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预后有关。 预后包括更高的发病率和更高的死亡率。我们最近的研究表明， 凋亡DNA酶/核酸内切酶的失活对AKI诱导的AKI具有保护作用 通过缺血或有毒的侮辱。这些观察得出的结论是，核酸内切酶是 参与死前DNA碎裂，这是细胞死亡之前和导致的。 这项建议是先前已完成的研究的延续，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得出了两个重要的结论 结论。其中之一是DNase I的失活具有更广泛的影响 可以应用于顺铂所致的中毒性肾损伤。 另一个观察结果是，DNase I可能与其他核酸内切酶协同作用。我们的 初步研究表明，另一种DNA酶--核酸内切酶G(Endog)在黄瓜中被诱导。 顺铂在体外和体内对肾小管上皮细胞的作用。这些研究表明，DNA酶I是 在几种体外和体内模型中诱导endog所必需的。Endog的调控 DNase I的表达是一个以前未知的途径。海流的假说 建议：(A)在顺铂肾损伤过程中，死前酶促DNA损伤是 由沿着DNase I启动的途径起作用的endog诱导，以及(B)失活 Endog对顺铂所致的肾脏损伤有保护作用。这一假设是 初步数据支持，这表明endog基因失活在 小鼠或原代肾小管上皮细胞，或肾小管上皮细胞内endog的沉默 对顺铂引起的DNA损伤和细胞死亡提供保护。 在特定的目标1中，我们计划确定DNase I在endog调节中的作用 表达、DNA损伤和细胞凋亡。具体目标2将集中于审查 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，来定义endog调节的中间机制 顺铂损伤时的损伤、膜通透性等。DNase I和Endog Null 小鼠，RNA干扰，DNase I过表达和endog，显性负突变 而抑制剂将被用来阻断体内或体外的特定途径，从而解决 以机械性和因果关系的方式实现目标。我们的终端将包括： 用实时定量RT-PCR、Western blotting和活性测定核酸内切酶的表达 Endog的免疫定位、TUNEL或Comet对DNA断裂的评估 检测和细胞存活率分析。 很可能是对endog在这一机制中的作用和调控的识别 死前DNA损伤和肾脏细胞死亡途径的研究将为 人类AKI的预防和治疗。

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.