Calcium and free radicals in hypoxia/reoxygenation injury of renal epithelial cells

Hypoxia and reoxygenation (H/R) gener-ate oxygen free radicals that result in renal cell injury. We tested the roles of calcium and calmodulin in mediating xanthine oxidase-derived oxygen free radical production during H/R. Lowering extracellular Ca²⁺ attenuated lethal cell injury. H/R increased Superoxide radical production over basal levels, whereas removing extracellular Ca²⁺ before hypoxia decreased Superoxide radical production to basal levels. Pretreatment with either 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride or thapsigargin, to inhibit release or deplete stores of intracellular Ca²⁺, did not affect injury following H/R. Ionomycin increased lactate dehydrogenase release during H/R but did not increase superoxide radical to levels greater than that observed for H/R alone. The calmodulin inhibitors trifluoperazine, calmidazolium, or N-(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide decreased cell injury to varying degrees. Trifluoperazine also decreased superoxide radical production during H/R and was shown to inhibit the conversion of xanthine dehydrogenase to xanthine oxidase. Cell injury and superoxide radical production correlated with cytosolic free Ca²⁺ during H/R as determined with the Ca²⁺-sensitive fluoroprobe indo 1. Cytosolic free Ca²⁺ increased slightly during hypoxia and showed a dramatic increase as soon as cells were reoxygenated. Cells incubated in a Ca²⁺-free medium actually showed a small decrease in intracellular Ca²⁺ despite H/R. In summary, Ca²⁺ derived from extracellular sources promoted superoxide radical production and renal cell injury by a calmodulin-dependent conversion of xanthine dehydrogenase to xanthine oxidase, a major source of oxygen free radicals during H/R.