P53在急性肾损伤小鼠肾脏的表达及其与细胞凋亡的关系

 目的：探讨缺血再灌注致急性肾损伤（AKI）小鼠肾脏不同部位P53的表达及其与细胞凋亡的关系。方法：采用随机对照动物实验方法，将18只小鼠随机分为假手术组、AKI组及P53抑制剂pifithrin-alpha（PFT-α）组，每组6只。采用双侧肾蒂夹闭45 min后松开动脉夹的方法建立小鼠AKI模型，PFT-α组于建模前5 min腹腔注射PFT-α  2.2 mg/kg，并于建模后48 h采血检测尿素氮和肌酐，取肾脏组织行HE染色观察肾组织病理学变化，采用Western blotting法测定P53蛋白含量，免疫荧光法确定肾脏不同部位P53的表达，TUNEL法检测肾脏细胞凋亡，免疫组化方法检测肿瘤坏死因子受体（TNFR）、caspase-3及Bcl-2蛋白水平 。结果：（1）PFT-α组和AKI组小鼠血尿素氮和肌酐水平均明显高于假手术组，而PFT-α组与AKI组比较血尿素氮和肌酐水平明显降低（P<0.05）；肾组织HE染色显示假手术组肾组织细胞形态完整，排列整齐，无明显病理改变，AKI组肾小管上皮细胞刷状缘脱落、空泡及滴状变性，皮髓质间有明显淤血带；PFT-α组肾小管上皮部分刷状缘脱落消失，空泡及滴状变性减轻，皮髓质间无明显淤血带；（2）假手术组小鼠肾脏有少量P53表达且未检测到凋亡细胞，而AKI组小鼠缺血再灌注48 h后P53蛋白水平及凋亡细胞明显增加（P<0.05），并且均主要定位在肾皮质，PFT-α组较AKI组小鼠肾脏P53蛋白含量及凋亡细胞指数均减少（P<0.05）；（3）与假手术组相比，AKI组小鼠肾脏TNFR蛋白及caspase-3蛋白水平升高（P<0.05），Bcl-2蛋白水平下降（P<0.05），PFT-α组较AKI组小鼠肾脏TNFR蛋白及caspase-3蛋白水平降低（P<0.05），Bcl-2蛋白水平升高（P<0.05）。结论：急性肾损伤时，肾组织P53表达增加，主要定位于皮质，通过调控凋亡蛋白Bcl-2和TNFR的水平，促进caspase-3的释放，从而介导细胞凋亡。

Relationship between apoptosis and change of P53 expression in mouse renal tissues after acute kidney injury

AIM: To investigate the relationship between renal cell apoptosis induced by ischemia/reperfusion injury and the activation of P53. METHODS: Eighteen mice were randomly divided into 3 groups: sham operation group, acute kidney injury (AKI) group and pifithrin-alpha(PFT-α) treatment group. The AKI model was established by clamping bilateral renal arteries for 45 min and then performing reperfusion. The mice in PFT-α group were intraperitoneally injected with PFT-α at dose of 2.2 mg/kg 5 min before AKI model was established. The changes of serum creatinine and urea nitrogen were determined and renal pathological changes were observed 48 h after AKI. The P53 expression in the kidney was evaluated by Western blotting and immunofluorescence methods. Apoptosis of the renal cells was observed by TUNEL assay. The protein expression of tumor necrosis factor receptor (TNFR), caspase-3 and Bcl-2 was detected by immunohistochemical method. RESULTS: The levels of serum creatinine and urea nitrogen in AKI group and PFT-α group were higher than those in sham operation group. Compared with AKI group, the levels of serum creatinine and urea nitrogen were significantly decreased in PFT-α group. No pathological change of the kidney was observed in sham operation group. In AKI group, the pathological changes such as shedding of brush border, vacuolus and dropwise degeneration in the renal tissues were observed. These pathological changes were attenuated in PFT-α group as compared with AKI group. The protein was expression level of P53 and the apoptotic cells were much higher in AKI group than those in sham operation group, and P53 protein was mainly expressed in the renal cortex, while those were significantly decreased in PFT-α group as compared with AKI group. Compared with sham operation group, the expression levels of TNFR and caspase-3 were increased and the Bcl-2 levels was decreased. Compared with AKI group, the expression level of TNFR and caspase-3 decreased and Bcl-2 expression was increased. CONCLUSION: P53 protein is mainly expressed in the renal cortex and induces apoptosis by increasing the expression of caspase-3 and regulating the expression of TNFR and Bcl-2 in the kidney following ischemia/reperfusion injury.