Direct evidence of free radical production after ischaemia and reperfusion and protective effect of desferrioxamine: ESR and vitamin E studies

Abstract

After surgical renal revascularisation, warm renal ischaemia due to renal artery cross-clamping contributes to postoperative renal dysfunction. After reperfusion, free radicals are thought to be a significant cause of injury. Nevertheless, indisputable proof of free radical production is scarce, partly because of their transient nature. In this study, electron paramagnetic resonance and vitamin E levels were used to demonstrate the free radical production after renal ischaemia and reperfusion. Rabbit kidneys were submitted either to 15 or 60 minutes of ischaemia followed by reperfusion. A spin trap agent (α-phenyl-N-tert-butyl nitrone (PBN), 20mg/ml, 1 ml/min) was infused during reperfusion directly into the left renal artery via an aortic catheter before declamping. Blood samples were selectively drawn from the left renal vein for ESR analysis (Varian spectrometer E109) of lipidic residues extracted from blood samples. The vitamin E content of the left renal cortex was determined by HPLC procedure. The right renal cortex was used as a control for the vitamin E values. In the venous effluent, ESR analysis revealed the formation of a spectrum consisting of a triplet of asymmetric doublets. This signal resulted from the spin trapping by PBN of a mixture of both oxygen- and carbon- centred lipidic radicals. The amplitude of the signal which is proportional to the amount of free radicals was significantly higher after 60 minutes ischaemia than after 15 minutes. When compared to the right control kidney, the vitamin E content of the left kidney was not modified by 15 minutes of ischaemia followed by 10 minutes of reperfusion, but was significantly decreased after 60 minutes of left renal artery occlusion followed by 10 minutes (46.7 ± 7.1%) or 30 minutes (29.5 ± 3.4%) of reperfusion. Infusion of the iron chelator desferrioxamine (50 mg/kg) prevented the decrease of the vitamin E concentration. These studies clearly demonstrate the existence of free radical production after warm renal ischaemia and reperfusion leading to the induction of lipid peroxidation and a reduction of the cortical concentration of the vitamin E, which is a major endogenous antioxidant.