NOD2在糖尿病肾病足细胞损伤中的作用及机制研究

发布时间：2018-07-01 07:46

本文选题：糖尿病肾病 + 免疫稳态　；参考：《山东大学》2014年博士论文

【摘要】：背景：糖尿病肾病是严重的糖尿病微血管并发症,是导致终末期肾病的最常见原因,也是导致糖尿病患者死亡的主要原因。虽然传统意义上认为糖尿病肾病是一种非免疫系统疾病,但是越来越多的临床和动物实验研究发现糖尿病肾病中存在大量浸润的免疫细胞、炎性介质、细胞因子,细胞外基质,并与肾脏固有细胞损伤相互关联,这表明固有免疫系统的激活和炎症机制在糖尿病肾病的发生发展中起重要作用。固有免疫是机体抵抗外来致病因子入侵的第一道防线。主要通过模式识别受体(pattern recognition receptors,PRR)识别进化上高度保守的病原相关分子模式(pathogen-associated molecular patterns,PAMP)或者损伤相关分子模式(damage-associated molecular patterns,DAMP),诱发组织处于持续炎性损伤状态。目前模式识别受体中的细胞内核苷酸结合寡聚化结构域(nucleotide-binding oligomerization domain,NOD)蛋白家族,NOD样受体(NOD-like receptors,NLR),是当前研究的热点,其中对NOD2的研究较多。NOD2包含2个CARD结构域,识别肽聚糖中的胞壁酰二肽(MDP),在炎症稳态中起重要作用。研究发现NOD2突变基因与克罗恩病和Blau综合征的易感性有关,这使NOD2在炎症稳态中的重要作用突显了出来。NOD2不仅分布在炎症细胞中,还广泛存在于其他细胞,例如脂肪细胞和上皮细胞等。病理条件下,NOD2在激活这些细胞的炎症反应的过程中起重要作用。胰岛素抵抗(IR)贯穿二型糖尿病的整个发病过程,能够导致机体高血糖反应,长期高血糖引发的固有免疫和炎症反应能够进一步加重组织的胰岛素抵抗状态。有研究表明胰岛素抵抗与固有免疫系统的激活以及慢性低程度的炎症反应有关。足细胞作为肾小球滤过膜的主要组成部分,也是胰岛素敏感细胞,且蛋白尿的生成与胰岛素抵抗导致的足细胞损伤有关。足细胞可以依赖于细胞骨架微丝蛋白易化葡萄糖转运子GLUT1和GLUT4,同时依赖足细胞特异蛋白肾病蛋白(nephrin)可以促使富含GLUT1和GLUT4的微泡与细胞膜融合。因而,nephrin在足细胞胰岛素敏感性上起着至关重要的作用。研究发现NOD2在人和小鼠的肾小管上皮细胞表达,Nod2敲除可以改善肾缺血再灌注引起的损伤,但有关NOD2在肾脏其他细胞中的表达分布以及是否在糖尿病肾病中发挥重要作用,迄今尚未见报道。目的：一、确定NOD2在糖尿病肾病活检样本和动物模型中的表达情况,并明确NOD2是否与糖尿病肾病的炎症病理过程有关。二、从炎性反应与足细胞的胰岛素抵抗角度深入探讨NOD2参与糖尿病肾病足细胞损伤中的作用及机制。方法：动物学研究实验采用选用8周野生型C57BL/6J小鼠和NOD2-/-小鼠各20只随机分成四组,即野生型正常饮食组,野生型高脂饮食/STZ刺激组,NOD2-/-小鼠正常饮食组和NOD2-/-小鼠高脂饮食/STZ刺激组(normal-diet wild-type mice, HFD/STZ-induced wild-type mice,normal-diet NOD2-/-mice, HFD/STZ-induced NOD2-/-mice)。正常组给以正常饮食,高脂饮食/STZ刺激组给予持续高脂饮食14周和STZ刺激以制备糖尿病小鼠模型。通过采用PAS染色和电镜分析两种办法,观察各组肾小球病理改变变化。Western blot检测NOD2在野生小鼠各器官中的表达以及野生糖尿病小鼠肾脏皮质NOD2表达变化。连续切片免疫组化染色检测糖尿病小鼠肾脏NOD2表达变化和浸润的单核巨噬细胞表达NOD2的情况。