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Rat reduced-folate carrier-1 is localized basolaterally in MDCK kidney epithelial cells and contributes to the secretory transport of methotrexate and fluoresceinated methotrexate

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Abstract

The reduced-folate carrier (Rfc-1), previously also called methotrexate carrier-1 (MTX-1), was recently identified as accounting for approximately 30% of the methotrexate (Mtx) uptake into rat kidney slices. The localization of the carrier and its contribution to secretory or reabsorptive flux of the drug was therefore evaluated in polarized epithelial layers of Madin Darby canine kidney (MDCK) cells. Confocal laser scanning microscopy revealed that the HA-epitope-tagged protein was sorted to the basolateral side. In flux assays, the basolateral-to-apical transport of fluoresceinated methotrexate (FMTX) was two-fold higher than in the apical-to-basolateral direction across rat Rfc-1 transfected, but not mock-transfected, monolayers. The same observation was made for unlabeled Mtx. This secretory transport of FMTX was inhibited by an excess of 1 mM Mtx and was saturable and temperature-dependent. No differences in directional flux were observed for the pure fluorescein label. Removal of sodium resulted in a marked decrease of directional FMTX flux. The pH profile of the active transport component showed a trough around 6.5 and a maximum at acidic pH, as reported for uptake into Rfc-1-expressing cells. Thus, rat Rfc-1 is sorted to the basolateral side in polarized MDCK epithelial cells and mediates the secretion of Mtx, probably in co-operation with efflux proteins, such as multidrug resistance associated proteins, which are also expressed in these cells.

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Acknowledgements

We wish to thank the Physics of Soft Matter Unit, University of Leipzig (Prof. Käs) and C.M. Lehr, Biopharmaceutics, Saarland University for use of their equipment. U. Knoll is acknowledged for her help with the setting up of the HPLC analysis.

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Authors and Affiliations

  1. Institute of Pharmacology, Pharmacy and Toxicology, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 15, 04103, Leipzig, Germany

    Carsten Kneuer & Walther Honscha

  2. Institute of Veterinary Physiology, Faculty of Veterinary Medicine, University of Leipzig, An den Tierkliniken 7, 04103, Leipzig, Germany

    Kerstin U. Honscha

Authors
  1. Carsten Kneuer
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  2. Kerstin U. Honscha
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  3. Walther Honscha
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Correspondence to Walther Honscha.

Additional information

This study was supported by the Deutsche Forschungsgemeinschaft (HO2103/1-2) and HO2512/1-1 (Kerstin U. Honscha).

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Kneuer, C., Honscha, K.U. & Honscha, W. Rat reduced-folate carrier-1 is localized basolaterally in MDCK kidney epithelial cells and contributes to the secretory transport of methotrexate and fluoresceinated methotrexate. Cell Tissue Res 320, 517–524 (2005). https://doi.org/10.1007/s00441-005-1092-x

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