Urate, a naturally occurring product of purine metabolism, is a scavenger of biological oxidants implicated in numerous disease processes, as demonstrated by its capacity of neuroprotection. It is present at higher levels in human blood (200 500 microM) than in other mammals, because humans have an effective renal urate reabsorption system, despite their evolutionary loss of hepatic uricase by mutational silencing. The molecular basis for urate handling in the human kidney remains unclear because of difficulties in understanding diverse urate transport systems and species differences. Here we identify the long-hypothesized urate transporter in the human kidney (URAT1, encoded by SLC22A12), a urate anion exchanger regulating blood urate levels and targeted by uricosuric and antiuricosuric agents (which affect excretion of uric acid). Moreover, we provide evidence that patients with idiopathic renal hypouricaemia (lack of blood uric acid) have defects in SLC22A12. Identification of URAT1 should provide insights into the nature of urate homeostasis, as well as lead to the development of better agents against hyperuricaemia, a disadvantage concomitant with human evolution.

ABCG2, also known as BCRP, is a high-capacity urate exporter, the dysfunction of which raises gout/hyperuricemia risk. Generally, hyperuricemia has been classified into urate 'overproduction type' and/or 'underexcretion type' based solely on renal urate excretion, without considering an extra-renal pathway. Here we show that decreased extra-renal urate excretion caused by ABCG2 dysfunction is a common mechanism of hyperuricemia. Clinical parameters, including urinary urate excretion, are examined in 644 male outpatients with hyperuricemia. Paradoxically, ABCG2 export dysfunction significantly increases urinary urate excretion and risk ratio of urate overproduction. Abcg2-knockout mice show increased serum uric acid levels and renal urate excretion, and decreased intestinal urate excretion. Together with high ABCG2 expression in extra-renal tissues, our data suggest that the 'overproduction type' in the current concept of hyperuricemia be renamed 'renal overload type', which consists of two subtypes—'extra-renal urate underexcretion' and genuine 'urate overproduction'—providing a new concept valuable for the treatment of hyperuricemia and gout.

Gout based on hyperuricemia is a common disease with a genetic predisposition, which causes acute arthritis. The ABCG2/BCRP gene, located in a gout-susceptibility locus on chromosome 4q, has been identified by recent genome-wide association studies of serum uric acid concentrations and gout. Urate transport assays demonstrated that ABCG2 is a high-capacity urate secretion transporter. Sequencing of the ABCG2 gene in 90 hyperuricemia patients revealed several nonfunctional ABCG2 mutations, including Q126X. Quantitative trait locus analysis of 739 individuals showed that a common dysfunctional variant of ABCG2, Q141K, increases serum uric acid. Q126X is assigned to the different disease haplotype from Q141K and increases gout risk, conferring an odds ratio of 5.97. Furthermore, 10% of gout patients (16 out of 159 cases) had genotype combinations resulting in more than 75% reduction of ABCG2 function (odds ratio, 25.8). Our findings indicate that nonfunctional variants of ABCG2 essentially block gut and renal urate excretion and cause gout.

