Urate Uptake Research Articles

Hereditary Renal Hypouricemia Type 1 and Autosomal Dominant Polycystic Kidney Disease

BackgroundRenal hypouricemia (RHUC) is a heterogeneous inherited disorder characterized by impaired tubular uric acid (UA) transport with severe complications, such as acute kidney injury. Type 1 is caused by a mutation in the SLC22A12 gene (URAT1) and type 2 in the SLC2A9 gene (GLUT9). In this article, the authors present a coexpression functional characterization of variants responsible for RHUC type 1 in a Czech family with polycystic kidney disease (PKD). MethodsThe serum UA concentration in the proband was 1.1 mg/dL and was expressed as an increase in the fractional excretion of UA (43%). The URAT1 allelic variants were prepared and their urate uptake and subcellular localization were studied by Xenopus oocytes expression system. Linkage analysis using a panel of 4 CA-repeat flanking markers for chromosome 16 and a panel of 3 CA-repeat flanking markers for chromosome 4 was performed, which carry the PKD1 and PKD2 genes, respectively. ResultsCoexpression results showed that p.G366R and p.R477H suppressed urate transport by wild types. Colocalization studies showed an accumulation of URAT1 in the endoplasmic reticulum of the p.G366R variant and mainly retention of wild-type protein by variants p.G366R and p.R477H. ConclusionsThe findings suggest that not only loss-of-function mutation of URAT1 but also the dominant-negative effect cause RHUC through loss of UA absorption, partly due to protein misfolding caused by accumulation of URAT1 protein in the endoplasmic reticulum.

Complex analysis of urate transporters SLC2A9, SLC22A12 and functional characterization of non-synonymous allelic variants of GLUT9 in the Czech population: no evidence of effect on hyperuricemia and gout.

ObjectiveUsing European descent Czech populations, we performed a study of SLC2A9 and SLC22A12 genes previously identified as being associated with serum uric acid concentrations and gout. This is the first study of the impact of non-synonymous allelic variants on the function of GLUT9 except for patients suffering from renal hypouricemia type 2.MethodsThe cohort consisted of 250 individuals (150 controls, 54 nonspecific hyperuricemics and 46 primary gout and/or hyperuricemia subjects). We analyzed 13 exons of SLC2A9 (GLUT9 variant 1 and GLUT9 variant 2) and 10 exons of SLC22A12 by PCR amplification and sequenced directly. Allelic variants were prepared and their urate uptake and subcellular localization were studied by Xenopus oocytes expression system. The functional studies were analyzed using the non-parametric Wilcoxon and Kruskall-Wallis tests; the association study used the Fisher exact test and linear regression approach.ResultsWe identified a total of 52 sequence variants (12 unpublished). Eight non-synonymous allelic variants were found only in SLC2A9: rs6820230, rs2276961, rs144196049, rs112404957, rs73225891, rs16890979, rs3733591 and rs2280205. None of these variants showed any significant difference in the expression of GLUT9 and in urate transport. In the association study, eight variants showed a possible association with hyperuricemia. However, seven of these were in introns and the one exon located variant, rs7932775, did not show a statistically significant association with serum uric acid concentration.ConclusionOur results did not confirm any effect of SLC22A12 and SLC2A9 variants on serum uric acid concentration. Our complex approach using association analysis together with functional and immunohistochemical characterization of non-synonymous allelic variants did not show any influence on expression, subcellular localization and urate uptake of GLUT9.

Hydrophobic residues and amino terminal phosphorylation site are essential for urate/urate exchange mediated by human glucose transporter 9, hSLC2A9 (908.10)

