The Experts below are selected from a list of 4494 Experts worldwide ranked by ideXlab platform

Hiroshi Kawachi - One of the best experts on this subject based on the ideXlab platform.

  • Nephrin ephrin b1 na h exchanger regulatory factor 2 ezrin actin axis is critical in podocyte injury
    American Journal of Pathology, 2021
    Co-Authors: Yoshiyasu Fukusumi, Ying Zhang, Hidenori Yasuda, Hiroshi Kawachi
    Abstract:

    Ephrin-B1 is one of the critical components of the slit diaphragm of kidney glomerular podocyte. However, the precise function of ephrin-B1 is unclear. To clarify the function of ephrin-B1, ephrin-B1–associated molecules were studied. RNA-sequencing analysis suggested that Na+/H+ exchanger regulatory factor 2 (NHERF2), a scaffolding protein, is associated with ephrin-B1. NHERF2 was expressed at the apical area and the slit diaphragm, and interacted with the Nephrin–ephrin-B1 complex at the slit diaphragm. The Nephrin–ephrin-B1–NHERF2 complex interacted with ezrin bound to F-actin. NHERF2 bound ephrin-B1 via its first postsynaptic density protein-95/disks large/zonula occludens-1 domain, and podocalyxin via its second postsynaptic density protein-95/disks large/zonula occludens-1 domain. Both in vitro analyses with human embryonic kidney 293 cells and in vivo study with rat nephrotic model showed that stimulaiton of the slit diaphragm, phosphorylation of Nephrin and ephrin-B1, and dephosphorylation of NHERF2 and ezrin, disrupted the linkages of ephrin-B1–NHERF2 and NHERF2-ezrin. It is conceivable that the linkage of Nephrin–ephrin-B1–NHERF2–ezrin–actin is a novel critical axis in the podocytes. Ephrin-B1 phosphorylation also disrupted the linkage of an apical transmembrane protein, podocalyxin, with NHERF2-ezrin-actin. The phosphorylation of ephrin-B1 and the consequent dephosphorylation of NHERF2 are critical initiation events leading to podocyte injury.

  • partitioning defective 6 ephrin b1 interaction is regulated by Nephrin mediated signal and is crucial in maintaining slit diaphragm of podocyte
    American Journal of Pathology, 2020
    Co-Authors: Sayuri Takamura, Ying Zhang, Yoshiyasu Fukusumi, Ichiei Narita, Hiroshi Kawachi
    Abstract:

    Ephrin-B1 plays a critical role at slit diaphragm. Partitioning-defective (Par)-6 is down-regulated in podocyte of ephrin-B1 knockout mouse, suggesting that Par-6 is associated with ephrin-B1. Par polarity complex, consisting of Par-6, Par-3, and atypical protein kinase C, is essential for tight junction formation. In this study, the expression of Par-6 was analyzed in the normal and nephrotic syndrome model rats, and the molecular association of Par-6, Par-3, ephrin-B1, and Nephrin was assessed with the human embryonic kidney 293 cell expression system. Par-6 was concentrated at slit diaphragm. Par 6 interacted with ephrin-B1 but not with Nephrin, and Par-3 interacted with Nephrin but not with ephrin-B1. The complexes of Par-6-ephrin-B1 and Par-3-Nephrin were linked via extracellular sites of ephrin-B1 and Nephrin. The Par-6-ephrin-B1 complex was delinked from the Par-3-Nephrin complex, and Par-6 and ephrin-B1 were clearly down-regulated already at early phase of nephrotic model. The alteration of Par-6/ephrin-B1 advanced that of Par-3/Nephrin. Stimulation to Nephrin phosphorylated not only Nephrin but also ephrin-B1, and consequently inhibited the interaction between ephrin-B1 and Par-6. Par-6 appeared at presumptive podocyte of early developmental stage and moved to basal area at capillary loop stage to participate in slit diaphragm formation at the final stage. Par-6-ephrin-B1 interaction is crucial for formation and maintenance of slit diaphragm of podocyte.

  • Nephrin binding ephrin b1 at the slit diaphragm controls podocyte function through the jnk pathway
    Journal of The American Society of Nephrology, 2018
    Co-Authors: Yoshiyasu Fukusumi, Ying Zhang, Ryohei Yamagishi, Kanako Oda, Toru Watanabe, Katsuyuki Matsui, Hiroshi Kawachi
    Abstract:

    Background B-type ephrins are membrane-bound proteins that maintain tissue function in several organs. We previously reported that ephrin-B1 is localized at the slit diaphragm of glomerular podocytes. However, the function of ephrin-B1 at this location is unclear.Methods We analyzed the phenotype of podocyte-specific ephrin-B1 knockout mice and assessed the molecular association of ephrin-B1 and Nephrin, a key molecule of the slit diaphragm, in HEK293 cells and rats with anti-Nephrin antibody-induced nephropathy.Results Compared with controls, ephrin-B1 conditional knockout mice displayed altered podocyte morphology, disarrangement of the slit diaphragm molecules, and proteinuria. Ephrin-B1 expressed in HEK293 cells immunoprecipitated with Nephrin, which required the basal regions of the extracellular domains of both proteins. Treatment of cells with an anti-Nephrin antibody promoted the phosphorylation of Nephrin and ephrin-B1. However, phosphorylation of ephrin-B1 did not occur in cells expressing a mutant Nephrin lacking the ephrin-B1 binding site or in cells treated with an Src kinase inhibitor. The phosphorylation of ephrin-B1 enhanced the phosphorylation of Nephrin and promoted the phosphorylation of c-Jun N-terminal kinase (JNK), which was required for ephrin-B1-promoted cell motility in wound-healing assays. Notably, phosphorylated JNK was detected in the glomeruli of control mice but not ephrin-B1 conditional knockout mice. In rats, the phosphorylation of ephrin-B1, JNK, and Nephrin occurred in the early phase (24 hours) of anti-Nephrin antibody-induced nephropathy.Conclusions Through interactions with Nephrin, ephrin-B1 maintains the structure and barrier function of the slit diaphragm. Moreover, phosphorylation of ephrin-B1 and, consequently, JNK are involved in the development of podocyte injury.

