Crosstalk between lipid droplets and other organelles in podocytes

摘要 研究支持脂滴（LD）蓄积在代谢和非代谢来源的肾小球疾病（包括Alport综合征（AS））发病机制中的线粒体功能障碍（MD）和足细胞损伤中的作用。LD调节细胞内TG的储存和稳态，并调节游离脂肪酸（FFA）的可用性，其可以在线粒体中氧化并用作能量来源。LD与线粒体的适当接触是由LD相关蛋白介导的，例如围脂蛋白5（PLIN 5）。PLIN 5是一种调节储存在LD中的TG的脂解、介导LD-线粒体接触形成并充当分子伴侣介导的自噬（CMA）的底物的蛋白质。然而，关于PLIN 5在足细胞中的功能知之甚少。 在初步数据中，我们证明了与对照相比，Col 4a 3 KO（AS小鼠）的肾皮质、AS足细胞和AS的斑马鱼模型中PLIN 5表达降低。在AS足细胞中，PLIN 5缺乏与增加的脂解、FFA积累和减少的LD-线粒体接触形成和线粒体膜电位相关，其通过PLIN 5过表达恢复。与WT足细胞相比，AS足细胞还具有增加的自噬体数量，通常在线粒体附近，这表明在AS中可能发生线粒体自噬增加。PLIN 5表达通过依折麦布（EZ）恢复，依折麦布是一种最初开发用于通过与NPC 1 L1结合来防止胆固醇吸收的化合物，表明该化合物具有脱靶效应。我们的研究结果表明PLIN 5缺乏在脂毒性诱导的MD和足细胞损伤以及AS的肾脏疾病进展中起重要作用。 该提案的总体目标是研究将PLIN 5缺陷与AS中足细胞损伤联系起来的新机制。我们假设PLIN 5缺乏导致在Col 4a 3敲除小鼠（AS的小鼠模型）中观察到的肾表型，并导致足细胞损伤和AS中的肾衰竭。 在目的1中，我们将研究PLIN 5缺乏导致足细胞损伤的机制以及依折麦布保护足细胞损伤的机制。在目的2中，我们将确定Col 4a 3 KO小鼠中PLIN 5的足细胞特异性过表达是否足以预防AS中的肾衰竭（目的2）。 如果成功，这项创新研究将把PLIN 5缺陷与脂毒性诱导的足细胞损伤的新途径联系起来，并可能揭示依折麦布的新MoA。

ABSTRACT Research supports a role for lipid droplet (LD) accumulation in mitochondrial dysfunction (MD) and podocyte injury in the pathogenesis of glomerular diseases of metabolic and non-metabolic origin, including Alport Syndrome (AS). LDs regulate the storage and homeostasis of intracellular TGs and regulate the availability of free fatty acids (FFAs), which can be oxidized in mitochondria and used as an energy source. Proper contact of LDs with mitochondria is mediated by LD-associated proteins, such as perilipin 5 (PLIN5). PLIN5 is a protein that regulates lipolysis of TGs stored in LDs, mediates LD-mitochondrial contact formation, and serves as a substrate for chaperone-mediated autophagy (CMA). However, little is known about the function of PLIN5 in podocytes. In preliminary data, we demonstrated decreased PLIN5 expression in kidney cortices of Col4a3 KO (AS mice), in AS podocytes and in a zebrafish model of AS when compared to controls. In AS podocytes, PLIN5 deficiency is associated with increased lipolysis, FFA accumulation, and reduced LD-mitochondrial contact formation and mitochondrial membrane potential, which was restored by PLIN5 overexpression. AS podocytes also have an increased autophagosome number compared to WT podocytes, often in proximity of mitochondria, suggesting increased mitophagy may occur in AS. PLIN5 expression is restored by Ezetimibe (EZ), a compound that was initially developed to prevent cholesterol absorption via its binding to NPC1L1, suggesting an off-target effect of this compound. Our results suggest an important role for PLIN5 deficiency in lipotoxicity-induced MD and podocyte injury and renal disease progression in AS. The overall goal of this proposal is to investigate a novel mechanism linking PLIN5 deficiency to podocyte injury in AS. We hypothesize that PLIN5 deficiency contributes to the renal phenotype observed in Col4a3 knockout mice, a mouse model of AS and causes podocyte injury and in renal failure in AS. In aim 1, we will investigate the mechanism by which PLIN5 deficiency contributes to podocyte injury and by which Ezetimibe protects from podocyte injury. In aim 2, we will determine if podocyte-specific overexpression of PLIN5 in Col4a3 KO mice is sufficient to prevent renal failure in AS (aim 2). If successful, this innovative study will link PLIN5 deficiency to a novel pathway of lipotoxicity-induced podocyte injury and may reveal a new MoA of Ezetimibe.