Progranulin保护糖尿病肾病足细胞损伤的作用机制

发布时间：2018-02-05 05:37

本文关键词： 糖尿病肾病颗粒蛋白前体足细胞自噬　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景:糖尿病肾病(diabetic nephropathy,DN)是导致终末期肾病(end stage renal disease,ESRD)的最重要的诱因之一,严重威胁人类的生命健康。糖尿病肾病的发病机制尤为复杂,主要包括糖脂代谢紊乱、血流动力学、氧化应激、炎症反应以及遗传因素等。足细胞(podocyte)是维持肾小球内环境稳态的重要组成部分,足细胞与肾小球内皮细胞和基底膜共同组成了肾小球滤过屏障,足细胞损伤是糖尿病肾病的早期事件,可导致滤过屏障遭到破坏,产生蛋白尿,最终加重肾脏损伤。但是,在糖尿病肾病中造成足细胞损伤的机制尚不完全明确。颗粒蛋白前体(progranulin,PGRN)是一种自分泌型生长因子,广泛表达分布于体内各种组织与器官,例如神经细胞、软骨细胞、免疫细胞以及上皮细胞等。PGRN具有诸多生物学功能,广泛参与到多种病理生理过程中,如肿瘤的发生发展、自身免疫类疾病、组织损伤修复、伤口愈合等。虽然课题组前期的研究证明PGRN可以通过负调控NOD2介导的信号通路在急性肾损伤中起到保护作用,但在糖尿病肾病中是否发挥作用尚不清楚。研究表明,自噬在糖尿病肾病的病理进程中发挥着重要的作用,正常生理条件下足细胞保持着较高的自噬水平,糖尿病肾病发生时足细胞自噬水平降低导致足细胞受损进而加重肾损伤。报道显示,AMPK是自噬的激活剂,AMPK可以通过直接磷酸化自噬启动蛋白ULK1从而促进自噬,进而在糖尿病肾病中起到保护作用。因此本实验主要为了探究PGRN在糖尿病肾病中的作用,以及是否与自噬,AMPK信号通路有关。研究方法与结果1.PGRN在糖尿病肾病中的表达变化选取8-12周龄雄性野生型(wild type,WT)C57BL/6J小鼠,单肾摘除后恢复一周,腹腔注射链脲佐霉素(streptozotocin,STZ)100mg/kg连续三天诱导糖尿病肾病。当小鼠模型构建成功后,使用蛋白免疫印迹法(Western blot,WB)、Real-time RT-PCR、免疫荧光(immunofluorescence,IF)和免疫组化(immunohistochemistry,IHC),检测PGRN在肾皮质中表达变化及分布情况,结果显示PGRN在糖尿病肾病模型组肾皮质中与对照组相比表达明显下降。同时体外高糖分别刺激人肾小球足细胞(humanpodocyte,HPC)、内皮细胞(glomerular endothelial cell,GEnC)和大鼠系膜细胞(rat messangial cell,RMC)结果显示,肾小球足细胞和内皮细胞中的PGRN均表达下降。2.PGRN在糖尿病肾病损伤中的作用适龄野生型雄性C57BL/6小鼠和Grn基因敲除鼠(B6(Cg)-Grntml lAidi/J)随机分组,分别构建糖尿病肾病模型和对照组。通过PAS对野生型和Grn基因敲除组肾脏石蜡切片染色,透射电镜观察小鼠足细胞损伤。结果显示,在糖尿病肾病组PGRN的缺失导致了肾皮质中肾小球系膜基质增生加重。透射电镜结果显示PGRN的缺失加重了肾小球足细胞损伤,导致足细胞足突的融合消失。IF标记足细胞特异性蛋白Nephrin、Podocin结果显示PGRN的缺失加重了足细胞功能蛋白的损伤。同时Real-time RT-PCR检测野生型与Grn基因敲除鼠肾皮质炎性因子与趋化因子的变化,结果显示,PGRN的缺失导致肾皮质中炎性因子与趋化因子较野生型糖尿病肾病组更高。体外流式细胞术检测足细胞凋亡水平,结果显示加入重组PGRN可以明显降低高糖引起的细胞凋亡,WB检测足细胞功能蛋白Nephrin,结果显示加入重组PGRN可恢复高糖诱导的Nephrin蛋白水平的降低。3.PGRN在糖尿病肾病损伤中的作用机制WB检测野生型与Grn基因敲除鼠肾皮质中LC3B等自噬相关分子,结果显示Grn基因敲除鼠糖尿病肾病组较野生型糖尿病肾病组的自噬水平更低。体外结果显示足细胞高糖条件下自噬水平显著降低,加入重组PGRN后自噬水平恢复,Tandem实验观察自噬流变化表明加入重组PGRN可以恢复足细胞高糖条件下自噬流水平。同时WB检测自噬启动蛋白ULK1磷酸化水平,结果表明高糖条件下ULK1磷酸化水平降低,加入重组PGRN可以恢复磷酸化水平。进一步WB检测糖尿病肾病肾皮质AMPK信号通路的水平,结果显示糖尿病肾病组中Grn基因敲除较野生型AMPK信号通路进一步降低。结论与创新性:1.糖尿病肾病小鼠模型中PGRN显著降低,PGRN的缺失加重糖尿病肾病足细胞损伤。2.PGRN可以通过激活AMPK信号通路促进ULK1磷酸化水平进而启动足细胞自噬。本课题对指导设计PGRN作为靶点治疗糖尿病肾病提供重要的依据。
[Abstract]:Background: diabetic nephropathy (diabetic, nephropathy, DN) is the leading cause of end-stage renal disease (end stage renal disease, ESRD) is the most important factor, a serious threat to human life and health. The pathogenesis of diabetic nephropathy is very complex, mainly including glucose and lipid metabolism disorders, hemodynamics, oxidative stress, inflammatory reaction and genetic factors podocyte. (podocyte) is an important part of maintaining homeostasis within the glomeruli, glomerular podocytes and endothelial cells and basement membrane composed of glomerular filtration barrier, podocyte injury is an early event of diabetic nephropathy, can lead to the destruction of the filtration barrier, resulting in proteinuria, eventually aggravate renal injury. But the mechanism caused by foot cell damage is still not completely clear in diabetic nephropathy. Progranulin (progranulin, PGRN) is an autocrine growth factor expression, widely divided The cloth in various tissues in the body and organs, such as nerve cells, cartilage cells, immune cells and epithelial cells,.PGRN has many biological functions, widely involved in the pathological process of many kinds, such as the occurrence and development of tumors, autoimmune diseases, tissue repair and wound healing. Although our previous studies have demonstrated that PGRN can signaling through negative regulation mediated by NOD2 in acute kidney injury plays a protective role in diabetic nephropathy, but the role is not clear. Studies suggest that autophagy plays an important role in the pathological process of diabetic nephropathy, normal physiological conditions of podocyte autophagy maintained higher levels of diabetic nephropathy. Occurs when the podocyte autophagy leading to podocyte damage and reduce the level of renal damage aggravated. Reports indicate that AMPK is the activator of autophagy, AMPK through direct phosphorylation of autophagy Start the ULK1 protein to promote autophagy, which play a protective role in diabetic nephropathy. So this experiment in order