  • avian podocytes which lack Nephrin use adherens junction proteins at intercellular junctions
    Journal of Histochemistry and Cytochemistry, 2016
    Co-Authors: Eishin Yaoita, Hiroshi Kawachi, Ying Zhang, Hiroko Nishimura, Masaaki Nameta, Yutaka Yoshida, Hiroki Takimoto, Hidehiko Fujinaka, Sameh Magdeldin, Tomizo Oyama
    Abstract:

    Nephrin, a major intercellular junction (ICJ) molecule of mammalian podocytes in the renal glomerulus, is absent in the avian genome. We hypothesized that birds use ICJ molecules other than Nephrin in their podocytes. Therefore, in the present study, we examined the possible involvement of adherens junction (AJ) proteins in the ICJs of avian podocytes. We found the AJ proteins N-cadherin and α- and β-catenins in podocytes of quail and chickens but not in those of rats, pigs or humans. The AJ proteins were prominent in avian glomerulus-rich fractions in immunoblot analyses, and in immunofluorescence microscopy analyses, they were localized along glomerular capillary walls appearing in at least two staining patterns: weakly diffuse and distinctly granular. Immunoelectron microscopy demonstrated that the significant accumulation of immunogold particles for the AJ proteins were especially evident in avian slit diaphragms and AJs. Furthermore, N-cadherin was found to be expressed in all nephron cells in the early developmental stage but became confined to podocytes during maturation. These results indicate that avian slit diaphragms clearly express AJ proteins as compared with that in the mammal-where AJ proteins are suppressed to an extremely low level-and that avian podocytes are interconnected by AJs per se in addition to slit diaphragms.

  • Nephrin mediates actin reorganization via phosphoinositide 3 kinase in podocytes
    Kidney International, 2008
    Co-Authors: Jianxin Zhu, Serge Lemay, Lamine Aoudjit, Hiroshi Kawachi, N Sun, Tomoko Takano
    Abstract:

    Nephrin is a slit diaphragm protein critical for structural and functional integrity of visceral glomerular epithelial cells (podocytes) and is known to be tyrosine phosphorylated by Src family kinases. We studied the role of phosphoinositide 3-kinase (PI3K), activated via the phosphorylation of Nephrin, in actin cytoskeletal reorganization of cultured rat podocytes. Phosphorylation of rat Nephrin by the Fyn kinase markedly increased its interaction with a regulatory subunit of PI3K. Stable transfection of rat Nephrin in the podocytes with podocin led to Nephrin tyrosine phosphorylation, PI3K-dependent phosphorylation of Akt, increased Rac1 activity, and an altered actin cytoskeleton with decreased stress fibers and increased lamellipodia. These changes were reversed with an inhibitor of PI3K and not seen when the Nephrin-mutant Y1152F replaced wild-type Nephrin. Rac1 and Akt1 contributed to lamellipodia formation and decreased stress fibers, respectively. Finally, in the rat model of puromycin aminonucleoside nephrosis, Nephrin tyrosine phosphorylation, Nephrin-PI3K association, and glomerular Akt phosphorylation were all decreased. Our results suggest that PI3K is involved in Nephrin-mediated actin reorganization in podocytes. Disturbed Nephrin–PI3K interactions may contribute to abnormal podocyte morphology and proteinuria.

Karl Tryggvason - One of the best experts on this subject based on the ideXlab platform.

  • the podocyte protein Nephrin is required for cardiac vessel formation
    Human Molecular Genetics, 2011
    Co-Authors: Nicole Wagner, Karl Tryggvason, Harris Morrison, Sophie Pagnotta, J F Michiels, Yannick Schwab, Andreas Schedl, Kaydietrich Wagner
    Abstract:

    Nephrin (NPHS1) has been described as an important structural protein of kidney podocytes. Mutations in this gene lead to the Finnish-type congenital nephrotic syndrome. More recently, a role of Nephrin as a signalling molecule in kidney podocytes has been identified. Here, we show that Nephrin not only has a function in kidney podocytes, but is also required for cardiovascular development. Nephrin is expressed in the epicardium and coronary vessels during human and mouse embryonic development. Nephrin knockout embryos showed abnormal epicardial cell morphology and, at later stages of development, a reduced number of coronary vessels due to increased apoptosis, and in addition, cardiac fibrosis. Connexin 43, which is required for coronary vessel formation, was downregulated in Nephrin knockout embryos. Expression of the p75NTR neurotrophin receptor, a known mediator of apoptosis, was increased in mutants. Furthermore, co-immunoprecipitation studies demonstrated a direct interaction of Nephrin with p75NTR. Primary Nephrin-deficient cardiac cells showed a 5-fold higher rate of apoptosis in response to progenitor of nerve growth factor compared with wild-type cells, which could be rescued by RNAi against p75NTR. Taken together, our data demonstrate that Nephrin directly interacts with p75NTR and reveal an important role for Nephrin in murine cardiac development by permitting survival of cardiovascular progenitor cells.

