Neph-Nephrin proteins bind the Par3-Par6-atypical protein kinase C (aPKC) complex to regulate podocyte cell polarity.
ABSTRACT: The kidney filter represents a unique assembly of podocyte epithelial cells that tightly enwrap the glomerular capillaries with their foot processes and the interposed slit diaphragm. So far, very little is known about the guidance cues and polarity signals required to regulate proper development and maintenance of the glomerular filtration barrier. We now identify Par3, Par6, and atypical protein kinase C (aPKC) polarity proteins as novel Neph1-Nephrin-associated proteins. The interaction was mediated through the PDZ domain of Par3 and conserved carboxyl terminal residues in Neph1 and Nephrin. Par3, Par6, and aPKC localized to the slit diaphragm as shown in immunofluorescence and immunoelectron microscopy. Consistent with a critical role for aPKC activity in podocytes, inhibition of glomerular aPKC activity with a pseudosubstrate inhibitor resulted in a loss of regular podocyte foot process architecture. These data provide an important link between cell recognition mediated through the Neph1-Nephrin complex and Par-dependent polarity signaling and suggest that this molecular interaction is essential for establishing the three-dimensional architecture of podocytes at the kidney filtration barrier.
Project description:Atypical protein kinase C (aPKC) is a central component of the evolutionarily conserved Par3-Par6-aPKC complex, one of the fundamental regulators of cell polarity. We recently demonstrated that these proteins interact with Neph-nephrin molecules at the slit diaphragm of the glomerular filtration barrier. Here, we report that podocyte-specific deletion of aPKClambda/iota in mice results in severe proteinuria, nephrotic syndrome, and death at 4 to 5 wk after birth. Podocyte foot processes of knockout mice developed structural defects, including mislocalization of the slit diaphragm. In the glomerulus, aPKClambda/iota was primarily expressed in developing glomerular epithelial cells and podocyte foot processes. Interestingly, under physiologic conditions, aPKClambda/iota translocated from the apical surface to the basolateral side of developing podocytes, and this translocation preceded the development of foot processes and formation of slit diaphragms. Supporting a critical role for aPKClambda/iota in the maintenance of slit diaphragms and podocyte foot processes, aPKClambda/iota associated with the Neph-nephrin slit diaphragm complex and localized to the tips of filopodia and leading edges of cultured podocytes. These results suggest that aPKC signaling is fundamental to glomerular maintenance and development.
Project description: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.
Project description:Neph1-deficient mice develop nephrotic syndrome at birth, indicating the importance of this protein in the development of a normal glomerular filtration barrier. While the precise subcellular localization of Neph1 remains unknown, its relationship with other components of the glomerular filtration barrier is of great interest in this field. In this paper, we localize the expression of Neph1 to the glomerular slit diaphragm by immunogold electron microscopy in rodents and describe its direct interaction with two other components of the slit diaphragm, nephrin and ZO-1. Both native and recombinant Neph1 associate with each other as dimers and multimers and interact with nephrin via their extracellular segments. Disruption of the Neph1-nephrin interaction in vivo by injecting combinations of individual subnephritogenic doses of anti-Neph1 and anti-nephrin results in complement- and leukocyte-independent proteinuria with preserved foot processes. This disruption modestly reduces Neph1 and nephrin protein expression in podocytes and dramatically reduces ZO-1 protein expression via the interaction of ZO-1 PDZ domains with the cytoplasmic tail of Neph1, independent of changes in mRNA expression of all three genes. The interaction between nephrin and Neph1 is specific and not shared by either protein with P-cadherin, another integral slit diaphragm protein. The interaction between nephrin and Neph1 therefore appears to be an important determinant of glomerular permeability.
Project description:MAGI-1 is a multidomain cytosolic scaffolding protein that in the kidney is specifically located at the podocyte slit diaphragm, a specialized junction that is universally injured in proteinuric diseases. There it interacts with several essential molecules, including nephrin and neph1, which are required for slit diaphragm formation and as an intracellular signaling hub. Here, we show that diminished MAGI-1 expression in cultured podocytes reduced nephrin and neph1 membrane localization and weakened tight junction integrity. Global magi1 knock-out mice, however, demonstrated normal glomerular histology and function into adulthood. We hypothesized that a second mild but complementary genetic insult might induce glomerular disease susceptibility in these mice. To identify such a gene, we utilized the developing fly eye to test for functional complementation between MAGI and its binding partners. In this way, we identified diminished expression of fly Hibris (nephrin) or Roughest (neph1) as dramatically exacerbating the effects of MAGI depletion. Indeed, when these combinations were studied in mice, the addition of nephrin, but not neph1, heterozygosity to homozygous deletion of MAGI-1 resulted in spontaneous glomerulosclerosis. In cultured podocytes, MAGI-1 depletion reduced intercellular contact-induced Rap1 activation, a pathway critical for proper podocyte function. Similarly, magi1 knock-out mice showed diminished glomerular Rap1 activation, an effect dramatically enhanced by concomitant nephrin haploinsufficiency. Finally, combined overexpression of MAGI-1 and nephrin increased Rap1 activation, but not when substituting a mutant MAGI-1 that cannot bind nephrin. We conclude that the interaction between nephrin and MAGI-1 regulates Rap1 activation in podocytes to maintain long term slit diaphragm structure.
