Download role of ABC and solute carrier transporters in the placental transport of

1
Role of ABC and solute carrier transporters
2
in the placental transport of lamivudine
3
Martina Ceckovaa , Josef Rezniceka, Zuzana Ptackovaa, Lukas Cervenya, Fabian Müllerb,*,
4
Marian Kacerovskyc, Martin F. Frommb, Jocelyn D. Glazierd, Frantisek Stauda,#
5
6
Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Department of
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Pharmacology and Toxicology, Hradec Kralove, Czech Republica; Institute of Experimental
8
and Clinical Pharmacology and Toxicology, Friedrich-Alexander-Universität Erlangen-
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Nürnberg, Erlangen, Germanyb; Department of Obstetrics and Gynecology, University
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Hospital, Charles University in Prague, Hradec Kralove, Czech Republicc; Maternal and Fetal
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Health Research Centre, Institute of Human Development, University of Manchester, St.
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Mary's Hospital, Central Manchester University Hospitals NHS Foundation Trust,
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Manchester Academic Health Science Centre, Manchester, UKd
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15
Running Head: Placental transport of lamivudine
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17
#
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*Present address: Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riß,
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Germany.
Address correspondence to Frantisek Staud, e-mail: [email protected]
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21
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ABSTRACT
23
Lamivudine is one of the antiretroviral drugs of choice for the prevention of mother-to-child
24
transmission (MTCT) in HIV-positive women. In this study, we investigated the relevance of
25
drug efflux transporters P-gp (MDR1, ABCB1), BCRP (ABCG2), MRP2 (ABCC2) and
26
MATE1 (SLC47A1) for the transmembrane transport and transplacental transfer of
27
lamivudine.
28
We employed in vitro accumulation and transport experiments on MDCK cells
29
overexpressing drug efflux transporters, in situ perfused rat term placenta and vesicular
30
uptake in microvillous plasma membrane (MVM) vesicles isolated from human term
31
placenta. MATE1 significantly accelerated lamivudine transport in MATE1-expressing
32
MDCK cells, whereas no transporter-driven efflux of lamivudine was observed in MDCK-
33
MDR1, MDCK-MRP2 and MDCK-BCRP monolayers. MATE1-mediated efflux of
34
lamivudine appeared to be a low affinity process (apparent Km = 4.21 mM, Vmax =
35
5.18 nmol/mg protein/min in MDCK-MATE1 cells). Consistent with in vitro transport
36
studies, the transplacental clearance of lamivudine was not affected by P-gp, BCRP or MRP2.
37
However, lamivudine transfer across dually perfused rat placenta and the uptake of
38
lamivudine into human placental MVM vesicles revealed pH dependency, indicating possible
39
involvement of MATE1 in the fetal-to-maternal efflux of the drug.
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To conclude, placental transport of lamivudine does not seem to be affected by P-gp, MRP2
41
or BCRP, but a pH-dependent mechanism mediates transport of lamivudine in the fetal-to-
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maternal direction. We suggest that MATE1 might be, at least partly, responsible for this
43
transport.
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2
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INTRODUCTION
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More than 36 million people are infected with HIV worldwide today (1). Half of them are
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women who, if pregnant, carry the risk of transferring the infection to their child in utero, at
48
delivery or during breastfeeding. Progress in preventing new HIV infections among children
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has been dramatic in recent years as the number of children becoming infected with HIV each
50
year dropped from 520.000 to less than 240.000 between 2000 and 2014 (1), clearly
51
demonstrating that well-timed antiretroviral prophylaxis can reduce the risk of mother-to-
52
child transmission (MTCT) of HIV. Current guidelines (2, 3) recommend lamivudine ([-]-b-
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L-2’,3’-dideoxy-3’-thiacytidine) as one of the antiretroviral drugs of choice in first line
54
therapy of HIV-positive pregnant women, including first trimester pregnancies. In addition to
55
diminishing the MTCT of HIV-1, lamivudine is also used to decrease the vertical
56
transmission of hepatitis B virus in pregnancy (4, 5). Lamivudine is known to cross placenta,
57
with the predicted fetal-to-maternal area under the concentration-time curve (AUC) reaching
58
86% in humans (6). However, detailed knowledge of mechanisms affecting lamivudine
59
transplacental transfer is still lacking.
60
The transplacental permeability of drugs predominantly depends on their physical-chemical
61
characteristics, which determine the rate of passive diffusion. However, it can also be
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extensively influenced by transporter proteins expressed in the apical microvillous plasma
63
membrane (MVM) of polarized trophoblasts (7). Among them the ATP-dependent (ABC)
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efflux transporters P-glycoprotein (P-gp, MDR1, ABCB1), breast cancer resistance protein
65
(BCRP, ABCG2) and multidrug resistance-associated protein 2 (MRP2, ABCC2) are well
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confirmed active components of the placental barrier providing fetal protection against
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potentially toxic compounds, including drugs (8-10). Besides ABC transport proteins, some
68
members of the SLC (solute carrier) transporter family can further modulate transplacental
69
drug transfer (7). MATE1 (SLC47A1), typically expressed in the apical membrane of
3
70
polarized cells, is an H+ - exchanger known to ensure the efflux of substrates that enter the
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cells via organic cation transporters (OCTs, SLC22A) located in the basolateral membrane
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(11). This OCT-MATE1 excretory pathway is typical for the kidneys and liver. Nevertheless,
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recent studies indicate that this vectorial transport mechanism might also be relevant for the
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placenta (12, 13). Expression of multidrug and toxin extrusion proteins MATE1/Mate1
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(SLC47A1/Slc47a1) and MATE2/Mate2 (SLC47A2/Slc47a2)) has been recently studied in
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human and rat first trimester and term placentas (13-15) and Mate1 was suggested as an
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efflux component of vectorial fetal-to-maternal drug transfer of metformin and cationic
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neurotoxin 1-methyl-4-phenylpyridinium (MPP+) in the dually perfused rat term placenta
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model (12, 16).
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Lamivudine was recently found to interact with MATE1 and its kidney variant MATE2-K
81
(17). Thus, we hypothesized that MATE1 could also affect the transplacental transport of
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lamivudine by mediating the efflux from polarized trophoblast cells back to the maternal
83
blood. All three subtypes of human organic cation uptake transporters, OCT1 (SLC22A1),
84
OCT2 (SLC22A2) and OCT3 (SLC22A3), were shown to contribute to lamivudine uptake into
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the cells, with decreasing efficacy in the following order: OCT1 > OCT2 > OCT3 (18, 19).
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OCT3 is considered the predominant placental OCT transporter (12-14, 20) nevertheless
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significant mRNA expression of OCT1 and OCT2 were also shown in first trimester as well
88
as term human placenta (13, 14).
