NHE3 - A NA+ H+ EXCHANGER ISOFORM OF RENAL BRUSH-BORDER/

Na+/H+ exchangers in the brush-border (luminal, apical) membrane of re nal proximal tubules are responsible for active, transcellular reabsor ption of NaHCO3 and NaCl. Although well characterized kinetically, the protein that mediates Na+/H+ exchange in the renal brush border has n ot been identified. Several Na+/H+ exchanger genes, including NHE1, NH E2, NHE3, and NHE4, are expressed in the kidney. To identify the NHE3 gene product and to determine its cellular and subcellular localizatio n in the rabbit kidney, an NHE3-isoform-specific antibody was prepared . Guinea pigs were immunized with purified fusion protein containing t he carboxy-terminal 40 amino acids of NHE3 (fpNHE3-C40). After affinit y purification, immune sera demonstrated specific reactivity to the NH E3 sequence within the fusion protein as well as to an 80-kDa polypept ide expressed in NHE3-transfected LAP1 cells. Western blot analysis sh owed that anti-fpNHE3-C40 specifically reacted with an 80-kDa protein that is relatively enriched in renal brush-border membrane compared wi th basolateral membrane. Immunocytochemical studies confirmed that the Na+/H+ exchanger isoform NHE3 is expressed along the microvillar memb rane of the brush border of proximal tubule cells in the rabbit kidney .