肾小管上皮细胞源性外泌体miRNA下调Klotho促进巨噬细胞M1极化在糖尿病肾脏病发病中的作用及分子机制

In our previously research, we found that Klotho was significantly downregulated in kidney macrophages in mice with DKD. Besides, knockout of Klotho promoted macrophages M1 polarization. Meanwhile, we also found that co-cultured albumin-stimulated renal tubular epithelial cells-derived exosomes with macrophages downregulating macrophage Klotho expression, and miR-199a may play a role. Therefore, we proposed that renal tubular epithelial cells down-regulated Klotho expression in macrophages through exosomes miRNA thus promoting macrophages M1 polarization during DKD process. We plan to use mice with conditional deletion of Rab27a in renal tubular epithelial cells which block exosomes secretion from renal tubular epithelial cells and the co-culture system to confirm the effect of renal tubular epithelial cells derived exosomes on macrophage polarization. Through using renal tubular epithelial cells with deletion of Dicer gene, bioinformatics analysis and injection exosomes through veil tail, we will confirm that exosomes down-regulated macrophage Klotho expression through miRNA. This research will clarify the mechanism of how renal tubular epithelial cells downregulate macrophage Klotho expression through exosomes thus promoting M1 polarization during DKD process. This research also provide a potential new target for the prevention and treatment of DKD.

课题组前期研究发现糖尿病肾脏病（DKD）小鼠肾脏巨噬细胞Klotho表达显著下调，敲除Klotho可促进巨噬细胞M1极化；进一步实验发现白蛋白刺激下肾小管上皮细胞来源的外泌体可下调巨噬细胞Klotho表达，且miR-199a可能发挥作用。据此，我们提出DKD过程中肾小管上皮细胞可能通过外泌体中的miRNA下调巨噬细胞Klotho进而促进巨噬细胞M1极化的假说。我们拟采用肾小管上皮细胞特异性外泌体分泌抑制的Rab27a敲除小鼠构建DKD模型，通过非接触共培养实验明确肾小管上皮细胞外泌体对巨噬细胞Klotho表达的调控作用；通过构建Dicer酶敲除细胞模型，生物信息学分析和外泌体注射技术验证miRNA是外泌体促进巨噬细胞Klotho表达下调的效应因子。本课题将阐明DKD过程中肾小管上皮细胞通过外泌体miRNA下调巨噬细胞Klotho进而促进其向M1极化的机制，为DKD防治提供潜在的新靶点。

糖尿病肾脏病（DKD）已成为目前导致终末期肾病的主要原因，如何有效延缓DKD进展是目前研究的热点和难点，然而目前尚无有效的治疗措施。因此，深入探讨DKD发病机制，寻找调控DKD进展的关键靶点，对于防治DKD进展具有十分重要的意义。白蛋白尿是DKD进展的独立危险因素，其被肾小管上皮细胞摄取后通过促进肾脏炎症导致肾脏纤维化。巨噬细胞是肾脏最重要的免疫细胞，其浸润及活化在DKD炎症反应过程中起重要作用。然而目前尚不清楚DKD过程中尿白蛋白如何通过肾小管上皮细胞影响巨噬细胞，从而促进巨噬细胞炎症因子的释放。本研究主要围绕肾小管上皮细胞来源的外泌体如何影响巨噬细胞极化开展相关实验。我们主要采用白蛋白诱导的人肾小管上皮细胞HK-2、佛波酯（PMA）诱导的THP-1细胞为细胞模型、db/db小鼠及高脂饮食联合STZ诱导的糖尿病小鼠为动物模型，并通过外泌体细胞共培养及体内注射、RT-PCR、western blot、免疫组化、染色质免疫共沉淀等方法进行相关实验。主要结果如下：1、DKD小鼠肾脏M1型巨噬细胞增加；2、白蛋白诱导的HK-2细胞通过分泌外泌体，促进巨噬细胞M1极化，敲减HK-2细胞Rab27a抑制外泌体的分泌可减少其对巨噬细胞极化的影响；3、白蛋白诱导的HK-2来源的外泌体中包含miR-199a-5p可靶向巨噬细胞Klotho促进其M1极化；4、尾静脉注射白蛋白诱导的HK-2来源的外泌体可促进db/db小鼠DKD进展及肾脏巨噬细胞M1极化，而抑制外泌体中miR-199a-5p逆转这一过程；5、通过体内外过表达Klotho可抑制肾脏巨噬细胞M1极化；5、Klotho通过TLR4影响巨噬细胞M1极化。本研究深入地阐明在DKD进展过程中白蛋白通过肾小管上皮细胞来源的外泌体对肾脏巨噬细胞极化的影响及其可能的机制，为防治DKD提供新的潜在靶点和治疗途径。