血管紧张素Ⅱ对大鼠肾小球内皮细胞炎性因子MCP-1表达和增殖、凋亡的影响及其AT_1受体拮抗剂替米沙坦作用的研究
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摘要
前言
肾内肾素-血管紧张素系统(renin-angiotensin system,RAS)的局部激活是多种慢性肾脏病变发生发展过程中的主要特点,血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)作为该系统的效应因子在机体血容量、血流动力学及内环境稳态的调节中发挥主导作用。已有研究表明肾内局部升高的AngⅡ水平与高血压、糖尿病时肾脏病变的发生发展关系密切。
研究发现肾内局部升高的AngⅡ不仅可以导致球内高压,同时还具有生长因子和炎症因子的作用,致使肾小球发生硬化病变。肾小球内皮细胞(GlomerularEndothelial Cells,GECs)又称肾脏微血管内皮细胞,是肾小球滤过屏障的重要组成部分,承受着远高于一般毛细血管的血压,是血液内的致病因子以及血液动力学变化易损伤的靶细胞,受损激活后可分泌单核细胞趋化因子(monocytechemoattractant protein-1,MCP-1)等多种炎症因子。目前有关AngⅡ是否会诱导肾小球内皮细胞发生炎症反应及其对增殖和凋亡作用方面的研究,尚未见报道。
血管活性肽AngⅡ可激活多条细胞内信号通路,刺激细胞内活性氧基团(reactive oxygen species,ROS)的生成。细胞内ROS的主要来源是通过还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶系统的活化,使活性氧基团ROS生成增加。p47phox蛋白是NADPH氧化酶的亚基,对NADPH氧化酶的活化起到重要作用。ROS作为极其重要的细胞内信使,与多条信号通路的活化相关。核因子-κB(Nuclear factor-κB,NF-κB)的活化,可上调多种相关炎性因子的表达。p38MAPK信号通路的激活,不仅在炎症、应激反应中具有重要作用,还参与细胞的存活、分化和凋亡等过程。AngⅡ的作用主要通过AT1和AT2两种类型受体所介导,在成人肾脏组织中,AT1受体分布广泛,而AT2受体分布很少。AngⅡ在心血管系统中的作用主要通过AT1受体介导。
目前有关AngⅡ对血管内皮细胞结构和功能影响的研究愈来愈受到重视,本实验主要从炎症、增殖和凋亡方面探讨AngⅡ对大鼠肾小球内皮细胞的作用及其相关机制,以及AT1受体拮抗剂替米沙坦对其作用的影响。
实验方法
1、大鼠肾小球内皮细胞的分离培养与鉴定
SD胎大鼠(20-30g)无菌条件下取双肾,剥去被膜,眼科剪剪下肾皮质部分于培养皿中,剪碎后经尼龙筛网滤过,采用胰蛋白酶消化法进行原代细胞培养。第3-5代细胞用于实验。细胞的鉴定采用形态学观察法和免疫荧光法检测第Ⅷ因子相关抗原。
2、Western Blot法检测RGECs的MCP-1、p47phox蛋白表达
收集各组细胞,经RIPA裂解液提取细胞总蛋白,以Lowry法检测总蛋白浓度。电泳后,将蛋白转印到PVDF膜上,5%脱脂奶粉室温封闭2 h,分别加入MCP-1、p47phox一抗(1:200)4℃孵育过夜,二抗(1:2000)37℃孵育2 h,DAB显色,观察结果。扫描PVDF膜并对其进行灰度值测定,对比各组间蛋白表达的差异,以β-actin作为对照。
3、RT-PCR法检测AT1受体mRNA水平
应用Trizol提取各组细胞总RNA,紫外分光光度计测定RNA的A260/A280比值,琼脂糖凝胶电泳证实所提RNA的完整性。RT和PCR反应按照说明书进行操作,PCR产物5μL用2%琼脂糖凝胶电泳进行检测,采用凝胶成像分析系统进行半定量分析,紫外灯下观察结果并拍照。
4、细胞内活性氧ROS的测定
采用2',7'二氯荧光黄双乙酸盐(DCFH-DA)荧光染色检测ROS。DCFH-DA本身没有荧光,但可以自由穿过细胞膜,进入细胞内后,细胞内的ROS可以氧化无荧光的DCFH生成有荧光的DCF。将培养的细胞接种于铺有盖玻片的24孔板中,给予AngⅡ(10~(-5)mol/L)作用不同时间(0 min、5 min、10 min、15 min、20min)后,用无血清的培养基清洗细胞两次,加入DCFH-DA(1:1000稀释)到24孔板中,37℃孵育箱中孵育20 min,荧光共聚焦显微镜观察细胞内ROS的荧光强度(激发波长488 nm,发射波长525 nm)。
5、免疫荧光法检测NF-κB活化
将培养的细胞接种于铺有盖玻片的24孔板中,给予AngⅡ(10~(-5)mol/L)作用不同时间(0 min、15 min、30 min、45 min)后,细胞用4%多聚甲醛固定10min,PBS洗后,滴加0.1%Triton—100“打孔”,室温10min;PBS洗;5%BSA封闭10 min,弃去不洗,滴一抗(1:300),4℃孵育过夜;PBS洗,滴加FITC标记二抗(1:100),室温1h,PBS洗,90%甘油封片。荧光显微镜下观察。
6、MTT法检测大鼠GECs的增殖率
GECs以2×10~3个/孔的密度接种至96孔板,80%融合后无血清培养液静置12h,分组继续培养。24h后每孔加入MTT溶液(5g/L)20μl,继续孵育4 h,终止培养。吸弃孔中的培养上清,每孔加入150μl DMSO,振荡10 min,酶联免疫检测分析仪测定各组吸光度(OD值),并计算细胞增值率。
7、AV/PI法流式细胞仪检测大鼠GECs的凋亡率
GECs以5×10~5个/瓶传代至25mL的培养瓶中,待融合后无血清培养液静置12h,分组继续培养24 h后,消化收集细胞,将其吹打成单细胞悬液,离心弃上清,用PBS漂洗,再离心,再漂洗后以100目筛网过滤,然后再离心加入细胞凋亡检测AV-PI(磷脂酰丝氨酸结合蛋白联合碘化丙啶双标记)工作液0.5ml,终浓度为10mg/L,室温避光30min后,进行流式细胞仪检测,计数凋亡细胞率。
8、统计学分析
采用SPSS17.0统计软件处理,计量资料以均数±标准差(x±s)表示,采用one-way ANOVA分析,组间比较采用LSD检验,P＜0.05为差异有统计学意
实验结果
1、大鼠肾小球内皮细胞的培养与鉴定
原代分离培养肾小球内皮细胞生长5-7天时,呈单层贴壁生长,互不重叠,细胞呈角形或短梭形,外观呈铺路石状,边界清楚。Ⅷ因子相关抗原免疫荧光鉴定,大鼠GECs胞膜和胞浆处呈红色荧光,证实为肾小球内皮细胞。
2、AngⅡ对大鼠肾小球内皮细胞MCP-1蛋白表达的影响
Western blot检测结果显示,浓度效应组中,正常对照组MCP-1蛋白表达量较低,施加AngⅡ刺激因素后MCP-1表达量明显高于正常对照组,且随着浓度的增加(10~(-7)-10~(-5)M)而表达增加,成剂量依赖效应;NADPH氧化酶抑制剂DPI(10~(-6)mol/L)和AT1受体拮抗剂TEL(10~(-6)mol/L)抑制AngⅡ的作用。
3、AngⅡ对大鼠肾小球内皮细胞p47phox蛋白表达的影响
Western blot检测结果显示,与正常对照组相比给予10~(-5)M AngⅡ刺激因素后,大鼠肾小球内皮细胞内p47phox蛋白表达明显增加,成时间依赖效应。AngⅡ的这一作用效果可分别被TEL(10~(-6)M)和DPI(10~(-6)M)所抑制。
4、AngⅡ对大鼠肾小球内皮细胞AT_1受体mRNA水平的影响
RT-PCR检测结果显示,与正常对照组相比给予10~(-5)mol/L的AngⅡ刺激因素后,大鼠肾小球内皮细胞AT1受体的mRNA水平明显增加,成时间依赖效应。
5、AngⅡ对大鼠肾小球内皮细胞ROS生成的影响
DCFH-DA荧光染色检测结果显示,与正常对照组相比,随着作用时间延长AngⅡ作用组细胞内ROS的生成量逐渐增加,15min时ROS生成量达到顶峰,后逐渐减少;加入TEL和DPI后DCFH荧光值降低,ROS生成量明显减少。
6、AngⅡ对大鼠肾小球内皮细胞NF-κB活化的影响
正常状态无AngⅡ刺激时,NF-κB主要表达在细胞浆中,细胞核内少见表达;给予AngⅡ刺激因素后NF-κB即开始活化,迅速核转移,荧光显微镜显示30 min时NF-κB核内表达达到高峰,后逐渐减弱。药物TEL和DPI抑制NF-κB的核转移。
7、AngⅡ对大鼠GECs增殖的影响
与对照组相比较,低浓度AngⅡ(10~(-8)M)作用组,大鼠GECs增殖率增加1.33倍左右,高浓度AngⅡ(10~(-6)M)作用组,细胞增殖率降低2.3倍左右,分别为1.33±0.03和0.43±0.03(与对照组比较,P＜0.01)。低浓度AngⅡ组和高浓度AngⅡ组之间增殖率有统计学差异。
8、AngⅡ对大鼠GECs凋亡的影响
与对照组比较,低浓度AngⅡ(10~(-8)M)组和高浓度AngⅡ(10~(-6)M)组大鼠GECs凋亡率分别降低和增加50%左右,与高浓度AngⅡ(10~(-6)M)组比较,AngⅡ+TEL和AngⅡ+SB组细胞凋亡率均下降,但两组间细胞凋亡率无统计学差异。
结论
1、AngⅡ促进RGECs的MCP-1表达,在一定范围内呈剂量依赖效应;
2、AngⅡ上调RGECs的AT1受体mRNA水平,在一定范围内呈时间依赖效应;
3、AngⅡ上调RGECs的p47phox蛋白表达,通过NADPH氧化酶依赖的ROS生成途径促进NF-κB的活化;
4、AT1受体拮抗剂替米沙坦可抑制AngⅡ的促炎性因子MCP-1表达作用;
5、低浓度AngⅡ促进RGECs的增殖,高浓度AngⅡ抑制RGECs增殖;
6、低浓度AngⅡ抑制RGECs的凋亡,高浓度AngⅡ促进RGECs凋亡;
7、p38MAPK信号通路参与AngⅡ对肾小球内皮细胞凋亡的调控;
8、AT_1受体拮抗剂替米沙坦抑制AngⅡ所诱导的RGECs凋亡发生。
Introduction
Activation of the intrarenal renin-angiotensin system(RAS) is a characteristic feature in the development and progression of chronic kidney disease.AngiotensinⅡ(AngⅡ),the main effector of RAS,is implicated in the pathogenesis of renal diseases. AngⅡinitiates its effects by interaction with at lease two pharmacologically distinct subtypes of cell-surface receptors,AT1 and AT2,The major functions of AngⅡin cardiovascular system are mediated through AT1 receptor.
