Phosphorylation of slit diaphragm proteins NEPHRIN and NEPH1 upon binding of HGF promotes podocyte repair
2021; Elsevier BV; Volume: 297; Issue: 3 Linguagem: Inglês
10.1016/j.jbc.2021.101079
ISSN1083-351X
AutoresAshish K. Solanki, Ehtesham Arif, Pankaj Srivastava, Christopher M. Furcht, Bushra Rahman, Pei Wen, Avinash Singh, Lawrence B. Holzman, Wayne R. Fitzgibbon, Milos N. Budisavljevic, Glenn P. Lobo, Sang‐Ho Kwon, Zhe Han, Matthew J. Lazzara, Joshua H. Lipschutz, Deepak Nihalani,
Tópico(s)Tuberous Sclerosis Complex Research
ResumoPhosphorylation (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. et al.Proteinuria and perinatal lethality in mice lacking NEPH1, a novel protein with homology to NEPHRIN.Mol. Cell. Biol. 2001; 21: 4829-4836Crossref PubMed Scopus (341) Google Scholar, 5Rantanen M. Palmén T. Pätäri A. Ahola H. Lehtonen S. Aström E. Floss T. Vauti F. Wurst W. Ruiz P. Kerjaschki D. Holthöfer H. Nephrin TRAP mice lack slit diaphragms and show fibrotic glomeruli and cystic tubular lesions.J. Am. Soc. Nephrol. 2002; 13: 1586-1594Crossref PubMed Scopus (100) Google Scholar). Although several studies suggest that the extracellular domains of NEPHRIN and NEPH1 have a structure-based function where their spatial arrangement provides integrity to the slit diaphragm, their intracellular domains were shown to initiate signaling cascades leading to actin cytoskeletal changes in podocytes (1Benzing T. Signaling at the slit diaphragm.J. Am. Soc. Nephrol. 2004; 15: 1382-1391Crossref PubMed Scopus (217) Google Scholar, 6Arif E. Rathore Y.S. Kumari B. Ashish F. Wong H.N. Holzman L.B. Nihalani D. Slit diaphragm protein Neph1 and its signaling: A novel therapeutic target for protection of podocytes against glomerular injury.J. Biol. Chem. 2014; 289: 9502-9518Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 7Huber T.B. Benzing T. The slit diaphragm: A signaling platform to regulate podocyte function.Curr. Opin. Nephrol. Hypertens. 2005; 14: 211-216Crossref PubMed Scopus (190) Google Scholar). This suggests that NEPHRIN and NEPH1 undergo activation that is driven by phosphorylation prior to initiation of downstream signaling. However, the mechanism(s) behind phosphorylation of these proteins remain unknown. Moreover, without the knowledge of a specific ligand that induces phosphorylation of these proteins, the primary function assigned to their extracellular domains remains structural organization of the slit diaphragm. In this study, a receptor-based phosphorylation mechanism was identified in which engagement of the extracellular domains of NEPHRIN and NEPH1 with the ligand hepatocyte growth factor (HGF) induced their phosphorylation. We identified SH2 domain–containing protein tyrosine phosphatase-2 (SHP-2) as a novel phosphatase for these proteins. Furthermore, functional studies using in vitro and ex vivo models of injury demonstrate that, in response to injury, recovery is initiated in an HGF-dependent manner, which involves ligand-based phosphorylation of NEPHRIN and NEPH1 leading to actin cytoskeletal reorganization and podocyte repair. To identify novel NEPH1-binding proteins we performed coimmunoprecipitation experiments. The proteins that immunoprecipitated with NEPH1 were analyzed by mass spectrometry. Analysis of the Neph1-binding proteins was performed using the Scaffold proteomics software, and 123 proteins were identified. SHP-2, a product of the tyrosine-protein phosphatase non-receptor type 11 (PTPN11) gene, was one of these proteins and had previously been linked to NEPH1 (8Verma R. Venkatareddy