CD2AP在足细胞中的生理功能研究
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摘要
第一部分CD2AP在肾脏细胞中的分布及其在足细胞中的作用初探
目的观察CD2相关蛋白(CD2AP)在肾脏不同细胞系中的表达分布,及其与足细胞裂孔隔膜分子nephrin和细胞骨架蛋白F-actin之间的联系。
方法以DMEM培养基培养人系膜细胞(HMC)和人肾小管上皮细胞系(HK-2),RPMI 1640培养基培养条件永生化小鼠足细胞系。以RT-PCR及Western blot方法检测足细胞内CD2AP和nephrin的表达。间接免疫荧光结合激光共聚焦方法观察CD2AP在HMC、HK-2、未分化及已分化足细胞中的表达情况,及CD2AP与nephrin在足细胞中的共存。直接免疫荧光结合激光共聚焦方法观察F-actin在足细胞的表达及其与CD2AP的共存。
结果CD2AP均匀分布于HK-2及未分化足细胞的核周及胞浆,而不表达于HMC细胞。在足细胞分化过程中,CD2AP的分布发生了变化,出现向周边聚集的现象。CD2AP与足细胞裂孔隔膜分子nephrin及细胞骨架蛋白F-actin在足细胞中存在共定位关系。
结论CD2AP表达于上皮来源的肾脏固有细胞。CD2AP在足细胞中的分布特点,提示CD2AP可能参与足细胞的分化过程,并与裂孔隔膜分子功能及细胞骨架调节有关。
目的本文旨在研究未分化和分化足细胞生物学性状及CD2相关蛋白(CD2AP)在足细胞分化过程中的作用。
方法体外33℃许可条件下培养由H-2Kb-tsA58转基因小鼠所建立的未分化足细胞系,并在37℃非许可条件下诱导其分化。观察足细胞分化后形态学改变;MTT法测定细胞的生长曲线;红色荧光染料PKH-26标记足细胞,追踪其在子代细胞中的分布,检测细胞增殖能力;流式细胞仪检测细胞周期的改变;Western印迹检测足细胞相关蛋白CD2AP、α-actinin和足细胞分化相关蛋白nephrin的表达;免疫荧光结合激光共聚焦方法检测CD2AP、nephrin、α-actinin、F-肌动蛋白和微管蛋白的表达变化。用CD2AP siRNA转染未分化足细胞后置于37℃非许可条件下培养,并设非许可条件下未转染组为对照组。检测足细胞的生长速度的变化以及足细胞分化标志物synaptopodin的表达。
结果与未分化足细胞相比,分化足细胞形态发生改变,生长速度减慢,增殖能力下降(P<0.05);细胞周期表现为G0/G1期细胞比例的增多和S期及G2/M期的细胞比例下降(P<0.05);CD2AP、nephrin和α-actinin的表达明显增高(P<0.05);CD2AP、nephrin、α-actinin、F-肌动蛋白和微管蛋白在表达分布上均发生明显的改变。synaptopodin仅表达于已分化足细胞,在未分化足细胞无表达。转染特异性siRNA下调CD2AP表达后,细胞生长速度明显减慢,synaptopodin mRNA表达下调(P<0.05),细胞分化迟滞。
结论足细胞分化过程中伴随细胞骨架的重新分布和细胞形态的改变;CD2AP可能作为足细胞裂孔隔膜分子与细胞骨架的连接蛋白,在足细胞分化过程中发挥重要作用。
目的观察敲低足细胞CD2相关蛋白(CD2AP)表达对细胞增殖和分裂的影响,探讨CD2AP在足细胞损伤和蛋白尿发病机理中的作用。
方法采用RPMI 1640培养基,在33℃许可条件下培养永生化小鼠足细胞系。以PKH-26红色荧光染料标记足细胞后,用Metafectene转染试剂转染针对CD2AP的小分子干扰RNA(siRNA)。转染24h后流式细胞仪检测转染效率;48h后RT-PCR和Western印迹检测转染后CD2AP mRNA和蛋白表达;72h后以流式细胞仪检测足细胞增殖指数及细胞周期;用Oregon Green? 488标记的Paclitaxel直接标记足细胞内的微管蛋白,激光共聚焦显微镜下检测双核及多核足细胞的比例。
