Autosomal dominant polycystic kidney disease (ADPKD) is a leading cause of renal failure for which there is no known treatment. The causal gene in 85% of all cases has recently been cloned, however the function of its protein product as well as the mechanisms whereby mutations in this gene lead to the pathogenesis of the disease remain a mystery. It is the aim of this proposal to combine cellular and biochemical approaches to further our understanding of the primary cause(s) of ADPKD. Numerous cellular functions have been reported to be affected in this disease, however, many of these might be explained by an alteration in the renal epithelial cell program specifying polarized membrane transport. This is a testable hypothesis which will be examined by measuring both protein and lipid sorting in primary normal or polycystic human kidney cells. The approaches will quantify any alterations in the transport kinetics as well as in the polarized delivery of these molecules. Thus, simple assays for measuring phenotypic alterations specifically associated with ADPKD and crucial for the evaluation of the disease gene function will be developed. In parallel, the protein compositions of normal and polycystic kidney cells will be compared by two-dimensional gel electrophoresis with the initial aim of establishing whether molecules known to be involved in membrane transport as well as those encoded by the PKD1 locus are affected in order to pinpoint the primary effectors. In the long-run, determining the extent of the changes in protein expression patterns manifested in ADPKD and using this as a functional assay for evaluating how genes at the PKD1 locus might influence these changes is expected to provide important clues concerning the mechanisms underlying the pathogenesis of the disease. The PKD1 locus contains as many as 20 genes, which are thought to be part of a genomic cluster of genes with important regulatory functions in kidney epithelial cell differentiation. Two of these genes are of particular interest because of their functions and their immediate proximity to the disease gene, PBP. One is the TSC2 gene which when mutated also causes renal disease and the second is the NIK7 gene which specifies a novel rab GTPase, not previously identified. The rab GTPases are known to be important regulators of intracellular membrane transport since dominant negative mutations have a drastic inhibitory effect on these processes. Thus far only one rab GTPase has been clearly demonstrated to be specifically expressed in epithelial cells and two others are currently under investigation. These facts suggest that the analysis of these two neighboring genes may shed some light on kidney epithelial development and/or on the disease process. Experiments will compare the expression of TSC2 and NIK7 in normal and polycystic kidney and investigate the subcellular localization of these proteins to reveal their possible functions. Coupled with the assays for quantifying ADPKD specific phenotypic changes it will be possible to evaluate directly the role of these genes in the disease process. In summary, the long-term goals of this work are to identify important regulators of specialized epithelial cell functions and to understand how these are subverted to give rise to ADPKD.
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