The Role And Underlying Mechanisms Of Nrf2 In Kidney Injury Induced By Cadmium

Objective:Cadmium（Cd）is a potentially toxic heavy metal,which possesses a destructive impact to most organs.Over the past few decades,the rapid development of industry and agriculture has led to excessive emission of cadmium,increasing the exposure of cadmium and causing more adverse health effects to the population.Kidney is recognized as a major target of cadmium-induced toxicity,approximately 50%of the accumulated dose is stored in the kidneys.Short-term and high-dose cadmium exposure can cause acute kidney damage,while long-term and low-dose cadmium exposure can lead to chronic kidney disease（CKD）.Dialysis or kidney transplantation is required in patients in the absence of timely interventions.Therefore,exploring novel therapeutic targets to prevent and treat kidney injury induced by cadmium is urgent.The production of reactive oxygen species（ROS）and inflammatory response induced by cadmium is the main cause of cell damage.Transcription factor E2-related factor 2（Nrf2）is the core transcription factor that regulates antioxidant defense genes and phase II detoxification enzymes,and plays an important role in regulating intracellular adaptive antioxidant response and redox homeostasis.Although in vitro studies have shown that Nrf2 silenced NRK-52E cells are more sensitive to Cd-induced apoptosis,while the overexpression of Nrf2 prevents Cd-induced apoptosis.However,there are no studies on the use of gene knockout mice to verify the key role of Nrf2 in Cd-induced renal injury.Moreover,renal tubular epithelial cells are extremely sensitive to cadmium toxicity,there are no studies to explore the role of Nrf2 in renal tubular epithelial cell human kidney-2（HK-2）injury induced by cadmium.Taken together,in this study,Nrf2-KO mice and NRF2 silenced HK-2 cells were used to explore the role and underlying mechanism of Nrf2 in kindey injury induced by subacute and subchronic cadmium exposure.The aim was to provide a new experimental basis for prevention and treatment of kidney injury induced by cadmium exposure.Methods:1.Experimental model of NRF2 silenced HK-2 cells exposed to cadmium in vitroThe aim was to obtain NRF2-KD HK-2 cells and Scramble cells by sh RNA lentivirus transfection technique.The cells were treated with 10μM CdCl₂ for 0,6,12,18 and 24 hours,and the cell survival rate was detected by trypan blue staining and CCK8 assay.Total RNA was extracted for measuring the transcript levels of genes associated with inflammatory responses,NRF2 and downstream of NRF2 pathway,Cd metabolism and detoxification.Total proteins were extracted to detect NRF2 and its downstream proteins expression.2.Experimental model of Nrf2-KO mice exposed to subacute and subchronic cadmium in vivoSubacute cadmium exposure model:adult Nrf2-WT and Nrf2-KO mice were intraperitoneally injected with normal saline and 1 mg/kg Cd or 2 mg/kg Cd,once a day for 7 days.They were divided into 6 groups:WT-Cont group,KO-Cont group,WT-1mg/kg Cd group,KO-1 mg/kg Cd group,WT-2 mg/kg Cd group and KO-2 mg/kg Cd group.Body weight was monitored every day.The mice were anesthetized after 7 days,blood,kidney and liver were collected for further analysis.Subchronic cadmium exposure model:adult Nrf2-WT and Nrf2-KO mice were provided normal drinking water or water containing 100 ppm or 200 ppm CdCl₂ for 24weeks.Mice were divided into 6 groups:WT-Cont group,KO-Cont group,WT-100 ppm group,KO-100 ppm group,WT-200 ppm group and KO-200 ppm group.The body weight,food and water consumption of mice were monitored every week,blood glucose and 24-hour fasting urine was collected through metabolic cages were monitored every month.The mice were anesthetized after 24 weeks,blood,liver and kidney were collected for further analysis.3.Mice samples treatmentThe contents of serum urea nitrogen,creatinine,neutrophil gelatinase associated lipid transport protein（NGAL）and kidney injury molecule 1（KIM1）,urine creatinine,NGAL and KIM1 were detected by corresponding kits.Periodate Schiff’s（PAS）,Terminal-deoxynucleoitidyl transferase mediated nick end labeling（TUNEL）and immunofluorescence staining were performed on the kidney fixed by 4%paraformaldehyde.The total RNA of kidney tissue was extracted to detect the transcript level of genes related to oxidative stress,cadmium metabolism and detoxification,inflammation and fibrosis response.Total proteins were extracted to detect fibrosis response,Nrf2 and its downstream proteins expression.Results:1.After CdCl₂ treatment,the expression of cadmium metabolism and detoxification,NRF2 and its downstream genes significantly decreased in NRF2-KD cells decreased,NRF2-KD cells showed more cell death in comparison to Scramble.Scramble and NRF2-KD cells were treated with 5,10,15 and 20μM CdCl₂ for 24h,NRF2-KD cells’viability was significantly lower than Scramble and trypan blue staining showed that the death rate of NRF2-KD cells was significantly higher than Scramble treated with 10μM CdCl₂ for 24 h.Treated with 10μM CdCl₂ for 0,2,6,12,18 and 24 h,there was no significant changes in protein expression levels of p-MLKL,p-RIP1,p-RIP3,MLKL,RIP1 and RIP3,but the basic transcript levels of HO-1 and GCLM decreased significantly in NRF2-KD cells compared with Scramble.The increase transcript levels of HO-1 induced by cadmium reached the peak at 6 h,but the increased levels of NRF2-KD was significantly lower than that of Scramble.The increase transcript