Extracellular acidosis and chloride channel inhibitors act in the late phase of cellular injury to prevent death

Extracellular acidosis is cytoprotective in several models against anoxia/hypoxia and a variety of toxicants. The goal of this study was to determine the temporal relationships among toxicant exposure, the initiation of extracellular acidosis, Cl^- influx, Cl^- channel inhibition and the onset of cellular death in rabbit renal proximal tubule suspensions. Extracellular acidosis was produced by adding HCl or H₂SO₄ to renal proximal tubule suspensions to decrease the extracellular buffer pH to 6.4 or by resuspending renal proximal tubules in a pH 6.4 buffer. The initiation of extracellular acidosis 15 min after the mitochondrial inhibitors antimycin A or carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone addition, a time point in which adenosine triphosphate levels are depleted and intracellular K⁺ is decreased, ameliorated lactate dehydrogenase release, a marker of necrotic cellular death. The initiation of extracellular acidosis 120 min after the addition of the toxicants tetrafluoroethyl-L-cysteine or t-butyl hydroperoxide decreased lactate dehydrogenase release 120 min later. Increased Cl^- influx is an important step during the late phase of toxicant- induced cellular injury. Therefore, we determined if extracellular acidosis cytoprotection was associated with inhibition of Cl^- influx and whether the Cl^- channel inhibitors indanyloxyacetic acid (1.0 mM), niflumic acid (100 μM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (100 μM) decreased Cl^- influx and cellular death in renal proximal tubules exposed to antimycin A. Indeed, all three Cl^- channel inhibitors significantly decreased ³⁶Cl^- influx and cellular death. In contrast, extracellular acidosis did not decrease ³⁶Cl^- influx but did prevent lactate dehydrogenase release. These results demonstrate that extracellular acidosis cytoprotection occurs during the late phase of cellular injury at a site distal to Cl^- influx. Furthermore, the Cl^- influx that occurs during the late phase of cellular injury and is critical for cellular swelling and lysis is sensitive to 5- nitro-2-(3-phenyl-propyl-amino)-benzoic acid, niflumic acid and indanyloxyacetic acid.