【作者】 姚芳；

【导师】 段惠军；

【作者基本信息】 河北医科大学 ， 病理学与病理生理学， 2009， 博士

【摘要】 目的:糖尿病肾病(diabetic nephropathy,DN)是糖尿病(diabetes mellitus,DM)最常见而严重的并发症,是终末期肾病的主要原因之一。糖尿病肾病的病变特征为肾小球系膜基质积聚和基底膜增厚。细胞外基质(extracellular matrix,ECM)的成分与含量取决于基质合成与降解两者之间的动态平衡。近年来的研究显示ECM降解能力下降在DN的发生、发展中起着重要的作用。基质金属蛋白酶(metalloproteinases,MMPs)是负责ECM降解的主要酶系,其中MMP-2(又称明胶酶A)主要参与IV型胶原的降解,而后者是DN中肾小球基底膜增厚和系膜基质堆积的主要成分,因此与DN的关系最为密切。关于MMP-2及其组织抑制物(tissue inhibitor of metalloproteinase-2, TIMP-2)在DN中的作用存有较多争议,而且关于MMP-2酶原的特异膜型激活因子—膜型基质金属蛋白酶-1(membrane type 1-matrix metalloproteinase,MT1-MMP)在DN中的表达及作用鲜见研究。结缔组织生长因子(connective tissue growth factor ,CTGF)是转化生长因子(transforming growth factor-β1, TGF-β1)下游重要效应分子,因其较强的致纤维化作用,且生物效应较单一成为DN研究的热点。目前有关CTGF在糖尿病肾病ECM代谢障碍中的作用机制鲜有深入研究报道。肾素-血管紧张素系统(renin-angiotensin system,RAS)的激活,血管紧张素Ⅱ(angiotensin II,AngII)的增加是DN发病机制中的关键环节,抑制AngII的产生并干扰其作用是DN治疗的关键。厄贝沙坦是血管紧张素Ⅱ1型受体拮抗剂(angiotensin II receptor I antagonist, AT1Ra),其肾脏保护作用是否涉及细胞外基质代谢紊乱尚未见报道。TGF-β1诱导ECM代谢失衡在组织纤维化进程中具有关键作用。TGF-β1的各种生物效应是依靠其信号转导网络来实现的,其中最重要的是Smads(Sma and Mad protein)蛋白转导途径。由于Smad2及Smad3具有较高同源性,在以往的研究中很难区分。RNA干扰(RNA interference, RNAi)是近年发展起来的一种新型基因静默技术,该技术具有高度的序列专一性,能简单、高效地阻抑特定基因的表达。本研究以糖尿病大鼠模型和体外培养的大鼠肾小球系膜细胞(glomerular mesangial cell, GMCs)为对象,从整体、细胞和分子水平,系统观察CTGF、MMP-2、TIMP-2和MT1-MMP表达的变化,并通过RNAi分别敲低系膜细胞Smad2和Smad3蛋白表达,以探讨TGF-β1/Smad信号通路在糖尿病性ECM代谢失衡中的作用。通过厄贝沙坦对动物及细胞的干预实验探讨AT1Ra对肾脏保护作用的机制。方法:1.糖尿病动物模型制备及相关指标观察雄性Wistar大鼠42只,戊巴比妥钠腹腔麻醉(40mg/kg)后行右肾切除术。2周后将大鼠随机分为三组:右肾切除对照组(n=18),糖尿病组(DM, n=18)和厄贝沙坦干预组(DM+Irb, n=6)。其中DM组和厄贝沙坦干预组大鼠腹腔注射链脲佐菌素(streptozotocin,STZ,65 mg/kg溶于0.1mol/L枸椽酸缓冲液中,pH值4.5),对照组只注射相同体积的枸橼酸缓冲液,48h后尾尖取血,血糖仪测定血糖,尿糖试纸测定尿糖,血糖≥16.7mmol/L,尿糖+++~++++者确定为糖尿病模型。厄贝沙坦干预组给予厄贝沙坦50mg·kg-1·d-1灌胃,连续给药12周。对照组和DM组只用等量生理盐水灌胃。厄贝沙坦干预组大鼠于给药12周后,对照组和DM组分别于注射STZ后4、8和12周每组取6只大鼠,收集血尿标本,切取肾脏,光镜观察肾脏一般病理改变,免疫组化检测肾组织MMP-2、TIMP-2和CTGF蛋白表达,以Western blot和RT-PCR分别检测肾组织MMP-2、TIMP-2、MT1-MMP和CTGF的蛋白和mRNA的表达。2.大鼠肾小球系膜细胞的培养和相关指标观察用含10%胎牛血清及100KU/L青霉素、100mg/L链霉素的DMEM培养基培养大鼠肾小球系膜细胞。细胞分为低糖组(LG组:5.5 mmol/L葡萄糖),渗透压对照组(LG+M组:5.5 mmol/L葡萄糖+24.5 mmol/L甘露醇),高糖组(HG组:30 mmol/L葡萄糖)和厄贝沙坦组处理组(HG+Irb组)。LG组、LG+M组和HG组分别刺激24h、48h、72h、96h。HG+Irb组设3个浓度,分别为:10-4mol/L,10-5mol/L,10-6mol/L,刺激时间为48h;然后选择浓度10-5mol/L,刺激24h、48h、72h、96h。于实验终点用免疫细胞化学、免疫荧光细胞化学、Western blot和RT-PCR检测MMP-2、TIMP-2、MT1-MMP和CTGF蛋白和mRNA的表达,并用ELISA法检测细胞上清中IV型胶原的含量。3. RNA干扰及相关指标观察针对大鼠Smad2和Smad3编码序列分别设计3条体外转录的小干扰RNA(small interference RNA, siRNA)。优化转染条件后将siRNA分别转染入系膜细胞,同时设正常和阴性对照,于转染结束后48小时收集细胞,提取蛋白,分别检测Smad2、Smad3和Smad4的蛋白表达量,确定敲低效果最佳的siRNA,并确认Smad2与Smad3的siRNA对相互间靶基因无交叉抑制,同时对Smad4也无抑制。随后将对数生长期细胞接种于6孔培养板中,24h后待细胞融合50%-60%开始转染。将细胞分为空白细胞对照孔、TGF-β1刺激孔、阴性对照+TGF-β1孔(转染阴性对照siRNA)、Smad21+TGF-β1孔(转染Smad21 siRNA)、Smad33+TGF-β1孔(转染Smad33 siRNA)、Smad21+Smad33+TGF-β1孔(转染Smad21和Smad33 siRNA)。转染后除空白对照组外,其余各组均经2ng/ml TGF-β1刺激48h后收集细胞提取蛋白和RNA,用Western blot和RT-PCR分别检测各组MMP-2、TIMP-2、MT1-MMP和CTGF的蛋白和mRNA表达。结果:1. MMP-2、TIMP-2、MT1-MMP和CTGF在糖尿病大鼠肾脏的表达①光镜下观察,糖尿病组大鼠从4周开始肾小球体积增大,8、12周系膜细胞增生,基质轻度增多,肾小管上皮细胞出现空泡变性,小灶状萎缩,间质水肿、炎症细胞浸润及间质纤维沉积。厄贝沙坦干预组大鼠肾组织病变较糖尿病组减轻。②免疫组化结果显示:与对照组相比,糖尿病组从4周开始MMP-2在肾小球的表达明显减少,而在肾小管则未有明显变化, 8周和12周糖尿病组肾小球和肾小管MMP-2表达均持续减少。