Hyperuricemia is a significant factor in a variety of diseases, including gout and cardiovascular diseases. Although renal excretion largely determines plasma urate concentration, the molecular mechanism of renal urate handling remains elusive. Previously, we identified a major urate reabsorptive transporter, URAT1 (SLC22A12), on the apical side of the renal proximal tubular cells. However, it is not known how urate taken up by URAT1 exits from the tubular cell to the systemic circulation. Here, we report that a sugar transport facilitator family member protein GLUT9 (SLC2A9) functions as an efflux transporter of urate from the tubular cell. GLUT9-expressed Xenopus oocytes mediated saturable urate transport (K_m: 365 ± 42 μm). The transport was Na⁺-independent and enhanced at high concentrations of extracellular potassium favoring negative to positive potential direction. Substrate specificity and pyrazinoate sensitivity of GLUT9 was distinct from those of URAT1. The in vivo role of GLUT9 is supported by the fact that a renal hypouricemia patient without any mutations in SLC22A12 was found to have a missense mutation in SLC2A9, which reduced urate transport activity in vitro. Based on these data, we propose a novel model of transcellular urate transport in the kidney; Remunurate is taken up via apically located URAT1 and exits the cell via basolaterally located GLUT9, which we suggest be renamed URATv1 (voltage-driven urate transporter 1).

Most tumor cell membranes overexpress l‐type amino acid transporter 1, while normal cell membranes contain l‐type amino acid transporter 2; both are Na⁺‐independent amino acid transporters. Therefore, compounds that selectively inhibit l‐type amino acid transporter 1 offer researchers with a novel cancer molecular target. Synthetic chemistry efforts and in vitro screening have produced a variety of novel compounds possessing high in vitrol‐type amino acid transporter 1 selectivity; KYT‐0353 was one such compound. The present studies illustrate that KYT‐0353 inhibited ¹⁴C‐leucine uptake and cell growth in human colon cancer‐derived HT‐29 cells; IC₅₀s were 0.06 μm and 4.1 μm, respectively. KYT‐0353 also inhibited ¹⁴C‐leucine uptake in mouse renal proximal tubule cells expressing l‐type amino acid transporter 1, and inhibited cell growth; IC₅₀s were 0.14 μm and 16.4 μm, respectively. Compared to control animals, intravenously administered KYT‐0353 (12.5 mg/kg and 25.0 mg/kg) showed statistically significant growth inhibition against HT‐29 tumors transplanted to nude mice with maximal inhibition ratios of 65.9% and 77.2%, respectively. Body weight increase with time – a safety indicator – was slightly depressed at 12.5 mg/kg and 25.0 mg/kg with maximal ratios of 3.7% (day 2) and 6.3% (day 11), respectively. Thus, KYT‐0353 showed significant growth inhibitory effects on HT‐29 cells both in vitro and in vivo, whereas it only caused a slight body weight depression. Therefore, KYT‐0353 appears to have potential as a novel anti‐tumor agent, presumably via selective in vivol‐type amino acid transporter 1 inhibition. (Cancer Sci 2009)

A cDNA that encodes a novel Na⁺-independent neutral amino acid transporter was isolated from FLC4 human hepatocarcinoma cells by expression cloning. When expressed in Xenopus oocytes, the encoded protein designated LAT3 (L-type amino acid transporter 3) transported neutral amino acids such as l-leucine, l-isoleucine, l-valine, and l-phenylalanine. The LAT3-mediated transport was Na⁺-independent and inhibited by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with the properties of system L. Distinct from already known system L transporters LAT1 and LAT2, which form heterodimeric complex with 4F2 heavy chain, LAT3 was functional by itself in Xenopus oocytes. The deduced amino acid sequence of LAT3 was identical to the gene product of POV1 reported as a prostate cancer-up-regulated gene whose function was not determined, whereas it did not exhibit significant similarity to already identified transporters. The Eadie-Hofstee plots of LAT3-mediated transport were curvilinear, whereas the low affinity component is predominant at physiological plasma amino acid concentration. In addition to amino acid substrates, LAT3 recognized amino acid alcohols. The transport of l-leucine was electroneutral and mediated by a facilitated diffusion. In contrast, l-leucinol, l-valinol, and l-phenylalaninol, which have a net positive charge induced inward currents under voltage clamp, suggesting these compounds are transported by LAT3. LAT3-mediated transport was inhibited by the pretreatment with N-ethylmaleimide, consistent with the property of system L2 originally characterized in hepatocyte primary culture. Based on the substrate selectivity, affinity, and N-ethylmaleimide sensitivity, LAT3 is proposed to be a transporter subserving system L2. LAT3 should denote a new family of organic solute transporters.