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To the best of our knowledge, the possible impact of drug efflux transporters on transplacental
90
pharmacokinetics of lamivudine has not previously been systematically evaluated. In the
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present work, we investigated the affinity of lamivudine to efflux transporters MDR1, BCRP,
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MRP2 and MATE1 and evaluated whether the maternal-to-fetal transfer of lamivudine could
93
be affected by any of these efflux transporters. To address these aims, we performed in vitro
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transport and accumulation assays on cellular monolayers and the in situ method of dually
4
95
perfused rat term placenta. Additionally, we verified the results in human placenta by uptake
96
assay in microvillous placental membrane (MVM) vesicles.
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METHODS
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Materials and reagents
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Lamivudine was kindly provided by the NIH as a part of the NIH AIDS Reagent Program.
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Radiolabelled lamivudine ([3H]-lamivudine, 21.3 or 5.2 Ci/mmol) was purchased from
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Moravek Biochemicals (California, USA). Mitoxantrone, ASP+ and fluorescein isothiocyanate
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labelled dextran (MW=40 kDa) were obtained from Sigma Aldrich (St. Louis, MO, USA).
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BCRP inhibitor Ko143 was purchased from Enzo Life Sciences AG (Lausen, Switzerland)
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and pentobarbital (Nembutal) was purchased from Abbott Laboratories (Abbott Park, Illinois,
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USA). Cell culture media and sera were obtained from Sigma-Aldrich (St. Louis, MO, USA)
107
and Gibco BRL Life Technologies (Rockville, MD, USA). All other chemicals were of
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analytical grade and obtained from Sigma-Aldrich (St. Louis, MO, USA). The bicinchoninic
109
acid assay kit (BCA assay) was purchased from Thermo Scientific (Rockford, USA).
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Cell lines and cell culture
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Madin–Darby canine kidney II (MDCKII) parental cell line and MDCKII cells stably
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transduced for expression of human transporters P-gp, BCRP or MRP2 further designed as
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MDCK-MDR1, MDCK-BCRP or MDCK-MRP2, were provided by Dr. Alfred Schinkel (The
115
Netherlands Cancer Institute, Amsterdam, The Netherlands). All the MDCK cell lines were
116
cultured in DMEM medium supplemented with 10% heat-inactivated FBS. Double-
117
transfected MDCKII cell lines stably expressing human OCT1 or OCT2 and MATE1
118
transporters (MDCK-OCT1-MATE1 and MDCK-OCT2-MATE1), as well as the respective
5
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mono-transfected cells (MDCK-OCT1, MDCK-OCT2, MDCK-MATE1) and control vector
120
cells (MDCK-Co), were established and characterized as described previously (17, 21, 22).
121
Cells were cultured in MEM containing 10% heat-inactivated FBS. All cells used in our
122
experiments were routinely cultivated in antibiotic-free medium and periodically tested for
123
mycoplasma contamination. Stable expression of all inserted human transporters was verified
124
by qRT-PCR and uptake assays with appropriate fluorescence substrates. Cells from passages
125
10 to 25 were used in all in vitro studies.
126
127
Animals
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Pregnant Wistar rats were purchased from MediTox s.r.o. (Konarovice, Czech Republic) and
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maintained under 12/12-hours day/night standard conditions with pellets and water provided
130
ad libitum. Experiments were carried out on day 21 of gestation. Overnight-fasted rats were
131
anesthetized with pentobarbital (40 mg/kg body weight) administered into the tail vein. All
132
experiments were approved by the Ethical Committee of the Faculty of Pharmacy in Hradec
133
Kralove (Charles University in Prague, Czech Republic) and were performed in accordance
134
with the Guide for the Care and Use of Laboratory Animals (1996) and the European
135
Convention for the Protection of Vertebrate Animals Used for Experimental and Other
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Scientific Purposes (Strasbourg, France, 1986).
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Human tissue samples
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All human tissue samples were obtained following written informed consent with the
140
approval of the Faculty hospital Research Ethics Committee. Placentas were collected from
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uncomplicated pregnancies at term (38–40 weeks of gestation) delivered by Caesarean section
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in the Department of Obstetrics and Gynaecology University Hospital in Hradec Kralove.
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Human normal kidney samples were obtained after written informed consent from human
144
biopsy in the Hemodialysis Center, University Hospital in Hradec Kralove.
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Transcellular transport assays in ABC transporter overexpressing MCDK cells
147
Transport assays employing MDCK parental and ABC transporter-expressing MDCK cells
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were performed on microporous polycarbonate membrane filters (3.0 µm pore size, 24 mm
149
diameter; Transwell 3414; Costar, Corning, New York) as described previously (23, 24).
150
MDCK-MDR1, MDCK-BCRP, MDCK-MRP2 or MDCK-parent cells were seeded at a
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density of 1.5 x 106 per insert and cultured 3-4 days until reaching confluency with daily
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replacement of cell culture medium. Before starting the transport experiment, cells were
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washed with pre-warmed phosphate buffered saline (PBS) on both the apical and basal sides
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of monolayers and Opti-MEM with radiolabelled lamivudine was added to either the apical or
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basolateral compartments. The lowest [3H]-lamivudine concentration used was 8 nM, as this
156
concentration achieved the minimal specific activity required for analysis (0.04 µCi/ml). The
157
experiments were run at 37 °C / 5% CO2, aliquots of 50 µl were collected at 2, 4 and 6 hours
158
from the opposite compartment and radioactivity was measured by liquid scintillation
159
counting (Tri-Carb 2900 TR Perkin Elmer). At the end of the experiment, leakage of
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fluorescein isothiocyanate labelled dextran (MW = 40 kDa) was analyzed and accepted at up
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to a rate of 1% per hour. The percentage of radioactivity appearing in the acceptor
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compartment relative to the stock solution initially added to the donor compartment was
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calculated. Transport ratios between basal-to-apical and apical-to-basal translocation after 6
164
hours’ incubation (rt) were calculated as described earlier (23) by dividing the percentage of
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drug transported in the basolateral-to-apical direction by the percentage of drug crossing the
166
monolayer in the apical-to-basolateral direction.