TIMP-2和CTGF在对照组的肾小球及间质中仅有少量的表达,而糖尿病组从4周至12周二者表达逐渐增强。与糖尿病组相比,厄贝沙坦干预组肾组织MMP-2表达升高,而TIMP-2和CTGF的表达则明显降低。③Western blot和半定量RT-PCR结果:与对照组相比,糖尿病组MMP-2和MT1-MMP表达从第4周开始下降,到第8周和12周显著下降,其中酶原型MMP-2(72kD )与活化型的MMP-2(63kD)的变化趋势基本一致;CTGF和TIMP-2则与之相反,从4周开始即表达明显增强,且表达水平随时间持续增高。糖尿病组CTGF的表达量与MMP-2/TIMP -2比值呈负相关(r=-0.7158, P<0.01)。MMP-2 mRNA和MT1-MMP mRNA的表达与各自蛋白的表达趋势一致。CTGF mRNA和TIMP-2 mRNA在正常大鼠肾脏中有低水平表达,而二者在糖尿病组从4周开始表达明显上调,其后直到12周表达持续增高。与糖尿病组相比,厄贝沙坦干预组大鼠肾脏MMP-2和MT1-MMP的表达均有不同程度提升,而TIMP-2和CTGF的表达水平则明显减低。2.高糖刺激及厄贝沙坦干预对大鼠肾小球系膜细胞MMP-2、TIMP-2、MT1-MMP和CTGF的表达的影响①免疫细胞化学检测显示MMP-2、TIMP-2和CTGF均表达于胞浆,在高糖刺激48h后系膜细胞TIMP-2和CTGF的表达明显上调,而MMP-2的表达则明显降低。免疫荧光检测显示MT1-MMP主要表达于肾小球系膜细胞胞膜。高糖组系膜细胞荧光强度较低糖组降低。与高糖组相比,10-5mol/L厄贝沙坦处理组在刺激48h后CTGF、TIMP-2表达减弱,而MMP-2和MT1-MMP的表达则增强。②蛋白印迹与半定量RT-PCR结果:高糖组MMP-2蛋白和mRNA表达在24h时较低糖组略有升高,至48h时则低于低糖组,其后随时间延长表达持续降低;MT1-MMP表达在24小时开始下降并随时间呈下降趋势;高糖组各时间点TIMP-2和CTGF表达上调,且随时间呈增高趋势。厄贝沙坦处理组在24h时MMP-2的表达低于高糖组,而与低糖组之间则无明显差异;从48h~96h厄贝沙坦处理组MMP-2表达均高于高糖组。厄贝沙坦处理组与高糖组相比各时间点CTGF、TIMP-2表达下调,MT1-MMP则表达上调。④细胞上清液检测显示,与低糖组相比,高糖组细胞上清中的IV型胶原于24 h即有增加,且持续增高至96 h;与高糖组相比,各时间点厄贝沙坦处理组IV型胶原含量明显下降(P<0.05, P<0.01)。3. SiRNA转染敲低Smad2和Smad3后系膜细胞ECM代谢相关调控因子的变化①Smad21 siRNA和Smad33 siRNA转染使系膜细胞Smad2和Smad3的表达减少约80%。Smad21 siRNA对Smad3和Smad33 siRNA对Smad2均无敲低效应,且二者对Smad4表达均无抑制。②系膜细胞在TGF-β1刺激48小时后MMP-2 mRNA和蛋白表达减少约60%-80%。Smad2敲低后,TGF-β1刺激对系膜细胞MMP-2的表达无明显影响,而Smad3敲低组和阴性对照组的系膜细胞在TGF-β1刺激后依然表现为MMP-2表达抑制。③在TGF-β1刺激48小时后系膜细胞MT1-MMP mRNA和蛋白表达减少超过60%,Smad2敲低后逆转了TGF-β1的作用,而Smad3敲低组和阴性对照组则未出现类似作用。④在TGF-β1刺激48小时后系膜细胞TIMP-2表达显著增加。与TGF-β1刺激组相比,Smad2敲低组TIMP-2表达减少约60%,Smad3敲低组TIMP-2表达减少约40%。同时敲低Smad2和Smad3则可完全抑制TGF-β1对TIMP-2的诱导作用。阴性对照siRNA对TGF-β1刺激下的系膜细胞TIMP-2的表达无影响。⑤TGF-β1刺激48小时使系膜细胞CTGF表达显著增加(蛋白增加4-5倍,mRNA增加6-7倍)。Smad3敲低可以逆转TGF-β1在系膜细胞诱导CTGF的作用,Smad2敲低和阴性对照siRNA对TGF-β1刺激下的系膜细胞CTGF的表达无影响。结论:①动物和细胞实验结果显示DN中MMP-2表达下调和活性下降伴有MT1-MMP表达减少和TIMP-2表达增多,提示MMP-2调控异常,引致其降解能力下降是导致ECM代谢障碍并发展为肾小球硬化的原因之一。②DN中CTGF表达增加,且与MMP-2/TIMP-2比值呈负相关,提示CTGF在DN中可能具有抑制ECM降解的作用。③厄贝沙坦能够减轻糖尿病大鼠肾脏病变,改善肾功能,同时减少糖尿病大鼠肾组织和系膜细胞CTGF和TIMP-2的表达,增加MMP-2和MT1-MMP的表达与活性。厄贝沙坦的肾脏保护作用可能是通过改善ECM合成/降解的平衡实现的。④TGF-β1调低系膜细胞MMP-2和MT1-MMP的表达水平的作用依赖于Smad2,对于CTGF的诱导则依赖于Smad3,而诱导TIMP-2的作用需要Smad2和Smad3的共同参与。更多还原

【Abstract】 Objectives: As a severe and most frequent complication of diabetes mellitus (DM), diabetic nephropathy is stated to be one of the most common causes of end-stage renal disease. Diabetic nephropathy is characterized by accumulation of extracellular matrix (ECM) and basement membrane thickening in the glomerulus. The components and volumes of ECM are determined by the dynamic balance between matrix synthesis and degradation. It has been gradually received that the reduced degradation of matrix plays an important role in the pathogenesis of diabetic nephropathy. Matrix metalloproteinases (MMPs) are the major physiologic determinants of ECM degradation and turnover in the glomerulus. With view to collagen IV mainly depositing in glomerular mesangium and basement membrane, MMP-2 (gelatinase A), which degradate collagen IV, is hypothetically quite associated with diabetic nephropathy. A