Hidetake Kurihara

Juntendo University, Edo, Tōkyō, Japan

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Publications (57)252.27 Total impact

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    ABSTRACT: The function of kidney podocytes is closely associated with actin cytoskeleton. Rho family small GTPase RhoA promotes stress fiber assembly through Rho-associated protein kinase (ROCK)-dependent myosin II phosphorylation and plays an important role in maintenance of actin stress fibers of podocytes. However, little is known how stress fiber assembly is regulated in podocytes. Here we found that afadin, an actin filament-binding protein, is required for RhoA/ROCK-dependent formation of actin stress fibers in rat podocyte C7 cells. We show that depletion of afadin in C7 cells induced loss of actin stress fibers. Conversely, forced expression of afadin increased the formation of actin stress fibers. Depletion of afadin inactivated RhoA and reduced the phosphorylation of myosin II. Moreover, the DIL domain of afadin appears to be responsible for actin stress fiber formation. Thus, afadin mediates RhoA/ROCK signaling and contributes to the formation of actin stress fibers in podocyte cells. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.
    Cytoskeleton 02/2015; 72(3). DOI:10.1002/cm.21211 · 3.01 Impact Factor
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    ABSTRACT: The slit diaphragm (SD), the specialized intercellular junction between renal glomerular epithelial cells (podocytes), provides a selective-filtration barrier in renal glomeruli. Dysfunction of the SD results in glomerular diseases that are characterized by disappearance of SD components, such as nephrin, from the cell surface. Although the importance of endocytosis and degradation of SD components for the maintenance of SD integrity has been suggested, the dynamic nature of the turnover of intact cell-surface SD components remained unclear. Using isolated rat glomeruli we show that the turnover rates of cell-surface SD components are relatively high; they almost completely disappear from the cell surface within minutes. The exocytosis, but not endocytosis, of heterologously expressed nephrin requires the kinase activity of the cell polarity regulator atypical protein kinase C (aPKC). Consistently, we demonstrate that podocyte-specific deletion of aPKCλ resulted in a decrease of cell-surface localization of SD components, causing massive proteinuria. In conclusion, the regulation of SD turnover by aPKC is crucial for the maintenance of SD integrity, and defects in aPKC signaling can lead to proteinuria. These findings not only reveal the pivotal importance of the dynamic turnover of cell-surface SD components but also suggest a novel pathophysiological basis in glomerular disease.
    Journal of Biochemistry 04/2014; DOI:10.1093/jb/mvu022 · 3.07 Impact Factor
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    ABSTRACT: Mesangial cell migration, regulated by several growth factors, is crucial after glomerulopathy and during glomerular development. Directional migration requires the establishment of a polarized cytoskeletal arrangement, a process regulated by coordinated actin dynamics and focal adhesion turnover at the peripheral ruffles in migrating cells. Here we found high expression of the actin cross-linking protein EPLIN (epithelial protein lost in neoplasm) in mesangial cells. EPLIN was localized in mesangial angles, which consist of actin-containing microfilaments extending underneath the capillary endothelium, where they attach to the glomerular basement membrane. In cultured mesangial cells, EPLIN was localized in peripheral actin bundles at focal adhesions and formed a protein complex with paxillin. The MEK-ERK (extracellular signal-regulated kinase) cascade regulated EPLIN-paxillin interaction and induced translocalization of EPLIN from focal adhesion sites to peripheral ruffles. Knockdown of EPLIN in mesangial cells enhanced platelet-derived growth factor-induced focal adhesion disassembly and cell migration. Furthermore, EPLIN expression was decreased in mesangial proliferative nephritis in rodents and humans in vivo. These results shed light on the coordinated actin remodeling in mesangial cells during restorative remodeling. Thus, changes in expression and localization of cytoskeletal regulators underlie phenotypic changes in mesangial cells in glomerulonephritis.Kidney International advance online publication, 2 April 2014; doi:10.1038/ki.2014.85.
