【作者】 聂静；

【导师】 刘伏友；

【作者基本信息】 中南大学 ， 内科学， 2009， 博士

【摘要】 研究背景糖尿病肾病(Diabetic nephropathy, DN)是终末期肾脏疾病(end stage renal disease, ESRD)的最常见原因之一。约30%-40%Ⅰ型糖尿病,15%Ⅱ型糖尿病最终将进展为ESRD。大量临床研究表明,未控制的高血糖是糖尿病肾病发生发展的关键因素,但其作用机制仍不清楚。有研究表明,活性氧簇(reactive oxygen species, ROS)在高血糖诱导的细胞损伤中起重要作用。高血糖造成肾脏ROS生成增加,ROS可活化信号转导级联效应(如PKC、MAPK、JAK/STAT等),又可作为胞内信号激活某些转录因子(如NFκB、内皮素-1等),导致TGF-p1等表达增加。上述信号分子进一步诱导ROS产生,不断放大高糖造成的细胞损伤,导致细胞外基质(extracellular matrix, ECM)合成增加、基底膜增厚、系膜扩张,最终导致进行性的肾小球硬化和肾小管萎缩,引起肾功能下降和衰竭。尽管高糖造成细胞ROS产生增加已得到公认,但ROS产生的具体机制至今仍未阐明。Src同源区2结构域蛋白C(SHC)家族成员ShcA基因编码3种蛋白,分子量分别为46,52和66kDa(p46、p52和p66)。p66Shc是哺乳动物生命周期相关的蛋白,也是近年来研究细胞氧化应激的主要蛋白之一,敲除p66Shc基因可以使小鼠的寿命延长30%。大量研究表明,p66Shc在衰老及其相关的疾病中起关键作用,其共同机制为p66Shc的缺失加强了机体对氧化应激的抵抗。研究表明,p66Shc主要定位于胞浆,小部分(10%-40%)与线粒体热休克蛋白70(mHSP70)及线粒体内、外膜转移酶(TIM/TOM)形成复合物,定位于线粒体膜间隙。在正常情况下,p66Shc是失活的,不影响线粒体的功能,线粒体内的p66Shc只有在某些信号的作用下(p53/H2O2/UV等)才能解聚形成有活性的单体。另外,这些信号又可活化PKCβ,使胞浆内的p66Shc磷酸化,磷酸化的p66Shc可被脯氨酰异构酶(prolyl isomerase 1, pin1)识别从而异构化,进而在蛋白磷酸酶2A (Protein Phosphatase 2A, PP2A)的作用下去磷酸化进入线粒体,在线粒体内氧化细胞色素C产生ROS,开放线粒体通透性转换孔(mitochondrial permeability transition pore, mPTP),增加膜通透性,使大量离子、溶质和水涌入线粒体,导致线粒体水肿及形态/功能改变(包括降低钙离子反应和线粒体3D结构的改变)；同时,促凋亡因子如细胞色素C(cytochrome C)通过开放的mPTP释放到胞浆,激活caspases,导致细胞凋亡。研究显示p66Shc与糖尿病产生的氧化应激及其造成的血管损伤密切相关。有学者发现Ⅱ型糖尿病病人外周血单核细胞p66Shc表达较正常人明显升高,其水平与血浆8-Isoprostane(一个氧化应激标记物)水平呈正相关。Menini等的体内实验也发现,在小鼠体内敲除p66Shc基因,可以减少STZ诱导的Ⅰ型糖尿病肾病肾组织产生的氧化应激,保护肾脏组织结构和功能,改善肾组织病理变化,降低蛋白尿,抑制NFκB的活性,降低AGE的形成。上述研究均提示p66Shc与糖尿病及糖尿病肾病进展密切相关。但具体的机制尚不清楚。基于以上分析,我们认为p66Shc可能与糖尿病肾病细胞凋亡及ROS产生有关,为此,本实验开展如下的研究。目的利用STZ诱导的糖尿病大鼠模型,观察肾组织中p66Shc的表达变化,并探讨p66Shc与肾组织氧化损伤的关系。方法本实验分糖尿病组(STZ组)和正常对照组(Con组),每组10只雄性SD大鼠,体重190-220克。采用一次性腹腔注射STZ60mg/kg的方法建立Sprague-Dawley (SD)大鼠糖尿病模型(对照组注射等量PBS)。实验前检查随机血糖>16.7mmol/L,入选为糖尿病组。第8周处死大鼠,取肾组织。采用HE、Masson染色观察肾脏病理改变；DHE染色检测组织活性氧簇(ROS)；免疫组化、Western blot方法检测肾脏p66Shc表达变化,并分析p66Shc表达与肾组织氧化损伤的关系。结果SD大鼠腹腔注射STZ后72小时,大鼠随机血糖>16.7mmol/L,4周后测尿蛋白在30mg/24h以上,表明DN模型制备成功。糖尿病组大鼠血糖升高,出现明显多饮、多食、多尿等症状,与对照组比较,体重明显减轻；同时,肾脏体积明显增大,肾重明显增加。与对照组比较,STZ组大鼠肾组织肾小球体积增大,部分小球可见细胞数目增多,肾小球系膜基质增多；近端小管上皮细胞刷状缘减少或消失,部分肾小管萎缩和脱落、肾小管上皮细胞空泡样变性。对照组大鼠肾组织仅部分细胞胞核内表达极微弱的DHE红色荧光,STZ组大鼠肾组织大量细胞胞核内可见明显的红色荧光,较对照组显著增强,提示糖尿病肾病肾组织ROS产生增加。免疫组化结果显示：p66Shc在对照组大鼠肾组织有少量表达；糖尿病组大鼠的肾组织特别是肾小管中p66Shc表达显著增强。Western blot结果显示：糖尿病组大鼠肾组织p66Shc蛋白的表达显著升高,与对照组比较有显著差异。结论糖尿病状态下,大鼠肾脏特别是肾小管p66Shc表达显著增加；p66Shc表达增加的同时伴随肾组织ROS的大量产生,提示p66Shc可能在糖尿病肾病ROS的产生过程中起重要作用。目的探讨高葡萄糖对肾小管上皮细胞p66Shc蛋白表达及其第36位丝氨酸磷酸化的影响。方法用不同浓度D-葡萄糖(5.5mmM,15mM,30mM,45mM)处理正常人近端肾小管上皮细胞株(D-甘露醇作为等渗对照组),在不同时间点提取细胞RNA和蛋白,采用realtime-PCR检测p66ShcmRNA的表达变化,Westem blot检测p66Shc蛋白、磷酸化p66ShcSer36蛋白的表达。结果HK-2细胞经不同浓度D-葡萄糖处理后,p66Shc蛋白表达逐渐增高,呈浓度依赖模式；其中45mM葡萄糖处理后,p66Shc蛋白表达最强；等渗对照组p66Shc表达与正常对照组比较无显著性差异。HK-2细胞经30mM葡萄糖处理后,p66Shc mRNA表达逐渐增高,呈时间依赖模式,p66Shc mRNA在24h表达最强；p66Shc蛋白表达的结果与mRNA的结果相一致,在48h表达最强。HK-2细胞经30mM葡萄糖处理后,15min磷酸化p66Shc Ser36的表达即明显增加,90min达最高峰,之后逐渐降低,但在180min仍高于0min组。结论人近端肾小管细胞株HK-2表达p66Shc；高葡萄糖呈时间和浓度依赖模式上调HK-2 p66Shc的表达；高葡萄糖促进p66Shc Ser36磷酸化。目的探讨衔接蛋白p66Shc对高糖诱导的肾小管上皮细胞氧化损伤及凋亡的影响及其作用机制。方法常规培养正常人近端肾小管上皮细胞株(HK-2),分别用质粒pcDNA3.1 his p66Shc和pcDNA3.1 his p66Shc S36A(第36位丝氨酸突变为丙氨酸)转染HK-2细胞,建立稳定表达的细胞株。