podocyte repair. To novel proteins we experiments. proteins that with NEPH1 were by of the proteins was using the and proteins were a of the was of these proteins and to NEPH1 R. M. A. P. with and tyrosine phosphorylation and is for in of podocyte Biol. PubMed Scopus Google recombinant purified proteins we demonstrated a the of NEPH1 and NEPHRIN in a R. M. A. P. with and tyrosine phosphorylation and is for in of podocyte Biol. PubMed Scopus Google we NEPH1 phosphorylation to phosphorylation of NEPH1 can be by the of with E. B. D. diaphragm protein and novel for of podocytes glomerular Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. R. D. D.B. with to a that induces actin Biol. 2007; PubMed Scopus Google or by with In we show that NEPH1 and To NEPH1 is a for we the binding of NEPH1 with a T. D. of to of protein tyrosine A. PubMed Scopus Google Scholar, M. D. tyrosine of through of Biol. PubMed Google Scholar). is a H. T. K. E. an receptor tyrosine signaling by and the Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google and the a higher to phosphorylated NEPH1 than the a the proteins. of the phosphorylated of a protein of the can be to the of to be a for we that the phosphorylation of NEPH1 be in the of We podocytes that are to NEPH1 and the of NEPH1 phosphorylation to growth using a E. B. D. diaphragm protein and novel for of podocytes glomerular Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. Arif E. Solanki A.K. Srivastava P. M.G. Lipschutz J.H. D. and in podocytes podocyte injury and glomerular filtration PubMed Scopus Google Scholar, A.K. E. Arif E. A. Srivastava P. H. M. C.E. in a slit diaphragm 2019; Full Text Full Text PDF PubMed Scopus Google Scholar). Phosphorylation of NEPH1 was in podocytes with HGF in binding of to NEPH1. We that and the binding of to NEPH1 and and Although a report that of with NEPHRIN phosphorylation to on a of on NEPHRIN was R. M. A. P. with and tyrosine phosphorylation and is for in of podocyte Biol. PubMed Scopus Google Scholar). is a we that NEPH1 and To we NEPH1 or NEPHRIN with in and we the and cultured podocytes (2Nihalani D. Solanki A.K. Arif E. Srivastava P. Rahman B. Zuo X. Dang Y. Fogelgren B. Fermin D. Gillies C.E. Sampson M.G. Lipschutz J.H. Disruption of the exocyst induces podocyte loss and dysfunction.J. Biol. Chem. 2019; 294: 10104-10119Abstract Full Text Full Text PDF PubMed Scopus (9) Google with and the proteins with their the phosphorylated NEPHRIN or NEPH1 were with purified recombinant in a dephosphorylation of NEPHRIN and NEPH1 were in the of we purified or domains with purified that was on phosphorylated NEPHRIN and NEPH1 proteins were by and with purified recombinant in the dephosphorylation of these proteins in a fashion and these demonstrate that is a for NEPHRIN and NEPH1. HGF is an of the mesenchymal epithelial transition receptor and J. growth factor (HGF) through to PubMed Scopus Google Scholar, M. M. tyrosine is for of extracellular and epithelial the receptor tyrosine Biol. PubMed Scopus Google Scholar). the of NEPH1 and NEPHRIN phosphorylation by HGF is novel and biological and clinical we this using NEPHRIN and NEPH1 were with HGF in and the were using with and that NEPHRIN and NEPH1 were phosphorylated in the of HGF In addition, we a were cultured on the and or were cultured on the of the phosphorylation of NEPHRIN and NEPH1 was the demonstrate that NEPHRIN and NEPH1 phosphorylation can be induced in the of HGF. HGF is to be a of the receptor K. H. H. K. in and Scholar, T. of hepatocyte growth signaling Google we was or involved in NEPHRIN and NEPH1 We first used a receptor a and of in vitro and with PubMed Scopus Google Scholar). to and was to the HGF-induced phosphorylation of NEPHRIN and NEPH1 and that was for NEPHRIN and NEPH1 To in NEPHRIN and NEPH1 we experiments. we NEPHRIN or NEPH1 and a of the by the of HGF to the in phosphorylation of NEPHRIN or NEPH1 and that HGF induces NEPHRIN and NEPH1 phosphorylation of the Finally, we used the to by of NEPHRIN and NEPH1 in these the we demonstrated that HGF phosphorylation of NEPHRIN or NEPH1 in the of the receptor and HGF as a it with NEPHRIN and NEPH1. To we first HGF with NEPHRIN or NEPH1 and their binding through This that NEPHRIN and NEPH1 with HGF To the is we experiments. using and a molecular modeling with the HGF-binding of the receptor as a we binding regions in the extracellular domains of NEPHRIN and NEPH1. in NEPH1 and in NEPHRIN as HGF domains regions were peptides these regions were and in a peptides were and with recombinant HGF. demonstrated that HGF with NEPH1 and NEPHRIN with NEPHRIN To these recombinant NEPHRIN and NEPH1 proteins were in we used an and the to and extracellular purified recombinant NEPHRIN and NEPH1 proteins were with purified HGF and surface plasma resonance was to their a of HGF with NEPHRIN and NEPH1 and Of the of HGF with NEPH1 was in the the with NEPHRIN was in the that HGF NEPHRIN with a higher and To the binding of HGF for NEPHRIN and NEPH1 with we a HGF was on in an and HGF binding to or NEPHRIN was in the of increasing of HGF-binding NEPH1 and NEPHRIN peptides and Although NEPH1 the binding of