内质网应激在糖尿病肾损害中的作用机制研究
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摘要
研究背景
糖尿病肾病(diabetic nephropathy, DN)是糖尿病患者中最常见且危害最大的合并症之一。众多的DN患者最终出现慢性肾功能不全,使得糖尿病成为目前全球终末期肾病(end-stage renal disease, ESRD)中最重要的原发病。DN的病理和病理生理发展是一个缓慢且复杂的过程,但是目前有众多的实验研究证据支持过度的细胞凋亡是其中重要的发生机制。
细胞凋亡是指在一定生理或病理条件下机体为维护内环境的稳定,通过有序的基因调控而诱导的细胞自杀。细胞凋亡和细胞增生共同调节各部分细胞总量的平衡。糖尿病状态下,促使细胞凋亡的诱导因素大大增多,使得凋亡的强度大于细胞增生,导致组织细胞数量减少,器官功能减退。目前认为有三条通路参与凋亡的发生：线粒体通路,死亡受体通路和内质网通路。
内质网通路是新近研究发现的凋亡途径,其核心内容是内质网应激(endoplasmic reticulum stress, ERS)反应。内质网在细胞中具有重要的功能,主要是参与膜／分泌性蛋白、氨基多糖、磷脂、胆固醇及钙信号等的代谢,特别是分泌性蛋白的合成与空间折叠、蛋白质糖基化修饰、蛋白质分泌等。当细胞内外的因素打破了蛋白质合成、成熟与降解之间的稳态,造成细胞内异常蛋白堆集,将诱发ERS。ERS通过未折叠蛋白反应(unfolded protein response, UPR)来适应细胞内外的应激,维持细胞的存活,主要的细胞反应包括暂时性中断细胞内总体蛋白合成、上调内质网分子伴侣和折叠酶的表达,诱导内质网相关性降解(ER-associated degradation, ERAD)处理蓄积的蛋白分子等,但是如果应激因素持续存在或刺激过强时,细胞功能不能恢复时,内质网相关的细胞凋亡(ER-associated apoptosis)就会激活,受损细胞通过凋亡通路消亡,从而达到维护整体器官功能的目的。但是如果凋亡的范围过大,速度过快,失去正常的调控,将导致组织细胞的大量丧失,同样导致器官功能严重减退。
肾脏细胞具有发达的内质网。电镜扫描显示,肾小球的系膜细胞(mesangialcell)、肾小囊的足细胞(podocyte)和肾小管的上皮细胞(tubular epithelial cell)的内质网结构丰富而复杂。从细胞功能上讲,肾脏细胞与蛋白的合成、分泌和降解关系密切：系膜细胞合成基膜和系膜基质成分,吞噬和降解沉积在系膜上的免疫复合物,分泌肾素等生物活性物质；足细胞是肾脏滤过屏障的组成部分,细胞膜外联结有丰富的糖蛋白,其完整性直接影响肾小球的滤过功能；肾小管上皮细胞除了重吸收管腔内的蛋白质并将其代谢掉,还可分泌激肽释放酶等活性分子。肾脏细胞的这些诸多功能很大程度上依赖于内质网来完成。因此,从细胞的结构和功能分析,肾脏具有发生ERS的条件和基础。
从ERS激活的刺激因素来讲,糖尿病状态下可能的刺激因素大大增加,包括高血糖,氧化应激,蛋白尿以及细胞内外电解质紊乱等,但目前关于ERS与糖尿病肾损害的具体试验证据尚不充分。因此,阐明糖尿病肾脏损害中ERS相关细胞凋亡的概况,明确触发凋亡的应激因素,了解凋亡通路的过程,从而对细胞凋亡做到适当的干预调节,挽救残存的肾脏细胞,对保护糖尿病患者的肾脏功能具有重要的积极意义。
研究目的
1.构建糖尿病大鼠模型,验证糖尿病大鼠肾脏细胞凋亡增加；
2.明确内质网应激在糖尿病大鼠肾脏细胞中是否激活；
3.明确内质网应激相关凋亡通路在糖尿病大鼠肾脏细胞凋亡中的信号通路。
研究方法
1.糖尿病大鼠模型的构建
7周左右的雄性Wistar大鼠30只,体重280±10g。随机分为2组：对照组10只,糖尿病组20只。标准大鼠饲料适应性喂养1周后,禁食12h,糖尿病组大鼠腹腔内注射链脲佐菌素(STZ)65mg/kg(溶于pH4.5的柠檬酸盐缓冲液),对照组大鼠则注射等量同pH值的柠檬酸盐缓冲液。糖尿病大鼠成模的诊断标准为：STZ注射7天后,尾静脉取血测定空腹血糖≥16.7mmol/L(300mg/d1)。未达此标准者则剔除。糖尿病组大鼠自血糖达到成模标准后,继续喂养16周,处死取材。
2.血糖监测和24小时尿白蛋白的测定
血糖每4周检测一次,造模后第1周和第16周利用代谢笼收集大鼠24小时尿液,测定尿白蛋白含量。
3.肾脏组织病理学检查
第16周末动物处死后,进行肾脏组织取材、固定、脱水、透明、浸蜡、石蜡包埋、切片,常规苏木素-伊红染色(H&E)和过碘酸雪夫染色(PAS),判断糖尿病大鼠是否出现DN的病理表现。
4.TUNEL染色法检测细胞凋亡
利用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测试剂盒,进行石蜡切片的TUNEL染色,观察并统计细胞凋亡水平。
5.免疫组织化学检测
取石蜡组织切片,进行GRP78, Caspase-12, p-JNK和CHOP免疫组织化学染色,观察各个指标阳性细胞的分布和强度。
6.免疫印迹Western blot检测
取-80℃保存肾脏组织,提取总蛋白,经过10%--14%SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离、转膜、抗体孵育、ECL显色等步骤,检测GRP78、Caspase-12、CHOP、Total-JNK和p-JNK的蛋白表达水平。
7.实时定量RT-PCR法检测
利用Trizol法提取两组肾脏组织RNA,经逆转录反应得到总cDNA,以β-actin作为参照,通过real-time PCR技术检测GRP78、Caspase-12、CHOP和JNK的mRNA表达水平。
研究结果
1.实验动物的基本情况
对照组大鼠精神状态良好,体重增加明显,反应敏捷,毛色白而光泽。糖尿病组大鼠精神萎靡,体重增加迟缓,出现多食、多饮、多尿和消瘦等症状,皮毛脏乱无光泽,部分出现烂尾、白内障等。整个实验过程中3只大鼠死亡,均为糖尿病组,死亡原因可能与糖尿病酮症酸中毒、感染或其他相关并发症有关。最终共27只完成实验,其中对照组10只,糖尿病组17只。
2.大鼠DN的确定
实验组大鼠DN的确定通过血糖和24小时尿白蛋白的测定以及肾脏组织切片病理检查共同确定。糖尿病大鼠组血糖自成模后第一次测量后,一直维持在16.7mmol/L以上,与对照组相比,具有显著性差异(P<0.05)。16周末24小时尿蛋白测定,与对照组相比,糖尿病组大鼠明显升高(P<0.05)。病理切片检查示与对照组大鼠肾脏比较,糖尿病大鼠肾小球体积增大,系膜基质增多,肾小管上皮细胞出现空泡变性,小灶样萎缩,小动脉管壁增厚,肾间质纤维化和玻璃样变。因此可判断糖尿病大鼠组出现DN改变。
3.糖尿病大鼠肾脏细胞凋亡增加
TUNEL染色检测肾脏细胞凋亡水平,结果示与对照组大鼠相比较,糖尿病大鼠肾脏细胞凋亡明显增加(P<0.05),凋亡细胞在肾小球和肾小管中均可观察到。
4.糖尿病肾脏中内质网分子伴侣GRP78表达上调
GRP78是位于内质网中重要的分子伴侣,帮助蛋白质的折叠和成熟,是广泛使用的ERS激活的标志分子。免疫组织化学、免疫印迹法和实时定量PCR法检测GRP78在两组肾脏组织中的分布范围和表达水平。结果显示,正常肾脏组织中,GRP78在肾小管细段和远曲肾小管上皮细胞中有轻度表达,而在糖尿病肾脏中,GRP78在肾小球和近端肾小管表达升高,在肾小管细段以及远端肾小管的表达则有显著的升高(P<0.05)。蛋白水平和mRNA水平的检测结果与免疫组化结果相一致,GRP78在糖尿病肾脏中表达明显上调(P<0.05)。ERS在糖尿病肾损害中激活。
5.内质网相关凋亡通路的检测
CHOP, JNK和Caspase-12分别是三条内质网相关凋亡通路中的关键分子。三条通路相对独立,任一通路的激活均可导致细胞的凋亡。Western blot检测结果示与对照组相比,糖尿病组CHOP, p-JNK和Caspase-12的表达水平均明显升高(P<0.05)。mRNA水平检测,糖尿病组JNK mRNA水平与对照组比较无显著性差异(P>0.05),CHOP, Caspase-12 mRNA表达水平则明显升高(P<0.05)。免疫组化结果显示,与对照组相比,在糖尿病大鼠肾脏中,三个分子的分布范围和表达强度明显升高,但三个分子之间相比,分布范围存在显著差异。Caspase-12在对照组肾脏组织中不能检测到,在糖尿病肾脏中,阳性细胞主要分布在肾小球中；p-JNK的分布主要是肾小球和肾小管区间质中,尤其是间质的小血管中,呈强阳性表达；CHOP的分布则主要是远曲小管上皮细胞中,在肾小球和近端小管中也可检测到颗粒样阳性表达。因此,3条内质网相关凋亡通路都可能参与DN的病理发生,但具体的激活途径和应激细胞存在差异。
