中华肾脏病杂志
中華腎髒病雜誌
중화신장병잡지
2013年
10期
754-760
,共7页
魏凯%李肖瑛%倪兆慧%陶花%顾乐怡
魏凱%李肖瑛%倪兆慧%陶花%顧樂怡
위개%리초영%예조혜%도화%고악이
足细胞%凋亡%环磷酸腺苷%蛋白激酶A%环磷酸腺苷直接激活的交换蛋白
足細胞%凋亡%環燐痠腺苷%蛋白激酶A%環燐痠腺苷直接激活的交換蛋白
족세포%조망%배린산선감%단백격매A%배린산선감직접격활적교환단백
Podocyte%Apoptosis%cAMP%PKA%Epac
目的 研究激活环磷酸腺苷(cAMP)信号对药物诱导的足细胞损伤的影响.方法 8周龄雄性BalB/C小鼠被随机分为对照组、阿霉素(ADR)组和腺苷酸环化酶激动剂(Forskolin) +ADR组,采用ADR尾静脉注射的方法制备肾病模型,Forskolin+ADR组小鼠给予腹腔内注射Forskolin.用免疫荧光激光共聚焦法检测小鼠肾组织环磷酸腺苷反应元件结合蛋白(CREB)磷酸化水平,透射电子显微镜观察足细胞形态改变,免疫组织化学法检测肾小球WT-1的表达.分别用嘌呤霉素氨基核苷(PAN),环磷酸腺苷直接激活的交换蛋白(Epac)信号激动剂8-pCPT-2-O-Me-cAMP (2Me),蛋白激酶A(PKA)信号激动剂pCPT-cAMP(pCPT)及其阻断剂H89处理条件永生化的足细胞,Western印迹法检测足细胞Epac、caspase 3及cleavedcaspase 3的表达水平,蛋白激酶检测系统检测足细胞PKA活性,CCK-8试剂盒法检测足细胞毒性,TUNEL法检测足细胞凋亡,JC-1染色法检测线粒体膜电位.结果 (1)与ADR组比较,Forskolin+ADR组小鼠尿白蛋白排泄量减少(P＜0.05),肾小球WT-1阳性细胞数增加(P＜0.05).(2)PAN刺激可致足细胞数量减少,作用呈时间依赖性(均P＜0.05);pCPT可减轻PAN诱导的足细胞数量减少(P＜ 0.05);PKA信号抑制剂H89有阻断pCPT的作用,并呈剂量依赖性(均P＜ 0.05).(3)对照组,PAN组和pCPT+PAN组绿色荧光细胞数所占比率分别是(12.67±2.15)％,(31.35±4.60)％和(16.96±2.51)％,P＜ 0.05.H89预处理有阻断pCPT的作用(P＜0.05).(4)PAN刺激足细胞凋亡细胞数和cleaved caspase3表达水平较对照组显著升高(均P＜0.05),pCPT预处理后足细胞凋亡细胞数和cleaved caspase3表达水平较PAN组显著下降(均P＜ 0.05).结论 激活cAMP信号可减轻ADR肾病小鼠足细胞损伤及PAN诱导的足细胞凋亡,cAMP/PKA信号通路可能介导了cAMP对药物诱导足细胞损伤的保护作用.
目的 研究激活環燐痠腺苷(cAMP)信號對藥物誘導的足細胞損傷的影響.方法 8週齡雄性BalB/C小鼠被隨機分為對照組、阿黴素(ADR)組和腺苷痠環化酶激動劑(Forskolin) +ADR組,採用ADR尾靜脈註射的方法製備腎病模型,Forskolin+ADR組小鼠給予腹腔內註射Forskolin.用免疫熒光激光共聚焦法檢測小鼠腎組織環燐痠腺苷反應元件結閤蛋白(CREB)燐痠化水平,透射電子顯微鏡觀察足細胞形態改變,免疫組織化學法檢測腎小毬WT-1的錶達.分彆用嘌呤黴素氨基覈苷(PAN),環燐痠腺苷直接激活的交換蛋白(Epac)信號激動劑8-pCPT-2-O-Me-cAMP (2Me),蛋白激酶A(PKA)信號激動劑pCPT-cAMP(pCPT)及其阻斷劑H89處理條件永生化的足細胞,Western印跡法檢測足細胞Epac、caspase 3及cleavedcaspase 3的錶達水平,蛋白激酶檢測繫統檢測足細胞PKA活性,CCK-8試劑盒法檢測足細胞毒性,TUNEL法檢測足細胞凋亡,JC-1染色法檢測線粒體膜電位.結果 (1)與ADR組比較,Forskolin+ADR組小鼠尿白蛋白排洩量減少(P＜0.05),腎小毬WT-1暘性細胞數增加(P＜0.05).(2)PAN刺激可緻足細胞數量減少,作用呈時間依賴性(均P＜0.05);pCPT可減輕PAN誘導的足細胞數量減少(P＜ 0.05);PKA信號抑製劑H89有阻斷pCPT的作用,併呈劑量依賴性(均P＜ 0.05).(3)對照組,PAN組和pCPT+PAN組綠色熒光細胞數所佔比率分彆是(12.67±2.15)％,(31.35±4.60)％和(16.96±2.51)％,P＜ 0.05.H89預處理有阻斷pCPT的作用(P＜0.05).(4)PAN刺激足細胞凋亡細胞數和cleaved caspase3錶達水平較對照組顯著升高(均P＜0.05),pCPT預處理後足細胞凋亡細胞數和cleaved caspase3錶達水平較PAN組顯著下降(均P＜ 0.05).結論 激活cAMP信號可減輕ADR腎病小鼠足細胞損傷及PAN誘導的足細胞凋亡,cAMP/PKA信號通路可能介導瞭cAMP對藥物誘導足細胞損傷的保護作用.
