阿拉善双峰驼ENaCα亚基基因克隆及激素对肾皮质细胞中该基因表达的影响
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摘要
为探究阿拉善双峰驼上皮细胞钠通道的作用特点,试验采用深圳华大公司提供的ENaCα亚基基因片段序列设计PCR引物,提取阿拉善双峰驼肾皮质总RNA,反转录合成cDNA,PCR扩增获得双峰驼ENaCα亚基基因,构建克隆质粒pMD19-T-ENaCα,对重组质粒进行双酶切和测序分析;采集双峰驼肾皮质部分,细胞体外培养到P3代后,加入不同浓度的醛固酮(aldosterone)和血管紧张素Ⅱ(angiotensinⅡ),实时荧光定量PCR技术检测细胞中ENaCα亚基基因的表达情况,以确定ENaCα对两种激素浓度变化的敏感性。结果显示,PCR扩增得到2.2kb的特异性条带,SmaⅠ和PstⅠ双酶切得到2.2和2.7kb的条带,表明重组质粒pMD19-T-ENaCα构建成功。实时荧光定量PCR结果显示,1×10~(-9) mol/L醛固酮和血管紧张素Ⅱ处理的双峰驼肾皮质细胞的ENaCα表达量显著高于对照组(P<0.05);1×10~(-6)、1×10~(-7)和1×10~(-8) mol/L醛固酮和血管紧张素Ⅱ表达量显著低于对照组(P<0.05)。本研究结果为揭示双峰驼水盐代谢的生理机制提供了试验依据。
This study was aimed to explore the effect of sodium channel in epithelial cells of Alax League Bactrian camel.PCR primers were designed using the ENaCαsubunit gene fragment which was provided by Shenzhen Huada company.Total RNA was extracted from the renal cortex of Alax League Bactrian camel,and cDNA was obtained by reverse transcription synthesis,then the renal cortex ENaCαsubunit gene fragments were amplified by PCR.The cloning plasmid pMD19-T-ENaCαwas constructed,the recombinant plasmid was identified by double enzyme digestion and squencing.The renal cortex of Alax League Bactrian camel was collected,after the cells were cultured in the P3 generation,different concentrations of aldosterone and angiotensinⅡwere added,the expression of ENaCαsubunit gene was detected by Real-time quantitative PCR to determine the sensitivity of ENaCαchanges in two hormone concentrations.The results showed that the ENaCαsubunit gene was successfully cloned with the length of 2.2 kb,double digestion with Sma Ⅰand PstⅠ gave 2.2 and 2.7 kb bands,the recombinant plasmid pMD19-T-ENaCαwas successfully constructed.Real-time quantitative PCR results confirmed that the expressionlevels of ENaCαtreated with 1×10~(-9) mol/L aldosterone and angiotensin Ⅱ were significantly higher than that of control group(P<0.05);The expression levels of ENaCαtreated with 1×10~(-6),1×10~(-7) and 1×10~(-8) mol/L aldosterone and angiotensin Ⅱ were significantly lower than that of control group(P<0.05).The results provided the experimental evidence for revealing the physiological mechanism of water and salt metabolism in Bactrian camel.
This study was aimed to explore the effect of sodium channel in epithelial cells of Alax League Bactrian camel.PCR primers were designed using the ENaCαsubunit gene fragment which was provided by Shenzhen Huada company.Total RNA was extracted from the renal cortex of Alax League Bactrian camel,and cDNA was obtained by reverse transcription synthesis,then the renal cortex ENaCαsubunit gene fragments were amplified by PCR.The cloning plasmid pMD19-T-ENaCαwas constructed,the recombinant plasmid was identified by double enzyme digestion and squencing.The renal cortex of Alax League Bactrian camel was collected,after the cells were cultured in the P3 generation,different concentrations of aldosterone and angiotensinⅡwere added,the expression of ENaCαsubunit gene was detected by Real-time quantitative PCR to determine the sensitivity of ENaCαchanges in two hormone concentrations.The results showed that the ENaCαsubunit gene was successfully cloned with the length of 2.2 kb,double digestion with Sma Ⅰand PstⅠ gave 2.2 and 2.7 kb bands,the recombinant plasmid pMD19-T-ENaCαwas successfully constructed.Real-time quantitative PCR results confirmed that the expressionlevels of ENaCαtreated with 1×10~(-9) mol/L aldosterone and angiotensin Ⅱ were significantly higher than that of control group(P<0.05);The expression levels of ENaCαtreated with 1×10~(-6),1×10~(-7) and 1×10~(-8) mol/L aldosterone and angiotensin Ⅱ were significantly lower than that of control group(P<0.05).The results provided the experimental evidence for revealing the physiological mechanism of water and salt metabolism in Bactrian camel.
