| Background:Diabetic nephropathy(DN)is currently the leading cause of end-stage renal disease worldwide.Although strict blood pressure and blood glucose control can effectively slow the disease process of diabetic nephropathy caused by type 1 or type 2 diabetes,it is often difficult to maintain it for a long time because intensive hypoglycemic treatment may increase the risk of hypoglycemia in patients.Therefore,new treatment strategies and methods for diabetic nephropathy are urgently needed.The micro-inflammatory state of the kidney has become an important basis for the development of diabetic nephropathy.Activation of inflammasome has been reported in a variety of renal intrinsic cells and has been confirmed in diabetic nephropathy.Pyroptosis is an inflammatory programmed cell death mediated by inflammatory caspase-1,caspase-4,and caspase-5(murine caspase-1,caspase-11).It is the immune response to infection and cell damage.This lytic cell death is distinguished from apoptosis by its characteristics of cytolysis and release of cytoplasmic contents.This is a form of cell death induced by gasdermin D(GSDMD)-mediated pore formation,causing cell swelling and rupture.As an innate immune response,pyroptosis was once thought to be a unique pathophysiological process of immune cells.However,it is unclear whether renal intrinsic cells can promote the occurrence of diabetic nephropathy through pyroptosis and how pyroptosis of these cells might be controlled.VX-765 is an effective,bioavailable,and non-toxic small molecule inhibitor.VX-765is safe and has been tested in a 6-week phase II human clinical trial for treatment of epilepsy.Therefore,VX-765 is a safe,effective and feasible drug.Recently,researchers have reported the therapeutic role of VX-765 in cardiac ischemia-reperfusion injury and neurological diseases,but the application of VX-765 in DN has not been reported.Previous studies have shown that caspase-1 gene knockout can play a renal protective role in diabetic mice.However,it is unclear whether caspase-1 silencing exerts its renal protective effect on DN by regulating pyroptosis.We propose the hypothesis that the clinical-grade drug VX-765 can be used to treat diabetic nephropathy,which can improve kidney injury and prognosis of DN by regulating pyroptosis.Methods:1.To study the role of pyroptosis in renal tubular epithelial cells of diabetic nephropathy,we used the human renal proximal tubule epithelial cell line(HK-2 cells).Cells were treated with 5.5 mM,15 mM,25 mM,35 mM D-glucose and mannitol as intervention conditions for 48 hours.Western blot was used to detect the expression of NLRC4,cleaved-caspase-1 and GSDMD p30,executioner of pyroptosis,to understand the effects of different concentration of glucose on the activation of inflammatory signaling pathways in HK-2 cells.To determine whether caspase-1 was activated by NLRC4-mediated mechanism in HK-2 cells,we performed co-immunoprecipitation analysis on high glucose-treated cells,and used small interfering RNA(siRNA)to knock down NLRC4 to observe the active caspase-1 p20 fragment expression.2.To further clarify whether pyroptosis occurred in HK-2 cells under high glucose conditions,we performed FAM-YVAD-FMK and PI fluorescence staining on the cells treated with high glucose,and used confocal microscopy combined with immunofluorescence staining to observe the morphological changes.Cells treated with 25mM D-glucose for 24 or 48 hours,and mannitol for 48 hours were analyzed with flow cytometry to quantify the ratio of pyroptosis(FAM-YVAD-FMK~+/PI~+cells).3.In order to determine whether caspase-1 plays an important role in pyroptosis in high glucose-stressed HK-2 cells,we used two selective caspase-1 inhibitors VX-765 and Z-YVAD-FMK to block caspase-1 activity.Western blot was used to detect the expression of pyroptosis-related proteins,NLRC4,GSDMD,and IL-1β,as well as the expression of collagen I and fibronectin.At the same time,the enzyme-linked immunosorbent assay(ELISA)was used to detect the level of IL-1βsecreted by the cells in the supernatant.Flow cytometry or LDH activity were then used to analyze the ratio of pyroptotic cells in each group.4.We administrated STZ injection in mice to obtain a diabetic animal model.In order to understand the role of inflammation and pyroptosis in the process of diabetic nephropathy,western blot was used to analyze the dynamic process of NLRC4,caspase-1,GSDMD,collagen I,and fibronectin expression in renal cortex of diabetic mice at different ages.Detection and analysis of hematuria biochemical indicators and renal pathological changes.5.To further clarify whether VX-765 can be an effective treatment for DN.We started to administrate DM intraperitoneally with 100 mg/kg VX-765 daily for 8 weeks,two weeks after STZ injection.The mice were monitored for weight and blood glucose.Serum and urine were collected.Serum creatinine,blood urea nitrogen,serum albumin,and urine albumin/creatinine ratios were measured.Western blot was used to