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，从而预防AKI。初步数据证实了这一点。 整体SIRT5基因敲除(SIRT5-/-，SIRT5+/-)小鼠对IRI和顺铂诱导的AKI的保护作用 在体外以及在原代人PTEC中SIRT5的siRNA被敲除。进一步的数据支持我们提出的 SIRT5-/-PTEC表现出一种以转移为特征的代谢适应的保护机制 从线粒体到过氧化体的脂肪酸氧化(FAO)的过程。过氧酶体以前被认为与 在其他动物模型中的肾脏保护作用，很可能是因为它们有能力消除ROS。在SIRT5/-肾脏中， 在AKI过程中，过氧化物体对损伤更具抵抗力。我们的核心假设将通过两个目标进行检验。目标 1将确定SIRT5在肾脏损伤过程中的具体作用部位，尤其是PTEC。而Aim 2将 深入研究代谢粮农组织的抑制和过氧化体脂肪的刺激相结合的肾脏保护作用 肾脏损伤时的酸氧化。这两个目标都将利用一种严格的、机械的方法，在体外结合 以及活体模型。在小鼠体内的研究也将使用全局SIRT5/-PTEC特异性SIRT5基因敲除 作为LCAD-/-(线粒体粮农组织关键酶)的全球和PTEC特异性敲除。体外研究将使用 分离的原代小鼠和人PTEC以及基因操纵的小鼠和人类细胞系。 采用单侧脑缺血再灌流损伤和单次大剂量给药的方法建立人急性肾损伤模型。 肾毒素顺铂。我们还优化了使用单侧缺血-再灌注损伤的CKD模型。 模特。该项目将通过开辟新的治疗途径显著推进该领域，SIRT5可以 被药物抑制或其肾脏保护机制可在AKI的背景下被利用以保护 防止损伤，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 protects against AKI. This is supported by preliminary data showing protection against both IRI and cisplatin-induced AKI in global Sirt5 knockout (Sirt5-/-, 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 adaptation 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, peroxisomes are more resistant to damage during AKI. Our central hypothesis will be tested with two 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 renoprotective role of metabolic FAO inhibition coupled with stimulation of peroxisomal fatty acid oxidation during kidney injury. Both 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-/- PTEC-specific knockout of Sirt5 as well as global and PTEC-specific knockout of 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. We have also optimized a CKD model using a unilateral ischemia-reperfusion injury model. This project will significantly advance the field by opening up new therapeutic avenues where Sirt5 can be pharmacologically inhibited or its renoprotective mechanism can be harnessed in the context of AKI to protect against injury and block the progression to chronic kidney disease.