Inflammatory Signaling in Kidney Stromal Cells Driving Interstitial Fibrosis

肾基质细胞中的炎症信号驱动间质纤维化

• 批准号: 10371183
• 负责人: Dario Lemos
• 金额: $ 26.85万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-09 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10371183
• 关键词: Ablation; Acute Renal Failure with Renal Papillary Necrosis; Alleles; Automobile Driving; Autophagocytosis; Biomedical Engineering; Blood Vessels; Bone Marrow; Cell Differentiation process; Cell Proliferation; Cells; Cellular biology; Chemistry; Cicatrix; Complex; Data; Development; Down-Regulation; End stage renal failure; Event; Fibroblasts; Fibrosis; Genetic; Genetic Transcription; Goals; Hematopoietic; Human; IL1R1 gene; IL6 gene; IRAK4 gene; Immune signaling; Impairment; In Vitro; Inflammation; Inflammatory; Injury to Kidney; Interleukin-1 Receptors; Interleukin-1 beta; Interleukins; Ischemia; Kidney; Knock-in; Laboratories; Ligands; LoxP-flanked allele; Mediating; Modeling; Molecular; Molecular Biology; Mus; Mutation; Myofibroblast; NF-kappa B; Names; Nephrons; Organoids; Pericytes; Pharmacology; Phosphotransferases; Process; Production; Profibrotic signal; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; Protocols documentation; Publishing; Receptor Signaling; Renal function; Reperfusion Injury; Reporting; Research; Rest; Role; Shapes; Signal Transduction; Stromal Cells; TLR2 gene; Testing; Therapeutic; Tissues; Toll-like receptors; Transcriptional Activation; Transgenic Mice; Work; base; cell type; comparative; cytokine; fibrous protein; in vivo; innovation; interstitial; ischemic injury; kidney fibrosis; mouse model; multicatalytic endopeptidase complex; negative affect; novel; novel therapeutics; pre-clinical; prevent; programs; rational design; reconstitution; renal damage; renal ischemia; response; scaffold; small molecule; small molecule inhibitor; synthetic protein; targeted treatment; therapeutically effective; tool

PROJECT SUMMARY/ABSTRACT A growing body of evidence indicates that molecular signaling mechanisms mediated by the myddosome complex in kidney stromal cells, drive fibrosis triggered by interleukin 1b (IL1b) and Toll-like receptor (TLR) ligands. Work from our laboratory and by others has shown that signaling mechanisms associated with IRAK4, a key component of the stromal cell myddosome, contributes to the development of renal fibrosis after acute kidney injury. IRAK4, therefore, emerges as a suitable target for much needed therapies targeting renal fibrosis. In order to effectively target IRAK4, however a deeper understanding of its mechanisms is needed. IRAK4 has been shown to possess two distinct functions, one as a serine/threonine kinase and the other as a structural scaffold necessary for myddosome formation. Importantly, the role of IRAK4 as a myddosome scaffold is necessary for its kinase activity, but the latter is not necessary for myddosome-mediated signaling. We have reported that pharmacologic inhibition of IRAK4 kinase activity with a selective small molecule significantly reduces pro-fibrotic stromal cell activity, including proliferation and differentiation into myofibroblasts, both ex vivo in response to IL1β stimulation, and in vivo after ischemic kidney injury. Our data further indicated that those profibrotic mechanisms depend on stabilization and activation of the transcriptional regulator MYC via a mechanism involving IL1R-driven autophagy. On the contrary, inhibition of IRAK4 kinase activity did not result in abrogation of synthesis and secretion of NF-κB-regulated inflammatory cytokines IL1β and IL6 in kidney stromal cells. Those results are in keeping with previous reports indicating that NF-kB activation by IRAK4 is myddosome-mediated and only partially dependent on IRAK4 kinase activity. The objective of this project is to dissect the molecular mechanisms through which IRAK4 mediates kidney fibrosis, by assessing the distinct contribution of kinase-dependent versus myddosome-dependent IRAK4 signaling mechanisms. The long-term goal of these studies is to set the pre-clinical basis for novel therapeutics that can ameliorate both renal fibrosis and local inflammation through blockage of stromal cell pro-fibrotic and pro- inflammatory activities. The central hypothesis is that following acute kidney injury IRAK4 kinase activity in stromal cells is necessary for pro-fibrotic mechanisms, while kinase-independent IRAK4-mediated myddosome assembly is necessary for inflammatory cytokine production by those cells during the process of kidney scarring. Our rationale for the research strategy proposed is based on a combination of genetic ablation strategies to study IRAK4 kinase domain-independent mechanisms, along with cutting-edge bioengineered human kidney organoids for the study of the nephron interstitial microenvironment in the absence of confusing hematopoietic immune signals. In addition, we propose the use of novel therapeutic chemistry shown to be effective for inducible targeted degradation of IRAK4 in vivo, abrogating myddosome formation. Our three specific aims will test three major hypotheses: (Aim 1) IRAK4 kinase function is necessary for profibrotic mechanisms of kidney stromal cells post-AKI; (Aim 2) signaling via IL1R/IRAK4 stabilizes MYC via a mechanism involving stromal cell autophagy and degradation of SQSTM1/P62; (Aim 3) pharmacologic abrogation of IRAK4 using a synthetic protein degrader molecule will impair stromal cell myddosome formation and ameliorate IRI- induced kidney fibrosis. Collectively, these studies will provide critical experimental and mechanistic basis for a rational design of therapies targeting IRAK4 in renal fibrosis.