结果流式细胞仪结果显示,CD2AP特异性siRNA转染效率为66.27%。转染siRNA 48h后,CD2AP mRNA和蛋白表达分别下降57%和39%。转染72h后,足细胞增殖指数明显下降(p<0.05),G2/M期的细胞数量明显增多(p<0.05)。共聚焦显微镜下可见转染CD2AP siRNA后,部分细胞有丝分裂后不能分离,双核及多核足细胞的比例明显增加(p<0.05)。
结论CD2AP表达降低可能通过阻碍足细胞增殖和有丝分裂,导致足细胞损伤。
目的观察抑制足细胞CD2相关蛋白(CD2AP)表达对细胞黏附和胞质伸展功能的影响,并初步探讨其机制。
方法用RPMI 1640培养基33℃下培养小鼠未分化足细胞系,转染针对CD2AP的小分子干扰RNA(siRNA),设无特异靶位点的Scrambing序列即Control siRNA转染组作对照。48h后将转染的足细胞制备成单细胞悬液,接种于预铺有Ⅳ型胶原蛋白的96孔板内,33℃下培养90min后检测足细胞的贴壁率和细胞的伸展面积,流式细胞仪检测抑制CD2AP后足细胞的凋亡率以及在不同PAN刺激下足细胞的凋亡率。激光共聚焦显微镜下检测微丝蛋白的分布变化。Western印迹和免疫共沉淀检测nephrin蛋白的表达及其磷酸化水平。
结果转染CD2AP siRNA足细胞的黏附率和细胞伸展的面积明显低于对照组(P<0.05);转染CD2AP siRNA后48h足细胞的凋亡率高于对照组,有统计学意义(P<0.05)。100μg/ml的PAN能明显诱导足细胞的凋亡,减少足细胞的黏附率(P<0.05)。敲低CD2AP的表达后足细胞微丝蛋白的分布发生明显的变化,nephrin蛋白表达和磷酸化水平下降(P<0.05)。
结论敲低CD2AP的表达使足细胞易于凋亡,影响细胞的黏附功能;足细胞骨架蛋白的紊乱和nephrin信号通路的抑制可能是足细胞黏附和伸展功能下降的机制。
目的观察足细胞对白蛋白的吞噬作用以及白蛋白过负荷对足细胞的损伤,并探讨CD2AP在其中的作用。
方法本研究采用化学方法制备FITC标记的白蛋白,在33℃许可条件下培养由H-2Kb-tsA58转基因小鼠所建立的未分化足细胞系,并模拟体内足细胞在肾小球鲍曼氏囊内所处的蛋白尿环境,免疫荧光结合激光共聚焦方法研究足细胞对白蛋白的吞噬功能以及CD2AP与FITC-BSA在足细胞中的共定位,Western印迹检测CD2AP的表达,流式细胞仪检测足细胞的凋亡,透射电镜检测细胞的超微结果变化。
结果在生理范围尿白蛋白浓度下(0.05mg/ml),即可发现足细胞对白蛋白具有吞噬功能。在大剂量白蛋白浓度下(1mg/ml),足细胞吞噬白蛋白存在时间效应,呈S型曲线,在2h后达到平衡状态。激光共聚焦显微镜证实在CD2AP与FITC-BSA在足细胞内存在共聚关系。BSA刺激12h后CD2AP的表达明显上调,凋亡的足细胞增多,细胞超微结构发生改变。
结论除了肾小球滤过屏障作用外,足细胞还对蛋白尿成分具有吞噬清除的功能,CD2AP参与了这一过程。
Objective To study the distribution of CD2-associated protein (CD2AP) in normal renal cell lines and its interaction with nephrin and F-actin in podocytes.
