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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08DK002723-02
Application #
6176763
Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Program Officer
Rankin, Tracy L
Project Start
1999-07-15
Project End
2001-06-30
Budget Start
2000-07-01
Budget End
2001-06-30
Support Year
2
Fiscal Year
2000
Total Cost
$116,370
Indirect Cost
Name
University of California San Francisco
Department
Pediatrics
Type
Schools of Medicine
DUNS #
073133571
City
San Francisco
State
CA
Country
United States
Zip Code
94143
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Rozansky, David J; Cornwall, Tonya; Subramanya, Arohan R et al. (2009) Aldosterone mediates activation of the thiazide-sensitive Na-Cl cotransporter through an SGK1 and WNK4 signaling pathway. J Clin Invest 119:2601-12
Wang, J; Barbry, P; Maiyar, A C et al. (2001) SGK integrates insulin and mineralocorticoid regulation of epithelial sodium transport. Am J Physiol Renal Physiol 280:F303-13