BACKGROUND: Liuwei dihuang pills is a famous Traditional Chinese Medicine with various anti-cancer properties. Over 50 pharmaceutical manufacturers produce Liuwei dihuang pills in China and an estimated millions of people around the world orally take it every day. D-glucaro-1,4-lactone (1,4-GL) was quantified to be about 12.0 mg/g in Liuwei dihuang pills and a primary bioactive component of it inhibiting the activity of β-glucuronidase in vivo. 1,4-GL can prevent and effectively inhibit various types of cancer. However, its exact mechanism of action remains unknown. The study would justify the traditional usage of Liuwei dihuang pills against cancers.
OBJECTIVE: 1,4-GL, a bioactive ingredient derived from Liuwei dihuang pills, a famous Traditional Chinese Medicine, could delay the progression of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in rats. The mechanism underpinning the effect, however, remains poorly understood.
METHODS: Healthy and HCC rats were treated with or without 1,4-GL (40.0 mg/kg) and 1HNMR-based metabonomic analysis was employed. 10 metabolites in uric acid pathway were quantitatively determined by UPLC-MS/MS. The expression of xanthine dehydrogenase (XDH), SLC2A9 mRNA, and SLC2A9 protein was determined using RT-qPCR and Western Blot. The effect of 1,4-GL on HCC-LM3 cells was verified in vitro. The alterations of ROS activity, SLC2A9 and XDH gene levels were observed in NCTC-1469 cells induced by lipopolysaccharide (LPS) after 1,4-GL treatment.
RESULTS: After the intervention of 1,4-GL, improved pathological morphology, liver lesions in HCC rats was observed with restored serum levels of AFP, AST, ALP, γ-GGT and Fisher\'s ratio. Hepatic metabonomics revealed that puring metabolism were significantly regulated by 1,4-GL in HCC rats. Uric acid, xanthine and hypoxanthine levels were quantified by UPLC-MS/MS and found to be nearly restored to control levels after 1,4-GL treatment in HCC rats. Changes in xanthine oxidase activity, XDH mRNA expression, and SLC2A9 mRNA and protein expression were also reversed. 1,4-GL treatment in LM3 HCC cells were consistent with the results in vivo. Furthermore, oxidative stress indicators such as T-SOD, GSH, CAT and MDA in serum and liver were improved after HCC rats treated with 1,4-GL. In vitro, 1,4-GL was observed to reduce lipopolysaccharide-induced ROS levels in NCTC-1469 cells with enhanced mRNA and protein expression of SLC2A9 and decreased mRNA level of XDH.
CONCLUSIONS: The protective effects of 1,4-GL against DEN-induced HCC by reducing uric acid and ROS levels due to down-regulation of uric acid production and up-regulation of SLC2A9 expressions. 1,4-GL may represent a novel treatment that improves recovery from HCC by targeting uric acid-ROS pathway.

Gout results from monosodium urate deposition caused by hyperuricemia, but most individuals with hyperuricemia remain asymptomatic. The pathogenesis of gout remains uncertain. To identify potential biomarkers distinguishing gout from asymptomatic hyperuricemia, we conducted a genetic analysis of urate transporters and metabolomic analysis as a proof-of-concept study, including 33 patients with gout and 9 individuals with asymptomatic hyperuricemia. The variant allele frequencies of rs72552713, rs2231142, and rs3733591, which are related to serum urate levels (SUA) and gout, did not differ between the gout and asymptomatic hyperuricemia groups. In metabolomic analysis, the levels of citrate cycle intermediates, especially 2-ketoglutarate, were higher in patients with gout than in those with asymptomatic hyperuricemia (fold difference = 1.415, p = 0.039). The impact on the TCA cycle was further emphasized in high-risk gout (SUA ≥ 9.0 mg/dL). Of note, urinary nicotinate was the most prominent biomarker differentiating high-risk gout from asymptomatic hyperuricemia (fold difference = 6.515, p = 0.020). Although urate transporters play critical roles in SUA elevation and promote hyperuricemia, this study suggests that the progression from asymptomatic hyperuricemia to gout might be closely related to other genetic and/or environmental factors affecting carbohydrate metabolism and urinary urate excretion.

