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Tenascin-C在急性肾损伤中的作用及机制研究
中文摘要

目的: 既往研究表明缺氧诱导因子(hypoxia-inducible factor,HIF)对急性肾损伤有保护作用,但其具体机制尚不明确。Tenascin-C(TNC)是一种细胞外基质蛋白,可在组织损伤后被诱导表达,可能参与细胞增殖、粘附及炎症反应等。本研究旨在探讨TNC在在急性肾损伤后的表达规律,探究TNC是否参与HIF对急性肾损伤的保护作用。 方法: 构建小鼠肾脏缺血-再灌注模型,检测肾组织TNC表达。原代分离TNC表达细胞并永生化体外培养,体外研究TNC表达调控机制。利用TNC-CreEReGFP〓(即TNC〓)小鼠,分别构建TNC〓和野生型小鼠单侧肾切除加对侧缺血再灌注模型,比较两组小鼠急性肾损伤严重程度。原代分离小管上皮细胞,利用低氧培养箱构建细胞缺氧模型,给予TNC干预,体外研究TNC作用及机制。 结果: 在小鼠肾脏缺血/再灌注损伤后早期(3-6小时),组织TNC即显著增加。TNC表达在24-48小时最高,此后逐渐下降。缺血/再灌注损伤后早期TNC主要表达于皮髓交界处间质中。肾组织TNC表达水平与损伤严重程度正相关。经软件预测发现TNC启动子区域存在低氧反应元件,利用低氧培养箱给予原代小鼠髓质间质细胞缺氧处理后TNC表达上调。HIF稳定剂DMOG及FG4592可上调小鼠肾组织及原代小鼠髓质间质细胞中TNC表达。HIF2 α抑制剂PT2385抑制缺氧后TNC增加。荧光素酶报告基因实验表明,Hif2α对启动子TNC有促进作用,突变TNC基因启动子区域HIF结合位点后这一作用不再存在。ChIP实验证实TNC可结合HIF-2 α启动子。因此,HIF2 α可直接结合TNC启动子序列,促进TNC转录,上调TNC表达。TNC基因缺失加重急性肾损伤,加重小管上皮细胞凋亡。TNC通过激活EGFR-STAT3信号通路,保护小管上皮细胞缺氧损伤,进而减轻急性缺血再灌注损伤时小管上皮细胞的凋亡/坏死,减轻AKI损伤。 结论: 在肾脏缺血再灌注损伤后,HIF-2 α可早期诱导肾组织Tenascin-C(TNC)表达。诱导表达的TNC在急性肾损伤中起保护作用。TNC可通过EGFRSTAT3通路减轻肾小管上皮细胞损伤。 关键词:Tenascin-C;急性肾损伤;HIF-2 α;STAT3 中图分类号:R5

英文摘要

Objective: Accumulating evidence suggests that the hypoxia-inducible factor (HIF) mediates cellular adaptions to hypoxia and has a protective effect in acute kidney injury, but the mechanism is not completely understood. The matricellular protein tenascin-C (TNC) which is transiently expressed during development can be reinduced after tissue injury and may be involved in creating a microenvironment that facilitates cell proliferation, adhesion and inflammation. The present study examined the role of TNC in mediating the protective effect of HIF in acute kidney injury using ischemia-reperfusion (IR) model. Methods: A tenascin-C promoter driven inducible CreER2 knock-in mouse line with an eGFP reporter was generated. TNC-CreER〓 (TNC〓) mice were used to examine the role of TNC in of AKI. Results: Following IR, TNC was markedly induced in the interstitium of corticomedullary junction of the kidney as early as 3 hours and peaked at 24-48 hours. Then the mechanism underlying TNC induction following injury was investigated. Since 4 hypoxic response elements (HRE) were identified in the promoter region of TNC, we examined the effect of HIF on TNC induction. Hypoxia induced TNC expression. DMOG and FG-4592, two HIF stabilizers, significantly induced TNC expression both in mice and in primary cultured renal interstitial cells. PT2385, a HIF-2α specific inhibitor, inhibited TNC induction following hypoxia. Luciferase reporter assay showed that hif-2α promoted the transcription of TNC, while mutation of the HRE of the TNC luciferase reporter abolished this effect. Chip assay proved the binding of HIF2a to the endogenous promoter of TNC gene. Deletion of TNC in mice significantly aggravated IR induced AKI, showing lower survival rate, higher BUN and more severe tubular injury after IR comparing to their wild type littermates. TNC protected primary tubular epithelial cells from hypoxic injury, and this protective effect might be mediated by EGFR-STAT3 pathway. Conculsion : After ischemia-reperfusion injury, HIF-2a induced expression of TNC, which might have a protective effect in ischemia-reperfusion injury by protecting tubular epithelial cells. Key words: Acute kidney injury; Tenascin-C (TNC) ; HIF-2a; STAT3 CLC : R5

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