| BackgroundNephrolithiasis is a common disease in urology,and it is the most common disease among inpatients in most hospitals in China,with an overall incidence rate of about 5%-10%.Among the kidney stones,80% are calcium oxalate stones.The treatment measures include natural stone-expelling,medical expulsive therapy,extracorporeal shock wave lithotripsy,flexible ureteroscopic lithotripsy,percutaneous nephrolithotomy,and laparoscopic nephrolithotomy.Treatment is becoming more and more minimally invasive,but due to the high recurrence rate of calcium oxalate stones,and some patients with complex kidney stones usually require multiple operations,which brings a great economic burden to the patient’s family and society.Therefore,a deep exploration of the occurrence,development,and related molecular mechanisms and signal pathways of renal calcium oxalate stones can help reduce the occurrence of calcium oxalate renal stones and prevent the recurrence of calcium oxalate renal stones.Previous studies have shown that the formation of calcium oxalate stones is a complex process,including crystal super saturation,renal tubular epithelial cell damage,lens adhesion,aggregation,nucleation and growth,etc.Among them,high oxalate stimulation damages renal tubular epithelial cell is currently considered as one of the key process in stone formation.However,the concrete mechanism of renal tubular epithelial cell injury remains unclear.ObjectiveThe effects of TNFR1/NF-κB signal pathway and ERS on the occurrence and development of renal calcium oxalate stones were investigated by in vitro cell experiments and in vivo animal models,molecular biology experiments and transcriptome sequencing.Materials and Methods1.Human renal tubular epithelial cells(HK-2)were used to establish an in vitro cell culture model stimulated by high oxalic acid.After gradient stimulation with different concentrations of oxalic acid,the expression of TNFR1 and downstream NF-κB pathway related proteins were detected.2.HK-2 cells were stimulated with normal saline and different concentrations of oxalic acid.The cell growth and the corresponding relationship with the concentration of oxalic acid were detected.The cell death and the formation of oxalic acid crystals were observed under microscope.3.Western Blot was used to detect the cleavage of apoptosis-related protein PARP in HK-2 cells after oxalic acid stimulation to verify the way of cell death.4.The calcium oxalate crystal model of mice was established by intraperitoneal injection of glyoxylic acid to increase the excretion of oxalic acid in mice urine and form high oxalic acid urine.The kidney tissue of mice was taken for HE staining to observe the calcium oxalate crystal deposition in kidney tissue.5.After the preliminary modeling was completed,30 mice were randomly divided into control group,oxalic acid stimulation model group and ERS inhibitor group.The mice in each group were given the same amount of normal saline,glyoxylic acid,and glyoxylic acid plus ERS inhibitor TUDCA,respectively.The changes in kidney appearance and body weight of mice in each group were observed.The kidneys of mice in each group were sliced and stained with HE,and then the degree of kidney damage was scored.6.TUNEL staining was used to detect the apoptosis level of kidney cells in three groups of mice,and WB was used to detect the expression of apoptosis-related proteins Caspase-3,Bcl2 and Bax.7.The expression levels and localization of NF-κB pathway protein and ERS protein in three groups of mice were detected by immunofluorescence,and WB was used for detection and verification.8.The number and level of autophagy lysosomes in each group were detected by autophagy lentivirus,and the apoptosis rate was detected by TUNEL staining and flow cytometry.9.Detection of mitochondrial membrane potential by JC-1 method and flow cytometry.10.Ultrastructural changes and the number of lysosomes observed by transmission electron microscope.11.The oxalic acid-stimulated NF-κB interference lentivirus group was constructed.WB and flow cytometry were used to detect the expression of NF-κB-related protein and ERS-related protein in each group and the apoptosis rate.12.Whole transcriptome sequencing of normal HK-2 cells and HK-2 cells stimulated by oxalic acid was performed,and all transcript information was measured,and statistical analysis was performed to explore transcriptional regulatory network.Results1.In vitro cell experiments showed that high oxalic acid