肾缺血再灌注损伤与核仁应激的相关性研究

Correlation between renal ischemia-reperfusion injury and nucleolar stress

目的:研究核仁应激是否参与了肾缺血再灌注损伤(ischemia-reperfusion injury,IRI),并进一步探索核仁应激的分子机制,为临床防治肾IRI提供新的靶点。方法:将48只雄性昆明小鼠根据肾脏缺血时间不同随机平均分为4组:对照(control)组(0 min)及IRI 30 min组、45 min组和60 min组,采用微创手术夹同时夹闭双侧肾动脉的方法建立急性肾IRI模型。24 h后取眼球血测定血尿素氮(blood urea nitrogen, BUN)和血清肌酐(serum creatinine, SCr)浓度;取双肾计算肾系数;并采用HE染色观察肾组织病理学改变;提取肾皮质组织总蛋白和RNA进行Western blot及RT-qPCR检测,待测指标包括p53蛋白、45S pre-rRNA、18S rRNA、Bax mRNA和Bcl2 mRNA。结果:与control组相比,IRI组BUN和SCr浓度上升(P<0.01),肾系数及病理损伤范围增大(P<0.01);肾组织结构和功能损害与缺血时间呈正相关(r>0.80,P<0.01)。与control组相比,IRI组皮质细胞内p53蛋白含量显著增加(P<0.05),Bax mRNA表达上调(P<0.01),同时45S pre-rRNA、18S rRNA和Bcl2 mRNA含量下降(P<0.01)。结论:IRI造成的肾组织结构和功能损害可能与核仁应激密切相关。IRI可能通过阻碍rDNA转录导致核仁应激,从而激活下游p53诱导的细胞凋亡。

AIM: To determine whether nucleolar stress is involved in renal ischemia-reperfusion injury(IRI), and further to explore the molecular mechanism of nucleolar stress for providing a new target for clinical prevention and treatment of renal IRI. METHODS: Male Kunming mice(n=48) were randomly divided into 4 groups according to the different time of renal ischemia: control(0 min) group, IRI 30 min group, IRI 45 min group and IRI 60 min group. The acute renal IRI model was established by clamping bilateral renal arteries with minimally invasive surgical clips. After 24 h, the concentrations of blood urea nitrogen(BUN) and serum creatinine(SCr) were measured by collecting eyeball blood. The kidney coefficient was calculated by weighing bilateral kidneys, and the renal histopathological changes were observed by HE staining. The total protein and RNA from the kidneys were obtained for determining the indicators including p53 protein, 45 S pre-rRNA, 18 S rRNA, Bax mRNA and Bcl2 mRNA by Western blot and RT-qPCR.RESULTS: Compared with control group, the BUN and SCr levels in IRI group were elevated(P<0.01), and the renal coefficient and the range of pathological damage were increased(P<0.01). There was a positive relationship between renal tissue structural and functional damage and ischemic time(r>0.80, P<0.01). Compared with control group, in the cortical cells, the p53 protein content in IRI group was significantly increased(P<0.05) and the Bax mRNA expression was up-regulated(P<0.01). At the same time, the levels of 45 S pre-rRNA, 18 S rRNA and Bcl2 mRNA were decreased(P<0.01). CONCLUSION: Renal tissue structural damage and dysfunction caused by IRI may be closely related to nucleolar stress. IRI may induce nucleolar stress by blocking rDNA transcription, thereby activating downstream p53-induced apoptosis.