Neutrophil extracellular traps induce glomerular endothelial cell dysfunction and pyroptosis in diabetic kidney disease

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease. Neutrophil extracellular traps (NETs) are a network structure composed of loose chromatin and embedded with multiple proteins. Here, we observed increased NETs deposition in the glomeruli of DKD patients and diabetic mice (streptozotocin-induced or db/db mice). After degrading NETs with DNase I, diabetic mice exhibited attenuated glomerulopathy and glomerular endothelial cell (GEC) injury. We also observed alleviated glomerulopathy and GEC injury in peptidylarginine deiminase 4 (PAD4)-knockout mice with streptozotocin-induced diabetes. In vitro, NET-induced GEC pyroptosis was characterized by pore formation in the cell membrane, dysregulation of multiple genes involved in cell membrane function, and high expression of pyroptosis-related proteins. Strengthening the GEC surface charge by oleylamine significantly inhibited NET-induced GEC pyroptosis. These results indicate that NET-induced alterations in GEC charge are associated with GEC pyroptosis in the pathogenesis of DKD and suggest that NETs are a potential therapeutic target for DKD. mRNA profiles of human renal glomerular endothelial cells (HRGECs) treated with neutrophil extracellular traps (NETs) or vehicle control, and the experiments were repeated five times.糖尿病肾病（Diabetic kidney disease, DKD）是终末期肾病的首要致病原因。中性粒细胞胞外陷阱（Neutrophil extracellular traps, NETs）是由松散染色质构成并嵌合多种蛋白质的网状结构。本研究观察到，DKD患者及糖尿病小鼠（链脲佐菌素诱导型或db/db小鼠）的肾小球内NETs沉积水平显著升高。采用脱氧核糖核酸酶I（DNase I）降解NETs后，糖尿病小鼠的肾小球病变与肾小球内皮细胞（glomerular endothelial cell, GEC）损伤均得到明显缓解。在链脲佐菌素诱导的糖尿病肽酰精氨酸脱亚氨酶4（peptidylarginine deiminase 4, PAD4）基因敲除小鼠中，同样观察到肾小球病变及GEC损伤得到改善。体外实验中，NETs诱导的GEC焦亡（pyroptosis）表现为细胞膜形成孔隙、多种参与细胞膜功能的基因表达失调以及焦亡相关蛋白高表达。通过油胺（oleylamine）增强GEC表面电荷，可显著抑制NETs诱导的GEC焦亡。上述结果表明，在DKD的发病机制中，NETs介导的GEC电荷改变与GEC焦亡密切相关，提示NETs可作为DKD潜在的治疗靶点。本数据集包含经NETs或溶剂对照处理的人肾肾小球内皮细胞（human renal glomerular endothelial cells, HRGECs）的mRNA表达谱，实验重复开展5次。