M. Kalinowski A. Patel S.R. Salant D.J. Garg P. Shp2 associates with and enhances nephrin tyrosine phosphorylation and is necessary for foot process spreading in mouse models of podocyte injury.Mol. Cell. Biol. 2016; 36: 596-614Crossref PubMed Scopus (25) Google Scholar) (Fig. 1A). By mixing recombinant purified proteins we demonstrated a direct interaction between the cytoplasmic domain of NEPH1 and SHP-2 (Fig. 1B). Since SHP-2 binds NEPHRIN in a phosphorylation-dependent manner (8Verma R. Venkatareddy M. Kalinowski A. Patel S.R. Salant D.J. Garg P. Shp2 associates with and enhances nephrin tyrosine phosphorylation and is necessary for foot process spreading in mouse models of podocyte injury.Mol. Cell. Biol. 2016; 36: 596-614Crossref PubMed Scopus (25) Google Scholar), we investigated whether NEPH1 phosphorylation also enhanced SHP-2 binding. Similar to NEPHRIN, phosphorylation of NEPH1 can be accomplished either by the treatment of cells (stable HEK293 NEPH1-overexpressing cells) with pervanadate (6Arif E. Rathore Y.S. Kumari B. Ashish F. Wong H.N. Holzman L.B. Nihalani D. Slit diaphragm protein Neph1 and its signaling: A novel therapeutic target for protection of podocytes against glomerular injury.J. Biol. Chem. 2014; 289: 9502-9518Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 9Garg P. Verma R. Nihalani D. Johnstone D.B. Holzman L.B. Neph1 cooperates with nephrin to transduce a signal that induces actin polymerization.Mol. Cell. Biol. 2007; 27: 8698-8712Crossref PubMed Scopus (118) Google Scholar) or by coexpressing with FYN kinase. In Figure 1C we show that FYN kinase significantly increases NEPH1 and SHP-2 binding. To further determine if NEPH1 is a substrate for SHP-2, we tested the binding of NEPH1 with a substrate trapping SHP-2DM mutant (10Flint A.J. Tiganis T. Barford D. Tonks N.K. Development of "substrate-trapping" mutants to identify physiological substrates of protein tyrosine phosphatases.Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (673) Google Scholar, 11Timmerman I. Hoogenboezem M. Bennett A.M. Geerts D. Hordijk P.L. van Buul J.D. The tyrosine phosphatase SHP2 regulates recovery of endothelial adherens junctions through control of beta-catenin phosphorylation.Mol. Biol. Cell. 2012; 23: 4212-4225Crossref PubMed Google Scholar). SHP-2 is a phosphatase (12Hanafusa H. Torii S. Yasunaga T. Matsumoto K. Nishida E. Shp2, an SH2-containing protein-tyrosine phosphatase, positively regulates receptor tyrosine kinase signaling by dephosphorylating and inactivating the inhibitor Sprouty.J. Biol. Chem. 2004; 279: 22992-22995Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), and the substrate trapping SHP-2DM mutant displayed a much higher ability to bind phosphorylated NEPH1 than the wildtype SHP-2 (Fig. 1C), indicating a functional interaction between the two proteins. Under physiologic conditions, detection of the phosphorylated form of a protein (typically only 5%–10% of the total protein) can be challenging owing to the presence of phosphatases. Since SHP-2 appeared to be a potent phosphatase for NEPH1, we hypothesized that the phosphorylation (ligand-induced) of endogenous NEPH1 would be suppressed in the presence of SHP-2. We therefore generated stable SHP-2 knockdown (KD) human podocytes that are known to endogenously express NEPH1 and tested the level of NEPH1 phosphorylation following exposure to various growth factors using a NEPH1-specific phosphoantibody (6Arif E. Rathore Y.S. Kumari B. Ashish F. Wong H.N. Holzman L.B. Nihalani D. Slit diaphragm protein Neph1 and its signaling: A