摘要
目的构建BALB/c小鼠免疫复合物性肾损伤模型,探讨观察此类肾损伤的早期、简便、灵敏检测方法和候选生物标志物,为生物技术药物非临床安全性评价毒性试验中肾损伤检测提供参考。方法BALB/c小鼠随机分为溶媒对照组和阳离子化牛血清白蛋白(C-BSA)低、中、高剂量造模组,每组10只。诱导免疫时,造模组动物给予2 mg·mL^(−1) C-BSA和弗氏不完全佐剂1∶1等体积混合乳剂(1 mg·mL^(−1)、5 mL·kg^(−1)),而溶媒对照组动物给予磷酸盐(PBS)缓冲液与弗氏不完全佐剂1∶1等体积混合乳剂,sc诱导免疫,每周1次,共2次;加强免疫时,各组动物均尾iv给予C-BSA,溶媒对照组和C-BSA低、中、高剂量组剂量分别为0、2.5、5.0、10.0 mg·kg^(−1),每3天给予1次,共5次。造模期末,检测造模期末小鼠24 h尿蛋白含量;检测凝血指标,包括凝血酶原时间(PT)、活化部分凝血活酶时间(APPT)以及纤维蛋白原含量(Fbg);检测血清血尿素氮(BUN)、血肌酐(SCr)以及三酰甘油(TG)水平;摘取主要脏器称质量并计算脏体比、脏脑比;HE染色和特殊染色[过碘酸-希夫(PAS)染色、Masson三色染色、过碘酸六胺银(PASM)染色]后,光学显微镜下检查肾组织形态学改变;免疫组化观察肾脏补体成分3(C3)、免疫球蛋白G(IgG)、结蛋白水平;免疫荧光观察肾脏C3水平;透射电子显微镜观察肾脏肾小球基底膜、足细胞足突的变化及毛细血管内皮细胞下有无电子致密物沉积。结果C-BSA各剂量组动物在尾iv给予C-BSA后出现不同程度精神萎靡、活动减少、皮肤发红和呼吸急促等过敏反应症状,溶媒对照组动物给药后无明显异常;在加强免疫2周后,与溶媒对照组比较,C-BSA低、中和高剂量组动物体质量增长显著缓慢(P<0.05);C-BSA低、中、高剂量组动物尿蛋白呈阳性。与溶媒对照组比较,C-BSA各组动物BUN、SCr、TG均未见规律改变,仅高剂量组动物的BUN升高,TG降低;C-BSA中、高剂量组动物PT显著降低(P<0.05),低、中剂量组动物APPT显著降低(P<0.05),低、中、高剂量组动物Fbg含量显著降低(P<0.001);C-BSA低、中、高剂量组动物肾脏的脏体比显著升高(P<0.01),且低、中和高剂量组的脾脏绝对质量和脏体比亦显著升高(P<0.05、0.01)。肾脏组织病理学检查及特殊染色检查发现C-BSA中、高剂量组动物肾小球系膜基质增加、基底膜增厚;免疫组化结果显示,与溶媒对照组比较,C-BSA低、中、高剂量组动物的C3、IgG、结蛋白表达水平均显著升高(P<0.001),高剂量组IgG水平显著高于低、中剂量组(P<0.05);免疫荧光结果显示,与溶媒对照组比较,C-BSA高剂量组C3水平显著升高(P<0.001);透射电镜结果显示C-BSA低、中、高剂量组动物肾脏肾小球基底膜增厚、足细胞足突部分融合,C-BSA中、高剂量组动物肾脏肾小球基底膜可见少量电子致密物沉积。结论成功建立了免疫复合物性肾损伤模型和早期、简便、灵敏的检测方法,C3、IgG和结蛋白可作为生物技术药物非临床安全性评价毒性试验中肾损伤检测的候选生物标志物。
Objective Construct an immune complex-induced renal injury model in BALB/c mice,explore early,simple,sensitive detection methods and candidate biomarkers for detecting such renal injury,and provide references for renal injury detection of biopharmaceuticals in toxicity studies during nonclinical safety evaluation.Methods BALB/c mice were randomly divided into solvent control group and C-BSA low,medium,and high dose modeling groups,with 10 mice in each group.During induction immunization,the animals of model groups were administrated with an emulsion made of a mixture of 2 mg·mL^(−1) C-BSA and equal volume of Freund's incomplete adjuvant via subcutaneous injection,while the animals of vehicle control group were given an emulsion made of a mixture of PBS buffer and equal volume of Freund's incomplete adjuvant.Once a week,twice in total.During booster immunization,all animals in each group were administered with 0,2.5,5.0,and 10.0 mg·kg^(−1) C-BSA via intravenous injection through tail vein for the vehicle control group and the low,medium,and high dose groups of C-BSA,respectively.Once every three days,five times in total.At the end of the modeling period,24-hour urine protein content of mice were detected.Coagulation indicators were also detected,including prothrombin time(PT),activated partial thromboplastin time(APPT),and fibrinogen(Fbg).Blood urea nitrogen(BUN),serum creatinine(SCr),and triglyceride(TG)levels were detected.The main organs were weighed and calculated the mass,organ weight to body weight ratio and organ weight to brain weight ratio.Slides of kidneys prepared with HE staining and special staining[periodic acid Schiff(PAS)staining,Masson trichrome staining,and periodic acid silver methenamine(PASM)staining],the morphological changes of renal tissue were examined under an optical microscope.Immunohistochemical examination of renal complement component 3(C3),immunoglobulin G(IgG),and desmin levels and immunofluorescence examination of renal C3 levels were carried out.Transmission electron microscopy(TEM)was used to observe the changes in the glomerular basement membrane