Down-Regulation of Hepatic CYP3A and CYP2C Mediated Metabolism in Rats with Moderate Chronic Kidney Disease

Abstract

ABSTRACT:Expression and activity of drug-metabolizing enzymes are decreased in severe kidney disease; however, only a small percentage of patients with chronic kidney disease (CKD) are at the final stage of the disease. This study aimed to determine the changes in drug-metabolizing enzyme function and expression in rats with varying degrees of kidney disease. Sprague-Dawley rats were subjected to surgical procedures that allowed the generation of three distinct models of kidney function: normal kidney function, moderate kidney function, and severe kidney disease. Forty-two days after surgery, rats were sacrificed and hepatic CYP3A and CYP2C expression was determined. In addition, enzymatic activity was determined in liver microsomes by evaluating midazolam (CYP3A), testosterone (CYP3A and CYP2C), and tolbutamide (CYP2C) enzyme kinetics. Both moderate and severe kidney disease were associated with a reduction in CYP3A2 and CYP2C11 expression (p < 0.05). Likewise, moderate kidney disease resulted in more than a 60% decrease in enzyme activity (V max ) for CYP2C11 and CYP3A, compared with controls (p < 0.05). When the degree of kidney disease was correlated with metabolic activity, an exponential decline in CYP2C-and CYP3A-mediated metabolism was observed. Our results demonstrate that CYP3A and CYP2C expression and activity are decreased in both moderate and severe CKD. Our data suggest that drug metabolism is significantly decreased in the earlier stages of CKD and imply that patients with moderate CKD may be subject to unpredictable pharmacokinetics.

Protein Restoration in Low-Birth-Weight Rat Offspring Derived from Maternal Low-Protein Diet Leads to Elevated Hepatic CYP3A and CYP2C11 Activity in Adulthood

“…Wistar rat liver microsomes were isolated by differential centrifugation using methods described previously by Velenosi et al (2012). Briefly, 0.9% NaCl solution was used to rinse liver tissue.…”

“…Metabolic activity of CYP3A and CYP2C11 in hepatic microsomes was 222 Sohi et al at ASPET Journals on May 12, 2018 dmd.aspetjournals.org Downloaded from determined using methods previously described by Velenosi et al (2012). Testosterone was selected as a probe for CYP3A and CYP2C11 enzyme activities based on previously documented selective metabolism by specific rat P450 isozymes (Souidi et al, 2005;Velenosi et al, 2012). We used 50 mM potassium phosphate buffer and 2 mM MgCl 2 (pH 7.4) with 1 mg/ml hepatic microsomal protein equating to a final volume of 250 ml for timed enzymatic reactions.…”

“…The reactions were initiated by an addition of 1 mM NADPH to microsomal samples containing varying concentrations of testosterone. The reaction was terminated by the addition of 50 ml of ice-cold acetonitrile followed by a 15-minute incubation on ice and centrifugation to pellet the precipitated protein (Velenosi et al, 2012).…”

“…Testosterone metabolite analysis was performed by solid-phase extraction followed by UPLC with photodiode array (PDA), using methods previously described elsewhere (Velenosi et al, 2012). The solid-phase extraction cartridge (C18, Strata-X Polymeric Reverse Phase 33 mm; Phenomenex, Torrance, CA) was conditioned according to the manufacturer's specifications.…”

“…The column was maintained at 40°C in a Waters Acquity UPLC HClass System (Waters Corporation, Milford, MA). The mobile phase flow and gradient used for each assay was the same as previously described elsewhere (Velenosi et al, 2012). An Acquity UPLC PDA detector (Waters) was used to detect testosterone (245 nm) and carbamazepine at (290 nm) for quantification.…”