血小板反应蛋白-1及其受体CD47在肾间质纤维化血管病变中的表达

Expression of thrombospondin-1 and its receptor CD47 in vascular lesions of renal interstitial fibrosis

目的观察血小板反应蛋白-1(TSP-1)及其受体CD47在单侧输尿管梗阻(UUO)大鼠模型中的表达,探讨TSP-1-CD47在肾周毛细血管(PTC)病变中的作用。方法将60只SD大鼠随机分为2组:UUO组45只和假手术组(SOR)15只。UUO组随机选取15只分别于术后3 d、7 d、14 d处死;SOR组随机选取5只于相同时间点处死。检测24 h尿蛋白定量,采用全自动生化分析仪检测各组大鼠的肾功能,通过Masson染色观察肾间质病理改变;免疫荧光双染色观察TSP-1及其受体CD47在UUO模型中是否存在共表达。Western印迹检测毛细血管病变指标CD34和血管内皮生长因子(VEGF)及TSP-1的表达;多组间均数比较使用方差分析;TSP-1与CD34、VEGF的相关性进行Pearson相关分析。结果与SOR组相比,UUO各组大鼠血清肌酐、尿素氮、24 h尿蛋白定量随梗阻时间延长无明显变化,但肾间质纤维化逐渐加重,肾小管损伤评分增加。免疫荧光双染色显示TSP-1及其受体CD47在肾小管间质存在共表达。Western印迹检测显示随UUO时间延长,TSP-1蛋白水平逐渐增加,而CD34及VEGF蛋白表达随梗阻时间延长逐渐下降。相关分析显示TSP-1蛋白水平与CD34及VEGF的表达呈负相关(r=-0.931,P<0.01;r=-0.953,P<0.01)。结论 TSP-1及其受体CD47在UUO大鼠模型肾间质存在共表达,且与PTC呈负相关。TSP-1可能通过CD47参与UUO肾脏PTC病变的发生发展。

Objective To observe the expression of TSP-1 and its receptor CD47 in unilateral ureteral obstruction （UUO） rats, and to investigate the role of TSP-1-CD47 in lesions of peritubular capillary （PTC）. Methods A total of 60 Sprague-Dawley rats were randomly divided into： sham-operation group （SOR） （n = 15） and UUO group （n =45）. UUO group sacrifice timing consisted of three time points： 3 day, 7 day, 14 day, with 15 animals for each time point. Detection of 24 h urinary protein and renal function was made by automatic biochemistry analyzer, and interstitial pathological changes were shown by Masson staining. The expression of TSP-1 and CD47 was measured by immunohistochemical and immunofluorescence double staining. Western blot was used to detect the expression of CD34, VEGF, TSP- 1 protein in rat kidneys. The correlations among CD34, VEGF, and TSP-1 protein expression were analyzed with Pearson method. Results Compared with the sham-operation group, the levels of serum creatinine （Scr）, blood urea nitrogen （BUN）, and 24h urinary protein did not change significantly in UUO group, but features of renal tubulointerstitial fibrosis were obvious, and the score of renal interstitial lesion was significantly higher in UUO group. Immunofluorescence double staining showed that TSP-1 and CD47 were coexpressed in UUO renal tubules. Western blot confirmed that TSP-1 protein level was signticantly higher in UUO group,while the expressions of CD34 and VEGF protein were decreased. Correlation analysis showed that TSP-1 protein was negatively associated with the expression of CD34 and VEGF（r = -0. 931, P 〈 0. 01 ; r = - 0. 953, P 〈 0. 01 ）. Conclusion TSP- 1 and its receptor CIM7 were coexpressed in UUO rats, and negatively associated with PTC. TSP-1 may be involved in the pathological changes of PTC through CD47 in UUO kidneys.