Novel endonuclease-targeted approaches to nephroprotection

新型核酸内切酶靶向肾保护方法

• 批准号: 8391580
• 负责人: Alexei G Basnakian
• 金额: --
• 依托单位: CENTRAL ARKANSAS VETERANS HLTHCARE SYS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-10-01 至 2013-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8391580
• 关键词: Acute; Acute Renal Failure with Renal Papillary Necrosis; Affect; Antioxidants; Apoptosis; Apoptotic; Autopsy; Biological Assay; Biology; Cell Death; Cell Nucleus; Cells; Ceramides; Chemicals; Cytoplasm; DNA; DNA Damage; DNA Fragmentation; DNA Methylation; Data; Deoxyribonuclease I; Dependovirus; Development; Disease; Down-Regulation; Ensure; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Epigenetic Process; Epithelial; Epithelial Cells; Epithelium; Event; Funding; Future; General Population; Glycerol; Goals; Grant Review; Health; Healthcare; Heat shock proteins; Heat-Shock Proteins 70; Hemin; Hemodialysis; Hemolysis; Hour; Human; In Situ Nick-End Labeling; In Vitro; Injury; Ionizing radiation; Kidney; Kidney Transplantation; Knockout Mice; Knowledge; Lead; Life; Measurement; Measures; Mediating; Medical; Methods; Military Personnel; Mitochondria; Molecular; Mus; Myoglobin; Necrosis; Organ; Oxidative Stress; Pathologist; Peptides; Pharmaceutical Preparations; Poison; Population; Production; Property; RNA Interference; Reactive Oxygen Species; Regulation; Reporting; Research; Resistance; Rhabdomyolysis; Scientist; Skeletal Muscle; Testing; Therapeutic; Time; Tissues; Toxic Environmental Substances; Toxic effect; Toxin; Trauma; Tubular formation; Veterans; Walkers; Zinc; adeno-associated viral vector; aminothiol; arm; base; cell injury; cell type; cytotoxic; design; disability; endonuclease; endonuclease G; enzyme activity; experience; improved; in vitro Model; in vitro testing; in vivo; in vivo Model; inhibitor/antagonist; innovation; kidney cell; knockout gene; novel; novel strategies; novel therapeutics; pathogen; pre-clinical; preclinical study; prevent; public health relevance; research study; small hairpin RNA; tool; trafficking; vector; weapons

DESCRIPTION (provided by applicant): ABSTRACT The kidney is an excreting organ that is affected by many toxic chemicals metabolized in the body, including products of rhabdomyolysis (skeletal muscle degradation), which are known to induce acute renal injury (AKI) through the production of reactive oxygen species (ROS). Resulting AKI is a life-threatening condition that requires hemodialysis or kidney transplantation. This proposal is a continuation of the previous research funded by VA Merit Review grant, which was focused on renal apoptotic endonucleases induced or activated by ROS during rhabdomyolysis. The aims in the previous project have been accomplished and the new goals described below are the logical extension of those aims. The present proposal is based on our recent observations that rhabdomyolysis and other tissue/cell injuries mediated by ROS are several times greater mainly because of the activation of apoptotic enzyme, endonuclease G (EndoG), which is induced by ROS. Non-toxic inhibitors of EndoG or other apoptotic endonucleases are not known, and there is virtually no protection against activated EndoG or other endonucleases during this period of time. This gap in knowledge can be explained, in part, by the fact that prior to our studies, the endonuclease activation was not recognized as essential for ROS injury and was considered as secondary postmortem event. We showed that knockout mice deficient in EndoG are protected against rhabdomyolysis-induced AKI, suggesting that EndoG acts premortem. However from a translational/therapeutic point of view a gene knockout is not an option. Therefore we focus this proposal on developing a set of new approaches to inhibit EndoG for nephroprotection during rhabdomyolysis. These approaches include: (a) targeting the broad spectrum of endonucleases and ROS, (b) targeting EndoG expression, and (c) targeting epigenetic regulation of EndoG and its trafficking. All these strategies have a likelihood of translational application to veterans and general public. We hypothesize that acute kidney injury due to rhabdomyolysis can be prevented by inhibiting EndoG expression, trafficking and activity before or, in some cases, after injury. We plan to use complimentary in vivo (glycerol-induced rhabdomyolysis) and in vitro (hemin or myoglobin) approaches to induce tubular epithelial cells injury. In vitro approaches will also be used for mechanistic studies, or to control the in vivo approach if the toxicity or stability of the inhibitor is unknown. Our specific aims are as follows. Specific Aim 1. Determining the efficiency of broad spectrum ROS/endonuclease- targeted kidney protection by zinc-aminothiol chelates. (1A) Testing antioxidant Zn- aminothiol chelates in vitro. (1B) Testing antioxidant Zn-aminothiol chelates in vivo. Specific Aim 2. Testing EndoG-targeted protection by specific shRNA delivery by AAV or naked DNA. (2A) Kidney protection from injury by shRNA in vitro. (2B) Kidney protection from injury by shRNA in vivo. Specific Aim 3. Defining mechanistic anti-EndoG approaches to nephroprotection. (3A) Epigenetic downregulation of EndoG. (3B) Interfering with EndoG trafficking. Potential Impact on Veterans Health Care. Successful completion of these studies can potentially lead to the development of new therapeutic tools to prevent AKI. Some of them (zinc-chelates) have strong translational value because they act even if administered after kidney injury, while others can become therapeutic options of the future. When applied to humans, the results of this study may allow saving human lives, improving the health of veterans, and decreasing the number of disabilities in the veteran population.

