Acute Renal Failure: An Endotoxin Hyper-Responsive State

急性肾衰竭：内毒素高反应状态

• 批准号: 7982459
• 负责人: KAROL BOMSZTYK
• 金额: $ 44.4万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7982459
• 关键词: Acute; Acute Kidney Failure; Acute Renal Failure with Renal Papillary Necrosis; Address; Bacterial Toxins; Bilateral; Binding; Binding Sites; Biological Assay; Biological Markers; Biology; Blood Circulation; CCL2 gene; Cause of Death; Cell model; Cells; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Cisplatin; Complement; Contralateral; DNA Modification Process; Data; Development; Docking; Endotoxemia; Endotoxins; Enzymes; Epigenetic Process; Event; Exposure to; Failure; Functional disorder; Future; Gene Activation; Gene Targeting; Genes; Genetic Transcription; Genomics; Goals; Hand; Histone Acetylation; Histones; Human; Hypersensitivity; In Vitro; Infection; Inflammation Mediators; Inflammatory; Inflammatory Response; Injury; Ischemia; Ischemic Preconditioning; Kidney; Knowledge; Laboratories; Lead; Left; Ligands; Ligation; Lipopolysaccharides; Maintenance; Mediating; Mediator of activation protein; Memory; Messenger RNA; Methylation; Modeling; Modification; Molecular; Nature; Nucleosomes; Organ; Organ failure; Pathway interactions; Patients; Phase; Polymerase; Polymerase Gene; Positioning Attribute; Preventive; Process; Production; RNA Polymerase II; Reagent; Recruitment Activity; Renal Tissue; Reperfusion Therapy; Right kidney; SMARCA4 gene; Sepsis; Sequence Analysis; Series; Severities; Small Interfering RNA; Specific qualifier value; Staging; Testing; Transcription Coactivator; Transcription Factor AP-1; Trauma; Tubular formation; Ureteral obstruction; Variant; Venous; base; bisulfite; cell injury; chemokine; chromatin remodeling; clinically relevant; cytokine; demethylation; histone modification; in vivo; kidney cortex; knock-down; mRNA Stability; memory process; mortality; novel; promoter; public health relevance; receptor; research study; response; response to injury; septic; spatiotemporal; transcription factor; translational approach

DESCRIPTION (provided by applicant): In the aftermath of acute renal injury (AKI), proximal tubular cells manifest dramatic hyper-responsiveness to Toll receptor ligands, most notably, endotoxin. This exaggerates renal cytokine (e.g., TNF-1) / chemokine (e.g., MCP-1) production which can worsen the severity of acute renal failure (ARF). With renal venous cytokine efflux, extra-renal injury may also result. This LPS hyper-responsiveness is transcriptionally regulated, and mediated, in part, by gene activating chromatin events. The overall goal of this proposal is to: i) further define the pathways which induce the underlying chromatin changes; and ii) delineate how they mediate an exaggerated LPS responsive state. To focus the application, one ARF model (ischemia/reperfusion) 1 exposure to one Toll ligand (LPS) will be tested in three Specific Aims: Aim#1: Define the temporal sequence of chromatin changes along the TNF-1 gene in AKI. We will characterize chromatin changes, including CpG methylation/demethylation, histone acetylation, methylation, histone variants, and alterations in nucleosome positions. Both renal injury, and proximal tubule-specific injury (in cultured tubular HK-2 cells), will be studied following reversible ischemia + LPS. These in vivo and in vitro experiments will set the stage for mechanistic assessments (Aim 3). Aim #2: Ascertain which transcription and chromatin remodeling factors / enzymes are recruited to the TNF-1 gene in AKI. We have already shown that the chromatin remodeler BRG1 is recruited to the TNF-1 gene and is required for injury-induced TNF-1 transcription. We have also demonstrated that NF-:B and AP-1 transcription factors are recruited to the TNF-1 gene in AKI. Using Matrix ChIP, we will more fully define the spatiotemporal sequence of chromatin and transcription events at the TNF-1 gene in response to reversible ischemia + LPS. This will help to ascertain which factors are hyper-recruited to the TNF-1 gene and may thus drive the LPS hyper-responsive state. Aim #3 Test the mechanistic relevance of the above defined changes in the induction of the LPS hyper-responsive state. The mechanistic relevance of the above defined changes in establishing LPS hyper-responsiveness will be tested both in vitro and in vivo using siRNA targeting (as we have already done to knock-down BRG1 in HK-2 cells). We will complement these in vivo and in vitro siRNA approaches with the use of pharmacologic reagents directed at the putative molecular mediators of the hyper-responsive chromatin state. The latter approach may lead to therapeutically relevant translational approaches for modulating experimental AKI and associated multiorgan failure. The proposed studies are highly novel in that, with the exception of data obtained by the PIs, virtually no information exists as to the nature, and consequences of, chromatin alterations in response to AKI. Hence, new perspectives on mechanisms of AKI and its downstream consequences should result. PUBLIC HEALTH RELEVANCE: Acute kidney injury (AKI) leads to renal production of inflammatory mediators and sensitizes/primes the kidney to bacterial toxins. These renal inflammatory mediators gain access to the systemic circulation and can induce extra-renal tissue damage, a common cause of mortality in patients with AKI, due to infections, trauma and other illness. We propose to define the chromatin/transcriptional mechanisms for these renal responses with the goal to develop preventive treatments in the future.

