While the mechanisms that regulate actin dynamics in cellular motility are intensively studied, relatively little is known about signaling events that transmit outside-in signals and direct assembly and regulation of actin polymerization complexes at the cell membrane. The kidney podocyte provides a unique model for investigating these mechanisms since deletion of Nephrin or Neph1, two interacting components of the specialized podocyte intercellular junction, results in abnormal podocyte morphogenesis and junction formation. We provide evidence that extends the existing model by which the Nephrin-Neph1 complex transduces phosphorylation-mediated signals that assemble an actin polymerization complex at the podocyte intercellular junction. Upon engagement, Neph1 is phosphorylated on specific tyrosine residues by Fyn, which results in the recruitment of Grb2, an event that is necessary for Neph1-induced actin polymerization at the plasma membrane. Importantly, Neph1 and Nephrin directly interact and, by juxtaposing Grb2 and Nck1/2 at the membrane following complex activation, cooperate to augment the efficiency of actin polymerization. These data provide evidence for a mechanism reminiscent of that employed by vaccinia virus and other pathogens, by which a signaling complex transduces an outside-in signal that results in actin filament polymerization at the plasma membrane.Precisely regulated actin polymerization provides the motive force necessary for intercellular junction formation and contributes to defining the shape and polarity of the cell. Similarly, directed actin polymerization proximate to the leading edge of the plasma membrane drives cell motility and is required in the complicated dynamics of lamellipodia, filopodia, and other specialized membrane structures including invadopodia and podosomes (6). While substantial progress has been made in understanding the cellular and molecular mechanisms that determine actin dynamics in these model systems (32), less is known about membrane-based proximal signaling events that transmit outside-in signals and direct assembly and regulation of actin polymerization complexes at these sites.Kidney glomerular visceral epithelial cells or podocytes are necessary for maintaining the glomerular filtration barrier (reviewed in reference 20). When mature, these cells have a unique octopus-like structure comprised of a central cell body that gives off branching primary, secondary, and tertiary processes. The tertiary processes or "foot processes" attach the podocyte to the glomerular capillary basement membrane, where they surround the glomerular capillary and where they interdigitate to form a specialized intercellular junction called the "slit diaphragm." Here foot processes provide a necessary element of the permeability-selective glomerular filter, allowing passage of water, solutes, and other small macromolecules from the capillary lumen to the urinary space while restricting the flux of cells and larger macromolecules.The podocyte provides a unique model for investiga...
Expression quantitative trait loci (eQTL) studies illuminate the genetics of gene expression and, in disease research, can be particularly illuminating when using the tissues directly impacted by the condition. In nephrology, there is a paucity of eQTL studies of human kidney. Here, we used whole-genome sequencing (WGS) and microdissected glomerular (GLOM) and tubulointerstitial (TI) transcriptomes from 187 individuals with nephrotic syndrome (NS) to describe the eQTL landscape in these functionally distinct kidney structures. Using MatrixEQTL, we performed cis-eQTL analysis on GLOM (n = 136) and TI (n = 166). We used the Bayesian "Deterministic Approximation of Posteriors" (DAP) to fine-map these signals, eQTLBMA to discover GLOM- or TI-specific eQTLs, and single-cell RNA-seq data of control kidney tissue to identify the cell type specificity of significant eQTLs. We integrated eQTL data with an IgA Nephropathy (IgAN) GWAS to perform a transcriptome-wide association study (TWAS). We discovered 894 GLOM eQTLs and 1,767 TI eQTLs at FDR < 0.05. 14% and 19% of GLOM and TI eQTLs, respectively, had >1 independent signal associated with its expression. 12% and 26% of eQTLs were GLOM specific and TI specific, respectively. GLOM eQTLs were most significantly enriched in podocyte transcripts and TI eQTLs in proximal tubules. The IgAN TWAS identified significant GLOM and TI genes, primarily at the HLA region. In this study, we discovered GLOM and TI eQTLs, identified those that were tissue specific, deconvoluted them into cell-specific signals, and used them to characterize known GWAS alleles. These data are available for browsing and download via our eQTL browser, "nephQTL."
The morphology of healthy podocyte foot processes is necessary for maintaining the characteristics of the kidney filtration barrier. In most forms of glomerular disease, abnormal filter barrier function results when podocytes undergo foot process spreading and retraction by remodeling their cytoskeletal architecture and intercellular junctions during a process known as effacement. The cell adhesion protein nephrin is necessary for establishing the morphology of the kidney podocyte in development by transducing from the specialized podocyte intercellular junction phosphorylation-mediated signals that regulate cytoskeletal dynamics. The present studies extend our understanding of nephrin function by showing that nephrin activation in cultured podocytes induced actin dynamics necessary for lamellipodial protrusion. This process required a PI3K-, Cas-, and Crk1/2-dependent signaling mechanism distinct from the previously described nephrin-Nck1/2 pathway necessary for assembly and polymerization of actin filaments. Our present findings also support the hypothesis that mechanisms governing lamellipodial protrusion in culture are similar to those used in vivo during foot process effacement in a subset of glomerular diseases. In mice, podocyte-specific deletion of Crk1/2 prevented foot process effacement in one model of podocyte injury and attenuated foot process effacement and associated proteinuria in a delayed fashion in a second model. In humans, focal adhesion kinase and Cas phosphorylation -markers of focal adhesion complex-mediated Crk-dependent signaling -was induced in minimal change disease and membranous nephropathy, but not focal segmental glomerulosclerosis. Together, these observations suggest that activation of a Cas-Crk1/2-dependent complex is necessary for foot process effacement observed in distinct subsets of human glomerular diseases. IntroductionWhen functioning properly in health, the kidney filtration barrier selectively prevents the passage of macromolecules from the blood compartment into the urinary space. Differentiated podocytes form a remarkable octopus-like morphology, extending numerous interdigitating foot processes defined by a unique 3-dimensional actin cytoskeletal architecture and requiring formation of a specialized intercellular junction. These foot processes adhere to and cover an extracellular matrix interposed between podocytes and an endothelium that creates the glomerular capillary wall. Podocytes undergo cytoskeletal remodeling to alter their morphology in nearly all forms of human glomerular disease, exhibiting what has been described as foot process spreading and retraction or as foot process effacement. This process by which podocytes change their cytoskeletal architecture appears to be a component of a common response of the podocyte to cellular injury, correlating with loss of normal filtration barrier selectivity and predicting the development of proteinuria in human disease and in experimental models (1, 2).
