NOD2在糖尿病肾病足细胞损伤中的作用及机制研究
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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, P<0.01)。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,P>0.05)。
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。
Background:
Diabetic nephropathy (DN) is one of the major micro-vascular complications of diabetes mellitus and is the most common cause of end-stage renal diseases. Although DN is traditionally considered as a non-immune disease, an increasing number of clinical and animal model studies have implicated that a lot of immune cells, inflammatory mediators, cytokines and extracellular basement in the kidney of the diabetic nephropathy was associated with the injury of renal cell showing that the activation of innate immune system and inflammatory mechanisms are of importance in the pathogenesis of DN.
Innate immunity is the first frontier of immunity system to fight with outside invasion, which induce tissue in a state of continuous injury by recognizing highly conserved pathogen-associated molecular patterns,(PAMPs) or damage-associated molecular patterns,(DAMPs). Now, intracellular PRRs, nucleotide-binding oligomerization domain,(NOD) protein family, called NOD-like receptors,(NLRs), is the center of research. Nucleotide-binding oligomerization domain containing2(NOD2) is a well-characterized member of the NLR family containing CARD domain that detects muramyl dipeptide (MDP) from peptidoglycan. It plays important role in inflammatory homeostasis. The importance of NOD2in inflammatory homeostasis is underscored by the observation that mutations in NOD2gene are associated with susceptibility to Crohn's disease and Blau syndrome.9In addition to being present in inflammatory cells, NOD2is also widely distributed in other cell types, such as adipocytes and epithelial cells, where it plays an important role in triggering the inflammatory response under pathological conditions.
Insulin resistance is a major defect underlying the development of type2diabetes and the innate immune activation. It can cause hyperglycemia reaction for a long time, which can induce the activation of innate immunity and inflammation promoting insulin resistance. Further studies showed that chronic low-grade inflammation was found to be associated with insulin resistance. Podocytes are important component in the filtration barrier and insulin sensitive of the kidney. The podocyte injury are associated with the albuminuria with states of insulin resistance. The insulin response of the podocyte occurs via the facilitative glucose transporters GLUT1and GLUT4, and this process is dependent on the filamentous actin cytoskeleton. At the same time, the specific protein of podocyte, nephrin, allows the GLUT1-and GLUT4-rich vesicles to fuse with the membrane of podocyte. Nephrin plays very important role in podocyte insulin responsiveness, which may have central relevance for understanding of DN and for the association of albuminuria with states of insulin resistance.The innate immunity and insulin resistance contribute to the development of diabetic nephropathy together.
It has been revealed that NOD2is highly expressed in the renal epithelial cells and that the absence of NOD2in mice results in protection from renal ischemia/reperfusion injury. However, so far, the expression patterns of NOD2in the kidney and the contribution of NOD2to the pathogenesis of DN is still unknown.
Objective:
First, to detect the expression of NOD2in human DN biopsies and HFD (high-fat diet)/STZ-induced diabetic mice, and the potential role of NOD2in the inflammatory pathogenesis.
Second, to reveal the mechanism that how NOD2paticipate in diabetic nephropathy by exacerbating inflammation or podocyte insulin resistance.
Methods:
Animal studies
Twenty eight-week wild male C57BL/6J mice and twenty male NOD2-/-mice were divided into four groups randomly, normal-diet wild-type mice, HFD/STZ-induced wild-type mice, normal-diet NOD2-/-mice, HFD/STZ-induced NOD2-/-mice.The normal-diet groups were given normal diet, while the HFD/STZ-induced groups were given high-fat diet for14weeks and injection of STZ to make diabetic mice model. After the blood glucose, albuminuria and pathological test to make sure the success models, the change of weight, blood pressure, plasma triglyceride and plasma free fatty acid were test, and samples were collected by heart perfusion by cold normal saline. The pathological changes were observed by PAS staining and electron microscopy analysis; the expression of NOD2in wild mice organs and the kidney of diabetic mice were tested by Western blotting; immunohistochemical staining of continuous slices were used to observe the change of NOD2in the kidney and the infiltrating macrophage cells of diabetic mice; enzyme-linked immunoabsorbent assay and real time RT-PCR were taken to detect the level of inflammatory factors; the differences of nephrin among four groups were observed by immunofluorescence staining and Western blotting.
Human renal biopsy samples studies
The patient samples were obtained from the Department of Pathology, Shandong University School of Medicine including normal subjects, diabetic nephropathy patients, diabetic patients without nephropathy, focal segmental glomerulosclerosis patients, IgA nephropathy patients, membranous glomerulonephritis patients, lupus nephritis patients and and minimal change disease patients, immunohistochemical staining of continuous slices were used to observe the change of NOD2in the kidney biopsies and the infiltrating macrophage cells of normal kidney, DN patients and diabetic patients without nephropathy. Relative mRNA levels of NOD2in the renal biopsies from all the subjects were examed by real time RT-PCR. The correlation between NOD2mRNA levels and estimated glomerular filtration rate (GFR) in all subjects and the correlation between NOD2mRNA levels and24-h urine protein excretion among all subjects with proteinuria were estimated.
