The Functional Implications of Common Genetic Variation in CYP3A5

Jan 15, 2015 - Background: Calcineurin inhibitors (CNIs) are the primary immunosuppressive drugs used in solid organ transplantation but are associate...
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The Functional Implications of Common Genetic Variation in CYP3A5 and ABCB1 in Human Proximal Tubule Cells Noel̈ Knops,*,†,§ Lambertus P. van den Heuvel,§,¶ Rosalinde Masereeuw,∥ Inge Bongaers,§ Henrieẗ te de Loor,⊥ Elena Levtchenko,†,§ and Dirk Kuypers⊥,‡ †

Department of Pediatric Nephrology and Solid Organ Transplantation and ‡Department of Nephrology and Renal Transplantation, University Hospitals Leuven, B-3000 Leuven, Belgium § Laboratory for Pediatrics, Department of Development & Regeneration and ⊥Laboratory of Nephrology, KU Leuven, 3000 Leuven, Belgium ¶ Laboratory for Genetic, Endocrine, and Metabolic Disorders and ∥Department of Pharmacology and Toxicology, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands S Supporting Information *

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. Conclusion: 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. KEYWORDS: pharmacogenetics, tacrolimus, CYP3A5, ABCB1, P-gp, proximal tubule cell

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INTRODUCTION

associated nephrotoxicity (CNIT) are encountered in nearly all

Calcineurin inhibitors (CNIs), such as cyclosporine A and tacrolimus, constitute the basis of immunosuppressive regimes in kidney and other fields of solid organ transplantation. However, CNIs are nephrotoxic, and prolonged treatment is associated with the development of histological lesions, which limit graft survival. Ultimately, histological signs of CNI© XXXX American Chemical Society

renal allograft recipients and are seen in nonrenal graft Received: September 2, 2014 Revised: December 30, 2014 Accepted: January 15, 2015

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Molecular Pharmaceutics recipients as well.1−3 Until now, the underlying mechanism for CNIT was unknown. Clinical studies have demonstrated a relation between the histological features of CNIT and factors related to CNI disposition. CNI disposition is for a large part determined by the interplay of metabolism by intracellular enzymes, such as the CYP3A family, and elimination via transporters, such as Pglycoprotein (P-gp), located at the cell membrane of epithelial cells located in the intestine, liver, and the kidney. The concerted effects of both metabolism and transport not only have an important impact on oral drug bioavailability, but also on local tissue parent and metabolite concentrations, drug interactions, and toxicity. There is a considerable amount of genetic variation in the genes encoding for enzymes and transporters involved in CNI metabolism, which may be associated with variation in CNI disposition.4 In particular, the carriers of a sequence variant in intron 3 of the CYP3A5 gene, designated CYP3A5*1, demonstrated to have up to two-fold higher tacrolimus dose requirements versus homozygous CYP3A5*3 carriers.5 This allele is dominant in people of African descent (allele frequency: 70−90%) but is also present in 5−20% of Caucasians. Thus far, more than 70 SNPs have been identified in the ABCB1 gene encoding for P-gp. Some of these SNPs (alone or in combination with others) have been associated with interpatient variability in CNI pharmacokinetics as well. The most common and extensively studied SNP in ABCB1 is a C to T transition at position 3435 within exon 26 (rs1045642; allele frequency: 40−60% of Caucasians vs 10−15% of people of African descent). In addition to the effect on CNI dose requirements, multiple studies pertaining to both renal and nonrenal allograft recipients have demonstrated an increased prevalence of CNIT in relation to in vivo renal CYP3A5 and P-gp expression and the carriership of the aforementioned genetic variants: CYP3A5*1 or ABCB1 3435C > T SNP.6−15 Proximal tubule cells (PTCs) are the key renal cells involved in CNI metabolism and are, in contrast to enterocytes and hepatocytes in which CYP3A4 activity predominates, largely dependent on CYP3A5 activity.16−20 Despite the aforementioned important nephrotoxic side effect, there is paucity in research aimed at studying renal CNI metabolism. A complicating factor for performing such studies is the lack of suitable human PTC models that express relevant transporters and enzymes.21,22 Furthermore, available cell lines fail to represent the pharmacogenetic variation present in the general population. In the past, our group established a cell model of human conditionally immortalized proximal tubular cells (ciPTCs) derived from cells in the urine. This in vitro model is able to constitute an impermeable monolayer and also possesses functional P-gp activity for functional testing.23 The aim of this study was to establish a model of ciPTCs incorporating common variation in genes associated with CNI metabolism and to study the functional implications of these pharmacogenetic variants with respect to protein function and CNI metabolism in the PTC. This model could be used for future experiments to investigate the role of renal CNI metabolism in CNIT development.

