Premortem enzymatic DNA damage in kidney injury

描述(由申请人提供)：人类急性肾损伤(AKI)的发生与较差的长期预后有关，包括较高的发病率和较高的死亡率。我们最近的研究表明，凋亡的DNA酶/核酸内切酶的失活对缺血或毒性损伤诱导的AKI具有保护作用。这些观察得出的结论是，内切酶参与了死前DNA的碎裂，这是细胞死亡之前和导致死亡的原因。这项建议是先前已完成的研究的继续，该研究的重点是DNase 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结论。其中之一是DNase I的失活具有比最初认为的更广泛的意义，它可以应用于顺铂引起的中毒性肾损伤。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，顺铂在体外和体内都能在肾小管上皮细胞中诱导另一种DNA酶，即核酸内切酶G(EnDog)。这些研究表明，在几种体外和体内模型中，DNase I是诱导endog所必需的。DNase I对endog表达的调控是一个未知的途径。目前的假设是：(A)在顺铂肾损伤过程中，Endog沿着DNase I启动的途径诱导死前的DNA酶损伤，并且(B)Endog的失活可能对顺铂引起的肾脏损伤具有保护作用。这一假说得到了初步数据的支持，这些数据表明，小鼠或原代肾小管上皮细胞中endog的遗传失活，或肾小管上皮细胞中endog的沉默，对顺铂诱导的DNA损伤和细胞死亡具有保护作用。在特定的目标1中，我们计划确定DNase I在调控endog表达、DNA损伤和细胞凋亡中的作用。具体目标2将集中于内切酶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，我们将确定顺铂损伤过程中通过细胞凋亡、DNA损伤、膜通透性等途径调节endog的中间机制。DNase I和endog缺失小鼠、RNA干扰、DNase I和endog的过表达、显性-阴性突变体和抑制剂将被用来在体内或体外阻断特定的通路，从而以机制和因果关系的方式解决这些目标。我们的终点将包括：实时RT-PCR定量内切酶的表达，Western blotting和活性，endog的免疫定位，TUNEL或彗星试验评估DNA片段化，以及细胞活力分析。Endog在死前DNA损伤和肾细胞死亡途径中的作用和调控机制的确定，很可能为人类AKI的预防和治疗提供新的途径。与公众健康相关本提案集中于毒性肾损伤引起的DNA损伤的机制，以及导致这种DNA损伤的两种酶：DNase I和核酸内切酶G的作用。我们认为这两种酶是相连的，其中DNase I激活核酸内切酶G，其中任何一种核酸内切酶的失活都可以保护肾脏免受毒性损伤。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE The proposal is focused on the DNA damage mechanisms induced during toxic kidney injury, and the role of two enzymes which are responsible for this DNA damage: DNase I and endonuclease G. We propose that these two enzymes are linked in a pathway, in which DNase I activates endonuclease G, and that inactivation of any of these endonuclease can protect kidney from toxic injury.