钠-葡萄糖协同转运蛋白2抑制剂对糖尿病肾病保护机制的研究进展

郭诗哲(综述)　张朝云(审校)

(复旦大学附属华山医院内分泌科-复旦大学内分泌糖尿病研究所　上海　200040)

糖尿病肾病(diabetic kidney disease,DKD)是糖尿病最严重的慢性并发症之一,是导致终末期肾病(end-stage renal disease,ESRD)的主要原因[1]。高血压、高血糖、遗传易感性和人种等多个因素与DKD的发展相关[2]。全球范围内,糖尿病患者的微量白蛋白尿发生率高达39%,蛋白尿的年发生率为3.1%[3]。在中国约30%的1型糖尿病患者和20%的2型糖尿病患者最终发展为DKD[4]。

近年来,一种新型降糖药钠-葡萄糖协同转运蛋白2 (sodium-glucose cotransporter 2,SGLT2)抑制剂受到越来越多的关注,其作为一类新型的口服降糖药,作用于肾小管,通过抑制肾小管对葡萄糖的重吸收而发挥降糖作用。此外,临床试验发现SGLT2抑制剂在肾脏保护方面也有作用,本文将就其基础及临床研究进展进行综述。

SGLT2抑制剂自从全球首个SGLT2抑制剂达格列净(Dapagliglozin)于2012年11月被欧盟批准上市以来,已经有多个SGLT2抑制剂相继上市。2013年3月,美国食品药品管理局(FDA)批准上市首个SGLT2抑制剂坎格列净(Canagliflozin),用于治疗2型糖尿病[6]。2014年FDA又批准了2个SGLT2抑制剂,分别是达格列净和恩格列净(Empagliflozin)。伊格列净(Ipragliflozin)、托格列净(Tofogliflozin)和鲁格列净(Luseogliglozin)在日本已批准上市[7]。Remogliflozin etabonate(BHV091009)和Ertugliflozin(PF-04971729/MK-8835)等SGLT2抑制剂已处于临床试验阶段。

作用机制体内葡萄糖转运经葡萄糖转运体实现,分为两类,即葡萄糖转运蛋白(glucose transporter,GLUT)和SGLT[8]。GLUT主要负责葡萄糖的被动转运,而SGLT则负责葡萄糖的主动转运。体内的SGLT被SLC5家族编码,分为5种:SGLT1～SGLT5,其中最主要的是SGLT1和SGLT2[9]。在肾脏中,SGLT1主要位于肾脏近端小管的S3段,经肾小球滤过的葡萄糖有10%经SGLT1重吸收,而SGLT2主要位于肾脏近端小管S1～S2段,90%葡萄糖的重吸收由SGLT2完成[10]。在1型和2型糖尿病状态下,肾脏近端小管的SGLT2表达增加,这一点在糖尿病患者与各种啮齿动物的糖尿病模型一致[11-15]。SGLT2表达水平升高,导致葡萄糖重吸收增加,这形成了糖尿病状态下一种代偿适应。而SGLT2 抑制剂通过抑制SGLT2、减少肾小管对葡萄糖的重吸收,使体内的葡萄糖从尿中排出,从而降低血糖水平。

SGLT2抑制剂对DKD的保护作用2016年发表于NEnglJMed的一项大型临床研究显示[16],SGLT2抑制剂恩格列净可以明显延缓DKD进展,这是除RAAS系统阻滞剂药物外,首项经循证医学证明可延缓DKD进展的药物。该研究对7 020例有高心脑血管风险的2型糖尿病患者进行随机分组,分为恩格列净治疗组与安慰剂治疗组,并进行了长达37个月的随访,结果发现与安慰剂治疗组相比,恩格列净治疗组的肾脏病发病率或恶化风险明显降低,尿白蛋白肌酐比值(urine albumin-to-creatinine,UACR)翻倍的相对危险率下降,肾脏替代治疗的发生率也明显减少[16]。Heerspink等[17]根据CANATA-SU研究结果报道,在1 450位使用二甲双胍以及RAAS系统阻滞剂作为基础治疗的2型糖尿病患者中,随机分为加用坎格列净和格列美脲治疗,并进行为期2年的随访。研究发现相较于格列美脲组,坎格列净组GFR估测值(estimated glomerular filtration rate,eGFR)明显降低,从而减缓DKD的发生发展。尽管如此,关于SGLT2抑制剂对于DKD的疗效及其安全性仍需要更多的临床证据来支持。目前注册的SGLT2抑制剂影响肾脏功能的临床研究已有数十项,例如临床试验NCT02065791正在招募2型糖尿病合并DKD的患者,旨在揭示坎格列净安慰剂是否可以降低DKD患者ESRD的发生率。

