【作者】 丁桂霞；

【导师】 陈荣华；

【作者基本信息】 南京医科大学 ， 儿科学， 2008， 博士

【摘要】 随着社会经济发展和人们生活方式的改变,人类疾病谱正在发生变化,慢性肾脏病(CKD)已呈现流行特征,并成为21世纪全球性的公共卫生问题。CKD一旦发展为终末期肾脏病(ESRD),则必须依赖肾脏替代治疗(RRT),即血液透析、腹膜透析、或肾移植来维持生命,给患者家庭及社会带来沉重的经济负担和社会压力。因此,早期诊断及防治慢性肾脏病有着特殊重要的意义。研究表明,肾素—血管紧张素—醛固酮系统(RAAS),尤其是血管紧张素Ⅱ(AngⅡ)在CKD的发生和进展中发挥重要作用。AngⅡ是RAAS系统最主要的效应分子,业已证实AngⅡ不仅通过改变肾小球血流动力学促进肾小球硬化,还可促进系膜细胞、内皮细胞和肾小管上皮细胞增生、生长因子表达及细胞外基质积聚。AngⅡ的作用是通过与Gq蛋白偶联的Ⅰ型血管紧张素受体(AT1受体)完成的,而AT1受体为跨膜糖蛋白,本身不具有酪氨酸激酶(此酶介导有丝分裂的细胞信号)活性,也不直接与酪氨酸激酶发生联系。本研究将深入探讨AngⅡ介导肾小球系膜细胞增殖及炎症介质的表达的信号转导机制,这不仅有助于加深人们对CKD发病机制的认识,同时也为未来CKD治疗策略的制定提供理论依据。本研究包括两部分内容:一、c-Jun氨基末端激酶介导血管紧张素Ⅱ诱导的人肾小球系膜细胞增殖及炎症介质表达目的:AngⅡ可诱导体外培养的系膜细胞c-Jun氨基末端激酶(JNK)活化,但其活化后的生物学意义仍不清楚。本研究选用新型的JNK特异性阻断剂SP-600125,探讨JNK-c-Jun/AP-1信号通路在AngⅡ诱导的肾小球系膜细胞增殖及炎症介质表达中的作用。方法:体外分离培养人肾小球系膜细胞,应用SP-600125预处理后加入AngⅡ刺激,应用Western Blot检测JNK、细胞外调节蛋白激酶(ERK1/2)和p38的活性以及c-Jun磷酸化;应用荧光素酶(Luciferase)活性检测法检测c-Jun转录活性及单核细胞趋化蛋白(MCP-1)启动子活性;凝胶电泳迁移率(EMSA)检测活化蛋白-1(AP-1)DNA结合活性;核酸酶保护法检测MCP-1 mRNA表达;ELISA检测培养上清中MCP-1、转化生长因子(TGF-β1)和纤维连接蛋白(FN)的分泌;~3H-胸腺嘧啶(~3H-TdR)掺入法和细胞计数法测定系膜细胞的增殖。结果:1.AngⅡ可呈时间依赖性和剂量依赖性的方式诱导JNK活化及c-Jun磷酸化,100 nmol/L AngⅡ刺激15 min后,c-Jun磷酸化显著增加,JNK活性明显上调,30分钟达到高峰,是对照组的6.2倍,1 h后几乎恢复到正常水平。2.JNK特异性抑制剂SP-600125呈剂量依赖性的方式抑制AngⅡ诱导的JNK活化和c-Jun磷酸化,当浓度为10μmol/L和20μmol/L时其抑制作用分别达75%和90%,SP-600125对ERK1/2和p38 MAPK磷酸化水平均无抑制作用。3.AngⅡ刺激后c-Jun转录活性及AP-1 DNA结合活性显著增加,SP-600125可呈剂量依赖的抑制AngⅡ诱导的c-Jun转录活性及AP-1 DNA结合活性的增加,20μmol/L SP-600125预处理几乎完全阻断c-Jun的反式激活。4.AngⅡ显著增加MCP-1启动子活性及MCP-1 mRNA表达,SP-600125可呈剂量依赖性的抑制AngⅡ诱导的MCP-1启动子活化和MCP-1 mRNA表达以及系膜细胞MCP-1、TGF-β和FN的分泌。5.1～20μmol/L SP-600125以剂量依赖的方式抑制AngⅡ促进的系膜细胞~3H-TdR掺入量和细胞计数的增加,而MEK1/2特异性抑制剂PD-98059和p38 MAPK特异性抑制剂SB-203580对AngⅡ诱导的系膜细胞数目的增加无抑制作用。结论:AngⅡ→JNK/SAPK→c-Jun/AP-1信号通路在系膜细胞增殖和炎症介质分泌中发挥一定的作用。JNK特异性抑制剂SP-600125能部分抑制AngⅡ诱导的系膜细胞增殖和炎症介质分泌。二、血管紧张素Ⅱ通过ROS/EGFR/JNK/AP-1信号通路诱导肾小球系膜细胞增殖目的:我们前期的研究发现AngⅡ可通过JNK/AP-1信号通路诱导肾小球系膜细胞增殖,但JNK/AP-1活化的上游通路尚不清楚。本研究探讨活性氧(ROS)和表皮生长因子受体(EGFR)在AngⅡ诱导的JNK/AP-1活化及系膜细胞增殖中的作用并探讨ROS释放的来源。方法:体外培养人肾小球系膜细胞,应用~3H-TdR掺入法和细胞计数测定系膜细胞增殖;荧光探针2,7—二氯二氢荧光素乙酰乙酸(DCFDA)检测细胞内ROS产生;化学发光法检测尼克酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶活性;实时定量RT-PCR检测NADPH亚基p47phox和p67phox mRNA表达;Western Blot检测p47phox和p67phox膜转位以及EGFR、JNK和c-Jun磷酸化。结果:1、AngⅡ呈时间依赖性和剂量依赖性促进肾小球系膜细胞ROS产生。AngⅡ刺激3 min,系膜细胞内ROS产生明显增加,至60 min达到高峰,系膜细胞ROS产生是对照组2.26倍;1、10、100nmol/L AngⅡ刺激60 min,ROS产生分别是对照组的1.82、2.92和4.08倍。AT1受体(AT1R)拮抗剂losartan完全阻断了AngⅡ诱导的ROS产生,而AT2受体(AT2R)拮抗剂PD123319无抑制作用。2、NADPH氧化酶抑制剂apocynin和DPI几乎完全阻断AngⅡ诱导的ROS产生,而线粒体complexⅠ抑制剂鱼藤酮(rotenone,ROT)、黄嘌呤氧化酶抑制剂别嘌呤醇(allopurinol,ALLO)、环氧化酶抑制剂吲哚美辛(indomethacin,INDO)、脂氧化酶抑制剂(nordihydroguiaretic acid,NDGA)、细胞色素P450氧化酶抑制剂酮康唑(ketoconazole,KETO)以及一氧化氮合成酶抑制剂N-硝基-L-甲酯精氨酸(N~G-nitro-L-arginine methyl ester,L-NAME)对AngⅡ诱导的ROS产生均无明显影响。