Changes of CLOCK gene and NRF2 gene rhythmic expression after renal denervation in mouse kidneys
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.
NETL
NSTL
万方数据
