Carboxyfluoroquinolones, such as ciprofloxacin, are used for the treatment of numerous infectious diseases. Renal secretion is a major determinant of their systemic and urinary concentration, but the specific transporters involved are virtually unknown. In vivo studies implicate the organic anion transporter (OAT) family as a pivotal component of carboxyfluoroquinolone renal secretion. Therefore, this study identified the specific renal basolateral OAT(s) involved, thereby highlighting potential sources of carboxyfluoroquinolone-drug interactions and variable efficacy. Two heterologous expression systems, Xenopus laevis oocytes and cell monolayers, were used to determine the roles of murine and human renal basolateral mOat1/hOAT1 and mOat3/hOAT3. Ciprofloxacin was transported by mOat3 in both systems (K m value, 70 Ϯ 6 M) and demonstrated no interaction with mOat1 or hOAT1. Furthermore, ciprofloxacin, norfloxacin, ofloxacin, and gatifloxacin exhibited concentration-dependent inhibition of transport on mOat3 in cells with inhibition constants of 198 Ϯ 39, 558 Ϯ 75, 745 Ϯ 165, and 941 Ϯ 232 M, respectively. Ciprofloxacin and gatifloxacin also inhibited hOAT3. Thereafter, in vivo elimination of ciprofloxacin was assessed in wild-type and Oat3 null mice [Oat3(Ϫ/Ϫ)]. Oat3(Ϫ/Ϫ) mice exhibited significantly elevated plasma levels of ciprofloxacin at clinically relevant concentrations (P Ͻ 0.05, male mice; P Ͻ 0.01, female mice). Oat3(Ϫ/Ϫ) mice also demonstrated a reduced volume of distribution (27%, P Ͻ 0.01, male mice; 14%, P Ͻ 0.01, female mice) and increased area under the concentration-time curve (25%, P Ͻ 0.05, male mice; 33%, P Ͻ 0.01, female mice). Female Oat3(Ϫ/Ϫ) mice had a 35% (P Ͻ 0.01) reduction in total clearance of ciprofloxacin relative to wild type. In addition, putative ciprofloxacin metabolites were significantly elevated in Oat3(Ϫ/Ϫ) mice. The present findings indicate that polymorphisms of and drug interactions on hOAT3 may influence carboxyfluoroquinolone efficacy, especially in urinary tract infections.Ciprofloxacin is a broad-spectrum antimicrobial that is used in the treatment of numerous infectious diseases, including those afflicting the skin (Lipsky et al., 1999) (Gogos et al., 1991). It is also a preferred agent for the prevention and treatment of anthrax (Meyerhoff et al., 2004). Its mechanism of action is through the effective inhibition of DNA gyrase, thus preventing DNA replication in susceptible bacteria (Gellert et al., 1977;Sugino et al., 1977). Ciprofloxacin is a carboxylic acidcontaining fluoroquinolone (carboxyfluoroquinolone) that undergoes renal and hepatic elimination, with ϳ50% (oral) or ϳ80% (intravenous) appearing in the urine as parent compound and metabolites 24 h after administration in humans (Höffken et al., 1985). Approximately 20 to 40% of circulating Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.042853.ABBREVIATIONS: LLC-PK 1 , porcine kidney cells; AUC, area under the concentration-t...

Abstract1. The transport of negatively charged drugs, xenobiotics, and metabolites by epithelial tissues, particularly the kidney, plays critical roles in controlling their distribution, concentration, and retention in the body. Thus, organic anion transporters (OATs) impact both their therapeutic efficacy and potential toxicity.2. This review summarizes current knowledge of the properties and functional roles of the cloned OATs, the relationships between transporter structure and function, and those factors that determine the efficacy of transport. Such factors include plasma protein binding of substrates, genetic polymorphisms among the transporters, and regulation of transporter expression.3. Clearly, much progress has been made in the decade since the first OAT was cloned. However, unresolved questions remain. Several of these issues -drug-drug interactions, functional characterization of newly cloned OATs, tissue differences in expression and function, and details of the nature and consequences of transporter regulation at genomic and intracellular sites -are discussed in the concluding Perspectives section.

Organic anion transporters (OATs) play a pivotal role in the clearance of small organic anions by the kidney, yet little is known about how their activity is regulated. A yeast twohybrid assay was used to identify putative OAT3-associated proteins in the kidney. Atypical protein kinase C (PKC) was shown to bind to OAT3. Binding was confirmed in immunoprecipitation assays. The OAT3/PKC interaction was investigated in rodent renal cortical slices from fasted animals. Insulin, an upstream activator of PKC, increased both OAT3-mediated uptake of estrone sulfate (ES) and PKC activity. Both effects were abolished by a PKC-specific pseudosubstrate inhibitor. Increased ES transport was not observed in renal slices from OAT3-null mice. Transport of the shared OAT1/OAT3 substrate, -aminohippurate, behaved similarly, except that stimulation was reduced, not abolished, in the OAT3-null mice. This suggested that OAT1 activity was also modified by PKC, subsequently confirmed using an OAT1-specific substrate, adefovir. Inhibition of PKC also blocked the increase in ES uptake seen in response to epidermal growth factor and to activation of protein kinase A. Thus, PKC acted downstream of the epidermal growth factor to protein kinase A signaling pathway. Activation of transport was accompanied by an increase in V max and was blocked by microtubule disruption, indicating that activation may result from trafficking of OAT3 into the plasma membrane. These data demonstrate that PKC activation up-regulates OAT1 and OAT3 function, and that protein-protein interactions play a central role controlling these two important renal drug transporters. Organic anion transporters (OATs)7 are members of the solute carrier 22A family and play a pivotal role in the renal clearance of small (Ͻ500 Dalton) anionic drugs, xenobiotics, and their metabolites. OAT substrates include a variety of drugs such as ␤-lactam antibiotics, non-steroidal anti-inflammatory drugs, diuretics, and chemotherapeutics (1). OATs are predominantly expressed in renal proximal tubule, with OATs 1-3 localized to the basolateral membrane and OAT4 and URAT1 on the apical membrane. OATs 1 and 3 are dicarboxylate exchangers, and are indirectly coupled to the sodium gradient maintained by Na,K-ATPase through sodium/dicarboxylate co-transport to drive the uphill basolateral step in renal organic anion secretion (2).Although the ionic gradients, electrophysiology, and underlying kinetics that drive transport by OATs 1 and 3 are well characterized, physiologically important interactions of these basolateral OATs with membrane or cytosolic proteins have yet to be identified (1). Nevertheless, there is clear evidence that other plasma membrane transporters do interact with protein partners, influencing a diverse array of functions including transport itself, cytoskeletal structure, vesicle formation, and trafficking, as well as signaling (3). Among the transporters with activity modulated by protein-protein interactions, particularly by the PDZ proteins, PDZK1 and NHERFs 1 and 2, are ap...