Mechanism Of Endoplasmic Reticulum Stress And Apoptosis On Kidney Injury Induced By Nephropathogenic Infectious Bronchitis Virus In Gout Chickens

The infection of nephropathogenic infectious bronchitis virus（NIBV）is one of the common causes of chicken gout in production.The purpose of this study was to investigate the correlation between endoplasmic reticulum（ER）stress and apoptosis in kidney injury induced by NIBV in gout chickens,and to clarify the regulatory roles and mechanisms of ER stress and apoptosis in kidney injury induced by NIBV in gout chickens.Three hundred Hy-line brown laying hens were randomly divided into Control group（Con）and Infected group.Each chicken in the Infected group was injected with SX9 strain(10^-5/0.2m L),and the Con group was inoculated with 0.2 m L sterile normal saline in eye drops for 28 days.Blood samples were collected at 1,5,11,18 and 28 days post infection（dpi）to detect serum uric acid and creatinine levels.The kidneys were collected and pathological tissue sections were made to observe the renal lesions.The expression of ATF-6 and Bcl-2 was detected by immunohistochemistry and immunofluorescence.TUNEL assay was used to detect renal apoptosis.After quantifying the viral load of the kidney at each time point,RT-q PCR was used to detect endoplasmic reticulum stress pathway related genes（IP3R,GRP78,PERK,e IF2α,ATF-4,ATF-6,IRE1,XBP-1,CHOP）and the m RNA expression levels of key apoptotic genes（Bax,Bcl-2,Caspase-3,Caspase-9,P53）,and the expression levels of kidney proteins（GRP78,PERK,e IF2α,ATF-4,IRE1,Bax,Caspase-3,Bcl-2）were detected by western blot.The results were as follows:1.NIBV infection can cause urate deposition and cause the classic"Mosaic kidney"symptoms in renal tissue.In addition,blood cells and inflammatory cells infiltrated outside the lumen of the renal tubules in the challenge group,resulting in serious glomerular hemorrhage.2.Compared with the Con group,serum uric acid in Infected group increased significantly at the 11 dpi（P<0.01）and gradually decreased over time.Creatinine content was significantly increased at 11 dpi,18 dpi and 28 dpi（P<0.01）,there was no significant difference at other time points.3.The morphological changes of ER in renal tissue infected by NIBV were observed by transmission electron microscopy.The results showed that NIBV infection led to the rupture of ER in kidney tissue cells,which preliminarily indicated that NIBV could induce ER stress.After chicks were infected with NIBV,the m RNA expression of glucose regulated protein 78（GRP78）was significantly increased at 18dpi（P<0.01）.Western blot results showed that NIBV infection up-regulated the expression of GRR78 protein,which further confirmed that NIBV infection induced ER stress in chicken kidneys.4.Unfolded protein response（UPR）pathway analysis showed that NIBV infection increased the m RNA and protein expressions of PERK and e IF2α（P<0.01）.In addition,the results showed that NIBV infection could increase the expression of downstream genes ATF-4 and CHOP（P<0.01 or P<0.05）.It indicated that NIBV virus infection could activate the UPR mediated by PERK-e IF2αsignaling axis.RT-q PCR and western blot results showed that NIBV infection up-regulated the expression of p-IRE1α,XBP-1,ATF-6（P<0.01 or P<0.05）,suggesting that NIBV infection induced the splicing of IRE1 substrate XBP-1 and promoted the expression of ATF-6.The above results indicated that NIBV infection can activate the PERK,IRE1 and ATF-6 pathways in the UPR.5.Compared with the Con group,apoptotic bodies appeared in the kidneys of the infected chickens by transmission electron microscopy.RT-q PCR showed that NIBV infection up-regulated the expression of apoptosis related genes（Bax,Caspase-3,Caspase-9,P53）（P<0.01 or P<0.05）,and down-regulated the expression of Bcl-2.Results of key proteins showed that NIBV infection increased the protein translation levels of Bax and Caspase-3（P<0.01 or P<0.05）,but decreased the protein level of Bcl-2（P<0.05）.In addition,the results of TUNEL staining confirmed that NIBV infection caused the increase of apoptotic cells,which further proved the occurrence of apoptosis.In conclusion,NIBV infection leads to the rupture of ER in chicken kidneys,which causes ER stress and activates the UPR,accompanied by the occurrence of renal cell apoptosis in the process,and ultimately leads to kidney damage.