小鼠肾脏失神经支配后局部时钟基因及NRF2基因节律性表达的变化

Changes of CLOCK gene and NRF2 gene rhythmic expression after renal denervation in mouse kidneys

目的:研究小鼠肾脏核心时钟基因和NRF2基因表达的昼夜节律性,及其在肾脏失神经支配(RDN)后昼夜节律性的变化。方法:将72只SPF级成年雄性C57BL/6小鼠随机分为两组(n=36):假手术组(Sham组)和去肾脏神经组(RDN组)。小鼠在12 h/12 h明暗交替的环境中饲养2周,早8:00开灯(时间记为ZT0)。苯酚溶液毁损双侧肾脏神经制备RDN模型,10 d后,分别间隔4 h(ZT0、4、8、12、16和20)收集血液、肾动脉、下丘脑视交叉上核(SCN)和肾脏组织标本。采用HE染色观察肾动脉周围神经毁损程度;ELISA法检测肾脏去甲肾上腺素(NE)水平;全自动生化分析仪检测血清肌酐(Scr)和尿素氮(BUN)水平;qRT-PCR和Western Blot法分别检测SCN和肾脏组织中BMAL1、CLOCK和NRF2基因mRNA转录及蛋白表达水平;免疫荧光法观察昼夜时点肾脏NRF2蛋白表达及核转位情况。通过尾静脉注射3种载有BMAL1短发卡RNA(shRNA1、2、3)的自互补腺相关病毒(pscAAV9-Arntl-EGFP)干扰小鼠体内BMAL1基因表达,探究肾脏组织中BMAL1基因与NRF2基因的相关性。结果:与Sham组比较,RDN组可见肾神经细胞空泡样变性,同时肾脏组织NE水平显著下降(P<0.05);血清Scr和BUN水平无明显变化(P>0.05);SCN中BMAL1基因mRNA表达的昼夜节律性无明显变化;而肾脏BMAL1、CLOCK和NRF2基因的mRNA转录和蛋白表达节律性消失,表现为24 h表达量中值下降、振幅减弱和峰值时间变化。免疫荧光进一步佐证RDN后肾脏NRF2蛋白表达的昼夜差异性消失,但各组均未观察到NRF2蛋白有明显的核转位。scAAV实验中,与对照组(NC_sh组)比较,BMAL1_sh1组肾脏BMAL1蛋白和NRF2蛋白表达均无明显变化(P>0.05);BMAL1_sh2和BMAL1_sh3组肾脏BMAL1蛋白表达明显下降,NRF2蛋白表达也相应下降(P<0.05)。结论:RDN后SCN失去了对肾脏生物钟的同步化调控,导致肾脏局部时钟基因BMAL1与CLOCK表达节律性消失。同时肾脏BMAL1基因可能通过对NRF2基因的调控,使得RDN后肾脏NRF2基因转录及蛋白表达的昼夜节律性亦消失,且呈现持续的低水平表达。

Objective:To investigate the rhythmic expression of the core CLOCK gene and NRF2 gene in mouse kidneys,and to evaluate the changes in their 24⁃h rhythmic expression after renal denervation.Methods:A total of seventy⁃two SPF adult male C57BL/6 mice were randomly divided into two groups(n=36):the sham operation group(Sham group)and the renal denervation group(RDN group).The mice were kept in the environment of 12 h⁃12 h alternating light and dark for 2 weeks,and the light was turned on at 8 am,which was recorded as ZT0.The model mice were fed for 10 days.The mice were sacrificed at time points of ZT0,4,8,12,16,and 20,and the blood,the renal artery,the hypothalamic suprachiasmatic nucleus(SCN),and the kidney tissue were collected.HE staining was used to observe the pathological damage of the renal nerve around the renal artery;the contents of norepinephrine(NE)in the kidney were detected by ELISA test;serum creatinine(Scr)and urea nitrogen(BUN)were detected by full⁃automatic biochemical analyzer;the mRNA expression of BMAL1,CLOCK,and NRF2 in SCN and kidney were detected by qRT⁃PCR;the protein expres⁃sion of BMAL1,CLOCK,and NRF2 in kidney was detected by Western Blot;the protein expression of NRF2 and nuclear translocation in kidney was observed by immunofluorescence.Three kinds of self⁃complementary adeno⁃associated viruses(pscAAV9⁃Arntl⁃EGFP)containing BMAL1 short hairpin RNA(shRNA1,2,3)were injected into the tail vein to interfere with the expression of the BMAL1 gene in mice,and to explore the correlation between BMAL1 gene and NRF2 gene in kidney tissue.Results:Compared with that respectively in the Sham group,the renal nerve cells in the RDN group showed vacuolar degeneration and the level of NE in renal tissue decreased significantly(P<0.05);no significant changes were found in the levels of Scr and BUN(P>0.05).There was no significant change in the circadian rhythmic expression of BMAL1 mRNA in SCN between the two groups;how⁃ever,in the kidney,the rhythmic expression of BMAL1,CLOCK,and NRF2 mRNA disappeared,and the rhythmic expression of BMAL1,CLOCK,and NRF2 proteins also disappeared,showing a decrease in mesor of 24⁃h expression and amplitude,and a change in acrophase.Immunofluorescence confirmed that the diurnal difference in NRF2 protein expression disappeared after RDN,but no obvi⁃ous nuclear translocation of NRF2 protein was observed in each group.In the scAAV experiment,compared with that in the control group(NC_sh group),no significant change was found in the protein expression of BMAL1 and NRF2 in the kidney in the BMAL1_sh1 group(P>0.05);in the BMAL1_sh2 and the BMAL1_sh3 group,the protein expression of BMAL1 and NRF2 in kidney de⁃creased significantly(P<0.05).Conclusion:After RDN in mice,SCN didn't synchronize with the re⁃nal biological clock,and the rhythmic expression of renal core CLOCK genes BMAL1 and CLOCK was disordered.Moreover,the BMAL1 gene in the kidney may regulate the NRF2 gene,so that the circadian rhythm of NRF2 gene transcription and protein expression also disappeared,showing a sus⁃tained low level of expression.