The Effect And Mechanism Of Fluoride-induced Kidney Injury Under Different Calcium Levels

Fluorine is an indispensable trace element in the human body.A proper intake of fluorine can maintain the normal development of bone.Once ingested in excess,it will enter the whole body causing systemic physiological and pathological changes in the body,which is called Endemic Fluorosis.Fluoride exposure can not only damage bone organs,but also cause damage to non-osseous organs such as brain,cardiovascular systems and kidney.Kidney is the main target organs of the body for fluoride exposure,and excessive intake of fluoride will change the histopathology and metabolic function of the kidney.In recent years,the molecular mechanism of fluoride-induced kidney damage has attracted widespread attention.Studies have shown that fluorine can induce kidney G0 / G1 block,free radical metabolism changes,cell lipid peroxidation,and membrane damage,thereby inducing cell apoptosis.Ca2+ is an important "second messenger" in the cells,and it participates in the physiological and pathological processes such as cell differentiation and apoptosis.In addition,There are also literatures showing that calcium supplementation can antagonize the body’s absorption of fluoride and its toxicity,and reduce the rate of cell apoptosis.However,the effect and mechanism of fluoride exposure to kidney damage under different calcium levels has not been reported.This experiment is divided into two parts,subchronic fluoride exposure and chronic fluoride exposure.A total of 120 first weaned ICR male mice are selected.After seven days of adaptation,they are randomly divided into 6 groups.After the fluoride exposure,The mice kidney organ coefficients and blood index related to kidney injury were measured first;observe the morphological structure and cell apoptosis rate of the kidney;observe the morphological structure of the kidney tissues with an optical microscope,and observe the apoptosis rate of kidney cells by TUNEL method.Then use Fluo-4 fluorescent probe to detect Ca2+ level in mouse kidney cells,and use DCFH-DA fluorescent probe load to detect the ROS levels in kidney cells.Finally,use RT-PCR and Western Blotmethods were used to detect the kidney cell membrane L-type calcium channels Cav1.2,intracellular death receptor signal transduction pathway molecules TNF-α,TNFR1,TRADD and downstream apoptosis regulators Caspase-8 and Caspase-3 gene / protein expression levels.It is planned to be the first to systematically investigate the role and molecular mechanism of death receptor pathway in fluoride-induced kidney injury under different calcium nutrition conditions.At the same time,screening safe doses of anti-fluoride agents to provide basic data for the prevention of fluorosis.The findings are as follows:(1)The results of body weight and organ coefficient in mice:There were no effect in body weight and organ coefficient,compared with subchronic /chronic fluoride exposure control group(P > 0.05).(2)Blood test results of kidney injury in mice:Compared with C group,urea nitrogen,creatinine and uric acid conten in mouse serum were significantly increased in subchronic /chronic fluorine exposure groups(P< 0.05 or P < 0.01).Compared with F group,urea nitrogen,creatinine and uric acid conten in mouse serum in LCa+F group were significantly increased in subchronic/chronic fluorine exposure(P < 0.05 or P < 0.01),while urea nitrogen,creatinine and uric acid conten in HCa+F group were significantly decreased(P < 0.05 or P < 0.01).Compared with subchronic fluoride exposure,urea nitrogen conten in mouse serum were significantly increased in F and LCa+F groups in chronic fluoride exposure(P <0.05 or P < 0.01),and LCa+F group creatinine conten were significantly increased(P< 0.05),and uric acid conten in F group and LCa+F group were significantly increased(P < 0.01).(3)Observation of morphological and structure of mouse KidneyIn control group,the glomerular renal vesicles structure is clear and the tubular epithelial cells were normal.In the F group,the glomerulus shrank as a whole,and renal tubular epithelial cells fell off to varying degrees,swelling and vacuolar changes.Compared with the F group,the renal tubule lumen of the LCa+F group become larger,and some renal tubular epithelium cells showed vacuolar degeneration,and the arrangement was sparseand disordered,while in the HCa+F group,renal tubular epithelial cells have complete structure,neatly arranged,and vacuolar degeneration reduced.Compared with subchronic fluoride exposure,chronic fluoride exposure group has significantly worsened the changes in kidney tissue morphology.