to explore the role of PGRN in diabetic nephropathy, and whether autophagy, AMPK signaling pathway. The research methods and results of 1.PGRN in diabetic nephropathy on the expression of selected 8-12 week old male wild type (wild type WT, C57BL/6J) mice, single kidney removed recovery after a week, intraperitoneal injection with streptozotocin (streptozotocin, STZ) 100mg/kg for three days induced diabetic nephropathy. When the successful construction of mouse model, using Western blotting (Western blot, WB), Real-time RT-PCR, immunofluorescence (immunofluorescence, IF) and immunohistochemistry (immunohistochemistry, IHC), the expression and distribution of PGRN was detected in renal cortex, showed that PGRN in the cortex of kidney in diabetic nephropathy model group compared with the control group was significantly decreased. At the same time were stimulated with high glucose in vitro human glomerular podocyte (humanpodocyte, HPC), endothelial cells (glomerular endothelial cell, GEnC) and rat mesangial cells (rat messangial cell, RMC) results showed that glomerular podocytes and endothelial cells in the expression of PGRN was decreased in diabetic nephropathy.2.PGRN injury age of wild type male C57BL/6 mice and Grn knockout mice (B6 (Cg) -Grntml lAidi/J) were randomly divided into diabetic nephropathy model were constructed and the control group. The PAS knockout group staining on paraffin sections of kidney and wild type Grn gene, observe the podocyte injury in mice by transmission electron microscope. The results showed that in the absence of PGRN in diabetic nephropathy group glomerular mesangial matrix hyperplasia in renal cortex increased. TEM results showed that deletion of PGRN increased glomerular podocyte injury, leading to podocyte footprocess fusion disappear podocyte.IF markers Specific protein Nephrin, Podocin results showed that PGRN deletion aggravated the injury of podocyte proteins. At the same time, Real-time RT-PCR detection of wild-type and Grn knockout rat renal cortex changes, inflammatory cytokines and chemokines showed that deletion of PGRN leads to inflammatory factors in renal cortex and chemotactic factor than the wild type diabetic nephropathy was higher in vitro. Flow cytometry was used to detect the apoptosis of podocyte levels, the results showed that the addition of recombinant PGRN can significantly reduce the cell apoptosis induced by high glucose, WB detection of podocyte protein Nephrin, the results showed that Nephrin protein levels with recombinant PGRN can restore glucose induced WB reduction mechanism of.3.PGRN in injury in diabetic nephropathy detection of wild-type and Grn knockout LC3B rats renal cortex autophagy related molecules, the results showed that Grn knockout mice diabetic nephropathy group compared with the wild type diabetic nephropathy The in vitro results showed lower levels of autophagy. Podocyte autophagy under high glucose decreased significantly and the recovery of autophagy with recombinant PGRN, Tandem experimental observation indicated that the addition of recombinant PGRN autophagy flow can restore podocytes under high glucose levels. At the same time WB for the detection of autophagy flow autophagy protein ULK1 phosphorylation level. The results show that the phosphorylation of ULK1 under the condition of high glucose levels decreased with recombinant PGRN can restore the phosphorylation level of WB. Further detection of diabetic nephropathy renal cortical AMPK signaling level, results showed that the diabetic nephropathy group compared to the wild type Grn gene knockout AMPK signal pathway is further reduced. The conclusion and Innovation: PGRN 1. diabetic nephropathy mice decreased significantly, PGRN the lack of increase of podocyte injury in diabetic nephropathy.2.PGRN can activate the AMPK signaling pathway to promote ULK1 phosphorylation and initiation of podocyte Autophagy. This topic provides an important basis for guiding the design of PGRN as a target for the treatment of diabetic nephropathy.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

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