Na+/H+ exchanger activity and phosphorylation in temperature-sensitive immortalized proximal tubule cell lines derived from the spontaneously hypertensive rat

Abstract

Freshly isolated proximal tubules from the spontaneously hypertensive rat (SHR) demonstrate elevated Na(+)/H(+) exchanger (NHE) activity, but the underlying mechanism is unclear. Because of the difficulties in preparing sufficient numbers of proximal tubule cells for detailed biochemical studies, we have generated cell lines from SHR and Wistar-Kyoto rat (WKY) proximal tubule cells. Cell lines were obtained by transforming the cells with an origin-defective mutant of simian virus 40 encoding a heat-labile T antigen (tsA58 transformant). Such cells proliferate at the permissive temperature of 33 degrees C, but growth is abolished at the restrictive temperature of 39 degrees C. The predominant NHE isoform expressed was isoform 1, as determined by sensitivity to HOE-694 (3-methylsulphonyl-4-piperidinobenzoyl guanidine) and Western blotting using specific polyclonal antisera to NHE-1. NHE-3 protein was also present. Northern blots of poly(A) mRNA extracts of the cell lines revealed a low abundance of transcripts for NHE-2, -3 and -4, with no systematic difference between the lines. Although the intracellular pH was similar in the SHR and WKY lines, HOE-694-sensitive H(+) efflux due to NHE-1 was substantially elevated in SHR lines compared with WKY lines (95.0+/-2.8 and 39. 9+/-5.7 mmol.min(-1).l(-1) respectively; P<0.001; n=6). H(+) efflux due to non-Na(+)-dependent mechanisms were similar in lines from the two strains. Western blotting revealed that NHE-1 density was also very similar in SHR and WKY lines, and subcellular fractionation of homogenates indicated that NHE-1 was localized predominantly to plasma membranes. Thus the turnover number of NHE-1 was increased. Immunoprecipitation of (32)P-labelled phosphoproteins from these lines demonstrated an approximately 2-fold higher degree of phosphorylation of NHE-1 in SHR compared with WKY lines. These cell lines form a useful model for defining the biochemical mechanisms leading to the NHE-1 phenotype in the SHR kidney, in addition to investigations of other SHR phenotypic markers.