黄芪总苷对实验性糖尿病动物肾脏保护作用及其机制研究
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摘要
糖尿病（Diabetes Mellitus, DM）是由于胰岛素分泌缺乏及（或）机体产生胰岛素抵抗所引起的以血糖升高为主要特征的常见内分泌代谢性疾病，现已成为继肿瘤、心脑血管疾病之后第三大危害人类健康的慢性疾病。糖尿病肾病（Diabeticnephropathy, DN）是DM最常见和最严重的并发症之一，约20%～30%的1型糖尿病（Type1diabetes mellitus, T1DM）或2型糖尿病（Type2diabetes mellitus, T2DM）患者最终并发DN，其中相当一部分可发展为终末期肾病（End-stage renal disease,ESRD）。DN是糖尿病患者死亡的重要原因。DN发病机制尚未完全阐明，但其发生和发展是以高糖状态为始动因子的多因素综合作用的结果，已有研究表明，高糖状态可引起机体的氧化应激反应增强，氧化应激可能是导致DN等并发症的主要原因，在DN的发生、发展中起着重要的作用。对DN的防治，目前亦尚缺乏理想的方法与药物。因此探讨DN发病机制，寻找有效防治DN的方法与药物具有重要的理论与实践意义。
祖国医学认为，糖尿病属于“消渴”、“水肿”病范畴，为“本虚标实、虚实夹杂”之证。治疗当以扶正与祛邪并举。临床以益气养阴、化瘀通络治疗为主。
黄芪为临床常用中药，具有补气升阳、益气固表、利水消肿、扶正固本和延缓衰老等多种药理作用，可用于气虚水肿，内热消渴等多种病症的防治。黄芪作为君药是防治心、肝、肾脏等疾病的常用药物。单味黄芪对肾病综合征患者即可产生明显的降低尿蛋白的作用，是临床常用防治肾病综合征的中药之一。黄芪总苷（Astragalosides，AST）是黄芪的主要有效部位群。本课题组多年的研究发现，AST有抗应激、抗氧化和免疫调节等多种药理作用。关于以黄芪组方或黄芪提取物治疗糖尿病的临床研究以及基础研究报道较多，但尚未见系统研究AST防治DM及其并发症DN的作用与机制的研究报道。本课题组对黄芪的有效组份AST及单体黄芪甲苷的药理学进行了多年的研究，在此基础上，我们拟在体内外，对AST治疗DM的作用及可能的作用机制进行探讨，寻找有效的防治DM与DN的药物，为提高临床治疗DM与DN并发症的疗效提供理论基础。
目的：
研究AST对链脲佐菌素（Streptozotocin, STZ）诱导的大鼠及小鼠糖尿病模型治疗作用，探讨AST对糖尿病动物肾脏保护作用及可能机制；采用高糖培养人肾小球系膜细胞（Human Mesangial Cell, HMC）作为目标细胞，进一步研究AST防治DM及DN的分子机制。
方法：
1. STZ（100mg·kg~(-1)）单次腹腔注射建立糖尿病小鼠模型。取造模成功的小鼠随机分为模型组（Model）、Tempol组（90mg·kg~(-1))、AST组（30,60,120mg·kg~(-1)）。每组20只动物。各组动物连续灌胃给药4-6W，正常组（Control）和模型组给予等容量的蒸馏水。分别在给药后4W、6W，各组随机取半数（10只）动物，取血检测空腹血糖（Fasting blood glucose, FBG）、甘油三酯（Triglyceride, TG)、总胆固醇（TotalCholesterol, TC)、糖化血清蛋白（Glycosylated serum protein, GSP）含量；取血后颈椎脱臼处死动物后迅速摘取肝脏、脾脏、肾脏，称重，计算脏器指数；制作10%肝脏组织匀浆，分离上清液检测T-SOD、MDA活性和GSH-Px含量；一侧肾脏制作病理切片，进行组织形态学检查，并用免疫组织化学方法检测肾脏TRPC6蛋白表达水平，TUNEL法检测STZ糖尿病小鼠肾小球细胞凋亡；另侧肾脏用于检测TGF-β_1mRNA、Col Ⅳ mRNA表达。
2. STZ（60mg·kg~(-1)）单次腹腔注射建立糖尿病大鼠模型。取造模成功大鼠随机分为模型组（Model group）、Tempol组（60mg·kg~(-1)）与AST组（40mg·kg~(-1)）。每组20只动物。各组动物每日灌胃给药1次，连续4-6W，正常组（Control）和模型组给予等容量蒸馏水。实验过程中每日观察大鼠生存状态与活动情况，并采用代谢笼法，在造模前（Before）、造模后（0W）、给药后1W、2W，4W、6W分别记录大鼠体重，日摄食量、日饮水量、24h尿量（urine output，UO）等生理指标。尿液静置后，采用渗透压摩尔浓度测定仪测定尿液渗透压。取适量尿液离心，用于检测尿液生化指标：尿液肌酐（urine creatinine，Ucr）、24h尿蛋白（UAE）、β2-微球蛋白（β2-MG）、肿瘤坏死因子α（TNF-α）、转化生长因子-β_1（TGF-β_1）含量。分别在给药后4W及6W，各组随机取半数（10只）动物麻醉后，腹主动脉取血，检测空腹血糖（FBG），血清糖化血清蛋白（GSP）、空腹血清胰岛素（Fasting insulin, FINS）、胰岛素敏感指数（Insulin sensitivity index, ISI）、总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白胆固醇（High density lipoprotein cholesterol, HDL-C）、低密度脂蛋白胆固醇（Lowdensity lipoprotein cholesterol, LDL-C）含量、T-SOD、GSH-Px活性和MDA含量、血清尿素氮（Blood urea nitrogen, BUN）、血肌酐（Serum creatinine, Scr）含量。取血后颈椎脱臼处死动物，迅速摘取肝脏、脾脏、肾脏，称重，计算脏器指数。一侧肾脏制作病理切片，进行组织形态学检查，并采用免疫组织化学方法检测肾脏TRPC6蛋白表达水平；另侧肾脏用于检测TRPC6mRNA、TGF-β_1mRNA、Col ⅣmRNA表达。
3.采用人肾小球系膜细胞（Human Mesangial Cell, HMC）作为目标细胞，分别设立正常对照组（NG）、甘露醇渗透压对照组（MA）、高糖组（HG）、Tempol组（100μmol·L~(-1)）、AST（50,100,200mg L~(-1)）组。NG组细胞予低糖DMEM培养液（含5.5mmol L~(-1)D-葡萄糖）培养，MA组予含24.5mmol L~(-1)甘露醇的低糖培养液培养，HG组予高糖DMEM培养液（含30mmol L~(-1)D-葡萄糖）培养，Tempol组予含100μmol L~(-1)Tempol的高糖培养液培养干预，AST各剂量组分别予含50,100,200mg L~(-1)AST高糖培养液培养。干预时间为24h。用MTT法检测AST（0-400mg L~(-1)）对高糖培养HMC细胞增殖与细胞活力影响；流式细胞仪检测各组HMC细胞中ROS含量；荧光实时定量PCR法检测各组HMC细胞内TRPC6mRNA、TGF-β_1mRNA、NOX4mRNA表达情况；Western blot法检测各组HMC细胞内TRPC6、TGF-β_1、NOX4蛋白表达情况。采用生物素双抗体夹心酶联免疫吸附法（Enzyme-linked immunosorbentassaym, ELISA）测定各组HMC细胞上清液内SOD、MDA、GSH-Px、TGF-β_1、FN、Col Ⅳ含量。
结果：
1黄芪总苷对实验性糖尿病小鼠肾脏保护作用
1.1AST可显著改善STZ糖尿病小鼠“三多一少”的症状，提高肝脏抗氧化能力，表现为升高T-SOD、GSH-Px活性，降低MDA含量；血清FBG、GSP、TC及TG水平亦有下降的趋势，但尚未能恢复至正常水平。
1.2AST可在一定程度上降低肾脏指数，对减轻肾小球损伤与凋亡呈剂量依赖性。
1.3AST可抑制肾脏Col Ⅳ mRNA、TGF-β_1mRNA高表达，显著提高糖尿病小鼠肾小球TRPC6蛋白表达水平。
2黄芪总苷对实验性糖尿病大鼠肾脏保护作用
2.1AST可显著改善STZ糖尿病大鼠“三多一少”症状，表现为体重降低减缓，日摄食量、日饮水量及尿量逐渐减少。
2.2AST可降低STZ糖尿病大鼠FBG、GSP，升高ISI；但大鼠FBG仍处于较高水平。
2.3AST可降低STZ糖尿病大鼠血清TC、TG及LDL-C水平，升高HDL-C含量，提示AST可在一定程度上纠正糖尿病大鼠的血脂代谢紊乱。
2.4AST可提高STZ糖尿病大鼠抗氧化能力，提高血清T-SOD、GSH-Px活性，降低MDA含量。
2.5AST可使STZ糖尿病大鼠UAE明显下降，显著降低糖尿病大鼠24h尿β2-MG、TNF-α、TGF-β_1排出量。改善STZ糖尿病大鼠肾功能，降低Scr、BUN含量，提高Ccr，降低肾脏指数，减轻肾小球损伤；降低肾脏Col Ⅳ mRNA、TGF-β_1mRNA表达，提高TRPC6mRNA表达量，同时也显著提高糖尿病大鼠肾小球TRPC6蛋白表达水平。这表明AST对STZ糖尿病大鼠具有较好的防治其肾脏功能损伤的作用。
3黄芪总苷对高糖培养人肾小球系膜细胞保护作用
3.1在0--400mg L~(-1)浓度范围内，AST可浓度依赖性的抑制高糖培养HMC细胞增殖。
3.2高糖培养HMC上清液内T-SOD、GSH-Px活性显著降低，MDA含量明显升高；AST可在一定程度上提高HMC T-SOD、GSH-Px活性，降低MDA含量。
3.3高糖培养HMC上清液TGF-β_1、FN、Col Ⅳ含量明显上升；各剂量AST均可明显降低TGF-β_1、FN、Col Ⅳ含量，除AST（50mg L~(-1)）组外，其他各剂量组已恢复至正常水平。
3.4高糖培养HMC内ROS含量明显上升；各剂量AST均可明显降低HMC细胞内ROS含量，其中AST（200mg L~(-1)）组HMC内ROS含量已降至正常水平。
3.5高糖培养HMC内TRPC6mRNA表达水平显著降低，TGF-β_1mRNA及NOX4mRNA表达水平明显上升； AST（100,200mg L~(-1)）可显著提高HMC细胞内TRPC6mRNA表达水平，降低TGF-β_1mRNA及NOX4mRNA表达水平。
3.6高糖培养HMC内TRPC6蛋白表达水平显著降低，TGF-β_1及NOX4蛋白表达水平明显升高；各剂量AST均可显著提高HMC细胞内TRPC6蛋白表达水平，降低TGF-β_1蛋白表达水平；除AST组（50mg L~(-1)）外，其他各剂量组NOX4蛋白表达水平也出现明显下降。
结论：
1AST对实验性糖尿病动物肾脏具有一定的保护作用，其机制可能与AST提高糖尿病动物的抗氧化能力、调节血脂代谢、抑制高糖状态所致的肾组织Col Ⅳ mRNA、TGF-β_1mRNA高表达及显著提高糖尿病动物肾组织TRPC6mRNA与TRPC6蛋白表达水平抑制肾小球细胞凋亡有关。
2AST对高糖培养HMC具有保护作用，其机制可能与AST提高HMC抗氧化能力、降低TGF-β_1、FN、Col Ⅳ含量、抑制TGF-β_1、NOX4高表达及提高TRPC6表达水平有关。
Background
Diabetes Mellitus (DM), a common endocrine-metabolic disease caused by defects ofinsulin secretion and/or insulin resistance, is chacterized by increased blood glucose. Ithas been the third major disease following tumor and cardio-cerebrovascular disease.Diabetic nephropathy (DN) is the most frequent and serious complications of DM andbecomes the main cause of deaths in DM. Approximately20to30persents of patientswith Type1diabetes mellitus (T1DM) or Type2diabetes mellitus (T2DM) have DN,most of which are further developed to end-stage renal disease (ESRD). Pathogenesy ofDN has not been clarified so far. It is believed to be influenced by multiple determintswith the high glucose level as the initiation factor. Strong oxidative stress is observed inDM and plays an important role in occurance and development of DM. Therefore,oxidative stress is regarded as the key reason of DM complications. As to the treatmentand prevention of DN, it is lack of ideal strategies and drugs. Thus, it would be of of greatsignificant to seek for effective methods for DN control.
In Traditional Chinese Medicine (TCM), it is considered that DM belongs to consumptivethirst and oedema and characterised with asthenia in origin, asthenia in superficiality andasthenia and sthenia. It treatment must focus on strengthening body resistance andeliminating pathogen. In clinical medication of TCM, supplementing qi and nourishingyin and disperse blood stasis and dredge collateral are suggested to be the predominanttreatments.
Astragalus membranaceus (AS) is one of the most commonly used herbal medicines in TCM, which possesses “Buqishengyang, Yiweigubiao, Tudushengji, Lishuituizhong”function. It can be used in deficiency of vital energy and calor internus. AS is a principaldrug to control diseases in heart, liver and kidney. Single AS can decrease urine protein innephritic syndrome and is one of the commonest traditional medicine to control nephriticsyndrome. Astragalosides (AST) is the effective fraction extracted from astragalus.Through many years of research by our team, AST is found to have many biologicaleffects, including anti-stress, anti-oxidation and immunological regulation. There havebeen lots of clinical and experimantal researches on DM treatment with astragalus orextract of astragalus, but the systematic study about the effect of AST on DM and itspotential mechanisms is still limited. In this thesis, we take the pharmacologic action ofastragalus effective component as the particular target and aim to understand the effect ofAST on DM and its potential mechanisms with both in vivo experiment and ex vivoexperiment. Findings generated from the exproments are expected to provide elementaryinformation on DM treatment and drug design for DN.
Objectives
To understand effect of AST on DM and its potential mechanisms by treating diabetic ratsand diabetic mice induced by streptozotocin (STZ) and the molecular mechanismsunderline the effect is investigated using high-glucose-incubated human mesangial cell(HMC) as the targeted cell.
Methods
1The diabetic mouse model was established by intraperitoneal injection with STZ (100mg·kg~(-1)) and the model mice were randomly divided into Model group, Tempol group (90mg·kg~(-1)), AST-L group (30mg·kg~(-1)), AST-M group (60mg·kg~(-1)), and AST-H group (120mg·kg~(-1)). The animals were given the drugs with successive intragastric administration for4-6weeks, the same volume of distilled water were admistered to Control group and Model group, respectively. After4weeks and6weeks of administration, half of the mice(10) in each group were randomly selected and blood sampkes were drawn to assessfasting blood glucose (FBG), triglyceride (TG), total cholesterol (TC), glycosylated serumprotein (GSP). The mice were sacrificed with cervical dislocation after blood sampleswere taken. And then, livers, spleens and kidneys were removed immediately, douchedwith ice physiological saline, dried with filter papers, weighed and organ index werecalculated. Liver tissue bomogenate (10%) was made by liver tissue of0.5g. Activities ofT-SOD and MDA and contents of GSH-Px were evaluated after isolating supernatants.One side of kidney was put into4%formaldehyde solution and made to paraffin sectionfor histomorphological examination. This side of kidney was also used to measure thelevel of TRPC6expression by immunohistochemistry as well as the apoptosis ofglomerular cells in glomeruli by TUNEL. The other side of kidney, however, was undercryopreservation at-80℃for the assessment of TGF-β_1mRNA、 Col Ⅳ mRNAexpression.
2The diabetic rat model was established by intraperitoneal injection with STZ (60mg·kg~(-1)) and the model rats were randomly divided into Model group, Tempol group (60mg·kg~(-1)) and AST group (40mg·kg~(-1)). The animals were given the drugs with successiveintragastric administration for4-6weeks, the same volume of distilled water wererespectively admistered to Control group and Model group. Survival status and activitiesof the animals were observed every day. Via metabolic cage, body weight, daily foodintake, daily water intake and daily urine output (UO) were recorded respectivelypre-therapy,0W,1W,4W and6W after administration. After urion prepared in standing,uric osmotic pressure was assessed by osmotic pressure molar concentration determinator.Proper amount of urion was centrifugated and prepared for measurement of biochemicalindicators under cryopreservation at-80℃, such as urine creatinine (Ucr),urinaryalbumin excretion (UAE) β2-microglobulin (β2-MG), tumor necrosis factor (TNF-α), transforming growth factor (TGF-β_1). After4weeks and6weeks of administration, halfof the rats (10) in each group were randomly selected and blood samples were drawn fromabdominal aorta after anesthesia to assess FBG, GSP, fasting serum insulin (FINS), insulinsensitivity index (ISI), TC, TG, high density lipoprotein (HDL), low density lipoprotein(LDL), as well as activities of T-SOD and GSH-Px and contents of MDA, blood ureanitrogen (BUN), serum creatinine (Sc). The rats were sacrificed with cervical dislocationafter blood samples were taken. And then, livers, spleens and kidneys were removedimmediately, douched with ice physiological saline, dried with filter papers, weighed andorgan index were calculated. Liver tissue bomogenate (10%) was made by liver tissue of0.5g. One side of kidney was put into4%formaldehyde solution and made to paraffinsection for histomorphological examination. This side of kidney was also used to measurethe level of TRPC6expression by immunohistochemistry. The other side of kidney,however, was under cryopreservation at-80℃for the assessment of TRPC6mRNA,TGF-β_1mRNA and Col ⅣmRNA expression.
3Human mesangial cells (HMC) was taken as target cells and divided into Normal group(NG), Mannitol (MA), high glucose group (HG), Tempol (100μmol·L~(-1)), AST-L(50mg L~(-1)), AST-M (100mg L~(-1)) and AST-H (200mg L~(-1)). NG group were incubated withlow glucose DMEM culture solution (5.5mmol L~(-1)D-glocose), MA group wereincubated with low glucose culture solution (24.5mmol L~(-1)Mannitol) and HG groupwere with high glucose DMEM culture solution (30mmol L~(-1)D-glocose). Similarly,Tempol group were incubated with high glucose culture solution (100μmol L~(-1)Tempol),and high glucose culture solution (50mg L~(-1)AST) in AST-L group, high glucose culturesolution (100mg L~(-1)AST) in AST-M group and high glucose culture solution (200mg L~(-1)AST) in AST-H group. The duration of administration was all defined as24hours. Then,different methods were adopted to assess various indicators. For instance, MTT Assay forthe effect of AST on proliferation and viability of high-glucose-incubated HMC, flow cytometry for ROS contents in HMC, fluorescent quantitative real time PCR for TRPC6mRNA、TGF-β_1mRNA、NOX4mRNA expression in HMC, Western blot for TRPC6、TGF-β_1、NOX4expression in HMC. In addition, enzyme-linked immunosorbent assaym(ELISA) was introduced to assess contents of SOD、MDA、GSH-Px、TGF-β_1、FN、Col Ⅳ in HMC supernatant in each group.
Results
1Protective effects of AST on experimental diabetic mice
1.1AST significantly improved the typical symptoms (polyuria, polydipsia, polyphagia,weight loss) and enhanced the anti-oxidative ability in liver of STZ diabetic mice. Indetail, it increased activities of T-SOD and GSH-Px and decreased MDA level. Althoughthe levels of serum FBG, GSP, TC and TG decreased, the reduction could not return tonormal level.
1.2AST decreased kidney index to some extent. The effects of alleviating glomerulusdefects and apoptosis were shown dose-dependent and this effect was most significant atthe dosage of60mg·kg~(-1).
1.3AST inhibited the high expression of collagen Type Ⅳ mRNA and TGF-β_1mRNA inkidney. AST raised the TRPC6expression in glomerulus in STZ diabetic mice.
2Protective effects of AST on experimental diabetic rats
2.1AST significantly improved the typical symptoms (polyuria, polydipsia, polyphagia,weight loss) in STZ diabetic rats. The body weights of rats didn’t continuously decrease,while daily food intake, daily water intake and daily urine output decreased gradually.
2.2AST decreased the levels of FBG and GSP and increase ISI. But the content of FBGstill kept at a relatively high level.