between December 2010 and December 2011. Further inclusion criteria were: age, >18 years; and primary or secondary kidney recipients. Patients planned for an indication biopsy due to acute graft dysfunction were excluded. Data regarding patient and graft characteristics were collected. The study protocol was approved by our hospital’s ethical committee, and all patients provided informed consent. PTC Isolation. An ultrasound-guided biopsy was performed to obtain two cortical samples (standard settings: 16 gauge, 22 mm). Half of one tissue core was put in a sterile physiological salt solution on ice and transported for immediate processing in a laminar flow cabinet. The sample was chopped into fine pulp. Then 20 mL of warm (37 °C) 0.07% collagenase-D (Roche) in culture medium was added, and the suspension was transferred to falcon tube for incubation (1.5 h at 37 °C) while gently shaking (culture medium: DMEM-HAM’s F-12, ITS (SigmaAldrich: 5 mL in 500 mL medium), hydrocortisone (SigmaAldrich: 36 ng mL−1), EGF (Sigma-Aldrich: 10 ng mL−1), triiodothyroine (Sigma-Aldrich: 40 pg mL−1), 10% FCS + Penicillin (Gibco; 100U ml−1), Streptomycin (Gibco: 100U ml−1)). The suspension was then passed through a cell sieve (Retsch, 125 um). After centrifugation (7′, 260g, 4 °C), the supernatant was removed. A second washing step with medium was performed. The “pellet” was then resuspended in 5 mL of medium and transferred to a 50 mL tube with a discontinuous Percoll gradient (GE healthcare; prod no: 17−0891−01; density 1.04 vs 1.07 g/mL). After centrifugation (25′, 1620g, 4 °C), the lower Percoll layer was “scraped” while aspirating using a 1 mL micropipette, and 5 mL of cell suspension was transferred to a new tube. Two additional washing steps followed. The pellet was then resuspended in 4 mL of medium (37 °C) and transferred to a T25 culture flask and put in a cell incubator (5% CO2, 37 °C). Culture medium was replaced for the first time after 5 days; later, 2−3 times per week. Primary cells were cultured until confluent and then split over two separate culture flasks. Cells from one flask were later stored in liquid nitrogen (10% DMSO in culture medium). At the same day of renal biopsy, we collected a sample of about 100−200 mL of midstream urine. Urinary samples were processed within several hours after collection as described previously.24 Immortalization and Subcloning. Primary cells were grown up to 50% confluence and then transduced with SV40T and hTERT vectors containing Geneticin and hygromycin B resistance. This transduction allows cells to proliferate at the low temperature of 33 °C, whereas a transfer to the nonpermissive temperature of 37 °C for 10 days will result in differentiation and expression of primary cell characteristics.23 Cells were then transferred to 33 °C and exposed to Geneticin (Sigma-Aldrich; 400 μg mL−1) and hygromycin B (SigmaAldrich; 25 μg mL−1) for 10 days. Remaining cells were designated as “conditionally immortalized” (prefix: ci-) and grown until confluence. Subsequently, to obtain a homogeneous cell culture, about 500 cells were seeded in a 10 cm Petri dish (Sarsted). Single cell derived colonies were collected with the help of cloning discs drained in trypsin/EDTA (Lonza) and transferred for further proliferation and subsequent characterization. Characterization of ciPTC. Transduced cells were selected based upon PTC phenotype determined by aquaporin-1 (AQP1) and aminopeptidase N (CD13) double expression. CD13 is expressed at the brushborder, and AQP1 is expressed at the apical and basolateral membrane of PTCs.25,26 Cells were

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METHODS Patient Selection. Subjects with a functioning kidney graft and scheduled for protocol biopsy (as a part of the standard clinical care program) were asked to participate in the period B

