【作者】 任小军；

【导师】 关广聚；

【作者基本信息】 山东大学 ， 内科学， 2009， 博士

【摘要】 背景随着世界范围内糖尿病患者的迅速增加,糖尿病并发症对患者生存质量和死亡率的影响也越来越重要。糖尿病肾病（diabetic nephropathy,DN）是糖尿病最常见的慢性并发症之一,也是导致终末期肾病（end-stage renal disease,ESRD）的主要原因。DN过去一直被认为是一种肾小球疾病,而新近的研究表明,小管损伤和进行性间质纤维化与DN的预后关系更为密切。为此,研究DN小管间质病变发生、发展的机制,对于寻找更加有效的治疗措施具有重要意义。DN小管间质病变的核心是细胞外基质（extracellular matrix,ECM）在损伤区的过量堆积。研究表明,由小管上皮细胞和间质成纤维细胞转分化而来的α-平滑肌肌动蛋白（α-smooth muscle actin,α-SMA）阳性的肌成纤维母细胞是ECM的主要来源。多种细胞因子和生长因子参与了ECM的生成和降解,其中转化生长因子-β（transforming growth factor-beta,TGF-β）超家族成员发挥了重要作用。根据结构和和分子生物学差异,TGF-β超家族可以细分为三个主要家族:TGF-β、骨形成蛋白（bone morphogenetic protein,BMP）和激活素（activin,ACT）。研究表明,在实验性DN早期,TGF-β在小管上皮表达即明显上调,并可促进小管上皮细胞向肌成纤维母细胞转分化。体外培养的小管上皮细胞,高糖和糖基化终末期产物（advanced glycation end products,AGEs）均可通过TGF-β依赖的方式促进ECM成分的产生。BMP-7与TGF-β作用正好相反,内源性BMP-7可阻止小管上皮细胞ECM的产生。在实验性DN模型中,BMP-7及其受体表达均明显下调。ACT最早是在性腺发现的糖蛋白,因特异性促进垂体细胞合成及分泌卵泡刺激素（FSH）而得名。ACT是由两个β亚单位借二硫键构成的二聚体蛋白,按照β亚基构成的不同,ACT分为三种形式:ACTA（βAβA）、ACT B（βBβB）、ACTAB（βAβB）,目前研究最多的是ACTA。类似于TGF-β超家族其它成员,ACT A主要通过Smad信号途径介导其生物学效应。卵泡抑素（follistatin,Fs）是由性腺分泌的单链多肽,可抑制垂体FSH的分泌。FS与ACT有很强的亲和力,并阻断其生物学效应;二者共同构成一个维持组织器官正常生长和代谢的自动平衡系统。目前对于ACT A在肾小管间质病变中的作用机制研究甚少,ACT与DN小管间质纤维化之间的潜在联系尚待进一步研究。目的本研究通过体内链脲佐菌素（STZ）诱导的糖尿病动物模型及体外高糖刺激的人近端肾小管上皮细胞株（HK-2）,探讨ACT A/Smad信号在糖尿病肾脏的表达及其与转分化标志物α-SMA和ECM成分纤维连接蛋白（FN）变化的关系。方法1.动物实验雄性Wistar大鼠,随机分成糖尿病组（DM）和对照组（NC）。DM组在禁食12小时后一次性腹腔注射STZ（60mg/kg）,NC组给予相同剂量的溶媒。给药72h后尾静脉采血测随机血糖（BG）＞16.7mmol/L为糖尿病大鼠。所有大鼠标准饲料饲养,自由饮水。各组分别于造模后4、8、12、16周处死大鼠6只。处死前1天,大鼠称重后单只置于代谢笼中,准确收集24h尿液。腹主动脉取血后分离左肾,部分肾组织固定于10%多聚甲醛供病理及免疫组化检查,另一部分置液氮待做PCR检测。肾脏肥大指数用左肾重/体重（KW/BW）表示;放免法测定尿白蛋白排泄率（AER）;BG、血肌酐（Scr）及尿肌酐（Ucr）用HITACHI-7150自动生化分析仪检测,肌酐清除率（Ccr）按公式:Ucr×每分钟尿量（ml）/Scr计算,并以体重校正。光镜观察肾组织形态学变化;免疫组化检测肾组织中ACTβA、FS、P-Smad2/3、α-SMA和FN蛋白表达;real time-PCR检测肾组织中ACTβA、FS、α-SMA和FNmRNA的表达。2.细胞培养HK-2细胞生长于含10%胎牛血清、100U/ml青霉素、100μg/ml链霉素的DMEM培养基,置37℃含5%CO₂的培养箱中孵育。待细胞生长至亚融合状态时,更换为无血清DMEM培养基使细胞生长同步化24h,然后将细胞分为以下5组:正常对照组（NG,5.5mmol/L葡萄糖）、甘露醇对照组（MG,5.5mmol/L葡萄糖+25mmol/L甘露醇）、高糖组（HG,25mmol/L葡萄糖）、HG+FS100组（25mmol/L葡萄糖+100ng/mlFS）、HG+FS500组（25mmol/L葡萄糖+500ng/mlFS）。12、24、48h后收集各组细胞及细胞上清液。Western blot检测各组细胞ACTβA和P-Smad2/3的表达;ELISA检测各组细胞上清液TGF-β、α-SMA和FN的含量。结果1.动物实验1.1一般生化指标的变化在各时间点,DM组大鼠血糖明显高于NC组。DM组大鼠KW/BW、AER及Ccr从第8周开始升高,并呈逐渐增高趋势,与NC组相比差异有统计学意义（P＜0.01）;DM组内16周末与8周末比较有统计学差别（P＜0.01）。1.2肾小管间质组织病理改变PAS染色结果显示,各时间点NC组肾小管结构正常,没有间质水肿和纤维化。DM组大鼠自实验第8周起可见部分肾小管上皮细胞空泡变性,肾小管肥大、扩张和萎缩,肾间质可见纤维化,随时间延长病变逐渐加重,肾小管间质损伤指数逐渐增加。1.3免疫组化检测肾小管间质ACTβA、FS、P-Smad2/3、α-SMA和FN的表达免疫组化结果显示,NC组大鼠在肾小球、肾小管及间质均未检测到ACTβA的表达;DM组大鼠肾小管上皮细胞ACTβA阳性表达自第4周起逐渐增加,并于12周时达峰值。与之相反,FS在NC组大鼠肾小管上皮细胞大量表达,在DM组其表达从第4周开始逐渐下降。P-Smad2/3和FN在NC组肾小管和间质有轻度表达,而在DM组大鼠从第8周起表达量逐渐增加,并一直持续到第16周。α-SMA在NC组仅表达于脉管系统,DM组除血管壁外,从第8周开始,肾小管和肾间质表达亦明显增加。1.4肾组织ACTβA、FS、α-SMA和FNmRNA的表达ACTβA mRNA在NC组只有极微量表达,而DM组大鼠肾组织ACTβAmRNA从第4周起即开始增加（NC组的3.3倍）,于12周时达峰值（NC组的7.7倍）,16周时表达量略有下降,但仍明显高于NC组（P＜0.01）;与之相反,FS在NC组大量表达,而DM组表达逐渐减少,实验第4周和12周时与NC组相比分别下降了32.5%和52.9%。与NC组相比,4周时DM组肾组织α-SMA和FN mRNA表达无明显改变,但从第8周起逐渐增加,并于第16周时达峰值。2.细胞培养2.1 ACTβA和P-Smad2/3在HK-2细胞的表达及FS的干预作用Western blot检测结果显示,ACTβA在NG组细胞有微量表达,而HG组ACTβA表达显著增加,呈时间依赖性。