The renal Na-K-Cl cotransporter mediates active reabsorption of NaCl in the thick ascending limb of Henle's loop and represents the primary site of action of the clinically important diuretics furosemide and bumetanide. The overall aims of this project are to identify the cis-acting regulatory elements and transcription factors that are responsible for kidney- specific expression of the renal Na-K-Cl cotransporter gene (NKCC2) and to test whether over-expression of the NKCC2 gene in the kidney causes arterial hypertension. Recently, cDNA and genomic clones that encode murine NKCC2 have been characterized, and an in vitro model that accurately recapitulates kidney-cell-specific expression of NKCC2 has been developed. The proposed studies will test the hypothesis that kidney- specific expression of NKCC2 is mediated by kidney-enriched transcription factors that bind to regulatory elements located within the proximal 5' flanking region of the gene. The NKCC2 gene promoter will be functionally expressed in cultured kidney epithelial cells, and evidence for cell-type- specificity will be obtained by expression in cells of differing lineages. Studies in cultured cells will also be used to explore whether hepatocyte nuclear factor 1 (HNF-1) is required for expression of the NKCC2 gene. Electrophoretic mobility-shift assays (EMSA) will be performed to determine whether HNF-1alpha or HNF-1beta bind to the putative HNF-1 site in the proximal 5' flanking region, and transient co-expression studies will be performed to determine whether HNF-1alpha (or HNF-1beta) transactivate the promoter. Studies in transgenic mice will be performed to test whether the proximal 5' flanking region contains regulatory elements that are sufficient for stage-dependent and kidney-specific expression of the NKCC2 gene in vivo. Deletion analysis will be performed to define the boundaries of the minimal DNA sequence that confers-kidney- specific expression. Essential regulatory elements that reside outside of the proximal 5' flanking region will be identified by nuclease hypersensitivity. Electrophoretic mobility-shift assays will be performed to determine whether these regulatory elements bind to kidney-enriched nuclear proteins, and specific sites of DNA-protein interaction will be identified using DNAse I footprinting. The effects of mutations of the elements on protein-binding and transcriptional activity will be assessed. Positive-acting elements that are identified in the preceding studies will be used to direct high-level, kidney-specific expression of the NKCC2 isoforms in transgenic mice. These studies will provide a useful animal model for evaluating whether enhanced NaCl reabsorption in the thick ascending limb causes arterial hypertension. In another approach, mutations of the human NKCC2 gene that are identified in hypertensive patients will be tested to ascertain whether they affect regulatory elements that control NKCC2 expression in vitro and in vivo. The proposed studies will examine the role of dysregulated Na-K-Cl cotransport in the pathogenesis of essential hypertension, provide novel insights into molecular mechanisms of kidney-specific gene expression, and generate a useful animal model with a well-defined primary defect in sodium homeostasis.
| Zhang, Junhui; Geller, David S (2008) Helix 3-helix 5 interactions in steroid hormone receptor function. J Steroid Biochem Mol Biol 109:279-85 |
| Mani, Arya; Radhakrishnan, Jayaram; Wang, He et al. (2007) LRP6 mutation in a family with early coronary disease and metabolic risk factors. Science 315:1278-82 |
| Alvarez de la Rosa, Diego; Gimenez, Ignacio; Forbush, Biff et al. (2006) SGK1 activates Na+-K+-ATPase in amphibian renal epithelial cells. Am J Physiol Cell Physiol 290:C492-8 |
| Zhang, Junhui; Tsai, Francis T F; Geller, David S (2006) Differential interaction of RU486 with the progesterone and glucocorticoid receptors. J Mol Endocrinol 37:163-73 |
| Zhang, Junhui; Simisky, Jessica; Tsai, Francis T F et al. (2005) A critical role of helix 3-helix 5 interaction in steroid hormone receptor function. Proc Natl Acad Sci U S A 102:2707-12 |
| Geller, David S (2005) Mineralocorticoid resistance. Clin Endocrinol (Oxf) 62:513-20 |
| Wilson, Frederick H; Hariri, Ali; Farhi, Anita et al. (2004) A cluster of metabolic defects caused by mutation in a mitochondrial tRNA. Science 306:1190-4 |
| Coric, Tatjana; Hernandez, Nelmary; Alvarez de la Rosa, Diego et al. (2004) Expression of ENaC and serum- and glucocorticoid-induced kinase 1 in the rat intestinal epithelium. Am J Physiol Gastrointest Liver Physiol 286:G663-70 |
| Geller, David S (2004) A genetic predisposition to hypertension? Hypertension 44:27-8 |
| Francis, Jean; Zhang, Junhui; Farhi, Anita et al. (2004) A novel SGLT2 mutation in a patient with autosomal recessive renal glucosuria. Nephrol Dial Transplant 19:2893-5 |
Showing the most recent 10 out of 21 publications