novel therapeutic target for protection of podocytes against glomerular injury.J. Biol. Chem. 2014; 289: 9502-9518Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 13Sagar A. Arif E. Solanki A.K. Srivastava P. Janech M.G. Kim S.H. Lipschutz J.H. Kwon S.H. Ashish Nihalani D. Targeting Neph1 and ZO-1 protein-protein interaction in podocytes prevents podocyte injury and preserves glomerular filtration function.Sci. Rep. 2017; 7: 12047Crossref PubMed Scopus (16) Google Scholar, 14Solanki A.K. Widmeier E. Arif E. Sharma S. Daga A. Srivastava P. Kwon S.H. Hugo H. Nakayama M. Mann N. Majmundar A.J. Tan W. Gee H.Y. Sadowski C.E. Rinat C. et al.Mutations in KIRREL1, a slit diaphragm component, cause steroid-resistant nephrotic syndrome.Kidney Int. 2019; 96: 883-889Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). Phosphorylation of endogenous NEPH1 was only visible in SHP-2 knockdown podocytes treated with HGF (Fig. 1D). As shown in Figure 1C, FYN kinase increases binding of SHP-2 to NEPH1. We next confirmed that FYN and pervanadate increased the binding of SHP-2 to both NEPH1 and NEPHRIN. (Fig. 2, A and B). Although a previous report suggested that coexpression of SHP-2 with NEPHRIN enhanced its phosphorylation owing to its effect on FYN activation, a direct effect of SHP-2 on NEPHRIN was not evaluated (8Verma R. Venkatareddy M. Kalinowski A. Patel S.R. Salant D.J. Garg P. Shp2 associates with and enhances nephrin tyrosine phosphorylation and is necessary for foot process spreading in mouse models of podocyte injury.Mol. Cell. Biol. 2016; 36: 596-614Crossref PubMed Scopus (25) Google Scholar). Since SHP-2 is a phosphatase, we hypothesized that SHP-2 directly dephosphorylates NEPH1 and NEPHRIN. To test this, we coexpressed NEPH1 or NEPHRIN with FYN in HEK293 cells and we also treated the NEPH1- and NEPHRIN-expressing stable 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 Scholar) with pervanadate and then immunoprecipitated the proteins with their respective antibodies. The immunoprecipitated complexes containing the phosphorylated NEPHRIN or NEPH1 were incubated with purified active recombinant SHP-2 in a phosphatase buffer. Significant dephosphorylation of both NEPHRIN and NEPH1 were noted in the presence of SHP-2 (Fig. 2, C–F). Subsequently, we incubated purified glutathione S-transferase (GST)-NEPH1 or GST-NEPHRIN cytoplasmic domains with purified active HIS-FYN (0.5 μg) that was immobilized on Ni-NTA beads. The resulting phosphorylated NEPHRIN and NEPH1 proteins were removed by centrifugation and incubated with purified active recombinant SHP-2 phosphatase, which resulted in the dephosphorylation of these proteins in a time-dependent fashion (Fig. 2, G and H). Collectively, these results demonstrate that SHP-2 is a phosphatase for NEPHRIN and NEPH1. HGF is an established activator of the mesenchymal epithelial transition (MET) receptor and SHP-2 (15Li J. Reed S.A. Johnson S.E. Hepatocyte growth factor (HGF) signals through SHP2 to regulate primary mouse myoblast proliferation.Exp. Cell. Res. 2009; 315: 2284-2292Crossref PubMed Scopus (25) Google Scholar, 16Maroun C.R. Naujokas M.A. Holgado-Madruga M. Wong A.J. Park M. The tyrosine phosphatase SHP-2 is required for sustained activation of extracellular signal-regulated kinase and epithelial morphogenesis downstream from the met receptor tyrosine kinase.Mol. Cell. Biol. 2000; 20: 8513-8525Crossref PubMed Scopus (235) Google Scholar). Since the concept of NEPH1 and NEPHRIN phosphorylation by HGF is novel and may have significant biological and clinical implications, we