结论
1.STZ诱导的糖尿病大鼠16周,出现DN的表现,肾脏细胞凋亡增加；
2.糖尿病大鼠肾脏GRP78的表达显著上调,ERS在糖尿病大鼠肾损害中激活；
3. CHOP, JNK和Caspase-12介导的3条内质网相关凋亡通路在DN中都激活,但各自激活的方式和存在的细胞可能存在差异。
研究背景
肾小球硬化是糖尿病肾病DN最重要的特征性病理改变,其主要形成原因是肾小球系膜区系膜细胞增生和系膜基质的大量增加。但是到了DN的中晚期,硬化的肾小球内过多的系膜基质仍在,但系膜细胞的数量却呈耗竭状态。系膜细胞从过度增生到异常减少的过程中,过度的细胞凋亡被认为是肾小球系膜细胞缺失的重要机制。
实验证据证明,在DN的发生发展过程中,伴随着肾小球内血管紧张素Ⅱ(angiotensin II, ANGⅡ)的高水平表达。无论是在生理还是病理状态下,ANGⅡ与肾脏系膜细胞都具有密切的关系。肾脏系膜细胞是肾小球主要的组成细胞之一,位于毛细血管球之间,不仅对血管球起到支持和连接的作用,而且是系膜基质的主要来源细胞,通过其收缩功能调节肾小球的滤过面积,可分泌多种细胞因子等,尤其是病理损害状态下。系膜细胞表面不仅有ANGⅡ的受体ATl,而且系膜细胞还可促进ANGⅡ的裂解生成。ANGⅡ与细胞表面的受体结合,从而产生收缩细胞,促进细胞外基质合成和分泌细胞因子等效应。除此以外,ANGⅡ还有导致细胞损伤,诱导细胞凋亡的效应。
超微结构研究显示,肾脏系膜细胞具有发达的内质网系统。内质网是蛋白质翻译后加工的场所,尤其是蛋白质的糖基化修饰,分泌蛋白的空间折叠等,而系膜细胞的重要产物是细胞外基质,其成分主要是糖蛋白和蛋白多糖。系膜细胞的蛋白合成十分活跃,尤其是在肾小球硬化发展过程中,系膜基质处于过度增多状态。而各种原因造成内质网内未折叠蛋白蓄积,将诱发内质网应激(ERS)。适度的刺激,内质网通过暂时性中断细胞内蛋白合成、上调内质网分子伴侣和折叠酶的表达,诱导内质网相关性降解(ERAD)处理蓄积的蛋白等措施维持细胞存活,而持续或过度强烈的刺激,将导致内质网相关凋亡通路的激活,诱导细胞凋亡。
近年来研究发现,ANGⅡ是多种细胞凋亡的诱发因素,其发生机制可能与ERS相关。但目前ANGⅡ诱导肾脏系膜细胞凋亡的ERS通路还缺乏明确的试验证据。体外实验证据证明,ANGⅡ可诱导系膜细胞活性氧自由基(ROS)的产生。而氧化应激可导致ERS发生的试验证据已经比较充足,因此ANGⅡ可能通过氧化应激激活ERS凋亡通路诱导系膜细胞凋亡的发生。除此以外,ANGⅡ的促进蛋白合成效应如果造成未折叠蛋白在内质网中的过度蓄积,也将诱发ERS。因此,本试验将进行ANGⅡ的系膜细胞体外诱导凋亡试验,验证ERS凋亡通路是否参与其中,从而为肾脏细胞的保护提供新的治疗靶点。
研究目的
1.观察ANGⅡ是否激活系膜细胞的ERS。
2.明确ERS相关凋亡通路是否参与ANGⅡ诱导的系膜细胞凋亡。
研究方法
1.实验设计
1)体外培养大鼠肾脏系膜细胞系HBZY-1,分别给与ANGⅡ不同浓度(0,1,10,100 nM)和不同时间梯度(0,12,24,48小时)刺激,利用Annexin V/PI试剂盒,在流式细胞仪上检测细胞凋亡率,筛选出ANGⅡ最佳刺激浓度和时间；
2)给予系膜细胞最佳刺激浓度和时间ANGⅡ处理,检测ERS标志蛋白GRP78和ERS凋亡相关分子CHOP, Caspase-12和p-JNK的表达；
3)根据步骤2的结果,对凋亡过程中表达上调的ERS凋亡分子,给予相对应的siRNA预处理,然后进行ANGⅡ最佳刺激浓度和时间处理,检测细胞凋亡率的变化。
2.系膜细胞系的体外培养
大鼠肾脏系膜细胞系HBZY-1购自中国武汉细胞库,在低糖DMEM培养基(葡萄糖浓度5.5mmol／L)中培养,取代数在10-20,处于生长对数期的细胞进行试验。
3.细胞免疫荧光染色
将系膜细胞种植在放有玻片的6孔板中,制作细胞爬片。待细胞生长至70%融合状态,给予ANGⅡ最佳浓度和时间刺激。刺激完毕后,取出爬片,清洗,固定,封闭,GRP78一抗孵育,FITC标记的二抗反应后,DAPⅠ染核,荧光显微镜下观察ERS标记分子GRP78的表达。
4. Annexin V/PI试剂盒检测细胞凋亡率
细胞刺激完毕,胰酶消化细胞,制作适当浓度细胞悬液,分别加入Annexin V-FITC和PI进行标记反应,随即进行流式细胞仪的检测。
5. Western blot检测蛋白质的表达变化
细胞刺激完毕,细胞刮刀收集细胞,提取总蛋白,进行SDS-PAGE电泳分离,转膜,蛋白印迹和免疫反应后检测GRP78, CHOP, Caspase-12和p-JNK的蛋白表达水平。
6.实时定量RT-PCR法检测mRNA表达水平
利用Trizol法提取刺激细胞总RNA,以β-actin作为参照,通过real-timeRT-PCR技术检测GRP78、Caspase-12、CHOP和JNK的mRNA表达水平。
研究结果
1.ANGⅡ呈浓度和时间梯度依赖性诱导系膜细胞的凋亡
给予系膜细胞不同浓度(0,1,10,100 nM)ANGⅡ刺激24小时后,流式细胞仪检测细胞凋亡率。结果显示,细胞凋亡率呈逐渐上升趋势,分别为1.37%±0.41%,3.72%±1.23%,9.27%±3.53%,17.74%±5.19%(P<0.05)。而使用100nM的ANGⅡ给予系膜细胞不同时间段(0,12,24,48小时)的刺激,细胞凋亡率的结果分别为1.53%±0.49%,5.72%±1.97%,19.14%±6.53%,24.74%±9.19%(P<0.05)。但是在48小时组,细胞坏死率明显上升,与24小时组相比,细胞坏死率为4.53%±1.69%VS.15.76%±6.87%(P<0.05)。因此可知ANGⅡ呈浓度和时间梯度依赖性诱导系膜细胞的凋亡。在本试验中,最佳的ANGⅡ浓度为100nM,而最佳的刺激时间为24小时。
2.ANGⅡ上调ERS标志分子GRP78的表达
细胞免疫荧光染色,Western blot和Realtime PCR三种方法检测100nmol／LANGⅡ刺激24小时后,系膜细胞内GRP78的表达。结果示与空白刺激组相比,GRP78的mRNA和蛋白表达水平显著上调(P<0.05)。
3. CHOP和Caspase-12在ANGⅡ的ERS凋亡诱导中表达上调
ANGⅡ100 nmol/L刺激系膜细胞24小时后,收集细胞,Western blot和Realtime PCR分别检测ERS相关凋亡分子的表达变化,结果示：与对照组相比,CHOP和Caspase-12的蛋白质和mRNA表达水平显著增加(P<0.05),而p-JNK的表达无显著性差异(P>0.05)。提示ANGⅡ的ERS凋亡诱导可能通过CHOP和Caspase-12介导。
4. CHOP和Caspase-12 siRNA转染降低ANGⅡ诱导的系膜细胞凋亡率
系膜细胞生长至30-50%融合状态,分别转染CHOP和Caspase-12 siRNA。转染后24小时,再给以ANGⅡ100 nmol／L24小时刺激,流式细胞仪检测细胞凋亡率。结果示与ANGⅡ+对照转染组相比,CHOP和Caspase-12 siRNA组均可显著降低细胞凋亡率,对照转染组,CHOPsiRNA组和Caspase-12 siRNA组的凋亡率分别为31.74%±8.57%,20.61%±6.33%,18.94%±5.49%(P<0.05)。
结论
1. ANGⅡ可诱导肾脏系膜细胞ERS的激活；
2. ANGⅡ可通过CHOP和Caspase-12通路介导诱导系膜细胞的ERS凋亡效应。
Background
Diabetic nephropathy is a common and serious complication of diabetes mellitus, which leads to renal failure in up to 30% of individuals with diabetes and thus becomes the most important cause of end-stage renal diseases (ESRD). The pathogenesis of diabetic nephropathy is a chronic and complicated process, and more and more evidences support that apoptosis plays pivotal role in the development of nephropathy.
