Treatment with ouabain led to massive death of principal cells from collecting ducts (C7-MDCK), indicated by cell swelling, loss of mitochondrial function, an irregular pattern of DNA degradation, and insensitivity to pan-caspase inhibitor. Equimolar substitution of extracellular Na+ by K+ or choline(+) sharply attenuated the effect of ouabain on intracellular Na+ and K+ content but did not protect the cells from death in the presence of ouabain. In contrast to ouabain, inhibition of the Na+/K+ pump in K+-free medium increased Na-i(+) content but did not affect cell survival. In control and K+-free medium, ouabain triggered half-maximal cell death at concentrations of similar to0.5 and 0.05 muM, respectively, which was consistent with elevation of Na+/K+ pump sensitivity to ouabain in K+-depleted medium. Our results show for the first time that the death of ouabain-treated renal epithelial cells is independent of the inhibition of Na+/K+ pump-mediated ion fluxes and the [Na+](i)]/[K+](i) ratio. (C) 2003 Elsevier Science (USA). All rights reserved.