    Kidney International 04/2014; 86(3). DOI:10.1038/ki.2014.85 · 8.52 Impact Factor
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    ABSTRACT: Previous studies have identified significant associations between the development of idiopathic focal segmental glomerulosclerosis (FSGS) and MYH9 encoding nonmuscle myosin heavy chain-IIA (NMMHC-IIA). However, these studies focused only on the linkage of MYH9 polymorphisms and development of FSGS. There have been no reports on pathological changes of NMMHC-IIA in human glomerular diseases. Here we report on the precise localization of NMMHC-IIA in podocytes and changes in NMMHC-IIA expression in pathological states in rats and humans. Immunocytochemical (immunofluorescence and immunoelectron microscopy) studies were performed to determine the precise localization of NMMHC-IIA. Expression levels of NMMHC-IIA were investigated in puromycin aminonucleoside (PAN)-treated rats; and expression levels of NMMHC-IIA and other podocyte-related proteins were investigated in glomeruli of patients with idiopathic FSGS and other heavy proteinuric glomerular diseases. NMMHC-IIA was located primarily at the cell body and primary processes of podocytes; this localization is distinct from other podocyte-related molecules causing hereditary FSGS. In PAN-treated rat kidneys, expression levels of NMMHC-IIA in podocytes decreased. Immunohistochemical analysis revealed that expression levels of NMMHC-IIA markedly decreased in idiopathic nephrotic syndrome, especially FSGS, whereas it did not change in other chronic glomerulonephritis showing apparent proteinuria. Changes in NMMHC-IIA expression were observed in glomeruli where expression of nephrin and synaptopodin was maintained. Considering previous genome-wide association studies and development of FSGS in patients with MYH9 mutations, the characteristic localization of NMMHC-IIA and the specific decrease in NMMHC-IIA expression in idiopathic nephrotic syndrome, especially FSGS, suggest the important role of NMMHC-IIA in the development of FSGS.
    Nephrology Dialysis Transplantation 09/2013; DOI:10.1093/ndt/gft350 · 3.49 Impact Factor
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    ABSTRACT: Vertebrate glomerular podocytes possess a highly sialylated transmembrane glycoprotein, Podocalyxin. In mammals, the sialic acid of Podocalyxin plays a crucial role in the formation of the characteristic podocyte architecture required for glomerular filtration. We examined the function of Podocalyxin in the developing zebrafish pronephros by disrupting the expression of podocalyxin through the use of morpholino antisense oligonucleotides. Podocalyxin was localized at the apical membrane of podocytes throughout pronephric glomerular development in zebrafish. Translational blocking of podocalyxin expression resulted in pericardial edema and a hypoplastic glomerulus. Whereas regular foot processes with a slit diaphragm covered 66.7 ± 7.8% of the urinary surface of glomerular basement membrane in control fish, only 14.4 ± 7.5% of this area was covered with regular foot processes in the translationally-blocked morphants. Splice blocking of podocalyxin exon 2, which partially encodes the bulky mucin domain with extensive sialic acid-containing sugar chains, resulted in the deletion of 53% of mucin domain-coding sequence from podocalyxin mRNA. Approximately 40% of these splice-blocked morphants had mild pericardial edema. Although the pronephric glomerulus in the splice-blocked morphants exhibited almost normal appearance with developed glomerular capillaries and mesangium, they had only 36.3 ± 6.9% of the area covered with regular foot processes. In conclusion, Podocalyxin is predominantly expressed in the podocytes and plays a distinct role in the formation of the podocyte foot processes with a slit diaphragm during zebrafish pronephric development.
    08/2013; 1(3). DOI:10.1002/phy2.74
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    ABSTRACT: Immunoglobulin A nephropathy (IgAN) is characterized by mesangial cell proliferation and mesangial expansion with mesangial depositions of IgA. We have found that electron-dense deposits (EDD) are often observed in areas other than paramesangial areas in glomeruli. To compare electron microscopic findings with light microscopic findings and clinical data, we examined the biopsies from 178 patients with IgAN. Patients were divided into two groups: group A had only paramesangial deposits and group B had deposits not only in paramesangial areas but also in other areas. All patients examined in this study had EDD in glomerular paramesangial areas. Thirty-six patients were included in group B. Cellular crescent formation in glomeruli and urinary protein in group B were significantly higher than those in group A (P < 0.01). Serum albumin and estimated glomerular filtration rate (eGFR) in group B were significantly lower than those in group A (P < 0.05). Group B showed a significant positive correlation with histological severity, which is defined in the Japanese Clinical Guidelines on IgAN. In patients with broad distribution of EDD, urinary protein was significantly increased (P < 0.05). Detailed observation of EDD distribution has an impact on evaluation of the disease activity of IgAN.
