Research About The Expression And Role Of Stc1/ucp2in The Fluorosis Renal Injury And The Effect Mechanism Of High-calcium Intervention

Fluoride intake for a long time or a lot content can cause damage tobody tissues and organs, and characteristic cause dental fluorosis, bone andjoint deformities, osteoporosis, bone and joint sclerosis, such diseasescaused by fluoride poisoning, and are collectively referred to as endemicfluorosis. Endemic fluorosis cover all continents, and affect millions ofpeople. In recent15years, impacts of fluorosis to non-phrenology systemhas gradually become active field of research. Kidney is the most importantorgan of the body to excrete the fluorine, and is also a poisoning target organby fluoride, so, the kidney is become focus of fluorosis research.Mitochondria is the sensitive target withstand attacking by fluoride, andkidneys is a organ rich in mitochondria. A large number of studies has beenconfirmed that structure and functions of mitochondria in renal tubularepithelial cells can be severely affected by fluorosis. Also, there is adose-degree of correlation of fluorosis renal injury, but the toxicologicalmechanism is not clear. Therefore, this research is work for the fluorosismechanisms of renal injury from the mitochondria level.The Stanniocalcin-1(STC1) is a glycoprotein hormone which regulate the balance of cellular calcium, is expressed in many organs of humanbody.STC1is high expression in the kidney, prostate, uterus, and thyroid.STC1is secreted by autocrine and paracrine,effects on mitochondria of cells,regulate the metabolism of calcium and phosphate. In recent years, someother functions of the the STC1were found, which relate to the damagerepair, cell signal transduction, angiogenesis, steroidogenesis, muscle andbone growth, tumor and so on. The latest researches have shown that STC1reduce the level of Ca²⁺which is an important second messager, binging onthe level of uncouple protein2(UCP2) is highly increased, resulting theinhibition to the mitochondrial membrane potential reduction and reduce theproduction of superoxide. This process is closely relate to the generation ofcellular ATP, NADH and FADH2balance and ROS production. UCP2wasfound on the inner mitochondrial membrane,which is a kind of uncouplingprotein that regulating the mitochondrial outer and inner membraneelectrochemical gradient. It is accounted for0.01-0.1%of the mitochondrialmembrane protein content. UCP2neither mediate mitochondrial membraneproton leak, nor produce too much impact on the mitochondrial membrane'sEMF. But when the cell is stimulated, especially the stimulation resultingoxidative stress or stimulation of lipid oxidative damage, it can contribute tothe UCP2-mediated proton leak in mitochondrial membrane, therebyreducing the EMF of mitochondrial inner/outer membrane, the membranepotential of reduce ROS production and production of superoxide which can damage to cells and tissue. UCP2-mediated proton leak is complete with theaid of [Ca²⁺] and of [Ca²⁺] related channel proteins, and the changes of Ca²⁺content can induce UCP2expression changes. Recent studies show thatcalcium and UCP2relations not only in the body to defend stimulation, butalso in the processes of a variety of tumor occurrence and development.UCP2as a tumor marker, by regulating the calcium content and calciumcorrelation channels affect tumor cell heterogeneity functions.Our subject sduties the molecular mechanisms as a breakthrough offluorosis renal injury, closely catch the weak point of fluorosis renal injury;We study on the mitochondrial uncoupling protein2and Stanniocalcinprotein in renal tubular epithelial cells as a research object, while the calciumis defined as the interference conditions to explore if UCP2and STC1isinvolved in the process of fluorosis renal injury. Find out the approximaterole of UCP2and STC1in the fluorosis kidney damage mechanism. In thisstudy, we conducted animal experiments firstly, to observe changes influorosis rat kidney of UCP2, STC1, mitochondrial solution couplingfunction changes and the level of oxidative stress parameters. And then ourteam give the rats high-calcium diet, observe if the expression of UCP2,STC1changed,dissuse the relationship between high-calcium and thechange; Constructing the recombinant the UCP2eukaryotic expressionplasmid vector, make sure the tubular epithelial cells (HK2) high expressionof UCP2, and then inject CaCl2to the cell cultures to study the results of different level of sodium fluoride effect on the cells, such the state,mitochondrial solution uncoupling index changes. Find out such changes ifhave relationship with the high calcium environment intervention, as well asthe relationship of UCP2, STC1[Ca²⁺] in the of fluorosis kidney injury; Thisexperiment studies the mechanisms of fluoride poisoning kidney damage insub-cells level, choosing the Ca2+, UCP2and STC1as a research point. Theexperimental design is logical, and the results can provide a basis for thestudy the molecular mechanisms of fluorosis kidney damage from themitochondrial level in the future. Objective: Establish fluorosis model and detect changes of UCP2,STC1and Ca²⁺, explore the role of UCP2and STC1in mechanisms