Crosstalk between lipid droplets and other organelles in podocytes

摘要 研究支持脂滴(LD)在线粒体功能障碍(MD)和足细胞损伤中的堆积在包括Alport综合征(AS)在内的代谢性和非代谢性肾小球疾病的发病机制中的作用。LDS调节细胞内TGS的储存和动态平衡，调节线粒体中可被氧化并用作能源的游离脂肪酸(FFA)的可获得性。LDS与线粒体的正确接触是由LD相关蛋白介导的，如Perilipin 5(PLIN5)。PLIN5是一种蛋白质，它调节储存在LDS中的TGS的脂解，介导LD-线粒体接触的形成，并作为伴侣介导的自噬(CMA)的底物。然而，对PLIN5在足细胞中的功能知之甚少。 在初步数据中，我们发现，与对照组相比，Col4A3 KO(AS小鼠)肾皮质、AS足细胞和斑马鱼模型中PLIN5的表达降低。在AS足细胞中，PLIN5缺乏与脂解增加、FFA积聚、LD-线粒体接触形成和线粒体膜电位降低有关，而PLIN5过表达可使其恢复。与WT足细胞相比，足细胞的自噬小体数量也增加，通常在线粒体附近，这表明AS可能发生了更多的有丝分裂。PLIN5的表达被Ezetimibe(EZ)恢复，EZ是一种最初开发的化合物，通过与NPC1L1结合来防止胆固醇吸收，这表明该化合物的非靶点效应。我们的结果提示PLIN5缺乏在脂毒性诱导的MD和AS的足细胞损伤和肾脏疾病进展中起重要作用。 这项建议的总体目标是研究PLIN5缺乏与AS足细胞损伤之间的联系的新机制。我们假设PLIN5缺乏有助于在Col4A3基因敲除小鼠中观察到的肾脏表型，这是一种AS的小鼠模型，并导致足细胞损伤和AS的肾衰竭。 在目标1中，我们将研究PLIN5缺乏导致足细胞损伤以及Ezetimibe保护足细胞损伤的机制。在目标2中，我们将确定在Col4A3KO小鼠中足细胞特异性过表达PLIN5是否足以预防AS的肾功能衰竭(目标2)。 如果成功，这项创新的研究将把PLIN5缺乏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Ezetimibe的一种新的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.