与HG组相比,FS干预组ACTβA表达显著减少,呈剂量依赖性;高糖培养48h后,HG+FS500组ACTβA表达与NG组相比无统计学差异（P＞0.05）。与NG组相比,P-Smad2/3在HG组培养24h后表达明显增加;FS可抑制P-Smad2/3的高表达,高糖培养48h后,HG+FS100组和HG+FS500组与HG组相比,P-Smad2/3表达分别下降了26%和41%。MG组细胞中ACTβA和P-Smad2/3表达与NG组相比无统计学差异（P＞0.05）。2.2 TGF-β、α-SMA和FN在HK-2细胞上清的表达及FS的干预作用ELISA检测结果显示,与NG组相比,HG组培养12h后TGF-β表达即显著增加,呈时间依赖性,FS干预对高糖诱导的TGF-β高表达没有影响。与NG组相比,HG组培养24h后α-SMA和FN表达显著增高（P＜0.01）,FS对此有明显抑制作用,呈剂量依赖性（P＜0.01）。MG组TGF-β、α-SMA和FN的表达与NG组相比无统计学差异（P＞0.05）。结论1.糖尿病大鼠肾小管间质存在ACT A/Smad信号蛋白的上调,同时转分化标志物α-SMA和ECM成分FN的合成增加。2.高糖能刺激HK-2细胞ACT A/Smad信号蛋白活化,并促进肾小管上皮细胞转分化标志物α-SMA和ECM成分FN的合成。3.FS可通过阻断高糖诱导的ACT A表达,从而阻抑高糖诱导的Smad信号活化,减轻肾小管上皮细胞转分化和FN的合成。更多还原

【Abstract】 BackgroundThe population of diabetic patients is dramatically increasing in the world.The development of the various complications of diabetes contributes to the high morbidity and mortality.Diabetic nephropathy （DN） is one of the most common chronic complications of diabetes mellitus and the main cause of end-stage renal disease（ESRD）.In the past,DN was chiefly recognized as a kind of glomerular disease. However,a recent study has indicated that tubular injury and progressive interstitial fibrosis are more closely related to DN prognosis.So the research on the mechanisms of the intiation and development of diabetic tubulointerstitial injuries will be benefit for development of an accurate treatment.The core of tubulointerstitial injuries in DN is the increased accumulation of extracellular matrix（ECM） in the injured region. Myofibroblasts are the main source of increased ECM deposition seen in tubulointerstitium.It has been demonstrated that not only interstitial fibroblasts,but also tubular epithelial cells can transform into myofibroblasts in the presense of high glucose levels. alpha-Smooth-muscle actin（α-SMA）,absent in normal tubular epithelial and fibroblasts,is a common mark used to localize myofibroblasts in vivo and in vitro.A variety of cytokines and growth factors are involved in ECM production and degradation,among which transforming growth factor beta（TGF-β） superfamily members play an important role.According to the differences of molecular structure and biology,the TGF-βsuperfamily can be divided into three main families:TGF-β,bone morphogenetic protein（BMP） and activin.Some studies have discovered that,in the early phase of DN,TGF-βexhibits upregulation in tubular epithelia and facilitates the transdifferentiation of tubular epithelia cells into myofibroblasts.In vitro cultured tubular epithelial cells,both high glucose and advanced glycation endproducts （AGEs） may promote ECM formation through a TGF-βdependent way.The roles of BMP-7 and TGF-βare exactly opposite.Endogenous BMP-7 may keep tubular epithelial cells from producing ECM.In an experimental DN model,the expression of BMP-7 and its receptor is obviously downregulated.Activin,a kind of glycoprotein,was first discovered in the gonads and named for