Effects Of Orai3-mediated Store-operated Calcium Entry On Parathyroid Hormone Release In Secondary Hyperparathyroidism

Background:Secondary hyperparathyroidism is a common complication of chronic kidney disease,mainly manifested as hypersecretion of parathyroid hormone and hypocalcemia.Phosphate accumulation,increased FGF23 levels,decreased production of active vitamin D,and hypocalcemia,increased PTH secretion are continuous stimulating factors for parathyroid hyperplasia.In the middle and late stages of chronic kidney disease,1,25（OH）D₃levels are reduced and parathyroid hormone is excessively secreted,which increases the risk of cardiovascular events and mortality.The mechanism of secondary hyperparathyroidism is complicated.Parathyroid glands and calcium affect each other,and 1,25（OH）D₃deficiency indirectly leads to increased PTH synthesis.PTH can directly or indirectly act on the bones,kidneys and intestines to mediate the increase of serum calcium;however,PTH also increases the production of FGF23,thereby enhancing phosphoruria and reducing the production of vitamin D in the kidneys.Orai3 is a member of the Orai family of Ca²⁺channel proteins and has received widespread attention due to its role in mediating store-operated calcium entry（SOCE）.The Orai family was first thought to be the constituent protein of SOC or calcium release-activated Ca²⁺channel(Ca²⁺release-activated Ca²⁺channel,CRAC).It is a four-pass transmembrane protein,including Orai1,Orai2 and Orai3,a selective calcium channel It plays an important role in the occurrence and development of tumors,cardiovascular diseases and autoimmune diseases,but the role of Orai3 in secondary hyperparathyroidism is unclear.Objective:（1）Observe whether Orai3 mediates store-operated calcium influx in parathyroid cells（2）Observe the effect of Orai3 on the secretion of parathyroid hormoneMethods:（1）RNA sequencing（RNA-seq）The obtained tissues of the control group and the experimental group were sequenced and analyzed using high-throughput sequencing technology to reflect the expression level of their RNA.（2）Western blottingIt is used to detect the expression level of Orai3 in the parathyroid tissue of SHPT patients;after transfecting specific Orai3 si RNA into primary parathyroid cells with lip3000,the knockdown efficiency of Orai3 si RNA on Orai3 protein is detected.（3）Calcium imagingIt is used to detect the influence of Orai3 channel on calcium signal in parathyroid cells,and the change of calcium signal after Orai3 si RNA knocks down the level of Orai3protein.（4）Enzyme-linked immunosorbent assayThe human parathyroid hormone-related protein（PTHr P）enzyme-linked immunosorbent assay kit uses a double antibody sandwich ELISA method to detect PTH levels.（5）Orai3 knockout mouseThe serum PTH level of Orai3 gene knockout rats was tested to verify the effect of reduced Orai3 expression on PTH levels.Results:（1）RNA sequencing,data analysis and western blot results showed that the expression of Orai3 in the parathyroid tissues of SHPT patients was significantly higher than that of the control group.（2）In parathyroid cells,transfection specific small interfering RNA knocks down Orai3,the expression level of Orai3 protein is significantly inhibited,and the calcium store-operated calcium influx is significantly inhibited.（3）In parathyroid cells,when Orai3 is knocked down by transfected specific small interfering RNA,the release of PTH is significantly reduced.（4）The level of PTH in the blood of Orai3 knockout mice was significantly reduced Conclusion:In the parathyroid tissues of SHPT patients,the expression level of Orai3 is elevated.Orai3 mediates store-operated calcium influx in parathyroid cells and affects the secretion of PTH.The expression level of Orai3 and the calcium signal mediated by it are the potential mechanism of parathyroid hormone release.Orai3 may play an important role in the occurrence and development of secondary hyperparathyroidism.