NOD2通过调控TRPC6离子通道的表达及活性参与高同型半胱氨酸血症引起的肾损伤

发布时间：2018-06-24 02:56

本文选题：NLR家族 + TRPC离子通道　；参考：《山东大学》2014年博士论文

【摘要】：研究目的：同型半胱氨酸是体内甲硫氨酸循环的中间产物,其代谢异常所导致的高同型半胱氨酸血症是终末期肾病(end stage renal disease, ESRD),以及心血管疾病并发症导致的终末期肾病发生发展中的一个独立危险因子,但是高同型半胱氨酸所致肾损伤的发生发展机制迄今尚未完全明确。NOD2(nucleotide binding oligomerzation domain2)属于模式识别受体(pattern-recognition receptors, PRRs)家族中的NLRs(NOD-1ike receptors, NLRs)亚家族成员,在免疫调控与炎性反应中起着重要的作用,前期研究证实其在肾脏多种细胞中表达并且有实验证明小鼠NOD2缺失对肾脏缺血再灌注和糖尿病肾病中具有保护作用。TRPC6(transient receptor potential cation channe6, TRPC6)是具有钙离子高选择性的瞬时电位阳离子通道,作为足细胞裂孔膜的重要组成蛋白之一,其过度活化可致足细胞内钙离子浓度增高,从而导致足细胞损伤和功能障碍,引起肾小球疾病发生。但是,高同型半胱氨酸血症中,NOD2是否参与了肾损伤的发生发展,及其是否与TRPC6存在调控关系并参与此病理过程,至今尚未见报道。为此,本课题分别从体内和体外两方面探讨在高同型半胱酸血症中,细胞内固有免疫受体NOD2在肾小球硬化中的作用以及对TRPC6的调控机制,为防治高同型半胱氨酸症导致的终末期肾病提供新的治疗策略。研究方法： 1.体内实验：NOD2在高同型半胱氨酸血症引起肾损伤中的作用及调控机制1.1高同型半胱氨酸血症引起的小鼠肾损伤以及NOD2表达变化：实验采用健康成年雄性CBS+/-(strain name:B6.129P2-CbstmIUnc/J)小鼠和野生型雄性C57BL/6J小鼠。随机分成四组,每组10只,野生型正常饮食组,野生型饮食叶酸缺乏组,CBS+／-小鼠正常饮食组和CBS+/-小鼠饮食叶酸缺乏组。高效液相色谱法(high performance liquid chromatography, HPLC)检测各组血浆中总同型半胱氨酸含量,以评价模型是否复制成功；酶联免疫吸附实验(enzyme-linked immunosorbent assay, ELISA)检测尿白蛋白／肌酐水平；采用PAS染色观察肾脏的形态学变化和电镜观察足细胞形态学变化；ELISA检测相关致炎因子和趋化因子的水平。分别采用定量RT-PCR、蛋白免疫印迹法和免疫组织荧光化学法,检测NOD2在小鼠肾皮质以及肾小球足细胞中的表达变化。 1.2NOD2在高同型半胱氨酸血肾损伤中的作用及对TRPC6通道的调控机制研究：采用健康成年雄性NOD2-/-(strain name:B6.129S1-Nod2tmlFlv/J)小鼠和野生型雄性C57BL/6J小鼠,随机分成四组,每组10只,野生型正常饮食组,野生型饮食叶酸缺乏组,NOD2-/-小鼠正常饮食组和NOD2-/-小鼠饮食叶酸缺乏组。采用免疫组化法观察肾小球TRPC6和nephrin的表达；其他生化与形态指标,包括血浆总同型半胱氨酸含量、尿白蛋白肌酐水平、肾小球和足细胞的形态学变化、致炎因子和趋化因子在小鼠肾皮质中的表达检测及TRPC6和nephrin的表达检测等,方法同上。 2.体外实验：NOD2对TRPC6通道的调控及小鼠足细胞损伤中的作用体外培养肾小球上皮细胞(足细胞),并经同型半胱氨酸刺激后,分别利用实时定量RT-PCR和蛋白免疫印迹法检测足细胞中NOD2、TRPC6和nephrin的表达变化；MDP诱导足细胞内NOD2活化,以及高同型半胱氨酸刺激转染shRNA-NOD2质粒获得的NOD2基因沉默足细胞后,重复检测上述指标。采用流式细胞术分析上述各实验组中足细胞的凋亡情况,激光共聚焦观察细胞骨架蛋白的结构变化,钙成像技术记录足细胞内钙离子浓度的改变,全细胞膜片钳技术分析TRPC6通道电流变化；为探讨TRPC6和nephrin在信号路径中的作用和相互关系,通过转染shRNA-TRPC6质粒基因沉默足细胞中的TRPC6,在同型半胱氨酸干预下,检测细胞凋亡情况和骨架蛋白的结构变化,质粒转染获得过表达nephrin的足细胞,检测其在Hcys刺激下TRPC6的蛋白表达水平的改变。结果 1.