Induced, In Vitro Proximal Tubular Injury

Intracellular iron reportedly mediates many forms of tissue injury, including ischemic and myohemoglobinuric acute renal failure. This action may be explained by the ability of iron to catalyze the formation of the highly toxic hydroxyl radical (.OH) from H202 via the Fenton/Haber-Weiss reactions. To assess whether renal tubular myoglobin/iron loading, induced by a physiological mechanism (endocytosis), alters its susceptibility to 02 deprivation/reoxygenationand H202-mediated injury, rats were infused with myoglobin or its vehicle (5% dextrose, control rats), and after 2 hours, proximal tubular segments (PTSs) were isolated for study. This infusion caused substantial myoglobin endocytic uptake (:25 ,tg/mg PTS protein), and it doubled PTS catalytic iron content (assessed by bleomycin assay). Nevertheless, PTS viability (percent lactate dehydrogenase release) was minimally affected (4% to 6% increase), and an increased -OH burden (assessed by the salicylate trap method) did not appear to result. Deferoxamine addition, reported to protect against in vivo acute renal failure, paradoxically increased OH levels (:25%) in myoglobin-loaded, but not control, PTSs. Conversely, dimethylthiourea (an *OH scavenger) depressed -OH (by z=:80%) in all PTSs. Myoglobin/iron loading modestly increased PTS vulnerability to exogenous H202 addition (P<.001). However, tubular susceptibility to hypoxia (15 and 30 minutes)/ reoxygenation injury was not affected. OH levels appeared to fall in response to both forms of injury, suggesting decreased -OH production and/or OH scavenging. To assess whether myoglobin decreases -OH levels in the presence of Fenton reactants, myoglobin and six other test proteins were incubated with Fe2+/H202. Myoglobin decreased *OH levels by =70%, a significantly greater decrement than was observed with the other proteins tested. Conclusions are as follows: (1) Myoglobin loading increases PTS catalytic iron content, predisposing to H202-mediated injury. (2) Myoglobin and deferoxamine can exert antioxidant and pro-oxidant effects, respectively. (3) PTS myoglobin loading does not worsen hypoxia/ reoxygenation injury, suggesting that myoglobinuria does not exacerbate in vivo ischemic acute renal failure by a direct proximal tubular cell effect. (Circ Res. 1993;73:926-934.)