An intriguing concept has emerged that AngⅡnot only mediates intraglomerular hypertension but also behaves as a pro-inflammatory and growth-stimulating factor contributing to the renal hypertension and sclerosis.AngⅡhas growth-stimulating properties on different renal cell types.However,possible inflammatory effects of this vasoactive peptide on endothelial cells isolated from the glomerular microvascularture have not formally been investigated.Glomerular endothelial cells(GECs),being exposed to the bloodstream,are the target of many different factors,that may alter the functional state of these cells and induce them to release inflammatory cytokines.
Inflammatory processes involve both the synthesis of inflammatory cytokines, such as monocyte chemoattractant protein-1(MCP-1),and the activation of their distinct signaling cascades.Recent findings suggest that AngⅡactivates intracellular signaling processes,including the polyol pathway and generation of reactive oxygen species(ROS).In cellular systems,a major source of ROS derives from the membrane-bound NAD(P)H oxidase system.Activation of NAD(P)H oxidase system requires the participation of p47phox,one of the NAD(P)H oxidase subunits,which plays a central role in the scenario of NAD(P)H oxidase activation.10Activation of nuclear factor -ΚB(NF-ΚB) is involved in the expression of pro-inflammatory genes.11 In this study,we investigated wether AngⅡactivates the NF-ΚB cascade by ROS and whether the ROS-dependent mechanism may be involved in AngⅡ-induced MCP-1 formation.Further,we tested whether telmisartan,an AT1 receptor blocker,and DPI,an inhibitor of NAD(P)H oxidase,modulate endothelial inflammation and oxidative cell damage induced by AngⅡ-dependent stimuli in cultured rat glomerular endothelial cells.
Methods
We isolated and characterized primary cultures in rat glomerular endothelial cells(GECs).We found that AngⅡinduced the synthesis of monocyte chemoattractant protein -1(MCP-1) in rat GECs determined by Western blot.AngⅡstimulation of rat GECs induced a rapid increase in reactive oxygen species(ROS) generation determined by laser fluoroscopy.The level of p47phox protein,a nicotinamide-adenine dinucleotide phosphate(NADPH) oxidase subunit,was also increased by AngⅡtreatment.These effects of AngⅡon GECs were all reduced by DPI,a NAD(P)H oxidase inhibitor.AngⅡstimulation also promoted the activation of Nuclear factor-k B (NF-ΚB).Telmisartan(TEL),an AT1 receptor blocker,blocked all the effect of AngⅡon rat GECs.We study the effect of AngⅡon the propagation and apoptosis of RGECs by the methods of MTT and AV/PI.
Results
We found that AngⅡinduced the synthesis of monocyte chemoattractant protein -1(MCP-1) in rat GECs determined by Western blot.AngⅡstimulation of rat GECs induced a rapid increase in reactive oxygen species(ROS) generation determined by laser fluoroscopy.The level of p47phox protein,a nicotinamide-adenine dinucleotide phosphate(NADPH) oxidase subunit,was also increased by AngⅡtreatment.These effects of AngⅡon GECs were all reduced by DPI,a NAD(P)H oxidase inhibitor.AngⅡstimulation also promoted the activation of Nuclear factor-k B(NF-ΚB).And the effect of AngⅡon the propagation and apoptosis of RGECs depends on its dose denstiny.Telmisartan(TEL),an AT1 receptor blocker,blocked all the effect of AugⅡon rat GECs.
Discussion
The present study demonstrates that AngⅡ-induced ROS generation in rat GECs depends on the P47phox subunit of NADPH Oxidase.ROS are additionally required for AngⅡ-induced activation of NF-ΚB.Blockade of the NADPH Oxidase by its inhibitor DPI significantly abolished AngⅡ-induced MCP-1 formation,indicating that NF-ΚB when activated by ROS,participates in the AngⅡ-induced MCP-1 production.
It's now well accepted that AngⅡmay function as a potent pro-inflammatory mediator and be implicated in the pathogenesis of chronic renal disease.13,14GECs are at the interface between blood and adjacent cell population and play a crucial role in preserving the kidney function.The monocyte-endothelium interaction induced by AngⅡmay contribute to the initiation of vascular inflammation.15 MCP-1,when expressed at the plasma membrane of endothelial cells,can mediate the initial capture of monocytes.16 Our results suggest that AngⅡcan induce MCP-1 formation in rat GECs and promote the monocyte adhesion to endothelial cells.
AngⅡacts via AT1 and AT2 receptors.Both receptor subtypes have been found in rat GECs.17 AngⅡexerts most of its already well-defined physiologic and pathophysiologic actions through AT1 receptors.18 Consistent with this concept,our results showed that the AngⅡeffect on MCP-1 formation was inhibited by the selective AT1 receptor antagonist telmisartan.
In mammalian cells,a major source of ROS derives from the membrane-bound NADPH Oxidase system,which exists in nonphagocytic cells of the vascular wall,for example,fibroblast,vascular smooth muscle cells and endothelial cells.19 Increased ROS generation can induce cell inflammation.20,21 The present study isolated rat glomerular endothelial cells and demonstrated that the expression of p47phox protein, one of the NADPH Oxidase subunits,was up-regulated by AngⅡtreatment in accordance with the increased ROS generation.Moreover,blockade of NADPH Oxidase by DPI reduced the P47phox protein synthesis and ROS generation.
Nuclear factor-ΚB normally exists in the cytoplasm in an inactive form bound to the inhibitory protein IΚB.Upon cellular activation,IΚB is rapidly degraded prior to the translocation of NF-ΚB into the nucleus and its subsequent activation,resulting in the transcriptional regulation of target genes encoding pro-inflammatory cytokines.NF-ΚB consists of two subunits,p50 and p65,with p65 containing a transcription domain.11,22 We here studied the possible involvement of NF-ΚB activation in AngⅡ-induced MCP-1 production in rat GECs.In the present study,AngⅡstimulation increased the translocation of NF-ΚB P65 subunit to the nucleus,which was reduced by DPI.The observation suggests that AngⅡ-induced NF-ΚB activation is partially mediated by ROS.
In summary,we demonstrated that AngⅡinduces MCP-1 production in rat GECs partially via the signaling of ROS-dependent NF-ΚB activation,which can be inhibited by the AT1 receptor antagonist telmisartan.Our finding may provide a mechanistic basis for the benefits of selective AT1 blockade in dealing with the chronic renal disease.
