The Role And Mechanism Of DcR2-mediated Renal Tubular Cell Senescence In Renal Interstitial Fibrosis Of Diabetic Nephropathy

BackgroundDiabetic nephropathy(DN)is the most common and serious complication of diabetes,and DN is one of the most common causes of end-stage renal disease.Tubulointerstitial fibrosis plays an important role in the progression of DN,which is closely correlated to renal malfunction,and determines renal prognosis.Current studies suggest that renal tubular epithelial cells(TECs)senescence is an important cellular biological event in interstitial fibrosis.On the one hand,senescent cells promote tissue inflammation and fibrosis by secreting a large number of pro-inflammatory,chemokine and other senescence-associated secretory phenotypes(SASP);on the other hand,senescent cells also have an apoptosis-resistant phenotype,which promote the survival of senescent cells and continuously secrete SASP,leading to the persistence and amplification of its damaging effects,and accelerating tissue inflammation and organ fibrosis.In DN,studies have demonstrated that renal tubular cell senescence is positively correlated with interstitial fibrosis and renal malfunction.However,the mechanism of TECs senescence and its role in the renal interstitial fibrosis is still unclear.Decoy receptor 2(DcR2)is a type II transmembrane receptor of tumor necrosis factor-related apoptosis-inducing ligand.It is currently regarded as a marker of tumor cell,and it is correlated to apoptosis resistance,differentiation degree and poor prognosis in tumor patients.In addition,DcR2 is also considered to be a biomarker of senescent fibroblasts,and it is correlated to apoptosis resistance and liver fibrosis.In renal tissue of DN patients,our previous study found that DcR2 was specifically expressed in senescent TEC,which was associated to interstitial fibrosis and renal function;DcR2-positive senescent TEC was highly expressed anti-apoptotic molecule FLIP and lowly expressed caspase-3,which possessed an apoptosis-resistant phenotype,resulting in survival and releasing SASP.However,the relationship between DcR2 and TECs senescence and apoptosis-resistant phenotype,and its role in renal interstitial fibrosis in DN remain unclear.This study will focus on the SASP and apoptosis-resistant phenotype of senescent renal tubular cells.Co-immunoprecipitation combined with quantitative proteomics and bioinformatics analysis were used to identify DcR2-interaction proteins from three dimensions of DN patients,STZ-DN mouse model,and high glucose-induced primary renal tubular cell senescence model.Subsequently,regulation of DcR2 expression in vitro and in vivo to explore the role and mechanism of DcR2 in senescent phenotype of TEC and renal interstitial fibrosis in DN.Materials and methods1.Subjects1.1 A total of 241 DN patients diagnosed by biopsy in our hospital from January 2012 to December 2019 were included.Patients with other kidney diseases,tumors,urinary tract infections,and other infections were excluded.Those using diuretics,traditional Chinese medicines,nephrotoxic drugs,those with severe hepatic or cardiac insufficiency were also excluded.The patients were divided into early DN(n=83)and advanced DN(n=158)according to urinary albumin/creatinine ratio(ACR)and renal function.1.2 The STZ-induced DN mouse model was constructed.DcR2-si RNA and DcR2 overexpression plasmids were transfected into the kidney through the mice tail vein using ultrasonic microbubble technology to build DcR2 low-expression and DcR2-overexpression mouse models.This method was used to achieve in vivo regulation of DcR2 expression.1.3 Primary TECs were culture in vitro,and a model of TECs senescence was constructed by high glucose(30 mmol/L)treatment.DcR2-si RNA and DcR2 overexpression plasmids were transfected with lentivirus in vitro to achieve regulation of DcR2 expression.2.Methods and indicators2.1 The expression of DcR2 in renal tissue of DN patients and STZ-DN were detected using immunohistochemistry.The co-localization relationship between DcR2 and senescent markers(p16、p21、SA-β-gal),SASP