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缺血预处理的肾小管细胞来源外泌体对肾缺血再灌注损伤大鼠PI3K/AKT/mTOR信号通路的调控机制
陈家辉, 张燕子, 张艾莎, 隋晓露, 许云鹏, 谢婷妃, 陈继红
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缺血预处理的肾小管细胞来源外泌体对肾缺血再灌注损伤大鼠PI3K/AKT/mTOR信号通路的调控机制
Regulation mechanism of ischemic preconditioning renal tubular cell-derived exosomes on PI3K/AKT/mTOR signaling pathway in rats with renal ischemia reperfusion injury
本研究拟通过建立大鼠肾缺血再灌注损伤（renal ischemia reperfusion injury，RIRI）模型，观察不同外泌体处理后磷脂酰肌醇3激酶（phosphatidylinositol⁃3⁃kinase，PI3K）/蛋白激酶B（protein kinase B，AKT）/雷帕霉素靶蛋白（mammalian target of rapamycin， mTOR）信号通路表达变化，并进行微RNA（microRNA，miRNA）差异表达分析，揭示缺血预处理（ischemic preconditioning，IPC）的肾小管细胞来源外泌体在减轻大鼠RIRI中的分子机制。首先取SD大鼠10只，全麻下切除双肾，制备原代肾小管细胞。第2代肾小管细胞分别予以下处理：常氧（38%O2、5%CO2）、低氧（1%O2、5%CO2）和低氧+灭活（65 ℃加热30 min）培养12 h，然后提取外泌体，分别得常氧外泌体、IPC外泌体和灭活外泌体。再取SD大鼠50只，随机分为5组：假手术组（Sham组）、RIRI组、RIRI+常氧外泌体组（NC组）、RIRI+IPC外泌体组（IPC组）、RIRI+灭活外泌体组（INA组），后4组均先建立RIRI模型，NC组、IPC组和INA组RIRI建模后24 h分别尾静脉注入200 μg常氧外泌体、IPC外泌体和灭活外泌体。6 d后取静脉血，摘取双肾，观察各组肾功能、肾脏组织病理及PI3K/AKT/mTOR信号通路变化，并对NC组和IPC组进行miRNA差异表达（P<0.05，|log2FC|≥1）分析，探查miRNA表达谱变化并进行GO分析和KEGG通路富集分析。结果发现，（1）与Sham组相比，RIRI组和INA组血肌酐、尿素氮水平升高（均P<0.01）；肾组织病理可见间质大量炎性细胞聚集伴不同程度水肿，肾小管结构变性肿胀，肾小管上皮细胞坏死脱落；TUNEL阳性细胞数明显升高，Ki67染色阳性细胞数明显减少；PI3K/AKT/mTOR信号通路的mRNA和蛋白表达较低。（2）与NC组相比，IPC组血肌酐和尿素氮水平较低（均P<0.01）；肾间质炎性细胞聚集显著减少、组织水肿明显改善；TUNEL 阳性细胞数减少，Ki67染色阳性细胞数增多；PI3K、PDK1、AKT、mTOR的mRNA和蛋白表达均较高（均P<0.05）。（3）与NC组相比，IPC组有56个miRNA表达上调，42个miRNA表达下调。GO富集分析发现，靶基因为PIK3C2A、PIK3CA、PIK3CB、PIK3CD、PIK3C2G、AKT1、mTOR、Rheb等；KEGG富集分析发现，在PI3K/AKT信号通路和mTOR信号通路均有显著性富集。本研究揭示，在RIRI病程中，IPC肾小管细胞来源外泌体可致肾组织miRNA差异性表达，使PI3K/AKT/mTOR信号通路表达增强，在减轻大鼠RIRI中发挥重要作用。
This study aims to establish a rat model of renal ischemia reperfusion injury (RIRI) to observe the alterations in the expression of phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway following various exosome treatments. Additionally, differential miRNA expression analysis will be conducted to elucidate the molecular mechanisms underlying the effects of exosomes derived from ischemic preconditioned (IPC) renal tubular cells in mitigating RIRI in rats. Initially, ten SD rats were subjected to bilateral nephrectomy under general anesthesia to prepare primary renal tubular cells. The second-generation renal tubular cells were then subjected to the following treatments for 12 hours: normoxia (38% O2, 5% CO2), hypoxia (1% O2, 5% CO2), and hypoxia plus inactivation (heated at 65 ℃ for 30 minutes). Following these treatments, exosomes were extracted, yielding normoxic exosomes, IPC exosomes, and inactivated exosomes, respectively. A subsequent cohort of 50 SD rats was randomly divided into five groups: Sham group, RIRI group, RIRI + normoxic exosome group (NC group), RIRI + IPC exosome group (IPC group), and RIRI + inactivated exosome group (INA group). RIRI model was established in the latter four