levels of GCLM in Scramble cells induced by cadmium reached the peak at 6 h,but was completely inhibited in NRF2-KD cells.The transcript levels of MT1 and MT2 were activated by cadmium at 6,12 and 24 h,but the level of NRF2-KD was significantly lower than Scramble.Treated with 10μM CdCl₂ for 6 h,the protein levels of NRF2,GCLC and GCLM increased significantly in Scramble cells compared with NRF2-KD cells.2.The deletion of Nrf2 in mice attenuate the expression of antioxidant genes and Cd metabolism related enzymes in kidney induced,aggravate renal oxidative stress,inflammatory response and renal injury induced by subacute cadmium exposure.There was no significant changes in body weight,liver and kidney weight and serum creatinine levels in all groups along the experimental period,but the levels of blood BUN and urine NGAL in KO-2 mg/kg group were significantly higher than WT-2mg/kg group（P<0.05）;PAS staining revealed a marked loss of brush border in proximal tubules and widespread desquamation/shedding of proximal convoluted tubules epithelial cells into tubular lumens in KO-1 mg/kg and 2 mg/kg groups.TUNEL staining showed that Nrf2-KO mice had more apoptosis of renal tubular epithelial cells under 2 mg/kg Cd exposure（P<0.05）.The immunofluorescence results of oxidative stress marker 4-HNE adduct showed that oxidative stress occurred in both genotypes of kidney under 2 mg/kg Cd exposure,but the levels of Nrf2-KO mice was higher than that of Nrf2-WT mice.The transcript levels of inflammatory cytokines Cd68 and Tnf in Nrf2-KO mice increased significantly after 2 mg/kg Cd exposure.After cadmium exposure,the levels of cadmium in blood,liver,kidney and urine were significantly increased in all groups,but there was no significant difference between Nrf2-KO and Nrf2-WT mice.Although the basic transcript levels of Gclc,Gclm,Ho-1,Mt1 and Mt2 were similar among different genotypes,the expression levels of these genes induced by cadmium was significantly decreased in Nrf2-KO mice.Consistent with mRNA levels,the protein levels of GCLC and GCLM induced by cadmium were decreased in Nrf2-KO kidney compared with Nrf2-WT.In order to eliminate the interference of anti-oxidation,cadmium metabolism and detoxification related genes expression induced by renal injury,cadmium significantly upregulated the mRNA levels of Nqo1,Ho-1 and Mt1 in the kidney of Nrf2-WT mice after 12 hours exposure,while the induction of these genes in Nrf2-KO mice decreased significantly.3.Nrf2-KO mice displayed massive renal fibrosis induced by subchronic cadmium exposure.The body weight,food and water consumption of mice decreased slightly after cadmium exposure,but there was no significant difference between two genotypes.Glucose sensitivity was not statistically different among groups.Under 200 ppm cadmium exposure,the relative weight of kidney and liver of Nrf2-KO mice was significantly lower than Nrf2-WT.Although there was no significant difference in the levels of urinary KIM1,NGAL and blood KIM1 levels among all groups,the levels of serum NGAL and BUN in 200 ppm cadmium treated group were significantly higher in Nrf2-KO mice than Nrf2-WT mice,and the level of NGAL in urine of Nrf2-KO mice was significantly higher than Nrf2-WT mice treated with the same treatment（P<0.05）.No matter in 100 ppm or 200 ppm cadmium exposed group,the level of NGAL in urine of Nrf2-KO mice was significantly higher than Nrf2-WT mice（P<0.05）.The results of PAS staining also showed compared with Nrf2-WT mice,the tight junction between renal tubules disappeared,a large number of epithelial cells died and the basement membrane thickened significantly in Nrf2-KO mice after 100 and 200 ppm cadmium exposure,and the levels of MEGALIN protein in Nrf2-KO mice decreased with increasing cadmium dose.There was no difference in the levels of WT1 protein among different groups.There was no significant difference in mRNA levels of Cd68,Il-6 and Tnf-αamong groups.Although there was no significant difference in the transcript levels of Fibronectin,Tgf-β1,α-Sma,Vimentin,E-cadherin,Col1α1,Col3α1 and Col4α1 among different groups,the protein levels of TGF-β,α-SMA and VIVIMENTIN in Nrf2-WT and Nrf2-KO mice exposed to 100 ppm and 200 ppm cadmium were significantly higher than those in control mice,and has a significant dose-response relationship,moreover,the expression levels of these proteins in Nrf2-KO mice was higher than Nrf2-WT mice.Although the basic mRNA levels of Gclc,Ho-1,Mt1 and Mt2 were similar among different genotypes,the expression of these genes induced by cadmium was significantly decreased in Nrf2-KO mice;In Nrf2-KO group,compared with the Nrf2-WT group,the expression of Nqo1 decreased significantly under cadmium exposure;Consistent with mRNA levels,the protein expression of GCLC,GCLM,NQO1 and HO-1 induced by100 ppm and 200 ppm cadmium exposure was significantly higher than Nrf2-KO in the kidney of Nrf2-WT mice.Conclusion:1.NRF2-KD HK-2 cells were more sensitive to cadmium toxicity.2.After subacute cadmium exposure,oxidative stress and inflammation in the kidney of Nrf2 deficient mice were more severe.Nrf2 plays a protective role by regulating the expression of downstream antioxidant genes and enzymes related to cadmium metabolism.3.After subchronic cadmium exposure,renal fibrosis was more severe in Nrf2deficient mice.Nrf2 plays a protective role by regulating the expression of downstream antioxidant genes and enzymes related to cadmium metabolism.