Role Of SETD6/ Nrf2 In The Mechanism Of Redox Homeostasis Disorder In Nephrotoxicity Induced By Cadmium And Intervented By Selenium

【Background】In recent years,the exploitation of cadmium ores is increasing,and cadmium has been widely used in industry and life.The cadmium environmental pollution aggravates year by year,which becomes serious public health problem.Despite the large number of reports on the treatment and mechanism of cadmium toxicity from the view of oxidative stress,lipid peroxidation,DNA damage,apoptosis and necrosis,effective strategies against cadmium poisoning are still lacking.The main problem lies in that the role of oxidative stress and how cadmium metabolism was regulated have not been fully understood.Our previous experiments showed that the expression of transcription factor Nuclear factor（erythroid-derived 2）-like 2（Nrf2）and methyltransferase SET domain containing protein 6（SETD6）both were significantly down-regulated in the rats treated with cadmium.Our further studies showed that Nrf2could be activated by its upstream signaling molecule protein deglycase DJ-1（DJ-1）and then regulated a series of downstream molecules to keep redox homeostasis.Under physiological conditions,SETD6 binds to chromatin DJ-1 and inhibits the activity of DJ-1.While under pathological conditions such as oxidative stress,the expression of SETD6 is down-regulated and DJ-1 is then activated.Therefore,we speculated that SETD6 and Nrf2 might play a key regulatory role in the imbalance of redox homeostasis in cells caused by cadmium.So now the questions are,can we reduce ROS levels by reguating SETD6 and Nrf2 to alleviate oxidative stress and protect cell function?How do SETD6/DJ-1/Nrf2 work on each other?We have previously studied the antagonism of selenium on cadmium toxicity and found that the treatment dose of selenium is the key point of its protection effect.Selenium can actually to be toxic with cadmium when beyond some certain doses.Therefore,what dose of selenium can antagonize cadmium toxicity without causing cell viability?How selenium affects SETD6 and Nrf2 expression?These are important scientific issues that need to be solved to study the pathogenic mechanism and therapeutic strategies of cadmium poisoning.It is also of great practical significance to clarify the key role of SETD6 and Nrf2 in cadmium poisoning and to study the dose effect and mechanism of selenium on antagonizing cadmium toxicity.【Aims】1.To investigate the mechanism of protein methyltransferase SETD6,oxidative stress receptor DJ-1,and transcription factor Nrf2 in the maintaining redox homeostasis and restoring cadmium-induced redox homeostasis disorder.2.To explore the antagonistic effect and mechanism of selenium supplementation on cadmium nephrotoxicity.【Methods】1.Cell models:By establishing a cadmium-induced redox imbalance model of renal tubular epithelial cells,dose and time effects of cadmium on ROS/SETD6/DJ-1/Nrf2were observed and relevant mechanisms were investigated.At the same time,the cell proliferation activity was measured,the redox status indicators such as ROS,SOD,MDA content in the cell homogenate were detected,and the changes of ROS,SETD6,DJ-1 and Nrf2 after selenium supplementation.To further study the role of SETD6,NRK-52E cells were transfected with SETD6-overexpressed lentivirus to determine the above indicators.2.Animal models:48 male SD rats weighing 120-150 g were randomly divided into control group,cadmium group,selenium group,and selenium+cadmium group,12 in each group.Animals were administrated by cadmium chloride and sodium selenite solutions by means of intragastric administration.The animals were treated with 3 mg Cd²⁺/kg body weight and 0.01 mg Se⁴⁺/kg body weight once a day and six times per week for 12 weeks.The body weight and kidney weight were measured.Serum,urine,and kidney tissues were collected to detect urea nitrogen,creatinine,NAG,SOD,MDA,GSH,and other biochemical indicators.Total kidney protein was extracted to determine the expression of redox homeostasis-regulatory proteins such as SETD6,DJ-1,and Nrf2.Renal cortical pathology was prepared and stained with HE.In total,the oxidative stress and redox system response in the cadmium-induced renal injury model were systematically studied,with emphasis on the effect of selenium on antagonizing cadmium toxicity.【Results】1.Cadmium exposure could dose-and time-dependently reduce renal cell viability,induce apoptosis and necrosis.With increasing dose and time of cadmium exposure,intracellular ROS level went up and the mRNA levels of SETD6 and DJ-1 decreased but that of Nrf2 increased.The mRNA level of Gpx1 was significantly inhibited but with an increase at 1-4 h.The detection of protein expression showed that with the increase of cadmium exposure time,SETD6 expression gradually decreased,Nrf2expression was upregulated at 0-6 h and gradually returned to basic level at 6-24 h.With the increase of cadmium concentration,the expression of SETD6 protein gradually decreased.Cadmium at a low concentration promoted the expression of DJ-1and Nrf2.But cadmium at a high concentration inhibited the expression of both.2.Selenium at low concentration had no significant effect on cell viability.CCK8tests showed that the cell viability did not change significantly after treatment with selenium below 2560 nM.Selenium could inhibit the cytotoxicity of cadmium and inhibit ROS production within a certain dose range.On the other hand,RT-PCR analysis showed that after treating cells with 10-40 nM selenium for 24 h,the mRNA levels of SETD6,DJ-1,Nrf2,Gpx1,and Gclc were significantly increased.The protein expression of SETD6,DJ-1,and Nrf2 increased