KLF4调控巨噬细胞极化对AKI后肾小管上皮细胞程序性坏死和NLRP3炎性小体活化的影响及机制研究

Necroptosis in kidney epithelial cells is known to be the key determinant for the pathogenesis of acute kidney injury (AKI). Thus, understanding the precise mechanisms regulating necroptosis is important for the prevention and treatment of AKI. In recent studies, KLF4 deficient macrophages exhibit enhanced M1 polarization, M1 macrophages infiltrate kidneys early after AKI and aggravate tubular epithelial cell injury, however, the exact mechanisms have not been fully elucidated. Our previous study suggested that KLF4 expression in macrophages is significantly inhibited after AKI, and necroptosis and NLRP3 inflammasome activation in tubular epithelial cells are augmented by ischemic AKI. Based on these findings, we hypothesized that suppressed KLF4 expression enhances M1 polarization after AKI, thereby stimulating necroptosis and NLRP3 inflammasome activation in tubular epithelial cells by secretion of TNF-α, thereby further aggravating renal inflammation and damage. In this study, we will investigate the efficacy of macrophage KLF4 on tubular necroptosis and NLRP3 inflammasome activation and the underlying mechanisms by in vivo and in vitro experiments. This study may provide novel understandings on AKI and therapeutic basis for the prevention of AKI.

肾小管上皮细胞程序性坏死（Necroptosis）是急性肾损伤（AKI）的关键环节，探讨其调控机制对临床防治急性肾损伤具有重要意义。近期研究发现，KLF4表达下降促使巨噬细胞向M1型分化，而AKI早期巨噬细胞浸润可加重肾小管上皮细胞损伤,但作用机制并不清楚。我们的前期研究发现，AKI后浸润的巨噬细胞内KLF4表达下降，肾小管上皮细胞程序性坏死及NLRP3炎性小体活化增强。基于以上发现，我们提出：AKI后巨噬细胞KLF4表达下降诱导其向M1型分化，并通过分泌TNF-α激活肾小管上皮细胞程序性坏死和NLRP3炎性小体通路，从而加重肾脏炎症和损伤。本课题拟在前期工作基础上，通过体内外实验进一步揭示KLF4调控巨噬细胞极化对AKI后肾小管上皮细胞Necroptosis及NLRP3炎性小体通路活化的影响，为临床防治AKI提供新的治疗靶点和理论依据。

急性肾损伤（acute kidney injury，AKI）是多种病因引起的危、急、重症，过重或反复的AKI损伤导致肾脏不完全性修复及持续的肾间质炎症反应，引起AKI向慢性肾脏病（chronic kidney disease，CKD）进展（AKI to CKD, AtoC）。目前，AKI 向 CKD 发展的确切机制尚不清楚，深入探讨 AtoC 发生机制对制定 AKI 防治新策略、改善 AKI 患者预后具有十分重要的意义。本项目通过构建巨噬细胞特异性 KLF4 敲除/过表达小鼠模型，探索肾脏缺血再灌注损伤后，KLF4 对巨噬细胞极化、肾脏损伤和炎症的影响，巨噬细胞来源TNF对肾小管上皮细胞内炎症信号通路的影响。（1）巨噬细胞KLF4敲除加重IRI-AKI肾脏损伤，在IRI-AKI的第7天，WT小鼠Cr与BUN基本恢复正常，而KLF4MKO鼠Cr与BUN则明显高于WT小鼠。PAS染色显示在I/R第7天，KLF4 MKO小鼠肾脏仍可见到大量管型，肾小管上皮细胞萎缩、坏死，伴有管腔扩张。（2）巨噬细胞KLF4敲除促使AKI向CKD进展，在I/R第30天，与WT小鼠相比，KLF4 MKO小鼠肾脏的纤维化模型，炎症细胞浸润增加。（3）KLF4调控巨噬细胞的极化，巨噬细胞KLF4敲除促进巨噬细胞向M1转化抑制巨噬细胞向M2转化，M1型细胞因子TNF-α、IL-1β、IL-6和IL-18的表达增加，其中TNF-α的表达差异最大，而M2标记Arg-1、Fizz和Ym-1的表达减少。（4）巨噬细胞KLF4敲除促进T细胞和中性粒细胞的激活，与WT小鼠相比，KLF4 MKO小鼠肾脏中浸润的CD44hiCD62LlowCD4+细胞、CD44hiCD62LlowCD8+细胞显著增加。（5）巨噬细胞TNF敲除缓解I/R第7天时的肾脏损伤，并能减轻I/R后第30天肾脏纤维化水平。（6）体内、体外实验均证实，巨噬细胞KLF4通过调控TNF释放，影响肾小管上皮细胞内炎性小体等炎症通路活化。我们的研究阐明巨噬细胞 KLF4介导的TNF释放在TEC损伤和炎症中的作用和机制，为创新 AtoC 的防治策略提供理论和实验依据。

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