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Transcellular transport assays in SLC transporter overexpressing MCDK cells
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Transport experiments employing mono-transfected MDCK-MATE1, MDCK-OCT1 and
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MDCK-OCT2 cells, double-transfected MDCK-OCT1-MATE1 and MDCK-OCT2-MATE1
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and the empty vector transfected control MDCK-Co cells were performed on Transwell 3402
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cell culture inserts (3.0 µm pore size, 24 mm diameter Costar, Corning, NY). For all
172
experiments, 0.5 x 106 cells per well were used and incubated for 3 days to confluence in
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standard cultivation medium MEM (Gibco) + 10% FBS. On the day of experiment, medium
174
was removed from both sides and the cellular monolayer was washed on both sides with pre-
175
warmed PBS. The experiment was started by addition of 0.8 ml HBSS buffer (pH 7.4) into the
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apical compartment and 0.8 ml of HBSS buffer (pH 7.4) with [3H]-lamivudine to the
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basolateral compartment. The [3H]-lamivudine concentration used was 100 nM, as this
178
concentration achieved the maximal sensitivity required for detection of interaction with drug
179
transporters and minimal specific activity required for final analysis in cell lysates. In the
180
inhibition experiments with mitoxantrone, the cellular monolayers were pre-incubated with
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HBSS medium (pH 7.4) containing 2 µM mitoxantrone in both compartments 10 minutes
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prior to initiation of the transport experiment. The experiments were run at 37 °C / 5% CO2
183
for 2 hours. Aliquots of 50 µl were sampled from the apical side at times 0.5, 1 and 2 hours.
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At the end of the incubation period, the medium was immediately removed and cells were
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washed twice with ice-cold PBS, then the inserts were excised and the cellular monolayer was
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dissolved in 0.02% SDS solution. The radioactivity of the collected samples and lysed
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monolayers was measured by liquid scintillation counting (Tri-Carb 2900 TR Perkin Elmer).
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Protein concentration in the cell lysates was quantified using a BCA assay. Net transport was
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obtained by subtraction of the transport by MDCK-Co cells from that by drug-transporter
190
overexpressing cells. The kinetics graph (transport velocity of lamivudine versus substrate
191
concentration) was fitted using the classic Michaelis-Menten equation, with Vmax representing
8
192
the maximal transport velocity (in nmol per mg of protein per minute) and Km representing
193
the substrate concentration at half-maximal transport velocity (micromolar), in GraphPad
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Prism 6 software (GraphPad Software, Inc., San Diego, California, USA).
195
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Quantitative RT-PCR
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The mRNA expression of MATE1 was quantified in the mono-transfected MDCK-MATE1
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cells, double-transfected MDCK-OCT1-MATE1 and MDCK-OCT2-MATE1 cell lines at the
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passages 10-25, in which the cells were used for transport experiments, in the human
200
placental villous tissue and in human kidney medulla used as the comparator sample due to
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the well confirmed expression of MATE1 (26). Total RNA was isolated from confluent
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monolayers of the cells or small pieces of fresh tissue samples using the TRI Reagent (Sigma-
203
Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Isolated RNA
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was dissolved in DEPC-treated water, and concentration and purity of each sample were
205
determined spectrophotometrically from A260/A280 measurements (NanoDrop, Thermo
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Scientific, Wilmington, DE, USA). Integrity of RNA was checked by agarose gel
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electrophoresis. cDNA was prepared from 1 μg extracted total RNA by MMLV reverse
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transcriptase using oligo(dT) VN nucleotides (gb Reverse Transcription Kit, Generi Biotech,
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Hradec Kralove, Czech Republic). PCR analysis was performed on QuantStudio 6 (Life
210
Technologies). cDNA (40 ng) was amplified using 2 × Probe Master Mix (Generi Biotech,
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Hradec Kralove, Czech Republic) and predesigned PCR assay (hSLC47A1_Q2) for SLC
212
transporter MATE1, Generi Biotech, Hradec Kralove, Czech Republic). All the samples were
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analysed three times in triplicate, Ct values were noted and averaged for each sample type.
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Since there was no other gene besides human MATE1 shared among the analysed samples we
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show the relevant Ct values and limit the comparison of the MATE1 expression to estimation
216
based on ΔCt to the comparator (kidney) and calculating the 2^ΔCt value.
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ASP+ uptake experiments
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Uptake experiments were performed using control MDCK-Co and OCT1-, OCT2- and
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MATE1- monotransfected MDCK cells in order to quantify the inhibitory effect of
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mitoxantrone to the particular SLC transporters. The aim of these experiments was to show
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that mitoxantrone could be used as a model inhibitor that selectively inhibits MATE1 in the
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subsequent transport studies using OCT-MATE double-transfected cells. Mitoxantrone was
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chosen as the model inhibitor because it has been shown to preferentially inhibit MATE1 over
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OCT1 and OCT2 according to the half-maximal inhibitory concentration (IC50) values in
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other cellular models (27). MATE1 is a pH-dependent carrier that is able to act as an uptake
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transporter in experimental settings (28) but mediates efflux in physiological ones. Hence,
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extracellular alkalinization was used to promote MATE1-mediated uptake (29, 30). The single
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SLC transporter-transfected MDCK cells were seeded on a 96-well plate at a density of 45 x
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103 cells per well and cultivated in standard cultivation medium (MEM + 10% FBS). Twenty-
231
four hours after seeding, uptake experiments with ASP+, a common fluorescence substrate of
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OCT1, OCT2 and MATE1, were performed. Cells were washed twice with 100 µl pre-
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warmed HBSS buffer (pH 7.4). Cell lines containing MATE1 were pre-incubated with 100 µl
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20 mM NH4Cl (pH 7.4) for 30 min. After washing the cells twice with 100 µl pre-warmed
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HBSS buffer (pH 8.0), solutions of mitoxantrone with 1µM ASP+ were added and incubated
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for 20 minutes. After washing the cells twice with 100 µl pre-warmed HBSS buffer pH 8.0,
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solutions of mitoxantrone with 1µM ASP+ were added for 20 minutes. At the end of the
238
incubation period, the medium was removed and cells were rinsed thrice with ice-cold HBSS
239
buffer (pH 7.4). Then, fluorescence was measured at a wavelength of 485 nm for excitation
240
and 585 nm for emission. Substrate uptake was normalized with to the protein concentration
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of the cell lysate measured by BCA assay.
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Dual perfusion of rat term placenta
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The method of dually perfused rat term placenta was used as described previously (31).
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Open-circuit perfusion system
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An open-circuit perfusion system was employed to study fetal-to-maternal (F→M) and
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maternal-to-fetal (M→F) lamivudine clearance. [3H]-lamivudine at 12 nM concentration was
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added to either the maternal (M→F studies) or fetal (F→M studies) reservoirs immediately
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after successful surgery. M→F transplacental clearance (Clmf) normalized to placenta weight
250
was calculated according to Equation. 1:
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𝐶𝑙mf = (𝐶fv . 𝑄f )/( 𝐶ma . 𝑊p )
(1)
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where Cfv is the drug concentration in the umbilical vein effluent [nmol/l], Qf is the umbilical
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flow rate [ml/min], Cma is concentration in the maternal reservoir [nmol/l], and Wp is the wet
254
weight of the placenta [g]. F→M transplacental clearance (Clfm) was calculated according to
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Equation 2.