Studies in vitro
Renal cells were cultured in vitro, and the relative level of NOD2mRNA were carried out by RT-PCR; Effects of different stimuli on the expression of NOD2in podocytes, such as high glucose, advanced glycation end-product (AGE), tumor necrosis factor-a (TNF-α), transforming growth factor-β (TGF-β) were carried out by Western blotting. MDP activate NOD2in podocytes, then, the activation of MAPKs and NF-κB signalings were examined by Western blotting, including phospho-ERK1/2, phospho-p38, phospho-JNK, IκBα Relative levels of proinflammatory factors in podocytes treated with MDP were carried out by real time RT-PCR and the podocyte apoptosis were determined by flow cytometric analysis. To understand the effect of NOD2activation on insulin signaling pathwathys, insulin-stimulated glucose uptake test was taken and confocal microscopic technology was used to show the different extent of GLUT4and Western blotting to show the relative NOD2protein levels in the membrane fraction of podocytes. Western blotting was used to show the level of phosphorylation of IR substrate-1(IRS-1) at Ser612in podocytes induced by MDP and immunoprecipitation was used to show the effect of MDP on a insulin-stimulated tyrosine phosphorylation of IRS-1and IRS-1/p85association. The effect of high glucose and MDP on the neprin expression in podocytes by Western blotting, and short hairpin RNA (shRNA)-NOD2transfection was used to show that gene silencing of NOD2attenuated HG-reduced nephrin expression.
Results:
Expression of NOD2in the renal cells
We first detected the expression of NOD2in the kidney and renal cells. Among adult murine kidney, lung, spleen, and small intestine, the kidney revealed a higher level of NOD2. These results are consistent with previous studies showing the organ-specific expression patterns of NOD2. Immuno-histochemical staining with anti-NOD2further revealed immuno-histochemical staining with anti-NOD2in the kidney and intestine from both wild-type (WT) and NOD2-/-mice, indicating the expression patterns of NOD2in the kidney and intestine from both wild-type (WT) and NOD2-/-mice, and the specificity of the NOD2immunostaining. Moreover, we found that NOD2was expressed in murine glomerular mesangial cells, murine podocytes, human glomerular endothelial cells, and human proximal tubule epithelial cells.
Renal cortical NOD2was significantly elevated in human DN biopsies and HFD/STZ-induced diabetic mice
Upregulation of NOD2was observed in paraffin-embedded sections of human diabetic kidney tissues by immunohistochemical staining. Heavy granular staining for NOD2was detected in the kidney from DN subjects, but weak staining in the kidney from normal controls and diabetic patients without nephropathy. Continuous slices were stained with CD68and NOD2individually, and CD68-positive infiltrating monocytes/macrophages in the interstitium and glomeruli were observed, which were colocalized with NOD2, indicating that except enhanced NOD2expression in renal parenchymal cells, infiltrating immune cells also contribute to the upregulation of NOD2expression in the kidney. By real-time, reverse transcriptase-PCR (RT-PCR) analysis, we further confirmed the upregulation of NOD2in renal biopsies from DN subjects. We also examined the expression of NOD2in other kidney diseases. Of note, among renal biopsies from patients with focal segmental glomerulosclerosis (n=7), IgA nephropathy (n=7), membranous glomerulonephritis (n=6), lupus nephritis (n=9), and minimal change disease (n=8), except for minimal change disease patients, NOD2mRNA levels were markedly increased in other patients compared with control subjects. In addition, the NOD2mRNA expression was negatively correlated with estimated glomerular filtration rate in all subjects (Spearman's r=-0.7274, P<0.01). No correlation was found between proteinuria and NOD2in all subjects with proteinuria (Spearman's r=0.1384, P>0.05).
In the HFD/STZ-induced DN mice, HFD/STZ treatment led to hyperglycemia and increase in plasma triglyceride and free fatty acids, and blood pressures were not significantly different among these groups. Consistent with the changes in DN patients, renal NOD2level was significantly increased by Western blot and immunochemical analyses from HFD/STZ mice. The infiltration of inflammatory cells into the kidney also contributed to the upregulation of NOD2expression in DN.
NOD2deficiency ameliorated renal injury in diabetic mice
Compared with WT diabetic mice, albuminuria was significantly less in NOD2-/-diabetic mice accompanied by decreased mesangial expansion and ameliorated podocyte injury. Furthermore, NOD2-/-diabetic mice showed decreased levels of proinflammatory cytokines and chemokines including IL-1β, IL-6, IL-8, TNF-α, MCP-1and ICAM-1in both kidney and serum. Moreover, the renal fibrosis-associated molecules including collagen IV and fibronectin were also decreased in the kidney from NOD2-/-diabetic mice.
NOD2was upregulated in podocytes in hyperglycemia
We found that high glucose concentration dependently enhanced NOD2expression in podocytes, and mannitol control had no effects on NOD2expression. We further found that podocytes treated with AGE, TNF-α or TGF-β significantly increased NOD2expression in a concentration-dependent manner.
MDP induced activation of MAPK signaling pathways, production of proinflammatory mediators, and podocyte apoptosis
We treated podocytes with MDP for the activation of NOD2, and we found that MDP induced activation of MAPKs as assessed by increasing the levels of phospho-specific extracellular signal-regulated kinase1/2, p38MAPK, and c-Jun N-terminal kinase, and the degradation of IκBα was mediated by MDP in a time-dependent manner, which is the key upstream event of NF-κB activation. We also observed that MDP enhanced the production of proinflammatory mediators in podoctes and the flow cytometry showed that MDP induced podocyte apoptosis.