NEPH1 to HGF and NEPHRIN the binding of NEPHRIN to HGF, these peptides to the binding of HGF to that the HGF-binding for NEPH1 and NEPHRIN with the HGF-binding for HGF-binding for with the HGF-binding for NEPH1 and for the HGF was on the of an and binding to and and in with NEPH1 and NEPHRIN was of the are with NEPH1 or NEPHRIN are as and were using a plasma of HGF in K. H. H. K. in and Scholar, T. of hepatocyte growth signaling Google Scholar, T. H. Y. H. T. E. hepatocyte growth factor in in PubMed Scopus Google Scholar, H. M. H. H. T. T. M. A. Y. of hepatocyte growth factor is a for the of in PubMed Scopus Google Scholar). HGF as an for we that HGF is involved in podocyte repair To cultured podocytes NEPH1 were injured with in actin E. Solanki A.K. Srivastava P. Rahman B. M.G. R. P. Lipschutz J.H. protein growth and in 2019; Full Text Full Text PDF PubMed Scopus Google Scholar). of recombinant HGF in of the podocyte actin cytoskeleton and the cultured podocytes of of the NEPH1 or of NEPH1 podocytes and the HGF-induced and In we a with podocytes, HGF-induced of podocytes injury that was by NEPHRIN and To study HGF-induced in an ex vivo model Drosophila were Drosophila were to and with to the Drosophila of to the of is a of the that is for and of a slit in H. R. E. and the Drosophila of and and of a slit in PubMed Scopus Google Scholar). We a in protein in in to was by HGF and Furthermore, the NEPH1 and NEPHRIN the of HGF that is in a and In NEPHRIN with HGF, the HGF. these demonstrate that HGF-induced NEPH1 and NEPHRIN phosphorylation in the of podocytes a report that the receptor is involved in podocyte P. Y. growth factor signaling podocyte injury and Full Text Full Text PDF PubMed Scopus Google we to HGF-induced NEPHRIN and NEPH1 phosphorylation as an for podocyte cultured podocytes and were with NEPH1 or NEPHRIN and injured with by HGF-induced Although was in podocytes, HGF was in podocytes NEPH1 or NEPHRIN was and in vitro and ex vivo that HGF, and in podocyte repair that podocyte repair are important for the of to NEPHRIN and NEPH1 are slit diaphragm proteins that are critical for podocyte function, and genetic in these proteins lead to renal in and T. slit signaling to podocyte Nephrol. PubMed Scopus Google Scholar, A.K. E. Arif E. A. Srivastava P. H. M. C.E. in a slit diaphragm 2019; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. M. Tryggvason K. K. A. is in and of and PubMed Google Scholar). that the extracellular domains of NEPHRIN and NEPH1 constitute the structural of the slit maintaining structural integrity J. J. M. Tryggvason K. K. A. is in and of and PubMed Google Scholar, E. Arif E. P. R. D. injury to kidney induces glomerular podocyte and of slit diaphragm proteins and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. A. D. K. engagement in and actin PubMed Scopus Google Scholar). is that NEPHRIN and NEPH1 signaling E. B. D. diaphragm protein and novel for of podocytes glomerular Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. R. D. D.B. with to a that induces actin Biol. 2007; PubMed Scopus Google Scholar, C.E. signaling in the of at the slit diaphragm and PubMed Scopus Google the mechanisms for remain unknown. In this study we that NEPHRIN and NEPH1 can be phosphorylated in a fashion by HGF and by the and genetic that tyrosine phosphorylation of the NEPHRIN and NEPH1 domains in podocytes to signaling events that to actin R. M. A. P. with and tyrosine phosphorylation and is for in of podocyte Biol. PubMed Scopus Google Scholar, M. K. R. P. by a protein that and PubMed Scopus Google Scholar). However, the that the phosphorylation of NEPHRIN and NEPH1 E. B. D. diaphragm protein and novel for of podocytes glomerular Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. R. D. D.B. with to a that induces actin Biol. 2007; PubMed Scopus Google Scholar, E. Arif E. P. R. D. injury to kidney induces glomerular podocyte and of slit diaphragm proteins and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). show that as a that NEPH1 and injury to podocytes is to NEPHRIN and NEPH1 phosphorylation J. Tryggvason K. 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Solanki A. D. D. of the and the of Biol. PubMed Scopus (9) Google Scholar). was the and the of the extracellular domains of NEPHRIN and NEPH1. of the was by and and were by of the to the podocytes and were with these and of and was by were in of for the of for the of and the of podocytes to (2Nihalani D. Solanki A.K. Arif E. Srivastava P. Rahman B. Zuo X. Dang Y. Fogelgren B. Fermin D. Gillies C.E. Sampson M.G. Lipschutz J.H. Disruption of the exocyst induces podocyte loss and dysfunction.J. Biol. Chem. 2019; 294: 10104-10119Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). To study the in vitro and of the interactions of NEPH1 and NEPHRIN with HGF, the was the was used to and recombinant the of NEPH1 or NEPHRIN was by events in the and was and was by and the was and the were used to for protein were to the protein and proteins were purified using a proteins were using on the as E. P. Solanki A. D. D. of the and the of Biol. 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