Wang Wenzhe, Li Yongxia, Zhu Fan, Huang Yunfang
Department of Nephrology, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, No. 26, Shengli Street, Jiang'an District, Wuhan, Hubei 430014, China.
Toxicol Res (Camb). 2024 May 31;13(3):tfae079. doi: 10.1093/toxres/tfae079. eCollection 2024 Jun.
Diabetic nephropathy (DN) is the most common microvascular complication of diabetes mellitus (DM), being the second cause of end-stage renal disease globally. Podocyte injury is closely associated with DN developmen. Our study aimed to investigate the role of long non-coding RNA (lncRNA) TTN-AS1 in DN-associated podocyte injury.
The mouse podocyte cell line (MPC5) and human primary podocytes were stimulated by high glucose (HG; 30 nM glucose) to establish the cellular model of DN. Before HG stimulation, both podocytes were transfected with sh-TTN-AS1#1/2 or pcDNA3.1/STAT3 to evaluate the influence of TTN-AS1 knockdown or STAT3 overexpression on HG-induced podocyte injury. TTN-AS1 and STAT3 expression in both podocytes was examined by RT-qPCR. Cell viability and death were assessed by CCK-8 and LDH release assay. ELISA was adopted for testing IL-6 and TNF-α contents in cell supernatants. The levels of oxidative stress markers (ROS, MDA, SOD, and GSH) in cell supernatants were determined by commercial kits. Western blotting was used for measuring the expression of fibrosis markers (fibronectin and α-SMA and podocyte function markers (podocin and nephrin) in podocytes.
HG stimulation led to decreased cell viability, increased cell death, fibrosis, inflammation, cell dysfunction and oxidative stress in podocytes. However, knockdown of TTN-AS1 ameliorated HG-induced podocyte injury. Mechanically, the transcription factor STAT3 interacted with TTN-AS1 promoter and upregulated TTN-AS1 expression. STAT3 overexpression offset the protective effect of TTN-AS1 silencing on HG-induced podocyte damage.
Overall, STAT3-mediated upregulation of lncRNA TTN-AS1 could exacerbate podocyte injury in DN through suppressing inflammation and oxidative stress.
糖尿病肾病（DN）是糖尿病（DM）最常见的微血管并发症，是全球终末期肾病的第二大病因。足细胞损伤与DN的发生密切相关。本研究旨在探讨长链非编码RNA（lncRNA）TTN-AS1在DN相关足细胞损伤中的作用。
用高糖（HG；30 nM葡萄糖）刺激小鼠足细胞系（MPC5）和人原代足细胞，建立DN细胞模型。在HG刺激前，将sh-TTN-AS1#1/2或pcDNA3.1/STAT3转染至两种足细胞中，以评估TTN-AS1敲低或STAT3过表达对HG诱导的足细胞损伤的影响。通过RT-qPCR检测两种足细胞中TTN-AS1和STAT3的表达。采用CCK-8和LDH释放试验评估细胞活力和死亡情况。采用ELISA检测细胞上清液中IL-6和TNF-α的含量。使用商业试剂盒测定细胞上清液中氧化应激标志物（ROS、MDA、SOD和GSH）的水平。采用蛋白质免疫印迹法检测足细胞中纤维化标志物（纤连蛋白和α-SMA）和足细胞功能标志物（足突蛋白和nephrin）的表达。
HG刺激导致足细胞活力降低、细胞死亡增加、纤维化、炎症、细胞功能障碍和氧化应激。然而，敲低TTN-AS1可改善HG诱导的足细胞损伤。机制上，转录因子STAT3与TTN-AS1启动子相互作用并上调TTN-AS1表达。STAT3过表达抵消了TTN-AS1沉默对HG诱导的足细胞损伤的保护作用。
总体而言，STAT3介导的lncRNA TTN-AS1上调可通过抑制炎症和氧化应激加重DN中的足细胞损伤。
