Premortem enzymatic DNA damage in kidney injury

摘要 人类急性肾损伤（AKI）的发生与不良的长期免疫反应有关。 预后包括较高的发病率和增加的死亡率。我们最近的研究表明， 凋亡DNA酶/核酸内切酶的失活提供了针对AKI诱导的保护， 缺血性或毒性损伤。这些观察结果得出结论，核酸内切酶是 参与死亡前DNA片段化，导致细胞死亡。 该建议是以前完成的研究的延续，该研究的重点是 DNA酶I在缺血性急性肾功能衰竭中的作用这项研究导致了两个重要的 结论.其中之一是DNA酶I的失活具有更广泛的意义 它比最初认为的要好，并且可以应用于顺铂诱导的毒性肾损伤。 另一个观察结果是DNA酶I可能与其他核酸内切酶协同作用。我们 初步的研究表明，另一种DNA酶，内切核酸酶G（EndoG），是诱导在 肾小管上皮细胞的顺铂在体外和体内。这些研究表明，DNase I是 在几种体外和体内模型中，EndoG诱导是必需的。EndoG的调节 通过DNA酶I的表达是以前未知的途径。电流的假设 建议是（a）在顺铂肾损伤过程中，死亡前酶促DNA损伤是 由EndoG诱导，EndoG沿着由DNA酶I起始的途径起作用，和（B）失活 结论：EndoG对顺铂诱导的肾脏损伤具有保护作用。这种假设是 初步数据支持，这些数据表明， 小鼠或原代肾小管上皮细胞，或肾小管上皮细胞中EndoG的沉默 提供了对顺铂诱导的DNA损伤和细胞死亡的保护。 在具体目标1中，我们计划确定DNase I在EndoG调节中的作用。 表达、DNA损伤和凋亡。具体目标2将侧重于审查 EndoG激活或失活对肾小管上皮细胞损伤的影响。在具体目标3中，我们 将通过细胞凋亡，DNA， 损伤、膜通透性等。DNase I和EndoG null 小鼠，RNA干扰，DNase I和EndoG过表达，显性失活突变 和抑制剂将用于在体内或体外中断特定途径， 以一种机械的、因果关系的方式实现目标。我们的终点将包括： 通过实时RT-PCR、Western印迹和活性定量的核酸内切酶的表达， EndoG的免疫定位，通过TUNEL或Comet评估DNA片段化 测定和细胞活力分析。 EndoG在这些机制中的作用和调节的确定很可能 死前DNA损伤和肾细胞死亡途径的研究将为 预防和治疗人类AKI。

Abstract The occurrence of acute kidney injury (AKI) in humans is associated with a poor long-term prognosis including higher morbidity and increased mortality. Our recent studies showed that the inactivation of apoptotic DNases/endonucleases provided protection against AKI induced by ischemic or toxic insults. These observations led to the conclusion that endonucleases are involved in premortem DNA fragmentation, which precedes and causes cell death. This proposal is a continuation of the previous accomplished study, which was focused on the role of DNase I in ischemic acute renal failure. This study resulted in two important conclusions. One of them is that the inactivation of DNase I has much broader implications than it was initially thought, and it can be applied to the toxic kidney injury induced by cisplatin. Another observation was that DNase I may act in concert with other endonucleases. Our preliminary studies showed that another DNase, endonuclease G (EndoG), is induced in the tubular epithelium by cisplatin in vitro and in vivo. These studies showed that DNase I is necessary for EndoG induction in several in vitro and in vivo models. The regulation of EndoG expression by DNase I is a previously unknown pathway. The hypothesis of the current proposal is that (a) during cisplatin kidney injury, premortem enzymatic DNA damage is induced by EndoG which acts along the pathway initiated by DNase I, and (b) the inactivation of EndoG may protect the kidney against injury induced by cisplatin. This hypothesis is supported by the preliminary data, which showed that the genetic inactivation of EndoG in mice or primary tubular epithelial cells, or the silencing of EndoG in tubular epithelial cells provided protection against DNA damage and cell death induced by cisplatin. In Specific Aim 1, we plan to determine the role of DNase I in the regulation of EndoG expression, DNA damage and apoptosis. Specific Aim 2 will be focused on examining the effects of EndoG activation or inactivation on tubular epithelial cell injury. In Specific Aim 3, we are going to define the intermediate mechanisms of EndoG regulation by apoptosis, DNA damage, membrane permeability and others during cisplatin injury. DNase I and EndoG null mice, RNA interference, overexpression of DNase I and EndoG, dominant-negative mutant and inhibitors will be used to interrupt specific pathways in vivo or in vitro and thus address the goals in a mechanistic and cause-effect relationship manner. Our endpoints will include: expression of endonucleases quantified by real-time RT-PCR, Western blotting and activity, immunolocalization of EndoG, the assessment of DNA fragmentation by TUNEL or Comet assays, and the analysis of cell viability. It is very likely that the identification of the role and regulation of EndoG in the mechanisms of premortem DNA damage and kidney cell death pathways will provide new modalities for the prevention and treatment of AKI in humans.