| Background: The incidence of Diabetic Kidney Disease(DKD)increases year by year,and its pathogenesis is very complex.With the progression of the disease,renal fibrosis will occur,and its treatment is very difficult.At present,the signaling pathway and molecular mechanism of fibrosis are not fully understood.The formation of renal fibrosis involves multiple processes,including tissue damage and repair,recruitment of inflammatory cells,production and release of fibrosis related factors,and deposition of collagen.Renal inflammation is one of the key factors leading to the continuous development of DKD and fibrosis.Pyroptosis is a highly inflammatory programmed cell death mode,which is mainly dependent on the Caspase-1 protein in the classical signal pathway and Caspase-11 protein in the non-classical signal pathway.After activation,it will cleaved Gasdermin D to form holes.Release of pro-inflammatory cytokines IL-1? and IL-18 into the extracellular environment,causing a cascading amplification of the inflammatory response.Current studies believe that Caspase-1-mediated pyroptosis,the classical pathway,promotes the progression of DKD.However,it is still unclear whether Caspase-11-mediated pyroptosis is involved in the progression of DKD and its role,and the regulatory mechanism has not been reported.Studies have shown that Caspase-11 is regulated by various E3 ubiquitin enzymes,such as NEDD4.NEDD4 L and NEDD4 belong to the same family and are highly homologous in genes.NEDD4 L is highly expressed in kidney,especially in renal tubular epithelial cells,and plays a renal protective role.However,how it changes in renal fibrosis of DKD,whether it plays a role and whether it can regulate Caspase-11 as NEDD4 does,remains to be clarified.Therefore,this study will observe whether the occurrence of cell pyroptosis mediated by Caspase-11 and the role it plays in the process of DKD.And explore whether NEDD4 L can interact with Caspase-11 to participate in DKD progress.To further elucidate the molecular mechanism of renal fibrosis in DKD,and to provide theoretical and experimental basis for seeking new intervention targets for prevention and treatment of DKD.Objective:1.To observe the occurrence of Caspase11-Gasdermin D induced cell pyroptosis in renal tissue of diabetic mouse and renal tubular epithelial cells cultured in high glucose,and to explore its role in DKD.2.To observe the effects of NEDD4 L on cell pyroptosis and fibrosis in diabetic renal tissue and renal tubular epithelial cells cultured with high glucose,and to clarify the role and possible mechanism of NEDD4 L in the pathogenesis of renal fibrosis in DKD.Methods:1.To observe whether Caspase11-Gasdermin D induced cell pyroptosis occurred in renal tissue of diabetic mouse and renal tubular epithelial cells(m RTEC)cultured in high glucose,and to explore its role in DKD:(1)In vivo experiment: mouse were taken as the research object and divided into wild control group(WT)and db/db group.The changes of biochemical indexes in mouse were detected by the kit.HE,Masson and PASM staining were used to observe the morphological changes of renal tissue.The expression of cell pyroptosis markers(Caspase-1,Caspase-11 and Gasdermin D-N)and fibrosis markers(Collagen-?,E-cadherin and Desmin)were detected by Western blot.The levels of IL-18 and IL-1?in kidney tissue and urine of mouse were determined by ELISA.