The Role Of L-type Calcium Channel In Fluoride-induced Kidney Injury And Its Mechanism

Fluorine(F)is one of the essential trace elements in human body.Long-term intake of excessive fluoride will cause systemic physiological and pathological changes,known as endemic fluorosis.Fluoride exposure can damage not only bone organs,but also non-bone organs such as nerve and urine.Kidney is one of the main target organs of fluoride exposure.The molecular mechanism of fluoride-induced kidney injury has gradually attracted attention.The damage of fluoride to kidney,especially to its epithelial cells,mainly lies in the inhibition of intracellular enzyme synthesis and metabolism and the destruction of intracellular membrane structure.Ca2+ is an important information molecule in neurons.L-type calcium channels play an important role in regulating the level of Ca2+ in the cytoplasm.Fluorosis can cause intracellular calcium overload,but how fluorosis causes intracellular calcium overload and the effect of calcium overload on apoptosis-related molecules downstream of L-type calcium channel in kidney cells are rarely reported.This study includes two parts: subchronic and chronic fluoride-induced kidney injury.140 ICR male mice were randomly divided into 7 groups: Control group(C),high fluoride group(HF),low fluoride group(LF),high fluoride injection calcium channel agonist group(FPL64176)(HF + F),high fluoride injection calcium channel antagonist group(Nifedipine)(HF + N),low fluoride injection calcium channel agonist group(LF + F),low fluoride injection calcium channel antagonist group(LF + N).The control group drank tap water freely,the high fluoride group and the low fluoride group drank 30 and 5 mg/L sodium fluoride solution respectively.The period of fluoride exposure was 90 days and 180 days.One week before the end of fluoride exposure,the control group was intraperitoneally injected with normal saline,and the fluoride-exposed groups were intraperitoneally injected with agonists or antagonists(5mg/kg d),respectively.After fluoride exposure,the kidney organ coefficient,blood index related to kidney injury,kidney tissue structure and antioxidant capacity of mice were measured.Then the renal cell apoptosis and the expression levels of Cav1.2 gene/protein of L-type calcium channel and Ca MKII,Ca M,Bax and Bcl-2 gene/protein of downstream renal cell apoptosis regulators were detected respectively.The above experiments were used to explore the molecular mechanism of kidney damage caused by drinking water type fluorine exposure,to preliminarily explore the role of l-type calcium channel in the process of kidney damage caused by drinking water type fluorosis,to further improve the contradiction theory of calcium caused by fluorine,and to explore new ways and new methods to treat fluorosis.Experimental results: 1.The results of body weight and organ coefficient in mice:There was no significant difference in body weight and organ coefficient between groups(P > 0.05).2.Blood test results of kidney injury in mice:Compared with the control group,urea nitrogen and uric acid content in HF,LF and HF+F groups were significantly increased at 3 months(P<0.01).Urea nitrogen in groups HF+N,LF+F and LF+N,and uric acid content in groups LF and HF+N were significantly increased(P<0.05).Urea nitrogen in HF and HF+F groups,creatinine in HF,HF+F,HF+N and LF+F groups,and uric acid content in HF,LF,HF+F,HF+N and LF+F groups were significantly increased at 6 months(P<0.01).Urea nitrogen in HF+N and LF+F groups and uric acid content in LF+N groups were significantly increased(P<0.05).Compared with HF group,creatinine content in HF+N group was significantly decreased at 3 months(P<0.05).Uric acid content in HF+F group was significantly increased(P<0.01),and uric acid content in HF+N group was significantly decreased(P<0.01).The contents of urea nitrogen,creatinine and uric acid in HF+F group were significantly increased at 6 months(P<0.01),and the contents of creatinine in HF+N group were significantly decreased(P<0.05).Compared with the LF group,the urea nitrogen content in the LF+N group was significantly decreased(P<0.01),and the creatinine and uric acid content in the LF+N group at 3 months and LF+F group at 6 months were significantly increased(P<0.05).Compared with the 3-month period,creatinine contents in groups of HF,LF,HF+F,HF+N and LF+F were significantly reduced in groups of 6-month period(P<0.05),while creatinine contents in groups of LF+N were significantly reduced in groups of 6-month period(P<0.01).Urea nitrogen in group HF+F,uric acid content in groups HF and HF+N significantly increased(P<0.05),and uric acid content in groups HF+F and LF+F significantly increased(P<0.01).3.Observation of Kidney Tissue Structure in MiceIn the control group,the structure of glomerular renal vesicles was clear and the epithelial cells of renal tubules were normal.In the fluoride-treated group,the renal vesicles were enlarged and some cells of renal tubular epithelium showed vacuolar changes.In HF group,the glomerulus shrank as a whole,the boundary of renal tubular epithelial cells was unclear,swelling,vacuolar