Project description:Although the slit diaphragm proteins in podocytes are uniquely organized to maintain glomerular filtration assembly and function, little is known about the underlying mechanisms that participate in trafficking these proteins to the correct location for development and homeostasis. Identifying these mechanisms will likely provide novel targets for therapeutic intervention to preserve podocyte function following glomerular injury. Analysis of structural variation in cases of human nephrotic syndrome identified rare heterozygous deletions of EXOC4 in two patients. This suggested that disruption of the highly-conserved eight-protein exocyst trafficking complex could have a role in podocyte dysfunction. Indeed, mRNA profiling of injured podocytes identified significant exocyst down-regulation. To test the hypothesis that the exocyst is centrally involved in podocyte development/function, we generated homozygous podocyte-specific Exoc5 (a central exocyst component that interacts with Exoc4) knockout mice that showed massive proteinuria and died within 4 weeks of birth. Histological and ultrastructural analysis of these mice showed severe glomerular defects with increased fibrosis, proteinaceous casts, effaced podocytes, and loss of the slit diaphragm. Immunofluorescence analysis revealed that Neph1 and Nephrin, major slit diaphragm constituents, were mislocalized and/or lost. mRNA profiling of Exoc5 knockdown podocytes showed that vesicular trafficking was the most affected cellular event. Mapping of signaling pathways and Western blot analysis revealed significant up-regulation of the mitogen-activated protein kinase and transforming growth factor-? pathways in Exoc5 knockdown podocytes and in the glomeruli of podocyte-specific Exoc5 KO mice. Based on these data, we propose that exocyst-based mechanisms regulate Neph1 and Nephrin signaling and trafficking, and thus podocyte development and function.
Project description:The nephron is the basic structural and functional unit of the vertebrate kidney. It is composed of a glomerulus, the site of ultrafiltration, and a renal tubule, along which the filtrate is modified. Although widely regarded as a vertebrate adaptation, 'nephron-like' features can be found in the excretory systems of many invertebrates, raising the possibility that components of the vertebrate excretory system were inherited from their invertebrate ancestors. Here we show that the insect nephrocyte has remarkable anatomical, molecular and functional similarity to the glomerular podocyte, a cell in the vertebrate kidney that forms the main size-selective barrier as blood is ultrafiltered to make urine. In particular, both cell types possess a specialized filtration diaphragm, known as the slit diaphragm in podocytes or the nephrocyte diaphragm in nephrocytes. We find that fly (Drosophila melanogaster) orthologues of the major constituents of the slit diaphragm, including nephrin, NEPH1 (also known as KIRREL), CD2AP, ZO-1 (TJP1) and podocin, are expressed in the nephrocyte and form a complex of interacting proteins that closely mirrors the vertebrate slit diaphragm complex. Furthermore, we find that the nephrocyte diaphragm is completely lost in flies lacking the orthologues of nephrin or NEPH1-a phenotype resembling loss of the slit diaphragm in the absence of either nephrin (as in human congenital nephrotic syndrome of the Finnish type, NPHS1) or NEPH1. These changes markedly impair filtration function in the nephrocyte. The similarities we describe between invertebrate nephrocytes and vertebrate podocytes provide evidence suggesting that the two cell types are evolutionarily related, and establish the nephrocyte as a simple model in which to study podocyte biology and podocyte-associated diseases.
Project description:Apical-basal polarity is a key feature of most epithelial cells and it is regulated by highly conserved protein complexes. In mammalian podocytes, which emerge from columnar epithelial cells, this polarity is preserved and the tight junctions are converted to the slit diaphragms, establishing the filtration barrier. In Drosophila, nephrocytes show several structural and functional similarities with mammalian podocytes and proximal tubular cells. However, in contrast to podocytes, little is known about the role of apical-basal polarity regulators in these cells. In this study, we used expansion microscopy and found the apical polarity determinants of the PAR/aPKC and Crb-complexes to be predominantly targeted to the cell cortex in proximity to the nephrocyte diaphragm, whereas basolateral regulators also accumulate intracellularly. Knockdown of PAR-complex proteins results in severe endocytosis and nephrocyte diaphragm defects, which is due to impaired aPKC recruitment to the plasma membrane. Similar, downregulation of most basolateral polarity regulators disrupts Nephrin localization but had surprisingly divergent effects on endocytosis. Our findings suggest that morphology and slit diaphragm assembly/maintenance of nephrocytes is regulated by classical apical-basal polarity regulators, which have distinct functions in endocytosis.
Project description: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.
Project description:Podocytes are specialized kidney cells that form the kidney filtration barrier through the connection of their foot processes. Nephrin and Neph family transmembrane molecules at the surface of podocytes interconnect to form a unique type of cell-cell junction, the slit diaphragm, which acts as a molecular sieve. The cytoplasmic tails of Nephrin and Neph mediate cytoskeletal rearrangement that contributes to the maintenance of the filtration barrier. Nephrin and Neph1 orthologs are essential to regulate cell-cell adhesion and Rac-dependent actin rearrangement during Drosophila myoblast fusion. We hypothesized here that molecules regulating myoblast fusion in Drosophila could contribute to signaling downstream of Nephrin and Neph1 in podocytes. We found that Nephrin engagement promoted recruitment of the Rac exchange factor Dock1 to the membrane. Furthermore, Nephrin overexpression led to lamellipodia formation that could be blocked by inhibiting Rac1 activity. We generated in vivo mouse models to investigate whether Dock1 and Dock5 contribute to the formation and maintenance of the kidney filtration barrier. Our results indicate that while Dock1 and Dock5 are expressed in podocytes, their functions are not essential for the development of the glomerular filtration barrier. Furthermore, mice lacking Dock1 were not protected from LPS-induced podocyte effacement. Our data suggest that Dock1 and Dock5 are not the important exchange factors regulating Rac activity during the establishment and maintenance of the glomerular barrier.
Project description: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 aPKC? 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 signalling 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.