title: Differential effects of glutathione and cysteine on Fe2+, Fe3+, H2O2 and myoglobin-induced proximal tubular cell attack.
abstract: Glutathione (GSH) is widely advocated as a cytoprotectant for preventing oxidant forms of renal damage. However, in the case of myoglobinuric tubular injury, both beneficial and adverse effects have been noted. The purpose of this study was to help elucidate this seeming paradox by assessing the impact on thiol supplementation on normal tubules and on tubules subjected to individual components of heme protein-induced oxidant attack (Fe2+, Fe3+, and H2O2). Isolated mouse proximal tubular segments (PTS) were exposed to either GSH or cysteine under normal conditions or in the presence of exogenous Fe2+, Fe3+, or H2O2. Lethal cell injury (LDH release) and lipid peroxidation (malondialdehyde) were then assessed. GSH and cysteine exerted iron dependent, H2O2 independent, pro-oxidant effects on normal PTS. Both were also pro-oxidant in the presence of an exogenous Fe3+ challenge. In contrast, each attenuated Fe2+ cytotoxicity. The importance of iron's redox status on the expression of tubular injury was further underscored by the fact that Fe3+ partially blocked Fe2+'s cytotoxic effects. GSH mitigated H2O2 toxicity (consistent with a fueling of GSH peroxidase activity). Conversely, cysteine promoted H2O2's injurious effects. To assess the impact of thiol supplementation on a fully integrated model of heme protein toxicity, proximal tubular (HK-2) cells were cultured with myoglobin x 24 hours +/- test reactants. Exogenous GSH worsened, while GSH depletion (BSO) protected, against myoglobin toxicity (indicating a predominance of GSH's pro-oxidant effects). Conversely, cysteine (but not homocysteine) decreased myoglobin toxicity. These GSH/cysteine effects were confirmed in LLC-PK1 cells subjected to iron attack. We conclude that: (1) GSH and cysteine can exert pro- and anti-oxidant effects, depending on the nature of the oxidant challenge and iron's redox status; (2) Fe3+ can function as a cytoprotectant, partially offsetting Fe2+ toxicity; and (3) cysteine, although potentially pro-oxidant, can mitigate heme protein-induced injury. Since the kidney rapidly catabolizes GSH to cysteine, the latter may be at least partially responsible for GSH's reported cytoprotective actions against myoglobinuric acute renal failure.
authors.affiliation: University of Washington, Seattle, USA. dzager@fhcrc.org
authors.forename: R A
authors.initials: RA
authors.lastname: Zager
authors.forename: K M
authors.initials: KM
authors.lastname: Burkhart
mesh_terms: D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003545:Cysteine / Q000494:pharmacology*; D005290:Ferric Compounds / Q000494:pharmacology; D005296:Ferrous Compounds / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D066298:In Vitro Techniques; D007668:Kidney / Q000166:cytology / Q000187:drug effects; D007687:Kidney Tubules, Proximal / Q000187:drug effects* / Q000473:pathology; D018374:LLC-PK1 Cells; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D009211:Myoglobin / Q000494:pharmacology*; D016877:Oxidants / Q000494:pharmacology*; D013552:Swine
chemical_list: D005290:Ferric Compounds; D005296:Ferrous Compounds; D009211:Myoglobin; D016877:Oxidants; D006861:Hydrogen Peroxide; D005978:Glutathione; D003545:Cysteine