Expression cloning in Xenopus laevisoocytes was used to isolate an organic anion transport protein from rat kidney. A cDNA library was constructed from size-fractionated poly(A)⁺ RNA and screened for probenecid-sensitive transport of p-aminohippurate (PAH). A 2,227-base pair cDNA clone containing a 1,656-base pair open reading frame coding for a peptide 551 amino acids long was isolated and named ROAT1. ROAT1-mediated transport of 50 μm [³H]PAH was independent of imposed changes in membrane potential. Transport was significantly inhibited at 4 °C, or upon incubation with other organic anions, but not by the organic cation tetraethylammonium, by the multidrug resistance ATPase inhibitor cyclosporin A, or by urate. External glutarate and α-ketoglutarate (1 mm), both counterions for basolateral PAH exchange, also inhibited transport, suggesting that ROAT1 is functionally similar to the basolateral PAH carrier. Consistent with this conclusion, PAH uptake was trans-stimulated in oocytes preloaded with glutarate, whereas the dicarboxylate methylsuccinate, which is not accepted by the basolateral exchanger, did nottrans-stimulate. Finally, ROAT1-mediated PAH transport was saturable, with an estimated K _m of 70 μm. Each of these properties is identical to those previously described for the basolateral α-ketoglutarate/PAH exchanger in isolated membrane vesicles or intact renal tubules.

Nephrotoxicity is the dose-limiting clinical adverse effect of cidofovir and adefovir, two potent antiviral therapeutics. Because renal uptake likely plays a role in the etiology of cidofovir- and adefovir-associated nephrotoxicity, we attempted to identify a renal transporter capable of interacting with these therapeutics. A cDNA clone was isolated from a human renal library and designated human organic anion transporter 1 (hOAT1). Northern analysis detected a specific 2.5-kilobase pair hOAT1 transcript only in human kidney. However, reverse transcription-polymerase chain reaction revealed hOAT1 expression in human brain and skeletal muscle, as well. Immunoblot analysis of human kidney cortex demonstrated that hOAT1 is an 80- to 90-kilodalton heterogeneous protein modified by abundantN-glycosylation. Xenopus laevis oocytes expressing hOAT1 supported probenecid-sensitive uptake of [³H]p-aminohippurate (K_m = 4 μM), which wastrans-stimulated in oocytes preloaded with glutarate. Importantly, both hOAT1 and rat renal organic anion transporter 1 (rROAT1) mediated saturable, probenecid-sensitive uptake of cidofovir, adefovir, and other nucleoside phosphonate antivirals. The affinity of hOAT1 toward cidofovir and adefovir (K_m = 46 and 30 μM, respectively) was 5- to 9-fold higher compared with rROAT1 (K_m = 238 and 270 μM, respectively). These data indicate that hOAT1 may significantly contribute to the accumulation of cidofovir and adefovir in renal proximal tubules and, thus, play an active role in the mechanism of nephrotoxicity associated with these antiviral therapeutics.

To begin to develop in vivo model systems for the assessment of the contributions of specific organic anion transporter (OAT) family members to detoxification, development, and disease, we carried out a targeted disruption of the murine organic anion transporter 3 (Oat3) gene. SurvivingOat3^−/− animals appear healthy, are fertile, and do not exhibit any gross morphological tissue abnormalities. NoOat3 mRNA expression was detected in kidney, liver, or choroid plexus (CP) of Oat3^−/− mice. A distinct phenotype manifested by a substantial loss of organic anion transport capacity in kidney and CP was identified. Uptake sensitive to inhibition by bromosulfophthalein or probenecid was observed for taurocholate, estrone sulfate, and para-aminohippurate in renal slices from wild-type mice, whereas inOat3^−/− animals transport of these substances was greatly reduced. No discernable differences in uptake were observed between hepatic slices from wild-type andOat3^−/− littermates, suggesting Oat3 does not play a major role in hepatic organic anion uptake. Cellular accumulation of fluorescein was reduced by ∼75% in CP fromOat3^−/− mice. However, capillary accumulation of fluorescein-methotrexate was unchanged, indicating the effects of Oat3 loss are restricted to the entry step and that Oat3 is localized to the apical membrane of CP. These data indicate a key role for Oat3 in systemic detoxification and in control of the organic anion distribution in cerebrospinal fluid.

To determine whether organic cation transporter (OCT) family members might mediate choline transport in choroid plexus (CP), the handling of choline by cloned transporters and by intact CP isolated from the adult rat was investigated. Expression of OCT1 and OCT2 in Xenopus oocytes increased hemicholinium-3-sensitive choline uptake. In contrast, OCT3 did not mediate choline transport. Estimated K(m) values for choline in rOCT1-, rOCT2-, and hOCT2-expressing oocytes were 346 +/- 50, 441 +/- 67, and 102 +/- 80 microm, respectively. Membrane potential was the major driving force for choline uptake in rat and human OCT2-expressing oocytes and in intact CP in vitro. Lowering of medium pH (6 versus 7.4) was equally effective at inhibiting choline uptake in CP, suggesting that there might be a non-OCT component of choline uptake that is responsive to an H(+) gradient. However, choline efflux from CP was not stimulated by a trans-applied H(+) gradient. Choline uptake by CP was Na(+)-independent with an estimated K(m) of 183 microm. Reverse transcriptase-polymerase chain reaction detected OCT2 and OCT3, but not OCT1, mRNA expression in CP. Transfection of intact CP with a rOCT2/green fluorescent protein fusion construct resulted in strong apical membrane fluorescence with no detectable signal in the basal and lateral plasma membranes. These data indicate that OCT2 mediates choline transport across the ventricular membrane of CP.