2.3AST decreased the level of serum TC, TG and LDL while enhance the level of HDL.This kind of effect could rectify lipid metabolic disorders in STZ diabetic rats.
2.4AST enhanced the anti-oxidative ability in STZ diabetic rats. It increased activities ofT-SOD and GSH-Px and decreased MDA level.
2.5AST decreased daily amount of urine protein excretion as well as daily amount ofurine β2-MG, TNF-α and TGF-β_1excretion. AST improved kidney function of STZdiabetic rats. It decreased serum levels of creatinine, urea nitrogen and enhanced thecreatinine clearance rate. It decreased kidney index and alleviate the defects of glomerulus.It also decreased expression of collagen Type Ⅳ mRNA and TGF-β_1mRNA, enhancedexpression of TRPC6mRNA and increased expression of TRPC6in glomerulus in STZdiabetic rats.
3Protective effect of AST on high glucose incubated glomerular mesangial cells
3.1At the range of0-400mg L~(-1), the cell proliferation inhibiting rate caused by ASTshowd dose-dependent in high glucose incubated incubated glomerular mesangial cells.
3.2Activities of T-SOD and GSH-Px in HMC supernatant decreased and increasedcontent of MDA. AST increased activities of T-SOD and GSH-Px in HMC and decreasedthe level of MDA.
3.3The level of TGF-β_1, FN and Col Ⅳ increased in high glucose incubated HMCsupernatant. AST with high, median and low dosage all decreased the contents of TGF-β_1,FN and Col Ⅳ. The three indicators returned to normal level in groups with high andmedian dosage.
3.4The level of ROS significantly increased in high glucose incubated HMC. AST withhigh, median and low dosage all decreased the contents of ROS in HMC. And ROSdecreased to normal level in group with high AST dosage.
3.5The expression of TRPC6mRNA decreased in high glucose incubated HMC while theexpression of TGF-β_1mRNA and NOX4mRNA significantly increased. AST with highand median dosage both increased the expression of TRPC6mRNA and decreased theexpression of TGF-β_1mRNA and NOX4mRNA.
3.6The expression of TRPC6protein decreased in high glucose incubated HMC while theexpression of TGF-β_1and NOX4protein significantly increased. AST with high, medianand low dosage all decreased the the expression of TRPC6protein in HMC and decreasedthe expression of TGF-β_1protein. The expression of NOX4protein significantlydecreased in group with high and median dosage.
Conclusions
1There is protective effect of AST on high glucose incubated glomerular mesangial cells.The potential mechanisms may include the enhancement of anti-oxidative ability of HMC,reduction of the levels of TGF-β_1, FN and Col Ⅳ, inhibitation in the high expression ofTGF-β_1and NOX4and promotion of the TRPC6expression by AST.
2There are some protective effects of AST on kidney of experimental diabetic rats andmice. The potential mechanisms may include improvement of anti-oxidative ability inserum and liver, regulation of lipid metabolism, protection of kidney function, inhibitationin apoptosis in glomerulus, inhibitation in the high expression of Col ⅣmRNA,TGF-β_1mRNA in kidney and enhancement of the expression of TRPC6mRNA andTRPC6protein in glomerulus by AST.
祖国医学认为，糖尿病属于“消渴”、“水肿”病范畴，为“本虚标实、虚实夹杂”之证。治疗当以扶正与祛邪并举。临床以益气养阴、化瘀通络治疗为主。
黄芪为临床常用中药，具有补气升阳、益气固表、利水消肿、扶正固本和延缓衰老等多种药理作用，可用于气虚水肿，内热消渴等多种病症的防治。黄芪作为君药是防治心、肝、肾脏等疾病的常用药物。单味黄芪对肾病综合征患者即可产生明显的降低尿蛋白的作用，是临床常用防治肾病综合征的中药之一。黄芪总苷（Astragalosides，AST）是黄芪的主要有效部位群。本课题组多年的研究发现，AST有抗应激、抗氧化和免疫调节等多种药理作用。关于以黄芪组方或黄芪提取物治疗糖尿病的临床研究以及基础研究报道较多，但尚未见系统研究AST防治DM及其并发症DN的作用与机制的研究报道。本课题组对黄芪的有效组份AST及单体黄芪甲苷的药理学进行了多年的研究，在此基础上，我们拟在体内外，对AST治疗DM的作用及可能的作用机制进行探讨，寻找有效的防治DM与DN的药物，为提高临床治疗DM与DN并发症的疗效提供理论基础。
目的：
研究AST对链脲佐菌素（Streptozotocin, STZ）诱导的大鼠及小鼠糖尿病模型治疗作用，探讨AST对糖尿病动物肾脏保护作用及可能机制；采用高糖培养人肾小球系膜细胞（Human Mesangial Cell, HMC）作为目标细胞，进一步研究AST防治DM及DN的分子机制。
方法：
1. STZ（100mg·kg~(-1)）单次腹腔注射建立糖尿病小鼠模型。取造模成功的小鼠随机分为模型组（Model）、Tempol组（90mg·kg~(-1))、AST组（30,60,120mg·kg~(-1)）。每组20只动物。各组动物连续灌胃给药4-6W，正常组（Control）和模型组给予等容量的蒸馏水。分别在给药后4W、6W，各组随机取半数（10只）动物，取血检测空腹血糖（Fasting blood glucose, FBG）、甘油三酯（Triglyceride, TG)、总胆固醇（TotalCholesterol, TC)、糖化血清蛋白（Glycosylated serum protein, GSP）含量；取血后颈椎脱臼处死动物后迅速摘取肝脏、脾脏、肾脏，称重，计算脏器指数；制作10%肝脏组织匀浆，分离上清液检测T-SOD、MDA活性和GSH-Px含量；一侧肾脏制作病理切片，进行组织形态学检查，并用免疫组织化学方法检测肾脏TRPC6蛋白表达水平，TUNEL法检测STZ糖尿病小鼠肾小球细胞凋亡；另侧肾脏用于检测TGF-β_1mRNA、Col Ⅳ mRNA表达。
2. STZ（60mg·kg~(-1)）单次腹腔注射建立糖尿病大鼠模型。取造模成功大鼠随机分为模型组（Model group）、Tempol组（60mg·kg~(-1)）与AST组（40mg·kg~(-1)）。每组20只动物。各组动物每日灌胃给药1次，连续4-6W，正常组（Control）和模型组给予等容量蒸馏水。实验过程中每日观察大鼠生存状态与活动情况，并采用代谢笼法，在造模前（Before）、造模后（0W）、给药后1W、2W，4W、6W分别记录大鼠体重，日摄食量、日饮水量、24h尿量（urine output，UO）等生理指标。尿液静置后，采用渗透压摩尔浓度测定仪测定尿液渗透压。取适量尿液离心，用于检测尿液生化指标：尿液肌酐（urine creatinine，Ucr）、24h尿蛋白（UAE）、β2-微球蛋白（β2-MG）、肿瘤坏死因子α（TNF-α）、转化生长因子-β_1（TGF-β_1）含量。分别在给药后4W及6W，各组随机取半数（10只）动物麻醉后，腹主动脉取血，检测空腹血糖（FBG），血清糖化血清蛋白（GSP）、空腹血清胰岛素（Fasting insulin, FINS）、胰岛素敏感指数（Insulin sensitivity index, ISI）、总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白胆固醇（High density lipoprotein cholesterol, HDL-C）、低密度脂蛋白胆固醇（Lowdensity lipoprotein cholesterol, LDL-C）含量、T-SOD、GSH-Px活性和MDA含量、血清尿素氮（Blood urea nitrogen, BUN）、血肌酐（Serum creatinine, Scr）含量。取血后颈椎脱臼处死动物，迅速摘取肝脏、脾脏、肾脏，称重，计算脏器指数。一侧肾脏制作病理切片，进行组织形态学检查，并采用免疫组织化学方法检测肾脏TRPC6蛋白表达水平；另侧肾脏用于检测TRPC6mRNA、TGF-β_1mRNA、Col ⅣmRNA表达。
3.采用人肾小球系膜细胞（Human Mesangial Cell, HMC）作为目标细胞，分别设立正常对照组（NG）、甘露醇渗透压对照组（MA）、高糖组（HG）、Tempol组（100μmol·L~(-1)）、AST（50,100,200mg L~(-1)）组。NG组细胞予低糖DMEM培养液（含5.5mmol L~(-1)D-葡萄糖）培养，MA组予含24.5mmol L~(-1)甘露醇的低糖培养液培养，HG组予高糖DMEM培养液（含30mmol L~(-1)D-葡萄糖）培养，Tempol组予含100μmol L~(-1)Tempol的高糖培养液培养干预，AST各剂量组分别予含50,100,200mg L~(-1)AST高糖培养液培养。干预时间为24h。用MTT法检测AST（0-400mg L~(-1)）对高糖培养HMC细胞增殖与细胞活力影响；流式细胞仪检测各组HMC细胞中ROS含量；荧光实时定量PCR法检测各组HMC细胞内TRPC6mRNA、TGF-β_1mRNA、NOX4mRNA表达情况；Western blot法检测各组HMC细胞内TRPC6、TGF-β_1、NOX4蛋白表达情况。采用生物素双抗体夹心酶联免疫吸附法（Enzyme-linked immunosorbentassaym, ELISA）测定各组HMC细胞上清液内SOD、MDA、GSH-Px、TGF-β_1、FN、Col Ⅳ含量。
结果：
1黄芪总苷对实验性糖尿病小鼠肾脏保护作用
1.1AST可显著改善STZ糖尿病小鼠“三多一少”的症状，提高肝脏抗氧化能力，表现为升高T-SOD、GSH-Px活性，降低MDA含量；血清FBG、GSP、TC及TG水平亦有下降的趋势，但尚未能恢复至正常水平。
1.2AST可在一定程度上降低肾脏指数，对减轻肾小球损伤与凋亡呈剂量依赖性。
1.3AST可抑制肾脏Col Ⅳ mRNA、TGF-β_1mRNA高表达，显著提高糖尿病小鼠肾小球TRPC6蛋白表达水平。
2黄芪总苷对实验性糖尿病大鼠肾脏保护作用
2.1AST可显著改善STZ糖尿病大鼠“三多一少”症状，表现为体重降低减缓，日摄食量、日饮水量及尿量逐渐减少。
2.2AST可降低STZ糖尿病大鼠FBG、GSP，升高ISI；但大鼠FBG仍处于较高水平。
2.3AST可降低STZ糖尿病大鼠血清TC、TG及LDL-C水平，升高HDL-C含量，提示AST可在一定程度上纠正糖尿病大鼠的血脂代谢紊乱。
2.4AST可提高STZ糖尿病大鼠抗氧化能力，提高血清T-SOD、GSH-Px活性，降低MDA含量。
2.5AST可使STZ糖尿病大鼠UAE明显下降，显著降低糖尿病大鼠24h尿β2-MG、TNF-α、TGF-β_1排出量。改善STZ糖尿病大鼠肾功能，降低Scr、BUN含量，提高Ccr，降低肾脏指数，减轻肾小球损伤；降低肾脏Col Ⅳ mRNA、TGF-β_1mRNA表达，提高TRPC6mRNA表达量，同时也显著提高糖尿病大鼠肾小球TRPC6蛋白表达水平。这表明AST对STZ糖尿病大鼠具有较好的防治其肾脏功能损伤的作用。
3黄芪总苷对高糖培养人肾小球系膜细胞保护作用
3.1在0--400mg L~(-1)浓度范围内，AST可浓度依赖性的抑制高糖培养HMC细胞增殖。
3.2高糖培养HMC上清液内T-SOD、GSH-Px活性显著降低，MDA含量明显升高；AST可在一定程度上提高HMC T-SOD、GSH-Px活性，降低MDA含量。
3.3高糖培养HMC上清液TGF-β_1、FN、Col Ⅳ含量明显上升；各剂量AST均可明显降低TGF-β_1、FN、Col Ⅳ含量，除AST（50mg L~(-1)）组外，其他各剂量组已恢复至正常水平。
3.4高糖培养HMC内ROS含量明显上升；各剂量AST均可明显降低HMC细胞内ROS含量，其中AST（200mg L~(-1)）组HMC内ROS含量已降至正常水平。
3.5高糖培养HMC内TRPC6mRNA表达水平显著降低，TGF-β_1mRNA及NOX4mRNA表达水平明显上升； AST（100,200mg L~(-1)）可显著提高HMC细胞内TRPC6mRNA表达水平，降低TGF-β_1mRNA及NOX4mRNA表达水平。
3.6高糖培养HMC内TRPC6蛋白表达水平显著降低，TGF-β_1及NOX4蛋白表达水平明显升高；各剂量AST均可显著提高HMC细胞内TRPC6蛋白表达水平，降低TGF-β_1蛋白表达水平；除AST组（50mg L~(-1)）外，其他各剂量组NOX4蛋白表达水平也出现明显下降。
结论：
1AST对实验性糖尿病动物肾脏具有一定的保护作用，其机制可能与AST提高糖尿病动物的抗氧化能力、调节血脂代谢、抑制高糖状态所致的肾组织Col Ⅳ mRNA、TGF-β_1mRNA高表达及显著提高糖尿病动物肾组织TRPC6mRNA与TRPC6蛋白表达水平抑制肾小球细胞凋亡有关。
2AST对高糖培养HMC具有保护作用，其机制可能与AST提高HMC抗氧化能力、降低TGF-β_1、FN、Col Ⅳ含量、抑制TGF-β_1、NOX4高表达及提高TRPC6表达水平有关。
Background
Diabetes Mellitus (DM), a common endocrine-metabolic disease caused by defects ofinsulin secretion and/or insulin resistance, is chacterized by increased blood glucose. Ithas been the third major disease following tumor and cardio-cerebrovascular disease.Diabetic nephropathy (DN) is the most frequent and serious complications of DM andbecomes the main cause of deaths in DM. Approximately20to30persents of patientswith Type1diabetes mellitus (T1DM) or Type2diabetes mellitus (T2DM) have DN,most of which are further developed to end-stage renal disease (ESRD). Pathogenesy ofDN has not been clarified so far. It is believed to be influenced by multiple determintswith the high glucose level as the initiation factor. Strong oxidative stress is observed inDM and plays an important role in occurance and development of DM. Therefore,oxidative stress is regarded as the key reason of DM complications. As to the treatmentand prevention of DN, it is lack of ideal strategies and drugs. Thus, it would be of of greatsignificant to seek for effective methods for DN control.
In Traditional Chinese Medicine (TCM), it is considered that DM belongs to consumptivethirst and oedema and characterised with asthenia in origin, asthenia in superficiality andasthenia and sthenia. It treatment must focus on strengthening body resistance andeliminating pathogen. In clinical medication of TCM, supplementing qi and nourishingyin and disperse blood stasis and dredge collateral are suggested to be the predominanttreatments.
Astragalus membranaceus (AS) is one of the most commonly used herbal medicines in TCM, which possesses “Buqishengyang, Yiweigubiao, Tudushengji, Lishuituizhong”function. It can be used in deficiency of vital energy and calor internus. AS is a principaldrug to control diseases in heart, liver and kidney. Single AS can decrease urine protein innephritic syndrome and is one of the commonest traditional medicine to control nephriticsyndrome. Astragalosides (AST) is the effective fraction extracted from astragalus.Through many years of research by our team, AST is found to have many biologicaleffects, including anti-stress, anti-oxidation and immunological regulation. There havebeen lots of clinical and experimantal researches on DM treatment with astragalus orextract of astragalus, but the systematic study about the effect of AST on DM and itspotential mechanisms is still limited. In this thesis, we take the pharmacologic action ofastragalus effective component as the particular target and aim to understand the effect ofAST on DM and its potential mechanisms with both in vivo experiment and ex vivoexperiment. Findings generated from the exproments are expected to provide elementaryinformation on DM treatment and drug design for DN.
Objectives
To understand effect of AST on DM and its potential mechanisms by treating diabetic ratsand diabetic mice induced by streptozotocin (STZ) and the molecular mechanismsunderline the effect is investigated using high-glucose-incubated human mesangial cell(HMC) as the targeted cell.
Methods
1The diabetic mouse model was established by intraperitoneal injection with STZ (100mg·kg~(-1)) and the model mice were randomly divided into Model group, Tempol group (90mg·kg~(-1)), AST-L group (30mg·kg~(-1)), AST-M group (60mg·kg~(-1)), and AST-H group (120mg·kg~(-1)). The animals were given the drugs with successive intragastric administration for4-6weeks, the same volume of distilled water were admistered to Control group and Model group, respectively. After4weeks and6weeks of administration, half of the mice(10) in each group were randomly selected and blood sampkes were drawn to assessfasting blood glucose (FBG), triglyceride (TG), total cholesterol (TC), glycosylated serumprotein (GSP). The mice were sacrificed with cervical dislocation after blood sampleswere taken. And then, livers, spleens and kidneys were removed immediately, douchedwith ice physiological saline, dried with filter papers, weighed and organ index werecalculated. Liver tissue bomogenate (10%) was made by liver tissue of0.5g. Activities ofT-SOD and MDA and contents of GSH-Px were evaluated after isolating supernatants.One side of kidney was put into4%formaldehyde solution and made to paraffin sectionfor histomorphological examination. This side of kidney was also used to measure thelevel of TRPC6expression by immunohistochemistry as well as the apoptosis ofglomerular cells in glomeruli by TUNEL. The other side of kidney, however, was undercryopreservation at-80℃for the assessment of TGF-β_1mRNA、 Col Ⅳ mRNAexpression.
2The diabetic rat model was established by intraperitoneal injection with STZ (60mg·kg~(-1)) and the model rats were randomly divided into Model group, Tempol group (60mg·kg~(-1)) and AST group (40mg·kg~(-1)). The animals were given the drugs with successiveintragastric administration for4-6weeks, the same volume of distilled water wererespectively admistered to Control group and Model group. Survival status and activitiesof the animals were observed every day. Via metabolic cage, body weight, daily foodintake, daily water intake and daily urine output (UO) were recorded respectivelypre-therapy,0W,1W,4W and6W after administration. After urion prepared in standing,uric osmotic pressure was assessed by osmotic pressure molar concentration determinator.Proper amount of urion was centrifugated and prepared for measurement of biochemicalindicators under cryopreservation at-80℃, such as urine creatinine (Ucr),urinaryalbumin excretion (UAE) β2-microglobulin (β2-MG), tumor necrosis factor (TNF-α), transforming growth factor (TGF-β_1). After4weeks and6weeks of administration, halfof the rats (10) in each group were randomly selected and blood samples were drawn fromabdominal aorta after anesthesia to assess FBG, GSP, fasting serum insulin (FINS), insulinsensitivity index (ISI), TC, TG, high density lipoprotein (HDL), low density lipoprotein(LDL), as well as activities of T-SOD and GSH-Px and contents of MDA, blood ureanitrogen (BUN), serum creatinine (Sc). The rats were sacrificed with cervical dislocationafter blood samples were taken. And then, livers, spleens and kidneys were removedimmediately, douched with ice physiological saline, dried with filter papers, weighed andorgan index were calculated. Liver tissue bomogenate (10%) was made by liver tissue of0.5g. One side of kidney was put into4%formaldehyde solution and made to paraffinsection for histomorphological examination. This side of kidney was also used to measurethe level of TRPC6expression by immunohistochemistry. The other side of kidney,however, was under cryopreservation at-80℃for the assessment of TRPC6mRNA,TGF-β_1mRNA and Col ⅣmRNA expression.
3Human mesangial cells (HMC) was taken as target cells and divided into Normal group(NG), Mannitol (MA), high glucose group (HG), Tempol (100μmol·L~(-1)), AST-L(50mg L~(-1)), AST-M (100mg L~(-1)) and AST-H (200mg L~(-1)). NG group were incubated withlow glucose DMEM culture solution (5.5mmol L~(-1)D-glocose), MA group wereincubated with low glucose culture solution (24.5mmol L~(-1)Mannitol) and HG groupwere with high glucose DMEM culture solution (30mmol L~(-1)D-glocose). Similarly,Tempol group were incubated with high glucose culture solution (100μmol L~(-1)Tempol),and high glucose culture solution (50mg L~(-1)AST) in AST-L group, high glucose culturesolution (100mg L~(-1)AST) in AST-M group and high glucose culture solution (200mg L~(-1)AST) in AST-H group. The duration of administration was all defined as24hours. Then,different methods were adopted to assess various indicators. For instance, MTT Assay forthe effect of AST on proliferation and viability of high-glucose-incubated HMC, flow cytometry for ROS contents in HMC, fluorescent quantitative real time PCR for TRPC6mRNA、TGF-β_1mRNA、NOX4mRNA expression in HMC, Western blot for TRPC6、TGF-β_1、NOX4expression in HMC. In addition, enzyme-linked immunosorbent assaym(ELISA) was introduced to assess contents of SOD、MDA、GSH-Px、TGF-β_1、FN、Col Ⅳ in HMC supernatant in each group.