Apoptosis is the process of gene-controlled programmed cell death that occurs under physiology or pathology condition to keep the homeostasis. Cell apoptosis with cell proliferation control the balance of total cell number in each organ. Under diabetic condition, factors inducing the apoptosis increase which result in the intense of apoptosis overwhelming proliferation, then the number of functional cells decreases and finally leads to the organ failure. At present 3 different pathways are considered that mediate the process of cell apoptosis:mitochondrial pathway, death receptor pathway and newly found endoplasmic reticulum (ER) stress associated pathway.
ER is the largest organelle in the cell and involved in the metabolism of membrane/secreted protein, glycosaminoglucan, phospholipid, cholesterol, and calcium, especially in the secreted protein maturation, folding and transport. Once the insulting factors destroy the balance of protein production, maturation and degrading, the ER stress is activated. Several complex homeostatic signaling pathways, known as the unfolded protein response (UPR), have evolved to cope with the ER stress. The processes of UPR include:temporary suspension the general protein synthesis, up regulation the expression of ER resident proteins and molecular chaperons, inducing the ER associated degradation (ERAD). Though moderate ER stress could alleviate the damage caused by stress, prolonged or severe stress response leads to apoptosis and removes the damaged cells. But if the intense or the extent of apoptosis is out of control, excess functional cells lose, which result in the premature organ failure.
Renal cells, including mesangial cell, podocyte, tubular epithelial cell and et al, have well-developed ER, which is tightly related to the cell physiological function. Mesangial cells are the primary manufacturers of basal membrane and mesangial matrix. They are also involved in the degrading of mesangial immune complex, and secreting the bioactive substances such as renin and angiotensin. Podocytes are the components of the glomerular filtration barrier. Glycoprotein connected to the outer membrane of the cells plays the vital role in the filtration. Besides re-absorbing and degrading the filtrated protein, tubular cells also produce the cytokines such as bradykinin. Above all, ER fulfills multiple protein related functions in the renal cells and ER stress might be easily activated in the renal disease.
Under diabetic condition, ER stress insulting factors are prone to increase, including hyperglycemia, oxidative stress, proteinuria, and cellular electrolyte disorder. However, fewer publications are found between ER stress and diabetic kidney injury. So the aim of this research is to investigate the role of ER stress in the diabetic nephropathy, and then provide possible strategies on the kidney protection.
Objectives
1. To establish the diabetic rat model, and demonstrate the increment of apoptotic renal cells.
2. To investigate the activation of ER stress in the diabetic kidney.
3. To explore the signaling pathways of ER associated apoptosis in the diabetic kidney injury.
Methods
1. Establishment of diabetic rat model
30 male Wistar rats (7 weeks of age; mean body weight 280±10 g) were used. After one week of acclimatization, of the 30 Wistar rats,20 were given a single intraperitoneal dose (65 mg/kg) of streptozotocin (STZ, dissolved in citrate buffer (pH 4.5)), while the remaining 10 rats were given the same dosage of citrate buffer (pH 4.5). Hyperglycemia was confirmed by measuring the venous circulating plasma concentrations of glucose. Seven days post-STZ injection, blood samples were obtained from the rat tail vein after 12 hours of fasting, and the rats with glucose concentration higher than 300 mg/dl were kept in diabetic group. All rats were killed at the 16th week after saline or STZ injection.
2. Blood glucose test and 24-hour proteinuria examination
Blood glucose was detected per 4 weeks. At the end of the 1st and 16th week,24-hour urine was collected with the metabolic cage and the levels of proteinuria were measured.
3. Renal histopathological staining
Kidneys were transversally sliced and fixed in 4% neutral formalin for over 24 hours, and then embedded in paraffin, cut into 4-μm thick sections for staining with H&E or PAS methods. Then slides were observed under the microscope to determine if the diabetic kidney injury occurred or not.
4. TUNEL staining
TUNEL staining was used to detect the expression of apoptotic cells in the diabetic kidney.
5. Immunohistochemical staining
Immunohistochemical staining was used to detect the protein expression of GRP78, CHOP, Caspase-12 and p-JNK. The intensity and extent of the positive area were calculated and compared.
6. Western blot analysis
Total proteins of the renal samples were extracted and conducted 10—14% SDS-PAGE. Then the protein expression levels of GRP78, CHOP, Caspase-12, Total-JNK and p-JNK were measured.
7. Realtime RT-PCR analysis
Realtime RT-PCR was used to detect the mRNA levels of GRP78, CHOP, Caspase-12, and JNK.
Results
1. General features of the experimental rats
In the whole experiment,3 diabetic rats died and might be caused by diabetic ketoacidosis, infection or other diabetic complications. Finally 27 rats survived and 10 from control group and 17 from diabetic group. Compared to the control, diabetic rats seemed to be inactive, and with symptoms of lower body weights, polyuria and polyphagia, which are normally associated with diabetic state.
2. The diagnosis of diabetic nephropathy
The diagnosis of diabetic nephropathy was determined by blood glucose monitoring,24-hour proteinuria detection and renal pathology. Compared to the control, concentrations of glucose blood in diabetic rats were always higher than 16.7 mmol/L (P<0.05). At the end of 16th week, the level of 24-hour proteinuria increased obviously in diabetic rats (P<0.05). And the examination of renal pathology showed glomerular hypertrophy, glomerular basement membrane thickness, glomerulosclerosis, renal tubular atrophy and interstitial fibrosis in diabetic kidney.
3. Enhanced apoptotic state in diabetic kidney
The results of TUNEL assay showed that apoptosis was observed in both the cortex and medulla of the diabetic kidney. Estimation of renal apoptosis revealed a nearly fivefold increase in TUNEL-positive nuclei in diabetic kidneys compared to the normal (P<0.05).
4. Up-regulation of ER chaperone GRP78 in kidneys of diabetes
GRP78, one important molecular chaperone localized in ER, has been used extensively as an indicator for the induction of ER stress. Immunohistochemistry study showed that GRP78 was abundantly expressed in the renal tissue from diabetic rats (P<0.05). In contrast, normal rats exhibited modest or weak immunoreactivity for this molecule. The result of western blot analysis confirmed the finding. Furthermore, real-time PCR analysis showed the same trend of grp78 in mRNA level (P <0.05). All these findings suggested the activation of ER stress in the kidneys of diabetic rats.
