The primary goals of this proposal are to define the structural biology of the podocyte and determine its role in glomerular permselectivity by an analysis of models of antibody- mediated glomerular injury. There are three aims. 1. Determine if and how nephrin, a major component of the podocyte slit diaphragm, is linked to the podocyte cytoskeleton. Preliminary findings suggest that nephrin may be anchored to the actin cytoskeleton, either directly or through various """"""""nephrin- associated proteins"""""""". Extracts of isolated rat glomerular cells will be prepared under defined conditions. High molecular weight complexes of nephrin will be isolated by co-immunoprecipitation or sucrose density ultracentrifugation and analyzed for interacting proteins using tandem mass spectroscopy. If putative nephrin-associated proteins are identified, heterologous expression, co-immunoprecipitation and blot overlay techniques will be used to confirm that they are able to bind the cytoplasmic tail of nephrin. An in vitro actin co-sedimentation system will be used to determine if nephrin or its partners are able to bind to actin. 2. Investigate how the slit diaphragm and its link to the cytoskeleton might be altered in models of antibody-mediated proteinuria.
This aim derives from observations that monoclonal antibody (mAb) 5-1-6 directly targets an extracellular epitope of nephrin in the slit diaphragm and causes severe proteinuria. Simultaneously, nephrin redistributes after cross-linking by mAb 5-1-6 and appears to be targeted to a lysosomal compartment and degraded. Nephrin also redistributes after complement-mediated podocyte injury in the passive Heymann nephritis (PHN) model of membranous nephropathy, in which the onset of proteinuria is associated with foot process effacement, collapse of the actin cytoskeleton and dislocation of the slit diaphragm. These two models will be compared and contrasted to determine if there is an alteration in nephrin association with the cytoskeleton and/or putative nephrin-associated proteins, and disruption of slit diaphragm architecture. Immunochemistry, immunohistology and electron microscopy will be used to analyze glomeruli from rats during the development of, and where relevant, during recovery of proteinuria. 3. Define the effect of mAb 5-1-6 on the fate of nephrin and putative nephrin associated proteins and their link to actin. Standard cell biological techniques will be employed to study the effect of mAb 5-1-6 on nephrin synthesis, cell surface expression, shedding, endocytosis and degradation. In the absence of a well-differentiated podocyte cell line, studies will be done with freshly dispersed glomerular cells using metabolic and cell surface labeling techniques combined with immunoprecipitation and western blotting. The normal state of phosphorylation of soluble and actin-bound fractions of nephrin and associated proteins, as well as the effect of mAb 5-1-6 will also be determined.
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