groups. Twenty⁃four hours after RIRI modeling, the NC, IPC, and INA groups received intravenous injections of 200 μg of normoxic exosomes, IPC exosomes, and inactivated exosomes via the tail vein, respectively. Six days later, venous blood samples were collected, and both kidneys were excised to observe renal function, histopathological changes in kidney tissue, and alterations in the PI3K/AKT/mTOR signaling pathway among the five groups. Furthermore, differential miRNA expression analysis [P<0.05, |log2(Fold Change)|≥1] was conducted between the NC and IPC groups to investigate the changes in the miRNA expression profile. Subsequently, GO analysis and KEGG pathway enrichment analysis were performed. The results revealed that: (1) Compared with the Sham group, the RIRI and INA groups exhibited elevated levels of serum creatinine and urea nitrogen (all P<0.01). Histopathological examination of kidney tissues showed substantial inflammatory cell infiltration in the interstitium accompanied by varying degrees of edema, degenerative swelling of tubular structures, necrosis, and detachment of tubular epithelial cells. Notably, the number of TUNEL⁃positive cells was significantly increased, while the number of Ki67⁃stained positive cells was markedly decreased. Additionally, the mRNA and protein expression of PI3K/AKT/mTOR signaling pathway in RIRI group and INA group were down-regulated. (2) Compared to the NC group, the IPC group demonstrated lower levels of serum creatinine and urea nitrogen (both P<0.01). Notably, there was a significant decrease in the accumulation of inflammatory cells in the renal interstitium, and tissue edema was markedly improved. Moreover, the number of TUNEL⁃positive cells was reduced, while the number of Ki67⁃stained positive cells was significantly increased. Additionally, the mRNA and protein expressions of PI3K, PDK1, AKT, and mTOR were all up-regulated (all P<0.05). (3) Compared to the NC group, 56 miRNAs were up-regulated and 42 miRNAs were down-regulated in the IPC group. The target genes of GO enrichment analysis were PIK3C2A, PIK3CA, PIK3CB, PIK3CD, PIK3C2G, AKT1, mTOR, Rheb, and KEGG enrichment analysis revealed significant enrichment in PI3K/AKT signal pathway and mTOR signal pathway. In conclusion, this study reveals that during the course of RIRI, exosomes derived from IPC renal tubular cells induce differential miRNA expression in kidney tissues, resulting in enhanced expression of the PI3K/AKT/mTOR signaling pathway, which plays a pivotal role in mitigating RIRI in rats.
外泌体 / 微RNAs / 再灌注损伤 / 磷酸肌醇3⁃激酶类 / PI3K/AKT/mTOR {{custom_keyword}} /
Exosomes / MicroRNAs / Reperfusion injury / Phosphatidylinositol 3⁃kinases / PI3K/AKT/mTOR {{custom_keyword}} /
表1 PCR引物序列 |
| 名称 | 引物 |
|---|---|
| β⁃actin | 上游引物5’⁃ CACCAGGGTGTGATGGTG⁃3’ |
| 下游引物5’⁃ GTACATGGCTGGGGTGTTG⁃3’ | |
