醛固酮对肾小球系膜细胞自噬的作用及机制研究

发布时间：2018-04-25 17:22

本文选题：自噬 + 系膜细胞　；参考：《第二军医大学》2014年博士论文

【摘要】：研究背景和目的慢性肾脏病（chronic kidney disease CKD）是全球范围内越来越严重的公共卫生问题。CKD患者大多数最终不得不接受肾脏替代治疗，并且持续占用大量的医疗资源。在CKD的治疗中，延缓肾功能持续进展是临床与科研工作者关注的重点。在肾脏固有细胞中，肾小球系膜细胞（mesangial cell MC）在CKD进展中的地位和作用越来越受到人们的重视。系膜细胞在不同的损伤因素下，可能表现出增殖肥大、凋亡、裂解、迁移、产生过量的系膜基质、产生过量的活性氧自由基（ROS）等等，造成肾损害加重及CKD持续的进展。所以，深入研究系膜细胞的生理功能以及在损伤条件下细胞生理功能的变化，有助于进一步了解系膜细胞的损伤应激系统，从而为监测与治疗CKD的进展提供更好的视角及切入点。自噬是体内最为基本的生理过程之一，真核细胞自噬活性的变化广泛参与了体内炎症、增殖、凋亡等复杂的生理及病理过程，并在其中发挥着极为重要甚至是决定性的作用。已有研究表明自噬的失调与肿瘤、神经退行性疾病、心血管疾病以及感染等疾病密切相关。而自噬在肾脏疾病中的作用正受到越来越多的关注。自噬活性的变化与肾小球系膜细胞应激损伤之间的关系现只有很少的研究报道。自噬是否参与了肾小球系膜细胞的损伤应激，这种损伤应激在CKD的进展中作用如何，值得进一步深入的研究。最新的研究表明，自噬的激活可以增加系膜细胞内I型胶原的降解，从而减少肾小球系膜基质的增多及抑制肾脏纤维化的进展。还有研究表明，引起肾脏纤维化最为重要的分子—TGF-β，能够诱导系膜细胞的自噬水平出现变化，影响着下游相关凋亡相关caspase-3分子的水平变化。可见，系膜细胞的自噬活性的变化参与了CKD进展中系膜细胞向成纤维细胞表型的转化，并可能在其中发挥了极为重要的作用。肾素-血管紧张素-醛固酮系统（RASS）的异常激活在慢性肾脏病的发生及发展进程中具有极为重要的意义。而近年来，醛固酮作为CKD的重要独立损伤因子，越来越受到人们的关注。在肾脏的炎症状态及纤维化发展过程中醛固酮发挥重要作用，影响着系膜细胞及其他肾脏固有细胞。国外研究结果表明，醛固酮通过激活活性氧（ROS）及表皮生长因子受体（EGFR），通过RAS/MAPK和PI3K/Akt信号通路诱使肾小球系膜细胞增殖。增殖的肾小球系膜细胞分泌系膜基质增加，并向纤维化的表型转化，促进了肾脏间质细胞的纤维化及CKD的进展。我们注意到，作为来自循环的系膜细胞损伤因子——醛固酮及维持系膜细胞自稳态及增加系膜基质清除的生理过程——自噬，都参与了系膜细胞的损伤应激及纤维化表型转化过程。醛固酮可以促进这一过程，系膜细胞自噬激活可以抑制这种转化。还有动物实验证实，醛固酮可以造成肾脏细胞的衰老，而自噬正是体内细胞对抗衰老的主要生理过程。综合上述的研究进展，醛固酮可能通过某些途径对系膜细胞的自噬产生负性调控，而这种自噬活性的变化可能参与了系膜细胞的损伤应激及CKD的进展过程。通过本研究，我们想进一步明确醛固酮对系膜细胞自噬的影响，初步的探讨醛固酮对自噬的调控作用产生的生理及病理意义，积极探索可能参与其中的信号通路及分子机制。细胞的自噬往往与线粒体功能的变化及细胞内ROS的水平具有很大的关联，而血管紧张素II及醛固酮都参与了系膜细胞线粒体的损伤及ROS的激活过程，我们也试图进一步研究二者与系膜细胞线粒体损伤的关系。实验方法 1．使用CCK8方法及流式细胞术测定醛固酮及血管紧张素对于系膜细胞增殖的影响。 2．通过western blot方法检测在饥饿、醛固酮、血管紧张素、表皮生长因子等不同干预的条件下系膜细胞自噬相关的蛋白标志物LC3、P62/SQSTM1的表达水平变化。检测信号通路蛋白EGFR、ERK、GAB1、PARP及剪切体PARP、AMPK及Beclin-1等蛋白表达水平的变化。 3.光镜下观察H2O2刺激系膜细胞在有无醛固酮长期干预下的凋亡变化。 4．通过向系膜细胞转入GFP-LC3的真核表达质粒，用激光共聚焦显微镜观察不同的刺激影响下系膜细胞荧光自噬点数量的变化并进行统计学分析。 5.通过电子显微镜观察不同的干预条件下系膜细胞自噬泡数量的变化，并进行统计学分析。 6.使用Mito-TrackerGreen探针（Invitrogen公司）检测不同的干预条件下系膜细胞线粒体的数量变化。使用Mito-ID细胞外液酸度检测试剂盒检测不同干预条件下细胞外液酸度变化情况，显示线粒体损伤后细胞糖酵解水平的变化。 7．免疫共沉淀的方法检测醛固酮干预对于Beclin-1-BCL2复合体的影响。 8. Real-timePCR方法检测了自噬相关基因及线粒体相关的基因在醛固酮及血管紧张素干预下的表达情况。结果 1.在体外培养的肾小球系膜细胞系HMCL及RMC中，，高于正常浓度的醛固酮与血管紧张素II不能使两种细胞系的增殖水平出现明显的变化。流式细胞仪检测干预后G2/M期细胞的比率没有明显的升高。 2.醛固酮可以抑制血清饥饿诱导的系膜细胞自噬活性的升高，且这种抑制表现为剂量依赖性。western blot检测发现醛固酮干预后EBSS培养的系膜细胞LC3II表达降低及P62/SQSTM1表达升高。共聚焦显微镜观察发现饥饿诱导的系膜细胞HMCL自噬点数量增加，而醛固酮可以抑制这种效应。