Effect Mechanisms Of Sodium Fluoride On The Renal Damage In Mouse

Fluorine,one of the necessary trace elements for the body,is widespread in the soil,atmosphere,rock and water.Also,fluoride is often used in the medicine,oral care products as well as a variety of industrial and agricultural practices such as the production of phosphate fertilizers,steel,aluminum,ceramic,etc.Fluoride in small quantity is useful for the formation of dental enamel and mineralization of bones.However,fluoride excessive intake may lead to serious lesions and/or damage in bone and soft tissues.As an organ involved in excretion and retention of fluoride,kidney is quite sensitive to the toxicity of fluoride.It has been shown that excessive intake of fluoride can cause a series of renal damage such as histopathological changes,cell cycle arrest and apoptosis.While,the mechanisms of fluoride-induced renal damage are not well understood at present.Therefore,the present study was conducted to explore the potential molecular mechanisms of fluoride-induced renal toxicity by observing the changes of the histological structure,function,antioxidant ability,cell cycle,apoptosis,autophagy,inflammatory responses as well as the mRNA and protein expression levels of related factors in PI3K-Akt signaling pathway in the mouse kidney with the methods of experimental pathology,flow cytometry,qRT-PCR,Western blotting,ELISA and TUNEL stain,providing new data reference and theoretical basis for further study on the toxicity effects of fluoride on kidney.The results are as follows:1.Effects of NaF on renal histological structure,function and antioxidant ability in miceTwo hundred and forty four-week-old ICR mice used in this study were randomized into four groups of 60 animals each.Animals were given intragastric administration of NaF at the dose of 0,12,24 and 48 mg/kg body weight,respectively.All animals were administered their respective doses daily by gavages for consecutively 42days,and the gavage volume was 1ml/100g body weight.During the experiment,clinical signs were observed daily.The pathological observation was conducted at 21 and 42 days of the experiment.The results showed that NaF in excess of 12 mg/kg decreased the renal volume and viscera index.Meanwhile,the histopathological lesions of kidney such as glomeruli swelling,degeneration and necrosis of the tubular cells,renal tubular hyaline casts and inflammatory cells infiltration were also observed.The renal histological structure was damaged.The abovementioned histopathological changes were presented in a time-and dose-dependent manner.The renal functional parameters were detected by the method of biochemistry at 21and 42 days of the experiment.The results showed that NaF in excess of 12 mg/kg increased the contents of serum UA,BUN and Cr as well as the activities of renal LDH and urinary NAG,and reducing the activities of renal Na+/K+-ATPase and ACP,demonstrating that the renal function was impaired or reduced.The parameters of oxidative damage,non-enzymatic antioxidant and antioxidant enzymes in the kidney were detected with the methods of biochemistry and qRT-PCR at 21and 42 days of the experiment.The results showed that NaF in excess of 12 mg/kg enhanced the renal oxidative damage by significantly increasing the levels of ROS production as well as the contents of MDA,PC and 8-OHdG,and reducing the abilities of ASA and AHR,the contents of GSH and the activities of antioxidant enzymes?SOD?CAT?GR and GSH-Px?,which finally caused renal lesions and dysfunctions.The decrease of antioxidant enzyme activities was consistent with the decrease of the gene expression levels of antioxidant enzymes?CuZn-SOD,CAT,GR and GSH-Px?.Oxidative damage is one of the mechanisms of fluoride-induced histological lesions and dysfunctions in the kidney.2.Effects of NaF on renal cell cycle in miceGrouping and treatment of the experimental animals were consistent with 1.Excessive ROS production can attack biological macromolecules such as DNA,leading to DNA damage,which then induce the changes of cell cycle progression.The cell cycle as well as the mRNA and protein expression levels of cell cycle regulatory molecules in the kidney were detected at 21 and 42 days of the experiment.The results detected by flow cytometry showed that the renal cells were arrested at G2/M phase.Western blotting and qRT-PCR results showed that the p-p53,p-CDK1?Tyr15?,p21,Gadd45a protein expression levels and p53,p21,Gadd45a mRNA expression levels were increased,and the CyclinB₁,PI3K,p-Akt?Ser473?,p-Akt?Thr308?,mdm2,PCNA protein expression levels and CyclinB₁,CDK1,PI3K,Akt,mdm2,PCNA mRNA expression levels were decreased.These results showed that NaF in excess of 12 mg/kg induced renal G2/M phase cell cycle arrest by suppressing PI3K-Akt-p53 signaling pathway,which finally inhibited the renal cell proliferation and renal development.3.Effects of NaF on renal apoptosis and autophagy in miceGrouping and treatment of the experimental animals were consistent with 1.In response to DNA damage,cell cycle checkpoints arrest the cell cycle progression in order to provide more time for DNA repair.If the repair is invalid,it will induce the death of cells?such as apoptosis and autophagy?.The apoptosis as well as the mRNA and protein expression levels of apoptosis and autophagy regulatory molecules in the kidney were detected at 21 and 42 days of the experiment.The results detected by TUNEL stain and flow cytometry showed that the percentages of apoptotic cell were increased.Western blotting and qRT-PCR results showed that the Foxo3a,Bim,Bax,caspase 9,caspase 3,Bad,Becline1,LC3?the marker of autophagy?mRNA expression levels,Foxo3a,and Bim,Bax,C-caspase 9,C-caspase 3,Bad,Becline1 protein expression levels as well as the ratios of LC3-II/LC3-I were increased.At the same time,the Bcl-2,Bcl-xL,mTOR,p70s6k,p62mRNA expression levels and Bcl-2,Bcl-xL,mTOR,p-p70s6k,p62 protein expression levels were decreased.The results showed that NaF in excess of 12 mg/kg induced renal apoptosis and autophagy by inhibiting PI3K-Akt-Foxo3a/caspase 9/Bad/mTOR signaling pathways.4.Effects of NaF on renal inflammatory responses in miceGrouping and treatment of the experimental animals were consistent with 1.The contents of NO and PGE₂,the activities and mRNA expression levels of iNOS as well as the mRNA and protein expression levels of inflammatory factors in the kidney were detected by the methods of ELISA,qRT-PCR and Western blotting at 21 and 42 days of the experiment.The results showed that NaF in excess of 12 mg/kg could induce the renal inflammatory responses by increasing the contents of NO and PGE₂,the activities and mRNA expression levels of iNOS as well as the COX-2,TNF-?,IL-1?,IL-6 and IL-8mRNA and protein expression levels via the inhibition of PI3K-Akt-NF-?B signaling pathway,and reducing the mRNA and protein expression levels of IL-4 and IL-10,which were consistent with the results of histopathological observation in the kidney.In conclusion,NaF in excess of 12 mg/kg can induce oxidative damage,G2/M phase cell cycle arrest,apoptosis,autophagy and inflammatory responses in the mouse kidney,which finally lead to pathological impairment and dysfunctions.Oxidative damage in the kidney is the pathological basis of NaF-induced renal damage.G2/M phase cell cycle arrest,apoptosis,autophagy and inflammatory responses are the pathways of NaF-induced renal damage.PI3K-Akt signaling pathway is the molecular mechanism of fluoride-induced renal damage.Meanwhile,this study is the first report to systematic investigate the renal histological structure,function and antioxidant ability induced by fluoride.Effects of NaF on cell cycle,apoptosis,autophagy and inflammatory responses in the kidney of mice were studied for the first time from the levels of cell,gene and protein.The effects of PI3K-Akt signaling pathway in renal damage,and the relationships between PI3K-Akt signaling pathway and cell cycle arrest,apoptosis,autophagy,inflammatory responses in mice were also discussed for the first time.