Genetic analysis for mechanism of underexcretion type hyperuricemia

尿酸盐在肾小管的顶膜两侧被重新吸收和分泌。我们克隆了尿酸转运蛋白1(URAT1，由SLC22A12编码)，位于近端小管的顶膜。尿酸盐的米氏常数(Km)为371±28 mm。我们发现，有机阴离子如乳酸、烟酸盐、乙酰乙酸酯、羟丁酸酯和琥珀酸选择性地抑制了尿酸盐通过URAT1的转运。在1 mM处，芳香族单羧酸盐，如吡嗪羧酸、烟酸盐和甲酸盐比脂族单羧酸盐(如乳酸盐)更有效地抑制尿酸盐的吸收。我们还测试了已知可降低高尿酸血症的尿酸物质的影响。顺式1 mM的丙磺舒、苯基丁酮、硫磺隆、非甾体抗炎药和利尿剂可抑制尿酸盐的摄取；苯溴马龙最强，在50 mM时完全抑制尿酸盐的摄取，其50%抑制浓度(IC_(50))小于0.1 mM。提示肾性低尿酸血症患者存在SLC22A12基因缺陷。肾性低尿酸血症是一种以肾小管尿酸盐转运受损为特征的异质性疾病。我们通过32名无血缘关系的患者阐明了肾性低尿酸血症的遗传特征以及URAT1在体内维持血尿酸水平的意义。32例患者中有30例存在SLC22A12突变(93.8%)，2例患者在URAT1编码区无突变。我们在54个等位基因中检测到SLC22A12突变(84.4%)。共发现SLC22A12基因的10个突变。突变包括2个无义突变、1个剪接点突变、1个5-碱基缺失和6个错义突变。54个SLC22A12等位基因中有40个发生G774A突变(74.1%)。所有错义突变和5-碱基缺失(1639-1643delGTCCT)在非洲爪哇卵母细胞中的体外表达分析检测尿酸转运活性。所有URAT1突变体的尿酸转运均显著降低，与未注射URAT1CRNA的卵母细胞相似。较少

Urate is reabsorbed and secreted bilaterally at the apical membrane in the tubules. We cloned urate transporter 1(URAT1 encoded by SLC22A12), located at the apical membrane in the proximal tubules. The estimated Michaelis constant (Km) for urate was 371±28mM. We found that the urate transport via URAT1 is inhibited selectively by organic anions such as lactate, nicotinate, acetoacetate, hydroxybutyrate and succinate. Aromatic monocarboxylates such as pyrazinecarboxylic acid, nicotinate and orotate at 1mM inhibited urate uptake more effectively than aliphatic monocarboxylates such as lactate. We also tested the influence of uricosuric substances known to reduce hyperuricemia. Probenecid, phenylbutazone, sulphinpyrazone, nonsteroidal anti-inflammatory drugs, and diuretic drugs at 1mM cis inhibited urate uptake ; benzbromarone was the most potent, completely inhibiting urate uptake at 50mM, with a 50%-inhibitory concentration(IC50) of less than 0.1mM.We also demonstrated that URAT1 regula … More tes serum urate levels by showing that patients with renal hypouricemia have defects in SLC22A12. Renal hypouricemia is a heterogeneous disorder characterized by impaired tubular urate transport. We elucidated genetic features of renal hypouricemia and the significance of URAT1 in maintaining serum urate levels in vivo using 32 unrelated patients. Thirty out of 32 patients had SLC22A12 mutations (93.8%), while 2 patients had none in the URAT1-coding region. We detected SLC22A12 mutations in 54 out of 64 alleles (84.4 %). Ten mutations in SLC22A12 were identified. Mutations included two nonsense mutations, one splice-site mutation, a 5-bp deletion and 6 missense mutations. The G774A mutation was identified in 40 out of 54 affected SLC22A12 alleles (74.1 %). All of the missense mutations and the 5-bp deletion (1639-1643delGTCCT) were tested for urate transport activity by in vitro expression analysis in Xenopus oocytes. The urate transports of all URAT1 mutants were significantly decreased and similar to that of oocytes that were not injected with URAT1 cRNA. Less