Sos2抑制NFATc1介导的糖尿病肾病足细胞损伤的机制研究
|
摘要
目的:观察高糖刺激对体外培养的小鼠肾足细胞鸟苷酸交换因子Sos2(Son of Sevenless homolog2)表达的影响,并初步探讨Sos2在高糖诱导足细胞损伤中的作用及其可能的分子机制。方法:通过免疫荧光染色及激光共聚焦显微镜观察Sos2在糖尿病肾病患者足细胞中的表达;体外培养小鼠永生化足细胞,以高糖(30 mmol/L葡萄糖)刺激足细胞48 h,采用RT-PCR、Western blot和免疫荧光检测高糖刺激下足细胞Sos2的mRNA及蛋白表达;采用Western bolt实验、免疫荧光及划痕实验检测Sos2过表达及沉默后podocin的表达、足细胞的活动性及NFATc1的入核情况;并采用RT-PCR检测NFATc1下游目的基因转录情况。结果:Sos2在糖尿病肾病患者的足细胞及高糖刺激体外培养的足细胞中表达显著降低(P <0. 05);沉默Sos2后,足细胞标志蛋白podocin表达显著降低,足细胞活动性增加,NFATc1入核增加,NFATc1下游目的基因转录增加(P <0. 05);与之相反,过表达Sos2组podocin表达显著增高,足细胞的活动性降低,NFATc1的入核减少,NFATc1下游目的基因转录降低(P <0. 05)。结论:Sos2可能通过抑制NFATc1入核而减轻糖尿病肾病足细胞损伤。
AIM: To observe the effect of high glucose( HG) stimulation on the expression of guanine nucleotide exchange factor Sos2( Son of Sevenless homolog 2) in mouse podocytes,and to explore the role of Sos2 in HG-induced podocyte damage and its possible molecular mechanisms. METHODS: The expression of Sos2 in the podocytes of diabetic nephropathy patients was observed by immunofluorescence staining and laser confocal microscopy. In vitro,the Sos2 expression at mRNA and protein levels in immortalized podocytes with HG( 30 mmol/L glucose) stimulation for 48 h was determined by the methods of RT-PCR,Western blot and immunofluorescence. Using Western blot,immunofluorescence and wound-healing assay,the expression of podocin,the translocation of NFATc1 into the nucleus and the podocyte migration with or without Sos2 silencing or overexpression were analyzed. The expression of downstream target genes for NFATc1 was detected by RT-PCR. RESULTS: The expression of Sos2 was significantly decreased in the podocytes of diabetic nephropathy patients and in vitro cultured podocytes with HG stimulation( P < 0. 05). When Sos2 was silenced,the expression of podocin was significantly decreased,the migration ability of podocytes was increased,and the translocation of NFATc1 into the nucleus was increased( P < 0. 05). In contrast,after overexpression of Sos2 in the podocytes with HG stimulation,the podocin expression level was obviously higher,and the podocyte migration ability and the translocation of NFATc1 into the nucleus were decreased( P < 0. 05). CONCLUSION: Sos2 may attenuate the diabetic nephropathy-induced podocyte injury by inhibiting NFATc1.
AIM: To observe the effect of high glucose( HG) stimulation on the expression of guanine nucleotide exchange factor Sos2( Son of Sevenless homolog 2) in mouse podocytes,and to explore the role of Sos2 in HG-induced podocyte damage and its possible molecular mechanisms. METHODS: The expression of Sos2 in the podocytes of diabetic nephropathy patients was observed by immunofluorescence staining and laser confocal microscopy. In vitro,the Sos2 expression at mRNA and protein levels in immortalized podocytes with HG( 30 mmol/L glucose) stimulation for 48 h was determined by the methods of RT-PCR,Western blot and immunofluorescence. Using Western blot,immunofluorescence and wound-healing assay,the expression of podocin,the translocation of NFATc1 into the nucleus and the podocyte migration with or without Sos2 silencing or overexpression were analyzed. The expression of downstream target genes for NFATc1 was detected by RT-PCR. RESULTS: The expression of Sos2 was significantly decreased in the podocytes of diabetic nephropathy patients and in vitro cultured podocytes with HG stimulation( P < 0. 05). When Sos2 was silenced,the expression of podocin was significantly decreased,the migration ability of podocytes was increased,and the translocation of NFATc1 into the nucleus was increased( P < 0. 05). In contrast,after overexpression of Sos2 in the podocytes with HG stimulation,the podocin expression level was obviously higher,and the podocyte migration ability and the translocation of NFATc1 into the nucleus were decreased( P < 0. 05). CONCLUSION: Sos2 may attenuate the diabetic nephropathy-induced podocyte injury by inhibiting NFATc1.