5. Involvement of the ER-associated apoptosis pathways
CHOP, JNK and caspase-12 are the key molecules in the three branches of ER-associated apoptosis pathways. Firstly, western blots were used to check the involvement of these pathways in diabetic kidneys. The densitometric analysis of bands for CHOP, caspase-12, and p-JNK revealed a significant increase in relative protein content in renal tissue from diabetic rats in comparison with those from normal rats(P<0.05). This means CHOP was induced, and caspase-12 and JNK pathway were activated in the diabetic kidney. Changes in RNA expression of jnk, chop, and caspase-12 were quantified by RT-PCR in control and diabetic rat kidneys. Expressions of chop, and caspase-12 were significantly increased in the diabetic kidneys (P<0.05), paralleled with their enhanced protein expression. However, the result of jnk did not show any differences between the diabetes and control (P>0.05). At last, the results of immunostaining provided more useful information. In diabetic kidney, Caspase-12 positive area only appeared in the glomerulus; p-JNK was mainly expressed in the glomerulus and interstitial area, especially the small vessels; CHOP could be observed in the distal convoluted tubule area, and focal expression in the glomerulus and proximal tubular area. The differences might be caused by the variance of involved cells and insulting factors.
Conclusion
1. The STZ induced diabetic rats showed classic diabetic nephropathy performances and enhanced apoptotic state.
2. The expression of ER chaperone GRP78 was up-regulated in diabetic kidney, which directly demonstrated the activation of ER stress.
3. CHOP, JNK and Caspase-12, three branches of ER associated apoptosis, were all stimulated in diabetic kidney. However, the activated modes or the cells involved may be different.
Background
Glomerulosclerosis, which is characterized by the expansion of mesangium, is the most important pathological change in the early stage of diabetic nephropathy. The expanded mesangium is comprised of a large number of mesangial cells and increased amount of matrix. However, in the later course of the disease, there is an abundance of matrix and a limited number of mesangial cells. And apoptosis is assumed to play the vital role in the mesangial cell loss.
Evidences show that intra-renal levels of angiotensinⅡ(ANGII) are elevated in the development of diabetic nephropathy. ANG II plays an important role in the function maintenance of mesangial cells in both normal and diseased states. Mesangial cells are specialized pericytes located among the glomerular capillaries within a renal corpuscle of a kidney. They are not only providing structural support for and regulate blood flow of the glomerular capillaries by their contractile activity, but also are the major contributors to the extracellular matrix which contains fibronectin, type IV collagen, perlecan, and laminin. All the functions mentioned above are tightly related to the ANGII regulation. However, ANGII could insult kinds of cells and induce the apoptosis or necrosis.
Ultrastructural analysis results show that endoplasmic reticulum (ER) in mesangial cell is well-developed, and in an active state. The ER is the site of biosynthesis for steroids, cholesterol and other lipids. It is also the sub-cellular entrance for a number of secretory and structural proteins and provides a unique environment for appropriate protein folding and assembly to produce functional, mature proteins. What's more, protein glycosylation is complicated in the ER. As is known, extracellular matrix is mainly comprised by glycoprotein and proteoglycan. And under the diabetic state, matrix is overloaded in the glomerular mesangium. Homeostasis in the ER is maintained by a coordinated adaptive program, so-called the unfolded protein response (UPR). A number of microenvironmental, developmental and pathophysiological insults as well as a wide range of chemical substances cause accumulation of unfolded proteins in the ER and activate ER stress. In response to accumulation of unfolded proteins, cells adapt themselves to the stress conditions via UPR:attenuation of general translation, induction of ER chaperones and activation of ER-associated degradation (ERAD) to eliminate immature proteins. If the stress is beyond the capacity of the adaptive machinery, cells undergo apoptosis Accumulated evidences showed that ANGII could induce the apoptosis of many cells, and the mechanisms might involve the ERS response. However, whether ANGII inducing apoptosis of mesangial cells is mediated by ERS is still unknown. On the one hand, ANGII has been demonstrated to stimulate the generations of reactive oxygen species in mesangial cells, and the relationship between oxidative stress and ERS is undeniable; On the other hand, ANGII promotes the protein synthesis in ER and might result in the unfolded protein accumulation,thus activate the ERS. Therefore the aim of this study is to investigate the role of ERS in the ANGII-induced apoptosis of mesangial cell.
Objectives
1. To observe the hallmark of ERS activation in ANGII-induced apoptosis of mesangial cell.
2. To explore the ERS associated apoptotic pathways in ANGII-induced apoptosis of mesangial cell.
Methods
1. Study design
1) The established rat renal mesangial cell line HBZY-1 was incubated with ANGII at different concentration (0,1,10,100 nmol/L) and duration (0,12,24,48 hours) to select the best treatment condition. After harvesting, apoptotic cells were labeled with Annexin V/PI kit, and the percentage of apoptosis was calculated with flow cytometer.
2) Cultured cells were treated with ANGII at the selected concentration and duration. Then Immunofluorescence staining, western blot and realtime PCR were used to measure the expression of GRP78, CHOP, Caspase-12 and p-JNK on protein and mRNA levels.
3) According to result of 2nd step, to synthesize the siRNA against ERS apoptotic molecules. Treated cells with siRNA before ANGII incubation, and then detected the apoptotic percentage with flow cytometer.
2. Cell culture
The established rat renal mesangial cell line HBZY-1, which was purchased from Wuhan cell center, was cultured in low-glucose DMEM. Cells in generation 10-20 were used in the treatment.
3. Immunofluorescence staining
Cells were grown on glass slides in the presence of selected concentration of ANGII for the proper duration. Then the cells were fixed with 4% paraformaldehyde for 15 minutes at 4℃and permeabilized in 0.1% Triton X-100 for 10 minutes at 4℃. The cells were then blocked in 10% rabbit blocking serum at 37℃for 30 minutes followed by incubation with GRP78 goat polyclonal IgG (1:50) overnight at 4℃. After washes with PBS three times, cells were incubated with FITC-conjugated rabbit anti-goat IgG (1:50) at 37℃for 1 hour. The results were evaluated under fluorescing microscope.
4. Annexin V/PI kit to measure the percentage of apoptosis When the stimulation was over, cells were digested with 0.25% trypsin and collected, then resuspended in binding buffer with annexin V-FITC and PI. After staining, cells were immediately analyzed with flow cytometer.
5. Western blot The total protein was extracted from the harvested cells. Western blot was used to detect the protein levels of GRP78, CHOP, Caspase-12 and p-JNK.
6. Realtime RT-PCR analysis The total RNA was extracted from the harvested cells with Trizol. Realtime RT-PCR was used to detect the mRNA levels of GRP78, CHOP, Caspase-12 and JNK.
Results
1. ANGII induced apoptosis of mesangial cells in a dose-and time-dependent manner
Treated mesangial cells with ANGII of different concentration (0,1,10,100 nmol/L) for 24 hours and measured the percentage of apoptosis, the results showed that the apoptotic ratio was increased gradually, were 1.37%±0.41%, 3.72%±1.23%,9.27%±3.53%,17.74%±5.19%respectively (P <0.05) Exposed cells with 100nmol/L ANGII for different duration (0, 12,24,48 hours) and the results of apoptosis tests are 1.53%±0.49%,5.72%±1.97%,19.14%±6.53%,24.74%±9.19% for each time point (P<0.05). Thus ANGII induced apoptosis of mesangial cells in a dose-and time-dependent manner. And the best ANG concentration and duration selected were 100nmol/L and 24 hours.
2. Up-regulation of ER chaperone GRP78
Immunofluorescence staining, Western blot and Realtime PCR, three methods were used to detect the expression of GRP78 after the ANGII treatment in the mesangial cell. And the results showed that GRP78 was obviously upregulated on both mRNA and protein levels compared to the control group (P<0.05).
3. The expressions of CHOP and Caspase-12 were elevated in ANGII
induced mesangial cell apoptosis Three key molecules of ERS associated apoptosis, CHOP, Caspase-12 and JNK, were examined with western blot and real time PCR after cell exposed to ANGII at selected concentration and duration. Results showed that the expressions of CHOP and Caspase-12 were increased dramatically compared to the control (P<0.05), but there was no change for JNK expression (P>0.05).
