To Explore The Role Of STIM1 And Orai1 In The Phenotype Transformation Of Renal Tubular Epithelial Cells And The Promotion Of Calcium Oxalate Stone Formation Calculi

Objective: To explore the change trend of STIM1 and Orai1 in the process of calcium oxalate nephrolithiasis caused by phenotype transformation of renal tubular epithelial cells,so as to provide theoretical basis for the etiological prevention of calcium oxalate nephrolithiasis.Methods: NRK-52 E cells were cultured in vitro and treated with calcium chloride to simulate high calcium environment in vitro.The highest concentration of calcium chloride solution which had no significant inhibition on the growth of NRK-52 E cells was selected through CCK-8 and flow cytometry apoptosis experiment,and the high calcium environment of NRK-52 E cells was simulated.NRK-52 E cells were treated in the above-mentioned high calcium medium for 24 hours and 48 hours.The calcium deposition on the cell surface was observed by alizarin red staining.RT-PCR and Western blot were used to detect the expression levels of E-cadherin,OPN,STIM1 and Orai1 in NRK-52 E cells under high calcium environment,and to verify whether STIM1 and Orai1 genes were differentially expressed in the phenotypic transformation of NRK-52 E cells.Subsequently,we explored the regulatory role of STIM1,a gene that was significantly differentially expressed during the phenotypic transformation of NRK-52 E cells,in phenotypic transformation and stone formation.The expression of NRK-52 E was up-regulated or down regulated by plasmid transfected normal NRK-52 E cells.Western blot and immunofluorescence were used to observe the change trend of phenotype transformation markers of NRK-52 E cells.Flow cytometry was used to detect the intracellular calcium concentration of NRK-52 E cells under different treatments to explore the direct effect of the gene expression.Finally,the microarray data of calcium oxalate plaque patients and normal people were analyzed by Gene Expression Omnibus(GEO)database in the United States.The expression of STIM1 and Orai1 in human renal papilla was collected and collated,so as to verify whether the change trend of STIM1 and Orai1 was consistent with that of the phenotype transformation of renal tubular epithelial cells in cell experiment.Results: Through CCK-8 and flow cytometry,we screened out the highest concentration of calcium chloride solution without significant inhibition on NRK-52 E cell growth,which was 0.7mg/ml.Alizarin red staining showed that the calcium deposition on the cell surface was more than that in the control group.The results of RT-PCR and Western blot showed that the expression of E-cadherin was significantly decreased and OPN was significantly increased in high calcium environment(P<0.05),which confirmed the osteogenesis(chondrogenesis)of NRK-52 E cells in high calcium environment,and the expression of STIM1 was significantly decreased(P<0.05).When the expression of STIM1 gene was down regulated by NRK-52 E cells transfected with plasmid,we found that the expression of E-cadherin protein decreased significantly,and the expression of OPN protein increased significantly(P<0.05).It is suggested that the low expression of STIM1 gene promotes the osteoblasts(chondroblast)transformation of NRK-52 E cells.The results were also verified by immunofluorescence.Flow cytometry was used to detect the intracellular calcium concentration from high to low in STIM1 gene up regulation group,control group,high calcium group and down regulation group.It was found that the expression level of STIM1 gene in the patients with calcium oxalate plaque was significantly lower than that in the normal control group,and the expression of Orai1 gene had no significant difference(P < 0.05).Conclusion: 1.High calcium microenvironment can effectively promote phenotype transformation and extracellular crystal adhesion of renal tubular epithelial cells 2.In the process of phenotype transformation of renal tubular epithelial cells promoted by high calcium microenvironment,the expression of STIM1 gene decreased significantly.3.The down-regulation of STIM1 gene can promote the phenotypic transformation of NRK-52 E to osteoblasts(chondroblast),and then induce the formation of calcium spots and lead to kidney stones.The results of population chip data analysis in GEO database also verify the above conclusions.