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. Within three hours, alterations in mitochondrial function are already apparent. 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 changes were observed in mitochondria studied at three hours, including the novel finding of degenerate mitochondria in autophagic vacuoles. Since the heme content in mitochondria was increased some tenfold within three hours, mitochondrial function was studied after exposure to concentrations of heme that reproduced such contents of heme: mitochondria initially displayed increased respiration, and subsequently, a persistent decline in oxygen consumption until oxygen consumption was virtually undetectable. With higher concentrations of heme, the early increase in oxygen consumption was blunted and the progressive decline in oxygen consumption was hastened. The antioxidant iron chelator, deferoxamine, prevented the early rise in oxygen consumption but did not prevent or delay the subsequent decline. We also assessed nuclear damage as a potential lesion in the glycerol model. DNA laddering was not observed at any time point. At 3 and 24 hours there was DNA injury by the TUNEL technique in the distal nephron but not in the proximal nephron. The 8-hydroxydeoxyguanosine/deoxyguanosine content was increased in the glycerol kidneys at 24 hours but not at three hours. At neither time point was evidence of apoptosis observed by light or electron microscopy. In studies undertaken in cell culture models, heme, at concentrations of 10 microM, failed to evince any such changes in LLC-PK1 cells, a cell line from the proximal tubule, or in MDCK cells, a cell line derived from the distal tubule. At concentrations of 50 microM, heme induced 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 failure. The presence of TUNEL-positive cells in the distal nephron but not at proximal sites in vivo underscores the increasing appreciation of the distinct responses of these nephron sites to nephrotoxic insults.

Renal oxidant injury and oxidant response induced by mercury.

The role of oxidative stress in mercuric chloride (HgCl2)-induced nephrotoxicity is uncertain and controversial. We demonstrate that I.L.C-PK1 cells, exposed to HgCl2, generate massive amounts of hydrogen peroxide, the latter completely quenched by the hydrogen peroxide scavenger, pyruvate. HgCl2 exerts a dose-dependent cytotoxicity which is attenuated by pyruvate and catalase. Cellular generation of hydrogen peroxide arises, at least in part, from mitochondria since mitochondrial rates of generation of hydrogen peroxide increase in response to HgCl2; HgCl2 also provokes a shift in absorbance spectra in rhodamine 123 loaded-mitochondria and stimulates mitochondrial state 4 respiration. HgCl2, applied for one hour, impairs cellular vitality as demonstrated by the MTT assay, an assay dependent in part on mitochondrial function. HgCl2 impairs function in other organelles such as lysosomes that maintain a transmembrane proton gradient; these latter effects are partially attenuated by pyruvate. We complement these in vitro findings with in vivo evidence demonstrating that HgCl2 stimulates renal generation of hydrogen peroxide. The functional significance of such generation of hydrogen peroxide was evaluated in rats deficient in selenium and vitamin E, a nutrient deficiency that impairs the scavenging of hydrogen peroxide and promotes the toxicity of this oxidant. In these rats serum creatinine values were significantly higher on sequential days following the administration of HgCl2. To probe the renal response to oxidative stress induced by HgCl2, we examined hydrogen peroxide-scavenging enzymes and redox-sensitive genes. Catalase activity was unaltered whereas glutathione peroxidase activity was decreased, effects that may contribute to the net renal generation of hydrogen peroxide. The redox sensitive enzyme, heme oxygenase, was markedly up-regulated in the kidney in response to HgCl2. HgCl2 also induced members of the bcl family, bcl2 and bclx, genes that protect against apoptosis and oxidant injury. In another model of oxidant-induced renal injury, the glycerol model, bcl2 mRNA was not induced at 6 and 24 hours after the administration of glycerol. In summary, we demonstrate that HgCl2 potently stimulates renal generation of hydrogen peroxide in vitro and in vivo and such generation of peroxide contributes to renal dysfunction in vitro and in vivo. We also demonstrate that in response to HgCl2, redox sensitive genes are expressed including heme oxygenase and members of the bcl family.