investigated this further using two independent techniques. First, NEPHRIN and NEPH1 were coexpressed with HGF in HEK293 cells and the cell lysates were probed using Western blot with NEPHRIN- and NEPH1-specific phosphoantibodies, which showed that NEPHRIN and NEPH1 were phosphorylated in the presence of HGF (Fig. 3A). In addition, we performed a Transwell assay where HGF-overexpressing HEK293 cells were cultured on the Transwell filter and NEPHRIN- or NEPH1-overexpressing HEK293 cells were cultured on the bottom of the wells (Fig. 3B). Significant phosphorylation of NEPHRIN and NEPH1 was noted only when the Transwell filters contained HGF-expressing cells (Fig. 3B). These results demonstrate that NEPHRIN and NEPH1 phosphorylation can be induced in the presence of HGF. Since HGF is known to be a potent activator of the MET receptor (17Matsumoto K. Funakoshi H. Takahashi H. Sakai K. HGF-met pathway in regeneration and drug discovery.Biomedicine. 2014; 2: 275-300Google Scholar, 18Kato T. Biological roles of hepatocyte growth factor-Met signaling from genetically modified animals.Biomed. Rep. 2017; 7: 495-503PubMed Google Scholar), we investigated if MET was directly or indirectly involved in NEPHRIN and NEPH1 phosphorylation. We first used a MET receptor inhibitor (Crizotinib) (19Ayoub N.M. Al-Shami K.M. Alqudah M.A. Mhaidat N.M. Crizotinib, a MET inhibitor, inhibits growth, migration, and invasion of breast cancer cells in vitro and synergizes with chemotherapeutic agents.Onco Targets Ther. 2017; 10: 4869-4883Crossref PubMed Scopus (16) Google Scholar). Crizotinib when added to NEPHRIN- and NEPH1-overexpressing HEK-293 cells was unable to attenuate the HGF-induced phosphorylation of NEPHRIN and NEPH1 (Fig. 4, A and B), indicating that MET was not required for NEPHRIN and NEPH1 phosphorylation. To further rule out MET involvement in NEPHRIN and NEPH1 phosphorylation, we performed two additional experiments. First, we generated HEK293 cells overexpressing NEPHRIN or NEPH1 and containing a stable knockdown of the MET receptor, followed by the addition of HGF to induce phosphorylation. As shown, the MET-KD cells displayed no change in HGF-mediated phosphorylation of NEPHRIN or NEPH1 (Fig. 4, C and D), suggesting that HGF induces NEPHRIN and NEPH1 phosphorylation independent of the MET receptor. Finally, we used the CRISPR-Cas system to generate stable MET knockout HEK293 cells (Fig. 4E) followed by overexpression of NEPHRIN and NEPH1 in these cells. Using the Transwell assay we demonstrated that HGF could still induce phosphorylation of NEPHRIN or NEPH1 even in the absence of the MET receptor (Fig. 4, F and G). If HGF acts as a ligand then it should interact extracellularly with NEPHRIN and NEPH1. To test this, we first coexpressed HGF with NEPHRIN or NEPH1 and evaluated their binding through coimmunoprecipitation. This showed that both NEPHRIN and NEPH1 interacted with HGF (Fig. 5A). To determine whether the interaction is direct, we performed two independent experiments. First, using sequence homology and a molecular modeling approach, with the HGF-binding site of the MET receptor as a template, we identified potential binding regions in the extracellular domains of NEPHRIN and NEPH1. These analyses revealed IgG3 in NEPH1 and IgG2 in NEPHRIN as putative HGF interacting domains (Fig. S1). These regions were highly conserved among multiple species (Fig. S2). The peptides from these regions were synthesized and tested in a dot blot assay, where peptides were immobilized and probed with recombinant HGF. The results