Conclusions
1 AngⅡincreases the expression of MCP- 1 and AT1mRNA;
2 AngⅡincreases the expression of p47phox in a certain ranges showing a time-dependent manner.
3 AngⅡinduces MCP-1 production in rat GECs partially via the signaling of ROS-dependent NF-ΚB activation,which can be inhibited by the AT1 receptor antagonist telmisartan.
4 the effect of AngⅡon the propagation and apoptosis of RGECs depends on its dose denstiny.
肾内肾素-血管紧张素系统(renin-angiotensin system,RAS)的局部激活是多种慢性肾脏病变发生发展过程中的主要特点,血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)作为该系统的效应因子在机体血容量、血流动力学及内环境稳态的调节中发挥主导作用。已有研究表明肾内局部升高的AngⅡ水平与高血压、糖尿病时肾脏病变的发生发展关系密切。
研究发现肾内局部升高的AngⅡ不仅可以导致球内高压,同时还具有生长因子和炎症因子的作用,致使肾小球发生硬化病变。肾小球内皮细胞(GlomerularEndothelial Cells,GECs)又称肾脏微血管内皮细胞,是肾小球滤过屏障的重要组成部分,承受着远高于一般毛细血管的血压,是血液内的致病因子以及血液动力学变化易损伤的靶细胞,受损激活后可分泌单核细胞趋化因子(monocytechemoattractant protein-1,MCP-1)等多种炎症因子。目前有关AngⅡ是否会诱导肾小球内皮细胞发生炎症反应及其对增殖和凋亡作用方面的研究,尚未见报道。
血管活性肽AngⅡ可激活多条细胞内信号通路,刺激细胞内活性氧基团(reactive oxygen species,ROS)的生成。细胞内ROS的主要来源是通过还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶系统的活化,使活性氧基团ROS生成增加。p47phox蛋白是NADPH氧化酶的亚基,对NADPH氧化酶的活化起到重要作用。ROS作为极其重要的细胞内信使,与多条信号通路的活化相关。核因子-κB(Nuclear factor-κB,NF-κB)的活化,可上调多种相关炎性因子的表达。p38MAPK信号通路的激活,不仅在炎症、应激反应中具有重要作用,还参与细胞的存活、分化和凋亡等过程。AngⅡ的作用主要通过AT1和AT2两种类型受体所介导,在成人肾脏组织中,AT1受体分布广泛,而AT2受体分布很少。AngⅡ在心血管系统中的作用主要通过AT1受体介导。
目前有关AngⅡ对血管内皮细胞结构和功能影响的研究愈来愈受到重视,本实验主要从炎症、增殖和凋亡方面探讨AngⅡ对大鼠肾小球内皮细胞的作用及其相关机制,以及AT1受体拮抗剂替米沙坦对其作用的影响。
实验方法
1、大鼠肾小球内皮细胞的分离培养与鉴定
SD胎大鼠(20-30g)无菌条件下取双肾,剥去被膜,眼科剪剪下肾皮质部分于培养皿中,剪碎后经尼龙筛网滤过,采用胰蛋白酶消化法进行原代细胞培养。第3-5代细胞用于实验。细胞的鉴定采用形态学观察法和免疫荧光法检测第Ⅷ因子相关抗原。
2、Western Blot法检测RGECs的MCP-1、p47phox蛋白表达
收集各组细胞,经RIPA裂解液提取细胞总蛋白,以Lowry法检测总蛋白浓度。电泳后,将蛋白转印到PVDF膜上,5%脱脂奶粉室温封闭2 h,分别加入MCP-1、p47phox一抗(1:200)4℃孵育过夜,二抗(1:2000)37℃孵育2 h,DAB显色,观察结果。扫描PVDF膜并对其进行灰度值测定,对比各组间蛋白表达的差异,以β-actin作为对照。
3、RT-PCR法检测AT1受体mRNA水平
应用Trizol提取各组细胞总RNA,紫外分光光度计测定RNA的A260/A280比值,琼脂糖凝胶电泳证实所提RNA的完整性。RT和PCR反应按照说明书进行操作,PCR产物5μL用2%琼脂糖凝胶电泳进行检测,采用凝胶成像分析系统进行半定量分析,紫外灯下观察结果并拍照。
4、细胞内活性氧ROS的测定
采用2',7'二氯荧光黄双乙酸盐(DCFH-DA)荧光染色检测ROS。DCFH-DA本身没有荧光,但可以自由穿过细胞膜,进入细胞内后,细胞内的ROS可以氧化无荧光的DCFH生成有荧光的DCF。将培养的细胞接种于铺有盖玻片的24孔板中,给予AngⅡ(10~(-5)mol/L)作用不同时间(0 min、5 min、10 min、15 min、20min)后,用无血清的培养基清洗细胞两次,加入DCFH-DA(1:1000稀释)到24孔板中,37℃孵育箱中孵育20 min,荧光共聚焦显微镜观察细胞内ROS的荧光强度(激发波长488 nm,发射波长525 nm)。
5、免疫荧光法检测NF-κB活化
将培养的细胞接种于铺有盖玻片的24孔板中,给予AngⅡ(10~(-5)mol/L)作用不同时间(0 min、15 min、30 min、45 min)后,细胞用4%多聚甲醛固定10min,PBS洗后,滴加0.1%Triton—100“打孔”,室温10min;PBS洗;5%BSA封闭10 min,弃去不洗,滴一抗(1:300),4℃孵育过夜;PBS洗,滴加FITC标记二抗(1:100),室温1h,PBS洗,90%甘油封片。荧光显微镜下观察。
6、MTT法检测大鼠GECs的增殖率
GECs以2×10~3个/孔的密度接种至96孔板,80%融合后无血清培养液静置12h,分组继续培养。24h后每孔加入MTT溶液(5g/L)20μl,继续孵育4 h,终止培养。吸弃孔中的培养上清,每孔加入150μl DMSO,振荡10 min,酶联免疫检测分析仪测定各组吸光度(OD值),并计算细胞增值率。
7、AV/PI法流式细胞仪检测大鼠GECs的凋亡率
GECs以5×10~5个/瓶传代至25mL的培养瓶中,待融合后无血清培养液静置12h,分组继续培养24 h后,消化收集细胞,将其吹打成单细胞悬液,离心弃上清,用PBS漂洗,再离心,再漂洗后以100目筛网过滤,然后再离心加入细胞凋亡检测AV-PI(磷脂酰丝氨酸结合蛋白联合碘化丙啶双标记)工作液0.5ml,终浓度为10mg/L,室温避光30min后,进行流式细胞仪检测,计数凋亡细胞率。
8、统计学分析
采用SPSS17.0统计软件处理,计量资料以均数±标准差(x±s)表示,采用one-way ANOVA分析,组间比较采用LSD检验,P＜0.05为差异有统计学意
实验结果
1、大鼠肾小球内皮细胞的培养与鉴定
原代分离培养肾小球内皮细胞生长5-7天时,呈单层贴壁生长,互不重叠,细胞呈角形或短梭形,外观呈铺路石状,边界清楚。Ⅷ因子相关抗原免疫荧光鉴定,大鼠GECs胞膜和胞浆处呈红色荧光,证实为肾小球内皮细胞。
2、AngⅡ对大鼠肾小球内皮细胞MCP-1蛋白表达的影响
Western blot检测结果显示,浓度效应组中,正常对照组MCP-1蛋白表达量较低,施加AngⅡ刺激因素后MCP-1表达量明显高于正常对照组,且随着浓度的增加(10~(-7)-10~(-5)M)而表达增加,成剂量依赖效应;NADPH氧化酶抑制剂DPI(10~(-6)mol/L)和AT1受体拮抗剂TEL(10~(-6)mol/L)抑制AngⅡ的作用。
3、AngⅡ对大鼠肾小球内皮细胞p47phox蛋白表达的影响
Western blot检测结果显示,与正常对照组相比给予10~(-5)M AngⅡ刺激因素后,大鼠肾小球内皮细胞内p47phox蛋白表达明显增加,成时间依赖效应。AngⅡ的这一作用效果可分别被TEL(10~(-6)M)和DPI(10~(-6)M)所抑制。
4、AngⅡ对大鼠肾小球内皮细胞AT_1受体mRNA水平的影响
RT-PCR检测结果显示,与正常对照组相比给予10~(-5)mol/L的AngⅡ刺激因素后,大鼠肾小球内皮细胞AT1受体的mRNA水平明显增加,成时间依赖效应。
5、AngⅡ对大鼠肾小球内皮细胞ROS生成的影响
DCFH-DA荧光染色检测结果显示,与正常对照组相比,随着作用时间延长AngⅡ作用组细胞内ROS的生成量逐渐增加,15min时ROS生成量达到顶峰,后逐渐减少;加入TEL和DPI后DCFH荧光值降低,ROS生成量明显减少。
6、AngⅡ对大鼠肾小球内皮细胞NF-κB活化的影响
正常状态无AngⅡ刺激时,NF-κB主要表达在细胞浆中,细胞核内少见表达;给予AngⅡ刺激因素后NF-κB即开始活化,迅速核转移,荧光显微镜显示30 min时NF-κB核内表达达到高峰,后逐渐减弱。药物TEL和DPI抑制NF-κB的核转移。
7、AngⅡ对大鼠GECs增殖的影响
与对照组相比较,低浓度AngⅡ(10~(-8)M)作用组,大鼠GECs增殖率增加1.33倍左右,高浓度AngⅡ(10~(-6)M)作用组,细胞增殖率降低2.3倍左右,分别为1.33±0.03和0.43±0.03(与对照组比较,P＜0.01)。低浓度AngⅡ组和高浓度AngⅡ组之间增殖率有统计学差异。
8、AngⅡ对大鼠GECs凋亡的影响
与对照组比较,低浓度AngⅡ(10~(-8)M)组和高浓度AngⅡ(10~(-6)M)组大鼠GECs凋亡率分别降低和增加50%左右,与高浓度AngⅡ(10~(-6)M)组比较,AngⅡ+TEL和AngⅡ+SB组细胞凋亡率均下降,但两组间细胞凋亡率无统计学差异。
结论
1、AngⅡ促进RGECs的MCP-1表达,在一定范围内呈剂量依赖效应;
2、AngⅡ上调RGECs的AT1受体mRNA水平,在一定范围内呈时间依赖效应;
3、AngⅡ上调RGECs的p47phox蛋白表达,通过NADPH氧化酶依赖的ROS生成途径促进NF-κB的活化;
4、AT1受体拮抗剂替米沙坦可抑制AngⅡ的促炎性因子MCP-1表达作用;
5、低浓度AngⅡ促进RGECs的增殖,高浓度AngⅡ抑制RGECs增殖;
6、低浓度AngⅡ抑制RGECs的凋亡,高浓度AngⅡ促进RGECs凋亡;
7、p38MAPK信号通路参与AngⅡ对肾小球内皮细胞凋亡的调控;
8、AT_1受体拮抗剂替米沙坦抑制AngⅡ所诱导的RGECs凋亡发生。
Introduction
Activation of the intrarenal renin-angiotensin system(RAS) is a characteristic feature in the development and progression of chronic kidney disease.AngiotensinⅡ(AngⅡ),the main effector of RAS,is implicated in the pathogenesis of renal diseases. AngⅡinitiates its effects by interaction with at lease two pharmacologically distinct subtypes of cell-surface receptors,AT1 and AT2,The major functions of AngⅡin cardiovascular system are mediated through AT1 receptor.
An intriguing concept has emerged that AngⅡnot only mediates intraglomerular hypertension but also behaves as a pro-inflammatory and growth-stimulating factor contributing to the renal hypertension and sclerosis.AngⅡhas growth-stimulating properties on different renal cell types.However,possible inflammatory effects of this vasoactive peptide on endothelial cells isolated from the glomerular microvascularture have not formally been investigated.Glomerular endothelial cells(GECs),being exposed to the bloodstream,are the target of many different factors,that may alter the functional state of these cells and induce them to release inflammatory cytokines.
