Cytochrome P450 2B1 mediates oxidant injury in puromycin-induced nephrotic syndrome

Abstract

Reactive oxygen metabolites (ROM) are important mediators of puromycin aminonucleoside (PAN) induced minimal change nephrotic syndrome (NS) in rats. We have recently shown that cytochrome P450 (CYP) is a significant source of catalytic iron in this model of glomerular injury. The current study was designed to identify the CYP isozyme(s) in the rat glomeruli and explore the role of the specific isozyme(s) in PAN-induced minimal change NS. NS was induced in rats by a single intravenous injection of PAN. Animals were sacrificed at different time points for variety of biochemical assays including Western blot, immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Ultrastructural histochemistry was utilized to study hydrogen peroxide (H2O2) generation in the kidney. Several CYP isozymes were tested and CYP2B1 was localized exclusively in the rat glomeruli but not in the tubules. Treatment with PAN resulted in the generation of H2O2 in the glomerular basement membrane with significant loss of CYP2B1 content accompanied by a marked increase in the catalytic iron. CYP2B1 inhibitors cimetidine and piperine significantly reduced H2O2 generation, and prevented the loss of CYP2B1 content and the increase in the catalytic iron. CYP2B1 inhibitors also provided significant protection against PAN induced proteinuria. The induction of heme oxygenase and ferritin also was observed in the glomeruli in PAN-treated rats. Both cimetidine and piperine reduced the up-regulation of these proteins. Our data indicate that CYP2B1 plays an important role in PAN induced NS by serving as a site for ROM generation and a significant source of catalytic iron.