Li Yuting, Tan Peng, Liu Qianpan, Liu Man, Wang Yue, Kong Weixin, Sun Huaixin, Shao Xiang
Department of Geriatrics, Suzhou Kowloon Hospital, Shanghai Jiaotong University School of Medcine, Suzhou, 215000, China.
Department of Nephrology, Suzhou Kowloon Hospital, Shanghai Jiaotong University School of Medcine, Suzhou, 215000, China.
Cell Biochem Biophys. 2024 Sep;82(3):2401-2411. doi: 10.1007/s12013-024-01351-4. Epub 2024 Jun 15.
Diabetic nephropathy (DN) is a serious microvascular complication of diabetes characterized by structural and functional changes of kidneys. Human renal tubular epithelial (HK-2) cells are important for kidney recovery post injury and usually used for establishment of DN cell models. The study explored the role of microRNA (miR)-133a-3p in DN cell model and animal model. A cell model for DN was established via high glucose (HG) stimulation to HK-2 cells. Cell viability and apoptotic rate were measured by cell counting kit 8 and flow cytometry. Polymerase chain reaction was performed to quantify levels of miR-133a-3p and targets. Luciferase reporter assay was conducted to verify the binding of miR-133a-3p and MAML1. After establishment of a mouse model of DN, levels of renal function indicators were measured by biochemical analysis. Hematoxylin-eosin and periodic acid-schiff staining of kidney samples were performed to analyze histological changes. Western blotting was conducted to quantify levels of apoptotic markers, MAML1, and factors related to Notch signaling. Results showed that HG induced HK-2 cell apoptosis and the reduction of cell viability. MiR-133a-3p was lowly expressed in HG-stimulated HK-2 cells. Overexpressed miR-133a-3p improved HK-2 cell injury by increasing cell viability and hampering apoptosis under HG condition. In addition, miR-133a-3p directly targets MAML1 3'-untranslated region. MAML1 overexpression countervailed the repressive impact of miR-133a-3p on cell apoptosis in the context of HG. Moreover, miR-133a-3p inhibited the activity of Notch pathway by downregulating MAML1. MiR-133a-3p inhibits DN progression in mice, as evidenced by reduced fasting blood glucose level, improved levels of renal function parameters, and alleviation of kidney atrophy. In conclusion, miR-133a-3p improves HG-induced HK-2 cell injury and inhibits DN progression by targeting MAML1 and inactivating Notch signaling.
糖尿病肾病（DN）是糖尿病一种严重的微血管并发症，其特征为肾脏的结构和功能改变。人肾小管上皮（HK-2）细胞对于损伤后的肾脏恢复很重要，常用于建立DN细胞模型。本研究探讨了微小RNA（miR）-133a-3p在DN细胞模型和动物模型中的作用。通过对HK-2细胞进行高糖（HG）刺激建立DN细胞模型。采用细胞计数试剂盒8和流式细胞术检测细胞活力和凋亡率。进行聚合酶链反应以定量miR-133a-3p及其靶标的水平。进行荧光素酶报告基因检测以验证miR-133a-3p与MAML1的结合。建立DN小鼠模型后，通过生化分析检测肾功能指标水平。对肾脏样本进行苏木精-伊红和过碘酸-希夫染色以分析组织学变化。进行蛋白质免疫印迹法以定量凋亡标志物、MAML1以及与Notch信号通路相关因子的水平。结果显示，HG诱导HK-2细胞凋亡并降低细胞活力。miR-133a-3p在HG刺激的HK-2细胞中低表达。过表达miR-133a-3p可通过提高细胞活力和抑制HG条件下的细胞凋亡来改善HK-2细胞损伤。此外，miR-133a-3p直接靶向MAML1的3'-非翻译区。在HG环境下，MAML1过表达抵消了miR-133a-3p对细胞凋亡的抑制作用。此外，miR-133a-3p通过下调MAML1抑制Notch信号通路的活性。miR-133a-3p抑制小鼠DN进展，表现为空腹血糖水平降低、肾功能参数水平改善以及肾脏萎缩减轻。总之，miR-133a-3p通过靶向MAML1并使Notch信号失活来改善HG诱导的HK-2细胞损伤并抑制DN进展。
