Immunolocalization of the electrogenic Na⁺- HCO 3 − cotransporter in mammalian and amphibian kidney

Abstract

Electrogenic cotransport of Na+ and[Formula: see text] is a crucial element of[Formula: see text] reabsorption in the renal proximal tubule (PT). An electrogenic Na+-[Formula: see text]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−/[Formula: see text]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.

Functional characterization of NBC4: a new electrogenic sodium-bicarbonate cotransporter

“…pNBC1 transcripts have also been detected in many organs, indicating the ubiquitous expression of this transporter (1). The second known NBC1 protein variant, kNBC1, is more restricted in its tissue expression and has thus far been detected in kidney and eye (8,29). Additional studies of the cellular/subcellular localization in various tissues, transport kinetics, and regulation of these electrogenic Na ϩ -HCO 3 Ϫ cotransporters are required to address the question as to why in the mammalian genome there are two distinct genes that encode electrogenic Na ϩ -HCO 3 Ϫ cotransporters.…”

“…In humans, NBC1 encodes two electrogenic Na ϩ -HCO 3 Ϫ cotransport variants, kNBC1 and pNBC1, which differ in their NH 2 terminus (3). kNBC1 mediates the majority of basolateral HCO 3 Ϫ efflux in the renal proximal tubule (2,9,26,29). pNBC1 is highly expressed in pancreas and various other tissues (1,8,14,20,21).…”

Acid-Base Transport by the Renal Proximal Tubule

“…This result probably explains why we and others failed in our attempts to expression clone NBCe1 using mammalian mRNA. Bernhard Schmitt raised the first antibodies to the electrogenic NBC, and he and his colleagues demonstrated that the protein is expressed heavily at the basolateral membranes of the S1 and likely the early S2 segments of the renal PT (70).…”

Molecular basis of ocular abnormalities associated with proximal renal tubular acidosis

“…It is therefore possible that mutations in the common NBC-1 region may affect the properties of all these isoforms, resulting in renal and extrarenal manifestations. For example, because kNBC-1 is thought to be responsible for a majority of HCO 3 -reabsorption from renal proximal tubules (10)(11)(12), its inactivation can readily explain the occurrence of pRTA. Recent studies (13,14) suggest that NBC-1 may also play a significant role in HCO 3 -transport by the corneal endothelium, which potentially could explain the occurrence of band keratopathy (4).…”