自噬在糖尿病肾病大鼠足细胞损伤中的作用研究

发布时间：2018-06-30 06:33

本文选题：糖尿病肾病 + 血管紧张素Ⅱ　；参考：《南方医科大学》2014年硕士论文

【摘要】：研究背景糖尿病(diabetes mellitus)的发病率逐年上升,我国已成为目前世界上DN患病人口最多的国家,由此导致的糖尿病肾病(diabetic nephropathy, DN)人数也越来越多,对DN的发病机制进行研究势在必行,但目前这一问题仍然悬而未决。足细胞(Podocyte)作为肾小球滤过屏障的重要组成部分,其在DN发生发展中占有的重要作用越来越受到关注,目前足细胞损伤已被公认为是DN进展的核心环节,且DN早期时就可观察到足细胞受损。虽然DN情况下造成足细胞损伤的机制尚不完全清楚,但有研究证明血管紧张素Ⅱ (angiotensin Ⅱ, Ang Ⅱ)是其可能的原因之一。自噬(autophagy)是细胞内清除蛋白质、受损及衰老细胞器的有效途径,被认为是维持细胞内环境稳定的细胞保护性机制。足细胞在正常情况下即具备高水平自噬,而病理情况下足细胞中的自噬水平紊乱可能将进一步加重足细胞损伤,故自噬对足细胞的存亡具有重要意义。自噬有望成为治疗早期DN的一个靶点,然而目前对于自噬在DN肾损伤中的作用机制还不清楚。目的本研究首先通过建立DN大鼠模型,研究了Ang Ⅱ水平在早期DN中的变化情况及自噬在足细胞损伤中的作用；然后用血管紧张素转换酶抑制剂(angiotensin converting enzyme inhibitor, ACEI)贝那普利和Ang Ⅱ受体拮抗剂(angiotensin II receptor blockers, ARBs)氯沙坦分别干预早期DN模型大鼠,观察两者对早期大鼠DN足细胞损伤的保护作用及对足细胞中自噬活性的影响,以探讨自噬在早期DN发生中的作用机制。方法 (1)将80只雄性Wistar大鼠随机分为正常对照组(n=6)、单肾切除组(n=6)、单肾切除+贝那普利组(n=6)、单肾切除+氯沙坦组(n=6)、糖尿病肾病组(n=10)、糖尿病肾病+胰岛素注射组(n=10)、糖尿病肾病+胰岛素注射+贝那普利组(n=12)和糖尿病肾病+胰岛素注射+氯沙坦组(n=12),共8组。 (2)大鼠适应性饲养1周后行单侧肾切除术,4周后腹腔注射中低剂量(45mg/kg)链脲佐菌素(streptozotocin, STZ)建立DN模型。模型建立后连续3天监测大鼠尾尖血糖,血糖持续超过16.7-33.3mmol/L则视为模型建立成功。此后每4周称量大鼠体重,检测鼠尾血糖,使模型组大鼠血糖始终高于16.7-33.3mmol/L,随时剔除造模失败及死亡实验对象。 (3)DN模型成功后第16周开始分别用贝那普利和氯沙坦对模型大鼠进行干预,单肾切除组实施相同干预,空白对照组给予等量安慰剂。 (4)实验终点前测量各组大鼠体重；干预8周后处死大鼠,分别留取血尿标本。 (5)测定血糖、血肌酐、血清白蛋白、放免法检测血清中AngⅡ水平、24小时尿蛋白等指标。 (6)留取肾脏组织,计算肾重／体重比值,一部分肾脏组织用于放免法测定肾脏组织中AngⅡ水平改变；一部分肾脏皮质组织分别经2.5%戊二醛和10%福尔马林溶液固定后分别行光镜及电镜检查肾脏组织病理改变；剩余肾脏组织用于提取肾小球,行Western Bloting检测足细胞标志蛋白Podocin和Nephrin表达情况的改变。 (7)电镜下分别观察各组大鼠肾小球足细胞中自噬体数目的变化情况；检测各组大鼠肾小球组织中自噬体标志物LC-Ⅱ、自噬相关蛋白Atg5及P62蛋白表达情况的变化。结果 (1)成功地建立DN大鼠模型。与正常对照组大鼠相比,模型组大鼠体重明显减轻(P0.05),经药物干预8周后体重明显增加(P0.05),单肾切除各组与正常对照组之间体重差异无统计学意义(P0.05)。 (2)与正常对照组大鼠相比,单肾切除各组和模型组大鼠肾质量/体质量比值均明显升高(P0.01)且模型组大鼠较单肾切除组大鼠升高更为明显(P0.01),经药物干预后模型大鼠的肾质量/体质量比值明显降低(P0.01)。 (3)模型大鼠血糖与正常对照组相比明显升高(P0.01),经胰岛素干预后血糖明显降低(P0.01),且始终维持在16～17mmol/L这一水平,单肾切除各组血糖与正常对照组相比差异无统计学意义。 (4)模型大鼠肌酐值较正常对照组明显升高(P0.01),经药物干预后肌酐值明显降低(P0.01),单肾切除各组肌酐值与正常对照组相比差异无统计学意义。 (5)血清白蛋白水平模型组及单肾切除组与正常对照组相比均明显降低(P0.01),其中模型组降低更为明显(P0.01),经药物干预后模型大鼠血清白蛋白水平明显升高(P0.01)。 (6)与正常对照组相比,模型大鼠24小时尿蛋白明显增加(P0.01),经贝那普利及氯沙坦治疗后模型大鼠蛋白尿明显减轻(P0.01),而单肾切除组大鼠24小时尿蛋白与正常对照组相比差异无统计学意义。 (7)接受氯沙坦治疗的大鼠(单肾切除+氯沙坦组及DN+胰岛素+氯沙坦组)血清中Ang Ⅱ水平与正常对照组相比明显升高(P0.05),后者升高更为明显(P0.05)其余各组与正常对照组相比差异无统计学意义。 (8)与正常对照组相比,单肾切除+氯沙坦组及DN各组大鼠肾组织中AngⅡ水平均明显升高(P0.01),其中DN+胰岛素+氯沙坦组大鼠肾组织Ang Ⅱ水平升高更为明显(P0.01)。 (9)光镜下,见模型组大鼠肾小球肥大,系膜膜细胞增生、基质增多,可见蛋白管型；电镜可见肾小球基底膜明显增厚、足细胞肥大、足突广泛融合。 (10)模型组及DN+胰岛素+贝那普利组大鼠肾小球组织中的Podocin及Nephrin与正常对照组相比明显升高(P0.05),其余各组与正常对照组相比差异无统计学意义。 (11)电镜下观察：模型组大鼠肾小球足细胞中自噬体数目较正常对照组明显增加(P0.05),经胰岛素及氯沙坦治疗后模型大鼠足细胞中自噬体数目明显减少(P0.05)。 (12)模型组大鼠肾小球组织中的LC3-Ⅱ、Atg5及P62蛋白表达较正常对照组大鼠明显增加(P0.01),氯沙坦干预后模型大鼠肾小球组织中的LC3-Ⅱ、Atg5及P62蛋白表达水平明显降低(P0.01)。讨论 (1)本研究中采用的建模方法成功模拟了DN早期病理特点。产生的蛋白尿在贝那普利及氯沙坦的干预作用下得到明显改善,但其对已产生的肾脏组织病理变化及足细胞损伤的改善并不十分明显,可能由于药物干预时间尚短。 (2)经氯沙坦干预的大鼠(单肾切除+氯沙坦和DN+胰岛素+氯沙坦)血清中及肾脏组织中Ang Ⅱ水平升高,是由于Ang Ⅱ与AT1受体的结合被氯沙坦阻滞后,血管阻力降低,醛固酮分泌减少,血浆AngⅡ水平增高。而作为血管紧张素转换酶抑制剂的贝那普利治疗的DN大鼠肾脏组织中的AngⅡ水平较正常对照组没有明显降低,则可能是由于其不能抑制由非血管紧张素转换酶路径合成的AngⅡ。 (3)本研究中DN大鼠肾小球组织中Podocin及Nephrin的表达明显增加,这可能是DN早期足细胞代偿性肥大、足突广泛融合所导致的。 (4)DN大鼠肾小球足细胞中自噬体数目增加、肾小球组织中自噬活性增加,其可能机制是a.DN状态下肾小球足细胞中的肾素-血管紧张素系统(renin-angiotensinsystem, RAS)活化,足细胞中的Ang Ⅱ水平升高激活了氧化应激产生活性氧族(reactive oxygen species, ROS),活性氧族的产生可诱导自噬增强；b.高糖状态直接激活氧化应激,产生活性氧族诱导自噬增强。 (5)氯沙坦干预后可减轻氧化应激诱导的自噬活性增强,降低蛋白尿,延缓DN进展。可能机制是通过选择性阻断血管紧张素受体1(AT1)阻断了AngⅡ的生物学效应。 (6)贝那普利干预后虽能减轻模型大鼠蛋白尿,但其肾小球组织中的自噬活性却依然升高,表明在DN状态下,除AngⅡ的作用外,还有其他机制影响足细胞中的自噬活性。本课题对于理解DN足细胞损伤的机制,及自噬在DN足细胞损伤中的作用,探索自噬活性在防治DN中的意义进行了一定程度的研究。
[Abstract]:Research background
The incidence of diabetes (diabetes mellitus) is increasing year by year. China has become the country with the largest population of DN in the world, and the number of diabetic nephropathy (diabetic nephropathy, DN) is also increasing. It is imperative to study the pathogenesis of DN, but the problem is still in pending. Podocyte (Podocyte) is still in the air. As an important part of the glomerular filtration barrier, its important role in the development of DN has attracted more and more attention. Podocyte injury is now recognized as the core of the progress of DN, and the damage of the Poda can be observed at the early stage of DN. Although the mechanism of the foot cell injury in the case of DN is not completely clear, there is a study Angiotensin II (angiotensin II, Ang II) is one of the possible causes. Autophagy (autophagy) is an effective way to remove protein, damage and senescence organelles in cells. It is considered to be a cellular protective mechanism to maintain the stability of the cell environment. Autophagy in podocytes may further aggravate foot cell damage, so autophagy is of great significance to the survival of podocytes. Autophagy is expected to be a target for the treatment of early DN. However, the mechanism of autophagy in DN renal injury is still unclear.