4. CHOP and Caspase-12 siRNA decreased the apoptosis induced by ANGII in mesangial cell Cells were transfected with CHOP and Caspase-12 siRNA 24 hours before ANGII treatment. And the results of apoptosis percentage showed that CHOP and Caspase-12 siRNA could decrease the apoptosis induced by ANGII in mesangial cell. The apoptotic percentages for control siRNA group, group CHOP siRNA and group Caspase-12 siRNA are 31.74%±8.57%, 20.61%±6.33%,18.94%±5.49%(P<0.05)。
Conclusion
1. ANGII could activate the ERS in renal mesangial cells.
2. ANGII-induced ERS associated apoptosis of mesangial cell might be mediated by CHOP and Caspase-12 pathways.
糖尿病肾病(diabetic nephropathy, DN)是糖尿病患者中最常见且危害最大的合并症之一。众多的DN患者最终出现慢性肾功能不全,使得糖尿病成为目前全球终末期肾病(end-stage renal disease, ESRD)中最重要的原发病。DN的病理和病理生理发展是一个缓慢且复杂的过程,但是目前有众多的实验研究证据支持过度的细胞凋亡是其中重要的发生机制。
细胞凋亡是指在一定生理或病理条件下机体为维护内环境的稳定,通过有序的基因调控而诱导的细胞自杀。细胞凋亡和细胞增生共同调节各部分细胞总量的平衡。糖尿病状态下,促使细胞凋亡的诱导因素大大增多,使得凋亡的强度大于细胞增生,导致组织细胞数量减少,器官功能减退。目前认为有三条通路参与凋亡的发生：线粒体通路,死亡受体通路和内质网通路。
内质网通路是新近研究发现的凋亡途径,其核心内容是内质网应激(endoplasmic reticulum stress, ERS)反应。内质网在细胞中具有重要的功能,主要是参与膜／分泌性蛋白、氨基多糖、磷脂、胆固醇及钙信号等的代谢,特别是分泌性蛋白的合成与空间折叠、蛋白质糖基化修饰、蛋白质分泌等。当细胞内外的因素打破了蛋白质合成、成熟与降解之间的稳态,造成细胞内异常蛋白堆集,将诱发ERS。ERS通过未折叠蛋白反应(unfolded protein response, UPR)来适应细胞内外的应激,维持细胞的存活,主要的细胞反应包括暂时性中断细胞内总体蛋白合成、上调内质网分子伴侣和折叠酶的表达,诱导内质网相关性降解(ER-associated degradation, ERAD)处理蓄积的蛋白分子等,但是如果应激因素持续存在或刺激过强时,细胞功能不能恢复时,内质网相关的细胞凋亡(ER-associated apoptosis)就会激活,受损细胞通过凋亡通路消亡,从而达到维护整体器官功能的目的。但是如果凋亡的范围过大,速度过快,失去正常的调控,将导致组织细胞的大量丧失,同样导致器官功能严重减退。
肾脏细胞具有发达的内质网。电镜扫描显示,肾小球的系膜细胞(mesangialcell)、肾小囊的足细胞(podocyte)和肾小管的上皮细胞(tubular epithelial cell)的内质网结构丰富而复杂。从细胞功能上讲,肾脏细胞与蛋白的合成、分泌和降解关系密切：系膜细胞合成基膜和系膜基质成分,吞噬和降解沉积在系膜上的免疫复合物,分泌肾素等生物活性物质；足细胞是肾脏滤过屏障的组成部分,细胞膜外联结有丰富的糖蛋白,其完整性直接影响肾小球的滤过功能；肾小管上皮细胞除了重吸收管腔内的蛋白质并将其代谢掉,还可分泌激肽释放酶等活性分子。肾脏细胞的这些诸多功能很大程度上依赖于内质网来完成。因此,从细胞的结构和功能分析,肾脏具有发生ERS的条件和基础。
从ERS激活的刺激因素来讲,糖尿病状态下可能的刺激因素大大增加,包括高血糖,氧化应激,蛋白尿以及细胞内外电解质紊乱等,但目前关于ERS与糖尿病肾损害的具体试验证据尚不充分。因此,阐明糖尿病肾脏损害中ERS相关细胞凋亡的概况,明确触发凋亡的应激因素,了解凋亡通路的过程,从而对细胞凋亡做到适当的干预调节,挽救残存的肾脏细胞,对保护糖尿病患者的肾脏功能具有重要的积极意义。
研究目的
1.构建糖尿病大鼠模型,验证糖尿病大鼠肾脏细胞凋亡增加；
2.明确内质网应激在糖尿病大鼠肾脏细胞中是否激活；
3.明确内质网应激相关凋亡通路在糖尿病大鼠肾脏细胞凋亡中的信号通路。
研究方法
1.糖尿病大鼠模型的构建
7周左右的雄性Wistar大鼠30只,体重280±10g。随机分为2组：对照组10只,糖尿病组20只。标准大鼠饲料适应性喂养1周后,禁食12h,糖尿病组大鼠腹腔内注射链脲佐菌素(STZ)65mg/kg(溶于pH4.5的柠檬酸盐缓冲液),对照组大鼠则注射等量同pH值的柠檬酸盐缓冲液。糖尿病大鼠成模的诊断标准为：STZ注射7天后,尾静脉取血测定空腹血糖≥16.7mmol/L(300mg/d1)。未达此标准者则剔除。糖尿病组大鼠自血糖达到成模标准后,继续喂养16周,处死取材。
2.血糖监测和24小时尿白蛋白的测定
血糖每4周检测一次,造模后第1周和第16周利用代谢笼收集大鼠24小时尿液,测定尿白蛋白含量。
3.肾脏组织病理学检查
第16周末动物处死后,进行肾脏组织取材、固定、脱水、透明、浸蜡、石蜡包埋、切片,常规苏木素-伊红染色(H&E)和过碘酸雪夫染色(PAS),判断糖尿病大鼠是否出现DN的病理表现。
4.TUNEL染色法检测细胞凋亡
利用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)检测试剂盒,进行石蜡切片的TUNEL染色,观察并统计细胞凋亡水平。
5.免疫组织化学检测
取石蜡组织切片,进行GRP78, Caspase-12, p-JNK和CHOP免疫组织化学染色,观察各个指标阳性细胞的分布和强度。
6.免疫印迹Western blot检测
取-80℃保存肾脏组织,提取总蛋白,经过10%--14%SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离、转膜、抗体孵育、ECL显色等步骤,检测GRP78、Caspase-12、CHOP、Total-JNK和p-JNK的蛋白表达水平。
7.实时定量RT-PCR法检测
利用Trizol法提取两组肾脏组织RNA,经逆转录反应得到总cDNA,以β-actin作为参照,通过real-time PCR技术检测GRP78、Caspase-12、CHOP和JNK的mRNA表达水平。
研究结果
1.实验动物的基本情况
对照组大鼠精神状态良好,体重增加明显,反应敏捷,毛色白而光泽。糖尿病组大鼠精神萎靡,体重增加迟缓,出现多食、多饮、多尿和消瘦等症状,皮毛脏乱无光泽,部分出现烂尾、白内障等。整个实验过程中3只大鼠死亡,均为糖尿病组,死亡原因可能与糖尿病酮症酸中毒、感染或其他相关并发症有关。最终共27只完成实验,其中对照组10只,糖尿病组17只。
2.大鼠DN的确定
实验组大鼠DN的确定通过血糖和24小时尿白蛋白的测定以及肾脏组织切片病理检查共同确定。糖尿病大鼠组血糖自成模后第一次测量后,一直维持在16.7mmol/L以上,与对照组相比,具有显著性差异(P<0.05)。16周末24小时尿蛋白测定,与对照组相比,糖尿病组大鼠明显升高(P<0.05)。病理切片检查示与对照组大鼠肾脏比较,糖尿病大鼠肾小球体积增大,系膜基质增多,肾小管上皮细胞出现空泡变性,小灶样萎缩,小动脉管壁增厚,肾间质纤维化和玻璃样变。因此可判断糖尿病大鼠组出现DN改变。
3.糖尿病大鼠肾脏细胞凋亡增加
TUNEL染色检测肾脏细胞凋亡水平,结果示与对照组大鼠相比较,糖尿病大鼠肾脏细胞凋亡明显增加(P<0.05),凋亡细胞在肾小球和肾小管中均可观察到。
4.糖尿病肾脏中内质网分子伴侣GRP78表达上调
GRP78是位于内质网中重要的分子伴侣,帮助蛋白质的折叠和成熟,是广泛使用的ERS激活的标志分子。免疫组织化学、免疫印迹法和实时定量PCR法检测GRP78在两组肾脏组织中的分布范围和表达水平。结果显示,正常肾脏组织中,GRP78在肾小管细段和远曲肾小管上皮细胞中有轻度表达,而在糖尿病肾脏中,GRP78在肾小球和近端肾小管表达升高,在肾小管细段以及远端肾小管的表达则有显著的升高(P<0.05)。蛋白水平和mRNA水平的检测结果与免疫组化结果相一致,GRP78在糖尿病肾脏中表达明显上调(P<0.05)。ERS在糖尿病肾损害中激活。
5.内质网相关凋亡通路的检测