number of literature support this hypothesis but with controversy on it. So far, there is little data available on the role of MT1-MMP (activator of pro-MMP-2) in the development of diabetic nephropathy.Connective tissue growth factor (CTGF) is thought to be a critical downstream mediator of TGF-βwith more simple pro-fibrotic effects. The role of CTGF on ECM dysregulation in diabetic nephropathy has not been thoroughly investigated.The renin-angiotensin system (RAS) is a pivotal contributor to the progress of diabetic renal disease. RAS blocking with type 1 receptor blockers (ARB) has been clearly demonstrated to have positive outcomes on diabetic complications. The precise mechanism of renoprotective effect of irbesartan (angiotensin-receptor 1 blocker) has not been fully understood.The imbalance between production and degradation of ECM induced by TGF-β1 is a main event in the progress of tissue fibrosis. Smads are important protein mediators in TGF-β1 signal transduction pathways. Owing to the marked similarity in their structure it was not possible to respectively study the role of Smad2 or Smad3 in TGF-β1 induced cellular responses in the past. RNA interference (RNAi) is an accurate and potent gene-silencing method, which can offer researchers a powerful tool to selectively silence genes.The present studies were performed to examine the expression of MMP-2, TIMP-2, MT1-MMP and CTGF in the renal tissue of STZ-induced diabetic model as well as glomerular mesangial cells (GMCs) cultured by high glucose medium. We also observed the effects of irbesartan on diabetic rats as well as high-glucose cultured GMCs and investigated the different role of Smad2 and Smad3 in TGFβ1-induced fibrotic responses by knocking them down respectively in GMCs.Methods:1. Diabetic model and examination of MMP-2, TIMP-2, MT1-MMP and CTGFUninephrectomized male Wistar rats were randomly divided into three groups: control group (n=18), DM group (n=18) and DM+Irb group (n=6). The rats of DM and DM+Irb group received a single intraperitoneal injection of STZ dissolved in 0.1mol/L sodium citrate buffer (pH 4.5) at a dose of 65mg/kg. The rats of control group only received an injection of the same volume of 0.1mol/L sodium citrate. The model of diabetes was considered to be successful when the blood glucose was≥16.7mmol/L and the glucose in urine was+++～++++after 48 hours of the injection. The rats in the DM+Irb group were treated with irbesartan (average dose of 50mg.kg-1.d-1) for 12 weeks. The rats of control and DM group were treated with the same volume of normal saline. Six rats from control or DM group were respectively sacrificed at week 4, 8, 12 after STZ injection. All rats in DM+Irb group were also sacrificed at week 12. The renal cortex were removed and used for histopathology, immohistochemistry, western blot and reverse transcription and polymerase chain reaction (RT-PCR). 