  • Nephrin a unique structural and signaling protein of the kidney filter
    Trends in Molecular Medicine, 2007
    Co-Authors: Jaakko Patrakka, Karl Tryggvason
    Abstract:

    Since the discovery of Nephrin, the first integral component of the slit diaphragm to be identified, the podocyte slit pore has become a major focus in research concerning the glomerular filtration barrier. Nephrin is a central component of the glomerular ultrafilter, with both structural and signaling functions. The extracellular domain of Nephrin and other components of the slit diaphragm seem to form a porous molecular sieve. The intracellular domain of Nephrin is associated with linker proteins, such as CD2-associated protein and Nck proteins that can connect Nephrin to the actin cytoskeleton. Alterations in Nephrin interactions with other proteins during development or injury can lead to complex signaling reactions aimed at establishing or restoring the filter function.

  • Nephrin promotes cell cell adhesion through homophilic interactions
    American Journal of Pathology, 2003
    Co-Authors: Jamshid Khoshnoodi, Kristmundur Sigmundsson, Larsgoran Ofverstedt, Ulf Skoglund, Bjorn Obrink, Jorma Wartiovaara, Karl Tryggvason
    Abstract:

    Nephrin is a type-1 transmembrane protein and a key component of the podocyte slit diaphragm, the ultimate glomerular plasma filter. Genetic and acquired diseases affecting expression or function of Nephrin lead to severe proteinuria and distortion or absence of the slit diaphragm. Here, we showed by using a surface plasmon resonance biosensor that soluble recombinant variants of Nephrin, containing the extracellular part of the protein, interact with each other in a specific and concentration-dependent manner. This molecular interaction was increased by twofold in the presence of physiological Ca 2+ concentration, indicating that the binding is not dependent on, but rather promoted by Ca 2+ . Furthermore, transfected HEK293 cells and an immortalized mouse podocyte cell line overexpressing full-length human Nephrin formed cellular aggregates, with cell-cell contacts staining strongly for Nephrin. The distance between plasma membranes at the Nephrin-containing contact sites was shown by electron microscopy to be 40 to 50 nm, similar to the width of glomerular slit diaphragm. The cell contacts could be dissociated with antibodies reacting with the first two extracellular Ig-like domains of Nephrin. Wild-type HEK293 cells were shown to express slit diaphragm components CD2AP, P-cadherin, FAT, and NEPH1. The results show that Nephrin molecules exhibit homophilic interactions that could promote cellular contacts through direct Nephrin-Nephrin interactions, and that the other slit diaphragm components expressed could contribute to that interaction.

  • altered ultrastructural distribution of Nephrin in minimal change nephrotic syndrome
    Nephrology Dialysis Transplantation, 2003
    Co-Authors: Annika Wernerson, Karl Tryggvason, Vesa Ruotsalainen, Fredrik Duner, Erna Pettersson, Silwa Mengarelli Widholm, U Berg, Kjell Hultenby, Magnus Soderberg
    Abstract:

    Background. Nephrin is a cell-adhesion protein that is defective in congenital nephrotic syndrome of the Finnish type (CNF). Nephrin is synthesized by the podocytes and is localized to the slit membrane between individual foot processes of the podocytes. Although Nephrin is apparently pivotal in the development of CNF, the role of Nephrin in other causes of nephrotic syndrome is not fully understood. Methods. Renal biopsy specimens from patients with minimal change nephrotic syndrome (MCNS) were investigated. Nephrin distribution was studied with immunohistochemical and semiquantitative immunoelectron microscopic techniques and the results were related to the degree of foot process effacement found under the electron microscope. Results. In normal kidney, immunofluorescence revealed a linear staining along the capillary basement membranes, corresponding to the localization of Nephrin in the slit membranes. In the biopsies from patients with MCNS, the Nephrin pattern had become granular. The degree of granularization corresponded to the degree of foot process effacement. Ultrastructural semiquantification showed the amount of Nephrin to be reduced both in areas with and without foot process effacement compared with the control specimens. The concentration of Nephrin was lowest in the areas with foot process effacement and there was redistribution from the slits into the cytoplasm. Conclusions. These findings demonstrate that Nephrin expression is altered in MCNS. Whether this reflects a pathogenetic role for Nephrin in MCNS or a phenomenon secondary to other causes of foot process effacement remains to be elucidated.

  • n linked glycosylation is critical for the plasma membrane localization of Nephrin
    Journal of The American Society of Nephrology, 2002
    Co-Authors: Kunimasa Yan, Vesa Ruotsalainen, Jamshid Khoshnoodi, Karl Tryggvason
    Abstract:

    The expression pattern, subcellular localization, and the role of glycosylation of the human Nephrin was examined in transfected cells. Stable cell lines, constitutively expressing a full-length human Nephrin cDNA construct, were generated from transfected immortalized mouse podocytes (IMP) and a human embryonic kidney cell line (HEK-293). Immunofluorescence confocal microscopy of transfected cells showed plasma membrane localization of the recombinant Nephrin. Immunoblotting showed that the recombinant Nephrin expressed in transfected cell lines migrated as a double band with a molecular weight of 185 kD. When cells were treated with the N-glycosylation inhibitor, tunicamycin, the molecular weight of Nephrin was decreased to a single immunoband of 150 kD, indicating that the shift in the electrophoretic migration of Nephrin is due to N-linked carbohydrate moieties. It was further shown that this glycosylation process is highly sensitive to inhibition by tunicamycin, which is a naturally occurring antibiotic, leading to retention of nonglycosylated Nephrin molecules in the endoplasmic reticulum. It was concluded that N-glycosylation of Nephrin is crucial for its proper folding and thereby plasma membrane localization; therefore, inhibition of this process might be an important factor in the onset of pathogenesis of some acquired glomerular diseases.

