核因子κB受体活化因子通过TRPC6?NFATc1信号通路介导足细胞损伤
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摘要
目的探究Rank通过TRPC6-NFATc1信号通路介导足细胞损伤。方法 (1)通过荧光定量PCR、Western印迹及免疫荧光染色检测Ionomycin刺激下足细胞Rank表达;(2)足细胞转染Rank-siRNA后,荧光定量PCR及Western印迹检测沉默效率,Western印迹检测足细胞标记蛋白Podocin表达;(3)通过Western印迹及免疫荧光染色检测沉默Podocin后足细胞核NFATc1表达;(4)Western印迹检测沉默Rank后TRPC6的表达,通过免疫共沉淀(Co-IP)检测Rank与TRPC6间关联。结果 (1)Ionomycin刺激下足细胞Rank表达增加;(2)沉默Rank表达后足细胞标记蛋白Podocin表达上调;(3)沉默Rank后足细胞核内NFATc1表达减少;(3)沉默Rank后足细胞TRPC6表达减少,Co-IP显示Rank结合TRPC6,且Ionomycin刺激下结合量表达增多。结论 Rank对足细胞具有损伤作用,其机制为Rank结合TRPC6调控NFATc1,从而介导足细胞损伤。
Objective To explore the role of Rank in TRPC6-NFATc1 axis induced podocyte injury.Methods(1)After podocyte was exposed to ionomycin,real-time quantity PCR,western blotting and immuno-fluorescence staining were used to analyze the expression of Rank;(2)Real-time quantity PCR and western blot-ting were used to analyze the effect of Rank-siRNA,and western blotting was used to detect the expression of Podo-cin in Rank knockdown podocytes;(3)Western blotting and immunofluorescence staining were used to evaluatethe change of nuclear localization of NFATc1 in Rank knockdown podocytes;(5)Co-immunoprecipitation assaywas used to observe the relationship between Rank and TRPC6.Results(1)The mRNA and protein expressionof Rank were markedly increased in injured podocyte after treatment with ionomycin;(2)Podocin protein expres-sion was up-regulated in Rank knockdown podocytes;(3)After knockdown Rank,nuclear localization of NFATc1 was decreased;(4)Co-IP assay demonstrated that Rank binds to TRPC6 and ionomycin enhance the binding ofRank to TRPC6.ConclusionRank mediates podocyte injure by binding to TRPC6,and enhances NFATc1signal-ling modulating podocyte injury.
Objective To explore the role of Rank in TRPC6-NFATc1 axis induced podocyte injury.Methods(1)After podocyte was exposed to ionomycin,real-time quantity PCR,western blotting and immuno-fluorescence staining were used to analyze the expression of Rank;(2)Real-time quantity PCR and western blot-ting were used to analyze the effect of Rank-siRNA,and western blotting was used to detect the expression of Podo-cin in Rank knockdown podocytes;(3)Western blotting and immunofluorescence staining were used to evaluatethe change of nuclear localization of NFATc1 in Rank knockdown podocytes;(5)Co-immunoprecipitation assaywas used to observe the relationship between Rank and TRPC6.Results(1)The mRNA and protein expressionof Rank were markedly increased in injured podocyte after treatment with ionomycin;(2)Podocin protein expres-sion was up-regulated in Rank knockdown podocytes;(3)After knockdown Rank,nuclear localization of NFATc1 was decreased;(4)Co-IP assay demonstrated that Rank binds to TRPC6 and ionomycin enhance the binding ofRank to TRPC6.ConclusionRank mediates podocyte injure by binding to TRPC6,and enhances NFATc1signal-ling modulating podocyte injury.
引文
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[17]TANOS T,SFLOMOS G,ECHEVERRIA P C,et al.Progesterone/RANKL is a major regulatory axis in the human breast[J].Sci Transl Med,2013,5(182):155r-182r.
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[20]PAN M G,XIONG Y,CHEN F.NFAT gene family in inflammation and cancer[J].Curr Mol Med,2013,13(4):543-554.
[21]WANG L,CHANG J H,PAIK S Y,et al.Calcineurin(CN)activation promotes apoptosis of glomerular podocytes both in vitro and in vivo[J].Mol Endocrinol,2011,25(8):1376-1386.
[22]FAUL C,DONNELLY M,MERSCHER-GOMEZ S,et al.The actin cytoskeleton of kidney podocytes is a direct target of the antiproteinuric effect of cyclosporine A[J].Nat Med,2008,14(9):931-938.
[23]NIJENHUIS T,SLOAN A J,HOENDEROP J G,et al.Angiotensin II contributes to podocyte injury by increasing TRPC6 expression via an NFAT-mediated positive feedback signaling pathway[J].Am J Pathol,2011,179(4):1719-1732.
