Abstract
BackgroundEnd-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.MethodsWe 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.ResultsRegarding 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 < 0.0001).ConclusionsReduced CYP expression was a direct evidence of transcriptional down-regulation of CYP genes in patients with impaired kidney function.
Highlights
The incidence of chronic kidney disease is 10–16% in European adult population, and approximately 0.1–0.2% of these patients suffers from end-stage renal disease [1, 2]
Hepatic enzymes belonging to CYP1–3 families are primarily responsible for the biotransformation of drugs with major contributions of CYP3A4/5, CYP2C9, CYP2C19, CYP2D6, CYP1A2 and CYP2B6; any information on hepatic cytochrome P450 (CYP) activities is of significant importance for the estimation of drug-metabolizing capacity [12]
CYP expression and activities in leukocytes are by magnitude much lower than in the liver, the leukocyte expression of several CYPs (CYP1A2, CYP2C9, CYP2C19 and CYP3A4) appears to reflect the hepatic activities [13]
Summary
The incidence of chronic kidney disease is 10–16% in European adult population, and approximately 0.1–0.2% of these patients suffers from end-stage renal disease [1, 2]. End-stage renal disease is an irreversible status, accompanied with proteinuria and substantial reduction of glomerular filtration rate; patients with kidney failure require renal replacement therapy, including dialysis or kidney transplantation, unless fatal outcome can be predicted [1, 2, 6]. Renal replacement therapy, the majority of patients receive dialysis because of the shortage of transplantable organs or contraindication to kidney transplantation [1]. 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. 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 < 0.0001). Conclusions Reduced CYP expression was a direct evidence of transcriptional down-regulation of CYP genes in patients with impaired kidney function
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