SIRT3调控肾小管上皮细胞线粒体动力学平衡在急性肾损伤发病中的机制研究

The imbalance of mitochondrial fusion and fission of tubular epithelial cells may play a pivotal role in the pathogenesis of acute kidney injury, but the thorough mechanism remains to be expounded. The SIRTs family has the same function as mitochondria on the regulation of energy metabolism and oxidative stress, among which SIRT3 is mitochondrial specific deacetylase. Our preliminary study has indicated that in ischemia/reperfusion (I/R) AKI model, SIRT3 expression has been decreased significantly along with the imbalance of mitochondrial dynamics. However, the causal relationship and molecular mechanism between them has not been identified yet. Therefore we propose a hypothesis that SIRT3 is able to deacetylate subunit 9 of oxidative respiratory electron-transport chain Complex I (NDUFA9), and such deacetylation is imperative for the balance of mitochondrial dynamics. In ischemia/reperfusion conditions, the deacetylation of SIRT3 on NDUFA9 decreases, which ultimately results in the imbalance of mitochondrial dynamics and the occurrence of AKI. In order to test the aforesaid hypothesis, we will use tubular epithelial cells in vitro and I/R-AKI animal model to explore the regulation of SIRT3 on mitochondrial dynamic balance of tubular epithelial cell and its possible mechanisms. If the hypothesis is established, the regulation of SIRT3 on the re-establishment of mitochondrial dynamic balance may become a new target for the prevention and treatment of AKI.

肾小管上皮细胞线粒体融合-分裂的动力学异常与急性肾损伤的病理生理进程密切相关，其深入机制尚未阐明。SIRTs家族和线粒体同样具有调节能量代谢和氧化应激的作用，其中SIRT3是线粒体特异性去乙酰化酶。我们的研究显示在缺血-再灌注AKI模型中，伴随着线粒体动力学失衡，SIRT3的表达显著下降，而二者的因果关系和分子机制还有待确定。为此我们提出假说：SIRT3能够使氧化呼吸电子传递链中的复合体I亚基9（NDUFA9）去乙酰化，这种去乙酰化状态是线粒体动力学平衡所必须的；在缺血/再灌注情况下，SIRT3对NDUFA9的去乙酰化作用减少，最终导致线粒体动力学失衡和AKI发生。为验证这一假说，我们利用体内外模型，通过分子生物学手段干预SIRT3和NDUFA9，深入探讨病理状态下SIRT3对肾小管上皮细胞线粒体动力学平衡的调控及其机制。调控SIRT3重建线粒体动力学平衡可能成为AKI防治的新靶点。

目的：肾小管上皮细胞线粒体融合-分裂的动力学异常与急性肾损伤的病理生理进程密切相关，其深入机制尚未阐明。SIRTs家族和线粒体同样具有调节能量代谢和氧化应激的作用，其中Sirt3是线粒体特异性去乙酰化酶。本研究探讨缺血再灌注（IR）肾损伤状态下Sirt3对肾小管上皮细胞线粒体动力学平衡的调控及其机制。.方法：入选60名复旦大学附属华山医院肾病科临床诊断为急性肾损伤（AKI）且肾活检证实为急性肾小管损伤或急性肾小管坏死的患者，及20名病理诊断为微小病变的患者作为对照，免疫组化检测肾组织Sirt3的表达。C57BL/6小鼠分别给予假手术、缺血再灌注（IR）处理以及AICAR预干预后IR处理，129sv-Sirt3敲除纯合子小鼠给予IR处理。检测肾功能，光镜和电镜检测肾小管以及线粒体损伤，免疫组化检测Sirt3表达，Western Blot和real time PCR检测Sirt3、NAMPT和Pgc1α表达。Co-IP LC-MS/MS分析筛选与Sirt3相互作用的蛋白，进一步Co-IP验证Sirt3与该蛋白的直接相互作用及其乙酰化水平。.结果：免疫组化结果显示急性肾小管损伤患者的肾脏Sirt3表达较微小病变患者明显减少。与正常小鼠相比，IR小鼠肾脏的Sirt3蛋白和mRNA表达均明显减少，伴有NAMPT和Pgc1α的mRNA表达降低。电镜提示IR小鼠的线粒体损伤加重，同时线粒体分裂蛋白Drp1蛋白和mRNA表达增加，线粒体融合蛋白OPA表达减少。AMPK激动剂AICAR可以通过增加Sirt3的表达改善IR引起的肾脏损伤和线粒体功能不全。Sirt3-/-小鼠IR后肾脏损伤和线粒体功能不全明显加重。通过Co-IP LC-MS/MS筛选出在IR条件下和Sirt3相互作用的蛋白，并进一步通过Co-IP在IR小鼠和Sirt3-/-小鼠中进行验证，证实Sirt3与苹果酸脱氢酶2（MDH2）存在直接相互作用，在IR条件下，Sirt3与MDH2的相互作用减少，MDH2的乙酰化水平增加。.结论：Sirt3能够与MDH2直接作用并使其去乙酰化，从而稳定线粒体动力学平衡；在IR状态下，Sirt3对MDH2的去乙酰化作用减少，最终导致线粒体动力学失衡和AKI发生。本研究有助于进一步揭示急性肾损伤的病理生理机制，为急性肾损伤的防控提供有意义的靶点，有可能防止终末器官损伤并降低死亡率。