Inflammatory Signaling in Kidney Stromal Cells Driving Interstitial Fibrosis

项目摘要/摘要 越来越多的证据表明，由Myddosome介导的分子信号机制 肾间质细胞中的复合体，推动白介素1b(IL1b)和Toll样受体(TLR)引发的纤维化 配基。我们实验室和其他人的研究表明，与IRAK4相关的信号机制， 基质细胞肌小体的关键成分，在急性肾纤维化的发生发展中起重要作用 肾损伤。因此，IRAK4成为针对肾脏的迫切需要的治疗的合适靶点 纤维化症。然而，为了有效地针对IRAK4，需要对其机制有更深入的了解。 IRAK4已被证明具有两种不同的功能，一种是丝氨酸/苏氨酸激酶，另一种是一种 形成多菌体所必需的结构支架。重要的是，IRAK4作为Myddosome的作用 支架是其激酶活性所必需的，但后者不是myddosome介导的信号转导所必需的。 我们已经报道了用选择性小分子药物抑制IRAK4激酶活性 显著降低促纤维化间质细胞的活性，包括增殖和分化为 肌成纤维细胞，无论是对IL1β刺激的体外反应，还是对缺血肾损伤后的体内反应。我们的数据 进一步表明，这些促纤维化机制依赖于转录的稳定和激活。 调节分子MYC通过一种涉及IL1R驱动的自噬机制。相反，抑制IRAK4激酶 活性不会导致抑制合成和分泌由NF-κB调节的炎性细胞因子 肾间质细胞表达IL-1、β和IL-6。这些结果与之前的报道一致，表明核因子-kB IRAK4的激活是由myddosome介导的，并且仅部分依赖于IRAK4的活性。这个 本项目的目的是剖析IRAK4介导肾脏纤维化的分子机制。 通过评估依赖于激酶的IRAK4信号与依赖于myddosome的IRAK4信号的不同贡献 机械装置。这些研究的长期目标是为新的疗法奠定临床前的基础，这些疗法可以 通过阻断促纤维化和促纤维化的间质细胞来减轻肾纤维化和局部炎症。 炎症活动。中心假说是在急性肾损伤后，IRAK4激酶活性在 基质细胞是促纤维化机制所必需的，而不依赖于激酶的IRAK4介导的肌小体 肾脏过程中这些细胞产生炎性细胞因子所必需的组装 伤痕累累。我们提出的研究策略的基本原理是基于基因消融的组合。 研究IRAK4激酶结构域非依赖性机制的策略，以及前沿的生物工程 人肾器官类物质在研究肾单位间质微环境中的无混淆 造血免疫信号。此外，我们建议使用新的治疗性化学物质 有效地在体内诱导IRAK4的靶向降解，消除粘菌体的形成。我们的三个 特定的AIMS将检验三个主要假说：(AIMS 1)IRAK4激酶功能是促纤维化所必需的 急性肾损伤后肾间质细胞的机制；(AIM 2)IL1R/IRAK4信号通路通过一种机制稳定MYC 涉及基质细胞自噬和SQSTM1/p62的降解；(目标3)药物消除IRAK4 使用人工合成的蛋白质降解物分子将损害基质细胞黏菌体的形成，并改善IRI。 导致肾脏纤维化。总的来说，这些研究将提供关键的实验和机制基础 针对IRAK4治疗肾纤维化的合理设计。

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.