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1 Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States
2 Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States; Charles and Jane Pak Center of Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas, United States
3 Department of Medicine/Nephrology, Yale School of Medicine, New Haven, Connecticut, United States
4 Department of Pediatrics, Univ. of Texas / Southwestern Med Ctr, Dallas, Texas, United States
* To whom correspondence should be addressed. E-mail: orson.moe{at}utsouthwestern.edu.
NHE8 is expressed in the apical membrane of the proximal tubule and is predicted to be a Na+/H+ exchanger based on its primary amino acid sequence. Functional characterization of native NHE8 in mammalian cells has not been possible to date. We screened a number of polarized renal cell lines for the plasma membrane Na+/H+ exchangers (NHE1, 2, 3, 4, and 8) and found only NHE1 and NHE8 transcripts in NRK cells by RT-PCR. NHE8 protein is expressed in the apical membrane of NRK cells based on immunoblots, confocal fluorescent immunocytochemistry, and immuno-electron microscopy. NHE1 on the other hand is expressed primarily in the basolateral membrane. Bilateral perfusion of NRK cells grown on permeable supports shows Na+/H+ exchange activity on both the apical and basolateral membranes. NHE8-specific siRNA knocks down NHE8 protein expression but does not affect NHE1 protein levels. Knock down of NHE8 protein is accompanied by a commensurate reduction in apical NHE activity, without altered basolateral NHE activity. Conversely, transfection of NHE1-specific siRNA knocks down NHE1 protein expression without affecting NHE8 protein levels, and reduces basolateral NHE activity without affecting apical NHE activity. NHE8 is the only apical membrane Na+/H+ exchanger in NRK cells. NHE8 activity is Na+-dependent displaying a cooperative sigmoidal relationship and is highly sensitive to 5-(N-ethyl-n-isopropyl)-amiloride (EIPA). NRK cells provide a useful system where NHE8 can be studied in its native environment.
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