Calcium and free radicals in hypoxia/reoxygenation injury of renal epithelial cells.
Hypoxia and reoxygenation (H/R) generate 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-1-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 Ca(2+)-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 Ca(2+)-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.
Greene
E L
EL
Department of Medicine, University of Minnesota, Minneapolis 55455.
Paller
M S
MS
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000502:physiology; D004848:Epithelium / Q000378:metabolism / Q000473:pathology; D000860:Hypoxia / Q000378:metabolism* / Q000473:pathology; D007425:Intracellular Membranes / Q000378:metabolism; D007668:Kidney / Q000378:metabolism* / Q000473:pathology*; D008297:Male; D010100:Oxygen / Q000494:pharmacology*; D010447:Peptide Hydrolases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014968:Xanthine Dehydrogenase / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism
D002147:Calmodulin; D017382:Reactive Oxygen Species; D014968:Xanthine Dehydrogenase; D014969:Xanthine Oxidase; D010447:Peptide Hydrolases; D010100:Oxygen; D002118:Calcium