Retinoid Mediated Protection Against Reactive Oxygen Species Induced Cytotoxicity

描述（由申请人提供）：我们已经证明，类维甲酸信号参与了11-脱氧-16,16-二甲基PGE2 （DDM-PGE2）介导的细胞保护，以对抗活性氧（ROS）诱导的坏死/肿瘤细胞死亡。蛋白质组学分析显示，细胞保护与一些特定蛋白质的合成增加有关，包括视黄醇结合蛋白（RBP）、肌动蛋白和葡萄糖调节蛋白78 （Grp78）。我们随后证实，全反式维甲酸（aTRA）在体外复制了ddm - pge2介导的细胞保护作用，更重要的是，单剂量aTRA （1 mg/kg，预处理6h）完全保护小鼠肾缺血/再灌注（I/R）损伤。此外，在这个治疗剂量下，aTRA诱导nrf2响应性抗氧化剂HO-1和NQO1基因，以及核维甲酸受体RAR？, RAR ?2, RAR ?2、类视黄醇X受体RXR？在肾脏。修订后的申请旨在确定aTRA在体外提供细胞保护的分子机制，以及这种细胞保护机制在体内重现的程度。我们的中心假设是atra诱导的细胞保护是由类似于缺血预处理的机制介导的。在Specific Aim 1中，我们建议确定aTRA在缺氧/再氧化损伤的体外模型（人肾上皮HK-2细胞）中提供细胞保护的能力，并优化aTRA在体内缺血/再灌注模型（IR）中介导的细胞保护方案。我们还将确定这种保护作用是否至少部分是通过抗氧化酶的上调介导的。类维甲酸的生物学效应通常是通过与其同源核受体，即视黄酸受体（RAR）和类维甲酸X受体（RXR）的相互作用来介导的。RAR和/或RXR参与atra介导的细胞保护的程度尚不清楚，特异性Aim 2将在体外和体内I/R模型中确定atra介导的细胞保护是否需要与RAR和/或RXR相互作用。因此，特异性Aims 1和2旨在确定类视黄醇信号的募集作为ROS在疾病病理中起重要作用的条件下的潜在治疗干预，例如涉及缺血再灌注损伤（Specific Aim 1），并初步表征这种作用的药理学基础（Specific Aim 2）。第三个也是最后一个特异性靶标旨在确定aTRA完成细胞保护的分子机制，每个子靶标都聚焦于在初步研究中已经确定的在细胞保护反应中起重要作用的靶标。具体来说，这些介质是Nrf2、Grp78和p38 MAPK，它们也是缺血预处理的关键介质。因此，特异性Aim 3将确定(i) aTRA介导的抗氧化应激反应诱导是否依赖于Nrf2；（ii） atra介导的细胞保护需要募集ER （Grp78）介导的应激反应通路；（iii） aTRA对Nrf2的募集依赖于p38 MAPK-Grp78的相互作用；（iv）通过在Nrf2-/-小鼠中测试aTRA诱导的肾保护，在体内模型中概括了在体外模型中确定的细胞保护机制。当前研究的意义在于，它们有可能在分子和细胞水平上增强我们对类维甲酸介导的细胞保护的理解，从而为化学诱导的组织损伤或缺氧/缺血-再灌注损伤的临床干预提供有效的新治疗策略。

DESCRIPTION (provided by applicant): We have shown that retinoid signaling is engaged during 11-deoxy-16,16-dimethyl PGE2 (DDM-PGE2) mediated cytoprotection against reactive oxygen species (ROS) induced necrotic/oncotic cell death. Proteomics analyses revealed that cytoprotection is associated with the increased synthesis of a select number of proteins, including retinol binding protein (RBP), actin, and glucose-regulated protein 78 (Grp78). We subsequently confirmed that all-trans-retinoic acid (aTRA) replicates DDM-PGE2-mediated cytoprotection in vitro, and more importantly, a single dose of aTRA (1 mg/kg, 6h pretreatment) completely protects mice from renal ischemia/reperfusion (I/R) injury. Furthermore, at this therapeutic dose, aTRA induces Nrf2-responsive antioxidant HO-1 and NQO1 genes, as well as nuclear retinoic acid receptors RAR?, RAR?2, RAR?2, and retinoid X receptors RXR? in the kidney. The revised application is designed to determine the molecular mechanisms by which aTRA affords cytoprotection in vitro, and the extent to which this mechanism(s) of cytoprotection is recapitulated in vivo. Our central hypothesis is that aTRA-induced cytoprotection is mediated by mechanisms similar to ischemic preconditioning. In Specific Aim 1 we propose to determine the ability of aTRA to offer cytoprotection in an in vitro model (human renal epithelial HK-2 cells) of hypoxia/reoxygenation injury, and to optimize protocols for aTRA-mediated cytoprotection in an in vivo ischemia/reperfuson model (IR). We will also ascertain whether the protective effects are mediated, at least in part, via the upregulation of anti-oxidant enzymes. The biological effects of retinoids are typically mediated via interaction with their cognate nuclear receptors, namely, retinoic acid receptors (RAR) and retinoid X receptors (RXR). The extent to which RAR and/or RXR participate in aTRA-mediated cytoprotection is not known, and Specific Aim 2 will determine, in both the in vitro and in vivo models of I/R, whether aTRA-mediated cytoprotection requires interaction with RAR and/or RXR. Specific Aims 1 and 2 are therefore designed to establish the recruitment of retinoid signaling as a potential therapeutic intervention in conditions where ROS play an important role in the pathology of the disease, such as those involving ischemia reperfusion injury (Specific Aim 1), and to initially characterize the pharmacological basis of this effect (Specific Aim 2). The third and final Specific Aim is designed to identify the molecular mechanisms by which aTRA accomplishes cytoprotection, with each sub- aim focusing on a target that has already been identified in preliminary studies as playing an important role in the cytoprotective response. Specifically, those mediators are Nrf2, Grp78, and p38 MAPK, each of which is also a key mediator of ischemia preconditioning. Specific Aim 3 will therefore determine whether (i) aTRA- mediated induction of the anti-oxidant stress response is dependent upon Nrf2; (ii) aTRA-mediated cytoprotection requires the recruitment of the ER (Grp78) mediated stress response pathway; (iii) the recruitment of Nrf2 by aTRA is dependent on p38 MAPK-Grp78 interactions; and (iv) the mechanism(s) of cytoprotection identified in the in vitro model are recapitulated in the in vivo model by testing aTRA induced renoprotection in Nrf2-/- mice. The significance of the current studies resides in their potential to enhance our understanding of retinoid mediated cytoprotection at the molecular and cellular level, which can subsequently provide insights into novel therapeutic strategies effective for clinical interventions during chemical induced tissue injury or hypoxia/ischemia-reperfusion injury. PUBLIC HEALTH RELEVANCE: Oxidative stress has been shown to cause or contribute to many diseases including different forms of renal disease such as acute renal failure, rhabdomyolysis, obstructive nephropathy, hyperlipidemia, glomerular damage, chronic renal failure, and ischemia/reperfusion. An understanding of the factors that regulate the cellular response to oxidative stress and of the molecular mechanisms, by which they interact with cellular constituents, and the consequences of such interactions, remain important fundamental goals of biomedical research. All-trans-retinoic acid (aTRA) may offer an alternative therapeutic strategy offering renoprotection through mechanisms similar to ischemia preconditioning, and therefore studies on the mechanism by which aTRA confers cytoprotection could have important therapeutic implications. Assessment of the beneficial effects of retinoids in animal model and human renal disease warrants further investigation.