描述（由申请人提供）：急性肾损伤（AKI）后，近端肾小管细胞对Toll受体配体表现出明显的高反应性，尤其是内毒素。这会增加肾细胞因子（如TNF-1） /趋化因子（如MCP-1）的产生，从而加重急性肾功能衰竭（ARF）的严重程度。肾静脉细胞因子外排也可能导致肾外损伤。这种LPS的高反应性是转录调控的，部分是由基因激活染色质事件介导的。本提案的总体目标是：i)进一步定义诱导潜在染色质变化的途径；ii)描述它们如何介导夸张的LPS反应状态。为了集中应用，一个暴露于一种Toll配体（LPS）的ARF模型（缺血/再灌注）1将在三个特定目的中进行测试：目的#1：确定AKI中沿TNF-1基因染色质变化的时间序列。我们将描述染色质的变化，包括CpG甲基化/去甲基化、组蛋白乙酰化、甲基化、组蛋白变异和核小体位置的改变。在可逆缺血+ LPS后，将研究肾损伤和近端小管特异性损伤（在培养的小管HK-2细胞中）。这些体内和体外实验将为机制评估奠定基础（目标3）。目的2：确定AKI中哪些转录和染色质重塑因子/酶被募集到TNF-1基因上。我们已经证明，染色质重塑器BRG1被招募到TNF-1基因上，并且是损伤诱导的TNF-1转录所必需的。我们也证明了NF-:B和AP-1转录因子在AKI中被募集到TNF-1基因上。利用Matrix ChIP，我们将更全面地定义在可逆性缺血+ LPS下，TNF-1基因染色质和转录事件的时空序列。这将有助于确定哪些因素被过度招募到TNF-1基因，从而可能驱动LPS的过度反应状态。目的#3测试上述定义的变化在诱导LPS超反应状态中的机制相关性。上述定义的变化在建立LPS超反应性中的机制相关性将在体外和体内使用siRNA靶向进行测试（正如我们已经在HK-2细胞中敲除BRG1一样）。我们将利用针对高反应性染色质状态的假定分子介质的药理学试剂来补充这些体内和体外siRNA方法。后一种方法可能导致治疗相关的转化方法来调节实验性AKI和相关的多器官衰竭。提议的研究非常新颖，因为除了pi获得的数据外，几乎没有关于AKI响应的染色质改变的性质和后果的信息。因此，对AKI的机制及其下游后果应该有新的看法。