Role Of Nrf2 Signaling Pathway In Cadmium-induced Nephrotoxicity

Cadmium(Cd)is a highly toxic heavy metal that can cause irreversible damage to multiple organs and systems.In recent years,cadmium environmental pollution and exposure to specific occupational environments have increased the threat of Cd exposure to human health.Studies have found that ingesting very small amounts of Cd could cause human kidney function damage.Oxidative stress is an important mechanism of Cd-induced nephrotoxicity,and Nrf2 signaling pathway is one of the key signaling pathways that regulate oxidative stress in the body.Previous researches showed that the autophagy enhancer trehalose and the antioxidant puerarin can exert their protective effects by inhibiting the Cd-activated Nrf2 signaling pathway,indicating Nrf2 signaling pathway could have negative effects on Cd-induced nephrotoxicity.However,there is currently no valid evidence to demonstrate the role of Nrf2 signaling pathway in Cd-induced nephrotoxicity.Therefore,this study was designed to clarify the role of Nrf2 signaling pathway in Cd-induced nephrotoxicity by in vitro and in vivo studies.In vitro studies,NRK-52 E cells were used as research objects to explore the role of Nrf2 signaling pathway in Cd-induced nephrotoxicity.Firstly,to explore the effect of Cd on Nrf2 signaling pathway,NRK-52 E cells were treated with 5 μM Cd at different time intervals(0,2,4,6,8,10,12 h).The results showed that Nrf2 protein levels in nuclear and cytoplasmic fractions and Nrf2 mRNA level reached a peak after 4-h Cd exposure,and then decreased.Then Cd treatment enriched nuclear Nrf2 protein levels and decreased Nrf2 mRNA levels while stabilized cytoplasmic Nrf2 protein levels from 6 to 12 h exposure.Simultaneously,Cd markedly decreased the nuclear and cytoplasmic Keap1 protein levels and Keap1 mRNA levels following a time-dependent manner.Also,protein levels of downstream target genes of Nrf2,i.e.,HO1 and NQO1,were determined.As expected,Cd treatment elevated protein levels and mRNA levels of HO1 and NQO1 in a time-dependent manner.These findings confirmed that Cd exposure can induce persistent activation of Nrf2 signaling pathway in NRK-52 E cells.Secondly,gene silencing technology was used to established the Nrf2-KDcells and then cells were treated with 5 μM Cd for 12 h.To test the efficiency of Nrf2 gene silencing,immunofluorescence and Western blotting were used to detect the nuclear translocation of Nrf2 and the levels of Nrf2 signaling pathway related proteins(Nrf2,Keap1,HO1 and NQO1)in NRK-52 E cells.It was confirmed that Nrf2 gene knockdown cells were successfully established.Thirdly,after the Nrf2-KD cells exposed to Cd,cell morphology observation,and cell survival rate,intracellular MDA levels,and ROS levels were measured.The results confirmed that persistent activation of Nrf2 signaling pathway exacerbated Cd-induced cytotoxicity and oxidative stress in NRK-52 E cells.Fourthly,Western blotting and GFP-LC3 plasmid transfection method were used to detect the changes of autophagy flow in NRK-52 E cells.The results showed that persistent activation of Nrf2 signaling pathway can be involved in Cd-induced autophagy inhibition.Fifthly,AO staining and LTR staining were used to detect the changes of lysosomal pH,and the protein levels of LAMP1 and LAMP2 and the mRNA levels of LAMP1 and LAMP2 in NRK-52 E cells were detected.It was found that persistent activation of Nrf2 signaling pathway can be involved in Cd-induced lysosomal dysfunction.Sixthly,the co-localization of lysosomal membrane protein LAMP1 and hydrolyzed CTSD was observed by immunofluorescence to detect the lysosomal membranes permeabilization.It was found that persistent activation of Nrf2 signaling pathway led to lysosomal membranes permeabilization.Seventhly,the protein levels of two major proteolytic enzymes(CTSB and CTSD)and the mRNA levels of four hydrolytic enzyme-encoding genes(CTSB,CTSD,CTSK,and CTSS)were detected in vivo and combined with DQ-BSA staining.The results confirmed that persistent activation of Nrf2 signaling pathway can lead to lysosomal degradation dysfunction.Finally,a combination of immunofluorescence and RFP-GFP-LC3 plasmid transfection method was used.It was confirmed that persistent activation of Nrf2 signaling pathway can be involved in Cd-induced autophagosome-lysosomal fusion defects.In vivo studies,SD rats were used as research objects to explore the role of Nrf2 signaling pathway in Cd-induced nephrotoxicity.Firstly,the protein levels of Nrf2,Keap1,HO1 and NQO1 were detected by Western blotting.The results showed that Bru can effectively inhibit Cd-induced persistent activation of Nrf2 signaling pathway in rat kidney tissue,indicating successful establishment of animal models.Secondly,the pathologicaldamage changes were detected by HE staining and the levels of MDA,CAT and GPx in rat serum were detected.The results showed that Bru can effectively relieve Cd-induced renal pathological damage and oxidative stress.Thirdly,Western blotting was used to detect the levels of p62 and LC3.The results showed that Bru can effectively relieve Cd-induced autophagy inhibition in rat kidney.Fifthly,the levels of lysosomal membrane proteins LAMP1 and LAMP2 were measured by Western blot to reflect the integrity of the lysosomal membrane,and the levels of CTSB and CTSD were measured to reflect the lysosomal degradation function.The results showed that Bru can effectively relieve Cd-induced lysosomal dysfunction.In summary,persistent activation of Nrf2 signaling pathway can participate in Cd-induced oxidative stress,and exacerbate Cd-induced kidney injury.The reasons for persistent activation of Nrf2 signaling pathway caused by Cd were Cd-induced Keap1 depletion and p62 accumulation.Persistent activation of Nrf2 signaling pathway can be involved in Cd-induced autophagy inhibition due to lysosomal damage,causing lysosomal membranes permeabilization,disrupting autophagosome-lysosome fusion and affecting autophagy flow.This study will open up new ideas for exploring the mechanisms of Cd-induced nephrotoxicity,and provide a new theoretical basis for the prevention and control of nephrotoxicity induced by Cd from the perspective of the Nrf2 signaling pathway.