尿酸盐转运体研究现状及其药物研发
2015-12-10张彩香祝开思
张彩香 祝开思
1南方医科大学研究生学院;2解放军第305医院内分泌科
体内嘌呤代谢紊乱或尿酸盐排泄失常,导致血尿酸水平升高,与痛风、高血压、心血管和肾脏疾病关系密切[1,2],而尿酸水平过低可出现肾尿酸结石以及与运动相关的急性肾损伤[2]。因此,维持适当的尿酸水平非常重要。在过去的10年里,关于尿酸盐转运机制取得了巨大进步,本文主要对尿酸盐转运体及降尿酸药物进行综述。
尿酸盐转运体
1. 尿酸盐转运子1(urate transporter,URAT1)
2 0 0 2 年首次发现了与肾脏尿酸盐转运的相关蛋白,并命名为URAT1[3]。编码人尿酸盐转运子1(human urate transporter,hURAT1)的基因SLC22A12位于染色体11q13.1区,该基因和编码有机阴离子转运子4(organic anion transporter,OAT4)的基因SLC22A11配对。URAT1蛋白是在近曲小管顶端膜侧介导管腔内尿酸盐重吸收入胞质(即管腔膜细胞)[4,5]。该蛋白由555氨基酸组成,有12段跨膜螺旋体,其羧基末端连接有支架蛋白结构域(PDZ),该蛋白可调节尿酸盐转运活性。人胚肾293细胞的PDZ中的蛋白(PDZK1)与URAT1相互作用可增加尿酸盐转运活性,若无PDZ结构域修饰,该增强作用消失[6]。URAT1中存在尿酸盐与无机氯离子转运机制,细胞内外的氯离子浓度梯度可驱动尿酸盐吸收[3,5]。通过基底膜有机阴离子转运子(OTAS)从滤液中重吸收和细胞本身代谢的阴离子,如乳酸盐或烟酸盐可作为URAT1底物,与尿酸盐交换促进转运。由此推测当与URAT1高亲和力的药物或化学物质在管腔中聚集,那么其就起促进尿酸盐排泄作用。相反,当其在细胞内浓度高于管腔时通过URAT1流出与尿酸盐交换,促进尿酸盐重吸收[3]。尿酸盐经URAT1的重吸收是一个三级激活过程,第一级是基底膜的Na+-K+ATP酶,维持细胞内外的钠离子梯度,是主要的激活系统;第二级是钠偶联单羧酸转运子(SMCT),利用钠离子梯度在细胞内积累单羧酸阴离子(如乳酸盐);URAT1则为第三级系统,由单羧酸阴离子(乳酸盐)形成驱动力促进尿酸盐吸收入细胞[7,8]。STCMs与URAT1表达在肾近曲小管细胞同侧(顶端膜)。当在爪蟾卵母细胞内预先加入STCMs的底物烟酸盐时,可激活URAT1介导的尿酸盐转运;将其底物移除,或加入STCMs抑制剂时,激活作用减弱。实验说明,STCMs通过提供单羧酸离子,加强了URAT1介导的尿酸盐转运。因此,STCMs可作为改变肾脏转运尿酸盐过程的间接作用靶点[9]。
2. 葡萄糖转运体9(GLUT9/ URATv1)
GLUT9因参与转运葡萄糖而得名,由SLC2A9编码,表达于肾小管上皮细胞基底膜侧[10]。部分URAT1功能正常的低尿酸血症患者存在SLC2A9基因突变,提示其编码的GLUT9参与肾脏尿酸盐的重吸收[11]。经过不断的研究,该结果得到了实验和临床证据的验证。Naohiko等[12]发现没有SLC22A12基因突变的患者存在SLC2A9错义突变,导致体内尿酸盐转运活性下降,并经实验首次证实 GLUT9(SLC2A9)参与尿酸盐重吸收过程。当清除细胞外钾离子(使得细胞膜去极化),GLUT9转运尿酸盐速率加快,说明GLUT9对膜电位敏感。因此,也将GLUT9称为电压驱动性尿酸盐转运子(URATv1)。小鼠SLC2A9基因表达增加,URATv1功能增强引起尿酸重吸收增加,最终导致高尿酸血症[13]。2013年Takeo等[12]发现转染了URAT1(顶端膜)和URATv1(基底膜)的细胞(UUv),尿酸盐从顶端膜到基底膜的渗透率增加7倍。尿酸盐的顶端膜摄取率取决于UUv和仅表达URAT1的细胞(U细胞)。细胞外有氯离子时,尿酸盐摄取率分别为对照细胞的1.63、1.72倍,细胞外缺乏氯离子时摄取率更高(5.23、9.61倍)。然而氯离子对GLUT9无影响,更说明URAT1是由一个向外的氯离子浓度梯度来驱动的。这些结果表明 URAT1与URATv1的协调作用对尿酸盐的重吸收必不可少[5]。
3. 多药耐药相关蛋白基 4(multidrugresistanceprotein,MRP4)
MRP4由ABCC4基因编码,是ATP结核框蛋白亚家族C的第四成员[14],位于肾近曲小管顶端膜,参与肾脏各种底物的转运,包括尿酸盐[15]。是第一个被发现的位于顶端膜介导尿酸盐分泌的转运子。MRP4主要表达于肾小管上皮细胞顶端膜[16]。Remon等[17]证实,MRP4为ATP依赖性单向性介导尿酸盐转运子。
