南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (05): 508-.doi: 10.12122/j.issn.1673-4254.2019.05.02

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阻断Pannexin-1 可减轻肾脏组织炎症细胞浸润和缓解顺铂诱导的急性肾损伤

刘崇斌，黄柳维，李彩珍，沈燕婷，王骏   

  • 出版日期:2019-05-20 发布日期:2019-05-20

Blocking pannexin-1 alleviates cisplatin-induced acute kidney injury in mice by reducing renal inflammatory cell infiltration

  • Online:2019-05-20 Published:2019-05-20

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摘要： 目的探讨阻断pannexin-1在顺铂致急性肾损伤中的作用。方法将26只6~8周龄雄性C57BL/6小鼠随机分为对照组、顺 铂模型组、顺铂+Carbenoxolone 处理组。顺铂模型组、顺铂+Carbenoxolone 处理组予顺铂20 mg/kg 腹腔注射1 次，顺铂+ Carbenoxolone处理组于顺铂处理前30 min、处理后24、48 h分别腹腔注射Carbenoxolone 20 mg/kg，所有组于顺铂处理72 h处 死小鼠，留取血浆及肾组织样品进行下一步检测。RT-qPCR及Western blot 检测肾组织内pannexin-1 的表达水平；检测血浆 BUN、Scr水平评估肾功能；PAS 染色评估肾组织病理学改变；免疫组化检测肾损伤分子1（KIM-1）表达水平及RT-qPCR检测肾 损伤分子1与中性粒细胞明胶酶相关脂质运载蛋白（NGAL）mRNA水平评估肾小管损伤情况；免疫荧光检测肾组织F4/80+巨噬 细胞及CD4+ T细胞浸润水平；RT-qPCR及Western blot检测肾组织pannexin-1的mRNA及蛋白表达水平；用终浓度50 μmol/L 顺铂刺激人近端小管上皮细胞株HK-2 细胞4、6、12、18、24 h 构建体外顺铂致急性肾损伤模型；RT-qPCR及Western blot 检测 HK-2细胞pannexin-1的表达水平。结果与对照组相比，顺铂模型组肾组织pannexin-1蛋白水平及mRNA水平表达上调（P< 0.005）；体外实验结果显示，与对照组相比，顺铂模型组的pannexin-1蛋白水平及mRNA水平表达水平上调（P<0.005）；与顺铂 模型组相比，体内应用pannexin-1抑制剂Carbenoxolone处理后，可减轻顺铂所致的肾脏损伤，肾小管损伤程度明显减轻，顺铂+ Carbenoxolone处理组的血浆BUN、Scr水平回降（P<0.01），肾组织KIM-1、NGAL表达减少（P<0.05），肾组织浸润的F4/80+巨噬 细胞（P<0.01）及CD4+ T 细胞（P<0.05）减少。结论在顺铂所致急性肾损伤小鼠模型中，Pannexin-1 表达显著上调，阻断 pannexin-1减少炎症细胞浸润，可减轻肾损伤。

Abstract: Objective To investigate the effect of blocking pannexin-1 against acute kidney injury induced by cisplatin. Methods Twenty-six male C57BL/6 mice aged 6-8 weeks were randomly divided into control group, cisplatin model (Cis) group and cisplatin + carbenoxolone treatment group (Cis + CBX). In Cis group and Cis +CBX group, the mice were injected intraperitoneally with 20 mg/kg of cisplatin and with CBX (20 mg/kg) at 30 min before and 24 and 48 h after cisplatin inhjection, respectively. All the mice were sacrificed at 72 h after cisplatin injection, and plasma and kidney samples were collected for testing mRNA and protein expression levels of pannexin-1 in the renal tissue using RT-qPCR and Western blotting and for detecting plasma creatinine and BUN levels; the pathological changes in the renal tissues were observed using Periodic Acid-Schiff staining. The expression of kidney injury molecule 1 (KIM-1) was examined using immunohistochemistry and the mRNA expressions of KIM-1 and neutrophil gelatinase- related lipid transport protein (NGAL) were detected by RT-qPCR to evaluate the injuries of the renal tubules. The infiltration of F4/80-positive macrophages and CD4-positive T cells were observed by immunofluorescence. In the in vitro experiment, human proximal tubule epithelial cell line HK-2 was stimulated with 50 μmol/L cisplatin to establish a cell model of acute kidney injury, and the mRNA and protein expressions of pannexin-1 were detected by RT-qPCR and Western blotting at 4, 6, 12, 18 and 24 h after the stimulation. Results Compared with the control mice, the cisplatin-treated mice showed significantly up-regulated protein levels (P<0.05) and mRNA levels (P< 0.005) of pannexin-1 in the kidney tissue. Cisplatin stimulation also caused significant increases in the protein levels (P<0.005) and mRNA levels (P<0.005) of pannexin-1 in cultured HK-2 cells. Compared with cisplatin-treated mice, the mice treated with both cisplatin and the pannexin-1 inhibitor CBX showed obviously lessened kidney pathologies and milder renal tubular injuries with significantly reduced plasma BUN and Scr levels (P<0.01), expressions of KIM-1 and NGAL in the kidney (P< 0.05), and infiltration of F4/80-positive macrophages (P<0.01) and CD4- positive T cells (P<0.05) in the kidney tissues. Conclusion In cisplatin induced acute kidney injury mice model, Pannexin-1 expression is up-regulated in the kidneys tissue, and blocking pannexin-1 alleviates the acute kidney injury via reducing renal inflammatory cell infiltration.