its specifically facilitating anterior pituitary gland to synthesize and release follicle stimulating hormone（FSH）.Activin consists of twoβ-subunits which are combined by disulfide bond to form a dimeric protein.Based on the constructive differences ofβ-subunit, activin is divided into three forms:activin A（βAβA）, activin B（βBβB） and activin AB（βAβB）.Among them,the studies on activin A are the most.Similar to other members of the TGF-βsuperfamily,activin A mediates its biological effects mainly through the Smad signal pathway.Follistatin is a single-chain polypeptide isolated from ovarian fluid,which inhibits pituitary FSH secretion. This protein binds to activin with high affinity and blocks its actions.Follistatin and activin together compose a balanced system of automatic control to maintain the normal growth and metabolism of tissues and organs.The underlying pathogenic link between activin A and the development of tubulointerstitial injury has not widely been investigated yet and the effect of activin A on tubulointerstitial fibrosis in DN is still waiting for elucidation.ObjectiveThe purpose of this study is to investigated the expression of activin A /Smad signal on tubulointerstitium of streptozotocin（STZ）-induced diabetic rats and high glucose-cultured HK-2 ceils and its relation withα-SMA and fibronectin.Methods1.Animal experimentMale Wistar rats were randomized into a normal control group（NC） and diabetes mellitus group（DM）.After fasting for 12 h,DM rats received an i.p.injection of STZ（60 mg/kg）,and NC rats received a corresponding amount of citrate buffer.Blood samples were collected from the tail vein 72 h after administration to determine random blood glucose（BG）.The rats with random BG more than 16.7 mmol/L were regarded as DM rats.The rats were given free access to tap water and standard chow.Six rats were respectively sacrificed 4,8,12 and 16 weeks after model establishment in each group.One day before execution,rats were weighed,and then put in individual metabolic cages for the exact collection of 24h urine samples.Blood samples were withdrawn from the abdominal aorta and the left kidneys were removed and dissected into two parts.One part was fixed in 10% paraformaldehyde for pathological and immunohistochemical detection, and another part was reserved in liquid nitrogen for polymerase chain reaction（PCR） assay.Renal hypertrophy index was monitored by left kidney weight/body weight（KW/BW,mg/g）.The urine albumin excretion rate（AER） was measured by radioimmunoassay according to the manufacturer’ s suggestions.BG,Urine and serum creatinine concentration were measured with a multiparametric analyzer（model 7150;HITACHI）.Creatinine clearance rate（Ccr） was calculated as urinary creatinine×urine volume/serum creatinine,and was expressed as mL/min·per kg.The morphology of tubulointerstitium was observed by light microscopy.Immunohistochemistry was applied to analyze the protein expression