Rock-Ⅰ激酶在肾间质纤维化大鼠不同阶段肾组织中的激活及意义

Contribution of small GTPase Rho effector—Rock-Ⅰin different stages of rat renal interstitial fibrosis

目的观察小G蛋白Rho相关激酶———Rock-Ⅰ在单侧输尿管结扎(UUO)大鼠肾组织的表达规律及其活化状态的改变,并探讨其在肾间质纤维化中的意义。方法采用免疫组织化学、RT-PCR、免疫印迹法,从基因、蛋白质水平观察UUO大鼠肾组织Rock-Ⅰ表达规律与定位;免疫印迹法检测其底物肌球蛋白磷酸酶的磷酸化状态,作为该激酶功能活化的标志。结果(1)Rock-ⅠmRNA在肾间质纤维化发生早期即明显上调(F=15.18,P<0.01),其表达较肌成纤维细胞标志α-SMAmRNA提前达到高峰,2者呈显著正相关(r=0.61,P<0.01)。(2)免疫印迹结果显示UUO模型大鼠肾组织中Rock-Ⅰ蛋白表达水平明显高于对照组(F=21.18,P<0.01),并随肾间质纤维化相对面积的扩大而逐渐增加,2者呈显著正相关(r=0.83,P<0.01)。(3)UUO模型组大鼠肾组织肌球蛋白磷酸酶磷酸化水平较对照组显著增高(F=48.87,P<0.01),并且与肾间质纤维化指数呈显著正相关(r=0.54,P<0.05)。结论UUO大鼠肾组织中存在小G蛋白Rho相关激酶Rock-Ⅰ的表达上调与功能活化,提示Rho/Rock信号转导通路在肾间质纤维化的发生机制中具有重要作用。

Objective To observe the expression of Rock Ⅰand its functional activation in renal tissue from a rat UUO model, and to investigate its role in the progression of renal interstitial fibrosis(RIF). Methods Expression of RockI mRNA and protein were examined by RT PCR and Western blotting, respectively.The phosphorylation of MBS(binding subunit of myosin phosphatase)—a substrate of Rock Ⅰwas detected by Western blotting and defined,as the mark of functional activation of the kinase. Results (1)The expression of Rock ⅠmRNA was increased before the onset of RIF(the 3rd day after the experiment) (F=15 18,P< 0 01).The expression peak of Rock ⅠmRNA was prior to that of α SMA, and there was a positive correlation between them(r=0 606,P< 0 01).(2)The expression of Rock Ⅰprotein was significantly increased in the UUO group than that in the vehicle control(F=21 18,P< 0 01), and its increase accompanied the fibrostic index(r=0 832,P< 0 01). (3)The phosphorylation of MBS was significantly upregulated in the UUO group than that in the vehicle control(F=48 87,P< 0 01)and had a positive correlation with the fibrostic index(r=0 544,P< 0 05). Conclusions Expression and functional activation of Rock Ⅰenhance in renal tissue from UUO rats. Rho/Rock signaling may play an important role in the progression of RIF.