title: The functional implications of common genetic variation in CYP3A5 and ABCB1 in human proximal tubule cells.
abstract: BACKGROUND
  Calcineurin inhibitors (CNIs) are the primary immunosuppressive drugs used in solid organ transplantation but are associated with the development of histological lesions leading to kidney failure. CNIs are metabolized by CYP3A and excreted by not only P-glycoprotein (P-gp) (ABCB1) in the gut and liver, but also by proximal tubule cells (PTCs) in the kidney. Multiple studies have demonstrated the importance of genetic variation in CYP3A5 and ABCB1 for CNI disposition and nephrotoxicity. The present study was designed to study the functional implication of variation in these two genes in human PTCs.
  
  
  METHODS
  A technique was developed to culture cells from renal tissue obtained from renal graft recipients by routine kidney biopsy. Primary cells were immortalized, subcloned, and then characterized for specific PTC markers (AQP1, CD13, brush border morphology) and donor CYP3A5(rs776746)/ABCB1(rs1045642) genotype. We then selected specific sets of confirmed conditionally immortalized PTCs (ciPTC) according to different combinations of the aforementioned genetic variants. Quantitative real-time polymerase chain reaction, Western blot, and immunohistochemistry were performed for studying CYP3A5 and ABCB1 expression. CYP3A5 activity was assessed by differential midazolam (MDZ) hydroxylation and P-gp (ABCB1 product) activity by a calcein efflux assay. Differential drug metabolism between cell lines was assessed by tacrolimus disappearance over 24 h.
  
  
  RESULTS
  Cell lines were generated from 27 out of 38 tissue samples. On the basis of genotype and PTC biomarkers, 11 subclones were selected. In vitro PTC morphology with brush border microvilli was confirmed. CYP3A5*1 carriers had increased 1-OH/4-OH MDZ formation versus homozygous *3 carriers (mean: 2.36 (95% CI:1.11-3.40) vs 0.88 (95% CI:0.48-1.27); p < 0.05). P-gp activity was confirmed by calcein accumulation (mean 38.6%; 95% CI:32.8-44.4%), which was higher in cell lines with the ABCB1 3435TT than the 3435CC/CT genotype (46.2% vs 35.5%; 95% CI:28.7-42.2%). Tacrolimus disappearance was about two-fold higher in cell lines with the combined CYP3A5*1/ABCB1 3435TT genotype versus other genotype combinations.
  
  
  CONCLUSIONS
  Biopsy-derived and immortalized human PTC cell lines demonstrate functional expression of genes involved in CNI metabolism. Differences in functional expression were detected according to common genetic variants in CYP3A5 and ABCB1. The studied genetic variants had a significant impact on in vitro tacrolimus metabolism. In particular, ciPTC with the combined CYP3A5*1/ABCB1 3435TT genotype demonstrated higher tacrolimus disappearance versus ciPTCs with a different pharmacogenetic profile. This in vitro model stresses the importance of the incorporation of pharmacogenetic variation in studies involved in (renal) drug disposition.
authors.affiliation: Department of Pediatric Nephrology and Solid Organ Transplantation and ‡Department of Nephrology and Renal Transplantation, University Hospitals Leuven , B-3000 Leuven, Belgium.
authors.forename: Noël
authors.initials: N
authors.lastname: Knops
authors.forename: Lambertus P
authors.initials: LP
authors.lastname: van den Heuvel
authors.forename: Rosalinde
authors.initials: R
authors.lastname: Masereeuw
authors.forename: Inge
authors.initials: I
authors.lastname: Bongaers
authors.forename: Henriëtte
authors.initials: H
authors.lastname: de Loor
authors.forename: Elena
authors.initials: E
authors.lastname: Levtchenko
authors.forename: Dirk
authors.initials: D
authors.lastname: Kuypers
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics / Q000378:metabolism; D000483:Alleles; D001702:Biopharmaceutics; D065095:Calcineurin Inhibitors / Q000493:pharmacokinetics; D002460:Cell Line; D051544:Cytochrome P-450 CYP3A / Q000235:genetics* / Q000378:metabolism*; D056726:Genetic Association Studies; D014644:Genetic Variation; D006801:Humans; D016030:Kidney Transplantation; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016559:Tacrolimus / Q000493:pharmacokinetics
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D065095:Calcineurin Inhibitors; D012333:RNA, Messenger; C510164:CYP3A5 protein, human; D051544:Cytochrome P-450 CYP3A; D016559:Tacrolimus
keywords: ABCB1; CYP3A5; P-gp; pharmacogenetics; proximal tubule cell; tacrolimus