degeneration,partial degeneration and necrosis,and inflammatory cells were found in the interstitium.The fluoride and agonist combined exposure group aggravated further,while the fluoride and antagonist combined exposure group reversed the above changes.The above changes were more severe at 6 months than at 3 months.4.Results of enzyme activity in mouse kidneyCompared with the control group,the SOD activity of GSH-PX,LF,HF+N,LF+F and LF+N groups significantly decreasedat 3 months(P<0.05),The content of MDA in LF and LF + N groups significantly increased(P < 0.05),GSH-PX,SOD activity in HF group and HF + F group significantly decreased(P < 0.01),MDA content in HF,HF+F,HF+N and LF+F groups significantly increased(P < 0.01);At 6 months,the activity of SOD in LF + N group,LF group and LF + N group significantly decreased(P < 0.05),the content of MDA in LF + N group significantly increased(P < 0.05),the activity of SOD in HF,LF,HF + F,HF + N and LF + F group significantly decreased(P < 0.01).The contents of MDA in HF,LF,HF+F,HF+N and LF+F groups significantly increased(P < 0.01);Compared with HF group,the activity of GSH-PX,SOD and MDA in LF + F group and HF + N group significantly decreasedat 3 months(P < 0.05).GSH-PX activity in HF+N group and MDA content in HF+F group significantly increased(P <0.05).The activity of GSH-PX significantly increasedin HF+N group at 6 months(P < 0.01).SOD activity in HF + N group and MDA content in HF + F group significantly increased(P < 0.05).SOD activity was significantly decreased in HF + F group(P < 0.05),and MDA content was significantly decreased in HF + N group(P < 0.01);Compared with LF group,the activity of GSH-PX in LF + F group significantly decreasedat 3 months(P < 0.05).GSH-PX activity significantly increasedin LF+N group at 6 months(P < 0.05);Compared with 3-month group,GSH-PX activity in LF+F and LF+N groups and MDA content in HF+F group significantly decreased(P < 0.05).GSH-PX activity was significantly decreased in HF,LF,HF+F and HF+N groups(P < 0.01).MDA contents in HF and HF+N groups were significantly increased(P < 0.05).5.Observation results of apoptosis of kidney cells in mice:Compared with the control group,the number of renal cell apoptosis significantly increasedin each group at 3 and 6 months(P < 0.01);Compared with HF group,the apoptotic rate of HF + N group significantly decreasedat 3 months(P < 0.05),and the apoptotic rate of HF + N group significantly decreasedat 6 months(P < 0.01);Compared with LF group,the apoptotic rate of LF + N group significantly decreasedafter 6 months(P < 0.05);Compared with 3-month group,the apoptotic rates of HF,LF,HF + F and LF + F groups significantly increased(P < 0.01)and HF + N groups significantly increased(P < 0.05).6.RT-PCR m RNA detection results:Compared with the control group,the expression levels of Cav1.2 genes in HF+F,LF+F and LF+N groups at 3 months and 6 months were significantly increased(P < 0.05);Compared with the HF group,the Cav1.2 gene expression level in the HF+F and HF+N groups was significantly increased(P < 0.05);Compared with the LF group,the Cav1.2 gene expression levels in the LF+F and LF+N groups were significantly increased at 3 months(P < 0.05),and the Cav1.2 gene expression levels in the LF+F and LF+N groups were significantly decreased at 6 months(P < 0.05);Compared with 3-month group,Cav1.2 gene expression level significantly increasedin HF,HF+F and HF+N groups at 6 months(P < 0.05),and significantly decreasedin LF+F and LF+N groups(P < 0.01).Compared with the control group,Ca MK II gene expression level significantly increasedat 3 months and 6 months(P < 0.05);Compared with HF group,Ca MK II gene expression level significantly decreasedat 3 months in HF + F group(P < 0.05),Ca MK II gene expression level significantly increasedin HF + N group(P < 0.01),Ca MK II gene expression level significantly increasedat 6 months in HF + N group(P < 0.05);Compared with LF group,Ca MK II gene expression level significantly increasedat 6 months in LF + F group(P < 0.05),Ca MK II gene expression level in LF + N group was significantly decreased(P < 0.05)because of the significant increase in expression level(P < 0.05);Compared with 3-month group,Ca MK II gene expression level in HF + F group was significantly increased(P < 0.05)in 6 months.Compared with the control group,the expression of Ca M gene in HF,HF+F and HF+N groups significantly decreased(P < 0.05),and the expression of Ca M gene in LF+F group significantly increased(P < 0.05);compared with 3-month group,the expression of Ca M gene in HF+N and LF+N groups significantly decreased(P < 0.05),and the expression of Ca M gene in LF+F group significantly increased(P < 0.01).Compared with the control group,the expression level of Bax gene in each group significantly increased(P < 0.05);Compared with HF group,Bax gene expression level in HF+N group was significantly decreased at 3 months and 6 months(P < 0.05);Compared with LF group,the expression level of Bax gene in LF + F and LF + N group significantly decreased(P < 0.01);Compared with 3-month group,the expression level of Bax gene in HF,LF and HF + F group