The Role Of NF-κB In Cadmium-induced Injury Of Rat Renal Tubular Epithelial Cells

Cadmium(Cd)is a heavy metal pollutant that exists widely in the environment.The kidney is the main target organ for Cd accumulation,and renal tubule is the main damage site.Long-term Cd exposure can cause renal tubular reabsorption dysfunction and eventually cause renal failure.Apoptosis and autophagy inhibition are important mechanisms of Cd-induced nephrotoxicity.As a transcription factor that balances cell survival and death,nuclear factor-kappaB(NF-κB)protein has a dual effect on apoptosis and autophagy,and then plays a pro-survival or pro-injury role in multiple disease processes.However,it is still unclear whether NF-κB is involved in Cd-induced nephrotoxicity and its role in Cd-induced rat renal tubular epithelial cells injury.Therefore,in this study,the normal rat kidney epithelial-like cells(NRK-52E cells)were used as the research object,and NF-κB-specific inhibitor BAY 11-7082,gene knockdown of NF-κB p65 siRNA(sip65)and NF-κB activator phorbol-12-myristate-13-acetate(PMA)were used to regulate the NF-κB activity in order to explore the changes of NF-κB protein after Cd exposure and the role of NF-κB in Cd-induced apoptosis and autophagy inhibition,and to provide a theoretical basis for revealing the mechanism of Cd-induced nephrotoxicity.1.Cd exposure activates NF-κBIn order to explore whether NF-κB is involved in Cd-induced nephrotoxicity,the NRK-52E cells were treated with varying concentrations of Cd(0,2.5,5,10 μM)for 12 h.Western blot was used to detect the phosphorylation levels of κB and NF-κB p65 in total protein and the expression levels of NF-κB p65 protein in nucleus and cytoplasm;GFP-p65 plasmid transfection method was used to detect the NF-κB p65 nuclear translocation.The results showed that compared with the control group,5 and 10 μM Cd treatment significantly increased the phosphorylation levels of IκB and NF-κB p65(P<0.01);5 and 10μM Cd treatment significantly increased expression of NF-κB p65 in the nucleus and decreased expression of NF-κB p65 in the cytoplasm(P<0.01).Thus,Cd promotes nuclear translocation of NF-κB in a concentration-dependent manner in NRK-52E cells.2.Activation of NF-κB antagonizes Cd-induced cytotoxicity in NRK-52E cells.To investigate the role of Cd-activated NF-κB in Cd-induced nephrotoxicity,NF-κB specific inhibitor BAY 11-7082,gene knockdown and NF-κB activator PMA were used to establish NF-κB inhibition and activation models,respectively.The treatment details were as follows:(1)NF-κB inhibitor BAY 11-7082:0.5 μM BAY 11-7082 was pretreated for 2 h,and then treated with 5 μM CdCl2 for 12 h.(2)NF-κB p65 gene knockdown:40 nM sip65 was transfected for 24 h,and then treated with 5 μM CdCl2 for 12 h.(3)NF-κB activator PMA:50 ng/mL PMA and 5 μM CdCl2 were co-treated for 12 h.After treatment,the cell morphology was observed under phase contrast microscope,the cell viability was detected by CCK-8 method,and the cell proliferation index was detected by RTCA technology;Western blot method was used to detect the phosphorylation levels of IκB and NF-κB p65 in total cell proteins,as well as the expression levels of NF-κB p65 in nuclear and cytoplasmic.The results showed:(1)Compared with the control group,the phosphorylation levels of IκB,NF-κB p65 and the nuclear translocation of NF-κB p65 were significantly increased in Cd group(P<0.01);compared with the Cd group,Cd+BAY 11-7082 group significantly inhibited the phosphorylation of IκB and NF-κB p65(P<0.05),and significantly inhibited Cd-induced nuclear translocation of NF-κB p65(P<0.01).Compared with the Cd+siCt group,the nuclear translocation of NF-κB was significantly inhibited in Cd+sip65 group(P<0.01).Compared with the Cd group,the Cd+PMA group significantly increased the expression levels of NF-κB p65 protein in nuclear and cytoplasmic(P<0.01).These results indicated that the activation and inhibition regulation of NF-κB under this treatment condition are effective,and subsequent experiments could be carried out.(2)Compared with the control group,the cells in the Cd group became shrunken and round,cell density decreased and cell viability significantly decreased(P<0.01);compared with the Cd group,the morphological demages in Cd+BAY 11-7082 group and Cd+sip65 group were more severe,and cell viability was further reduced(P<0.05),as well as the cell proliferation index in the Cd+sip65 group was significantly decreased;compared with the Cd group,shrunken cells,decreased cell density,and decreased cell viability were relieved by PMA(P<0.05).The above results suggest that the activation of NF-κB may play an active role in Cd-induced nephrotoxicity.3.NF-κB is involved in the regulation of apoptosis and autophagy in Cd-induced cytotoxicity.In order to explore the role of NF-κB protein in Cd-induced apoptosis and autophagy inhibition,we continued to use NF-κB inhibition model and activation model.Annexin V-FITC/PI double staining flow cytometry was used to detect cell apoptosis rate,GFP-RFP-LC3 plasmid transfection was used to detect autophagy flux,and Western blot was used to detect the expression levels of apoptosis marker proteins Bax,Bcl-2,cleaved caspase-3,and autophagy marker proteins LC3-Ⅱ and p62.The results showed:(1)Compared with the control group,the ratio of Bax/Bcl-2 and the expression of cleaved caspase-3 were significantly increased(P<0.01),and the apoptosis rate was significantly increased in the Cd group(P<0.01),indicating that Cd induced apoptosis in NRK-52E cells;compared with the Cd group,the ratio of Bax/Bcl-2 and the expression of cleaved caspase-3 were significantly increased in Cd+BAY 11-7082 group and Cd+sip65 group(P<0.01),and the apoptosis rate was significantly increased(P<0.05);in Cd+PMA group,the ratio of Bax/Bcl-2 and the expression of cleaved caspase-3 were significantly decreased(P<0.01),and the apoptosis rate was significantly decreased(P<0.01).(2)Compared with the control group,the expression levels of LC3-Ⅱ and p62 proteins were significantly increased,autophagosomes accumulated and autophagic flow was blocked in Cd group,indicating that Cd induced autophagy inhibition in NRK-52E cells.Compared with the Cd group,the expressions of LC3-Ⅱ and p62 proteins were further significantly increased(P<0.01),number of autophagosomes were increased,and autophagic flow was further blocked in the Cd+BAY 11-7082 group and the Cd+sip65 group;in Cd+PMA group,the expression of LC3-Ⅱ protein was significantly decreased(P<0.05),the expression of p62 protein was significantly decreased(P<0.01),number of autophagosomes was decreased,and autophagic flux was restored.The above results indicate that activation of NF-κB plays an active role in Cd-induced NRK-52E cells injury by inhibiting apoptosis and promoting autophagy.In conclusion,different concentrations of Cd exposure can promote NF-κB phosphorylation and subsequent activation of nuclear translocation.Activation of NF-κB can protect NRK-52E cells from Cd-induced cytotoxicity,which is associated with the anti-apoptotic and pro-autophagy effects of NF-κB.