    Medical Molecular Morphology 12/2012; 45(1):29-34. DOI:10.1007/s00795-011-0538-3 · 1.07 Impact Factor
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    ABSTRACT: The glomerulus of the vertebrate kidney links the vasculature to the excretory system and produces the primary urine. It is a component of every single nephron in the complex mammalian metanephros and also in the primitive pronephros of fish and amphibian larvae. This systematic work highlights the benefits of using teleost models to understand the pronephric glomerulus development. The morphological processes forming the pronephric glomerulus are astoundingly different between medaka and zebrafish. (1) The glomerular primordium of medaka - unlike the one of zebrafish - exhibits a C-shaped epithelial layer. (2) The C-shaped primordium contains a characteristic balloon-like capillary, which is subsequently divided into several smaller capillaries. (3) In zebrafish, the bilateral pair of pronephric glomeruli is fused at the midline to form a glomerulus, while in medaka the two parts remain unmerged due to the interposition of the interglomerular mesangium. (4) Throughout pronephric development the interglomerular mesangial cells exhibit numerous cytoplasmic granules, which are reminiscent of renin-producing (juxtaglomerular) cells in the mammalian afferent arterioles. Our systematic analysis of medaka and zebrafish demonstrates that in fish, the morphogenesis of the pronephric glomerulus is not stereotypical. These differences need be taken into account in future analyses of medaka mutants with glomerulus defects.
    PLoS ONE 09/2012; 7(9):e45286. DOI:10.1371/journal.pone.0045286 · 3.53 Impact Factor
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    ABSTRACT: The slit diaphragm (SD) is an intercellular junction between renal glomerular epithelial cells (podocytes) that is essential for permselectivity in glomerular ultrafiltration. The SD components, nephrin and Neph1, assemble a signaling complex in a tyrosine phosphorylation dependent manner, and regulate the unique actin cytoskeleton of podocytes. Mutations in the NPHS1 gene that encodes nephrin cause congenital nephrotic syndrome (CNS), which is characterized by the loss of the SD and massive proteinuria. Recently, we have identified the expression of the transmembrane glycoprotein signal regulatory protein α (SIRPα) at the SD. In the present study, we analyzed the expression of SIRPα in developing kidneys, in kidneys from CNS patients and in proteinuric rat models. The possibility that SIRPα interacts with known SD proteins was also investigated. SIRPα was concentrated at the SD junction during the maturation of intercellular junctions. In the glomeruli of CNS patients carrying mutations in NPHS1, where SD formation is disrupted, the expression of SIRPα as well as Neph1 and nephrin was significantly decreased, indicating that SIRPα is closely associated with the nephrin complex. Indeed, SIRPα formed hetero-oligomers with nephrin in cultured cells and in glomeruli. Furthermore, the cytoplasmic domain of SIRPα was highly phosphorylated in normal glomeruli, and its phosphorylation was dramatically decreased upon podocyte injury in vivo. Thus, SIRPα interacts with nephrin at the SD, and its phosphorylation is dynamically regulated in proteinuric states. Our data provide new molecular insights into the phosphorylation events triggered by podocyte injury.
    FEBS Journal 07/2012; 279(17):3010-21. DOI:10.1111/j.1742-4658.2012.08682.x · 3.99 Impact Factor
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    ABSTRACT: RASSF6, a member of RASSF tumour suppressor proteins, binds to mammalian Ste20-like kinases (MST1/2), core kinases of the proapoptotic Hippo pathway and cooperates with the Hippo pathway to induce apoptosis. We originally identified RASSF6 as a putative interactor of membrane-associated guanylate kinase inverted (MAGI)-1 by the yeast two-hybrid screening. We used human kidney cDNA library for the screening. MAGI-1 is abundantly expressed in kidney and is a core component of the slit diaphragm. These findings suggest that RASSF6 is expressed in kidney. However, the function of RASSF6 in kidney is not yet studied. We performed this study to confirm the interaction of RASSF6 with MAGI-1, to analyse the expression of RASSF6 in kidney and to gain insight into the function of RASSF6 in kidney. RASSF6 binds to PDZ domains of MAGI-1 through its C-terminal PDZ-binding motif and is coimmunoprecipitated with MAGI-1 from rat liver. RASSF6 is localized in normal kidney glomerulus but disappears when the slit diaphragm is disrupted in nephrotic kidney. RASSF6 is also localized on apical membranes in renal proximal tubular epithelial cells. We demonstrated that RASSF6 as well as the Hippo pathway are involved in the sorbitol-induced apoptosis in immortalized human proximal renal tubular epithelial HK-2 cells.