objective
In this study, the changes of the level of Ang II in the early DN and the role of autophagy in the foot cell injury were studied by establishing the DN rat model, and then the angiotensin converting enzyme inhibitor (angiotensin converting enzyme inhibitor, ACEI) and Ang II receptor antagonists (angiotensin II receptor) were used. S) the effect of losartan on early DN model rats and the protective effect of both of them on DN podocyte injury in early rats and the effect on autophagy in podocytes were observed in order to explore the mechanism of autophagy in early DN.
Method
(1) 80 male Wistar rats were randomly divided into normal control group (n=6), single nephrectomy group (n=6), single nephrectomy + benazepril group (n=6), single nephrectomy + losartan group (n=6), diabetic nephropathy group (n=10), diabetic nephropathy + insulin injection group (n=10), diabetic nephropathy + insulin injection + benazepril group (n=12) and diabetic nephropathy + insulin injection. There were 8 groups in the group of shoot + losartan (n=12).
(2) the rats were fed with unilateral nephrectomy after 1 weeks of adaptive feeding, and the DN model was established by intraperitoneal injection of middle and low dose (45mg/kg) streptozotocin (streptozotocin, STZ) after 4 weeks. After the establishment of the model, the rat tail tip blood glucose was monitored continuously for 3 days, and the blood sugar continued to exceed 16.7-33.3mmol/L as a model. After the model, the rat weight was weighed every 4 weeks and the tail of the rat was detected. The blood glucose level of the model group was higher than that of 16.7-33.3mmol/L, and the model failure and death were excluded at any time.
(3) the model rats were intervened with Benner Pury and losartan respectively at sixteenth weeks after the DN model was successful. The same intervention was carried out in the single nephrectomy group, and the blank control group was given the same amount of placebo.
(4) the weight of rats in each group was measured before the end of the experiment. After 8 weeks of intervention, rats were sacrificed and blood and urine samples were kept.
(5) determination of blood glucose, creatinine, serum albumin, radioimmunoassay, serum Ang II level, 24 hour urine protein and other indicators.
(6) the renal tissue was retained and the ratio of kidney weight / weight was calculated. Some renal tissues were used for radioimmunoassay to determine the change of Ang II in renal tissue. A part of renal cortical tissue was fixed by 2.5% glutaraldehyde and 10% formalin solution respectively after fixation by light microscopy and electron microscopy to examine the pathological changes of kidney tissue, and the remaining renal tissue was used for extraction. Glomerular Western Bloting was used to detect the expression of podocyte marker protein Podocin and Nephrin.
(7) the changes in the number of autophagosomes in the glomerular podocytes were observed under the electron microscope, and the autophagosomes LC- II, the autophagy related protein Atg5 and the expression of P62 protein in the glomerular tissues of the rats were detected.
Result
(1) the DN rat model was successfully established. Compared with the normal control group, the body weight of the model rats was significantly reduced (P0.05), and the weight increased significantly after 8 weeks of drug intervention (P0.05). There was no significant difference in weight difference between the single nephrectomy group and the normal control group (P0.05).
(2) compared with the normal control group, the renal mass / body mass ratio of the rats in the single nephrectomy group and the model group were significantly increased (P0.01) and the rats in the model group were more significantly higher than those in the single nephrectomy group (P0.01), and the renal mass / body mass ratio of the rats after the drug dry prognosis was significantly decreased (P0.01).
(3) the blood glucose in the model rats was significantly higher than that in the normal control group (P0.01), and the blood sugar decreased significantly (P0.01) after the intervention of insulin, and remained at the level of 16 to 17mmol/L. There was no significant difference in blood sugar in the single nephrectomy group compared with the normal control group.
