Murine and human type I Na-phosphate cotransporter genes: structure and promoter activity

Na-phosphate (P-i) cotransporters in the apical membrane of renal proximal tubular cells play a major role in the maintenance of P-i homeostasis. Alth ough two such cotransporters, Npt1 and Npt2, have been identified, little i s known about the function and regulation of Npt1. We cloned and characteri zed the murine (Npt1) and human (NPT1) genes, isolated the 5'-flanking regi on of Npt1, and analyzed its promoter activity. Npt1 is similar to 29 kb wi th 12 exons, whereas NPT1 is similar to 49 kb with one additional exon. The Npt1 promoter has a TATA-like box but no CAAT box, and the transcription s tart site was identified by primer extension and 5'-rapid amplification of cDNA ends. Transfection of opossum kidney cells with Npt1 promoter-reporter gene constructs demonstrated significant activity in a 570-bp fragment tha t was completely inhibited by cotransfection with the transcription factor, hepatocyte nuclear factor (HNF)-3 beta. Deletion of 200 bp from the 3'-end of the 570-bp fragment abrogated its promoter activity. In addition, promo ter activity of a 4.5-kb fragment, but not the 570-bp fragment, was stimula ted fourfold by cotransfection with HNF-1 alpha. Other well-characterized c is-acting elements were identified in the Npt1 promoter. We suggest that Np t1 expression is transcriptionally regulated and provide a basis for the in vestigation of Npt1 function by targeted mutagenesis.