Cold storage induces time-dependent F₂-isoprostane formation in renal tubular cells and rat kidneys

Background. Previous findings suggest a possible role for free radicals in cold-storage-associated tissue injury. Because free radical-induced lipid peroxidation catalyzes the cyclooxygenase-independent formation of vasoconstrictive F₂-isoprostanes, the hypothesis that isoprostanes are produced during cold storage was tested in this study. Methods. Total isoprostanes (free and esterified) in renal tubular epithelial (LLC-PK₁) cells or whole kidneys, subjected to cold storage, were quantitated employing the gas chromatographic-mass spectroscopic method. LLC-PK₁ cells were stored at 4°C in a University of Wisconsin (UW) solution for 0, 24, 48, and 72 hours or 48 hours with desferrioxamine (DFO) or the lazaroid compound 2- methyl aminochroman (2-MAC). In the rat model, kidneys were perfused and stored for 48 hours in the UW solution with or without added DFO or 2-MAC. Results. Isoprostanes in LLC-PK₁ cells increased by fivefold following 24 hours of cold storage (36 ± 2 pg/well to 185 ± 6, mean ± SE, following 24 hours of cold storage, P < 0.0001), and the levels continued to increase significantly at 48 and 72 hours. DFO and 2-MAC caused significant suppression of isoprostane formation. Cold storage of the kidneys in UW solution for 48 hours was accompanied by an eightfold increase in isoprostanes compared with control kidneys not subjected to cold storage (25.0 ± 3.0 vs. 2.9 ± 0.1 ng/g, P < 0.0001). The addition of 2-MAC or DFO to the UW solution was associated with a near complete suppression of 48- hour cold-induced isoprostane formation. Conclusion. Our findings provide evidence for the formation of large quantities of antioxidant-suppressible isoprostanes in kidney cells and whole kidney during cold preservation. Based on this, it is hypothesized that (a) isoprostanes, which are potent renal vasoconstrictors, may contribute to immediate post-transplant vasoconstriction and dysfunction in kidneys subjected to extended cold storage, and that (b) the addition of 2-MAC or DFO to a UW solution in such circumstances may attenuate these alterations partly by suppressing isoprostane formation.