随后将细胞分为4组：1)未转染HK-2+5.5mM葡萄糖(正常对照组)；2)未转染HK-2+30mM葡萄糖(高糖组)；3)转染p66Shc WT的HK-2+30mM葡萄糖(p66Shc组)；4)转染p66Shc S36A的HK-2+30mM葡萄糖(S36A组)。采用Annexin V和Hoechst33258染色检测细胞凋亡；MitoSox Red染色检测细胞线粒体ROS含量；TMRE染色检测线粒体膜电位；Westem blot检测procaspase9、线粒体cytochromec及线粒体p66Shc在HK-2细胞的表达；提取线粒体DNA, PCR检测线粒体DNA的损伤；LDH检测试剂盒检测细胞上清液LDH的水平；TBA法检测细胞上清液MDA的含量。结果1.p66Shc细胞株鉴定：转染p66Shc WT和p66Shc S36A质粒的HK-2细胞p66Shc的表达较未转染的HK-2细胞显著增高。2.在高葡萄糖作用下,p66Shc在线粒体内聚集；p66Shc过表达增加高糖导致的细胞凋亡和线粒体ROS的产生；加重高糖导致的细胞线粒体膜电位下降,进一步激活线粒体凋亡通路激活：促进线粒体cyto c的释放和caspase 9的活化；p66Shc过表达促进高糖导致的细胞氧化损伤和线粒体DNA损伤。转染突变质粒p66Shc S36A对高糖导致的细胞损伤和凋亡无明显促进作用。结论p66Shc过表达加重高糖诱导的HK-2氧化损伤和细胞凋亡；p66Shc通过线粒体参与高糖诱导的肾小管上皮细胞氧化损伤和凋亡,可能在糖尿病肾病早期肾小管萎缩和细胞凋亡过程中发挥重要作用。更多还原

【Abstract】 Background Diabetic nephropathy (DN) is one of the leading causes to end stage renal disease(ESRD). Approximately 30-40% of patients with type I and 15% with type II DM develop renal dysfunction. In both instances, poor glycemic control contributes to the development of diabetic nephropathy, but the mechanisms underlying hyperglycemia induced injury are not fully understood. Intracellular ROS is increased in kidney in diabetes and the overproduction of ROS is a direct consequence of hyperglycemia. ROS mediates hyperglycemia-induced activation of signal transduction cascades (PKC, mitogen-activated protein kinases, and janus kinase/signal transducers and activators of transcription) and transcription factors (NF-κB, activated protein-1, and specificity protein-1) leading to upregulation of TGF-(31 and ECM accumulation, and finally result in cellular injury in kidney. However, the mechanism of ROS generation in DN is still unknown.The ShcA gene encodes three isoforms:p46Shc, p52Shc and p66Shc and the p66Shc controls cellular oxidative stress responses and life span. Recently, studies showed that the mice lacking p66Shc reduced intracell-ular ROS and live 30% longer than control animals, suggesting that dele-tion of p66Shc increased oxidative resistance and prolonged a life span. It has been demonstrated that p66Shc is localized partly in the cytoplasm, while almost 10% to 40% within the mitochondrial intermembrane space. In the mitochondria, p66Shc binds to an inhibitory complex which inclu-des members of the Hsp70 and TIM-TOM import. system. Furthermore, it has been shown that the p66Shc protein might be a mediator traducing oxidative stress to injury and apoptosis in MEFs. It is inactive in normal condition, and activated by oxidative stress mediated by the phosphoryl-ation of Ser36. As recently reported, oxidative stress activates PKC(3, induces phosphorylation of p66Shc in the cytoplasm and triggers mitoc-hondria accumulation of the protein after it is recognized by Pinl; On the other hand, oxidative stress also induces the dissociation of the inhibitory complex and the release of monomeric p66Shc. Once it activated, p66Shc generates ROS through oxidation of cytochrome c, which then induces opening of the mitochondrial permeability transition pore (mPTP), with subsequent increase of mitochondrial membrane