Methods The HMC and HK-2 were cultured in DMEM. Conditionally immortalized murine podocyte cell line was cultured in RPMI 1640. The expressions of CD2AP and nephrin in podocytes were examined by RT-PCR and Western blot. The distribution of CD2AP in HMC, HK-2, differentiated and undifferentiated podocytes was observed by laser scanning confocal microscope. The colocalizations of CD2AP with nephrin and F-actin in undifferentiated podocytes were also detected.
Results CD2AP was distributed within the cytoplasm and perinulcear region of HK-2 and undifferentiated podocytes, but was absent in HMC cells. Its distribution profile changed and presented as peripheral accumulation when podocytes were put into differentiation-permissive conditions. CD2AP colocalized with nephrin and F-actin in podocytes.
Conclusion CD2AP can be detected in epithelial-originated renal cells. The alteration of distribution profile of CD2AP indicates it may participate in the process of podocytes differentiation and be involved in the regulation of slit diaphragm and cytoskeleton.
Objective To study the changes of biological characters and the potential role of CD2AP in podocyte differentiation.
Methods Conditionally immortalized murine podocyte cell line, which was established from transgenic H-2Kb-tsA58 mice, was cultured in RPMI 1640 medium at 33℃permissive conditions and shifted to 37℃non-permissive conditions to induce differentiation. Podocytes morphology changes, growth curve, proliferation ability and cell cycle were measured respectively in undifferentiated and differentiated podocytes. The expressions of CD2AP,α-actinin, nephrin and cytoskeletal proteins (i.e. F-actin and tubulin) were detected by laser scanning confocal microscope. After transfection with CD2AP small interfering RNA (siRNA) the cells were shifted to 37℃non-permissive conditions. Simultaneously, untransfected cells were taken as differentiation control. The changes of cell proliferation and the expression of synaptopodin were examined respectively.
Results In differentiated podocytes, cell morphology changed along with secondary foot process formation and reproductive activity descended. Cells in G0/G1 period accumulated and in S, G2/M period decreased. The expressions of CD2AP, nephrin andα-actinin were elevated in differentiated podocytes. The distribution profiles of podocyte associated proteins and cytoskeletal proteins apparently altered. After transfection with CD2AP siRNA, the expression of CD2AP was partially inhibited and cell growth arrested. Synaptopodin, the differentiation podocyte marker, were apparently down-regulated and cells showed delayed to differentiate.
Conclusion These results demonstrated that podocyte differentiation was accompanied by cytoskeleton rearrangement and cell morphology change. CD2AP might play an essential role in it.
Objective To study the effect of CD2-associated protein (CD2AP) in podocyte proliferation and cell division.
Methods Conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium at 33℃permissive conditions. After labeling with red fluorescent dye PKH-26, podocytes were transfected with CD2AP small interfering RNA (siRNA) using transfection reagent Metafectene. Transfection efficiency was measured by flow cytometer 24h later and inhibition effect of CD2AP siRNA was determined by RT-PCR and Western blot 48h after transfection. Proliferation index and cell cycle of podocytes were examined by flow cytometer 72h later. Microbule of podocyte was detected by Oregon Green? 488-conjugated paclitaxel. The ratio of binuclear and polynuclear podocytes were counted under laser scanning confocal microscope (LSCM).
Results The transfection efficiency of CD2AP siRNA is 66.27%. 48h after transfection with specific siRNA, the expressions of CD2AP mRNA and protein were down-regulated by 57% and 39% respectively. The podocyte proliferation index apparently descended (p<0.05) and the cells in the phase of G2/M accumulated significantly when detected at 72h (p<0.05). With the help of LSCM, some podocytes that could not separate after M-period were easily visible. The ratio of binuclear and polynuclear podocytes in CD2AP siRNA transfeced group was markedly higher than that in control group (p<0.05).
