Utilizing cytochrome P450 inhibitors we have recently demonstrated that P450 2B1 can serve as a site for reactive oxygen species generation in puromycin aminonucleoside (PAN)-induced nephrotic syndrome, which mimics minimal change disease in humans. In the current study, overexpression of P450 2B1 in glomerular epithelial cells significantly increased PAN-induced reactive oxygen species generation, cytotoxicity, cell death and collapse of the actin cytoskeleton. Silencing of P450 2B1 markedly attenuated reactive oxygen species generation, cytotoxicity, cell death and preserved the actin cytoskeleton. P450 2B1 protein content was significantly decreased while its mRNA level was markedly increased in the PAN-treated glomerular epithelial cells, indicating that the P450 2B1 protein decrease resulted from protein degradation rather than transcriptional inhibition. The degradation of P450 2B1 was accompanied by induction of heme oxygenase-1, an important indicator of heme-induced oxidative stress. This induction was significantly decreased in the P450 2B1-silenced cells treated with PAN. Treatment of the P450 2B1-silenced cells with PAN prevented cleavage of the endoplasmic reticulum-specific procaspase 12 and significantly decreased caspase 3 activity. Our data strongly suggests a pivotal role of P450 2B1 as an important site for reactive oxygen species production in PAN-induced cytotoxicity through an endoplasmic reticulum mediated pathway.

Read more »