NOD2-mediated glucose uptake, GLUT4translocation, and insulin signaling in podocytes
We found that podocytes significantly increased glucose uptake in response to insulin and MDP selectively reduced insulin stimulation of2-deoxyglucose uptake. Immunofluorescent staining and Western blot showed that MDP disrupted insulin-induced GLUT4translocation to the plasma membrane, which was consistent with a loss of insulin-induced glucose uptake in podocytes. We further found that NOD2activation induced serine phosphorylation of IRS-1, followed by a reduction in IRS-1tyrosine phosphorylation in response to insulin. By immunoprecipitation, it was found that MDP reduced insulin-stimulated binding of p85to IRS-1.
NOD2activation reduced nephrin expression in hyperglycemia
Finally, nephrin staining was detected as a fine, linear-like pattern along the glomerular capillary loop in normal-diet mice and nephrin level was significantly reduced in WT diabetic mice, which was improved in NOD2-/-diabetic mice by immunefluorescent and Western bolt analysis. In vitro, we found that high glucose decreased nephrin expression in podocytes and MDP reduced nephrin expression, which was also confirmed by immunofluorescent staining. Moreover, we found that the effect of MDP on nephrin was associated with changes in cytoskeleton distribution as evidenced by the loss of actin filaments and a granular cytoplasmic pattern of actin distribution. Further studies showed that silencing of NOD2expression by shRNA-NOD2transfection blocked high glucose-reduced nephrin expression.
Conclusion:
1. For the first time, the upregulation of NOD2was explored in the human diabetic nephropathy and diabetic mice. The expression of NOD2in renal cells and higher in diabetic mouse models and diabetic nephropathy patients, while the markedly attenuated severity of the glomerular changes, improved albuminuria and less proinflammatory mediators in NOD2-/-diabetic mice indicated that NOD2is important in diabetic nephropathy.
2. The higher expression in kidney diseases and the negative correlation between NOD2expression and estimated glomerular filtration rate in inflammation-associated kidney diseases, and no correlation between NOD2and urine albumin indicated that the upregulation of NOD2could be a common feature of human inflammation-associated kidney diseases.
3. CD68-positive infiltrating monocytes/macrophages in the interstitium and glomeruli, which were colocalized with NOD2, indicated that except enhanced NOD2expression in renal parenchymal cells, infiltrating immune cells also contributed to the upregulation of NOD2expression in the kidney.
4. A significant positive correlation between NOD2expression and hyperglycemia in podocytes, along with increased proinflammatory cytokines; the NOD2-dependent decrease in insulin signaling coincided with a reduction in insulin-dependent GLUT4translocation and glucose uptake indicated the contribution of NOD2in the regulation of inflammation and the insulin resistance in podocyte.
5. The blockade of hyperglycemia-reduced nephrin expression was observed in NOD2-/-diabetic mice, and the silence of Nod2gene reduce the high glucose-induced nephrin expression in podocytes indicated that NOD2mediated high glucose-induced podocyte dysfunction by nephrin.
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, P<0.01)。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,P>0.05)。
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。
Background:
Diabetic nephropathy (DN) is one of the major micro-vascular complications of diabetes mellitus and is the most common cause of end-stage renal diseases. Although DN is traditionally considered as a non-immune disease, an increasing number of clinical and animal model studies have implicated that a lot of immune cells, inflammatory mediators, cytokines and extracellular basement in the kidney of the diabetic nephropathy was associated with the injury of renal cell showing that the activation of innate immune system and inflammatory mechanisms are of importance in the pathogenesis of DN.
Innate immunity is the first frontier of immunity system to fight with outside invasion, which induce tissue in a state of continuous injury by recognizing highly conserved pathogen-associated molecular patterns,(PAMPs) or damage-associated molecular patterns,(DAMPs). Now, intracellular PRRs, nucleotide-binding oligomerization domain,(NOD) protein family, called NOD-like receptors,(NLRs), is the center of research. Nucleotide-binding oligomerization domain containing2(NOD2) is a well-characterized member of the NLR family containing CARD domain that detects muramyl dipeptide (MDP) from peptidoglycan. It plays important role in inflammatory homeostasis. The importance of NOD2in inflammatory homeostasis is underscored by the observation that mutations in NOD2gene are associated with susceptibility to Crohn's disease and Blau syndrome.9In addition to being present in inflammatory cells, NOD2is also widely distributed in other cell types, such as adipocytes and epithelial cells, where it plays an important role in triggering the inflammatory response under pathological conditions.
Insulin resistance is a major defect underlying the development of type2diabetes and the innate immune activation. It can cause hyperglycemia reaction for a long time, which can induce the activation of innate immunity and inflammation promoting insulin resistance. Further studies showed that chronic low-grade inflammation was found to be associated with insulin resistance. Podocytes are important component in the filtration barrier and insulin sensitive of the kidney. The podocyte injury are associated with the albuminuria with states of insulin resistance. The insulin response of the podocyte occurs via the facilitative glucose transporters GLUT1and GLUT4, and this process is dependent on the filamentous actin cytoskeleton. At the same time, the specific protein of podocyte, nephrin, allows the GLUT1-and GLUT4-rich vesicles to fuse with the membrane of podocyte. Nephrin plays very important role in podocyte insulin responsiveness, which may have central relevance for understanding of DN and for the association of albuminuria with states of insulin resistance.The innate immunity and insulin resistance contribute to the development of diabetic nephropathy together.
