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.
Highlights
Focal segmental glomerulosclerosis (FSGS)2 is an important cause of end-stage renal disease worldwide, accounting for ϳ20% of all dialysis patients [1]
We demonstrate that the homophilic adhesion molecule sidekick-1 is up-regulated in podocytes in FSGS both in rodent models and in human kidney biopsy samples
We show that sdk-1 associates with the slit diaphragm linker protein MAGUK with inverted domain structure-1 (MAGI-1), which is already known to interact with several critical podocyte proteins including synaptopodin, ␣-actinin-4, nephrin, JAM4, and -catenin
Summary
Specificity of the antiserum was confirmed by Western blotting on lysates of 293T cells transfected (Effectene, Qiagen) with mouse sdk-1 or sdk-2 expression plasmids. Immunoprecipitation—FLAG-sdk and FLAG-sdk-1-C-del expression plasmids were generated by PCR amplifying the intracellular (including the transmembrane domain) portion of sdk-1 and subcloning the products into pFLAG-CMV-2 (Sigma-Aldrich). Bound proteins were analyzed by Western blotting using anti-MAGI-1 antibody (Sigma-Aldrich). Lysates were incubated with either anti-sdk-1 (antibody number 3534) [17] or normal rabbit serum fixed on protein A-agarose. A, expression of sdk-1 RNA is significantly up-regulated in podocytes in cell culture when exposed to either HIV-1 infection or PAN as determined by quantitative PCR. D, upper, a novel rabbit anti-human sdk-1 antibody interacts with lysate from cells transfected with an sdk-1, but not an sdk-2, expression plasmid. Results were considered significant at p values Ͻ 0.05
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