In the previous grant cycle, we have demonstrated that the development of polycystic kidney disease in the Han:SPRD rat is accompanied by abnormalities in redox metabolism, that the acid content of the diet and/or experimental modifications of the redox state have profound effects on renal cystogenesis in Han:SPRD rats and pcy mice and on renal carcinogenesis in Eker rats, and that extracellular acidification increases the production of oxidant species and the rate of somatic mutations in cultured tubular epithelial cells. We hypothesize that oxidative stress resulting from acid administration, glutathione depletion, genetically determined deficiencies of CuZn superoxide dismutase (SOD1), Mn superoxide dismutase (SOD2) or selenium dependent cellular glutathione peroxidase (GPX1), or a combination of these factors will promote cystogenesis in Pkd1 del34/+ and Pkd2 -/+ mice by increasing the rate of mutation of the wild type Pkd1 and Pkd2 genes. Conversely we hypothesize that base administration will reduce the rate of intragenic homologous recombination between tandemly repeated portions of the wild-type and mutant exons 1 within the WS25 allele and will reduce the severity of polycystic kidney disease in compound heterozygous Pkd2 -/WS25 mice. Finally, we hypothesize that the generation of oxidant species in tubular epithelial cells in response to acidification or glutathione depletion is a lipid peroxyl or alkoxyl radical and that the rate of production of this radical in response to acidification or glutathione depletion in vitro and in vivo depends on the cell fatty acid composition and affects the development of polycystic kidney disease. Measurements of tissue levels of glutathione and activities of SOD1, SOD2, and GPX1 will be performed to control for the experimental conditions. The severity of the polycystic kidney disease will be assessed by histomorphometric analysis and determinations of plasma creatinine and urea. Immunohistology, together with laser capture microdissection of individual cyst cell populations, PCR amplification, and DNA sequencing will be used to assess the molecular mechanism of cyst formation. The effect of cell fatty acid composition on the generation of oxidant species, lipid peroxyl and alkoxyl radicals, mutagenesis and cystogenesis will be studied in cell culture systems using a DCF fluorometric assay, electron paramagnetic resonance, and an SV40-based shuttle vector containing suppressor tyrosyl t-RNA as a target for mutagenesis, as well as in Pkd1 -/+ and Pkd2 -/+ mice. The main thrust of the proposed studies is directed towards the identification of pathogenetic mechanisms than can be subject to preventive and therapeutic interventions.
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