引文
[1]柏丽,冯登侦.中国双峰驼血液生理生化特性研究[J].畜牧与兽医,2015,14(1):103-105.BAI L,FENG D Z.Study on blood physiology and biochemical characteristics of Alax League Bactrian camel[J].Animal Husbandry and Veterinary Medicine,2015,14(1):103-105.(in Chinese)
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[15]KELLENBERGER S,GAUTSCHI I,ROSSIER B C,et al.Mutations causing Liddle syndrome reduce sodium-dependent downregulation of the epithelial sodium channel in the Xenopus oocyte expression system[J].Journal of Clinical Investigation,1998,101(12):2741-2750.
[16]HANUKOGLU A.TypeⅠpseudohypoaldosteronism includes two clinically and genetically distinct entities with either renal or multiple target organ defects[J].Journal of Clinical Endocrinology and Metabolism,1991,73(5):936-944.
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[18]SHERIDAN M B,FONG P,GROMAN J D,et al.Mutations in the beta-subunit of the epithelial Na+channel in patients with a cystic fibrosis-like syndrome[J].Human Molecular Genetics,2005,14(22):3493-3498.
[19]CLARK K L,HUGHES S A,BULSARA P,et al.Pharmacological characterization of a novel ENaCαsiRNA(GSK2225745)with potential for the treatment of cystic fibrosis[J].Molecular Therapy Nucleic Acids,2013,2(1):e65.
[20]AMBROSIUS W T,BLOEM L J,ZHOU L,et al.Genetic variants in the epithelial sodium channel in relation to aldosterone and potassium excretion and risk for hypertension[J].Hypertension,1999,34(4Pt1):631-637.
[21]陈秋生,刘仪.双峰驼肾脏产生高浓缩尿的形态学基础[J].中国农业科学,2002,35(3):331-334.CHEN Q S,LIU Y.Morphological investigation on high concentrated urine production in kidney of Bactrian camel[J].Scientia Agricultura Sinica,2002,35(3):331-334.(in Chinese)
[22]OSHAUGHNESSY K M.The genetics of essential hypertension[J].Human Heredity,2015,7(1):120.
[23]TANIRA M O M,BALUSHI K A A,Genetic variations related to hypertension:A review[J].Journal of Human Hypertension,2005,19(1):7.
[24]MCDONALD F J,PRICE M P,SNYDER P M,et al.Cloning and expression of the beta-and gamma-subunits of the human epithelial sodium channel[J].The American Journal of Physiology,1995,268(5Pt1):C1157-C1163.
[25]何玉琴,吴安国,刘志学,等.双峰驼血清中抗利尿素和醛固酮含量的动态研究[J].畜牧兽医学报,1999,30(2):117-120.HE Y Q,WU A G,LIU Z X,et al.The dynamic study of antidiuretic hormone(ADH)and aldosterone(AL-DO)contients in blood serum under the different conditions of water drinking on Bactrian camels[J].Acta Veterinaria et Zootechnica Sinica,1999,30(2):117-120.(in Chinese)
[26]LI X C,COOK J L,RUBERA I,et al.Intrarenal transfer of an intracellular fluorescent fusion of angiotensinⅡselectively in proximal tubules increases blood pressure in rats and mice[J].American Journal of Physiology-Renal Physiology,2011,300(5):F1076-F1088.
[27]NOWICKI T S,ZHAO H,DARZYNKIEWICZ Z,et al.Downregulation of uPAR inhibits migration,invasion,proliferation,FAK/PI3K/Akt signaling and induces senescence in papillary thyroid carcinoma cells[J].Cell Cycle,2011,10(1):100-107.