significantly increasedat 6 months(P < 0.05).Compared with the control group,the expression level of Bcl-2 gene in HF,HF+F and LF+F groups significantly decreasedat 3 months and 6 months(P < 0.05);Compared with HF group,the expression level of Bcl-2 gene in HF+F group significantly decreasedat 3 months(P < 0.05),the expression level of Bcl-2 gene in HF+F group significantly increasedat 6 months(P < 0.01);Compared with LF group,the expression level of Bcl-2 gene in LF+F group significantly decreasedat 6 months(P < 0.05);Compared with HF+F group,the expression level of Bcl-2 gene significantly decreasedat 6 months(P < 0.05).Compared with 3-month group,Bcl-2 gene expression level significantly decreasedat 6 months(P < 0.05).7.Western blot protein detection results:Compared with the control group,the expression level of Cav1.2 protein in each group significantly increasedat 3 months and 6 months(P < 0.05);Compared with HF group,the expression level of Cav1.2 protein in HF + F group significantly increasedat 3 months(P < 0.01),the expression level of Cav1.2 protein in HF + N group significantly decreasedat 6 months(P < 0.05);Compared with LF group,the expression level of Cav1.2 protein in LF + N group significantly decreasedat 3 months(P < 0.05);Compared with HF + F group,the expression level of Cav1.2 protein significantly decreasedat 6 months(P < 0.05).Compared with 3-month group,the expression of Cav1.2 protein in LF,HF+F,LF+F and LF+N groups significantly decreasedat 6 months(P < 0.05).Compared with the control group,the expression of Ca MK II protein in HF,LF,HF+F and LF+F groups significantly decreasedat 3 months and 6 months(P < 0.05),and the expression of Ca MK II protein in LF group significantly increased(P < 0.01);Compared with HF group,the expression of Ca MK II protein in HF+N group significantly increasedat 3 months(P < 0.05);Compared with LF group,the expression of Ca MK II protein in LF+N group significantly increasedat 3 months(P < 0.05),and in LF+N group at 6 months(P < 0.05).Compared with 3-month group,the expression of Ca MK II protein in LF and LF+F groups significantly increasedat 6 months(P < 0.01),and the expression of Ca MK II protein in LF+N group significantly increased(P < 0.05).Compared with the control group,the expression level of Ca M protein in each group significantly increased(P < 0.05);Compared with HF group,the expression level of Ca M protein in HF + F group significantly increased(P < 0.05);Compared with LF group,the expression level of Ca M protein in LF + N group significantly decreased(P < 0.05),the expression level of Ca M protein in LF + F group significantly increasedat 6 months(P < 0.05);Compared with 3-month group,the expression level of Ca M protein in each group had no significant difference at 6 months(P > 0.05).Compared with the control group,the expression level of Bax protein in each group significantly increased(P < 0.05);Compared with HF group,the expression level of Bax protein in HF + F group significantly increased(P < 0.05);Compared with LF group,the expression level of Bax protein in LF + N group significantly decreased(P < 0.05);Compared with 3-month group,the expression level of Bax protein in LF + N group significantly decreasedat 6 months(P < 0.05).Compared with the control group,the expression of Bcl-2 protein in each group significantly decreased(P < 0.05);Compared with HF group,the expression of Bcl-2 protein in HF + F group significantly decreased(P < 0.05);Compared with LF group,the expression of Bcl-2 protein in LF + F group significantly decreased(P < 0.05);Compared with 3-month group,the expression of Bcl-2 protein in HF group significantly decreasedat 6 months(P < 0.05).In summary,fluoride exposure can lead to kidney damage in mice,mainly as follows: decreasing antioxidant capacity of kidney tissue,causing pathological changes of renal tissue structure,aggravating renal cell apoptosis.The molecular mechanism may be related to the increase of Cav1.2,Ca MK II and Bax expression,the decrease of Ca M and Bcl-2 expression and the increase of Bax/Bcl-2 ratio in renal cells induced by fluoride exposure,and the expression levels of Cav1.2 and downstream apoptotic regulators in these kidney cells were correlated with the dose and time of fluoride exposure.Injection of L-type calcium channel agonist FPL64176 has synergistic toxicity with fluoride exposure,i.e.aggravating renal damage caused by fluorosis,and making the molecular changes in the above-mentioned renal cells more significant.While injection of L-type calcium channel antagonist Nifedipine has antagonistic effect and can reverse the above-mentioned indicators of kidney damage caused by fluorosis.Among the above indicators,only L-type calcium channel Cav1.2 gene/protein and downstream Bax/Bcl-2 gene/protein showed regular changes,suggesting that L-type calcium channel Cav1.2 may be the key link of fluoride-induced kidney injury.Abnormal expression of downstream Bax/Bcl-2 gene/protein may lead to apoptosis,which may be one of the causes of fluoride-induced kidney injury.L-type calcium channel inhibitor N Ifedipine may be a new effective anti-fluoride drug.