Department of Nephrology, The First Hospital of China Medical University, No. 155 Nanjing Bei Street, Heping District, Shenyang, 110001, China.
Department of Nephrology, The Fourth Hospital of China Medical University, Shenyang, 110000, China.
Mol Cell Biochem. 2024 Sep;479(9):2483-2496. doi: 10.1007/s11010-023-04843-8. Epub 2023 Oct 13.
The current study aimed to explore the role and underpinning molecular mechanisms of metformin in renal cellular injury induced by high glucose levels. Male C57BL/KsJ (db/db) and (db/m +) mice were utilized in this study. The experimental group was administered 1 mg/mL of metformin through drinking water. Renal tissues were harvested for hematoxylin and eosin (HE) staining, superoxide dismutase (SOD) activity detection, biochemical indices analysis, Western blotting, and qRT-PCR. HK-2 cells were utilized for Nrf2 siRNA transfection and to establish a high level of glucose-induced cell models. Metformin was administered at a concentration of 1 mmol/L in the experimental group. Cellular viability was assessed using CCK-8, whereas acridine orange (AO) staining and LC3-mitotracker co-localization staining were employed to evaluate autophagy. The expression of Nrf2, P21, LC3, PTEN-induced putative kinase 1 (PINK1), translocase of outer mitochondrial membrane 20 (TOMM20), and Kelch-like ECH-associated protein 1 (Keap1) were determined through Western blotting and qRT-PCR. Metformin mitigated renal tissue inflammatory damage in diabetic mice, as indicated by upregulated expression of Nrf2, PINK1, LC3, and TOMM20, and downregulated expression of Keap1 and P21. High level of glucose treatment in HK-2 cells resulted in decreased autophagy, and reduced expression of Nrf2, PINK1, LC3, and TOMM20 alongside elevated the expression of Keap1 and P21. Notably, metformin treatment partially counteracted these effects. Nrf2 knockdown intensified these phenomena in the high level of glucose-induced model. Protein-protein interaction network analysis indicated that Nrf2 could regulate the majority autophagy-related proteins via Keap1. Metformin modulates mitochondrial autophagy in high glucose-induced renal tubular epithelial senescence via the Keap1/Nrf2 pathway.
本研究旨在探讨二甲双胍在高糖诱导的肾细胞损伤中的作用及其潜在的分子机制。该研究使用雄性 C57BL/KsJ（db/db）和（db/m+）小鼠。实验组通过饮用水给予 1mg/ml 的二甲双胍。采集肾组织进行苏木精和伊红（HE）染色、超氧化物歧化酶（SOD）活性检测、生化指标分析、Western blot 和 qRT-PCR。使用 Nrf2 siRNA 转染 HK-2 细胞并建立高糖诱导的细胞模型。实验组给予 1mmol/L 二甲双胍。使用 CCK-8 评估细胞活力，使用吖啶橙（AO）染色和 LC3-mitotracker 共定位染色评估自噬。通过 Western blot 和 qRT-PCR 检测 Nrf2、P21、LC3、PTEN 诱导的假定激酶 1（PINK1）、外膜线粒体转位酶 20（TOMM20）和 Kelch 样 ECH 相关蛋白 1（Keap1）的表达。二甲双胍减轻了糖尿病小鼠的肾组织炎症损伤，表现为 Nrf2、PINK1、LC3 和 TOMM20 的表达上调，Keap1 和 P21 的表达下调。HK-2 细胞高糖处理导致自噬减少，Nrf2、PINK1、LC3 和 TOMM20 的表达下调，Keap1 和 P21 的表达上调。值得注意的是，二甲双胍处理部分逆转了这些效应。高糖诱导模型中 Nrf2 敲低加剧了这些现象。蛋白质-蛋白质相互作用网络分析表明，Nrf2 可以通过 Keap1 调节大多数自噬相关蛋白。二甲双胍通过 Keap1/Nrf2 通路调节高糖诱导的肾小管上皮细胞衰老中的线粒体自噬。