Abstract. Renal hypouricemia is an inherited and heterogeneous disorder characterized by increased urate clearance (CUA). The authors recently established that urate was reabsorbed via URAT1 on the tubular apical membrane and that mutations in SLC22A12 encoding URAT1 cause renal hypouricemia. This study was undertaken to elucidate and correlate clinical and genetic features of renal hypouricemia. The SLC22A12 gene was sequenced in 32 unrelated idiopathic renal hypouricemia patients, and the relationships of serum urate levels, and CUA/creatinine clearance (Ccr) to SLC22A12 genotype were examined. Uricosuric (probenecid and benzbromarone) and anti-uricosuric drug (pyrazinamide) loading tests were also performed in some patients. Three patients had exercise-induced acute renal failure (9.4%), and four patients had urolithiasis (12.5%). The authors identified eight new mutations and two previously reported mutations that result in loss of function. Thirty patients had SLC22A12 mutations; 24 homozygotes and compound heterozygotes, and 6 heterozygotes. Mutation G774A dominated SLC22A12 mutations (74.1% in 54 alleles). Serum urate levels were significantly lower and CUA/Ccr was significantly higher in heterozygotes compared with healthy subjects; these changes were even more significant in homozygotes and compound heterozygotes. These CUA/Ccr relations demonstrated a gene dosage effect that corresponds with the difference in serum urate levels. In contrast to healthy subjects, the CUA/Ccr of patients with homozygous and compound heterozygous SLC22A12 mutations was unaffected by pyrazinamide, benzbromarone, and probenecid. The findings indicate that SLC22A12 was responsible for most renal hypouricemia and that URAT1 is the primary reabsorptive urate transporter, targeted by pyrazinamide, benzbromarone, and probenecid in vivo.Approximately 90% of all urate that is filtered through the glomerulus is eventually reabsorbed. A four-component hypothesis has been proposed to explain the renal urate transport mechanisms; it includes glomerular filtration, presecretory reabsorption, secretion, and postsecretory reabsorption (1,2). Renal hypouricemia is a common inherited and heterogeneous disorder characterized by impaired tubular urate transport (3). The incidence of renal hypouricemia has been reported to be 0.12 to 0.72% (4,5), and exercise-induced acute renal failure and nephrolithiasis have been reported as complications (6).Renal hypouricemia has been classified into the following five types according to responses to the anti-uricosuric drug pyrazinamide, and the uricosuric drug, probenecid: (a) a presecretory reabsorptive defect with an attenuated response to both pyrazinamide and probenecid (3); (b) a post-secretory reabsorptive defect when pyrazinamide suppressible urate clearance (CUA) is not influenced by probenecid (7); (c) total inhibition of urate reabsorption when pyrazinamide induces elimination of CUA exceeding the rate of glomerular filtration (8); (d) enhanced secretion when the pyrazinamide suppressible CUA ...

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 M). 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).Urate (uric acid), an end product of purine metabolism in humans because of the genetic silencing of hepatic uricase, is now recognized as a natural antioxidant that has neuroprotective properties (1). Despite its beneficial role, elevation of the serum urate level is correlated with gout, hypertension, and cardiovascular and renal diseases (1, 2). The kidney plays a dominant role in maintaining plasma urate levels through the excretion process; it eliminates ϳ70% of the daily urate production (3). Therefore, it is important to understand the mechanism of renal urate handling because underexcretion of urate has been demonstrated in the majority of hyperuricemia patients (4).Since urate is a weak acid at physiological pH (pK a , 5.75), it hardly permeates the plasma membrane of cells in the absence of transport proteins (3). In 2002, we identified a long hypothesized urate-anion exchanger, URAT1, 2 encoded by SLC22A12, that localized on the apical side of the renal proximal tubule (5). Despite several potential candidate proteins for urate transport such as UAT (uric acid transporter), OAT1 (organic anionic transporter 1), OAT3, OAT4, OATv1/NPT1 (sodium phosphate transporter 1), MRP4 (multidrug resistance-associated protein), and OAT10 (6 -10), URAT1 is the sole transporter whose physiological role in renal urate reabsorption is established, based on the fact that lossof-function mutations in URAT1 cause renal hypouricemia (5). However, it is not known how urate taken up via URAT1 exits from the tubular cell (11). Moreover, there are patients with renal hypouricemia who had no...

Xanthine oxidase (oxidoreductase; XOR) and aldehyde oxidase (AO) are similar in protein structure and prosthetic group composition, but differ in substrate preference. Here we show that mutation of two amino acid residues in the active site of human XOR for purine substrates results in conversion of the substrate preference to AO type. Human XOR and its Glu803-to-valine (E803V) and Arg881-to-methionine (R881M) mutants were expressed in an Escherichia coli system. The E803V mutation almost completely abrogated the activity towards hypoxanthine as a substrate, but very weak activity towards xanthine remained. On the other hand, the R881M mutant lacked activity towards xanthine, but retained slight activity towards hypoxanthine. Both mutants, however, exhibited significant aldehyde oxidase activity. The crystal structure of E803V mutant of human XOR was determined at 2.6 A resolution. The overall molybdopterin domain structure of this mutant closely resembles that of bovine milk XOR; amino acid residues in the active centre pocket are situated at very similar positions and in similar orientations, except that Glu803 was replaced by valine, indicating that the decrease in activity towards purine substrate is not due to large conformational change in the mutant enzyme. Unlike wild-type XOR, the mutants were not subject to time-dependent inhibition by allopurinol.