APOL1 variants have been associated with renal phenotypes in blacks. To refine clinical outcomes and discover mechanisms of APOL1-associated kidney injury, we analyzed clinical and genomic datasets derived from 90 black subjects in the Nephrotic Syndrome Study Network (NEPTUNE), stratified by APOL1 risk genotype. Ninety subjects with proteinuria $0.5 g/d were enrolled at first biopsy for primary nephrotic syndrome and followed. Clinical outcomes were determined, and renal histomorphometry and sequencing of Mendelian nephrotic syndrome genes were performed. APOL1 variants were genotyped, and glomerular and tubulointerstitial transcriptomes from protocol renal biopsy cores were analyzed for differential and correlative gene expression. Analyses were performed under the recessive model (high-risk genotype defined by two risk alleles). APOL1 high-risk genotype was significantly associated with a 17 ml/min per 1.73 m 2 lower eGFR and a 69% reduction in the probability of complete remission at any time, independent of histologic diagnosis. Neither APOL1 risk group was enriched for Mendelian mutations. On renal biopsy, high-risk genotype was associated with increased fractional interstitial area, interstitial fibrosis, and tubular atrophy. Risk genotype was not associated with intrarenal APOL1 mRNA expression levels. Differential expression analysis demonstrated an increased steady-state level of five genes associated with the high-risk genotype (CXCL9, CXCL11, and UBD in glomerulus; SNOR14B and MUC13 in tubulointerstitium). APOL1 tubulointerstitial coexpression analysis showed coexpression of APOL1 mRNA levels with a group of intrarenal transcripts that together were associated with increased interstitial fibrosis and tubular atrophy. These data indicate the high-risk APOL1 genotype confers renal risk across histopathologic diagnoses.
The podocyte proteins Neph1 and nephrin organize a signaling complex at the podocyte cell membrane that forms the structural framework for a functional glomerular filtration barrier. Mechanisms regulating the movement of these proteins to and from the membrane are currently unknown. This study identifies a novel interaction between Neph1 and the motor protein Myo1c, where Myo1c plays an active role in targeting Neph1 to the podocyte cell membrane. Using in vivo and in vitro experiments, we provide data supporting a direct interaction between Neph1 and Myo1c which is dynamic and actin dependent. Unlike wild-type Myo1c, the membrane localization of Neph1 was significantly reduced in podocytes expressing dominant negative Myo1c. In addition, Neph1 failed to localize at the podocyte cell membrane and cell junctions in Myo1c-depleted podocytes. We further demonstrate that similarly to Neph1, Myo1c also binds nephrin and reduces its localization at the podocyte cell membrane. A functional analysis of Myo1c knockdown cells showed defects in cell migration, as determined by a wound assay. In addition, the ability to form tight junctions was impaired in Myo1c knockdown cells, as determined by transepithelial electric resistance (TER) and bovine serum albumin (BSA) permeability assays. These results identify a novel Myo1c-dependent molecular mechanism that mediates the dynamic organization of Neph1 and nephrin at the slit diaphragm and is critical for podocyte function.Glomerular filtration assembly involves three layers, a fenestrated endothelium, a glomerular basement membrane, and specialized epithelial cells termed podocytes. Studies of various glomerular diseases, including nephrotic syndromes, diabetic nephropathy, and focal segmental glomerulosclerosis (FSGS), suggest that podocytes are a major target of these insults and that their dysfunction is associated with proteinuria and decreased kidney function. The identification of podocyte proteins such as nephrin, Neph1, podocin, synaptopodin, CD2AP, and ␣-actinin-4 that are localized specifically at the podocyte filtration barrier or slit diaphragm has provided greater insight into the mechanisms that mediate podocyte structure and function. Recent analyses of various glomerular disorders, including FSGS, membranous nephropathy, and minimal-change nephrotic syndrome, have reported alterations in the expression and localization of the slit diaphragm proteins nephrin, podocin, CD2ap, and Neph1 (20,45). These data provide further support for the hypothesis that alterations in the molecular arrangement of the slit diaphragm contribute to the development of proteinuria in several glomerular diseases.In contrast to nephrin, Neph1 is widely expressed in numerous cell types, including podocytes, where it localizes at the insertion site of the slit diaphragm (2, 11). Structurally, the extracellular region of Neph1 contains five immunoglobulinlike repeats, followed by a transmembrane domain and a cytoplasmic domain of ϳ198 to 235 amino acids (40). Knockout studies with mice su...
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