Role of NLRP3 signals in ischemia/reperfusion-induced organ injury

项目总结：这项建议评估了细胞质固有的细胞内信号通路是如何产生的 免疫受体NLRP3在肾小管上皮细胞中有调节作用。肾小管上皮细胞在 缺血性肾损伤，我们的实验室和其他人已经证明，全局阻断NLRP3可以防止实验性肾脏 缺血/再灌注损伤。NLRP3信号典型地导致细胞死亡(下垂)，然而替代的是NLRP3 最近发现的信号通路可以导致IL1b/IL18的分泌，而不会导致细胞死亡。因此，有选择地 靶向NLRP3介导的细胞死亡信号可以防止肾小管损伤，同时仍然保持宿主的能力 分泌IL1b/IL18并安装所需的宿主防御反应(例如，抗菌反应)。该项目具有很高的 对于住院期间经常发生的缺血性肾损伤的靶向治疗的开发具有重要意义 患者和所有获得用于移植的器官。 广泛/长期目标：拟议研究的长期目标是确定NLRP3如何有助于 肾脏的损伤组织反应及其信号如何有效和选择性地被靶向。 具体目标：这项提议的具体目标是检验细胞质模式识别 受体NLRP3可特异性靶向于肾小管上皮细胞，选择性阻断NLRP3介导的肾脏 小管细胞死亡，同时保留IL1b/IL18反应。目标1定义了驱动NLRP3信令的机制 近端肾小管细胞的通路。AIM 2确定NLRP3信号如何在 近端肾小管细胞。AIM 3决定NLRP3信号及其选择性阻断对肾脏的影响 当NLRP3表达分离到肾小管上皮细胞时，肾小管损伤/IL1b/IL18在体内分泌。 研究设计和实现上述目标的方法：目标1比较和对比NLRP3的激活 在人和小鼠近端肾小管上皮细胞中的表达，并将确定NLRP3激活如何促进下垂 和/或生产IL1b/IL18。AIM 2将确定如何针对特定的NLRP3激活通路 这两个物种可以防止细胞死亡和保存细胞因子的分泌。目标3将重点了解NLRP3如何 限制在体内的肾小管上皮细胞可靶向预防肾小管损伤，而不损害IL1b/IL18。 在第三个目标中，将使用两种不同的NLRP3表达模型；一种是肾小管表达条件性 NLRP3的功能丧失以及存在超激活形式的NLRP3的另一种。这里的研究将确定 规范的NLRP3介导的细胞死亡信号是否可以从非规范信号中分离出来以防止细胞 在保留IL-1b/IL-18分泌的同时死亡。 项目的健康相关性：如果达到本提案的目标，我们将了解NLRP3(被视为 可调节为针对一种常见疾病，即肾脏缺血/再灌流损伤。这 知识对于开发合理的靶向治疗预防或改善肾脏疾病至关重要。 预期缺氧的临床情况下的缺血/再灌流损伤。

Project Summary: This proposal evaluates how intracellular signaling pathways generated by the cytoplasmic innate immune receptor NLRP3 are regulated in renal tubular epithelial cells. Renal tubule epithelial cells play a central role in ischemic kidney injury, and our lab and others have shown that global NLRP3 blockade can prevent experimental renal ischemia/reperfusion injury. NLRP3 signaling classically results in cell death (pyroptosis), however alternative NLRP3 signaling pathways have recently been identified that lead to secretion of IL1b/IL18 without cell death. Thus, selectively targeting NLRP3-mediated cell death signaling might prevent renal tubule injury while still preserving the host’s ability to secrete IL1b/IL18 and mount needed host defense responses (e.g., antimicrobial responses). The project is highly significant for the development of targeted therapeutics for ischemic kidney injury, which occurs frequently in hospitalized patients and in all organs procured for transplantation. Broad/long-term objectives: The long-term goals of the proposed research are to define how NLRP3 contributes to injurious tissue responses in the kidney and how its signaling can be effectively and selectively targeted. Specific Aims: The specific objective of this proposal is to test the hypothesis that the cytoplasmic pattern recognition receptor NLRP3 can be specifically targeted in renal tubular epithelial cells to selectively block NLRP3-mediated renal tubule cell death, while preserving IL1b/IL18 responses. Aim 1 defines the mechanisms that drive NLRP3 signaling pathways in proximal renal tubule cells. Aim 2 determines how NLRP3 signaling can be selectively targeted in the proximal renal tubule cells. Aim 3 determines how NLRP3 signaling, and its selective blockade, influences renal tubular injury/IL1b/IL18 secretion in vivo, when NLRP3 expression is isolated to the renal tubule epithelium. Research Design and Methods for Achieving the Stated Goals: Aim 1 compares and contrasts activation of NLRP3 in human and murine proximal renal tubular cells and will determine how NLRP3 activation contributes to pyroptosis and/or production of IL1b/IL18. Aim 2 will determine how specific NLRP3 activation pathways can be targeted in the two species to prevent cell death and preserve cytokine secretion. Aim 3 will focus on understanding how NLRP3 restricted to the renal tubule epithelium in vivo can be targeted to prevent renal tubular injury without impairing IL1b/IL18. In the third aim, two different models of NLRP3 expression will be used; one where the renal tubules express a conditional loss-of-function of NLRP3 and another where there is a hyper-activatable form of NLRP3. The studies here will determine whether canonical NLRP3-mediated cell death signaling can be uncoupled from noncanonical signaling to prevent cell death while preserving secretion of IL1b/IL18. Health Relatedness of Project: If the aims of this proposal are met, we will learn how activation of NLRP3 (deemed a central cellular stress detector) can be regulated to target a common disease, renal ischemia/reperfusion injury. This knowledge is crucial for the development of rational targeted therapies for prevention or amelioration of renal ischemia/reperfusion injury in clinical situations where hypoxia is anticipated.