Conclusion Lowered CD2AP expression may play a crucial role in podocytes injury during the pathogenesis of proteinuria by interfering cell separation after mitosis and podocytes proliferation.
Objective To study the effect of CD2-associated protein (CD2AP) in podocyte adhesion and extension ability and explore its possible mechanism.
Methods Conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium at 33℃permissive conditions. The podocytes were transfected with CD2AP small interfering RNA (siRNA) and scrambing sequences labeled with fluorescein were taken as control. The transfected podocytes were trypsinized and seed into collagenⅣcoated plates. The relative cell adhesion and cell area were examined 90min later. Apoptotic rates of CD2AP siRNA transfected podocytes and different PAN concentrations incubated podocytes were detected by flow cytometer. The distribution of F-actin was observed under laser scanning confocal microscope. Nephrin protein expression and its phosphorylation level were examined by immunofluorescence and Western blot.
Results The relative cell adhesion and cell area of CD2AP siRNA transfected podocytes were apparently less than that of control group(P<0.05). The apoptotic rate in CD2AP siRNA transfected podocytes was significantly higher than control podocytes(P<0.05). 100μg/ml PAN could markedly induce podocytes to apoptosis and impaired cell adhesion ability(P<0.05). Nevertheless, no obvious difference was founed to cell body spreading(P>0.05). The distribution of F-actin in CD2AP depletion podocytes was apparently altered. The expression of nephrin protein and its phosphorylation level were conspicuous descended to some degree(P<0.05).
Conclusion CD2AP depletion facilitated podocytes to apoptosis and impaired the cell adhesion function. Cytoskeleton confusion and nephrin signaling weakness that caused by CD2AP depletion might be responsible for the decline of cell adhesion and spreading.
Objective To observe the proteinuria endocytosis of podocytes and the part the CD2AP plays in it.
Methods In this study, conditionally immortalized murine podocytes cell line, which was established from transgenic H-2Kb-tsA58 mice, was cultured in RPMI 1640 medium at 33℃permissive conditions. FITC conjugated bovine serum albumin (BSA) was prepared to mimic the podocytes floating in Bowman's capsule under proteinuria circumstance in vivo. The endocytosis of BSA in podocytes was investigated by flow cytometer and laser scanning confocal microscope (LSCM). The co-localization of CD2AP and FITC-BSA was detected during endocytosis. CD2AP expression profile was measured by Western blot.
Results The results demonstrated that podocytes had endocytosis ability to albumin even under physiological threshold (0.05mg/ml). Endocytosis curve of FITC-BSA with time was made at large concentration (1mg/ml) and exhibited an“S”shape. A dynamic equilibrium of endocytosis was observed at 2h or so. Co-localization of CD2AP and FITC-BSA was conformed by LSCM in podocytes. CD2AP protein expression was markedly up-regulated 24h later. Apoptosis podocytes and ultrastructure changes induced by BSA were observed.
Conclusion Beyond acting as the filtration barrier component, podocytes also exert its endocytosis to proteinuria in microalbuminuria and macroalbuminuria individual. CD2AP is involved in the urine protein endocytosis of podocytes.