(2)In vitro experiment: a)m RTEC was taken as the research object and divided into normal glucose(NG)group and high glucose(HG)group.AO/EB staining and ELISA were used to detect LDH,the integrity of cell membrane was observed under high glucose condition.The expression of cell pyroptosis markers(Caspase-1,Caspase-11 and Gasdermin D-N)and fibrosis markers(Collagen-?,E-cadherin and Desmin)were detected by Western blot.The contents of IL-18 and IL-1? in the medium were detected by ELISA.b)m RTEC was cultured with normal glucose and high glucose respectively,and transfected with knockdown or overexpression of Caspase-11 plasmid.Grouping: NG,NG+Vector,NG+ Caspase-11plasmids(knockdown or overexpression),HG,HG+Vector and HG+Caspase-11plasmids(knockdown or overexpression)were used to observe the effects of Gasdermin D-N,fibrosis indicators and the release of inflammatory cytokines IL-18 and IL-1?.C)m RTEC was cultured with normal glucose and high glucose respectively,and transfected with knockdown or overexpression of Gasdermin D plasmid.Grouping: NG,NG+Vector,NG+Gasdermin D plasmids(knockdown or overexpression),HG,HG+Vector and HG+Gasdermin D plasmids(knockdown or overexpression)were used to observe the effects on fibrosis indexes and the release of inflammatory cytokines IL-18 and IL-1?.2.To observe the effects of NEDD4 L on cell pyroptosis and fibrosis in diabetic renal tissue and renal tubular epithelial cells cultured with high glucose,and to clarify the role and possible mechanism of NEDD4 L in the pathogenesis of renal fibrosis in DKD:(1)In vitro experiment: a)The expression of NEDD4 L in NG group and HG group was detected by Western blot.b)m RTEC was cultured with normal glucose and high glucose,respectively,and the NEDD4 L plasmids were transfected with the following groups: NG,NG+Vector,NG+NEDD4L plasmids(knockdown or overexpression),HG,HG+Vector and HG+ NEDD4 L plasmids(knockdown or overexpression).Western blot was used to detect the changes of cell pyroptosis and fibrosis indexes.The contents of IL-18 and IL-1? in each group were determined by ELISA.(2)In vivo experiment: a)The expression of NEDD4 L in the kidney of mouse in WT group and db/db group was detected by Western blot.b)Adeno-associated virus overexpressing NEDD4L(p AAV-ITR-CMV-Nedd4L)was injected through tail vein and divided into WT group,db/db group,db/db+Vector group and db/db+p AAV-ITR-CMV-Nedd4 L intervention group.After verifying the intervention efficiency of NEDD4 L,biochemical indexes of each group were detected by the kit.HE,Masson and PASM staining were used to observe the morphological changes of renal tissues in each group.The fibrosis indexes(FN,PDGFR-?,Collagen-?,Collagen-?,E-cadherin,Desmin and ?-SMA),the cell pyroptosis indexes(Caspase-1,Caspase-11 and Gasdermin D-N)were detected by Western blot,Inflammatory markers(NLRP3,Mcp1 and TNF-?).The levels of IL-18 and IL-1? in kidney tissue and urine of mouse were determined by ELISA.3.Take Caspase-11 as the entry point to further explore the molecular mechanism of NEDDL regulation of Caspase-11:(1)In vitro experiment: a)The co-localization region between NEDD4 L and Caspase-11 was observed by double staining with cell immunofluorescence.b)m RTEC was taken as the research object and divided into the following groups: Input,Ig G,NG,HG,HG+overexpressed NEDD4 L.Caspase-11 ubiquitination was detected by immunoprecipitation assay.The protein levels of NEDD4 L and Caspase-11 were detected by Western blot.(2)In vivo experiment: a)The interaction between NEDD4 L and Caspase-11 and Caspase-1 was detected by immunoprecipitation assay.b)mouse were taken as the research object,divided them into as Input group,Ig G group,WT group,db/db group and db/db+p AAV-ITR-CMV-Nedd4 L group.Caspase-11 ubiquitination was detected by immunoprecipitation assay.The changes of NEDD4 L and Caspase-11 protein levels were detected by Western blot.Results:1.Both classical(caspase-1)and nonclassical(caspase-11)signaling pathways were activated during the course of diabetic nephropathy: a)Compared with WT group,BG,T-CHO,TG,CRE,BUN,MAU and ACR were increased and UCR was decreased in db/db group,accompanied by fibrosis.It indicated that pathological damage in mouse kidney from db/db group.And the protein expressions of Caspase-1,Caspase-11 and Gasdermin D-N in db/db group were significantly increased,the contents