Results
1Protective effects of AST on experimental diabetic mice
1.1AST significantly improved the typical symptoms (polyuria, polydipsia, polyphagia,weight loss) and enhanced the anti-oxidative ability in liver of STZ diabetic mice. Indetail, it increased activities of T-SOD and GSH-Px and decreased MDA level. Althoughthe levels of serum FBG, GSP, TC and TG decreased, the reduction could not return tonormal level.
1.2AST decreased kidney index to some extent. The effects of alleviating glomerulusdefects and apoptosis were shown dose-dependent and this effect was most significant atthe dosage of60mg·kg~(-1).
1.3AST inhibited the high expression of collagen Type Ⅳ mRNA and TGF-β_1mRNA inkidney. AST raised the TRPC6expression in glomerulus in STZ diabetic mice.
2Protective effects of AST on experimental diabetic rats
2.1AST significantly improved the typical symptoms (polyuria, polydipsia, polyphagia,weight loss) in STZ diabetic rats. The body weights of rats didn’t continuously decrease,while daily food intake, daily water intake and daily urine output decreased gradually.
2.2AST decreased the levels of FBG and GSP and increase ISI. But the content of FBGstill kept at a relatively high level.
2.3AST decreased the level of serum TC, TG and LDL while enhance the level of HDL.This kind of effect could rectify lipid metabolic disorders in STZ diabetic rats.
2.4AST enhanced the anti-oxidative ability in STZ diabetic rats. It increased activities ofT-SOD and GSH-Px and decreased MDA level.
2.5AST decreased daily amount of urine protein excretion as well as daily amount ofurine β2-MG, TNF-α and TGF-β_1excretion. AST improved kidney function of STZdiabetic rats. It decreased serum levels of creatinine, urea nitrogen and enhanced thecreatinine clearance rate. It decreased kidney index and alleviate the defects of glomerulus.It also decreased expression of collagen Type Ⅳ mRNA and TGF-β_1mRNA, enhancedexpression of TRPC6mRNA and increased expression of TRPC6in glomerulus in STZdiabetic rats.
3Protective effect of AST on high glucose incubated glomerular mesangial cells
3.1At the range of0-400mg L~(-1), the cell proliferation inhibiting rate caused by ASTshowd dose-dependent in high glucose incubated incubated glomerular mesangial cells.
3.2Activities of T-SOD and GSH-Px in HMC supernatant decreased and increasedcontent of MDA. AST increased activities of T-SOD and GSH-Px in HMC and decreasedthe level of MDA.
3.3The level of TGF-β_1, FN and Col Ⅳ increased in high glucose incubated HMCsupernatant. AST with high, median and low dosage all decreased the contents of TGF-β_1,FN and Col Ⅳ. The three indicators returned to normal level in groups with high andmedian dosage.
3.4The level of ROS significantly increased in high glucose incubated HMC. AST withhigh, median and low dosage all decreased the contents of ROS in HMC. And ROSdecreased to normal level in group with high AST dosage.
3.5The expression of TRPC6mRNA decreased in high glucose incubated HMC while theexpression of TGF-β_1mRNA and NOX4mRNA significantly increased. AST with highand median dosage both increased the expression of TRPC6mRNA and decreased theexpression of TGF-β_1mRNA and NOX4mRNA.
3.6The expression of TRPC6protein decreased in high glucose incubated HMC while theexpression of TGF-β_1and NOX4protein significantly increased. AST with high, medianand low dosage all decreased the the expression of TRPC6protein in HMC and decreasedthe expression of TGF-β_1protein. The expression of NOX4protein significantlydecreased in group with high and median dosage.
Conclusions
1There is protective effect of AST on high glucose incubated glomerular mesangial cells.The potential mechanisms may include the enhancement of anti-oxidative ability of HMC,reduction of the levels of TGF-β_1, FN and Col Ⅳ, inhibitation in the high expression ofTGF-β_1and NOX4and promotion of the TRPC6expression by AST.
2There are some protective effects of AST on kidney of experimental diabetic rats andmice. The potential mechanisms may include improvement of anti-oxidative ability inserum and liver, regulation of lipid metabolism, protection of kidney function, inhibitationin apoptosis in glomerulus, inhibitation in the high expression of Col ⅣmRNA,TGF-β_1mRNA in kidney and enhancement of the expression of TRPC6mRNA andTRPC6protein in glomerulus by AST.
引文
[1]刘烨,张琳,洪天配.2011年糖尿病学领域的研究进展和热点回顾[J].中国医学前沿杂志（电子版）,2011;3(6):27-31
[2]中华医学会糖尿病学分会．中国2型糖尿病防治指南（2010年版）[J]．中国医学前沿杂志（电子版），2011;3(6):54-109．
[3] Bloomgarden ZT. Diabetic nephropathy [J]. Diabetes Care,2008;31(4):823-827.
[4] Beisswenger PJ, Drummond KS, Nelson RG, Howell SK, Szwergold BS, Mauer M.Susceptibility to diabetic nephropathy is related to dicarbonyl and oxidative stress [J].Diabetes,2005;54(11):3274-3281.
[5] Brownlee M. Biochemistry and molecular cell biology of diabetic complications [J]. Nature,2001;414(6865):813-820.
[6] Banday AA, Fazili FR, Lokhandwala MF. Oxidative stress causes renal dopamine D1receptordysfunction and hypertension via mechanisms that involve nuclear factor-kB and protein kinase C[J]. J Am Soc Nephrol,2007;18(5):1446-1457.
[7] Gill PS, Wilcox CS. NADPH oxidases in the kidney [J]. Antioxid Redox signal,2006;8(9-10):1597-1607.
[8] Nauseef WM. Biological roles for the NOX family NADPH oxidases [J]. J Biol Chem,2008;283(25):16961-16965.
[9] Gorin Y, Block K, Hernandez J, Bhandari B, Wagner B, Barnes JL, Abboud HE. Nox4NAD(P)H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney[J]. J BiolChem.2005;280(47):39616-39626.
[10] Houstis N, Rosen ED, Lander ES. Reactive oxygen species have a causal role in multiple formsof insulin resistance [J]. Nature,2006;440(7086):944-948．
[11]王宇翎,李卫平,张艳.益肾复方对大鼠阿霉素肾病的作用[J].安徽医科大学学报,2007;42(1):51-53.
[12]余凌,李惊子,王海燕.黄芪、当归在肾脏疾病中的应用及其机制研究进展[J].中国中西医结合杂志,2001;21(5):396-398.
[13] Motomura K, Fujiwara Y, Kiyota N, Tsurushima K, Takeya M, Nohara T, Nagai R, Ikeda T.Astragalosides isolated from the root of astragalus radix inhibit the formation of advanced glycationend products[J]. J Agric Food Chem.2009;57(17):7666-7672.
[14] Lei H, Wang B, Li WP, Yang Y, Zhou AW, Chen MZ. Anti-aging effect of astragalosides and itsmechanism of action [J]. Acta Pharmacol Sin,2003;24(3):230-234.
[15]牟娜,张庆怡,倪兆慧,童菊芳.黄芪对高糖作用下肾间质成纤维细胞表达HGF的影响[J].中国中西医结合肾病杂志,2002;3(1):7-9.
[16] Xiang FL, Lu X, Strutt B, Hill DJ, Feng Q.NOX2deficiency protects againststreptozotocin-induced beta-cell destruction and development of diabetes in mice [J]. Diabetes.2010;59(10):2603-2611.
[17] Liu WH, Hei ZQ, Nie H, Tang FT, Huang HQ, Li XJ, Deng YH, Chen SR, Guo FF, Huang WG,Chen FY, Liu PQ. Berberine ameliorates renal injury in streptozotocin-induced diabetic rats bysuppression of both oxidative stress and aldose reductase [J]. Chin Med J (Engl).2008;121(8):706-712.
[18] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitativePCR and the2(-Delta Delta C(T)) Method[J]. Methods.2001;25(4):402-408.
[19]周琚,张汝学,贾正平.不同糖尿病动物模型的特点比较[J].国际内分泌代谢杂志,2010;30(4):273-276.
[20] Ktorza A, Bernard C, Parent V, Penicaud L, Froguel P, Lathrop M, Gauguier D. Are animalmodels of diabetes relevant to the study of the genetics of non-insulin-dependent diabetes in humans[J]．Diabetes Metab，1997;23(Suppl2):38-46．
[21]王欣,海春旭.糖尿病动物模型研究进展[J].毒理学杂志，2011;25(5):387-390
[22]冯烈,罗璐,卢筱华.福辛普利对糖尿病大鼠肾脏的保护作用[J].暨南大学学报(医学版),2001,22(4):35-39.
[23]李伟,张红,殷松楼,褚璇.不同剂量链脲佐菌素诱导SD大鼠糖尿病肾病模型的研究[J].徐州医学院学报,2006;26(1):52-55.
[24]陈秋,夏永鹏,邱宗荫.2型糖尿病大鼠模型的建立与评价[J].天津医药,2006;34(1):33-35.
[25]黄波,刘学政,庞东渤.不同途径注射链脲佐菌素致大鼠糖尿病模型的研究[J].锦州医学院学报,2003;24(1):19-21.
[26]沈亚非,徐焱成.链脲佐菌素诱导实验性糖尿病大鼠模型建立的研究[J].实用诊断和治疗杂志,2005;19(2):79-80.
[27]赵芳,蒋朝晖,杨国珍,李兴,朱丽英,潘卫.不同剂量链脲佐菌素建立1型糖尿病大鼠模型[J].贵阳医学院学报,2010;35(1):22-25.
[28]农慧盛,庆寿,梁健,闫福曼,罗荣敬,李小英. STZ诱导糖尿病大鼠模型的研究[J].广西医科大学学报,2010;27(1):69-72.
[29]刘志民,孙亮亮.氧化应激在糖尿病发病机制中作用的认识[J].内科理论与实践,2007;2(3):153-155.
[30] Brownlee M. The Pathobiology of diabetic complications:a unifying mechanism[J].Diabetes，2005;54(6):1615-1625.
[31]宿世震,邢冬杰.氧化应激与糖尿病肾病[J].中国实用医药,2005;3(3)531-532.
[32] Ha H, Yu MR, Choi YJ, Kitamura M, Lee HB. Role of high glucose-in-duced nuclearfactor-kappaB activation in monocyte che-moattractant protein-1expression by mesangial cells [J].J AmSoc Nephrol,2002;13(4):894-902.
[33] Waldre EN. Cellular oxidative processes in relation to renal disease [J]. Am J NePhrol,2005;25(1):13-22.
[34] Maritim AC, Sanders RA, Watkins JB. Effects of alpha-lipoic acid onbiomarkers of oxidativestress in streptozotocin-induced diabetic rats [J]. J Nutr Biochem,2003;14(5):288-294.
[35] Shokoji T, Nishiyama A, Fujisawa Y, Hitomi H, Kiyomoto H, Takahashi N, Kimura S, KohnoM, Abe Y. Renal sympathetic nerve responses to tempol in spontaneously hypertensive rats[J].Hypertension,2003;41(2):266-273.
[36] Meng S, Cason GW, Gannon AW, Racusen LC, Manning RD Jr. Oxidative stress in Dalaisalt-sensitive hypertension [J]. Hypertension,2003;41(6):1346-1352.
[37] Kopkan L, Majid DS. Enhanced superoxide activity modulates renal function in NO-deficientliypertensive rats [J]. Hypertension,2006;47(3):568-572.
[38] Laight DW, Andrews TJ, Haj-Yehia AI, Carrier MJ, Angg rd EE. Microssoy of superoxideanion sctivity in vitro [J]. Environmental Toxicology and Pharmacology,1997;3(1):65-68.
[39]Mitchell JB, Samuni A, Krishna MC, Krishna MC, DeGraff WG, Ahn MS, Samuni U, Russo A.Biologcally active metal-indismutase mimincs[J].Biochemistry,1990;29(11):2802-2807.
[40]孙风兰,王国英,李其平,吴延芳.氮氧化物对人角质形成细胞氧化损伤的保护作用[J].中华皮肤科杂志,2002;35(6):429-431.
[41] Bernstein EF, Kong SK, Brown DB, Kwak BC, Takeuchi T, Gasparro FP, Uitto J. The nitroxideTempol affords protection against ultraviolet radiation in a transgenic murine fibroblast culturemodel of cutaneous photoaging [J]. Exp Dermatol,200l;10(1):55-61.
[42] Yan SX, Hong XY, Hu Y, Liao KH. Tempol, one of nitroxides,is a novel ultraviolet—A1radiation protector for human dermal fibroblasts[J]. Journal of Dermatological Science,2005;37(3):137-143.
[43]严淑贤,徐昱,胡跃,廖康煌.氮氧化物对中波紫外线照射下包皮成纤维细胞的保护作用[J].中华老年医学杂志,2005;24(10):753-756.
[44] Yanes L, Romero D, Iliescu R, Cucchiarelli VE, Fortepiani LA, Santacruz F, Bell W, Zhang H,Reckelhoff JF. Systemic arterial pressure response to two weeks of tempol therapy in SHR:involvement of NO, the RAS, and oxidative stress [J]. Am J Physiol Regul Integr Comp Physiol,2005;288(4):903-908.
[45] Bayorh MA, Mann G, Walton M, Eatman D. Effects of enalapril,tempol,and eplerenone onsalt-induced hypertension in Dahl salt-sensitive rats[J]. Clini and Expe Hypertens,2006;28(2):121-132.
[46]刘文军,贾一韬,夏照帆,付晋凤,马兵,卫伟,吕开阳,徐豫,王钰,孙瑜. Tempol对浸水束缚应激大鼠胃黏膜p38MAPK活化的影响[J].解放军医学杂志,2008;33(2):163-165.
[47] Wilcox CS. Effects of tempol and redox-cycling nitroxides in models of oxidative stress[J].Pharmacol Ther,2010;126(2):119-45.
[48] Chatterjee PK, Cuzzocrea S, Brown PA, Zacharowski K, Stewart KN, Mota-Filipe H,Thiemermann C.Tempol, a membrane-permeable radical scavenger, reduces oxidant stress-mediatedrenal dysfunction and injury in the rat[J]. Kidney Int.2000;58(2):658-73.
[49] Asaba K, Tojo A, Onozato ML, Goto A, Fujita T. Double-edged action of SOD mimetic indiabetic nephropathy[J]. J Cardiovasc Pharmacol,2007;49(1):13-9.
[50]中华中医药学会.糖尿病中医防治指南(2007版)[M].北京:中国中医药出版社,2007:5.
[51]黄可儿,赵敏,王建华.黄芪总苷的药理研究进展[J].中药新药与临床药理,2005;16(6):461-463.
[52]宋锦叶,李深,孟立强,屈磊,李晓玫.黄芪当归合剂对5/6肾切除大鼠肾组织损伤的治疗作用[J].北京大学学报（医学版）,2009;41(2):196-202.
[53] Gilbert RE, Tsalamandris C, Bach LA, Panagiotopoulos S, O'Brien RC, Allen TJ, Goodall I,Young V, Seeman E, Murray RM Long-term glycemic control and the rate of progression of earlydiabetic kidney disease[J].Kidney Int,1993;44(4):855-861.
[54]马燕,张晶,王亚,喇孝瑾,张晓鹏,吕建东,喇万英.黄芪降糖颗粒降糖作用实验研究[J].中国实验方剂学杂志,2011;17(8):157-160
[55]徐郁杰,张庆怡,吴青伟.黄芪对糖尿病大鼠早期肾肥大和蛋白尿的影响[J].上海第二医科大学学报,1997；17(5):357-359.
[56]王法德,王兆淦,王立琴,田立,王朝霞,刘雪君.参芪克糖灵治疗糖尿病的实验研究[J].山东中医杂志,2997;16(7):316-319.
[57]刘道芳,袁敏.黄芪研究进展[J].中医药信息,1998;15(2):13-14.
[58]郝玉美,刘洪琪.苦瓜、黄芪、黄芩苷对2型糖尿病模型大鼠胰岛素抵抗的影响[J].中药药理与临床,2007;23(5):145-147.
[59] Aso Y, Yoshida N, Okumura K, Wakabayashi S, Matsutomo R, Takebayashi K, Inukai T.Coagulation and inflammation in overt diabetic nephropathy:association withhyperhonoeysteinemia[J]. Clin Chem Aeta,2004;348(1-2):139-145.
[60]刘洪凤,郭新民,王桂云,冯芹喜,包海花,崔荣军.黄芪多糖对2-DM胰岛素抵抗大鼠血糖及血脂的影响[J].牡丹江医学院学报,2007;28(5):18-20.
[61]廖苇萍,石元刚.黄芪多糖和大豆异黄酮对糖尿病大鼠糖代谢的影响[J].第三军医大学学报,2007;29(5):416-418.
[62]阮耀,岳兴如,李晓明,王珍珍,阮翘.黄芪对糖尿病大鼠心肌MDA及SOD, GSH-PX,Na+-k+ATP酶活性的影响[J].时珍国医国药,2007;18(3):593-594.
[63]王艳桥,徐向进,陈频.氧化应激研究进展及其在糖尿病肾病发病中的作用[J].医学综述,2010;16(11):1681-1684.
[64] Hwang SY, Siow YL, Au-Yeung KK, House J, OK.Folic acid supplementation inhibits NADPHoxidase-mediated superoxide anion production in the kidney[J]. Am J Physiol Renal Physiol,2011;300(1):F189-198.
[65] Montezano AC, Burger D, Ceravolo GS, Yusuf H, Montero M, Touyz RM. Novel Noxhomologues in the vasculature: focusing on Nox4and Nox5[J]. Clin Sci (Lond).2011;120(4):131-41.
[66] Kashihara N, Haruna Y, Kondeti VK, Kanwar YS. Oxidative stress in diabetic nephropathy [J].Curr Med Chem.2010;17(34):4256-69.
[67] Geiszt M, Kopp JB, Varnai P, Leto TL. Identification of Renox, an NADPH oxidase in kidney[J]. Proc Natl Acad Sci U S A,2000;97(14):8010-8014.
[68] Gorin Y, Ricono JM, Kim NH, Bhandari B, Choudhury GG, Abboud HE. Nox4mediatesangiotensin Ⅱ-induced activation of Akt/protein kinase B in mesangial cells [J]. Am J Physiol RenalPhysiol,2003;285(2):F219-F229.
[69] Block K, Eid A, Griendling KK, Lee DY, Wittrant Y, Gorin Y. Nox4NADPH oxidase mediatesSrc-dependent tyrosine phosphorylation of PDK-1in response to angiotensin Ⅱ.Role in mesangialcell hypertrophy and fibronectin expression[J]. J Biol Chen,2008;283(35):24061-24076.
[70]王有德.糖尿病肾病发病机制的研究进展[J].当代医学,2010;16(7):36-41.
[71] Aksun SA, Ozmen D, Ozmen B, Parildar Z, Mutaf I, Turgan N, Habif S, Kumanlio luc K,Bayindir O. Beta2microglobulin and cystatin C in type2diabetes：Assessment of diabeticdephropathy[J]. Exp Clin Endocrinol Diabetes,2004;112(4):195-200.
[72]吴志美.糖尿病肾病尿β2-微球蛋白、转铁蛋白的测定[J].检验医学与临床,2007;4(1):32.
[73]蒋最明,顾敏,任风琴.糖尿病肾损伤早期诊断的尿液联合检测[J]．实用全科医学，2008;6(1):85-86．
[74] Dalla Vestra M., Fioretto P. Diabetic nephropathy: renal struetural studies in typel and2diabetiepatients [J]. International Congress Series,2003;1253:163-169.
