N. Knops, Y. Ramazani, L. van den Heuvel, R. Goldschmeding, T. Nguyen, E. Levtchenko, D. Kuypers
Thursday 15 march 2018
16:25 - 16:35h
at Van Weelde Zaal
Categories: Basic / translational research, Session (parallel)
Parallel session: Parallel session 7: Basic / translational research
Background & Aim
Clinical studies have demonstrated the importance of genetic variation in CYP3A5 and ABCB1 for tacrolimus disposition and suggested a role in the development of renal fibrosis associated with long-term tacrolimus treatment. Our aim was to explore the implications of tacrolimus exposure in a model of human proximal tubule cells incorporating genetic variation in CYP3A5 and ABCB1 on the expression of the key profibrotic cytokine: CTGF and correlate these findings with CTGF expression in kidney allograft biopsies.
Methods
We selected 8 clones of human conditional immortalized PTC (ciPTC) with 4 different combinations of CYP3A5 (rs776746) and ABCB1 (rs1045642) Cells were incubated with vehicle, 50 ng/ml and 300 ng/ml tacrolimus (=tissue concentration range in allografts). Quantitative RT-PCR and western blot were performed to study CTGF expression. In addition, CTGF staining was performed on protocol biopsies with a known pharmacogenetic background derived from 17 allograft recipients over a period of 2 years.
Results
CTGF mRNA and protein expression increased with tacrolimus concentration (CTGF vs. β-actin vs. vehicle at 50ng/ml: + 34.1% (95% CI: 22.3 - 45.9) and at 300 ng/ml: +45.0% (95% CI: 35.2 - 54.8); p<0.001). Subgroup analysis demonstrated 46% higher CTGF protein expression in CYP3A5 *3/*3 allele carriers vs. *1 allele carriers (p=0.047) and more than 2-fold higher CTGF expression in ABCB1 3435 TTs, while in the genetic CC/CT counterparts CTGF expression decreased (p=0.01). Immunohistochemical studies of protocol biopsies demonstrated a 38.3% increase in tubular cell CTGF staining between 3 to 24 months in kidneys from 3435 TT genotype donors, while in CC/CT donor grafts the percentage of CTGF positive tubuli remained stable (p=0.046).
Conclusions
Tacrolimus exposure in human PTCs results in a concentration-dependent increase in CTGF expression. Tacrolimus exposure for 72 hours resulted in increased CTGF expression in PTC derived from CYP3A5 *3/*3 allele carriers, and in particular with the ABCB1 3435TT genotype. Immunohistochemical studies on protocol biopsies confirm increasing CTGF expression over time in donor kidneys with the ABCB1 3435TT genotype.