significantly after cells were treated with 10 nM selenium and 10μM cadmium.3.The effect or co-effect of cadmium and selenium had no significant effect on body weight and kidney coefficient in rats.Biochemical indicators showed that cadmium exposure can significantly increase the level of urea nitrogen and creatinine in blood and urine and increase the total protein concentration in urine.Selenium intervention can significantly reduce the biochemical indicators such as urea nitrogen,creatinine,urinary total protein concentration.DHE staining of cryosections in the renal cortex showed that cadmium stimulated ROS production in kidney tissue.Whereas after selenium intervention,ROS level was effectively decreased.Detection of renal oxidative stress markers showed that cadmium exposure can significantly increase MDA,GSH,GSSG,NAG levels,inhibit SOD activity,and induce oxidative damage.After selenium intervention,the above indicators were effectively improved.Protein detection revealed that cadmium exposure inhibited the expression of SETD6,DJ-1,and Nrf2.Selenium intervention increased the expression of DJ-1 and Nrf2,but further decreased the expression of SETD6..Histopathological results showed that cadmium exposure caused severe glomerular atrophy,disordered and swelled renal tubules,narrow lumen,severe renal interstitial congestion,and severe inflammatory cell infiltration.Selenium intervention significantly improved the above pathological changes.4.SETD6 over-expressed cells were tolerant to cadmium toxicity compared to wild-type cells as comparing by cell viability.However,there was no significant difference in ROS production.【Conclusions】1.In kidney cells,cadmium can cause oxidative stress and promote ROS production.Low concentrations of cadmium can produce low levels of ROS,activate DJ-1,Nrf2and downstream antioxidant enzymes to improve oxidative stress injury.Comparatively high concentrations of cadmium can produce high levels of ROS,downregulate DJ-1,Nrf2 and downstream antioxidant enzymes to induce oxidative stress.Thess results indicated that the activation of Nrf2 pathway is closely related to cadmium induced oxidative level.Once ROS was overproduced,the antioxidant system regulated by Nrf2 would be damaged and resulted in oxidative damage.It has been reported that SETD6 expression is significantly higher in tumor cells than that in normal cells,which prevents cells from apoptosis by inhibiting NF-κB.In this study,with the increase of cadmium treatment dose and time,SETD6 expression decreased,apoptosis and necrosis were observed,and cell viability decreaesd.Therefore,we speculated that under physiological conditions,the binding of SETD6 to Nrf2 was blocked and the transcription of DJ-1 was inhibited,resulting in a low level of antioxidant system.Under pathological conditions such as oxidative stress,downregulated SETD6 casused the release of DJ-1 and activate transcription of Nrf2,which raises the level of the antioxidative system to combat oxidative stress and maintain cellular redox homeostasis.2.Selenium activates SETD6 and Nrf2 expression.On the one hand,selenium enhances intracellular antioxidant levels by activating Nrf2 expression and Gpx1.Beacause the maintenance of redox homeostasis requires a dynamic balance between oxidative and antioxidative levels,excessive antioxidant levels can activate cellular adaptive mechanisms by upregulating SETD6.Therefore,the activity of DJ-1 and Nrf2is inhibited so as to keep the redox homeostasis.Selenium,on the other hand,is an essential element for the synthesis of selenoproteins in cells.When the maximum amount of selenium required for selenoprotein synthesis is reached,excess selenium will cause toxicity.Therefore,the role of selenium in antagonizing the toxicity of cadmium is limited within a certain range.In this study,selenium can exert a significant protective effect in the range of 10-20 nM.As the concentration increases,the protective effect gradually decreased.3.Long-term low-dose cadmium exposure can significantly induce kidney damage in rats.In this study,cadmium was administered at a dose of 3 mg/kg body weight for12 weeks to establish a chronic renal oxidative injury model in rats.Serum and urine biochemical indicators showed that urea nitrogen,creatinine,and urinary total protein concentrations were significantly elevated and renal function was impaired.The detection of oxidative stress indicators showed that MDA,ROS,NAG content increased and the activities of antioxidant enzymes SOD and Gpx were significantly decreased in renal tissue.These results suggested that oxidative stress and lipid peroxidation occured in the kidney.However,the expression of SETD6,DJ-1,and Nrf2 proteins were significantly down-regulated,suggesting that the kidneys were in a state of severe oxidative stress and SETD6 was heavily depleted,DJ-1 and Nrf2 were inhibited,and redox homeostasis was seriously deregulated.Obvious glomerular and tubular lesions can be observed in pathological findings.After intervention with selenium,the biochemical indexes such as urea nitrogen,creatinine,and urinary total protein decreased significantly.The redox indicators such as MDA,ROS content and the activities of SOD and Gpx in the kidney were also improved.The pathological changes of the glomerulus and renal tubules were alleviated.NAG content and activity also declined.The above results together indicated that 0.01 mg/kg body weight of selenium can effectively reverse renal oxidative damage caused by cadmium exposure.4.Overexpression of SETD6 can reduce the cytotoxic effect of cadmium on cells but has no significant effect on cadmium-induced ROS production.