慢性低氧状态对肾小球足细胞高通量钙激活钾通道表达和功能的影响
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摘要
目的探讨慢性低氧对人肾小球足细胞高通量钙激活钾通道(BK通道)表达和功能的影响,及其在慢性肾脏病进展中的作用。方法分化完全的条件永生性人类足细胞分别置于常氧(21%O_2)和低氧(10%或2%O_2)条件下培养6~48 h,采用实时聚合酶链反应(real-time PCR)和Western blot检测足细胞BK通道α、β3及β4亚基的表达;用电生理膜片钳技术记录全细胞模式下足细胞BK通道功能的变化。结果电生理研究结果显示低氧环境(2%O_2 24 h)中,足细胞BK通道在+80 mV电压刺激下的电流水平由(14.45±2.06)pS下降至(4.78±1.12)pS,差异有统计学意义(P<0.05),且通道激活的时间常数由(4.59±1.67)增加至(25.16±11.04)(P<0.05);分子生物学研究提示在低氧条件(2%O_2 24 h)下足细胞BK通道的β4亚基水平升高,且表现为时间依赖性和剂量依赖性。结论慢性低氧通过上调β4亚基表达抑制BK通道活性。
Objective To explore the influence of chronic hypoxia on expression and function of high-flux Ca~(2+)-activated K~+channel(BK channel)in human glomerular podocytes,and to evaluate its roles in the progression of chronic kidney diseases(CKD).Methods A fully-differentiated and conditionally-immortalized human podocyte cell line was placed into the normoxic(21%O_2)or hypoxic(2%O_2 or 10%O_2)environment in a duration of 6 to 48h.The expressions ofα,β3 andβ4 subunits of BK channels were detected with real-time PCR and Western blot.The changes of BK channel function in podocytes under whole-cell model were examined by the patch clamp technique.Results Exposure of podocytes to 2%O_2 for 24 h caused a significant reduction in currents at+80 mV from(14.45±2.06)pS to(4.78±1.12)pS(P<0.05).Chronic hypoxia also caused a marked increase in the time constant for BK channel activation from(4.59±1.67)to(25.16±11.04)with significant difference(P<0.05).The total expression ofβ4 subunit of BK channel significantly increased in a time-and dose-dependent manner.Conclusions Chronic hypoxia inhibits BK channel current by upregulatingβ4 subunit expression.
Objective To explore the influence of chronic hypoxia on expression and function of high-flux Ca~(2+)-activated K~+channel(BK channel)in human glomerular podocytes,and to evaluate its roles in the progression of chronic kidney diseases(CKD).Methods A fully-differentiated and conditionally-immortalized human podocyte cell line was placed into the normoxic(21%O_2)or hypoxic(2%O_2 or 10%O_2)environment in a duration of 6 to 48h.The expressions ofα,β3 andβ4 subunits of BK channels were detected with real-time PCR and Western blot.The changes of BK channel function in podocytes under whole-cell model were examined by the patch clamp technique.Results Exposure of podocytes to 2%O_2 for 24 h caused a significant reduction in currents at+80 mV from(14.45±2.06)pS to(4.78±1.12)pS(P<0.05).Chronic hypoxia also caused a marked increase in the time constant for BK channel activation from(4.59±1.67)to(25.16±11.04)with significant difference(P<0.05).The total expression ofβ4 subunit of BK channel significantly increased in a time-and dose-dependent manner.Conclusions Chronic hypoxia inhibits BK channel current by upregulatingβ4 subunit expression.
引文
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[19]NAVARRO-ANTOLIN J,LEVITSKY K L,CALDERON E,et al.Decreased expression of maxi-K+channel beta1-subunit and altered vasoregulation in hypoxia[J].Circulation,2005,112(9):1309-1315.
[20]CORNFIELD D N.Developmental regulation of oxygen sensing and ion channels in the pulmonary vasculature[J].Adv Exp Med Biol,2010,661:201-220.
[21]TANG X D,XU R,REYNOLDS M F,et al.Haem can bind to and inhibit mammalian calcium-dependent Slo1 BK channels[J].Nature,2003,425(6957):531-535.
[22]SAHOO N,HOSHI T,HEINEMANN S H.Oxidative modulation of voltage-gated potassium channels[J].Antioxid Redox Signal,2014,21(6):933-952.
[23]KEMP P J,TELEZHKIN V,WILKINSON W J,et al.Enzymelinked oxygen sensing by potassium channels[J].Ann N Y Acad Sci,2009,1177:112-118.
[24]KHAVANDI K,BAYLIE R L,SUGDEN S A,et al.Pressureinduced oxidative activation of PKG enables vasoregulation by Ca2+sparks and BK channels[J].Sci Signal,2016,9(449):ra100.
[25]ZHAO G,ADEBIYI A,XI Q,et al.Hypoxia reduces KCa channel activity by inducing Ca2+spark uncoupling in cerebral artery smooth muscle cells[J].Am J Physiol Cell Physiol,2007,292(6):C2122-2128.
[26]LEWIS A,PEERS C,ASHFORD M L,et al.Hypoxia inhibits human recombinant large conductance,Ca2+-activated K+(maxi-K)channels by a mechanism which is membrane delimited and Ca2+sensitive[J].J Physiol,2002,540(3):771-780.
[2]OW C P,ABDELKADER A,HILLIARD L M,et al.Determinants of renal tissue hypoxia in a rat model of polycystic kidney disease[J].Am J Physiol Regul Integr Comp Physiol,2014,307(10):R1207-1215.