Inflammatory processes involve both the synthesis of inflammatory cytokines, such as monocyte chemoattractant protein-1(MCP-1),and the activation of their distinct signaling cascades.Recent findings suggest that AngⅡactivates intracellular signaling processes,including the polyol pathway and generation of reactive oxygen species(ROS).In cellular systems,a major source of ROS derives from the membrane-bound NAD(P)H oxidase system.Activation of NAD(P)H oxidase system requires the participation of p47phox,one of the NAD(P)H oxidase subunits,which plays a central role in the scenario of NAD(P)H oxidase activation.10Activation of nuclear factor -ΚB(NF-ΚB) is involved in the expression of pro-inflammatory genes.11 In this study,we investigated wether AngⅡactivates the NF-ΚB cascade by ROS and whether the ROS-dependent mechanism may be involved in AngⅡ-induced MCP-1 formation.Further,we tested whether telmisartan,an AT1 receptor blocker,and DPI,an inhibitor of NAD(P)H oxidase,modulate endothelial inflammation and oxidative cell damage induced by AngⅡ-dependent stimuli in cultured rat glomerular endothelial cells.
Methods
We isolated and characterized primary cultures in rat glomerular endothelial cells(GECs).We found that AngⅡinduced the synthesis of monocyte chemoattractant protein -1(MCP-1) in rat GECs determined by Western blot.AngⅡstimulation of rat GECs induced a rapid increase in reactive oxygen species(ROS) generation determined by laser fluoroscopy.The level of p47phox protein,a nicotinamide-adenine dinucleotide phosphate(NADPH) oxidase subunit,was also increased by AngⅡtreatment.These effects of AngⅡon GECs were all reduced by DPI,a NAD(P)H oxidase inhibitor.AngⅡstimulation also promoted the activation of Nuclear factor-k B (NF-ΚB).Telmisartan(TEL),an AT1 receptor blocker,blocked all the effect of AngⅡon rat GECs.We study the effect of AngⅡon the propagation and apoptosis of RGECs by the methods of MTT and AV/PI.
Results
We found that AngⅡinduced the synthesis of monocyte chemoattractant protein -1(MCP-1) in rat GECs determined by Western blot.AngⅡstimulation of rat GECs induced a rapid increase in reactive oxygen species(ROS) generation determined by laser fluoroscopy.The level of p47phox protein,a nicotinamide-adenine dinucleotide phosphate(NADPH) oxidase subunit,was also increased by AngⅡtreatment.These effects of AngⅡon GECs were all reduced by DPI,a NAD(P)H oxidase inhibitor.AngⅡstimulation also promoted the activation of Nuclear factor-k B(NF-ΚB).And the effect of AngⅡon the propagation and apoptosis of RGECs depends on its dose denstiny.Telmisartan(TEL),an AT1 receptor blocker,blocked all the effect of AugⅡon rat GECs.
Discussion
The present study demonstrates that AngⅡ-induced ROS generation in rat GECs depends on the P47phox subunit of NADPH Oxidase.ROS are additionally required for AngⅡ-induced activation of NF-ΚB.Blockade of the NADPH Oxidase by its inhibitor DPI significantly abolished AngⅡ-induced MCP-1 formation,indicating that NF-ΚB when activated by ROS,participates in the AngⅡ-induced MCP-1 production.
It's now well accepted that AngⅡmay function as a potent pro-inflammatory mediator and be implicated in the pathogenesis of chronic renal disease.13,14GECs are at the interface between blood and adjacent cell population and play a crucial role in preserving the kidney function.The monocyte-endothelium interaction induced by AngⅡmay contribute to the initiation of vascular inflammation.15 MCP-1,when expressed at the plasma membrane of endothelial cells,can mediate the initial capture of monocytes.16 Our results suggest that AngⅡcan induce MCP-1 formation in rat GECs and promote the monocyte adhesion to endothelial cells.
AngⅡacts via AT1 and AT2 receptors.Both receptor subtypes have been found in rat GECs.17 AngⅡexerts most of its already well-defined physiologic and pathophysiologic actions through AT1 receptors.18 Consistent with this concept,our results showed that the AngⅡeffect on MCP-1 formation was inhibited by the selective AT1 receptor antagonist telmisartan.
In mammalian cells,a major source of ROS derives from the membrane-bound NADPH Oxidase system,which exists in nonphagocytic cells of the vascular wall,for example,fibroblast,vascular smooth muscle cells and endothelial cells.19 Increased ROS generation can induce cell inflammation.20,21 The present study isolated rat glomerular endothelial cells and demonstrated that the expression of p47phox protein, one of the NADPH Oxidase subunits,was up-regulated by AngⅡtreatment in accordance with the increased ROS generation.Moreover,blockade of NADPH Oxidase by DPI reduced the P47phox protein synthesis and ROS generation.
Nuclear factor-ΚB normally exists in the cytoplasm in an inactive form bound to the inhibitory protein IΚB.Upon cellular activation,IΚB is rapidly degraded prior to the translocation of NF-ΚB into the nucleus and its subsequent activation,resulting in the transcriptional regulation of target genes encoding pro-inflammatory cytokines.NF-ΚB consists of two subunits,p50 and p65,with p65 containing a transcription domain.11,22 We here studied the possible involvement of NF-ΚB activation in AngⅡ-induced MCP-1 production in rat GECs.In the present study,AngⅡstimulation increased the translocation of NF-ΚB P65 subunit to the nucleus,which was reduced by DPI.The observation suggests that AngⅡ-induced NF-ΚB activation is partially mediated by ROS.
