CerS6介导的线粒体神经酰胺蓄积在DKD足细胞炎症性损伤中的作用及机制

天然免疫激活与DKD发生发展密切相关，但其机制仍不明确。申请者前期测序发现DKD模型肾小球脂代谢及天然免疫通路异常激活，提示两者活化是DKD状态的主要生物学事件。结合脂质蓄积可诱发微炎症以及DKD病人临床及病理特点，本项目提出脂质蓄积可激活天然免疫炎症。前期实验发现高糖刺激引起特异表达于足细胞的神经酰胺合成关键酶CerS6上调；高糖刺激促进足细胞mtDNA胞质泄漏，导致天然免疫信号cGAS-STING激活。鉴此，本项目拟以mtDNA泄漏为切入点，通过糖尿病动物模型、双荧光报告小鼠分离的原代足细胞、足细胞系和DKD肾活检标本，明晰CerS6在DKD足细胞炎性损伤中的作用；通过条件性基因敲除小鼠、体外基因调节，揭示CerS6介导神经酰胺蓄积引起mtDNA泄漏进而激活天然免疫炎症的分子机制。本项目旨在从天然免疫角度揭示DKD脂代谢与微炎症状态的关系，为防治DKD提供新的理论依据和治疗靶点。

Innate immune activation is closely related to the occurrence and development of DKD, but its pathogenic mechanism is still unclear. Through transcriptomics analysis, applicant found that innate immune pathways and lipid metabolism pathways were abnormally activated in DKD models, suggesting that the activation of both is the main biological event in the DKD state. Previous studies have demonstrated that lipid accumulation can induce micro-inflammation. Combining with the clinical and pathological characteristics of DKD patients, we propose that lipid accumulation can activate innate immune inflammation. Preliminary experiments have found that high glucose can stimulate the up-regulation of CerS6, which is a key enzyme for ceramide synthesis specifically expressed in podocytes; high glucose stimulation promotes mtDNA leakage from mitochondria to the cytoplasm and leads to the activation of the innate immune signal cGAS-STING. Two diabetic models, primary podocytes isolated from dual-fluorescence reporter mice, podocyte lines, and kidney biopsy specimens from DKD patients will be used to evaluate the relationship between CerS6 and inflammatory damage; Conditional gene knockout mice and gene regulation intervention methods in vitro will be used to reveal the molecular mechanism that CerS6-mediated ceramide accumulation can promote the leakage of mtDNA and subsequently lead to the activation of innate immunity. This project aims to reveal the relationship between DKD lipid metabolism and microinflammation from the perspective of innate immunity，and to provide new theoretical basis and therapeutic target for the prevention and treatment of DKD.