It has been revealed that NOD2is highly expressed in the renal epithelial cells and that the absence of NOD2in mice results in protection from renal ischemia/reperfusion injury. However, so far, the expression patterns of NOD2in the kidney and the contribution of NOD2to the pathogenesis of DN is still unknown.
Objective:
First, to detect the expression of NOD2in human DN biopsies and HFD (high-fat diet)/STZ-induced diabetic mice, and the potential role of NOD2in the inflammatory pathogenesis.
Second, to reveal the mechanism that how NOD2paticipate in diabetic nephropathy by exacerbating inflammation or podocyte insulin resistance.
Methods:
Animal studies
Twenty eight-week wild male C57BL/6J mice and twenty male NOD2-/-mice were divided into four groups randomly, normal-diet wild-type mice, HFD/STZ-induced wild-type mice, normal-diet NOD2-/-mice, HFD/STZ-induced NOD2-/-mice.The normal-diet groups were given normal diet, while the HFD/STZ-induced groups were given high-fat diet for14weeks and injection of STZ to make diabetic mice model. After the blood glucose, albuminuria and pathological test to make sure the success models, the change of weight, blood pressure, plasma triglyceride and plasma free fatty acid were test, and samples were collected by heart perfusion by cold normal saline. The pathological changes were observed by PAS staining and electron microscopy analysis; the expression of NOD2in wild mice organs and the kidney of diabetic mice were tested by Western blotting; immunohistochemical staining of continuous slices were used to observe the change of NOD2in the kidney and the infiltrating macrophage cells of diabetic mice; enzyme-linked immunoabsorbent assay and real time RT-PCR were taken to detect the level of inflammatory factors; the differences of nephrin among four groups were observed by immunofluorescence staining and Western blotting.
Human renal biopsy samples studies
The patient samples were obtained from the Department of Pathology, Shandong University School of Medicine including normal subjects, diabetic nephropathy patients, diabetic patients without nephropathy, focal segmental glomerulosclerosis patients, IgA nephropathy patients, membranous glomerulonephritis patients, lupus nephritis patients and and minimal change disease patients, immunohistochemical staining of continuous slices were used to observe the change of NOD2in the kidney biopsies and the infiltrating macrophage cells of normal kidney, DN patients and diabetic patients without nephropathy. Relative mRNA levels of NOD2in the renal biopsies from all the subjects were examed by real time RT-PCR. The correlation between NOD2mRNA levels and estimated glomerular filtration rate (GFR) in all subjects and the correlation between NOD2mRNA levels and24-h urine protein excretion among all subjects with proteinuria were estimated.
Studies in vitro
Renal cells were cultured in vitro, and the relative level of NOD2mRNA were carried out by RT-PCR; Effects of different stimuli on the expression of NOD2in podocytes, such as high glucose, advanced glycation end-product (AGE), tumor necrosis factor-a (TNF-α), transforming growth factor-β (TGF-β) were carried out by Western blotting. MDP activate NOD2in podocytes, then, the activation of MAPKs and NF-κB signalings were examined by Western blotting, including phospho-ERK1/2, phospho-p38, phospho-JNK, IκBα Relative levels of proinflammatory factors in podocytes treated with MDP were carried out by real time RT-PCR and the podocyte apoptosis were determined by flow cytometric analysis. To understand the effect of NOD2activation on insulin signaling pathwathys, insulin-stimulated glucose uptake test was taken and confocal microscopic technology was used to show the different extent of GLUT4and Western blotting to show the relative NOD2protein levels in the membrane fraction of podocytes. Western blotting was used to show the level of phosphorylation of IR substrate-1(IRS-1) at Ser612in podocytes induced by MDP and immunoprecipitation was used to show the effect of MDP on a insulin-stimulated tyrosine phosphorylation of IRS-1and IRS-1/p85association. The effect of high glucose and MDP on the neprin expression in podocytes by Western blotting, and short hairpin RNA (shRNA)-NOD2transfection was used to show that gene silencing of NOD2attenuated HG-reduced nephrin expression.
Results:
Expression of NOD2in the renal cells
We first detected the expression of NOD2in the kidney and renal cells. Among adult murine kidney, lung, spleen, and small intestine, the kidney revealed a higher level of NOD2. These results are consistent with previous studies showing the organ-specific expression patterns of NOD2. Immuno-histochemical staining with anti-NOD2further revealed immuno-histochemical staining with anti-NOD2in the kidney and intestine from both wild-type (WT) and NOD2-/-mice, indicating the expression patterns of NOD2in the kidney and intestine from both wild-type (WT) and NOD2-/-mice, and the specificity of the NOD2immunostaining. Moreover, we found that NOD2was expressed in murine glomerular mesangial cells, murine podocytes, human glomerular endothelial cells, and human proximal tubule epithelial cells.
Renal cortical NOD2was significantly elevated in human DN biopsies and HFD/STZ-induced diabetic mice
Upregulation of NOD2was observed in paraffin-embedded sections of human diabetic kidney tissues by immunohistochemical staining. Heavy granular staining for NOD2was detected in the kidney from DN subjects, but weak staining in the kidney from normal controls and diabetic patients without nephropathy. Continuous slices were stained with CD68and NOD2individually, and CD68-positive infiltrating monocytes/macrophages in the interstitium and glomeruli were observed, which were colocalized with NOD2, indicating that except enhanced NOD2expression in renal parenchymal cells, infiltrating immune cells also contribute to the upregulation of NOD2expression in the kidney. By real-time, reverse transcriptase-PCR (RT-PCR) analysis, we further confirmed the upregulation of NOD2in renal biopsies from DN subjects. We also examined the expression of NOD2in other kidney diseases. Of note, among renal biopsies from patients with focal segmental glomerulosclerosis (n=7), IgA nephropathy (n=7), membranous glomerulonephritis (n=6), lupus nephritis (n=9), and minimal change disease (n=8), except for minimal change disease patients, NOD2mRNA levels were markedly increased in other patients compared with control subjects. In addition, the NOD2mRNA expression was negatively correlated with estimated glomerular filtration rate in all subjects (Spearman's r=-0.7274, P<0.01). No correlation was found between proteinuria and NOD2in all subjects with proteinuria (Spearman's r=0.1384, P>0.05).
