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Am J Physiol Renal Physiol 279: F901-F909, 2000;
0363-6127/00 $5.00
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Vol. 279, Issue 5, F901-F909, November 2000

Immunolocalization of electroneutral Na-HCO3minus cotransporter in rat kidney

Henrik Vorum1,*, Tae-Hwan Kwon1,2,*, Christiaan Fulton3, Brian Simonsen3, Inyeong Choi4, Walter Boron4, Arvid B. Maunsbach2, Søren Nielsen2, and Christian Aalkjær3

1 Department of Medical Biochemistry, 2 Department of Cell Biology, Institute of Anatomy, and 3 Department of Physiology, University of Aarhus, DK-8000 Aarhus C, Denmark; and 4 Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520

An electroneutral Na-HCO3- cotransporter (NBCN1) was recently cloned, and Northern blot analyses indicated its expression in rat kidney. In this study, we determined the cellular and subcellular localization of NBCN1 in the rat kidney at the light and electron microscopic level. A peptide-derived antibody was raised against the COOH-terminal amino acids of NBCN1. The affinity-purified antibody specifically recognized one band, ~180 kDa, in rat kidney membranes. Peptide-N-glycosidase F deglycosylation reduced the band 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 NBCN1 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 NBCN1 immunolabeling. Immunoelectron microscopy demonstrated that NBCN1 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 NBCN1. The localization of NBCN1 in medullary TAL cells and medullary collecting duct intercalated cells suggests that NBCN1 may be important for electroneutral basolateral HCO3- transport in these cells.

bicarbonate handling; immunohistochemistry; immunoelectron microscopy; intracellular pH; ion transport

* H. Vorum and T.-H. Kwon contributed equally to this study.




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