ObjectiveGout, caused by hyperuricaemia, is a multifactorial disease. Although genome-wide association studies (GWASs) of gout have been reported, they included self-reported gout cases in which clinical information was insufficient. Therefore, the relationship between genetic variation and clinical subtypes of gout remains unclear. Here, we first performed a GWAS of clinically defined gout cases only.MethodsA GWAS was conducted with 945 patients with clinically defined gout and 1213 controls in a Japanese male population, followed by replication study of 1048 clinically defined cases and 1334 controls.ResultsFive gout susceptibility loci were identified at the genome-wide significance level (p<5.0×10−8), which contained well-known urate transporter genes (ABCG2 and SLC2A9) and additional genes: rs1260326 (p=1.9×10−12; OR=1.36) of GCKR (a gene for glucose and lipid metabolism), rs2188380 (p=1.6×10−23; OR=1.75) of MYL2-CUX2 (genes associated with cholesterol and diabetes mellitus) and rs4073582 (p=6.4×10−9; OR=1.66) of CNIH-2 (a gene for regulation of glutamate signalling). The latter two are identified as novel gout loci. Furthermore, among the identified single-nucleotide polymorphisms (SNPs), we demonstrated that the SNPs of ABCG2 and SLC2A9 were differentially associated with types of gout and clinical parameters underlying specific subtypes (renal underexcretion type and renal overload type). The effect of the risk allele of each SNP on clinical parameters showed significant linear relationships with the ratio of the case–control ORs for two distinct types of gout (r=0.96 [p=4.8×10−4] for urate clearance and r=0.96 [p=5.0×10−4] for urinary urate excretion).ConclusionsOur findings provide clues to better understand the pathogenesis of gout and will be useful for development of companion diagnostics.

Abstract. Mouse renal-specific transporter (RST) cDNA, the amino acid sequence of which has 74% identity with that of human urate transporter 1 (hURAT1), is potentially the mouse homologue of hURAT1, the gene responsible for hereditary renal hypouricemia. The aim of this study is to determine the location and characteristics of RST molecule in mouse kidney and investigate urate transport by RST using the Xenopus oocyte expression system. RST transported 14 C-urate in a Michaelis-Menten manner. The K m and the V max values of RST-dependent urate transport were 1213 Ϯ 222 M and 268.8 Ϯ 38.0 pmol/oocyte per hr, respectively (n ϭ 3). RSTdependent urate transport was cis-inhibited significantly by 1 mM probenecid (68.7 Ϯ 9.4%), 50 M benzbromarone (67.9 Ϯ 6.4%), and 10 mM lactate (50.9 Ϯ 9.5%). However, 1 mM p-aminohippurate (PAH), 1 mM xanthine, and 1 mM oxonate did not inhibit RST-dependent urate transport. Substitution of Cl anion with gluconate in the external solution enhanced RST-dependent urate transport. Pre-injected pyrazinoic acid (PZA) or L-lactate trans-stimulated RST-dependent urate transport. Using immunohistochemistry for mouse kidney, the brush border or intracellular membrane of proximal tubules was stained by an affinity-purified antibody that recognized mouse URAT1 (mURAT1) expressed on Xenopus oocyte. Using Western blotting, anti-mURAT1 antibody detected 70-kD and 62-kD protein bands. The 70-kD protein was N-glycosylated and was identified as a Triton X-100 insoluble brush border membrane protein. RST mRNA and protein levels were higher in male kidneys than female. RST transported urate similar to hURAT1 and, therefore, appears to be mURAT1-the mouse homologue of hURAT1.Urate is metabolized by uricase for most mammals, and is an intermediate product of purine metabolism. Urate becomes the end product of purine metabolism for higher primates who have lost uricase activity. Therefore, it is important to understand urate handling mechanisms in the kidney because the underexcretion of urate has been implicated in the development of hyperuricemia that leads to gout. However, urate handling mechanisms are complicated; in that, urate is transported bidirectionally, being both reabsorbed and secreted in the kidney. Moreover, the differences in renal urate transport among different species have made it difficult to analyze urate handling mechanisms in the kidney. Nevertheless, renal urate transport in various animals has been investigated for comparison and to elucidate the evolution of renal urate handling mechanisms. Pigs and rabbits excrete more urate than is filtered through the glomerulus. Birds, like humans, have lost uricase activity. However, birds don't reabsorb urate in the kidney (1). In contrast, rats and mice reabsorb urate in their kidney, like humans, although uricase maintains their plasma urate at a lower level. The cloning and characterization of the urate transporter from mice is significant for understanding urate handling in the human kidney because the renal transport system of urate i...