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𝐶𝑙fm = (𝐶fa − 𝐶fv )𝑄f /(𝐶fa . 𝑊p )
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where Cfa is the drug concentration [nmol/l] in the fetal reservoir entering the perfused
258
placenta via the umbilical artery.
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Closed-circuit (recirculation) perfusion system
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A closed circuit (recirculation) perfusion system was employed to study the effect of pH on
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the fetal/maternal lamivudine concentration ratio at equilibrium. Both the maternal and fetal
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sides of the placenta were infused with 9 nM [3H]-lamivudine and after a short-time
263
stabilization period, the fetal perfusate (10 ml) was recirculated for 60 minutes. In the fetal
(2)
11
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recirculating reservoir, a pH of 7.4 was maintained throughout the experiments, whereas the
265
pH in the maternal reservoir was adjusted by HCl/NaOH to 6.5, 7.4 or 8.5. Samples (250 µl)
266
were collected every 10 minutes from the maternal and fetal reservoirs and the
267
[3H]-lamivudine concentration was measured. This experimental setup ensured a steady
268
concentration on the maternal side of the placenta and enabled investigation of the
269
fetal/maternal ratio; any net transfer of the substrate would imply transfer against a
270
concentration gradient and provide evidence of active transport.
271
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Uptake assay in human placental microvillous plasma membrane vesicles
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MVM vesicles were isolated from human term placentas using Mg2+ precipitation and
274
differential centrifugation as described previously (32). The final MVM pellet was
275
resuspended in an intravesicular buffer (IVB) at two different pH values adjusted by changing
276
the HEPES to Tris ratio (IVB 7.4: 290 mM sucrose, 5 mM HEPES and 5 mM Tris, pH 7.4 or
277
IVB 6.2: 290 mM sucrose, 9.5 mM HEPES and 0.5 mM Tris, pH 6.2), vesiculated by passing
278
15 times through a 25-gauge needle and stored at -70 °C until use in the uptake experiments.
279
MVM protein concentration was determined using the BCA assay and purity was confirmed
280
by measuring the enrichment of MVM alkaline phosphatase activity compared with the
281
placental homogenate. The alkaline phosphatase enrichment factor was 21.8 ± 5.6 (mean ±
282
SD, n = 7).
283
284
Uptake of [3H]-lamivudine into MVM vesicles was measured at room temperature using rapid
285
vacuum filtration (33). MVM vesicles (10 mg protein/ml) were equilibrated to room
286
temperature (21–25 °C) prior to uptake. Uptake of [3H]-lamivudine was initiated by mixing
287
10 µl MVM vesicles with 10 µl 100 nM [3H]-lamivudine in extravesicular buffer (EVB) at pH
12
288
7.4 or 8.4 (145 mM KCl, 10 mM Na+-HEPES/HCl-Tris, pH 7.4 or IVB 8.4). After 1 minute,
289
uptake was stopped by addition of 2 ml ice-cold stop buffer (130 mM NaCl, 10 mM
290
Na2HPO4, 4.2 mM KCl, 1.2 mM MgSO4, and 0.75 mM CaCl2, pH 7.4) and filtered through a
291
0.45 µM mixed cellulose ester filter (MF-Millipore membrane filter HAWP02500) under
292
vacuum. Filters were washed with 10 ml of stop buffer and the filter-associated radioactivity
293
was determined by liquid scintillation counting. No protein controls (replacement of MVM
294
vesicle protein by relevant IVB) were included in parallel to determine tracer binding to the
295
filter, which was subtracted from the total vesicle count.
296
297
Statistical analysis
298
All data were assessed and statistically analyzed using GraphPad Prism 6.0 software
299
(GraphPad Software, Inc., San Diego, California, USA). Statistical significance was
300
investigated using Student’s t-test, the Kruskal-Wallis test or two-way ANOVA followed by
301
Bonferroni’s multiple comparison post-test as applicable and described in the figure legends.
302
A P value ≤ 0.05 was taken to be statistically significant. Data are presented as the mean ±
303
standard deviation (SD) or with the 95% confidence interval (CI) where appropriate. Half-
304
maximal inhibitory concentration (IC50) values for mitoxantrone were calculated by non-
305
linear regression using sigmoidal Hill kinetics using GraphPad Prism 6.0 software.
306
307
RESULTS
308
Transcellular transport of lamivudine across ABC transporter expressing MDCK
309
monolayers
310
Transcellular transport of [3H]-lamivudine (8 nM) across the polarized monolayers of MDCK
311
parental and MDR1-, BCRP- and MRP2-overexpressing cells was studied using the
13
312
conventional bi-directional (concentration gradient) transport assay. No difference between
313
basal to apical (BA) and apical to basal (AB) transfer of the drug was observed at time points
314
2, 4 hours and transport ratios (rt) close to 1 were observed after 6 hours of transport
315
(Table 1). The rt value obtained in MDCK-BCRP cells (1.25 ± 0.0277) was significantly
316
higher than that measured in MDCK-parent cells (0.923 ± 0.195). However, the cut-off value
317
for BCRP substrates (rt ≥ 2) set by the International Transporter Consortium was not reached
318
(34). These data indicate that lamivudine is not a substrate of P-gp or MRP2, whereas BCRP
319
might to some extent be responsible for acceleration of lamivudine transport in the BA
320
direction.
321
322
Expression of MATE1 mRNA in the relevant MDCK cell lines and in human placentas
323
In order to confirm the expression of MATE1 mRNA in the cell lines and human placentas
324
used in the subsequent functional studies, RT-PCR approach was employed. Since there was
325
no other gene shared among the analysed samples we limited the comparison of the
326
expression to showing the mean Ct values (Table 2). The MATE1 expression was confirmed
327
in MDCK-MATE1 and MDCK-OCT2-MATE1 cell lines with the same level of MATE1
328
transcripts (Ct values), while lower level (higher average Ct) was detected in MDCK-OCT1-
329
MATE1 cells (Table 2). Nevertheless, the expressions in all the cell lines exceeded
330
significantly that of kidney mainly due to the lack of non-MATE1 expressing cells that are
331
present in the whole tissue samples. MATE1 expression in human placental villous tissue was
332
highly variable and was found only in 7 out of 10 analysed human placentas. The average Ct
333
for the MATE1-expressing placentas was about 10 cycles higher (Table 2) than that in kidney
334
medulla tissue indicating approximately 2^10 = 1000 times lower MATE1 expression.