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Molecular Pharmaceutics grown until 80−90% confluence at 33 °C and then incubated at 37 °C for a minimum of 10 days to allow differentiation. AQP1 expression was analyzed by Western blot using 12% SDS-PAGE and rabbit anti-AQP1 (1/3000; polyclonal, Millipore). Cellular homogenates were made by lysis in a RIPA buffer (1% igepal CA630, 0.5% nadeoxycholate, 0.1% SDS, 0.01% phenylmethane sulphonylfluoride, 3% aprotinin, and 1 mM Na-orthovanadate). CD13 expression was assessed using FACS analysis. Pellets were collected using Trypsin/EDTA (Lonza, BE17−161E/12) after a wash step with PBS (Lonza, BE17−512F/12). Cells were divided over two tubes following a next washing step. Pellets were then resuspended in 300 μL of PBS. Ten microliters of monoclonal mouse antihuman CD13-FITC (Dako, F0831) was added to one sample. After 1 h at room temperature protected from light, 10 mL of PBS was added, and the cell suspension was centrifuged (5000g at 4 °C, 5′). After a second washing step, cells were resuspended in 300 μL of PBS. The cell suspension was then transferred to the flow cytometer (BD, FACS Canto II). The increase in mean fluorescence intensity of FITC-labeled versus unlabeled cells was used to identify CD13-positive clones. When a specific donor yielded more than one PTC subclone, we chose the cell line with the highest AQP1/CD13 expression. Scanning Electron Microscopy. Cells were grown for a minimum of 10 days at 37 °C in culture slides (BD Falcon). Medium was removed and then washed twice with PBS and subsequently fixed using 2.5% glutaraldehyde in 0.1 M Nacacodylate buffer pH 7.2 for 90−120 min. The cell pellets were washed in cacodylate buffer, postfixed in 1% osmiumtetroxide (2 h, 0 °C, protected from light), rinsed with dH2O, and dehydrated in a graded ethanol series (50−100%) followed by hexamethyldisilazane. Samples were left to dry overnight in a vacuum desiccator, mounted and sputter coated with platinum, and finally observed, and images were recorded in a JEOL JSF7401 field emission scanning electron microscope. Genotyping for CYP3A5 and ABCB1 Variants. Genomic DNA was isolated from a confluent T25 culture flask of primary kidney cells after isolation. Cell pellets were lysed in 700 μL of buffer (0.1 M Tris-HCl; 5 mM EDTA, 0.2% SDS; 0.2 M NaCl in AD) with 7 μL of Proteinase K (10 mg mL−1; Invitrogen: AM2542). DNA was collected after the addition of 700 μL of isopropanolol, washed with 70% ethanol, and then resuspended in TE buffer (10 mM TrisHcl, 1 mM EDTA in AD). Genotyping for CYP3A5 and ABCB1 SNPs was performed using polymerase chain reaction (PCR) restriction fragment length polymorphism methods. Forward and reverse primers were, respectively, 5′-ATGGAGAGTGGCATAGGAGATA-3′ and 5′-TGTGGTCCAAACAGGGAAGAAATA-3′ for the CYP3A5*3 A6986G SNP (rs776746); and 5′TGTTTTCAGCTGCTTGATGG-3′ and 5′AAGGCATGTATGTTGGCCTC-3′ for the ABCB1 C3435T SNP (rs1045642). The PCR mixture contained per DNA sample: 0.5 μL of forward (10 pmol ml−1) and 0.5 μL of reverse (10 pmol ml −1) primer, 0.5 μL of (10 μmol ml −1 ) deoxyribonucleoside triphosphate (Sigma), 0.2 μL of Tac DNA polymerase (Invitrogen; 10342−020) together with 2.5 μL of the accompanying 10× PCR buffer and 1 μL of (50 μmol ml−1) MgCl2 for ABCB1 C3435T, and 0.5 μL for the CYP3A5 A6986G SNP. PCR conditions were as follows: 3′ at 95 °C; 40 cycles of 30″ at 95 °C, 35″ at 60 °C, 30″ at 72 °C; and finally, 7″ at 72 °C and 12′ at 95.1 °C for both genes. After amplification, the PCR products were digested with restriction