AngⅡ显著刺激NADPH氧化酶活化及p47phox和p67phox膜转位。3、AngⅡ可呈时间依赖性和剂量依赖性诱导系膜细胞EGFR磷酸化,100 nmol/L AngⅡ刺激30 min,EGFR磷酸化达到高峰,是对照组的3.96倍;AT1受体阻断剂losartan、抗氧化剂乙酰半胱氨酸(NAC)以及NADPH氧化酶抑制剂apocynin和DPI显著抑制AngⅡ诱导的EGFR磷酸化,同时EGFR拮抗剂AG-1478几乎完全阻断AngⅡ诱导的系膜细胞增殖。4、Losartan、NAC、apocynin、DPI和AG-1478阻断AngⅡ诱导的JNK/AP-1活化和系膜细胞增殖。结论:ROS/EGFR/JNK/AP-1信号通路参与AngⅡ诱导的肾小球系膜细胞增殖。NADPH氧化酶抑制剂和EGFR受体拮抗剂能显著抑制AngⅡ诱导的系膜细胞增殖,可能具有一定的治疗作用。本研究创新之处在於:1.首次发现:AngⅡ可通过JNK—c-Jun/AP-1信号通路诱导肾小球系膜细胞增殖和MCP-1、TGF-β以及FN表达(参见:Zhang A,Ding G,Huang S,Wu Y,Pan X,Guan X,Chen R,Yang T.c-Jun NH2-terminal kinase mediationof angiotensinⅡ-induced proliferation of human mesangial cells.Am J PhysiolRenal Physiol.2005;288:F1118-1124)。2.首次发现:活性氧依赖的表皮细胞生长因子受体磷酸化介导AngⅡ诱导JNK—c-Jun/AP-1信号通路活化及肾小球系膜细胞增殖(参见:Ding G,Zhang A,Huang S,Pan X,Zhen G,Chen R,Yang T.ANGⅡinduces c-JunNH2-terminal kinase activation and proliferation of human mesangial cells viaredox-sensitive transactivation of the EGFR.Am J Physiol Renal Physiol.2007;293:F1889-1897)。更多还原

【Abstract】 Chronic kidney disease（CKD）is an emerging top public health problem affecting 1,300,000,000 Chinese.CKD arises from a wide variety of etiologies and develops into a common manifestation of end-stage renal disease characterized by glomerulosclerosis and tubulointerstitial fibrosis and the complete loss of renal function,which require dialysis or kidney transplantation to maintain life.The use of inhibitors of the components of the renin-angiotensin axis,namely, angiotensin-converting enzyme inhibitors（ACEi）and angiotensin type 1（AT1） receptor antagonists（ARBs）,has become a standard clinical practice for treatment of CKD.Despite intensive investigation,the mechanism of the renal beneficial effects of ACEi and ARBs is incompletely understood.It is clear,however,that these effects of ACEi and ARBs cannot be entirely attributed to their hemodynamic effects.Consistent with this notion,AngiotensinⅡ（AngⅡ）exerts a direct detrimental effect in the renal cells.In particular,AngⅡstimulates activation of mesangial cells（MCs）,a hallmark of the pathological changes in CKD.Therefore,it is imperative to understand the signaling mechanism of the AngⅡ-elicited activation-promoting effect in the MCs.The elucidation the signaling pathway involved in AngⅡ-induced mesangial cells activation provides insights into the mechanisms of CKD and may also help identify new targets for treatment of CKD.The present study consists two