1.西南医科大学附属医院肾病内科, 四川 泸州 646000;2.四川省肾疾病临床医学研究中心, 四川 泸州 646000;3.代谢性血管疾病四川省重点实验室, 四川 泸州 646000
1. Department of Nephrology, Affiliated Hospital of Southwest Medical University, Luzhou 646000, China. 2. Sichuan Clinical Research Center for Nephropathy, Luzhou 646000. 3. Sichuan Key Laboratory of Metabolic Vascular Diseases, Luzhou 646000
目的 观察腺嘌呤诱导慢性肾病大鼠模型肾铁死亡相关因子的变化并探讨铁死亡参与慢性肾病进展的机制。 方法 20 只大鼠随机分为对照组(Con)和模型组(CKD),CKD 组予以2. 5%腺嘌呤灌胃诱导,所有大鼠于6 周末处死。留取大鼠血样检测血清尿素氮(BUN)、肌酐(Scr);HE 染色观察肾病理改变;Masson 染色观察肾小管间质纤维化情况;检测肾组织铁含量、丙二醛(MDA)和谷胱甘肽(GSH)含量;免疫组化染色观察过氧化物酶4(GPX4)以及4-羟基壬烯醛(4-HNE)蛋白的表达;Western blot 法检测铁死亡相关蛋白胱氨酸转运蛋白系统Xc- 亚基SLC7A11、过氧化物酶4(GPX4)以及铁代谢相关蛋白转铁蛋白受体1(TFR-1)、铁蛋白重链(FTH)、铁蛋白轻链(FTL)、膜铁转运蛋白(FPN)的蛋白表达情况。分析铁含量、MDA 含量、GSH 水平与肾间质纤维化的相关性。 结果 与Con 组相比,CKD 组大鼠血清 BUN、Scr 均明显增加(P<0. 05),HE 染色和Masson 染色见明显肾小管扩张及间质纤维化;铁含量以及MDA 含量明显升高(P<0. 05),GSH 含量明显降低(P<0. 05);免疫组化显示,与Con 组相比,CKD 组4-HNE 蛋白表达明显增加,GPX4 蛋白表达明显减少。Western blot结果显示,与Con 组相比,CKD 组的SLC7A11、GPX4、TFR-1、FPN 蛋白表达明显减少,而FTH、FTL 蛋白表达明显增加。相关性分析提示铁含量、MDA含量与肾纤维化相对面积(%)呈正相关、GSH 含量与肾纤维化相对面积(%)呈负相关。 结论 在腺嘌呤诱导的CKD 大鼠模型中存在铁死亡,并可能通过GSH-GPX4 轴参与CKD 肾纤维化进展。
Objective To observe expression of ferroptosis-related factors in an adenine-induced chronic kidney disease model, and investigate the mechanism of ferroptosis in the pathological process of chronic kidney disease. Methods Twenty rats were randomly divided into a control group (Con) and model group (CKD). The CKD model was established by 2. 5% adenine gavage. All rats were sacrificed after 6 weeks. Blood samples were collected to measure serum urea nitrogen (BUN) and serum creatinine (Scr). HE staining was performed to observe renal pathological changes. Masson staining was performed to observe interstitial fibrosis of renal tubules. Iron, malondialdehyde (MDA), and glutathione(GSH) concentrations were measured. Western blot was performed to detect the protein expression of iron ferroptosisrelated proteins including systemic Xc- subunit SLC7A11, peroxidase 4 (GPX4), and iron metabolism-related proteins including transferrin receptor 1 (TFR-1), ferritin heavy chain (FTH), ferritin light chain (FTL), and membrane iron transporter protein (FPN). Correlations between iron, MDA and GSH levels and interstitial renal fibrosis were analyzed. Results Compared with the Con group, serum urea nitrogen and serum creatinine were significantly increased in the CKD group (P<0. 05). HE and Masson staining showed significant tubular dilatation and interstitial fibrosis. Iron and MDA concentrations were significantly increased (P<0. 05), and the GSH concentration was significantly decreased (P<0. 05). Immunohistochemistry showed that, 4-HNE protein expression was significantly increased and GPX4 protein expression was significantly decreased in the CKD group compared with the Con group. Western blot showed that SLC7A11, GPX4, TFR-1, and FPN protein expression was significantly decreased, while FTH and FTL expression was significantly increased compared with the Con group. Correlation analysis indicated that iron and MDA concentrations were positively correlated to the relative area (%) of renal fibrosis, and the GSH concentration was negatively correlated with them. Conclusions Ferroptosis is present in the adenine-induced CKD rat model and may be involved in renal fibrosis progression in CKD via the GSH-GPX4 axis.
LIU Yiqin, ZHU Tingting, MAO Haixia, KANG Ting, ZHANG Liling, WU Weihua, OU Santao.腺嘌呤诱导慢性肾病大鼠模型肾纤维化过程中铁死亡相关因子的变化[J].中国比较医学杂志,2023,33(7):85~91.复制