Inflammatory Signaling in Kidney Stromal Cells Driving Interstitial Fibrosis

项目总结/摘要 越来越多的证据表明，由myddosome介导的分子信号机制， 肾基质细胞中的复合物，驱动由白细胞介素1b（IL 1b）和Toll样受体（TLR）触发的纤维化 配体。我们实验室和其他人的工作表明，与IRAK 4相关的信号机制， 基质细胞mydosome的关键成分，有助于急性肾纤维化后的发展， 肾损伤因此，IRAK 4成为急需的肾脏治疗的合适靶点 纤维化然而，为了有效地靶向IRAK 4，需要对其机制有更深入的了解。 IRAK 4已被证明具有两种不同的功能，一种是丝氨酸/苏氨酸激酶，另一种是丝氨酸/苏氨酸激酶。 myddosome形成所必需的结构支架。重要的是，IRAK 4作为myddosome的作用 支架是其激酶活性所必需的，但后者不是myddosome介导的信号传导所必需的。 我们已经报道了用选择性小分子药物抑制IRAK 4激酶活性， 显著降低促纤维化基质细胞活性，包括增殖和分化为 肌成纤维细胞，在离体响应IL 1 β刺激和在体内缺血性肾损伤后。我们的数据 进一步表明，这些促纤维化机制依赖于转录因子的稳定和激活， 调节MYC通过涉及IL 1 R驱动的自噬机制。相反，抑制IRAK 4激酶 活性并不导致NF-κ B调节的炎性细胞因子的合成和分泌的消除 肾间质细胞IL 1 β和IL 6。这些结果与以前的报告一致，表明NF-κ B IRAK 4的激活是myddosome介导的，并且仅部分依赖于IRAK 4激酶活性。的 该项目的目的是剖析IRAK 4介导肾纤维化的分子机制， 通过评估激酶依赖性与线粒体依赖性IRAK 4信号传导的不同贡献， 机制等这些研究的长期目标是为新的治疗方法建立临床前基础， 通过阻断基质细胞促纤维化和促炎症， 炎症活动。中心假设是在急性肾损伤后，IRAK 4激酶活性降低。 基质细胞是促纤维化机制所必需的，而激酶非依赖性IRAK 4介导的mydosome 组装是必要的炎症细胞因子的生产过程中，这些细胞的肾脏 疤痕我们提出的研究策略的基本原理是基于基因切除和基因治疗的结合， 研究IRAK 4激酶结构域独立机制的策略，沿着尖端的生物工程 人肾类器官用于在无混淆情况下研究肾单位间质微环境 造血免疫信号。此外，我们建议使用新的治疗化学显示， 有效用于体内IRAK 4的诱导型靶向降解，消除多粒体形成。我们的三 具体目的将测试三个主要假设：（目的1）IRAK 4激酶功能是促纤维化所必需的。 AKI后肾基质细胞的机制;（目的2）通过IL 1 R/IRAK 4的信号传导通过一种机制稳定MYC 涉及基质细胞自噬和SQSTM 1/P62的降解;（目的3）IRAK 4的药理学废除 使用合成的蛋白质降解剂分子将损害基质细胞mydosome的形成并改善IRI， 诱发肾纤维化。总的来说，这些研究将提供关键的实验和机制基础， 合理设计针对肾纤维化中IRAK 4的疗法。

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.