电镜下自噬泡的的计数观察同样证实醛固酮可以抑制饥饿诱导的系膜细胞自噬活化。 3.通过western blot、荧光显微镜观察自噬点的形成以及电镜结果均可证实雷帕霉素可以激活肾小球系膜细胞的自噬，醛固酮不能够有效抑制这种自噬的激活效应。 4.在醛固酮导致的长期的自噬抑制状态下，系膜细胞在面临氧化应激时更容易发生凋亡。表现为在相同的时间点，凋亡细胞的比率明显增加，western blot检测发现PARP蛋白的剪切体的表达上升。 5.醛固酮、血管紧张素II能够在5、15、60分钟内轻度激活EGFR，促使其磷酸化的水平提高，但这种激活作用远远弱于EGF的直接刺激，三者激活EGFR的效应按强度大小排列为EGFAngIIAld。但EGF刺激EGFR磷酸化的强度随时间很快衰减，而血管紧张素II与醛固酮对EGFR磷酸化则在1小时内缓慢增强，至48小时仍有持续的EGFR磷酸化及ERK的磷酸化。提示细胞在高浓度的醛固酮及血管紧张素II持续刺激下可表现为EGFR及ERK的持续激活状态，且同时具有时间依赖性和剂量依赖性。 6.醛固酮可以降低在饥饿条件下AMPK的磷酸化水平，通过降低AMPK的磷酸化来抑制Beclin-1在Ser93/96位点的磷酸化，进而增加了其与BCL2的结合，导致Beclin-1与VPS34复合体结合减少，而抑制自噬。 7.与对照组及血清饥饿组相比，醛固酮干预24小时对于系膜细胞线粒体的数量有明显的增加，血管紧张素II效果更为显著。血管紧张素II不仅可以增加线粒体的数量，更影响了线粒体的形态，荧光显微镜下可见大量线粒体融合成拉丝状，电镜下线粒体肿胀，宽大畸形，部分融合，线粒体嵴排列紊乱。血管紧张素II及醛固酮均能降低线粒体的储备功能，削弱系膜细胞线粒体对于应激的反应性，但血管紧张素II对其影响更大。 8.醛固酮及血管紧张素II均可以造成部分自噬相关基因的表达发生变化，其中血管紧张素II多造成这些基因表达的下调，而醛固酮则造成这些基因的表达上调。醛固酮对于部分线粒体相关的基因的表达影响较轻，而血管紧张素II的影响较明显。特别是血管紧张素II对线粒体相关基因的影响程度大于醛固酮。结论 1.本实验第一次发现了醛固酮对于系膜细胞饥饿诱导的自噬激活的抑制效应。这种抑制效应参与了醛固酮在氧化应激中对系膜细胞的协同损伤。 2.醛固酮抑制自噬效应的靶点可能在雷帕霉素的上游。有可能是通过抑制AMPK磷酸化及下游的Beclin-1Ser93/96位点磷酸化。 3.血管紧张素II及醛固酮都能不同程度的损伤系膜细胞线粒体，激活ROS及造成EGFR的持续激活，前者较后者更为严重。
[Abstract]:Background and purpose of research
Chronic kidney disease (chronic kidney disease CKD) is an increasingly serious public health problem worldwide. Most of the patients with.CKD have to accept renal replacement therapy and continue to occupy a large number of medical resources. In the treatment of CKD, the continued progress of renal function is the focus of attention of clinical and scientific researchers. In the cells, the role and role of mesangial cell MC in the progression of CKD has been paid more and more attention. Under different damage factors, mesangial cells may show proliferation, apoptosis, cracking, migration, excessive mesangial matrix, excessive reactive oxygen free radical (ROS) and so on, resulting in renal damage plus renal damage. Therefore, it is important to further study the physiological functions of the mesangial cells and the changes in the physiological functions of the cells under the condition of damage. It is helpful to further understand the damage stress system of mesangial cells, and thus provide a better perspective and breakthrough point for the monitoring and treatment of the progress of CKD.
