The Mechanism Of Autophagy And Pyroptosis Co-induced By Molybdenum And Cadmium In Duck Kidneys

Molybdenum（Mo）is an essential trace element for animals,but excess Mo has toxic effects on the body.Cadmium（Cd）is a highly toxic,cumulative industrial and environmental heavy metal pollutant.The combined pollution of Mo and Cd to the environment is often caused by industrial and agricultural production,and ultimately endangers animal and human health through the food chain.The kidney is a crucial target organ for the toxicity of Mo and Cd,and the renal tubule is the target site of nephrotoxic damage.Autophagy and pyroptosis are two kinds of programmed cell death that are completely different in morphology and mechanism,but they are closely related and affect each other.At present,there is no report on the occurrence mechanism of autophagy and pyroptosis in the process of kidney injury caused by the combination of Mo and Cd as well as the regulation relationship between the two metals.Therefore,in this study,duck was used as the experimental animal to explore the mechanism of autophagy and pyroptosis co-induced by Mo and Cd in duck kidneys.240 healthy 8-day-old Shaoxing ducks（Anas platyrhyncha）were randomly divided into 4 groups（60 birds/group）,and Cd or/and Mo were added to basic diet per kilogram（kg）in vivo:control group（0 mg Mo and 0 mg Cd）,Mo group[100 mg Mo,（NH₄）₆Mo₇O₂₄·4H₂O],Cd group（4 mg Cd,3Cd SO₄·8H₂O）,and Mo+Cd group（100 mg Mo and 4 mg Cd）.At the 4^th,8^th,12^th,and 16^thweeks of the experiment,10 ducks were randomly collected from each group,blood and kidneys of each duck were collected,respectively.Transmission electron microscopy,real-time fluorescent quantitative PCR and Western blotting and so on were used to detect the contents of trace elements,oxidative stress indexes,and the levels of autophagy and pyroptosis in kidneys.Additionally,duck primary renal tubular epithelial cells were used as the research object in vitro.The IC₅₀of cells treated with Mo or Cd alone for 12 h was combined with a series of concentrations of Cd or Mo for 12 h,or cells were treated with 500μM Mo,4μM Cd and500μM Mo+4μM Cd for 12 h,respectively.Finally,antioxidant 100μM BHA,pyroptosis inhibitors（10μM MCC950 and 10μM Z-YVAD-FMK）and autophagy inhibitors（2.5μM3-MA and 10μM CQ）were applied for the intervention of Mo and Cd co-exposure for 12h,respectively.Cells and supernatant samples were collected,respectively.The cell viability,Mo and Cd contents,oxidative stress indexes,the levels of autophagy and pyroptosis were detected by using flow cytometry,real-time fluorescent quantitative PCR,Western blotting and so on.The results were as following:1.Mo or/and Cd obviously increased their contents in kidneys,decreased Cu,Fe,Zn,Se contents and CAT,T-SOD,GSH-Px activities,and elevated MDA,H₂O₂,UA and CREA contents,resulting in renal tubular epithelial cells degeneration,necrosis,interstitial congestion,bleeding and inflammatory cell infiltration as well as mitochondrial swelling,cristae fragmentation,even vacuolization,nuclear deformation and pyknosis;increased the number of autophagosomes and LC3II puncta,evidently upregulated autophagy-related factors（AMPKα-1,Atg5,Beclin-1,LC3A,LC3B and LC3II/LC3I）expression levels,markedly downregulated autophagy-related factors（m TOR,Dynein and p62）expression levels;obviously elevated pyroptosis-related factors（NLRP3,Caspase-1,ASC,NEK7,GSDMD,GSDME,GSDMA,IL-1βand IL-18）expression levels and the contents of IL-1βand IL-18,and showed time-effects.The above results showed that Mo and/or Cd could interfere with the homeostasis of trace elements in duck kidneys,thereby causing oxidative stress,ultimately leading to pathological damage to renal tissue,and induced autophagy and pyroptosis.2.Mo or/and Cd caused shrink,round and vacuolation of cells,and significantly reduced cell viability.Toxicity of non-equitoxic binary mixtures of Mo and Cd presented synergic effects.3.Mo or/and Cd markedly increased the contents of Mo and Cd in cells,induced oxidative stress and autophagy,and MCC950 remarkably decreased pyroptosis caused by Mo or/and Cd,which demonstrated that Mo or/and Cd triggered autophagy and pyroptosis and the combination of Mo and Cd induced pyroptosis through the NLRP3/Caspase-1pathway..4.BHA markedly reduced oxidative stress,autophagy and pyroptosis co-induced by Mo and Cd,which indicated that autophagy and pyroptosis co-induced by Mo and Cd were mediated by oxidative stress.5.Z-YVAD-FMK dramatically alleviated autophagy co-induced by Mo and Cd,indicating that inhibiting pyroptosis notably reduced autophagy co-induced by Mo and Cd.6.3-MA（or CQ）distinctly enhanced oxidative damage and pyroptosis co-induced by Mo and Cd,illustrating that inhibiting autophagy obviously promoted pyroptosis co-induced by Mo and Cd.In conclusion,Mo or/and Cd could interfere with the homeostasis of trace elements in duck kidneys and cause oxidative stress,thereby leading to abnormal renal function and renal pathological damage.Toxicity of non-equitoxic binary mixtures of Mo and Cd presented synergic effects.Mo and Cd co-exposure induced autophagy and pyroptosis mediated by oxidative stress in duck kidneys.Inhibition of pyroptosis remarkably reduced autophagy co-induced by Mo and Cd in duck renal tubular epithelial cells,and inhibition of autophagy markedly increased pyroptosis co-induced by Mo and Cd.