Immunolocalization of the electrogenic na/hco3 cotransporter in
mammalian and amphibian kidney.
Schmitt, Bernhard M., Daniel Biemesderfer, Michael F. Romero, Emile L.
Boulpaep, and Walter F. Boron.
Department of Cellular and Molecular Physiology, and 1Department of
Internal Medicine, Section of Nephrology, Yale University School of
Medicine, New Haven, CT 06520, USA
APStracts 5:0163F, 1998.
Electrogenic cotransport of Na+ and HCO_3 is a crucial element of
HCO_3 reabsorption in the renal proximal tubule (PT). An electrogenic
Na/HCO3 cotransporter (NBC) has recently been cloned from salamander
and rat kidney. In the present study, we generated polyclonal
antibodies (pAbs) to NBC, and used them to characterize NBC on the
protein level by immunochemical methods. We generated pAbs in guinea
pigs and rabbits by immunizing with a fusion protein containing the
carboxy-terminal 108 amino acids (a.a. 928 _ 1035) of rat kidney NBC
(rkNBC). By indirect immunofluorescence microscopy, the pAbs strongly
labeled HEK-293 cells transiently expressing NBC, but not in
untransfected cells. By immunoblotting, the pAbs recognized a 130-kDa
band in Xenopus laevis oocytes expressing rkNBC, but not in control
oocytes injected with water or cRNA for the Cl-HCO3 exchanger AE2. In
immunoblotting experiments on renal microsomes, the pAbs specifically
labeled a major band at 130 kDa in both rat and rabbit, and a single
160-kDa band in salamander kidney. By indirect immunofluorescence
microscopy on 0.5-_m cryosections of rat and rabbit kidneys fixed in
paraformaldehyde-lysine-periodate (PLP), the pAbs produced a strong
and exclusively basolateral staining of the PT. In the salamander
kidney, the pAbs labeled only weakly the basolateral membrane of the
proximal tubule. In contrast, we observed strong basolateral labeling
in the late distal tubule, but not in the early distal tubule,. The
specificity of the pAbs for both immunoblotting and
immunohistochemistry was confirmed in antibody pre-absorption
experiments using either the fusion protein used for immunization, or
similarly prepared control fusion proteins. In summary, we have
developed antibodies specific for NBC, determined the apparent
molecular weights of rat, rabbit and salamander kidney NBC proteins,
and described the localization of NBC within the kidney of these
mammalian and amphibian species.
Received 17 April 1998; accepted in final form 11 September 1998.
APS Manuscript Number F88-8.
Article publication pending Am. J. Physiol. (Renal Physiology).
ISSN 1080-4757 Copyright 1998 The American Physiological Society.
Published in APStracts on 20 October 1998