of IL-1? and IL-18 in kidney tissue and urine of mouse were significantly increased,accompanied by the decreased expression of E-cadherin,Collagen-III and Desmin expression increased.These results indicated that both Caspase-1 and Caspase-11 were activated in renal tissue of diabetic mice.b)EB fluorescence staining was enhanced after HG stimulation of m RTEC,and the content of LDH in the medium of HG group was significantly increased,indicating that the integrity of cell membrane was damaged.Compared with NG group,the expression of Casapse-1,Casapse-11 and Gasdermin D-N increased after high glucose stimulation,and the secretion of IL-1? and IL-18 increased.With the occurrence of EMT,the expression of E-cadherin decreased,and the expression of Collagen-III and Desmin increased.These results indicated that both caspase-1 and caspase-11 were activated in renal tubular epithelial cells(m RTEC)cultured with high glucose.2.Cell pyroptosis mediated by caspase11-Gasdermin-D promoted the progression of fibrosis: a)Down-regulation of Caspase-11 expression inhibited cell pyrotosis,manifested by decreased expression of Gasdermin D-N,decreased release of IL-18 and IL-1?,and EMT process was inhibited.up-regulation of Caspase-11 promoted cell pyroptosis and EMT.b)After Gasdermin D was knockeddown,the release of IL-18 and IL-1? was reduced,and EMT is inhibited.While Gasdermin D was up-regulated,the release of IL-18 and IL-1? was increased,and the occurrence of EMT was promoted.3.NEDD4 L inhibited the occurrence of cell pyroptosis and delayed the progression of renal fibrosis: a)NEDD4L was decreased in renal tubular epithelial cells cultured with high glucose and in renal tissue of diabetic mice.b)Downregulation of NEDD4 L promoted the occurrence of cell pyrotosis,which was manifested by significantly increased expressions of Caspase-11 and Gasdermin D-N,and increased release of IL-18 and IL-1?,and the occurrence of EMT was promoted.The process of cell pyroptosis and EMT were inhibited after up-regulation of NEDD4 L.c)Six weeks after the injection of with NEDD4 L overexpression adeno-associated virus through the tail vein,T-CHO,TG,CRE,BUN,MAU,ACR decreased and UCR increased,and the fibrosis was relieved.With the increase of NEDD4 L expression,FN,PDGFR-?,Collagen-IV,Collagen-III,Desmin and ?-SMA were all decreased,and E-cadherin expression was recovered.It is suggested that the up-regulation of NEDD4 L can delay the progression of fibrosis.Moreover,compared with the db/db group,the up-regulation of NEDD4 L could inhibit cell pyroptosis and the expression of inflammation-related factors.4.NEDD4 L promoted the ubiquitination degradation of Caspase-11 by binding with it: a)NEDD4L protein was negatively correlated with Caspase-11 protein expression.In addition,NEDD4 L and Caspase-11 overlap each other in space,and there is an interaction between them.b)The expression of NEDD4 L was decreased in renal tubular epithelial cells stimulated by HG and in renal tissues of db/db mouse,and the expression of Caspase-11 protein was significantly increased while the ubiquitination level was decreased.The up-regulation of NEDD4 L by exogenous intervention promoted Caspase-11 ubiquitination,thus significantly reduced Caspase-11 protein level.Conclusions:(1)In renal tubular epithelial cells cultured with high glucose and renal tissue of diabetic mouse,cell pyrotosis,accompanied by inflammation and fibrosis were observed.Caspase11-Gasdermin D mediates cell pyroptosis,which promotes the progression of inflammation and fibrosis.(2)NEDD4L plays a protective role in the process of DKD.By binding to Caspase-11 protein,NEDD4 L degrades Caspase-11 ubiquitin,resulting in a low level of Caspase-11,thereby reducing cell pyroptosis and renal inflammation,and finally delaying the progression of renal fibrosis. |
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