Folate transport and binding by cultured human proximal tubule cells

Because mechanisms for the renal regulation of folate excretion are poorly understood, a cell culture system representative of the human proximal tubule (HPT) was used for studies of renal folate transport. After confluent cultures of HPT cells were incubated with 3H-labeled folic acid (PteGlu), binding to the apical membrane was determined by an acid removal procedure, and transport was subsequently measured in solubilized cells. Although PteGlu binding was almost all specific (suppressed by excess unlabeled PteGlu), HPT cells transported PteGlu by specific and nonspecific processes. Specific PteGlu binding and transport were both saturable processes, reaching maxima of 0.5 and 0.1 pmol/mg protein, respectively, with half-maximal constants of 12 and 50 nM, respectively. The PteGlu analogues methotrexate and 5-methyltetrahydrofolic acid (5-CH3-H4PteGlu) inhibited both the binding and transport of PteGlu, with 5-CH3-H4PteGlu being more potent (lower half-maximal inhibitory concentration). In contrast, 5-formyltetrahydrofolic acid significantly reduced PteGlu transport at concentrations (100-250 nM) that had no effect on binding. These data suggest that the HPT cells will serve as a good model for studies of renal folate reabsorption. Initial characterization of the transport of folate by HPT cells suggests two distinct processes, binding to a high-affinity membrane folate-binding protein followed by a structurally specific transfer into the cell.