    Journal of Biochemistry 05/2012; 152(1):111-9. DOI:10.1093/jb/mvs056 · 3.07 Impact Factor
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    ABSTRACT: Connexin43 (Cx43), a gap junction protein, mediates cell-cell communication via electrical and chemical coupling. Ischemic stress of the cardiac muscle interrupts intercellular communication by changing the distribution and phosphorylation status of Cx43. This may be a factor contributing to reentrant arrhythmia. The calcium channel blocker diltiazem is known for its protective and anti-arrhythmogenic effect in ischemic heart disease. In this study, we assess the effect of diltiazem pretreatment upon ischemia-induced phosphorylation change of Cx43. Langendorff preparations of isolated Wistar rat hearts were performed. After stabilization, hearts were treated with (D+) or without diltiazem (D⁻), then subjected to hypoxia-reoxygenation. After perfusion, the left ventricle was prepared for immunocytochemistry and immunoblot analysis. During perfusion, left ventricular function was better in the D+ group than the D⁻ group. Immunostaining of the heart indicated that dephosphorylated Cx43 (dpCx43) signal was increased after hypoxic perfusion, and this finding was confirmed by immunoblot data. The quantitative area analysis of dpCx43 using the immunohistochemical approach showed that the dpCx43-positive area was enlarged, as the hypoxic perfusion time was longer, and it was reduced by pretreatment of diltiazem. There was a negative correlation between the dpCx43 area and %RPP (rate-pressure product), calculated by heart rate and contraction force. Pretreatment of diltiazem could protect the heart against hypoxia-reoxygenation injury by attenuation of dephosphorylation of Cx43. The anti-arrhythmic mechanism of diltiazem may include the preservation of phosphorylation status of Cx43 after hypoxia-reoxygenation injury.
    Histology and histopathology 03/2011; 26(3):315-22. · 2.24 Impact Factor
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    ABSTRACT: The renal glomerulus consists of endothelial cells, podocytes, and mesangial cells. These cells cooperate with each other for glomerular filtration; however, the intercellular signaling molecules between glomerular cells are not fully determined. Tyrosine phosphorylation of slit diaphragm molecules is a key to the detection of the signal to podocytes from other cells. Although src kinase is involved in this event, the molecules working for dephosphorylation remain unclear. We demonstrate that signal-inhibitory regulatory protein (SIRP)-alpha, which recruits a broadly distributed tyrosine dephosphorylase SHP-2 to the plasma membrane, is located in podocytes. SIRP-alpha is a type I transmembrane glycoprotein, which has three immunoglobulin-like domains in the extracellular region and two SH2 binding motifs in the cytoplasm. This molecule functions as a scaffold for many proteins, especially the SHP-2 molecule. SIRP-alpha is concentrated in the slit diaphragm region of normal podocytes. CD47, a ligand for SIRP-alpha, is also expressed in the glomerulus. CD47 is located along the plasma membrane of mesangial cells, but not on podocytes. CD47 is markedly decreased during mesangiolysis, but increased in mesangial cells in the restoration stage. SIRP-alpha is heavily tyrosine phosphorylated under normal conditions; however, tyrosine phosphorylation of SIRP-alpha was markedly decreased during mesangiolysis induced by Thy1.1 monoclonal antibody injection. It is known that the cytoplasmic domain of SIPR-alpha is dephosphorylated when CD47 binds to the extracellular domain of SIRP-alpha. The data suggest that the CD47-SIRP-alpha interaction may be functionally important in cell-cell communication in the diseased glomerulus.