进行Elisa和实时定量RT-PCR方法检测炎症因子表达。通过组织免疫荧光染色和Western blot两种方法检测各组肾小球nephrin表达变化。相关疾病病人样本研究获取正常人、糖尿病肾病病人、糖尿病非肾病病人、局灶性节段性肾小球硬化病人、IgA膜性肾病病人、膜性肾小球肾炎病人、红斑狼疮肾炎病人、肾微小病变病人活检样木。进行免疫组化染色确定糖尿病肾病样本NOD2表达变化和浸润的巨噬细胞NOD2的表达情况。实时定量RT-PCR检测NOD2mRNA含量,判断NOD2mRNA与估算肾小球滤过率和24小时尿蛋白量的关系。体外实验研究体外培养肾脏固有细胞,进行RT-PCR检测肾脏固有细胞NOD2mRNA的表达。体外模拟糖尿病肾病病理状态,Western blot检测足细胞在高糖、糖基化终末产物(AGE)、肿瘤刺激因子-α(TNF-a)和转化生长因子-β(TGF-p,糖尿病肾病常见损伤因子)刺激下NOD2的表达变化。MDP激活足细胞NOD2后,Western blot检测phospho-ERK1/2、phospho-p38、phospho-JN、IκBα的变化评估MAPKs通路和NF-κB通路激活情况；实时定量RT-PCR检测促炎因子的变化；流式细胞术测足细胞的凋亡；MDP刺激足细胞,葡萄糖摄取实验评估足细胞对葡萄糖的摄取情况：细胞免疫荧光观察GLUT4在胞膜的分布变化；Western blot检测GLUT4在细胞膜的表达变化；通过Western blot检测MDP激活NOD2诱导胰岛素受体底物-1丝氨酸残基的磷酸化,免疫共沉淀法评估MDP影响胰岛素诱导的胰岛素受体底物-1酪氨酸残基与p85亚基的结合从而判断胰岛素信号通路的变化情况。通过Western blot检测高糖和MDP刺激对足细胞nephrin表达情况的影响；通过shRNA干扰技术检测Nod2基因沉默对高糖条件下足细胞nephrin表达情况的影响。结果： NOD2在肾脏细胞的表达本课题首先检测NOD2在肾脏组织和肾脏细胞中的表达。与成年小鼠小肠组织、脾脏、肺脏相比较,肾脏的表达量较高。这与以往研究NOD2器官特异性表达的结果相一致。NOD2在野生型和NOD2-/-小鼠肾脏和小肠石蜡切片的免疫组化染色进一步显示了NOD2的表达模式以及NOD2抗体免疫组化染色的特异性。小鼠肾小球系膜细胞、小鼠足细胞、人肾小球内皮细胞以及人远端肾小管上皮细胞均表达NOD2。人糖尿病肾病活检样本和HFD/STZ诱导的糖尿病小鼠的肾皮质NOD2的表达升高人糖尿病肾病活检样本石蜡切片的免疫组化染色发现NOD2表达上调。针对CD68和NOD2的连续切片染色可以在间质和肾小球中观察到CD68阳性的单核巨噬细胞浸润,并且与NOD2共定位,这说明NOD2在肾脏固有细胞和浸润的免疫细胞中表达增强共同导致了肾脏中NOD2的表达上调。实时定量RT-PCR分析进一步证实糖尿病肾病活检样本NOD2mRNA水平升高。同时,本课题也检测了NOD2在其他类型的肾脏疾病中的表达。发现与正常对照相比,除肾微小病变病人(n=8)外,局灶性节段性肾小球硬化病人(n=7)、IgA膜性肾病病人(n=7)、膜性肾小球肾炎病人(n=6)和红斑狼疮肾炎病人(n=9)的肾活检样本的NOD2mRNA水平明显升高。所有样本中NOD2mRNA水平与估算肾小球滤过率成负相关(Spearman's r=-0.7274, P0.01)。在有蛋白尿的样本中,NOD2mRNA水平与蛋白尿无明显相关性(Spearman's r=-0.1384,P0.05)。 HFD/STZ诱导的糖尿病肾病小鼠模型中发现,HFD/STZ诱导产生高血脂,增加血浆甘油三酯和游离脂肪酸含量,而血压无明显变化。Western blot和免疫组化分析发现HFD/STZ诱导的糖尿病肾病小鼠模型中肾NOD2的水平明显升高,而且浸润的炎症细胞也同样导致了肾脏NOD2的升高,这与糖尿病肾病病人的检测结果是一致的。 