CHOP, JNK和Caspase-12分别是三条内质网相关凋亡通路中的关键分子。三条通路相对独立,任一通路的激活均可导致细胞的凋亡。Western blot检测结果示与对照组相比,糖尿病组CHOP, p-JNK和Caspase-12的表达水平均明显升高(P<0.05)。mRNA水平检测,糖尿病组JNK mRNA水平与对照组比较无显著性差异(P>0.05),CHOP, Caspase-12 mRNA表达水平则明显升高(P<0.05)。免疫组化结果显示,与对照组相比,在糖尿病大鼠肾脏中,三个分子的分布范围和表达强度明显升高,但三个分子之间相比,分布范围存在显著差异。Caspase-12在对照组肾脏组织中不能检测到,在糖尿病肾脏中,阳性细胞主要分布在肾小球中；p-JNK的分布主要是肾小球和肾小管区间质中,尤其是间质的小血管中,呈强阳性表达；CHOP的分布则主要是远曲小管上皮细胞中,在肾小球和近端小管中也可检测到颗粒样阳性表达。因此,3条内质网相关凋亡通路都可能参与DN的病理发生,但具体的激活途径和应激细胞存在差异。
结论
1.STZ诱导的糖尿病大鼠16周,出现DN的表现,肾脏细胞凋亡增加；
2.糖尿病大鼠肾脏GRP78的表达显著上调,ERS在糖尿病大鼠肾损害中激活；
3. CHOP, JNK和Caspase-12介导的3条内质网相关凋亡通路在DN中都激活,但各自激活的方式和存在的细胞可能存在差异。
研究背景
肾小球硬化是糖尿病肾病DN最重要的特征性病理改变,其主要形成原因是肾小球系膜区系膜细胞增生和系膜基质的大量增加。但是到了DN的中晚期,硬化的肾小球内过多的系膜基质仍在,但系膜细胞的数量却呈耗竭状态。系膜细胞从过度增生到异常减少的过程中,过度的细胞凋亡被认为是肾小球系膜细胞缺失的重要机制。
实验证据证明,在DN的发生发展过程中,伴随着肾小球内血管紧张素Ⅱ(angiotensin II, ANGⅡ)的高水平表达。无论是在生理还是病理状态下,ANGⅡ与肾脏系膜细胞都具有密切的关系。肾脏系膜细胞是肾小球主要的组成细胞之一,位于毛细血管球之间,不仅对血管球起到支持和连接的作用,而且是系膜基质的主要来源细胞,通过其收缩功能调节肾小球的滤过面积,可分泌多种细胞因子等,尤其是病理损害状态下。系膜细胞表面不仅有ANGⅡ的受体ATl,而且系膜细胞还可促进ANGⅡ的裂解生成。ANGⅡ与细胞表面的受体结合,从而产生收缩细胞,促进细胞外基质合成和分泌细胞因子等效应。除此以外,ANGⅡ还有导致细胞损伤,诱导细胞凋亡的效应。
超微结构研究显示,肾脏系膜细胞具有发达的内质网系统。内质网是蛋白质翻译后加工的场所,尤其是蛋白质的糖基化修饰,分泌蛋白的空间折叠等,而系膜细胞的重要产物是细胞外基质,其成分主要是糖蛋白和蛋白多糖。系膜细胞的蛋白合成十分活跃,尤其是在肾小球硬化发展过程中,系膜基质处于过度增多状态。而各种原因造成内质网内未折叠蛋白蓄积,将诱发内质网应激(ERS)。适度的刺激,内质网通过暂时性中断细胞内蛋白合成、上调内质网分子伴侣和折叠酶的表达,诱导内质网相关性降解(ERAD)处理蓄积的蛋白等措施维持细胞存活,而持续或过度强烈的刺激,将导致内质网相关凋亡通路的激活,诱导细胞凋亡。
近年来研究发现,ANGⅡ是多种细胞凋亡的诱发因素,其发生机制可能与ERS相关。但目前ANGⅡ诱导肾脏系膜细胞凋亡的ERS通路还缺乏明确的试验证据。体外实验证据证明,ANGⅡ可诱导系膜细胞活性氧自由基(ROS)的产生。而氧化应激可导致ERS发生的试验证据已经比较充足,因此ANGⅡ可能通过氧化应激激活ERS凋亡通路诱导系膜细胞凋亡的发生。除此以外,ANGⅡ的促进蛋白合成效应如果造成未折叠蛋白在内质网中的过度蓄积,也将诱发ERS。因此,本试验将进行ANGⅡ的系膜细胞体外诱导凋亡试验,验证ERS凋亡通路是否参与其中,从而为肾脏细胞的保护提供新的治疗靶点。
研究目的
1.观察ANGⅡ是否激活系膜细胞的ERS。
2.明确ERS相关凋亡通路是否参与ANGⅡ诱导的系膜细胞凋亡。
研究方法
1.实验设计
1)体外培养大鼠肾脏系膜细胞系HBZY-1,分别给与ANGⅡ不同浓度(0,1,10,100 nM)和不同时间梯度(0,12,24,48小时)刺激,利用Annexin V/PI试剂盒,在流式细胞仪上检测细胞凋亡率,筛选出ANGⅡ最佳刺激浓度和时间；
2)给予系膜细胞最佳刺激浓度和时间ANGⅡ处理,检测ERS标志蛋白GRP78和ERS凋亡相关分子CHOP, Caspase-12和p-JNK的表达；
3)根据步骤2的结果,对凋亡过程中表达上调的ERS凋亡分子,给予相对应的siRNA预处理,然后进行ANGⅡ最佳刺激浓度和时间处理,检测细胞凋亡率的变化。
2.系膜细胞系的体外培养
大鼠肾脏系膜细胞系HBZY-1购自中国武汉细胞库,在低糖DMEM培养基(葡萄糖浓度5.5mmol／L)中培养,取代数在10-20,处于生长对数期的细胞进行试验。
3.细胞免疫荧光染色
将系膜细胞种植在放有玻片的6孔板中,制作细胞爬片。待细胞生长至70%融合状态,给予ANGⅡ最佳浓度和时间刺激。刺激完毕后,取出爬片,清洗,固定,封闭,GRP78一抗孵育,FITC标记的二抗反应后,DAPⅠ染核,荧光显微镜下观察ERS标记分子GRP78的表达。
4. Annexin V/PI试剂盒检测细胞凋亡率
细胞刺激完毕,胰酶消化细胞,制作适当浓度细胞悬液,分别加入Annexin V-FITC和PI进行标记反应,随即进行流式细胞仪的检测。
5. Western blot检测蛋白质的表达变化
细胞刺激完毕,细胞刮刀收集细胞,提取总蛋白,进行SDS-PAGE电泳分离,转膜,蛋白印迹和免疫反应后检测GRP78, CHOP, Caspase-12和p-JNK的蛋白表达水平。
6.实时定量RT-PCR法检测mRNA表达水平
利用Trizol法提取刺激细胞总RNA,以β-actin作为参照,通过real-timeRT-PCR技术检测GRP78、Caspase-12、CHOP和JNK的mRNA表达水平。
研究结果
1.ANGⅡ呈浓度和时间梯度依赖性诱导系膜细胞的凋亡
给予系膜细胞不同浓度(0,1,10,100 nM)ANGⅡ刺激24小时后,流式细胞仪检测细胞凋亡率。结果显示,细胞凋亡率呈逐渐上升趋势,分别为1.37%±0.41%,3.72%±1.23%,9.27%±3.53%,17.74%±5.19%(P<0.05)。而使用100nM的ANGⅡ给予系膜细胞不同时间段(0,12,24,48小时)的刺激,细胞凋亡率的结果分别为1.53%±0.49%,5.72%±1.97%,19.14%±6.53%,24.74%±9.19%(P<0.05)。但是在48小时组,细胞坏死率明显上升,与24小时组相比,细胞坏死率为4.53%±1.69%VS.15.76%±6.87%(P<0.05)。因此可知ANGⅡ呈浓度和时间梯度依赖性诱导系膜细胞的凋亡。在本试验中,最佳的ANGⅡ浓度为100nM,而最佳的刺激时间为24小时。
2.ANGⅡ上调ERS标志分子GRP78的表达
细胞免疫荧光染色,Western blot和Realtime PCR三种方法检测100nmol／LANGⅡ刺激24小时后,系膜细胞内GRP78的表达。结果示与空白刺激组相比,GRP78的mRNA和蛋白表达水平显著上调(P<0.05)。
3. CHOP和Caspase-12在ANGⅡ的ERS凋亡诱导中表达上调
ANGⅡ100 nmol/L刺激系膜细胞24小时后,收集细胞,Western blot和Realtime PCR分别检测ERS相关凋亡分子的表达变化,结果示：与对照组相比,CHOP和Caspase-12的蛋白质和mRNA表达水平显著增加(P<0.05),而p-JNK的表达无显著性差异(P>0.05)。提示ANGⅡ的ERS凋亡诱导可能通过CHOP和Caspase-12介导。
4. CHOP和Caspase-12 siRNA转染降低ANGⅡ诱导的系膜细胞凋亡率
系膜细胞生长至30-50%融合状态,分别转染CHOP和Caspase-12 siRNA。转染后24小时,再给以ANGⅡ100 nmol／L24小时刺激,流式细胞仪检测细胞凋亡率。结果示与ANGⅡ+对照转染组相比,CHOP和Caspase-12 siRNA组均可显著降低细胞凋亡率,对照转染组,CHOPsiRNA组和Caspase-12 siRNA组的凋亡率分别为31.74%±8.57%,20.61%±6.33%,18.94%±5.49%(P<0.05)。
结论
1. ANGⅡ可诱导肾脏系膜细胞ERS的激活；
2. ANGⅡ可通过CHOP和Caspase-12通路介导诱导系膜细胞的ERS凋亡效应。
Background
Diabetic nephropathy is a common and serious complication of diabetes mellitus, which leads to renal failure in up to 30% of individuals with diabetes and thus becomes the most important cause of end-stage renal diseases (ESRD). The pathogenesis of diabetic nephropathy is a chronic and complicated process, and more and more evidences support that apoptosis plays pivotal role in the development of nephropathy.