offluorosis kidney damage and the their ralationships with calcium.Methods: According to the weight24male SD rats divided averagelyinto4groups, such as control group, low dose group, the middle fluoridegroup and the high fluoride group; The control group injected physiologicalsaline, and the experimental group was given the NAF intraperitonealinjection5mg/kg body weight,10mg/kg,20mg/kg respectively. We inject1time every48hours, and fed distilled water and regular rat feedingstufffreely; After establishing modle16weeks, took out and immobilize the leftkidney. Detecte the UCP2, STC1protein's localization and expression byimmunohistochemistry. Extract50mg right kidney organization's totalRNA,and detecte expression levels of UCP2, STC1by RT-PCR. and finallydetect content of renal cells calcium.Results: immunohistochemistry prompted UCP2highly expressed inkidney skin medullary junction of the proximal convoluted tubule.STC1ismainly localized in the proximal tubule and a small amount of expression inthe medulla of the distal convoluted tubule. Immunohistochemistry andRT-PCR's results suggest that the UCP2, STC1expression in fluorosis rats were significantly higher than the control group. And the Ca²⁺is increased in3groups while the fluoride increased(pairwise comparisons, P<0.05); ratsrenal cells calcium content in each experimental group were significantlyhigher than the control group and it increase while the fluoride increased(pairwise comparisons, P <0.05).Conclusion: UCP2and STC1maybe involved in mechanism offluorosis injury of rat kidney in which calcium imbalance also play animportant part. Objective: To observe of UCP2and STC1changes in the kidneys ofrats with fluoride with high calcium against rats renal injury, and explorethe relationship of UCP2, STC1, and high calcium in the renal injury offluorosis rats.Methods:24male SD rats were divided into four groups according tobody weight, control group low fluoride plus calcium group, middle fluorideplus calcium group, high fluoride plus calcium group. The control groupwith normal saline injection, low, middle and high fluoride plus calciumgroups were given5mg/kg,10mg/kg and20mg/kg of sodium fluoridesolution by intraperitoneal injection according to body weight ratios,1injection every48hours. Low, meddle and high fluoride plus calcium groupswere given high calcium diet for16weeks. Ways of immunohistochemistryand RT-PCR were used to detect location and expression of STC1and UCP2.Content of renal cell [Ca2+]i was detect by fluorescent probes, and we alsodetected the lipid peroxides activity of rats renal tissue.Results: Confirmed that UCP2/STC1was mainly localized in renalcortex and a very small amount expressed in the renal medulla distalconvoluted tubule. Expression of UCP2and STC1was increasing with the contents of fluoride compared with the control group and high calciumcould increase the expression of UCP2and STC1, the difference wassignificant (P<0.05). Lateral compared with the dose simply exposed tofluoride group of UCP2expression in the high fluoride plus calcium groupwas significantly higher than the high fluoride group (P<0.05). UCP2in thelow, low fluoride plus calcium group compared exposed to fluoride grouphad significantly changes in expression also showed an upward trend. STC1in the expression of each exposed to fluoride plus calcium group and thecorresponding dose simply exposed to fluoride group were not significantlychanged (P>0.05), but expression also showed an upward trend; in eachexperimental group of rat kidney cells of [Ca²⁺] i levels were significantlyhigher than that control group and exposed to fluoride dose increaseselevated, high calcium and inhibit renal cell [Ca²⁺]i level.Conclusion: The UCP2/STC1of fluorosis renal injury can beincreased reactivity, high calcium can antagonize the the fluorosis renalinjury, and promoting of UCP2, STC1expression and inhibition of renal cell[Ca²⁺] level. UCP2, STC1may be involved in fluorosis rat renal injurymechanism, and the participation process may be related to the calcium ion. Objective: To detect the changes of mitochondrial uncoupling-relatedindicators and oxygen free radical in the tissue of SD rats and compare withexpression of UCP2. Explore whether the uncoupling function of UCP2isinvolved in mechanism of fluorosis kidney injury.Method: Extract of rat kidney mitochondria and detect mitochondrialrespiratory function. By the principle that mitochondrial membrane poreopening make the mitochondria swelling, detect the swelling level ofmitochondria by fluorescence spectrophotometer. By the principle thatrhodamine123will fluorescence quench in mitochondria rapidly, detectmitochondrial membrane EMF by fluorescence spectrophotometer (△Ψm).Measure mitochondrial superoxide content by coelosphere reader becauseunpaired electrons and superoxide anion from mitochondria react withtetrazolium salt (NBT) and the production will be waken in570nmlight-wave.Results: Compared with the control group, mitochondrial III staterespiration rate raise mildly in the low fluoride group. Tended to decline inthe medium fluoride group, but low fluoride group was no significantdifference with medium group (P>0.05); significantly decreased in the