목적 연구격활배린산선감(cAMP)신호대약물유도적족세포손상적영향.방법 8주령웅성BalB/C소서피수궤분위대조조、아매소(ADR)조화선감산배화매격동제(Forskolin) +ADR조,채용ADR미정맥주사적방법제비신병모형,Forskolin+ADR조소서급여복강내주사Forskolin.용면역형광격광공취초법검측소서신조직배린산선감반응원건결합단백(CREB)린산화수평,투사전자현미경관찰족세포형태개변,면역조직화학법검측신소구WT-1적표체.분별용표령매소안기핵감(PAN),배린산선감직접격활적교환단백(Epac)신호격동제8-pCPT-2-O-Me-cAMP (2Me),단백격매A(PKA)신호격동제pCPT-cAMP(pCPT)급기조단제H89처리조건영생화적족세포,Western인적법검측족세포Epac、caspase 3급cleavedcaspase 3적표체수평,단백격매검측계통검측족세포PKA활성,CCK-8시제합법검측족세포독성,TUNEL법검측족세포조망,JC-1염색법검측선립체막전위.결과 (1)여ADR조비교,Forskolin+ADR조소서뇨백단백배설량감소(P＜0.05),신소구WT-1양성세포수증가(P＜0.05).(2)PAN자격가치족세포수량감소,작용정시간의뢰성(균P＜0.05);pCPT가감경PAN유도적족세포수량감소(P＜ 0.05);PKA신호억제제H89유조단pCPT적작용,병정제량의뢰성(균P＜ 0.05).(3)대조조,PAN조화pCPT+PAN조록색형광세포수소점비솔분별시(12.67±2.15)％,(31.35±4.60)％화(16.96±2.51)％,P＜ 0.05.H89예처리유조단pCPT적작용(P＜0.05).(4)PAN자격족세포조망세포수화cleaved caspase3표체수평교대조조현저승고(균P＜0.05),pCPT예처리후족세포조망세포수화cleaved caspase3표체수평교PAN조현저하강(균P＜ 0.05).결론 격활cAMP신호가감경ADR신병소서족세포손상급PAN유도적족세포조망,cAMP/PKA신호통로가능개도료cAMP대약물유도족세포손상적보호작용.
Objective To investigate the role of activated cylic AMP(cAMP) signaling in chemical-induced podocyte injury.Methods Eight-weeks-old male BalB/C mice were randomly divided into three groups:control group,Adriamycin (ADR) group and Forskolin+ADR group.ADR nephropathy models were established by tail intravenous injection,and part of them were injected Forskolin,an agonist of adenylate cyclase,intraperitoneally.Phosphorylation of cAMP response element binding protein (CREB) was detected by laser confocal microscopy,morphology of foot processes were determined with transmission electron microscope,and WT-1 expression in glomeruli were detected by immunohistochemistry.Conditionally immortalized podocytes were treated with puromycin aminonucleoside (PAN),Exchange protein directly activated by cAMP (Epac) agonist 8-pCPT-2-O-Me-cAMP (2Me),protein kinase A (PKA) antagonist H89 and its agonist pCPT-cAMP(pCPT).Western blot was used to detect the expression levels of Epac,caspase3 and cleaved caspase3.PKA activity was assayed using cAMP-dependent protein kinase detection system.Cell viability was determined by a cell count kit and podocyte apoptosis was estimated by TUNEL staining.Mitochondrial membrane potential was evaluated by JC-1 staining.Results (1)Compared with ADR group,the urine albumin decreased significantly (P ＜ 0.05) among Forskolin + ADR group and the WT-1 positive cells per glomerulus increased obviously (P ＜ 0.05).(2)PAN decreased podocyte number in a time-dependent manner (P ＜ 0.05),pre-treatment with pCPT obviously inhibited PAN induced podocyte decrease (P ＜0.05),but H89 prevented the effect of pCPT in a dose-dependent manner (P ＜ 0.05).(3)JC-1 staining showed that the percentage of podocyte with green fluorescence for control,PAN and pCPT+PAN group were (12.67±2.15)％,(31.35±4.60)％ and (16.96 ± 2.51)％ respectively (P ＜ 0.05),and pretreatment with H89 inhibited the effect of pCPT (P ＜ 0.05).(4) PAN promoted podocyte apoptosis and cleaved caspase3 expression (P ＜ 0.05),and pretreatment with pCPT significantly prevented PAN-induced podocyte apoptosis and cleaved caspase3 expression (P ＜ 0.05).Conclusions cAMP signaling activation ameliorated podocyte injury in ADR nice and PAN-induced podocyte apoptosis,and cAMP/ PKA pathway may mediate these processes.
