Premortem enzymatic DNA damage in kidney injury

肾损伤中的死前酶促 DNA 损伤

• 批准号: 7596422
• 负责人: Alexei G Basnakian
• 金额: $ 29万
• 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 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7596422
• 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; Classification; Code; Collaborations; Comet Assay; DNA; DNA Damage; DNA Double Strand Break; DNA Fragmentation; DNA Single Strand Break; 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; 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; TP53 gene; TdT-Mediated dUTP Nick End Labeling Assay; Testing; Time; Toxic effect; Transfection; Tubular formation; Western Blotting; abstracting; 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）的发生与长期预后不良有关，包括更高的发病率和死亡率增加。我们最近的研究表明，凋亡DN酶/核酸酶的失活提供了针对缺血或有毒损伤引起的AKI的保护。这些观察结果得出的结论是，核酸内切酶参与前体DNA碎片，这是在细胞死亡之前并导致细胞死亡的。该提议是先前的研究的延续，该研究的重点是DNase I在缺血性急性肾衰竭中的作用。这项研究得出了两个重要的结论。其中之一是，DNase I的失活比最初想象的要广泛，并且可以应用于顺铂诱导的有毒肾脏损伤。另一个观察结果是，DNase我可能会与其他核心裂合作。我们的初步研究表明，另一种DNase核酸内切酶G（内og）是由顺铂在体外和体内诱导的。这些研究表明，在几种体外和体内模型中，DNase I对于内og诱导是必不可少的。 DNase I对内og表达的调节是以前未知的途径。当前建议的假设是（a）在顺铂肾损伤期间，前体DNA损伤是由沿DNase I引发的途径的内生诱导的，（b）内OG灭活可能会保护肾脏可保护肾脏免受顺铂诱导的损伤。该假设得到了初步数据的支持，该数据表明，在小鼠或原发性管状上皮细胞中内og的遗传失活，或者在肾小管上皮细胞中内og沉默的遗传灭活，可保护避免异脑肽诱导的DNA损伤和细胞死亡。在特定的目标1中，我们计划确定DNase I在调节内og表达，DNA损伤和凋亡中的作用。具体目标2将集中于检查内生激活或失活对管状上皮细胞损伤的影响。在特定的目标3中，我们将通过凋亡，DNA损伤，膜渗透性等在顺铂损伤期间来定义内组调节的中间机制。 DNase I和内og无效小鼠，RNA干扰，DNase I和内og的过表达，主导性突变体和抑制剂将用于体内或体外中断特定的特定途径，从而以机械和因果关系方式的方式解决目标。我们的终点将包括：通过实时RT-PCR，蛋白质印迹和活性，内生的免疫定位，通过TUNEL或COMET分析评估DNA碎片的内切核酸的表达，以及细胞生存能力的分析。在前体DNA损伤和肾脏细胞死亡途径的机理中，鉴定内OG的作用和调节很可能会为人类预防和治疗AKI提供新的方式。该提案的公共卫生相关性集中在毒性肾脏损伤期间引起的DNA损伤机制上，以及两种造成这种DNA损害的酶的作用：DNase I和核酸内切酶G。我们建议这两种酶在途径中连接在途径中，在这种途径中，DNase I从该核酸内酶造成了这些启发性的启发，并培养了这些型号的能力，并将其与这些无效的效果联系起来。