Premortem enzymatic DNA damage in kidney injury

描述（由申请方提供）：人类急性肾损伤（阿基）的发生与不良的长期预后相关，包括较高的发病率和死亡率。我们最近的研究表明，凋亡DNA酶/核酸内切酶的失活提供了对缺血性或毒性损伤诱导的阿基的保护。这些观察结果导致的结论是，核酸内切酶参与死前DNA片段化，这之前，并导致细胞死亡。本研究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，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。我们的初步研究表明，另一种DNA酶，内切核酸酶G（EndoG），诱导在肾小管上皮细胞顺铂在体外和体内。这些研究表明，在几种体外和体内模型中，DNA酶I是EndoG诱导所必需的。通过DNA酶I调节EndoG表达是以前未知的途径。当前建议的假设是：（a）在顺铂肾损伤期间，死亡前酶促DNA损伤由EndoG诱导，EndoG沿着由DNA酶I启动的途径起作用，以及（B）EndoG的失活可以保护肾脏免受顺铂诱导的损伤。这一假设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的沉默提供了针对顺铂诱导的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表达、DNA损伤和凋亡调节中的作用。具体目标2将集中于检查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，我们将通过顺铂损伤期间的细胞凋亡、DNA损伤、膜通透性等来确定EndoG调节的中间机制。DNase I和EndoG缺失小鼠、RNA干扰、DNase I和EndoG的过表达、显性负突变体和抑制剂将用于在体内或体外中断特定途径，从而以机制和因果关系的方式解决目标。我们的终点将包括：通过实时RT-PCR定量的核酸内切酶的表达、蛋白质印迹和活性、EndoG的免疫定位、通过TUNEL或彗星测定评估DNA片段化以及分析细胞活力。EndoG在死前DNA损伤和肾细胞死亡途径机制中的作用和调节的鉴定很可能为预防和治疗人类阿基提供新的模式。该提案的重点是在中毒性肾损伤过程中诱导的DNA损伤机制，以及负责这种DNA损伤的两种酶的作用：DNA酶I和核酸内切酶G。我们认为，这两种酶是连接在一个途径，其中DNA酶I激活核酸内切酶G，这些核酸内切酶的任何失活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.