Colocalization of H-ATPase and H,K-ATPase immunoreactivity in the rat kidney

Abstract

Urinary acidification in the collecting duct (CD) is via V-type H-ATPase and P-type H,K-ATPase. The localization and polar distribution of H-ATPase in intercalated cells (IC) have been well studied. Tne localization of H,K-ATPase to IC has been reported, but its intracellular distribution has not been defined. To colocalize these pumps, a murine monoclonal antibody (E11) to the 31-kd subunit of the H-ATPase and a rabbit antiserum (HKαN2) to a synthetic peptide based on the N terminus of the hog gastric H,K-ATPase α-subunit were used. In immunocytochemical staining of rat kidney, H,K-ATPase was present only in the IC of the CD with the same polar distribution as H-ATPase. The preabsorption of HKαN2 with affinity-purified bovine H-ATPase did not affect the H,K-ATPase staining, whereas preabsorption with the immunizing synthetic peptide eliminated immunoreactivity. HKαN2 stained only parietal cells in the rat gastric mucosa, whereas preimmune serum and E11 showed no immunoreactivity. On immunoblots of rat gastric mucosal microsomes, HKαN2 labeled a single 94-kd band, and this staining disappeared after preabsorption with the immunizing synthetic peptide. HKαN2 labeled no bands on immunoblots of affinity-purified bovine H-ATPase. All immunochemistry was negative with preimmune serum. The finding that gastric parietal cells only stained with HKαN2 and not with E11, that HKαN2 identified the typical 94-kd band on immunoblots of rat gastric mucosal microsomes, and the immunoabsorption studies with purified H-ATPase protein versus the immunizing synthetic peptide all indicate that HKαN2 specifically recognizes the H,K-ATPase pump. It was concluded that H,K-ATPase is present only in the IC of the cortical, outer medullary, and early inner medullary CD, with a polar distribution identical to that of the H-ATPase pump.