促红细胞生成素在斑马鱼胚胎发育中抑制肾脏细胞凋亡
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摘要
目的:研究促红细胞生成素(Epo)在斑马鱼早期胚胎肾脏细胞发育中的凋亡和坏死方面起到的作用。方法:使用吗啉反义寡核苷酸技术通过胚胎注射使Epo及其受体(EpoR)的表达下调,并用real-time PCR及血红蛋白染色进行基因下调的有效性验证。在胚胎受精48 h观察肾脏形态并进行TUNEL凋亡染色,使用流式细胞术分析Annexin V凋亡染色,并用Western blot进行Akt磷酸化验证。结果:Epo及EpoR基因表达下调的斑马鱼胚胎在受精48 h可见明显肾小球囊样改变,且肾小管颈部缩短消失。TUNEL及Annexin V染色结果提示受精48 h后,Epo及EpoR基因表达下调的斑马鱼胚胎中肾脏凋亡细胞增多,但EpoR基因下调的斑马鱼胚胎内主要表现为晚期凋亡细胞及坏死细胞增多。Western blot实验结果提示Epo及EpoR基因表达下调的斑马鱼胚胎较对照组Akt磷酸化减少(P<0.05)。结论:促红细胞生成素可引起Akt磷酸化。促红细胞生成素通过保护肾脏细胞免于凋亡和坏死而在斑马鱼胚胎肾脏发育中发挥重要作用。
AIM: To analyze zebrafish embryos and to identify erythropoietin( Epo) as an active renal antiapoptotic factor in an Epo receptor( EpoR)-dependent manner. METHODS: For transient knockdown of Epo and EpoR in renal Tg( wt1b: EGFP) zebrafish reporter lines,the morpholino antisense oligonucleotide technique was used. Morphant zebrafish embryos were phenotypically analyzed using fluorescence microscopy. Apoptosis was determined by TUNEL assay and Annexin V staining. Western blot was used to identify Akt phosphorylation in Epo and EpoR knockdown zebrafish. RESULTS: Epo and EpoR zebrafish morphants exhibited pathophysiological changes within the pronephros,adversely affecting pronephric structure. Zebrafish embryos upon silencing of Epoa and EpoR showed a significant increase in the apoptosis within the zebrafish pronephros,consequently leading to renal pathophysiological effects. Decreased p-Akt was identified in Epo and EpoR knockdown zebrafish embryos. CONCLUSION: Epo is an essential regulator of renal development and function by interacting with its receptor EpoR and thereby repressing apoptosis,mechanistically by promoting Akt phosphorylation.
AIM: To analyze zebrafish embryos and to identify erythropoietin( Epo) as an active renal antiapoptotic factor in an Epo receptor( EpoR)-dependent manner. METHODS: For transient knockdown of Epo and EpoR in renal Tg( wt1b: EGFP) zebrafish reporter lines,the morpholino antisense oligonucleotide technique was used. Morphant zebrafish embryos were phenotypically analyzed using fluorescence microscopy. Apoptosis was determined by TUNEL assay and Annexin V staining. Western blot was used to identify Akt phosphorylation in Epo and EpoR knockdown zebrafish. RESULTS: Epo and EpoR zebrafish morphants exhibited pathophysiological changes within the pronephros,adversely affecting pronephric structure. Zebrafish embryos upon silencing of Epoa and EpoR showed a significant increase in the apoptosis within the zebrafish pronephros,consequently leading to renal pathophysiological effects. Decreased p-Akt was identified in Epo and EpoR knockdown zebrafish embryos. CONCLUSION: Epo is an essential regulator of renal development and function by interacting with its receptor EpoR and thereby repressing apoptosis,mechanistically by promoting Akt phosphorylation.
引文
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[11]Wingert RA,Davidson AJ.The zebrafish pronephros:a model to study nephron segmentation[J].Kidney Int,2008,73(10):1120-1127.
[12]Chu CY,Cheng CH,Chen GD,et al.The zebrafish erythropoietin:functional identification and biochemical characterization[J].FEBS Lett,2007,581(22):4265-4271.
