Characterization of Na⁺/H⁺exchanger NHE8 in cultured renal epithelial cells

Abstract

NHE8 is expressed in the apical membrane of the proximal tubule and is predicted to be a Na+/H+ exchanger on the basis of 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 as demonstrated by immunoblots, confocal fluorescent immunocytochemistry, and immunoelectron 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 small interfering RNA knocks down NHE8 protein expression but does not affect NHE1 protein levels. Knockdown of NHE8 protein is accompanied by a commensurate reduction in apical NHE activity, without altered basolateral NHE activity. Conversely, transfection of NHE1-specific small interfering RNA 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.

Characterization of the Sodium/Hydrogen Exchanger NHA2

“…It has been documented for several "intracellular" NHE that they can also reside and function on the plasma membrane under certain circumstances, [31][32][33] and dual mitochondrial and plasmalemmal localization has also been reported for the voltage-dependent anion channel VDAC. 34 For the intracellular NHE isoform NHE6, the scaffolding protein RACK1 was recently shown to regulate the balance between plasmalemmal and intracellular localization.…”

“…Monoclonal anti-NHE1 antibody (clone 4E9) was obtained from Calbiochem (Temecula, CA); monoclonal anti-NHE8 antibody has been described before. 32 …”

Traditional and emerging roles for the SLC9 Na+/H+ exchangers

“…NRK cells endogenously express NHE8 at the plasma 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 membrane. Using the latter cells, Moe et al demonstrated that NHE8 is sensitive to amiloride [190].…”

Decreased NHE8 isoform expression and defective acidification in proximal convoluted tubules of senile rats

“…Goyal et al (2003Goyal et al ( , 2005 described a new member of the family of mammalian NHE exchangers, the NHE8 isoform. This new isoform mediates Na + -dependent acid extrusion across the apical membrane of proximal tubule cells (Zhang et al 2007). NHE8-mediated transport is retained in NHE3/NHE2 null mice and can be inhibited by EIPA (Choi et al 2000;Zhang et al 2007).…”

“…NHE8 is a recently identified renal Na + /H + antiporter isoform (Goyal et al 2003) that has been localised to the apical membrane of the rat proximal tubule (Goyal et al 2005) and is highly EIPA sensitive (Zhang et al 2007). Interestingly, expression of the NHE8 isoform diminishes with age (Becker et al 2007).…”