【作者】 白寿军；

【导师】 刘晓城；

【作者基本信息】 华中科技大学 ， 肾脏病学， 2011， 博士

【摘要】 慢性肾脏病(chronic kidney disease, CKD)不可逆的最终结局是终末期肾功能衰竭(end stage renal disease, ESRD),各种进行性慢性肾脏病发展为终末期肾功能衰竭的共同的病理特征是肾小管间质纤维化(tubular interstitial fibrosis,TIF),其对慢性肾脏病的预防和治疗与肾小球硬化相比更为重要。在肾脏疾病进展的过程中,肾间质纤维化程度与肾功能的减退密切相关。其中,肾小管上皮细胞-间充质细胞转分化(Epithelial to mesenchymal transition, EMT)是肾小管间质纤维化过程中的关键事件。EMT的完成需要数天时间,经过一系列复杂的过程,主要包括以下四个经典的步骤：早期细胞间粘附功能的丧失,a-SMA开始表达和肌动蛋白重组,肾小管基底膜的破坏和细胞转移和侵袭力增加。TGF-β1是参与EMT发生、发展的重要因子,TGF-β1可通过诱导EMT参与肾间质纤维化的进展,研究发现其信号传导需要多种介质的参与,包括Smads和非Smads途径,后者主要有PI3K/Akt、MAPK、ERK、p38MAPK、JNK、MEK、PKA、PKC和RhoGTPase等,而RhoGTPase分子机制研究较少。Cdc42相关蛋白4(Cdc42 interacting protein-4,CIP4)是属于RhoGTPase蛋白,含Bin/amphiphysin/Rvs (BAR)结构域蛋白超家族,主要调节肌动蛋白的聚合和细胞膜的动力学、稳定张力纤维丝,调节细胞内胞吞和胞吐等胞内、外物质的转运,故在细胞骨架的构建、细胞黏附、细胞内运输、信号转导、细胞周期等多种生物学方面发挥重要作用。过量表达的CIP4会引起细胞黏附功能的丧失、细胞转化、癌细胞的浸袭和转移。由于纤维化疾病与肿瘤一样都存在细胞转分化过程,因此我们推测在肾间质纤维化过程中CIP4高表达可能促进了肾小管EMT,进而加速了慢性纤维化的发展。本课题探讨了CIP4在TGF-β1诱导的肾小管EMT中的作用及其机制：通过构建5/6肾大部切除和TGF-β1诱导的肾小管上皮细胞-间充质转分化模型,拟构建高表达CIP4质粒稳定转染至肾肾小管上皮细胞,设计CIP4-siRNA,敲除CIP4基因,研究CIP4在慢性肾脏病肾小管间质纤维化中的表达及作用,以及CIP4与极性蛋白Par6的关系。第一部分Cdc42相关蛋白4(CIP4)与肾间质纤维化之间的关系目的观察5/6肾大部切除大鼠肾脏中和TGF-β1诱导的人肾小管上皮细胞(HK-2)Cdc42相关蛋白4(CIP4)的表达,并探讨内源性CIP4表达与肾脏间质纤维化的关系,为研究CIP4在肾小管上皮细胞-间充质转分化(EMT)过程中的作用提供基础。方法选择雄性SD大鼠20只(含正常组10只),采用5/6肾大部切除建立慢性肾脏病肾间质纤维化模型,按常规把SD大鼠饲养12周后,用自动生化仪检测大鼠血清尿素氮(BUN)、肌酐(Scr),Masson染色检测各组SD大鼠肾小球硬化和小管间质纤维化的程度,应用免疫组化SABC法检测CIP4在各组大鼠肾脏组织的分布变化,蛋白质免疫印迹法测定CIP4蛋白在肾脏组织中表达水平。用RT-PCR和Western blot法检测TGF-β1刺激的HK-2细胞12-72h后,各组细胞内CIP4的mRNA和蛋白表达的变化。结果CIP4蛋白在假手术组表达较少,仅分布于肾小管,而5/6肾大部切除组CIP4表达明显增多,几乎所有小管细胞均有表达。RT-PCR和Westernblot示,肾衰组CIP4蛋白的表达较假手术组明显增加(P<0.05)。体外实验CIP4在TGF-β1刺激的HK-2细胞内表达上调,24h时升高有显著意义(P<0.05),一直到72h仍在升高。结论通过体内和体外实验研究发现：CIP4在肾间质纤维化中表达增高,TGF-β1可能是其增高的原因。第二部分Cdc42相关蛋白4对TGF-β1诱导的肾小管上皮细胞-间充质转分化的影响目的构建CIP4稳定转染的HK-2细胞系,观察CIP4对HK-2细胞上皮细胞-间充质转分化(EMT)的影响,设计一条特异性沉默CIP4表达的RNA片段(siRNA-CIP4),将其转染TGF-β1刺激的HK-2细胞,观察CIP4对TGF-β1刺激的HK-2细胞上皮细胞-间充质转分化(EMT)的影响,并探讨PI3K/Akt信号通路在CIP4产生中的作用。方法根据GenBank里人CIP4 cDNA序列,设计一对含Not I酶切位点的特异引物,从人肾脏组织中通过RT-PCR技术得到的全长CIP4-cDNA,利用Not I酶切位点将全长CIP4-cDNA克隆到pcDNA3.1载体上,经过限制性酶切、生物公司测序、鉴定正确后,将pcDNA3.1/CIP4稳定转染至HK-2细胞。蛋白质免疫印迹法检测转染细胞的CIP4、E-cadherin和a-SMA的蛋白表达变化,共聚焦显微镜检测各组细胞内E-cadherin和a-SMA蛋白分布的改变。根据GenBank里人CIP4 cDNA序列,设计一条CIP4-siRNA片段,特异性沉默CIP4基因,然后将其用Lipofactamine2000转染TGF-β1刺激的HK-2细胞,Western blot检测各组细胞内CIP4、E-cadherin和a-SMA蛋白的表达变化,共聚焦显微镜检测E-cadherin和a-SMA蛋白的分布,通过P13K/Akt特异性抑制剂’Wortmannin 1μM干预TGF-β1刺激的HK-2细胞48h, Western blot检测各组细胞内CIP4表达的变化。结果肾小管上皮细胞经CIP4高表达的质粒转染后,上皮细胞形态明显发生改变,长梭形细胞替代了类圆形的肾小管上皮细胞,其细胞标志物E-cadherin蛋白表达减少,肌成纤维细胞标志a-SMA的表达增多(P<0.05), CIP4-siRNA转染TGF-β1刺激的HK-2细胞,上述相同的细胞形态改变出现逆转,细胞逐渐恢复成类圆形正常小管细胞,与TGF-β1刺激组相比,E-cadherin蛋白表达明显增多,a-SMA蛋白表达显著减少(P<0.05)。CIP4基因敲除逆转了TGF-β1诱导的肾小管EMT。以上结果证实CIP4能够促进肾小管上皮细胞—间充质细胞转分化。Wortmannin抑制TGF-β1刺激的HK-2细胞48小时后,CIP4蛋白表达显著减少。结论TGF-β1通过PI3K/Akt途径产生大量的CIP4,CIP4进一步参与TGF-β1诱导的肾小管上皮细胞EMT。第三部分Cdc42相关蛋白4对TGF-β1诱导的大鼠肾小管上皮细胞-间充质转分化的影响及其与极性蛋白Par6关系目的观察CIP4对TGF-β1诱导的大鼠肾小管上皮细胞-间充质转分化(EMT)的影响,并探讨极性蛋白Par6在TGF-β1诱导的肾小管EMT中的作用。方法TGF-β1 20μg/L作用72h诱导大鼠肾小管上皮细胞(NRK-52E)转分化,用Western blot法检测细胞内CIP4、E-cadherin和α-SMA的蛋白表达及Par6蛋白及其磷酸化水平,免疫共沉淀检测CIP4与Par6蛋白的结合状况,免疫荧光共聚焦检测Par6、E-cadherin和α-SMA的分布。