NOD2缺失减轻糖尿病肾病小鼠肾脏损伤与野生型糖尿病肾病小鼠相比,NOD2-/-的糖尿病肾病小鼠的蛋白尿明显减少,同时伴随系膜细胞增生和足细胞损伤减轻。进一步实验表明,NOD2-/-糖尿病肾病小鼠双肾和血清的促炎细胞因子和趋化因子水平减低,包括IL-1β、IL-6、 IL-8、TNF-α、单核细胞趋化蛋白-1(MCP-1)以及细胞内粘附分子-1(ICAM-1)。糖尿病肾病小鼠肾脏组织中肾脏纤维化相关分子包括胶原IV和纤连蛋白的水平也降低。高糖环境各因子刺激足细胞NOD2表达上调足细胞NOD2表达上调具有葡萄糖浓度依赖性,其中甘露醇对照组NOD2的表达没有明显变化,说明可以排除渗透压对NOD2的影响。进一步检测发现足细胞在糖基化终末产物、TNF-a和TGF-p的刺激下,NOD2的表达均显著升高并呈浓度依赖性。MDP诱导MAPK信号通路激活、促炎递质的生成和足细胞凋亡 MDP刺激足细胞激活NOD2,检测发现特异性细胞外液信号调节激酶(ERK)1/2.p38MAPK和c-Jun N-端激酶(JNK)磷酸化水平升高,且MDP以时间依赖的方式调控NF-κB信号通路的关键成分IκBα的降解。同时,MDP增加足细胞促炎递质的生成,进一步利用流式细胞术检测发现MDP诱导足细胞凋亡。NOD2介导足细胞的葡萄糖吸收、GLUT4转位和胰岛素信号通路在胰岛素刺激下,足细胞葡萄糖摄取明显增加,而MDP可以选择性的减低胰岛素诱导的2-脱氧葡萄糖的吸收。进而免疫荧光染色和Western blot证明MDP破坏胰岛素诱导的GLUT4转位到细胞膜,这与NOD2激活减少胰岛素诱导的足细胞葡萄糖吸收的实验结果相一致。进一步检测发现NOD2的激活引起IRS-1丝氨酸残基的磷酸化,胰岛素刺激后IRS-1酪氨酸残基的磷酸化水平减轻。同时通过免疫共沉淀发现,MDP降低胰岛素刺激引起的p85亚基与磷酸化的IRS酪氨酸残基之间的相互结合,影响下游P13K通路。高血糖时NOD2的激活降低nephrin的表达免疫荧光分析和Western blot检测发现,在正常饮食小鼠nephrin染色沿着肾小球血管袢呈现流畅线型,但在糖尿病肾病小鼠nephrin明显减少,在NOD2-/-糖尿病肾病小鼠又有所改善。在体外实验研究中,高糖降低足细胞nephrin的表达,而MDP也降低足细胞nephrin的表达。更重要的是,本课题发现MDP引起足细胞actin微丝减少且胞浆actin呈颗粒状分布说明MDP降低nephrin的表达与细胞骨架蛋白的改变相关。进一步通过shRNA-NOD2转染后发现,Nod2沉默可以使高糖诱导的nephrin表达降低的程度减轻。结论： 1.首次明确NOD2在人糖尿病肾病肾组织及糖尿病肾病小鼠模型中的表达上调。而NOD2-/-糖尿病小鼠的肾小球改变明显减轻,蛋白尿情况得到改善,炎症介质的生成减少,提示NOD2在糖尿病肾病中可能发挥重要作用。 2.NOD2在肾脏疾病中均表达升高且NOD2水平与肾小球率过滤之间存在负相关,与蛋白尿之间不存在明显的相关关系,提示NOD2的表达上调很可能是人类炎症相关肾脏疾病的共同特征。 3.糖尿病肾病病人和小鼠糖尿病动物模型肾脏组织连续切片中NOD2与单核巨噬细胞标记蛋白CD68共定位,表明NOD2在肾脏固有细胞和浸润的免疫细胞中表达增强共同导致了肾脏中NOD2的表达上调。 4.足细胞中NOD2表达增高呈现葡萄糖浓度依赖性,同时伴随促炎因子的增多和MAPKs及NF-κB信号通路的激活；NOD2激活后胰岛素信号通路的水平降低,GLUT4转位减少以及糖摄取量降低,提示NOD2通过调控炎症机制参与足细胞的胰岛素抵抗。 5.NOD2-/-糖尿病小鼠肾脏组织以及Nod2沉默的足细胞中高糖诱导的nephrin降低得到改善,提示NOD2通过影响nephrin的表达调控高糖诱导的足细胞功能失调。
[Abstract]:Background :