[75] Ding DF, You N, Wu XM, Xu JR, Hu AP, Ye XL, Zhu Q, Jiang XQ, Miao H, Liu C, Lu YB.Resveratrol attenuates renal hypertrophy in early-stage diabetes by activating AMPK [J]. Am JNephrol,2010,31(4):363-374.
[76] Zhang HJ, Zhao JB, Li P. Progress in the study on animal model of diabeticnephropathy[J]．Chin Pharmacol Bull，2008;24(7):845-848．
[77] Yu M，Zhou HH，Liu ZQ．Progress in related genes of diabetic nephropathy[J]．Chin PharmacolBull，2009;24(11):1419-1422．
[78] Mauer SM, Steeffs MW, Borwn DM. Structure functional relation in diabetic nephropathy[J]. JClin Invest,1984,74(4):1143-1154.
[79]孙晋芳,焦金菊,宋其蔓,白玉红.黄芪皂甙对大鼠肾小球系膜细胞增殖及周期的影响[J].中国组织工程研究与临床康复,2008;12(15):2926-2928.
[80] Li X, Pabla N, Wei Q, Dong G, Messing RO, Wang CY, Dong Z. PKC-delta Promotes RenalTubular Cell Apoptosis Associated with Proteinuria[J].J Am Soc Nephrol,2010;21(7):1115-24.
[81] Watson S, Cailhier JF, Hughes J, Savill J. Apoptosis and Glomerulonephritis[J].Curr DirAutoimmun,2006;9:188-204.
[82]张艳玲,段惠军,李春香,王燕,史永红,李英敏.糖尿病大鼠肾脏细胞凋亡与Bax和Bcl-2基因表达[J].中国糖尿病杂志,2002;10(3):159-162.
[83]苏君梅,董峰,易华,董传仁,欧阳静萍,杨海鹭.糖尿病大鼠肾脏细胞凋亡的研究[J].武汉大学学报(医学版),2003;24(1):17-20.
[84] Niranjan T, Bielesz B, Gruenwald A, Ponda MP, Kopp JB, Thomas DB, Susztak K. The Notchpathway in podocytes plays a role in the development of glomerular disease [J]. NatMed,2008;14(3):290-298.
[85] Susztak K, Raff AC, Schiffer M, B ttinger EP. Glucose induced reactive oxygen species causeapoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy [J]. Diabetes,2006;55(1):225-233.
[86]桂定坤,陈建国,陈宜方,黄建华.黄芪甲苷抑制高糖诱导的足细胞凋亡作用及机制[J].中华中医药学刊,2010;28(4):822-824.
[87] Huang Y, Chen Y, Xu M, Gu W, Bi Y, Li X, Ning G. Low-grade albuminuria is associated withcarotid intima-media thickness in Chinese type2diabetic patients [J]. J Clin Endocrinol Metab.2010;95(11):5122-5128.
[88] Ding G, Pesek-Diamond I, Diamond JR. Cholesterol, macrophages and gene expression ofTGF-β1and fibronectin during nephrosis [J]. Am J Physiol,1993;264(4Pt2):F577-84.
[89]苏进,田浩明.纤维连接蛋白与糖尿病肾病[J].医学综述,2001;7(11):654-655
[90] Doi K, Okamoto K, Tokunaga K, Fujita T, Noiri E. Genome study of kidney disease in the ageof post genome-sequencing[J]. Endocr Metab Immune Disord Drug Targets.2008;8(3):173-183.
[91] Xu YF，Wu YL，Wan JX，WANG CY．Effects of BMP-7on the bagpression of extracellularmatrix in human renal proximal epithelial cells induced by TGF-β1[J]．Chin Pharmacol Bull，2008;24(11):1453-1457．
[92] Rovin BH, Tan LC. LDL stimulates mesangial fibronectin production and chemoattractantexpression [J]. Kidney Int,1993;43(1):218-25.
[93] Chandrasekar B, Streitman JE, Colston JT, Freeman GL. Inhibition of nuelear factor kappa Battemiates proinflammatory cytokine and inducible nitric-oxide synthase expression in postisehemicmyoeardium [J]. Biochim Biophys Acta,1998;1406(l):91-106.
[94] Lubin FD, Reingold SC. Defining the clinical course of multiple sclerosis: results of aninternational survey. National Multiple Sclerosis Society (USA) Advisory Committee on ClinicalTrials of New Agents in Multiple Sclerosis [J]. Neurology,1996;46(4):907-911.
[95]范兴忠,李宏,赵建强,王建英,李新东.黄芪对肾病综合征患者血清和尿肿瘤坏死因子水平的影响[J].免疫学杂志,2005;21(5):421-422.
[96] Watanabe H, Sanada H, Shigetomi S, Katoh T, Watanabe T. Urinary excretion of type IVcollagen as a specific indicator of the progression of diabetic nephropathy[J]. Nephron,2000;86(1):27-35
[97]尹得海，梁晓春.从肾小球系膜细胞表型转化谈中医药防治糖尿病肾病机制研究通路[J].中国中医药信息杂志,2005;15(6):90-91.
[98]叶太生,周必发,张莹雯.百令胶囊对大鼠肾小球系膜细胞增殖、Ⅳ型胶原及TGF-β1mRNA表达的影响[J].中国中西医结合肾病杂志,2010;11(1):41-43.
[99] Lam S, van der Geest RN, Verhagen NA, Daha MR, van Kooten C. Secretion of collagen typeIV by human renal fibroblasts is increased by high glucose via a TGF-β1-independent pathway[J].Nephrol Dia Transplant,2004;19(7):1694-1701.
[100] Chen S, Hong SW, Iglesias-de la Cruz MC, Isono M, Casaretto A, Ziyadeh FN. The key roleof the transforming growth factor beta system in the pathogenesis of diabetic nephropathy [J]. RenFail,2001;23(3-4):471-481.
[101]杨政,白云凯. Ⅰ、Ⅳ型胶原和肾脏疾病[J].国外医学·泌尿系统分册,2005;25(5):78-83.
[102] Wang S, Denichilo M, Brubaker C, Hirschberg R. Conneetive tissue growth faetorintubuloin,terstitial injury of diabetic nephropathy[J]. Kidney Int,2001;60(1):96.
[103] Inomata S, Itoh M, Sato T. Difference of serum levels of type IV collagen7S between early orovert nephropathy and nondiabetic renal disease in diabetic patients with micro-ormacroalbuminuria.[J]. Nephron,1996;73(3):601-605.
[104] Woodrow D, Moss J, Shore I, Spiro RG. Diabetic glomerulosclerosis immunogoldultrastructural studieson the glomerular distribution oftypeⅣcollagen and sulphate proteoglycan [J].J Pathol,1992;167(1):49-58.
[105] Sharma K, Jin Y, Guo J, Ziyadeh FN. Neutralization of TGF-beta by anti-TGF-beta antibodyattenuates kidney hypertrophy and the enhanced extracellular matrix gene expression inSTZ-induced diabetic mice[J]. Diabetes,1996;45(4):522-530.
[106] Isaka Y, Fujiwara Y, Ueda N, Kaneda Y, Kamada T, Imai E. Glomerulosclerosis by in vivotransforming growth factor-β1and platelet derived growth factor gene into the rat kidney[J]. J ClinInvest,1993;92(6):2597-2601.
[107] Ziyadeh FN, Sharma K, Ericksen M, Wolf G. Stimulation of collagen gene expression andprotein synthesis in murine mesangial cells by high glucose is mediated by autocrine of transforminggrowth factor beta[J]. J Clin Invest,1994:93(2):536-542.
[108]陶松桔,尹支农,徐自强,宋卫红,谭辉.黄芪注射液对糖尿病肾病患者血Ⅳ型胶原与转化生长因子-β1水平的影响及其意义[J].中国综合临床,2004;20(3):217-219.
[109]陶少平,陈学峰,孙艳,杭宇,周蓓.黄芪注射液对糖尿病肾病患者血转化生长因子-β1及Ⅳ型胶原水平的影响及其意义[J].中国中西医结合肾病杂志,2006;7(3):156-157.
[110]薛艳芸,张立新.黄芪、灯盏花素对实验性糖尿病大鼠肾脏内皮素-1、TGF-β1的作用[J].细胞与分子免疫学杂志,2010;26(9):935-936.
[111]徐郁杰,张庆怡,陆敏,赵涵芳,陈诗书.黄芪对糖尿病大鼠肾皮质TGF-β1表达的影响[J].中华内分泌代谢杂志,1998;14(5):312-314.
[112] Bank N. Mechanisms of diabetic hyperfiltration [J]. Kidney Int.1991;40(4):792-807.
[113] Wei P, Lane PH, Lane JT, Padanilam BJ, Sansom SC. Glomerular structural and functionalchanges in a high-fat diet mouse model of early-stage type2diabetes[J]. Diabetologia,2004;47(9):1541-1549.
[114] Brindeiro CMT, Fallet RW, Lane PH, Carmines PK. Potassium channel contributions toafferent arteriolar tone in normal and diabetic rat kidney[J]. Am J Physiol Renal Physiol.,2008;295(1): F171-178.
[115] Kikkawa R, Kitamura E, Fujiwara Y, Arimura T, Haneda M, Shigeta Y. Impaired contractileresponsiveness of diabetic glomeruli to angiotensin II: a possible indication of mesangialdysfunction in diabetes mellitus[J]. Biochem Biophysic Res Commun,1986;136(3):1185-1190.
[116] Chiarelli F, Gaspari S, Marcovecchio ML. Role of growth factors in diabetic kidney disease[J].Horm Metab Res,2009;41(8):585-593.
[117] Nutt LK, O'Neil RG. Effect of elevated glucose on endothelin-induced store-operated andnon-store-operated calcium influx in renal mesangial cells [J]. J Am Soc Nephrol,2000;11(7):1225-1235.
[118] Whiteside CI, Hurst RD, Stevanovic ZS. Calcium signaling and contractile response ofdiabetic glomerular mesangial cells [J]. Kidney Int Suppl.1995;51: S28-S33.
[119] Schl ndorff D, Banas B. The mesangial cell revisited: no cell is an island [J]. J Am SocNephrol.2009;20(6):1179-1187.
[120] Hurst RD, Whiteside CI, Thompson JC. Diabetic rat glomerular mesangial cells displaynormal inositol trisphosphate and calcium release [J]. Am J Physiol.1992;263(4pt2): F649-F655.
[121] Li WP, Tsiokas L, Sansom SC, Ma R. Epidermal growth factor activates store-operated Ca2+channels through an IP3independent pathway in human glomerular mesangial cells[J]. J Biol Chem.2004;279(6):4570-4577.
[122] Ma R, Sansom SC. Epidermal growth factor activates store-operated calcium channels inhuman glomerular mesangial cells [J]. J Am Soc Nephrol.2001;12(1):47-53.
[123] Ma R, Smith S, Child A, Carmines PK, Sansom SC. Store-operated Ca2+channels in humanglomerular mesangial cells[J]. Am J Physiol.2000;278(6): F954-F961.
[124] Mené P, Pugliese G, Pricci F, Di Mario U, Cinotti GA, Pugliese F. High glucose inhibitscytosolic calcium signaling in cultured rat mesangial cells[J]. Kidney Int.1993;43(3):585-591.
[125] Hsu YJ, Hoenderop JG, Bindels RJ. TRP channels in kidney disease [J]. Biochim Biophys Acta.2007;1772(8):928-936.
[126] Dietrich A, Gudermann T. TRPC6[J]. Handb Exp Pharmacol.2007;179:125-141.
[127] Winn MP, Daskalakis N, Spurney RF, Middleton JP. Unexpected role of TRPC6channel infamilialnephrotic syndrome: does ithave clinicalImplications [J]. J Am SocNephrol.2006;17(2):378-387.
[128] Plant TD, Schaefer M. TRPC4andTRPC5receptor-operatedCa2+-permeable nonselectivecation channels [J]. Cell Calcium.2003;33(5-6):441-450.
[129] Thebault S, Zholos A, Enfissi A, Slomianny C, Dewailly E, Roudbaraki M, Parys J,Prevarskaya N.Receptor-operated Ca2+entry mediated by TRPC3/TRPC6proteins in rat prostatesmooth muscle (PS1) cell line[J]. J Cell Physiol.2005;204(1):320-328.
[130] Parekh AB, Putney JW Jr.. Store-operated calcium channels [J]. Physiol Rev,2005;85(2):757-810.
[131] Patrakka J, Tryggvason K. New insights into the role of podocytes in proteinuria [J]. Nat RevNephrol.2009;5(8):463-468.
[132] Inoue R, Okada T, Onoue H, Hara Y, Shimizu S, Naitoh S, Ito Y, Mori Y. The transientreceptor potential protein homologue TRP6is the essential component of vascularα1-adrenoceptor-activated Ca2+-permeable cation channel [J]. Circ Res.2001;88(3):325-332.
[133] Weissmann N, Dietrich A, Fuchs B, Kalwa H, Ay M, Dumitrascu R, Olschewski A, Storch U,Mederos Schnitzler M, Ghofrani HA, Schermuly RT, Pinkenburg O, Seeger W, Grimminger F,Gudermann T. Classical transient receptor potential channel6(TRPC6) is essential for hypoxicpulmonary vasoconstriction and alveolar gas exchange [J]. Proc Natl Acad Sci U S A,2006;103(50):19093-19098.
[134] Saleh SN, Albert AP, Peppiatt CM, Large WA. Angiotensin II activates two cationconductances with distinct TRPC1and TRPC6channel properties in rabbit mesenteric arterymyocytes [J]. J Physiol.2006;577(pt2):479-495.
[135] Winn MP, Conlon PJ, Lynn KL, Farrington MK, Creazzo T, Hawkins AF, Daskalakis N, KwanSY, Ebersviller S, Burchette JL, Pericak-Vance MA, Howell DN, Vance JM, Rosenberg PB.Amutation in theTRPC6cation channel causes familial focal segmental glomerulosclerosis [J].Science,2005,308(5729):1801-1804.
[136] Reiser J, Polu KR, M ller CC, Kenlan P, Altintas MM, Wei C, Faul C, Herbert S, Villegas I,Avila-Casado C, McGee M, Sugimoto H, Brown D, Kalluri R, Mundel P, Smith PL, Clapham DE,Pollak MR. TRPC6is a glomerular slit diaphragm-associated channel required for normal renalfunction[J]. Nat Genet,2005,37(7):739-744.
[137] Facemire CS, Mohler PJ, Arendshorst WJ. Expression and relative abundance of shorttransient receptor potential channels in the rat renal microcirculation [J]. Am J Physiol Renal Physiol,2004;286(3):F546-551.
[138] Bandyopadhyay BC, Swaim WD, Liu X, Redman RS, Patterson RL, Ambudkar IS. Apicallocalization of a functional TRPC3/TRPC6-Ca2＋-signaling complex in polarized epithelial cellsRole in apical Ca2＋influx[J]．J Biol Chem,2005；280(13):12908-12916．
[139] Goel M, Sinkins WG, Zuo CD, Estacion M, Schilling WP. Identifcation and localization ofTRPC channels in the rat kidney [J]. Am J Physiol Renal Physiol,2006；290(5):F1241-F1252．
[140]范青锋,邢燕,刘淑芳,张涵,丁洁.瞬时受体电位阳离子通道蛋白6在肾脏的表达[J].实用儿科临床杂志,2007;22(17):1293-1295．
[141] Dryer SE, Reiser J. TRPC6channels and their binding partners in podocytes: role inglomerular filtration and pathophysiology [J]. Am J Physiol Renal Physiol.2010;299(4):F689-701.
[142] M ller CC, Wei C, Altintas MM, Li J, Greka A, Ohse T, Pippin JW, Rastaldi MP, Wawersik S,Schiavi S, Henger A, Kretzler M, Shankland SJ, Reiser J. Induction of TRPC6channel in acquiredforms of proteinuric kidney disease[J]. J Am Soc Nephrol.2007;18(1):29-36.
[143] Ma R, Du J, Sours S, Ding M. Store-operated Ca2+channel in renal microcirculation andglomeruli[J]. Exp Biol Med (Maywood).2006;231(2):145-153.
[144] Schlondorff JS, Pollak MR. TRPC6in glomerular health and disease: what we know and whatwe believe [J]. Semin Cell Dev Biol.2006;17(6):667-674.
[145] Sours S, Du J, Ma R, Ding M, Zhou XJ, Ma R. Expression of canonical transient receptorpotential (TRPC) proteins in human glomerular mesangial cells[J]. Am J Physiol Renal Physiol.2006;290(6): F1507-F1515.
[146] Du J, Ding M, Ma R, Graham S, Ma R. Mediation of angiotensin II-induced Ca2+signalingby polycystin2in glomerular mesangial cells[J]. Am J Physio Renal Physiol.2008;294(4):F909-F918.
[147] Du J, Sours-Brothers S, Coleman R Ding M, Graham S, Kong DH, Ma R. Canonical transientreceptor potential1channel is involved in contractile function of glomerular mesangial cells[J]. JAm Soc Nephrol.2007;18(5):1437-1445.
[148] Jiang L, Ding J, Tsai H, Li L, Feng Q, Miao J, Fan Q. Over-expressing transient receptorpotential cation channel6in podocytes induces cytoskeleton rearrangement through increases ofintracellular Ca2+and RhoA activation[J]. Exp Biol Med (Maywood).2011;236(2):184-193.
[149] Estacion M, Li S, Sinkins WG, Gosling M, Bahra P, Poll C, Westwick J, Schilling WP.Activation of human TRPC6channels by receptor stimulation [J]. J Biol Chem,2004;279(21):22047-22056.
[150] Gottlieb P, Folgering J, Maroto R, Raso A, Wood TG, Kurosky A, Bowman C, Bichet D, PatelA, Sachs F, Martinac B, Hamill OP, Honoré E. Revisiting TRPC1and TRPC6mechanosensitivity[J].Pflugers Arch,2008;455(6):1097-1103.
[151] Graham S, Gorin Y, Abboud HE, Ding M, Lee DY, Shi H, Ding Y, Ma R. Abundance ofTRPC6protein in glomerular mesangial cells is decreased by ROS and PKC in diabetes[J]. Am JPhysiol Cell Physiol,2011;301(2):C304-15.
[152] Graham S, Ding M, Sours-Brothers S, Yorio T, Ma JX, Ma R. Downregulation of TRPC6protein expression by high glucose, a possible mechanism for the impaired Ca2+signaling inglomerular mesangial cells in diabetes. Am J Physiol Renal Physiol,2007;293(4):F1381-1390.
[153] Du J, Sours-Brothers S, Coleman R, Ding M, Graham S, Kong DH, Ma R. Canonical transientreceptor potential channel is involved in contractile function of glomerular measangial cells[J]. J AmSoc Nephrol,2007;18(5):1446-1457.
[154]吴华勋,严尚学,魏伟.糖尿病肾病动物模型[M]//魏伟,吴希美,李元建主编.药理实验方法学.第4版.北京:人民卫生出版社,2010:1238-1239.
[1] Haraldsson B, Nystrom J, Deen W M. Properties of the glomerular barrier and mechanisms ofproteinuria [J]. Physiol Rev,2008;88(2):451-487.
[2] Dalla Vestra M, Masier OA, Roiter AM, et al. Is podocyte injury relevant in diabetic nephropathyStudies in patients with type2diabtes [J]. Diabetes,2003;52(4):1031-1035.
[3] Bank N. Mechanisms of diabetic hyperfiltration [J]. Kidney Int,1991;40:792–807.
[4] Kikkawa R, Kitamura E, Fujiwara Y, Arimura T, Haneda M, Shigeta Y. Impaired contractileresponsiveness of diabetic glomeruli to angiotensin II: a possible indication of mesangialdysfunction in diabetes mellitus[J]. Biochem Biophys Res Commun,1986;136:1185–1190.
[5] Mene′P, Pascale C, Teti A, Bernardini SV, Cinotti GA, Pugliese F. Effects of advanced glycationend products on cytosolic Ca2+signaling of cultured human mesangial cells[J]. J Am SocNephrol,1999;10:1478–1486.
[6] Mene′P, Pugliese G, Pricci F, DiMario U, Cinotti GA, Pugliese F. High glucose level inhibitscapacitative Ca2+influx in cultured rat mesangial cells by a protein kinase C-dependentmechanism[J]. Diabetologia,1997;40:521–527.
