Ceramides as Novel Mediators of Tubular Metabolic Dysfunction Driving Kidney Injury

项目总结/摘要。急性肾损伤（阿基）是一种常见的疾病， 患者死亡率和医疗保健支出的负担。存在一个显著的未满足的需求，以阐明肾脏特异性 驱动肾损伤的发作和进展，以便开发能够 直接针对肾脏病变令人信服的证据表明，改变代谢内的 肾近端小管与阿基有关。在这个奖学金申请中提出的工作将批判性地评估 一类脂毒性脂质物质，称为神经酰胺，作为线粒体功能障碍，氧化应激， 应激和多种肾脏病理学特征的脂质积累。人类相关数据表明 改变的管状神经酰胺代谢与临床肾脏疾病终点相关，如肾脏 纤维化和估计的肾小球滤过率。以前的报告表明，肾神经酰胺升高， 肾损伤后或慢性疾病背景下的临床前模型，以及提供的初步数据 本文证明了神经酰胺全身消耗成功地预防了急性肾损伤， 肾缺血再灌注或梗阻性损伤后的组织病理学。本申请旨在解决 关于局部肾脏神经酰胺是否在疾病机制中发挥作用的知识仍存在差距， 细胞类型。具体而言，拟议的项目将确定神经酰胺是否是 肾小管上皮细胞代谢功能障碍。在目标一中，我们将探索保护肾脏免受损伤 在肾小管上皮细胞中神经酰胺基因缺失的新型小鼠品系中发生。此外，委员会认为， 将评估肾小管内神经酰胺遗传获得的动物的肾脏发育 功能障碍和组织病理学。目标二中提出的研究将采用体外和离体方法， 表征神经酰胺驱动线粒体生物能量学和脂质损伤的新机制 在肾小管和原代细胞中蓄积。初步研究结果表明， 神经酰胺损害培养的永生化近端肾小管的线粒体呼吸和ATP产生 上皮细胞这项工作将是第一次调查，以直接评估，如果管源性神经酰胺是 与肾小管损伤和组织病理学之前的代谢紊乱有关， 了解降低神经酰胺干预对急性肾脏病主要病因的治疗潜力 损伤此外，完成拟议的研究将大大丰富申请人的博士前培训， 掌握技术技能（例如，肾损伤和疾病模型的实施和评价，评价 线粒体代谢和底物利用，以及基于质谱的神经酰胺分析 水平），并作为一个年轻的科学家追求独立的研究生涯的发展。

PROJECT SUMMARY/ABSTRACT. Acute kidney injury (AKI) is a prevalent condition which elicits an enormous burden on patient mortality and healthcare spending. A significant unmet need exists to elucidate kidney-specific insults driving the onset and progression of kidney injury in order to develop novel therapeutic strategies capable of directly targeting renal pathology. Compelling evidence has suggested that altered metabolism within the kidney proximal tubule is implicated in AKI. The work proposed in this fellowship application will critically evaluate the role of a class of lipotoxic lipid species, termed ceramides, as drivers of mitochondrial dysfunction, oxidative stress, and lipid accumulation characteristic of multiple kidney pathologies. Human correlational data suggest that altered tubular ceramide metabolism correlates with clinical kidney disease endpoints, such as kidney fibrosis and estimated glomerular filtration rate. Previous reports indicate that renal ceramides are elevated in pre-clinical models following kidney injury or in the setting of chronic disease, and preliminary data presented herein demonstrate that whole-body depletion of ceramides successfully prevents acute kidney injury and histopathology following renal ischemia reperfusion or obstructive injury. This application intends to address the remaining gap in knowledge regarding whether local kidney ceramides play a role in disease mechanisms and in which cell types. Specifically, the proposed project will determine if ceramides are candidate mediators of metabolic dysfunction in tubular epithelial cells. In Aim One, we will probe for protection from kidney injury incurred in novel mouse lines with genetic depletion of ceramides in the kidney tubular epithelium. Furthermore, animals with genetic gain-of-ceramide within kidney tubules will be assessed for development of kidney dysfunction and histopathology. Studies proposed in Aim Two will employ in vitro and ex vivo methods to characterize novel mechanisms of ceramides driving impairment of mitochondrial bioenergetics and lipid accumulation in kidney tubules and primary cells. Preliminary findings demonstrate that accumulation of ceramides impairs mitochondrial respiration and ATP production in cultured immortalized proximal tubular epithelial cells. This work will be the first investigation to directly assess if tubule-derived ceramides are implicated in the metabolic perturbations preceding tubular injury and histopathology and will provide valuable insight into the therapeutic potential of ceramide-lowering interventions for dominant causes of acute kidney injury. Furthermore, completion of the proposed studies will greatly enrich the applicant’s pre-doctoral training, mastery of technical skills (e.g., implementation and evaluation of kidney injury and disease models, evaluation of mitochondrial metabolism and substrate utilization, and mass spectrometry-based analysis of ceramide levels), and development as a young scientist pursuing an independent research career.