Tomoko Takano - One of the best experts on this subject based on the ideXlab platform.

  • multivalent Nephrin nck interactions define a threshold for clustering and tyrosine dependent Nephrin endocytosis
    Journal of Cell Science, 2020
    Co-Authors: Claire E Martin, Tomoko Takano, Laura A New, Ava Keyvani Chahi, Noah J Phippen, Alexander E Mitro, Tony Pawson, Ivan M Blasutig
    Abstract:

    Assembly of signaling molecules into micrometer-sized clusters is driven by multivalent protein-protein interactions, such as those found within the Nephrin/Nck complex. Phosphorylation on multiple tyrosines within the tail of the Nephrin transmembrane receptor induces recruitment of the cytoplasmic adaptor protein Nck, which binds via its triple SH3 domains to various effectors, leading to actin assembly. The physiological consequences of Nephrin clustering are not well understood. Here we demonstrate that Nephrin phosphorylation regulates the formation of membrane clusters in podocytes. We also reveal a connection between clustering and endocytosis, which appears to be driven by threshold levels of Nephrin tyrosine phosphorylation and Nck SH3 domain signaling. Finally, we expose an in vivo correlation between transient changes in Nephrin tyrosine phosphorylation, Nephrin localization and integrity of the glomerular filtration barrier during podocyte injury. Altogether, our results suggest that Nephrin phosphorylation determines the composition of effector proteins within clusters to dynamically regulate Nephrin turnover and podocyte health.

  • shca adaptor protein promotes Nephrin endocytosis and is upregulated in proteinuric nephropathies
    Journal of The American Society of Nephrology, 2017
    Co-Authors: Claire E Martin, Lamine Aoudjit, Tomoko Takano, Vera Eremina, Kelly A Petersen, Manali Tilak, Rod W Hardy, Susan E Quaggin, Nina Jones
    Abstract:

    Nephrin is a key structural component of the podocyte slit diaphragm, and proper expression of Nephrin on the cell surface is critical to ensure integrity of the blood filtration barrier. Maintenance of Nephrin within this unique cell junction has been proposed to require dynamic phosphorylation events and endocytic recycling, although the molecular mechanisms that control this interplay are poorly understood. Here, we investigated the possibility that the phosphotyrosine adaptor protein ShcA regulates Nephrin turnover. Western blotting and immunostaining analysis confirmed that ShcA is expressed in podocytes. In immunoprecipitation and pulldown assays, ShcA, via its SH2 domain, was associated with several phosphorylated tyrosine residues on Nephrin. Overexpression of ShcA promoted Nephrin tyrosine phosphorylation and reduced Nephrin signaling and cell surface expression in vitro In a rat model of reversible podocyte injury and proteinuria, phosphorylated Nephrin temporally colocalized with endocytic structures coincident with upregulation of ShcA expression. In vivo biotinylation assays confirmed that Nephrin expression decreased at the cell surface and correspondingly increased in the cytosol during the injury time course. Finally, immunostaining in kidney biopsy specimens demonstrated overexpression of ShcA in several human proteinuric kidney diseases compared with normal conditions. Our results suggest that increases in ShcA perturb Nephrin phosphosignaling dynamics, leading to aberrant Nephrin turnover and slit diaphragm disassembly.

  • at2r deficiency mediated podocyte loss via activation of ectopic hedgehog interacting protein hhip gene expression
    The Journal of Pathology, 2017
    Co-Authors: Minchun Liao, Tomoko Takano, Xinping Zhao, Shiaoying Chang, Isabelle Chenier, Julie R Ingelfinger, Shaoling Zhang
    Abstract:

    Angiotensin II type 2 receptor (AT2 R) deficiency in AT2 R knockout (KO) mice has been linked to congenital abnormalities of the kidney and urinary tract; however, the mechanisms by which this occurs are poorly understood. In this study, we examined whether AT2 R deficiency impaired glomerulogenesis and mediated podocyte loss/dysfunction in vivo and in vitro. Nephrin-cyan fluorescent protein (CFP)-transgenic (Tg) and Nephrin/AT2 RKO mice were used to assess glomerulogenesis, while wild-type and AT2 RKO mice were used to evaluate maturation of podocyte morphology/function. Immortalized mouse podocytes (mPODs) were employed for in vitro studies. AT2 R deficiency resulted in diminished glomerulogenesis in E15 embryos, but had no impact on actual nephron number in neonates. Pups lacking AT2 R displayed features of renal dysplasia with lower glomerular tuft volume and podocyte numbers. In vivo and in vitro studies demonstrated that loss of AT2 R was associated with elevated NADPH oxidase 4 levels, which in turn stimulated ectopic hedgehog interacting protein (Hhip) gene expression in podocytes. Consequently, ectopic Hhip expression activation either triggers caspase-3 and p53-related apoptotic processes resulting in podocyte loss, or activates TGFβ1-Smad2/3 cascades and α-SMA expression to transform differentiated podocytes to undifferentiated podocyte-derived fibrotic cells. We analyzed HHIP expression in the kidney disease database (Nephroseq) and then validated this using HHIP immunohistochemistry staining of human kidney biopsies (controls versus focal segmental glomerulosclerosis). In conclusion, loss of AT2 R is associated with podocyte loss/dysfunction and is mediated, at least in part, via augmented ectopic Hhip expression in podocytes. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  • Nephrin tyrosine phosphorylation is required to stabilize and restore podocyte foot process architecture
    Journal of The American Society of Nephrology, 2016
    Co-Authors: Laura A New, Claire E Martin, Rizaldy P Scott, Mathew J Platt, Ava Keyvani Chahi, Colin D Stringer, Bozena Samborska, Vera Eremina, Tomoko Takano
    Abstract:

    Podocytes are specialized epithelial cells of the kidney blood filtration barrier that contribute to permselectivity via a series of interdigitating actin-rich foot processes. Positioned between adjacent projections is a unique cell junction known as the slit diaphragm, which is physically connected to the actin cytoskeleton via the transmembrane protein Nephrin. Evidence indicates that tyrosine phosphorylation of the intracellular tail of Nephrin initiates signaling events, including recruitment of cytoplasmic adaptor proteins Nck1 and Nck2 that regulate actin cytoskeletal dynamics. Nephrin tyrosine phosphorylation is altered in human and experimental renal diseases characterized by pathologic foot process remodeling, prompting the hypothesis that phosphoNephrin signaling directly influences podocyte morphology. To explore this possibility, we generated and analyzed knockin mice with mutations that disrupt Nephrin tyrosine phosphorylation and Nck1/2 binding (Nephrin(Y3F/Y3F) mice). Homozygous Nephrin(Y3F/Y3F) mice developed progressive proteinuria accompanied by structural changes in the filtration barrier, including podocyte foot process effacement, irregular thickening of the glomerular basement membrane, and dilated capillary loops, with a similar but later onset phenotype in heterozygous animals. Furthermore, compared with wild-type mice, Nephrin(Y3F/Y3F) mice displayed delayed recovery in podocyte injury models. Profiling of Nephrin tyrosine phosphorylation dynamics in wild-type mice subjected to podocyte injury indicated site-specific differences in phosphorylation at baseline, injury, and recovery, which correlated with loss of Nephrin-Nck1/2 association during foot process effacement. Our results define an essential requirement for Nephrin tyrosine phosphorylation in stabilizing podocyte morphology and suggest a model in which dynamic changes in phosphotyrosine-based signaling confer plasticity to the podocyte actin cytoskeleton.

  • increased shp 1 protein expression by high glucose levels reduces Nephrin phosphorylation in podocytes
    Journal of Biological Chemistry, 2015
    Co-Authors: Benoit Denhez, Tomoko Takano, Farah Lizotte, Marieodile Guimond, Nina Jones, Pedro Geraldes
    Abstract:

    Nephrin, a critical podocyte membrane component that is reduced in diabetic nephropathy, has been shown to activate phosphotyrosine signaling pathways in human podocytes. Nephrin signaling is important to reduce cell death induced by apoptotic stimuli. We have shown previously that high glucose level exposure and diabetes increased the expression of SHP-1, causing podocyte apoptosis. SHP-1 possesses two Src homology 2 domains that serve as docking elements to dephosphorylate tyrosine residues of target proteins. However, it remains unknown whether SHP-1 interacts with Nephrin and whether its elevated expression affects the Nephrin phosphorylation state in diabetes. Here we show that human podocytes exposed to high glucose levels exhibited elevated expression of SHP-1, which was associated with Nephrin. Coexpression of Nephrin-CD16 and SHP-1 reduced Nephrin tyrosine phosphorylation in transfected human embryonic kidney 293 cells. A single tyrosine-to-phenylalanine mutation revealed that rat Nephrin Tyr(1127) and Tyr(1152) are required to allow SHP-1 interaction with Nephrin. Overexpression of dominant negative SHP-1 in human podocytes prevented high glucose-induced reduction of Nephrin phosphorylation. In vivo, immunoblot analysis demonstrated that Nephrin expression and phosphorylation were decreased in glomeruli of type 1 diabetic Akita mice (Ins2(+/C96Y)) compared with control littermate mice (Ins2(+/+)), and this was associated with elevated SHP-1 and cleaved caspase-3 expression. Furthermore, immunofluorescence analysis indicated increased colocalization of SHP-1 with Nephrin in diabetic mice compared with control littermates. In conclusion, our results demonstrate that high glucose exposure increases SHP-1 interaction with Nephrin, causing decreased Nephrin phosphorylation, which may, in turn, contribute to diabetic nephropathy.

Lawrence B Holzman - One of the best experts on this subject based on the ideXlab platform.

  • phosphorylation of slit diaphragm proteins Nephrin and neph1 upon binding of hgf promotes podocyte repair
    Journal of Biological Chemistry, 2021
    Co-Authors: Ashish K Solanki, Lawrence B Holzman, Pankaj Srivastava, Ehtesham Arif, Christopher M Furcht, Bushra Rahman, Pei Wen, Avinash Singh, Wayne R Fitzgibbon, Milos N Budisavljevic
    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.