保护肾脏机制

降低血糖高血糖主要通过以下4种途径引起DKD:多元醇通路的激活,晚期糖基化终末产物(advanced glycation end-product,AGE)的形成,蛋白激酶C (protein kinase C,PKC)活化和己糖胺通路的激活[18]。SGLT2抑制剂可以通过减少葡萄糖的重吸收降低血糖,大大减少了慢性高血糖导致的肾脏代谢紊乱。SGLT2抑制剂仅在血糖水平得到有效改善时,才能显示出肾脏保护作用[15,19-23]。Komala等[24]用链脲佐菌素(streptozocin,STZ)诱导eNOS-/-的小鼠成为糖尿病模型,分别给予恩格列净和替米沙坦治疗,并用胰岛素使各组的血糖水平均一化,不同于替米沙坦,恩格列净治疗组并未表现出肾脏保护作用,这项试验表明DKD早期SGLT2抑制剂对肾脏的保护作用可能依赖于循环中葡萄糖水平的改善。

改善肾脏血流动力学稳态在很多糖尿病研究模型中,高灌注、肾小球内高压以及代偿升高的GFR都被证实是肾脏功能异常的早期表现[25]。在1型和2型糖尿病患者中,高灌注的发生率近50%[26],因此成为预测糖尿病最终是否发展为DKD的一项重要指标。肾小球的高灌注通常涉及神经血管机制和管球反射(tubuloglomeruler feedback,TGF)机制。COX-2抑制剂与ACEI都是通过调节肾脏血管节律即神经血管机制来改善高灌注的病理情况。然而,使用这些药物并不能完全改善高灌注的现象,这提示应该关注管球反射机制在疾病进展过程中的作用。

在糖尿病患者慢性高血糖情况下,近曲小管SGLT2重吸收葡萄糖增加,随之共转运的Na+增加,在远端尿液经过致密斑时Na+/K+/2Cl-浓度降低,致密斑就会接受等同于循环血量不足导致电解质浓度降低的信号调控,产生入球小动脉扩张的效应,引起肾小球高灌注、高球内压及高滤过率[27]。在一项为期7天的研究中,给予STZ诱导的大鼠糖尿病模型以非选择性抑制SGLT1和SGLT2受体的根皮甙(phlorizin)可以有效改善肾小球高灌注[28]。在STZ诱导的SGLT-/-小鼠糖尿病模型中肾小球高滤过的状态也有明显的改善[19]。另有实验证明,特异性抑制SGLT2的药物达格列净可通过调节致密斑来降低糖尿病大鼠的高GFR[29]。

减轻氧化应激水平氧化应激是活性氧类(reactive oxygen species,ROS)和抗氧化防御之间的失衡,ROS的来源包括高糖诱导线粒体呼吸链来源的ROS产生增多、糖自动氧化及还原性烟酰胺腺嘌呤二核苷酸氧化酶氧化途径等,而抗氧化系统的改变包括非酶性抗氧化分子减少和抗氧化酶的活性改变等[33]。在DKD中,ROS产生增加,清除减少,导致肾脏的损伤[34]。研究证明,AGE与氧化应激密切相关,AGE与巨噬细胞、血管内皮细胞、肾小球系膜细胞上的特异性受体(AGE recptor,RAGE)相结合,介导ROS产生等一系列反应。研究发现,STZ诱导大鼠产生高血糖和低体重的症状后,肾脏皮质和髓质中过氧化氢酶(catalase,CAT)的活性降低,谷胱甘肽过氧化物酶(glutathione peroxidase,GPx)活性上升,3-硝基酪氨酸(3-nitrotyrosine)相对于对照组有所增加,而根皮甙皮下注射有抗氧化应激的作用[35]。另一项研究发现,恩格列净可以减少STZ诱导的糖尿病大鼠产生AGE、降低RAGE表达及氧化应激指标[36]。

