Retinoid Mediated Protection Against Reactive Oxygen Species Induced Cytotoxicity

描述（由申请人提供）：我们已经表明，类维生素A信号传导参与了11-脱氧-16，16-二甲基PGE 2（DDM-PGE 2）介导的细胞保护作用，以对抗活性氧（ROS）诱导的坏死/坏死细胞死亡。蛋白质组学分析表明，细胞保护与增加合成的选择数量的蛋白质，包括视黄醇结合蛋白（RBP），肌动蛋白，和葡萄糖调节蛋白78（Grp 78）。我们随后证实，全反式维甲酸（aTRA）复制DDM-PGE 2介导的细胞保护作用在体外，更重要的是，一个单一的剂量的aTRA（1毫克/公斤，6小时预处理）完全保护小鼠免受肾缺血/再灌注（I/R）损伤。此外，在该治疗剂量下，aTRA诱导Nrf 2响应性抗氧化剂HO-1和NQO 1基因，以及核视黄酸受体RAR？，RAR？2、RAR？2、维甲酸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诱导的细胞保护作用是由类似于缺血预处理的机制介导的。在具体目标1中，我们提出确定aTRA在缺氧/复氧损伤的体外模型（人肾上皮HK-2细胞）中提供细胞保护的能力，并优化在体内缺血/再灌注模型（IR）中aTRA介导的细胞保护的方案。我们还将确定保护作用是否是介导的，至少部分是通过上调抗氧化酶。类维生素A的生物学效应通常通过与其同源核受体（即视黄酸受体（RAR）和类维生素A X受体（RXR））的相互作用介导。RAR和/或RXR参与aTRA介导的细胞保护的程度尚不清楚，具体目标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，特定目的1和2旨在建立类维生素A信号传导的募集，作为ROS在疾病病理学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的条件（特定目的1），并初步表征这种作用的药理学基础（特定目的2）。第三个也是最后一个特定目标旨在确定aTRA实现细胞保护的分子机制，每个子目标都侧重于在初步研究中已被确定为在细胞保护反应中发挥重要作用的靶标。具体而言，这些介质是Nrf 2、Grp 78和p38 MAPK，它们中的每一个也是缺血预处理的关键介质。因此，具体目标3将确定（i）aTRA介导的抗氧化应激反应的诱导是否依赖于Nrf 2;（ii）aTRA介导的细胞保护是否需要ER（Grp 78）介导的应激反应途径的募集;（iii）aTRA对Nrf 2的募集是否依赖于p38 MAPK-Grp 78相互作用;和（iv）通过在Nrf 2-/-小鼠中测试aTRA诱导的肾保护作用，在体内模型中重现了在体外模型中鉴定的细胞保护作用机制。目前的研究的意义在于他们的潜力，以提高我们的理解维甲酸介导的细胞保护在分子和细胞水平上，这可以随后提供洞察到新的治疗策略，有效的化学诱导的组织损伤或缺氧/缺血-再灌注损伤的临床干预。 公共卫生相关性：氧化应激已显示引起或促成许多疾病，包括不同形式的肾病，如急性肾衰竭、横纹肌溶解、阻塞性肾病、高脂血症、肾小球损伤、慢性肾衰竭和缺血/再灌注。了解调节细胞对氧化应激反应的因素以及它们与细胞成分相互作用的分子机制，以及这种相互作用的后果，仍然是生物医学研究的重要基本目标。全反式维甲酸（aTRA）可能提供一种替代的治疗策略，通过类似于缺血预处理的机制提供肾保护，因此研究aTRA赋予细胞保护的机制可能具有重要的治疗意义。类维生素A在动物模型和人类肾脏疾病中的有益作用评估需要进一步研究。

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.