(4) the creatinine value of the model rats was significantly higher than that in the normal control group (P0.01), and the creatinine value decreased significantly after the drug intervention (P0.01). The creatinine value of the single nephrectomy group was not statistically significant compared with the normal control group.
(5) the serum albumin level model group and the single nephrectomy group were significantly lower than the normal control group (P0.01), in which the model group decreased more significantly (P0.01), and the serum albumin level of the rat model was significantly increased (P0.01) after the drug dry prognosis model.
(6) compared with the normal control group, the 24 hour urine protein in the model rats increased significantly (P0.01). The proteinuria of the model rats after the treatment of Benner Pury and losartan was significantly reduced (P0.01), but there was no significant difference between the 24 hours urine protein in the single nephrectomy group and the normal control group.
(7) the level of Ang II in the serum of rats treated with losartan (single nephrectomy plus losartan group and DN+ insulin + losartan group) was significantly higher than that of the normal control group (P0.05), the latter was more obvious (P0.05) and the difference between the other groups was not statistically significant compared with the normal control group.
(8) compared with the normal control group, the levels of Ang II in the renal tissue of the single nephrectomy + losartan group and the DN rats were all significantly increased (P0.01), and the level of Ang II in the renal tissue of the rats of DN+ insulin + losartan group was more obvious (P0.01).
(9) under the light microscope, the glomerular hypertrophy, the proliferation of mesangial cells, the increase of the matrix and the protein tube type were seen in the model group. The glomerular basement membrane was obviously thickened, the podocytes were hypertrophy, and the foot process was widely fused.
(10) the Podocin and Nephrin in the glomerular tissue of the model group and the DN+ insulin + benazepril group were significantly higher than that in the normal control group (P0.05), and the other groups had no statistical difference compared with the normal control group.
(11) observation under electron microscope: the number of autophagosomes in the glomerular podocytes of rat model group was significantly higher than that in the normal control group (P0.05). The number of autophagosomes in the rat foot cells after the treatment of insulin and losartan was significantly decreased (P0.05).
(12) the expression of LC3- II, Atg5 and P62 protein in the glomerular tissue of the model rats was significantly higher than that in the normal control group (P0.01). The expression level of LC3- II, Atg5 and P62 protein in the glomerular tissue of rats with losartan dry prognosis was significantly decreased (P0.01).
discuss
(1) the modeling method used in this study successfully simulated the early pathological characteristics of DN. The proteinuria produced by the intervention of Benner Pury and losartan was obviously improved, but the improvement of the pathological changes of kidney tissue and the improvement of podocyte injury was not very obvious, which may be due to the short time of drug intervention.
(2) the levels of Ang II in the serum and renal tissues of rats treated with losartan intervention (mononrennephrectomy + losartan and DN+ insulin + Losartan) were increased in the serum and renal tissues, due to the decrease of vascular resistance, the decrease of aldosterone secretion and the level of Ang II in plasma after the combination of the combination of Ang II and AT1 receptor by losartan, and the level of plasma Ang II. The level of Ang II in the renal tissue of the DN rats treated with nalpril was not significantly lower than that in the normal control group, perhaps because it could not inhibit the Ang II synthesized by the non angiotensin converting enzyme pathway.
(3) the expression of Podocin and Nephrin in the glomerular tissue of DN rats increased significantly in this study, which may be caused by the compensatory hypertrophy of the early podocytes in DN and the extensive fusion of the poddate.
(4) the number of autophagosomes in the glomerular podocytes of DN rats increased and the autophagic activity in the glomerular tissue increased. The possible mechanism was the activation of the renin angiotensin system (renin-angiotensinsystem, RAS) in the glomerulopodocyte (a.DN), and the increase of Ang II in the podocytes activated the oxidative stress to produce the living oxygen group (reactive oxyge). N species (ROS), the production of reactive oxygen species can induce autophagy enhancement; B. high glucose state directly activates oxidative stress and induces reactive oxygen species to induce autophagy enhancement.
(5) the intervention of losartan can reduce the enhancement of autophagy induced by oxidative stress, reduce proteinuria and delay the progress of DN. The possible mechanism is to block the biological effect of Ang II by selectively blocking angiotensin receptor 1 (AT1).
(6) although Benner Pury intervention can reduce the proteinuria of model rats, the autophagy activity in the glomerular tissue is still elevated, indicating that in the state of DN, there are other mechanisms that affect the autophagy in the podocytes except for the role of Ang II. This topic is the mechanism for understanding the damage of DN podocytes and the role of autophagy in DN podocyte injury. The significance of autophagy activity in the prevention and treatment of DN has been explored to some extent.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R587.2;R692

【参考文献】