体外研究证实,人肾脏近曲小管上皮细胞系HK-2在高糖环境(30 mmol/L)暴露后,ROS的产生显著增加。用RNA干扰的方法敲除肾小管SGLT2基因后,高糖诱导的ROS的产生则受到抑制[37]。 SGLT2抑制剂托格列净与抗氧化剂N-乙酰半胱氨酸产生相似作用,都可以减少高糖暴露下的HK-2细胞中ROS的产生[38]。

降低尿酸水平一项日本的研究显示,在校正了年龄、性别、血压及eGFR后,血尿酸水平高于7.2 mg/dL与肾血管壁增厚、透明变性及肾血管病相关[39]。一项中国的2型糖尿病横断面研究也显示,高尿酸血症和尿酸升高的人群中DKD发病率明显增加[40]。流行病学资料显示,无论在糖尿病还是非糖尿病人群中,尿酸都是肾脏疾病进展的独立危险因素[40-41]。

在Ⅲ期临床试验中,与安慰剂相比,坎格列净可显著降低血尿酸水平[42]。一项Meta分析也显示,达格列净治疗可以降低血尿酸水平[43]。一项试验将鲁格列净给正常受试者服用,发现血中尿酸水平下降,尿中尿酸水平升高。研究发现,鲁格列净对尿酸在肾脏中的重吸收并没有直接的作用[16]。肾脏中尿酸主要在近段肾小管重吸收,经尿酸转移载体(uric acid transporter1,URAT1,SLC22A12)、有机阴离子转移载体 4(organic anion transporter 4,OAT4,SLC22A11)及OAT10(SLC22A13)转移入近段肾小管,经葡萄糖转移载体9的亚型1(GLUT9 isoform 1)转移入血[44-48]。最近,有细胞实验发现高糖(葡萄糖10 mmol/L)可以增加近段肾小管尿酸经GLUT9亚型2外流。SGLT2抑制剂使肾小管局部葡萄糖浓度升高,使肾小管细胞顶端膜上发生的尿素-葡萄糖交换率提高,更多的尿酸从血液中排泄到尿液,最终降低了血尿酸水平[44,49]。这提示SGLT2抑制剂可通过降低尿酸发挥肾脏保护作用。

减轻肾小管间质损伤除了肾小球损伤,近年来肾小管损伤引起的肾脏功能恶化也受到越来越多的重视[50]。在2型糖尿病初期,肾小管的改变包括HK-2细胞肥大和基底膜增厚以及随着病程进展引起的相应肾小管结构萎缩[50]。STZ诱导的糖尿病大鼠经过4周的恩格列净治疗,尿液中的肾小管间质损伤指标L-FABP下降,这提示SGLT2抑制剂可减轻肾小管损伤[36]。高糖环境下HK-2细胞中ROS生成增加,RAGE表达升高,当使用siRNA干扰SGLT2表达时可以减少肾小管细胞的凋亡[37]。同样,恩格列净也可以对高糖下HK-2起到抗氧化应激的作用[51]。

减轻肾脏炎症及纤维化肾脏内的炎症是糖尿病肾损伤的重要机制。在肾损伤早期,巨噬细胞浸润并产生一系列的细胞毒性产物,包括促炎症因子(如肿瘤坏死因子α、干扰素-γ、IL-1β)及促纤维化因子(如转化生长因子β1)[52]。

在db/db 2型糖尿病小鼠中,用达格列净治疗12周后肾脏炎症反应显著减轻,减少巨噬细胞浸润和促炎因子产生。同时,达格列净还可以抑制促细胞凋亡的因子(如Caspase-12和Bax)的产生[23]。恩格列净可通过抑制Akita小鼠的NF-κB从而抑制其下游激活转录的一系列炎症因子(如CCL2、CD14、IL-6)[15]。

其他高血压在2型糖尿病患者中十分常见,也是糖尿病微血管和大血管病变的重要的危险因素。因此,血压控制对于肾脏保护必不可少[53-54]。SGLT2抑制剂增加肾脏对葡萄糖排泄的同时,也增加肾脏对水的排泄,从而降低体液容量,降低血压[55]。因此,SGLT2可能通过有效调控血压来保护肾脏。

结语根据现有的临床和基础研究,SGLT2抑制剂对于DKD病变保护的效果与机制尚不完全明确。SGLT2抑制剂作为一种新型降血糖药物,除了改善血糖,还具有心脏、肾脏保护作用,还需要通过更多的长期临床研究加以验证。

[1]BYRNE C,FORD D,GILG J,etal.UK Renal Registry 12th Annual Report (December 2009):chapter 3:UK ESRD incident rates in 2008:national and centre-specific analyses[J].NephronClinPract,2010,115(Suppl 1):9-39.

