Abstract

17α-Ethinylestradiol (EE2), a synthetic and potent estrogen receptor agonist, is extensively metabolized in both intestine and liver and is largely excreted in bile and urine as the 3-O-sulfate (EE2-Sul) and 3-O-glucuronide. In the present study, EE2-Sul was evaluated as a substrate of various transporters known to be expressed in the kidney. Uptake studies were performed with human epithelial cells [human embryonic kidney (HEK)-293] that contained individually expressed organic cation transporter 2 (OCT2), organic anion transporter (OAT) forms 3 and 4, and multidrug and toxin extrusion 1 (MATE1). The transporter phenotyping studies were extended to include insect cell (Sf9) membrane vesicles that expressed multidrug resistance-associated protein 4 (MRP4) and Madin-Darby canine kidney cells that expressed OAT1. Based on the results obtained, we concluded that EE2-Sul serves as a substrate of OAT3 and OAT4, but not OCT2, OAT1, MATE1, and MRP4. First, EE2-Sul uptake was highly increased in OAT3/HEK-293 cells (versus mock/HEK-293 cells) and was inhibited by OAT3 inhibitors such as bromosulfophthalein (BSP), cimetidine, and probenecid. OAT3-mediated uptake also conformed to single-K_m (Michaelis constant) kinetics (K_m = 21.1 μM). Second, EE2-Sul uptake was also significantly higher in OAT4/HEK-293 cells and was inhibited by BSP, methotrexate, and probenecid. In contrast to OAT3, OAT4-dependent uptake was characterized by a two-K_m model (K_m1 = 1.6 μM; K_m2 = 195 μM). Based on the results of this study, we hypothesize that EE2-Sul is taken up into renal proximal tubule cells by OAT3, and OAT4 plays a role in its secretion into the renal brush border lumen.