[28]DOOLEY R,HARVEY B J,THOMAS W.Non-genomic actions of aldosterone:From receptors and signals to membrane targets[J].Molecular and Cellular Endocrinology,2012,350(2):223-234.
[29]陈学伟.醛固酮快速调控上皮钠通道作用的研究[D].北京:中国人民解放军军事医学科学院,2012.CHEN X W.Rapid regulation of aldosterone on epithelia sodium channel[D].Beijing:Academy of Military Medical Sciences,2012.(in Chinese)
[30]孙红周,卞钊.血管紧张素Ⅱ2型受体对血管和肾脏功能的影响[J].医学综述,2007,13(5):382-384.SUN H Z,BIAN Z.Effects of anglotensinⅡtype 2reception on vascular and renal action[J].Medical Recapitulate,2007,13(5):382-384.(in Chinese)
[2]CANESSA C M,HORISBERGER J D,TOSSIER B C.Epithelial sodium channel related to proteins involved in neurodegeration[J].Nature,1993,361(6411):467-470.
[3]HORISBERGE J D,CANNESSA C,ROSSIER B C.The epithelial sodium channel:Recent developments[J].Cellular Physiology and Biochemistry,2008,3(5-6):283-294.
[4]BARBRY P,HOMFMAN P.Molecular biology of Na+absorption[J].The American Journal of Physiology,1997,273(3Pt1):G571-G585.
[5]ESCOUBET B,COUREAU C,BONVALET J P,et al.Noncoordinate regulation of epithelial Na+channel and Na pump subunit mRNA in kidney and colon by aldosterone[J].The American Journal of Physiology,1997,272(5Pt1):C1482-C1491.
[6]CHOW Y H,WANG Y,PLUMB J,et al.Hormonal regulation and genomic organization of the human amiloride-sensitive epithelial sodium channel alpha subunit gene[J].Pediatric Research,1999,46(2):208-214.
[7]MICK V E,ITANI O A,LOFTUS R W,et al.The alpha-subunit of the epithelial sodium channel is an aldosterone-induced transcript in mammalian collecting ducts,and this transcriptional response is mediated via distinct cis-elements in the 5′-flanking region of the gene[J].Molecular Endocrinology,2001,15(4):575-588.
[8]TAKAHASHI H,YOSHIKA M,KOMIYAMA Y,et al.The central mechanism underlying hypertension:A review of the roles of sodium ions,epithelial sodium channels,the renin-angiotensin-aldosterone system,oxidative stress and endogenous digitalis in the brain[J].Hypertension Research,2011,34(11):1147-1160.
[9]ZAIKA O,MAMENKO M,STARUSCHENKO A,et al.Direct activation of ENaC by AngiotensinⅡ:Recent advances and new insights[J].Current Hypertension Reports,2013,15(1):17-24.
[10]LOFFING J,KAISSLING B.Sodium and calcium transport pathways along the mammalian distal nephron:From rabbit to human[J].American Journal of Physiology-Renal Physiology,2003,284(4):F628-F643.
[11]LOFFING J,PIETRI L,AREGGER F,et al.Differential subcellular localization of ENaC subunits in mouse kidney in response to high-and low-Na diets[J].American Journal of Physiology-Renal Physiology,2000,279(2):F252-F258.
[12]MASILAMANI S,KIM G H,MITCHELL C,et al.Aldosterone-mediated regulation of ENaCα,β,andγsubunit proteins in rat kidney[J].Journal of Clinical Investigation,1999,104(7):19-25.
[13]EPPLE H J,AMASHEH S,MANKERTZ J,et al.Early aldosterone effect in distal colon by transcriptional regulation of ENaC subunits[J].American Journal of Physiology Gastrointestinal and Liver Physiology,2014,278(5):718-724.
[14]NICCO C,WITTNER M,DISTEGANO A,et al.Chronic exposure to vasopressin upregulates ENaCand sodium transport in the rat renal collecting duct and lung[J].Hypertension,2001,38(5):1143-1149.
