肾脏单核-巨噬细胞系统中IKKα-p52:RelB途径活化对促进肾脏缺血再灌注损伤后修复的作用及机制

The recovery and regeneration usually occurs shortly (between 24 and 72 hours) post-ischemia, during which cellular dedifferentiation and tubular epithelial cell proliferation occur. There is compelling evidence that the renal mononuclear phagocytic system (Moph) actively participates in the resolution of injury and promotes tissue restoration in the phase after renal ischemia reperfusion (IR) injury. Several lines of evidence suggest that the IKKα-driven noncanonical NFκB p52:RelB signaling pathway is critical in the regression of acute inflammation and the process of keratinocyte and endothelium proliferation and differentiation. Our previous studies revealed that IKKα activation in tubular epithelial cells mediated kidney self repair by infiltrating T regular cells, promoting anti-inflammatory cytokine IL-10, chemotactic factor CXCL12 ( also be named as stromal cell-derived factor 1, SDF-1) synthesis. To further investigate whether KKα-driven noncanonical NFκB p52:RelB signaling pathway is activated in Moph in the repair phase. We hypothesized that activation of this signaling pathway in Moph would participate in the survived tubular epithelial cells proliferation and kidney repair after renal IR injury by two possible ways: 1. to mediate Treg cells developing, maturation and releasing IL-10 ; 2. to initiate and sustain the expression of CXCL12, which result in the survived tubular epithelial cells migration, dedifferentiation, proliferation and repairs the injured tubular cells. To test this hypothesis, monocytes, na?ve T cells and tubular epithelial cells will be isolated and co-cultured in vitro and Moph adoptive transfer will be used in vivo in this study, in order to elucidate the molecular mechanism of IKKα-driven noncanonical NFκB p52:RelB signaling pathway in Moph in promoting tubular epithelial cell regeneration and kidney repair , and further to provide a novel clue for preventing and treating ischemic acute kidney injury.

肾脏在缺血-再灌注损伤后不久就开始修复，此时肾组织中单核-巨噬细胞系统( MoPh )发生表型转化，参与了抑制炎症和促进修复的过程；而IKKα-p52:RelB途径在抑制炎症反应和促进细胞增殖过程中起重要的作用。我们的前期研究表明，肾脏IR损伤后修复时， M2型MoPh和Treg 浸润明显，间质中IL-10、CXCL12和肾小管上皮细胞内IKKα表达升高。但MoPh内IKKα是否活化？是否通过IKKα-p52:RelB途径活化参与修复的？尚不清楚。我们假设：MoPh内IKKα-p52:RelB途径被激活后，一方面介导Treg成熟并释放IL-10；另一方面，启动并维持CXCL12的表达，最终促进细胞迁移，增殖，修补丢失的肾小管上皮细胞，恢复肾脏功能。本研究使用基因敲除小鼠，采用体外细胞共培养，体内细胞过继转移等技术，阐明Mpoh参与肾脏修复的机制，为探索急性肾损伤治疗的新靶点提供理论依据。

目前发现急性肾损伤可导致慢性肾脏病，这种转变过程我们称之为急性肾损伤慢性肾脏病转化，导致肾功能的持续恶化。目前急性肾损伤的慢性转化机制仍不完全明确，肾小管上皮细胞向间质细胞的转化是重要因素，而巨噬细胞的浸润起到重要的调节作用。我们之前的研究显示肾小管上皮细胞中IKKα可减少炎症因子的释放并调节T细胞的功能，有其他研究显示巨噬细胞中IKKα的缺乏可导致细胞的凋亡。我们推测IKKα参与了巨噬细胞的激活与极化，影响到急性肾损伤慢性肾脏病转化。通过建立缺血性肾损伤恢复期修复模型，纪录3天、7天、14天、21天四个时间点的各组小鼠肾脏的形态、重量以及血清肌酐、尿素氮水平。结果显示野生型小鼠损伤的肾脏较敲除型萎缩，血清肌酐尿素氮水平明显高于敲除小鼠。HE染色结果显示与同期野生型小鼠肾组织相比，敲除小鼠肾脏存留肾小管上皮细胞较多，肾小管间质增生较少。对肾脏IRI后的肾小管上皮细胞EMT进行研究，发现EMT的标记性分子如collagen I，MMP9，vimentin and α-SMA在野生型小鼠IRI后肾脏中的表达要不同程度高于敲除型小鼠，说明野生型小鼠的肾损伤后的更多的肾小管上皮细胞发生EMT。通过马松染色和Collagen III的免疫组化染色，可以直观的观察到野生组的肾纤维化程度更加严重。通过腹腔诱导小鼠腹腔巨噬细胞的产生并流式细胞仪检测发现野生型小鼠经诱导后巨噬细胞中M2亚型的比例更高，通过Westernblot检测巨噬细胞的分泌型Marker，结果显示M1型巨噬细胞的分泌型Marker在野生型与敲除型小鼠中表达无明显差异；M2型巨噬细胞分泌型Marker在野生型中较敲除型更多。对损伤的肾脏进行免疫荧光染色发现M2巨噬细胞所占比例在野生型小鼠肾中要明显高于敲除型小鼠。我们分离小鼠肾小管上皮细胞并建立缺氧损伤模型，分别与野生型巨噬细胞和敲除型巨噬细胞共培养发现： 缺氧损伤可导致肾小管上皮细胞发生EMT，野生型巨噬细胞相较敲除型巨噬细胞，可明显促进缺氧损伤后肾小管上皮细胞的EMT，而敲除型巨噬细胞无明显的促进EMT作用。对肾小管上皮细胞中Wnt信号通路分子检测发现，野生型巨噬细胞可促进肾小管上皮细胞Wnt信号分子的上调，而敲除型巨噬细胞则无此作用。本课题提示巨噬细胞中的IKKα可作为治疗急性肾损伤慢性转化的治疗靶点。