ABSTRACT. We recently demonstrated that endothelial cells are more susceptible than renal tubular epithelial cells to oxidant injury and that renal tubular epithelial cells with proximal tubular characteristics including porcine proximal tubular epithelial cells, opossum kidney proximal tubular epithelial cells, and normal human kidney cortical epithelial cells are more susceptible to oxidant injury than the distal nephron-derived Madin Darby canine kidney cell line. To determine the basis of this differential response, we evaluated several antioxidant defenses in the five cell lines. Glutathione levels were not significantly different among the five cell lines, but catalase and glutathione reductase levels were significantly (p < 0.01) lower in endothelial cells compared to all renal tubular epithelial cells. Among renal tubular epithelial cells, Madin Darby canine kidney cells had significantly (p < 0.05) higher glutathione peroxidase activity. To further evaluate the role of antioxidant defenses in limiting oxidant injury, we determined two responses to oxidant injury (ATP depletion and 51Cr release) when glutathione was depleted with buthionine sulfoxamine and when catalase was inhibited with aminotriazole. Oxidant-induced ATP depletion was accentuated when catalase was inhibited as well as when glutathione was depleted with buthionine sulfoxamine. In contrast, inhibition of catalase had little or no effect on 51Cr release, whereas glutathione depletion resulted in accentuated 5'Cr release. We conclude that the increased susceptibility of endothelial cells to oxidant injury as compared with epithelial cells is associated with lower antioxidant defenses. Disruption of the glutathione redox cycle results in accentuated ATP depletion and lytic injury, whereas inhibition of catalase results in accentuated ATP depletion with little effect on lytic injury. Augmented oxidant-induced ATP depletion without augmented cell lysis suggests that ATP depletion alone may not be a critical mediator of cell death in oxidant stress.
To determine the mechanism(s) of oxidant-mediated cell lysis in renal tubular epithelial cells, we determined ATP depletion, DNA damage, lipid peroxidation, and cytotoxicity in LLC-PK1 cells exposed to 500 microM hydrogen peroxide for 1 h with and without inhibitors of lipid peroxidation including a lazaroid compound, 2-methylaminochroman (2-MAC), and Trolox, a vitamin E analog. ATP levels were determined by luciferin-luciferase, DNA damage by the alkaline unwinding technique, lipid peroxidation by the generation of malondialdehyde, and early cytotoxicity (5 h) by the release of 51Cr, whereas late cytotoxicity (24 h) was determined by release of [3H]leucine from prelabeled cells. Cells exposed to 500 microM hydrogen peroxide demonstrated significant (P < 0.01) ATP depletion, DNA damage, and lipid peroxidation, followed by cell death at 5 h. Concentrations of 0.1-25 microM 2-MAC or 25-500 microM Trolox each markedly and significantly (P < 0.01) inhibited lipid peroxidation and early cytotoxicity but had little to no effect on ATP depletion or DNA damage. Thus oxidant-stressed cells remained intact for several hours despite significant ATP depletion and DNA damage when lipid peroxidation was inhibited with the antioxidant compounds. At 24 h, 2-MAG and Trolox had lost their protective effect, suggesting that mechanisms other than lipid peroxidation play a role in later cytotoxicity. We conclude that ATP depletion and DNA damage are not the primary mediators of early cytotoxicity following oxidant stress, whereas lipid peroxidation plays an central role in mediating early cytotoxicity following oxidant injury.
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