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

Freshly isolated proximal tubules from the spontaneously hypertensive rat ( SHR) demonstrate elevated Na+/H+ exchanger (NHE) activity, but the underlyi ng mechanism is unclear. Because of the difficulties in preparing sufficien t numbers of proximal tubule cells for detailed biochemical studies. we hav e generated cell lines from SHR and Wistar-Kyoto rat (WKY) proximal tubule cells. Cell lines were obtained by transforming the cells with an origin-de fective mutant of simian virus 40 encoding a heat-labile T antigen (tsA58 t ransformant). Such cells proliferate at the permissive temperature of 33 de grees C, but growth is abolished at the restrictive temperature of 39 degre es C. The predominant NHE isoform expressed was isoform was determined by s ensitivity to HOE-694 (3-methylsulphonyl-4-piperidinobenzoyl guanidine) and Western blotting using specific polyclonal antisera to NHE-1. NHE-3 protei n was also present. Northern blots of poly(A) mRNA extracts of the cell lin es revealed a low abundance of transcripts for NHE-2, -3 and -4, with no sy stematic difference between the lines. Although the intracellular pH was si milar in the SHR and WKY lines, HOE-694-sensitive H+ efflux due to NHE-1 wa s substantially elevated in SHR lines compared with WKY lines (95.0+/-2.8 a nd 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 str ains. 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 turnov er number of NHE-1 was increased. Immunoprecipitation of P-32-labelled phos phoproteins from these lines demonstrated an approximately 2-fold higher de gree 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.