目的观察CD2相关蛋白(CD2AP)在肾脏不同细胞系中的表达分布,及其与足细胞裂孔隔膜分子nephrin和细胞骨架蛋白F-actin之间的联系。
方法以DMEM培养基培养人系膜细胞(HMC)和人肾小管上皮细胞系(HK-2),RPMI 1640培养基培养条件永生化小鼠足细胞系。以RT-PCR及Western blot方法检测足细胞内CD2AP和nephrin的表达。间接免疫荧光结合激光共聚焦方法观察CD2AP在HMC、HK-2、未分化及已分化足细胞中的表达情况,及CD2AP与nephrin在足细胞中的共存。直接免疫荧光结合激光共聚焦方法观察F-actin在足细胞的表达及其与CD2AP的共存。
结果CD2AP均匀分布于HK-2及未分化足细胞的核周及胞浆,而不表达于HMC细胞。在足细胞分化过程中,CD2AP的分布发生了变化,出现向周边聚集的现象。CD2AP与足细胞裂孔隔膜分子nephrin及细胞骨架蛋白F-actin在足细胞中存在共定位关系。
结论CD2AP表达于上皮来源的肾脏固有细胞。CD2AP在足细胞中的分布特点,提示CD2AP可能参与足细胞的分化过程,并与裂孔隔膜分子功能及细胞骨架调节有关。
目的本文旨在研究未分化和分化足细胞生物学性状及CD2相关蛋白(CD2AP)在足细胞分化过程中的作用。
方法体外33℃许可条件下培养由H-2Kb-tsA58转基因小鼠所建立的未分化足细胞系,并在37℃非许可条件下诱导其分化。观察足细胞分化后形态学改变;MTT法测定细胞的生长曲线;红色荧光染料PKH-26标记足细胞,追踪其在子代细胞中的分布,检测细胞增殖能力;流式细胞仪检测细胞周期的改变;Western印迹检测足细胞相关蛋白CD2AP、α-actinin和足细胞分化相关蛋白nephrin的表达;免疫荧光结合激光共聚焦方法检测CD2AP、nephrin、α-actinin、F-肌动蛋白和微管蛋白的表达变化。用CD2AP siRNA转染未分化足细胞后置于37℃非许可条件下培养,并设非许可条件下未转染组为对照组。检测足细胞的生长速度的变化以及足细胞分化标志物synaptopodin的表达。
结果与未分化足细胞相比,分化足细胞形态发生改变,生长速度减慢,增殖能力下降(P<0.05);细胞周期表现为G0/G1期细胞比例的增多和S期及G2/M期的细胞比例下降(P<0.05);CD2AP、nephrin和α-actinin的表达明显增高(P<0.05);CD2AP、nephrin、α-actinin、F-肌动蛋白和微管蛋白在表达分布上均发生明显的改变。synaptopodin仅表达于已分化足细胞,在未分化足细胞无表达。转染特异性siRNA下调CD2AP表达后,细胞生长速度明显减慢,synaptopodin mRNA表达下调(P<0.05),细胞分化迟滞。
结论足细胞分化过程中伴随细胞骨架的重新分布和细胞形态的改变;CD2AP可能作为足细胞裂孔隔膜分子与细胞骨架的连接蛋白,在足细胞分化过程中发挥重要作用。
目的观察敲低足细胞CD2相关蛋白(CD2AP)表达对细胞增殖和分裂的影响,探讨CD2AP在足细胞损伤和蛋白尿发病机理中的作用。
方法采用RPMI 1640培养基,在33℃许可条件下培养永生化小鼠足细胞系。以PKH-26红色荧光染料标记足细胞后,用Metafectene转染试剂转染针对CD2AP的小分子干扰RNA(siRNA)。转染24h后流式细胞仪检测转染效率;48h后RT-PCR和Western印迹检测转染后CD2AP mRNA和蛋白表达;72h后以流式细胞仪检测足细胞增殖指数及细胞周期;用Oregon Green? 488标记的Paclitaxel直接标记足细胞内的微管蛋白,激光共聚焦显微镜下检测双核及多核足细胞的比例。
结果流式细胞仪结果显示,CD2AP特异性siRNA转染效率为66.27%。转染siRNA 48h后,CD2AP mRNA和蛋白表达分别下降57%和39%。转染72h后,足细胞增殖指数明显下降(p<0.05),G2/M期的细胞数量明显增多(p<0.05)。共聚焦显微镜下可见转染CD2AP siRNA后,部分细胞有丝分裂后不能分离,双核及多核足细胞的比例明显增加(p<0.05)。
结论CD2AP表达降低可能通过阻碍足细胞增殖和有丝分裂,导致足细胞损伤。
目的观察抑制足细胞CD2相关蛋白(CD2AP)表达对细胞黏附和胞质伸展功能的影响,并初步探讨其机制。