Renal hypouricemia (RHUC) is a rare hereditary disorder caused by loss-of-function mutations in the SLC22A12 (RHUC type 1) or SLC2A9 (RHUC type 2) genes, encoding urate transporters URAT1 and GLUT9, respectively, that reabsorb urate in the renal proximal tubule. The characteristics of this disorder are low serum urate levels, high renal fractional excretion of urate, and occasional severe complications such as nephrolithiasis and exercise-induced acute renal failure. In this study, we report two Spanish (Caucasian) siblings and a Pakistani boy with clinical characteristics compatible with RHUC. Whole-exome sequencing (WES) analysis identified two homozygous variants: a novel pathogenic SLC22A12 variant, c.1523G>A; p.(S508N), in the two Caucasian siblings and a previously reported SLC2A9 variant, c.646G>A; p.(G216R), in the Pakistani boy. Our findings suggest that these two mutations cause RHUC through loss of urate reabsorption and extend the SLC22A12 mutation spectrum. In addition, this work further emphasizes the importance of WES analysis in clinical settings.

High uric acid is associated with gout, hypertension, metabolic syndrome, cardiovascular disease, and kidney disease. URAT1 (SLC22A12), originally discovered in mice as Rst, is generally considered a very selective uric acid transporter compared to other closely-related kidney uric acid transporters such as OAT1 (SLC22A6, NKT) and OAT3 (SLC22A8). While the role of URAT1 in regulating human uric acid is well-established, in recent studies the gene has been linked to redox regulation in flies as well as progression of renal cell carcinoma. We have now identified over twenty metabolites in the Urat1 knockout that are generally distinct from metabolites accumulating in the Oat1 and Oat3 knockout mice, with distinct molecular properties as revealed by chemoinformatics and machine learning analysis. These metabolites are involved in seemingly disparate aspects of cellular metabolism, including pyrimidine, fatty acid, and amino acid metabolism. However, through integrative systems metabolic analysis of the transcriptomic and metabolomic data using a human metabolic reconstruction to build metabolic genome-scale models (GEMs), the cellular response to loss of Urat1/Rst revealed compensatory processes related to reactive oxygen species handling and maintaining redox state balances via Vitamin C metabolism and cofactor charging reactions. These observations are consistent with the increasingly appreciated role of the antioxidant properties of uric acid. Collectively, the results highlight the role of Urat1/Rst as a transporter strongly tied to maintaining redox homeostasis, with implications for metabolic side effects from drugs that block its function.

This study aimed to evaluate whether the single nucleotide polymorphisms of ATP-binding cassette subfamily G member 2 (ABCG2) and solute carrier family 2 member 9 (SLC2A9) affect individual blood uric acid levels using pyrosequencing. ABCG2 (rs2231142, rs72552713, rs2231137), SLC2A9 (rs3734553, rs3733591, rs16890979), and individual uric acid levels were prospectively analyzed in 250 healthy young Korean male participants. Prominent differences in uric acid levels of the alleles were observed in the SLC2A9 rs3733591 polymorphism: wild-type (AA) vs. heterozygote (AG), 0.7 mg/dL (p < 0.0001); AA vs. mutant type (GG), 1.32 mg/dL (p < 0.0001); and AG vs. GG, 0.62 mg/dL (p < 0.01). In ABCG2 single nucleotide polymorphisms (SNPs), the statistically significant differences in uric acid levels were only found in rs2231142 between CC vs. AA (1.06 mg/dL; p < 0.001), and CC vs. CA (0.59 mg/dL; p < 0.01). Serum uric acid levels based on the ABCG2 and SLC2A9 diplotype groups were also compared. The uric acid levels were the lowest in the CC/AA diplotype and highest in the AA/AG diplotype. In addition, the SNP SLC2A9 rs3733591 tended to increase the uric acid levels when the ABCG2 rs2231142 haplotypes were fixed. In conclusion, both the ABCG2 rs2231142 and SLC2A9 rs3733591 polymorphisms may additively elevate blood uric acid levels.

Gout is a common inflammatory arthritis caused by the deposition of sodium urate crystals in the joints. Hyperuricemia is the fundamental factor of gout. The onset of hyperuricemia is related to purine metabolism disorders or uric acid excretion disorders. Current studies have shown that the intestine is an important potential organ for the excretion of uric acid outside the kidneys. The excretion of uric acid of gut is mainly achieved through the action of uric acid transporters and the catabolism of intestinal flora, which plays an important role in the body\'s uric acid balance. Here we reviewed the effects of intestinal uric acid transporters and intestinal flora on uric acid excretion, and provide new ideas for the treatment of hyperuricemia and gout.

