基于糖原合成酶激酶-3β及内质网应激探讨Renalase抗肾脏纤维化的作用机制

Role of GSK-3β in Renalase inhibition of renal fibrosis

目的 探讨糖原合成酶激酶-3β(GSK-3β)在Renalase抗肾脏纤维化过程中的作用及其可能的机制。方法 选用C57BL/6小鼠,予以下分组:(1)Sham+Ad-β-gal组;(2)Sham+Ad-Renalase组;(3)UUO+Ad-β-gal组;(4)UUO+Ad-Renalas组。观察GSK-3β表达的变化,其中UUO组为单侧输尿管结扎致肾脏纤维化模型组,Sham组为假手术对照组,Ad-Renalase为过表达Renalase腺病毒,Ad-β-gal为对照腺病毒。然后应用病毒转染技术上调(UUO+Ad-Renalase+AAV-GSK-3β组)及下调(UUO+Ad-Renalase+Ad-GSK-3β-RNAi组)GSK-3β后观察Renalase抗纤维化作用的变化。结果 与假手术(Sham)组相比,UUO组GSK-3β表达增加;与UUO+Ad-β-gal组相比,UUO+Ad-Renalase组纤维组织沉积减少、纤维化标志蛋白Col-Ⅰ和FN表达下降的同时GSK-3β表达下调;当上调GSK-3β后,Renalase抗纤维化作用被抵消,而当下调GSK-3β后,与UUO+Ad-Renalase组相比,UUO+Ad-Renalase+Ad-GSK-3β-RNAi组纤维化没有进一步改善。同时观察到与Sham组相比,UUO组内质网应激激活明显,过表达Renalase后,内质网应激被抑制。当加入内质网应激激动剂TM后,与UUO+Ad-Renalase组相比,UUO+Ad-Renalase+TM组纤维化加重,GSK-3β表达升高;但无论GSK-3β升高或者降低,内质网应激均无明显变化。结论 本研究证实在单侧输尿管结扎致肾脏纤维化过程中,Renalase通过抑制内质网应激下调GSK-3β表达从而延缓肾脏纤维化。

Objective To explore the role of glycogen synthase kinase-3β(GSK-3β)in Renalase's anti-renal fibrosis process and its possible mechanism.Methods C57BL/6 mice were divided into(1)Sham+Ad-β-gal,(2)Sham+Ad-Renalase,(3)UUO+Ad-β-gal,(4)UUO+Ad-Renalas groups to observe the change of GSK-3βexpression.Among them,UUO group was a model group of renal fibrosis caused by unilateral ureteral ligation,Sham group was a sham operation control group,Ad-Renalase was an over-expressed Renalase adenovirus,and Ad-β-gal was a control adenovirus.After overexpressing(UUO+Ad-Renalase+AAV-GSK-3β)or knocking down(UUO+Ad-Renalase+Ad-GSK-3β-RNAi)GSK-3βby virus transfection.Results Compared with Sham group,the expression of GSK-3βin UUO group was significantly increased.Compared with UUO+Ad-β-gal group,the deposition of fiber tissue decreased,the expression of Col-Ⅰ,fibronectin(FN)and GSK-3βwas down-regulated in UUO+Ad-Renalase group.After overexpression of GSK-3β,the anti-fibrosis effect of Renalase was counteracted,but when GSK-3βwas knocked down,compared with UUO+Ad-Renalase group,the fibrosis did not further improve in UUO+Ad-Renalase+Ad-GSK-3β-RNAi group.Meanwhile,compared with Sham group,endoplasmic reticulum stress in UUO group was obviously activated,and the activated endoplasmic reticulum stress was obviously inhibited after overexpression of Renalase.When endoplasmic reticulum stress agonist was added,the anti-fibrosis effect of Renalase was obviously weakened in UUO+Ad-Renalase+TM group compared with UUO+Ad-Renalase group,and down-regulated GSK-3βwas obviously recovered.However,the expression of endoplasmic reticulum stress markers did not change significantly whether GSK-3βwas overexpressed or knocked down.Conclusion The study confirmed that Renalase can reduce the expression of GSK-3βby inhibiting endoplasmic reticulum stress,thus delaying renal fibrosis inUUO.