Submitted on July 18, 2003
Accepted on November 8, 2003
The Sodium-Dependent Methotrexate Carrier-1 is Expressed in Rat Kidney: Cloning and Functional Characterization
Carsten Kneuer1, Kerstin U. Honscha2, and Walther Honscha1*
1 Institute of Veterinary Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, University of Leipzig, Leipzig, Saxony, Germany
2 Institute of Veterinary Physiology, Faculty of Veterinary Medicine, University of Leipzig, Leipzig, Saxony, Germany
* To whom correspondence should be addressed. E-mail: honscha{at}vetmed.uni-leipzig.de.
Previous Northern blot studies suggested strong expression of a homologue to the sodiumdependent 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 MDCK cells. A sodium-dependent transport of methotrexate with a Km of 41 µM and a Vmax of 337 pmol/mg protein and minute 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. Further, expression of RK-MTX-1 in MDCK cells enhanced methotrexate toxicity in these cells 5-fold. Analysis of a fusion protein of RK-MTX-1 and the
influenza virus HA-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.
