Study On Mechanisms Of Nickel Chloride-damaged Kidney In Broilers

Nickel（Ni）is widely present in the environment due to its abundance in the earth’s crust.Ni and its compounds are widely used in modern industry.The widespread extraction and use increase Ni concentrations in biogeochemical cycles and enhance human exposure to Ni and Ni compounds through environmental contamination and occupational exposure.And,Ni has been recognized as a toxicological and oncogenic heavy metal.It has been reported that excess exposure to Ni can induce oxidative stress,inflammatory responses,apoptosis and necrosis,which then impaire the histological structure and function.It is known that kidney plays a principal role in Ni excretion and is the major target organ of Ni toxicology.NiCl₂ is one of the most common Ni compounds in nature.Up to now,there have no systematic studies on the NiCl₂-damaged kidney in animals and human.Therefore,the present study was conducted to explore the potential molecular mechanisms of NiCl₂-damaged kidney by observing the changes of the histological structure,function,oxidative stress,cell cycle,ER stress,apoptosis,and inflammatory responses with the methods of experimental pathology,flow cytometry,qRT-PCR,immunohistochemistry,ELISA and TUNEL,which provide new data reference and theoretical basis for further study on the toxicity effects of Ni on kidney.The results are as follows:1.Effects of NiCl₂ on renal histological structure,function and oxidative damage in broilersA total of 280 one-day-old healthy broilers were divided into four groups（N=70）.Broilers were feed with the corn-soybean basal diet containing 0,300,600 and 900 mg/kg NiCl₂,respectively.Broilers were housed in cages with electrical heaters and were provided with feed and water as well as above-mentioned experimental diets ad libitum for 42 days.During the experiment,clinical signs were observed daily.The pathological observation was conducted at 14,28 and 42 days of the experiment.The results showed that NiCl₂ in excess of 300 mg/kg decreased the renal volume and viscera index.NiCl₂ resulted in dose-and time-dependent histopathological changes in the kidney,including tubular granular degeneration,vacuolar degeneration,necrosis and apoptosis.The renal histological structure was impaired.The renal functional parameters were detected by the method of biochemistry at 14,28and 42 days of the experiment.The results showed that NiCl₂ in excess of 300 mg/kg elevated ALP activity,and reduced activities of Na⁺/K⁺-ATPase,Ca²⁺-ATPase,LDH,SDH and ACP in the kidney.The abovementioned results showed that the renal function was impaired,which was consistent with the impairment of the renal histological structure,and Ni accumulation in kidney.The parameters of oxidative stress in the kidney were detected with the methods of biochemistry and qRT-PCR at 14,28 and 42 days of the experiment.NiCl₂ in excess of 300mg/kg elevated the MDA,NO,8-OHdG contents,and reduced the ability to inhibit hydroxy radical.The T-AOC and activities of antioxidant enzymes including CuZn-SOD,Mn-SOD,CAT,GSH-Px,GR and GST were decreased as well as the GSH contents were decreased in the kidney in the NiCl2-treated groups.Concurrently,mRNA expression levels of the renal CuZn-SOD,Mn-SOD,CAT,GSH-Px,GST and GR were decreased,and the renal iNOS activity and mRNA expression levels were increased.The above-mentioned results showed that NiCl₂ caused renal oxidative damage,which was pathological basis of NiCl2-damaged renal structure and function.2.Effects of NiCl₂ on renal cell cycle in broilersGrouping and treatment of the experimental animals were consistent with 1.At 14,28 and42 days of the experiment,flow cytometry showed that NiCl₂ in excess of 300 mg/kg arrested G₂/M cell cycle and reduced cell percentages at G₀/G₁ phase.The qRT-PCR and immunochemistry results showed that NiCl₂ increased p-ATM protein expression and ATM mRNA expression.NiCl₂-induced the G₂/M cell cycle arrest via ATM-mediated p53-dependent and–independent pathways.In p53-dependent pathway,p53,p21 protein expression levels