oa Cytochrome P450 Enzymes And Their Role In Diabetic Nephropathy: A Novel Mechanistic Pathway Leading To Kidney Complications

Diabetic nephropathy (DN), a serious complication of diabetes, is characterized by hyperfiltration, hypertrophy, extracellular matrix accumulation, fibrosis and proteinuria leading to loss of renal function. In renal hypertrophy, tubules increase in size and accumulate extracellular matrix and are also associated with alterations in renal sodium handling as well as hypertension; processes linked by involvement of the arachidonic acid (AA) metabolites 20-HETE and EETs. This study aims to determine the specific AA-metabolizing CYP450 isoforms present in proximal tubules (PT) that are altered by high glucose (HG) in cultured PTs, and in an animal model of diabetes. It intends to investigate the effects of alterations in CYP isoforms and/or AA-metabolite levels in DN. This work will investigate the mechanism of PTs injury and the effect of inhibition of AA-metabolites in vitro and will also get insight onto the cross-talk between CYP450 isoforms and other sources of Reactive Oxygen Species (ROS). Immunohistochemistry, hypertrophy, apoptosis, fibrosis, ROS generation, 20-HETE and EET formation, CYP4A and Nox protein expression, and mRNA levels were measured in vitro and in vivo. In our study, we show that exposure of rats proximal tubular epithelial cells to high glucose (HG) resulted in increased extracellular matrix accumulation and hypertrophy. HG treatment increased ROS production and was associated with alteration in CYPs 4A and 2C11 expression concomitant with alteration in 20-HETE and EETs formation. HG-induced tubular injury were blocked by HET0016, an inhibitor of CYPs 4A. In contrast, inhibition of EETs promoted the effects of HG on cultured proximal tubular cells. Our results also show that alteration in CYPs 4A and 2C expression and 20HETE and EETs formation regulates the activation of the mTOR/p70S6Kinase pathway, known to play a major role in the development of DN. To assess the significance of our in vitro findings, in vivo experiments were performed. Type 1 diabetic rats were used to assess the levels of different cytochromes as well as the levels of injury in these rats. In this study, we demonstrate that rats with streptozotocin-induced diabetes develop renal hypertrophy and increased fibronectin expression concomitant with an increase in CYP4A expression and a decrease in CYP2C expression. These observations were paralled by an alteration in 20-HETE and EETs productions. These results were also paralleled by an increase in reactive oxygen species (ROS) production and NADPH oxidase activity. Treatment of diabetic rats with HET0016, selective inhibitor of CYP 4A, prevented all these changes. In contrast, treatment of diabetic rats with MsPPOH, a potent inhibitor of EETs formation worsens the injury seen in the kidneys of the diabetic rats. Our results indicate that hyperglycemia in diabetes has a significant effect on the expression of AA-metabolizing CYPs, manifested by increased AA metabolism, and might thus alter kidney function through alteration of type and amount of AA metabolites; this pathway is through an oxidative stress-dependant mechanism.