Macrophage Function in Kidney Repair

急性损伤后肾小管的修复需要清除死细胞和增殖细胞。 通过那些在最初的损伤中存活下来的细胞使小管重新增殖。我们通过跟踪个人获得的数据 在缺血-再灌注损伤后第1天和第3天使用高分辨率多光子Z-堆栈图像的肾小管 证实了近端小管的S3段是大多数小管细胞丢失和管腔内管型的部位 上皮增生和管型清除在外条的组织学表现中占主导地位 损伤后2-5天肾髓质的变化。在此期间，巨噬细胞（称为单核细胞） 巨噬细胞（MNP）的均匀性）逐渐积累在受损的肾脏，并特异性诱导 在缺血再灌注损伤（IRI）后2-3天开始表达修复基因。我们现在展示一个 在这些促修复标记中，精氨酸酶1（Arg 1）仅在外条中的MNP中被选择性地直接诱导 靠近受伤的S3节段使用脂质体氯膦酸盐或 使用LysM-Cre选择性敲除MNP Arg 1表达; Arg 1fl/fl小鼠减少增殖性S3修复 损伤肾脏对管型的清除率增加，死亡率增加，BUN和肌酐升高，肾小管功能恶化 在MNP Arg 1敲除小鼠中的修复。对IRI后第3天分离的细胞进行单细胞RNA测序，或 假手术表明这些Arg 1+细胞组成了MNP的一个独特亚群， 归巢受体Ccr 2和CD 74，而受损的PT细胞表达同源配体Ccl 2和Mif。的 特异性诱导OS MNP中的Arg 1，结合这些细胞促进增殖小管的能力， 修复和石膏清除，使我们假设，受伤的OS形成了一个利基招聘， 选择性激活MNP以促进S3修复。 在这个提议中，我们将使用体外OS模拟物来定义关键的细胞组分和分泌因子 促进OS中MNP归巢和修复性激活，确定一种药理学方法， 这种修复性激活，然后使用这种修复性MNP的药理学激活以及成像质量 细胞计数和免疫荧光（IF）分析，以验证IRI后小鼠肾脏中的这些体外发现 (SA 1）。然后，我们将探讨修复性巨噬细胞诱导上皮细胞增殖的机制， 使用候选因子筛选、scRNA-seq和蛋白质组学分析的组合进行管型清除， MNP分泌的刺激肾小管细胞增殖的因子，以及修复性MNP在 解毒PT细胞氨基酸代谢产物，促进S3-thin管型降解 降支连接处（SA 2）。本提案的综合输出将提供机械的理解 MNP如何促进修复性增殖和管型清除，我们将利用这些信息， 开发药理学方法来模拟这些反应并促进肾小管修复。

Repair of the kidney tubules after an acute insult requires the clearance of dead cells and proliferative repopulation of the tubules by those cells that survive the initial insult. Our data obtained by tracking individual tubules using high resolution multiphoton Z-stack images on days 1 and 3 after ischemia-reperfusion injury confirms that the S3 segment of the proximal tubule is the site of most tubular cell loss and intraluminal cast formation, and that epithelial proliferation and cast clearance dominate the histologic findings in the outer stripe of the renal medulla from days 2-5 after injury. During this exact time, macrophages (termed mononuclear phagocytes (MNPs) for uniformity) progressively accumulate in the injured kidney and are specifically induced to express reparative genes beginning on day 2-3 after ischemia reperfusion injury (IRI). We now show that one of those pro-repair markers, arginase 1 (Arg1), is selectively induced only in MNPs in the outer stripe directly adjacent to the injured S3 segment. General depletion of reparative MNPs using liposomal clodronate or selective knock-out of MNP Arg1 expression using LysM-Cre;Arg1fl/fl mice reduces both proliferative S3 repair and cast clearance by the injured kidney, with increased mortality, higher BUN and creatinine, and worse tubule repair in the MNP Arg1 null mice. Single cell RNA sequencing performed on cells isolated on day 3 after IRI or sham operation demonstrates that these Arg1+ cells make up a distinct subset of MNP that selectively express the homing receptors Ccr2 and CD74, while injured PT cells express the cognate ligands Ccl2 and Mif. The specific induction of Arg1 in the OS MNP, combined with the ability of these cells to promote proliferative tubule repair and cast clearance, has led us to hypothesize that the injured OS forms a niche for recruiting and selectively activating MNP to facilitate S3 repair. In this proposal we will use an in vitro OS mimetic to define the critical cellular components and secreted factors that promote MNP homing and reparative activation in the OS, identify a pharmacologic approach to recreate this reparative activation, and then use this pharmacologic activation of reparative MNP as well as imaging mass cytometry and immunofluorescence (IF) analysis to validate these in vitro findings in the mouse kidney after IRI (SA 1). We will then pursue the mechanism by which reparative macrophages induce epithelial proliferation and cast clearance using a combination of candidate factor screening, scRNA-seq and proteomic analysis to identify the MNP-secreted factors that stimulate tubule cell proliferation, as well as the role of reparative MNP in detoxifying metabolites of PT cell amino acid catabolism and promoting cast degradation at the S3-thin descending limb junction (SA 2). The combined output of this proposal will provide mechanistic understanding of how MNPs promote both reparative proliferation and cast clearance, and we will use this information to develop pharmacologic approaches to mimic these responses and facilitate kidney tubule repair.