XIST Promoted Calcium Oxalate Crystal Induced Renal Tubular Epithelial Cell Inflammatory Responses And Oxidative Injury Via MiRNA-223/NLRP3 Pathway

Objective:Calcium oxalate nephrocalcinosis and renal calculus can induce inflammation and damage to the kidney,thereby further aggravating the formation and development of kidney stones and even impairing kidney function.Therefore,we study the effects of XIST on inflammation and oxidative stress damage to reveal its potential role in the development of kidney stones.Methods:This study first established a glyoxylate-induced calcium oxalate nephrocalcinosis mouse model.Male C57 mice were injected with 0,50 mg/kg or 100mg/kg glyoxylate every day via intraperitoneal injection.Kidney specimens were collected after one week,and HE staining and Pizzolato Staining was used to detect renal crystal deposition.PAS staining and TUNEL were performed to detect renal tubular epithelial cell damage,and immunohistochemical experiments were adopted to detect the levels of downstream protein expression.RNA of kidney samples was extracted for q PCR.We co-cultured HK-2 cells exposed to calcium oxalate monohydrate（COM）.After transfection with si-XIST or miR-223 mimics and miRNA-223 inhibitor,we measured the expression of RNA and protein and detected the generations of LDH,SOD,MDA and H₂O₂at the cell level.The apoptosis and the level of ROS were detected.The dual-luciferase reporter assay was used to determine the interaction between miR-223 and XIST or m RNA of NLRP3.A glyoxylate-induced calcium oxalate nephrocalcinosis mouse model was constructed by daily intraperitoneal injection of 100 mg/kg glyoxylate,and three groups were injected with r AAV-sh-NC,r AAV-sh-XIST,antagomiRNA-223 and r AAV-sh-XIST+antagomiRNA-223into the tail vein of mice on day 1 and day 4,respectively.After collecting mouse kidney specimens,we detected calcium oxalate crystal deposition,inflammatory protein expression and renal tubular damage.Outcomes:In a glyoxylate-induced calcium oxalate nephrocalcinosis mouse model,we found that the deposition of calcium oxalate crystals in the kidney,the expression of inflammatory proteins and renal tubular damage increased with the increase of the dose of glyoxylate.The RNA level of XIST and NLRP3,Caspase-1,IL-1βwere significantly higher than the control group.In cell experiments,XIST silence can significantly inhibit inflammation and oxidative stress damage,reduce the expression of NLRP3,Caspase-1 and IL-1β.The knockdown of XIST significantly reduced cell necrosis and ROS products.We found that miRNA-223 directly targeted to the 3’-UTR of XIST and NLRP3.MiRNA-223mimics inhibited the expression of downstream molecules,thereby reversing the promoting effect of XIST on calcium oxalate crystal-induced inflammation and oxidative stress injury.In addition,we verified in a mouse model that XIST silencing alleviated the inflammatory response and oxidative stress injury induced by calcium oxalate nephrocalcinosis through the ce RNA mechanism of XIST/miRNA-223/NLRP3.Conclusion:our results suggested that Lnc RNA XIST promoting calcium oxalate crystal induced inflammatory response and ROS via interplaying with miR-223 and NLRP3/Caspase-1/IL-1βpathway,and further aggravated calcium oxalate deposition.