Smad6、7蛋白表达与梗阻性肾病小管间质损伤关系的实验研究

Relationship between expression of Smad6 and Smad7 and renal tubulointerstitial injury following unilateral ureteral obstruction in rats

目的 研究Smad6,7蛋白表达与梗阻性肾病小管间质损伤的关系。方法 32只Wistar大鼠随机分为单侧输尿管梗阻组和假手术组 (对照组 ) ,于术后 3、7、14、2 1d处死大鼠。采用逆转录多聚酶链反应 (RT PCR)检测TGF β1 mRNA表达水平 ;用免疫组化和Western杂交分别检测Smad6,Smad7蛋白的定位和表达 ;DNA片断末端标记原位检测肾小管细胞凋亡数 ;图像分析小管细胞和肾小球毛细血管球囊平均卡规直径的变化。判断纤维化程度通过测定肾组织羟脯氨酸含量。结果 与对照组相比 ,梗阻肾组织TGF β1 mRNA表达显著增高 ,并伴有明显的肾小管和肾小球毛细血管球囊萎缩、小管细胞的凋亡 ,以及肾组织羟脯氨酸含量增高。Smad6、7蛋白主要在肾小球和皮质肾小管上皮细胞内表达。输尿管梗阻后 ,Smad6、7蛋白表达明显下降 ,该Anti Smad蛋白下调与小管间质损伤呈明显相关。结论 TGF β1 信号通路参与了梗阻性肾病小管间质损伤的进程 ,Anti Smad蛋白表达下降可能是该模型TGF β1 致伤作用加重的主要原因。

Objective To investigate the relationship between the expressions of Smad6 and Smad7 and renal tubulointerstitial injury in rats with unilateral ureteral obstruction. Methods Thirty two rats were subjected to unilateral ureteral obstruction (UUO) or sham operation and then sacrificed on day 3, 7, 14 and 21 postoperatively. The levels of TGF β 1 mRNA were measured with RT PCR. The expression sites and levels of Smad6 and Smad7 were examined with immunohistochemical staining and western blot respectively. The apoptosis of renal tubular cell(RTC)was measured with in situ detection of nuclear DNA fragmentation. The diameters of tubular cell and glomerular capillary capsule(GCC)were quantitated with histomorphometry. Renal fibrosis was assessed by the determination of tissue hydroxyproline content. Results Compared with controls, the expression of TGF β 1 mRNA was significantly increased in UUO rats, and progressive tubular cell apoptosis, tubular and glomerular atrophy, and increased hydroxyproline content were observed at the same time. Immunohistochemistry indicated that Smad6 and Smad7 were expressed in both the glomeruli and proximal renal tubular cells. The expressions of Smad6 and Smad7 were significantly reduced from day 3 to 21 after UUO, which was significantly correlated to the down regulation of Anti-Smad and renal tubulointerstitial injury. Conclusion These results indicate that TGF β 1 signaling is involved in the process of renal tubulointerstitial injury after obstruction, the down regulation of these anti Smads may be the major cause of the exacerbation of renal injury in this model