The Study About The Role And Mechanism Of TGFβ/smads Pathway In Kidney EMT Induced By Calcium Oxalate Crystal

Chronic kidney disease has become a global public health problem.With the increase of patients with end-stage renal failure year by year,it has formed a huge burden on China’s medical and economic.Obstructive nephropathy is an important cause of renal failure.The most common disease is urinary calculi,the main component of which is calcium oxalate calculi.As the course of disease is prolonged and repeated,obstructive nephropathy caused by stones has become one of the main causes of chronic kidney disease.Calcium oxalate is the main component of urinary calculi,accounting for more than 80% of urinary calculi.However,the mechanism of renal calcium oxalate stone formation is still not very clear.There may be many factors involved in the formation of renal calcium oxalate stone,including immunity,oxidative stress,renal papilla calcium plaque,and many other factors.These mechanisms suggest that renal tubular injury caused by many factors may further promote the nucleation and deposition of crystal.Therefore,renal tubular injury may become a new direction in the study of renal stone formation.Our previous research results suggest that as early as in the calcium oxalate crystallization stage,oxidative stress,apoptosis and autophagy are involved,as well as the occurrence of tubular epithelial mesenchymal transition(EMT).These factors interact with each other,which can further lead to renal interstitial fibrosis and aggravate the progression of kidney disease.On this basis,this project intends to further study the role of TGF β / Smad signaling pathway in the process of renal tubular epithelial cell injury in the calcium oxalate crystallization stage,whether it can inhibit the occurrence of EMT through the regulation of acetylation / deacetylation,delay the process of renal fibrosis through TGF β / Smad signaling pathway,and provide new ideas and theoretical basis for clinical treatment Foundation.Part I: Analysis of the effect of TGF-β on renal tubular epithelial cells by gene chipObjective:The gene chip technology was used to analyze h K2 cells stimulated by TGF-β,and the differentially expressed genes were screened.Then the downstream bioinformatics analysis was carried out to screen and analyze the effect of TGF-β 1 on renal tubular epithelial cells.Methods:Geo’s tool was used to analyze the differentially expressed genes of HK-2 cells in 48 hour control group and TGF-β 1 treatment group(three independent duplicate samples in each group),and R statements were downloaded for further analysis in r4.0.3environment to screen DEGs.2.Using Benjamin and Hochberg(BH)method to adjust the p value to false discovery rate(FDR),and selecting FDR < 0.05 and log fold change absolute value(| logfc |)> 2 as the threshold,we get the DEGs of the dataset.3.Using string11.0(https://string-db.org/)The interaction network of DEG protein was analyzed.Results : 1.Using microarray technology,269 DEGs were screened from the gse20247 dataset of h K2 cells stimulated by TGF-β,of which 112 were up-regulated and157 were down regulated in TGF-β 1 treatment group.2.The gene expression profile of h K2 cells stimulated by TGF-β was analyzed by microarray,DEGs analysis and GSEA analysis.It was found that 24 functional signaling pathways related to TGF-β 1 / Smad pathway and EMT were enriched in HK-2 cells treated with TGF-β 1.Conclusion: Based on the microarray analysis of the two groups of gene expression data,the functional signaling pathways related to TGF β 1 / Smad pathway and EMT were enriched in HK-2 cells after TGF-β 1 treatment.Therefore,TGF β 1 is an important stimulator of EMT in HK-2 cells,and TGF β 1 / Smad pathway is the key pathway.Part II: Establishment of calcium oxalate crystal kidney injury modelObjective:Objective to investigate the role of TGF β 1 / Smad in the process of renal tubular epithelial cell injury and whether TGF β 1 / Smad pathway can inhibit EMT and delay the process of chronic renal fibrosis.Methods:1.The effects of different concentrations of sodium oxalate on HK-2 cell apoptosis,mitochondrial membrane potential and intracellular ROS were detected by flow cytometry;2.The effects of sodium oxalate on HK-2 cell apoptosis under different time conditions;3.The expressions of EMT pathway related proteins in HK-2 cells were detected under time and concentration gradients.4.To detect the expression of TGF β /Smad pathway protein in sodium oxalate crystal kidney injury cell model.Results:1.The results of flow cytometry showed that with the gradual increase of different concentrations of sodium oxalate(0 mm,0.5 mm,1 mm,2 mm,4 mm),the apoptosis increased gradually,compared with the blank control group(P < 0.05).2.When the concentration of sodium oxalate was 1 mm,the apoptosis of h K2 cells was significantly increased with the prolongation of sodium oxalate treatment time,and the difference was statistically significant compared with the control group(P < 0.05).3.The difference of gene expression of Smad2,Smad3,Smad7,HDAC1,TGF β 1,tgfbr.Compared with HK-2 cells in blank control group,Smad2 was up-regulated,Smad3 was up-regulated,and Smad7 was up-regulated in a concentration gradient dependent manner.HDAC1,TGF β and tgfbr were up-regulated in a concentration dependent manner.4.After cultured in 1 m M sodium oxalate medium for different time,Smad7 was down regulated compared with the blank control group.After 12 hours,Smad7 was up-regulated,but it was still down regulated compared with the blank control group(P < 0.05).HDAC1 was up-regulated,while TGF β and tgfbr were up-regulated in a time-dependent manner(P <0.05).E-cadherin was up-regulated in a time-dependent manner.Vimentin was down regulated in a time-dependent manner.4.When h K2 cells were treated with 1 mm oxalic acid for 24 hours,the protein levels of Smad2 / 3 and TGF β increased gradually with the prolongation of oxalic acid stimulation time,and the epithelial cell marker ZO-1 decreased gradually with the prolongation of oxalic acid stimulation time.Conclusion: Medium concentration of oxalic acid(1 mm)for 24 hours may be a suitable condition for the model of crystalline kidney injury.In this process,the expression of TGF β / Smads signaling pathway related proteins increased and EMT occurred.In the model,the expression of HDAC1 increased with the increase of oxalate treatment time.Part III: Saha regulates TGF β / Smad signaling pathway and affects EMT expression in calcium oxalate crystallized kidney injury modelObjective: Objective to investigate the regulation of Saha,inhibit TGF-β / Smad signal transduction,reduce EMT and protect renal fibrosis.Methods: flow cytometry was used to detect the effect of deacetylase Saha on cell apoptosis in the model of crystalline kidney injury.The effect of SAHA on TGF β-Smad pathway was detected by Western blot.Western blot and immunofluorescence were used to detect the effect of SAHA on EMT of crystalline kidney injury model.Results:1.Compared with the blank control group,the TGF β / Smads signaling pathway protein was significantly up-regulated in the oxalic acid stimulated h K2 cell crystal kidney injury model,which led to the loss of epithelial cell morphology of renal tubular cells,the EMT phenomenon of down-regulation of epithelial markers and upregulation of interstitial markers.2.The deacetylase inhibitor Saha improved EMT in the model group by down regulating TGF β / Smads signaling pathway.Conclusion: Saha can improve EMT and renal fibrosis by down regulating TGF β /Smads signaling pathway.