INTRACELLULAR TARGETS IN HEME PROTEIN-INDUCED RENAL INJURY

We examined two potential intracellular targets in the glycerol model of acute renal failure, namely, the mitochondrion and the nucleus. Wit hin three hours, alterations in mitochondrial function are already app arent. With either glutamate/malate or succinate/rotenone, state 3 and uncoupled respirations were decreased at three hours, and at 24 hours , such decrements were quite pronounced: in the presence of glutamate/ malate, state 2 respiration was also depressed at 24 hours, while with succinate/rotenone state 2 was increased. Marked ultrastructural chan ges were observed in mitochondria studied at three hours, including th e novel finding of degenerate mitochondria in autophagic vacuoles. Sin ce the heme content in mitochondria was increased some tenfold within three hours, mitochondrial function was studied after exposure to conc entrations of heme that reproduced such contents of heme: mitochondria initially displayed increased respiration, and subsequently, a persis tent decline in oxygen consumption until oxygen consumption was virtua lly undetectable. With higher concentrations of heme, the early increa se in oxygen consumption was blunted and the progressive decline in ox ygen consumption was hastened. The antioxidant iron chelator, deferoxa mine, prevented the early rise in oxygen consumption but did not preve nt or delay the subsequent decline. We also assessed nuclear damage as a potential lesion in the glycerol model. DNA laddering was not obser ved at any time point. At 3 and 24 hours there was DNA injury by the T UNEL technique in the distal nephron but not in the proximal nephron. The 8-hydroxydeoxyguanosine/deoxyguanosine content was increased in th e glycerol kidneys al 24 hours but not at three hours. At neither rime point was evidence of apoptosis observed by light or electron microsc opy. In studies undertaken in cell culture models, heme, at concentrat ions of 10 mu M, failed to evince any such changes in LLC-PK1 cells, a cell line from the proximal tubule, or in MDCK cells, a cell line der ived from the distal tubule. At concentrations of 50 mu M, heme induce d approximately 20% positivity in MDCK cells but none in LLC-PK1 cells by the TUNEL technique. We conclude that mitochondria and nuclei are prominent targets for injury in the glycerol model of acute renal fail ure. The presence of TUNEL-positive cells in the distal nephron but no t at proximal sites in vivo underscores the increasing appreciation of the distinct responses of these nephron sites to nephrotoxic insults.