2. Cell culture and examination of MMP-2, TIMP-2, MT1-MMP and CTGFGlomerular mesangial cells were cultured at 37℃in 95% air and 5% CO2 in DMEM media containing 10% fetal calf serum, 100U/ml penicillin and 100μg/ml streptomycin. Confluent mesangial cells were divided into four groups: LG group (media containing 5.5mM glucose), HG group (media containing 30mM glucose), LG+M group (media containing 5.5mM glucose and 24.5mM mannitol), HG+Irb group (media containing 30mM glucose and irbesartan). Cells cultured in the mixture of 5.5 mM glucose and 24.5 mM mannitol were used as an osmotic control. The incubation time extends to 24h, 48h, 72h and 96h. At the end of incubation, the supernatant and cells were collected. The expression of MMP-2, TIMP-2, MT1-MMP and CTGF in the cells were assessed by immunochemistry, immunofluorescence, western blot and RT-PCR. ELISA was used to detect the level of collagen IV in supernatant.3. RNA interference and examination of MMP-2, TIMP-2, MT1-MMP and CTGF in GMCs.In this study RNA interference was used to achieve selective and specific knockdown of Smad2 and Smad3. After optimizing transfection conditions the preliminary experiment was performed to determine the most effective siRNA of Smad2 or Smad3 and make sure that siRNA treatment results in selective and specific knockdown of respective Smad protein. After siRNA transfection GMCs were treated with either vehicle (0.1%BSA) or TGF-β1 (2ng/ml) for 48h under serum-free condition. Cellular MMP-2, TIMP-2, MT1-MMP and CTGF were assessed by Wertern blot and RT-PCR.Results:1. The expression of MMP-2, TIMP-2, MT1-MMP and CTGF in the renal tissue of the diabetic rats①The diabetic rats showed slightly enlarged glomeruli at week 4. Thickened glomerular basement membrane and expanded mesangium were observed with tubular epithelial vacuolar degeneration, minor focal atrophy as well as inflammatory cell infiltration were also observed at week 8 and week 12.②Compared with those of control group immunohistochemical positive staining for MMP-2 decreased notably in glomeruli at week 4 but without obvious changes in tubuli of diabetic kidney. The positive stainings for MMP-2 decreased both in glomeruli and tubuli from 8 weeks to 12 weeks in diabetic rats. The control rats showed weak stainings for TIMP-2 and CTGF either in glomeruli or in interstitium whereas markedly enhancing positive signals were detected in diabetic rats from 4 weeks to 12 weeks.③The Western blot and RT-PCR results for TIMP and CTGF are consistent with the immunohistochemical results. The expression and activity of MMP-2 were decreasing from 4 weeks to 12 weeks in diabetic group versus control group. The expression of CTGF was correlated with MMP-2/TIMP-2 negatively (r=-0.7158, p<0.01).④The morphological and functional impairment of the kidneys induced by STZ was ameliorated in irbesartan group. Pro-MMP-2 and active MMP-2 as well as MT1-MMP were up-regulated in irbesartan group, while on the contrary the expression of TIMP-2 and CTGF was down-regulated in irbesartan group.2. The expression of MMP-2, TIMP-2, MT1-MMP and CTGF in GMCs cultured in high glucose medium①The immunochemical positive stainings for MMP-2, TIMP-2, CTGF appeared in cytoplasm. The immunofluorescence positive stainings for MT1-MMP were mainly anchored at the cell membrane of GMCs.