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.
Clinical trials using direct administration of antioxidants such as Vitamins C and E to treat oxidant stress in human disease have yielded inconsistent and disappointing results;and there is now increasing interest in therapeutic approaches that can directly increase the body's oxidant defenses. One potential target for these therapies is the protein Nrf2, which is a master antioxidant and cell protective protein that increases the expression of genes that combat oxidant stress. This project will use genetic techniques to eliminate Nrf2 in rats, in order to understand the function of this crucial protective protein and o evaluate the potential of directly activating Nrf2 as a treatment to combat oxidant stress in human diseases.