of activinβA,follistatin,P-Smad2/3,α-SMA and fibronectin and real-time PCR was used to detect the mRNA expression of activinβA,follistatin,α-SMA and fibronectin in renal tissue.2.Cell cultureHK-2 cells,were maintained in Dulbecco’ s modified Eagle’ s medium（DMEM） which was supplemented with 10%fetal bovine serum（FBS）, penicillin（100 U/mL） and streptomycin（100μg/mL） and cultured in an atmosphere of 5%CO₂ and 95%air at 37℃.When 80%confluent,the cells were incubated in a serum-free medium for 24 h and then exposed to the following experimental conditions for different time periods （12,24 and 48 h）:5 mmol/L glucose（normal glucose group,NG）, 5 mmol/L glucose plus 25 mmol/L mannitol（osmotic control,mannitol group,MG）,25 mmol/L glucose（high glucose group,HG）,25 mmol/L glucose plus 100 ng/mL rh-follistatin（100 ng/mL rh-follistatin intervention group,HG + FS100） or 25 mmol/L glucose plus 500 ng/mL rh-follistatin（500 ng/mL rh-follistatin intervention group, HG + FS500）.Then,the cells and cellular supernatant were collected for further detection.ActivinβA and P-Smad2/3 expression were determined by Western blotting analysis.TGF-β,α-SMA and fibronectin released into media were quantitatively analyzed by enzyme-linked immunosorbent assay（ELISA）.Results1.Animal experiment1.1 General biochemical parametersAt each time point,BG in the DM group rats was higher than that in the NC group.In the DM group,KW/BW,AER,and Ccr began to rise from 8 weeks and showed a gradually increasing trend.There were statistically significant differences between DM and NC groups and between 8 and 16 weeks in the DM group（P＜0.01,respectively）.1.2 Pathological changes of tubulointerstitiumThe PAS stain showed that at each time point,the renal tubular epithelial cells in the NC group rats were intact,and there were neither interstitial oedema nor interstitial fibrosis.Vacuolar degeneration in tubular epithelial cells,tubular hypertrophy, dilation and atrophy,inflammatory cell infiltration and renal interstitial fibrosis occurred from 8 weeks and worsened over time in the DM group rats.By semiquantitative analysis,the tubulointerstitial injury index was gradually elevated in the DM group from 8 to 16 weeks.1.3 Immunohistochemistry staining for activinβA,follistatin,P-Smad2/3,α-SMA and fibronectin in tubulointerstitiumThe immunohistochemical results demonstrated that activinβA staining was prominent in tubular epithelial cells in the DM group rats,but no staining was detected in the NC group rats.By semiquantitative analysis,significantly elevated activinβA staining was observed in the DM group rats at 4 weeks and reached peak values at 12 weeks.By contrast,follistatin staining was abundant in tubular epithelial cells in the NC group rats.However,in the DM group rats, its staining began to decrease gradually from 4 weeks to 16 weeks.P-Smad2/3 and fibronectin showed slight staining in tubulointerstitium in the NC group rats.But in the