The Mechanism Of Podocyte Injury Mediated By C/EBPβ In The Pathogenesis Of LN Through Pim-1

BackgroundLupus nephritis(LN)is a serious complication of systemic lupus erythematosus(SLE),which can cause renal damage by producing autoantibodies to form circulation and in situ immune complex.The clinical manifestations are nephritis syndrome,nephrotic syndrome,simple hematuria and albuminuria.Some of them progressed to end-stage renal disease(ESRD).Renal biopsy showed inherent cell injury and inflammatory cell infiltration,and then gradually developed into renal fibrosis.Thus it can be seen that autoimmune dysfunction and inflammation play a key role in the pathogenesis of lupus nephritis.Podocyte is an important part of glomerular filtration barrier,which can use its foot process and charge to prevent the filtration of useful components in blood through mechanical barrier and charge barrier.Its damage is related to massive albuminuria,which leads to hypoproteinemia,induce complications such as thrombotic tendency,malnutrition and so on.At the same time,podocytes are terminal differentiated cells,and the non-regeneration after injury makes it necessary to study its injury mechanism.Inflammatory cell infiltration and the secretion of inflammatory factors in kidney can lead to podocyte injury,but the regulatory mechanism of inflammatory response is not clear.Pyroptosis is a kind of programmed cell death.Pattern recognition receptors(PRRs)recognize pathogen-related molecular pattern(PAMP)or injury-related molecular pattern(DAMP)which induces cell assembly to form inflammasome through a series of activation pathways,releases inflammatory cytokines through membrane pores and leads to cell death.It can be seen that it participates in the regulation of inflammatory response.At present,many studies have proved that pyroptosis is related to the pathogenesis of lupus nephritis,but its upstream regulation mechanism is not clear.C/EBPβ,a member of CCAAT enhancer binding protein,shows an elevated level in lupus,but its mechanism is not clear.Pim-1,is a member of the serine/threonine kinase family,which can participate in cell cycle regulation.More importantly,its elevated level in a variety of autoimmune diseases such as rheumatoid arthritis,and proved that its abnormal expression is related to the secretion of inflammatory factors.Recent experiments in lupus nephritis have also shown that Pim-1 can participate in the regulation of pyroptosis by regulating the level of intracellular Ca2+.Therefore,we assume that C/EBPβ can mediate pyroptosis by interacting with pim-1,thus participating in the inflammatory injury of podocytes in lupus nephritis.ObjectiveTo investigate the role and mechanism of C/EBPβ in lupus nephritis,and to further explain the effect of C/EBPβ/pim-1/NLRP3 on podocyte inflammatory injury in lupus nephritis.Methods(1)9-week-old female MRL/lpr(LN group)and MRL/MPJ(Control group)mice were acclimated to the environment for one week(10 weeks old).At the 10,14 and 18 weeks,the kidney tissue and blood were collected,and the expression of C/EBPβ in kidney tissue was detected by Real-time PCR and Western blot,and the levels of serum creatinine and blood urea nitrogen were detected by kit.(2)C/EBPβ silencing lentivirus vector(shC/EBPβ-LV)and empty vector lentivirus(NC-LV)were constructed.After adapting to 9-week-old mice for 1 week,MRL/lpr mice were injected with NC-LV or shC/EBPβ-LV via tail vein(2×108TU),and at the end of 18 weeks,the kidney tissue was collected.The level of C/EBPβ was measured by qPCR.The levels of C/EBPβ,NLRP3,Caspase-1,IL-1β and GSDMD were detected by Western blot.The expression of C/EBPβ was detected by immunohistochemistry.(3)The Pim-1 level after knocking down C/EBPβ was measured by Western blot and immunofluorescence.(4)The mouse podocytes were cultured in vitro.The C/EBPβ silencing lentivirus was constructed,and the interference fragment was transfected into differentiated glomerular podocytes with high efficiency transfection reagent.72 hours after transfection,the expression level of C/EBPβ mRNA was detected by Real-time PCR and the expression of C/EBPβ protein was detected by Western blot to verify the interference efficiency.Podocytes were stimulated with LPS(200ng/ml)for 4 hours and ATP(5mM)for 1 hour.The expression of molecules related with pyroptosis(NLRP3,Caspase-1,IL-1β)was detected by Western blot,and the levels of IL-6 and IL-1β were detected by ELISA.FAM-FLICA Caspase-1 detection kit was used to detect Caspase-1 activity by flow cytometry.(5)C/EBPβ interference fragments(siC/EBPβ-1 and siC/EBPβ-2)were transfected into differentiated glomerular podocytes.The expression of Pim-1 mRNA in differentiated podocytes was detected by Real-time PCR.The binding between C/EBPβ and pim-1 was detected by chromatin co-immunoprecipitation assay and double luciferase reporter assay.(6)The overexpression vector of Pim-1 was constructed and the expression of Pim-1 mRNA was detected by Real-time PCR 72 hours after transfection to verify the transfection effect.After that co-transfection of mouse podocytes with siC/EBPβ vector,the levels of GSDMD,NLRP3,Caspase-1 and IL-1β were detected by Western blot assay after LPS+ATP stimulation,and the levels of IL-6 and IL-1β were detected by ELISA to confirmed that C/EBPβ mediated pyroptosis was mediated by pim-1.Results(1)There was no significant difference in BUN between the Control group and the LN group at the 10 and 14 weeks(P=0.5206 and 0.0712,respectively),but at the 18 weeks,there were significant difference between the two groups(P<0.0001).The level of serum creatinine in the LN group was higher than that in the Control group at the 14 weeks(P<0.05),and the difference was more significant at the 18 weeks(P<0.0001).At the same time,the levels of C/EBPβ protein and mRNA in the LN group were significantly higher than those in the Control group at the 10 weeks(P<0.001),and were more significantly different from those in the Control group at the 14 and 18 weeks(P<0.0001).(2)After knocking down C/EBPβ,the protein expression levels of NLRP3,Caspase-1,IL-1β and GSDMD in kidney of mice decreased significantly(P<0.0001).(3)The expression of Pim-1 in shC/EBPβ group was down-regulated(P<0.0001),and the immunofluorescence intensity was significantly decreased in shC/EBPβ group.(4)After C/EBPβ lentivirus transfection into podocytes,the protein expression of NLRP3,Caspase-1 and IL-1β decreased significantly in C/EBPβ silent group(P<0.0001).ELISA test showed that the levels of IL-6 and IL-1β in C/EBPβ silence group were significantly decreased(P<0.01).At the same time,the activity of Caspase-1 decreased significantly by flow cytometry.(5)The expression of Pim-1 in podocytes transfected with C/EBPβ silencing lentivirus was significantly lower than that in Control group(P<0.001).Compared with IgG group,obvious bands were observed in anti-C/EBPβ group by chromatin co-immunoprecipitation assay,and luciferase activity was significantly increased in C/EBPβ-OE group(P<0.01).(6)After co-transfection of podocytes with Pim-1 overexpression and C/EBPβsilencing lentivirus,compared with the C/EBPβ silencing lentivirus group,the protein expression levels of GSDMD,NLRP3,Caspase-1 and IL-1β were significantly increased(P<0.0001).The levels of IL-1β and IL-6 in the supernatant were also significantly higher than those in the C/EBPβ silencing lentivirus group(P<0.05).ConclusionC/EBPβ is involved in the inflammatory injury of podocytes in lupus nephritis,which may be achieved by combining with pim-1 and mediating pyroptosis.