[2]ROSSING P,HOUGAARD P,PARVING HH.Risk factors for development of incipient and overt diabetic nephropathy in type 1 diabetic patients:a 10-year prospective observational study[J].DiabetesCare,2002,25(5):859-864.

[3]REUTENS AT,ATKINS RC.Epidemiology of diabetic nephropathy[J].ContribNephrol,2011,170:1-7.

[4]YANG G,ZHANG M,ZHANG M,etal.Effect of Huangshukuihua (Flos Abelmoschi Manihot) on diabetic nephropathy:a meta-analysis[J].JTraditChinMed,2015,35(1):15-20.

[5]MAJEWSKI C,BAKRIS GL.Has RAAS blockade reached its limits in the treatment of diabetic nephropathy?[J].CurrDiabRep,2016,16(4):24.

[6]GHOSH RK,BANDYOPADHYAY D,HAJRA A,etal.Cardiovascular outcomes of sodium-glucose cotransporter 2 inhibitors:a comprehensive review of clinical and preclinical studies[J].IntJCardiol,2016,212:29-36.

[7]SCHEEN AJ.Pharmacokinetics,pharmacodynamics and clinical use of SGLT2 inhibitors in patients with type 2 diabetes mellitus and chronic kidney disease[J].ClinPharmacokinet,2015,54(7):691-708.

[8]GERICH JE.Role of the kidney in normal glucose homeostasis and in the hyperglycaemia of diabetes mellitus:therapeutic implications[J].DiabetMed,2010,27(2):136-142.

[9]WRIGHT EM,LOO DD,HIRAYAMA BA.Biology of human sodium glucose transporters[J].PhysiolRev,2011,91(2):733-794.

[10]BALEN D,LJUBOJEVIC M,BRELJAK D,etal.Revised immunolocalization of the Na+-D-glucose cotransporter SGLT1 in rat organs with an improved antibody[J].AmJPhysiolCellPhysiol,2008,295(2):475-489.

[11]VESTRI S,OKAMOTO MM,DE FREITAS HS,etal.Changes in sodium or glucose filtration rate modulate expression of glucose transporters in renal proximal tubular cells of rat[J].JMembrBiol,2001,182(2):105-112.

[12]RAHMOUNE H,THOMPSON PW,WARD JM,etal.Glucose transporters in human renal proximal tubular cells isolated from the urine of patients with non-insulin-dependent diabetes[J].Diabetes,2005,54(12):3427-3434.

[13]TABATABAI NM,SHARMA M,BLUMENTHAL SS,etal.Enhanced expressions of sodium-glucose cotransporters in the kidneys of diabetic Zucker rats[J].DiabetesResClinPract,2009,83(1):27-30.

[14]VALLON V.The mechanisms and therapeutic potential of SGLT2 inhibitors in diabetes mellitus[J].AnnuRevMed,2015,66:255-270.

[15]VALLON V,GERASIMOVA M,ROSE MA,etal.SGLT2 inhibitor empagliflozin reduces renal growth and albuminuria in proportion to hyperglycemia and prevents glomerular hyperfiltration in diabetic Akita mice[J].AmJPhysiolRenal,2014,306(2):194-204.

[16]WANNER C,INZUCCHI SE,LACHIN JM,etal.Empagliflozin and progression of kidney disease in type 2 diabetes[J].NEnglJMed,2016,375(4):323-334.

[17]HEERSPINK HJ,DESAI M,JARDINE M,etal.Canagliflozinslows progression of renal function decline independently of glycemic effects[J].JAmSocNephrol,2017,28(1):368-375.

[18]杨雁,余学锋.糖尿病肾病机制的研究进展[J].临床肾脏病杂志,2012,12(5):196-198.

[19]VALLON V,ROSE M,GERASIMOVA M,etal.Knockout of Na-glucose transporter SGLT2 attenuates hyperglycemia and glomerular hyperfiltration but not kidney growth or injury in diabetes mellitus[J].AmJPhysiolRenalPhysiol,2013,304(2):156-167.