| PI3K | 上游引物5’⁃ CCCGGGTAGGTTTGAATTCGTT⁃3’ |
| 下游引物5’⁃ TGCCCTAGGTGACCTGACACA⁃3’ | |
| PDK1 | 上游引物5’⁃ AGGCGTTTATCCCCCGATTC⁃3’ |
| 下游引物5’⁃ CTCCCCGGTCACTCATCTTC⁃3’ | |
| AKT | 上游引物5’⁃ CGCTTCTTTGCCAACATCGT⁃3’ |
| 下游引物5’⁃ CACTCCATGCTGTCATCTTGA⁃3’ | |
| mTOR | 上游引物5’⁃ TGGAGGTTACCGGTCTCGAT⁃3’ |
| 下游引物5’⁃ GGCATTTGTGTCCATCAGCC⁃3’ |
图1 各组大鼠肾功能和PI3K/AKT/mTOR信号通路表达比较（全自动生化分析仪或实时定量PCR或Western印迹） |
图3 各组大鼠肾脏病理改变（HE ×400） |
图5 肾小管细胞外泌体形态（电镜 ×20 000） |
表2 肾缺血再灌注损伤+缺血预处理外泌体组肾小管细胞外泌体颗粒粒径⁃浓度分布比例 |
| 峰值直径（nm） | 浓度（个/ml） | 半峰宽（nm） | 百分比（%） |
|---|---|---|---|
| 96.8 | 3.5×106 | 67.9 | 98.2 |
| 272.5 | 9.1×104 | 15.2 | 0.5 |
| 337.4 | 3.6×104 | 17.9 | 0.4 |
| 385.2 | 1.6×104 | 15.1 | 0.2 |
| 466.0 | 1.2×104 | 10.9 | 0.1 |
表3 肾缺血再灌注损伤+缺血预处理外泌体组和肾缺血再灌注损伤+常氧外泌体组肾小管细胞外泌体GO富集分析 (与肾缺血再灌注损伤+常氧外泌体组比较) |
| 编号 | 词条 | 差异表达基因数 | P值 | 部分靶基因 |
|---|---|---|---|---|
| GO：0008152 | 代谢过程 | 5 177 | <1.0×10-30 | AKT1、mTOR、PIK3C2A、PIK3C2G、PIK3C3、PIK3CA、PIK3CB、PIK3CD |
| GO：0071704 | 有机物代谢过程 | 4 961 | <1.0×10-30 | AKT1、mTOR、PIK3C2A、PIK3C2G、PIK3C3、PIK3CA、PIK3CB、PIK3CD |
| GO：0005622 | 细胞内 | 6 458 | <1.0×10-30 | AKT1、mTOR、PIK3C2A、PIK3C2G、Rheb、PIK3CA、PIK3CB、PIK3CD |
| GO：0044424 | 细胞内部分 | 6 455 | <1.0×10-30 | AKT1、mTOR、PIK3C2A、PIK3C2G、Rheb、PIK3CA、PIK3CB、PIK3CD |
| GO：0005488 | 结合物 | 5 857 | 3.7×10-20 | AKT1、mTOR、PIK3C2A、PIK3C2G、Rheb、PIK3CA、PIK3CB、PIK3CD |
| GO：0005515 | 蛋白结合物 | 3 845 | 2.4×10-20 | AKT1、mTOR、PIK3C2A、PIK3C2G、Rheb、PIK3CA、PIK3CB、PIK3CD |
图10 肾缺血再灌注损伤+缺血预处理外泌体组和肾缺血再灌注损伤+常氧外泌体组肾小管细胞外泌体差异性靶基因KEGG分析（P值最小的20个通路条目） |
表4 肾缺血再灌注损伤+缺血预处理外泌体组和肾缺血再灌注损伤+常氧外泌体组肾小管细胞外泌体差异表达miRNA靶基因KEGG分析（与肾缺血再灌注损伤+常氧外泌体组比较） |
| 编号 | 说明 | Q值 | 基因（部分） |
|---|---|---|---|
| rno04151 | PI3K/AKT信号通路 | 1.05×10-10 | PIK3R5、PIK3CA、PIK3CB、AKT1 |
| rno04150 | mTOR信号通路 | 6.12×10-7 | PIK3CA、AKT1、Rheb、PIK3CD、PIK3R2 |
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黄世雪, 杨定平. 肾缺血-再灌注损伤的致病机制及治疗方法的研究进展[J]. 临床肾脏病杂志, 2019, 19(7): 529-533. DOI: 10.3969/j.issn.1671-2390.2019.07.013 .
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http://journal.yiigle.com/LinkIn.do?linkin_type=cma&DOI=10.3760/cma.j.cn441217-20231006-01004
所有作者均声明不存在利益冲突
陈家辉、张燕子：研究设计与实施、论文撰写；张艾莎、隋晓露、许云鹏、谢婷妃：数据整理、统计学分析；陈继红：研究设计与实施、研究指导、论文修改、经费支持
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表1 PCR引物序列
图1 各组大鼠肾功能和PI3K/AKT/mTOR信号通路表达比较（全自动生化分析仪或实时定量PCR或Western印迹）图3 各组大鼠肾脏病理改变（HE ×400）图4 各组大鼠细胞凋亡和增殖情况比较（TUNEL ×400）图5 肾小管细胞外泌体形态（电镜 ×20 000）图6 肾缺血再灌注损伤+缺血预处理外泌体组肾小管细胞外泌体颗粒粒径⁃浓度分布图和分布比例表2 肾缺血再灌注损伤+缺血预处理外泌体组肾小管细胞外泌体颗粒粒径⁃浓度分布比例图7 肾小管细胞外泌体CD63和TSG101蛋白表达图8 肾缺血再灌注损伤+缺血预处理外泌体组和肾缺血再灌注损伤+常氧外泌体组肾小管细胞外泌体差异表达miRNA分析图9 缺血预处理组和肾缺血再灌注损伤+常氧外泌体组肾小管细胞外泌体GO富集分析表3 肾缺血再灌注损伤+缺血预处理外泌体组和肾缺血再灌注损伤+常氧外泌体组肾小管细胞外泌体GO富集分析 (与肾缺血再灌注损伤+常氧外泌体组比较)图10 肾缺血再灌注损伤+缺血预处理外泌体组和肾缺血再灌注损伤+常氧外泌体组肾小管细胞外泌体差异性靶基因KEGG分析（P值最小的20个通路条目）表4 肾缺血再灌注损伤+缺血预处理外泌体组和肾缺血再灌注损伤+常氧外泌体组肾小管细胞外泌体差异表达miRNA靶基因KEGG分析（与肾缺血再灌注损伤+常氧外泌体组比较）/
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