    AJP Renal Physiology 09/2010; 299(3):F517-27. DOI:10.1152/ajprenal.00571.2009 · 4.42 Impact Factor
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    ABSTRACT: Focal segmental glomerulosclerosis (FSGS) is a leading cause of nephrotic syndrome and end-stage renal disease worldwide. Although the mechanisms underlying this important disease are poorly understood, the glomerular podocyte clearly plays a central role in disease pathogenesis. In the current work, we demonstrate that the homophilic adhesion molecule sidekick-1 (sdk-1) is up-regulated in podocytes in FSGS both in rodent models and in human kidney biopsy samples. Transgenic mice that have podocyte-specific overexpression of sdk-1 develop gradually progressive heavy proteinuria and severe FSGS. We also show that sdk-1 associates with the slit diaphragm linker protein MAGI-1, which is already known to interact with several critical podocyte proteins including synaptopodin, alpha-actinin-4, nephrin, JAM4, and beta-catenin. This interaction is mediated through a direct interaction between the carboxyl terminus of sdk-1 and specific PDZ domains of MAGI-1. In vitro expression of sdk-1 enables a dramatic recruitment of MAGI-1 to the cell membrane. Furthermore, a truncated version of sdk-1 that is unable to bind to MAGI-1 does not induce podocyte dysfunction when overexpressed. We conclude that the up-regulation of sdk-1 in podocytes is an important pathogenic factor in FSGS and that the mechanism involves disruption of the actin cytoskeleton possibly via alterations in MAGI-1 function.
    Journal of Biological Chemistry 08/2010; 285(33):25677-85. DOI:10.1074/jbc.M110.133959 · 4.60 Impact Factor
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    Koichiro Ichimura, Hidetake Kurihara, Tatsuo Sakai
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    ABSTRACT: Most tubular epithelial cell types express primary cilia, and mutations of primary-cilium-associated proteins are well known to cause several kinds of cystic renal disease. However, until now, it has been unclear whether mammalian podocytes express primary cilia in vivo. In this study, we determined whether primary cilia are present in the podocytes of rat immature and mature glomeruli by means of transmission electron microscopy of serial ultrathin sections. In immature glomeruli of fetal rats, podocytes express the primary cilia with high percentages at the S-shaped body (88 ± 5%, n = 3), capillary loop (95 ± 4%, n = 4), and maturing glomerulus (76 ± 13%, n = 5) stages. The percentage of ciliated podocytes was significantly lower at the maturing glomerulus stage than at the former two stages. In mature glomeruli of adult rats, ciliated podocytes were not found at all (0 ± 0%, n = 11). These findings indicate that the primary cilia gradually disappear in rat podocytes during glomerular development. Since glomerular filtration rate increases during development, the primary cilia on the podocytes are subjected to a stronger bending force. Thus, the disappearance of the primary cilia presumably prevents the entry of excessive calcium-ions via the cilium-associated polycystin complexes and the disturbance of intracellular signaling cascades in mature podocytes. Electronic supplementary material The online version of this article (doi:10.1007/s00441-010-0983-7) contains supplementary material, which is available to authorized users.
    Cell and Tissue Research 07/2010; 341(1):197-209. DOI:10.1007/s00441-010-0983-7 · 3.33 Impact Factor
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    ABSTRACT: Proper localization of nephrin determines integrity of the glomerular slit diaphragm. Slit diaphragm proteins assemble into functional signaling complexes on a raft-based platform, but how the trafficking of these proteins coordinates with their signaling function is unknown. Here, we demonstrate that a raft-mediated endocytic (RME) pathway internalizes nephrin. Nephrin internalization was slower with raft-mediated endocytosis than with classic clathrin-mediated endocytosis. Ultrastructurally, the RME pathway consisted of noncoated invaginations and was dependent on cholesterol and dynamin. Nephrin constituted a stable, signaling-competent microdomain through interaction with Fyn, a Src kinase, and podocin, a scaffold protein. Tyrosine phosphorylation of nephrin triggered its own RME-mediated internalization. Protamine-induced hyperphosphorylation of nephrin led to noncoated invaginations predominating over coated pits. These results demonstrate that an RME pathway couples nephrin internalization to its own signaling, suggesting that RME promotes proper spatiotemporal assembly of slit diaphragms during podocyte development or injury.