根据Genbank里大鼠Par6的完全cDNA序列,设计一条Par6-siRNA片段,特异性沉默Par6的表达,通过Lipofactamine2000将Par6-siRNA转染至TGF-β1刺激的NRK-52E细胞,检测正常细胞、TGF-β1刺激细胞、par6-siRNA转染的TGF-β1刺激细胞内CIP4、E-cadherin和α-SMA的蛋白改变；同时设计根据Genbank里大鼠CIP4的完全cDNA序列,设计CIP4-siRNA,利用Lipofactamine2000(?)将其转染至TGF-β1刺激的NRK-52E细胞,检测正常细胞、TGF-β1刺激细胞、CIP4-siRNA转染的TGF-β1刺激细胞内Par6、P-Par6、E-cadherin和α-SMA的蛋白改变；免疫荧光共聚焦检测E-cadherin和α-SMA的分布。结果TGF-β1刺激72h后NRK-52E细胞上皮细胞标志E-cadherin(?)减少,并出现肌成纤维细胞标志α-SMA,提示TGF-β1 20μg/L能够促进肾小管上皮细胞向间充质转分化。随着NRK-52E发生EMT, CIP4蛋白表达增高,CIP4与Par6结合增强,Par6磷酸化水平显著增强。Par6-siRNA、CIP4-siRNA分别转染TGF-β1刺激细胞,磷酸化Par6均减少,细胞内E-cadherin蛋白表达增加,恢复到NRK-52E细胞膜和细胞连接处,α-SMA相应减少,Par6-siRNA和CIP4-siRNA转染逆转了TGF-β1诱导的NRK-52E细胞EMT。结论Cdc42相关蛋白4(CIP4)在TGF-β1诱导的肾小管EMT细胞内表达增高,通过极性蛋白Par6,促进了TGF-β1诱导大鼠肾小管上皮细胞-间充质细胞转分化。更多还原

【Abstract】 Tubular interstitial fibrosis（TIF） is a common pathological characteristics and an inevitable outcome of all kinds of chronic kidney disease （CKD） progress to end-stage renal failure. Tubular epithelial-mesenchymal transition （TEMT） contributes significantly to the onset and pathogenesis of renal fibrosis. This phenotype has been shown to a complicated multistep process instead of simply a morphological transition, which consists of four key steps:loss of epithelial cell adhesion, de novo a-SMA expression and actin reorganization, disruption of tubular basement membrane, and enhanced cell migration and invasion, transforming growth factor-β1（TGF-β1）, as a sole one, initiates and completes the entire course of EMT. TGF-β1 mediates the EMT process via intracellular pathways, including the canonical Smad pathway, phosphatidylinositol-3-kinase/Akt （PI3K/AKT） pathway, mitogen-activted protein kinase （MAPK） pathway, integrinlinked kinases（ERK） pathway, p38MAPK pathway, c-Jun N-terminal kinases （JNK） pathway and Rho-like GTPase signaling pathway. While the Smad, MAPK and PI3K/AKT pathways have been well described, less is known about Rho GTPase signaling molecular mechanism.Cdc42 interacting protein-4 （CIP4） is a small Rho GTPase, belongs to the Bin/ amphiphysin/Rvs （BAR） domain protein superfamily, which involved in membrane remodeling in various cellular pathways ranging from endocytosis and cytokinesis, T-tubule formation to cell migration and neuromorphogenesis. Thus, CIP4 plays important roles in the many aspects of biological fuctions such as the construction of cytoskeleton, cell adhesion, intracellular transportation, signal transduction and cell cycles. In human renal tumor cells, large amount of CIP4 splicing variant has been produced to promote the (3-catenin tyrosine phosphorylation and lead to renal tumor cells metastasis. The activated CIP4 can also induce cellular transformation and cell-cell interactions, the overexpression of CIP4 in pancreatic tumor cells and breast cancer cells have been demonstrated to be able to promote their migration and invasion. As the migration and invasion of cancer cells is a process analogous to that observed during renal epithelial-mesenchymal transition （EMT）, the data above suggests that CIP4 may play as an important Rho GTPase signaling molecule to promote renal tubular EMT. In present study, we examined whether TGF-β1-induced EMT is mediated by CIP4. For this purpose, we measured CIP4 in the fibrotic renal tissue in a model of 5/6 nephrectomy, this model