Myoglobin toxicity in proximal human kidney cells: Roles of Fe, Ca²⁺, H₂O₂, and terminal mitochondrial electron transport

Myoglobin toxicity in proximal human kidney cells: Roles of Fe, Ca²⁺, H₂O₂, and terminal mitochondrial electron transport. The purpose of this study was to gain direct insights into mechanisms by which myoglobin induces proximal tubular cell death. To avoid confounding systemic and hemodynamic influences, an in vitro model of myoglobin cytotoxicity was employed. Human proximal tubular (HK-2) cells were incubated with 10 mg/ml myoglobin, and after 24 hours the lethal cell injury was assessed (vital dye uptake; LDH release). The roles played by heme oxygenase (HO), cytochrome p450, free iron, intracellular Ca²⁺, nitric oxide, H₂O₂, hydroxyl radical (·OH), and mitochondrial electron transport were assessed. HO inhibition (Sn protoporphyrin) conferred almost complete protection against myoglobin cytotoxicity (92% vs. 22% cell viability). This benefit was fully reproduced by iron chelation therapy (deferoxamine). Conversely, divergent cytochrome p450 inhibitors (cimetidine, aminobenzotriazole, troleandomycin) were without effect. Catalase induced dose dependent cytoprotection, virtually complete, at a 5000 U/ml dose. Conversely, ·OH scavengers (benzoate, DMTU, mannitol), xanthine oxidase inhibition (oxypurinol), superoxide dismutase, and manipulators of nitric oxide expression (L-NAME, L-arginine) were without effect. Intracellular (but not extracellular) calcium chelation (BAPTA-AM) caused ∼50% reductions in myoglobin-induced cell death. The ability of Ca²⁺ (plus iron) to drive H₂O₂ production (phenol red assay) suggests one potential mechanism. Blockade of site 2 (antimycin) and site 3 (azide), but not site 1 (rotenone), mitochondrial electron transport significantly reduced myoglobin cytotoxicity. Inhibition of Na,K-ATPase driven respiration (ouabain) produced a similar protective effect. We conclude that: (1) HO-generated iron release initiates myoglobin toxicity in HK-2 cells; (2) myoglobin, rather than cytochrome p450, appears to be the more likely source of toxic iron release; (3) H₂O₂ generation, perhaps facilitated by intracellular Ca²⁺/iron, appears to play a critical role; and (4) cellular respiration/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.