This study assessed the impacts of iron, heme oxygenase (HO), hydroxyl radical (.OH), and glutathione (GSH) on the initiation phase of myohemoglobinuric proximal tubular injury using a novel model system. Rhabdomyolysis was induced in rats by glycerol injection and four hours later proximal tubular segments (PTS) were isolated. They were incubated for 0 to 90 minutes either in the presence or absence of an iron chelator (deferoxamine; DFO), .OH scavengers, an .OH trapping agent (salicylate; to gauge .OH production), GSH, or catalase. In selected experiments, an HO inhibitor (Sn protoporphyrin) was given at the time of glycerol injection to assess HO's acute effects on the evolving injury. Cell death and lipid peroxidation were quantified by % LDH release and malondialdehyde (MDA) generation, respectively. PTS from normal rats served as controls. Post-glycerol PTS manifested progressive LDH release (47 +/- 2%) and 20-fold MDA increments during the incubations, whereas only 11 +/- 1% LDH release and no MDA generation was observed in the normal PTS. DFO completely prevented both parameters of glycerol-induced injury. HO inhibition exerted an acute protective effect, despite previous in vivo data suggesting that HO is a cytoprotectant. Neither .OH scavengers nor catalase mitigated post-glycerol injury, the latter correlating with reduced, not increased, .OH production. GSH slightly decreased LDH release while causing a paradoxical threefold MDA increment. The latter was iron dependent (blocked by DFO), was expressed in normal PTS, and it could be reproduced by equimolar cysteine. That GSH increased iron-dependent lipid peroxidation in a cell free system (exogenous phosphatidylcholine) indicated that GSH metabolism to cysteine was not a requirement for this reaction.
In conclusion: (1) chelatable iron can fully account for heme protein-triggered proximal tubular injury; (2) HO contributes to this injury, presumably by causing iron release; (3) the heme-induced injury appears to be mediated by non-.OH oxidizing intermediates; (4) GSH can exert both anti- and pro-oxidant effects; and (5) i.m. glycerol injection, followed by proximal tubular isolation, represents a new and highly useful model for studying direct determinants of heme protein cytotoxicity.