We hypothesize that basement membrane protein isoform switching is critical for development and maintenance of the differentiated state for both endothelial ceils and podocytes. Further, we speculate that both cell types monitor their evolving GBM substrates and attempt to compensate for defects in part by reversing/altering their normal isoform substitution programs. In turn, these temporally abnormal GBMs thwart normal endothelial cell and podocyte differentiation/maturation. Support for these hypotheses come from gene targeting studies in mice that disrupt laminin alpha 5 or collagen alpha 3(IV) chain synthesis. In the latter case, the resulting phenotype closely resembles Alport congenital nephritis. Moreover, accumulating evidence from experimental and human disease shows that molecular defects in type IV collagen isotype switching leads to GBM laminin synthetic errors as well. Experiments addressing these hypotheses will be carried out using metanephric allografting and molecular genetic technologies wherein expression of certain laminin and collagen IV protein chains are selectively amplified or repressed in endothelial cells and podocytes specifically. We predict that deregulated expression of GBM laminin/collagen IV isotypes will disrupt normal glomerular morphogenesis and result in endothelial/podocyte deficits. Additionally, we expect that abnormal phenotype development can be partially rescued through the restoration of temporally correct GBM components.
Our specific aims are: (1) We will undertake metanephric grafting experiments in which kidneys from embryos with targeted alpha 3(IV) collagen or alpha 5 laminin deletions are implanted into kidney cortices of wild type host mice. Hybrid glomeruli consisting of host (wild type) endothelial ceils and graft (mutant) podocytes will be examined for structural and functional correction of the GBM phenotype. (2) We will create new transgenic mice carrying the alpha 3(IV) collagen deletion in which inducible promoters drive selective expression of alpha 3(IV) collagen by either endothelial cells or podocytes, respectively, and by both cells, to rescue the mutant phenotype. (3) Using high resolution scanning confocal image acquisition and analysis, the relative abundance and turnover rates of laminins and type IV collagen within normal and diseased GBMs will be quantified. These studies will provide fundamentally new information on glomerular development and maintenance.
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