335
14
336
MATE1-mediated transfer of lamivudine across cellular monolayers
337
After addition of 100 nM lamivudine to the basolateral compartment, all three MATE1
338
expressing cell lines, i.e. MDCK-MATE1, MDCK-OCT1-MATE1 and MDCK-OCT2-
339
MATE1, showed significantly higher transcellular transfer of [3H]-lamivudine from the basal
340
to the apical compartment compared with MDCK-Co cells (increase to 262.4% , 196.8% and
341
250.9%, respectively, P ≤ 0.01, Student´s t-test) and their respective non-MATE1 expressing
342
control cells (MDCK-Co, MDCK-OCT1 and MDCK-OCT2, respectively, Fig. 1 A,B,C). The
343
rate of lamivudine transcellular transport was lowest in the MDCK-OCT1/MATE1
344
monolayers among MATE1-expressing cells, in agreement with the lowest gene expression of
345
hMATE1 in this cell line shown by qRT-PCR (Table 2).
346
Intracellular concentrations of lamivudine in the monolayers of MDCK–MATE1, MDCK-
347
OCT1-MATE1 and MDCK-OCT2-MATE1 cells were significantly lower than in the
348
respective MATE1-free control cell lines (decrease to 7.31%, 62.1% and 24.6% compared to
349
MDCK-Co, MDCK-OCT1 and MDCK-OCT2, respectively, P < 0.001, Student´s t-test, Fig. 1
350
D,E,F). As expected, monolayers of MDCK-OCT1 and MDCK-OCT2 in the transport
351
experiment accumulated significantly higher amount of lamivudine compared to MDCK-Co
352
cells (P < 0.05, Student´s t-test). Interestingly, the transcellular transfer of lamivudine across
353
MDCK-MATE1 cells was not significantly different from that in double-transfected MDCK-
354
OCT2-MATE1 cells that express the same level of hMATE1 mRNA (Table 2) but
355
accumulated a higher amount of lamivudine due to OCT2-mediated uptake (Fig 2 D, F).
356
357
The apparent affinity of lamivudine to the MATE1 transporter was further evaluated by
358
transport assays in MDCK-MATE1 monolayers using lamivudine concentrations ranging
359
from 100 nM to 10 mM. The Km value for transfer of lamivudine across MDCK-MATE1
15
360
monolayers was 4213 ± 558.2 µM (Fig. 2), indicating that MATE1-mediated transfer of
361
lamivudine is a low affinity process.
362
363
Mitoxantrone-mediated MATE1 inhibition of lamivudine transport
364
To further test the contribution of MATE1 to the transcellular transfer of lamivudine, a potent
365
inhibitor of MATE1, mitoxantrone, was employed. First, ASP+ uptake was investigated in
366
MDCK-OCT1, MDCK-OCT2 and MDCK-MATE1 cells to assess the inhibitory potency of
367
mitoxantrone to the different SLC transporters (accumulation buffer of pH 7.4 was used in
368
both OCT-expressing cells, whereas pH 8.4 was used for the MDCK-MATE1 cells to reverse
369
the direction of the ASP+ transport to uptake). When comparing IC50 values, the observed
370
selectivity for MATE1 inhibition was 4.8 and 9.1 times higher than that for OCT1 and OCT2
371
(P < 0.001), respectively (Fig. 3A). Based on these results and the dose dependency inhibition
372
curve of ASP+ uptake, 2 µM mitoxantrone was chosen for the subsequent transport assay to
373
predominantly inhibit MATE1 over OCTs in MATE1/OCT transporters expressing cellular
374
monolayers (Fig. 3B,C).
375
Addition of mitoxantrone (2 µM) to the cellular monolayers reduced the basolateral to apical
376
transport of lamivudine in all the MATE1 expressing cells (MDCK-MATE1, MDCK-OCT1-
377
MATE1 and MDCK-OCT2-MATE1) to the level of MDCK-Co control cell line (P < 0.001,
378
two-way ANOVA, Fig. 3B) and showed no statistically significant difference in lamivudine
379
transport among the mitoxantrone inhibited cell lines. No effect of mitoxantrone on the
380
transcellular transport across MDCK-OCT1 and MDCK-OCT2 monolayers was observed
381
(data not shown). Consistent with these data, addition of mitoxantrone significantly increased
382
lamivudine accumulation in the monolayers of MATE1-, OCT1-MATE1- and OCT2-MATE1
16
383
expressing cells (P < 0.01, two-way ANOVA), whereas no increase was observed in MDCK-
384
Co cells (Fig. 3C).
385
386
Lamivudine transport across the perfused rat placenta; effect of pH
387
The ratio between F→M and M→F clearances found in the perfusion studies, in which [3H]-
388
lamivudine ( 12 nM) was applied from maternal or fetal side of the placenta was 1.8,
389
indicating possible active transplacental transport of lamivudine from the fetal to maternal
390
side. Nevertheless, the differences between M→F and F→M clearances did not reach
391
statistical significance (P = 0.096, Student´s t-test). Less than 1% of the lamivudine dose was
392
detected in the placenta after the perfusion experiments, suggesting limited tissue binding or
393
accumulation in trophoblasts and negligible effect on the clearance calculation. To further
394
study lamivudine transplacental transport, both sides of the placenta were perfused with the
395
same concentration of [3H]-lamivudine (12 nM) in a closed circuit experimental setup. We
396
observed a slight decrease of lamivudine concentration in the fetal perfusate, achieving 95.3 ±
397
2.46 % of the initial concentrations over 60 min perfusion. This decline might indicate a small
398
contribution of an active transport against the concentration gradient from the fetal to the
399
maternal side of the placenta. To study the effect of pH on lamivudine fetal-to-maternal
400
transport, which could reflect involvement of MATE1-mediated efflux, the pH in the maternal
401
reservoir was set to 6.5 or 8.5, while the pH in the fetal reservoir was set to 7.4. The fetal
402
lamivudine concentration changed significantly between the experiments with pH 6.5 and 8.5
403
(Fig. 4), suggesting involvement of proton-dependent transport of lamivudine across the
404
placenta.
405
17
406
Uptake of [3H]-lamivudine into human placenta MVM vesicles: effect of pH
407
To determine the relevance of MATE1-mediated transport for the transplacental transfer of
408
lamivudine in human placenta, and, more specifically, MATE1 involvement in the transport
409
of lamivudine across the MVM of human placenta, uptake of lamivudine into MVM vesicles
410
isolated from human term placenta of uncomplicated pregnancies was measured. The
411
lamivudine accumulation into the vesicles was stimulated by a higher extravesicular pH.
