Acute Renal Failure: An Endotoxin Hyper-Responsive State

描述(申请人提供)：在急性肾损伤(AKI)后，近端肾小管上皮细胞对Toll受体配体表现出显著的高反应性，最显著的是内毒素。这会夸大肾脏细胞因子(例如，肿瘤坏死因子-1)/趋化因子(例如，单核细胞趋化因子-1)的产生，从而加重急性肾功能衰竭(ARF)的严重程度。随着肾静脉细胞因子外流，也可能导致肾外损伤。这种内毒素的高反应性是由转录调节的，部分是通过基因激活染色质事件来调节的。这项建议的总体目标是：i)进一步定义导致潜在染色质变化的途径；以及ii)描述它们如何调节被夸大的内毒素反应状态。为了集中应用，将在三个特定目标下测试一种ARF模型(缺血/再灌注)1暴露于一种Toll配体(LPS)：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、组蛋白变体和核小体位置的变化。肾损伤和近端小管特异性损伤(在培养的肾小管HK-2细胞中)将在可逆性缺血脂多糖后进行研究。这些体内和体外实验将为机械学评估(目标3)奠定基础。目的#2：确定AKI中哪些转录和染色质重塑因子/酶被招募到肿瘤坏死因子-1基因。我们已经证明染色质重构体BRG1被招募到肿瘤坏死因子-1基因中，并且是损伤诱导的肿瘤坏死因子-1转录所必需的。我们还证明了在AKI中，核转录因子-B和AP-1转录因子被招募到肿瘤坏死因子-1基因中。利用Matrix芯片，我们将更全面地定义可逆性缺血脂多糖对肿瘤坏死因子-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子被过度招募到肿瘤坏死因子-1基因，从而可能驱动内毒素的过度反应状态。目的#3测试上述定义的变化在诱导内毒素高反应状态中的机制相关性。我们将在体外和体内使用siRNA靶向(就像我们已经在HK-2细胞中敲除BRG1所做的那样)来测试上述定义的变化在建立内毒素高反应性方面的机制相关性。我们将通过使用药理试剂来补充这些体内和体外的siRNA方法，这些药物针对的是可能的高反应性染色质状态的分子介质。后一种方法可能导致治疗相关的翻译方法，用于调节实验性AKI和相关的多器官衰竭。拟议的研究具有很高的新颖性，因为除了PI获得的数据外，几乎不存在关于AKI反应中染色质变化的性质和后果的信息。因此，应该对AKI的机制及其下游后果产生新的观点。 公共卫生相关性： 急性肾损伤(AKI)导致肾脏产生炎症介质，使肾脏对细菌毒素敏感。这些肾炎性介质可进入体循环，并可导致肾外组织损伤，这是由于感染、创伤和其他疾病而导致AKI患者死亡的常见原因。我们建议确定这些肾脏反应的染色质/转录机制，以期在未来开发预防性治疗。

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.