体内实验：NOD2在高同型半胱氨酸血症引起的肾损伤中的作用及调控机制 1.1高同型半胱氨酸血症引起的小鼠肾损伤以及NOD2表达变化：首先,HPLC法检测结果显示野生叶酸缺乏组和CBS+／-叶酸缺乏组血浆总同型半胱氨酸水平均明显高于正常饮食组(10μmol/L),表明高同型半胱氨酸血症小鼠模型复制成功；尿白蛋白／肌酐比率与血浆总同型半胱氨酸水平呈正性相关；PAS染色可见正常饮食小鼠肾小球,基底膜清晰,粗细均匀,肾小球血管袢薄而清晰,而叶酸缺乏组小鼠肾小球均不同程度地呈现基底膜粗细不均,系膜增生,PAS阳性物质沉积；通过电镜观察,足细胞超微结构呈现基底膜厚薄不均,局部膨出,足细胞次级足突融合,CBS+／-叶酸缺乏组小鼠肾小球基底膜内容物外漏,肾小球滤过屏障功能受损。以上结果表明,高同型半胱氨酸血症引起了肾小球损伤。同时,我们发现在高同型半胱氨酸血症中,肾皮质中小鼠NOD2蛋白和mRNA表达明显增加；采用足细胞marker蛋白S ynaptopodin和NOD2共定位,免疫荧光共聚焦显示高同型半胱氨酸血症中,肾小球足细胞中NOD2蛋白高表达；并且小鼠肾皮质白介素-1β(IL-1β).白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α).单核细胞趋化蛋白-1(MCP-1).细胞间粘附分子-1(ICAM-1)的水平增高,表明高同型半胱氨酸血症中肾皮质的炎症反应增强。 1.2NOD2在高同型半胱氨酸血症所致肾损伤中的作用及对TRPC6的调控机制研究：血浆总同型半胱氨酸水平的检测结果证实,叶酸缺乏饮食复制高同型半胱氨酸血症模型成功,虽然叶酸缺乏饮食导致NOD2-/-小鼠与野生小鼠的血浆tHcys无显著性差异,但是NOD2基因敲除明显降低了高同型半胱氨酸血症中的尿白蛋白／肌酐比率,一定程度上缓解了小鼠肾功能障碍；形态学观察结果表明NOD2基因敲除显著减轻了高同型半胱氨酸血症导致的肾小球损伤,并且发现肾小球基底膜膨出少见,足细胞次级足突排列较整齐,逆转了高同型半胱氨酸血症导致的小鼠肾小球足细胞损伤。我们进一步发现,高同型半胱氨酸血症诱导小鼠肾小球TRPC6表达升高,而NOD2基因敲除能够有效地抑制高同型半胱氨酸导致的TRPC6高表达和致炎因子以及趋化因子的产生；相反的,NOD2基因敲除一定程度上恢复了高同型半胱氨酸血症所致的nephrin表达。上述结果说明,高同型半胱氨酸血症导致的肾损伤过程中,NOD2参与了对TRPC6和nephrin的表达的调控。 2.体外实验：NOD2对TRPC6通道的调控及小鼠足细胞损伤中的作用同型半胱氨酸诱导了体外培养足细胞中NOD2.TRPC6的表达,导致OAG作用下的足细胞内钙浓度的增加,并且通过全细胞膜片钳技术检测,发现TRPC6通道电流幅度的显著升高。全细胞膜片钳技术具有高灵敏度和高分辨率的特点,电流可精确至1pA,空间和时间分辨率能达到1μm和10μs,实验采用特异性通道激动剂OAG开放足细胞膜上TRPC6通道,记录全细胞电流,能够精确地反应足细胞膜上TRPC6的通道数和开放概率。同样,MDP活化足细胞NOD2后检测到上述各指标相同的变化趋势,而NOD2基因沉默后,同型半胱氨酸导致足细胞的上述变化被有效的抑制,表明同型半胱氨酸导致的足细胞TRPC6表达是NOD2依赖性的,并且NOD2参与调控同型半胱氨酸引起的TRPC6通道依赖性的钙信号通路；激光共聚焦观察进一步发现NOD2活化和同型半胱氨酸均导致了足细胞骨架蛋白结构重构,而TRPC6基因沉默与NOD2基因沉默结果一致性地改善了同型半胱氨酸导致的足细胞骨架重构,并且足细胞凋亡也明显受到抑制；同时,nephrin在足细胞中的表达由于NOD2活化或同型半胱氨酸被抑制,这与体内实验中高同型半胱氨酸NOD2-/-小鼠肾皮质nephrin水平高于其他实验组相呼应；质粒转染获得nephrin过表达的足细胞则有效降低了Hcys诱导的TRPC6的表达。结果证实,NOD2介导的nephrin依赖性的TRPC6信号路径参与了同型半胱氨酸引起的足细胞损伤过程。结论与创新性： 1.本课题在高同型半胱氨酸血症中,首次表明NOD2参与了对TRPC6离子通道的调控,提示TRPC6介导的钙离子信号路径是连接固有免疫受体NOD2和肾损伤的一条重要信号传导途径,针对NOD2信号路径中各级分子的靶向治疗有可能成为临床防治高同型半胱氨酸血症相关的终末期肾病的新的治疗策略。 2.我们进一步就NOD2调控TRPC6的机制及作用进行深入研究,表明NOD2对nephrin的表达的调节可能是引起TRPC6变化的重要因素之一
[Abstract]:Objective : Homocysteine is an intermediate product of methionine circulation in vivo . The hyperhomocysteinemia caused by metabolic abnormalities is an independent risk factor in the development of renal ischemia reperfusion and diabetic nephropathy .