  • hgf induced activation of Nephrin and neph1 serves as a novel mechanism for recovery of podocytes from injury
    bioRxiv, 2020
    Co-Authors: Ashish K Solanki, Lawrence B Holzman, Pankaj Srivastava, Ehtesham Arif, Christopher M Furcht, Bushra Rahman, Pei Wen, Avinash Singh, Wayne R Fitzgibbon, Glenn P Lobo
    Abstract:

    When activated, slit diaphragm proteins Nephrin and NEPH1 enable signaling pathways leading to podocyte actin cytoskeleton reorganization, which is critical for podocyte recovery from injury. However, the mechanisms through which these proteins are activated remain unknown. This study presents a novel concept showing ligand-induced activation of Nephrin and NEPH1. We first identified phosphatase SHP-2, which directly dephosphorylated these proteins. We next identified HGF, a known SHP-2 modulator, as a rapid inducer of Nephrin and NEPH1 phosphorylation. Using baculovirus expressed recombinant purified proteins, SPR (surface plasma resonance), molecular modeling and peptide binding approaches, we show that HGF directly binds Nephrin and NEPH1 extracellular domains. Further, using cultured podocytes and Drosophila nephrocytes, we demonstrate that while HGF treatment repaired injured podocytes, the addition of inhibitory NEPH1 or Nephrin peptides blocked HGF-induced recovery. Overall, this study shows novel activation and deactivation mechanisms for Nephrin and NEPH1 that are required for their function.

  • Nephrin Preserves Podocyte Viability and Glomerular Structure and Function in Adult Kidneys
    Journal of the American Society of Nephrology : JASN, 2015
    Co-Authors: Peter Y. Chuang, Vivette D. D'agati, Yan Dai, Rabi Yacoub, Oltjon Taku, Prem K. Premsrirut, Lawrence B Holzman
    Abstract:

    Nephrin is required during kidney development for the maturation of podocytes and formation of the slit diaphragm junctional complex. Because Nephrin expression is downregulated in acquired glomerular diseases, Nephrin deficiency is considered a pathologic feature of glomerular injury. However, whether Nephrin deficiency exacerbates glomerular injury in glomerular diseases has not been experimentally confirmed. Here, we generated mice with inducible RNA interference–mediated Nephrin knockdown. Short-term Nephrin knockdown (6 weeks), starting after the completion of kidney development at 5 weeks of age, did not affect glomerular structure or function. In contrast, mice with long-term Nephrin knockdown (20 weeks) developed mild proteinuria, foot process effacement, filtration slit narrowing, mesangial hypercellularity and sclerosis, glomerular basement membrane thickening, subendothelial zone widening, and podocyte apoptosis. When subjected to an acquired glomerular insult induced by unilateral nephrectomy or doxorubicin, mice with short-term Nephrin knockdown developed more severe glomerular injury compared with mice without Nephrin knockdown. Additionally, Nephrin-knockdown mice developed more exaggerated glomerular enlargement when subjected to unilateral nephrectomy and more podocyte apoptosis and depletion after doxorubicin challenge. AKT phosphorylation, which is a slit diaphragm–mediated and Nephrin-dependent pathway in the podocyte, was markedly reduced in mice with long-term or short-term Nephrin knockdown challenged with uninephrectomy or doxorubicin. Taken together, our data establish that under the basal condition and in acquired glomerular diseases, Nephrin is required to maintain slit diaphragm integrity and slit diaphragm–mediated signaling to preserve glomerular function and podocyte viability in adult mice.

  • vascular endothelial growth factor receptor 2 direct interaction with Nephrin links vegf a signals to actin in kidney podocytes
    Journal of Biological Chemistry, 2011
    Co-Authors: Claudia Bertuccio, Lawrence B Holzman, Delma Veron, P K Aggarwal, Alda Tufro
    Abstract:

    The transmembrane protein Nephrin is an essential component of slit diaphragms, the specialized cell junctions that link podocyte foot processes. Podocytes are epithelial cells that surround the glomerular capillaries in the kidney and are necessary for the organ-filtering function. Nephrin signaling complex transduces extracellular cues to the podocyte cytoskeleton and regulates podocyte shape and function. Vascular endothelial growth factor A (VEGF-A) is a required growth factor produced and secreted by podocytes. Accumulating evidence suggests a cross-talk between VEGF-A and Nephrin signaling pathways. We previously showed that in vivo Nephrin associates with VEGF receptor-2 (VEGFR2), the signaling receptor for VEGF-A. In the present work, we characterized the interaction between Nephrin and VEGFR2 in cultured cells and in vitro. We demonstrate that Nephrin-VEGFR2 interaction is direct using mass spectrometry, immunoprecipitation, GST-binding assays, and blot overlay experiments. This interaction occurs through VEGFR2 and Nephrin cytoplasmic domains. Nephrin-VEGFR2 interaction is modulated by tyrosine phosphorylation of both cytoplasmic domains. Furthermore, the Nephrin-VEGFR2 complex involves Nck and actin. VEGF-A signaling via this complex results in decreased cell size. We provide evidence that this multiprotein interaction occurs in cultured podocytes. We propose that the Nephrin-VEGFR2 complex acts as a key mediator to transduce local VEGF-A signals to the podocyte actin cytoskeleton, regulating the foot process structure and glomerular filter integrity.

