Thrombospondin-1 expression in the kidney of a rat model of fibrosis

doi:10.3969/j.issn.2095-4344.2016.27.021

Abstract

Abstract:

BACKGROUND: Thrombospondin-1 is an important endogenous activator of transforming growth factor beta 1 in this experimental inflammatory kidney disease model. Transforming growth factor beta 1 is considered the major cytokine that causes tissue fibrosis in many different inflammatory disease processes, in particular in renal disease.
OBJECTIVE: To investigate the expression of thrombospondin-1 on renal fibrosis in rats.
METHODS: Healthy male Sprague-Dawley rats were randomly divided into sham surgery group and model group. In the model group, right ureters of rats were ligated to construct models of renal fibrosis. 3 weeks after surgery, blood and urine were obtained weekly. Enzyme linked immunosorbent assay and Bradford method were used to detect the contents of serum creatinine, blood urea nitrogen and urinary protein. After rats were sacrificed, kidneys were fixed. Western blot assay was utilized to identify the expression of vascular endothelial growth factor, transforming growth factor beta 1 and thrombospondin-1 protein. Hematoxylin-eosin staining was applied to detect the changes in pathological structure of the kidney after surgery.
RESULTS AND CONCLUSION: (1) One week after model induction, urinary protein, serum creatinine and urea nitrogen levels were significantly higher in the model group than in the sham surgery group (P < 0.05). Three weeks later, the difference in each index was significant (P < 0.01), which showed that the injury of the kidney was aggravated. (2) Transforming growth factor beta 1 protein and thrombospondin-1 expression was significantly higher in the model group than in the sham surgery group, but vascular endothelial growth factor protein expression was significantly lower in the model group than in the sham surgery group. (3) Hematoxylin-eosin staining results demonstrated that severe pathological changes of renal tissue in rats were detected after ligation of renal tubule. (4) These results confirmed that thrombospondin-1 expression increased in renal tissue, and its expression was strongly associated with vascular endothelial growth factor protein and transforming growth factor beta 1, which may play an important role in the renal fibrosis.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

Key words: Fibrosis, Nephrosis, Vascular Endothelial Growth Factors, Transforming Growth Factor beta 1;Tissue Engineering

CLC Number:

R318

Du Jun-wen, Wu Tao, Zhang Kun, Su Bai-yu, Lu Cai-ping, Wang Wei-chao, Lei Lin, Guo Jing-xia. Thrombospondin-1 expression in the kidney of a rat model of fibrosis[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(27): 4098-4104.

Figures/Tables 3

2.1 造模成功动物数量及过程 30只大鼠均完成实验进入结果分析，实验造模过程中无死亡。造模流程图见图1。 2.2 模型更接近人类或临床实践/模型稳定性该模型各项生化指标变化显著，间质纤维化病变突出，与临床肾纤维化的发生发展过程及其相似。使用该模型的一些研究结果与临床患者的观察结果相似。同时，此方法成模率高，重复性好，啮齿类动物模型可反映人类肾脏纤维化的疾病过程。所以该模型是一种研究肾纤维化发生机制、肾脏细胞转化和评价肾纤维化治疗方法的理想模型。 2.3 肾功能检测 2.3.1 尿蛋白大鼠造模后1，2，3周，利用Bradford方法检测尿蛋白含量。造模后1周，与假手术组相比，模型组大鼠的尿蛋白水平显著升高(P < 0.05)；造模后2周起，模型组大鼠尿蛋白升高更明显，与假手术组相比差异有非常显著性意义(P < 0.01)，且呈上升趋势。见表1。 2.3.2 血清肌酐与尿素氮大鼠造模后1，2，3周，利用ELISA方法检测大鼠血清中肌酐与尿素氮水平。造模后1周，模型组大鼠血清中肌酐与尿素氮较假手术组显著升高(P < 0.05)；造模后3周，模型组大鼠血清中肌酐与尿素氮升高更明显，与假手术组相比差异有非常显著性意义(P < 0.01)，见表2。尿蛋白、血清肌酐与尿素氮表达量的增加表明肾功能损伤加重。 2.4 大鼠肾脏组织中血小板反应蛋白1、血管内皮生长因子和转化生长因子β1蛋白的表达 Western blot方法检测大鼠手术后，结扎侧肾脏组织中血小板反应蛋白1、血管内皮生长因子和转化生长因子β1蛋白的表达水平。结果显示，造模1周后，模型组大鼠肾脏组织中转化生长因子β1与血小板反应蛋白1的蛋白表达量开始升高，且模型组中血管内皮生长因子蛋白的表达量与假手术组相比也开始降低，显著低于假手术组；造模后3周，模型组转化生长因子β1与血小板反应蛋白1的蛋白表达量升高更明显，血管内皮生长因子蛋白的表达量与假手术组相比亦显著降低。见图2、表3。 2.5 大鼠肾组织纤维化的病理变化苏木精-伊红染色观察大鼠术后肾组织病理结构变化。假手术组大鼠肾组织结构完整，没有变性和坏死，细胞核周围没有炎性细胞浸润，在间质中没有纤维细胞增殖、腺嘌呤结晶沉积及肾小管上皮细胞空泡化(图3A-C)；模型组大鼠肾组织中，肾小管沉积很多结晶且上皮细胞空泡化严重，肾小球内皮细胞、上皮细胞、系膜细胞完全消失萎缩，肾包膜与肾小管部分细胞全部消失，间质中纤维细胞显著增殖，并伴有大量的炎性细胞浸润，并随时间的推移越加严重(图3D-F)。"

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