Human urate homeostasis is achieved primarily in liver and kidney, where urate is significantly regulated by human SLC2A9a and 9b. These transporters show trans‐acceleration between urate and hexoses, and urate/urate; however, the mechanism has not been identified.Our preliminary data suggested that phosphorylation of hSLC2A9 is an important process for urate transport regulation and serine 9 has been postulated to be an important phosphorylation site in the N‐terminus of 9a. Objective:To investigate urate/urate trans‐acceleration mechanism mediated by9a,9b and their mutants. Methods: hSLC2A9 isoforms and mutants were over expressed in X. laevis oocytes. Urate/urate trans‐stimulation activities were determined by radiolabelled urate uptake and efflux measurements. Membrane protein expression levels were determined by Biotinylation and Western Blot analysis. Results:Urate trans‐stimulated urate flux and efflux in wild type hSLC2A9a and 9b and most of their mutants, except S9A and W110A in the predicted N‐terminal phosphorylation site and TM7 of 9a, respectively. This result suggests that substitution of alanine at S9 and W110, removes the asymmetry of the transporter, which normally allows for trans‐acceleration. These finding advance our understanding of how trans‐stimulation contributes to the regulation of urate and hexoses by hSLC2A9.Grant Funding Source: CIHR

How Does Whisky Lower Serum Urate Level?

Clinical studies have shown that moderate whisky consumption increased renal excretion of urate into urine and decreased serum urate level, but the mechanism involved has not been established. Because renal reabsorption influences serum urate level, the effects of the whisky congeners on urate transporters, urate transporter 1 (URAT1), and voltage-driven urate transporter (URATv1) involved in reabsorptive transport of urate were examined. In transporter-expressing Xenopus oocytes, 12-year-old and 18-year-old whisky congeners inhibited urate uptake by URAT1 with IC50 values of 0.08 ± 0.01 and 0.04 ± 0.01 mg/mL, respectively, while urate uptake by URATv1 was inhibited only at 1 mg/mL. Decreased serum urate level after whisky consumption may be mainly due to inhibition of URAT1 by the congeners.

Hydrophobic residues as a key determinant in hexoses exchange urate mechanism mediated by human glucose transporter 9, hSLC2A9

Human GLUT9 (hSLC2A9) is a urate/hexose transporter expressed predominantly in the liver and kidney where human urate homeostasis is achieved. hSLC2A9 has two splice variants: 9a and 9b differing in their N‐termini and targeted differentially within polarized epithelia. The transporter shows trans‐acceleration between urate and hexoses, however, the mechanism is unknown. Hydrophobic residues in TM7 of GLUTs have been shown to play a role in hexose selectivity.ObjectiveTo investigate urate/hexose exchange mediated by SLC2A9 isoforms and their mutants.MethodshSLC2A9 isoforms and I335V mutants were overexpressed in X. laevis oocytes. Urate induced currents and I‐V curves were obtained using Two Micro‐Electrode Voltage Clamp (TEVC) recordings. Urate transport and hexose trans‐stimulation activities were determined by radiolabelled urate uptake and efflux measurements.ResultsTEVC studies demonstrated that the membrane potential plays a major role in urate transport mediated by hSLC2A9. Fructose was found to trans‐stimulate urate uptake and efflux in wild type hSLC2A9a; whereas it stimulates only urate efflux in the I335V mutant. These finding advance our understanding of how fructose intake is linked with serum urate levels and implicates hSLC2A9 as an important transporter that may contribute to pathophysiological conditions involving urate or fructose. Supported by CIHR.

Novel allelic variants and evidence for a prevalent mutation in URAT1 causing renal hypouricemia: biochemical, genetics and functional analysis

Renal hypouricemia (RHUC) is a heterogeneous inherited disorder characterized by impaired tubular uric acid (UA) transport with severe complications, such as acute kidney injury (AKI). Type 1 is caused by a loss-of-function mutation in the SLC22A12 gene (URAT1), type 2 in the SLC2A9 gene (GLUT9). This article describes three Czech families with RHUC type 1. The serum UA in the probands was 0.9, 1.1 and 0.5 mg/dl and expressed as an increase in the fractional excretion of UA (48, 43 and 39%). The sequencing analysis of SLC22A12 revealed three novel variants: p.G366R, p.T467M and a deletion p.L415_G417del. A detailed metabolic investigation in proband C for progressive visual failure supported suspicion of neuronal ceroid lipofuscinosis type 7 conditioned by the mutation in the MFSD8 gene. Functional studies showed significantly decreased urate uptake and a mis-localized URAT1 signal in p.G366R, p.L415_G417del and p.T467M. Furthermore, colocalization studies showed accumulation of URAT1 protein in the endoplasmic reticulum. The findings suggest that loss-of-function mutations cause RHUC via loss of UA absorption partly by protein misfolding. However, they do not necessarily lead to AKI and a possible genotype-phenotype correlation was not proposed. Furthermore, results confirm an uneven geographical and ethnic distribution of SLC22A12 variants; the p.L415_G417del mutation predominates in the Roma ethnic group in the Czech Republic.

