CLONING AND KIDNEY CELL-SPECIFIC ACTIVITY OF THE PROMOTER OF THE MURINE RENAL NA-K-CL COTRANSPORTER GENE

The murine Nkcc2/Slc12a1 gene encodes a bumetanide sensitive Na-K-Cl c otransporter that is expressed exclusively in the kidney in the thick ascending limb of the loop of Henle, Nuclear run-off assays demonstrat ed that kidney-specific expression of Nkcc2 was due, at least in part, to kidney-specific gene transcription, To begin study of the gene pro moter, a genomic clone that contained 13.5 kilobases of the 5'-flankin g region of Nkcc2 was isolated. A single transcription initiation site was located 1330 base pairs (bp) upstream of the start codon. The seq uence of the proximal 5'-flanking region contained typical eukaryotic promoter elements including a TATA box, two CCAAT boxes, and an initia tor, A (G-A)(28) (C-T)(28) microsatellite and consensus binding sites for hepatocyte nuclear factor 1, cAMP-response element binding protein , CCAAT/enhancer-binding proteins, and basic helix-loop-helix proteins , were also identified, To functionally express the promoter, 2255 bp of the proximal 5'-flanking region was ligated to a luciferase reporte r gene and transfected into thick ascending limb (TAL) cells, a stable cell line derived from microdissected loops of Henle of the Tg(SV40E) Bri7 mouse. TAL cells exhibited furosemide-sensitive Na-K(NH4+)-Cl cot ransport activity and endogenously expressed the 5.0-kilobase Nkcc2 tr anscript. Luciferase activity was 130-fold greater following transfect ion into TAL cells compared with transfection into cells that did not express Nkcc2 (NIH 3T3 fibroblasts). Deletion analysis revealed that p romoter activity in TAL cells was similar in constructs extending from the transcription initiation site to -1529 to -469, whereas further d eletion to -190 resulted in a 76% decrease in activity, We conclude th at the Nkcc2 promoter exhibits kidney cell-specific activity. Regulato ry elements required for maximal promoter activity are located in a 28 0-bp DNA segment that contains consensus binding sites for several tra nscription factors expressed in the kidney.