[15]KELLENBERGER S,GAUTSCHI I,ROSSIER B C,et al.Mutations causing Liddle syndrome reduce sodium-dependent downregulation of the epithelial sodium channel in the Xenopus oocyte expression system[J].Journal of Clinical Investigation,1998,101(12):2741-2750.
[16]HANUKOGLU A.TypeⅠpseudohypoaldosteronism includes two clinically and genetically distinct entities with either renal or multiple target organ defects[J].Journal of Clinical Endocrinology and Metabolism,1991,73(5):936-944.
[17]BONNY O,ROSSIER B C.Disturbances of Na/K balance:Pseudohypoaldosteronism revisited[J].Journal of the American Society of Nephrology,2002,13(9):2399-2414.
[18]SHERIDAN M B,FONG P,GROMAN J D,et al.Mutations in the beta-subunit of the epithelial Na+channel in patients with a cystic fibrosis-like syndrome[J].Human Molecular Genetics,2005,14(22):3493-3498.
[19]CLARK K L,HUGHES S A,BULSARA P,et al.Pharmacological characterization of a novel ENaCαsiRNA(GSK2225745)with potential for the treatment of cystic fibrosis[J].Molecular Therapy Nucleic Acids,2013,2(1):e65.
[20]AMBROSIUS W T,BLOEM L J,ZHOU L,et al.Genetic variants in the epithelial sodium channel in relation to aldosterone and potassium excretion and risk for hypertension[J].Hypertension,1999,34(4Pt1):631-637.
[21]陈秋生,刘仪.双峰驼肾脏产生高浓缩尿的形态学基础[J].中国农业科学,2002,35(3):331-334.CHEN Q S,LIU Y.Morphological investigation on high concentrated urine production in kidney of Bactrian camel[J].Scientia Agricultura Sinica,2002,35(3):331-334.(in Chinese)
[22]OSHAUGHNESSY K M.The genetics of essential hypertension[J].Human Heredity,2015,7(1):120.
[23]TANIRA M O M,BALUSHI K A A,Genetic variations related to hypertension:A review[J].Journal of Human Hypertension,2005,19(1):7.
[24]MCDONALD F J,PRICE M P,SNYDER P M,et al.Cloning and expression of the beta-and gamma-subunits of the human epithelial sodium channel[J].The American Journal of Physiology,1995,268(5Pt1):C1157-C1163.
[25]何玉琴,吴安国,刘志学,等.双峰驼血清中抗利尿素和醛固酮含量的动态研究[J].畜牧兽医学报,1999,30(2):117-120.HE Y Q,WU A G,LIU Z X,et al.The dynamic study of antidiuretic hormone(ADH)and aldosterone(AL-DO)contients in blood serum under the different conditions of water drinking on Bactrian camels[J].Acta Veterinaria et Zootechnica Sinica,1999,30(2):117-120.(in Chinese)
[26]LI X C,COOK J L,RUBERA I,et al.Intrarenal transfer of an intracellular fluorescent fusion of angiotensinⅡselectively in proximal tubules increases blood pressure in rats and mice[J].American Journal of Physiology-Renal Physiology,2011,300(5):F1076-F1088.
[27]NOWICKI T S,ZHAO H,DARZYNKIEWICZ Z,et al.Downregulation of uPAR inhibits migration,invasion,proliferation,FAK/PI3K/Akt signaling and induces senescence in papillary thyroid carcinoma cells[J].Cell Cycle,2011,10(1):100-107.
[28]DOOLEY R,HARVEY B J,THOMAS W.Non-genomic actions of aldosterone:From receptors and signals to membrane targets[J].Molecular and Cellular Endocrinology,2012,350(2):223-234.
[29]陈学伟.醛固酮快速调控上皮钠通道作用的研究[D].北京:中国人民解放军军事医学科学院,2012.CHEN X W.Rapid regulation of aldosterone on epithelia sodium channel[D].Beijing:Academy of Military Medical Sciences,2012.(in Chinese)
[30]孙红周,卞钊.血管紧张素Ⅱ2型受体对血管和肾脏功能的影响[J].医学综述,2007,13(5):382-384.SUN H Z,BIAN Z.Effects of anglotensinⅡtype 2reception on vascular and renal action[J].Medical Recapitulate,2007,13(5):382-384.(in Chinese)