Mechanism and prevention of cold storage-induced human renal tubular cell injury

Background. The recent observation that cold storage of kidneys and tubular cells causes marked increase in free radical-catalyzed F2-isoprostanes sug gests that radicals might be formed during cold storage. As cold temperatur e is associated with reduced metabolic and enzymic activity, the notion tha t cold temperature causes free radical production appeared less tenable. Th e objective was, therefore, to seek direct evidence for the free radical pr oduction during the cold storage of human renal tubular cells, and to defin e the roles of extrinsic and intrinsic antioxidants in cold-induced cell in jury. Methods. Human renal tubular cells were cold-stored at 4 degreesC for varyi ng duration in University of Wisconsin solution and subjected to mRNA analy sis, biochemical measurements, and cytoprotective studies. Results: Cold storage caused a time-dependent reduction in glutathione leve ls, and an increase in the formation superoxide, hydrogen peroxide, and hyd roxyl radicals, Cold-induced lactate dehydrogenase (LDH) release, ATP deple tion, DNA damage, and membrane degradation were suppressed with the inclusi on of antioxidant 2-methyl aminochroman or deferroxamine. The cells that we re structurally protected with antioxidants mere also intact functionally, as they had significantly improved cell proliferation. To examine the effec t of cold on intrinsic antioxidant gene expression, antioxidant mRNA levels were analyzed using reverse transcription-polymerase chain reaction. The g ene expression of mitochondrial Mn-superoxide dismutase (SOD), but not of c ytosolic Cu,Zn-SOD or of glutathione peroxidase expression increased with c old exposure. The oxidant-sensitive gene heme oxygenase I increased slightl y with 48-hr cold storage. Conclusions. Cold storage of human tubular cells causes marked increase in free radicals. These are likely of mitochondrial origin as thence is a diff erential inducement of Mn-SOD gene, and are causal to cold-induced cell inj ury as extrinsic antioxidants abrogated the injury. Our findings support th e strategy of adding antioxidants to preservation solutions or the strategy of preconditioning the organs to oxidative stress to minimize cold storage -induced organ damage.