引文
[1]Yang W,Lu J,Weng J,et al.Prevalence of diabetes among men and women in China[J].N Engl J Med,2010,362(12):1090-1101.
[2]谈晓凡,陈源汉,俞春萍,等.晚期糖基化终产物通过m TORC1/u PAR途径促进足细胞移动[J].中国病理生理杂志,2014,30(12):2232-2237.
[3]Rask-Madsen C,King GL.Diabetes:Podocytes lose their footing[J].Nature,2010,468(7320):42-44.
[4]Reidy K,Kang HM,Hostetter T,et al.Molecular mechanisms of diabetic kidney disease[J].J Clin Invest,2014,124(6):2333-2340.
[5]Wang Y,Jarad G,Tripathi P,et al.Activation of NFATsignaling in podocytes causes glomerulosclerosis[J].JAm Soc Nephrol,2010,21(10):1657-1666.
[6]Nijenhuis T,Sloan AJ,Hoenderop JG,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.
[7]陈恩平,杜丽根,邬银伟,等.高糖通过下调PGC-1α激活NFAT并促进足细胞凋亡[J].中国病理生理杂志,2017,33(4):620-626.
[8]Zhang B,Shi W,Ma J,et al.The calcineurin-NFATpathway allows for urokinase receptor-mediated beta3 integrin signaling to cause podocyte injury[J].J Mol Med(Berl),2012,90(12):1407-1420.
[9]Müller MR,Rao A.NFAT,immunity and cancer:a transcription factor comes of age[J].Nat Rev Immunol,2010,10(9):645-656.
[10]Li R,Zhang L,Shi W,et al.NFAT2 mediates high glucose-induced glomerular podocyte apoptosis through increased bax expression[J].Exp Cell Res,2013,319(7):992-1000.
[11]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.
[12]Zhang B,Xie S,Shi W,et al.Amiloride off-target effect inhibits podocyte urokinase receptor expression and reduces proteinuria[J].Nephrol Dial Transplant,2012,27(5):1746-1755.
[13]Bonfini L,Karlovich CA,Dasgupta C,et al.The Son of sevenless gene product:a putative activator of Ras[J].Science,1992,255(5044):603-606.
[14]Guittard G,Kortum RL,Balagopalan L,et al.Absence of both Sos-1 and Sos-2 in peripheral CD4+T cells leads to PI3K pathway activation and defects in migration[J].Eur J Immunol,2015,45(8):2389-2395.
[15]Li M,Li Y,Weeks O,et al.SOS2 and ACP1 loci identified through large-scale exome chip analysis regulate kidney development and function[J].J Am Soc Nephrol,2017,28(3):981-994.
[16]Levin A,Tonelli M,Bonventre J,et al.Global kidney health 2017 and beyond:a roadmap for closing gaps in care,research,and policy[J].Lancet,2017,390(10105):1888-1917.
[17]Reiser J,Altintas MM.Podocytes[J].F1000Res,2016,5:114.
[18]Nimnual A,Bar-Sagi D.The two hats of SOS[J].Sci STKE,2002,2002(145):pe36.
[19]Uhlén M,Fagerberg L,Hallstr9m BM,et al.Proteomics.Tissue-based map of the human proteome[J].Science,2015,347(6220):1260419.
[20]Roselli S,Heidet L,Sich M,et al.Early glomerular filtration defect and severe renal disease in podocin-deficient mice[J].Mol Cell Biol,2004,24(2):550-560.
[21]Cellesi F,Li M,Rastaldi MP.Podocyte injury and repair mechanisms[J].Curr Opin Nephrol Hypertens,2015,24(3):239-244.
[22]Reiser J,Oh J,Shirato I,et al.Podocyte migration during nephrotic syndrome requires a coordinated interplay between cathepsin L and alpha3 integrin[J].J Biol Chem,2004,279(33):34827-34832.
[23]Zhang B,Shi W.Is the antiproteinuric effect of cyclosporine a independent of its immunosuppressive function in Tcells[J].Int J Nephrol,2012,2012:809456.
