Elsevier

Translational Research

Volume 162, Issue 6, December 2013, Pages 398-409
Translational Research

Original Article
Interaction of immunosuppressive drugs with human organic anion transporter (OAT) 1 and OAT3, and multidrug resistance-associated protein (MRP) 2 and MRP4

Citations of meeting abstracts where the work was previously presented: El-Sheikh AAK, van den Heuvel JJMW, Koenderink JB, Russel FGM. Interaction of immunosuppressants with methotrexate transport mediated by human multidrug resistance transporters MRP2 and MRP4. Naunyn- Schmiedebergs Arch Pharmacol 2009;379:202 [abstract]. Greupink R, El-Sheikh AA, Wortelboer H, van den Heuvel JJMW, Schreurs M, Koenderink JB, Masereeuw R, Russel FGM. Interaction of immunosuppressants with human organic anion transporters 1 and 3 and multidrug resistance proteins 2 and 4. Presented at the 52nd annual meeting of the Society of Toxicology, March 10–14, 2013, San Antonio, Texas, abstract no. 499.
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Renal proximal tubule transporters can play a key role in excretion, pharmacokinetic interactions, and toxicity of immunosuppressant drugs. Basolateral organic anion transporters (OATs) and apical multidrug resistance-associated proteins (MRPs) contribute to the active tubular uptake and urinary efflux of these drugs, respectively. We studied the interaction of 12 immunosuppressants with OAT1- and OAT3-mediated [3H]-methotrexate (MTX) uptake in cells, and adenosine triphosphate-dependent [3H]-MTX transport in membrane vesicles isolated from human embryonic kidney 293 cells overexpressing human MRP2 and MRP4. Our results show that at a clinically relevant concentration of 10 μM, mycophenolic acid inhibited both OAT1- and OAT3-mediated [3H]-MTX uptake. Cytarabine, vinblastine, vincristine, hydrocortisone, and mitoxantrone inhibited only OAT1, whereas tacrolimus, azathioprine, dexamethasone, cyclosporine, and 6-mercaptopurine had no effect on both transporters. Cyclophosphamide stimulated OAT1, but did not affect OAT3. With regard to the apical efflux transporters, mycophenolic acid, cyclophosphamide, hydrocortisone, and tacrolimus inhibited MRP2 and MRP4, whereas mitoxantrone and dexamethasone stimulated [3H]-MTX transport by both transporters. Cyclosporine, vincristine, and vinblastine inhibited MRP2 only, whereas 6-mercaptopurine inhibited MRP4 transport activity only. Cytarabine and azathioprine had no effect on either transporter. In conclusion, we charted comprehensively the differences in inhibitory action of various immunosuppressive agents against the 4 key renal anion transporters, and we provide evidence that immunosuppressant drugs can modulate OAT1-, OAT3-, MRP2-, and MRP4-mediated transport of MTX to different extents. The data provide a better understanding of renal mechanisms underlying drug-drug interactions and nephrotoxicity concerning combination regimens with these compounds in the clinic.

Section snippets

Materials and Methods

We conformed to all relevant guidelines for human and animal research and adhered to national guidelines and laws for working with genetically modified organisms when overexpressing recombinant human proteins in vitro.

[3H]-MTX transport and inhibition in OAT1-expressing cells

Fig 1A shows that [3H]-MTX uptake was increased in the OAT1-expressing cells compared with controls. Moreover, MTX uptake in HEK-OAT1 cells could be inhibited by the prototypical OAT inhibitor probenecid (Fig 1, B). Transporter-mediated uptake of [3H]-MTX over time in HEK-OAT1 cells is presented in Fig 1, C and was linear up to 5 minutes.

Next, HEK-OAT1 cells were incubated with 0.5 μM [3H]-MTX in the presence or absence of 10 μM of different immunosuppressants for 5 minutes. Our results (Fig 1,

Discussion

We have previously investigated the role of MRP2 and MRP4 in the clinically reported interaction between MTX and nonsteroidal anti-inflammatory drugs, and from these results we suggested that both transporters have allosteric binding sites.14 Here, we studied the interaction of different immunosuppressant drugs with [3H]-MTX, which is a common substrate of renal basolateral OAT1 and OAT3,16 as well as apical efflux MRP217 and MRP418 transporters. Our results show that several immunosuppressants

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    Conflict of Interest: All authors have read the journal’s policy on disclosure of potential conflicts of interest and have none to declare.

    This work was supported in part by a scholarship granted by the Egyptian Ministry of Higher Education to A. A. K. El-Sheikh.

    A. A. K. El-Sheikh and R. Greupink contributed equally to this work. A. A. K. El-Sheikh, R. Greupink, J. B. Koenderink, R. Masereeuw, and F. G. M. Russel participated in research design; A. A. K. El-Sheikh, J. J. M. W. van den Heuvel, R. Greupink, and M. Schreurs conducted experiments; H. M. Wortelboer contributed organic anion transporter 3 overexpressing cells; A. A. K. El-Sheikh and R. Greupink performed data analysis; and A. A. K. El-Sheikh, R. Greupink, J. B. Koenderink, R. Masereeuw, and F. G. M. Russel Wrote or contributed to the writing of the manuscript.

    This work was performed in part during the PhD research of A. A. K. El-Sheikh, conducted at the Department of Pharmacology and Toxicology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen, Netherlands, and supervised by J. B. Koenderink, R. Masereeuw, and F. G. M. Russel. The thesis was titled “Renal Transport and Drug Interactions of Immunosuppressants,” printed by Printpartners Ipskamp, Enschede, Netherlands, 2008 (ISBN: 978-90-9023458-8).

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