Autophagy is one of the most basic physiological processes in the body. The changes in autophagic activity of eukaryotic cells are widely involved in the complex physiological and pathological processes, such as inflammation, proliferation, apoptosis and other complex processes in the body, and play a very important and even decisive role in it. The role of autophagy in renal diseases is becoming more and more concerned. The relationship between the changes of autophagy and the stress damage of glomerular mesangial cells is only rarely reported. Is autophagy involved in the damage stress of glomerular mesangial cells; this damage stress is progresses in the progress of CKD The latest research shows that the activation of autophagy can increase the degradation of I type collagen in mesangial cells, thus reducing the increase of mesangial matrix and the progress in inhibiting renal fibrosis. And the study shows that the most important molecule TGF- beta, which causes renal fibrosis, can induce mesangial cells. The change of autophagy affects the level of Caspase-3 molecules associated with apoptosis in the downstream. It is seen that the changes in autophagy of mesangial cells are involved in the transformation of mesangial cells to the phenotype of fibroblasts in the progress of CKD, and may play an important role in it.
The abnormal activation of the renin angiotensin aldosterone system (RASS) is of great importance in the development and development of chronic renal disease. In recent years, aldosterone has attracted more and more attention as an important independent damage factor of CKD. Aldosterone plays an important role in the inflammatory state of the kidney and the development of fibrosis. The results showed that aldosterone induced the proliferation of glomerular mesangial cells by activating reactive oxygen species (ROS) and epidermal growth factor receptor (EGFR) by activating the active oxygen (ROS) and epidermal growth factor receptor (EGFR). The transformation promoted the fibrosis of renal interstitial cells and the progress of CKD.
It is noted that aldosterone as a damage factor from the circulatory mesangial cells, the self homeostasis of aldosterone and the maintenance of mesangial cells, and the physiological process of increasing the removal of the mesangial matrix - autophagy, is involved in the damage stress and phenotypic transformation of the mesangial cells. Aldosterone can promote this process and the autophagy activation of mesangial cells can be suppressed. And animal experiments have proved that aldosterone can cause the aging of renal cells, and autophagy is the main physiological process of cell aging in vivo. Synthesis of aldosterone may be a negative regulation of autophagy in mesangial cells through some ways, and the changes in autophagy may be involved in the system. Damage stress of membrane cells and the progress of CKD.
Through this study, we want to further clarify the effect of aldosterone on autophagy in mesangial cells, preliminarily explore the physiological and pathological significance of aldosterone in the regulation of autophagy, and actively explore the signaling pathways and molecular mechanisms involved in it. The autophagy of the cells is often associated with the changes in the function of the grain body and the level of ROS in the cell. The relationship between angiotensin II and aldosterone is involved in the mitochondrial damage of mesangial cells and the activation of ROS. We also try to further study the relationship between the two and the mitochondrial damage in mesangial cells.
Experimental method
1. the effects of aldosterone and angiotensin on proliferation of mesangial cells were measured by CCK8 and flow cytometry.
2. Western blot method was used to detect the changes in the expression level of the autophagy related protein markers, LC3, P62/SQSTM1, in the conditions of starvation, aldosterone, angiotensin, epidermal growth factor, etc., and to detect the changes in the expression level of the protein EGFR, ERK, GAB1, PARP and the PARP, AMPK and Beclin-1 protein of the signaling pathway protein, ERK, GAB1, PARP and shear.
3. the apoptosis of mesangial cells stimulated by H2O2 was observed under light microscope.