项目摘要/摘要 越来越多的证据表明分子信号传导机制是由myddosome介导的 肾脏基质细胞中的复合物，驱动纤维化，由白介素1b（IL1B）和Toll样受体（TLR）触发 配体。我们实验室和其他实验室的工作表明，与Irak4相关的信号传导机制， 基质细胞myddosom体的关键组成部分，有助于急性后肾纤维化的发展 肾脏受伤。因此，IRAK4成为针对肾脏急需疗法的合适靶标 纤维化。为了有效地瞄准irak4，需要对其机制有更深入的了解。 irak4已显示具有两个不同的功能，一个功能是丝氨酸/苏氨酸激酶，另一个是 Myddosome形成所需的结构支架。重要的是，irak4作为myddosome的角色 支架对于其激酶活性是必需的，但是后者对于myddosome介导的信号传导并不是必需的。 我们报道了用选择性小分子对IRAK4激酶活性的药物抑制 显着降低了促纤维性基质细胞活性，包括增殖和分化 肌成纤维细胞，均在体内响应IL1β刺激以及缺血性肾脏损伤后体内。我们的数据 进一步表明这些纤维化机制取决于转录的稳定和激活 调节器MYC通过一种机制涉及IL1R驱动的自噬。形成鲜明对比的是抑制IRAK4激酶 活性并没有导致NF-κB调节的炎症细胞因子的合成和分泌 肾基质细胞中的IL1β和IL6。这些结果与以前的报告一致，表明NF-KB IRAK4激活是由myddosome介导的，仅部分取决于IRAK4激酶活性。这 该项目的目的是剖析IRAK4介导肾纤维化的分子机制， 通过评估激酶依赖性与myddosome依赖性IRAK4信号的不同贡献 机制。这些研究的长期目标是为可以的新疗法设定临床前基础 通过阻塞基质细胞促纤维化和促疾病，可以改善肾纤维化和局部炎症 炎症活动。中心假设是急性肾脏损伤IRAK4激酶活性 基质细胞对于促纤维化机制是必需的，而非激酶独立于IRAK4介导的myddosome则是必需的 组装对于这些细胞在肾脏过程中的炎性细胞因子的产生是必需的 疤痕。我们提出的研究策略的理由是基于遗传消融的组合 研究IRAK4激酶域独立机制的策略，以及尖端的生物工程 人类肾脏类器官在没有混乱的情况下研究肾肾质子间微环境 造血免疫信号。此外，我们提出了显示的新型热化学的使用 有效地诱导靶向irak4在体内降解，从而消除了myddosome组的形成。我们的三个 具体目的将检验三个主要假设：（目标1）IRAK4激酶功能对于专业 Aki后肾脏基质细胞的机制； （AIM 2）通过IL1R/IRAK4通过机制稳定MYC的信号 涉及SQSTM1/p62的基质细胞自噬和降解； （AIM 3）IRAK4的药理学废除 使用合成蛋白质降解子分子会损害基质细胞肌体组的形成并改善iri- 诱导肾纤维化。总的来说，这些研究将为A提供关键的实验和机械基础 针对肾纤维化中IRAK4的疗法的合理设计。