The Effect And Mechanism Of Pantothenate Kinase 1 In The Formation Of Calcium Oxalate Crystals

Objective Using the gene chip technology to analyze the changes of gene expression profile in mouse models of renal calcium oxalate crystal,we screen out the target genes of research value and carry on the preliminary clinical verifications,test expression changes of the target genes through zoological and cytological experiments and the influence on apoptosis of renal tubular epithelial cells,and explore its role and mechanism in the crystallization of renal calcium oxalate.Methods The model of renal calcium oxalate crystallization in mice was established by abdominal injection of glyoxalic acid.Using Von Kossa stain to detect the calcium salt deposition on the kidney tissue in mice.Analyzing the changes of gene spectrum of kidney tissues in model group using gene chip technology and select out the target gene.Collecting the kidney tissues of kidney stone and the normal part of kidney cancer respectively and using the scanning electron microscope to observe the ultrastructure of renal tubular epithelial cells,and then using the Immunohistochemical to detect the difference expression of target gene.Reconstructing the renal model of calcium oxalate crystallization in mice and detecting the expression levels of target gene by qPCR and Western blot,and then using the HE stains and TUNEL test to detect the changes of ultrastructure and apoptosis of kidney tissues cells.Using the Immunohistochemical to detect the expression of target gene in the kidney tissues of mouse,and detect the level of apoptosis-related proteins.Constructing the calcium oxalate crystal cell model by the stimulation of COM and building the PANK1 overexpression and PANK1-siRNA slow virus and using fluorescence to observe the infection status.Then using Western blot to detect the expression levels of target gene and the apoptosis of kidney tissues cells,and finally,using qPCR to detect the expression of downstream related key proteins.Results The model of renal calcium oxalate crystallization in mice was successfully established.The Von Kossa stain resluts showed that there was obvious deposition of calcium salt on the kidney tissue sections of the model group.Through gene chip,the most obvious target gene PANK1 was screened out in the crystal model(p<0.05).The results of scanning electron microscope showed that the renal ultrastructure of who suffered from calcium oxalate nephrolithiasis had obvious damage and apoptotic changes.Immunohistochemical test showed that the expression level of PANK1 of calcium oxalate nephrolithiasis patients was significantly lower than that in the control group.In the mice model,qPCR results showed that the mRNA level of PANK1 in the kidney tissue of the model group was significantly lower than that in the control group(p<0.05).Western blot results also showed that the expression level of PANK1 protein in kidney tissues of the model group was significantly lower than that in the control group.Immunohistochemical test showed that the expression of PANK1 was significantly reduced in the model group,and the expression of PANK1 was mainly concentrated in renal tubular epithelial cells.HE stain and TUNEL test results showed that,compared with the control group,the renal tissue cells of mice in the model group had obvious apoptotic changes,and the apoptosis mainly occurred in the renal tubular epithelial cells.Western blot results also showed that the pro-apoptotic protein Bax increased in the kidney tissues of the model group,and the anti-apoptotic protein bcl-2 decreased.In the calcium oxalate crystal cell model,Western blot results showed that the expression level of PANK1 in renal tubular epithelial cells was significantly reduced through calcium oxalate crystal stimulation.The results of flow cytometry showed that the apoptosis level of the group with calcium oxalate crystal stimulation was significantly higher than that of the control group.After successfully building PANK1 overexpression and PANK1-siRNA slow virus and they infected cells,the results of flow cytometry detection showed that the apoptosis of PANK1 overexpression group obviously decreased,while the apoptosis of PANK1-siRNA group cells increased significantly.The results of qPCR showed that the expression level of downstream protein SIRT3 and FOXO3 a in the signal pathway associated with energy metabolism apoptosis was positively correlated with the expression of PANK1.Conclusions Calcium oxalate crystal stimulus will reduce the expression of PANK1 in renal tubular epithelial cells and cause renal tubular epithelial cells apoptotic changes.It is likely that by affecting cell energy metabolism,PANK1 activates energy receptors and affects the expression of downstream key protein SIRT3 and the transcriptional activity of transcription factor FOXO3,leading to apoptosis of renal tubular epithelial cells,thus promoting the formation of renal calcium oxalate crystals.