Diabetic nephropathy is a serious diabetic microvascular complication , which is the most common cause of end - stage renal disease and is the main cause of death in diabetic patients . Although it is traditionally considered that diabetic nephropathy is a non - immune system disease , more and more clinical and animal experimental studies have found that there are a large number of infiltrating immune cells , inflammatory mediators , cytokines , extracellular matrix in diabetic nephropathy , and correlated with the damage of the natural cells of the kidney . This suggests that the activation of the innate immune system and the mechanism of inflammation play an important role in the development of diabetic nephropathy .

In this paper , we find that NOD2 is associated with the susceptibility to Crohn ' s disease and Blau ' s syndrome . The NOD2 is not only distributed in inflammatory cells but also in other cells , such as fat cells and epithelial cells . NOD2 plays an important role in the activation of inflammatory responses of these cells .

Insulin resistance ( IR ) penetrates the whole pathogenesis of type 2 diabetes mellitus , can lead to hyperglycemia reaction , and the innate immunity and inflammatory response induced by long - term hyperglycemia can further aggravate the insulin resistance state of the tissues . The study shows that insulin resistance is related to the activation of the innate immune system and the chronic low degree of inflammatory response .

It was found that NOD2 was expressed in human and mouse renal tubular epithelial cells . Nod2 knock - out could improve renal ischemia - reperfusion injury , but the distribution of NOD2 in other cells of kidney and whether it plays an important role in diabetic nephropathy has not been reported to date .

Purpose :

1 . To determine the expression of NOD2 in diabetic nephropathy biopsy samples and animal models , and to determine whether NOD2 is related to the inflammatory pathological process of diabetic nephropathy .

Second , the role and mechanism of NOD2 in diabetic nephropathy induced by diabetic nephropathy were discussed in detail from the viewpoint of inflammatory response and insulin resistance of the foot cells .

Method :

Zoology Studies

Twenty - two randomly divided into four groups , i.e . wild - type normal diet group , wild type high - fat diet / insulin - stimulated group , NOD2 - / - mouse normal diet group and NOD2 - / - mouse high - fat diet - type mice ( normal - diet - induced wild - type mice , normal - diet NOD2 - / - mice , HFD - induced NOD2 - / - mice ) were randomly divided into four groups : wild - type normal diet group , wild - type high - fat diet - type mice , NOD2 - / - mouse normal diet group and NOD2 - / - mouse normal - diet wild - type mice . The expression of NOD2 in various organs of diabetic mice and the expression of NOD2 in kidney cortex of wild diabetic mice were observed by immunohistochemistry staining and Western blot .

The expression of NOD2 mRNA in diabetic nephropathy patients , diabetic nephropathy patients , diabetic nephropathy patients , focal segmental glomerulosclerosis patients , IgA membranous nephropathy patients , membranous glomerulonephritis patients , lupus nephritis patients and renal microlesions were determined .

The expression of NOD2 mRNA in kidney was detected by RT - PCR in vitro . Western blot was used to detect the expression of NOD2 in diabetic nephropathy . Western blot was used to detect the expression of NOD2 in high - sugar , glycosylated terminal products ( AGE ) , tumor - stimulating factor - 伪 ( TNF - a ) and transforming growth factor - 尾 ( TGF - p , diabetic nephropathy ) .
Real - time quantitative RT - PCR for the detection of pro - inflammatory factors ;
Flow cytometry was used to measure the apoptosis of foot cells .
The uptake of glucose was assessed by MDP stimulation of the foot cells and glucose uptake assay : the distribution of GLUT4 in the membrane was observed by immunofluorescence assay .
Western blot was used to detect the expression of GLUT4 in the cell membrane .
The expression of insulin receptor substrate - 1 tyrosine residue and p85 subunit was assessed by Western blot . The effect of high glucose and MDP stimulation on the expression of nephrin was detected by Western blot .
The effect of silencing of Nod2 gene on the expression of nephrin under high glucose condition was investigated by shRNA interference technique .

Results :

Expression of NOD2 in renal cells

In this study , the expression of NOD2 in renal tissue and kidney cells was first examined . The expression of NOD2 was higher than that of NOD2 in the small intestine tissues , spleen and lungs of adult mice . The expression pattern of NOD2 and the specificity of NOD2 antibody immunohistochemical staining were further demonstrated in both wild type and NOD2 - / - mouse kidney and small intestine paraffin sections . NOD2 was expressed in mouse glomerular mesangial cells , mouse foot cells , human glomerular endothelial cells , and human distal tubular epithelial cells .

