The Role Of Ferroptosis In NIBV Induced Kidney Damage In Gouty Chickens

Renal infectious bronchitis virus（NIBV）infection is one of the important pathogenic factors of avian gout,which can cause serious economic losses to the poultry industry.Recent studies have shown that ferroptosis plays an important role in the progression of kidney injury-related diseases and viral infectious diseases.Therefore,the hypothesis of this study was proposed:ferroptosis may be a potential mechanism in the process of NIBV-induced kidney injury.The purpose of this study was to investigate the relationship between NIBV-induced kidney injury and ferroptosis in gout chickens,and to elucidate the role and regulatory mechanism of ferroptosis in NIBV-induced kidney injury in gout chickens.The main contents of this experiment are as follows:（1）Effect of ferroptosis on Kidney Damage in Chickens Infected with NIBVThree hundred 28-day-old Hy-Line brown chicks were random Ly divided into control group（100）and pathological group（200）.The pathological group was given0.2 m L of NIBV strain SX9(10^-5/0.2 m L)by eye drops,and the control group was given the same amount of normal saline.The chickens were observed daily after challenge,and the kidney tissues of chickens were collected at 1 dpi,5 dpi and 11 dpi.Kidney pathological damage was observed by pathological tissue section staining and transmission electron microscopy.The contents of Fe²⁺,GSH and LPO in kidney tissue were measured.The m RNA transcription levels of ferroptosis-related genes（DMT1,FTH1,FTL1,TFR1,NCOA4,GPX4,SLC7A11,ACSL4,LPCAT3,PTSG2,DHFR and FSP1）in kidney were detected by RT-qPCR and the expression levels of some ferroptosis-related proteins in renal tissue were detected by Western blotting.The results are as follows:1.In the pathological group,the chicken kidneys were swollen and chalky,with obvious urate deposits.Microscopic examination of pathological sections showed renal inflammatory cell infiltration and tubular necrosis.Under transmission electron microscopy,it was observed that the normal structure of mitochondria in the kidney was severely damaged,with specific manifestations of reduced volume,increased density of the bilayer membrane,and blurred or disappeared mitochondrial cristae.Compared with the control group,the levels of Fe²⁺and LPO in the kidneys of chickens in the pathological group were significantly increased（P<0.01 or P<0.05）,while the content of GSH was significantly decreased（P<0.01）.2.Compared with the control group,NIBV infection significantly decreased the m RNA expression levels of ferroptosis related genes FTH1,FTL1,SLC7A11,GPX4,DHFR,and FSP1（P<0.01 or P<0.05）;Significantly upregulated the m RNA expression levels of TFR1,NCOA4,ACSL4,LPCAT3,and PTSG2（P<0.01 or P<0.05）;There was no significant change in the m RNA expression level of DMT1gene（P>0.05）.3.Compared with the control group,the protein expression of NCOA4,ACSL4,and PTSG2 in the kidney of chickens in the pathological group significantly increased,while GPX4 significantly decreased,consistent with the trend of m RNA transcription level expression（P<0.01 or P<0.05）.The immunofluorescence results confirmed that the fluorescence intensity of GPX4 and PTSG2 conformed to the protein expression trend.（2）Effect of ferroptosis on primary renal tubular epithelial cells infected by NIBVPrimary renal tubular epithelial cells were prepared from 1-7 day old Hy-Line brown chicks and random Ly divided into a control group and a pathological group.The pathological group was challenged with 50%lethal dose.Samples were collected at 12h,24h,36h and 48h of the experiment to determine protein and m RNA levels.Compared with the control group,the m RNA expression levels of DMTI,TFR1,FTH1,FTL1,GPX4,and DFHR genes in renal tubular epithelial cells after challenge decreased（P<0.01 or P<0.05）;The m RNA expression levels of NCOA4,ACSL4,FSP1,LPCAT3,and PTSG2 increased（P<0.01 or P<0.05）;The trend of protein expression levels of NCOA4,ACSL4,PTSG2,and GPX4 was consistent with m RNA levels.Conclusion:NIBV infection can cause iron metabolism disorder and GSH depletion in chicken renal tubular epithelial cells,which in turn causes lipid peroxide accumulation and cell ferroptosis,which is one of the key pathways to induce chicken kidney damage.