[3]NARITA I,SHIMADA M,YAMABE H,et al.NF-kappaB-dependent increase in tissue factor expression is responsible for hypoxic podocyte injury[J].Clin Exp Nephrol,2016,20(5):679-688.
[4]NEUSSER M A,LINDENMEYER M T,MOLL A G,et al.Human nephrosclerosis triggers a hypoxia-related glomerulopathy[J].Am J Pathol,2010,176(2):594-607.
[5]GRAHAMMER F,BENZING T,and HUBER T B.New insights into mechanisms of glomerular injury and repair from the 10th International Podocyte Conference 2014[J].Kidney Int,2015,87(5):885-893.
[6]CELLESI F,LI M,and RASTALDI M P.Podocyte injury and repair mechanisms[J].Curr Opin Nephrol Hypertens,2015,24(3):239-244.
[7]BAUMANN B,HAYASHIDA T,LIANG X,et al.Hypoxiainducible factor-1alpha promotes glomerulosclerosis and regulates COL1A2 expression through interactions with Smad3[J].Kidney Int,2016,90(4):797-808.
[8]LIU H,MOCZYDLOWSKI E,and HADDAD G G.O2 deprivation inhibits Ca2+-activated K+channels via cytosolic factors in mice neocortical neurons[J].J Clin Invest,1999,104(5):577-588.
[9]RIESCO-FAGUNDO A M,PEREZ-GARCIA M T,GONZALEZ C,et al.O2 modulates large-conductance Ca2+-dependent K+channels of rat chemoreceptor cells by a membrane-restricted and CO-sensitive mechanism[J].Circ Res,2001,89(5):430-436.
[10]MCCARTNEY C E,MCCLAFFERTY H,HUIBANT J M,et al.A cysteine-rich motif confers hypoxia sensitivity to mammalian large conductance voltage-and Ca-activated K(BK)channel alpha-subunits[J].Proc Natl Acad Sci U S A,2005,102(49):17870-17876.
[11]吴曙辉,刘丹,阎勇,等.大电导钙激活钾通道参与缺氧致前庭内侧核神经元兴奋性异常[J].听力学及言语疾病杂志,2017,25(1):44-48.
[12]朱艳,侯晓钰,傅晓冬.重度子痫前期胎盘小动脉血管平滑肌细胞BKCa通道的改变[J].泸州医学院学报,2016,39(5):471-475.
[13]HARVEY T W,ENGEL J E,and CHADE A R.Vascular endothelial growth factor and podocyte protection in chronic hypoxia:Effects of endothelin-a receptor antagonism[J].Am JNephrol,2016,43(2):74-84.
[14]梁立峰,张惠莉,罗朋立,等.低氧、高糖对肾小球内皮细胞膜表面多糖-蛋白复合物厚度影响的分析[J].中国糖尿病杂志,2015,23(12):1103-1105.
[15]王妍君,罗朋立.低氧环境下小鼠肾小球内皮细胞通透性的变化及其机制[J].山东医药,2016,56(31):31-33.
[16]KNAUS H G,MCMANUS O B,LEE S H,et al.Tremorgenic indole alkaloids potently inhibit smooth muscle high-conductance calcium-activated potassium channels[J].Biochemistry,1994,33(19):5819-5828.
[17]CASTILLO K,CONTRERAS G F,PUPO A,et al.Molecular mechanism underlying beta1 regulation in voltage-and calciumactivated potassium(BK)channels[J].Proc Natl Acad Sci USA,2015,112(15):4809-4814.
[18]BAUTISTA L,CASTRO M J,LOPEZ-BARNEO J,et al.Hypoxia inducible factor-2 alpha stabilization and maxi-K+channel beta1-subunit gene repression by hypoxia in cardiac myocytes:role in preconditioning[J].Circ Res,2009,104(12):1364-1372.
[19]NAVARRO-ANTOLIN J,LEVITSKY K L,CALDERON E,et al.Decreased expression of maxi-K+channel beta1-subunit and altered vasoregulation in hypoxia[J].Circulation,2005,112(9):1309-1315.
[20]CORNFIELD D N.Developmental regulation of oxygen sensing and ion channels in the pulmonary vasculature[J].Adv Exp Med Biol,2010,661:201-220.
[21]TANG X D,XU R,REYNOLDS M F,et al.Haem can bind to and inhibit mammalian calcium-dependent Slo1 BK channels[J].Nature,2003,425(6957):531-535.
[22]SAHOO N,HOSHI T,HEINEMANN S H.Oxidative modulation of voltage-gated potassium channels[J].Antioxid Redox Signal,2014,21(6):933-952.
[23]KEMP P J,TELEZHKIN V,WILKINSON W J,et al.Enzymelinked oxygen sensing by potassium channels[J].Ann N Y Acad Sci,2009,1177:112-118.
[24]KHAVANDI K,BAYLIE R L,SUGDEN S A,et al.Pressureinduced oxidative activation of PKG enables vasoregulation by Ca2+sparks and BK channels[J].Sci Signal,2016,9(449):ra100.
[25]ZHAO G,ADEBIYI A,XI Q,et al.Hypoxia reduces KCa channel activity by inducing Ca2+spark uncoupling in cerebral artery smooth muscle cells[J].Am J Physiol Cell Physiol,2007,292(6):C2122-2128.
[26]LEWIS A,PEERS C,ASHFORD M L,et al.Hypoxia inhibits human recombinant large conductance,Ca2+-activated K+(maxi-K)channels by a mechanism which is membrane delimited and Ca2+sensitive[J].J Physiol,2002,540(3):771-780.