enzymes SspI and Sau3AI (New England Biolabs, Westburg, Leusden, The Netherlands) for CYP3A5*3 and ABCB1 C3435T, respectively. Digested products were separated on 2 and 3.5% agarose gels and visualized with GelRed (Biotium). Quantitative Real-Time PCR (qPCR) for mRNA Expression of CYP3A5 and ABCB1. RNA was isolated from ciPTC grown for at least 10 days at 37 °C in a T25. Cells were collected with trypsin/EDTA as described before. RNA was isolated with the RNeasy Plus minikit (Quiagen) according to the manufacturer’s instructions. RNA concentration was determined with the Nanodrop2000 spectrophotometer (Thermo Scientific). cDNA was made by incubating 100 ng of the RNA solution with 0.5 μL of (0.5 μg μL−1) Oligo(dT) primers, 1 μL of (200 ng μL−1) random primers, and 1 μL of dNTP mix (10 mM) (all Invitrogen) with DEPC-treated water at 65 °C during 5′ in the thermal cycler and then cooled for at least 1′ on ice. Then, 4 μL of 5× First Strand buffer, 1 μL of 0.1 M DTT, 1 μL of Superscript III RT(Invitrogen), and 1 μL of DEPC-treated water was added and again placed in the thermocycler (5′ at 25 °C, 60′ at 50 °C and 15′ at 70 °C) and stored at −20 °C. qPCR was performed with the CFX96 Real Time system on a C1000 thermal cycler (Biorad). Specific forward and reverse primers (10 μM) for CYP3A5 and ABCB1 were made (IDT Technologies) and added to the qPCR mix (Platinum SYBR green; Invitrogen) together with the cDNA solution and subsequently run on the cycler according to the manufacturer’s protocol. Primer sequences are given in S-Table I of the Supporting Information. Relative expression of the gene of interest versus GAPDH or HMBS expression was calculated with the comparative Ct method. Immunocytochemistry. Cells were grown in four chamber culture slides (BD Falcon) at 33 °C until confluence and then transferred to an incubator at 37 °C for a minimum of 10 days. Medium was removed, and the slides were left to dry for at least 30′. Then slides were immersed in cooled acetone for fixation. After washing and drying, the slides were incubated for 60 min with primary antibody in diluent (Dako) at the following dilutions: AQP1 (Millipore; AB2219), 1/200; CYP3A5 (Abcam: ab76667), 1/20; MDR1 (Monosan: mon9011−1), 1/20. This was followed by incubation with a secondary antibody (poly-HRP-GAM/R/R IgG ImmunoLogic, RTU) for 30′ and AEC Chromogen (Dako, RTU) for 15′. Counterstaining was performed with Mayer’s hematoxilline for 10−15′. Transplant kidney biopsy samples were used as a positive control. Western Blot CYP3A5. Protein samples of ciPTC were prepared as described above. The Bradford protein assay (BioRad) was used to determine protein concentrations in cell homogenates. CYP3A5 expression in the subclones was analyzed by Western blot using 7.5% SDS-PAGE and rabbit anti-CYP3A5 (1/100; polyclonal rabbit, Abcam; ab76667). Human kidney homogenate was used as control. We were unsuccessful in demonstrating P-gp protein expression with a set of different commercially available antibodies against human P-gp in both control samples (human kidney and HeLa cell lysate) and ciPTC lysates by following the protocol described by the manufacturers. P-gp Activity. The activity of the ABC efflux transporter Pgp was assessed by measuring the accumulation of calcein in cell lysates.27 ciPTCs (24-well plate, 10−21 days at 37 °C) were inspected before the assay. Subclones demonstrating T genotype. Panel A: relative ABCB1 mRNA expression versus GAPDH according to genotype (n = 11, bars represent median). Panel B: functional expression of P-gp assessed by calcein accumulation in ciPTC according to genotype (n = 10, bars represent mean).

0.000, R2 = 0.57 with p = 0.000 for the 3435C > T SNP). This was also confirmed in the analysis of the cell lines according to their combined CYP3A5/ABCB1 3435C > T genotype and time where CYP3A5*1/3435TT demonstrated increased tacrolimus disappearance versus the other combinations, but no difference was demonstrated between the other variants (F(2.53) = 11.03; p = 0.000; R2 = 0.29; Figure 4D) .

protein concentration and is depicted in Figure 4, panel A. Tacrolimus disappearance in ciPTC clones was significantly determined by CYP3A5 genotype, p = 0.001 (Figure 4B). Tacrolimus disappearance corrected for protein mass was about 1.6-times higher in ciPTCs from CYP3A5*1 carriers, albeit with a large variation (at original concentration of 50 ng tacrolimus μL−1 medium; mean disappearance, 64.7 pg μg−1; 95% CI:53.4−76.0 vs 40.8 pg μg−1; 95% CI:32.5−49.3 in CYP3A5*3 carriers). The incubation time period tested appeared less important than CYP3A5 genotype. Analysis of these data according to ABCB1 genotype irrespective of CYP3A5 genotype demonstrated a trend toward increased mean tacrolimus disappearance in cell lines with the 3435TT genotype; however, this was not significant (64.0 pg μg−1 (TT), 45.5 pg μg−1 (CT), 38.4 (CC); p = 0.08 (Figure 4C). Subgroup analysis for the relation of the ABCB1 3435C > T polymorphism with tacrolimus disappearance and time according to CYP3A5 genotype revealed no effect of the 3435C > T polymorphism within homozygote CYP3A5*3 allele carriers. However, CYP3A5*1 allele carriers with the 3435TT genotype demonstrated an almost two-fold increased tacrolimus disappearance versus cell lines with the CYP3A5*1/ 3435CT combination (mean, 82.2 pg μg−1; 95% CI:68.1−96.3 (TT) vs 47.2 pg μg−1; 95% CI:35.6−58.9; F(2.21) = 14.29 ; p =

