【作者】 朱雪婧；

【导师】 刘伏友；

【作者基本信息】 中南大学 ， 内科学， 2011， 博士

【摘要】 研究背景糖尿病肾病(Diabetic Nephropathy, DN)是糖尿病最常见并发症,也是引起糖尿病病人死亡的最主要原因。近年来,糖尿病肾病(Diabetic nephropathy, DN)发病率呈逐年上升的趋势,从而致使慢性肾脏疾病(Chronic kidney disease, CKD)的发病率和患病率不断攀升,已成为糖尿病患者的主要死亡原因之一。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。因此,明确糖尿病肾病的发病机制,预防或延缓其发生及发展有着重要的科学和社会意义,符合我国国情,并有一定的迫切性。大量研究证实肾小管损伤在糖尿病肾损伤中具有重要的意义,在糖尿病肾病的早期即可观察到肾小管细胞的氧化损伤和凋亡。肾小管上皮细胞的凋亡可导致肾小管再吸收和分泌功能异常,促进肾小管上皮细胞的萎缩和间质纤维化,是糖尿病肾病进展为肾衰竭的主要因素。因此,人们开始重视肾小管上皮细胞氧化损伤和凋亡在糖尿病肾病发病机制中的作用。有研究表明,活性氧簇(reactive oxygen species, ROS)在高血糖诱导的小管细胞损伤中起重要作用。高血糖造成肾脏小管上皮细胞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-β1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(extracellularmatrix, 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。糖尿病过度产生ROS是糖尿病合并症时细胞损伤的首要和原始启动因子。因此,研究高葡萄糖诱导ROS产生的调控机制是国际肾脏研究领域的热点。Rap1 (Ras相关蛋白1)是原癌基因Ras超家族成员之一。Rap1具有广泛的细胞生物学功能,包括细胞生长、分化、肥大、增殖,参与EMT的形成,介导肾小管离子的转运和吸收。已知Ras GTP酶与细胞内ROS产生有关；Ha-Ras-Val12突变可诱导细胞内ROS的产生；C-Raf,一个Rap1b下游蛋白,通过控制线粒体ROS的产生和Ca2+平衡,调节细胞的凋亡[54]；Ras/cAMP信号通路调节线粒体膜电位和线粒体ROS的产生,而ROS对Ras/cAMP信号又有反馈调节作用；特别是发现Epac1,一个Rap1的激活蛋白可转移至线粒体并表达在线粒体基质,而Rap1及其下游蛋白Raf也被发现在细胞线粒体,说明Rap1在线粒体ROS产生过程中可能起重要的调节作用。本实验室既往研究显示,原癌基因Ras超家族成员之一的Rap1b,可以通过逆转肾小管上皮细胞中高糖引起的线粒体ROS的生成增加以及细胞色素C的释放,抑制细胞凋亡。在之前的动物模型研究中我们发现大鼠肾组织表达Rap1b,在糖尿病状态下大鼠肾脏表达ROS明显升高,同时伴有肾小管Rap1b的表达显著增高。但具体机制尚不明确。本研究旨在初步探讨糖尿病肾病患者肾脏Rap1b的表达情况,为进一步研究其在糖尿病肾病发病过程中的作用提供实验依据。基于以上分析,我们认为Rap1b可能与糖尿病肾病小管上皮细胞线粒体氧化损伤及细胞凋亡的产生有关,为此,本实验开展如下的研究。第一章Rap1b在糖尿病肾病患者肾组织中的表达及其与肾问质纤维化的关系目的观察糖尿病肾病患者肾组织中Rap1b的表达,初步探讨Rap1b与糖尿病肾病肾间质纤维化的关系。方法随机选取经临床和肾脏病理确诊为糖尿病肾病及微小病变患者(原发性肾病)各6人。采用Masson、PASM、PAS染色观察肾脏普通病理改变；免疫组化检测肾脏组织Rap1b表达变化。结果肾脏普通病理结果显示肾活检组织呈典型的糖尿病结节性硬化样改变：肾小球系膜基质中-重度增生,多个节段呈结节状硬化,基底膜弥漫性或节段性增厚僵硬,部分节段毛细血管袢扩张,多灶性肾小管萎缩并伴部分小管代偿性扩张,肾间质多灶性纤维化；对照组患者可见系膜细胞及基质仅节段性轻度增生,基底膜不厚,个别肾小管上皮细胞颗粒样变性,肾间质和肾血管未见明显异常。糖尿病肾病患者肾小管间质损伤程度明显高于对照组。免疫组化显示：对照组患者肾小管上皮细胞有明显的Rap1b表达；而糖尿病肾病患者Rap1b表达上调,尤其在肾小管上皮细胞萎缩及发生肾间质纤维化的区域。结论糖尿病肾病患者肾组织,特别是肾小管,Rap1b表达上调；Rap1b表达上调的同时伴有肾小管间质损伤,提示Rap1b可能与糖尿病肾病肾间质纤维化有关。第二章Raplb通过线粒体途径调控高葡萄糖诱导的肾小管上皮细胞氧化损伤和凋亡目的探讨高葡萄糖对肾小管上皮细胞线粒体氧化应激的损伤机制,以及肾小管上皮细胞Raplb蛋白对高糖诱导的线粒体氧化损伤的保护作用。方法用不同浓度D-葡萄糖(5.5mM, 10mM,20mM,30mM)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mRNA和蛋白,通过细胞免疫荧光以及WesteM blot检测高糖对Rap1b蛋白的影响。通过Mito SOX荧光染色、线粒体呼吸链复合物活性检测、线粒体ATP检测、线粒体抗氧化酶活性检测及Real-time PCR,评价高糖对线粒体相关功能的抑制作用。通过细胞免疫荧光以及Westem blot检测高糖对小管上皮细胞的caspase-9、caspase-3的表达以及细胞凋亡的影响。质粒转染cDNA3.1/Rap1b质粒及两个失活性的Rap1b真核表达质粒(Rap1bS17N,第17位丝氨酸突变为天门冬氨酸；Rap1bT61R,第61位苏氨酸突变为精氨酸)使HK2细胞过表达Rap1b,观察Rap1b对高糖诱导的上述指标的变化。结果HK-2细胞中高糖导致Rap1b蛋白表达明显下降,尤其是Rap1bGTP呈浓度及时间依赖模式；等渗对照组Rap1b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,高糖诱导线粒体呼吸链活性受抑,ATP生成减少,抗氧化酶GSH Px及CAT的活性受抑,MnSOD2的mRNA水平表达升高,线粒体ROS的生成增加,procaspase-9、procaspase-3的表达下降,细胞发生细胞凋亡。而过表达Rap1b蛋白表达,可以逆转上述高糖诱导的线粒体的氧化应激损伤以及细胞凋亡。结论Rap1b可以特异性逆转高葡萄糖诱导的线粒体呼吸链活性受抑、ATP生成减少以及抗氧化酶的活性受抑,进而抑制线粒体ROS的生成增加,促进清除,保护细胞,减轻氧化应激引起的细胞凋亡的发生。第三章Rap1b通过调节线粒体动力蛋白及线粒体生源基因表达介导高葡萄糖诱导的肾小管上皮细胞氧化损伤和凋亡的调控目的探讨Rap1b蛋白对高糖诱导的肾小管上皮细胞氧化损伤及凋亡的作用机制。方法常规培养正常人近端肾小管上皮细胞株(HK-2), cDNA3.1/Rap1b质粒转染HK-2细胞,建立稳定表达的细胞株。采用realtime-PCR及Westem blot检测线粒体融合分裂蛋白Drp-1 Mfn2以及线粒体生源基因PGC-1αNRF-1的mRNA水平及蛋白水平的表达。结果过表达的Rap1b可以特异性的明显下调高糖诱导的Drp-1的mRNA及蛋白的表达水平的升高,上调Mfn2的表达水平,逆转上述高糖诱导的线粒体融合分裂的失衡状态,加强线粒体融合与分裂过程的协调性。不仅如此,过表达的Rap1b还可以特异性上调由于高糖诱导的线粒体生源基因PGC-1α、NRF-1的表达下降。结论Rap1b可能通过调节PGC-1α以及NRF等线粒体生源基因,稳定线粒体融合分裂蛋白的平衡,保护高糖诱导肾小管上皮细胞氧化损伤和凋亡。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 typeⅠand 15% with typeⅡDM develop renal dysfunction. High glycemic level contributes to the development of diabetic nephropathy; however, the mechanisms underlying hyperglycemia induced injury are not fully understood. Recent observations indicate that hyperglycemia triggers the generation of free radicals and oxidant stress in renal tubular epithelial cells, which might be one of the important mechanisms in the development of renal dysfunction in diabetic nephropathy. 