ウリカーゼ融合タンパク質を用いた細胞内尿酸検出法の開発

近年同定された腎臓に存在する尿酸トランスポーターは，尿酸の分泌・再吸収に関係することから，血清尿酸値を規定する存在として注目されている．既存のトランスポーター評価法には[14C]標識尿酸を用いた方法があるが，細胞を破砕した試料を使用するため，経時的な尿酸輸送評価などは不可能である．そこで本研究では，生細胞において経時的に細胞内尿酸を検出可能なプローブを開発することを目的として，uricase-HyPer融合タンパク質を構築した．ここでは，尿酸が尿酸代謝酵素uricaseにより酸化される際の副生成物である過酸化水素に着目した．過酸化水素と反応し蛍光を変化させるタンパク質HyPerとuricaseを融合させることにより，uricaseによる尿酸の酸化時にHyPerが発する蛍光によって尿酸を検出することが可能なツールを構築した．結果，URAT1を恒常的に発現するCOS7細胞にuricase-HyPer融合タンパク質を強制発現させると，10-200μM尿酸添加に伴い有意に蛍光強度比が上昇することが示された．さらに尿酸と共にトランスポーター阻害薬ベンズブロマロンを0.1-10μMとなるよう添加すると，その蛍光強度比は有意に低下した．これらのことから，uricase-HyPer融合タンパク質によって尿酸トランスポーターを介した尿酸取り込みの蛍光検出が可能であることが示された．本手法は経時的な尿酸量の変化を検出でき，かつ簡便に多検体処理が可能であることから，トランスポーター阻害薬の新規スクリーニングなどに有用であると考えられる．

Human SLC2A9a and SLC2A9b isoforms mediate electrogenic transport of urate with different characteristics in the presence of hexoses

Human SLC2A9 (GLUT9) is a novel high-capacity urate transporter belonging to the facilitated glucose transporter family. In the present study, heterologous expression in Xenopus oocytes has allowed us to undertake an in-depth radiotracer flux and electrophysiological study of urate transport mediated by both isoforms of SLC2A9 (a and b). Addition of urate to SLC2A9-producing oocytes generated outward currents, indicating electrogenic transport. Urate transport by SLC2A9 was voltage dependent and independent of the Na(+) transmembrane gradient. Urate-induced outward currents were affected by the extracellular concentration of Cl(-), but there was no evidence for exchange of the two anions. [(14)C]urate flux studies under non-voltage-clamped conditions demonstrated symmetry of influx and efflux, suggesting that SLC2A9 functions in urate efflux driven primarily by the electrochemical gradient of the cell. Urate uptake in the presence of intracellular hexoses showed marked differences between the two isoforms, suggesting functional differences between the two splice variants. Finally, the permeant selectivity of SLC2A9 was examined by testing the ability to transport a panel of radiolabeled purine and pyrimidine nucleobases. SLC2A9 mediated the uptake of adenine in addition to urate, but did not function as a generalized nucleobase transporter. The differential expression pattern of the two isoforms of SLC2A9 in the human kidney's proximal convoluted tubule and its electrogenic transport of urate suggest that these transporters play key roles in the regulation of plasma urate levels and are therefore potentially important participants in hyperuricemia and hypouricemia.