412
However, the magnitude of the increase in response to the imposed outwardly directed proton
413
gradient was rather variable between MVM vesicle isolates and reached the statistical
414
significance only with the pH gradient of 2.2 units (pH 6.2-8.4), but not with the 1 unit (pH
415
7.4-8.4) gradient (Fig. 5).
416
417
DISCUSSION
418
Lamivudine is considered a first line antiretroviral drug to prevent MTCT in HIV-positive
419
pregnant women (35). The concentrations of lamivudine found in cord blood at delivery have
420
been reported to reach maternal levels, suggesting that the drug can freely cross the placenta
421
by passive diffusion (36, 37). Nevertheless, a fetal-to-maternal area under the concentration-
422
time curve (AUC) ratio of 0.86 (6) indicates that transplacental crossing of lamivudine is not
423
entirely passive. One explanation is that a fetal-to-maternal directed transport mechanism is
424
involved which diminishes the transplacental passage of lamivudine from mother to fetus.
425
Such information would be of considerable importance for optimizing the pharmacotherapy of
426
pregnant women because placental drug transporters could be involved in pharmacokinetic
427
drug-drug interactions (DDI) affecting fetal drug exposure of concomitantly administered
428
drugs and leading to impaired treatment outcome or adverse effects (7, 38). This issue is of
18
429
particular importance in anti-HIV therapy, where combination of two or more antiretroviral
430
drugs is recommended (35).
431
Several drug efflux transporters are functionally expressed in the apical microvillous plasma
432
membrane of human syncytiotrophoblast (8-10). In the present project, we aimed to assess
433
whether lamivudine is a substrate of placental drug transport proteins and address whether
434
transplacental transfer of lamivudine is affected by drug efflux transporters.
435
Interaction of lamivudine with P-gp was evaluated by de Souza et al (40), using transport
436
assay in P-gp expressing monolayers. Based on the lack of interaction of lamivudine with a
437
potent P-gp inhibitor (GG 918), the authors concluded that P-gp does not significantly affect
438
the transport of lamivudine (40). Correspondingly, we did not observe any P-gp-accelerated
439
transport of lamivudine (8 nM) across the MDCK-MDR1 monolayers, confirming the lack of
440
relevant P-gp involvement in lamivudine transport.
441
Transport of lamivudine by BCRP was suggested by Kim et al., who observed decreased
442
lamivudine uptake in MDCK-BCRP cells and saturable lamivudine transport across BCRP
443
expressing monolayers in a 60 minutes-experiment (41). Nevertheless, functionally relevant
444
polymorphic variants of BCRP had no effect on lamivudine disposition in healthy volunteers,
445
indicating that BCRP is unlikely to make a relevant contribution to lamivudine
446
pharmacokinetics. To further address this issue, we performed transport study with a low
447
lamivudine concentration in MDCK-BCRP cells in a bidirectional concentration gradient
448
setup. The lamivudine transport ratio (rt) in MCDK-BCRP significantly exceeded that in the
449
parental MDCK cells (Table 1), however, it did not reach the value of 2, which is considered
450
by the International Transporter Consortium guidelines as a cut-off level for classifying a drug
451
as a transporter substrate (34). We therefore hypothesize that lamivudine might be only very
452
low affinity BCRP substrate questioning the transport of lamivudine by BCRP observed by
19
453
Kim et al in a short time set up assay (41). Similarly to the results in MDCK-MDR1 and
454
MDCK-BCRP cells, no active transporter-driven transfer of lamivudine was observed in
455
MDCK-MRP2 cells, showing that lamivudine is not significantly transported by any of the
456
three main placental ABC transporters.
457
In situ dually perfused rat term placenta represents a valuable alternative model to study
458
placental pharmacology and physiology. Using this approach, we have previously identified
459
ABC transporter-mediated placental passage of several compounds, including antiretrovirals
460
(23, 24, 31, 43). In the present study, we showed that the fetal-to-maternal clearance of
461
lamivudine was not significantly higher than the maternal-to-fetal clearance and that the
462
decrease of lamivudine concentration in fetal compartment during recirculation experiment
463
was insignificant (Fig. 4). These observations are in agreement with the above-discussed in
464
vitro transport experiments and indicate no, or only minor involvement of active transporter-
465
mediated fetus-to-mother directed efflux of lamivudine.
466
Lamivudine has recently been shown as a substrate of human MATE1 and the kidney-specific
467
MATE2-K transporters and MATEs-mediated pH-dependent efflux was suggested to
468
contribute to renal tubular excretion of lamivudine (17). This antiretroviral has also been
469
shown to be a substrate of all three subtypes of human organic cation transporter, with
470
efficacy dependent on kinetic parameters and Vmax/Km ratio decreasing in the following order:
471
OCT1 > OCT2 > OCT3 (18, 19). In the present study, we evaluated the affinity of lamivudine
472
to the MATE1 transporter and aimed to address in detail the role of OCTs in the transcellular
473
transfer of lamivudine using double-transfected MDCK-OCT1-MATE1 and MDCK-
474
OCT2/MATE2 cell lines and relevant mono-transfected and vector control MDCK cells. By
475
applying low concentrations of lamivudine to increase the sensitivity of the assay, we
476
confirmed that MATE1 significantly increases the transcellular passage of lamivudine whilst
477
decreasing intracellular accumulation of the drug (Fig. 1). Monolayers of OCT1- and OCT220
478
single-transfected cells accumulated higher levels of lamivudine but retained a similar
479
transcellular transport efficacy to that in vector control cells, confirming that OCT
480
transporters affect the influx of lamivudine into the cells. In agreement with previous
481
observations with 10 µM lamivudine (17), lamivudine transfer across MDCK-OCT2-MATE1
482
monolayers did not differ significantly from that in MDCK-MATE1 cells expressing the same
483
level of MATE1 mRNA (Table 2). Mitoxantrone used at a concentration preferentially
484
inhibiting MATE1 over OCT1 and OCT2 (2 µM) significantly decreased the lamivudine
485
transcellular transfer in all the MATE1 expressing cells to the level of control cells but
486
increased the cellular accumulation in all the MATE1-expressing cell lines (Fig. 3). Our data
487
thereby suggest that lamivudine transcellular transfer is controlled mainly by MATE1-
488
mediated efflux and is not significantly affected by OCT-mediated uptake.
