摘要
目的构建乙二醇(EG)中毒急性肾损伤(AKI)大鼠模型,分析发病机制,评价模型的特点,为今后进一步研究EG中毒AKI的病理生理学特征及药物干预措施提供实验动物数据参考。方法采用单次灌胃不同剂量(4和8g/kg)EG构建急性中毒模型。观察各组大鼠的存活情况,于造模后48h留取标本,计算肾脏指数。ELISA法检测各组存活大鼠血清中肾损伤生物标志肾损伤分子1(Kim-1)、中性粒细胞明胶酶相关脂质运载蛋白(NGAL)及肌酐(Cr)的含量;苏木素-伊红(HE)、PAS及Masson染色观察肾损伤病理表现;Pizzolato染色及偏振光显微镜观察肾草酸钙结晶沉积情况;Westernblot检测肾组织中凋亡相关蛋白Bax、cleaved Caspase-3及抗凋亡蛋白Bcl-2的相对表达;高效液相色谱法检测血清中EG代谢产物乙醇酸及草酸的含量。结果EG(4g/kg)及EG(8g/kg)组大鼠48h累积生存率分别为86.7%和53.3%,与对照组及EG(4g/kg)组比较,EG(8g/kg)组48h累积生存率明显下降(χ^(2)_(对照组)=8.898.P<0.01;χ^(2)_(EG(4g/kg))组=4.020,P<0.05)。ELISA结果显示,EG中毒后血清肾损伤标志物Kim-1、NGAL、Cr及肾脏指数均明显升高且具有剂量依赖性(F_(kim-1)=68.386,F_(NGAL)=449.157,F_(Cr)=24.967,F_(肾脏指数)=56.103,P<0.01)。病理结果显示,EG中毒后呈现出不同程度的肾小管、肾小球病理损伤,EG(8g/kg)组肾小管损伤指数评分及草酸钙结晶沉积明显增加(F_(肾小管损伤指数评分)=284.747,F_(pualao)=113.645,F_(偏振光)=154.788,P<0.01)。Westen blot结果显示,EG中毒组肾组织中凋亡相关因子Bax、cleaved Caspase-3明显升高(F_(Bax)=21.149,Fcleaved C_(aspase-3)=63.101,P<0.01),抗凋亡因子Bcl-2明显下降(F_(Bcl-2)=343.673,P<0.01),且均具有剂量依赖性。高效液相色谱结果显示,EG中毒组大鼠血清EG毒性代谢产物乙醇酸及草酸的含量显著增加且具有剂量依赖性(F_(乙醇酸)=21.615,F_(草酸)=44.994,P<0.01)。结论单次大剂量EG 8g/kg经口暴露可成功构建急性EG中毒AKI并发大量草酸钙结晶肾内沉积大鼠模型,其致AKI的机制可能包括肾组织细胞的凋亡、肾小管及肾小球的损伤坏死以及草酸钙结晶的小管内沉积。
Objective To establish a rat model of acute kidney injury(AKI)induced by ethylene glycol(EG)poisoning,analyze the pathogenesis,and evaluate the characteristics of the model.This will provide experimental animal data as a reference for further research on the pathophysiological characteristics and drug intervention measures of EG-induced AKI.Methods An acute poisoning model was established by a single intragastric administration of ethylene glycol(EG)at different doses(4 g/kg,8 g/kg).The survival of rats in each group was observed,and the samples were collected 48 hours after modeling.The kidney index was calculated,and the serum levels of kidney injury biomarkers Kidney Injury Molecule 1(Kim-1),Neutrophil Gelatinase Associated Lipocalin(NGAL),and creatinine(Cr)were detected using ELISA.The pathological manifestations of renal injury were observed using HE,PAS,and Masson staining.Calcium oxalate crystal deposition in the kidney was observed using Pizzolato staining and polarized light microscopy.Western blot was used to detect the relative expressions of apoptosis-related proteins Bax,cleaved Caspase-3,and anti-apoptotic protein Bcl-2 in kidney tissues.Additionally,the contents of glycolic acid and oxalic acid EG metabolites in serum were determined using high-performance liquid chromatography.Results The 48-hour cumulative survival rate of rats in EG(4 g/kg)and EG(8 g/kg)groups was 86.7%and 53.3%,respectively.Compared with the control group and EG(4 g/kg)group,the 48-hour cumulative survival rate of rats in EG(8 g/kg)group was significantly decrease(χ^(2)_(control group)=8.898.P<0.01;χ^(2)_(EG(4g/kg))group=4.020,P<0.05)ELISA result showed that the serum markers of kidney injury Kim-1,NGAL,Cr and kidney index were significantly increased in a dose-dependent way after EG poisoning(F_(kim-1)=68.386,F_(NGAL)=449.157,F_(Cr)=24.967,F_(kidney index)=56.103,P<0.01)。Pathological resul showed that renal tubule and glomerular lesions of different degrees were present after EC poisoning.The renal tubule injury index score and calcium oxalate crystal deposition in EC(8 g/kg)group were significantly increased(F_(renal tubule injury index seore)=284.747,F_(pizzolato)=113.645,F_(polarizing)=154.788,P<0.01),Western blot result showed that the apoptosis-related factors Bax and cleaved Caspase-3 in renal tissues of the EG poisoning group were significantly inereased(F_(Bax)=21.149,Fcleaved C_(aspase-3)=63.101,P<0.01).The anti-aptosis factor Bel-2 was sigificantly decreased (F_(Bcl-2)=343.673,P<0.01)in a dose-dependent manner.The result of high performance liquid chromatography showed that the contents of glycolic acid and oxalic acid in serum of EG toxic metabolites were significantly increased in a dose-dependent manner(F_(glycolic acid)=21.615,F_(oxalic acid)=44.994,P<0.01).Conclusion 1The rat model of acute EG intoxication AKI complicated with massive calcium oxalate crystal renal deposition can be successfully established by a single high-dose EG exposure of 8 g/kg orally.The mechanism of AKI may include apoptosis of renal tissue cells,injury and necrosis of renal tubules and glomeruli,and tubular deposition of calcium oxalate crystals.
作者
杨凯
彭鹏
杨建中
张晓霞
迟骋
李鑫鹏
肖克来提·霍加合买提
YANG Kai;PENG Peng;YANG Jian-zhong;ZHANG Xiao-xia;CHI Cheng;LI Xin-peng;XIAOKELAITI Huojiahemaiti(Emergency Trauma Center,First Affiliated Hospital of Xinjiang Medical University,Urumqi,Xinjiang 830011,China;Emergency Department of the People's Hospital of Peking University,Beijing 100044,China)
出处
《毒理学杂志》
CAS
2024年第5期352-359,共8页
Journal of Toxicology
基金
新疆维吾尔自治区自然科学基金面上项目(2022D01C226)
新疆维吾尔自治区研究生实践创新项目(XJ2023G164)。
关键词
乙二醇
急性中毒
急性肾损伤
大鼠
Ethylene glycol
Acute poisoning
Acute kidney injury
Rat
作者简介
杨凯,博士研究生在读,研究方向:急危重症与中毒;通讯作者:肖克来提·霍加合买提,主治医师,研究方向:急危重症与中毒。