stimulation of HK-2 cells could induce the expression of TNFR1 protein and activate the downstream NF-κB signal pathway of TNFR1,and the downstream JNK signal pathway was not activated.2.High oxalic acid can inhibit the growth of renal tubular epithelial cells in vitro,and the higher the concentration,the stronger the inhibition.WB confirmed the cleavage of PARP,the substrate of Caspase-3,suggesting that high oxalic acid inhibits cell growth by inducing apoptosis.3.Mouse model experiment in vivo confirmed that compared with the control group,the appearance swelling,kidney weight and kidney injury grade score of the model group were significantly increased(p < 0.05).Compared with the model group,the renal quality of the inhibitor group was significantly improved(p < 0.05).4.TUNEL staining was used to detect the apoptosis of renal tissues in the three groups,and it was found that the apoptosis level in the oxalic acid-stimulated model group and the inhibitor group was significantly increased.But,the apoptosis level in the inhibitor group was significantly decreased compared with that in the oxalic acid-stimulated model group,and the differences were statistically significant(p < 0.05),indicating that high oxalic acid stimulation could induce apoptosis of HK-2 cells.After inhibiting ERS,the apoptosis level decreased,indicating that endoplasmic reticulum stress was involved in this process.5.Immunofluorescence and WB were used to detect the ERS related protein GRP78,CHOP,NF-κB and its inhibitor protein levels.The results showed that the levels of GRP78,CHOP and NF-κB(nucleus)proteins in the oxalic acid-stimulated model group and the inhibitor group were increased,while the levels of IκBα and NF-κB(cytoplasm)proteins were decreased,and the differences were statistically significant(p < 0.05).The protein levels of GRP78,CHOP,and NF-κB(nucleus)in the inhibitor group were significantly lower than those in the oxalate group,while the protein levels of IκBα and NF-κB(cytoplasm)were significantly higher than those in the oxalate group(p < 0.05).The results showed that oxalic acid could activate ERS and NF-κB signaling pathways in renal tissue.6.JC-1 method and flow cytometry confirmed that compared with the normal control group,MMP in oxalic acid stimulation model group and inhibitor group decreased significantly,while MMP in inhibitor group and model group increased significantly.The differences were statistically significant(p<0.05).WB verified the expression of mitochondrial fusion proteins OPA1 and Mfn2 in each group,which was consistent with the results of JC-1 and flow cytometry,suggesting that ERS was involved in the mitochondrial imbalance of HK-2 cells induced by oxalic acid.7.The lentivirus interference group of NF-κB stimulated by oxalic acid(sh RNA group)was constructed,and the ERS and NF-κB signal pathway related proteins in each group were detected by WB.The results showed that compared with the control group,the protein expression levels of GRP78,XBP1,p-EIF2 and IκBα in the model group and sh RNA group were significantly increased,and the protein expression levels of NF-κB(cytoplasm)and IκBα were significantly decreased,and the difference was statistically significant(p < 0.05).The expression levels of GRP78,XBP1,p-EIF2 and IκBα in sh RNA group were consistent with those in model group.However,after lentivirus interference down-regulated the expression of NF-κB,the levels of NF-κB(cytoplasm)and NF-κB(nucleus)proteins in sh RNA group were decreased.The results showed that although the cells in sh RNA group were still at high ERS level,the NF-κB signaling pathway was significantly inhibited.It proved that oxalic acid activates NF-κB pathway by stimulating ERS in HK-2 cells.8.In the whole transcriptome sequencing,pathway enrichment analysis of genes in key RNA was carried out,and the first five pathways with the most significant enrichment were selected.The results showed that the key genes of NF-κB pathway and ERS pathway such as ATF6β in this study were enriched,and the conclusions of in vitro cell experiments and in vitro animal models were verified at the transcriptional level.ConclusionsHigh concentration oxalic acid stimulation can induce mitochondrial dysfunction in renal tubular epithelial cells by activating ERS,enhance autophagy,activate TNFR1/NF-κB signal pathway,induce renal tubular epithelial cell damage and apoptosis,and promote renal calcium oxalate stone formation. |
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