Cold preservation of isolated rabbit proximal tubules induces radical-mediated cell injury

Background. Reactive oxygen species (ROS) are involved in reperfusion injury after preservation, Recent studies in isolated endothelial cells and hepatocytes suggested the occurrence of ROS-mediated injury during the period of cold incubation, In the present study, formation of ROS and subsequent cell injury were studied in freshly isolated rabbit proximal tubules (PTs). Methods, PTs were incubated in University of Wisconsin (UW) solution, Euro-Collins solution, or a modified Krebs-Henseleit buffer under aerobic conditions for up to 94 hr at 4 degrees, ROS formation and cell death were assessed as lipid peroxidation (formation of thiobarbituric acid-reactive substances [TBARS]) and release of lactate dehydrogenase, respectively, The involvement of ROS was further investigated in UW solution using compounds that might interfere with ROS formation, In addition, tubules were studied under anaerobic conditions (gassing with 95% N-2/5% CO2). Results. Cold preservation of rabbit PTs in any of the solutions under aerobic conditions caused progressive lipid peroxidation and concomitant cell injury, Addition to UW solution of inhibitors of ROS formation, in particular 2,2'-dipyridyl, or removal of oxygen by gassing with 95% N-2/5% CO2, prevented lipid peroxidation and protected rabbit PTs against cold injury, Both the nitric oxide (NO) synthase inhibitor L-NAME and dexamethasone, which blocks the inducible NO synthase, were ineffective, The cytoprotectant glycine affected neither TEARS formation nor lactate dehydrogenase release, Conclusions, Cold preservation of renal PTs under aerobic conditions caused cell injury even in the specially designed preservation solution UW. Cell injury is caused by iron-dependent, NO synthase-independent ROS formation.