[7] Nutt LK, O’Neil RG. Effect of elevated glucose on endothelin-induced store-operated andnon-store-operated calcium influx in renal mesangial cells [J]. J Am Soc Nephrol,2000;11:1225–1235.
[8] Whiteside CI, Hurst RD, Stevanovic ZS. Calcium signaling and contractile response of diabeticglomerular mesangial cells [J]. Kidney Int,1995;8:S28–S33.
[9] Guan N, Ding J, Zhang J, et al. Expression of nephrin, podocin, alpha-actinin, and WT1inchildren with nephrotic syndrome[J]. Pediatr Nephrol,2003;18(11):1122-1127.
[10]范青锋,丁洁,张敬京，等. Nephrin、podocin及α-actinin在小鼠肾小球足细胞表达与分布[J].肾脏病与透析肾移植杂志,2003;12(5):407-411.
[11] Guan N, Ding J, Deng J, et al. Key molecular events in puromycin aminonucleoside nephrosisrats [J]. Pathol Int,2004;54(9):703-711.
[12] Fan Q, Ding J, Zhang J,et al. Effect of the knockdown of podocin mRNA on nephrin andalpha-actinin in mouse podocyte[J]. Exp Biol Med (Maywood),2004,229(9):964-970.
[13]邓江红,丁洁,管娜,等.嘌呤霉素肾病大鼠肾小球足细胞相关分子表达的共定位分布[J].肾脏病与透析肾移植杂志,2004,13(3):205-210.
[14] Winn MP, Conlon PJ, Lynn KL, et al. A mutation in the trpc6cation channel causes familialfocal segmental glomerulosclerosis [J]. Science,2005,308(5729):1801-1804.
[15] Eiser J, Polu KR, M ller CC, et al.TRPC6is a glomerular slit diaphragm-associated channelrequired for normal renal function [J]. Nat Genet,2005,37(7):739-744.
[16] Sours S, Du J, Chu S, Ding M, Zhou XJ, Ma R.Expression of canonical transient receptorpotential (TRPC) proteins in human glomerular mesangial cells[J]. Am J Physiol RenalPhysiol.,2006,290(6):F1507-15.
[17] Ockand JD, Sansom SC. Glomerularmesangial cells: Electophysiology and regulation ofcontraction [J]. Physiol Rev,1998,78(3):723-743.
[18] Avenstadt H. Roles of the podocyte in glomerular function [J]. Am J Physiol Renal Physiol,2000,278(2): F173-F179.
[19] Tanaka H, Smogorzewski M, Koss M,et al. Pathways involved in PTH induced rise in cytosolicCa2+concentration of rat renal proximal tubule[J]. Am J Physiol,1995,268(2Pt2): F330-F337.
[20] Van Baal J, Raber G, de Slegte J,et al. Vasopressin stimulated Ca2+reabsorption in rabbitcortical collecting system: effects on cAMP and cytosolic Ca2+[J]. Pflugers Arch,1996,433(1-2):109-115.
[21] Mller CC, Wei C, Altintas MM, et al.Induction of TRPC6channel in acquired forms ofproteinuric kidney disease [J]. J Am Soc Nephrol,2007,18(1):29-36.
[22] Sarabeth Graham, Min Ding, Sherry Sours-Brothers. Downregulation of TRPC6proteinexpression by high glucose, a possible mechanism for the impaired Ca2+signaling in glomerularmesangial cells in diabetes [J]. Am J Physiol Renal Physiol,2007,293:1381-1390.
[23] Stgaard J, Qvortrup K. Sieve plugs in fenestrae of glomerular capillaries site of filtration barrier[J]. Cell Tissues Organs,2002,170(2-3):132-138.
[24] Luft J H. Fine structures of capillary and endocapillary layer as revealed by ruthenium red [J].Fed Proc,1966,25(6):1773-1783.
[25] Montesano R, Mossaz A, Ryser JE, et al. Leukocyte in terleukins induces cultured endothelialcells to produce a highly organized, glycosam inoglycan rich pericellular matrix [J]. J Cell Bio, l1984,99(5):1706-1715.
[26] Jeansson M, Haraldsson B.Morphological and functional evidence for an important role of theendothelial cell glycocalyx in the glomerular barrier [J]. Am J Physiol Renal Physiol,2006,290:F111–116.
[27] Deckert T, Feldt-Rasmussen B, Borch-Johnsen K, et al. Albuminuria reflects wide spreadvascular damage: The Steno hypothesis [J]. Diabetologia,1989,32(4):219-226.
[28] Algenstaedt P, Schaefer C, Biermann T, et al. Microvascular alterations in diabetic mice correlate with level of hyperglycemia [J]. Diabetes,2003,52(2):542-549.
[29] Erdely A, Freshour G, Maddox DA, et al. Renal disease in rats with type2diabetes is associatedwith decreased renal nitric oxide production [J]. Diabetologia,2004,47(10):1672-1676.
[30] Koo J R, Vaziri ND. Effects of diabetes, insulin and antioxidants on NO synthase abundanceand NO interaction with reactive oxygen species [J]. Kidney Int,2003,63(1):195-201.
[31] Piqueras L, Kubes P, Alvarez A, et al. Angiotensin II induces leukocyte-endothelial cell interactions in vivo via AT (1) and AT (2) receptor-mediated P-selectin upregulation [J]. Circulation,2000;102(17):2118-2123.
[32] Levidiotis V, Power DA. New insights into the molecular biology of the glomerular filtrationbarrier and associated disease [J]. Nephrology (Carlton),2005;10:157–166.
[33] Barker DF, Hostikka SL, Zhou J, et al. Identification of mutations in the COL4A5collagen genein Alport syndrome [J]. Science,1990,248:1224–1227.
[34] Zenker M, Aigner T, Wendler O, et al. Human laminin beta2deficiency causes congenitalnephrosis with mesangial sclerosis and distinct eye abnormalities [J]. Hum Mol Genet,2004,13:2625–2632.
[35] Tamsma JT, vanden Born J, Bruijn JA, et al. Expression of glomerular extracellularm atrix components in human diabetic nephropathy: Decrease of heparan sulphate in the glomerular basementmembrane [J]. Diabetologia,1994;37(3):313-320.
[36] Raats C J, vanden Born J, Berden J H. Glomerular heparan sulfate alterations: Mechanisms andrelevance for proteinuria [J]. Kidney Int,2000,57(2):385-400.
[37] Yard BA, Kahlert S, Engelleiter R, et al. Decreased glomerular expression of agrin in diabeticnephropathy and podocytes, cultured in high glucose medium [J].Exp Nephrol,2001;9(3):214-222.
[38] Maxhimer JB, Somenek M, Rao G, et al. Heparanase-1gene expression and regulation by highglucose in renal epithelial cells: A potential role in the pathogenesis of proteinuria in diabeticpatients [J]. Diabetes,2005,54(7):2172-2178.
[39]刘莉莉,刘志红,郑春霞,等.足细胞损伤对糖尿病肾病患者预后的影响[J].肾脏病与透析移植杂志,2007,16(6):509-515.
[40] Rodewald R, Karnovsky MJ. Porous substructure of the glomerular slit diaphragm in the rat andmouse [J]. J Cell Biol,1974;60:423–433.
[41] Pavenstadt H, Kriz W, Kretzler M. Cell biology of the glomerular podocyte [J]. Physiol Rev,2003,83(1):253-307.
[42]陈惠萍.糖尿病肾病患者肾小球滤过膜结构和功能及代谢指标的再认识[J].医学研究生学报,2010;23(8):785-791.
[43] Glazebrook PA, SchillingWP, Kunze DL. TRPC channels as signal transducers [J].PflugersArch,2005,451(1):125-130.
[44] Hsu YJ, Hoenderop JG, BindelsRJ. TRP channels in kidney disease [J]. Biochim BiophysActa,2007,1772(8):928-936.
[45] Weissmann N, DietrichA, FuchsB, et al Classical transient receptor potential channel6(TRPC6)is essential forhypoxic pulmonary vasoconstriction and alveolar gas exchange [J]. Proc Natl AcadSciUSA,2006,103(50):19093-190938.
[46] PlantTD, SchaeferM. TRPC4andTRPC5receptor-operated Ca2+permeable nonselective cationchannels [J]. Cell Calcium,2003;33(5-6):441-450.
[47] Cayouette S, LussierMP, Mathieu EL, et a.l Exocytotic insertion of TRPC6channel into theplasmamembrane upon Gq protein-coupled receptor activation [J]. J Biol Chem,2004;279(8):7241-7246.
[48] Li S, Gosling M, Poll C. Determining the functional role of TRPC channels in primary cells [J].PflugersArch,2005,451(1):43-52.
[49]BaeYM, Kim A, Lee YJ, et al. Enhancement of receptor-operated cation current and TRPC6expression in arterial smooth muscle cells of deoxy corticosterone acetate-salthy pertensive rats [J]. JHypertens,2007,25(4):809-817.
[50] Dietrich A, Gudermann T. TRPC6[J]. Handb Exp Pharmacol,2007,(179):125-141.
[51] Dietrich A, Kalwa H, Rost BR, et al.The diacylgylcerol-sensitive TRPC3/6/7subfamily ofcation channels: functional characterization and physiological relevance[J]. Pflugers Arch,2005,451(1):72-80.
[52] Winn MP, Daskalakis N, Spurney RF, et al Unexpected role of TRPC6channel in familialnephrotic syndrome: does it have clinical Implications [J]. J Am Soc Nephrol,2006,17(2):378-387.
[53] Plant TD, Schaefer M. TRPC4and TRPC5receptor-operated Ca2+permeable nonselectivecation channels [J]. Cell Calcium,2003,33(5-6):441-450.
[54]匡新宇,黄文彦. TRPC6与肾脏疾病[J].国际病理科学与临床杂志,2008;28(5):456-460.
[55] DE' sposito M, Strazzullo M, Cuccurese M. Identification and assignment of the humantransient receptor potential channel6gene TRPC6to chromosome11q21-->q22[J].Cytogenet CellGenet,1998,83(1-2):46-47.
[56] Estacion M, Li S, Sinkins WG, et al. Activation of human TRPC6channels by receptorstimulation [J]. J Biol Chem,2004,279(21):22047-22056.
[57] DE Clapham, LW Runnels, C Strubing. The TRP ion channel family [J]. Nat.Rev Neurosci,2001,2:387–396.
[58] Hofmann T，Obukhov AG，Schaefer M，et al. Direct activation of humanTRPC6and TRPC3channels by diacylglyeerol [J].Nature,1999;397:259-263.
[59] Okada T, Inoue R, Yamazaki K, Maeda A, Kurosaki T, Yamakuni T, Tanaka I, Shimizu S,Ikenaka K, Imoto K, Mori Y. Molecular and functional characterization of a novel mouse transientreceptor potential protein homologue TRP7. Ca(2+)-permeable cation channel that is constitutivelyactivated and enhanced by stimulation of G protein-coupled receptor[J]. J Biol Chem,1999;274(39):27359-70.
[60]邢燕,范青锋,丁洁.导致家族性局灶节段性肾小球硬化新基因-TRPC6[J].肾脏病与透析肾移植杂志,2006;5(2):140-143.
[61] Beech DJ. Emerging functions of10types of TRP cationic channel in vascular smoothmuscle[J].Clin Exp Pharmacol Physiol2005,32(8):597-603.
[62] Facemire CS, Mohler PJ, Arendshorst WJ. Expression and relative abundance of short transientreceptor potential channels in the rat renal vasculature [J]. Am J Physiol Renal Physiol,2004,286(3):F546-F551.
[63] Bandyopadhyay BC, Swaim WD, Liu X,et al. Apical localization of a functional TRPC3/TRPC6-Ca2+-signaling complex in polarized epithelial cells: role in apical Ca2+influx[J]. J BiolChem,2005,280(13):12908-12916.
[64] Goel M, Sinkins WG, Zuo CD,et al. Identification and Localization of TRPC Channels in RatKidney[J].Am J Physiol Renal Physiol,2006,290(5):F1241-52.
[65] Dietrich A, MederosY, Schnitzler M,et al. Increased vascular smooth muscle contractility inTRPC6-/-mice [J]. Mol Cell Biol,2005,25(16):6980-6989.
[66] Reiser J, Polu KR, Mller CC, et al. TRPC6is a glomerular slit diaphragm-associated channelrequired for normal renal function [J]. Nat Genet,2005,37(7):739-744.
[67]Ahmmed GU, Malik AB. Functional role of TRPC channels in the regulation of endothelialpermeability [J].Pflugers Arch.,2005,451(1):131-42.
[68] Eeremina, et al. VEGF inhibition and renal thrombotic microangiopathy[J]. N. Engl. J. Med,2008,358:1129–1136.
[69] H.W. Cheng, A.F. James, R.R. Foster, J.C. Hancox, D.O. Bates. VEGF activatesreceptor-operated cation channels in human microvascular endothelial cells, Arterioscler [J]. Thromb.Vasc. Biol,2006,26:1768–1776.
[70] T.M. Pocock, R.R. Foster, D.O. Bates. Evidence of a role for TRPC channels inVEGF-mediated increased vascular permeability in vivo[J].Am. J. Physiol. Heart. Circ. Physiol,2004,286:H1015–1026.
[71] Foster RR，Satchell SC，Seckley J，et al. VEGF—CP romotes survival in Podoeytes[J].Am JPhysiol Renal Physiol,2006;291:196-207.
[72] Bieke F, Schrij vers, Allan F, et al. The role of vascular endothelial growth factor (VEGF) inrenal pathophysiology [J]. Kidney Int,2004,65(6):2003-2017.
[73] Veron D, Reidy KJ, Bertuccio C, et al. Overexpression of VEGF-A in podocytes of adult micecauses glomerular disease [J]. Kidney Int,2010,77(11):989-999.
[74] Müller-Deile J, Worthmann K, Saleem M, et al. The balance of autocrine VEGF-A and VEGF-Cdetermines podocyte survival [J].Am J Physiol Renal Physiol,2009,297(6):F1656-67.
[75]Lin SY, Corey DP. TRP channels in mechanosensation [J]. Curr Opin Neurobio,2005;15:350–357.
[76] Duh E, Nello LP. Vascular endothelial growth factor and diabetes: the agonist versus antagonistparadox [J]. Diabetes,1999,48(10):1899-1909.
[77]柳飞,黄颂敏.肾小球内皮细胞损害与糖尿病肾病[J].华西医学,2007,22(2):446-447.
[78]范青锋,邢燕,刘淑芳,张涵,丁洁.瞬时受体电位阳离子通道蛋白6在肾脏的表达[J].实用儿科临床杂志,2007,22(17):1293-1295.
[79] Ly J,Alexander M,Quaggin SE.A podocentric view of nephrology[J].Curr Opin NephrolHypertens,2004;13:299–305.
[80] L.Barisoni, W.Kriz, P.Mundel, V.D'Agati. The dysregulated podocyte phenotype: a novelconcept in the pathogenesis of collapsing idiopathic focal segmental glomerulosclerosis andHIV-associated nephropathy [J]. J.Am.Soc. Nephrol,1999,10:51–61.
[81] Wada T, Pippin JW, Marshall CB, et al. Dexamethasone prevents podocyte apoptosis induced bypuromycin aminonucleoside: role of p53and Bcl-2related family proteins[J]. J Am Soc Nephrol,2005,16:2615-2625.
[82] Kim YH, Goyal M, Kurnit D, et al.Podocyte depletion and glomerulosclerosis have a directrelationship in the PAN-treated rat [J].Kidney Int,2002,60:957-968.
[83] Sun X, Fang Z, Zhu Z, Yang X, He F, Zhang C. Effect of down-regulation of TRPC6on thepuromycin aminonucleoside-induced apoptosis of mouse podocytes[J]. J Huazhong Univ SciTechnolog Med Sci.,2009,29(4):417-22.
[84] Moller CC，Wei C，Altintas MM, et al. Induction of TRPC6channel in acquired forms ofproteinuric kidney disease [J]. J Am Soc NePhrol,2007,18:29-36.
[85] P.Mundel, S.J.Shankland. Podocyte biology and response to injury [J]. J.Am.Soc.Nephrol,2002,13:3005–3015.
[86] Verma R, Wharram B, Kovari I,et al. Fyn binds to and phosphorylates the kidney slit diaphragmcomponent nephrin [J]. J Biol Chem,2003,278(23):20716-20723.
[87] Li H, Lemay S, Aoudjit L,et al. SRC-family kinase Fyn phosphorylates the cytoplasmic domainof nephrin and modulates its interaction with podocin[J]. J Am SocNephrol,2004,15(12):3006-3015.
[88] M.A.Spassova, T. Hewavitharana, W.Xu, J.Soboloff, D.L.Gill. A common mechanism underliesstretch activation and receptor activation of TRPC6channels [J]. Proc.Natl.Acad.Sci,2006,103:16586-16591.
[89] M.Kestila, M.Mannikko, C.Holmberg, et al. Congenital nephrotic syndrome of the Finnish typemaps to the long arm of chromosome19[J]. Am.J.Hum.Genet,1994,54:757–764.
[90] Spassova MA,Hewavitharana T,Xu W,et al.A common mechanism underlies stretch activationand receptor activation of TRPC6channels[J].Proc Natl Acad Sci USA,2006,103,16586-16591.
[91] Huber TB,Schermer B,Muller RU,et al.Podocin and MEC-2bind cholesterol to regulate theactivity of associated ion channels[J].Proc Natl Acad Sci USA,2006,103:17079-17086.
[92] Y.H.Kim, M.Goyal, D.Kurnit,et al. Podocyte depletion and glomerulosclerosis have a directrelationship in the PAN-treated rat [J]. Kidney Int,2001,60:957–968.
[93] Mukerji N，Damodaran TV, Winn MP. TRPC6and FSGS: the latest TRP channelopathy,2007,1772:859-868.
[94] Schiffer M, Bitzer M, Roberts IS, et al.Apoptosis in podocytes induced by TGF-beta and Smad7[J]. J Clin Invest,2001,108:807-816.
[95] Liu SF, Ding J, Fan QF, et al.Establishment of a podocyte cell injury model induced bypuromycin aminonucleoside [J]. Beijing Da Xue Xue Bao,2008,40:586-589.
[96] Jia J, Ding G, Zhu J, et al.Angiotensin Ⅱ infusion induces nephrin expression changes andpodocyte apoptosis [J].Am J Nephrol,2008,28:500-507.
[97] Hara M, Yanagihara T, Kihara L. Urinary podocytes in primary focal segmentalglomerulosclerosis [J]. Nephron,2001,89:342-347.
[98] Tryggvason K, Patrakka J,Wartiovaara J.Hereditary proteinuria syndromes and mechanisms ofproteinuria[J]. N Engl J Med,2006,354:1387-401.
[99] Shankland SJ.The podocytes response to injury: role in proteinuria and glomerulosclerosis [J].Kidney Int,2006,69:2131-3147.
[100] Ma R, Pluznick JL, and Sansom SC. Ion channels in mesangial cells: function, malfunction, orfiction [J]. Physiology,2005,20:102–111.
[101] Sherry Sours, Juan Du, Shaoyou Chu.Expression of canonical transient receptor potential(TRPC) proteins in human glomerular mesangial cells [J]. Am J Physiol Renal Physiol,2006,290:F1507–F1515.
[102] Stockand JD, Sansom SC. Glomerularmesangial cells: Electophysiology and regulation ofcontraction [J]. PhysiolRev,1998,78(3):723-743.
[103] Saleh SN, Albert AP, Peppiatt CM, Large WA. Angiotensin II activates two cationconductances with distinct TRPC1and TRPC6channel properties in rabbit mesenteric arterymyocytes [J]. J Physiol,2006,577:479-495.
[104] Marrero MB, Schieffer B, ma HP, et al. ANG II-induced tyrosine phosphorylation stimulatesphospholipase C and C1-channels in mesangial cells [J]. Am J Physiol Cell Physiol,1996,270:C1834–C1842.
[105] Ha H, Yu M, Kim KH. Melatonin and taurine reduce early glomerulopathy in diabetic rats [J].Free Radic Biol Med,1999,26:944–950.
[106] The Diabetes Control and Complications Trial Research Group. The effect of intensivetreatment of daibetes on the development and progression of long-term complications ininsulin-dependent diabetes mellitus [J]. N Engl J Med,1993,329(14):977–986.
