Role of Anti-aging Klotho Gene on Adaptation to Oxidative Stress

描述（由申请人提供）：Klotho最初被确定为抗衰老基因，但Klotho蛋白具有抗氧化等多种作用。缺氧诱导线粒体活性氧（reactive O2 species， ROS）泄漏，是肺泡生长重塑和细胞寿命的生理信号。相反，高氧会产生过量的活性氧，诱导肺泡变性，类似肺气肿。因此，缺氧和高氧都会对肺造成氧化应激。有趣的是，Klotho缺乏症会导致寿命短、生长迟缓、动脉粥样硬化、器官变性和肺气肿等早衰综合征。Klotho缺失纯合子（KL-/-）小鼠在年轻时死亡，并伴有广泛的异常。杂合子小鼠（KL）发生肺气肿，但其他方面正常。目前尚不清楚产生过量Klotho蛋白（Tg-KL）的转基因小鼠是否受到氧化损伤的保护。我们假设肺的生长和衰老代表了由一组共享基因调节的氧化应激适应谱的不同末端，而Klotho处于这些途径的中心阶段。目的是探讨Klotho在出生后暴露于不同氧浓度的动物肺生长和功能中的作用。我将确定氧化应激对Klotho基因缺乏或过表达小鼠肺结构和功能的影响，并验证Klotho基因缺乏损害低氧诱导的肺生长并加速高氧诱导的肺损伤，而Klotho基因过表达增强低氧诱导的肺生长并减轻高氧诱导的肺损伤的假设。将转基因小鼠和匹配的对照组暴露于13%、21%或40%的氧气中3周，然后测量肺功能、超微结构和氧化损伤的生物标志物。最后，我将在体外测试Klotho对肺上皮细胞氧化损伤反应能力的直接影响。这些研究扩大了我的训练范围和科学探索。Drs。Kuro-o和Moe一直在研究Klotho在多器官衰老和疾病相互作用中的代谢作用。他们之所以对肺感兴趣，是因为肺是KL小鼠中唯一表现出基线异常的器官。我将在Moe博士的实验室负责Klotho工作小组的肺部部分。我将把我在生物工程博士培训中学到的知识应用到这个新模型上。我将在一个新的环境中，除了生理学之外，我将更加重视细胞和分子生物学。这些结果将促进我们对连接肺生长和衰老的基本机制的理解，为操纵Klotho的产生是否促进肺生长或防止退化提供新的见解，并为探索Klotho蛋白的潜在用途奠定基础，可能通过吸入输送，以增加肺生长或减轻慢性肺病治疗中的破坏。由于Klotho缺乏症也存在于人类中，并且Klotho疗法正在被深入研究，因此该主题具有广泛的生物学和临床相关性，将成为我作为生物医学研究人员开展职业生涯的理想平台。

DESCRIPTION (provided by applicant): Klotho was first identified as an anti-aging gene but Klotho protein has multiple effects including anti-oxidation. Hypoxia induces mitochondrial leak of reactive O2 species (ROS), which can serve as physiologic signals for alveolar growth and remodeling and cell longevity. Conversely, hyperoxia produces excess ROS that induce alveolar degeneration resembling emphysema. Hence, both hypoxia and hyperoxia impose oxidative stress on the lung. Interestingly Klotho deficiency causes a premature aging syndrome with short lifespan, growth retardation, atherosclerosis, organ degeneration and pulmonary emphysema. Mice homozygous for Klotho deletion (KL-/-) die at a young age with widespread abnormalities. Heterozygous mice (KL) develop pulmonary emphysema but are otherwise normal. It is not known if transgenic mice producing excess Klotho protein (Tg-KL) are protected from oxidative damage. We postulate that growth and aging of the lung represent different ends in the spectrum of adaptation to oxidative stress regulated by a shared set of genes, and that Klotho is at the center stage of these pathways. The Aim is to explore the role of Klotho on lung growth and function in animals exposed to different O2 tensions during postnatal life. I will determine the effects of oxidative stress on lung structure and function in mice bearing deficiency or overexpression of the Klotho gene, and test the hypothesis that Klotho deficiency impairs hypoxia-induced lung growth and accelerates hyperoxia- induced lung damage while Klotho overexpression augments hypoxia-induced lung growth and mitigates hyperoxia-induced lung damage. Genetically modified mice and matched controls will be exposed to 13, 21 or 40% O2 for 3 wk, followed by measurement of lung function, ultrastructure, and biomarkers of oxidative damage. Finally, I will test the direct effects of Klotho on the ability of lung epithelial cells to react to oxidative insult in vitro. These studies expand the scope of my training as well as the scientific quest. Drs. Kuro-o and Moe have been investigating the metabolic effects of Klotho on the interaction of aging and disease in multiple organs. They became interested in the lung since it is the only organ in the KL mice that exhibits baseline abnormalities. I will take on the pulmonary part of this Klotho working group in Dr. Moe's laboratory. I will apply what I have learned in my doctorate training as a bioengineer to this new model. I will be in a new environment with greater emphasis on cell and molecular biology in addition to physiology. The results will advance our understanding of fundamental mechanisms that link lung growth and aging, offer new insight into whether manipulation of Klotho production enhances lung growth or protects against degeneration, and lay the foundation for exploring potential uses of Klotho protein, perhaps by inhalational delivery, to augment lung growth or mitigate destruction in the treatment of chronic lung disease. Since Klotho deficiency also exists in humans and Klotho therapy is being intensely studied, this topic has broad biologic and clinical relevance and will be an ideal platform to launch my career as a biomedical researcher. PUBLIC HEALTH RELEVANCE: Klotho is an anti-aging protein with multiple effects including anti-oxidative actions. My goal is to understand how Klotho modulates the adaptation in lung structure and function induced by exposure to different oxygen levels. Studies will examine whether Klotho-deficient lungs are more susceptible to oxidative damage using transgenic mice that do not produce Klotho, whether the lungs from transgenic mice producing excess Klotho are protected from oxidative damage, and whether Klotho directly modulates the ability of lung cells to resist oxidative damage in a cell culture model. The studies will build on the strengths of my doctoral training and expand my technical and intellectual skills. The results help us understand the mechanisms that regulate lung growth and aging, and explore a potential use of Klotho in the protection or treatment of chronic lung diseases. Since Klotho deficiency exists in humans, this topic has immediate and broad biologic and clinical relevance.