parts:PartⅠ:c-Jun NH₂-terminal kinase mediation of angiotensinⅡ-induced proliferation and inflammatory mediators expression in human mesangial cells Objective:The octapeptide hormone AngⅡhas been shown to activate c-Jun NH2-terminal kinase（JNK）in cultured mesangial cells,but the functional implication of this phenomenon remains to be determined,largely due to the lack of an effective approach to block JNK.Therefore,the present study was carried out to examine whether JNK is involved in AngⅡ-induced cell proliferation and expression of inflammatory mediators,such as monocyte chemoattractant protein-1 （MCP-1）,transforming growth factor-β（TGF-β）and fibronectin（FN）,in cultured human mesangial cells（HMCs）with the use of a newly developed JNK-selective blocker,SP-600125.Methods:Normal-appearing portions of human kidneys that were surgically removed for renal carcinoma were used to culture MCs from outgrowths of collagenase-treated glomeruli.The incorporation of ³H-thymidine（³H-TdR）and cell count were used as the measure of mesangial cell proliferation.MCP-1 mRNA expression was determined by ribonuclease protection assay.MCP-1,TGF-β1,and FN excretion was examined by ELISA.JNK and c-Jun phosphorylation was detected by Western Blot.Transient luciferase reporter assay and electrophoretic mobility shift assay（EMSA）were used to detect the transcription activity of c-Jun and the activity of AP-1.Results:1.The increase in JNK activity and c-Jun phosphorylation in HMCs was detected at 15 minutes after the addition of AngⅡ.Maximal activity occurred at 30 minutes（6.2-fold increase）and reducing thereafter.AngⅡactivated JNK in a dose-dependent manner,with a maximal stimulation seen at 100 nmol/L.2. SP600125 countered the AngⅡ-induced Ser63 phosphorylation of c-Jun in a dose-dependent manner.The reduction in JNK activity was 75%at 10μmol/L concentration and 90%at 20μmol/L concentration.SP600125 had no significant effect on the activation of ERK1/2 and p38 MAPK.3.AngⅡ-induced transcriptional activity of the c-Jun was inhibited by SP600125.Nuclear extract from HMCs treated with AngⅡshowed increased binding to the AP-1 motif in a time-dependent manner.SP600125 effectively countered the increased AP-1 binding in a dose-dependent manner.4.AngⅡstrongly increased MCP-1 promoter activity and MCP-1 mRNA expression in a time-dependant manner.SP600125 had a concentration-dependent inhibitory effect on AngⅡ-induced MCP-1 promoter activity and mRNA expression at a concentration range of 1 to 20μmol/L. SP600125 inhibited AngⅡ-induced