In the HFD/STZ-induced DN mice, HFD/STZ treatment led to hyperglycemia and increase in plasma triglyceride and free fatty acids, and blood pressures were not significantly different among these groups. Consistent with the changes in DN patients, renal NOD2level was significantly increased by Western blot and immunochemical analyses from HFD/STZ mice. The infiltration of inflammatory cells into the kidney also contributed to the upregulation of NOD2expression in DN.
NOD2deficiency ameliorated renal injury in diabetic mice
Compared with WT diabetic mice, albuminuria was significantly less in NOD2-/-diabetic mice accompanied by decreased mesangial expansion and ameliorated podocyte injury. Furthermore, NOD2-/-diabetic mice showed decreased levels of proinflammatory cytokines and chemokines including IL-1β, IL-6, IL-8, TNF-α, MCP-1and ICAM-1in both kidney and serum. Moreover, the renal fibrosis-associated molecules including collagen IV and fibronectin were also decreased in the kidney from NOD2-/-diabetic mice.
NOD2was upregulated in podocytes in hyperglycemia
We found that high glucose concentration dependently enhanced NOD2expression in podocytes, and mannitol control had no effects on NOD2expression. We further found that podocytes treated with AGE, TNF-α or TGF-β significantly increased NOD2expression in a concentration-dependent manner.
MDP induced activation of MAPK signaling pathways, production of proinflammatory mediators, and podocyte apoptosis
We treated podocytes with MDP for the activation of NOD2, and we found that MDP induced activation of MAPKs as assessed by increasing the levels of phospho-specific extracellular signal-regulated kinase1/2, p38MAPK, and c-Jun N-terminal kinase, and the degradation of IκBα was mediated by MDP in a time-dependent manner, which is the key upstream event of NF-κB activation. We also observed that MDP enhanced the production of proinflammatory mediators in podoctes and the flow cytometry showed that MDP induced podocyte apoptosis.
NOD2-mediated glucose uptake, GLUT4translocation, and insulin signaling in podocytes
We found that podocytes significantly increased glucose uptake in response to insulin and MDP selectively reduced insulin stimulation of2-deoxyglucose uptake. Immunofluorescent staining and Western blot showed that MDP disrupted insulin-induced GLUT4translocation to the plasma membrane, which was consistent with a loss of insulin-induced glucose uptake in podocytes. We further found that NOD2activation induced serine phosphorylation of IRS-1, followed by a reduction in IRS-1tyrosine phosphorylation in response to insulin. By immunoprecipitation, it was found that MDP reduced insulin-stimulated binding of p85to IRS-1.
NOD2activation reduced nephrin expression in hyperglycemia
Finally, nephrin staining was detected as a fine, linear-like pattern along the glomerular capillary loop in normal-diet mice and nephrin level was significantly reduced in WT diabetic mice, which was improved in NOD2-/-diabetic mice by immunefluorescent and Western bolt analysis. In vitro, we found that high glucose decreased nephrin expression in podocytes and MDP reduced nephrin expression, which was also confirmed by immunofluorescent staining. Moreover, we found that the effect of MDP on nephrin was associated with changes in cytoskeleton distribution as evidenced by the loss of actin filaments and a granular cytoplasmic pattern of actin distribution. Further studies showed that silencing of NOD2expression by shRNA-NOD2transfection blocked high glucose-reduced nephrin expression.
Conclusion:
1. For the first time, the upregulation of NOD2was explored in the human diabetic nephropathy and diabetic mice. The expression of NOD2in renal cells and higher in diabetic mouse models and diabetic nephropathy patients, while the markedly attenuated severity of the glomerular changes, improved albuminuria and less proinflammatory mediators in NOD2-/-diabetic mice indicated that NOD2is important in diabetic nephropathy.
2. The higher expression in kidney diseases and the negative correlation between NOD2expression and estimated glomerular filtration rate in inflammation-associated kidney diseases, and no correlation between NOD2and urine albumin indicated that the upregulation of NOD2could be a common feature of human inflammation-associated kidney diseases.
3. CD68-positive infiltrating monocytes/macrophages in the interstitium and glomeruli, which were colocalized with NOD2, indicated that except enhanced NOD2expression in renal parenchymal cells, infiltrating immune cells also contributed to the upregulation of NOD2expression in the kidney.
4. A significant positive correlation between NOD2expression and hyperglycemia in podocytes, along with increased proinflammatory cytokines; the NOD2-dependent decrease in insulin signaling coincided with a reduction in insulin-dependent GLUT4translocation and glucose uptake indicated the contribution of NOD2in the regulation of inflammation and the insulin resistance in podocyte.