and foot process of podocytes of the kidneys,as well as the presence of electron dense deposits under capillary endothelial cells.Results The animals in each modeling group showed varying degrees of allergic reaction symptoms such as lethargy,decreased activity,skin redness,and short breath after administration of C-BSA via intravenous injection,whereas the animals in the solvent control group showed no significant abnormalities after the administration.After two weeks of booster immunization,compared with the vehicle control group,the animals in the low,medium,and high dose modeling group showed significant slow body mass growth(P<0.05).The low,medium,and high dose modeling groups of C-BSA showed positive urine protein.Compared with the solvent control group,there were no regular changes in BUN,SCr,and TG in the animals of modeling groups.Only the animals of high dose modeling group showed an increase in BUN and a decrease in TG.PT of animals in the medium and high dose modeling groups was significantly reduced(P<0.05),APPT of animals in the low and medium dose modeling groups was significantly reduced(P<0.05),and the Fbg of animals in the low,medium,and high dose modeling groups was significantly reduced(P<0.001).The organ weight to body weight ratio of the kidneys in the low,medium,and high dose modeling groups was significantly increased(P<0.01),and the absolute mass and organ weight to body weight ratio of the spleen in the low,medium,and high dose modeling groups were also significantly increased(P<0.05,0.01).Histopathological examination of kidneys showed that the increased mesangial matrix and thickened basement membrane of animals in the middle and high dose modeling groups.The immunohistochemical examination results showed that compared with the vehicle control group,the expression levels of C3,IgG,and desmin in the low,medium,and high dose modeling groups were significantly increased(P<0.001),while the IgG levels in the high dose modeling group were significantly higher than those in the low and medium dose modeling groups(P<0.05).The immunofluorescence examination results showed that compared with the vehicle control group,the high dose modeling group had a significant increase in C3 levels(P<0.001).The TEM results showed that animals in the low,medium,and high dose modeling groups had thickened glomerular basement membrane and partially fused foot processes of podocytes.A small amount of electron dense material was observed in the glomerular basement membrane of animals in the medium and high dose modeling groups.Conclusion An immune complex-induced renal injury model,as well as early,simple and sensitive detection methods were successfully established.C3,IgG and desmin might be candidate biomarkers for detecting immune complexinduced renal injury in the toxicity studies during nonclinical safety evaluation of biopharmaceuticals.
作者
郑少秋
张亚群
缪成贤
李一昊
王娅
刘军
王先浩
王明
孙璐瑶
吕建军
汪溪洁
ZHENG Shaoqiu;ZHANG Yaqun;MIAO Chengxian;LI Yihao;WANG Ya;LIU Jun;WANG Xianhao;WANG Ming;SUN Luyao;LYU Jianjun;WANG Xijie(Jiangxi University of Traditional Chinese Medicine,Nanchang 330103,China;Innostar Biotechnology Nantong Co.,Ltd.,Nantong 226133,China;Yangtze Delta Advanced Research Institute,Yangtze Delta Pharmaceutical College Nantong,Nantong 226133,China;Shanghai Innostar Biotechnology Co.,Ltd.,Shanghai 201203,China)
出处
《药物评价研究》
CAS
2023年第5期934-943,共10页
Drug Evaluation Research
基金
江苏省新药一站式高效非临床评价公共服务平台建设(BM2021002)。