[2]KONG Y Y,YOSHIDA H,SAROSI I,et al.OPGL is a key regulator of osteoclastogenesis,lymphocyte development and lymph-node organogenesis[J].Nature,1999,397(6717):315-323.
[3]SIGL V,JONES L P,PENNINGER J M.RANKL/RANK:from bone loss to the prevention of breast cancer[J].Open Biol,2016,6(11),pii:160230.
[4]LIU S,SHI W,XIAO H,et al.Receptor activator of NF-kappaB and podocytes:towards a function of a novel receptor-ligand pair in the survival response of podocyte injury[J].PLoSOne,2012,7(7):e41331.
[5]CHEN X W,LIU W T,WANG Y X,et al.Cyclopropanyldehydrocostunolide LJ attenuates high glucose-induced podocyte injury by suppressing RANKL/RANK-mediated NF-kappaB and MAPK signaling pathways[J].J Diabetes Complications,2016,30(5):760-769.
[6]ZHANG H,LIANG S,DU Y,et al.Inducible ATF3-NFAT axis aggravates podocyte injury[J].J Mol Med(Berl),2018,96(1):53-64.
[7]ZHANG B,SHI W,MA J,et al.The calcineurin-NFAT pathway allows for urokinase receptor-mediated beta3 integrin signaling to cause podocyte injury[J].J Mol Med(Berl),2012,90(12):1407-1420.
[8]WANG Y,JARAD G,TRIPATHI P,et al.Activation of NFAT34signaling in podocytes causes glomerulosclerosis[J].J Am Soc Nephrol,2010,21(10):1657-1666.
[9]ZHANG L,LI R,SHI W,et al.NFAT2 inhibitor ameliorates diabetic nephropathy and podocyte injury in db/db mice[J].Br J Pharmacol,2013,170(2):426-439.
[10]SCHLONDORFF J,DEL C D,CARRASQUILLO R,et al.TRPC6 mutations associated with focal segmental glomerulosclerosis cause constitutive activation of NFAT-dependent transcription[J].Am J Physiol Cell Physiol,2009,296(3):C558-C569.
[11]CELLESI F,LI M,RASTALDI M P.Podocyte injury and repair mechanisms[J].Curr Opin Nephrol Hypertens,2015,24(3):239-244.
[12]HARALDSSON B.A new era of podocyte-targeted therapy for proteinuric kidney disease[J].N Engl J Med,2013,369(25):2453-2454.
[13]陈洁茹,李辉远,温跃强.钙调磷酸酶结合蛋白1在足细胞线粒体损伤中的表达[J].实用医学杂志,2017,33(23):3864-3867.
[14]WARREN J T,ZOU W,DECKER C E,et al.Correlating RANK ligand/RANK binding kinetics with osteoclast formation and function[J].J Cell Biochem,2015,116(11):2476-2483.
[15]HANADA R,HANADA T,SIGL V,et al.RANKL/RANK-beyond bones[J].J Mol Med(Berl),2011,89(7):647-656.
[16]SHIMAMURA M,NAKAGAMI H,OSAKO M K,et al.OPG/RANKL/RANK axis is a critical inflammatory signaling system29in ischemic brain in mice[J].Proc Natl Acad Sci U S A,2014,111(22):8191-8196.
[17]TANOS T,SFLOMOS G,ECHEVERRIA P C,et al.Progesterone/RANKL is a major regulatory axis in the human breast[J].Sci Transl Med,2013,5(182):155r-182r.
[18]LI R,ZHANG L,SHI W,et al.NFAT2 mediates high glucoseinduced glomerular podocyte apoptosis through increased Bax expression[J].Exp Cell Res,2013,319(7):992-1000.
[19]ZHANG B,SHI W.Is the antiproteinuric effect of cyclosporine a independent of its immunosuppressive function in T cells?[J].Int J Nephrol,2012,2012:809456.
[20]PAN M G,XIONG Y,CHEN F.NFAT gene family in inflammation and cancer[J].Curr Mol Med,2013,13(4):543-554.
[21]WANG L,CHANG J H,PAIK S Y,et al.Calcineurin(CN)activation promotes apoptosis of glomerular podocytes both in vitro and in vivo[J].Mol Endocrinol,2011,25(8):1376-1386.
[22]FAUL C,DONNELLY M,MERSCHER-GOMEZ S,et al.The actin cytoskeleton of kidney podocytes is a direct target of the antiproteinuric effect of cyclosporine A[J].Nat Med,2008,14(9):931-938.
[23]NIJENHUIS T,SLOAN A J,HOENDEROP J G,et al.Angiotensin II contributes to podocyte injury by increasing TRPC6 expression via an NFAT-mediated positive feedback signaling pathway[J].Am J Pathol,2011,179(4):1719-1732.