| Objective Diabetic nephropathy(DN)is the most common and serious microvascular complication secondary to diabetes mellitus,accounting for more than 40% of end-stage renal disease(ESRD)worldwide,and is an important cause of death of diabetic patients.Recent studies have shown that pyroptosis was involved in the pathogenesis of diabetes mellitus and diabetic cardiomyopathy.Moreover,Pyroptosis occurred in infectious disease and tumor was an inflammatory programmed cell death mediated by Gasdermin family members.Meanwhile,Gasdermin E(GSDME)was highly expressed in kidney tissues.We wonder whether GSDME mediated pyroptosis plays a role in inflammatory injury during DN.This study aims to clarify the expression and interaction of GSDME,GSDME-N,caspase-3,p17,caspase-1,p20,IL-1? and m IL-1? in diabetic nephropathy,and to explore the role of caspase-3cleaved GSDME-induced pyroptosis in the pathogenesis of DN,in order to provide new targets for the treatment and diagnosis of DN.Methods 1.The SD rat model of type 1 diabetic nephropathy were constructed by intraperitoneal injection of streptozotocin(1% STZ,65 mg/kg).Blood glucose concentrations,body weight,24-hour urine volume and the quantitation of24-hour urine protein were measured in control group(Ctrl group)and diabetic nephropathy group(DN group)at 0,4,8,12,16 and 20 weeks;2.HE and PAS staining were used to observe the pathological changes of kidney tissue under optical microscope;3.Western Blot was used to detect the protein expression levels of GSDME,GSDME-N,caspase-3,p17,caspase-1,p20,IL-1? and m IL-1? in kidney tissues of Ctrl rats and DN rats at 12,16 and 20 weeks.Immunohistochemical staining for GSDME,caspase-3,caspase-1 and m IL-1? protein expression levels;4.ELISA was used to detect the protein concentratin of m IL-1? in venous blood of Ctrl rats and DN rats at 12,16 and 20 weeks;5.TUNEL staining was used to detect the positive rate of pyroptosis cells in kidney tissues of Ctrl rats and DN rats at 12,16 and 20 weeks;6.Morphological changes of HK2 cell lines and RGMCs cells cultured in high glucose were observed by phase contrast microscopy;7.HK2 cells and RGMCs cells were cultured with high glucose for 0,12,24,36 and 48 hours.The expression level of GSDME,caspase-3,caspase-1 and IL-1?gene was detected by Real-Time PCR;Western Blot was used to detect the protein expression levels of GSDME,GSDME-N,caspase-3,p17,caspase-1,p20,IL-1? and m IL-1?;ELISA was used to detect the protein concentratin of m IL-1?in cell culture supernatant;8.HK2 cells and RGMCs cells were transfected with GSDME-si RNA,caspase-3-si RNA or caspase-1-si RNA or both caspase1/3 si RNA,subsequently stimulated with high glucose for 48 hours.The protein expression levels of GSDME?GSDME-N?caspase-3?p17?caspase-1?p20?IL-1? and m IL-1? were detected by Western Blot.The protein concentration of m IL-1? in cell culture supernatant was detected by ELISA.Results 1.SD rat type 1 diabetic nephropathy animal model was successfully constructed:After injection of STZ for 72 h,the random blood glucose of rats was higher than16.7 m M,showing an increase in urine output.The random blood glucose concentration and 24-hour urine volume of rats in DN group were significantly higher than those in Ctrl group,and the quantitation of 24-hour urinary protein was significantly increased after 8 weeks of STZ injection,while body weight was significantly lower than that of rats in Ctrl group;2.PAS staining showed that mesangial dilatation and extracellular matrix accumulation were observed in DN group compared with those in Ctrl group.HE staining results showed that the renal tissue structure of DN group was disordered,the renal tubule hypertrophy and tubulointerstitial injury were observed in DN group;3.Western Blot showed that the cleavage of GSDME in kidney tissue of DN group increased significantly after 20 weeks of STZ injection,the activation of caspase-3 and caspase-1 increased significantly,and the expression of m IL-1?was decreased at 16 and 20 weeks after a significant increase in 12 weeks;Immunohistochemical staining revealed that DN group kidney tissue total GSDME,caspase-3 and caspase-1 protein expression increased,and m IL-1?reduced at 16 weeks and 20 weeks after a significant increase in 12 weeks;4.At the same time,ELISA assay showed that the protein concentratin of m IL-1? in venous blood of DN group was significantly higher than that of Ctrl group at 16 and 20 weeks;5.The results of TUNEL staining showed that the number of TUNEL-positive cells in kidney tissue of DN group was higher than that of Ctrl group at 20 weeks;6.The results of phase contrast microscopy showed that HK2 cells and RGMCs cells in HG group were significantly swollen and characteristic vesicles appeared on the plasma membrane after culture 48 hours compared with NG group;7.The m RNA levels of caspase-3,GSDME,caspase-1 and IL-1? in HK2 cells and RGMCs cells of HG group were significantly higher than those in NG group;Western Blot assay showed that the protein expression levels of GSDME-N,p17 and P20 in HK2 cells and RGMCs cells induced by high glucose after 48 h in HG group were significantly higher than those in NG group;Meanwhile,the concentration of m IL-1? in the supernatant of related cells increased in a time-dependent manner from 36 hours;Compared with the NC-si RNA group,the intracellular m IL-1? protein level in GSDME-si RNA group was significantly increased,while the concentraion of m IL-1? in the corresponding supernatant of GSDME-si RNA group decreased;8.The results showed that the protein levels of GSDME-N,caspase-1 and p20,caspase-3 and p17 in si RNA-caspase-3 and si RNA-caspase-1 groups cells were significantly lower than that in NC-si RNA group,while the protein levels of m IL-1? in cells were significantly higher than those in NC-si RNA group and si RNA-caspase-1 group,and lower than those in si RNA-caspase-3 group;The concentration of m IL-1? in cell supernatant of si RNA-caspase-3 and si RNA-caspase-1 groups was lower than that of other groups.Conclusions 1.The SD rat model of type 1 diabetes nephropathy was successfully constructed;2.In diabetic nephropathy,pyroptosis occurs in kidney cells,and the increasing release of m IL-1? caused by pyroptosis plays an important role in the inflammatory pathogenesis of diabetic nephropathy;3.Caspase-3 cleaved GSDME-induced pyroptosis and increase the release of m IL-1? is one of the pathogenesis of diabetic nephropathy. |
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