highfluoride group (P<0.05); Compared with the control group, the respiratory state III rate decrease in the low fluoride plus calcium group and the mediumfluoride plus calcium group, but no significant differences (P>0.05)In thehigh fluoride plus calcium group, a certain extent enhance of the rate isfound. The â…£ state respiration rate increases in the low fluoride pluscalcium group and the medium fluoride plus calcium group and the highfluoride group's decrease, but there was no significant difference (P>0.05).Calcium to produce a certain degree of inhibition of state IV respiration rateof fluorosis in the rat kidney mitochondria, but the difference is not obvious.Mitochondrial respiratory control ratio (RCR) as exposed to fluorideincreased gradually to reduce and the high fluoride group was significantlylower (P<0.05). Although a significant reduction was found in the highfluoride plus calcium group, but the RCR recovery,because calcium canantagonize this change. Mitochondrial respiratory control ratio raise influoride plus calcium groups.Conclusion: When fluorosis kidney injury happened, themitochondrial uncoupling-related indicators, EMF, mitochondrial swellinglevel changing significantly, suggest that in the mitochondrial membranechannel transition pore opening degree of a significant change. Thesesuggest that the uncoupling function maybe involve in the mechanism offluorosis renal injury. With fluoride concentration increased, the degree ofswelling of mitochondria and the level and oxygen free radicals content willincrease, but high calcium can inhibit this change. The experimental results confirmed previous studies that mitochondrial damage and fluorosis oxygenfree radical damage caused by fluorosis, and high calcium can antagonizethis damage. Objective: To determine sodium fluoride effect on the renal tubularepithelial cells (HK2), and detect UCP2, STC1, mitochondrialuncoupling-related indicators changes and their relation. Make HK2highlyexpress UCP2, and observe UCP2, STC1, uncoupling indicators' changesand calcium content. Explore if UCP2have antagonistic effect to fluorosis.Discuss the relation between UCP2, STC1, Ca²⁺ and calcium.Method: To construct plasmid containing human UCP2genefragments, transfect HK2. Infect the normal HK2, transfected UCP2,transfected HK2low(0.8mmol/L), medium(1.6mmol/L), high (2.4mmol/L),three levels of sodium fluoride. Use calcium chloride to adjust the calciumconcentration in the medium. Detect cell proliferation by the MTT assay;Detect the expression of STC1by RT-PCR. Extract mitochondria,detectmitochondrial swelling level, mitochondrial membrane EMF andintracellular calcium ion content.Results: The proliferative activity of renal tubular epithelial cells withthe dye fluorine increase in the degree of increase and in the fluoride group,the high fluoride group was significantly decreased (P<0.05). High calciumcontent can antagonize the negative effect of fluorosis to HK2's proliferation. HK2's change is different in three groups which UCP2is highly expressed.Although there is no significant different, in the low fluoride group and themedium fluoride group, the HK2transfected with UCP2's proliferation israise. Compare the high fluoride group with HK2high expression of UCP2,the transfected HK2's proliferation activity was further reduced.(P<0.05).High calcium can antagonize the effect of fluorosis to the HK2celltransfected but the difference is not significant (P>0.05). STC1mRNAexpression in renal tubular epithelial cells of fluoride groups is significantlydifferent with the control group (P<0.05); High calcium content can promotethe expression of STC1. Transfected HK2's STC1expression is significantlyhigher than the normal of HK2group (P<0.05). The high calcium contentcan enhance the expression of STC1, but the difference is no significant(P>0.05). The fluoride groups' calcium content is higher than the controlgroup(p<0.05). Calcium content in HK2cells from fluoride groups issignificantly higher than control groups, by pairwise compared (P<0.05).When given high calcium handling, calcium content in normal renal tubularepithelial cells appear significantly reduced, and pairwise compared, thedifference was significant (P<0.05); Intracellular calcium content intransfected HK2cells from the high fluoride group is less than the normalHK2cells while the low fluoride group and the medium group have nosignificant change (P>0.05). Give transfected HK2cells high calciumhandling the calcium content becoming lower than the normal HK2cells. The high fluoride group and the medium fluoride group is reducedsignificantly.(P<0.05).Conclusions: Fluorosis have negative effect on the proliferation ofHK2cells and it can enhance the expression of UCP2and STC1.the changeabove is more significant with the fluoride content. Fluorosis affect theuncouple function of HK2cells' mitochondria and the result have some doserelation with fluoride content. So the result prove that UCP2, STC1and theuncouple function of mitochondria involve the mechanism of kidneyfluorosis. The present findings provided that UCP2is a factor to stimulateSTC1expression and reduce the calcium content of UCP2, that have effectson cell in many side.