  • Nephrin ectodomain engagement results in src kinase activation Nephrin phosphorylation nck recruitment and actin polymerization
    Journal of Clinical Investigation, 2006
    Co-Authors: Rakesh Verma, Iulia Kovari, Deepak Nihalani, Abdul Soofi, Kevin M Patrie, Lawrence B Holzman
    Abstract:

    A properly established and maintained podocyte intercellular junction, or slit diaphragm, is a necessary component of the selective permeability barrier of the kidney glomerulus. The observation that mutation or deletion of the slit diaphragm transmembrane protein Nephrin results in failure of podocyte foot process morphogenesis and concomitant proteinuria first suggested the hypothesis that Nephrin serves as a component of a signaling complex that directly integrates podocyte junctional integrity with cytoskeletal dynamics. The observations made herein provide the first direct evidence to our knowledge for a phosphorylation-mediated signaling mechanism by which this integrative function is derived. Our data support the model that during podocyte intercellular junction formation, engagement of the Nephrin ectodomain induces transient Fyn catalytic activity that results in Nephrin phosphorylation on specific Nephrin cytoplasmic domain tyrosine residues. We found that this Nephrin phosphorylation event resulted in recruitment of the SH2-SH3 domain-containing adapter protein Nck and assembly of actin filaments in an Nck-dependent fashion. Considered in the context of the role of Nephrin family proteins in other organisms and the integral relationship of actin dynamics and junction formation, these observations establish a function for Nephrin in regulating actin cytoskeletal dynamics.

Vesa Ruotsalainen - One of the best experts on this subject based on the ideXlab platform.

  • clustering induced tyrosine phosphorylation of Nephrin by src family kinases1
    Kidney International, 2003
    Co-Authors: Juhani Lahdenpera, Pekka Kilpelainen, Xiao Li Liu, Timo Pikkarainen, Paula Reponen, Vesa Ruotsalainen
    Abstract:

    Clustering-induced tyrosine phosphorylation of Nephrin by Src family kinases. Background Nephrin is a recently discovered protein of the immunoglobulin (Ig) superfamily. In the kidney, it is located at the slit diaphragm, which forms the decisive size-selective filter of glomerular ultrafiltration barrier and locates between the interdigitating foot processes of podocytes. Nephrin is mutated in congenital nephrosis of the Finnish type (NPHS1) and has been demonstrated to be an essential component of the slit diaphragm. Based on its domain structure, Nephrin is likely to be a cell-cell or cell-matrix adhesion protein that may have a signaling function. In this study, we hypothesized that the clustering of Nephrin with antibodies on cell surface mimics the situation where the interaction between Nephrin and its extracellular ligand(s) is altered. Methods Nephrin was clustered on the surface of stably transfected HEK293 cells by a monoclonal antiNephrin antibody and polyclonal secondary antibody. Clusters were visualized by immunofluorescence microscopy. Changes in protein phosphorylation were studied employing immunoprecipitations and Western blot analysis. A specific inhibitor and cotransfection experiments were used to investigate role of Src family kinases in Nephrin phosphorylation. Results Clustering of Nephrin induced its own tyrosine phosphorylation. This phosphorylation was inhibited by PP2, an inhibitor of Src family kinases. Several members of Src family kinases were able to induce Nephrin phosphorylation when cotransfected to HEK293 cells with Nephrin. Moreover, the Src family kinase Fyn was consistently found to be coimmunoprecipitated with Nephrin. Interestingly, clustering of Nephrin induced also tyrosine phosphorylation of a 46 kD protein that was as well found to be coimmunoprecipitated with Nephrin. Conclusion Nephrin is a signaling protein phosphorylated by Src family kinases.

  • altered ultrastructural distribution of Nephrin in minimal change nephrotic syndrome
    Nephrology Dialysis Transplantation, 2003
    Co-Authors: Annika Wernerson, Karl Tryggvason, Vesa Ruotsalainen, Fredrik Duner, Erna Pettersson, Silwa Mengarelli Widholm, U Berg, Kjell Hultenby, Magnus Soderberg
    Abstract:

    Background. Nephrin is a cell-adhesion protein that is defective in congenital nephrotic syndrome of the Finnish type (CNF). Nephrin is synthesized by the podocytes and is localized to the slit membrane between individual foot processes of the podocytes. Although Nephrin is apparently pivotal in the development of CNF, the role of Nephrin in other causes of nephrotic syndrome is not fully understood. Methods. Renal biopsy specimens from patients with minimal change nephrotic syndrome (MCNS) were investigated. Nephrin distribution was studied with immunohistochemical and semiquantitative immunoelectron microscopic techniques and the results were related to the degree of foot process effacement found under the electron microscope. Results. In normal kidney, immunofluorescence revealed a linear staining along the capillary basement membranes, corresponding to the localization of Nephrin in the slit membranes. In the biopsies from patients with MCNS, the Nephrin pattern had become granular. The degree of granularization corresponded to the degree of foot process effacement. Ultrastructural semiquantification showed the amount of Nephrin to be reduced both in areas with and without foot process effacement compared with the control specimens. The concentration of Nephrin was lowest in the areas with foot process effacement and there was redistribution from the slits into the cytoplasm. Conclusions. These findings demonstrate that Nephrin expression is altered in MCNS. Whether this reflects a pathogenetic role for Nephrin in MCNS or a phenomenon secondary to other causes of foot process effacement remains to be elucidated.

