MITOCHONDRIAL FREE-RADICAL PRODUCTION INDUCES LIPID-PEROXIDATION DURING MYOHEMOGLOBINURIA

Iron catalyzed free radical formation and lipid peroxidation are accep ted mechanisms of heme protein-induced acute renal failure. However, t he source(s) of those free radicals which trigger lipid peroxidation i n proximal tubular cells remains unknown, This study tested the potent ial involvement of mitochondrial electron transport, xanthine oxidase activity, and arachidonic acid metabolism in the heme-induced peroxida tive state. The impact of cytosolic Ca2+ loading also was assessed. Rh abdomyolysis was induced in mice by glycerol injection, and two hours later heme-laden proximal tubular segments (PTS) were isolated for stu dy. PTS from normal mice served as controls. During 30 to 60 minute in cubations, heme loaded PTS developed progressive cytotoxicity (LDH rel ease) and iron-dependent lipid peroxidation (malondialdehyde, MDA, gen eration; inhibited by deferoxamine). Site 2 (antimycin A) or site 3 (c yanide, hypoxia) mitochondrial respiratory chain inhibition completely blocked lipid peroxidation, whereas site 1 inhibition (rotenone) doub led its extent (presumably by shunting NADH through NADH dehydrogenase , a free radical generating system). Conversely, these agents did not substantially alter MDA in normal PTS. Normal and heme loaded PTS deve loped comparable degrees of LDH release during respiratory blockade ir respective of increased or decreased MDA production (indicating that l ipid peroxidation was not a critical determinant of cell death). Neith er increasing free arachidonic acid (PLA(2) treatment) nor adding cycl ooxygenase/lipoxygenase/cytochrome p450 inhibitors conferred a consist ent protective effect. Altering free Ca2+ status (chelators; ionophore addition) and xanthine oxidase inhibition had no discernible impacts. Despite mitochondrial free radical production, mitochondrial function , as assessed by the ATP/ADP ratio, seemingly remained intact. In conc lusion, (1) the terminal mitochondrial respiratory chain is the domina nt source of free radicals which trigger PTS lipid peroxidation; (2) i ron is a required secondary factor; (3) although mitochondria fuel lip id peroxidation, they do not appear to be critical targets of the heme -induced oxidant attack.