方法用RPMI 1640培养基33℃下培养小鼠未分化足细胞系,转染针对CD2AP的小分子干扰RNA(siRNA),设无特异靶位点的Scrambing序列即Control siRNA转染组作对照。48h后将转染的足细胞制备成单细胞悬液,接种于预铺有Ⅳ型胶原蛋白的96孔板内,33℃下培养90min后检测足细胞的贴壁率和细胞的伸展面积,流式细胞仪检测抑制CD2AP后足细胞的凋亡率以及在不同PAN刺激下足细胞的凋亡率。激光共聚焦显微镜下检测微丝蛋白的分布变化。Western印迹和免疫共沉淀检测nephrin蛋白的表达及其磷酸化水平。
结果转染CD2AP siRNA足细胞的黏附率和细胞伸展的面积明显低于对照组(P<0.05);转染CD2AP siRNA后48h足细胞的凋亡率高于对照组,有统计学意义(P<0.05)。100μg/ml的PAN能明显诱导足细胞的凋亡,减少足细胞的黏附率(P<0.05)。敲低CD2AP的表达后足细胞微丝蛋白的分布发生明显的变化,nephrin蛋白表达和磷酸化水平下降(P<0.05)。
结论敲低CD2AP的表达使足细胞易于凋亡,影响细胞的黏附功能;足细胞骨架蛋白的紊乱和nephrin信号通路的抑制可能是足细胞黏附和伸展功能下降的机制。
目的观察足细胞对白蛋白的吞噬作用以及白蛋白过负荷对足细胞的损伤,并探讨CD2AP在其中的作用。
方法本研究采用化学方法制备FITC标记的白蛋白,在33℃许可条件下培养由H-2Kb-tsA58转基因小鼠所建立的未分化足细胞系,并模拟体内足细胞在肾小球鲍曼氏囊内所处的蛋白尿环境,免疫荧光结合激光共聚焦方法研究足细胞对白蛋白的吞噬功能以及CD2AP与FITC-BSA在足细胞中的共定位,Western印迹检测CD2AP的表达,流式细胞仪检测足细胞的凋亡,透射电镜检测细胞的超微结果变化。
结果在生理范围尿白蛋白浓度下(0.05mg/ml),即可发现足细胞对白蛋白具有吞噬功能。在大剂量白蛋白浓度下(1mg/ml),足细胞吞噬白蛋白存在时间效应,呈S型曲线,在2h后达到平衡状态。激光共聚焦显微镜证实在CD2AP与FITC-BSA在足细胞内存在共聚关系。BSA刺激12h后CD2AP的表达明显上调,凋亡的足细胞增多,细胞超微结构发生改变。
结论除了肾小球滤过屏障作用外,足细胞还对蛋白尿成分具有吞噬清除的功能,CD2AP参与了这一过程。
Objective To study the distribution of CD2-associated protein (CD2AP) in normal renal cell lines and its interaction with nephrin and F-actin in podocytes.
Methods The HMC and HK-2 were cultured in DMEM. Conditionally immortalized murine podocyte cell line was cultured in RPMI 1640. The expressions of CD2AP and nephrin in podocytes were examined by RT-PCR and Western blot. The distribution of CD2AP in HMC, HK-2, differentiated and undifferentiated podocytes was observed by laser scanning confocal microscope. The colocalizations of CD2AP with nephrin and F-actin in undifferentiated podocytes were also detected.
Results CD2AP was distributed within the cytoplasm and perinulcear region of HK-2 and undifferentiated podocytes, but was absent in HMC cells. Its distribution profile changed and presented as peripheral accumulation when podocytes were put into differentiation-permissive conditions. CD2AP colocalized with nephrin and F-actin in podocytes.