(4)Results of apoptosis detection in mouse kidney cellsThe nucleus observed under the light microscope was TUNEL stained and the tan was apoptotic cells.Compared with C group,the number of kidney cell apoptosis of mice in subchronic / chronic fluoride exposure treatment groups was significantly increased(P < 0.01).Compared with F group,the number of kidney cell apoptosis of mice in subchronic / chronic fluoride exposure LCa+F group was significantly increased(P < 0.01);while HCa+F group was significantly decreased(P < 0.01).Compared with subchronic fluoride exposure,chronic fluorine exposure in F group and LCa+F group kidney cell apoptosis was significantly increased(P < 0.01).(5)Measurement results of intracellular Ca2+ levels in mouse kidneyCompared with C group,Ca2+ levels in the kidney cells of mice were significantly increased in subchronic fluorine exposure treatment groups(P < 0.05 or P < 0.01),and the levels of Ca2+ in the kidney cells of mice in F group,LCa group,HCa+F group and LCa+F group were significantly increased in chronic fluorine exposure(P < 0.05 or P < 0.01).Compared with F group,the levels of Ca2+ in the kidney cells of mice in LCa+F group were significantly increased in subchronic /chronic fluoride exposure(P < 0.05 or P < 0.01);while in HCa+F group Ca2+ levels were significantly decreased in the kidney cells of mice(P < 0.05 or P < 0.01).Compared with subchronic fluoride exposure,the levels of Ca2+ in the kidney cells of mice in F group,LCa+F group and HCa+F group in chronic fluorine exposure were significantly increased(P < 0.05 or P < 0.01).(6)Measurement results of ROS levels in mouse kidneyCompared with C group,the levels of ROS in the kidney cells of mice were significantly increased in subchronic / chronic fluorine exposure treatment groups(P< 0.05 or P < 0.01).Compared with F group,subchronic / chronic fluoride exposure ROS levels in the kidney cells of mice in LCa+F group were significantly increased(P < 0.01);while in HCa+F group ROS levels were significantly decreased in the kidney cells of mice(P < 0.05 or P < 0.01).Compared with subchronic fluoride exposure,the levels of ROS in the kidney cells of mice in F and LCa+F groups in chronic fluoride exposure were significantly increased(P < 0.05 or P < 0.01).(7)gene /protein detection results:Compared with C group,Cav1.2、TNF-α、TNFR1、TRADD、Caspase-8、Caspase-3 gene /protein expression level in subchronic / chronic fluorine exposure treatment groups were significantly increased(P < 0.05 or P < 0.01).Compared with F group,in the LCa+F group Cav1.2、TNF-α、TNFR1、TRADD、Caspase-8、Caspase-3 gene /protein expression level was significantly increased in subchronic/chronic fluorine exposure(P < 0.05 or P < 0.01);while in the HCa+F group Cav1.2、TNF-α、TNFR1、Caspase-3 gene /protein expression level was significantly decreased in subchronic /chronic fluorine exposure(P < 0.05 or P < 0.01),TRADD protein expression level in the HCa+F group was significantly decreased in subchronic fluorine exposure(P < 0.01),TRADD gene /protein expression level in the HCa+F group was significantly decreased in chronic fluorine exposure(P < 0.05 or P < 0.01),Caspase-8 gene expression level in the HCa+F group was significantly decreased in subchronic fluorine exposure(P < 0.05),Caspase-8 gene /protein expression level in the HCa+F group was significantly decreased in chronic fluorine exposure(P < 0.01).Compared with subchronic fluoride exposure,the expression level of TNF-α、TNFR1 、Caspase-3 gene /protein in F group and LCa+F group were significantly increased in chronic fluorine exposure(P < 0.05).Cav1.2、TRADD gene /protein expression level were significantly increased in F group,LCa+F group and HCa+F group in chronic fluorine exposure(P < 0.05).chronic fluorine exposure Caspase-8gene /protein expression level in the kidney cells of the mice exposed to the F group and HCa+F group were significantly increased(P < 0.05).In conclusion,the molecular mechanism of kidney damage caused by fluorine exposure may be: the level of Cav1.2 gene / protein expression of L-type calcium channel in mouse kidney cells caused by fluorosis,resulting in intracellular calcium increased,and the level of ROS increased,which decreased the antioxidant capacity of kidney tissue;meanwhile,excessive ROS will activates the death receptor TNF-αin the kidney cell membrane,and further activates the TNF-α /TNFR1 signaling pathway,and up-regulates the TNF-α,TNFR1,TRADD,Caspase-8,and Caspase-3gene /protein expression level,induce abnormal apoptosis of kidney cells,and eventually damage kidney cells.And dietary high calcium(2%)can reverse Cav1.2gene /protein expression level in kidney cell and inhibit Ca2+ overload in the cell,and antagonize the abnormal expression of death receptor pathway,eventually reduce the rate of apoptosis.The results suggest that the death receptor pathway is involved in fluorine-induced abnormal apoptosis,and plays an important role in the process of kidney damage caused by fluorine exposure,and dietary high calcium may be a economical and cost-effective anti-fluoride agent.