Role of Autophagy in Maladaptive Renal Repair Following Acute Kidney Injury

摘要 急性肾损伤（AKI）后的适应不良肾修复可导致慢性肾脏疾病 (CKD)引起肾小管萎缩、毛细血管稀疏和间质纤维化。缺氧是一种已知的 CKD发展中的致病因子，并可引发自噬，一种溶酶体降解 使细胞内成分分解以进行能量再利用的途径。我们已经证明， 损伤肾脏中的长期代谢紊乱导致长期的自噬反应， 导致肾小管萎缩和血管脱落。我们现建议将这些研究结果加以扩展， 在以下目标中进行研究。目的1将检查代谢紊乱和肾小管 缺血-再灌注损伤（IRI）导致CKD发展过程中的上皮细胞自噬。 我们将使用我们的新型自噬报告小鼠来量化自噬水平，并监测自噬水平。 自噬过程与代谢紊乱的关系。小鼠将用以下前体处理： 乙酰辅酶A直接测试是否补充代谢物防止肾小管自噬。 接下来，我们将通过遗传学方法来测试持续的上皮细胞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，并检查它们对肾小管上皮细胞的影响。 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方法。 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。我们发现压力反应 转录因子FoxO3a在具有适应不良修复的肾的肾小管中被激活。 腺病毒感染原代培养的肾上皮细胞 激活的FoxO3a导致自噬途径的激活。的影响和调节 通过进行缺失来研究患病肾脏中FoxO3a的持续自噬， 过量表达和拯救实验。生物化学和遗传学方法将被应用于 了解FoxO3a脯氨酰羟基化通过PHD介导的反应，需要氧气和α- 酮戊二酸。在目标3中，我们将检验具有持续自噬的小管具有 Vefga表达减少，这有助于毛细血管稀疏。血管脱落进一步造成 代谢干扰小管，从而建立一个自我延续的恶性循环。我们将删除 vegfa特异性地在肾小管中使用强力霉素诱导系统， 肾小管和管周毛细血管的相互依赖性。此外，我们亦会研究是否- 肾小管衍生的VEGF的调节是由长期的细胞分裂引起的一般分解代谢的结果。 自噬和/或由于FoxO3a的转录抑制。该项目的目标是双重的。 第一个目标是了解从AKI向CKD转变过程中的发病机制， 肾小管自噬伴代谢紊乱。第二个目标是了解 通过研究缺氧诱导的FoxO3a激活来进行上皮细胞自噬的分子调节。

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.