[20]KOJIMA N,WILLIAMS JM,TAKAHASHI T,etal.Effects of a new SGLT2 inhibitor,luseogliflozin,on diabetic nephropathy in T2DN rats[J].JPharmacolExpTher,2013,345(3):464-472.

[21]LIN B,KOIBUCHI N,HASEGAWA Y,etal.Glycemic control with empagliflozin,a novel selective SGLT2 inhibitor,ameliorates cardiovascular injury and cognitive dysfunction in obese and type 2 diabetic mice[J].CardiovascDiabetol,2014,13:148.

[22]NAGATA T,FUKUZAWA T,TAKEDA M,etal.Tofogliflozin,a novel sodium-glucose co-transporter 2 inhibitor,improves renal and pancreatic function in db/db mice[J].BritJPharmacol,2013,170(3):519-531.

[23]TERAMI N,OGAWA D,TACHIBANA H,etal.Long-term treatment with the sodium glucose cotransporter 2 inhibitor,dapagliflozin,ameliorates glucose homeostasis and diabetic nephropathy in db/db mice[J].PLoSOne,2014,9(6):e100777.

[24]GANGADHARAN KOMALA M,GROSS S,MUDALIAR H,etal.Inhibition of kidney proximal tubular glucose reabsorption does not prevent against diabetic nephropathy in type 1 diabetic eNOS knockout mice[J].PLoSOne,2014,9(11):e108994.

[25]HOSTETTER TH,RENNKE HG,BRENNER BM.The case for intrarenal hypertension in the initiation and progression of diabetic and other glomerulopathies[J].AmJMed,1982,72(3):375-380.

[26]SASSON AN,CHERNEY DZ.Renal hyperfiltration related to diabetes mellitus and obesity in human disease[J].WorldJDiabetes,2012,3(1):1-6.

[27]GILBERT RE.Sodium-glucose linked transporter-2 inhibitors:potential for renoprotection beyond blood glucose lowering?[J].KidneyInt,2014,86(4):693-700.

[28]MALATIALI S,FRANCIS I,BARAC-NIETO M.Phlorizin prevents glomerular hyperfiltration but not hypertrophy in diabetic rats[J].ExpDiabetesRes,2008,2008:305403.

[29]THOMSON SC,RIEG T,MIRACLE C,etal.Acute and chronic effects of SGLT2 blockade on glomerular and tubular function in the early diabetic rat[J].AmJPhysiolRegulIntegrCompPhysiol,2012,302(1):R75-R83.

[30]CHERNEY DZ,PERKINS BA,SOLEYMANLOU N,etal.Renal hemodynamic effect of sodium-glucose cotransporter 2 inhibition in patients with type 1 diabetes mellitus[J].Circulation,2014,129(5):587-597.

[31]YALE JF,BAKRIS G,CARIOU B,etal.Efficacy and safety of canagliflozin in subjects with type 2 diabetes and chronic kidney disease[J].DiabetesObesMetab,2013,15(5):463-473.

[32]KOHAN DE,FIORETTO P,TANG W,etal.Long-term study of patients with type 2 diabetes and moderate renal impairment shows that Dapagliflozin reduces weight and blood pressure but does not improve glycemic control[J].KidneyInt,2014,85(4):962-971.

[33]TAN AL,FORBES JM,COOPER ME.AGE,RAGE,and ROS in diabetic nephropathy[J].SeminNephrol,2007,27(2):130-143.

[34]HA H,LEE HB.Reactive oxygen species amplify glucose signalling in renal cells cultured under high glucose and in diabetic kidney[J].Nephrology(Carlton),2005,10 (Suppl):S7-S10.

[35]OSORIO H,CORONEL I,ARELLANO A,etal.Sodium-glucose cotransporter inhibition prevents oxidative stress in the kidney of diabetic rats[J].OxidMedCellLongev,2012,2012:542042.

[36]OJIMA A,MATSUI T,NISHINO Y,etal.Empagliflozin,an inhibitor of sodium-glucose cotransporter 2 exerts anti-inflammatory and antifibrotic effects on experimental diabetic nephropathy partly by suppressing AGEs-receptor axis[J].HormMetabRes,2015,47(9):686-692.