5. The blockade of hyperglycemia-reduced nephrin expression was observed in NOD2-/-diabetic mice, and the silence of Nod2gene reduce the high glucose-induced nephrin expression in podocytes indicated that NOD2mediated high glucose-induced podocyte dysfunction by nephrin.
引文
1 Navarro-Gonzalez JF, Mora-Fernandez C, Muros de Fuentes M et al. Inflammatory molecules and pathways in the pathogenesis of diabetic nephropathy. Nat Rev Nephrol 2011; 7:327-340.
2 Lin M, Yiu WH, Wu HJ et al. Toll-like receptor 4 promotes tubular inflammation in diabetic nephropathy. J Am Soc Nephrol 2012; 23:86-102.
3 Tamrakar AK, Schertzer JD, Chiu TT et al. NOD2 activation induces muscle cell-autonomous innate immune responses and insulin resistance. Endocrinology 2010; 151:5624-5637.
4 Anders HJ. Toll-like receptors and danger signaling in kidney injury. J Am Soc Nephrol 2010; 21:1270-1274.
5 Gluba A, Banach M, Hannam S et al. The role of Toll-like receptors in renal diseases. Nat Revs Nephrol 2010; 6:224-235.
6 Kuwabara T, Mori K, Mukoyama M et al. Exacerbation of diabetic nephropathy by hyperlipidaemia is mediated by Toll-like receptor 4 in mice. Diabetologia 2012; 55: 2256-2266.
7 Anders HJ, Muruve DA. The inflammasomes in kidney disease. JAmSoc Nephrol 2011; 22:1007-1018.
8 Schroder K, Tschopp J. The inflammasomes. Cell 2010; 140:821-832.
9 Ting JP, Duncan JA, Lei Y. How the noninflammasome NLRs function in the innate immune system. Science 2010; 327:286-290.
10 Zhao L, Hu P, Zhou Y et al. NODI activation induces proinflammatory gene expression and insulin resistance in 3T3-L1 adipocytes. Am J Physiol Endocrinol Metab 2011; 301:E587-E598.
11 Lech M, Avila-Ferrufino A, Skuginna V et al. Quantitative expression of RIG-like helicase, NOD-like receptor and inflammasome-related mRNAs in humans and mice. Int Immunol 2010; 22:717-728.
12 Shigeoka AA, Kambo A, Mathison JC et al. Nod1 and nod2 are expressed in human and murine renal tubular epithelial cells and participate in renal ischemia reperfusion injury. J Immunol 2010; 184:2297-2304.
13 Coward RJ, Welsh GI, Yang J et al. The human glomerular podocyte is a novel target for insulin action. Diabetes 2005; 54:3095-3102.
14 Welsh GI, Coward RJ. Podocytes, glucose and insulin. Curr Opin Nephrol Hypertens 2010; 19:379-384.
15'Schertzer JD, Tamrakar AK, Magalhaes JG et al. NODI activators link innate immunity to insulin resistance. Diabetes 2011;60:2206-2215.
16 Mu J, Woods J, Zhou YP et al. Chronic inhibition of dipeptidyl peptidase-4 with a sitagliptin analog preserves pancreatic beta-cell mass and function in a rodent model of type 2 diabetes. Diabetes 2006; 55:1695-1704.
17 Yi F, Jin S, Zhang F et al. Formation of lipid raft redox signalling platforms in glomerular endothelial cells:an early event of homocysteine-induced glomerular injury. J Cell Mol Med 2009; 13:3303-3314.
18 Yi F, Zhang AY, Janscha JL et al. Homocysteine activates NADH/NADPH oxidase through ceramide-stimulated Rac GTPase activity in rat mesangial cells. Kidney Int 2004; 66:1977-1987.
19 Zhang S, Zhang Y, Wei X et al. Expression and regulation of a novel identified TNFAIP8 family is associated with diabetic nephropathy. Biochim Biophys Acta 2010; 1802:1078-1086.
20 Candolfi M, Yagiz K, Foulad D et al. Release of HMGB1 in response to proapoptotic glioma killing strategies:efficacy and neurotoxicity. Clin Cancer Res 2009; 15:4401-4414.
21 Lennon R, Pons D, Sabin MA et al. Saturated fatty acids induce insulin resistance in human podocytes:implications for diabetic nephropathy. Nephrol Dial Transplant 2009; 24:3288-3296.
22 Wang Z, Wei X, Zhang Y et al. NADPH oxidase-derived ROS contributes to upregulation of TRPC6 expression in puromycin aminonucleosideinduced podocyte injury. Cell Physiol Biochem 2009; 24:619-626.
23 Galkina E, Ley K. Leukocyte recruitment and vascular injury in diabetic nephropathy. J Am Soc Nephrol 2006; 17:368-377.
24 Magalhaes JG, Sorbara MT, Girardin SE et al. What is new with Nods? Curr Opin Immunol 2011; 23:29-34.
25 Dagenais M, Dupaul-Chicoine J, Saleh M. Function of NOD-like receptors in immunity and disease. Curr Opin Investig Drugs 2010; 11:1246-1255.
26 Franchi L, Warner N, Viani K et al. Function of Nod-like receptors in microbial recognition and host defense. Immunol Rev 2009; 227:106-128.
27 Kohan DE. Progress in gene targeting:using mutant mice to study renal function and disease. Kidney Int 2008;74:427-437.
28 Brosius FC 3rd, Alpers CE, Bottinger EP et al. Mouse models of diabetic nephropathy. J Am Soc Nephrol 2009; 20:2503-2512.