This review focuses on the post-genome-wide association study (GWAS) era in gout, i.e., the translation of GWAS genetic association signals into biologically informative knowledge. Analytical and experimental follow-up of individual loci, based on the identification of causal genetic variants, reveals molecular pathogenic pathways. We summarize in detail the largest GWAS in urate to date, then we review follow-up studies and molecular insights from ABCG2, HNF4A, PDZK1, MAF, GCKR, ALDH2, ALDH16A1, SLC22A12, SLC2A9, ABCC4, and SLC22A13, including the role of insulin signaling. One common factor in these pathways is the importance of transcriptional control, including the HNF4α transcription factor. The new molecular knowledge reveals new targets for intervention to manage urate levels and prevent gout.

Circulation of urate levels is determined by the balance between urate production and excretion, homeostasis regulated by the function of urate transporters in key epithelial tissues and cell types. Our understanding of these physiological processes and identification of the genes encoding the urate transporters has advanced significantly, leading to a greater ability to predict risk for urate-associated diseases and identify new therapeutics that directly target urate transport. Here, we review the identified urate transporters and their organization and function in the renal tubule, the intestinal enterocytes, and other important cell types to provide a fuller understanding of the complicated process of urate homeostasis and its role in human diseases. Furthermore, we review the genetic tools that provide an unbiased catalyst for transporter identification as well as discuss the role of transporters in determining the observed significant gender differences in urate-associated disease risk.

Renal hypouricemia is a rare genetic disorder. Hypouricemia can present as renal stones or exercise-induced acute renal failure, but most cases are asymptomatic. Our previous study showed that two recessive variants of SLC22A12 (p.Trp258*, pArg90His) were identified in 90% of the hypouricemia patients from two independent cohorts: the Korean genome and epidemiology study (KoGES) and the Korean Cancer Prevention Study (KCPS-II). In this work, we investigate the genetic causes of hypouricemia in the rest of the 10% of unsolved cases. We found a novel non-synonymous mutation of SLC2A9 (voltage-sensitive uric acid transporter) in the whole-exome sequencing (WES) results. Molecular dynamics prediction suggests that the novel mutation p.Met126Val in SLCA9b (p.Met155Val in SLC2A9a) hinders uric acid transport through a defect of the outward open geometry. Molecular analysis using Xenopus oocytes confirmed that the p.Met126Val mutation significantly reduced uric acid transport but does not affect the SLC2A9 protein expression level. Our results will shed light on a better understanding of SLC2A9-mediated uric acid transport and the development of a uric acid-lowering agent.

Replication studies showed conflicting effects of ABCG2 and SLC2A9 polymorphisms on gout and serum urate. This meta-analysis therefore aimed to pool their effects across studies.
Studies were located from MEDLINE and Scopus from inception to 17th June 2018. Observational studies in adults with any polymorphism in ABCG2 or SLC2A9, and outcome including gout, hyperuricemia, and serum urate were included for pooling. Data extractions were performed by two independent reviewers. Genotype effects were pooled stratified by ethnicity using a mixed-effect logistic model and a multivariate meta-analysis for dichotomous and continuous outcomes.
Fifty-two studies were included in the analysis. For ABCG2 polymorphisms, mainly studied in Asians, carrying 1-2 minor-allele-genotypes of rs2231142 and rs72552713 were respectively about 2.1-4.5 and 2.5-3.9 times higher odds of gout than non-minor-allele-genotypes. The two rs2231142-risk-genotypes also had higher serum urate about 11-18 μmol/l. Conversely, carrying 1-2 minor alleles of rs2231137 was about 36-57% significantly lower odds of gout. For SLC2A9 polymorphisms, mainly studied in Caucasians, carrying 1-2 minor alleles of rs1014290, rs6449213, rs6855911, and rs7442295 were about 25-43%, 31-62%, 33-64%, and 35-65% significantly lower odds of gout than non-minor-allele-genotypes. In addition, 1-2 minor-allele-genotypes of the latter three polymorphisms had significantly lower serum urate about 20-49, 21-51, and 18-54 μmol/l than non-minor-allele-genotypes.
Our findings should be useful in identifying patients at risk for gout and high serum urate and these polymorphisms may be useful in personalized risk scores.
PROSPERO registration number: CRD42018105275 .