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-β1 and ECM accumulation, and finally result in cellular injury in kidney.Ras superfamily of small molecular weight G-proteins was reported to be as an important factor participating in mitochondrial dynamics, resulting in mitochondrial dysfunction. To be a member of Ras superfamily, Ras-proximate-1b (Rap1b) is a homolog of a well-characterized small GTPase and is known to antagonize the mitogenic and transforming activity of Ras. Recently, Raplb signal has received more and more attention in nephrology because it was found to be involved in progression of renal diseases such as diabetic nephrology and glomerulonephritis. However, very limited literature information is available that documents the effects of Rap1b in HG-induced intracellular ROS generation in the mitochondria of tubular cells in hyperglycemia-induced injury models. Researches have shown that Rap1b is upregulated in the hyperglycemic state and is known to increase B-Raf, an antiapoptotic effector protein. And in addition, our previous studies observed that Raplb ameliorates glucose-induced mitochondrial dysfunction against oxidase stress and thus prevents the apoptosis in renal tubular cells in diabetic nephrology (DN). However, the mechanism is unclear. To this end, experimental research is carried out as follow. ChapterⅠThe expression of Raplb in the kidney of diabetic nephropathy patientsObjective To observe the expression of Rap1b in the kidney in order to investigate the relationship of Rap1b and high gluose ambience in diabetic nephropathy patients.Methods 12 patients were enrolled into the study, they were diagnosed as DN (n=6) or minimal changes disease (primary kidney disease, control group, n=6) based on clinical manifestations and renal pathology. Renal pathological changes were observed by Masson, PASM and PAS staining. Interstitial damage score and collagen deposition score were evaluated. The expression of Rap1b was tested by immunohistochemistry and the relationship of Rap1b and tubular interstitial damage in DN was analyzed.Results Masson, PASM and PAS staining showed that there were many lesions in the kidneys of DN patients such as moderate to severe proliferation of mesangial matrix, nodular sclerosis, diffuse or segmental thickening of the basement membrane, tubular atrophy and compensatory expansion, local interstitial fibrosis. Meanwhile, the lesions in the kidneys of minimal change disease patients were less severe. There were mild mesangial cell and matrix proliferation, basement membrane is not thick, mild granular degeneration and vacuolar degeneration in renal tubular epithelial cells and no significant abnormalities in interstitial and renal vascular. Interstitial damage score and collagen deposition score in the kidneys of DN patients were higher than that in control group. Immunohistochemistry showed that the expression of Raplb was significantly up-regulated in DN patients compared with that in control group.Conclusion The expression of Raplb is significantly up-regulated in the kidney of DN patients and Raplb is negatively related with renal interstitial fibrosis. ChapterⅡ. The expression of Raplb and its role in the process of high glucose induced mitochondrial oxidative injury in human renal tubular epithelial cellsObjective To detect the expression of Raplb in human renal proximal epithelial cell lines (HK-2) and investigate the role of Raplb