[107] Dietrich A, Gudermann T. TRPC6[J]. Handb Exp Pharmacol,2007,179:125–141.
[108] Young BA, Johnson RJ, Alpers C, et al. Cellular events in the evolution of experimentaldiabetic nephropathy[J]. Kidney Int,1995,479(3):935-944.
[109] Gorin Y, Block K, Hernandez J, Bhandari B, Wagner B, Barnes JL, Abboud HE. Nox4NADPH oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney [J]. J BiolChem,2005,280:39616–39626.
[110] Hinokio Y, Hirai M, Chiba M, Hirai A, Toyota T. Oxidative DNA damage in diabetes mellitus:its association with diabetic complications [J]. Diabetologia,1999,42:995–998.
[111] Kim NH, Rincon-Choles H, Bhandari B, Choudhury GG, Abboud HE, Gorin Y. Redoxdependence of glomerular epithelial cell hypertrophy in response to glucose[J]. Am J Physiol RenalPhysiol,2006,290: F741–F751.
[112] Schnackenberg CG. Oxygen radicals in cardiovascular-renal disease [J]. Curr Opin Pharmacol,2002,2:121–125.
[113] Shah SV, Baliga R, Rajapurkar M, Fonseca VA. Oxidants in chronic kidney disease [J]. J AmSoc Nephrol,2007,18(1):16–28.
[114] Ha H, Yu MR, Choi YJ, Kitamura M, Lee HB. Role of high glucose-induced nuclear factor-κBactivation in monocyte chemoattractant protein-1expression by mesangial cells [J]. J Am SocNephrol,2002,13:894–902.
[115] Gorin Y, Ricono JM, Wagner B, Kim NH, Bhandari B, Choudhury GG. Angiotensin II-inducedERK1/ERK2activation and protein synthesis are redox-dependent in glomerular mesangial cells [J].Biochem J,2004,381:231–239.
[116] Iglesias La, Cruz MC-DE, Ruiz-Torres P, Alcami J, Diez-Marques L, Ortega-Velazquez R,Chen S, Rodriguez-Puyol M, Ziyadeh FN, Rodriguez-Pyol D. Hydrogen peroxide increasesextracellular matrix mRNA through TGF-βin human mesangial cells[J]. Kidney Int,2001,59:87–95.
[117] Dandona P, Thusu K, Cook S, Snyder B, Makowski J, Armstrong D, Nicotera T. Oxidativedamage to DNA in diabetes mellitus[J]. Lancet,1996;347:444-445.
[118] Tryggvason K, Wartiovaara J.How does the kidney filter plasma [J]. Physiology（Bethesda）,2005;20:96-101.
[2]中华医学会糖尿病学分会．中国2型糖尿病防治指南（2010年版）[J]．中国医学前沿杂志（电子版），2011;3(6):54-109．
[3] Bloomgarden ZT. Diabetic nephropathy [J]. Diabetes Care,2008;31(4):823-827.
[4] Beisswenger PJ, Drummond KS, Nelson RG, Howell SK, Szwergold BS, Mauer M.Susceptibility to diabetic nephropathy is related to dicarbonyl and oxidative stress [J].Diabetes,2005;54(11):3274-3281.
[5] Brownlee M. Biochemistry and molecular cell biology of diabetic complications [J]. Nature,2001;414(6865):813-820.
[6] Banday AA, Fazili FR, Lokhandwala MF. Oxidative stress causes renal dopamine D1receptordysfunction and hypertension via mechanisms that involve nuclear factor-kB and protein kinase C[J]. J Am Soc Nephrol,2007;18(5):1446-1457.
[7] Gill PS, Wilcox CS. NADPH oxidases in the kidney [J]. Antioxid Redox signal,2006;8(9-10):1597-1607.
[8] Nauseef WM. Biological roles for the NOX family NADPH oxidases [J]. J Biol Chem,2008;283(25):16961-16965.
[9] Gorin Y, Block K, Hernandez J, Bhandari B, Wagner B, Barnes JL, Abboud HE. Nox4NAD(P)H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney[J]. J BiolChem.2005;280(47):39616-39626.
[10] Houstis N, Rosen ED, Lander ES. Reactive oxygen species have a causal role in multiple formsof insulin resistance [J]. Nature,2006;440(7086):944-948．
[11]王宇翎,李卫平,张艳.益肾复方对大鼠阿霉素肾病的作用[J].安徽医科大学学报,2007;42(1):51-53.
[12]余凌,李惊子,王海燕.黄芪、当归在肾脏疾病中的应用及其机制研究进展[J].中国中西医结合杂志,2001;21(5):396-398.
[13] Motomura K, Fujiwara Y, Kiyota N, Tsurushima K, Takeya M, Nohara T, Nagai R, Ikeda T.Astragalosides isolated from the root of astragalus radix inhibit the formation of advanced glycationend products[J]. J Agric Food Chem.2009;57(17):7666-7672.
[14] Lei H, Wang B, Li WP, Yang Y, Zhou AW, Chen MZ. Anti-aging effect of astragalosides and itsmechanism of action [J]. Acta Pharmacol Sin,2003;24(3):230-234.
[15]牟娜,张庆怡,倪兆慧,童菊芳.黄芪对高糖作用下肾间质成纤维细胞表达HGF的影响[J].中国中西医结合肾病杂志,2002;3(1):7-9.
[16] Xiang FL, Lu X, Strutt B, Hill DJ, Feng Q.NOX2deficiency protects againststreptozotocin-induced beta-cell destruction and development of diabetes in mice [J]. Diabetes.2010;59(10):2603-2611.
[17] Liu WH, Hei ZQ, Nie H, Tang FT, Huang HQ, Li XJ, Deng YH, Chen SR, Guo FF, Huang WG,Chen FY, Liu PQ. Berberine ameliorates renal injury in streptozotocin-induced diabetic rats bysuppression of both oxidative stress and aldose reductase [J]. Chin Med J (Engl).2008;121(8):706-712.
[18] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitativePCR and the2(-Delta Delta C(T)) Method[J]. Methods.2001;25(4):402-408.
[19]周琚,张汝学,贾正平.不同糖尿病动物模型的特点比较[J].国际内分泌代谢杂志,2010;30(4):273-276.
[20] Ktorza A, Bernard C, Parent V, Penicaud L, Froguel P, Lathrop M, Gauguier D. Are animalmodels of diabetes relevant to the study of the genetics of non-insulin-dependent diabetes in humans[J]．Diabetes Metab，1997;23(Suppl2):38-46．
[21]王欣,海春旭.糖尿病动物模型研究进展[J].毒理学杂志，2011;25(5):387-390
[22]冯烈,罗璐,卢筱华.福辛普利对糖尿病大鼠肾脏的保护作用[J].暨南大学学报(医学版),2001,22(4):35-39.
[23]李伟,张红,殷松楼,褚璇.不同剂量链脲佐菌素诱导SD大鼠糖尿病肾病模型的研究[J].徐州医学院学报,2006;26(1):52-55.
[24]陈秋,夏永鹏,邱宗荫.2型糖尿病大鼠模型的建立与评价[J].天津医药,2006;34(1):33-35.
[25]黄波,刘学政,庞东渤.不同途径注射链脲佐菌素致大鼠糖尿病模型的研究[J].锦州医学院学报,2003;24(1):19-21.
[26]沈亚非,徐焱成.链脲佐菌素诱导实验性糖尿病大鼠模型建立的研究[J].实用诊断和治疗杂志,2005;19(2):79-80.
[27]赵芳,蒋朝晖,杨国珍,李兴,朱丽英,潘卫.不同剂量链脲佐菌素建立1型糖尿病大鼠模型[J].贵阳医学院学报,2010;35(1):22-25.
[28]农慧盛,庆寿,梁健,闫福曼,罗荣敬,李小英. STZ诱导糖尿病大鼠模型的研究[J].广西医科大学学报,2010;27(1):69-72.
[29]刘志民,孙亮亮.氧化应激在糖尿病发病机制中作用的认识[J].内科理论与实践,2007;2(3):153-155.
[30] Brownlee M. The Pathobiology of diabetic complications:a unifying mechanism[J].Diabetes，2005;54(6):1615-1625.
[31]宿世震,邢冬杰.氧化应激与糖尿病肾病[J].中国实用医药,2005;3(3)531-532.
[32] Ha H, Yu MR, Choi YJ, Kitamura M, Lee HB. Role of high glucose-in-duced nuclearfactor-kappaB activation in monocyte che-moattractant protein-1expression by mesangial cells [J].J AmSoc Nephrol,2002;13(4):894-902.
[33] Waldre EN. Cellular oxidative processes in relation to renal disease [J]. Am J NePhrol,2005;25(1):13-22.
[34] Maritim AC, Sanders RA, Watkins JB. Effects of alpha-lipoic acid onbiomarkers of oxidativestress in streptozotocin-induced diabetic rats [J]. J Nutr Biochem,2003;14(5):288-294.
[35] Shokoji T, Nishiyama A, Fujisawa Y, Hitomi H, Kiyomoto H, Takahashi N, Kimura S, KohnoM, Abe Y. Renal sympathetic nerve responses to tempol in spontaneously hypertensive rats[J].Hypertension,2003;41(2):266-273.
[36] Meng S, Cason GW, Gannon AW, Racusen LC, Manning RD Jr. Oxidative stress in Dalaisalt-sensitive hypertension [J]. Hypertension,2003;41(6):1346-1352.
[37] Kopkan L, Majid DS. Enhanced superoxide activity modulates renal function in NO-deficientliypertensive rats [J]. Hypertension,2006;47(3):568-572.
[38] Laight DW, Andrews TJ, Haj-Yehia AI, Carrier MJ, Angg rd EE. Microssoy of superoxideanion sctivity in vitro [J]. Environmental Toxicology and Pharmacology,1997;3(1):65-68.
[39]Mitchell JB, Samuni A, Krishna MC, Krishna MC, DeGraff WG, Ahn MS, Samuni U, Russo A.Biologcally active metal-indismutase mimincs[J].Biochemistry,1990;29(11):2802-2807.
[40]孙风兰,王国英,李其平,吴延芳.氮氧化物对人角质形成细胞氧化损伤的保护作用[J].中华皮肤科杂志,2002;35(6):429-431.
[41] Bernstein EF, Kong SK, Brown DB, Kwak BC, Takeuchi T, Gasparro FP, Uitto J. The nitroxideTempol affords protection against ultraviolet radiation in a transgenic murine fibroblast culturemodel of cutaneous photoaging [J]. Exp Dermatol,200l;10(1):55-61.
[42] Yan SX, Hong XY, Hu Y, Liao KH. Tempol, one of nitroxides,is a novel ultraviolet—A1radiation protector for human dermal fibroblasts[J]. Journal of Dermatological Science,2005;37(3):137-143.
[43]严淑贤,徐昱,胡跃,廖康煌.氮氧化物对中波紫外线照射下包皮成纤维细胞的保护作用[J].中华老年医学杂志,2005;24(10):753-756.
[44] Yanes L, Romero D, Iliescu R, Cucchiarelli VE, Fortepiani LA, Santacruz F, Bell W, Zhang H,Reckelhoff JF. Systemic arterial pressure response to two weeks of tempol therapy in SHR:involvement of NO, the RAS, and oxidative stress [J]. Am J Physiol Regul Integr Comp Physiol,2005;288(4):903-908.
[45] Bayorh MA, Mann G, Walton M, Eatman D. Effects of enalapril,tempol,and eplerenone onsalt-induced hypertension in Dahl salt-sensitive rats[J]. Clini and Expe Hypertens,2006;28(2):121-132.
[46]刘文军,贾一韬,夏照帆,付晋凤,马兵,卫伟,吕开阳,徐豫,王钰,孙瑜. Tempol对浸水束缚应激大鼠胃黏膜p38MAPK活化的影响[J].解放军医学杂志,2008;33(2):163-165.
[47] Wilcox CS. Effects of tempol and redox-cycling nitroxides in models of oxidative stress[J].Pharmacol Ther,2010;126(2):119-45.
[48] Chatterjee PK, Cuzzocrea S, Brown PA, Zacharowski K, Stewart KN, Mota-Filipe H,Thiemermann C.Tempol, a membrane-permeable radical scavenger, reduces oxidant stress-mediatedrenal dysfunction and injury in the rat[J]. Kidney Int.2000;58(2):658-73.
[49] Asaba K, Tojo A, Onozato ML, Goto A, Fujita T. Double-edged action of SOD mimetic indiabetic nephropathy[J]. J Cardiovasc Pharmacol,2007;49(1):13-9.
[50]中华中医药学会.糖尿病中医防治指南(2007版)[M].北京:中国中医药出版社,2007:5.
[51]黄可儿,赵敏,王建华.黄芪总苷的药理研究进展[J].中药新药与临床药理,2005;16(6):461-463.
[52]宋锦叶,李深,孟立强,屈磊,李晓玫.黄芪当归合剂对5/6肾切除大鼠肾组织损伤的治疗作用[J].北京大学学报（医学版）,2009;41(2):196-202.
[53] Gilbert RE, Tsalamandris C, Bach LA, Panagiotopoulos S, O'Brien RC, Allen TJ, Goodall I,Young V, Seeman E, Murray RM Long-term glycemic control and the rate of progression of earlydiabetic kidney disease[J].Kidney Int,1993;44(4):855-861.
[54]马燕,张晶,王亚,喇孝瑾,张晓鹏,吕建东,喇万英.黄芪降糖颗粒降糖作用实验研究[J].中国实验方剂学杂志,2011;17(8):157-160
[55]徐郁杰,张庆怡,吴青伟.黄芪对糖尿病大鼠早期肾肥大和蛋白尿的影响[J].上海第二医科大学学报,1997；17(5):357-359.
[56]王法德,王兆淦,王立琴,田立,王朝霞,刘雪君.参芪克糖灵治疗糖尿病的实验研究[J].山东中医杂志,2997;16(7):316-319.
[57]刘道芳,袁敏.黄芪研究进展[J].中医药信息,1998;15(2):13-14.
[58]郝玉美,刘洪琪.苦瓜、黄芪、黄芩苷对2型糖尿病模型大鼠胰岛素抵抗的影响[J].中药药理与临床,2007;23(5):145-147.
[59] Aso Y, Yoshida N, Okumura K, Wakabayashi S, Matsutomo R, Takebayashi K, Inukai T.Coagulation and inflammation in overt diabetic nephropathy:association withhyperhonoeysteinemia[J]. Clin Chem Aeta,2004;348(1-2):139-145.
[60]刘洪凤,郭新民,王桂云,冯芹喜,包海花,崔荣军.黄芪多糖对2-DM胰岛素抵抗大鼠血糖及血脂的影响[J].牡丹江医学院学报,2007;28(5):18-20.
[61]廖苇萍,石元刚.黄芪多糖和大豆异黄酮对糖尿病大鼠糖代谢的影响[J].第三军医大学学报,2007;29(5):416-418.
[62]阮耀,岳兴如,李晓明,王珍珍,阮翘.黄芪对糖尿病大鼠心肌MDA及SOD, GSH-PX,Na+-k+ATP酶活性的影响[J].时珍国医国药,2007;18(3):593-594.
[63]王艳桥,徐向进,陈频.氧化应激研究进展及其在糖尿病肾病发病中的作用[J].医学综述,2010;16(11):1681-1684.
[64] Hwang SY, Siow YL, Au-Yeung KK, House J, OK.Folic acid supplementation inhibits NADPHoxidase-mediated superoxide anion production in the kidney[J]. Am J Physiol Renal Physiol,2011;300(1):F189-198.
[65] Montezano AC, Burger D, Ceravolo GS, Yusuf H, Montero M, Touyz RM. Novel Noxhomologues in the vasculature: focusing on Nox4and Nox5[J]. Clin Sci (Lond).2011;120(4):131-41.
[66] Kashihara N, Haruna Y, Kondeti VK, Kanwar YS. Oxidative stress in diabetic nephropathy [J].Curr Med Chem.2010;17(34):4256-69.
[67] Geiszt M, Kopp JB, Varnai P, Leto TL. Identification of Renox, an NADPH oxidase in kidney[J]. Proc Natl Acad Sci U S A,2000;97(14):8010-8014.
[68] Gorin Y, Ricono JM, Kim NH, Bhandari B, Choudhury GG, Abboud HE. Nox4mediatesangiotensin Ⅱ-induced activation of Akt/protein kinase B in mesangial cells [J]. Am J Physiol RenalPhysiol,2003;285(2):F219-F229.
[69] Block K, Eid A, Griendling KK, Lee DY, Wittrant Y, Gorin Y. Nox4NADPH oxidase mediatesSrc-dependent tyrosine phosphorylation of PDK-1in response to angiotensin Ⅱ.Role in mesangialcell hypertrophy and fibronectin expression[J]. J Biol Chen,2008;283(35):24061-24076.
[70]王有德.糖尿病肾病发病机制的研究进展[J].当代医学,2010;16(7):36-41.
[71] Aksun SA, Ozmen D, Ozmen B, Parildar Z, Mutaf I, Turgan N, Habif S, Kumanlio luc K,Bayindir O. Beta2microglobulin and cystatin C in type2diabetes：Assessment of diabeticdephropathy[J]. Exp Clin Endocrinol Diabetes,2004;112(4):195-200.
[72]吴志美.糖尿病肾病尿β2-微球蛋白、转铁蛋白的测定[J].检验医学与临床,2007;4(1):32.
[73]蒋最明,顾敏,任风琴.糖尿病肾损伤早期诊断的尿液联合检测[J]．实用全科医学，2008;6(1):85-86．
[74] Dalla Vestra M., Fioretto P. Diabetic nephropathy: renal struetural studies in typel and2diabetiepatients [J]. International Congress Series,2003;1253:163-169.
[75] Ding DF, You N, Wu XM, Xu JR, Hu AP, Ye XL, Zhu Q, Jiang XQ, Miao H, Liu C, Lu YB.Resveratrol attenuates renal hypertrophy in early-stage diabetes by activating AMPK [J]. Am JNephrol,2010,31(4):363-374.
[76] Zhang HJ, Zhao JB, Li P. Progress in the study on animal model of diabeticnephropathy[J]．Chin Pharmacol Bull，2008;24(7):845-848．
[77] Yu M，Zhou HH，Liu ZQ．Progress in related genes of diabetic nephropathy[J]．Chin PharmacolBull，2009;24(11):1419-1422．
[78] Mauer SM, Steeffs MW, Borwn DM. Structure functional relation in diabetic nephropathy[J]. JClin Invest,1984,74(4):1143-1154.
[79]孙晋芳,焦金菊,宋其蔓,白玉红.黄芪皂甙对大鼠肾小球系膜细胞增殖及周期的影响[J].中国组织工程研究与临床康复,2008;12(15):2926-2928.
[80] Li X, Pabla N, Wei Q, Dong G, Messing RO, Wang CY, Dong Z. PKC-delta Promotes RenalTubular Cell Apoptosis Associated with Proteinuria[J].J Am Soc Nephrol,2010;21(7):1115-24.
[81] Watson S, Cailhier JF, Hughes J, Savill J. Apoptosis and Glomerulonephritis[J].Curr DirAutoimmun,2006;9:188-204.
[82]张艳玲,段惠军,李春香,王燕,史永红,李英敏.糖尿病大鼠肾脏细胞凋亡与Bax和Bcl-2基因表达[J].中国糖尿病杂志,2002;10(3):159-162.
[83]苏君梅,董峰,易华,董传仁,欧阳静萍,杨海鹭.糖尿病大鼠肾脏细胞凋亡的研究[J].武汉大学学报(医学版),2003;24(1):17-20.
[84] Niranjan T, Bielesz B, Gruenwald A, Ponda MP, Kopp JB, Thomas DB, Susztak K. The Notchpathway in podocytes plays a role in the development of glomerular disease [J]. NatMed,2008;14(3):290-298.
[85] Susztak K, Raff AC, Schiffer M, B ttinger EP. Glucose induced reactive oxygen species causeapoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy [J]. Diabetes,2006;55(1):225-233.
[86]桂定坤,陈建国,陈宜方,黄建华.黄芪甲苷抑制高糖诱导的足细胞凋亡作用及机制[J].中华中医药学刊,2010;28(4):822-824.
[87] Huang Y, Chen Y, Xu M, Gu W, Bi Y, Li X, Ning G. Low-grade albuminuria is associated withcarotid intima-media thickness in Chinese type2diabetic patients [J]. J Clin Endocrinol Metab.2010;95(11):5122-5128.