demonstrated that HGF interacted with NEPH1 Peptide-1 and NEPHRIN Peptide-1 but not with NEPHRIN Peptide-2 (Fig. 5B). To further validate these results recombinant mammalian NEPHRIN and NEPH1 proteins were generated. As shown in Figure 5C, we used an SF9 insect cell line and the Baculoviral Expression system to express mammalian His-FLAG-NEPH1- full length (FL) and His-FLAG-NEPHRIN- extracellular domain (ECD). The purified recombinant NEPHRIN and NEPH1 proteins were mixed with commercially obtained purified HGF (Fig. 5C), and surface plasma resonance was employed to evaluate their binding. The results showed a concentration-dependent direct interaction of HGF with NEPHRIN and NEPH1 (Fig. 5, D and E). Of interest, the interaction of HGF with NEPH1 was in the mid nanomolar range (KD = 278 nM) (steady-state analysis, Fig. 5D, lower panel), whereas the interaction with NEPHRIN was in the low nanomolar range (KD = 2.4 nM) (steady-state analysis, Fig. 5E, lower panel), indicating that HGF binds NEPHRIN with a much higher affinity (Fig. 5, D and E). To further test if the binding sites of HGF for NEPHRIN and NEPH1 overlap with MET we performed a competitive ELISA. HGF was immobilized on individual wells in an ELISA plate and HGF binding to MET, NEPH1, or NEPHRIN was evaluated in the presence of increasing concentrations of HGF-binding NEPH1 and NEPHRIN peptides (Fig. 6, A and B). Although NEPH1 Peptide-1 inhibited the binding of NEPH1 to HGF and NEPHRIN Peptide-1 inhibited the binding of NEPHRIN to HGF, these peptides failed to inhibit the binding of HGF to MET. These data confirm that the HGF-binding sites for NEPH1 and NEPHRIN do not overlap with the HGF-binding site for MET.Figure 6The HGF-binding site for MET does not overlap with the HGF-binding sites for NEPH1 and NEPHRIN. A, schematic for the competitive ELISA. HGF was immobilized on the wells of an ELISA plate, and its binding to NEPH1, NEPHRIN, and MET alone and in combination with NEPH1 peptide-1 and NEPHRIN peptide-1 was analyzed. B, quantification of the data. All comparisons are with NEPH1 or NEPHRIN alone. Data are presented as mean ± SEM, and p-values were calculated using a two-tailed Student's t test. ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p < 0.0001.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Increased plasma levels of HGF have been reported in various diseases (17Matsumoto K. Funakoshi H. Takahashi H. Sakai K. HGF-met pathway in regeneration and drug discovery.Biomedicine. 2014; 2: 275-300Google Scholar, 18Kato T. Biological roles of hepatocyte growth factor-Met signaling from genetically modified animals.Biomed. Rep. 2017; 7: 495-503PubMed Google Scholar, 20Okamoto T. Takatsuka H. Fujimori Y. Wada H. Iwasaki T. Kakishita E. Increased hepatocyte growth factor in serum in acute graft-versus-host disease.Bone Marrow Transplant. 2001; 28: 197-200Crossref PubMed Scopus (38) Google Scholar, 21Yamagamim H. Moriyama M. Matsumura H. Aoki H. Shimizu T. Saito T. Kaneko M. Shioda A. Tanaka N. Arakawa Y. Serum concentrations of human hepatocyte growth factor is a useful indicator for predicting the occurrence of hepatocellular carcinomas in C-viral chronic liver diseases.Cancer. 