4. the changes in the number of autophagic points of the mesangial cells under the influence of different stimuli were observed and analyzed by a laser confocal microscope through the eukaryotic expression plasmid transferred into the mesangial cells to GFP-LC3.
5. the number of autophagic vacuoles in mesangial cells under different intervention conditions was observed by electron microscopy and analyzed statistically.
6. Mito-TrackerGreen probe (Invitrogen) was used to detect the changes in the number of mitochondria in mesangial cells under different intervention conditions. Mito-ID extracellular acidity detection kit was used to detect the change of acidity of extracellular fluid under different intervention conditions, and the changes of the level of fine cell glycolysis after mitochondrial injury were shown.
7. co immunoprecipitation was used to detect the effect of aldosterone intervention on Beclin-1-BCL2 complex.
8. the expression of autophagy related genes and mitochondrial associated genes under aldosterone and angiotensin were detected by Real-timePCR.
Result
1. in cultured glomerular mesangial cell lines HMCL and RMC, higher levels of aldosterone and angiotensin II were not significantly higher than normal concentrations of aldosterone and angiotensin, and the rate of G2/M cells was not significantly increased after flow cytometry.
2. aldosterone could inhibit the increase of autophagy in the mesangial cells induced by serum starvation, and this inhibition showed that the dose dependent.Western blot detection was used to detect the decrease of LC3II expression and the increase of P62/SQSTM1 expression in the mesangial cells cultured by EBSS. The confocal microscope observed the autophagy point of HMCL in the mesangial cells induced by starvation. The number of aldosterone could inhibit the effect. The observation of autophagic vesicles under electron microscope also confirmed that aldosterone could inhibit the activation of autophagy in the mesangial cells induced by starvation.
3. by Western blot, the formation of autophagic points and the results of electron microscopy can prove that rapamycin can activate autophagy in glomerular mesangial cells. Aldosterone can not effectively inhibit the activation of this autophagy.
4. in the state of long-term autophagy induced by aldosterone, mesangial cells are more prone to apoptosis in the face of oxidative stress. The percentage of apoptotic cells increased significantly at the same time point, and the expression of the shear body of PARP protein increased by Western blot detection.
5. aldosterone, angiotensin II, can activate EGFR slightly within 5,15,60 minutes to increase the level of phosphorylation, but this activation is far weaker than the direct stimulation of EGF. The effect of the three activates EGFR on the intensity of EGFAngIIAld. but EGF stimulates EGFR phosphorylation rapidly with time, and angiotensin II and Aldosterone was enhanced slowly for EGFR phosphorylation in 1 hours, and continued EGFR phosphorylation and phosphorylation of ERK to 48 hours. It was suggested that the cells exhibited persistent activation of EGFR and ERK at high concentrations of aldosterone and angiotensin II, and were dependent on the time dependent and dose-dependent manner.
6. aldosterone can reduce the phosphorylation level of AMPK under starvation, inhibit phosphorylation of AMPK to inhibit the phosphorylation of Beclin-1 at the Ser93/96 site, and then increase its binding with BCL2, resulting in a decrease in the binding of Beclin-1 to the VPS34 complex and inhibition of autophagy.
7. compared with the control group and the serum starvation group, the number of mitochondria in the mesangial cells increased significantly for 24 hours, and the effect of angiotensin II was more significant. Angiotensin II could not only increase the number of mitochondria, but also affect the morphology of mitochondria. Mitochondrial swelling, broad deformities, partial fusion and mitochondrial crista disorder. Angiotensin II and aldosterone both reduce the mitochondrial reserve function and weaken the response to stress in the mesangial cell mitochondria, but angiotensin II has a greater impact on it.
8. aldosterone and angiotensin II can cause changes in the expression of partial autophagy related genes, in which angiotensin II causes the downregulation of these gene expressions, and aldosterone causes the up-regulated expression of these genes. Aldosterone has a light influence on the expression of some mitochondrial related genes, and the effect of angiotensin II The effect of angiotensin II on mitochondrial related genes was greater than that of aldosterone.
1. the inhibition effect of aldosterone on the activation of autophagy induced by the starvation of mesangial cells was first discovered in this experiment. This inhibitory effect was involved in the synergistic damage of aldosterone to mesangial cells during oxidative stress.
2. aldosterone may inhibit the autophagy effect upstream of rapamycin, probably by inhibiting AMPK phosphorylation and downstream Beclin-1Ser93/96 site phosphorylation.
3. angiotensin II and aldosterone can damage the mitochondria of mesangial cells in different degrees, activate ROS and cause EGFR to continue to activate. The former is more serious than the latter.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R692

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