Study method :

1 . In vivo experiment : The effect of NOD2 in kidney injury induced by homocysteinemia and the expression of NOD2 in mice induced by hyperhomocysteinemia were studied . The experimental results showed that healthy adult male CBS + / - ( strain name : B6 . 129P2 - CbstmIUnc / J ) mice and wild - type male C57BL / 6J mice were randomly divided into four groups : 10 in each group , wild type normal diet group , wild type diet folic acid deficiency group , CBS + / - mouse normal diet group and CBS + / - mouse diet folic acid deficiency group .
Enzyme - linked immunosorbent assay ( ELISA ) was used to detect urinary albumin / creatinine levels .
The morphological changes of the kidney were observed by PAS staining and the morphological changes of the foot cells were observed by electron microscope .
Quantitative RT - PCR , Western blotting and immunohistochemistry were used to detect the expression of NOD2 in renal cortex of mice and glomerulus .

The effects of 1.2NOD2 on the renal injury of hyperhomocysteinemia and the control mechanism of TRPC6 pathway were studied : healthy adult male NOD2 - / - ( strain name : B6 . 129S1 - 2 tmlFlv / J ) mouse and wild - type male C57BL / 6J mice were randomly divided into four groups : 10 in each group , wild type normal diet group , wild type diet folic acid deficiency group , NOD2 - / - mouse normal diet group and NOD2 - / - mouse diet folic acid deficiency group .
Other biochemical and morphological indexes , including plasma total homocysteine levels , urinary albumin creatinine levels , morphological changes of glomerular and foot cells , inflammatory factors and chemokine expression in renal cortex of mice , and expression of TRPC6 and nephrin .

2 . In vitro experiment : The effect of NOD2 on TRPC6 channel regulation and mouse foot cell injury

The expression of NOD2 , TRPC6 and nephrin was detected by real - time quantitative RT - PCR and Western blotting respectively after the cultured glomerular epithelial cells ( foot cells ) were cultured in vitro and stimulated with homocysteine .
The expression of NOD2 gene was detected by flow cytometry . The changes of calcium ion concentration in the cells were observed by flow cytometry , and the changes of calcium ion concentration in the cells were recorded by calcium imaging technique .
In order to investigate the role and correlation of TRPC6 and nephrin in signal path , TRPC6 was transfected with shRNA - TRPC6 plasmid gene to silence TRPC6 . Under the intervention of homocysteine , the apoptosis of cells and the structural changes of skeletal proteins were detected . The expression level of TRPC6 was detected by plasmid transfection .