489
Kinetic analysis of the MATE1-mediated transport of lamivudine revealed a rather low
490
affinity and transport capacity of MATE1 to the antiretroviral (Km = 4.21 mM, Vmax = 5.18
491
nmol/mg protein/min). The transport efficacies of OCT1 and OCT2 transporters to
492
lamivudine have been reported as Vmax/Km (OCT1) = 8.03 (18) or 8.0(19) µl/mg protein/min and
493
Vmax/Km (OCT2) = 4.1 (18) or 4.4(19) µl/mg protein/min, respectively, exceeding the efficacy of
494
MATE1 in the transport of lamivudine found in the present study (Vmax/Km = 1.23 µl/mg
495
protein/min). We therefore suggest that MATE1-mediated efflux might be the rate-limiting
496
step in the transcellular transfer of lamivudine, which is consistent with the results of our
497
transport studies showing that the transport rate of lamivudine across MDCK-MATE1 cells
498
did not differ from that across MDCK-OCT2-MATE1 cells (Results 3.1). Nevertheless, the in
499
vitro determined Km values do not necessary preclude kinetic impacts of the transporter in
500
vivo. For instance, some drugs of high Km values determined in vitro play a significant role in
501
MATE1 excretory pathways in vivo (44).
21
502
Moreover, the concentration of half maximal velocity lamivudine transport by MATE1 is
503
about three orders of magnitude higher than the maximal therapeutic concentration (≈ 3-11
504
µM) achieved in the plasma of pregnant women (37, 45), thus making saturation of MATE1
505
in vivo unlikely.
506
The involvement of pH-dependent transport in the transplacental pharmacokinetics of
507
lamivudine was further evaluated in situ by employing dually perfused rat term placenta.
508
Whereas expression of Mate1 appears to be absent in murine placenta (46, 47) abundant
509
placental expression has been found in rats (12, 14, 15) and the transporter was found to
510
mediate the pH-dependent fetal-to-maternal transfer of MPP+ (12) and metformin (16) in the
511
perfused rat placenta(12, 14). Using the same placental model the ratio between F→M and
512
M→F clearances of 12 nM lamivudine reached the value of 1.8, indicated possible active
513
transplacental transport of lamivudine from the fetal to maternal side. However, this value
514
was much lower than that measured for 10 nM MPP+ (123) and 100 nM metformin (7.3) in
515
previous studies (12, 16) and did not reach statistical significance. We observed that
516
decreasing the maternal pH increased the fetal-to-maternal transfer of lamivudine in the
517
closed circuit setup, indicating involvement of a pH-dependent transport mechanism on the
518
maternal facing side of trophoblasts in the transplacental transfer of lamivudine.
519
In contrast to rat, human placenta seems to express only low and highly variable levels of
520
hMATE1 mRNA (12, 14). Accordingly, we observed hMATE1 mRNA expression only in 7
521
of 10 analyzed placentas showing the level of MATE1 mRNA transcripts about 1000 times
522
lower when compared with human kidney medulla tissue (Table 2). To address the relevance
523
of MATE1 for transplacental transfer of lamivudine in humans, we additionally evaluated the
524
pH-dependent transport of lamivudine directly on microvillous plasma membrane (MVM)
525
vesicles isolated from the MATE1-expressing human term placental trophoblast. The uptake
526
of lamivudine across MVM appears to be sensitive to the imposed H+ gradient, suggesting
22
527
that MATE1-mediated H+/lamivudine exchange contribute to the transfer of lamivudine
528
across human placenta. However, the results were variable and achieved statistical
529
significance only with higher pH gradient (2.2 pH units) but not with lower pH gradient (1 pH
530
unit). It is possible that temporal dissipation of the H+ gradient across the MVM plasma
531
membrane contributed to the variability in the capacity of MATE1 to accumulate lamivudine
532
as substrate (32). The imposition of a steeper transmembrane H+ gradient across the MVM
533
plasma membrane may help clarify this further.
534
The maternal-fetal interface does not offer such a steep pH gradient as proximal tubules in the
535
kidney favouring the MATE1-mediated efflux, but it cannot be excluded that MATE1-
536
mediated transport in the placenta is linked also to another H+ transferring transporter(s)
537
providing a sufficient H+ ion gradient to drive the efflux such as Na+/H+ exchanger and ATP-
538
dependent H+ pump that were found functionally expressed in human placenta (48-50). It has
539
been also suggested that MATE-driven organic cation excretion may occur independently of a
540
pH gradient across the brush-border membrane (51) supporting the possible physiological role
541
of MATE1 in the placenta. Nevertheless, contribution of other pH-dependent transport
542
mechanism, such as MATE2 (13) in transfer of lamivudine across the MVM cannot be
543
excluded. In addition, higher portion of unionized form of lamivudine available for passive
544
diffusion into the vesicles provided by the pH gradient should be also taken into account. We
545
therefore suggest that MATE1 might represent one of the mechanisms mediating the
546
maternal-to-fetal transfer of lamivudine in human placenta; however its contribution to this
547
transfer is probably rather limited. The low impact of the MATE1 efflux transporters in the
548
transplacental transfer of lamivudine may be also caused by high transplacental passage of
549
lamivudine, driven by passive diffusion or any concentration gradient-driven mechanisms,
550
such nucleoside transport proteins (52-54). Nevertheless, since the human placenta tends to
551
express higher levels of MATE1 in the first trimester compared with term (13) it is feasible to
23
552
hypothesize that the MATE1-mediated transplacental transport of lamivudine might be of
553
higher importance in earlier phases of pregnancy.
554
To conclude, we have shown here that P-gp, MRP2 and BCRP do not affect the transplacental
555
transfer of lamivudine, making the risk of pharmacokinetic DDI between lamivudine and
556
other antiretroviral substrates of the ABC transporters in the placenta unlikely. On the other
557
hand, we demonstrated a low affinity efflux of lamivudine by MATE1, which seems to act
558
independently of the OCT-mediated cellular uptake of lamivudine. We further showed that a
559
pH-dependent mechanism mediates transport of lamivudine in the fetal-to-maternal direction
560
in rat as well as human placenta and concluded that MATE1 might be at least partly
561
responsible for this transport. However, further research is needed to address the role MATE1
562
may play in human placenta during pregnancy and to elucidate the risk of MATE1-mediated
563
DDI in the transplacental pharmacokinetics of lamivudine in humans.
564
565
ACKNOWLEDGEMENT
566
We wish to thank Dana Souckova and Renata Exnarova for their skilful assistance with the
567
perfusion experiments and Martina Hudeckova for her help with the collection and sampling
568
of human placentas. We also thank Dr. Roman Safranek, Ph.D. for providing us with the
569
kidney biopsy samples.
570
571
FUNDING INFORMATION
572
This work was supported by the Czech Science Foundation (Projects GACR P303-120850
573
and GACR 13-31118P) and the Grant Agency of Charles University in Prague
574
(SVV/2016/260 293).