Increased expression of NOD2 in renal cortex of diabetic mice induced by human diabetic nephropathy biopsy samples and HFD / induced diabetic mice

The expression of NOD2 mRNA in renal biopsy specimens of diabetic nephropathy patients ( n = 7 ) , IgA membranous nephropathy ( n = 7 ) , membranous glomerulonephritis ( n = 6 ) and lupus nephritis ( n = 9 ) were detected by RT - PCR . In the samples with proteinuria , the level of NOD2mRNA was not significantly correlated with that of proteinuria ( spearman ' s r = - 0.1384 , P0.05 ) .

The results of Western blot and immunohistochemistry showed that the levels of renal NOD2 in diabetic nephropathy mice were significantly higher than those of diabetic nephropathy .

NOD2 deletion reduces kidney damage in diabetic nephropathy mice

Compared with the wild - type diabetic nephropathy mice , the proteinuria of NOD2 - / - diabetic nephropathy mice was significantly decreased , accompanied by decreased proliferation of mesangial cells and injury of the foot cells . Further experiments showed that the levels of pro - inflammatory cytokines and chemokine levels in both kidney and serum of NOD2 - / - diabetic nephropathy mice were reduced , including IL - 1尾 , IL - 6 , IL - 8 , TNF - 伪 , monocyte chemoattractant protein - 1 ( MCP - 1 ) , and intracellular adhesion molecule - 1 ( ICAM - 1 ) . The levels of renal fibrosis - related molecules in kidney tissues of diabetic nephropathy mice , including collagen IV and fibronectin , were also reduced .

Up - regulation of NOD2 expression in human cells stimulated by high - sugar environment

There was no significant change in the expression of NOD2 in the expression of NOD2 , and the expression of NOD2 was significantly increased and the concentration - dependent manner was observed in the expression of NOD2 in the control group . The activation of MAPK signal pathway , the formation of pro - inflammatory and the apoptosis of the cells were induced by MDP .

MDP stimulates the activation of NOD2 , and the detection of extracellular fluid signal regulated kinase ( ERK ) 1 / 2.p38MAPK and c - Jun N - terminal kinase ( ERK ) 1 / 2 . p38MAPK and c - Jun N - terminal kinase have increased phosphorylation level , and MDP regulates the degradation of NF - 魏B signaling pathway in time - dependent manner .

It was found that the activation of NOD2 induced phosphorylation of IRS - 1 serine residue and decreased phosphorylation of IRS - 1 tyrosine residue after insulin stimulation .

Immunofluorescence analysis and Western blot analysis showed that nephrin staining in normal diet mice was streamlined along the glomerular vascular loop , but the nephrin expression in diabetic nephropathy mice was improved . In vitro experiments , high glucose decreased the expression of nephrin and MDP also decreased the expression of nephrin .

Conclusion :

1 . The expression of NOD2 was up - regulated in kidney tissues and diabetic nephropathy mice . NOD2 - / - diabetic mice had a marked decrease in glomerulus alteration , and proteinuria was improved , and the formation of inflammatory mediators decreased , suggesting that NOD2 might play an important role in diabetic nephropathy .

2 . There was a negative correlation between NOD2 level and glomerular filtration rate in renal disease . There was no obvious correlation between NOD2 and proteinuria , suggesting that the up - regulation of NOD2 was probably the common characteristic of human inflammation - related kidney disease .

3 . NOD2 co - located between NOD2 and single - core macrophage marker protein CD68 in diabetic nephropathy patients and diabetic animal models of diabetic rats , suggesting that the expression of NOD2 in kidney - specific cells and infiltrating immune cells together leads to an up - regulation of NOD2 in the kidney .

4 . The expression of NOD2 in foot cells showed glucose concentration - dependent , accompanied by increased pro - inflammatory factors and activation of MAPKs and NF - 魏B signaling pathways .
The level of insulin signaling pathway decreased , GLUT4 translocation decreased and glucose uptake decreased after NOD2 activation , suggesting that NOD2 was involved in the insulin resistance of the foot cells by regulating the inflammatory mechanism .

5 . The nephrin - induced decrease in nephrin induced by NOD2 - / - diabetic mice kidney tissues and Nod2 - silenced foot cells was improved , suggesting that NOD2 regulates the dysregulation of high glucose - induced foot cell dysfunction by influencing the expression of nephrin .
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R587.2;R692

【共引文献】