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DISCUSSION In this study, a novel model of biopsy-derived and immortalized human PTC cell lines is described with functional expression of CYP3A5 and ABCB1 genes involved in CNI metabolism. We demonstrate differences in functional expression according to common variants in these genes. In addition, we demonstrate that this genetic variation has a significant effect on in vitro tacrolimus metabolism, in particular for ciPTC with the combined CYP3A5*1/ABCB1 3435TT genotype exhibiting a two-fold increase in drug disappearance versus cells with a different genotype. First, we developed a method to culture renal cells from a reduced biopsy core through modifications in a protocol for deriving renal cells from nephrectomized kidneys.31 Notwithstanding the small tissue sample, proliferation arrest, and loss due to infections, the final success rate for obtaining primary F

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Figure 4. Total tacrolimus disappearance in ciPTC corrected for cell mass over a 24 h time period (pg/μg protein; error bars, ± 1 SEM). Panel A: for individual subclones (in duplo). Panel B: according to CYP3A5 genotype (p = 0,001*). Panel C: according to ABCB1 3435C > T genotype. Panel D: according to the CYP3A5/ABCB1 3435C > T genotype combination (p < 0.000#).

the histological background in the corresponding donor kidney of successfully cultured urine-derived renal cells revealed a relatively higher prevalence of fibrosis and tubular atrophy versus the entire cohort of cells derived directly from the biopsy core. This suggests that allografts with more histological damage have an intrinsic higher potential for culturing renal cells from the urine possibly due to an increased shedding of epithelial cells. To our knowledge there are no studies quantifying renal cell shedding in stable renal allograft recipients. A recent paper did describe increased “leukocyturia” in relation to IF/TA in protocol biopsies.35 However, the authors did not perform characterization of the urinary cells, and these might be, at least partially, epithelial cells instead. Increased shedding has been reported in response to renal ischemia and BK nephropathy36−38 and may thus be associated with kidney injury. We, therefore, chose not to use these exfoliated cells for our cell study since the cells obtained directly from a biopsy are more representative for the functioning PTCs in the kidney.

cells was 71%. After the process of immortalization and subcloning, 41% of clones tested demonstrated molecular and histological PTC characteristics. The relatively high efficacy of this method suggests potential for other fields of research in kidney disease. More elaborate characterization of PTC phenotype of cells obtained with similar protocols for immortalizing renal cells was described earlier (including different transporter proteins such as OCT-2, MRP4, BCRP).23,32,33 We additionally aimed to culture PTCs from the urine. In the paper published by Wilmer et al., the success rate for obtaining renal cells from urine samples in pediatric cystinosis patients was high (9/9).24 In contrast, only 19% of samples from healthy pediatric controls (4−13 yrs) yielded cells, a number very similar to this study (17%). The majority of urine samples collected in our study were lost early due to bacterial overgrowth in samples collected from a population with an increased risk for urinary tract infections,34 which might have had a negative effect on the success rate. Statistical analysis of G