29 Breyer MD, Bottinger E, Brosius FC 3rd et al. Mouse models of diabetic nephropathy. J Am Soc Nephrol 2005; 16:27-45.
30 Soler MJ, Riera M, Batlle D. New experimental models of diabetic nephropathy in mice models of type 2 diabetes:efforts to replicate human nephropathy. Exp Diabetes Res 2012; 2012:616313.
31 Tan AL, Forbes JM, Cooper ME. AGE, RAGE, and ROS in diabetic nephropathy. Semin Nephrol2007;27:130-143.
32 Shimoike T, Inoguchi T, Umeda F, Nawata H, Kawano K, Ochi H. The meaning of serum levels of advanced glycosylation end products in diabetic nephropathy. Metabolism2000; 49:1030-1035.
33 Huebschmann AG, Regensteiner JG, Vlassara H, Reusch JE. Diabetes and advanced glycoxidation end products. Diabetes Care2006; 29:1420-1432.
34 Busch M, Franke S, Ruster C, Wolf G. Advanced glycation end-products and the kidney. Eur J Clin Invest2010; 40:742-755.
35 Nakamura T, Fukui M, Ebihara I, Osada S, Nagaoka I, Tomino Y, Koide H. mRNA expression of growth factors in glomeruli from diabetic rats. Diabetes1993; 42: 450-456.
36 Sugimoto H, Shikata K, Wada J, Horiuchi S, Makino H. Advanced glycation end products-cytokine-nitric oxide sequence pathway in the development of diabetic nephropa-thy:aminoguanidine ameliorates the overexpression of tumour necrosis factor-alpha and inducible nitric oxide syn-thase in diabetic rat glomeruli. Diabetologial999; 42:878-886.
37 Royall JA, Berkow RL, Beckman JS, Cunningham MK, Ma-talon S, Freeman BA. Tumor necrosis factor and interleukin 1 alpha increase vascular endothelial permeability. Am J Physiol1989; 257:L399-L410.
38 Hasegawa G,Nakano K, Kondo M. Role of TNF and IL-1 in the development of diabetic nephropathy. Nefrologia1995;15:1-4.
39 Ban CR, Twigg SM. Fibrosis in diabetes complications:pathogenic mechanisms and circulating and urinary markers. Vasc Health Risk Manag2008; 4:575-596.
40 Ziyadeh FN. Different roles for TGF-beta and VEGF in the pathogenesis of the cardinal features of diabetic nephropathy. Diabetes Res Clin Pract2008; 82 Suppl 1: S38-S41.
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44 Steinberg GR. Inflammation in obesity is the common link between defects in fatty acid metabolism and insulin resistance. Cell Cycle 2007; 6:888-894.
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2 Lin M, Yiu WH, Wu HJ et al. Toll-like receptor 4 promotes tubular inflammation in diabetic nephropathy. J Am Soc Nephrol 2012; 23:86-102.
3 Tamrakar AK, Schertzer JD, Chiu TT et al. NOD2 activation induces muscle cell-autonomous innate immune responses and insulin resistance. Endocrinology 2010; 151:5624-5637.
4 Anders HJ. Toll-like receptors and danger signaling in kidney injury. J Am Soc Nephrol 2010; 21:1270-1274.
5 Gluba A, Banach M, Hannam S et al. The role of Toll-like receptors in renal diseases. Nat Revs Nephrol 2010; 6:224-235.
6 Kuwabara T, Mori K, Mukoyama M et al. Exacerbation of diabetic nephropathy by hyperlipidaemia is mediated by Toll-like receptor 4 in mice. Diabetologia 2012; 55: 2256-2266.
7 Anders HJ, Muruve DA. The inflammasomes in kidney disease. JAmSoc Nephrol 2011; 22:1007-1018.
8 Schroder K, Tschopp J. The inflammasomes. Cell 2010; 140:821-832.
9 Ting JP, Duncan JA, Lei Y. How the noninflammasome NLRs function in the innate immune system. Science 2010; 327:286-290.
10 Zhao L, Hu P, Zhou Y et al. NODI activation induces proinflammatory gene expression and insulin resistance in 3T3-L1 adipocytes. Am J Physiol Endocrinol Metab 2011; 301:E587-E598.
11 Lech M, Avila-Ferrufino A, Skuginna V et al. Quantitative expression of RIG-like helicase, NOD-like receptor and inflammasome-related mRNAs in humans and mice. Int Immunol 2010; 22:717-728.
12 Shigeoka AA, Kambo A, Mathison JC et al. Nod1 and nod2 are expressed in human and murine renal tubular epithelial cells and participate in renal ischemia reperfusion injury. J Immunol 2010; 184:2297-2304.
13 Coward RJ, Welsh GI, Yang J et al. The human glomerular podocyte is a novel target for insulin action. Diabetes 2005; 54:3095-3102.
14 Welsh GI, Coward RJ. Podocytes, glucose and insulin. Curr Opin Nephrol Hypertens 2010; 19:379-384.
15'Schertzer JD, Tamrakar AK, Magalhaes JG et al. NODI activators link innate immunity to insulin resistance. Diabetes 2011;60:2206-2215.
16 Mu J, Woods J, Zhou YP et al. Chronic inhibition of dipeptidyl peptidase-4 with a sitagliptin analog preserves pancreatic beta-cell mass and function in a rodent model of type 2 diabetes. Diabetes 2006; 55:1695-1704.
