Expression and cellular localization of mRNA encoding the "gastric" isoform of H(+)-K(+)-ATPase alpha-subunit in rat kidney

Abstract

The distribution of transcripts encoding the gastric H(+)-K(+)-adenosinetriphosphatase (ATPase) alpha-subunit in the normal rat kidney was studied by reverse transcription-polymerase chain reaction (RT-PCR), combined with DNA sequence analysis and renal microdissection, and by nonradioactive in situ hybridization of fixed kidney sections using highly specific molecular probes. RT-PCR products corresponding to the gastric H(+)-K(+)-ATPase alpha-subunit were detected in the cortex, outer and inner medulla, and in isolated cortical (CCD) and inner medullary collecting ducts (IMCD). With digoxigenin-labeled cRNAs derived from the 5' and 3' ends of the gastric H(+)-K(+)-ATPase alpha-subunit cDNA, specific hybridization signal was detected prominently in all the cells of the connecting segment and CCD, the intercalated cells of the outer medullary collecting duct, the IMCD, and the renal pelvic epithelium lining the secondary pouches. Weak labeling was noted in the S3 segment of the proximal tubule, the distal convoluted tubule, and the cortical thick ascending limb of Henle. Hybridization with the sense probes produced no cellular labeling. These data provide the first direct demonstration for the expression and cellular distribution of mRNA encoding the gastric H(+)-K(+)-ATPase alpha-subunit in the normal, potassium-replete kidney, and they provide essential tools for the molecular analysis of renal acid base and potassium transport under physiological and pathophysiological conditions.