Methods to Evaluate the Effect of Ethanol on the Folate Analogue: Fluorescein Methotrexate Uptake in Human Proximal Tubular Cells

Abstract

Ethanol-induced folate deficiency is due to effects of ethanol on folate metabolism and absorption. We have already shown by using different methods that ethanol interferes with reabsorption of folate from the proximal tubule. In this study, we have used the folate analogue, the fluorescein methotrexate (FL-MTX), in order to evaluate effects of ethanol on FL-MTX uptake by the human proximal tubular (HPT) cells by using a confocal microscope and fluoroskan microplate reader. Since endothelins (ETs) play a major role in a number of diseases and also in the damage induced by a variety of chemicals, we have used endothelin-B (ET-B) and protein kinase-C (PKC) inhibitors to evaluate the role of endothelin in ethanol-mediated FL-MTX uptake by using fluoroskan microplate reader. Confocal microscope and fluoroskan studies reveal that cellular absorption of FL-MTX is concentration-dependent. Moreover, ethanol concentration has an impact on FL-MTX uptake. Fluoroskan studies reveal that the ethanol-induced decrease in FL-MTX uptake is reversed by adding the ET-B receptor antagonist (RES-701-1) or PKC-selective inhibitor (BIM). Thus, we can conclude that ethanol may act via ET and ET in turn may act via ET-B receptor and the PKC signaling pathway to impair FL-MTX transport.