in the development of high glucose induced induced mitochondrial oxidative injury and apoptosis in HK-2.Methods The cells were exposed respectively in different concentrations of D-glucose(5.5,10,20,30 mM). The expression of Raplb protein was examined by Western Blot. The mitochondrial ROS was detected by Mitosox staining using confocal microscopy. Respiratory Chain Complex Activities assay, ATP assay, Catalase and GSH-Px assay were detected for mitochondrial function. Western blot and Realtime PCR were examined for procaspase-9, procaspase-3 and MnSOD2. Cell apoptosis was measured by Hoechst 33258 staining using fluorescence microscopy. Wild-Type Rap1b plasmid (cDNA3.1/Rap1b) and the Rap1b mutant plasmid (Rap1bS17N and Rap1bT61R) 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).And the detections above were examined in order to investigate the effect of Rap1b on the high glucose-induced mitochondrial injury.Results Stimulation of HK-2 with 30mM D-glucose resulted in a significant decrease in the expression of Raplb protein, compared with medium control group. However, there was no significant difference between the control and D-mannitol group. Decrease of Raplb GTP protein in HK-2 treated by different concentrations of high glucose in a time-dependent manner. In additon, HK-2 treated with 30mM D-glucose resulted in a time-dependent decrease in the expression of Raplb GTP protein, with the peak at 2h. Compared to control (5.5 mM),30 mM D-gluocse (HG) induced mitochondrial dysfunction, including decreased activity of respiratory chain complexⅢactivity, the level of ATP and the decreased capacity of the cellular antioxidant defense system(Catalase, GSH Px and MnSOD2). HG increases overproduction of mitochondrial superoxide. Pro-caspase-3 and pro-caspase-9 were both decreased accompanied by increasing cellular apoptosis. However, over-expression of Rap1b partially reversed such abnormalities.Conclusion Raplb could also protect mitochondria from high glucose-induced injury by reversing the decreased activity of respiratory chain complexⅢactivity, the level of ATP, the decreased capacity of the cellular antioxidant defense system, alteration of expression of Pro-caspase-3 and pro-caspase-9. These data show Rap1b contributes for the inhibition of mitochondria-mediated pathway of apoptosis induced by high glucose. ChapterⅢRap1b regulates high glucose induced mitochondrial oxidative injury via modulation of balance of fusion and fission in mitochondria in renal tubular epithelial cellsObjective To investigate the mechanism by which Rap1b modulates the process of high glucose induced mitochondrial oxidative injury in renal tubular epithelial cells (HK-2).Methods Wild-Type Raplb plasmid (cDNA3.1/Rap1b) and the Rap1b mutant plasmid (Rap1bS17N and Rap1bT61R) 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). Real-time PCR and Western blot were detected for mitochondrial fusion and fission proteins(Drpl,Mfh2) and mitochondria-related transcription factors PGC-1αand NRF-1.ResultsAfter 48h of treatment, high glucose triggers the development of imbalance of fusion and fission in mitochondria, including increased Drp1 and decreased Mfh2 in mRNA levels and protein levels. In addition, mitochondria-related transcription factors PGC-1a and NRF-1 were both decreased. While the effect was significantly reversed in Raplb transfected HK-2 cells, compared with that in Rap1b mutant plasmid.Conclusion Overexpression of Raplb might reverse the effects of oxidative injury induced by 30mM D-glucose in HK-2 via maintaining the PGC-1a/NRF-1-regulated fusion and fission in mitochondria.更多还原