permeability to ions, solutes and water, swelling and disruption of the organelle, release of cytochrome c into the cytosol, and consequent apoptosis.Recently, p66Shc has been involved in diabetes mellitus and its complications. Pagnin et al found that p66Shc mRNA expression was increased in human peripheral blood mononuclear cells in type II diabetic patients compared with healthy subjects, and it is positively correlated with total plasma 8-isoprostanes, a validated marker of oxidative stress. It is also reported by Menini that Streptozotocin-induced diabetic p66Shc knockout (KO) mice showed less marked changes in renal function and structure, as indicated by the significantly lower levels of proteinuria, albuminuria, glomerular sclerosis index, and glomerular and mesangial areas.Serum and renal tissue advanced glycation end products and activa-tion of NF-κB were also lower in diabetic KO than in wild-type mice. These data suggest that p66Shc might be involved in the pathogenesis of diabetic nephropathy. However, the mechanism is unknown. Therefore, we think that p66Shc may play a key role of ROS generation and cell apoptosis in diabetic nephropathy. To this end, experimental research is carried out as follow. Objective To investigate the expression of both adaptor protein p66Shc and reactive oxygen species (ROS) in the kidneys in STZ-induced diabetic nephropathy.Methods Twenty SD male rats, weight ranged from 190g to 220g, were random divided into 2 groups(n=10 each group):Normal control group(Con group) and diabetes model group(STZ group). Diabetes was induced by the injection of STZ (The control group only with injection of PBS). Rats with blood glucose levels> 250mg/dl were selected for studies. Rats were killed 8 weeks after STZ injection. Kidney tissues were harvested for HE and MASSON test and DHE staining. The expression of p66Shc was examined by immunohistochemically staining and Western blot, respectively.Results Compared with control, the decrease of body weight, increase of kidney weight and urine protein were seen in STZ group; Pathologic changes of renal tubule were observed by HE and MASSON staining. Severe oxidative stress was seen by DHE staining in kidneys of STZ rats. The expression of p66Shc significantly increased in STZ rats in comparison to that of control group.Conclusion The expression of p66Shc was significantly up-regulated in rat kidneys with diabetes which associated with severe oxidative stress. Objective To detect the expression of p66Shc and the phosphorylation of p66shc-Ser36 induced by high glucose in the human renal proximal epithilial cell line(HK-2).Methods The cells were exposed respectively in different concentrations of D-glucose(5.5mM D-glucose as normal control and D-mannitol as the isoosmotic control). The expression of p66Shc mRNA was examined by Real-time PCR. Expressions of p66Shc and phospho-Ser36 protein was detected by Western blot.Results A single band with a predicted molecular weight (66kD) was seen in HK-2 cell by Western blot, which