[24]Wang L,Chang JH,Paik SY,et al.Calcineurin(CN)activation promotes apoptosis of glomerular podocytes both in vitro and in vivo[J].Mol Endocrinol,2011,25(8):1376-1386.
[25]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.
[2]谈晓凡,陈源汉,俞春萍,等.晚期糖基化终产物通过m TORC1/u PAR途径促进足细胞移动[J].中国病理生理杂志,2014,30(12):2232-2237.
[3]Rask-Madsen C,King GL.Diabetes:Podocytes lose their footing[J].Nature,2010,468(7320):42-44.
[4]Reidy K,Kang HM,Hostetter T,et al.Molecular mechanisms of diabetic kidney disease[J].J Clin Invest,2014,124(6):2333-2340.
[5]Wang Y,Jarad G,Tripathi P,et al.Activation of NFATsignaling in podocytes causes glomerulosclerosis[J].JAm Soc Nephrol,2010,21(10):1657-1666.
[6]Nijenhuis T,Sloan AJ,Hoenderop JG,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.
[7]陈恩平,杜丽根,邬银伟,等.高糖通过下调PGC-1α激活NFAT并促进足细胞凋亡[J].中国病理生理杂志,2017,33(4):620-626.
[8]Zhang B,Shi W,Ma J,et al.The calcineurin-NFATpathway allows for urokinase receptor-mediated beta3 integrin signaling to cause podocyte injury[J].J Mol Med(Berl),2012,90(12):1407-1420.
[9]Müller MR,Rao A.NFAT,immunity and cancer:a transcription factor comes of age[J].Nat Rev Immunol,2010,10(9):645-656.
[10]Li R,Zhang L,Shi W,et al.NFAT2 mediates high glucose-induced glomerular podocyte apoptosis through increased bax expression[J].Exp Cell Res,2013,319(7):992-1000.
[11]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.
[12]Zhang B,Xie S,Shi W,et al.Amiloride off-target effect inhibits podocyte urokinase receptor expression and reduces proteinuria[J].Nephrol Dial Transplant,2012,27(5):1746-1755.
[13]Bonfini L,Karlovich CA,Dasgupta C,et al.The Son of sevenless gene product:a putative activator of Ras[J].Science,1992,255(5044):603-606.
[14]Guittard G,Kortum RL,Balagopalan L,et al.Absence of both Sos-1 and Sos-2 in peripheral CD4+T cells leads to PI3K pathway activation and defects in migration[J].Eur J Immunol,2015,45(8):2389-2395.
[15]Li M,Li Y,Weeks O,et al.SOS2 and ACP1 loci identified through large-scale exome chip analysis regulate kidney development and function[J].J Am Soc Nephrol,2017,28(3):981-994.
[16]Levin A,Tonelli M,Bonventre J,et al.Global kidney health 2017 and beyond:a roadmap for closing gaps in care,research,and policy[J].Lancet,2017,390(10105):1888-1917.
[17]Reiser J,Altintas MM.Podocytes[J].F1000Res,2016,5:114.
[18]Nimnual A,Bar-Sagi D.The two hats of SOS[J].Sci STKE,2002,2002(145):pe36.
[19]Uhlén M,Fagerberg L,Hallstr9m BM,et al.Proteomics.Tissue-based map of the human proteome[J].Science,2015,347(6220):1260419.
[20]Roselli S,Heidet L,Sich M,et al.Early glomerular filtration defect and severe renal disease in podocin-deficient mice[J].Mol Cell Biol,2004,24(2):550-560.
[21]Cellesi F,Li M,Rastaldi MP.Podocyte injury and repair mechanisms[J].Curr Opin Nephrol Hypertens,2015,24(3):239-244.
[22]Reiser J,Oh J,Shirato I,et al.Podocyte migration during nephrotic syndrome requires a coordinated interplay between cathepsin L and alpha3 integrin[J].J Biol Chem,2004,279(33):34827-34832.
[23]Zhang B,Shi W.Is the antiproteinuric effect of cyclosporine a independent of its immunosuppressive function in Tcells[J].Int J Nephrol,2012,2012:809456.
[24]Wang L,Chang JH,Paik SY,et al.Calcineurin(CN)activation promotes apoptosis of glomerular podocytes both in vitro and in vivo[J].Mol Endocrinol,2011,25(8):1376-1386.
[25]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.