Oxidant Stress in the Nrf2 Knockout Rat

描述（由申请人提供）：转录因子核因子红系2相关因子2 （Nrf2）是抗氧化反应的“主调节剂”，调节数百个与氧化状态和其他生理过程相关的基因。nrf2调控基因的失调可能为氧化应激与涉及多个生理过程、多个器官和多个基因网络的大约200种人类疾病之间的联系提供了一个合乎逻辑的解释。由于直接使用抗氧化剂治疗的临床试验结果令人失望，人们对直接激活Nrf2作为上调人类内源性抗氧化防御的治疗方法也有浓厚的兴趣。他的研究目标是开发一种缺乏Nrf2基因的突变大鼠品系，并在Nrf2（-/-）大鼠中进行广泛的血压、血管反应性和肾功能表型分析，作为验证Nrf2基因缺失具有生理影响的原理证明。由于创始大鼠已经被开发出来，表型方案目前已经制定，本项目的目标是在利用TALEN技术敲除Nrf2基因的新大鼠品系中进行繁殖，基因分型和表征关键表型。本项目有两个具体目的：1)表征NRF2（-/-）突变大鼠NRF2蛋白功能和下游基因调控；2)对Nrf2（-/-）突变大鼠和野生型对照进行初始表型分析，以验证基因敲除的有效性以及Nrf2等位基因缺失是否存在功能后果。待评估的表型包括有意识动物的动脉血压；在Nrf2诱导剂Protandim （60.5 mg/kg/day）存在和不存在的情况下，用正常盐（0.4% NaCl）或高盐（4% NaCl）喂养Nrf2（-/-）突变大鼠和野生型对照，观察其内皮功能、离体抵抗动脉NO水平和超氧化物水平、输注血管活性药物后的全身血压反应、血浆和尿液氧化应激标志物以及全动物和肾脏功能的组织学评价。除了提供Nrf2系统在调节微循环功能、肾功能和最终血压方面的生理作用的重要基础信息外，该项目还将使我们能够确定该模型是否复制预测的疾病状况，并可用于开发基于激活Nrf2上调内源性氧化防御的治疗方法。

DESCRIPTION (provided by applicant): The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a "master regulator" of the antioxidant response that regulates hundreds of genes related to oxidant state and other physiological processes. Dysregulation of Nrf2-regulated genes may provide a logical explanation for the connections between oxidative stress and perhaps 200 human diseases involving multiple physiological processes, multiple organs, and multiple gene networks. There is also keen interest in direct activation of Nrf2 as a therapeutic approach to upregulate endogenous antioxidant defenses in humans in light of disappointing results of clinical trials involving direct treatment with antioxidants. The goal of his study is to develop a mutant rat strain lacking the Nrf2 gene, and conduct extensive phenotyping of blood pressure, vascular reactivity, and renal function in the Nrf2(-/-) rats as proof of principle to verify that loss of the Nrf2 gene has physiological effects. Because the founder rats have been developed and phenotyping protocols are currently in place, the goal of the present project is to breed, genotype, and characterize crucial phenotypes of interest in a novel rat strain in which the Nrf2 gene has been knocked out utilizing TALEN technology. This project has two specific aims: 1) to characterize NRF2 protein function and downstream gene regulation in the Nrf2(-/-) mutant rat; and 2), to conduct initial phenotyping of Nrf2(-/-) mutant rats and wild type controls to verify the efficacy of the knockout and the presence of functional consequences of loss of the Nrf2 allele. Phenotypes to be evaluated include arterial blood pressure in conscious animals; endothelial function, NO levels, and superoxide levels in isolated resistance arteries, whole body blood pressure responses to infusion of vasoactive drugs, plasma and urinary markers of oxidant stress, and whole animal and histological evaluation of renal function in Nrf2(-/-) mutant rats and wild type controls fed normal salt (0.4% NaCl) or high salt (4% NaCl) diet in the presence and absence of the Nrf2 inducer Protandim (60.5 mg/kg/day). In addition to providing important fundamental information on the physiological roles of the Nrf2 system in regulating microcirculatory function, renal function, and ultimately blood pressure, this project will enable us to determine whether the model replicates predicted disease conditions and can be employed to develop therapeutic approaches based on upregulation of endogenous oxidant defenses by activation of Nrf2.