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3 cotransporter
in mammalian and amphibian kidney
Departments of 1 Cellular and Molecular Physiology and 2 Internal Medicine, Section of Nephrology, Yale University School of Medicine, New Haven, Connecticut 06520
Electrogenic
cotransport of Na+ and
HCO
3 is a crucial element of
HCO3 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 (amino acids 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, as well as a single ~160-kDa band in salamander kidney. By
indirect immunofluorescence microscopy on 0.5-µm 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 PT. 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 preabsorption
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.
fluid and electrolytes; renal bicarbonate reabsorption; acid-base; polyclonal antibody; immunofluorescence; immunoblotting; rat; rabbit; salamander
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