and mRNA expression levels were increased.In p53-independent pathway,p-Chk1,p-Chk2 protein expression and Chk1,Chk2 mRNA expression levels were increased,and p-cdc25C protein expression levels and cdc25 mRNA expression levels were decreased.Meanwhile,the results showed that the G₂ phase to M phase cell cycle regulatory molecules including cdc2,cyclinB and PCNA protein expression levels and mRNA expression levels were decreased.The above-mentioned results show that NiCl₂-induced G₂/M cell cycle arrest via ATM-p53-p21 and ATM-Chk1/Chk2-cdc25pathways in the kidney,which finally inhibited the renal cell proliferation and renal development.3.Effects of NiCl₂ on renal ER stress in broilersGrouping and treatment of the experimental animals were consistent with 1.At 14,28 and42 days of the experiment,NiCl₂ in excess of 300 mg/kg induced ER stress,which was characterized by increasing protein levels and mRNA expression levels of ER stress markers,e.g.,GRP78 and GRP94.The UPR pathway responses were detected in the ER stress by qRT-PCR.All the three UPR pathways were activated by NiCl₂.Firstly,the PERK pathway was activated by increasing eIF2a and ATF4 mRNA expression levels.Secondly,the IRE1 pathway was activated due to increase in IRE1 and XBP1 mRNA expression levels.And thirdly,the increase of ATF6 mRNA expression levels suggested that ATF6 pathway was activated.4.Effects of NiCl₂ on renal apoptosis in broilersGrouping and treatment of the experimental animals were consistent with 1.At 14,28 and42 days of the experiment,the results of TUNEL stain and flow cytometry showed that the percentages of apoptotic cell were increased at the NiCl₂-treated groups,which was induced through mitochondria-mediated,fas-mediated and PI3K/Akt anti-apoptosis pathway pathways.At 14,28 and 42 days of the experiment,NiCl₂ reduced the protein and mRNA expressionlevels of anti-apoptotic Bcl-2 and Bcl-xL and increased the protein and mRNA expression levels of pro-apoptotic Bax and Bak.NiCl₂ in excess of 300 mg/kg resulted in a significant decrease in MMP,and increase in cyt c,AIF,Smac,EndoG,HtrA2,Apaf1,caspase-9,caspase-3,caspase-6,caspase-7,and PARP mRNA expression levels,and decrease in XIAP mRNA expression levels.And,the immunochemistry results showed that cyt c,caspase-9,caspase-3 and PARP protein expression levels were increased.Abovementioned results demonstrated that NiCl₂ activated mitochondria-mediated apoptosis pathway.At 14,28 and 42 days of the experiment,mRNA expression levels of Fas,FasL,caspase-8,caspase-10 and Bid,and protein expression levels of caspase-8,caspase-10 were increased in the NiCl₂-treated groups.Abovementioned results demonstrated that NiCl₂ activated Fas-mediated apoptosis pathway.Concurrently,NiCl₂ inhibited the PI3K/Akt anti-apoptosis pathway,which was characterized by decreasing PI3K,Akt1 and Akt2 mRNA expression levels.5.Effects of NiCl₂ on renal inflammatory responses in broilersGrouping and treatment of the experimental animals were consistent with 1.At 14,28 and42 days,NiCl₂ in excess of 300 mg/kg increased NF-κB and pro-inflammatory mediators including TNF-α,COX-2,IL-1β,IL-6,IL-8 and IL-18 mRNA expression levels and p-NF-κB protein expression levels.The results indicated that NiCl₂ induces renal inflammation through activating NF-κB pathway.Meanwhile,NiCl₂ also decreased mRNA expression levels of the anti-inflammatory mediators including IL-2,IL-4 and IL-13.In conclusion,NiCl₂ in excess of 300 mg/kg could induce histological lesions and dysfunctions,oxidative damage,G₂/M phase cell cycle arrest,ER stress,inflammatory responses and apoptosis in the kidney.Oxidative damage was the pathological basis of NiCl₂-induced renal damage.G₂/M phase cell cycle arrest,ER stress,apoptosis and inflammatory responses were the pathways of NiCl₂-induced renal damage.The present study was firstly to systematically investigate the renal lesions,dayfunction and oxidative damage,G2/M phase cell cycle arrest,ER stress,apoptosis and inflammatory responses induced by NiCl₂ from the levels of cell,gene expression and protein expression.