Apoptosis is the process of gene-controlled programmed cell death that occurs under physiology or pathology condition to keep the homeostasis. Cell apoptosis with cell proliferation control the balance of total cell number in each organ. Under diabetic condition, factors inducing the apoptosis increase which result in the intense of apoptosis overwhelming proliferation, then the number of functional cells decreases and finally leads to the organ failure. At present 3 different pathways are considered that mediate the process of cell apoptosis:mitochondrial pathway, death receptor pathway and newly found endoplasmic reticulum (ER) stress associated pathway.
ER is the largest organelle in the cell and involved in the metabolism of membrane/secreted protein, glycosaminoglucan, phospholipid, cholesterol, and calcium, especially in the secreted protein maturation, folding and transport. Once the insulting factors destroy the balance of protein production, maturation and degrading, the ER stress is activated. Several complex homeostatic signaling pathways, known as the unfolded protein response (UPR), have evolved to cope with the ER stress. The processes of UPR include:temporary suspension the general protein synthesis, up regulation the expression of ER resident proteins and molecular chaperons, inducing the ER associated degradation (ERAD). Though moderate ER stress could alleviate the damage caused by stress, prolonged or severe stress response leads to apoptosis and removes the damaged cells. But if the intense or the extent of apoptosis is out of control, excess functional cells lose, which result in the premature organ failure.
Renal cells, including mesangial cell, podocyte, tubular epithelial cell and et al, have well-developed ER, which is tightly related to the cell physiological function. Mesangial cells are the primary manufacturers of basal membrane and mesangial matrix. They are also involved in the degrading of mesangial immune complex, and secreting the bioactive substances such as renin and angiotensin. Podocytes are the components of the glomerular filtration barrier. Glycoprotein connected to the outer membrane of the cells plays the vital role in the filtration. Besides re-absorbing and degrading the filtrated protein, tubular cells also produce the cytokines such as bradykinin. Above all, ER fulfills multiple protein related functions in the renal cells and ER stress might be easily activated in the renal disease.
Under diabetic condition, ER stress insulting factors are prone to increase, including hyperglycemia, oxidative stress, proteinuria, and cellular electrolyte disorder. However, fewer publications are found between ER stress and diabetic kidney injury. So the aim of this research is to investigate the role of ER stress in the diabetic nephropathy, and then provide possible strategies on the kidney protection.
Objectives
1. To establish the diabetic rat model, and demonstrate the increment of apoptotic renal cells.
2. To investigate the activation of ER stress in the diabetic kidney.
3. To explore the signaling pathways of ER associated apoptosis in the diabetic kidney injury.
Methods
1. Establishment of diabetic rat model
30 male Wistar rats (7 weeks of age; mean body weight 280±10 g) were used. After one week of acclimatization, of the 30 Wistar rats,20 were given a single intraperitoneal dose (65 mg/kg) of streptozotocin (STZ, dissolved in citrate buffer (pH 4.5)), while the remaining 10 rats were given the same dosage of citrate buffer (pH 4.5). Hyperglycemia was confirmed by measuring the venous circulating plasma concentrations of glucose. Seven days post-STZ injection, blood samples were obtained from the rat tail vein after 12 hours of fasting, and the rats with glucose concentration higher than 300 mg/dl were kept in diabetic group. All rats were killed at the 16th week after saline or STZ injection.
2. Blood glucose test and 24-hour proteinuria examination
Blood glucose was detected per 4 weeks. At the end of the 1st and 16th week,24-hour urine was collected with the metabolic cage and the levels of proteinuria were measured.
3. Renal histopathological staining
Kidneys were transversally sliced and fixed in 4% neutral formalin for over 24 hours, and then embedded in paraffin, cut into 4-μm thick sections for staining with H&E or PAS methods. Then slides were observed under the microscope to determine if the diabetic kidney injury occurred or not.
4. TUNEL staining
TUNEL staining was used to detect the expression of apoptotic cells in the diabetic kidney.
5. Immunohistochemical staining
Immunohistochemical staining was used to detect the protein expression of GRP78, CHOP, Caspase-12 and p-JNK. The intensity and extent of the positive area were calculated and compared.
6. Western blot analysis
Total proteins of the renal samples were extracted and conducted 10—14% SDS-PAGE. Then the protein expression levels of GRP78, CHOP, Caspase-12, Total-JNK and p-JNK were measured.
7. Realtime RT-PCR analysis
Realtime RT-PCR was used to detect the mRNA levels of GRP78, CHOP, Caspase-12, and JNK.
Results
1. General features of the experimental rats
In the whole experiment,3 diabetic rats died and might be caused by diabetic ketoacidosis, infection or other diabetic complications. Finally 27 rats survived and 10 from control group and 17 from diabetic group. Compared to the control, diabetic rats seemed to be inactive, and with symptoms of lower body weights, polyuria and polyphagia, which are normally associated with diabetic state.
2. The diagnosis of diabetic nephropathy
The diagnosis of diabetic nephropathy was determined by blood glucose monitoring,24-hour proteinuria detection and renal pathology. Compared to the control, concentrations of glucose blood in diabetic rats were always higher than 16.7 mmol/L (P<0.05). At the end of 16th week, the level of 24-hour proteinuria increased obviously in diabetic rats (P<0.05). And the examination of renal pathology showed glomerular hypertrophy, glomerular basement membrane thickness, glomerulosclerosis, renal tubular atrophy and interstitial fibrosis in diabetic kidney.
3. Enhanced apoptotic state in diabetic kidney
The results of TUNEL assay showed that apoptosis was observed in both the cortex and medulla of the diabetic kidney. Estimation of renal apoptosis revealed a nearly fivefold increase in TUNEL-positive nuclei in diabetic kidneys compared to the normal (P<0.05).
4. Up-regulation of ER chaperone GRP78 in kidneys of diabetes
GRP78, one important molecular chaperone localized in ER, has been used extensively as an indicator for the induction of ER stress. Immunohistochemistry study showed that GRP78 was abundantly expressed in the renal tissue from diabetic rats (P<0.05). In contrast, normal rats exhibited modest or weak immunoreactivity for this molecule. The result of western blot analysis confirmed the finding. Furthermore, real-time PCR analysis showed the same trend of grp78 in mRNA level (P <0.05). All these findings suggested the activation of ER stress in the kidneys of diabetic rats.
