ESPN 50th Annual Meeting

ESPN 2017


 
IN VITRO TACROLIMUS EXPOSURE IN HUMAN PROXIMAL TUBULE CELLS RESULTS IN DIFFERENTIALLY INCREASED CTGF EXPRESSION IN RELATION TO THE PHARMACOGENETIC BACKGROUND FOR CYP3A5 AND ABCB1.
YASAMAN RAMAZANI 1 BERT VAN DEN HEUVEL 1 ELENA LEVTCHENKO 1 DIRK KUYPERS 1 NOEL KNOPS 1

1- KU Leuven
 
Introduction:

The interplay between CYP3A enzymes and the P-gp pump (ABCB1) is important for drug metabolism and toxicity. Genetic variation in CYP3A5 and ABCB1 are important in tacrolimus (Tac) nephrotoxicity. We show for the first time, the implications of Tac exposure in a novel human PTC model incorporating the genetic variation in CYP3A5 and ABCB1 on the function of these genes and the production of pro-fibrotic cytokines.

Material and methods:

Eight clones of conditionally immortalized PTC (ciPTC) with different combinations of CYP3A5 (rs776746) and ABCB1(rs1045642) genotypes were incubated with vehicle, 50ng/mL and 300ng/mL Tac for 24 and 72h. qRT-PCR and western blot were performed for CYP3A5, ABCB1 and CTGF expression. Functional CYP3A5 expression was assessed by differential midazolam (MDZ) hydroxylation using LC-MS and P-gp activity by calcein efflux.

  

 

Results:

-Baseline mRNA, protein and functional expression of CYP3A5 was higher in ciPTC with the *1 versus *3/*3 allele. Increasing Tac concentration resulted in decreasing 1’OH MDZ hydroxylation (p=0.01).

-Baseline mRNA and calcein-AM efflux was higher in ABCB1 3435 TT vs. CC/CTs (mean ΔCt ABCB1: 2,25 vs. 2,97 and Δfluorescence: 23.4% vs. 45.3%, respectively; p= 0.001). A progressive decrease in calcein efflux (50ng/mL: 79.1%; 300ng/mL: 68.8%; p<0.001) was observed for both variants. Prolonged incubation resulted in decreased mRNA and protein expression (p=0.01) in CC/CTs only. Calcein-AM efflux remained higher in TTs (29.90% vs. 41.26%; p=0.016).

-Both relative CTGF mRNA and protein expression increased with Tac concentration (50 ng/mL: 0.73; 300ng/mL 0.79; p=0.02). Prolonged exposure nor CYP3A5 genotype had a significant effect on mean relative CTGF expression. However, subgroup analysis demonstrated lower CTGF expression after 72h in *1 carriers vs. non-carriers (0.58 vs. 0.81; p=0.03). CC/CTs demonstrated a significant decrease after 72h, while CTGF expression in the TTs was significantly higher than CC/CTs.

 

Conclusions:

 

-Tac exposure in human PTC has no direct effect on the regulation of gene expression but results in decreasing functions of CYP3A5 and P-gp.

-Tac exposure in human PTC results in a concentration-dependent increase in CTGF expression (pro-fibrotic cytokine).

-Prolonged Tac exposure results in increased CTGF expression in PTC with lower CYP3A5 functional expression and higher P-gp function which might correspond with an increased risk for renal fibrosis in patients.