4. 乳腺癌抵抗蛋白(BCRP/ABCG2蛋白)
BCRP的编码基因ABCG2位于染色体4q的痛风易感性位点上,基因组研究显示该基因位点与痛风相关[18]。BCRP表达在许多组织的顶端膜,包括肾脏[19]、肝脏和肠道[20],肠道是肾脏以外排泄尿酸的主要器官[21]。相关基因研究发现ABCG2基因与血尿酸水平密切相关[22-24]。2012年Kimiyoshi等[25]发现,高尿酸血症合并尿尿酸排泄分数升高患者发生频率与ABCG2基因突变密切相关(P=3.60×10-10)。ABCG2基因突变导致BCRP功能失常程度越严重,尿尿酸排泄分数越高。敲除Abcg2的小鼠肾脏转运子Urit1表达下降,说明肾脏尿酸盐重吸收减少是根本原因。因此,ABCG2功能失常引起的尿酸盐排泄下降是经肠道途径,而非肾脏。建议将目前分型中的“尿酸生成过多型”改为“肾脏过负荷型”,这样就包括了肾脏外尿酸排泄下降和真正“产生过多”的原因。
5. 磷酸钠盐转运蛋白1(NPT1)
NPT1由SLC17A1基因编码,介导钠离子和有机磷酸盐共转运[26]。NPT1位于近曲小管细胞顶端膜侧[27]。人群基因研究[28,29]发现,SLC17A1基因多态性与高尿酸血症及痛风密切相关。细胞实验证实,NPT1在肾脏具有介导尿酸盐排泄的功能。SLC17A1突变型I269T属于功能获得型变异,可增加尿酸盐排出,减少肾脏尿酸盐排泄下降型(RUE)痛风风险[27]。Rosa等[30]发现RUE型痛风与肾小管重吸收转运体基因多态性相关,尿酸盐排泄正常型(非下降型)痛风与肾小管分泌转运体基因多态性相关,说明尿酸盐排泄下降或升高的高尿酸血症或痛风发病机制不同。推测NPT1和ABCG2相似,其功能失常可导致肠道排泄尿酸盐下降。这一机制可推进痛风的分类,将其分为肠道排泄下降型和肾脏排泄下降型。因此,持续性高尿酸血症,尤其是痛风患者,应该常规检测24h尿尿酸排泄,明确其分型,对治疗方案选择意义重大。
6. 磷酸钠盐转运蛋白4(NTP4)
NTP4属于I型磷酸钠盐协同转运蛋白家族,由SLC17A3基因编码,有hNPT4-L和hNPT4-S两种mRNA类型[31]。人NTP4蛋白表达于肾近曲小管顶端膜侧[32]。GWAS研究显示,SLC17A3基因多核苷酸多态性与尿酸水平及患痛风风险相关[24],SLC17A3基因突变引起的高尿酸血症,尿尿酸排泄分数下降[32]。在肾小管细胞尿酸盐排泄模型上,成功模拟了体内血循环中的尿酸盐经OAT1和OAT3进入肾小管细胞,并经hNPT4介导排入管腔的过程[32]。
总 结
临床上常用的促进尿酸排泄药物苯溴马隆是经其肝脏代谢产物6-羟基苯溴马隆滤过尿液抑制URAT1尿酸盐重吸收,从而促进尿酸排泄。苯溴马隆可引起肝功能损伤,因此6-羟基苯溴马隆可能为更好的药物选择,但还需进一步研究[33]。调脂药非诺贝特也是通过其主要代谢产物非诺贝特酸作用于URAT1抑制尿酸重吸收[34]。丙磺舒、吲哚美锌、水杨酸对于URAT1的半数抑制浓度均高于它们的血药治疗浓度,因此认为它们在体内也可通过抑制URAT1功能促进尿酸排泄[33]。水杨酸盐在低剂量时激活而高剂量时抑制尿酸盐的转运,这可能与其药物动力学相关[5]。沙坦类药物与URAT1有高亲和力,但只有氯沙坦和普拉沙坦达到足够浓度能从肾小球滤过进入管腔中,与管腔内的尿酸盐竞争URAT1从而抑制尿酸盐的重吸收,起到促进尿酸盐排泄的作用[35]。氯沙坦中等程度抑制GLUT9功能,抑制尿酸盐重吸收。因此,GLUT9也可作为降尿酸药物的作用靶点[12]。髓袢(丁苯氧酸、呋塞米)及噻嗪类利尿剂通过抑制近曲小管基底膜OAT1和OAT3,以及顶端膜NPT4分泌尿酸盐,导致高尿酸血症[36]。每天早晨和氢氯噻嗪随服一定剂量的丙磺舒可以抵消其引起的高尿酸血症[37]。
目前临床上降尿酸药物主要是通过抑制体内尿酸盐合成和促进肾脏尿酸盐排泄两方面起作用,主要的不良反应有嘌呤代谢紊乱和尿酸性肾结石。有研究表明,通过增加肠道BCRP功能促进尿酸盐的肠道分泌,此可作为靶点研制降尿酸药物。该类药物主要促进肠道尿酸盐的排泄,既不干扰体内嘌呤代谢,也无肾结石风险。了解更多关于尿酸盐的转运机制,对发现药物的作用靶点和更好地应用目前已有的降尿酸药物非常有益。
[1] Becker MA, Jolly M. Hyperuricemia and associated diseases[J]. Rheum Dis Clin North Am, 2006, 32(2): 275-293.
