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 LLC-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.