DM group rats,an abruptly increased staining could be detected at 8 weeks and lasted until 16 weeks.In the NC group rats,immunostaining forα-SMA was essentially confined to the media of arteries and arterioles.By contrast,in the DM group rats,besides the vascular wall,the expression ofα-SMA was detected in the tubular epitheliar cells and interstitium from 8 weeks.1.4 6ene expression of activinβA,follistatin,α-SMA and fibronectin in renal tissueCompared with NC group rats,ActivinβA mRNA was markedly elevated to 3.3-fold higher at 4 weeks and 7.7-fold at 12 weeks,and then decreased slightly at 16 weeks in the DM group rats.On the contrary, follistatin mRNA was decreased 32.5%at 4 weeks and 52.9%at 12 weeks in the DM group rats.No significant change inα-SMA and fibronectin mRNA was detected at 4 weeks in the DM group rats,but a gradually increase was found at 8 weeks and lasted until the end of the study.2.Cell culture2.1 ActivinβA and P-Smad2/3 expression in HK-2 cells and the intervention of follistatinWestern blotting results demonstrated that ActivinβA exhibited very slight activation in the NG group,but its production was enhanced in the HG group after 12 h culture and increased gradually until study completion.Follistatin blocked its production in a dosedependent fashion.Treatment with 500 ng/mh rh-follistatin reduced activinβA production to NG group levels after 48 h culture（P＞0.05）. P-Smad2/3 was significantly increased after 24 h culture in the HG group compared with the NG group and follistatin treatment decreased its overexpression in the HG group.The fall averaged 26%in the HG + FSIO0 group and 41%in the HG + FS500 group after 48 h culture. There were no statistical differences in the expression of activinβA and P-Smad2/3 between the MG and NG groups（p＞0.05）.2.2 TGF-β,α-SMA and fibronectin protein in cellular supernatant and the intervention of follistatinResults of ELISA disclosed that TGF-βwas markedly higher in the HG group than that in the NG group throughout the 48 h experimental periods and both doses of follistatin failed to modify the overexpression of TGF-βin HG.Compared with the NG group,α-SMA and fibronectin in the HG group were obviously upregulated after 24h culture（p＜0.01）.Follistatin reduced their overexpression in a dosedependent manner（p＜0.01）.There were no statistical differences in the expression of TGFβ,α-SMA and fibronectin between the MG and NG groups（p＞0.05）.Conclusion1.Activin A/Smad pathway activation,transdifferentiation and fibronectin production are involved in renal tubulointerstitial injuries of STZ-induced diabetic rats.2.High glucose can activate Activin A/Smad pathway,induce tubular epithelial cells-myofibroblast transdiffrentiation and promote fibronectin production in HK-2 cells.3.Inhibition of high glucose stimulated transdifferentiation and fibronectin production in HK-2 cells by follistatin is associated with blocking of Activin A/Smad pathway.更多还原