反复注射顺铂诱导小鼠慢性肾功能不全模型的建立

Establishment of a mouse model of chronic renal insufficiency induced by repeated administration of cisplatin

目的构建慢性肾脏病(chronic kidney disease,CKD)C57BL/6小鼠模型,探索其小管损伤指标和间质纤维化程度随顺铂造模剂量的改变,为研究从AKI到CKD进展过程提供动物实验依据。方法将24只8周龄雄性C57BL/6小鼠随机平均分为对照组和低、中、高剂量顺铂模型组。模型组小鼠按5、7、10 mg/kg顺铂腹腔注射给药,每周1次,连续注射4周构建模型。处死小鼠后,留取标本进行相关检测。检测血浆肌酐和24 h尿蛋白排泌量来评估小鼠肾功能;PAS染色观察肾脏病理学变化;免疫组化检测肾损伤分子1(KIM-1)和尿液检测N-乙酰-β-D氨基葡萄糖苷酶(NAG)水平以评估肾小管损伤情况;免疫组化法检测肾脏CD3阳性T细胞和免疫荧光法检测F4/80阳性巨噬细胞浸润情况;天狼星红染色、免疫组化法检测胶原I和α-平滑肌肌动蛋白(α-SMA)表达以评估肾脏纤维化情况。结果与正常对照组相比,随着注射顺铂浓度的升高,小鼠肾脏损伤越明显,其中10 mg/kg顺铂高剂量组最为显著。与对照组相比,顺铂高剂量组小鼠肾功能下降,表现为血浆肌酐浓度和24 h尿蛋白排泌量显著升高(P<0.05和P<0.001);肾小管上皮细胞坏死、空泡变性等病理学改变显著,肾组织KIM-1表达显著上升(P<0.05),尿NAG水平升高;肾组织浸润的CD3阳性T细胞和F4/80阳性巨噬细胞增多;肾组织天狼星红染色阳性胶原纤维区域显著增多(P<0.001),胶原I和α-SMA表达也明显增多(P<0.01),肾小管-间质发生纤维化。结论反复注射4周10 mg/kg顺铂可诱导小鼠慢性肾功能不全模型,可为研究AKI向CKD的转化机制提供了新的实验模型。

Objective To observe the changes of renal tubular injury and the extent of interstitial fibrosis in the C57BL/6 mouse models of chronic kidney disease（ CKD）,and provide experimental animal evidence for study of the progression of acute kidney injury（ AKI） to chronic kidney disease as well as its mechanisms. Methods Twenty-four 8-week-old male C57BL/6 mice were randomly and equally divided into control group,low-dose,medium-dose,and highdose cisplatin groups,6 mice in each group. Mice in the cisplatin groups were administrated with 5,7 or 10 mg/kg cisplatin by intraperitoneal injection once a week for 4 weeks. Plasma creatinine and 24-hour urinary protein were detected to assess the renal function. The mice were sacrificed,and plasma and kidney samples were collected for subsequent tests.Pathological changes were observed using periodic acid-Schiff（ PAS） staining. To evaluate renal tubules injury,the expression of kidney injury molecule 1（ KIM-1） was examined by immunohistochemistry and the level of urinary N-Acetyl-β-D-glucosaminidase was detected with a commercial kit. The infiltration of CD3-positive T cells and F4/80-positive macrophages was observed by immunohistochemistry（ IHC） and immunofluorescence. The expression of collagen I and α-smooth muscle actin（ α-SMA） were tested by immunohistochemistry to assess the renal fibrosis,while total kidney collagen was detected by Picrosirius red staining. Results In contrast to the normal control group,the kidney injury became more serious in the cisplatin-treated mice as cisplatin concentration increased. Particularly,significant kidney damage was observed in the high-dose cisplatin group. Compared with the control group,the plasma creatinine and 24-hour urinary protein were significantly increased in the high-dose cisplatin group（ P〈0. 05 and P〈0. 001） indicating impaired renal function. Morphologically,numerous clear vacuoles and necrosis were present in renal tubule epithelial cells in the high-dose cisplatin group. The expression of KIM-1 was markedly up-regulated and the level of urinary NAG was elevated. Infiltration of CD3-positive T cells and F4/80-positive macrophages was enhanced in the mice of high-dose cisplatin group. Data from immunohistochemistry and picrosirius red staining showed that mice of the high-dose cisplatin group developed renal fibrosis evidenced by markedly up-regulated expression of collagen I and α-SMA. Conclusions Repeated administration of 10 mg/kg cisplatin for 4 weeks can induce chronic renal insufficiency in mice,which may serve as a novel model for the research on underlying mechanisms of progression from acute kidney injury to chronic kidney disease.