[2] Kutzing MK, Firestein BL. Altered uric acid levels and disease states[J]. J Pharmacol Exp Ther, 2008, 324(1): 1-7.
[3] Enomoto A, Kimura H, Chairoungdua A, et al. Molecular identification of a renal urateanion exchanger that regulates blood urate levels[J]. Nature, 2002, 417(6887): 447-452.
[4] Eraly SA, Hamilton BA, Nigam SK.Organic anion and cation transporters occur in pairs of similar and similarly expressed genes[J]. Biochem Biophys Res Com mun, 2003, 300(2): 333-342.
[5] Nakanishi T, Ohya K, Shimada S, et al. Functional cooperation of URAT1 (SLC22A12) and URATv1(SLC2A9) in renal reabsorption of urate[J].Nephrol Dial Transplant, 2013, 28(3): 603-611.
[6] Anzai N, Miyazaki H, Noshiro R, et al. The multivalent PDZ domaincontaining protein PDZK1regulates transport activity of renal urateanion exchanger URAT1 via its C terminus[J]. J Biol Chem, 2004, 279(44): 45942-45950.
[7] Gopal E, Fei YJ, Sugawara M, et al. Expression of slc5a8 in kidney and its role in Na(+) -coupled transport of lactate[J]. J Biol Chem, 2004, 279(43): 44522-44532.
[8] Cha SH, Sekine T, Kusuhara H, et al. Molecular cloning and characterization of multispecific organic anion transporter 4 expressed in the placenta[J]. J Biol Chem, 2000, 275(6): 4507-4512.
[9] LuY, Nakanihsi T, Tamai I. Funtional cooperation of STCM and URAT1 for renal reabsorption transport of urate[J]. Drug Metab Pharmacokinet, 2013, 28(2): 153-158.
[10] Augustin R, Carayannopoulos MO, Dowd LO, et al. Identification and characterization of human glucose transporter-like protein-9(GLUT9): alternative splicing alters trafficking[J]. J Biol Chem, 2004, 279(16): 16229-16236.
[11] Matsuo H, Chiba T, Nagamori S, et al. Mutations in glucose transporter 9 gene SLC2A9 cause renal hypouricemia[J]. Am J Hum Gene, 2008, 83(6): 744-751.
[12] Anzai N, Ichida K, Jutabha P, et al. Plasma urate level is directly regulated by a voltage-driven urate efflux transporter URATv1(SLC2A9) in humans[J].J Biol Chem, 2008, 283(40): 26834-26838.
[13] Kimura T, Amonpatumrat S, Tsukada A, et al. Increased expression of SLC2A9 decreases urate excretion from the kidney[J].NucleosidesNucleotidesand NucleicAcids, 2011, 30(12): 1295-1301.
[14] Dean M, Rzhetsky A, Allikmets R. The human ATP-binding cassette(ABC) transporter superfamily[J]. Genome Res, 2001, 11(7): 1156-1166.
[15] Van Aubel RA, Smeets PH, van den Heuvel JJ, et al. Human organic anion transporter MRP4(ABCC4) is an effluxpump for the purine end metabolite urate with multiple allosteric substrate binding sites[J]. Am J Physiol Renal Physiol, 2005, 288(2): F327-333.
[16] Van Aubel RA, Smeets PH, Peters JG, et al. The MRP4/ABCC4 gene encodes a novel apical organic anion transporter in human kidney proximal tubules: putative efflux pump for urinary cAMP and cGMP[J]. Am Soc Nephrol, 2002, 13(3): 595-603.
