Role of Autophagy in Maladaptive Renal Repair Following Acute Kidney Injury

自噬在急性肾损伤后肾适应不良修复中的作用

• 批准号: 9355626
• 负责人: FANGMING LIN
• 金额: $ 24万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-21 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9355626
• 关键词: Acute Renal Failure with Renal Papillary Necrosis; Address; Adenoviruses; Affect; Atrophic; Autophagocytosis; Biochemical Genetics; Blood Vessels; Blood capillaries; Catabolism; Cells; Chimeric Proteins; Chronic Kidney Failure; Degradation Pathway; Development; Digestion; Disease Progression; Down-Regulation; Doxycycline; Dropout; Enzymes; Epithelial; Epithelial Cells; FOXO3A gene; FRAP1 gene; Feedback; Fibrosis; Future; Genetic; Goals; Health; Hydroxylation; Hypoxia; Kidney; Kidney Diseases; Kidney Failure; Lead; Link; Maintenance; Mediating; Metabolic; Molecular; Monitor; Mus; Nutrient; Organelles; Oxygen; Pathogenesis; Pathway interactions; Pharmacology; Phase; Primary Infection; Process; Reaction; Regulation; Renal tubule structure; Reperfusion Injury; Reporter; Role; Starvation; Stress; System; Testing; Tetanus Helper Peptide; Tissues; Transcriptional Activation; Transgenic Organisms; Tubular formation; Vascular Endothelial Growth Factors; Virulence Factors; alpha ketoglutarate; biological adaptation to stress; capillary; density; design; experimental study; gene repression; genetic approach; injured; interstitial; novel; overexpression; prevent; repaired; response; tool; transcription factor

Abstract Maladaptive renal repair following acute kidney injury (AKI) can lead to chronic kidney disease (CKD) causing tubular atrophy, capillary rarefaction, and interstitial fibrosis. Hypoxia is a known pathogenic factor in the development of CKD and can trigger autophagy, a lysosomal degradation pathway that recycles intracellular constituents for energy reutilization. We have showed that protracted metabolic perturbation in the injured kidney leads to a prolonged autophagic response and contributes to tubular atrophy and vascular dropout. We now propose to extend these findings by performing studies in the following aims. Aim 1 will examine metabolic perturbation and tubular epithelial autophagy during the development of CKD resulting from ischemia-reperfusion injury (IRI). We will use our novel autophagy reporter mice to quantify autophagy levels and monitor the autophagic process in relationship with metabolic perturbation. Mice will be treated with a precursor of acetyl co-enzyme A to directly test whether replenishing metabolites prevents tubular autophagy. Next, we will test whether sustained epithelial autophagy can lead to tubular atrophy by taking genetic and pharmacological approaches to alter autophagy levels and examine their effects on tubular atrophy. In Aim 2, we will study molecular regulation of autophagy by FoxO3a and further explore our newly discovered mechanism that links hypoxia to autophagy via activation of FoxO3a through inhibition of prolyl hydroxylation and degradation of FoxO3a. We find that the stress-responsive transcription factor FoxO3a is activated in renal tubules of the kidney with maladaptive repair. Infection of primary cultures of renal epithelial cells with adenoviruses expressing constitutively activated FoxO3a results in activation of the autophagic pathway. The effect and regulation of sustained autophagy by FoxO3a in the diseased kidney will be investigated by performing deletion, overexpression, and rescue experiments. Biochemical and genetic approaches will be applied to understand FoxO3a prolyl hydroxylation via a PHD-mediated reaction that requires oxygen and α- ketoglutarate. In Aim 3, we will test the hypothesis that tubules with sustained autophagy have reduced Vefga expression, which contributes to capillary rarefaction. Vascular dropout creates further metabolic perturbation to tubules, thus setting up a self-perpetuating, vicious cycle. We will delete Vegfa specifically in renal tubules using a doxycycline-inducible system and examine the interdependence of tubules and peritubular capillaries. Furthermore, we will study whether down- regulation of tubule-derived Vegf is a result of general catabolic consequence from prolonged autophagy and/or due to transcriptional repression by FoxO3a. The goals of this project are two-fold. The first goal is to understand the pathogenesis during the transition from AKI to CKD by focusing on tubular autophagy in the kidneys with metabolic disturbance. The second goal is to understand the molecular regulation of epithelial autophagy by investigating hypoxia-induced FoxO3a activation.

抽象的 急性肾损伤（AKI）后的适应不良肾脏修复可导致慢性肾脏疾病 (CKD) 导致肾小管萎缩、毛细血管稀疏和间质纤维化。缺氧是众所周知的 CKD 发展的致病因素，可引发自噬（一种溶酶体降解） 回收细胞内成分以进行能量再利用的途径。我们已经证明 受损肾脏的长期代谢紊乱会导致自噬反应延长， 导致肾小管萎缩和血管脱落。我们现在建议通过以下方式扩展这些发现 为了以下目标进行研究。目标 1 将检查代谢扰动和肾小管 缺血再灌注损伤（IRI）引起的 CKD 发展过程中的上皮自噬。 我们将使用我们的新型自噬报告小鼠来量化自噬水平并监测 自噬过程与代谢扰动的关系。将用前体治疗小鼠 乙酰辅酶 A 直接测试补充代谢物是否会阻止肾小管自噬。 接下来，我们将通过基因检测来测试持续的上皮自噬是否会导致肾小管萎缩。 和药理学方法来改变自噬水平并检查其对肾小管的影响 萎缩。在目标2中，我们将研究FoxO3a对自噬的分子调控，并进一步探索我们的研究成果。 新发现的机制通过激活 FoxO3a 将缺氧与自噬联系起来 抑制脯氨酰羟基化和 FoxO3a 降解。我们发现应激反应 转录因子 FoxO3a 在肾脏肾小管中被激活，导致适应不良修复。 用组成型表达的腺病毒感染肾上皮细胞的原代培养物 激活的 FoxO3a 会导致自噬途径的激活。作用及调节 将通过删除来研究患病肾脏中 FoxO3a 的持续自噬， 过度表达和救援实验。生物化学和遗传学方法将应用于 通过 PHD 介导的需要氧气和 α- 的反应了解 FoxO3a 脯氨酰羟基化 酮戊二酸。在目标 3 中，我们将检验以下假设：具有持续自噬作用的肾小管具有 Vefga 表达减少，导致毛细血管稀疏。血管脱落进一步造成 肾小管的代谢紊乱，从而形成自我延续的恶性循环。我们将删除 使用多西环素诱导系统特异性地在肾小管中检测 Vegfa 并检查 肾小管和肾小管周围毛细血管的相互依赖性。此外，我们将研究是否向下 肾小管源性 Vegf 的调节是长期分解代谢结果的结果 自噬和/或由于 FoxO3a 的转录抑制。该项目的目标有两个。 第一个目标是通过关注从 AKI 向 CKD 转变过程中了解发病机制 肾脏中的肾小管自噬伴有代谢紊乱。第二个目标是了解 通过研究缺氧诱导的 FoxO3a 激活对上皮自噬的分子调控。