2002; 95: 824-834Crossref PubMed Scopus (80) Google Scholar). Since HGF functions as an injury-induced effector for tissue repair, we hypothesized that HGF is involved in podocyte repair following injury. To test this, cultured human podocytes overexpressing NEPH1 were injured with protamine sulfate (PS), which results in severe actin cytoskeletal disorganization (22Arif E. Solanki A.K. Srivastava P. Rahman B. Tash B.R. Holzman L.B. Janech M.G. Martin R. Knolker H.J. Fitzgibbon W.R. Deng P. Budisavljevic M.N. Syn W.K. Wang C. Lipschutz J.H. et al.The motor protein Myo1c regulates transforming growth factor-beta-signaling and fibrosis in podocytes.Kidney Int. 2019; 96: 139-158Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Addition of recombinant HGF resulted in significant recovery of the podocyte actin cytoskeleton (Fig. 7, A and B). Since the cultured podocytes express measurable amounts of endogenous NEPH1, but not NEPHRIN, addition of the NEPH1 peptide (that binds HGF), or use of NEPH1 knockdown podocytes (Fig. S3, A and B), blocked the HGF-induced recovery (Fig. 7, A and B). In parallel, we performed a similar experiment with NEPHRIN-overexpressing podocytes, which also showed HGF-induced recovery of podocytes from injury that was similarly blocked by NEPHRIN Peptide-1 (Fig. S4, A and B). To further study HGF-induced recovery in an ex vivo model system, Drosophila nephrocytes were used. Nephrocytes isolated from Drosophila were subjected to PS treatment and stained with antibody to sticks-and-stones (SNS, the Drosophila ortholog of NEPHRIN) to evaluate the extent of injury. SNS is a member of the immunoglobulin superfamily that is essential for myoblast fusion and formation of a slit diaphragm-like structure in insect nephrocytes (23Zhuang S. Shao H. Guo F. Trimble R. Pearce E. Abmayr S.M. Sns and Kirre, the Drosophila orthologs of Nephrin and Neph1, direct adhesion, fusion and formation of a slit diaphragm-like structure in insect nephrocytes.Development. 2009; 136: 2335-2344Crossref PubMed Scopus (113) Google Scholar). We observed a significant decrease in SNS protein in nephrocytes in response to injury, which was rescued by HGF treatment (Fig. 7, C and D). Furthermore, the HGF-interacting peptides, NEPH1 Peptide-1 and NEPHRIN Peptide-1, blocked the rescue effect of HGF indicating that recovery is mediated in a NEPH1- and NEPHRIN-dependent manner. In contrast, NEPHRIN Peptide-2, which does not interact with HGF, did not block the recovery following HGF. Collectively, these results demonstrate that HGF-induced NEPH1 and NEPHRIN phosphorylation participate in the recovery of podocytes following injury. Since a previous report suggested that the MET receptor is involved in podocyte recovery (24Dai C. Saleem M.A. Holzman L.B. Mathieson P. Liu Y. Hepatocyte growth factor signaling ameliorates podocyte injury and proteinuria.Kidney Int. 2010; 77: 962-973Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar), we wanted to know if HGF-induced NEPHRIN and NEPH1 phosphorylation could serve as an alternate mechanism for podocyte recovery from injury. Therefore, CRISPR-Cas9-mediated MET knockout cultured podocytes (Fig. 8, A and B) were transfected with lentiviral particles expressing either NEPH1 or NEPHRIN and injured with PS followed by HGF-induced recovery. Although recovery was minimal in MET knockout podocytes, significant recovery following HGF addition was observed in MET knockout podocytes where either NEPH1 or NEPHRIN was overexpressed (Fig. 8, C and D). Thus, our in vitro and ex vivo results indicate that NEPHRIN, NEPH1, HGF, and SHP-2 participate in podocyte repair following injury. Determining factors that regulate podocyte repair are critically important for the identification of much needed therapies to treat podocytopathies. NEPHRIN and NEPH1 are slit diaphragm proteins that are critical for podocyte function, and genetic defects in these proteins lead to renal dysfunction in mice and humans (7Huber T.B. Benzing T. The slit diaphragm: A signaling platform to regulate podocyte function.Curr. Opin. Nephrol. Hypertens. 