Gou X-Y, Li Y, Fan X-P
Department of Radiology, Suining Central Hospital, Suining, Sichuan Province, China.
Physiol Res. 2025 Mar 24;74(1):79-92. doi: 10.33549/physiolres.935445.
To explore the effects and underlying mechanisms of Mdivi-1 on three common clinical models of acute kidney injury (AKI). Three common AKI cell models were constructed, classified into the control group (human renal tubular epithelial cells [HK-2] cells), the Iohexol group (HK-2 cells treated with Iohexol), the Genta group (HK-2 cells treated with Gentamicin), and the Cis group (HK-2 cells treated with Cisplatin). To explore the optimal protective concentration of Mdivi-1 for each AKI cell model, the experimental design consisted of the following seven groups: the control group (HK-2 cells cultured in medium), three injury groups (HK-2 cells subjected to Iohexol, Gentamicin, or Cisplatin), and the corresponding protection groups (with a certain concentration of Mdivi-1 added to each injury group). Cellular survival and apoptosis, reactive oxygen species (ROS) levels, and the expression of recombinant Sirtuin 3 (SIRT3) in each group were measured. Mitochondrial fission and fusion dynamics in cells were observed under an electron microscope. To explore relevant pathways, the changes in relevant pathway proteins were analyzed through Western blotting. The half maximal inhibitory concentration (IC50) values were 150.06 mgI/ml at 6 h in the Iohexol group, 37.88 mg/ml at 24 h in the Gentamicin group, and 13.48 microM at 24 h in the Cisplatin group. Compared with the control group, the three injury groups showed increased cell apoptosis rates and higher expressions of apoptotic proteins in HK-2 cells, with an accompanying decrease in cell migration. After the addition of corresponding concentrations of Mdivi-1, the optimal concentrations were 3 µM in the Iohexo-3 group, 1 microM in the Genta-1 group, and 5 µM in the Cis-5 group, HK-2 cells showed the highest survival rate, reduced apoptosis, decreased mitochondrial ROS and SIRT3 expression, and reduced mitochondrial fission and autophagy when compared with each injury group. Further verification with Western blot analysis after the addition of Mdivi-1 revealed a reduction in the expressions of mitochondrial fission proteins DRP1, Nrf2, SIRT3, Caspase-3, Jun N-terminal Kinase (JNK)/P-JNK, NF-kappaB, Bcl2, and autophagic protein P62, as well as reduced ROS levels. Mdivi-1 had protective effects on the three common AKI cell models by potentially reducing mitochondrial fission in cells and inhibiting the production of ROS through the mediation of the NF- B/JNK/SIRT3 signaling pathway, thereby exerting protective effects. Key words AKI, Cisplatin, Gentamicin, Iohexol, Mdivi-1.
探讨Mdivi-1对三种常见急性肾损伤（AKI）临床模型的作用及潜在机制。构建三种常见的AKI细胞模型，分为对照组（人肾小管上皮细胞[HK-2]细胞）、碘海醇组（用碘海醇处理的HK-2细胞）、庆大霉素组（用庆大霉素处理的HK-2细胞）和顺铂组（用顺铂处理的HK-2细胞）。为探究Mdivi-1对每种AKI细胞模型的最佳保护浓度，实验设计包括以下七组：对照组（在培养基中培养的HK-2细胞）、三个损伤组（分别用碘海醇、庆大霉素或顺铂处理的HK-2细胞）以及相应的保护组（在每个损伤组中添加一定浓度的Mdivi-1）。检测每组细胞的存活率和凋亡率、活性氧（ROS）水平以及重组沉默调节蛋白3（SIRT3）的表达。在电子显微镜下观察细胞中的线粒体分裂和融合动态。为探究相关途径，通过蛋白质免疫印迹法分析相关途径蛋白的变化。碘海醇组在6小时时的半数抑制浓度（IC50）值为150.06mgI/ml，庆大霉素组在24小时时为37.88mg/ml，顺铂组在24小时时为13.48μM。与对照组相比，三个损伤组的HK-2细胞凋亡率增加，凋亡蛋白表达升高，同时细胞迁移减少。添加相应浓度的Mdivi-1后，碘海醇-3组的最佳浓度为3μM，庆大霉素-1组为1μM，顺铂-5组为5μM，与各损伤组相比，HK-2细胞的存活率最高，凋亡减少，线粒体ROS和SIRT3表达降低，线粒体分裂和自噬减少。添加Mdivi-1后通过蛋白质免疫印迹分析进一步验证发现，线粒体分裂蛋白DRP1、Nrf2、SIRT3、半胱天冬酶-3、应激活化蛋白激酶（JNK）/磷酸化JNK、核因子κB、Bcl2以及自噬蛋白P62的表达降低，同时ROS水平降低。Mdivi-1可能通过减少细胞中的线粒体分裂并通过核因子κB/JNK/SIRT3信号通路介导抑制ROS的产生，从而对三种常见的AKI细胞模型发挥保护作用。关键词：急性肾损伤；顺铂；庆大霉素；碘海醇；Mdivi-1
