INTRACELLULAR MYOGLOBIN LOADING WORSENS H2O2-INDUCED, BUT NOT HYPOXIAREOXYGENATION-INDUCED, IN-VITRO PROXIMAL TUBULAR INJURY

Intracellular iron reportedly mediates many forms of tissue injury, in cluding ischemic and myohemoglobinuric acute renal failure. This actio n may be explained by the ability of iron to catalyze the formation of the highly toxic hydroxyl radical (.OH) from H2O2 via the Fenton/Habe r-Weiss reactions. To assess whether renal tubular myoglobin/iron load ing, induced by a physiological mechanism (endocytosis), alters its su sceptibility to O2 deprivation/reoxygenation- and H2O2-mediated injury , rats were infused with myoglobin or its vehicle (5% dextrose, contro l rats), and after 2 hours, proximal tubular segments (PTSs) were isol ated for study. This infusion caused substantial myoglobin endocytic u ptake (almost-equal-to 25 mug/mg PTS protein), and it doubled PTS cata lytic iron content (assessed by bleomycin assay). Nevertheless, PTS vi ability (percent lactate dehydrogenase release) was minimally affected (4% to 6% increase), and an increased .OH burden (assessed by the sal icylate trap method) did not appear to result. Deferoxamine addition, reported to protect against in vivo acute renal failure, paradoxically increased .OH levels (almost-equal-to 25%) in myoglobin-loaded, but n ot control, PTSs. Conversely, dimethylthiourea (an .OH scavenger) depr essed .OH (by almost-equal-to 80%) in all PTSs. Myoglobin/iron loading modestly increased PTS vulnerability to exogenous H2O2 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 level s in the presence of Fenton reactants, myoglobin and six other test pr oteins were incubated with Fe2+/H2O2. Myoglobin decreased .OH levels b y almost-equal-to 70%, a significantly greater decrement than was obse rved with the other proteins tested. Conclusions are as follows: (1) M yoglobin loading increases PTS catalytic iron content, predisposing to H2O2-mediated injury. (2) Myoglobin and deferoxamine can exert antiox idant and pro-oxidant effects, respectively. (3) PTS myoglobin loading does not worsen hypoxia/reoxygenation injury, suggesting that myoglob inuria does not exacerbate in vivo ischemic acute renal failure by a d irect proximal tubular cell effect.