Identification of selective substrates and inhibitors of the major human renal uptake transporters

Renal clearance of drugs mediated by transporters can be affected by diseases (eg, inflammation due to infections), physiological changes (eg, pregnancy), or drug-drug interactions. To elucidate the transporters involved, the magnitude of effect, and the underlying mechanisms, human proximal tubular epithelial cells could be exposed to potential perpetrators (eg, cytokines, pregnancy-related hormones or the interacting drug), and the activity of transporters quantified. A crucial prerequisite for such studies is the identification of selective substrates or substrate-inhibitor pairs for each renal transporter. Using transporter-transfected mammalian cells and membrane vesicles, we systematically evaluated the selectivity of 6 substrates (or substrate-inhibitor pairs) for the major uptake and efflux renal transporters. Cidofovir, levocetirizine, and ergothioneine were found to be selective substrates of the organic anion transporter (OAT) 1, 4, and organic cation/carnitine transporter 1, respectively. Nicotinic acid was transported by OAT2, but also by OAT1 and 3, though to a lesser extent. Probenecid did not selectively inhibit OAT1/3-mediated uptake of nicotinic acid, but quercetin did, allowing selective measurement of OAT2 activity. Interestingly, nicotinic acid was also transported by the endogenous monocarboxylate transporter 1 in HEK293 cells. Glycochenodeoxycholic acid sulfate was transported by OAT3 and multidrug resistance-associated protein 2 (MRP2), with MRP2 selectively inhibited by cyclosporine A, allowing selective measurement of OAT3 activity. Atenolol was transported by organic cation transporter 2 and multidrug and toxin extrusion proteins 1 and 2-K, with multidrug and toxin extrusion proteins activity selectively inhibited by mitoxantrone, allowing selective measurement of organic cation transporter 2 activity. SIGNIFICANCE STATEMENT: These findings provide a framework for measuring the in vitro activity of individual uptake transporters in primary human proximal tubular epithelial cells. By applying our proposed methodology, researchers can quantify how various factors (eg, cytokines, pregnancy-related hormone, drug interactions) modulate individual renal uptake transporter activity in proximal tubular epithelial cells.