associated with EMT induced by over-expression of TGF-beta, and in vitro, we detected CIP4 in HK-2 cells treated with TGF-β1, and examined the effect of CIP4-siRNA on TGF-β1-induced EMT. As the polarity protein Par6 was identified as a key regulator in epithelial cell polarity and tight-junction, the interaction between CIP4 and Par6 was subsequently detected.Par I Relationship of CIP4 and Tubulointerstitial fibrosisObjective To study the expression and significance of Cdc42 interacting protein-4 （CIP4） in the model of chronic renal failure of 5/6 nephrectomized rats and the model of EMT of HK-2 cells induced by TGF-(31.Methods 20 SD rats were involved in this experiment cotaining 10 in the normal group. The model of chronic kidney disease was induced by a operation of 5/6 nephrectomy in rat. The expression of CIP4 was detected by Immunohistochemical SABC method at 12 weeks, and Western Blot was used to evaluate the proteins of CIP4. In vitro, CIP4 in HK-2 cells induced by TGF-β1 for 12,24,48,72 h were also detected by Western blot and RT-PCR.Results Immunohistochemical assay showed that rat CIP4 （rCIP4） as extensively expressed in renal tubules in 5/6-nephrectomized rats, whereas there was little staining in sham-operated rats at 12 weeks. The mRNA and proteins of CIP4 was also notably up-regulated in HK-2 cells induced by TGF-β1.Conclusion TGF-β1 could significantly improve the level of CIP4 in renal tubular epithelial cells both in vivo and in vitro.PartⅡCIP4 Regulates TGF-β1-Induced Renal Epithelial to Mesenchymal TransiformationObjective To observe the effect of CIP4 on human renal tubular epithelial to mesenchymal transition induced by TGF-β1 and to study the mechanism of its generation.Methods According to CIP4（human Cdc42 interacting protein-4） cDNA sequence in genebank （NM₀04240）, Reverse transcription polymerase chain reaction （RT-PCR） was used to amplify the full-length CIP4 by utilizing two primers including restriction sites Not I. After purification by RT-PCR, the amplified cDNA was inserted into the pcDNA 3.1 plasmid at the Not I site. To carry on, the recombined plasmids pcDNA 3.1/CIP4 were transfected into HK-2 cells. Western blot or confocal microscope was performed to detect the phenotypic changes correlated to EMT in pcDNA3.1/CIP4 transfected HK-2 cells, as indicated the changes in epithelial and mesenchymal protein E-cadherin and a-SMA respectively. One set of siRNA oligos specific for CIP4 were designed based on the full CIP4 sequence in Genbank, Then HK-2 cells induced by TGF-β1 were transfected with CIP4-siRNA via Lipofactamine 2000.Western blot were used to evaluate the protein expressions of CIP4, E-cadherin andα-SMA respectively in control cells, TGF-β1 treated cells, siRNA transfected cells. The distribution of E-cadherin and a-SMA was observed by confocal microscope. After we interferenced HK-2 cells stimulated with TGF-β1 with specific inhibitor of PI3K/Akt （Wortmannin） 1μM for 48 hours, Western blotting was used to detect the CIP4 protein in control cells and Interferenced cells.Results HK-2 cells acquired phenotypic changes correlated with EMT by transfection of CIP4. The normal HK-2 and pcDNA3.1/Zeo transfected