探究中性粒细胞胞外诱捕网加重特发性膜性肾病内皮功能紊乱并促进高凝状态形成

The Exploration of Neutrophil Extracellular Traps in Aggravating Endothelial Dysfunction and Promoting Hypercoagulability in Idiopathic Membranous Nephropathy

目的:探究中性粒细胞胞外诱捕网(NETs)在加重特发性膜性肾病(IMN)内皮功能紊乱并促进高凝形成、疾病进展中的作用。方法:根据KDIGO指南诊断标准,筛选年龄在18~80岁之间且于2019年3月—2020年12月在哈尔滨医科大学附属第一医院肾内科行肾活检病理诊断为IMN,病理分期为Ⅱ、Ⅲ期的患者,行双下肢彩超或双肾血管彩超,根据彩超对于急慢性血栓的诊断标准排除陈旧血栓影响,分为有血栓组、无血栓组各20例为IMN组,再选取健康者20例为对照组。采集实验对象空腹外周静脉血,分离血浆测定各组血浆中细胞外DNA(cell free-DNA,cf-DNA)、骨髓过氧化物酶(MPO)-DNA复合物、甲肾上腺素(NE)的含量。用IMN组患者及对照组血浆刺激正常中性粒细胞进行NETs生成实验,通过免疫荧光显微镜观察NETs结构,并进行NETs提取,进一步进行内皮刺激实验,采用人脐静脉内皮细胞(HUVECs),观察内皮结构以及促凝表型转变。进行DNA酶(DNase)及抗组织因子抑制实验,观察NETs的生成与凝血间的关系。结果:与对照组相比,IMN组外周血NETs相关指标明显上升,有血栓组较无血栓组上升更明显。IMN组较对照组血浆更易刺激中性粒细胞生成NETs,有血栓组较无血栓组生成NETs更明显。与对照组相比,IMN组表现为凝血时间明显缩短,凝血酶-抗凝血酶复合体(TAT)明显增加,有血栓组较无血栓组更明显,对有血栓组进行抑制试验,发现anti-TF、DNaseI能明显逆转这一现象。当内皮受到NETs刺激后,内皮间正常连接消失,间隙增大,同时内皮表达TF明显升高,表现为促凝表型。结论:NETs加剧内皮功能紊乱并驱动血栓倾向。针对NETs形成的策略可能为IMN提供一种潜在的治疗方法。

Objective:To explore the role of neutrophil extracellular traps(NETs)in aggravating endothelial dysfunction and promoting hypercoagulability and disease progression in idiopathic membranous nephropathy(IMN).Methods:According to the diagnostic criteria of the KDIGO guideline,patients between the ages of 18 and 80 who underwent renal biopsy in the hospital from March 2019 to December 2020 with a pathological diagnosis of IMN and the pathological stage of stage Ⅱ and Ⅲ were screened for double lower extremity color Doppler ultrasound or double kidney Angiography.According to the diagnostic criteria of color Doppler ultrasound for acute and chronic thrombus,the influence of old thrombus was excluded,and the patients were divided into the thrombosis group and the non-thrombotic group,20 cases in each group were the IMN group,and 20 cases were selected as the healthy control group.The fasting peripheral venous blood was collected from the experimental subjects,and the plasma was separated to determine the contents of extracellular DNA(cf-DNA),MPO-DNA complex and NE in the plasma of each group.Normal neutrophils were stimulated with plasma from the IMN group and the control group to conduct NETs generation experiments.The structure of NETs was observed by immunofluorescence microscope,and NETs were extracted,and further endothelial stimulation experiments were performed.Human umbilical vein endothelial cells(HUVECs)were used to observe endothelial structure and procoagulant phenotype transition.DNase and anti-tissue factor inhibition experiments were performed to observe the relationship between the formation of NETs and coagulation.Results:Compared with the control group,the related indexes of peripheral blood NETs in the IMN group increased significantly,and the increase in the thrombosis group was more obvious than that in the nonthrombotic group.The plasma of the IMN group was more likely to stimulate neutrophils to generate NETs than the control group,and the generation of NETs was more obvious in the thrombosis group than in the non-thrombotic group.Compared with the control group,the coagulation time(CT)was significantly shortened in the IMN group,and the thrombin-antithrombin complex(TAT)was significantly increased,and the thrombosis group was more obvious than the nonthrombotic group.The inhibition test was performed on the thrombosis group,and it was found that anti-TF and DNase I could obviously reverse this phenomenon.When the endothelium was stimulated by NETs,the normal connection between the endothelium disappeared,the gap increased,and the expression of TF in the endothelium was significantly increased,showing a procoagulant phenotype.Conclusion:NETs exacerbate endothelial dysfunction and drive thrombophilia.Strategies targeting the formation of NETs may offer a potential therapeutic approach for IMN.