Aldosterone plays an important regulatory role in blood pressure control and electrolyte balance through actions initiated by mineralocorticoid receptors on gene transcription. Little is known, however, about the specific molecular basis of the hormone's target genes that regulate sodium transport in the kidney collecting duct. Though subtractive hybridization techniques, a compelling target gene-a kinase- has been identified. This gene has been shown to be rapidly induced by aldosterone and to increase by seven-fold epithelial sodium channel (ENaC) activity. The five year plan detailed in this proposal seeks to clarify if this early response gene to aldosterone plays a critical role in the regulation of ENaC-mediated sodium transport in the kidney collecting duct (CD). The proposal's methodology employs A6 CD-like cells grown on monolayer in a well-characterized system for the study of transcriptional regulation and of sodium transport. Corticosteroid- induced transcriptional regulation of the kinase gene will be studied according to established protocols for identifying hormone response elements in the gene's promoter: transient transfection assays on promoter-luciferase reporter constructs, gel mobility shift assays, and nuclear runoffs. Antisense technology and establishment of a stable transfected cell line expressing the kinase gene will be utilized to study the gene's functional relevance to affecting sodium transport. A6 cells targeted with antisense oligonucleotides and cells constitutively expressing the kinase gene will be compared with their appropriate controls and mutants for their electrophysiological effect and significance. How these kinase modifies specific subunits of the ENaC will be an additional important goal of this investigation. In vivo and in vitro kinase assays will thus be performed, and immunoprecipitation, gel electrophoresis, and phosphorimaging of this product will help delineate the subunits' modifications. Data from this study may help define a gene that not only bridges the mechanistic gap between aldosterone and its effects' regulating sodium influx, but it may additionally provide insight into mechanisms involved in natriuresis, diuresis, and the pathogenesis of hypertension.
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