575
24
576
TRANSPARENCY DECLARATIONS
577
FM holds a minor share of stock in Novartis, received research funding from Sanofi-Aventis
578
Deutschland and is an employee of Boehringer Ingelheim Pharma GmbH & Co. KG.
579
580
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FIGURE LEGENDS AND TABLES
808
30
809
Figure 1. Transport of lamivudine in single-transfected MDCK cells overexpressing OCT1,
810
OCT2 and MATE1, double-transfected MDCK-cells overexpressing OCT1 or OCT2 and
811
MATE1 (MDCK-OCT1-MATE1, MDCK-OCT2-MATE1) and vector control cells MDCK-
812
Co. Cells were seeded on Transwell semipermeable supports dividing a basal compartment
813
and an apical compartment. Lamivudine (100 nM) was added to the basal compartment and
814
sampled at time points 0.5, 1 and 2 h from the apical side of monolayers (A, B, C).
815
Intracellular accumulation of lamivudine in the monolayers was determined in cell lysates at
816
the end of the experiment (D, E, F). Data were analyzed by Student´s t-test (***P < 0.001 vs.
817
respective control, Co, OCT1 or OCT2) and are shown as means ± SD (n ≥ 3).
818
819
820
Figure 2. Concentration-dependent net transcellular transport of lamivudine by MATE1.
821
Basolateral-to-apical transport of lamivudine across MDCK-MATE1 and MDCK-Co cells
822
cultured as monolayers on Transwell membranes was investigated for increasing
823
concentrations of non-radiolabelled lamivudine (1 x 10-4; 1 x 10-3; 0.01; 0.1; 1.0; 2.0; 5.0 and
824
10 mM) with addition of tracer [3H]-lamivudine (16.7 nM) applied to the basolateral
825
compartment. The transcellular transport of lamivudine across MDCK-Co monolayers was
826
subtracted from that in MDCK-MATE1 cells at each concentration point. Kinetic parameters
827
(Km and Vmax) were estimated by fitting MATE-specific transport rates to a Michaelis-Menten
31
828
non-linear equation. Data (nmol/mg protein/min) represent the mean ± SD from three
829
independent experiments.
830
831
832
Figure 3. Inhibitory effect of mitoxantrone on lamivudine transport by MATE1. A: IC50
833
values reflecting inhibition of ASP+ uptake into OCT1-, OCT2-, and MATE1-expressing
834
MDCK cells by mitoxantrone. The IC50 values with 95% confidential interval were calculated
835
from three independent measurements. B, C: Effect of 2 µM mitoxantrone on transcellular
836
transport (B) and intracellular accumulation (C) of lamivudine (100 nM) in monolayers of
837
MATE1-expressing and control cells. Data were analyzed by two-way ANOVA with multiple
838
comparisons (**P < 0.01, ***P < 0.001 vs. respective non-inhibited controls) and are shown
839
as mean ± SD (n ≥ 3).
840
841
Figure 4. Effect of maternal pH on elimination of lamivudine from the fetal circulation. In the
842
closed-circuit perfusion setup, both the fetal and maternal sides of the placenta were
843
simultaneously infused with 12 nM [3H]-lamivudine. The fetal pH was set to 7.4, whereas the
32
844
pH in the maternal reservoir was set to 6.5, 7.4 or 8.5. The fetal perfusate was recirculated for
845
60 min and then, fetal and maternal concentrations of lamivudine were compared. A:
846
Lamivudine fetal concentration over 60 min perfusion at pH 6.5 and 8.5 applied on the
847
maternal site. B: Final ratio between fetal and maternal concentrations at equilibrium showing
848
statistically significant difference between ratios calculated for perfusions at pH 6.5 and pH
849
8.5 (P < 0.05; Kruskal-Wallis test), suggesting involvement of a proton-cation antiporter
850
system in lamivudine transplacental transport. Data are presented as means ± SD (n ≥ 3).
851
852
Figure 5. Effect of H+ gradient on [3H]-lamivudine uptake by MVM vesicles from human
853
term placentas. MVM vesicles were prepared in intravesicular buffer (IVB) at pH 6.2 or 7.4.
854
A: One minute uptake of [3H]-lamivudine was examined in extravesicular buffer (EVB)
855
containing 100 nM [3H]-lamivudine at pH 7.4 or 8.4. B: Paired measurements for pH 6.2 IVB
856
MVM vesicles showing stimulation of [3H]-lamivudine uptake in the presence of an increased
857
pH gradient (change = 422 ± 276%, mean ± SD, n=6; P=0.044, paired t-test). Data are
858
presented as means ± SD of experiments obtained from 5-7 placentas.
859
33
860
Table 1. Lamivudine transport across monolayers of MDCK parent and human MDR1-,
861
BCRP- and MRP2-transporter expressing MDCK cells
Cell line
Transport ratio (rt)
(basal-to-apical/apical-to-basal)
MDCK parent
0.923 ± 0.195
MDCK-MDR1
0.930 ± 0.140
MDCK-BCRP
1.25* ± 0.0277
MDCK-MRP2
1.03 ± 0.131
862
Cells were seeded on Transwell semipermeable supports and grown as monolayers dividing,
863
similarly to a polarized trophoblast layer, the basal compartment (corresponding to fetal side)
864
and apical compartments (corresponding to maternal compartment). The transport ratios (rt)
865
for translocation of lamivudine (8 nM) across monolayers of ABC-transporter expressing cells
866
and parent MDCK cells after 6 h are shown. Data are presented as means ± SD of ratios
867
obtained in at least four independent experiments (n ≥ 4). Student´s t-test was employed to
868
compare the ratios of particular ABC-transporter expressing cells to the respective ratio in
869
MDCK parent control cells and results were considered statistically significant when P ≤
870
0.05(*).
871
872
Table 2. qRT-PCR for human MATE1 (SLC47A1) mRNA expression in MATE1 transfected
873
MDCK cell lines, placenta and kidney
MDCK-MATE1
Ct
17.4 ± 0.277
MDCK-OCT1/MATE1
21.3 ± 0.364
MDCK-OCT2/MATE1
MDCK-Co
17.3 ± 0.294
ND
Human placenta
Human kidney
33.4 ± 5.86
23.6 ± 1.00
874
RNA isolated from cells and tissues was reverse-transcribed and resulting cDNA (40 ng) was
875
quantified for expression of hMATE1 (SLC47A1) using the QuantStudio 6 detector. Mean Ct
876
values of two independent experiments performed in triplicate for each sample are shown for
34
877
cell lines, human term placenta villous tissue (n = 7) and human kidney medulla samples (n =
878
2). ND no expression detected.
879
880
881
882
35