5. Involvement of the ER-associated apoptosis pathways
CHOP, JNK and caspase-12 are the key molecules in the three branches of ER-associated apoptosis pathways. Firstly, western blots were used to check the involvement of these pathways in diabetic kidneys. The densitometric analysis of bands for CHOP, caspase-12, and p-JNK revealed a significant increase in relative protein content in renal tissue from diabetic rats in comparison with those from normal rats(P<0.05). This means CHOP was induced, and caspase-12 and JNK pathway were activated in the diabetic kidney. Changes in RNA expression of jnk, chop, and caspase-12 were quantified by RT-PCR in control and diabetic rat kidneys. Expressions of chop, and caspase-12 were significantly increased in the diabetic kidneys (P<0.05), paralleled with their enhanced protein expression. However, the result of jnk did not show any differences between the diabetes and control (P>0.05). At last, the results of immunostaining provided more useful information. In diabetic kidney, Caspase-12 positive area only appeared in the glomerulus; p-JNK was mainly expressed in the glomerulus and interstitial area, especially the small vessels; CHOP could be observed in the distal convoluted tubule area, and focal expression in the glomerulus and proximal tubular area. The differences might be caused by the variance of involved cells and insulting factors.
Conclusion
1. The STZ induced diabetic rats showed classic diabetic nephropathy performances and enhanced apoptotic state.
2. The expression of ER chaperone GRP78 was up-regulated in diabetic kidney, which directly demonstrated the activation of ER stress.
3. CHOP, JNK and Caspase-12, three branches of ER associated apoptosis, were all stimulated in diabetic kidney. However, the activated modes or the cells involved may be different.
Background
Glomerulosclerosis, which is characterized by the expansion of mesangium, is the most important pathological change in the early stage of diabetic nephropathy. The expanded mesangium is comprised of a large number of mesangial cells and increased amount of matrix. However, in the later course of the disease, there is an abundance of matrix and a limited number of mesangial cells. And apoptosis is assumed to play the vital role in the mesangial cell loss.
Evidences show that intra-renal levels of angiotensinⅡ(ANGII) are elevated in the development of diabetic nephropathy. ANG II plays an important role in the function maintenance of mesangial cells in both normal and diseased states. Mesangial cells are specialized pericytes located among the glomerular capillaries within a renal corpuscle of a kidney. They are not only providing structural support for and regulate blood flow of the glomerular capillaries by their contractile activity, but also are the major contributors to the extracellular matrix which contains fibronectin, type IV collagen, perlecan, and laminin. All the functions mentioned above are tightly related to the ANGII regulation. However, ANGII could insult kinds of cells and induce the apoptosis or necrosis.
Ultrastructural analysis results show that endoplasmic reticulum (ER) in mesangial cell is well-developed, and in an active state. The ER is the site of biosynthesis for steroids, cholesterol and other lipids. It is also the sub-cellular entrance for a number of secretory and structural proteins and provides a unique environment for appropriate protein folding and assembly to produce functional, mature proteins. What's more, protein glycosylation is complicated in the ER. As is known, extracellular matrix is mainly comprised by glycoprotein and proteoglycan. And under the diabetic state, matrix is overloaded in the glomerular mesangium. Homeostasis in the ER is maintained by a coordinated adaptive program, so-called the unfolded protein response (UPR). A number of microenvironmental, developmental and pathophysiological insults as well as a wide range of chemical substances cause accumulation of unfolded proteins in the ER and activate ER stress. In response to accumulation of unfolded proteins, cells adapt themselves to the stress conditions via UPR:attenuation of general translation, induction of ER chaperones and activation of ER-associated degradation (ERAD) to eliminate immature proteins. If the stress is beyond the capacity of the adaptive machinery, cells undergo apoptosis Accumulated evidences showed that ANGII could induce the apoptosis of many cells, and the mechanisms might involve the ERS response. However, whether ANGII inducing apoptosis of mesangial cells is mediated by ERS is still unknown. On the one hand, ANGII has been demonstrated to stimulate the generations of reactive oxygen species in mesangial cells, and the relationship between oxidative stress and ERS is undeniable; On the other hand, ANGII promotes the protein synthesis in ER and might result in the unfolded protein accumulation,thus activate the ERS. Therefore the aim of this study is to investigate the role of ERS in the ANGII-induced apoptosis of mesangial cell.
Objectives
1. To observe the hallmark of ERS activation in ANGII-induced apoptosis of mesangial cell.
2. To explore the ERS associated apoptotic pathways in ANGII-induced apoptosis of mesangial cell.
Methods
1. Study design
1) The established rat renal mesangial cell line HBZY-1 was incubated with ANGII at different concentration (0,1,10,100 nmol/L) and duration (0,12,24,48 hours) to select the best treatment condition. After harvesting, apoptotic cells were labeled with Annexin V/PI kit, and the percentage of apoptosis was calculated with flow cytometer.
2) Cultured cells were treated with ANGII at the selected concentration and duration. Then Immunofluorescence staining, western blot and realtime PCR were used to measure the expression of GRP78, CHOP, Caspase-12 and p-JNK on protein and mRNA levels.
3) According to result of 2nd step, to synthesize the siRNA against ERS apoptotic molecules. Treated cells with siRNA before ANGII incubation, and then detected the apoptotic percentage with flow cytometer.
2. Cell culture
The established rat renal mesangial cell line HBZY-1, which was purchased from Wuhan cell center, was cultured in low-glucose DMEM. Cells in generation 10-20 were used in the treatment.
3. Immunofluorescence staining
Cells were grown on glass slides in the presence of selected concentration of ANGII for the proper duration. Then the cells were fixed with 4% paraformaldehyde for 15 minutes at 4℃and permeabilized in 0.1% Triton X-100 for 10 minutes at 4℃. The cells were then blocked in 10% rabbit blocking serum at 37℃for 30 minutes followed by incubation with GRP78 goat polyclonal IgG (1:50) overnight at 4℃. After washes with PBS three times, cells were incubated with FITC-conjugated rabbit anti-goat IgG (1:50) at 37℃for 1 hour. The results were evaluated under fluorescing microscope.
4. Annexin V/PI kit to measure the percentage of apoptosis When the stimulation was over, cells were digested with 0.25% trypsin and collected, then resuspended in binding buffer with annexin V-FITC and PI. After staining, cells were immediately analyzed with flow cytometer.
5. Western blot The total protein was extracted from the harvested cells. Western blot was used to detect the protein levels of GRP78, CHOP, Caspase-12 and p-JNK.
6. Realtime RT-PCR analysis The total RNA was extracted from the harvested cells with Trizol. Realtime RT-PCR was used to detect the mRNA levels of GRP78, CHOP, Caspase-12 and JNK.
Results
1. ANGII induced apoptosis of mesangial cells in a dose-and time-dependent manner
Treated mesangial cells with ANGII of different concentration (0,1,10,100 nmol/L) for 24 hours and measured the percentage of apoptosis, the results showed that the apoptotic ratio was increased gradually, were 1.37%±0.41%, 3.72%±1.23%,9.27%±3.53%,17.74%±5.19%respectively (P <0.05) Exposed cells with 100nmol/L ANGII for different duration (0, 12,24,48 hours) and the results of apoptosis tests are 1.53%±0.49%,5.72%±1.97%,19.14%±6.53%,24.74%±9.19% for each time point (P<0.05). Thus ANGII induced apoptosis of mesangial cells in a dose-and time-dependent manner. And the best ANG concentration and duration selected were 100nmol/L and 24 hours.
2. Up-regulation of ER chaperone GRP78
Immunofluorescence staining, Western blot and Realtime PCR, three methods were used to detect the expression of GRP78 after the ANGII treatment in the mesangial cell. And the results showed that GRP78 was obviously upregulated on both mRNA and protein levels compared to the control group (P<0.05).
3. The expressions of CHOP and Caspase-12 were elevated in ANGII
induced mesangial cell apoptosis Three key molecules of ERS associated apoptosis, CHOP, Caspase-12 and JNK, were examined with western blot and real time PCR after cell exposed to ANGII at selected concentration and duration. Results showed that the expressions of CHOP and Caspase-12 were increased dramatically compared to the control (P<0.05), but there was no change for JNK expression (P>0.05).
4. CHOP and Caspase-12 siRNA decreased the apoptosis induced by ANGII in mesangial cell Cells were transfected with CHOP and Caspase-12 siRNA 24 hours before ANGII treatment. And the results of apoptosis percentage showed that CHOP and Caspase-12 siRNA could decrease the apoptosis induced by ANGII in mesangial cell. The apoptotic percentages for control siRNA group, group CHOP siRNA and group Caspase-12 siRNA are 31.74%±8.57%, 20.61%±6.33%,18.94%±5.49%(P<0.05)。
Conclusion
1. ANGII could activate the ERS in renal mesangial cells.
2. ANGII-induced ERS associated apoptosis of mesangial cell might be mediated by CHOP and Caspase-12 pathways.
引文
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