Immunohistochemical localization of colonic H-K-ATPase to the apical membrane of connecting tubule cells

Previous studies indicate that the colonic H-K-ATPase mRNA is expressed as the distal nephron. However, the exact intrarenal localization of the colon ic H-K-ATPase protein is still unclear. The goal of the present study was t o determine the cellular and subcellular localization of the colonic H-K-AT Pase protein in the rabbit kidney. We used three monoclonal antibodies (MAb s) directed against different epitopes of the rabbit colonic H-K-ATPase alp ha -subunit (HK alpha (2)) to localize HK alpha (2) protein by immunofluore scence labeling of kidney sections and laser-scanning confocal microscopy. The specificity of the MAbs was confirmed by reaction with a single similar to 100-kDa band on Western blots of distal colon. Specific immunohistochem ical reaction with the apical membrane of surface epithelial cells was obse rved with all three MAbs on distal colon sections. In rabbit kidney, immuno fluorescence was detected only on the apical membrane of connecting tubule cells. Immunofluorescence was not detected in the cortical-, outer-, and in ner-medullary collecting ducts. Furthermore, costaining with principal- and intercalated cell-specific MAbs and a MAb against the thick ascending limb suggests that these cell types express HK alpha2 protein at levels that ar e below the detection limit with this method. We conclude that in the rabbi t kidney, under normal dietary conditions, the HK alpha2 protein is express ed in the apical membrane of connecting tubule cells.