In summary,we demonstrated that AngⅡinduces MCP-1 production in rat GECs partially via the signaling of ROS-dependent NF-ΚB activation,which can be inhibited by the AT1 receptor antagonist telmisartan.Our finding may provide a mechanistic basis for the benefits of selective AT1 blockade in dealing with the chronic renal disease.
Conclusions
1 AngⅡincreases the expression of MCP- 1 and AT1mRNA;
2 AngⅡincreases the expression of p47phox in a certain ranges showing a time-dependent manner.
3 AngⅡinduces MCP-1 production in rat GECs partially via the signaling of ROS-dependent NF-ΚB activation,which can be inhibited by the AT1 receptor antagonist telmisartan.
4 the effect of AngⅡon the propagation and apoptosis of RGECs depends on its dose denstiny.
引文
1 Hiroyuki K,Yuri O,Yuki S.Young scholars award lecture:intratubular angiotensinogen in hypertension and kidney diseases.Am J.2008;19:541-550.
2 Eduard NL,Anne ME,Kafait UM.Mechanism of high glucose induced Angiotensin Ⅱ production in rat vascular smooth muscle cells.Circ.Res.2007;101:455-464.
3 Lee HB,JY Seo,MR Yu,S-t Uh,H Ha.Radical approach to diabetic nephropathy.Kidney International.2007;00:1-4.
4 Mario BM,Amy KB,David MS,Douglas CE.Role of the JAK/STAT signaling pathway in diabetic nephropathy.AJP-Renal Phsiol.2006;290:F762-F768.
5 Lassila M,Cooper ME,and Jandeleit-Dahm K.Antiproteinuric effect of RAS blockade:new mechanism.Curr Hypertens Rep.2004;6:383-392.
6 Bernhard S,Maren L,Sabine B,Andres H,et al.Role of NAD(P)H oxidase in Angiotensin Ⅱ-induced JAK/STAT signaling and cytokine induction.Circ.Res.2008;87:1195-1201.
7 Kou BJ,Manu V,Donald RJ.Effects of Angiotension Ⅱ on human endothelial cells survival signaling pathways and its angiogenic response.Vascular Pharmacology.2007;47:199-208.
8 Lee HB,Yu MR,Yang YQ,et al.Reactive oxygen species-regulated signaling pathways in diabetic nephropathy.J Am Soc Nephrol.2003;14:S241-S245.
9 Barnes PJ,Karin M.Nuclear factor-κB:a pivotal transcription factor in chronic inflammatory disease.New England Journal of Medicine.1997;15:1066-1071.
10 Amy KB,S e an S,John Js,Heather R,et al.Angiotensin Ⅱ blockade prevents hyperglycemia-induced activation of JAK and STAT proteins in diabetic rat kidney glomeruli.AJP-Renal Physiol.2008;286:F653-F659.
11 Liu HQ,Wei XB,Sun R,Cai YW.Angiotensin Ⅱ stimulates intercellular adhesion molecular-1 via an ATI receptor/nuclear factor-KB pathway in brain microvascular endothelial cells.Life Science.2006;78:1293-1298.
12 Gunter W,Fuad NZ,Gunter Z,Rolf AK.Angiotensin Ⅱ is mitogenic for cultured rat glomerular endothelial cells.Kidney Int.1994;45:314-319.
13 Amy KB,Sean S,Ma GC,et al.Effects of simvastatin on high glucose-and Angiotension-induced activation of the JAK/STAT pathway in mesangial cells.AJP-Renal Physiol.2006;291:F116-F121.
14 Farhad A,Sean S,Wang XD,Tang J,et al.Angiotensin Ⅱ activation of the JAK/STAT pathway in mesangial cells is altered by high glucose.Kidney International.2007;61:1605-1616.
15 Katherine R.Turtle.Linking metabolism and immunology:diabetic nephropathy is an inflammatory disease.J Am Soc Nephrol.2007;16:1537-1538.
16.Evis H,Bu DX,Barry IH,Chang JS,et al.Vascular and inflammatory stresses mediate atherosclerosis via RAGE and its ligands in apoE-/-mice.J Clin Invest.2008;118:183-194.
17 Marc de G.Angiotensin Ⅱ and Nitric Oxide Interaction.Heart Failure Reviews.2002;7:347-358.
18 Griendling KK,Soresou D,Ushio-Fukai M.NAD(P)H oxidase:role in cardiovascular biology and a)disease.Circ Res.2006;86:494-501.
19 HA HJ,LEE HB.Reactive oxygen species and matrix remodeling in diabetic nephropathy.J Am Soc Nephrol.2007;14:S246-S249.
20 Liang XB,Ma LJ,Takashi N,Wang YH,et al.Angiotensin type 1 receptor blocker restores podocyte potential to promote glomerular endothelial cell growth.J Am Soc Nephrol.2006;17:1886-1895.
21 Merlin CT,Christos T,Wendy MB,Katarzyna B,et al.Interactions between rennin angiotensin system and advanced glycation in the kidney.J Am Soc Nephrol.2005;16:2976-2984.
22 Alber S,Baldwin J.The NF-K B and 1KB proteimnew discoveries and insights.Annual a)Review of Immunology.1996;14:549-681.
23 Song JH,Cha SH,Lee HJ,et al.Effect of low-dose dual blockade of renin-angiotensin system on urinary TCF-{beta} in type 2 diabetic patients with advanced kidney disease.Nephrol Dial Transplant.2008;21:683-89.
24 Lewis EJ,Hunsicke LG,Clarke WR,et al.Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes.N Engl j Med.2001; 345:851-860.
25 Eduard NL,Anne ME,Kafair UM.Mechanism of high glucose-induced angiotensin Ⅱ production in rat vascular smooth muscle cells.CR.2007;16:455-464.
26 Hu CP,Abhijit D,Jawahar LM.Angiotensin Ⅱ induces capillary formation from endothelial cells via LOX-1 dependent redox-sensitive pathway.Hypertension.2007;50:952-957.
27 Mehta PK,Griendling KK.Angiotensin Ⅱ cell signaling:physiological and pathological effects in cardiovascular system.Am J Physiol Cell Physiol.2007:292:C82-C97.
28 Mii A,Shimizu A,Masuda Y,Ishizaki M,Kawachi H,lino Y,Katayama Y,Fukuda Y.Angiotensin Ⅱ receptor blockade inhibits acute glomerular injuries with the alteration of receptor expression.Lab Invest.2009;89(2):164-77.
29 Jia L.Zhuo,Xiao C.Li.Novel roles of intracrine angiotensin Ⅱ and signaling mechanisms in kidney cells.Journal of the Renin-Angiotensin-Aldosterone System.2007;8:23-33.
30 Ling BN,Matsunaga H,Ma H,Eaton DC.Role of growth factors in mesangial cell ion channel regulation.Kidney Int.1993;48:1158-1166.
31 Park HC,Choi HY,Kim BS,An HR,Lee JS,Ha SK,Han DS.Urinary TGF-betal as an indicator of antiproteinuric response to angiotensin Ⅱ receptor blocker in proteinuric renal diseases.Biomed Pharmacother.2009;28:145-156.
32 Hunsicker LG.Emerging trends for prevention and treatment of diabetic nephropathy:blockade of the RAAS and BP control.J Manag Care Pharm.2004;10:s12-17.
33 Qian C,Schoemaker RG,van Gilst WH,Roks AJ.The role of the renin-angiotensin-aldosterone system in cardiovascular progenitor cell function.Clin Sci(lond).2009;116(4):301-314.
34 Flegal-Demotta MA,Doghu S,Banks WA.Angiotensin Ⅱ modulates BBB permeability via activation of the AT(1)receptor in brain endothelial cells.J Cereb Blood Flow Metab.i.2009;29(3):640-647.
35 Kim JH,Yu YS,Cho CS,Kim KW.Blockade of angiotensin Ⅱ attenuates VEGF-mediatedblood-retinal barrier breakdown in diabetic retinopathy.J Cereb Blood Flow Metab.2009;29(3):621-628.
36 Kou BJ,Manu V,Donald R.J.S.Effects of angiotensin Ⅱ on human endothelial cells survival signaling pathways and its angiogenic response.Vascular Pharmacology.2007;47:199-208.
37 Liu HQ,Wei XB,Sun R,Cai YW,Lou HY,Wang JW.Chen AF,Zhang XM.Angiotensin Ⅱ stimulates intercellular adhesion molecule-1 via an ATI receptor/nuclear factor-Kb pathway in brain microvascular endothelial cells.Life Science.2006;78:1293-1298.
