Molecular Mechanism Of Ammonia-induced Inflammatory Injury Based On RIP/MLKL Pathway In Chicken Kidneys

Ammonia（NH₃）is an environmental pollutant in livestock and poultry houses.Excess NH₃ has adverse effects on the health of livestock,poultry,and farm worker.Kidney is the most important excretory organ of the body.Previous studies have reported that NH₃ exposure caused structure alterations in kidneys,but the molecular mechanism of NH₃-induced kidney injury remains unclear.In order to explore the mechanism,we established a chicken model of NH₃ exposure,collected the kidney tissues,observed the microstructure and ultrastructure,detected oxidative stress parameters and ATPase activities by assay kits,then performed q RT-PCR and Western blot to determine the m RNA and protein expression of mitochondrial dynamics-related genes,necroptosis-related genes,DAMPs,cytokines,inflammatory factors,mi R-6615-5p,and Smad7.A total of 108 broiler chicks（Ross 308）at one day of age were randomly allocated into three groups:the control group,the NH₃group 1,and the NH₃ group 2.In the control group,NH₃ concentration was held below 5 mg/m³during the entire experimental period.In the NH₃ group 1 and the NH₃ group 2,NH₃ concentrations were 10±0.5 mg/m³ and 20±0.5 mg/m³ from 0 to 21 days of age,and 15±0.5 mg/m³ and 45±0.5 mg/m³ from 22 to 42 days of age,respectively.On the 14^th,28^th,and 42^nd days of the experiment,kidney samples were collected.The light microscope and transmission electron microscope were used to observe the microstructure and ultrastructure of chicken kidney tissue.The assay kits were used to measure oxidative stress parameters（SOD,CAT,GSH-Px,and T-AOC activities;H₂O₂ and MDA contents）,ATPase(Na⁺-K⁺-ATPase,Mg²⁺-ATPase,Ca²⁺-ATPase,and Ca²⁺-Mg²⁺-ATPase activities),and inflammatory factors（i NOS activity and NO content）.The q RT-PCR and Western blot were performed to determine the m RNA and protein expression of mitochondrial dynamics-related genes（drp1,opa1,mfn1,mfn2,and mff）,necroptosis-related genes（TNF-α,RIP1,RIP3,MLKL,and JNK）,DAMPs（HMGB1,HSP70,and HSP90）,cytokines（IL-1β,IL-2,IL-4,IL-6,IL-12β,IL-17,IFN-γ,and TGF-β₁）,inflammatory factors（IκBα,NF-κB,COX-2,PGEs,and i NOS）,mi R-6615-5p,and Smad7.The results showed that:（1）After NH₃ exposure,the microstructure of chicken kidneys showed evident alterations,including a large number of red blood cells in renal interstitium,the infiltrations of mononuclear cells in renal glomeruli and interstitium,and granular cast formation.The result demonstrated that NH₃ exposure induced kidney injury in chickens.The ultrastructure of chicken kidneys showed typical necrotic features,including mitochondrial swelling,the disruption of mitochondrial cristae,plasma membrane rupture,and the release of intracellular contents.The result demonstrated that NH₃ exposure led to nephrocyte necrosis in chickens.（2）NH₃ exposure inhibited the activities of SOD,CAT,GSH-Px,and T-AOC,increased the contents of H₂O₂ and MDA,caused oxidative stress in chicken kidneys.Mitochondrial dynamics-related genes drp1 and mff were up-regulated;opa1,mfn1,and mfn2 were down-regulated;and ATPase activities were reduced,suggesting mitochondrial dysfunction occurred.Necroptosis-related genes TNF-α,RIP1,RIP3,MLKL,and JNK increased,indicating that necrosis type was necroptosis in chicken nephrocyte.These results showed that oxidative stress and mitochondrial dysfunction were involved in NH₃-induced nephrocyte ncroptosis in chickens.（3）With the increase of NH₃ exposure time and concentration,the expression of HMGB1,HSP70 and HSP90 elevated,which indicated the secretion of DAMPs increased.Th2 and Th17cytokines up-regulated,Th1 and Treg cytokines down-regulated,which revealed that Th1/Th2 and Th17/Treg imbalance occurred.NF-κB was activated and inflammatory factors COX-2,PGEs,i NOS,and NO expression increased,which indicated NH₃ caused demonstrated inflammatory response in chicken kidneys.These results showed that NH₃ exposure induced inflammatory injury in chicken kidneys through DAMPs secretion,immune imbalance,and NF-κB pathway.（4）Through the detection and analysis of Smad7 and mi R-6615-5p,it is preliminarily confirmed that Smad7 was a target gene of mi R-6615-5p.The result revealed that mi R-6615-5p/Smad7 axis participated in NH₃ exposure-induced kidneys injury in chickens.In summary,NH₃ exposure induced oxidative stress and mitochondrial dysfunction,activated the RIP/MLKL pathway,and resulted in nephrocyte necroptosis in chickens.NH₃ exposure promoted the secretion of DAMPs,induced Th1/Th2 and Th17/Treg imbalances,triggered NF-κB pathway,regulated mi R-6615-5p and Smad7,and caused inflammatory injury in chicken kidneys.This study is helpful for understanding the nephrotoxicity mechanism of NH₃,provides new insight into toxic effects of NH₃,and is meaningful for poultry health and the occupational safety of farm workers.