[88] Ding G, Pesek-Diamond I, Diamond JR. Cholesterol, macrophages and gene expression ofTGF-β1and fibronectin during nephrosis [J]. Am J Physiol,1993;264(4Pt2):F577-84.
[89]苏进,田浩明.纤维连接蛋白与糖尿病肾病[J].医学综述,2001;7(11):654-655
[90] Doi K, Okamoto K, Tokunaga K, Fujita T, Noiri E. Genome study of kidney disease in the ageof post genome-sequencing[J]. Endocr Metab Immune Disord Drug Targets.2008;8(3):173-183.
[91] Xu YF，Wu YL，Wan JX，WANG CY．Effects of BMP-7on the bagpression of extracellularmatrix in human renal proximal epithelial cells induced by TGF-β1[J]．Chin Pharmacol Bull，2008;24(11):1453-1457．
[92] Rovin BH, Tan LC. LDL stimulates mesangial fibronectin production and chemoattractantexpression [J]. Kidney Int,1993;43(1):218-25.
[93] Chandrasekar B, Streitman JE, Colston JT, Freeman GL. Inhibition of nuelear factor kappa Battemiates proinflammatory cytokine and inducible nitric-oxide synthase expression in postisehemicmyoeardium [J]. Biochim Biophys Acta,1998;1406(l):91-106.
[94] Lubin FD, Reingold SC. Defining the clinical course of multiple sclerosis: results of aninternational survey. National Multiple Sclerosis Society (USA) Advisory Committee on ClinicalTrials of New Agents in Multiple Sclerosis [J]. Neurology,1996;46(4):907-911.
[95]范兴忠,李宏,赵建强,王建英,李新东.黄芪对肾病综合征患者血清和尿肿瘤坏死因子水平的影响[J].免疫学杂志,2005;21(5):421-422.
[96] Watanabe H, Sanada H, Shigetomi S, Katoh T, Watanabe T. Urinary excretion of type IVcollagen as a specific indicator of the progression of diabetic nephropathy[J]. Nephron,2000;86(1):27-35
[97]尹得海，梁晓春.从肾小球系膜细胞表型转化谈中医药防治糖尿病肾病机制研究通路[J].中国中医药信息杂志,2005;15(6):90-91.
[98]叶太生,周必发,张莹雯.百令胶囊对大鼠肾小球系膜细胞增殖、Ⅳ型胶原及TGF-β1mRNA表达的影响[J].中国中西医结合肾病杂志,2010;11(1):41-43.
[99] Lam S, van der Geest RN, Verhagen NA, Daha MR, van Kooten C. Secretion of collagen typeIV by human renal fibroblasts is increased by high glucose via a TGF-β1-independent pathway[J].Nephrol Dia Transplant,2004;19(7):1694-1701.
[100] Chen S, Hong SW, Iglesias-de la Cruz MC, Isono M, Casaretto A, Ziyadeh FN. The key roleof the transforming growth factor beta system in the pathogenesis of diabetic nephropathy [J]. RenFail,2001;23(3-4):471-481.
[101]杨政,白云凯. Ⅰ、Ⅳ型胶原和肾脏疾病[J].国外医学·泌尿系统分册,2005;25(5):78-83.
[102] Wang S, Denichilo M, Brubaker C, Hirschberg R. Conneetive tissue growth faetorintubuloin,terstitial injury of diabetic nephropathy[J]. Kidney Int,2001;60(1):96.
[103] Inomata S, Itoh M, Sato T. Difference of serum levels of type IV collagen7S between early orovert nephropathy and nondiabetic renal disease in diabetic patients with micro-ormacroalbuminuria.[J]. Nephron,1996;73(3):601-605.
[104] Woodrow D, Moss J, Shore I, Spiro RG. Diabetic glomerulosclerosis immunogoldultrastructural studieson the glomerular distribution oftypeⅣcollagen and sulphate proteoglycan [J].J Pathol,1992;167(1):49-58.
[105] Sharma K, Jin Y, Guo J, Ziyadeh FN. Neutralization of TGF-beta by anti-TGF-beta antibodyattenuates kidney hypertrophy and the enhanced extracellular matrix gene expression inSTZ-induced diabetic mice[J]. Diabetes,1996;45(4):522-530.
[106] Isaka Y, Fujiwara Y, Ueda N, Kaneda Y, Kamada T, Imai E. Glomerulosclerosis by in vivotransforming growth factor-β1and platelet derived growth factor gene into the rat kidney[J]. J ClinInvest,1993;92(6):2597-2601.
[107] Ziyadeh FN, Sharma K, Ericksen M, Wolf G. Stimulation of collagen gene expression andprotein synthesis in murine mesangial cells by high glucose is mediated by autocrine of transforminggrowth factor beta[J]. J Clin Invest,1994:93(2):536-542.
[108]陶松桔,尹支农,徐自强,宋卫红,谭辉.黄芪注射液对糖尿病肾病患者血Ⅳ型胶原与转化生长因子-β1水平的影响及其意义[J].中国综合临床,2004;20(3):217-219.
[109]陶少平,陈学峰,孙艳,杭宇,周蓓.黄芪注射液对糖尿病肾病患者血转化生长因子-β1及Ⅳ型胶原水平的影响及其意义[J].中国中西医结合肾病杂志,2006;7(3):156-157.
[110]薛艳芸,张立新.黄芪、灯盏花素对实验性糖尿病大鼠肾脏内皮素-1、TGF-β1的作用[J].细胞与分子免疫学杂志,2010;26(9):935-936.
[111]徐郁杰,张庆怡,陆敏,赵涵芳,陈诗书.黄芪对糖尿病大鼠肾皮质TGF-β1表达的影响[J].中华内分泌代谢杂志,1998;14(5):312-314.
[112] Bank N. Mechanisms of diabetic hyperfiltration [J]. Kidney Int.1991;40(4):792-807.
[113] Wei P, Lane PH, Lane JT, Padanilam BJ, Sansom SC. Glomerular structural and functionalchanges in a high-fat diet mouse model of early-stage type2diabetes[J]. Diabetologia,2004;47(9):1541-1549.
[114] Brindeiro CMT, Fallet RW, Lane PH, Carmines PK. Potassium channel contributions toafferent arteriolar tone in normal and diabetic rat kidney[J]. Am J Physiol Renal Physiol.,2008;295(1): F171-178.
[115] Kikkawa R, Kitamura E, Fujiwara Y, Arimura T, Haneda M, Shigeta Y. Impaired contractileresponsiveness of diabetic glomeruli to angiotensin II: a possible indication of mesangialdysfunction in diabetes mellitus[J]. Biochem Biophysic Res Commun,1986;136(3):1185-1190.
[116] Chiarelli F, Gaspari S, Marcovecchio ML. Role of growth factors in diabetic kidney disease[J].Horm Metab Res,2009;41(8):585-593.
[117] Nutt LK, O'Neil RG. Effect of elevated glucose on endothelin-induced store-operated andnon-store-operated calcium influx in renal mesangial cells [J]. J Am Soc Nephrol,2000;11(7):1225-1235.
[118] Whiteside CI, Hurst RD, Stevanovic ZS. Calcium signaling and contractile response ofdiabetic glomerular mesangial cells [J]. Kidney Int Suppl.1995;51: S28-S33.
[119] Schl ndorff D, Banas B. The mesangial cell revisited: no cell is an island [J]. J Am SocNephrol.2009;20(6):1179-1187.
[120] Hurst RD, Whiteside CI, Thompson JC. Diabetic rat glomerular mesangial cells displaynormal inositol trisphosphate and calcium release [J]. Am J Physiol.1992;263(4pt2): F649-F655.
[121] Li WP, Tsiokas L, Sansom SC, Ma R. Epidermal growth factor activates store-operated Ca2+channels through an IP3independent pathway in human glomerular mesangial cells[J]. J Biol Chem.2004;279(6):4570-4577.
[122] Ma R, Sansom SC. Epidermal growth factor activates store-operated calcium channels inhuman glomerular mesangial cells [J]. J Am Soc Nephrol.2001;12(1):47-53.
[123] Ma R, Smith S, Child A, Carmines PK, Sansom SC. Store-operated Ca2+channels in humanglomerular mesangial cells[J]. Am J Physiol.2000;278(6): F954-F961.
[124] Mené P, Pugliese G, Pricci F, Di Mario U, Cinotti GA, Pugliese F. High glucose inhibitscytosolic calcium signaling in cultured rat mesangial cells[J]. Kidney Int.1993;43(3):585-591.
[125] Hsu YJ, Hoenderop JG, Bindels RJ. TRP channels in kidney disease [J]. Biochim Biophys Acta.2007;1772(8):928-936.
[126] Dietrich A, Gudermann T. TRPC6[J]. Handb Exp Pharmacol.2007;179:125-141.
[127] Winn MP, Daskalakis N, Spurney RF, Middleton JP. Unexpected role of TRPC6channel infamilialnephrotic syndrome: does ithave clinicalImplications [J]. J Am SocNephrol.2006;17(2):378-387.
[128] Plant TD, Schaefer M. TRPC4andTRPC5receptor-operatedCa2+-permeable nonselectivecation channels [J]. Cell Calcium.2003;33(5-6):441-450.
[129] Thebault S, Zholos A, Enfissi A, Slomianny C, Dewailly E, Roudbaraki M, Parys J,Prevarskaya N.Receptor-operated Ca2+entry mediated by TRPC3/TRPC6proteins in rat prostatesmooth muscle (PS1) cell line[J]. J Cell Physiol.2005;204(1):320-328.
[130] Parekh AB, Putney JW Jr.. Store-operated calcium channels [J]. Physiol Rev,2005;85(2):757-810.
[131] Patrakka J, Tryggvason K. New insights into the role of podocytes in proteinuria [J]. Nat RevNephrol.2009;5(8):463-468.
[132] Inoue R, Okada T, Onoue H, Hara Y, Shimizu S, Naitoh S, Ito Y, Mori Y. The transientreceptor potential protein homologue TRP6is the essential component of vascularα1-adrenoceptor-activated Ca2+-permeable cation channel [J]. Circ Res.2001;88(3):325-332.
[133] Weissmann N, Dietrich A, Fuchs B, Kalwa H, Ay M, Dumitrascu R, Olschewski A, Storch U,Mederos Schnitzler M, Ghofrani HA, Schermuly RT, Pinkenburg O, Seeger W, Grimminger F,Gudermann T. Classical transient receptor potential channel6(TRPC6) is essential for hypoxicpulmonary vasoconstriction and alveolar gas exchange [J]. Proc Natl Acad Sci U S A,2006;103(50):19093-19098.
[134] Saleh SN, Albert AP, Peppiatt CM, Large WA. Angiotensin II activates two cationconductances with distinct TRPC1and TRPC6channel properties in rabbit mesenteric arterymyocytes [J]. J Physiol.2006;577(pt2):479-495.
[135] Winn MP, Conlon PJ, Lynn KL, Farrington MK, Creazzo T, Hawkins AF, Daskalakis N, KwanSY, Ebersviller S, Burchette JL, Pericak-Vance MA, Howell DN, Vance JM, Rosenberg PB.Amutation in theTRPC6cation channel causes familial focal segmental glomerulosclerosis [J].Science,2005,308(5729):1801-1804.
[136] Reiser J, Polu KR, M ller CC, Kenlan P, Altintas MM, Wei C, Faul C, Herbert S, Villegas I,Avila-Casado C, McGee M, Sugimoto H, Brown D, Kalluri R, Mundel P, Smith PL, Clapham DE,Pollak MR. TRPC6is a glomerular slit diaphragm-associated channel required for normal renalfunction[J]. Nat Genet,2005,37(7):739-744.
[137] Facemire CS, Mohler PJ, Arendshorst WJ. Expression and relative abundance of shorttransient receptor potential channels in the rat renal microcirculation [J]. Am J Physiol Renal Physiol,2004;286(3):F546-551.
[138] Bandyopadhyay BC, Swaim WD, Liu X, Redman RS, Patterson RL, Ambudkar IS. Apicallocalization of a functional TRPC3/TRPC6-Ca2＋-signaling complex in polarized epithelial cellsRole in apical Ca2＋influx[J]．J Biol Chem,2005；280(13):12908-12916．
[139] Goel M, Sinkins WG, Zuo CD, Estacion M, Schilling WP. Identifcation and localization ofTRPC channels in the rat kidney [J]. Am J Physiol Renal Physiol,2006；290(5):F1241-F1252．
[140]范青锋,邢燕,刘淑芳,张涵,丁洁.瞬时受体电位阳离子通道蛋白6在肾脏的表达[J].实用儿科临床杂志,2007;22(17):1293-1295．
[141] Dryer SE, Reiser J. TRPC6channels and their binding partners in podocytes: role inglomerular filtration and pathophysiology [J]. Am J Physiol Renal Physiol.2010;299(4):F689-701.
[142] M ller CC, Wei C, Altintas MM, Li J, Greka A, Ohse T, Pippin JW, Rastaldi MP, Wawersik S,Schiavi S, Henger A, Kretzler M, Shankland SJ, Reiser J. Induction of TRPC6channel in acquiredforms of proteinuric kidney disease[J]. J Am Soc Nephrol.2007;18(1):29-36.
[143] Ma R, Du J, Sours S, Ding M. Store-operated Ca2+channel in renal microcirculation andglomeruli[J]. Exp Biol Med (Maywood).2006;231(2):145-153.
[144] Schlondorff JS, Pollak MR. TRPC6in glomerular health and disease: what we know and whatwe believe [J]. Semin Cell Dev Biol.2006;17(6):667-674.
[145] Sours S, Du J, Ma R, Ding M, Zhou XJ, Ma R. Expression of canonical transient receptorpotential (TRPC) proteins in human glomerular mesangial cells[J]. Am J Physiol Renal Physiol.2006;290(6): F1507-F1515.
[146] Du J, Ding M, Ma R, Graham S, Ma R. Mediation of angiotensin II-induced Ca2+signalingby polycystin2in glomerular mesangial cells[J]. Am J Physio Renal Physiol.2008;294(4):F909-F918.
[147] Du J, Sours-Brothers S, Coleman R Ding M, Graham S, Kong DH, Ma R. Canonical transientreceptor potential1channel is involved in contractile function of glomerular mesangial cells[J]. JAm Soc Nephrol.2007;18(5):1437-1445.
[148] Jiang L, Ding J, Tsai H, Li L, Feng Q, Miao J, Fan Q. Over-expressing transient receptorpotential cation channel6in podocytes induces cytoskeleton rearrangement through increases ofintracellular Ca2+and RhoA activation[J]. Exp Biol Med (Maywood).2011;236(2):184-193.
[149] Estacion M, Li S, Sinkins WG, Gosling M, Bahra P, Poll C, Westwick J, Schilling WP.Activation of human TRPC6channels by receptor stimulation [J]. J Biol Chem,2004;279(21):22047-22056.
[150] Gottlieb P, Folgering J, Maroto R, Raso A, Wood TG, Kurosky A, Bowman C, Bichet D, PatelA, Sachs F, Martinac B, Hamill OP, Honoré E. Revisiting TRPC1and TRPC6mechanosensitivity[J].Pflugers Arch,2008;455(6):1097-1103.
[151] Graham S, Gorin Y, Abboud HE, Ding M, Lee DY, Shi H, Ding Y, Ma R. Abundance ofTRPC6protein in glomerular mesangial cells is decreased by ROS and PKC in diabetes[J]. Am JPhysiol Cell Physiol,2011;301(2):C304-15.
[152] Graham S, Ding M, Sours-Brothers S, Yorio T, Ma JX, Ma R. Downregulation of TRPC6protein expression by high glucose, a possible mechanism for the impaired Ca2+signaling inglomerular mesangial cells in diabetes. Am J Physiol Renal Physiol,2007;293(4):F1381-1390.
[153] Du J, Sours-Brothers S, Coleman R, Ding M, Graham S, Kong DH, Ma R. Canonical transientreceptor potential channel is involved in contractile function of glomerular measangial cells[J]. J AmSoc Nephrol,2007;18(5):1446-1457.
[154]吴华勋,严尚学,魏伟.糖尿病肾病动物模型[M]//魏伟,吴希美,李元建主编.药理实验方法学.第4版.北京:人民卫生出版社,2010:1238-1239.
[1] Haraldsson B, Nystrom J, Deen W M. Properties of the glomerular barrier and mechanisms ofproteinuria [J]. Physiol Rev,2008;88(2):451-487.
[2] Dalla Vestra M, Masier OA, Roiter AM, et al. Is podocyte injury relevant in diabetic nephropathyStudies in patients with type2diabtes [J]. Diabetes,2003;52(4):1031-1035.
[3] Bank N. Mechanisms of diabetic hyperfiltration [J]. Kidney Int,1991;40:792–807.
[4] Kikkawa R, Kitamura E, Fujiwara Y, Arimura T, Haneda M, Shigeta Y. Impaired contractileresponsiveness of diabetic glomeruli to angiotensin II: a possible indication of mesangialdysfunction in diabetes mellitus[J]. Biochem Biophys Res Commun,1986;136:1185–1190.
[5] Mene′P, Pascale C, Teti A, Bernardini SV, Cinotti GA, Pugliese F. Effects of advanced glycationend products on cytosolic Ca2+signaling of cultured human mesangial cells[J]. J Am SocNephrol,1999;10:1478–1486.
[6] Mene′P, Pugliese G, Pricci F, DiMario U, Cinotti GA, Pugliese F. High glucose level inhibitscapacitative Ca2+influx in cultured rat mesangial cells by a protein kinase C-dependentmechanism[J]. Diabetologia,1997;40:521–527.
[7] Nutt LK, O’Neil RG. Effect of elevated glucose on endothelin-induced store-operated andnon-store-operated calcium influx in renal mesangial cells [J]. J Am Soc Nephrol,2000;11:1225–1235.
[8] Whiteside CI, Hurst RD, Stevanovic ZS. Calcium signaling and contractile response of diabeticglomerular mesangial cells [J]. Kidney Int,1995;8:S28–S33.
[9] Guan N, Ding J, Zhang J, et al. Expression of nephrin, podocin, alpha-actinin, and WT1inchildren with nephrotic syndrome[J]. Pediatr Nephrol,2003;18(11):1122-1127.
[10]范青锋,丁洁,张敬京，等. Nephrin、podocin及α-actinin在小鼠肾小球足细胞表达与分布[J].肾脏病与透析肾移植杂志,2003;12(5):407-411.
[11] Guan N, Ding J, Deng J, et al. Key molecular events in puromycin aminonucleoside nephrosisrats [J]. Pathol Int,2004;54(9):703-711.
[12] Fan Q, Ding J, Zhang J,et al. Effect of the knockdown of podocin mRNA on nephrin andalpha-actinin in mouse podocyte[J]. Exp Biol Med (Maywood),2004,229(9):964-970.
[13]邓江红,丁洁,管娜,等.嘌呤霉素肾病大鼠肾小球足细胞相关分子表达的共定位分布[J].肾脏病与透析肾移植杂志,2004,13(3):205-210.
[14] Winn MP, Conlon PJ, Lynn KL, et al. A mutation in the trpc6cation channel causes familialfocal segmental glomerulosclerosis [J]. Science,2005,308(5729):1801-1804.
[15] Eiser J, Polu KR, M ller CC, et al.TRPC6is a glomerular slit diaphragm-associated channelrequired for normal renal function [J]. Nat Genet,2005,37(7):739-744.
[16] Sours S, Du J, Chu S, Ding M, Zhou XJ, Ma R.Expression of canonical transient receptorpotential (TRPC) proteins in human glomerular mesangial cells[J]. Am J Physiol RenalPhysiol.,2006,290(6):F1507-15.
[17] Ockand JD, Sansom SC. Glomerularmesangial cells: Electophysiology and regulation ofcontraction [J]. Physiol Rev,1998,78(3):723-743.
[18] Avenstadt H. Roles of the podocyte in glomerular function [J]. Am J Physiol Renal Physiol,2000,278(2): F173-F179.
[19] Tanaka H, Smogorzewski M, Koss M,et al. Pathways involved in PTH induced rise in cytosolicCa2+concentration of rat renal proximal tubule[J]. Am J Physiol,1995,268(2Pt2): F330-F337.