[17] Van Aubel RA, Smeets PH, van den Heuvel JJ, et al. Human organic anion transporter MRP4(ABCC4) is an efflux pump for the purine end metabolite urate with multiple allosteric substrate binding sites[J]. Am J Physiol Renal Physiol, 2005, 288(2): F327-333.
[18] Cheng LS, Chiang SL, Tu HP, et al. Genomewide scan for gout in taiwanese aborigines reveals linkage to chromosome 4q25[J]. Am J Hum Genet, 2004, 75(3): 498-503.
[19] Huls M, Brown CD, Windass AS, et al. The breast cancer resistance protein transporter ABCG2 is expressed in the human kidney proximal tubule apical membrane[J]. Kidney Int, 2008, 73(2): 220-225.
[20] Maliepaard M, Scheffer GL, Faneyte IF, et al. Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues[J]. Cancer Res, 2001, 61(8): 3458-3464.
[21] HosomiA, Nakanishi T, Fujita T, et al. Extra-renal elimination of uric acid via intestinal efflux transporter BCRP/ABCG2[J]. Intestinal Secretion of Uric Acid, 2012, 7(2): 30456.
[22] Kolz M, Johnson T, Sanna S, et al. Meta-analysis of 28,141 individuals identifies common variants within five new loci that influence uric acid concentrations[J]. PLoS Genet, 2009, 5(6): e1000504.
[23] Matsuo H, Takada T, Ichida K, et al. Common defects of ABCG2, a high-capacity urate exporter, cause gout: a function-based genetic analysis in a Japanese population[J]. Sci Transl Med, 2009, 1(5): 5ra11.
[24] Dehghan A, Kottgen A, Yang Q, et al. Association of three genetic loci with uric acid concentration and risk of gout: a genome-wide association study[J]. Lancet, 2008, 372(19): 1953-1961.
[25] Ichida K, Matsuo H, TakadaT, et al. Decreased extra-renal urate excretion is a common cause of hyperuricemia[J]. Nat Commun, 2012, 3: 764.
[26] Busch AE, Schuster A, Waldegger S, et al. Expression of a renal type I sodium/phosphate transporter(NaPi-1) induces a conductance in Xenopus oocytes permeable for organic and inorganic anions[J]. Proc Natl Acad Sci USA, 1996, 93: 5347-5351.
[27] ChibaT, MatsuoH, Kawamura Y. NPT1/SLC17A1 is a renal urate exporter in humans and its common gain-of-function variant decreases the risk of renal underexcretion gout[J]. Arthritis Rheumatol, 2015, 67(1): 281-287.
[28] Urano W, Taniguchi A, Anzai N, et al. Sodium-dependent phosphate cotransporter type 1 sequence polymorphisms in male patients with gout[J]. Ann Rheum Dis, 2010, 69(6): 1232-1234.
[29] Yang Q, Kottgen A, Dehghan A, et al. Multiplegenetic loci influence serum urate and their relationship with gout and cardiovascular disease risk factors[J]. Circ Cardiovasc Genet, 2010, 3(6): 523-530.
[30] Torres RJ, de Miguel E, Bailen R, et al. Tubular urate transporter gene polymorphisms differentiate patients with gout who have normal and decreased urinary uric acid excretion[J]. The J Rheumatol, 2014, 41( 9): 1863-1870.
[31] Jutabha P, Anzai N, Wempe MF, et al. Apical voltage-driven urate efflux transporter NPT4 in renal proximal tubule[J]. Nucleosides Nucleotides Nucleic Acids, 2011, 30(12): 1302-1311.
[32] JutabhaP, Anzai N, Kitamura K, et al. Human Sodium Phosphate Transporter 4(hNPT4/SLC17A3) as a common renal secretory pathway for drugs and urate[J]. J Biol Chem, 2010, 285(45): 35123-35132.
[33] Shin HJ, Takeda M, Enomoto A, et al. Interactions of urate transpor ter URAT1 in human kidney with uricosuric drugs[J]. Nephrology(Carlton), 2011, 16(2): 156-162.
[34] Uetake D, Ohno I, Ichida K. Effect of fenofibrate on uric acid metabolism and urate transporter [J]. Inter Med, 2010, 49(2): 89-94.
[35] Iwanaga T, Sato M, Maeda T, et al. Concentration-dependent mode of interaction of angiotensin II receptor blockers with uric acid transporter[J]. J Pharmacol Exp Ther, 2007, 320: 211-217.
[36] Nakamura M, Anzai N, Jutabha P, et al. Concentration-dependent inhibitory effect of irbesartan on renal uric acid transporters[J]. J Pharmacol Sci, 2010, 114(1): 115-118.
[37] Bach MH, Simkin PA. Simkin.Uricosuric drugs: the once and future therapy for hyperuricemia?[J]. Curr Opin Rheumatol, 2014, 26(2): 169-175.