ESPN 53rd Annual Meeting

ESPN 2021


 
Exploring the effects of CYP3A5 and ABCB1 genetic variations in functional differences, tacrolimus metabolism and CTGF production in human proximal tubule cells
YASAMAN RAMAZANI 1 NOEL KNOPS 1 JETTY DE LOOR 1 ROEL GOLDSCHMEDING 2 TRI Q NGUYEN 2 BERT P. VAN DEN HEUVEL 1 ELENA LEVTCHENKO 3 DIRK KUYPERS 3

1- KU LEUVEN
2- UMC UTRECHT
3- KU LEUVEN AND UZ LEUVEN
 
Introduction:

The mechanisms of calcineurin inhibitor nephrotoxicity (interstitial fibrosis and hyalinosis), is largely unknown. The interplay between CYP3A5 enzymes and the P-glycoprotein (P-gp) pump is important for tacrolimus (tac) metabolism and toxicity. There is a large genetic variation in the genes encoding CYP3A5 and P-gp. Our objectives are: 1) exploring the tacrolimus-induced profibrotic signature of the only cells in the kidney responsible for tacrolimus metabolism, the proximal tubule cells 2) exploring the underlying mechanisms for the genetic-dependent tacrolimus-induced profibrotic responses.

Material and methods:

Eleven clones of conditionally immortalized PTC (ciPTC) from donor kidneys with different combinations of CYP3A5 (rs776746) and ABCB1(rs1045642) genotypes were incubated with vehicle, 50 and 300 ng/mL tac (tissue range) for 72 h. qRT-PCR and western blot were performed for CYP3A5, ABCB1 (gene encoding P-gp) and connective tissue growth factor (CTGF) expression. Functional CYP3A5 expression was assessed by midazolam hydroxylation using LC-MS and P-gp activity by calcein efflux. Quantitative metabolite analysis was performed by HPLC. Immunohistochemistry of CTGF expression in protocol biopsies was performed blindly by two pathologists.

Results:

1) ciPTC, produce CTGF, a clinically relevant profibrotic protein, when exposed to increasing concentration of tacrolimus in a dose-dependent manner. This increase is significantly higher in ciPTC with CYP3A5 *1 ABCB1 3435 TT genotype. 2) increasing concentration of tacrolimus, decreases the function of CYP3A5 and P-gp without altering their expression. 3) P-gp activity is significantly higher in carriers of ABCB1 3435 TT vs CC/CT. 4) Proximal tubule cells carrying CYP3A5*1 allele, metabolize tacrolimus faster than the carriers of the *3/*3 allele with a preferential generation of 13-O-demethyl (M1). 5) ciPTC with CYP3A5 *1 ABCB1 3435 TT genotype has significantly higher production of M1 compared to other genetic combinations. 6) Staining in protocol biopsies, in a 2-year follow-up period, demonstrates a higher CTGF accumulation in donor kidneys with ABCB1 3435 TT allele vs the CC/CT.

Conclusions:

We show for the first time, the direct genetic-dependent profibrotic effect of tacrolimus in a novel human PTC model incorporating the genetic variation in CYP3A5 and ABCB1. The increase of CTGF in PTC in cells with the highest M1 production and altered P-gp activity, is suggestive for a genetic-dependent interplay of tacrolimus metabolism/efflux in fibrotic processes and long-term kidney function.