cells were similar in morphology, presenting typical morphology of epithelial cells of round or ovoid shape, and they grew by adhering to the flask wall. As compared with normal HK-2 and pcDNA3.1/Zeo transfected cells, the cells transfected with hCIP4 were altered in morphology, presenting long fusiform and widened inter-cellular space, which was similar to the morphology of myofibroblast. A cell-cell adhesion molecule, E-cadherin present in the plasma membrane of most epithelial cells, was decreased and formed zipper-like patterns at cell borders in CIP4-transfected cells. a-SMA, markers of mesenchymal cells, were increased in CIP4-transfected cells. It show that TGF-β1 can induce EMT, after transfected with CIP4-siRNA, the protein expression of E-cadherin was increased, and a-SMA was decreased, the EMT induced by TGF-β1 was effectively reversed. With treatment of Wortmannin, the expression of CIP4 was decreased.Conclusion TGF-β1 produced a large number of CIP4 via PI3K/Akt pathway in renal tubular EMT and CIP4 further participate in TGF-β1 induced EMT.PartⅢCdc42 Interacting Protein-4 Regulates Transforming Growth Factor-β1-induced Renal Tubular Epithelial-Mesenchymal Transition through the Polarity Protein Par6Objective To observe the effect of CIP4 on the transdifferentiation of rat renal tubular epithelial cells （NRK-52E） induced by TGF-β1, and to search for the influence of the polarity protein Par6 on the TGF-β1-mediated transdifferentiation of NRK-52E cells.Methods In vitro, the rat tubular epithelial cells （NRK-52E） were cultured with 20μg/ L TGF-β1 for 72 h. Western blot or confocal microscope was used to detected the phenotypic changes associated with EMT in TGF-(31-treated NRK-52E cells, as illustrated by alterations in epithelial and mesenchymal protein E-cadherin and a-SMA, also as demonstrated the level of protein CIP4 and Par6 phosphorylation. To investigate whether CIP4 interacts with Par6, immunoprecipitation were used to evaluate the combination CIP4 and Par6. To further demonstrate the CIP4-induced EMT related to the activation of the polarity protein Par6, According to Par6 cDNA sequence （NM₀01003653） and CIP4 cDNA （NM 053920.1） in genebank, a Par6-siRNA and a CIP4-siRNA were devised and synthesised by Shanghai Invitrogen company. The Par6-siRNA and CIP4-siRNA were transfected into TGF-β1-treated cells respectively, and the phenotype changes associated with TGF-β1-induced EMT were then detected by confocal microscope or Western blot.Results TGF-β1 induced phenotypic alternations correlate with EMT in NRK-52E cells. E-cadherin molecule, a cell-cell adhesiontake on in the plasma membrane of most epithelial cells, was decreased and shaped zipper-like patterns at cell borders in TGF-β1-treated cells. a-SMA, markers of mesenchymal cells, were increased in TGF-β1-treated NRK-52E cells. With treatment of Par6-siRNA and CIP4-siRNA respectively, the phosphorylated Par6 was reduced and the losting E-cadherin was re-expressed and re-localized at cell boundary in TGF-β1-treated cells, on the other hand a-SMA were deceased.Conclusion The overexpression of CIP4 is directly involved in TGF-β1-induced tubular EMT through the polarity Par6.更多还原