analyze the expression of NLRC4,GSDMD,IL-1β,collagen I and fibronectin in the renal cortex of mice in each group.The level of IL-1βin the tissue homogenate of renal cortex was detected by ELISA. Immunohistochemistry/immunofluorescence were used to analyze the expression of GSDMD,CD45,collagen I and fibronectin in kidney tissues.HE and Masson staining were used to analyze the kidney pathological changes of mice in each group.6.We previously screened a series of abnormally regulated circRNAs through microarray analysis,which may be involved in renal tubular injury by regulating pyroptosis.In this study,we used RT-qPCR to validate 10 differentially expressed circRNAs.circACTR2 shRNA was transfected into glucose-stressed HK-2 cells using plasmid transfection.IL-1βsecreted by cells was detected by ELISA.Flow cytometry and LDH activity analysis were used to detect the ratio of pyroptotic cells.Collagen IV and fibronectin expression were detected by western blot.Results:1.High glucose stimulated up-regulation of NLRC4 expression in tubular epithelial cells,and promotes the activation of caspase-1 and GSDMD.Co-immunoprecipitation analysis showed that high glucose stimulated the assembly of NLRC4/caspase-1 inflammasome in HK-2 cells,and knockdown of NLRC4 could significantly inhibit caspase-1 activation.2.We used confocal microscopy to locate the pyroptotic cells(FAM-YVAD-FMK~+/PI~+cells)and observed the cell morphology under transmitted light.We found that the cells exhibit pyroptotic-like morphological features,ballooning of the cell membrane.The results of flow cytometry analysis showed that high glucose treatment can significantly increase the rate of pyroptosis,and it is more obvious after 48 hours of treatment.The change in osmotic pressure caused by mannitol is not the main factor to promote pyroptosis.3.Caspase-1 inhibitor treatment can down-regulate the high expression of GSDMD p30and mature-IL-1βin high glucose,and inhibit the release of IL-1βby cells.Both VX-765and Z-YVAD-FMK significantly inhibited the rate of pyroptosis and the release of LDH in HK-2 cells treated with high glucose.High glucose promotes the expression of collagen I and fibronectin,molecular indicators of fibrosis.While caspase-1 inhibitors can significantly down-regulate their high expression.4.The expression of NLRC4,caspase-1 and GSDMD in 10-week-old DM mice started to increase,indicating that inflammation and pyroptosis were involved in the development of diabetic nephropathy in the early stages.However,collagen I and fibronectin began to be highly expressed in DM mice at 13 or 16 weeks,suggesting that inflammation and pyroptosis precede and are potentially linked with renal fibrosis in DN.Pathological analysis revealed significant extracellular matrix aggregation and tubulointerstitial fibrosis in the kidney of DM mice aged 16-18 weeks.5.VX-765 treatment can significantly reduce proteinuria in diabetic mice,effectively improve renal function in mice,and reduce the high expression of GSDMD p30,mature- IL-1β,collagen I,and fibronectin in renal cortex.Immunohistochemical staining showed that GSDMD was highly expressed in the tubules in DM,and VX-765 treatment could down-regulate its expression.Immunohistochemistry/immunofluorescence staining analysis showed that VX-765 treatment can inhibit CD45~+inflammatory cell infiltration in the kidney of DM,and down-regulate the high expression of collagen I and fibronectin.Analysis of HE and Masson staining showed that tubular injury and interstitial fibrosis were alleviated after VX-765 treatment in DM.Its therapeutic effects are also independent of changes in body weight and blood glucose.6.Among the candidate circRNAs,we found that circACTR2 was significantly up-regulated in HK-2 cells treated with high glucose,while VX-765 treatment inhibited its high expression.Knockdown of circACTR2 can significantly reduce pyroptosis,IL-1βsecretion,cytoplasmic content release and fibrosis indicators,suggesting that circACTR2can effectively regulate pyroptosis and inflammation.Conclusions:1.In high glucose conditions,renal tubular epithelial cells undergo pyroptosis,which is characterized by membrane ballooning,caspase-1 activation,GSDMD cleavage,pro-inflammatory cytokine release,and cytoplasmic content effusion.NLRC4 inflammasome participates in caspase-1-mediated activation of inflammatory signaling pathways in renal tubular cells in high glucose.2.VX-765 can effectively inhibit caspase-1 enzyme activity,GSDMD cleavage,IL-1βmaturation and release,LDH release,reduce the ratio of pyroptosis,effectively improve proteinuria and renal function in diabetic mice,down-regulate the expression of inflammatory indicators,and reduce renal tubulointerstitial fibrosis.3.VX-765 plays a protective role in tubular cells by down-regulating the abnormally high expression of circACTR2 in high glucose,thus significantly reducing pyroptosis,IL-1βsecretion and cytoplasmic content release. |
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