17 Yi F, Jin S, Zhang F et al. Formation of lipid raft redox signalling platforms in glomerular endothelial cells:an early event of homocysteine-induced glomerular injury. J Cell Mol Med 2009; 13:3303-3314.
18 Yi F, Zhang AY, Janscha JL et al. Homocysteine activates NADH/NADPH oxidase through ceramide-stimulated Rac GTPase activity in rat mesangial cells. Kidney Int 2004; 66:1977-1987.
19 Zhang S, Zhang Y, Wei X et al. Expression and regulation of a novel identified TNFAIP8 family is associated with diabetic nephropathy. Biochim Biophys Acta 2010; 1802:1078-1086.
20 Candolfi M, Yagiz K, Foulad D et al. Release of HMGB1 in response to proapoptotic glioma killing strategies:efficacy and neurotoxicity. Clin Cancer Res 2009; 15:4401-4414.
21 Lennon R, Pons D, Sabin MA et al. Saturated fatty acids induce insulin resistance in human podocytes:implications for diabetic nephropathy. Nephrol Dial Transplant 2009; 24:3288-3296.
22 Wang Z, Wei X, Zhang Y et al. NADPH oxidase-derived ROS contributes to upregulation of TRPC6 expression in puromycin aminonucleosideinduced podocyte injury. Cell Physiol Biochem 2009; 24:619-626.
23 Galkina E, Ley K. Leukocyte recruitment and vascular injury in diabetic nephropathy. J Am Soc Nephrol 2006; 17:368-377.
24 Magalhaes JG, Sorbara MT, Girardin SE et al. What is new with Nods? Curr Opin Immunol 2011; 23:29-34.
25 Dagenais M, Dupaul-Chicoine J, Saleh M. Function of NOD-like receptors in immunity and disease. Curr Opin Investig Drugs 2010; 11:1246-1255.
26 Franchi L, Warner N, Viani K et al. Function of Nod-like receptors in microbial recognition and host defense. Immunol Rev 2009; 227:106-128.
27 Kohan DE. Progress in gene targeting:using mutant mice to study renal function and disease. Kidney Int 2008;74:427-437.
28 Brosius FC 3rd, Alpers CE, Bottinger EP et al. Mouse models of diabetic nephropathy. J Am Soc Nephrol 2009; 20:2503-2512.
29 Breyer MD, Bottinger E, Brosius FC 3rd et al. Mouse models of diabetic nephropathy. J Am Soc Nephrol 2005; 16:27-45.
30 Soler MJ, Riera M, Batlle D. New experimental models of diabetic nephropathy in mice models of type 2 diabetes:efforts to replicate human nephropathy. Exp Diabetes Res 2012; 2012:616313.
31 Tan AL, Forbes JM, Cooper ME. AGE, RAGE, and ROS in diabetic nephropathy. Semin Nephrol2007;27:130-143.
32 Shimoike T, Inoguchi T, Umeda F, Nawata H, Kawano K, Ochi H. The meaning of serum levels of advanced glycosylation end products in diabetic nephropathy. Metabolism2000; 49:1030-1035.
33 Huebschmann AG, Regensteiner JG, Vlassara H, Reusch JE. Diabetes and advanced glycoxidation end products. Diabetes Care2006; 29:1420-1432.
34 Busch M, Franke S, Ruster C, Wolf G. Advanced glycation end-products and the kidney. Eur J Clin Invest2010; 40:742-755.
35 Nakamura T, Fukui M, Ebihara I, Osada S, Nagaoka I, Tomino Y, Koide H. mRNA expression of growth factors in glomeruli from diabetic rats. Diabetes1993; 42: 450-456.
36 Sugimoto H, Shikata K, Wada J, Horiuchi S, Makino H. Advanced glycation end products-cytokine-nitric oxide sequence pathway in the development of diabetic nephropa-thy:aminoguanidine ameliorates the overexpression of tumour necrosis factor-alpha and inducible nitric oxide syn-thase in diabetic rat glomeruli. Diabetologial999; 42:878-886.
37 Royall JA, Berkow RL, Beckman JS, Cunningham MK, Ma-talon S, Freeman BA. Tumor necrosis factor and interleukin 1 alpha increase vascular endothelial permeability. Am J Physiol1989; 257:L399-L410.
38 Hasegawa G,Nakano K, Kondo M. Role of TNF and IL-1 in the development of diabetic nephropathy. Nefrologia1995;15:1-4.
39 Ban CR, Twigg SM. Fibrosis in diabetes complications:pathogenic mechanisms and circulating and urinary markers. Vasc Health Risk Manag2008; 4:575-596.
40 Ziyadeh FN. Different roles for TGF-beta and VEGF in the pathogenesis of the cardinal features of diabetic nephropathy. Diabetes Res Clin Pract2008; 82 Suppl 1: S38-S41.
41 M.E. Pagtalunan, P.L. Miller, S. Jumping-Eagle, R.G. Nelson, B.D. Myers, H.G. Rennke, N.S. Coplon, L. Sun, T.W. Meyer Podocyte loss and progressive glomerular injury in type II diabetes J. Clin. Invest.,99 (1997), pp.342-348.
42 G. Wolf, S. Chen, F.N. Ziyadeh From the periphery of the glomerular capillary wall toward the center of disease:podocyte injury comes of age in diabetic nephropathy Diabetes,54 (2005), pp.1626-1634.
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