Detection of hydroxyl and carbon-centred radicals by EPR spectroscopy after ischaemia and reperfusion of the rat kidney

Recent studies suggest that oxygen-derived free radicals are involved in mediating renal reperfusion injury. EPR spectroscopy and spin trapping with the spin traps DMPO and PEN, were used to detect and quantitate the formation of hydroxyl radicals in rat kidney after ischaemia-reperfusion in vivo and in vitro in the isolated rat kidney perfused in the absence of leucocytes. EPR analysis of homogenised kidneys and of venous samples did not detect radical adducts with either spin trap. With PEN, radical adducts were not detected in vitro. When DMPO was used as the spin trap in kidneys perfused without albumin in the perfusate, EPR signals characteristic of hydroxyl and carbon-centred radical adducts were detected during early reperfusion following ischaemia. These studies confirm the generation of hydroxyl radicals during ischaemia-reperfusion in kidney. During reperfusion the total DMPO adduct concentration reached 4.35 +/- 1.05 nmol/g kidney/3 min, p < 0.05. In control kidneys total adduct were present at lower concentration (2.55 +/- 1.1 nmol/g kidney/3 min). Addition of 15 mM dimethylthiourea abolished formation of these adducts following ischaemia-reperfusion but did not prevent a reduction in glomerular filtration rate. These results indicate that significant levels of hydroxyl and carbon-centred radicals are formed in the absence of circulating neutrophils during early renal reperfusion following ischaemia.