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porter assays.45,46 Indeed, Gow et al. demonstrated that certain ABCB1 polymorphism demonstrated inhibitor-related differences in calcein accumulation in response to cyclosporin A in HEK293 cells. Unfortunately, they did not look specifically at the 3435C > T variant.47 Taken together, these data suggest a substrate and inhibitor-specific altered P-gp transport activity for homozygous carriers of the ABCB1 3435TT SNP irrespective of quantitative mRNA or protein expression. Finally, we confirmed in vitro tacrolimus metabolism in our ciPTC clones and demonstrated a significant effect of aforementioned genetic variants and their combination on drug metabolism. Precise assessment of tacrolimus disappearance due to metabolism by the ciPTC clones was hampered by nonspecific drug adsorption to the plastic surface of culture plates and storage vials. This is a well-recognized property of hydrophobic drugs (i.e., tacrolimus)48 that was anticipated in the current study by optimizing tacrolimus recovery from the culture plates through washing with an amphiphilic organic solvent (methanol). Nonetheless, supernatant and lysates derived from ciPTC with a different genetic background already demonstrated a broad variation in drug disappearance after the first hour, which indicates important differences in drug metabolism between the cell lines manifesting shortly after incubation. In accordance with the previously established higher CYP3A5 expression in ciPTC of CYP3A5*1 allele carriers, we demonstrated a two-fold higher tacrolimus disappearance rates in cell lines with this genotype. To our knowledge, there are no other studies that have looked into in vitro tacrolimus metabolism in PTCs. However, a study by Dai et al. described that kidney-derived microsomes from CYP3A5*1 allele carriers generated 13.5-fold higher concentrations of tacrolimus metabolites than microsomes from noncarriers.19 Further analysis of our data according to ABCB1 3435C > T variants demonstrated a trend toward increased tacrolimus disappearance for ciPTC with the 3435TT genotype, but this was not statistically significant. However, combination of CYP3A5 and ABCB1 genotypes in a multiple regression analysis proved to be the best predictive model for tacrolimus disappearance over 24 h and demonstrated increased drug clearance in cell lines with the CYP3A5*1/3435TT genotype combination. Interestingly, although ciPTC with the CYP3A5*1/3435CC-CT genotype combination had slightly higher tacrolimus disappearance than non-CYP3A5*1 carriers, this was not significant. This is the first report demonstrating in vitro data on the effect of ABCB1 SNPs on tacrolimus metabolism. A recent report demonstrated increased tacrolimus accumulation in transfected HEK293 cells with the ABCB1 1199G > A variant, which indeed confirmed possible decreased P-gp activity due to genetic variation in ABCB1.49 Next to an intrinsic genetic defect in P-gp function, additional and differential potentiation of P-gp inhibition by the CNI could be important as well, as discussed earlier.47 Decreased functioning of the membrane efflux pump will lead to an increased exposure of the drug to intracellular enzymes and increased drug metabolism. This phenomenon of protein interplay was described previously in a paper by Wu et al. in which they demonstrated increased tacrolimus clearance in an isolated perfused rat liver after administration of a selective P-gp inhibitor.50 The effect of the functional capacity of the efflux pump on the metabolism of a dual substrate for enzyme and transporter will be more pronounced in cells with a genetic higher capacity for enzymatic drug metabolism and is in

Our model of immortalized PTCs demonstrated increased expression of CYP3A5 in heterozygous CYP3A5*1/*3 versus homozygous CYP3A5*3 allele carriers. CYP3A5 RNA expression in ciPTC proved similar to previously published data by Bolbrinker et al., who demonstrated a 6.2-fold increased expression in primary kidney tissue derived from CYP3A5*1/ *3 allele carriers.20 Also, Givens et al. demonstrated an eightfold increase in renal microsomal content of CYP3A5*1 allele carriers derived from kidney donors.39 In analogy with these data, differential CYP3A5 function was demonstrated by an increased 1′−OH over 4′−OH MDZ ratio in ciPTC with the CYP3A5*1/*3 genotype versus CYP3A5*3/*3. The oxidation of midazolam by CYP3A4 and CYP3A5 generates two important metabolites: 1′hydroxymidazolam (1′−OH MDZ) and 4′ hydroxymidazolam (4′−OH MDZ). CYP3A5 is less efficient in the formation of the 4′−OH versus the 1′−OH metabolite in comparison to CYP3A4. Indeed, earlier studies using kidney and liver microsomes demonstrated an increased formation of 1′−OH MDZ and an increased 1′−OH over 4′− OH MDZ ratio in function of microsomal CYP3A5 protein content17 and CYP3A5 genotype.29 Vincristine might be preferred as a probe for future “in-depth” metabolic phenotyping of proximal tubular cells for CYP3A5 enzyme activity since intrinsic clearance of this drug by CYP3A5 was demonstrated to be 9−14-fold higher versus CYP3A4 in a model of recombinant CYP3A in insect microsomes40 (vs the 2.2−2.5 higher intrinsic clearance for midazolam in human hepatic microsomes reported by Kuehl et al.29). However, we chose midazolam over vincristine because of concerns about the intrinsic cytotoxic profile (e.g., microtubule disassembling agent; MDA) of the latter, which could interfere (confound) with future “repeat experiments” exploring the role of functional CYP3A5 expression in CNIT. We confirmed functional ABCB1/P-gp expression in our ciPTC model, reflected best by calcein accumulation in all subclones after addition of a specific P-gp inhibitor. We were not able to detect differences in quantitative expression of ABCB1 mRNA for variants of the 3435C > T genotype. However, subclones with the ABCB1 3435 TT variant demonstrated higher relative calcein accumulation after inhibition than the “wild-type” CC/CT carriers. Comparable data on in vivo ABCB1/P-gp expression in normal human kidney tissue are scarce. Brown et al. confirmed ABCB1 mRNA expression in primary PTCs.31 Other studies demonstrated similar calcein accumulation in urinary and kidney-derived human ciPTC.33,41 The role of genetic variation in functional ABCB1 expression in human renal cells was not studied before. The functional capacity of P-gp depends on both the amount of the membrane efflux pump and the interaction between the transporter and its substrate. A study in recombinant LLC-PK1 cells (porcine) demonstrated that ABCB1 variant alleles 3435C > T, 1236C > T, and 2677G > T/A, whether present individually or in linkage disequilibrium, significantly minimize P-gp activity.42 Publications on “nonrenal” ABCB1 mRNA expression in relation to genotype in humans are equivocal. Wang et al. demonstrated lower mRNA expression in liver samples with the 3435T allele.43 On the other hand, Goto et al. could not detect any difference in intestinal ABCB1 mRNA expression in tissue obtained during liver transplantation.44 In line with our data, previous in vitro studies using manipulated cell lines with a different genetic background but with similar mRNA and P-gp protein expression demonstrated altered druginhibitor interactions for ABCB1 C3435T variants in transH