描述（由申请人提供）： 摘要肾脏是一个排泄器官，受到体内代谢的许多有毒化学物质的影响，包括横纹肌溶解（骨骼肌降解）的产物，已知其通过产生活性氧（ROS）诱导急性肾损伤（阿基）。阿基是一种危及生命的疾病，需要血液透析或肾移植。 该提案是由VA Merit Review资助的先前研究的延续，该研究专注于横纹肌溶解症期间ROS诱导或激活的肾凋亡核酸内切酶。上一个项目的目标已经实现，下文所述的新目标是这些目标的合理延伸。目前的建议是基于我们最近的观察，即由ROS介导的横纹肌溶解和其他组织/细胞损伤是几倍大，主要是因为细胞凋亡酶，内切核酸酶G（EndoG），这是由ROS诱导的激活。EndoG或其他凋亡核酸内切酶的无毒抑制剂是未知的，并且在这段时间内几乎没有针对活化的EndoG或其他核酸内切酶的保护。这种知识上的差距可以部分地解释为，在我们的研究之前，核酸内切酶的激活并不被认为是ROS损伤所必需的，而是被认为是继发性死后事件。我们发现，EndoG缺陷的敲除小鼠可以保护其免受横纹肌溶解诱导的阿基，这表明EndoG在死前起作用。然而，从翻译/治疗的角度来看，基因敲除不是一种选择。 因此，我们将这一建议集中在开发一套新的方法来抑制EndoG在横纹肌溶解症的肾保护。这些方法包括：（a）靶向广谱核酸内切酶和ROS，（B）靶向EndoG表达，和（c）靶向EndoG及其运输的表观遗传调节。所有这些策略都有可能转化为退伍军人和普通公众的应用。 我们假设，横纹肌溶解症引起的急性肾损伤可以通过在损伤前或在某些情况下在损伤后抑制EndoG表达、运输和活性来预防。我们计划使用体内（甘油诱导的横纹肌溶解）和体外（氯化血红素或肌红蛋白）方法诱导肾小管上皮细胞损伤。如果抑制剂的毒性或稳定性未知，体外方法也将用于机制研究，或用于控制体内方法。我们的具体目标如下。 具体目标1.通过锌-氨基硫醇螯合物确定广谱ROS/核酸内切酶靶向的肾保护的效率。(1A)抗氧化剂锌-氨基硫醇螯合物的体外测试。(1B)测试抗氧化剂锌-氨基硫醇螯合物在体内。 具体目标2。通过AAV或裸DNA的特异性shRNA递送测试EndoG靶向保护。(2A)shRNA在体外保护肾脏免受损伤。(2B)肾保护免受损伤的shRNA在体内。 具体目标3。定义肾保护的机制性抗EndoG方法。(3A)EndoG的表观遗传下调。(3B)干扰EndoG的交易 对退伍军人医疗保健的潜在影响。这些研究的成功完成可能会导致开发新的治疗工具来预防阿基。其中一些（锌螯合物）具有很强的转化价值，因为即使在肾损伤后给药，它们也会起作用，而另一些则可以成为未来的治疗选择。当应用于人类时，这项研究的结果可能会挽救人类生命，改善退伍军人的健康状况，并减少退伍军人中的残疾人数。