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Am J Physiol Renal Physiol 286: F564-F571, 2004. First published November 11, 2003; doi:10.1152/ajprenal.00257.2003
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Sodium-dependent methotrexate carrier-1 is expressed in rat kidney: cloning and functional characterization

Carsten Kneuer,1,* Kerstin U. Honscha,2,* and Walther Honscha1

1Institute of Veterinary Pharmacology, Pharmacy and Toxicology, and 2Institute of Veterinary Physiology, Faculty of Veterinary Medicine, University of Leipzig, 04103 Leipzig, Germany

Submitted 18 July 2003 ; accepted in final form 8 November 2003

Previous Northern blot studies suggested strong expression of a homolog to the sodium-dependent hepatocellular methotrexate transporter in the kidneys. Here, we report on the cloning of the cDNA for the renal methotrexate carrier isoform-1 (RK-MTX-1) and its functional characterization. Sequencing revealed 97% homology to the rat liver methotrexate carrier with an identical open reading frame. Differences were located in the 5'-untranslated region and resulted in the absence of putative regulatory elements (Barbie box, Ah/ARNT receptor) identified in the cDNA for the hepatocellular carrier. For functional characterization, MTX-1 cDNA was stably expressed in Madin-Darby canine kidney (MDCK) cells. A sodium-dependent transport of methotrexate with a Km of 41 µM and a Vmax of 337 pmol·mg protein-1·min-1 was observed. This uptake was blocked by the reduced folates dihydro- and tetrahydrofolate as well as by methotrexate itself. Folate was inhibiting only weakly, whereas 5-methyltetrahydrofolate was a strong inhibitor. Further inhibitors of the methotrexate transport included the bile acids cholate and taurocholate and xenobiotics like bumetanide and BSP. PAH, ouabain, bumetanide, cholate, taurocholate, and acetyl salicylic acid were tested as potential substrates. However, none of these substances was transported by MTX-1. Furthermore, expression of RK-MTX-1 in MDCK cells enhanced methotrexate toxicity in these cells fivefold. Analysis of a fusion protein of RK-MTX-1 and the influenza virus hemagglutinin epitope by immunoblotting revealed a major band at 72 kDa within the cell membrane but not in the soluble fraction of transfected MDCK. Indirect immunofluorescence staining revealed an exclusive localization of the carrier in the plasma membrane, and by confocal laser-scanning microscopy we were able to demonstrate that the protein is expressed in the serosal region of MDCK tubules grown in a morphogenic collagen gel model.

renal organic anion transporter; drug elimination; nephrotoxicity


Address for reprint requests and other correspondence: W. Honscha, Institute of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, Univ. of Leipzig, An den Tierkliniken 15, D-04103 Leipzig, Germany (E-mail: honscha{at}vetmed.uni-leipzig.de).






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