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Molecular Pharmaceutics line with our finding in cell lines with the CYP3A5*1/3435TT genotype. Whether this increased tacrolimus metabolism is associated with more cellular injury is the subject of our ongoing investigation into the mechanisms of CNIT. In conclusion, we developed a new model of biopsy-derived human PTCs that demonstrates the importance of incorporating common pharmacogenetic variants in studies of in vitro CNI disposition. In particular, the combination of polymorphisms that leads to an increased enzymatic activity and a decreased efflux pump capacity will lead to a significantly increased metabolism of dual substrates such as tacrolimus.

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renal failure after transplantation of a nonrenal organ. N. Engl. J. Med. 2003, 349 (10), 931−40. (4) Knops, N.; Levtchenko, E.; van den Heuvel, B.; Kuypers, D. From gut to kidney: Transporting and metabolizing calcineurin inhibitors in solid organ transplantation. Int. J. Pharm. 2013, 452 (1− 2), 14−35. (5) Macphee, I. A.; Fredericks, S.; Mohamed, M.; Moreton, M.; Carter, N. D.; Johnston, A.; Goldberg, L.; Holt, D. W. Tacrolimus pharmacogenetics: The CYP3A5*1 allele predicts low dosenormalized tacrolimus blood concentrations in whites and South Asians. Transplantation 2005, 79 (4), 499−502. (6) Metalidis, C.; Lerut, E.; Naesens, M.; Kuypers, D. R. Expression of CYP3A5 and P-glycoprotein in renal allografts with histological signs of calcineurin inhibitor nephrotoxicity. Transplantation 2011, 91 (10), 1098−102. (7) Kuypers, D. R.; Naesens, M.; de Jonge, H.; Lerut, E.; Verbeke, K.; Vanrenterghem, Y. Tacrolimus dose requirements and CYP3A5 genotype and the development of calcineurin inhibitor-associated nephrotoxicity in renal allograft recipients. Ther. Drug Monit. 2010, 32 (4), 394−404. (8) Kuypers, D. R.; Claes, K.; Evenepoel, P.; Maes, B.; Vanrenterghem, Y. Clinical efficacy and toxicity profile of tacrolimus and mycophenolic acid in relation to combined long-term pharmacokinetics in de novo renal allograft recipients. Clin. Pharmacol. Ther. 2004, 75 (5), 434−47. (9) Kuypers, D. R.; de Jonge, H.; Naesens, M.; Lerut, E.; Verbeke, K.; Vanrenterghem, Y. CYP3A5 and CYP3A4 but not MDR1 singlenucleotide polymorphisms determine long-term tacrolimus disposition and drug-related nephrotoxicity in renal recipients. Clin. Pharmacol. Ther. 2007, 82 (6), 711−25. (10) Naesens, M.; Lerut, E.; de Jonge, H.; Van Damme, B.; Vanrenterghem, Y.; Kuypers, D. R. Donor age and renal Pglycoprotein expression associate with chronic histological damage in renal allografts. J. Am. Soc. Nephrol. 2009, 20 (11), 2468−80. (11) Hauser, I. A.; Schaeffeler, E.; Gauer, S.; Scheuermann, E. H.; Wegner, B.; Gossmann, J.; Ackermann, H.; Seidl, C.; Hocher, B.; Zanger, U. M.; Geiger, H.; Eichelbaum, M.; Schwab, M. ABCB1 genotype of the donor but not of the recipient is a major risk factor for cyclosporine-related nephrotoxicity after renal transplantation. J. Am. Soc. Nephrol. 2005, 16 (5), 1501−11. (12) Hawwa, A. F.; McKiernan, P. J.; Shields, M.; Millership, J. S.; Collier, P. S.; McElnay, J. C. Influence of ABCB1 polymorphisms and haplotypes on tacrolimus nephrotoxicity and dosage requirements in children with liver transplant. Br. J. Clin. Pharmacol. 