Functional and structural regulation of urate handling by human SLC2A9 isoforms in the Xenopus laevis oocyte expression system

Human GLUT9 (SLC2A9) is a high capacity urate transporter, backed by genome‐wide association scans. This gene's SNPs coincide with urate pathology1. The protein is predominant in liver and kidney, main organs in human urate homeostasis. Two splice variants of the carrier, SLC2A9a (A) and SLC2A9b (B), which differ in their residue composition in the N‐terminus, target differently to cell membranes of polarized epithelium2. Hydrophobic residues in this family of transporters were shown to play a role in hexose selectivity3.OBJECTIVESTo investigate differences in urate transport function and selectivity by SLC2A9 variants, for uptake and efflux modes.METHODhSLC2A9 isoforms and appropriate mutants were overexpressed in X. laevis oocytes. Radiolabelled urate uptake and efflux was measured under varied conditions.PKC agonist affects (A)‐ but not (B)‐mediated urate uptake and efflux. Kinetic experiments reveal that both affinity and capacity of the transporter are altered, without changes in funcional surface expression of the protein. Elevation of intracellular cAMP altered (B)‐ but not (A)‐mediated urate uptake under specific conditions. These observations suggest that urate reabsorption (A) and urate secretion (B) may be differentially regulated in polarized epithelia. Furthermore, hydrophobic residues around exofacial vestibule may act as hexose and urate selectivity determinants.

Interactions of urate transporter URAT1 in human kidney with uricosuric drugs

Hyperuricaemia is a significant factor in a variety of diseases, including gout and cardiovascular diseases. The kidney plays a dominant role in maintaining plasma urate levels through the excretion process. Human renal urate transporter URAT1 is thought to be an essential molecule that mediates the reabsorption of urate on the apical side of the proximal tubule. In this study the pharmacological characteristics and clinical implications of URAT1 were elucidated. Madin-Darby canine kidney (MDCK) cells stably expressing URAT1 (MDCK-URAT1) were established and examined the interactions of URAT1 with various drugs such as benzbromarone and its metabolites including 6-hydroxybenzbromarone, angiotensin-converting enzyme inhibitors, non-steroidal anti-inflammatory drugs and urate transport inhibitors including E3040 and probenecid. MDCK-URAT1 cells exhibited a time- and dose-dependent increase in urate uptake, with a Km value of 570.7 µmol/L. When an URAT1-green fluorescent protein fusion protein construct was expressed in MDCK cells, the protein was sorted mainly to the apical side of the membrane. The drugs except for captoril dose-dependently inhibited urate uptake mediated by URAT1, with half maximal inhibitory concentration (IC(50) ) values ranging 0.05-716 µmol/L. Comparing these IC(50) values with intratubular concentrations of unbound drugs in humans, it is thought that URAT1 is a target molecule of uricosuric drugs, including 6-hydroxybenzbromarone, probenecid, indomethacin and salicylate, to inhibit urate reabsorption in vivo. In addition, a cell line that stably expressing URAT1 could be a useful tool for the development of uricosuric drugs.

URAT1 mutations cause renal hypouricemia type 1 in Iraqi Jews

Hereditary renal hypouricemia may be complicated by nephrolithiasis or exercise-induced acute renal failure. Most patients described so far are of Japanese origin and carry the truncating mutation W258X in the uric acid transporter URAT1 encoded by SLC22A12. Recently, we described severe renal hypouricemia in Israeli patients with uric acid transporter GLUT9 (SLC2A9) loss-of-function mutations. Renal hypouricemia in Iraqi Jews has been previously reported, but its molecular basis has not been ascertained. Three Jewish Israeli families of Iraqi origin with hereditary hypouricemia and hyperuricosuria were clinically characterized. DNA was extracted and the URAT1 gene was sequenced. Transport studies into Xenopus laevis oocytes were utilized to evaluate the function of URAT1 mutants found. A missense URAT1 mutation, R406C, was detected in all three families. Two affected siblings were found to carry in addition a homozygous missense URAT1 mutation, G444R. Both mutations dramatically impaired urate uptake into X. laevis oocytes. Moreover, we demonstrate for the first time that URAT1 facilitates urate efflux, which was abolished in the mutants, indicating also a secretion defect. Homozygous patients had serum uric acid concentrations of 0.5-0.8 mg% and a fractional excretion of uric acid of 50-85%. Most individuals studied were asymptomatic, two had nephrolithiasis and none developed exercise-induced acute renal failure. The URAT1 R406C mutation detected in all three families is likely to be the founder mutation in Iraqi Jews. Our findings contribute to a better definition of the different types of hereditary renal hypouricemia and suggest that the phenotype of this disorder depends mainly on the degree of inhibition of uric acid transport.

Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.

Characterization of Cellular Uptake of Perfluorooctanoate via Organic Anion-Transporting Polypeptide 1A2, Organic Anion Transporter 4, and Urate Transporter 1 for Their Potential Roles in Mediating Human Renal Reabsorption of Perfluorocarboxylates

It has been hypothesized that human renal apical membrane transporters play a key role in human renal reabsorption of perfluorooctanoate (PFO), which contributes to the long half-life of PFO in humans. In the present study, PFO uptake kinetics of human organic anion-transporting polypeptide (OATP) 1A2, organic anion transporter (OAT) 4, and urate transporter 1 (URAT1) in stably transfected cell lines was investigated. OAT4 and URAT1, but not OATP1A2, were shown to mediate saturable PFO cellular uptake. OAT4-mediated PFO uptake was stimulated by a low extracellular pH, which was evidenced as a lower Michaelis constant (K(m)) at pH 6 (172.3 ± 45.9μM) than that at pH 7.4 (310.3 ± 30.2μM). URAT1-mediated PFO uptake was greatly enhanced by an outward Cl(-) gradient, and its K(m) value was determined to be 64.1 ± 30.5μM in the absence of extracellular Cl(-). The inhibition of OATP1A2- or OAT4-mediated estrone-3-sulfate uptake or URAT1-mediated urate uptake has been compared for linear perfluorocarboxylates (PFCs) with carbon chain lengths from 4 to 12. A clear chain length-dependent inhibition was observed, suggesting that PFCs in general are substrates of OAT4 and URAT1 but with different levels of affinities to the transporters depending on their chain length. Our results suggest that OAT4 and URAT1 are key transporters in renal reabsorption of PFCs in humans and, as a result, may contribute significantly to the long half-life of PFO in humans.

Mouse GLUT9: evidences for a urate uniporter

GLUT9 (SLC2A9) is a newly described urate transporter whose function, characteristics, and localization have just started to be elucidated. Some transport properties of human GLUT9 have been studied in the Xenopus laevis oocyte expression system, but the type of transport (uniport, coupled transport system, stoichiometry ... .) is still largely unknown. We used the same experimental system to characterize in more detail the transport properties of mouse GLUT9, its sensitivity to several uricosuric drugs, and the specificities of two splice variants, mGLUT9a and mGLUT9b. [(14)C]urate uptake measurements show that both splice variants are high-capacity urate transporters and have a K(m) of approximately 650 microM. The well-known uricosuric agents benzbromarone (500 microM) and losartan (1 mM) inhibit GLUT9-mediated urate uptake by 90 and 50%, respectively. Surprisingly, phloretin, a glucose-transporter blocker, inhibits [(14)C]urate uptake by approximately 50% at 1 mM. Electrophysiological measurements suggest that urate transport by mouse GLUT9 is electrogenic and voltage dependent, but independent of the Na(+) and Cl(-) transmembrane gradients. Taken together, our results suggest that GLUT9 works as a urate (anion) uniporter. Finally, we show by RT-PCR performed on RNA from mouse kidney microdissected tubules that GLUT9a is expressed at low levels in proximal tubules, while GLUT9b is specifically expressed in distal convoluted and connecting tubules. Expression of mouse GLUT9 in the kidney differs from that of human GLUT9, which could account for species differences in urate handling.

Plasma Urate Level Is Directly Regulated by a Voltage-driven Urate Efflux Transporter URATv1 (SLC2A9) in Humans

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 microm). 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; urate [corrected] 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).

