Immunoelectron microscopic localization of NBC3 sodium-bicarbonate cotransporter in rat kidney

In the present study, we produced a rabbit peptide-derived polyclonal COOH-terminal antibody that selectively recognizes NBC3, to determine the cellular and subcellular localization of NBC3 in rat kidney, using immunocytochemistry and immunoelectron microscopy. Immunocytochemistry with cryostat sections and semithin cryosections revealed specific staining of intercalated cells (ICs) in the connecting tubule and in cortical, outer medullary, and initial inner medullary collecting ducts. In the connecting tubule and in the cortical and medullary collecting duct, the labeling was associated with both type A and type B ICs. In type A ICs, labeling was confined to the apical and subapical domains, whereas in type B ICs, basal domains were exclusively labeled. In contrast, collecting duct principal cells were consistently unlabeled, and this was confirmed using anti-aquaporin-2 antibodies, which labeled principal cells in parallel semithin cryosections. Glomeruli, proximal tubules, descending thin limbs, ascending thin limbs, thick ascending limbs, distal convoluted tubules, and vascular structures were unlabeled. For immunoelectron microscopy, tissue samples were freeze-substituted, and immunolabeling was performed on ultrathin Lowicryl HM20 sections. Immunoelectron microscopy demonstrated that NBC3 labeling was very abundant in the apical plasma membrane, in intracellular vesicles, and in tubulocisternal profiles in the subapical domains of type A ICs. In type B ICs, NBC3 was mainly present in the basolateral plasma membrane. Immunolabeling controls using peptide-absorbed antibody were consistently negative. In conclusion, NBC3 is highly abundant in the apical plasma membrane of type A ICs and in the basolateral plasma membrane of type B ICs. This suggests that NBC3 plays an important role in modulating bicarbonate transport in the connecting tubule and collecting duct.