[20] Van Baal J, Raber G, de Slegte J,et al. Vasopressin stimulated Ca2+reabsorption in rabbitcortical collecting system: effects on cAMP and cytosolic Ca2+[J]. Pflugers Arch,1996,433(1-2):109-115.
[21] Mller CC, Wei C, Altintas MM, et al.Induction of TRPC6channel in acquired forms ofproteinuric kidney disease [J]. J Am Soc Nephrol,2007,18(1):29-36.
[22] Sarabeth Graham, Min Ding, Sherry Sours-Brothers. Downregulation of TRPC6proteinexpression by high glucose, a possible mechanism for the impaired Ca2+signaling in glomerularmesangial cells in diabetes [J]. Am J Physiol Renal Physiol,2007,293:1381-1390.
[23] Stgaard J, Qvortrup K. Sieve plugs in fenestrae of glomerular capillaries site of filtration barrier[J]. Cell Tissues Organs,2002,170(2-3):132-138.
[24] Luft J H. Fine structures of capillary and endocapillary layer as revealed by ruthenium red [J].Fed Proc,1966,25(6):1773-1783.
[25] Montesano R, Mossaz A, Ryser JE, et al. Leukocyte in terleukins induces cultured endothelialcells to produce a highly organized, glycosam inoglycan rich pericellular matrix [J]. J Cell Bio, l1984,99(5):1706-1715.
[26] Jeansson M, Haraldsson B.Morphological and functional evidence for an important role of theendothelial cell glycocalyx in the glomerular barrier [J]. Am J Physiol Renal Physiol,2006,290:F111–116.
[27] Deckert T, Feldt-Rasmussen B, Borch-Johnsen K, et al. Albuminuria reflects wide spreadvascular damage: The Steno hypothesis [J]. Diabetologia,1989,32(4):219-226.
[28] Algenstaedt P, Schaefer C, Biermann T, et al. Microvascular alterations in diabetic mice correlate with level of hyperglycemia [J]. Diabetes,2003,52(2):542-549.
[29] Erdely A, Freshour G, Maddox DA, et al. Renal disease in rats with type2diabetes is associatedwith decreased renal nitric oxide production [J]. Diabetologia,2004,47(10):1672-1676.
[30] Koo J R, Vaziri ND. Effects of diabetes, insulin and antioxidants on NO synthase abundanceand NO interaction with reactive oxygen species [J]. Kidney Int,2003,63(1):195-201.
[31] Piqueras L, Kubes P, Alvarez A, et al. Angiotensin II induces leukocyte-endothelial cell interactions in vivo via AT (1) and AT (2) receptor-mediated P-selectin upregulation [J]. Circulation,2000;102(17):2118-2123.
[32] Levidiotis V, Power DA. New insights into the molecular biology of the glomerular filtrationbarrier and associated disease [J]. Nephrology (Carlton),2005;10:157–166.
[33] Barker DF, Hostikka SL, Zhou J, et al. Identification of mutations in the COL4A5collagen genein Alport syndrome [J]. Science,1990,248:1224–1227.
[34] Zenker M, Aigner T, Wendler O, et al. Human laminin beta2deficiency causes congenitalnephrosis with mesangial sclerosis and distinct eye abnormalities [J]. Hum Mol Genet,2004,13:2625–2632.
[35] Tamsma JT, vanden Born J, Bruijn JA, et al. Expression of glomerular extracellularm atrix components in human diabetic nephropathy: Decrease of heparan sulphate in the glomerular basementmembrane [J]. Diabetologia,1994;37(3):313-320.
[36] Raats C J, vanden Born J, Berden J H. Glomerular heparan sulfate alterations: Mechanisms andrelevance for proteinuria [J]. Kidney Int,2000,57(2):385-400.
[37] Yard BA, Kahlert S, Engelleiter R, et al. Decreased glomerular expression of agrin in diabeticnephropathy and podocytes, cultured in high glucose medium [J].Exp Nephrol,2001;9(3):214-222.
[38] Maxhimer JB, Somenek M, Rao G, et al. Heparanase-1gene expression and regulation by highglucose in renal epithelial cells: A potential role in the pathogenesis of proteinuria in diabeticpatients [J]. Diabetes,2005,54(7):2172-2178.
[39]刘莉莉,刘志红,郑春霞,等.足细胞损伤对糖尿病肾病患者预后的影响[J].肾脏病与透析移植杂志,2007,16(6):509-515.
[40] Rodewald R, Karnovsky MJ. Porous substructure of the glomerular slit diaphragm in the rat andmouse [J]. J Cell Biol,1974;60:423–433.
[41] Pavenstadt H, Kriz W, Kretzler M. Cell biology of the glomerular podocyte [J]. Physiol Rev,2003,83(1):253-307.
[42]陈惠萍.糖尿病肾病患者肾小球滤过膜结构和功能及代谢指标的再认识[J].医学研究生学报,2010;23(8):785-791.
[43] Glazebrook PA, SchillingWP, Kunze DL. TRPC channels as signal transducers [J].PflugersArch,2005,451(1):125-130.
[44] Hsu YJ, Hoenderop JG, BindelsRJ. TRP channels in kidney disease [J]. Biochim BiophysActa,2007,1772(8):928-936.
[45] Weissmann N, DietrichA, FuchsB, et al Classical transient receptor potential channel6(TRPC6)is essential forhypoxic pulmonary vasoconstriction and alveolar gas exchange [J]. Proc Natl AcadSciUSA,2006,103(50):19093-190938.
[46] PlantTD, SchaeferM. TRPC4andTRPC5receptor-operated Ca2+permeable nonselective cationchannels [J]. Cell Calcium,2003;33(5-6):441-450.
[47] Cayouette S, LussierMP, Mathieu EL, et a.l Exocytotic insertion of TRPC6channel into theplasmamembrane upon Gq protein-coupled receptor activation [J]. J Biol Chem,2004;279(8):7241-7246.
[48] Li S, Gosling M, Poll C. Determining the functional role of TRPC channels in primary cells [J].PflugersArch,2005,451(1):43-52.
[49]BaeYM, Kim A, Lee YJ, et al. Enhancement of receptor-operated cation current and TRPC6expression in arterial smooth muscle cells of deoxy corticosterone acetate-salthy pertensive rats [J]. JHypertens,2007,25(4):809-817.
[50] Dietrich A, Gudermann T. TRPC6[J]. Handb Exp Pharmacol,2007,(179):125-141.
[51] Dietrich A, Kalwa H, Rost BR, et al.The diacylgylcerol-sensitive TRPC3/6/7subfamily ofcation channels: functional characterization and physiological relevance[J]. Pflugers Arch,2005,451(1):72-80.
[52] Winn MP, Daskalakis N, Spurney RF, et al Unexpected role of TRPC6channel in familialnephrotic syndrome: does it have clinical Implications [J]. J Am Soc Nephrol,2006,17(2):378-387.
[53] Plant TD, Schaefer M. TRPC4and TRPC5receptor-operated Ca2+permeable nonselectivecation channels [J]. Cell Calcium,2003,33(5-6):441-450.
[54]匡新宇,黄文彦. TRPC6与肾脏疾病[J].国际病理科学与临床杂志,2008;28(5):456-460.
[55] DE' sposito M, Strazzullo M, Cuccurese M. Identification and assignment of the humantransient receptor potential channel6gene TRPC6to chromosome11q21-->q22[J].Cytogenet CellGenet,1998,83(1-2):46-47.
[56] Estacion M, Li S, Sinkins WG, et al. Activation of human TRPC6channels by receptorstimulation [J]. J Biol Chem,2004,279(21):22047-22056.
[57] DE Clapham, LW Runnels, C Strubing. The TRP ion channel family [J]. Nat.Rev Neurosci,2001,2:387–396.
[58] Hofmann T，Obukhov AG，Schaefer M，et al. Direct activation of humanTRPC6and TRPC3channels by diacylglyeerol [J].Nature,1999;397:259-263.
[59] Okada T, Inoue R, Yamazaki K, Maeda A, Kurosaki T, Yamakuni T, Tanaka I, Shimizu S,Ikenaka K, Imoto K, Mori Y. Molecular and functional characterization of a novel mouse transientreceptor potential protein homologue TRP7. Ca(2+)-permeable cation channel that is constitutivelyactivated and enhanced by stimulation of G protein-coupled receptor[J]. J Biol Chem,1999;274(39):27359-70.
[60]邢燕,范青锋,丁洁.导致家族性局灶节段性肾小球硬化新基因-TRPC6[J].肾脏病与透析肾移植杂志,2006;5(2):140-143.
[61] Beech DJ. Emerging functions of10types of TRP cationic channel in vascular smoothmuscle[J].Clin Exp Pharmacol Physiol2005,32(8):597-603.
[62] Facemire CS, Mohler PJ, Arendshorst WJ. Expression and relative abundance of short transientreceptor potential channels in the rat renal vasculature [J]. Am J Physiol Renal Physiol,2004,286(3):F546-F551.
[63] Bandyopadhyay BC, Swaim WD, Liu X,et al. Apical localization of a functional TRPC3/TRPC6-Ca2+-signaling complex in polarized epithelial cells: role in apical Ca2+influx[J]. J BiolChem,2005,280(13):12908-12916.
[64] Goel M, Sinkins WG, Zuo CD,et al. Identification and Localization of TRPC Channels in RatKidney[J].Am J Physiol Renal Physiol,2006,290(5):F1241-52.
[65] Dietrich A, MederosY, Schnitzler M,et al. Increased vascular smooth muscle contractility inTRPC6-/-mice [J]. Mol Cell Biol,2005,25(16):6980-6989.
[66] Reiser J, Polu KR, Mller CC, et al. TRPC6is a glomerular slit diaphragm-associated channelrequired for normal renal function [J]. Nat Genet,2005,37(7):739-744.
[67]Ahmmed GU, Malik AB. Functional role of TRPC channels in the regulation of endothelialpermeability [J].Pflugers Arch.,2005,451(1):131-42.
[68] Eeremina, et al. VEGF inhibition and renal thrombotic microangiopathy[J]. N. Engl. J. Med,2008,358:1129–1136.
[69] H.W. Cheng, A.F. James, R.R. Foster, J.C. Hancox, D.O. Bates. VEGF activatesreceptor-operated cation channels in human microvascular endothelial cells, Arterioscler [J]. Thromb.Vasc. Biol,2006,26:1768–1776.
[70] T.M. Pocock, R.R. Foster, D.O. Bates. Evidence of a role for TRPC channels inVEGF-mediated increased vascular permeability in vivo[J].Am. J. Physiol. Heart. Circ. Physiol,2004,286:H1015–1026.
[71] Foster RR，Satchell SC，Seckley J，et al. VEGF—CP romotes survival in Podoeytes[J].Am JPhysiol Renal Physiol,2006;291:196-207.
[72] Bieke F, Schrij vers, Allan F, et al. The role of vascular endothelial growth factor (VEGF) inrenal pathophysiology [J]. Kidney Int,2004,65(6):2003-2017.
[73] Veron D, Reidy KJ, Bertuccio C, et al. Overexpression of VEGF-A in podocytes of adult micecauses glomerular disease [J]. Kidney Int,2010,77(11):989-999.
[74] Müller-Deile J, Worthmann K, Saleem M, et al. The balance of autocrine VEGF-A and VEGF-Cdetermines podocyte survival [J].Am J Physiol Renal Physiol,2009,297(6):F1656-67.
[75]Lin SY, Corey DP. TRP channels in mechanosensation [J]. Curr Opin Neurobio,2005;15:350–357.
[76] Duh E, Nello LP. Vascular endothelial growth factor and diabetes: the agonist versus antagonistparadox [J]. Diabetes,1999,48(10):1899-1909.
[77]柳飞,黄颂敏.肾小球内皮细胞损害与糖尿病肾病[J].华西医学,2007,22(2):446-447.
[78]范青锋,邢燕,刘淑芳,张涵,丁洁.瞬时受体电位阳离子通道蛋白6在肾脏的表达[J].实用儿科临床杂志,2007,22(17):1293-1295.
[79] Ly J,Alexander M,Quaggin SE.A podocentric view of nephrology[J].Curr Opin NephrolHypertens,2004;13:299–305.
[80] L.Barisoni, W.Kriz, P.Mundel, V.D'Agati. The dysregulated podocyte phenotype: a novelconcept in the pathogenesis of collapsing idiopathic focal segmental glomerulosclerosis andHIV-associated nephropathy [J]. J.Am.Soc. Nephrol,1999,10:51–61.
[81] Wada T, Pippin JW, Marshall CB, et al. Dexamethasone prevents podocyte apoptosis induced bypuromycin aminonucleoside: role of p53and Bcl-2related family proteins[J]. J Am Soc Nephrol,2005,16:2615-2625.
[82] Kim YH, Goyal M, Kurnit D, et al.Podocyte depletion and glomerulosclerosis have a directrelationship in the PAN-treated rat [J].Kidney Int,2002,60:957-968.
[83] Sun X, Fang Z, Zhu Z, Yang X, He F, Zhang C. Effect of down-regulation of TRPC6on thepuromycin aminonucleoside-induced apoptosis of mouse podocytes[J]. J Huazhong Univ SciTechnolog Med Sci.,2009,29(4):417-22.
[84] Moller CC，Wei C，Altintas MM, et al. Induction of TRPC6channel in acquired forms ofproteinuric kidney disease [J]. J Am Soc NePhrol,2007,18:29-36.
[85] P.Mundel, S.J.Shankland. Podocyte biology and response to injury [J]. J.Am.Soc.Nephrol,2002,13:3005–3015.
[86] Verma R, Wharram B, Kovari I,et al. Fyn binds to and phosphorylates the kidney slit diaphragmcomponent nephrin [J]. J Biol Chem,2003,278(23):20716-20723.
[87] Li H, Lemay S, Aoudjit L,et al. SRC-family kinase Fyn phosphorylates the cytoplasmic domainof nephrin and modulates its interaction with podocin[J]. J Am SocNephrol,2004,15(12):3006-3015.
[88] M.A.Spassova, T. Hewavitharana, W.Xu, J.Soboloff, D.L.Gill. A common mechanism underliesstretch activation and receptor activation of TRPC6channels [J]. Proc.Natl.Acad.Sci,2006,103:16586-16591.
[89] M.Kestila, M.Mannikko, C.Holmberg, et al. Congenital nephrotic syndrome of the Finnish typemaps to the long arm of chromosome19[J]. Am.J.Hum.Genet,1994,54:757–764.
[90] Spassova MA,Hewavitharana T,Xu W,et al.A common mechanism underlies stretch activationand receptor activation of TRPC6channels[J].Proc Natl Acad Sci USA,2006,103,16586-16591.
[91] Huber TB,Schermer B,Muller RU,et al.Podocin and MEC-2bind cholesterol to regulate theactivity of associated ion channels[J].Proc Natl Acad Sci USA,2006,103:17079-17086.
[92] Y.H.Kim, M.Goyal, D.Kurnit,et al. Podocyte depletion and glomerulosclerosis have a directrelationship in the PAN-treated rat [J]. Kidney Int,2001,60:957–968.
[93] Mukerji N，Damodaran TV, Winn MP. TRPC6and FSGS: the latest TRP channelopathy,2007,1772:859-868.
[94] Schiffer M, Bitzer M, Roberts IS, et al.Apoptosis in podocytes induced by TGF-beta and Smad7[J]. J Clin Invest,2001,108:807-816.
[95] Liu SF, Ding J, Fan QF, et al.Establishment of a podocyte cell injury model induced bypuromycin aminonucleoside [J]. Beijing Da Xue Xue Bao,2008,40:586-589.
[96] Jia J, Ding G, Zhu J, et al.Angiotensin Ⅱ infusion induces nephrin expression changes andpodocyte apoptosis [J].Am J Nephrol,2008,28:500-507.
[97] Hara M, Yanagihara T, Kihara L. Urinary podocytes in primary focal segmentalglomerulosclerosis [J]. Nephron,2001,89:342-347.
[98] Tryggvason K, Patrakka J,Wartiovaara J.Hereditary proteinuria syndromes and mechanisms ofproteinuria[J]. N Engl J Med,2006,354:1387-401.
[99] Shankland SJ.The podocytes response to injury: role in proteinuria and glomerulosclerosis [J].Kidney Int,2006,69:2131-3147.
[100] Ma R, Pluznick JL, and Sansom SC. Ion channels in mesangial cells: function, malfunction, orfiction [J]. Physiology,2005,20:102–111.
[101] Sherry Sours, Juan Du, Shaoyou Chu.Expression of canonical transient receptor potential(TRPC) proteins in human glomerular mesangial cells [J]. Am J Physiol Renal Physiol,2006,290:F1507–F1515.
[102] Stockand JD, Sansom SC. Glomerularmesangial cells: Electophysiology and regulation ofcontraction [J]. PhysiolRev,1998,78(3):723-743.
[103] Saleh SN, Albert AP, Peppiatt CM, Large WA. Angiotensin II activates two cationconductances with distinct TRPC1and TRPC6channel properties in rabbit mesenteric arterymyocytes [J]. J Physiol,2006,577:479-495.
[104] Marrero MB, Schieffer B, ma HP, et al. ANG II-induced tyrosine phosphorylation stimulatesphospholipase C and C1-channels in mesangial cells [J]. Am J Physiol Cell Physiol,1996,270:C1834–C1842.
[105] Ha H, Yu M, Kim KH. Melatonin and taurine reduce early glomerulopathy in diabetic rats [J].Free Radic Biol Med,1999,26:944–950.
[106] The Diabetes Control and Complications Trial Research Group. The effect of intensivetreatment of daibetes on the development and progression of long-term complications ininsulin-dependent diabetes mellitus [J]. N Engl J Med,1993,329(14):977–986.
[107] Dietrich A, Gudermann T. TRPC6[J]. Handb Exp Pharmacol,2007,179:125–141.
[108] Young BA, Johnson RJ, Alpers C, et al. Cellular events in the evolution of experimentaldiabetic nephropathy[J]. Kidney Int,1995,479(3):935-944.
[109] Gorin Y, Block K, Hernandez J, Bhandari B, Wagner B, Barnes JL, Abboud HE. Nox4NADPH oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney [J]. J BiolChem,2005,280:39616–39626.
[110] Hinokio Y, Hirai M, Chiba M, Hirai A, Toyota T. Oxidative DNA damage in diabetes mellitus:its association with diabetic complications [J]. Diabetologia,1999,42:995–998.
[111] Kim NH, Rincon-Choles H, Bhandari B, Choudhury GG, Abboud HE, Gorin Y. Redoxdependence of glomerular epithelial cell hypertrophy in response to glucose[J]. Am J Physiol RenalPhysiol,2006,290: F741–F751.
[112] Schnackenberg CG. Oxygen radicals in cardiovascular-renal disease [J]. Curr Opin Pharmacol,2002,2:121–125.
[113] Shah SV, Baliga R, Rajapurkar M, Fonseca VA. Oxidants in chronic kidney disease [J]. J AmSoc Nephrol,2007,18(1):16–28.
[114] Ha H, Yu MR, Choi YJ, Kitamura M, Lee HB. Role of high glucose-induced nuclear factor-κBactivation in monocyte chemoattractant protein-1expression by mesangial cells [J]. J Am SocNephrol,2002,13:894–902.
[115] Gorin Y, Ricono JM, Wagner B, Kim NH, Bhandari B, Choudhury GG. Angiotensin II-inducedERK1/ERK2activation and protein synthesis are redox-dependent in glomerular mesangial cells [J].Biochem J,2004,381:231–239.
[116] Iglesias La, Cruz MC-DE, Ruiz-Torres P, Alcami J, Diez-Marques L, Ortega-Velazquez R,Chen S, Rodriguez-Puyol M, Ziyadeh FN, Rodriguez-Pyol D. Hydrogen peroxide increasesextracellular matrix mRNA through TGF-βin human mesangial cells[J]. Kidney Int,2001,59:87–95.
[117] Dandona P, Thusu K, Cook S, Snyder B, Makowski J, Armstrong D, Nicotera T. Oxidativedamage to DNA in diabetes mellitus[J]. Lancet,1996;347:444-445.
[118] Tryggvason K, Wartiovaara J.How does the kidney filter plasma [J]. Physiology（Bethesda）,2005;20:96-101.
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