Abstract: SA-PO640

The Effect of Tacrolimus Exposure on CYP3A5 and P-gp Expression in a Model of Human Proximal Tubule Cells for Studying the Role of Pharmacogenetic Variation in Renal Drug Metabolism and Toxicity

Session Information

Category: Pharmacokinetics, Pharmacodynamics, and Pharmacogenetics

  • 1601 Pharmacokinetics, Pharmacodynamics, Pharmacogenomics

Authors

  • Knops, Noel, University Hospitals Leuven, Leuven, Belgium
  • Ramazani, Yasaman, KU Leuven, Leuven, Belgium
  • Levtchenko, Elena N., University Hospitals Leuven, Leuven, Belgium
  • Kuypers, Dirk R, University Hospitals Leuven, Leuven, Belgium
Background

Tacrolimus (Tac) constitutes the mainstay of immunosuppressive therapy and is metabolized through the interplay between CYP3A enzymes and the P-gp transporter (ABCB1). Clinical and fundamental studies have demonstrated the importance of genetic variation for the expression of corresponding proteins in relation to drug metabolism and toxicity. The effect of Tac on their expression in renal cells with a variable pharmacogenetic background common in the general population is unknown.

Methods

Human immortalized proximal tubule cells (PTC) with 4 combinations of CYP3A5(rs776746) and ABCB1(rs1045642) were selected. Tac exposure during experiments was based on WST-1 assay and in vivo data on tissue levels in allograft recipients, i.e. vehicle, 50 ng/ml and 300 ng/ml for 24 and 72hrs. Quantitative and functional expression was assessed by RT-PCR, WB, midazolam (MDZ) hydroxylation (for CYP3A5) and calcein efflux (P-gp).

Results

Only very high Tac concentrations (45,000ng/ml) resulted in cell death. Baseline mRNA, protein and functional expression of CYP3A5 was higher in ciPTC with the *1 versus *3/*3 allele. Increasing Tac conc. within the range of tissue levels had no effect on CYP3A5 mRNA or protein expression but resulted in decreasing 1’OH MDZ hydroxylation (p<0.001). Quantitative expression of ABCB1mRNA and Pgp was similar between variants of ABCB1 3435C>T, but calcein-AM efflux was higher in TT vs. CC/CT (delta fluorescence: 45.3% vs. 27.1%; p= 0.001). Tac conc. did not affect quantitative ABCB1/P-gp expression but resulted in decreasing calcein efflux (p<0.001) in both variants.

Conclusion

Tacrolimus exposure associated with in vivo tissue levels are not lethal and have no direct effect on the regulation of gene/protein expression for CYP3A5 and P-gp in PTC with a variable pharmacogenetic background. However, these concentrations do result in decreasing functional expression of both actors involved in Tac metabolism on top of the differences associated with the genetic background. The potential nephrotoxic effect ascribed to Tac might therefore be the result of basal variation in functional expression due to underlying genotype for CYP3A5 or ABCB1, and/or in combination with the inhibitory effect of tacrolimus on the enzyme and pump function.