Novel endonuclease-targeted approaches to nephroprotection

摘要 肾脏是一个排泄器官，会受到体内代谢的许多有毒化学物质的影响 身体，包括横纹肌溶解（骨骼肌降解）的产物， 已知通过产生活性氧诱导急性肾损伤（阿基） 物种（ROS）。导致的阿基是一种危及生命的疾病，需要血液透析或 肾移植 这项提案是由VA Merit Review资助的先前研究的延续 格兰特，这是集中在肾细胞凋亡核酸内切酶诱导或激活的 横纹肌溶解症期间的ROS。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。本 该提议是基于我们最近的观察，即横纹肌溶解和其他组织/细胞 由ROS介导的损伤是几倍大，主要是因为 凋亡酶，内切核酸酶G（EndoG），其由ROS诱导。无毒 EndoG或其他凋亡核酸内切酶的抑制剂是未知的， 在此过程中，几乎没有针对活化的EndoG或其他核酸内切酶的保护作用。 段时间这种知识上的差距可以部分地解释为， 在我们的研究中，核酸内切酶的激活并不被认为是ROS损伤所必需的 被认为是继发性死后事件。我们发现基因敲除小鼠 缺乏EndoG的人受到保护，免受横纹肌溶解诱导的阿基，这表明， EndoG在死前就行动了然而，从翻译/治疗的角度来看， 击倒是不可能的 因此，我们的建议集中在开发一套新的方法来抑制 EndoG用于横纹肌溶解症期间的肾保护。这些办法包括： 靶向广谱核酸内切酶和ROS，（B）靶向EndoG 表达，和（c）靶向EndoG的表观遗传调控及其运输。所有这些 战略有可能转化为退伍军人和一般公众的应用。 我们假设横纹肌溶解症引起的急性肾损伤可以通过以下方法预防： 抑制EndoG表达、运输和活性之前或在某些情况下之后， 损伤我们计划在体内（甘油诱导的横纹肌溶解症）和体内使用补充药物。 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。体外 方法也将用于机制研究，或控制体内方法，如果 抑制剂的毒性或稳定性是未知的。我们的具体目标如下。 具体目标1。确定广谱ROS/核酸内切酶的效率- 锌-氨基硫醇螯合物的靶向肾脏保护作用。(1A)测试抗氧化剂Zn- 氨基硫醇在体外螯合。(1B)测试抗氧化剂锌-氨基硫醇螯合物在体内。 具体目标2。通过特异性shRNA递送测试EndoG靶向保护， AAV或裸DNA。(2A)shRNA在体外保护肾脏免受损伤。(2B)肾 在体内通过shRNA保护免受损伤。 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。成功完成这些研究 可能会导致新的治疗工具的开发，以防止阿基。一些 它们（锌螯合物）具有很强的转化价值，因为它们即使在 在肾损伤后给予，而其他药物可以成为治疗选择。 未来当应用于人类时，这项研究的结果可能会拯救人类的生命， 改善退伍军人的健康，减少残疾人数， 老百姓。

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.