Identifying Selective Substrates and/or Inhibitors of Renal Drug Uptake Transporters

Abstract ID 90435 Poster Board 227 Proximal tubular epithelial cells (PTECs) of the human kidney express multiple drug transporters that participate in the renal tubular secretion and reabsorption of drug molecules. Primary PTECs, isolated from human kidneys, can be useful to study the in vitro effect of treatment (e.g., cytokines or pregnancy-related hormones) on individual renal drug transporter activity. To do so, selective substrates of each transporter must be identified. Many commonly used in vitro probe substrates for renal drug transporters, including those recommended by the FDA (e.g., furosemide), lack selectivity. Furthermore, existing literature data on the selectivity of many renal drug transporter substrates are inconsistent or incomplete. Therefore, the goal of this study was to identify and/or confirm selective substrates and/or inhibitors of renal drug uptake transporters. The uptake transporters tested were the organic anion transporters 1 to 4 (OAT1-4)and the organic cation transporter 2 (OCT2). The efflux transporters tested were the multidrug and toxin extrusion proteins 1 and 2K (MATE1/2-K), multidrug resistance proteins 1-4 (MRP1-4), breast cancer resistance protein (BCRP), and P-glycoprotein (P-gp). Substrates tested for OATs were cidofovir, nicotinic acid, glycochenodeoxycholate sulfate (GCDCA-S), and levocetirizine, while for OCT2, atenolol was tested. Uptake assays were conducted using transporter-overexpressing human embryonic kidney (HEK293) cells and vesicles. To measure passive diffusion, the uptake of the substrates by OATs, OCT2, and MATE1/2-K was measured in the absence and presence of classic inhibitors (probenecid for OAT1/3, bromsulfthalein for OAT2, and pyrimethamine for OCT2 and MATE1/2-K). For OAT4, passive diffusion of the substrate was measured by uptake into non-transfected cells. For vesicular transporter assays, passive diffusion of the substrate was measured by replacing ATP with adenosine monophosphate (AMP). All experiments were performed in triplicate. Our results showed that cidofovir and levocetirizine were selective substrates of OAT1 and OAT4, respectively, and none of the other tested transporters. GCDCA-S was a selective substrate for the uptake transporter OAT3 among the OATs but was also transported by MRP1, 2, and 4. Nicotinic acid, though previously reported to be a selective OAT2 substrate, was shown to also be transported by OAT1 and 3, albeit to a lesser extent than by OAT2. Atenolol was confirmed to be a substrate of OCT2 and MATE1/2-K. In order to establish selectivity for OAT2 and OCT2-mediated uptake of nicotinic acid and atenolol, respectively, potential selective inhibitors of OAT1/3 and MATE1/2-K were tested in a concentration-dependent manner. Quercetin (25 μM) selectively inhibited OAT1/3-mediated nicotinic acid uptake, while minimally impacting OAT2 activity. Similarly, mitoxantrone (25 μM) selectively inhibited MATE1/2-K-mediated atenolol uptake but not OCT2. In summary, we identified and confirmed selective substrates and substrate/inhibitor combinations for the above renal uptake transporters. These data can now be applied to determine which uptake transporters are affected when PTECs are treated with cytokines or pregnancy-related hormones. This work was supported by the NIH Grant R01HD102786.