The Key Effect Of Nox4 And GSH On Redox Regulation Imbalance And Kidney Injuries Induced By Cadmium

BackgroundCadmium（Cd）is a heavy metal toxic element and its exposure can cause serious damage on multiple organs.Cd has been listed as a human carcinogen by the international Agency for Research on Cancer（IARC）.The harm of Cd pollution to human beings and the ecological environment is highly concerned all over the world.There are many reports on Cd poisoning and its injury mechanism.Acute and chronic Cd exposure can cause multiple organ damage,but the mechanism of Cd injury is unclear.The research progress shows that Cd poisoning is closely related to the imbalance of the Redox system.However,some reports showed that Cd is not an active element and generally can not act with other material in the Redox reaction.Therefore,effect of Cd on oxidative damage is still a mystery.Some researchers think that Cd may damage antioxidant defense system by activating other factors,so it can induce oxidative stress.It is unclear which molecules play a critical regulatory role due to the many factors involved in the redox balance.We need further studied on it.Kidney is most import organ where Cd accumulated.Oxidative stress can damage the filtration function of renal tubules,and then lead to the accumulation of metabolin,including toxic substances,such as Cd²⁺compounds.Therefore,we need future study on the mechanism of Cd poisoning based on our previous research.It is very important for the discovery of the intervention measures of Cd toxicity and public health.ObjectiveThe risk of chronic injury induced by Cd increase because Cd pollution,the opportunity of people intake Cd increase and long biology half-life at the present.Cd toxicity is closely related to redox imbalance and no effective measurement for Cd toxicity is found.Based on the above reasons,we did the following research.We screened the different expression genes between Cd exposure and non-cd exposure by gene chip data and data mining technology in order to find the crucial genes closely related to Cd.We established animal models of renal tissue oxidative damage induced by subchronic Cd exposure,studied early biomarkers,verify the role of key genes suggested by gene chip,tried to find the key molecules of Redox imbalance,and search for effective anti-Cd drugs.At last,based on our results from animal,we established the low expression stable strain of Nox4 by lentivirus transfection and investigated the interaction of Nox4 and GSH on Cd toxicity.Methods1.Differential expression gene（DEGs）between Cd exposure and non-cd exposure were screened by GSE9951 gene chip data,and the DEGs was conducted to evaluate the role of Cd in oxidative damage by genomic analysis.The hub genes closely related to Cd were screened and evaluated and pave to mechanism of Cd poisoning on animal and cell level.（1）Screening of DEGs.（2）Analysis of gene expression profile.（3）PCA analysis of DEGs.（4）Construct protein-protein interaction（PPI）network of DEGs.（5）GO function and KEGG enrichment analysis of DEGs.（6）Analysis of the interaction between Cdcl2 and proteins.2.We established animal models of renal tissue oxidative damage induced by subchronic Cd exposure,studied early biomarkers,verify the role of key genes suggested by gene chip,try to find the key molecules of Redox imbalance,and search for effective anti-Cd drugs.（1）Establishment of animal model on renal tissue oxidative damage induced by subchronic Cd exposure.（2）Selection and determination of early biomarkers for renal injury.（3）Determination of key ions in rat serum,urine and renal cortex.（4）Determination of enzyme activity in the Redox system.（5）Determination of cell apoptosis in kidney.（6）Effect of Apocynin on Cd nephrotoxicity.3.Based on the results from animal,we established the low expression stable strain of Nox4（sh-Nox4-NRK-52E）by lentivirus transfection in rat renal tubular epithelial cells（NRK-52E）and investigated the interaction of Nox4 and GSH on Cd toxicigy.（1）Oxidative damage of induced by Cd in sh-Nox4 NRK-52E cells.（2）The effects of Cd on NADP⁺/NADPH and GSSG/GSH in sh-Nox4 NRK-52E cells.（3）The apoptosis of sh-Nox4 NRK-52E cells induced by Cd.Results1.Genomic analysis suggested that Cd exposure caused changes in gene transcription spectrum at the initial stage.All hub genes closely related to Cd were related to mitochondrial respiratory chain complex and NADPH electron transfer.and oxidative phosphorylation of key proteins and signal pathway disorder of NADPH electron transfer on mitochondrial are the key factors of damage induced by Cd.2.We confirmed on animal studies thatα₁-MG andβ₂-MG were better biomarkers for early renal injury.Cd competitively inhibited divalent metal ions such as Ca²⁺,Fe²⁺and Zn²⁺,and increased the free Fe²⁺and Zn²⁺in serum,but reduced the contents of Ca²⁺,Fe²⁺and Zn²⁺in renal cortex.Cd reduced the activity of the antioxidant enzymes,such as SOD,CAT,GPx which contained metal ions and induced ocidative damage.This was the main reason of renal toxicity for Cd exposure.3.Both Nox and GSH were closely related to electron transport of NADPH.Cd inhibited the antioxidant activity of GSH and enhance the role of Nox4.This effect increased the accumulation of ROS in renal tissues and induced Redox imbalance mainly caused by oxidative damage.4.For the first time,We found that Apo can effectively antagonize the oxidative damage of renal tissue induced by subchronic Cd.Apo is a Nox inhibitor.It can effectively improve the activity of antioxidant enzymes,reduce the production of ROS and relieve the oxidative damage caused by Cd.5.By Knockdown of Nox4 gene at the cell level,we verified that Cd can deplete GSH,reduce the NADPH content,increase the NADP⁺/NADPH ratio,result in NADPH electron transport disorder involving in GSH and Nox.It showed the activity of all antioxidant enzyme decreased.Our results proved that Nox4 plays an important regulatory role on redox imbalance induced by Cd in renal tubular epithelial cells.ConclusionsBased on the study of biological information,our results showed that the damage of NADPH electron transport chain was an important reason for the redox imbalance caused by Cd in renal in animal and cell level.NADPH oxidase（Nox）and GSH,which are closely related to the NADPH electron transport,play important regulatory roles in Cd nephrotoxicity.Cd can cause the damage of antioxidant repair system by increasing expression of Nox4 and inhibiting the effect of antioxidant enzymes such as GSH.Our study provides experimental basis for further searching for effective intervention measures and prevention Cd poisoning.