Premortem enzymatic DNA damage in kidney injury

肾损伤中的死前酶促 DNA 损伤

• 批准号: 8213651
• 负责人: Alexei G Basnakian
• 金额: $ 28.42万
• 依托单位: UNIV OF ARKANSAS FOR MED SCIS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8213651
• 关键词: Acute; Acute Kidney Failure; Acute Renal Failure with Renal Papillary Necrosis; Address; Affect; Agonist; Animals; Apoptosis; Apoptosis DNA Damage Pathway; Apoptotic; Autopsy; Biological Assay; Breast Cancer Cell; Camptothecin; Cell Death; Cell Membrane Permeability; Cell Nucleus; Cell Survival; Cells; Cisplatin; Code; Collaborations; Comet Assay; DNA; DNA Damage; DNA Double Strand Break; DNA Fragmentation; DNA Single Strand Break; DNA biosynthesis; DNA-dependent protein kinase; Data; Deoxyribonuclease I; Deoxyribonucleases; Dominant-Negative Mutation; Enzymes; Epigenetic Process; Epithelial; Epithelial Cells; Epithelium; Etoposide; G Cells; Gene Silencing; Genes; Genetic; Goals; Health; Human; Hypoxia; Immunohistochemistry; In Situ Nick-End Labeling; In Vitro; Induction of Apoptosis; Injury; Injury to Kidney; Ischemia; Kidney; Knock-out; Knockout Mice; Label; Laboratories; Link; Location; Malignant neoplasm of prostate; Measures; Mediating; Messenger RNA; Methylation; Mitochondria; Modality; Modeling; Morbidity - disease rate; Mus; Nuclear; Nuclear Import; Nuclear Pore; Nuclear Translocation; Participant; Pathway interactions; Patients; Peptide Signal Sequences; Peroxisome Proliferator-Activated Receptors; Play; Prevention; Process; RNA Interference; RNA chemical synthesis; Regulation; Renal function; Reperfusion Injury; Reperfusion Therapy; Reporting; Rest; Reverse Transcriptase Polymerase Chain Reaction; Role; Site; Small Interfering RNA; TdT-Mediated dUTP Nick End Labeling Assay; Testing; Time; Toxic effect; Transfection; Tubular formation; Western Blotting; apoptosis inducing factor; cancer cell; caspase-2; caspase-9; cell injury; cytotoxic; endonuclease; endonuclease G; extracellular; immunocytochemistry; in vitro Model; in vivo; in vivo Model; inhibitor/antagonist; kidney cell; mortality; outcome forecast; overexpression; passive transport; programs; promoter; tubular necrosis; uptake

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.

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相关，包括较高的发病率和死亡率增加。我们最近的研究表明，凋亡dna酶/内切酶的失活对缺血性或毒性损伤引起的AKI提供了保护。这些观察得出的结论是，内切酶参与了死前DNA的断裂，这先于并导致细胞死亡。该建议是先前完成的研究的延续，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失活的意义比最初认为的要广泛得多，它可以应用于顺铂诱导的中毒性肾损伤。另一个观察结果是DNase I可能与其他内切酶协同作用。我们的初步研究表明，顺铂在体外和体内诱导小管上皮中存在另一种dna酶，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的必要条件。DNase I对EndoG表达的调控是一个以前未知的途径。当前提案的假设是：(a)在顺铂肾损伤期间，死前酶促DNA损伤是由EndoG诱导的，EndoG沿着DNase I启动的途径起作用，(b) EndoG的失活可能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，EndoG在小鼠或原代小管上皮细胞中的基因失活，或EndoG在小管上皮细胞中的沉默，对顺铂诱导的DNA损伤和细胞死亡具有保护作用。在Specific Aim 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。特异性目标2将重点研究EndoG激活或失活对小管上皮细胞损伤的影响。在Specific Aim 3中，我们将明确顺铂损伤过程中细胞凋亡、DNA损伤、膜通透性等对EndoG调控的中间机制。DNase I和EndoG缺失小鼠、RNA干扰、DNase I和EndoG过表达、显性阴性突变体和抑制剂将被用来中断体内或体外的特定途径，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：实时RT-PCR定量的内切酶表达，Western blotting和活性，EndoG的免疫定位，TUNEL或Comet检测的DNA片段评估，以及细胞活力分析。发现EndoG在死前DNA损伤和肾细胞死亡途径中的作用和调控，很可能为人类AKI的预防和治疗提供新的模式。该建议侧重于中毒性肾损伤期间诱导的DNA损伤机制，以及负责这种DNA损伤的两种酶的作用：DNase I和内切酶G。我们提出这两种酶在一条途径中连接，其中DNase I激活内切酶G，并且任何这些内切酶的失活都可以保护肾脏免受毒性损伤。