38 Lshidoya S,Mocracken R,et al.Angiotensin Ⅱ recepor antagonist ameliorates renal tubulointerstitial fibrosis caused by anilatcral ureternal obstruction.Kidney Int.1995;47:1285-1291.
39 Moudgil R,Musat2Marcu S,Xu Y,et al.Increased AT2 R protein expression but notincreased apoptosis during cardioprotection induced by ATI R blockade.Can J Cardiol.2002;18:1107-1116.
40 Wassmann S,Nickenig G.Pathophysiological regulation of the ATI receptor and implications for vascular disease.J Hypertension Suppl.2006;24:S15-S21.
41 Dimmeler S,Rippmann V,Weiland U,et al.Angiotensin Ⅱ induces apoptosis of human endot helial cells protective effect of nitric oxide.Circ Res.1997;81:970-976.
42 Goldsmith ZG,Dhanasekaran DN.G protein regulation of MAPK networks.Oncogene.2007;26(22):3122-3142.
1 Song JH,Cha SH,Lee HJ,et al.Effect of low-dose dual blockade of renin-angiotensin system on urinary TCF-{beta} in type 2 diabetic patients with advanced kidney disease.Nephrol Dial Transplant.2008;21:683-689.
2 DJ F,AO P.Diabetic nephropathy.Medicine.2007;35:9:503-506.
3 Lewis EJ,Hunsicke LG,Clarke WR,et al.Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes.N Engl j Med.2001;345:851-860.
4 Eduard NL,Anne M E,Kafair UM.Mechanism of high glucose-induced angiotensin Ⅱ production in rat vascular smooth muscle cells.CR.2007;455-464
5 Wcodmar ZL,Oppong SY,Cook S,Hooper NM,et al.Shedding o f somatic angiotensin converting enzyme ACE is inefficient.Biochem J.2007;347:711-718
6 Koka V.Wang,Huang XP,Kim Viitsuyama S.Truong LD,et al:Fuctional evidence of a role of vascular chymase in the production of angiotensin Ⅱ in isolated human arteries.Circulation.2008;104:750-752
7 Mario B.Marrero,Amy K.Banes-Berceli,et al.:Role of the JAK/STAT signaling in diabetic nephropathy.Am j physiol Renal physiol.2006;290:F762-F768.
8 Ling BN.Regulation of mesangial chloride channels by insulin and glucose:role in diabetic nephropathy.Clin Exp Pharmacol Physiol.1996;23:89-94.
9 Ling BN,Matsunaga H,Ma H,and Eaton DC.Role of growth factors in mesangial cell ion channel regulation.Kidney Int.1993;48:1158-1166.
10 Jia L.Zhuo,Xiao C.Li.Novel roles of intracrine angiotensin Ⅱ and signaling mechanisms in kidney cells.Journal of the Renin-Angiotensin-Aldosterone System.2007;8:23-33.
11 Hiroyuki Kobori,Yuri Ozawa,Yuki Suzai,et al:Young Scholars Award Lecture:Intratular Angiotensiongen in Hypertension and Kidney Diseases.Diagnostics.2008;19:541-550.
12 Tusty-jiua Hsieh,Shao ling Zhang,etal.:High glucose stimulats angiotensinogen gene expression via Reactive Oxygen Species Generation in Rat Kidney Proximal Tubular Cells.Endocrinology 2008;143 (8):2975-2985.
13 Soo Hyun Park,HO Zae Han.:The mechanism of angiotensin Ⅱ binding downregulation by high glucose in primary renal proximal tubule cells.Am J Physiol Renal Physiol.2007;282:F228-F237.
14 Suzaki Y,Koori H,Ozawa Y.Intrarenal angiotensinogen augmentation is precedent to diabetic nephropathy in Zucker diabetic fatty obese rats.J Am Soc Nephrol.2008;16:202-210.
15 Atsuhiro Ichihara,Fumiaki Suzuki,et al.:Prorenin Receptor Blokade Inhibits Development of Glomerulosclerosis in Diabetic Angiotensin Ⅱ Type 1a Receptor-Deficient Mice.American Society of Nephrology.2006;17:1950-1961.
16 陈祥新,傅国胜.血管紧张素Ⅱ及其受体与血管内皮功能的关系(J) 1临床心血管病杂志.2003;19(11):698-700.
17 符史干,刘培庆,鲁伟,等.蛋白激酶C在血管紧张素Ⅱ抑制心肌细胞一氧化氮合成中的作用(J)生理学报.2000;52(4):318-322.
18 李皓,尹鸿操,张华,等.血管紧张素Ⅱ对人内皮细胞转录因子NF-κB的激活机制及其对血小板源生长因子B链基因转录的影响(J)中华病理学杂志.2001;30(4):276-280.
19 Xiaona Ge,Brad Low,Mei Liang,Jian Fu.:Angiotensin Ⅱ Directly Triggers Endothelial Exocytosis via Protein Kinase C-Dependent Protein Kinase D2 Activation.J Pharmacol Sci.2007;105:168-176.
20 Baijun Kou,Manu Vatish,Donald R.J.Singer.:Effects ofAngiotensin Ⅱ on human endothelial cells survival signalling pathways and its angiogenic response.Vascular Pharmcology.2007;47:199-208.
21 Xiu-Bin Liang,Li-Jun Ma,et al.:Agiotensin Type 1 receptor Blocker Restores Podocyte Potential to Promote Glomerular Endothelial Cell Growth.American Society of Nephrology.2006;17:1886-1895.
22 Bonavita F,Stefanelli C,Giordano E,et al.H9c2 cardiac myoblast s undergo apoptosis in a model of ischemia consisting of serum deprivation and hypoxia:inhibition by PMA[J].FEBS Lett.2003;536(123):85-91.
23 He M,He X,Xie Q,et al.Angiotensin Ⅱ induces t he expression of tissue factor and it s mechanism in human monocytes[J].Thromb Res.2006;117:579-590.
24 Mahrouf M,Ouslimani N,Peynet J,et al.Metformin reduces angiotensin-mediated intracellular production of reactive oxygen species in endot helial cells through t he inhibition a) of protein kinase C[J].Biochem Pharmacol.2008;72:176-183.
25 Walter DH,Haendeler J,Galle J,et al.Cyclosporin A inhibit apoptosis of human endothelial cells by preventing release of cytochrome C from mitochondria[J].Circulation.1998;98:1153-1157.
26 Buccellato LJ,Tso M,Akinci OI,et al.Reactive oxygen species are required for hyperoxia2induced Bax activation and cell death in alveolar epithelial cells[J].J Biol Chem.2007;279:6753-6760.
27 Distelhorst CW,Shore GC.Bc122 and calcium:controversy beneath t he surface[J].Oncogene.2004;23:2875-2880.
28 Ueda S,Masutani H,Nakarnura H,et al.Redox cont rol of cell death[J].Antioxid Redox Signal.2002;4:405-414.
29 Rossig L,Hoffmann J,Hugle B,et al.Vitamin C inhibits endothelial cell apoptosis in congestive heart failure[J].Circulation.2001;104:2182-2187.
30 Lshidoya S,Mocracken R,et al.Angiotensin Ⅱ recepor antagonist ameliorates renal tubulointerstitial fibrosis caused by anilatcral ureternal obstruction.Kidney Int.1995;47:1285-1291.
31 冯江敏,吴靖川,张国娟,等.慢性间质性肾炎炎症细胞浸润的特点中华肾脏杂志.1999;15(6):3-4.
32 Short AD,Bian J,Chosh TK,et al.intracellular Ca2 + pool content is linked to control of cell growth.Proc Natl AcadSci USA.1993;90:79-86.
33 Lixia Zeng,Hanshi XU,Teng-Leong Chew,et al.:HMG-CoA reductase inhibition modulates VEGF-induced endothelial cell hyperpermeability by preventing RhoA activation and myosi regularory light chain phosphorylation.The FASEB Journal.2005;17:235-240.
34 Masao Takemoto,James K.Liao.:Pleiotropic Effects of 3-Hydroxy-3-Methylglutarl Coenzyme A Reductase Inhibitors.Arterioscler.Thromb.Vasc.Biol.2008;21:1712-1719.
35 Hideaki Sawaki,Jungo Terasaki,et al.:A renoprotective effect of low dose losartan in patients with type 2 diabetes.Diabetes Research and Clinical Practice.2007;08:01-04.
36 Ahmet M.Sengul,Yukel Altuntas,et al:Beneficial effect of lisinopril plus telmisartan in patients with type 2 diabetes,nicroalbuninuria and hypertension.Diabetes Research and Clinical Practice.2006;71:210-219.
37 Moudgil R,Musat2Marcu S,Xu Y,et al.Increased AT2R protein expression but notincreased apoptosis during cardioprotection induced by AT1R blockade.Can J Cardiol.2002;18:873-883.