markers(IL-1β、IL-6、TGF-β1),apoptosis-related markers(FLIP,Bcl-2,caspase-3,caspase-8,Tunel)and fibrotic markers(a-SMA,collagen I,collagen IV,fibronectin)were analyzed by immunofluorescence co-staining.In vitro,magnetic activated cell sorting(MACS)was used to sort out DcR2-positive TECs,and detect the activity of caspase-3,the expression of apoptosis-related markers with western blotting,the percentage of apoptotic cells with flow cytometry and the level of SASP in the supernatant of cells with ELISA.2.2 After regulating the expression of DcR2 in vivo,renal function was assessed by blood and urine markers(serum creatinine,blood urea nitrogen,urine microalbumin/creatinine)and the area of renal interstitial fibrosis was detected by Masson staining.Quantitative RT-PCR,immunohistochemistry,WB and other methods were used to assess the expression of fibrotic markers,cell senescence,SASP and apoptosis-related markers.The levels of the above markers were detected after regulating the expression of DcR2 in vitro.2.3 The co-localization relationship of DcR2 with TRAIL and DR5 was detected by immunofluorescence co-staining in renal tissue of DN patients,and the binding relationship of DcR2 with TRAIL and DR5 in STZ-DN renal tissue was analyzed by co-immunoprecipitation.2.4 In renal tissue from DN patients and TECs samples,DcR2 interacting proteins were screened and analyzed by co-immunoprecipitation combined with quantitative proteomics and bioinformatics.The interaction between DcR2 and PRDX1,DcR2 and GRP78 was verified by co-immunoprecipitation and immunofluorescence co-staining from of DN patients,STZ-DN mouse model and primary TECs.2.5 The expression and distribution of PRDX1 were observed in DN patients and STZ-DN renal tissues;PRDX1-si RNA and PRDX1 overexpression plasmid were transfected in vitro to observe the effects on the expression of cell cycle-related proteins(CDK6,cyclin D1,p16).The expression of GRP78 and the co-localization relationship between GRP78 and cell senescence and apoptosis-related markers were detected in renal tissue of DN patients;GRP78-si RNA and GRP78 overexpression plasmid were transfected in vitro to observe the effect on the expression of apoptosis-related markers.2.6 DcR2 and PRDX1-related plasmids were co-transfected in vitro to observe the effects on TECs senescence and SASP.DcR2 and GRP78-related plasmids were co-transfected in vitro to observe the effects on the expression of apoptosis-related markers(caspase-3,7)in TECs.2.7 After regulating the expression of DcR2 in vitro and in vivo,the levels of PRDX1 m RNA,protein and phosphorylation,the peroxidase activity of PRDX1 and ROS levels were measured;the expression levels of Akt and p-Akt were detected.ResultsPart 1 DcR2-PRDX1 mediates TECs senescence and SASP,leading to accelerate renal interstitial fibrosis in DN1.1 DcR2 promotes renal interstitial fibrosis in DN1.1.1 DcR2 was specifically expressed in tubular cells,and the levels of DcR2 gradually increased with the progression of DN,and it was positively correlated with the scores of renal interstitial fibrosis.Furthermore,DcR2 was co-localized with fibrotic markers.1.1.2 After up-regulation of DcR2 expression in vivo,the area of renal interstitial fibrosis was increased in STZ-DN mice and the expression levels of fibrotic markers were elevated;down-regulation of DcR2 expression effectively inhibited renal interstitial fibrosis,indicating that DcR2 regulated the renal interstitial fibrosis in DN.1.2 DcR2 mediates TECs senescence and SASP in DNAfter overexpression of DcR2 in vitro and in vivo,the expression levels of TEC senescent markers and SASP were increased;after transfection of DcR2-si RNA,the opposite results were shown,indicating that DcR2 mediated TECs senescence and SASP,thereby aggravating renal interstitial fibrosis in DN.1.3 DcR2 mediates TECs senescence independent of TRAIL/DR5 signalingDcR2 was not co-localize with TRAIL and DR5 in renal tissue of DN patients.In STZ-DN,DcR2 did not bind with TRAIL and DR5.The results indicated that DcR2 mediated TECs senescence independent of TRAIL/DR5 signaling.1.4 Screening and validation