PROMOTER ACTIVITY OF CARBONIC-ANHYDRASE II REGULATORY REGIONS IN CULTURED RENAL PROXIMAL, TUBULAR CELLS

Carbonic anhydrase II (CAII) plays an important role in the acid-base homeostasis of the body and its deficiency results in renal tubular ac idosis. In order to identify the regulatory regions in the CATI gene f or the future development of kidney-targeted gene therapy, we investig ated the 5' region of the gene for its promoter activity. Deletion con structs with various lengths of the 5' flanking region of the human CA II promoter were ligated to the CAT reporter gene and lipofected in pr imary cultures of mouse proximal renal tubular cells and in cells of t he established porcine proximal tubular cell line, LLC-PK1. The CAT ac tivity was measured 48 hours after gene transfection. The -12000/CAT a nd -1300/CAT constructs expressed the highest CAT activity in both typ es of renal tubular cells (143- and 180-fold increase, respectively, i n mouse proximal tubular cells; 50- and 70-fold increase, respectively , in LLC-PK, cells) but not the -420/CAT, -270/CAT, or -180/CAT constr ucts (9, 12, and 9% of that of -1300/CAT construct, respectively, in m ouse proximal tubular cells and, 23, 9, and 8%, respectively, in LLC-P K1 cells, all p < 0.01 vs. -1300/CAT construct). No cytotoxicity was d etected in the transfected cells. A computer search identified multipl e putative transcription factor binding elements including Ap1 and Ap2 binding elements, which are present in the -1300/CAT construct but no t in the shorter constructs. In conclusion, we demonstrate that the hu man CAII 5' sequence of proximal 1.3 kb contains strong promoter seque nce(s) for renal tubular cells.