  • n linked glycosylation is critical for the plasma membrane localization of Nephrin
    Journal of The American Society of Nephrology, 2002
    Co-Authors: Kunimasa Yan, Vesa Ruotsalainen, Jamshid Khoshnoodi, Karl Tryggvason
    Abstract:

    The expression pattern, subcellular localization, and the role of glycosylation of the human Nephrin was examined in transfected cells. Stable cell lines, constitutively expressing a full-length human Nephrin cDNA construct, were generated from transfected immortalized mouse podocytes (IMP) and a human embryonic kidney cell line (HEK-293). Immunofluorescence confocal microscopy of transfected cells showed plasma membrane localization of the recombinant Nephrin. Immunoblotting showed that the recombinant Nephrin expressed in transfected cell lines migrated as a double band with a molecular weight of 185 kD. When cells were treated with the N-glycosylation inhibitor, tunicamycin, the molecular weight of Nephrin was decreased to a single immunoband of 150 kD, indicating that the shift in the electrophoretic migration of Nephrin is due to N-linked carbohydrate moieties. It was further shown that this glycosylation process is highly sensitive to inhibition by tunicamycin, which is a naturally occurring antibiotic, leading to retention of nonglycosylated Nephrin molecules in the endoplasmic reticulum. It was concluded that N-glycosylation of Nephrin is crucial for its proper folding and thereby plasma membrane localization; therefore, inhibition of this process might be an important factor in the onset of pathogenesis of some acquired glomerular diseases.

  • recurrence of nephrotic syndrome in kidney grafts of patients with congenital nephrotic syndrome of the finnish type role of Nephrin
    Transplantation, 2002
    Co-Authors: Jaakko Patrakka, Karl Tryggvason, Vesa Ruotsalainen, Paula Reponen, Christer Holmberg, Erik Qvist, Jarmo Laine, Hannu Jalanko
    Abstract:

    Background. Congenital nephrotic syndrome of the Finnish type (CNF, NPHS1) is caused by mutations in the NPHS1 gene. NPHS1 codes for Nephrin, a cell adhesion protein located at the glomerular slit diaphragm. Renal transplantation is the only treatment option for most patients with NPHS1. We have previously described recurrence of severe proteinuria in grafts transplanted to children with NPHS1. Here we studied the pathophysiology of this proteinuria. Methods. Clinical data, light and electron microscopic findings as well as the expression of Nephrin in the proteinuric grafts were studied. The patients' sera were screened for antibodies against kidney glomerulus and Nephrin molecule using indirect immunofluorescence and ELISA. Results. Fifteen episodes of recurrent nephrotic syndrome occurred in 13 (25%) of 51 grafts transplanted to 45 Finnish children with NPHS1. All nine patients with recurrence had a Fin-major/Fin-major genotype, which leads to absence of Nephrin in the native kidney. Rescue therapy (cyclophosphamide) was successful in seven episodes, but six kidneys were lost due to this process. Antibodies reacting against glomerulus were found in eight, and high anti-Nephrin antibody levels were dectected in four of the nine patients. In electron microscopy, the fusion of the foot process and decreases in the detectable slit diaphragms in th podocyte pores were observed. The expression of Nephrin mRNA was markedly reduced in two, and granular staining for Nephrin was seen in three of five grafts. Conclusions. Circulating anti-Nephrin antibodies seem to have a pathogenic role in the development of heavy proteinuria in kidney grafts of NPHS1 patients with Fin-major/Fin-major genotype.

  • recurrence of nephrotic syndrome in kidney grafts of patients with congenital nephrotic syndrome of the finnish type role of Nephrin
    Transplantation, 2002
    Co-Authors: Jaakko Patrakka, Karl Tryggvason, Vesa Ruotsalainen, Paula Reponen, Christer Holmberg, Erik Qvist, Jarmo Laine, Hannu Jalanko
    Abstract:

    Background Congenital nephrotic syndrome of the Finnish type (CNF, NPHS1) is caused by mutations in the NPHS1 gene. NPHS1 codes for Nephrin, a cell adhesion protein located at the glomerular slit diaphragm. Renal transplantation is the only treatment option for most patients with NPHS1. We have previously described recurrence of severe proteinuria in grafts transplanted to children with NPHS1. Here we studied the pathophysiology of this proteinuria. Methods Clinical data, light and electron microscopic findings as well as the expression of Nephrin in the proteinuric grafts were studied. The patients' sera were screened for antibodies against kidney glomerulus and Nephrin molecule using indirect immunofluorescence and ELISA. Results Fifteen episodes of recurrent nephrotic syndrome occurred in 13 (25%) of 51 grafts transplanted to 45 Finnish children with NPHS1. All nine patients with recurrence had a Fin-major/Fin-major genotype, which leads to absence of Nephrin in the native kidney. Rescue therapy (cyclophosphamide) was successful in seven episodes, but six kidneys were lost due to this process. Antibodies reacting against glomerulus were found in eight, and high anti-Nephrin antibody levels were detected in four of the nine patients. In electron microscopy, the fusion of the foot process and decreases in the detectable slit diaphragms in the podocyte pores were observed. The expression of Nephrin mRNA was markedly reduced in two, and granular staining for Nephrin was seen in three of five grafts. Conclusions Circulating anti-Nephrin antibodies seem to have a pathogenic role in the development of heavy proteinuria in kidney grafts of NPHS1 patients with Fin-major/Fin-major genotype.