    Journal of the American Society of Nephrology 10/2009; 20(12):2534-45. DOI:10.1681/ASN.2009010011 · 9.47 Impact Factor
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    ABSTRACT: Morphological change of podocytes is closely correlated with the development of proteinuria. Podocalyxin is a major sialoglycoprotein of the podocytes and is thought to be involved in the maintenance of the foot processes structure. Our aim was to examine the mechanism by which podocalyxin contributes to the morphological change of podocytes. We searched protein(s) which coprecipitate with podocalyxin using rat glomerular lysate. Localization of podocalyxin and the coprecipitated protein, cortactin, was studied in a model of puromycin aminonucleoside (PAN) nephrosis by immunocytochemistry. Tyrosine phosphorylation of cortactin was examined. Association of the podocalyxin/cortactin complex with the actin cytoskeleton was evaluated by extraction with Triton-X and immunoblotting. Cortactin was found to be co-immunoprecipitated with podocalyxin. Immunocytochemical analysis revealed that these 2 proteins colocalized in the apical side of podocytes. In PAN nephrosis, localization of cortactin was altered after the onset of proteinuria, with increased tyrosine phosphorylation. Simultaneously, the dissociation of the podocalyxin/cortactin complex from the actin cytoskeleton was induced. These results indicate that cortactin mediates interaction between podocalyxin and actin filaments in podocytes and that alteration of this interaction may play a role in the process of morphological change of podocytes.
    Nephron Experimental Nephrology 09/2009; 113(3):e89-96. DOI:10.1159/000235245 · 1.65 Impact Factor
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    ABSTRACT: CXC chemokine ligand 12 (CXCL12; stromal cell-derived factor 1) is a unique homeostatic chemokine that signals through its cognate receptor, CXCR4. CXCL12/CXCR4 signaling is essential for the formation of blood vessels in the gastrointestinal tract during development, but its contribution to renal development remains unclear. Here, we found that CXCL12-secreting stromal cells surround CXCR4-positive epithelial components of early nephrons and blood vessels in the embryonic kidney. In glomeruli, we observed CXCL12-secreting podocytes in close proximity to CXCR4-positive endothelial cells. Both CXCL12- and CXCR4-deficient kidneys exhibited identical phenotypes; there were no apparent abnormalities in early nephrogenesis or in differentiation of podocytes and tubules, but there was defective formation of blood vessels, including ballooning of the developing glomerular tuft and disorganized patterning of the renal vasculature. To clarify the relative importance of different cellular defects resulting from ablation of CXCL12 and CXCR4, we established endothelial cell-specific CXCR4-deficient mice, which recapitulated the renal phenotypes of conventional CXCR4-deficient mice. We conclude that CXCL12 secreted from stromal cells or podocytes acts on endothelial cells to regulate vascular development in the kidney. These findings suggest new potential therapeutic targets for remodeling the injured kidney.
    Journal of the American Society of Nephrology 06/2009; 20(8):1714-23. DOI:10.1681/ASN.2008060640 · 9.47 Impact Factor
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    ABSTRACT: A specialized intercellular junction between podocytes, known as the slit diaphragm (SD), forms the essential structural frame-work for glomerular filtration in the kidney. In addition, mounting evidence demonstrates that the SD also plays a crucial role as a signaling platform in physiological and pathological states. Nephrin, the major component of the SD, is tyrosine-phosphorylated by a Src family tyrosine kinase, Fyn, in developing or injured podocytes, recruiting Nck to Nephrin via its Src homology 2 domain to regulate dynamic actin remodeling. Dysregulated Ca2+ homeostasis has also been implicated in podocyte damage, but the mechanism of how podocytes respond to injury is largely unknown. Here we have identified phospholipase C-γ1 (PLC-γ1) as a novel phospho-Nephrin-binding protein. When HEK293T cells expressing a chimeric protein consisting of CD8 and Nephrin cytoplasmic domain (CD) were treated with anti-CD8 and anti-mouse antibodies, clustering of Nephrin and phosphorylation of Nephrin-CD were induced. Upon this clustering, PLC-γ1 was bound to phosphorylated Nephrin Tyr-1204, which induced translocation of PLC-γ1 from cytoplasm to the CD8/Nephrin cluster on the plasma membrane. The recruitment of PLC-γ1 to Nephrin activated PLC-γ1, as detected by phosphorylation of PLC-γ1 Tyr-783 and increase in inositol 1,4,5-trisphosphate level. We also found that Nephrin Tyr-1204 phosphorylation triggers the Ca2+ response in a PLC-γ1-dependent fashion. Furthermore, PLC-γ1 is significantly phosphorylated in injured podocytes in vivo. Given the profound effect of PLC-γ in diverse cellular functions, regulation of the Ca2+ signaling by Nephrin may be important in modulating the glomerular filtration barrier function.