38 Dimmeler S,Rippmann V,Weiland U,et al.Angiotensin Ⅱ induces apoptosis of human endot helial cells protective effect of nitric oxide[J].Circ Res.2007;81:970-976.
39 Kim H,You S,Farris J,et al.Post2t ranscriptional inactivation of p53 in immortalized murine embryo fibroblast cells [J].Oncogene.2008;20:3306-3310.
40 Kren BT,Trembley J H,Steer CJ.Alterations in mRNA stability during rat liver regeneration[J].Am J Physiol.2008;270:G763-G777.
2 Eduard NL,Anne ME,Kafait UM.Mechanism of high glucose induced Angiotensin Ⅱ production in rat vascular smooth muscle cells.Circ.Res.2007;101:455-464.
3 Lee HB,JY Seo,MR Yu,S-t Uh,H Ha.Radical approach to diabetic nephropathy.Kidney International.2007;00:1-4.
4 Mario BM,Amy KB,David MS,Douglas CE.Role of the JAK/STAT signaling pathway in diabetic nephropathy.AJP-Renal Phsiol.2006;290:F762-F768.
5 Lassila M,Cooper ME,and Jandeleit-Dahm K.Antiproteinuric effect of RAS blockade:new mechanism.Curr Hypertens Rep.2004;6:383-392.
6 Bernhard S,Maren L,Sabine B,Andres H,et al.Role of NAD(P)H oxidase in Angiotensin Ⅱ-induced JAK/STAT signaling and cytokine induction.Circ.Res.2008;87:1195-1201.
7 Kou BJ,Manu V,Donald RJ.Effects of Angiotension Ⅱ on human endothelial cells survival signaling pathways and its angiogenic response.Vascular Pharmacology.2007;47:199-208.
8 Lee HB,Yu MR,Yang YQ,et al.Reactive oxygen species-regulated signaling pathways in diabetic nephropathy.J Am Soc Nephrol.2003;14:S241-S245.
9 Barnes PJ,Karin M.Nuclear factor-κB:a pivotal transcription factor in chronic inflammatory disease.New England Journal of Medicine.1997;15:1066-1071.
10 Amy KB,S e an S,John Js,Heather R,et al.Angiotensin Ⅱ blockade prevents hyperglycemia-induced activation of JAK and STAT proteins in diabetic rat kidney glomeruli.AJP-Renal Physiol.2008;286:F653-F659.
11 Liu HQ,Wei XB,Sun R,Cai YW.Angiotensin Ⅱ stimulates intercellular adhesion molecular-1 via an ATI receptor/nuclear factor-KB pathway in brain microvascular endothelial cells.Life Science.2006;78:1293-1298.
12 Gunter W,Fuad NZ,Gunter Z,Rolf AK.Angiotensin Ⅱ is mitogenic for cultured rat glomerular endothelial cells.Kidney Int.1994;45:314-319.
13 Amy KB,Sean S,Ma GC,et al.Effects of simvastatin on high glucose-and Angiotension-induced activation of the JAK/STAT pathway in mesangial cells.AJP-Renal Physiol.2006;291:F116-F121.
14 Farhad A,Sean S,Wang XD,Tang J,et al.Angiotensin Ⅱ activation of the JAK/STAT pathway in mesangial cells is altered by high glucose.Kidney International.2007;61:1605-1616.
15 Katherine R.Turtle.Linking metabolism and immunology:diabetic nephropathy is an inflammatory disease.J Am Soc Nephrol.2007;16:1537-1538.
16.Evis H,Bu DX,Barry IH,Chang JS,et al.Vascular and inflammatory stresses mediate atherosclerosis via RAGE and its ligands in apoE-/-mice.J Clin Invest.2008;118:183-194.
17 Marc de G.Angiotensin Ⅱ and Nitric Oxide Interaction.Heart Failure Reviews.2002;7:347-358.
18 Griendling KK,Soresou D,Ushio-Fukai M.NAD(P)H oxidase:role in cardiovascular biology and a)disease.Circ Res.2006;86:494-501.
19 HA HJ,LEE HB.Reactive oxygen species and matrix remodeling in diabetic nephropathy.J Am Soc Nephrol.2007;14:S246-S249.
20 Liang XB,Ma LJ,Takashi N,Wang YH,et al.Angiotensin type 1 receptor blocker restores podocyte potential to promote glomerular endothelial cell growth.J Am Soc Nephrol.2006;17:1886-1895.
21 Merlin CT,Christos T,Wendy MB,Katarzyna B,et al.Interactions between rennin angiotensin system and advanced glycation in the kidney.J Am Soc Nephrol.2005;16:2976-2984.
22 Alber S,Baldwin J.The NF-K B and 1KB proteimnew discoveries and insights.Annual a)Review of Immunology.1996;14:549-681.
23 Song JH,Cha SH,Lee HJ,et al.Effect of low-dose dual blockade of renin-angiotensin system on urinary TCF-{beta} in type 2 diabetic patients with advanced kidney disease.Nephrol Dial Transplant.2008;21:683-89.
24 Lewis EJ,Hunsicke LG,Clarke WR,et al.Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes.N Engl j Med.2001; 345:851-860.
25 Eduard NL,Anne ME,Kafair UM.Mechanism of high glucose-induced angiotensin Ⅱ production in rat vascular smooth muscle cells.CR.2007;16:455-464.
26 Hu CP,Abhijit D,Jawahar LM.Angiotensin Ⅱ induces capillary formation from endothelial cells via LOX-1 dependent redox-sensitive pathway.Hypertension.2007;50:952-957.
27 Mehta PK,Griendling KK.Angiotensin Ⅱ cell signaling:physiological and pathological effects in cardiovascular system.Am J Physiol Cell Physiol.2007:292:C82-C97.
28 Mii A,Shimizu A,Masuda Y,Ishizaki M,Kawachi H,lino Y,Katayama Y,Fukuda Y.Angiotensin Ⅱ receptor blockade inhibits acute glomerular injuries with the alteration of receptor expression.Lab Invest.2009;89(2):164-77.
29 Jia L.Zhuo,Xiao C.Li.Novel roles of intracrine angiotensin Ⅱ and signaling mechanisms in kidney cells.Journal of the Renin-Angiotensin-Aldosterone System.2007;8:23-33.
30 Ling BN,Matsunaga H,Ma H,Eaton DC.Role of growth factors in mesangial cell ion channel regulation.Kidney Int.1993;48:1158-1166.
31 Park HC,Choi HY,Kim BS,An HR,Lee JS,Ha SK,Han DS.Urinary TGF-betal as an indicator of antiproteinuric response to angiotensin Ⅱ receptor blocker in proteinuric renal diseases.Biomed Pharmacother.2009;28:145-156.
32 Hunsicker LG.Emerging trends for prevention and treatment of diabetic nephropathy:blockade of the RAAS and BP control.J Manag Care Pharm.2004;10:s12-17.
33 Qian C,Schoemaker RG,van Gilst WH,Roks AJ.The role of the renin-angiotensin-aldosterone system in cardiovascular progenitor cell function.Clin Sci(lond).2009;116(4):301-314.
34 Flegal-Demotta MA,Doghu S,Banks WA.Angiotensin Ⅱ modulates BBB permeability via activation of the AT(1)receptor in brain endothelial cells.J Cereb Blood Flow Metab.i.2009;29(3):640-647.
35 Kim JH,Yu YS,Cho CS,Kim KW.Blockade of angiotensin Ⅱ attenuates VEGF-mediatedblood-retinal barrier breakdown in diabetic retinopathy.J Cereb Blood Flow Metab.2009;29(3):621-628.
36 Kou BJ,Manu V,Donald R.J.S.Effects of angiotensin Ⅱ on human endothelial cells survival signaling pathways and its angiogenic response.Vascular Pharmacology.2007;47:199-208.
37 Liu HQ,Wei XB,Sun R,Cai YW,Lou HY,Wang JW.Chen AF,Zhang XM.Angiotensin Ⅱ stimulates intercellular adhesion molecule-1 via an ATI receptor/nuclear factor-Kb pathway in brain microvascular endothelial cells.Life Science.2006;78:1293-1298.
38 Lshidoya S,Mocracken R,et al.Angiotensin Ⅱ recepor antagonist ameliorates renal tubulointerstitial fibrosis caused by anilatcral ureternal obstruction.Kidney Int.1995;47:1285-1291.
39 Moudgil R,Musat2Marcu S,Xu Y,et al.Increased AT2 R protein expression but notincreased apoptosis during cardioprotection induced by ATI R blockade.Can J Cardiol.2002;18:1107-1116.
40 Wassmann S,Nickenig G.Pathophysiological regulation of the ATI receptor and implications for vascular disease.J Hypertension Suppl.2006;24:S15-S21.
