Perturbation of Cell pH Regulation by H2O2 in Renal Epithelial Cells

The purpose of these studies was to define the effect of oxidant stress on intracellular pH in renal tubular epithelial cells. Cell pH was therefore quantitated from the fluorescence ratio of 2′, 7′-bis- (2-carboxyethyl)-5(6)-carboxyfluorescein in the BSC-1 and opossum kidney (OK) established cell lines, both of which are known to be susceptible to oxidant injury. Exposure to 1 mM H2O2 acidified cytosolic pH in both cell types, and this acidification reversed on withdrawal of the H2O2. Half-maximal acidification was seen at 10 μM H2O2. Effects on Na/H antiport were first measured as the recovery from an NH4Cl-pulse acid load: 1 mM H2O2 inhibited Na/H anti-port-mediated pH recovery by approximately 40% in both cell types. H2O2 substantially increased the rate of passive Na-independent H+ flux in the OK cell but under the conditions used did not effect this flux in the BSC-1 cell. When both the Na/H antiport and Na-independent transport systems were blocked by perfusion with a 0-Na depolarizing buffer, the H2O2-induced acidification was totally abolished. Measured from the rate of H+ appearance in the medium, H2O2 decreased the rates of endogenous TV production in both cell types. As measured from the fluorescence of endocytosed fluorescein isothiocyanate-dextran, H2O2 alkalinized lysosomes/late-endosomes, but at a rate much slower than the rate of cytosolic acidification. The results indicate that H2O2 acidifies cell pH in these cell lines by inhibiting Na/H antiport, by accelerating the passive influx of TV, and to some extent by releasing H+ from subcellular compartments. The acidification may have important effects in modulating the toxicity of H2O2.