MYOGLOBIN TOXICITY IN PROXIMAL HUMAN KIDNEY-CELLS - ROLES OF FE, CA2-TRANSPORT(, H2O2, AND TERMINAL MITOCHONDRIAL ELECTRON)

The purpose of this study was to gain direct insights into mechanisms by which myoglobin induces proximal tubular cell death. To avoid confo unding systemic and hemodynamic influences, an in vitro model of myogl obin cytotoxicity was employed. Human proximal tubular (HK-2) cells we re incubated with 10 mg/ml myoglobin, and after 24 hours the lethal ce ll injury was assessed (vital dye uptake; LDH release). The roles play ed by heme oxygenase (HO), cytochrome p450, free iron, intracellular C a2+, nitric oxide, H2O2, hydroxyl radical (.OH), and mitochondrial ele ctron transport were assessed. HO inhibition (Sn protoporphyrin) confe rred almost complete protection against myoglobin cytotoxicity (92% vs . 22% cell viability). This benefit was fully reproduced by iron chela tion therapy (deferoxamine). Conversely, divergent cytochrome p450 inh ibitors (cimetidine, amino-benzotriazole, troleandomycin) were without effect. Catalase induced dose dependent cytoprotection, virtually com plete, at a 5000 U/ml dose. Conversely, .OH scavengers (benzoate, DMTU , mannitol), xanthine oxidase inhibition (oxypurinol), superoxide dism utase, and manipulators of nitric oxide expression (L-NAME, L-arginine ) were without effect. Intracellular (but not extracellular) calcium c helation (BAPTA-AM) caused similar to 50% reductions in myoglobin-indu ced cell death. The ability of Ca2+ (plus iron) to drive H2O2 producti on (phenol red assay) suggests one potential mechanism. Blockade of si te 2 (antimycin) and site 3 (azide), but not site 1 (rotenone), mitoch ondrial electron transport significantly reduced myoglobin cytotoxicit y. Inhibition of Na,K-ATPase driven respiration (ouabain) produced a s imilar protective effect. We conclude that: (1) HO-generated iron rele ase initiates myoglobin toxicity in HK-2 cells; (2) myoglobin, rather than cytochrome p450, appears to be the more likely source of toxic ir on release; (3) H2O2 generation, perhaps facilitated by intracellular Ca2+/iron, appears to play a critical role; and (4) cellular respirati on/terminal mitochondrial electron transport ultimately helps mediate myoglobin's cytotoxic effect. Formation of poorly characterized toxic iron/H2O2-based reactive intermediates at this site seems likely to be involved.