②High-glucose incubation resulted in down-regulation of MT1-MMP and up-regulation of TIMP-2 and CTGF along with the increasing secretion of Col IV. All of those regulations mediated by high-glucose are of time-dependent manners. The regulation of MMP-2 seems a little complicated. Either mRNA or protein of MMP-2 slightly increased after 24h-incubation in high-glucose media, then the expression of MMP-2 decreased up to 96h. The changes of active MMP-2 were consistent with changes of pro-MMP-2.③Immunochemistry and immunofluorescence showed decreased expression of TIMP-2 and CTGF, meanwhile increased expression of MMP-2 and MT1-MMP in irbesartan treatment group versus high-glucose stimulating group.3. The different role of Smad2 and Smad3 in TGFβ1-induced fibrotic responses in GMCs①Smad2 and Smad3 siRNA treatment resulted in approx. 80% reduction in band density of respective Smad protein. The knockdown of respective Smad proteins was selective, as Smad2 protein levels were reduced by Smad21 siRNA, but not by Smad33 siRNA. Similarly, Smad3 protein levels were reduced by Smad33 siRNA and not by Smad21 siRNA. Neither of the transfections had any effect on Smad4 protein levels.②TGF-β1 treatment for 48h resulted in about 60%-80% decrease in MMP-2 expression. The TGF-β1-induced down-regulation of MMP-2 was prevented by Smad2 knockdown, but not by smad3 knockdown. TGF-β1 and Smad siRNA had the similar effects on the expression of MT1-MMP.③TGF-β1 treatment for 48h resulted in significant increasing expression of TIMP-2. Inhibition of either Smad2 or Smad3 resulted in partial reduction of TGF-β1-induced TIMP-2 expression. This induction was abolished by simultaneous knockdown of both Smad2 and Smad3.④TGF-β1 treatment for 48h resulted in 4-5-fold increases in CTGF protein expression as well as 6-7-fold increases in its mRNA expression. This induction was markedly attenuated in the presence of Smad3 knockdown. However Smad2 knockdown had no effect on TGF-β1-induced up-regulation of CTGF.Conclusion:①Studies in vivo as well as in vitro reveal that the decrease of expression and proteolytic activity of MMP-2 are accompanied with the decreased expression of MT1-MMP as well as the increased expression of TIMP-2. Dysregulation of MMP-2 might be one of the causes of ECM accumulation which in turn results in diabetic glomerularsclerosis.②The increased expression of CTGF and its correlation with MMP-2/TIMP-2 suggest that CTGF might inhibit the degradation of ECM in DN.③Renoprotective effects of irbesartan might be partially by recovering the tilting balance between synthesis and degradation of ECM.④TGF-β1-induced decreases in expression of MMP-2 and MT1-MMP were Smad2-dependent, whereas increases of CTGF expression were Smad3-dependent. Increases of TIMP-2 expression were dependent on both Smad2 and Smad3.更多还原