of DcR2-interacting proteins-PRDX1Co-immunoprecipitation combined with quantitative proteomics screening,bioinformatics analysis DcR2-interaction proteins.It was confirmed that DcR2 interacted with PRDX1 in renal tissue of DN patients,STZ-DN and cells.1.5 The role of PRDX1 in TECs senescence and SASPPRDX1 was co-localized with cell cycle-related proteins.In vitro,regulation the expression of PRDX1 affected the levels of cell cycle-related proteins,indicating that PRDX1 mediated TECs senescence by regulating cell cycle.1.6 Molecular mechanism of DcR2-PRDX1 mediates TECs senescence and SASP in DN1.6.1 DcR2 inhibits the peroxidase activity of PRDX1,which promotes TECs senescence and SASPAfter over-expression of DcR2 in vitro,the peroxidase activity of PRDX1 was decreased and cellular ROS level was increased,which promoted TECs senescence;while low-expression of DcR2 showed the opposite results.1.6.2 DcR2 mediates TECs senescence and SASP by regulating the phosphorylation level of PRDX1.The expression of DcR2 and PRDX1 mediated TECs senescence and SASP by affecting cell cycle-related proteins in vitro.Up/down-expression of DcR2 did not affect the levels of PRDX1 m RNA and protein,but affected the phosphorylation level of PRDX1,indicating that DcR2 affected the expression of PRDX1 at the post-translational level.Part Ⅱ DcR2-GRP78 mediates the apoptosis-resistant phenotype of senescent TECs and accelerates renal interstitial fibrosis in DN2.1 DcR2 mediates the apoptosis-resistant phenotype of senescent TECsAfter overexpression of DcR2 in vitro and in vivo,the expression of senescent markers and anti-apoptosis-related indicators in TECs were increased,while the expression of pro-apoptosis-related indicators were decreased.After transfection of DcR2-si RNA,the opposite results were shown.The results indicated that DcR2 mediated the apoptosis-resistant phenotype of TECs in DN.2.2 Screening and validation of DcR2-interacting proteins-GRP78Co-immunoprecipitation combined with quantitative proteomics screening,bioinformatics analysis DcR2-interaction proteins.It was confirmed that DcR2 interacted with GRP78 in renal tissue of DN patients,STZ-DN mice and cells.2.3 The role of GRP78 in the apoptotic resistance of senescent TECsGRP78 was co-localized with senescent markers,and also high expression of anti-apoptotic markers and low expression of pro-apoptotic markers.After inhibiting the expression of GRP78 in vitro,the expression level of apoptotic markers and the apoptotic rate were increased,indicating that GRP78 regulated the apoptotic resistance of senescent TECs.2.4 Molecular mechanism of DcR2-GRP78 mediates the apoptosis-resistant phenotype of senescent TECs2.4.1 DcR2 mediates the apoptosis-resistant phenotype of senescent TECs by enhancing the anti-apoptotic activity of GRP78The expression of DcR2 and GRP78 affected the expression of apoptosis-related markers such as caspase-7 in vitro.The expression of DcR2 affected the binding between GRP78 and caspase-7 and the phosphorylation level of Akt.The above suggested that DcR2 enhanced the binding between GRP78 and caspase-7 through p-Akt,thereby leading to apoptotic resistance in senescent TECs.Conclusions1.This study demonstrates that the interaction of DcR2 with PRDX1 mediates TECs senescence and SASP from three dimensions of DN patients,STZ-DN mouse model,and high glucose-induced primary renal tubular cell senescence model,thereby accelerating renal interstitial fibrosis in DN.The mechanism is that DcR2 promotes the phosphorylation of PRDX1 and regulates the expression of cell cycle-related proteins,which ultimately leads to cell cycle arrest,cellular senescence and SASP secretion.2.DcR2 mediates the apoptosis-resistant phenotype of senescent TECs by interacting with GRP78,thereby accelerating renal interstitial fibrosis in DN.Mechanistic studies have shown that DcR2 enhances the anti-apoptotic activity of GRP78,disrupts the balance of anti-apoptotic and pro-apoptotic protein expression,and ultimately leads to apoptotic resistance,survival accumulation,sustained secretion of SASP and renal interstitial fibrosis.