41 Dimmeler S,Rippmann V,Weiland U,et al.Angiotensin Ⅱ induces apoptosis of human endot helial cells protective effect of nitric oxide.Circ Res.1997;81:970-976.
42 Goldsmith ZG,Dhanasekaran DN.G protein regulation of MAPK networks.Oncogene.2007;26(22):3122-3142.
1 Song JH,Cha SH,Lee HJ,et al.Effect of low-dose dual blockade of renin-angiotensin system on urinary TCF-{beta} in type 2 diabetic patients with advanced kidney disease.Nephrol Dial Transplant.2008;21:683-689.
2 DJ F,AO P.Diabetic nephropathy.Medicine.2007;35:9:503-506.
3 Lewis EJ,Hunsicke LG,Clarke WR,et al.Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes.N Engl j Med.2001;345:851-860.
4 Eduard NL,Anne M E,Kafair UM.Mechanism of high glucose-induced angiotensin Ⅱ production in rat vascular smooth muscle cells.CR.2007;455-464
5 Wcodmar ZL,Oppong SY,Cook S,Hooper NM,et al.Shedding o f somatic angiotensin converting enzyme ACE is inefficient.Biochem J.2007;347:711-718
6 Koka V.Wang,Huang XP,Kim Viitsuyama S.Truong LD,et al:Fuctional evidence of a role of vascular chymase in the production of angiotensin Ⅱ in isolated human arteries.Circulation.2008;104:750-752
7 Mario B.Marrero,Amy K.Banes-Berceli,et al.:Role of the JAK/STAT signaling in diabetic nephropathy.Am j physiol Renal physiol.2006;290:F762-F768.
8 Ling BN.Regulation of mesangial chloride channels by insulin and glucose:role in diabetic nephropathy.Clin Exp Pharmacol Physiol.1996;23:89-94.
9 Ling BN,Matsunaga H,Ma H,and Eaton DC.Role of growth factors in mesangial cell ion channel regulation.Kidney Int.1993;48:1158-1166.
10 Jia L.Zhuo,Xiao C.Li.Novel roles of intracrine angiotensin Ⅱ and signaling mechanisms in kidney cells.Journal of the Renin-Angiotensin-Aldosterone System.2007;8:23-33.
11 Hiroyuki Kobori,Yuri Ozawa,Yuki Suzai,et al:Young Scholars Award Lecture:Intratular Angiotensiongen in Hypertension and Kidney Diseases.Diagnostics.2008;19:541-550.
12 Tusty-jiua Hsieh,Shao ling Zhang,etal.:High glucose stimulats angiotensinogen gene expression via Reactive Oxygen Species Generation in Rat Kidney Proximal Tubular Cells.Endocrinology 2008;143 (8):2975-2985.
13 Soo Hyun Park,HO Zae Han.:The mechanism of angiotensin Ⅱ binding downregulation by high glucose in primary renal proximal tubule cells.Am J Physiol Renal Physiol.2007;282:F228-F237.
14 Suzaki Y,Koori H,Ozawa Y.Intrarenal angiotensinogen augmentation is precedent to diabetic nephropathy in Zucker diabetic fatty obese rats.J Am Soc Nephrol.2008;16:202-210.
15 Atsuhiro Ichihara,Fumiaki Suzuki,et al.:Prorenin Receptor Blokade Inhibits Development of Glomerulosclerosis in Diabetic Angiotensin Ⅱ Type 1a Receptor-Deficient Mice.American Society of Nephrology.2006;17:1950-1961.
16 陈祥新,傅国胜.血管紧张素Ⅱ及其受体与血管内皮功能的关系(J) 1临床心血管病杂志.2003;19(11):698-700.
17 符史干,刘培庆,鲁伟,等.蛋白激酶C在血管紧张素Ⅱ抑制心肌细胞一氧化氮合成中的作用(J)生理学报.2000;52(4):318-322.
18 李皓,尹鸿操,张华,等.血管紧张素Ⅱ对人内皮细胞转录因子NF-κB的激活机制及其对血小板源生长因子B链基因转录的影响(J)中华病理学杂志.2001;30(4):276-280.
19 Xiaona Ge,Brad Low,Mei Liang,Jian Fu.:Angiotensin Ⅱ Directly Triggers Endothelial Exocytosis via Protein Kinase C-Dependent Protein Kinase D2 Activation.J Pharmacol Sci.2007;105:168-176.
20 Baijun Kou,Manu Vatish,Donald R.J.Singer.:Effects ofAngiotensin Ⅱ on human endothelial cells survival signalling pathways and its angiogenic response.Vascular Pharmcology.2007;47:199-208.
21 Xiu-Bin Liang,Li-Jun Ma,et al.:Agiotensin Type 1 receptor Blocker Restores Podocyte Potential to Promote Glomerular Endothelial Cell Growth.American Society of Nephrology.2006;17:1886-1895.
22 Bonavita F,Stefanelli C,Giordano E,et al.H9c2 cardiac myoblast s undergo apoptosis in a model of ischemia consisting of serum deprivation and hypoxia:inhibition by PMA[J].FEBS Lett.2003;536(123):85-91.
23 He M,He X,Xie Q,et al.Angiotensin Ⅱ induces t he expression of tissue factor and it s mechanism in human monocytes[J].Thromb Res.2006;117:579-590.
24 Mahrouf M,Ouslimani N,Peynet J,et al.Metformin reduces angiotensin-mediated intracellular production of reactive oxygen species in endot helial cells through t he inhibition a) of protein kinase C[J].Biochem Pharmacol.2008;72:176-183.
25 Walter DH,Haendeler J,Galle J,et al.Cyclosporin A inhibit apoptosis of human endothelial cells by preventing release of cytochrome C from mitochondria[J].Circulation.1998;98:1153-1157.
26 Buccellato LJ,Tso M,Akinci OI,et al.Reactive oxygen species are required for hyperoxia2induced Bax activation and cell death in alveolar epithelial cells[J].J Biol Chem.2007;279:6753-6760.
27 Distelhorst CW,Shore GC.Bc122 and calcium:controversy beneath t he surface[J].Oncogene.2004;23:2875-2880.
28 Ueda S,Masutani H,Nakarnura H,et al.Redox cont rol of cell death[J].Antioxid Redox Signal.2002;4:405-414.
29 Rossig L,Hoffmann J,Hugle B,et al.Vitamin C inhibits endothelial cell apoptosis in congestive heart failure[J].Circulation.2001;104:2182-2187.
30 Lshidoya S,Mocracken R,et al.Angiotensin Ⅱ recepor antagonist ameliorates renal tubulointerstitial fibrosis caused by anilatcral ureternal obstruction.Kidney Int.1995;47:1285-1291.
31 冯江敏,吴靖川,张国娟,等.慢性间质性肾炎炎症细胞浸润的特点中华肾脏杂志.1999;15(6):3-4.
32 Short AD,Bian J,Chosh TK,et al.intracellular Ca2 + pool content is linked to control of cell growth.Proc Natl AcadSci USA.1993;90:79-86.
33 Lixia Zeng,Hanshi XU,Teng-Leong Chew,et al.:HMG-CoA reductase inhibition modulates VEGF-induced endothelial cell hyperpermeability by preventing RhoA activation and myosi regularory light chain phosphorylation.The FASEB Journal.2005;17:235-240.
34 Masao Takemoto,James K.Liao.:Pleiotropic Effects of 3-Hydroxy-3-Methylglutarl Coenzyme A Reductase Inhibitors.Arterioscler.Thromb.Vasc.Biol.2008;21:1712-1719.
35 Hideaki Sawaki,Jungo Terasaki,et al.:A renoprotective effect of low dose losartan in patients with type 2 diabetes.Diabetes Research and Clinical Practice.2007;08:01-04.
36 Ahmet M.Sengul,Yukel Altuntas,et al:Beneficial effect of lisinopril plus telmisartan in patients with type 2 diabetes,nicroalbuninuria and hypertension.Diabetes Research and Clinical Practice.2006;71:210-219.
37 Moudgil R,Musat2Marcu S,Xu Y,et al.Increased AT2R protein expression but notincreased apoptosis during cardioprotection induced by AT1R blockade.Can J Cardiol.2002;18:873-883.
38 Dimmeler S,Rippmann V,Weiland U,et al.Angiotensin Ⅱ induces apoptosis of human endot helial cells protective effect of nitric oxide[J].Circ Res.2007;81:970-976.
39 Kim H,You S,Farris J,et al.Post2t ranscriptional inactivation of p53 in immortalized murine embryo fibroblast cells [J].Oncogene.2008;20:3306-3310.
40 Kren BT,Trembley J H,Steer CJ.Alterations in mRNA stability during rat liver regeneration[J].Am J Physiol.2008;270:G763-G777.