    Journal of Biological Chemistry 03/2009; 284(13):8951-8962. · 4.60 Impact Factor
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    ABSTRACT: A specialized intercellular junction between podocytes, known as the slit diaphragm (SD), forms the essential structural frame-work for glomerular filtration in the kidney. In addition, mounting evidence demonstrates that the SD also plays a crucial role as a signaling platform in physiological and pathological states. Nephrin, the major component of the SD, is tyrosine-phosphorylated by a Src family tyrosine kinase, Fyn, in developing or injured podocytes, recruiting Nck to Nephrin via its Src homology 2 domain to regulate dynamic actin remodeling. Dysregulated Ca(2+) homeostasis has also been implicated in podocyte damage, but the mechanism of how podocytes respond to injury is largely unknown. Here we have identified phospholipase C-gamma1 (PLC-gamma1) as a novel phospho-Nephrin-binding protein. When HEK293T cells expressing a chimeric protein consisting of CD8 and Nephrin cytoplasmic domain (CD) were treated with anti-CD8 and anti-mouse antibodies, clustering of Nephrin and phosphorylation of Nephrin-CD were induced. Upon this clustering, PLC-gamma1 was bound to phosphorylated Nephrin Tyr-1204, which induced translocation of PLC-gamma1 from cytoplasm to the CD8/Nephrin cluster on the plasma membrane. The recruitment of PLC-gamma1 to Nephrin activated PLC-gamma1, as detected by phosphorylation of PLC-gamma1 Tyr-783 and increase in inositol 1,4,5-trisphosphate level. We also found that Nephrin Tyr-1204 phosphorylation triggers the Ca(2+) response in a PLC-gamma1-dependent fashion. Furthermore, PLC-gamma1 is significantly phosphorylated in injured podocytes in vivo. Given the profound effect of PLC-gamma in diverse cellular functions, regulation of the Ca(2+) signaling by Nephrin may be important in modulating the glomerular filtration barrier function.
    Journal of Biological Chemistry 02/2009; 284(13):8951-62. DOI:10.1074/jbc.M806851200 · 4.60 Impact Factor
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    ABSTRACT: Glomerular visceral epithelial cells (podocytes) contain interdigitated processes that form specialized intercellular junctions, termed slit diaphragms, which provide a selective filtration barrier in the renal glomerulus. Analyses of disease-causing mutations in familial nephrotic syndromes and targeted mutagenesis in mice have revealed critical roles of several proteins in the assembly of slit diaphragms. The nephrin-podocin complex is the main constituent of slit diaphragms. However, the molecular mechanisms regulating these proteins to maintain the slit diaphragms are still largely unknown. Here, we demonstrate that the PAR3-atypical protein kinase C (aPKC)-PAR6beta cell polarity proteins co-localize to the slit diaphragms with nephrin. Furthermore, selective depletion of aPKClambda in mouse podocytes results in the disassembly of slit diaphragms, a disturbance in apico-basal cell polarity, and focal segmental glomerulosclerosis (FSGS). The aPKC-PAR3 complex associates with the nephrin-podocin complex in podocytes through direct interaction between PAR3 and nephrin, and the kinase activity of aPKC is required for the appropriate distribution of nephrin and podocin in podocytes. These observations not only establish a critical function of the polarity proteins in the maintenance of slit diaphragms, but also imply their potential involvement in renal failure in FSGS.
    PLoS ONE 02/2009; 4(1):e4194. DOI:10.1371/journal.pone.0004194 · 3.53 Impact Factor

Publication Stats

2k Citations
252.27 Total Impact Points

Institutions

  • 2000–2015
    • Juntendo University
      • • Department of Anatomy and Life Structure
      • • Department of Medicine
      Edo, Tōkyō, Japan
  • 2012
    • Tokyo Medical and Dental University
      • Department of Medical Biochemistry
      Edo, Tōkyō, Japan
  • 2009
    • The University of Tokyo
      • Institute of Medical Science
      Tokyo, Tokyo-to, Japan
  • 1997–2009
    • Shionogi & Co., Ltd.
      Ōsaka, Ōsaka, Japan