The role of Sirtuin 5 in acute kidney injury

摘要 急性肾损伤(AKI)发生在近五分之一的住院患者中，并与发病率增加有关 以及各个年龄段的死亡率。许多AKI患者在受伤后会恢复肾功能，但随后进展为 慢性肾病(CKD)。人们对这种机制知之甚少，目前还没有有效的 预防、限制或逆转组织损伤的疗法。迫切需要确定所涉及的机制 在AKI的发病机制中起重要作用。我们的长期目标是阐明这些机制，并利用它们来创造新的 限制AKI和防止过渡到CKD的治疗。近端小管上皮细胞(PTEC)，一个主要的部位 AKI期间的损伤，是非常活跃的代谢和丰富的线粒体。线粒体新陈代谢引起 与缺血再灌注损伤(IRI)有关的ROS升高 顺铂引起的肾毒性。在急性心肌梗死期间调节线粒体功能是一个有吸引力的，但到目前为止 无法实现的战略。我们的中心假设是线粒体sirtuin赖氨酸脱酰酶SIRT5的缺失 导致PTEC代谢的变化，从而防止AKI。初步数据证实了这一点。 整体SIRT5基因敲除(SIRT5-/-)小鼠对IRI和顺铂诱导的AKI的保护作用 在SIRT5 siRNA敲除的原代人PTEC中也是如此。进一步的数据支持我们提出的 SIRT5-/-PTEC表现出一种代谢预适应的保护机制 脂肪酸氧化(FAO)从线粒体到过氧化体的转变。过氧酶体以前被连接在一起 在其他动物模型中进行肾脏保护，很可能是因为它们有能力消除ROS。在SIRT5/-肾脏中， 在AKI期间，过氧化体数量在基线时增加，并且对损伤具有更强的抵抗力。我们的 核心假说将通过三个目标进行检验。目标1将定义SIRT5在肾脏中的具体作用部位 损伤，特别是PTEC。而目标2将深入研究过氧异构体脂肪酸氧化的作用 在肾脏损伤期间。最后，Aim 3将从机制上确定SIRT5在肾脏损伤过程中的分子靶点。 所有这三个目标都将利用一种严格的、机械的方法，结合体外和体内模型。在……里面 在小鼠体内的研究将同时使用全局SIRT5-/-和可诱导的、PTEC特异性的SIRT5敲除以及全局 LCAD-/-(粮农组织线粒体关键酶)。体外研究将使用分离的原代小鼠和人PTEC作为 以及基因操纵的小鼠和人类细胞系。人AKI将在小鼠体内单侧复制 缺血再灌注损伤和肾毒素顺铂的单次大剂量治疗。在两种损伤模型中， 将研究SIRT5在调节从急性肾脏疾病到慢性肾脏疾病进展过程中的作用。这个项目 将通过开辟新的治疗途径来显著推进该领域，其中SIRT5可以在药理学上 在AKI的背景下被抑制，以保护免受伤害和阻止进展为慢性肾脏疾病。

ABSTRACT Acute kidney injury (AKI) occurs in nearly 1 of 5 hospitalized patients and is associated with increased morbidity and mortality across all ages. Many AKI patients will recover kidney function post-injury but then progress to chronic kidney disease (CKD). The mechanisms are poorly understood and there are currently no effective therapies to prevent, limit, or reverse the tissue damage. There is a critical need to identify mechanisms involved in the pathogenesis of AKI. Our long-term goal is to elucidate these mechanisms and leverage them for new therapies to limit AKI and prevent the transition to CKD. Proximal tubule epithelial cells (PTEC), a major site of damage during AKI, are very metabolically active and rich in mitochondria. Mitochondrial metabolism causes increased reactive oxygen species (ROS) which has been implicated in both ischemia-reperfusion injury (IRI) and cisplatin-induced nephrotoxicity. Modulating mitochondrial function during AKI is an attractive, but thus far unachievable, strategy. Our central hypothesis is that loss of the mitochondrial sirtuin lysine deacylase Sirt5 leads to shifts in PTEC metabolism that protect against AKI. This is supported by preliminary data showing protection against both IRI and cisplatin-induced AKI in global Sirt5 knockout (Sirt5-/-) mice in vivo and in vitro as well as in primary human PTEC with siRNA knockdown of Sirt5. Further data support our proposed mechanism of protection in which Sirt5-/- PTEC exhibit a form of metabolic preconditioning characterized by a shift of fatty acid oxidation (FAO) from mitochondria to peroxisomes. Peroxisomes have previously been linked to renoprotection in other animal models, most likely due to their ability to eliminate ROS. In Sirt5-/- kidneys, peroxisome number is increased at baseline and the peroxisomes are more resistant to damage during AKI. Our central hypothesis will be tested with three aims. Aim 1 will define the specific site of Sirt5 action during kidney injury with a particular focus on PTEC. While aim 2 will drill down on the role of peroxisomal fatty acid oxidation during kidney injury. Finally, aim 3 will mechanistically define the molecular targets of Sirt5 during kidney injury. All of the three aims will utilize a rigorous, mechanistic approach that combines in vitro and in vivo models. In vivo studies in mice will use both global Sirt5-/- and inducible, PTEC-specific knockout of Sirt5 as well as global LCAD-/- (key mitochondrial FAO enzyme). In vitro studies will use isolated primary mouse and human PTEC as well as genetically manipulated mouse and human cell lines. Human AKI will be modeled in mice by unilateral ischemia-reperfusion injury and single high dose treatment with the nephrotoxin cisplatin. In both models of injury, the role of Sirt5 in mediating the progression from acute to chronic kidney disease will be studied. This project will significantly advance the field by opening up new therapeutic avenues where Sirt5 can be pharmacologically inhibited in the context of AKI to protect against injury and block the progression to chronic kidney disease.