Immunocytochemical localization of Na,K-ATPase gamma subunit and CHIF in inner medulla of rat kidney.

The gamma subunit of Na,K-ATPase and CHIF both belong to the FXYD single-membrane-spanning protein family and have been suggested to have regulatory functions in kidney tubules. CHIF is known to be present in the collecting duct, and gamma has been demonstrated in several segments of the rat kidney tubule, but never clearly in the inner medullary collecting duct (IMCD). Here, we demonstrate the cellular and subcellular localization of the gamma subunit and CHIF in the IMCD in inner medulla by using Western blotting, laser-scanning confocal immunofluorescence, and immunoelectron microscopy. In the initial quarter of the IMCD (next to the outer medulla), antibodies against the C-terminal of gamma as well as splice variant gammaa labeled the basolateral surface of intercalated cells (ICs), while principal cells (PCs) remained unlabeled. In the middle segment of the IMCD, all PCs exhibited distinct basolateral staining for the gammaC-terminal as well as gammaa and CHIF. Immunoelectron microscopy showed that the gammaC-terminal and CHIF were associated with the inner leaflet of the basolateral plasma membrane in the labeled cells. Immunoblotting demonstrated the presence of both the gammaC-terminal and gammaa in inner medullary tissue. However, splice variant gammab was not detected in inner medulla by immunocytochemistry or immunoblotting. The present observations demonstrate that the Na,K-ATPase gamma subunit and CHIF are strategically located in the inner medulla to participate in the fine-tuning of urine ion composition through the regulation of the Na,K-ATPase activity in the IMCD.