Elevated serum urate levels cause gout, and correlate with cardio-metabolic diseases via poorly understood mechanisms. We performed a trans-ethnic genome-wide association study of serum urate among 457,690 individuals, identifying 183 loci (147 novel) that improve prediction of gout in an independent cohort of 334,880 individuals. Serum urate showed significant genetic correlations with many cardio-metabolic traits, with genetic causality analyses supporting a substantial role for pleiotropy. Enrichment analysis, fine-mapping of urateassociated loci and co-localization with gene expression in 47 tissues implicated kidney and liver as main target organs and prioritized potentially causal genes and variants, including the transcriptional master regulators in liver and kidney, HNF1A and HNF4A. Experimental validation showed that HNF4A trans-activated the promoter of the major urate transporter ABCG2 in kidney cells, and that HNF4A p.Thr139Ile is a functional variant. Transcriptional coregulation within and across organs may be a general mechanism underlying the observed pleiotropy between urate and cardio-metabolic traits.

The pathophysiological nature of the common ABCG2 gout and hyperuricemia associated variant Q141K (rs2231142) remains undefined. Here, we use a human interventional cohort study (ACTRN12615001302549) to understand the physiological role of ABCG2 and find that participants with the Q141K ABCG2 variant display elevated serum urate, unaltered FEUA, and significant evidence of reduced extra-renal urate excretion. We explore mechanisms by generating a mouse model of the orthologous Q140K Abcg2 variant and find male mice have significant hyperuricemia and metabolic alterations, but only subtle alterations of renal urate excretion and ABCG2 abundance. By contrast, these mice display a severe defect in ABCG2 abundance and function in the intestinal tract. These results suggest a tissue specific pathobiology of the Q141K variant, support an important role for ABCG2 in urate excretion in both the human kidney and intestinal tract, and provide insight into the importance of intestinal urate excretion for serum urate homeostasis.

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Elevated urate levels in the serum can contribute to the development of many diseases, including gout, the most prevalent form of inflammatory arthritis. Our previous work identified ABCG2 as a key urate transporter of the kidney and gut epithelium and its dysfunction significantly increases risk of hyperuricemia and gout. A new collaborative meta analysis of genome wide association studies, integrating the results from 457,690 individuals, identified 183 loci associated with serum urate. A further analysis of associated loci to identify independent and potentially causal variants created a credible set of variants for experimental follow up including variants in ABCG2, HNF1A, and HNF4A. HNF1A and HNF4A are transcription factors that are key regulators of liver, renal, and gut transport proteins. Here we sought to examine if HNF1A or HNF4A regulated the expression of ABCG2, and the effects of the identified HNF1A and HNF4A missense variants. To determine any direct regulation of ABCG2 by HNF1A or HNF4A, we used a luciferase assay reporter system where the ABCG2 promoter was used to drive expression of a luciferase reporter. HEK293T cells were co‐transfected with this ABCG2 construct, and plasmids expressing either HNF1A or HNF4A, as well as a GFP reporter used to determine cell number and transfection efficiency. HNF4A increases expression of ABCG2 driven luciferase in a dose dependent and significantly reproducible manner. No increase is observed with either HNF1A co‐transfection, or in the empty vector negative control. Next, the missense mutations were examined in the same luciferase assay for both HNF1A (A98V) and HNF4A (T139I). HNF4A‐T139I demonstrated an increase in ABCG2 luciferase expression to a significantly greater extent than the wild‐type protein, again in a dose dependent and reproducible manner. The increased ABCG2 promoter activation observed with the HNF4A‐T139I was not attributable to increased abundance of the mutant protein. In addition, the HNF4A‐T139I amino acid resides in an area critical for interacting with target DNA promoter regions. Our findings that HNF4A‐T139I is a gain of function variant resulting in increased abundance of ABCG2, the primary secretory transporter, is consistent with our observation that this variant associates with decreased serum urate levels in humans. Thus, we conclude that HNF4A is likely directly regulating expression of ABCG2, and thereby influencing serum urate levels.Support or Funding InformationNIDDK R01DK114091This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hyperuricemia causes gout and is implicated in metabolic syndrome, impaired glucose tolerance/type 2 diabetes, and hypertension. Individuals carrying a common loss of function variant in the dominant secretory transporter, Q141K ABCG2, have significantly increased serum urate and gout risk yet their renal fractional excretion of urate (FEUA) is largely unchanged. However, the role in ABCG2 and other urate transporters in extra‐renal secretion remains mostly unexplored. Here we sought to identify the role of ABCG2 in trans‐epithelial urate transport in the intestines and contribution of intestinal ABCG2 dysfunction to hyperuricemia and urate related phenotypes. First, we established a genetic mouse model of the human ABCG2 Q141K variant (Q140K in mouse) using CRISPR Cas9 gene editing techniques on a C57BL6 mouse background. As compared to control mice, mice possessing the Q140K Abcg2 allele were profoundly hyperuricemic, yet exhibited only modest changes in renal excretion of urate, similar to what has been reported previously in humans. To explore mechanisms, we first mapped prominent urate transporters ABCG2 and SLC2A9 along the mouse intestine and found the highest levels in the jejunum and ileum. Localization of the wild type ABCG2 protein was exclusive to the brush border (apical membrane) of the villus cells, seemingly optimized to facilitate urate secretion(JUAsm). Interestingly, we also found evidence that SLC2A9 localized predominantly to the basolateral enterocyte membrane. In Ussing chamber experiments, we functionally confirmed basolateral localization of the electrogenic SLC2A9 transporter by observing basolateral but not apical addition of 0.5 mM urate significantly stimulated Isc (24 ± 3μA/cm2) in comparison with control (10± 2.2 μA/cm2). Further, treatment of the serosal membrane with SLC2A9 inhibitors tranilast and pCMBS partially inhibited JUAsm flux of urate in an intestinal loop model. In contrast to the WT controls, Q140K mice demonstrated a significant decrease of apical ABCG2 protein abundance, with commiserate, significant loss of unidirectional urate transport (JUAsm) in jejunal loops (Q140K, 24 ± 3.38 μM/cm2/min; WT, 46 ± 1.50 μM/cm2/min), resulting in conversion of net urate secretion (−18 ± 4 μM/cm2/min) to net urate absorption (+5.99 ± 2.2 μM/cm2/min). Interestingly, these significant alterations in intestinal urate secretion in the Q140K mice correlated with evidence of altered serum insulin and non‐fasting glucose levels, not unlike what has been previously reported in mice lacking intestinal SLC2A9 strongly suggesting the establishment of SLC2A9 and ABCG2 as the intestinal urate excretion pathway.CONCLUSIONSOur findings demonstrate the pathophysiology of one of the most common hyperuricemia and gout risk variants, Q141K ABCG2, primarily affects the urate secretion in the gut, causing hyperuricemia and alterations in insulin and glucose levels with important implications for human disease.Support or Funding InformationNIDDK RO1DK114091AHA14SDG18060004This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.