Human organic anion transporter OAT4 is expressed in the kidney and placenta and mediates high-affinity transport of estrone-3-sulfate (E1S). Because a previous study demonstrated no trans-stimulatory effects by E1S, the mode of organic anion transport via OAT4 remains still unclear. In the present study, we examined the driving force of OAT4 using mouse proximal tubular cells stably expressing OAT4 (S2 OAT4). OAT4-mediated E1S uptake was inhibited by glutarate (GA) (IC50: 1.25 mM) and [14C]GA uptake via S2 OAT4 was significantly trans-stimulated by unlabeled GA (5 mM) (P<0.001). [3H]E1S uptake via S2 OAT4 was significantly trans-stimulated by preloaded GA (P<0.001) and its [14C]GA efflux was significantly trans-stimulated by unlabeled E1S in the medium (P<0.05). In additon, both the uptake and efflux of [14C]p-aminohippuric acid (PAH) and [14C]GA via S2 OAT4 were significantly trans-stimulated by unlabeled GA or PAH. The immunoreactivities of OAT4 were observed in the apical membrane of proximal tubules along with those of basolateral organic anion/dicarboxylate exchangers such as hOAT1 and hOAT3 in the same tubular population. These results indicate that OAT4 is an apical organic anion/dicarboxylate exchanger and mainly functions as an apical pathway for the reabsorption of some organic anions in renal proximal tubules driven by an outwardly directed dicarboxylate gradient.

The urate-anion exchanger URAT1 is a member of the organic anion transporter (OAT) family that regulates blood urate level in humans and is targeted by uricosuric and antiuricosuric agents (Enomoto, A., Kimura, H., Chairoungdua, A., Shigeta, Y., Jutabha, P., Cha, S. H., Hosoyamada, M., Takeda, M., Sekine, T., Igarashi, T., Matsuo, H., Kikuchi, Y., Oda, T., Ichida, K., Hosoya, T., Shimotaka, K., Niwa, T., Kanai, Y., and Endou, H. (2002) Nature 417, 447–452). URAT1 is expressed only in the kidney, where it is thought to participate in tubular urate reabsorption. We found that the multivalent PDZ (PSD-95, Drosophila discs-large protein, Zonula occludens protein 1) domain-containing protein, PDZK1 interacts with URAT1 in a yeast two-hybrid screen. Such an interaction requires the PDZ motif of URAT1 in its extreme intracellular C-terminal region and the first, second, and fourth PDZ domains of PDZK1 as identified by yeast two-hybrid assay, in vitro binding assay and surface plasmon resonance analysis (K_D = 1.97–514 nm). Coimmunoprecipitation studies revealed that the wild-type URAT1, but not its mutant lacking the PDZ-motif, directly interacts with PDZK1. Colocalization of URAT1 and PDZK1 was observed at the apical membrane of renal proximal tubular cells. The association of URAT1 with PDZK1 enhanced urate transport activities in HEK293 cells (1.4-fold), and the deletion of the URAT1 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by a significant increase in the V_max of urate transport via URAT1 and was associated with the increased surface expression level of URAT1 protein from HEK293 cells stably expressing URAT1 transfected with PDZK1. Taken together, the present study indicates the novel role of PDZK1 in regulating the functional activity of URAT1-mediated urate transport in the apical membrane of renal proximal tubules.