[37]MAEDA S,MATSUI T,TAKEUCHI M,etal.Sodium-glucose cotransporter 2-mediated oxidative stress augments advanced glycation end products-induced tubular cell apoptosis[J].DiabetesMetabRes,2013,29(5):406-412.

[38]ISHIBASHI Y,MATSUI T,YAMAGISHI S.Tofogliflozin,a highly selective inhibitor of SGLT2 blocks proinflammatory and proapoptotic effects of glucose overload on proximal tubular cells partly by suppressing oxidative stress generation[J].HormMetabRes,2016,48(3):191-195.

[39]MENDE C.Management of chronic kidney disease:the relationship between serum uric acid and development of nephropathy[J].AdvTher,2015,32(12):1177-1191.

[40]YAN D,TU Y,JIANG F,etal.Uric Acid is independently associated with diabetic kidney disease:a cross-sectional study in a Chinese population[J].PLoSOne,2015,10(6):e0129797.

[41]LI L,YANG C,ZHAO Y,etal.Is hyperuricemia an independent risk factor for new-onset chronic kidney disease?:a systematic review and meta-analysis based on observational cohort studies[J].BMCNephrol,2014,15:122.

[42]DAVIES MJ,TRUJILLO A,VIJAPURKAR U,etal.Effect of canagliflozin on serum uric acid in patients with type 2 diabetes mellitus[J].DiabetesObesMetab,2015,17(4):426-429.

[43]MUSSO G,GAMBINO R,CASSADER M,etal.A novel approach to control hyperglycemia in type 2 diabetes:sodium glucose co-transport (SGLT) inhibitors:systematic review and meta-analysis of randomized trials[J].AnnMed,2012,44(4):375-393.

[44]CAULFIELD MJ,MUNROE PB,O'NEILL D,etal.SLC2A9 is a high-capacity urate transporter in humans[J].PLoSMed,2008,5(10):e197.

[45]ANZAI N,ICHIDA K,JUTABHA P,etal.Plasma urate level is directly regulated by a voltage-driven urate efflux transporter URATv1 (SLC2A9) in humans[J].JBiolChem,2008,283(40):26834-26838.

[46]VITART V,RUDAN I,HAYWARD C,etal.SLC2A9 is a newly identified urate transporter influencing serum urate concentration,urate excretion and gout[J].NatGenet,2008,40(4):437-442.

[47]LI S,SANNA S,MASCHIO A,etal.The GLUT9 gene is associated with serum uric acid levels in Sardinia and Chianti cohorts[J].PLoSGenet,2007,3(11):e194.

[48]KOLZ M,JOHNSON T,SANNA S,etal.Meta-analysis of 28,141 individuals identifies common variants within five new loci that influence uric acid concentrations[J].PLoSGenet,2009,5(6):e1000504.

[49]CHINO Y,SAMUKAWA Y,SAKAI S,etal.SGLT2 inhibitor lowers serum uric acid through alteration of uric acid transport activity in renal tubule by increased glycosuria[J].BiopharmDrugDispos,2014,35(7):391-404.

[50]GILBERT RE,COOPER ME.The tubulointerstitium in progressive diabetic kidney disease:more than an aftermath of glomerular injury?[J].KidneyInt,1999,56(5):1627-1637.

[51]PANCHAPAKESAN U,PEGG K,GROSS S,etal.Effects of SGLT2 inhibition in human kidney proximal tubular cells--renoprotection in diabetic nephropathy?[J].PLoSOne,2013,8(2):e54442.

[52]BARUTTA F,BRUNO G,GRIMALDI S,etal.Inflammation in diabetic nephropathy:moving toward clinical biomarkers and targets for treatment[J].Endocrine,2015,48(3):730-742.

[53]XIE X,ATKINS E,LV J,etal.Effects of intensive blood pressure lowering on cardiovascular and renal outcomes:updated systematic review and meta-analysis[J].Lancet(London,England),2016,387(10017):435-443.

[54]LV J,NEAL B,EHTESHAMI P,etal.Effects of intensive blood pressure lowering on cardiovascular and renal outcomes:a systematic review and meta-analysis[J].PLoSMed,2012,9(8):e1001293.

[55]VASILAKOU D,KARAGIANNIS T,ATHANASIADOU E,etal.Sodium-glucose cotransporter 2 inhibitors for type 2 diabetes:a systematic review and meta-analysis[J].AnnInternMed,2013,159(4):262-274.