MCP-1,TGF-βand FN production in a concentration-dependent manner.5.SP600125（1 20μmol/L）inhibited AngⅡ-induced mesangial cell proliferation as determined by ³H-thymidine incorporation and cell count in a concentration-dependent manner,the incorporation was decreased by 1,5,10,and 20μmol/L of SP600125,respectively.Conclusion:These results show that the JNK/AP-1 pathway is involved in the cell proliferation,MCP-1 and TGF-βexpression,and ECM production and JNK is an important therapeutic target for glomerulonephritis and glomerulosclerosis.PartⅡ:NADPH oxidase-derived ROS mediated AngiotensinⅡ-induced Human Mesangial Cells ProliferationObjective:We have previously shown that AngⅡinduces mesangial cell proliferation via JNK/AP-1 pathway.The present study attempted to determine the upstream mediators of the JNK activation with emphasis on reactive oxygen species（ROS）.Methods:The incorporation of ³H-thymidine（³H-TdR）and cell count were used as the measure of mesangial cell proliferation.ROS production was determined by DCFDA fluorescence.Nicotinamide adenine dinucleotide phosphate（NADPH） oxidase activity was examined by lucigenin chemiluminescence.JNK activation was assayed by Western Blot.Results:1.In cultured human mesangial cells,AngⅡtime-dependently and dose-dependently increased intracellular ROS production as early as 3 min and peak at 60 min.Incubation with different dose of AngⅡ（1,10,100 nmol/L AngⅡ） for 60 min,ROS production increased for 1.82-,2.92-,and 4.08-fold,respectively. 2.AngⅡ-induced ROS generation was sensitive to diphenyleneiodonium sulfate （DPI）（10μmol/L）and apocynin（500μmol/L）,two structurally distinct NADPH oxidase inhibitors.In contrast,inhibitors of other oxidant-producing enzymes, including the mitochondrial complexⅠinhibitor rotenone,the xanthine oxidase inhibitor allopurinol,the cyclooxygenase inhibitor indomethacin,the lipoxygenase inhibitor nordihydroguiaretic acid,the cytochrome P450 oxygenase inhibitor ketoconazole and the nitric oxide synthase inhibitor G-nitro-L-arginine methyl ester were without an effect.3.AngⅡ-induced ROS generation was inhibited by the AT1 antagonist losartan（10μmol/L）but not the AT2 antagonist PD 123319（10μmol/L）.4.AngⅡtreatment induced translocation of cytosolic of p47phox and p67phox to the membrane.The antioxidants almost abolished AngⅡ-mitogenic response,associated with a remarkable blockade of the activation of JNK（83% inhibition）.Conclusions:NADPH oxidase-derived ROS involved in AngⅡ-induced JNK/AP-1 activation and mesangial cell proliferation.更多还原