Immunolocalization of NAVH exchanger isoform NHE-2 in rat kidney with confocal fluorescence microscopy

K. P. Yip, C. M. Tse, M. Donowitz, D. J. Marsh

Research output: Contribution to journalArticlepeer-review

Abstract

Na+/H+ exchanger activity was found in many segments of nephron. Four distinct isoforms of the mammalian Na+/H+ exchanger (NHE) have been identified by molecular cloning, but in situ immunolocalization of these isoforms has not been fully established. In the present study, we used a polyclonal anti-NHE-2 antibody (Ab597) to immunolocalize NHE-2 isoform in the various segments of nephron in rat kidney. Ab597 was raised against the fusion protein of glutathione-S-transferase and the last 87 amino acids of NHE-2 peptide. The specificity of this antibody was established by immunoblotting membranes from NHE deficient PS 120 cells that had been transfected with either NHE-1, 2 and 3. The antibody also labeled NHE-2 as a 85-kDa protein in rat kidney cortical brush-border membranes during western analysis. Confocal fluorescence images were acquired from frozen sections of rat kidney after indirect immunostaining for NHE-2 isoform. Double labelling was performed when appropriate to differentiate various segments of the nephron. Strong fluorescence signal was found in the apical membrane of distal convoluted tubule and thick ascending tubule of Henle's loop. Weaker staining was found in the brush border of proximal tubule, apical membrane of thin limb of Henle's loop and medullary collecting duct. These observations suggest that NHE-2 is extensively expressed along luminal surface of the nephron, and the apical Na+/H+ exchange activity observed in kidney might involve both NHE-2 and NHE-3 isoforms.

Original languageEnglish (US)
Pages (from-to)A370
JournalFASEB Journal
Volume10
Issue number3
StatePublished - Dec 1 1996

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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