A critical role for oxygen free radicals in cold storage-induced renal tubular epithelial cell injury

We recently made a novel observation that cold storage of renal tubular epithelial cells and kidneys are associated with increased formation of free radical-catalyzed vasoconstrictive prostaglandins, F2-isoprostanes (JASN 9:656A, 1998). Whether free radicals play a critical role in cold storage-induced cell injury is, however, not clearly defined. Cold storage (4°C) of LLC-PK1 cells and primary human proximal tubular epithelial in University of Wisconsin (UW) solution were associated with a time-dependent (0-72 hr) increase in cell injury (%LDH release), lipid peroxidation (thiobarbituric acid method, nmoles/mg protein), ATP depletion (luciferin-luciferase method, nmoles/mg protein), and DNA damage (alkaline-unwinding assay, % residual double stranded DNA). These were effectively suppressed with the inclusion of antioxidant 2-methyl aminochroman (2-MAC, 1.56 μM) or deferroxamine (DFO, 2.5 mM). Cold storage of cells was also associated with a time-dependent reduction in glutathione and increase in catalase-inhibitable H2O2 production. These were also suppressed with antioxidant inclusion. To verify the functional significance of the antioxidant-afforded cytoprotection, cells cold stored for 48-hr in UW solution with or without the addition of antioxidants were plated, and the rate of cell proliferation by ³H-thymidine incorporation was determined. Proliferation was markedly suppressed in cell stored without antioxidants compared to control cells, i.e., not subjected to cold storage (130±4 vs. 3287±113 DPM/hr, n = 4, m±SE, p<0.0001). Inclusion of 2-MAC and DFO significantly improved cell proliferation (2487±88 and 763±36, respectively, both p<0.0001 vs. 48-hr cold without antioxidants). Thus, we provide several lines of evidence to suggest that storage of renal epithelial cells in the cold is associated with free radical-induced damage, and that addition of antioxidants further enhances the storage potential of UW solution. Studies are required to determine the importance of our findings in the in-vivo setting.