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A novel organic anion transporter 8 (OAT8) is localized to the intercalated cells in the rat kidney collecting duct

The mRNA for a recently cloned organic anion transporter 8 (OAT8) was found to be expressed only in the mammalian kidney. Previous immunocytochemical studies in rats indicated that OAT8 protein may be present in the apical domain of the kidney collecting duct (CD). However, detailed localization along the nephron and intracellular distribution of OAT8 in the CD cells, represented by the water-transporting principal cells (PC) and various types of proton-transporting intercalated cells (IC), has not been reported, and is the aim of this study. Studies were performed with polyclonal antibodies (immune rabbit sera and affinity purified antibodies) to the C- and N-terminals of the OAT8 peptide, and rabbit- or chicken-raised affinity purified antibodies to the water channel aquaporin 2 (AQP2) and 31 kDa subunit of the proton-transporting V-ATPase. The abundance of the protein was studied by Western blotting (WB) in total cell membranes (TCM) from the rat kidney cortex, outer stripe, and pooled inner stripe&inner medulla, whereas localization of the protein along the nephron was studied by immunocytochemistry in tissue slices of the same zones. In WB of TCM from various tissue zones, the anti-OAT8 antibodies labeled two major protein bands of MW ~50 and ~55 kDa. The labeling of both bands was protected by the respective immunizing peptides. In immunocytochemical studies, the immune serum to the OAT8 C-terminal domain exhibited an exclusive, peptide-inhibitable staining of the distinct cells along the CD, whereas other anti-OAT8 antibodies showed no specific staining. In the OAT8-positive cells, the staining was either apical or basolateral, or diffuse, and was blocked by the immunizing peptide. The double staining experiments with the anti-AQP2 (principal cells stained) and anti-V-ATPase antibodies (intercalated cells stained) indentified these cells as various types of intercalated cells. We conclude that OAT8 is present in the intercalated cells along the rat CD, with intracellular distribution that resembles that of the V-ATPase.

organic anions; mammalian kidney; immunocytochemistry; renal tubules

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