ObjectiveA genome-wide association study (GWAS) of gout and its subtypes was performed to identify novel gout loci, including those that are subtype-specific.MethodsPutative causal association signals from a GWAS of 945 clinically defined gout cases and 1213 controls from Japanese males were replicated with 1396 cases and 1268 controls using a custom chip of 1961 single nucleotide polymorphisms (SNPs). We also first conducted GWASs of gout subtypes. Replication with Caucasian and New Zealand Polynesian samples was done to further validate the loci identified in this study.ResultsIn addition to the five loci we reported previously, further susceptibility loci were identified at a genome-wide significance level (p<5.0×10−8): urate transporter genes (SLC22A12 and SLC17A1) and HIST1H2BF-HIST1H4E for all gout cases, and NIPAL1 and FAM35A for the renal underexcretion gout subtype. While NIPAL1 encodes a magnesium transporter, functional analysis did not detect urate transport via NIPAL1, suggesting an indirect association with urate handling. Localisation analysis in the human kidney revealed expression of NIPAL1 and FAM35A mainly in the distal tubules, which suggests the involvement of the distal nephron in urate handling in humans. Clinically ascertained male patients with gout and controls of Caucasian and Polynesian ancestries were also genotyped, and FAM35A was associated with gout in all cases. A meta-analysis of the three populations revealed FAM35A to be associated with gout at a genome-wide level of significance (pmeta=3.58×10−8).ConclusionsOur findings including novel gout risk loci provide further understanding of the molecular pathogenesis of gout and lead to a novel concept for the therapeutic target of gout/hyperuricaemia.

Riboflavin transporter (RFT) 2 has recently been identified as a transporter that may be, mainly based on the functional characteristics of its rat ortholog (rRFT2), involved in the intestinal absorption of riboflavin. The present study was conducted to further examine such a possible role of RFT2, focusing on the functional characteristics of its human ortholog (hRFT2) and the response of rRFT2 expression in the small intestine to deprivation of dietary riboflavin. When transiently expressed in human embryonic kidney 293 cells, hRFT2 could transport riboflavin efficiently in a pH-sensitive manner, favoring acidic pH and without requiring Na(+). Riboflavin transport by hRFT2 was saturable with a Michaelis constant of 0.77 μmol/L at pH 6.0, and inhibited by some riboflavin derivatives, such as lumiflavin. It was also inhibited, to a lesser extent, by some cationic compounds, such as ethidium. Thus, hRFT2 was suggested to, together with a finding that its mRNA is highly expressed in the small intestine, have characteristics as an intestinal RFT. Furthermore, feeding rats a riboflavin-deficient diet caused an upregulation of the expression of rRFT2 mRNA in the small intestine, presumably as an adaptive response to enhance riboflavin absorption, which would involve rRFT2, and its apically localized characteristic was suggested by the observation of rRFT2 tagged with green fluorescent protein stably expressed in polarized Madin-Darby canine kidney II cells. All these results combined indicate that RFT2 is a transporter involved in the epithelial uptake of riboflavin in the small intestine for its nutritional utilization.

Licorice-induced pseudoaldosteronism is a common adverse effect in traditional Japanese Kampo medicine, and 3-monoglucuronyl glycyrrhetinic acid (3MGA) was considered as a causative agent of it. Previously, we found 22α-hydroxy-18β-glycyrrhetyl-3-O-sulfate-30-glucuronide (1), one of the metabolites of glycyrrhizin (GL) in the urine of Eisai hyperbilirubinuria rats (EHBRs) treated with glycyrrhetinic acid (GA), and suggested that it is also a possible causative agent of pseudoaldosteronism. The discovery of 1 also suggested that there might be other metabolites of GA as causal candidates. In this study, we found 22α-hydroxy-18β-glycyrrhetyl-3-O-sulfate (2) and 18β-glycyrrhetyl-3-O-sulfate (3) in EHBRs’ urine. 2 and 3 more strongly inhibited rat type 2 11β-hydroxysteroid dehydrogenase than 1 did in vitro. When EHBRs were orally treated with GA, GA and 1–3 in plasma and 1–3 in urine were detected; the levels of 3MGA were quite low. 2 and 3 were shown to be the substrates of organic anion transporter (OAT) 1 and OAT3. In the plasma of a patient suffering from pseudoaldosteronism with rhabdomyolysis due to licorice, we found 8.6 µM of 3, 1.3 µM of GA, and 87 nM of 2, but 1, GL, and 3MGA were not detected. These findings suggest that 18β-glycyrrhetyl-3-O-sulfate (3) is an alternative causative agent of pseudoaldosteronism, rather than 3MGA and 1.

The purine salvage pathway plays a major role in the nucleotide production, relying on the supply of nucleobases and nucleosides from extracellular sources. Although specific transporters have been suggested to be involved in facilitating their transport across the plasma membrane in mammals, those which are specifically responsible for utilization of extracellular nucleobases remain unknown. Here we present the molecular and functional characterization of SLC43A3, an orphan transporter belonging to an amino acid transporter family, as a purine-selective nucleobase transporter. SLC43A3 was highly expressed in the liver, where it was localized to the sinusoidal membrane of hepatocytes, and the lung. In addition, SLC43A3 expressed in MDCKII cells mediated the uptake of purine nucleobases such as adenine, guanine, and hypoxanthine without requiring typical driving ions such as Na+ and H+, but it did not mediate the uptake of nucleosides. When SLC43A3 was expressed in APRT/HPRT1-deficient A9 cells, adenine uptake was found to be low. However, it was markedly enhanced by the introduction of SLC43A3 with APRT. In HeLa cells, knock-down of SLC43A3 markedly decreased adenine uptake. These data suggest that SLC43A3 is a facilitative and purine-selective nucleobase transporter that mediates the cellular uptake of extracellular purine nucleobases in cooperation with salvage enzymes.

Pseudoaldosteronism is a common adverse effect associated with traditional Japanese Kampo medicines. The pathogenesis is mainly caused by 3-monoglucuronyl glycyrrhetinic acid (3MGA), one of the metabolites of glycyrrhizin (GL) contained in licorice. We developed an anti-3MGA monoclonal antibody (MAb) and an ELISA system to easily detect 3MGA in the plasma and urine of the patients. However, we found that some metabolites of GL cross-reacted with this MAb. Mrp2-deficient Eisai Hyperbilirubinemia rats (EHBRs) were administered glycyrrhetinic acid (GA), and we isolated 22α-hydroxy-18β-glycyrrhetyl-3-O-sulfate-30-glucuronide (1) from the pooled urine with the guidance of positive immunostaining of eastern blot as the new metabolite of GL. The IC50 of 1 for type 2 11β-hydroxysteroid dehydrogenase (11β-HSD2) was 2.0 µM. Similar plasma concentrations of 1 and GA were observed 12 h after oral administration of GA to EHBR. Compound 1 was eliminated via urine, whereas GA was not. In Sprague–Dawley (SD) rats orally treated with GA, compound 1 was absent from both the plasma and the urine. Compound 1 was actively transported into cells via OAT1 and OAT3, whereas GA was not. Compound 1, when produced in Mrp2-deficiency, represents a potential causative agent of pseudoaldosteronism, and might be used as a biomarker to prevent the adverse effect.