represents p66shc expression. Stimulation of HK-2 with 30mM D-glucose resulted in a significant increase in the expression of p66Shc protein, compared with medium control group. However, there was no significant difference between the control and D-mannitol group. HK-2 treated with 30mM D-glucose resulted in a time-dependent increase in the expression of p66shc mRNA, with the peak at 24h. In additon, an increase of p66Shc protein in HK-2 treated by 30mM D-glucose was also seen, compared to that of control group. Interestingly, after treatment with 30mM D-glucose, the level of phospho p66Shc-Ser36 protein began to increase at 15min, and reached the peak at 90min in HK-2.Conclusion It was demonstrated by first time that expression of p66Shc was seen and high glucose up-regulated p66shc expression in a time and dosage dependent manner in HK-2. Moreover, high glucose induces phosphorylation of p66shc-Ser36 in HK-2. Objective To study the role of adaptor protein p66Shc in high glucose induced oxidative injury and apoptosis in the human renal proximal epithilial cell line(HK-2).Methods Wild-Type p66Shc plasmid (p66Shc WT) and the p66Shc mutant plasmid(Ser36 was mutated to Ala, called p66Shc S36A) were transfected into HK-2 cells using lipofectamine 2000, respectively. After selection of stable transfectants, cells were maintained in the defined medium.30mM D-glucose was added (5.5mM D-glucose as control). Cell apoptosis was measured by AnnexinⅤstaining using flow cytometry and Hoechst 33258 staining using fluorescence microscopy. The mitochondrial ROS was detected by Mitosox staining using confocal microscopy. Mitochondrial membrane potential was detected by TMRE, then measured by confocal microscopy. The mtDNA damage was evaluated by amplifying of mitochondrial long and short DNA. The expression of cytochrome c and p66Shc in mitochondria and caspase9 were by Western blot. Cell lactate dehydrogenase (LDH) activity was examined by Colorimetric assay and Malondialdehyde (MDA) was examined by TBA assay. Results1. p66Shc protein is overexpressed with p66Shc constructs(p66Shc WT/S36A) transfection in HK-2 cells2. Overexpression of p66Shc in HK-2, which then treated with 30mM D-glucose induced an increase of mitochondrial ROS, LDH activity, MDA level and apoptotic rates, in comparison to HG group. A decrease of mitochondrial membrane potential, release of cytochrome c, accumulation of p66Shc in mitochondrial, activation of caspase9 and damage of mitochondrial DNA was also observed in p66Shc overexpression group, compared with HG group. While the effect was significantly abolished in p66Shc mutant plasmid (p66Shc S36A) transfected HK-2 cells, compared with p66Shc transfected HK-2 cells.Conclusion Overexpression of p66Shc intensified the effects induced by 30mM D-glucose in HK-2, p66Shc mediates oxidative injury and cell apoptosis induced by high glucose via mitochondria dependent pathway in HK-2. p66Shc may play a key role in tubular atrophy and cell apoptosis during early stage of diabetic nephropathy.更多还原