SLC2A9 Is a High-Capacity Urate Transporter in Humans

BackgroundSerum uric acid levels in humans are influenced by diet, cellular breakdown, and renal elimination, and correlate with blood pressure, metabolic syndrome, diabetes, gout, and cardiovascular disease. Recent genome-wide association scans have found common genetic variants of SLC2A9 to be associated with increased serum urate level and gout. The SLC2A9 gene encodes a facilitative glucose transporter, and it has two splice variants that are highly expressed in the proximal nephron, a key site for urate handling in the kidney. We investigated whether SLC2A9 is a functional urate transporter that contributes to the longstanding association between urate and blood pressure in man.Methods and FindingsWe expressed both SLC2A9 splice variants in Xenopus laevis oocytes and found both isoforms mediate rapid urate fluxes at concentration ranges similar to physiological serum levels (200–500 μM). Because SLC2A9 is a known facilitative glucose transporter, we also tested whether glucose or fructose influenced urate transport. We found that urate is transported by SLC2A9 at rates 45- to 60-fold faster than glucose, and demonstrated that SLC2A9-mediated urate transport is facilitated by glucose and, to a lesser extent, fructose. In addition, transport is inhibited by the uricosuric benzbromarone in a dose-dependent manner (K i = 27 μM). Furthermore, we found urate uptake was at least 2-fold greater in human embryonic kidney (HEK) cells overexpressing SLC2A9 splice variants than nontransfected kidney cells. To confirm that our findings were due to SLC2A9, and not another urate transporter, we showed that urate transport was diminished by SLC2A9-targeted siRNA in a second mammalian cell line. In a cohort of men we showed that genetic variants of SLC2A9 are associated with reduced urinary urate clearance, which fits with common variation at SLC2A9 leading to increased serum urate. We found no evidence of association with hypertension (odds ratio 0.98, 95% confidence interval [CI] 0.9 to 1.05, p > 0.33) by meta-analysis of an SLC2A9 variant in six case–control studies including 11,897 participants. In a separate meta-analysis of four population studies including 11,629 participants we found no association of SLC2A9 with systolic (effect size −0.12 mm Hg, 95% CI −0.68 to 0.43, p = 0.664) or diastolic blood pressure (effect size −0.03 mm Hg, 95% CI −0.39 to 0.31, p = 0.82).ConclusionsThis study provides evidence that SLC2A9 splice variants act as high-capacity urate transporters and is one of the first functional characterisations of findings from genome-wide association scans. We did not find an association of the SLC2A9 gene with blood pressure in this study. Our findings suggest potential pathogenic mechanisms that could offer a new drug target for gout.

Identification of a New Urate and High Affinity Nicotinate Transporter, hOAT10 (SLC22A13)

The orphan transporter hORCTL3 (human organic cation transporter like 3; SLC22A13) is highly expressed in kidneys and to a weaker extent in brain, heart, and intestine. hORCTL3-expressing Xenopus laevis oocytes showed uptake of [(3)H]nicotinate, [(3)H]p-aminohippurate, and [(14)C]urate. Hence, hORCTL3 is an organic anion transporter, and we renamed it hOAT10. [(3)H]Nicotinate transport by hOAT10 into X. laevis oocytes and into Caco-2 cells was saturable with Michaelis constants (K(m)) of 22 and 44 microm, respectively, suggesting that hOAT10 may be the molecular equivalent of the postulated high affinity nicotinate transporter in kidneys and intestine. The pH dependence of hOAT10 suggests p-aminohippurate(-)/OH(-), urate(-)/OH(-), and nicotinate(-)/OH(-) exchange as possible transport modes. Urate inhibited [(3)H]nicotinate transport by hOAT10 with an IC(50) value of 759 microm, assuming that hOAT10 represents a low affinity urate transporter. hOAT10-mediated [(14)C]urate uptake was elevated by an exchange with l -lactate, pyrazinoate, and nicotinate. Surprisingly, we have detected urate(-)/glutathione exchange by hOAT10, consistent with an involvement of hOAT10 in the renal glutathione cycle. Uricosurics, diuretics, and cyclosporine A showed substantial interactions with hOAT10, of which cyclosporine A enhanced [(14)C]urate uptake, providing the first molecular evidence for cyclosporine A-induced hyperuricemia.