Results

1 . In vivo experiment : The effect and mechanism of NOD2 in renal injury induced by homocysteinemia

1.1 Changes in renal impairment and NOD2 expression in mice due to hyperhomocysteinemia :

First , the levels of plasma total homocysteine in the wild folate deficiency group and CBS + / - folate deficiency group were significantly higher than those in the normal diet group ( 10 渭mol / L ) .
Urinary albumin / creatinine ratio was positively correlated with the level of plasma homocysteine ;
PAS staining showed that the glomeruli of normal diet mice , the basement membrane was clear , the thickness was uniform , the glomerulus vascular loop was thin and clear , and the glomerulus of the folic acid deficient group presented the basement membrane coarse and fine unevenness , mesangial hyperplasia and PAS positive substance deposition .
The results showed that the expression of NOD2 protein and mRNA in renal cortex was significantly increased in hyperhomocysteinemia .
The high expression of NOD2 protein in hyperhomocysteinemia was demonstrated by co - localization of the foot cell marker protein S ynaptopodin and NOD2 .
IL - 1尾 ( IL - 1尾 ) , interleukin - 6 ( IL - 6 ) , tumor necrosis factor - 伪 ( TNF - 伪 ) , monocyte chemoattractant protein - 1 ( MCP - 1 ) . The level of intercellular adhesion molecule - 1 ( ICAM - 1 ) increased .

The role of 1.2NOD2 in renal injury induced by homocysteinemia and its mechanism of regulation of TRPC6 :

The result of detection of plasma total homocysteine level confirmed that folate deficiency diet induced the success of hyperhomocysteinemia model , although folate deficiency diet resulted in no significant difference between NOD2 - / - mice and wild mice , but NOD2 knockout significantly reduced urinary albumin / creatinine ratio in hyperhomocysteinemia , to a certain extent the impairment of renal dysfunction in mice ;
The results of morphological observation showed that the knock - out of NOD2 gene significantly alleviated the glomerular injury caused by hyperhomocysteinemia , and it was found that the expression of TRPC6 in mice induced by homocysteinemia was reversed , while the NOD2 gene knocked out the high expression of TRPC6 induced by homocysteinemia and the production of inflammatory factor and chemokine .
In contrast , the NOD2 gene knockout to some extent restores nephrin expression resulting from hyperhomocysteinemia . The above results suggest that NOD2 is involved in regulating the expression of TRPC6 and nephrin in the course of renal injury resulting from hyperhomocysteinemia .

2 . In vitro experiment : The effect of NOD2 on TRPC6 channel regulation and mouse foot cell injury

The expression of NOD2 and TRPC6 was induced by homocysteine , which resulted in the increase of intracellular calcium concentration in the cells under the action of OAG , and it was found that TRPC6 channel current amplitude was significantly increased by whole cell patch clamp technique .
Laser cofocus observation further found that both NOD2 activation and homocysteine resulted in the structural remodeling of the scaffold protein of the foot cells , while the silencing of TRPC6 gene and the silencing of NOD2 gene significantly improved the remodeling of the foot cytoskeletal structure caused by homocysteine , and the apoptosis of the foot cells was also significantly inhibited ;
At the same time , the expression of nephrin in full - term cells was inhibited by NOD2 activation or homocysteine , which was correlated with the level of nephrin in renal cortex of high homocysteine NOD2 - / - mice in vivo experiments than in other experimental groups ;
The results showed that the TRPC6 signal pathway mediated by NOD2 mediated nephrin - dependent TRPC6 signal pathway was involved in the process of foot cell injury induced by homocysteine .

Conclusion and Innovation :

1 . In hyperhomocysteinemia , NOD2 is shown to be involved in the regulation of TRPC6 ion channel . It is suggested that TRPC6 - mediated calcium ion signal pathway is an important signal transduction pathway linking innate immune receptor NOD2 and kidney injury . Targeting therapy for all levels of NOD2 signaling pathway may be a new treatment strategy for the treatment of end - stage renal disease associated with hyperhomocysteinemia .

2 . We further study the mechanism and function of NOD2 regulation TRPC6 , indicating that the regulation of NOD2 ' s expression of nephrin may be one of the important factors that cause TRPC6 change
【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】：R692.5;R589

【共引文献】