Role of intracellular complement activation in kidney fibrosis

项目概要/摘要 在美国，慢性肾脏病 (CKD) 的患病率很高，并且随着人口老龄化，患病率持续上升。 与 CKD 的病因无关，肾小管间质纤维化和肾小球硬化代表了 CKD 的主要途径。 然而，目前还没有批准的药物可以治疗肾纤维化 改善 CKD 的进展。在最近的研究中，我们证明了局部合成和激活的肾脏 补体成分驱动肾纤维化的发病机制。补体系统的主要作用是 在先天性和适应性免疫中很重要，但失调的补体已被证明发挥着重要作用 在肾脏损伤中。我们的试验数据利用单核 RNA 测序 (snRNAseq)，表明细胞特异性 补体成分的区室，特别是 RTE 细胞中 C3 和 C5 的区室。我们的数据还表明 这些成分配位小鼠肾脏巨噬细胞上的同源受体 C3aR 和 C5aR1 肾纤维化模型。在初步数据集中，以细胞特异性方式遗传删除这些因子 对肾脏疤痕具有显着的保护作用。因此，本申请要检验的中心假设 是肾细胞补体成分的内在表达，它们被加工成活性片段 其同源受体的激活是肾功能逐渐丧失和恐惧的关键。这个 假设将通过以下独立但互补的具体目标进行调查： 目标 1：描绘 肾细胞补体成分的细胞内表达。我们将在小鼠肾脏中使用 snRNAseq 纤维化来描绘补体成分及其受体的细胞类型特异性表达。我们将确认 通过蛋白质组学方法获得数据并识别产生活性分泌片段的加工机制 补充成分。目标 2. 确定 RTE 衍生的 C3 对肾脏生理和纤维化的重要性。 我们将使用在 RTE 细胞中专门缺乏 C3 的小鼠来确定细胞内 补充肾脏生理学和损伤引起的纤维化。我们将研究缺乏 C3ar1 受体的小鼠 肾细胞或骨髓细胞以确定是否需要自分泌或旁分泌 C3 发信号。目标 3. 确定 C5a/C5aR1 轴激活对巨噬细胞诱导肾功能的影响 纤维化或修复。我们将验证我们的初步观察结果，即巨噬细胞特异性删除 C5ar1 减少纤维化。我们将确定 C5ar1 是否直接影响巨噬细胞表型和功能，或者 C5ar1 巨噬细胞是否通过招募和激活炎症 T 细胞来促进疤痕形成。总的来说， 该提案将建立健康肾细胞补体成分表达的综合图谱 和受伤的肾脏，建立这些途径在肾脏疤痕形成中的关键机制，并实现临床靶向 这些途径与治疗相关的策略。

PROJECT SUMMARY / ABSTRACT The prevalence of Chronic Kidney Disease (CKD) in the US is high and continues to rise in our aging population. Independent of the cause of CKD, tubulointerstitial fibrosis and glomerulosclerosis represent major pathways of progression of kidney disease, however, there are no approved drugs to treat kidney fibrosis that could ameliorate progression of CKD. In recent studies we demonstrated that locally synthesized and activated kidney complement components drive the pathogenesis of kidney fibrosis. The complement system is of major importance in innate and adaptive immunity, but dysregulated complement has been shown to play a major role in kidney injury. Our pilot data, leveraging single nuclear RNA sequencing (snRNAseq), indicate cell-specific compartmentation of complement components, particularly of C3 and C5 in RTE cells. Our data also indicate that these components ligand their cognate receptors, C3aR and C5aR1, on kidney macrophages in murine models of kidney fibrosis. In preliminary datasets, genetic deletion of these factors in a cell-specific manner imparted significant protection against renal scarring. Thus, the central hypothesis to be tested in this application is that intrinsic expression of complement components from kidney cells, their processing to active fragments and activation of their cognate receptors are key to the progressive loss of kidney function and scaring. This hypothesis will be investigated via the following independent, but complementary, specific aims: Aim 1: Delineate the intracellular expression of complement components by kidney cells. We will use snRNAseq in murine kidney fibrosis to delineate cell type-specific expression of complement components and their receptors. We will confirm data by proteomic approaches and identify the processing machinery generating active secretory fragments from complement components. Aim 2. Determine the importance of RTE-derived C3 for renal physiology and fibrosis. We will use mice engineered to lack C3 specifically in RTE cells to determine the relevance of intracellular complement for renal physiology and injury-induced fibrosis. We will study mice lacking the C3ar1 receptor on either kidney or myeloid cells to establish whether there is an obligate requirement for autocrine or paracrine C3 signaling. Aim 3. Determine the impact of C5a/C5aR1 axis activation on macrophages for the induction of kidney fibrosis or repair. We will validate our preliminary observations that macrophage-specific deletion of C5ar1 reduces fibrosis. We will determine whether C5ar1 directly affects macrophage phenotype and function, or whether C5ar1 macrophages promote scarring by recruiting and activating inflammatory T cells. Collectively, this proposal will establish a comprehensive atlas of complement component expression in kidney cells in healthy and injured kidneys, establish key mechanisms of these pathways in kidney scarring and enable clinical targeting of these pathways with therapeutically relevant strategies.