Functional analysis of phenolsulfonphthalein transport system in Long–Evans Cinnamon rats

It has been reported that the transport function for organic anions on the kidney is maintained in multidrug resistance-associated protein 2 (Mrp2)-deficient rats. Different from Mrp2-deficient rats, Long–Evans Cinnamon (LEC) rats have impaired urinary excretion of Mrp2-substrate, phenolsulfonphthalein (PSP). PSP is transported by the potential-sensitive urate transport system in rat brush-border membranes. We analyzed the function of PSP transport system in LEC rats. Unlike Long–Evans Agouti (LEA) rats, the initial uptake of PSP and urate into the renal brush-border membrane vesicles of LEC rats were not significantly enhanced in the presence of positive intravesicular potential, suggesting that the potential-sensitive urate transport system is impaired in LEC rats. LEC rats should be useful for elucidating the potential-sensitive urate transport system in rats at the molecular level.

New insights into renal transport of urate

This review focuses on recent progress in the understanding of various aspects of renal transport of urate. Since the molecular cloning of the renal apical urate/anion exchanger URAT1 (SLC22A12), several membrane proteins relevant to the transport of urate have been identified. The molecular identification of two sodium-coupled monocarboxylate transporters, SMCT1(SLC5A8) and SMCT2(SLC5A12), and the emerging role of PDZ (PSD-95, DglA, and ZO-1) scaffold for renal apical transporters have led to a new concept of renal urate transport: urate-transporting multimolecular complex, or 'urate transportsome', that may form an ultimate functional unit including the sodium-coupled urate transport system by linking URAT1 and sodium-coupled monocarboxylate transporters or the coordinated apical urate uptake system by balancing reabsorptive (URAT1) and efflux (NPT1/OATv1 and MRP4) transporters. In addition, genetic variations of the URAT1 gene are associated not only with idiopathic renal hypouricemia but also with reduced renal urate excretion. Although our knowledge of renal urate handling has been increased by the molecular identification of urate transport proteins and by results of genetic studies on patients with serum urate disorders, current evidence is insufficient to fully understand the precise mechanism governing the bi-directional transport of urate. Further studies are still necessary.

Morin (3,5,7,2′,4′-Pentahydroxyflavone) Exhibits Potent Inhibitory Actions on Urate Transport by the Human Urate Anion Transporter (hURAT1) Expressed in Human Embryonic Kidney Cells

In allopurinol-allergic patients, uricosuric agents are often used in the treatment of hyperuricemia. The existing uricosuric agents are not without problems and the availability of better and safer alternatives is highly desirable. Our previous study (J Pharmacol Exp Ther (2006) 316:169-175) has demonstrated that morin (3,5,7,2',4'-pentahydroxyflavone), which occurs in the twigs of Morus alba L. documented in traditional Chinese medicinal literature for treatment of conditions akin to gout, is a potent inhibitor of urate uptake in rat renal brush-border membrane vesicles. It is also effective in lowering uric acid level in a hyperuricemic rat model in vivo. Whether morin is an equally effective uricosuric agent in human requires verification. The human urate anion transporter (hURAT1) has recently been cloned and identified to be the organic anion transporter that mediates renal urate reabsorption in the human kidney. In the present investigation, human embryonic kidney cells were transfected with hURAT1 and the expression was validated by reverse transcription-polymerase chain reaction and subcellular distribution of the exogenously introduced transporter by confocal microscopy. The inhibitory actions of morin on human renal urate reabsorption were demonstrated using this system. The IC50 value of the inhibition by morin was determined to be 2.0 microM, compared with 50 microM for probenecid, 100 microM for sulfinpyrazone, and 0.3 microM for benzbromarone. Kinetic analysis of the uptake inhibition by morin indicates that this compound is a competitive inhibitor of urate uptake on the human urate transporter with a K(i) value of 5.74 microM.