Immunocytochemical characterization of Na(+)-H+ exchanger isoform NHE-1 in rabbit kidney.
We have recently isolated cDNAs encoding a Na(+)-H+ exchanger isoform, referred to as NHE-1, from rabbit kidney and LLC-PK1 cells. To identify the NHE-1 protein and to establish its cellular and subcellular localization in the rabbit kidney, we prepared antibodies to a NHE-1 fusion protein. cDNA encoding the COOH-terminal 41 amino acids of NHE-1 was subcloned into a maltose-binding protein vector and the purified fusion protein (FP347A) used to immunize guinea pigs. To identify the NHE-1 protein, we performed Western blot analysis against membrane fractions prepared from rabbit renal cortex. Anti-FP347A antibody specifically reacted with a polypeptide with an apparent molecular mass of 100-110 kDa that was enriched in basolateral membrane fractions. When indirect immunofluorescence was performed on semithin (0.5 micron) cryosections of paraformaldehyde-lysine-periodate-fixed rabbit kidney, anti-FP347A specifically stained the basolateral plasma membrane of cells of the proximal tubule, thick ascending limb, and distal convoluted tubule. Anti-FP347A similarly stained connecting tubule cells and principal cells. No staining was detected on the apical membrane of any cells of the rabbit nephron. We conclude that NHE-1 is a 100- to 110-kDa protein expressed on the basolateral membrane of multiple nephron segments.
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism*; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D009399:Nephrons / Q000378:metabolism; D011817:Rabbits; D017923:Sodium-Hydrogen Exchangers; D013194:Staining and Labeling; D014018:Tissue Distribution
D002352:Carrier Proteins; D008565:Membrane Proteins; D017923:Sodium-Hydrogen Exchangers; C069139:growth factor-activatable Na-H exchanger NHE-1