End-stage renal disease reduces the expression of drug-metabolizing cytochrome P450s

Background End-stage renal disease is an irreversible status of kidney dysfunction that reduces both renal and non-renal drug clearance. Accumulation of uremic toxins seems to modify the activities of drug-metabolizing cytochrome P450 (CYP) enzymes. The aim of the present work was to refine gene expression analysis for efficient and accurate quantification of CYP mRNAs in patients’ leukocytes. Methods We compared six liquid–liquid extraction reagents for RNA isolation and five reverse transcriptase kits for RNA-to-cDNA conversion, and developed quantitative polymerase chain reaction methods for duplex measurements of CYP target genes and the reference gene. The expression of CYP1A2, CYP2C9, CYP2C19 and CYP3A4 in patients with end-stage kidney disease ( N = 105) and in organ donors with healthy kidney function ( N = 110) was compared. Results Regarding the RNA yield and purity, TRIzol, Trizolate and TRI reagents were equal; however, TRI reagent was the most advantageous in terms of financial cost. Reverse transcription using Maxima First Strand cDNA Synthesis kit appeared to be the most efficient with the widest range for quantification of the target transcript. The refined method with the detection of various CYPs and the reference gene in duplex PCR efficiently quantified even the low-level CYP expression. In leukocytes of patients with end-stage renal disease, all four CYPs were expressed at significantly lower level than in organ donors with normal kidney function ( p