[13]Paffett-Lugassy N,Hsia N,Fraenke L PG,et al.Functional conservation of erythropoietin signaling in zebrafish[J].Blood,2007,110(7):2718-2726.
[14]Sharma KR,Heckler K,Stoll SJ,et al.ELMO1 protects renal structure and ultrafiltration in kidney development and under diabetic conditions[J].Sci Rep,2016,6:37172.
[15]Jelkmann W.Erythropoietin:back to basics[J].Blood,2010,115(21):4151-4152.
[16]张新金,马业新,文渊,等.促红细胞生成素通过PI3-K/Akt信号通路抑制血管紧张素Ⅱ诱导的新生大鼠心脏成纤维细胞增殖[J].中国病理生理杂志,2009,25(2):293-298.
[17]Ahlqvist E,Van Zuydam NR,Groop LC,et al.The genetics of diabetic complications[J].Nat Rev Nephrol,2015,11(5):277-287.
[2]Jelkmann W.Biosimilar epoetins and other"follow-on"biologics:update on the European experiences[J].Am J Hematol,2010,85(10):771-780.
[3]Garimella PS,Katz R,Patel KV,et al.Association of serum erythropoietin with cardiovascular events,kidney function decline,and mortality:the health aging and body composition study[J].Circ Heart Failure,2016,9(1):e002124.
[4]罗梓垠,陈海娥,宋冬,等.促红细胞生成素通过JNK通路对再灌注损伤肺细胞凋亡的影响[J].中国应用生理学杂志,2016,32(4):382-384.
[5]陈远寿,潘贵书,秦伟,等.促红细胞生成素上调海马p CREB表达和改善脑缺血小鼠认知功能[J].中国病理生理杂志,2011,27(4):722-726.
[6]Echigoya m H,Obikane K,Nakashima T,et al.Glomerular localization of erythropoietin receptor mRNA and protein in neonatal and mature mouse kidney[J].Nephron Exp Nephrol,2005,100(1):e21-e29.
[7]Tong Z,Yang Z,Patel S,et al.Promoter polymorphism of the erythropoietin gene in severe diabetic eye and kidney complications[J].Proc Natl Acad Sci U S A,2008,105(19):6998-7003.
[8]Menne J,Park JK,Shushakova N,et al.The continuous erythropoietin receptor activator affects different pathways of diabetic renal injury[J].J Am Soc Nephrol,2007,18(7):2046-2053.
[9]Yu X,Lin CS,Costantini F,et al.The human erythropoietin receptor gene rescues erythropoiesis and developmental defects in the erythropoietin receptor null mouse[J].Blood,2001,98(2):475-477.
[10]Howe DG,Bradford YM,Conlin T,et al.ZFIN,the Zebrafish Model Organism Database:increased support for mutants and transgenics[J].Nucleic Acids Res,2013,41(Database issue):D854-D860.
[11]Wingert RA,Davidson AJ.The zebrafish pronephros:a model to study nephron segmentation[J].Kidney Int,2008,73(10):1120-1127.
[12]Chu CY,Cheng CH,Chen GD,et al.The zebrafish erythropoietin:functional identification and biochemical characterization[J].FEBS Lett,2007,581(22):4265-4271.
[13]Paffett-Lugassy N,Hsia N,Fraenke L PG,et al.Functional conservation of erythropoietin signaling in zebrafish[J].Blood,2007,110(7):2718-2726.
[14]Sharma KR,Heckler K,Stoll SJ,et al.ELMO1 protects renal structure and ultrafiltration in kidney development and under diabetic conditions[J].Sci Rep,2016,6:37172.
[15]Jelkmann W.Erythropoietin:back to basics[J].Blood,2010,115(21):4151-4152.
[16]张新金,马业新,文渊,等.促红细胞生成素通过PI3-K/Akt信号通路抑制血管紧张素Ⅱ诱导的新生大鼠心脏成纤维细胞增殖[J].中国病理生理杂志,2009,25(2):293-298.
[17]Ahlqvist E,Van Zuydam NR,Groop LC,et al.The genetics of diabetic complications[J].Nat Rev Nephrol,2015,11(5):277-287.