Immunolocalization of electroneutral Na-HCO3- cotransporter in rat kidney

An electroneutral Na-HCO3- cotransporter (NBC(N)1) was recently cloned, and Northern blot analyses indicated its expression in rat kidney. In this study, we determined the cellular and subcellular localization of NBC(N)1 in the rat kidney at the light and electron microscopic level. A peptide-derived antibody was raised against the COOH-terminal amino acids of NBC(N)1. The affinity-purified antibody specifically recognized one band, similar to 180 kDa, in rat kidney membranes. Peptide-N-glycosidase F deglycosylation reduced the band to similar to 140 kDa. Immunoblotting of membrane fractions from different kidney regions demonstrated strong signals in the inner stripe of the outer medulla (ISOM), weaker signals in the outer stripe of the outer medulla and inner medulla, and no labeling in cortex. Immunocytochemistry demonstrated that NBC(N)1 immunolabeling was exclusively observed in the basolateral domains of thick ascending limb (TAL) cells in the outer medulla (strongest in ISOM) but not in the cortex. In addition, collecting duct intercalated cells in the ISOM and in the inner medulla also exhibited NBC(N)1 immunolabeling. Immunoelectron microscopy demonstrated that NBC(N)1 labeling was confined to the basolateral plasma membranes of TAL and collecting duct type A intercalated cells. Immunolabeling controls were negative. By using 2,7-bis-carboxyethyl-5,6-caboxyfluorescein, intracellular pH transients were measured in kidney slices from ISOM and from mid-inner medulla. The results revealed DIDS-sensitive, Na- and HCO3--dependent net acid extrusion only in the ISOM but not in mid-inner medulla, which is consistent with the immunolocalization of NBC(N)1 . The localization of NBC(N)1 in medullary TAL cells and medullary collecting duct intercalated cells suggests that NBC(N)1 may be important for electroneutral basolateral HCO3- transport in these cells.