基于肾脏类器官研究镁离子减轻顺铂诱导的急性肾损伤的作用和机制  

The effect and mechanism of magnesium ion alleviates cisplatin induced acute kidney injury based on kidney organoids

作　　者：吴欢 嵇姞 鲁敏 宁一纯 孙肇星 丁小强 俞小芳 WU Huan;JI Ji;LU Min;NING Yi-chun;SUN Zhao-xing;DING Xiao-qiang;YU Xiao-fang(Department of Nephrology,Zhongshan Hospital,Fudan University,Shanghai 200032,China)

机构地区：[1]复旦大学附属中山医院肾内科,上海200032

出　　处：《复旦学报（医学版）》2024年第4期455-464,483,共11页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金面上项目(82270772,81970667)。

摘　　要：目的利用肾脏类器官和HK-2细胞研究镁离子(Mg2+)对顺铂诱导的急性肾损伤(cisplatin-induced acute kidney injury,Cis-AKI)的作用,并探究可能的机制。方法首先利用人源性诱导多能干细胞(induced pluripotent stem cells,iPSCs)构建肾脏类器官,在此基础上构建Cis-AKI模型,利用HE染色观察肾脏类器官结构,通过免疫荧光染色观察标志物定位及cleaved caspase-3表达,通过qRT-PCR检测肾小管和肾小球标志物及炎症因子mRNA水平。随后将肾脏类器官及HK-2细胞随机分为对照组、顺铂组(Cis组)和Mg2+预处理组(Cis+Mg2+组)。通过CCK-8和ATP含量评估肾小管上皮细胞的活力;利用TUNEL染色检测肾小管上皮细胞凋亡情况;通过Western blot检测细胞凋亡通路的关键蛋白Bcl-2、Bax、cleaved caspase-3及有机阳离子转运体2(organic cation transporter 2,OCT2)表达;通过免疫荧光检测OCT2的定位与表达。结果肾脏类器官培养第10天的肾小管结构清晰,肾脏标志物大量表达;10μmol/L顺铂导致肾脏类器官结构破坏,cleaved caspase-3表达量和炎症因子mRNA水平显著升高,ATP含量显著下降。与Cis组相比,Cis+Mg2+组肾脏类器官ATP含量升高,TUNEL阳性细胞数减少,细胞凋亡相关蛋白表达显著下降,OCT2表达显著下降;而Cis+Mg2+组HK-2细胞活力、TUNEL阳性细胞数及凋亡相关蛋白均无明显改善,且几乎不表达OCT2。结论肾脏类器官是研究Cis-AKI发病与治疗的理想体外模型;Mg2+预处理可显著减轻顺铂所致肾脏类器官的损伤,其机制可能与OCT2的下调有关。Objective To investigate the role of magnesium ion(Mg2+)in cisplatin-induced acute kidney injury(Cis-AKI)in kidney organoids and HK-2 cells,as well as the potential mechanism.Methods Initially,we utilized human-derived induced pluripotent stem cells(iPSCs)to construct kidney organoids,and then built a Cis-AKI model based on kidney organoids.HE staining was used to observe the structure of kidney organoids,and immunofluorescence staining was used to observe the localization of markers and the expression of cleaved caspase-3.qRT-PCR was conducted to detect mRNA levels of tubular and glomerular markers,as well as inflammatory factors.Subsequently,the kidney organoids were randomly divided into control group,cisplatin group(Cis group),and Mg2+pretreatment group(Cis+Mg2+group).CCK-8 and ATP content assays were employed to evaluate the cell viability of renal tubular epithelial cells.TUNEL staining was performed to detect the apoptosis of renal tubular epithelial cells.Western blot was utilized to detect the expression of apoptosis-associated proteins(Bcl-2,Bax,cleaved caspase-3)and organic cation transporter 2(OCT2).Immunofluorescence was used to detect the localization and expression of OCT2.Results On the 10th day,the tubular structure in kidney organoids was visible,with abundant expression of renal markers.Treatment with 10μmol/L cisplatin resulted in structural damage to kidney organoids,significantly increased expression of cleaved caspase-3 and mRNA levels of inflammatory factors,and significantly decreased ATP content.Compared with the Cis group,the Cis+Mg2+group showed increased ATP content in kidney organoids,reduced number of TUNEL-positive cells,significantly decreased expression of apoptosis-associated proteins,and significantly decreased expression of OCT2.However,there was no significant improvement in HK-2 cell viability,the number of TUNEL-positive cells,or apoptosis-associated proteins in the Cis+Mg2+group,and HK-2 cells did not express OCT2.Conclusion Kidney organoid is an ideal in vitro model to s

关 键 词：顺铂 急性肾损伤(AKI) 镁离子(Mg2+) 有机阳离子转运体2(OCT2) 肾脏类器官 

分 类 号：R692.5[医药卫生—泌尿科学]