2005; 14: 211-216Crossref PubMed Scopus (190) Google Scholar, 14Solanki A.K. Widmeier E. Arif E. Sharma S. Daga A. Srivastava P. Kwon S.H. Hugo H. Nakayama M. Mann N. Majmundar A.J. Tan W. Gee H.Y. Sadowski C.E. Rinat C. et al.Mutations in KIRREL1, a slit diaphragm component, cause steroid-resistant nephrotic syndrome.Kidney Int. 2019; 96: 883-889Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 25Hulkko J. Patrakka J. Lal M. Tryggvason K. Hultenby K. Wernerson A. Neph1 is reduced in primary focal segmental glomerulosclerosis, minimal change nephrotic syndrome, and corresponding experimental animal models of adriamycin-induced nephropathy and puromycin aminonucleoside nephrosis.Nephron Extra. 2014; 4: 146-154Crossref PubMed Google Scholar). The prevailing dogma indicates that the extracellular domains of NEPHRIN and NEPH1 constitute the structural framework of the slit diaphragm, thereby maintaining its structural integrity (25Hulkko J. Patrakka J. Lal M. Tryggvason K. Hultenby K. Wernerson A. Neph1 is reduced in primary focal segmental glomerulosclerosis, minimal change nephrotic syndrome, and corresponding experimental animal models of adriamycin-induced nephropathy and puromycin aminonucleoside nephrosis.Nephron Extra. 2014; 4: 146-154Crossref PubMed Google Scholar, 26Wagner M.C. Rhodes G. Wang E. Pruthi V. Arif E. Saleem M.A. Wean S.E. Garg P. Verma R. Holzman L.B. Gattone V. Molitoris B.A. Nihalani D. Ischemic injury to kidney induces glomerular podocyte effacement and dissociation of slit diaphragm proteins Neph1 and ZO-1.J. Biol. Chem. 2008; 283: 35579-35589Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 27Verma R. Kovari I. Soofi A. Nihalani D. Patrie K. Holzman L.B. Nephrin ectodomain engagement results in Src kinase activation, nephrin phosphorylation, Nck recruitment, and actin polymerization.J. Clin. Invest. 2006; 116: 1346-1359Crossref PubMed Scopus (269) Google Scholar). It is known that NEPHRIN and NEPH1 transduce outside-in signaling (6Arif E. Rathore Y.S. Kumari B. Ashish F. Wong H.N. Holzman L.B. Nihalani D. Slit diaphragm protein Neph1 and its signaling: A novel therapeutic target for protection of podocytes against glomerular injury.J. Biol. Chem. 2014; 289: 9502-9518Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 9Garg P. Verma R. Nihalani D. Johnstone D.B. Holzman L.B. Neph1 cooperates with nephrin to transduce a signal that induces actin polymerization.Mol. Cell. Biol. 2007; 27: 8698-8712Crossref PubMed Scopus (118) Google Scholar, 28Martin C.E. Jones N. Nephrin signaling in the podocyte: An updated view of signal regulation at the slit diaphragm and beyond.Front. Endocrinol. (Lausanne). 2018; 9: 302Crossref PubMed Scopus (60) Google Scholar); however, the mechanisms for such transduction remain unknown. In this study we present evidence that NEPHRIN and NEPH1 have receptor-like properties, where they can be phosphorylated (activated) in a ligand-based fashion by HGF and dephosphorylated (deactivated) by the phosphatase SHP-2. Previous biochemical and genetic studies showed that Src family kinases mediated tyrosine phosphorylation of the NEPHRIN and NEPH1 cytoplasmic domains in podocytes to induce subsequent downstream signaling events that led to actin cytoskeletal reorganization (8Verma R. Venkatareddy M. Kalinowski A. Patel S.R. Salant D.J. Garg P. Shp2 associates with and enhan
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