The long-term objectives of this project are to identify the cis-acting regulatory elements and transcription factors that are responsible for kidney-specific gene expression. The proposed studies will examine the renal absorptive Na-K-Cl cotransporter (Nkcc2/Slc12a1) as a model kidney- specific gene. In the kidney, NKCC2 mediates active reabsorption of NaCl in the thick ascending limb of the loop of Henle and represents the site of action of the clinically important loop diuretics furosemide and bumetanide. Importantly, expression of Nkcc2 transcripts is only observed in the kidney. Recent studies demonstrate that the Nkcc2 gene is under transcriptional control and that the Nkcc2 promoter exhibits cell-lineage- specific activity. The proposed studies will test the hypothesis that kidney-specific transcription of Nkcc2 is mediated by the interaction between kidney-enriched transcription factors and regulatory elements of the Nkcc2 gene. To identify regulatory elements that are responsible for kidney-specific promoter activity, fragments of the proximal 5' flanking region of the Nkcc2 gene will be ligated to a promoterless reporter gene and transiently expressed in cultured kidney cells. Evidence for cell- type-specific promoter activity will be obtained by transfection into cells of differing lineages. It will be important to verify that regulatory elements identified in vitro also confer kidney-specificity upon expression of a lacZ reporter gene in transgenic mice. Specific enhancer and negative regulatory elements within the 5' flanking region will be identified by deletion analysis and phylogenetic sequence conservation. Regulatory elements located elsewhere in the gene will be identified on the basis of DNase I hypersensitivity. Whether candidate regions contain kidney-specific regulatory elements will be determined by evaluating their effects on transcription from a heterologous promoter both in vitro and in vivo. Electrophoretic mobility-shift assays will be performed to determine whether cis-acting elements bind to kidney-enriched nuclear proteins. Specific sites of DNA-protein interaction will be defined using in vitro DNA footprinting, and the effects of mutations of the elements on protein-binding and transcriptional activity will be assessed. Transcription factors that bind to relevant regulatory elements will be identified by Southwestern blotting or UV crosslinking, and cDNAs encoding these proteins will be cloned by screening expression libraries with recognition site probes. Identification of the cis-acting regulatory elements that are required for transcription of Nkcc2 will provide general insights into mechanisms of kidney-specific gene expression: will be important for future studies of dysregulated expression of Nkcc2 in states of abnormal renal salt handling, such as Bartter's syndrome and essential hypertension; and will provide reagents that may be useful for future in vivo gene therapy of the kidney. Characterization of the transcription factors involved in expression of Nkcc2 may also be clinically important since mutations affecting such proteins may cause developmental, neoplastic, or cystic disorders of the kidney.
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