2009, 68 (3), 413−21. (13) Moore, J.; McKnight, A. J.; Dohler, B.; Simmonds, M. J.; Courtney, A. E.; Brand, O. J.; Briggs, D.; Ball, S.; Cockwell, P.; Patterson, C. C.; Maxwell, A. P.; Gough, S. C.; Opelz, G.; Borrows, R. Donor ABCB1 variant associates with increased risk for kidney allograft failure. J. Am. Soc. Nephrol. 2012, 23 (11), 1891−9. (14) de Denus, S.; Zakrzewski, M.; Barhdadi, A.; Leblanc, M. H.; Racine, N.; Belanger, F.; Carrier, M.; Ducharme, A.; Dube, M. P.; Turgeon, J.; White, M. Association between renal function and CYP3A5 genotype in heart transplant recipients treated with calcineurin inhibitors. J. Heart Lung Transplant. 2011, 30 (3), 326−31. (15) Woillard, J. B.; Rerolle, J. P.; Picard, N.; Rousseau, A.; Guillaudeau, A.; Munteanu, E.; Essig, M.; Drouet, M.; Le Meur, Y.; Marquet, P. Donor P-gp polymorphisms strongly influence renal function and graft loss in a cohort of renal transplant recipients on cyclosporine therapy in a long-term follow-up. Clin. Pharmacol. Ther. 2010, 88 (1), 95−100. (16) Schuetz, E. G.; Schuetz, J. D.; Grogan, W. M.; Naray-Fejes-Toth, A.; Fejes-Toth, G.; Raucy, J.; Guzelian, P.; Gionela, K.; Watlington, C. O. Expression of cytochrome P450 3A in amphibian, rat, and human kidney. Arch. Biochem. Biophys. 1992, 294 (1), 206−14. (17) Haehner, B. D.; Gorski, J. C.; Vandenbranden, M.; Wrighton, S. A.; Janardan, S. K.; Watkins, P. B.; Hall, S. D. Bimodal distribution of renal cytochrome P450 3A activity in humans. Mol. Pharmacol. 1996, 50 (1), 52−9.

ASSOCIATED CONTENT

S Supporting Information *

Primer sequences and donor/recipient characteristics of successfully biopsy-derived renal cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +32 16 34 38 22. Fax: +32 16 34 38 42. E-mail: noel. [email protected]. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare the following competing financial interest(s): This project was supported by an IDS grant from Astellas Pharmaceuticals. Astellas was not involved in the experimental setup, data analysis, or writing of this manuscript.

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ACKNOWLEDGMENTS We would like to thank S. Van Aerschot from our laboratory for pediatrics, P. Baatsen from the Electron Microscopy Core Facility of the KU Leuven, K. Van den Eynde from the laboratory for Translational Cell and Tissue research, and N. Holmstock from the Laboratory for Drug Delivery and Disposition, KU Leuven for their technical support and advice. This work was supported by an IDS research grant from Astellas Pharmaceuticals. N.K. was supported by the “KOF” grant provided by the University Hospitals Leuven and the fund for Scientific Research, Flanders (F.W.O.: Grant No. 1701515N). Both E.L. and D.K. were supported by the fund for Scientific Research, Flanders (F.W.O.) as well (Grant No. 1801110N and G.0195.07, respectively).

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ABBREVIATIONS CNI, calcineurin inhibitor; PTC, proximal tubule cell; MDZ, midazolam; CNIT, CNI-associated nephrotoxicity; P-gp, Pglycoprotein; ciPTC, human conditionally immortalized proximal tubular cell

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REFERENCES

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