小鼠单侧输尿管梗阻模型肾损伤随时间变化的研究

Study about renal injury of unilateral ureteral obstruction model with time

目的建立小鼠单侧(左侧)输尿管梗阻(UUO)模型,探究肾损伤随时间的变化情况和相关机制。方法50只小鼠随机分为两组:假手术组及UUO模型组(结扎左侧输尿管制成UUO模型),观察不同时间点两组生化指标、左肾重量/终末体重(LR/BW)、右肾重量/终末体重(RR/BW),并计算左肾重量/右肾重量比值(LR/RR)。采用HE、Masson及PAS染色法检测小鼠的肾脏病理学变化。免疫荧光染色观察小鼠肾小管周围毛细血管(PTC)缺失、肾实质细胞增殖(Ki67^(+)细胞)以及巨噬细胞(CD68^(+)标记)、成纤维细胞浸润及肾脏Wnt/β-catenin表达情况。结果UUO组小鼠体重在造模第3天迅速下降[(18.2±1.1)g vs(22.4±1.2)g],此后体重缓慢增加直至第28天,在第60天体重显著下降[(17.5±0.8)g];LR/RR和LR/BW在第3天显著上升,随后逐渐下降;第60天时肾功能明显恶化[血清肌酐(0.89±0.09)mg/dl,尿素氮(41.26±5.65)mg/dl];UUO组梗阻侧肾脏光镜显示肾小管间质纤维化和肾小球硬化,随着时间的推移,PTC缺失逐渐加重;巨噬细胞最初在左肾实质内显著增多,但在28 d后开始减少;成纤维细胞数量在梗阻侧(左侧)肾脏前14 d显著增多,此后下降至正常水平;UUO组非梗阻侧肾脏的细胞数量与假手术组差异无统计学意义;UUO组梗阻侧(左侧)Wnt/β-catenin免疫荧光强度在肾脏损伤后前14 d的表达显著上调,28 d后表达下降。结论UUO肾脏纤维化的发展涉及很多方面的变化,包括PTC缺失、巨噬细胞的浸润、成纤维细胞激活以及表达,但是这些变化随着时间而减弱。

Objective The unilateral(left)ureteral obstruction(UUO)model was established in mice to explore the changes of renal injury with time and the related mechanisms.Methods Fifty mice were randomly divided into two groups:sham group and UUO group(UUO model was made by unilateral ureteral ligation).The biochemical indexes,left kidney weight/final weight(LR/BW)and right kidney weight/final weight(RR/BW)of the two groups at different time points were observed,and the left kidney weight/right kidney weight ratio(LR/RR)was calculated.Hematoxylin-eosin(HE),Masson and periodic acid-Schiff(PAS)staining were used to detect the pathological changes of the kidney in mice.Immunofluorescence staining was used to observe the loss of peritubular capillaries(PTC),proliferation of renal parenchymal cells(Ki67^(+)cells),macrophages(CD68^(+)markers),infiltration of fibroblasts and expression of Wnt/β-catenin in the kidney of mice.Results The weight of mice in UUO group decreased rapidly[(18.2±1.1)g vs(22.4±1.2)g]on the third day of modeling,then slowly increased until the 28th day,and significantly decreased[(17.5±0.8)g]on the 60th day;LR/RR and LR/BW increased significantly in the third day,and then decreased gradually;Renal function of mice in UUO group deteriorated significantly on the 60th day[serum creatinine(0.89±0.09)mg/dl,urea nitrogen(41.26±5.65)mg/dl].In UUO group,renal tubulointerstitial fibrosis and glomerulosclerosis were observed under light microscope in the obstructed kidney;with the passage of time,PTC loss gradually increased;macrophages increased significantly in the left renal parenchyma at first,but began to decrease 28 days later;the number of fibroblasts increased significantly in the first 14 days of the obstructed side(left side)kidney,and then decreased to the normal level;There was no significant difference in the cell number of the non obstructive kidney between UUO group and sham group;The immunofluorescence intensity expression of Wnt/β-catenin of obstructive side(left side)in UUO group was significantly up-regulated in the first 14 days after renal injury,and decreased after 28 days.Conclusions The development of UUO renal fibrosis involves many changes,including PTC loss,macrophage infiltration,fibroblast activation and expression,but these changes weaken with time.