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. 2011 Jun;163(3):546-55.
doi: 10.1111/j.1476-5381.2010.01052.x.

Double-transfected MDCK cells expressing human OCT1/MATE1 or OCT2/MATE1: determinants of uptake and transcellular translocation of organic cations

J König  1 , O Zolk, K Singer, C Hoffmann, M F Fromm
Affiliations

Double-transfected MDCK cells expressing human OCT1/MATE1 or OCT2/MATE1: determinants of uptake and transcellular translocation of organic cations

J König et al. Br J Pharmacol. 2011 Jun.

Abstract

Background and purpose: The organic cation transporters 1 (OCT1) and 2 (OCT2) mediate drug uptake into hepatocytes and renal proximal tubular cells, respectively. Multidrug and toxin extrusion protein 1 (MATE1) is a major component of subsequent export into bile and urine. However, the functional interaction of OCTs and MATE1 for uptake and transcellular transport of the oral antidiabetic drug metformin or of the cation 1-methyl-4-phenylpyridinium (MPP(+)) has not fully been characterized.

Experimental approach: Single-transfected Madin-Darby canine kidney (MDCK) cells as well as double-transfected MDCK-OCT1-MATE1 and -OCT2-MATE1 cells were used to study metformin and MPP(+) uptake into and transcellular transport across cell monolayers, along with their concentration and pH dependence.

Key results: Cellular accumulation of MPP(+) and metformin was significantly reduced by 31% and 46% in MDCK-MATE1 single-transfected cells compared with MDCK control cells (10 µM; P < 0.01). Over a wide concentration range (10-2500 µM) metformin transcellular transport from the basal into the apical compartment was significantly higher in the double-transfected cells compared with the MDCK control and MDCK-MATE1 monolayers. This process was not saturated up to metformin concentrations of 2500 µM. In MDCK-OCT2-MATE1 cells basal to apical MPP(+) and metformin transcellular translocation decreased with increasing pH from 6.0 to 7.5.

Conclusions and implications: Our data demonstrate functional interplay between OCT1/OCT2-mediated uptake and efflux by MATE1. Moreover, MATE1 function in human kidney might be modified by changes in luminal pH values.

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Figures

Figure 1
Figure 1
A. RT-PCR analysis of SLC22A1 (encoding OCT1), SLC22A2 (encoding OCT2) and SLC47A1 (encoding MATE1) mRNA expression in control cells (MDCK-Co), single- (MDCK-OCT1, MDCK-OCT2, MDCK-MATE1) and double-transfected (MDCK-OCT1-MATE1, MDCK-OCT2-MATE1) cell lines, used in this study, and in human liver and kidney. B. Immunofluorescence of hMATE1 in MDCK-OCT1-MATE1 and MDCK-OCT2-MATE1 cells. hMATE1 is localized to the apical membrane of both double-transfected cell lines. No positive staining was observed in MDCK control cells (MDCK-Co; data not shown).
Figure 2
Figure 2
MPP+ (10 and 50 µM) was administered to the basal side of monolayers of MDCK-control (Co), MDCK-OCT1 (OCT1), MDCK-OCT2 (OCT2), MDCK-MATE1 (MATE1), MDCK-OCT1-MATE1 (OCT1-MATE1) and MDCK-OCT2-MATE1 (OCT2-MATE2) cells. Uptake of MPP+ into the cells (A, B) and transcellular transport of MPP+ from the basal to the apical compartment after 60 min (C, D) are shown. Data are shown as mean value ± standard deviation. ***P < 0.001 versus MDCK-control; #P < 0.05, ###P < 0.001 versus MDCK-OCT1-MATE1; §§P < 0.01, §§§P < 0.001 versus MDCK-OCT2-MATE1 cells.
Figure 3
Figure 3
Metformin (10 and 50 µM) was administered to the basal side of monolayers of MDCK-control (Co), MDCK-OCT1 (OCT1), MDCK-OCT2 (OCT2), MDCK-MATE1 (MATE1), MDCK-OCT1-MATE1 (OCT1-MATE1) and MDCK-OCT2-MATE1 (OCT2-MATE1) cells. Uptake of metformin into the cells (A, B) and translocation of metformin from the basal to the apical compartment after 60 min (C, D) are shown. Data are shown as mean value ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001 versus MDCK-control; ###P < 0.001 versus MDCK-OCT1-MATE1; §§P < 0.01, §§§P < 0.001 versus MDCK-OCT2-MATE1 cells.
Figure 4
Figure 4
Concentration-dependent uptake (A) and transcellular transport (B) of metformin in monolayers of MDCK-control (MDCK-Co), MDCK-MATE1, MDCK-OCT1-MATE1 and MDCK-OCT2-MATE1 cells. (C) Net metformin transcellular transport calculated from the differences in basal to apical transport in the double-transfected cells minus the corresponding transport in the vector control cells. Metformin (10, 25, 50, 100, 250, 500, 1000, 2500 µM) was administered to the basal side of the monolayers and uptake and transcellular transport into the apical compartment were determined after 60 min. Data are shown as mean value ± standard deviation. *P < 0.05, ***P < 0.001 versus MDCK-control; ##P < 0.01, ###P < 0.001 versus MDCK-MATE1; §P < 0.05, §§§P < 0.001 versus MDCK-OCT1-MATE1 cells.
Figure 5
Figure 5
pH dependence of MPP+ (50 µM) and metformin (50 µM) basal to apical (b-a) and apical to basal (a-b) transcellular transport in monolayers of MDCK-OCT2-MATE1 cells after administration of MPP+ or metformin to either the basal or the apical side of the cell monolayers. pH in the apical compartment was 6.0 (A), 6.5 (B), 7.0 (C) and 7.5 (D). pH in the basal compartment was 7.4 in all experiments. There was no pH dependent change both in basal to apical (b-a) as well as apical to basal (a-b) transcellular translocation of MPP+ and metformin in monolayers of MDCK control cells (data not shown). The transport ratio (R) was calculated as the quotient of the mean of the apically directed transport and the mean of the basally directed transport at 3 h. Data are shown as mean value ± standard deviation.
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