Tissue iron deposition secondary to PEO+ induced hemolysis. (A) High levels of cell free hemoglobin result in macroscopic discoloration of kidneys after exposure to IV PEO+. (B) Quantitation of T24h urine hemoglobin. (C) Quantitation of nonheme bound iron accumulation in the renal cortex of PEO+ injected animals measured by a colorimetric ferrozine-based assay at the study end point. (D) Iron deposition within the renal cortex highlighted by Perls DAB staining. Renal cortical areas are shown from control, single (1×), and multidosed (5×) animals (scale bar, 50 µm), demonstrating the tendency for iron to deposit within the proximal tubule. (E) Western blot of HO-1 expression in kidney tissue and its quantitative densitometric analysis. β-actin was probed as a loading control. (F) Splenic sections stained with H&E and Perls DAB from a multidosed (5×) and control animal (scale bar, 50 µm), demonstrating increased hemosiderin deposition and red cell engorgement. Data displayed as mean values ± SEM (n = 4 per group). *P < .05 (1-way ANOVA).