Conclusion CD2AP can be detected in epithelial-originated renal cells. The alteration of distribution profile of CD2AP indicates it may participate in the process of podocytes differentiation and be involved in the regulation of slit diaphragm and cytoskeleton.
Objective To study the changes of biological characters and the potential role of CD2AP in podocyte differentiation.
Methods Conditionally immortalized murine podocyte cell line, which was established from transgenic H-2Kb-tsA58 mice, was cultured in RPMI 1640 medium at 33℃permissive conditions and shifted to 37℃non-permissive conditions to induce differentiation. Podocytes morphology changes, growth curve, proliferation ability and cell cycle were measured respectively in undifferentiated and differentiated podocytes. The expressions of CD2AP,α-actinin, nephrin and cytoskeletal proteins (i.e. F-actin and tubulin) were detected by laser scanning confocal microscope. After transfection with CD2AP small interfering RNA (siRNA) the cells were shifted to 37℃non-permissive conditions. Simultaneously, untransfected cells were taken as differentiation control. The changes of cell proliferation and the expression of synaptopodin were examined respectively.
Results In differentiated podocytes, cell morphology changed along with secondary foot process formation and reproductive activity descended. Cells in G0/G1 period accumulated and in S, G2/M period decreased. The expressions of CD2AP, nephrin andα-actinin were elevated in differentiated podocytes. The distribution profiles of podocyte associated proteins and cytoskeletal proteins apparently altered. After transfection with CD2AP siRNA, the expression of CD2AP was partially inhibited and cell growth arrested. Synaptopodin, the differentiation podocyte marker, were apparently down-regulated and cells showed delayed to differentiate.
Conclusion These results demonstrated that podocyte differentiation was accompanied by cytoskeleton rearrangement and cell morphology change. CD2AP might play an essential role in it.
Objective To study the effect of CD2-associated protein (CD2AP) in podocyte proliferation and cell division.
Methods Conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium at 33℃permissive conditions. After labeling with red fluorescent dye PKH-26, podocytes were transfected with CD2AP small interfering RNA (siRNA) using transfection reagent Metafectene. Transfection efficiency was measured by flow cytometer 24h later and inhibition effect of CD2AP siRNA was determined by RT-PCR and Western blot 48h after transfection. Proliferation index and cell cycle of podocytes were examined by flow cytometer 72h later. Microbule of podocyte was detected by Oregon Green? 488-conjugated paclitaxel. The ratio of binuclear and polynuclear podocytes were counted under laser scanning confocal microscope (LSCM).
Results The transfection efficiency of CD2AP siRNA is 66.27%. 48h after transfection with specific siRNA, the expressions of CD2AP mRNA and protein were down-regulated by 57% and 39% respectively. The podocyte proliferation index apparently descended (p<0.05) and the cells in the phase of G2/M accumulated significantly when detected at 72h (p<0.05). With the help of LSCM, some podocytes that could not separate after M-period were easily visible. The ratio of binuclear and polynuclear podocytes in CD2AP siRNA transfeced group was markedly higher than that in control group (p<0.05).
Conclusion Lowered CD2AP expression may play a crucial role in podocytes injury during the pathogenesis of proteinuria by interfering cell separation after mitosis and podocytes proliferation.
Objective To study the effect of CD2-associated protein (CD2AP) in podocyte adhesion and extension ability and explore its possible mechanism.
Methods Conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium at 33℃permissive conditions. The podocytes were transfected with CD2AP small interfering RNA (siRNA) and scrambing sequences labeled with fluorescein were taken as control. The transfected podocytes were trypsinized and seed into collagenⅣcoated plates. The relative cell adhesion and cell area were examined 90min later. Apoptotic rates of CD2AP siRNA transfected podocytes and different PAN concentrations incubated podocytes were detected by flow cytometer. The distribution of F-actin was observed under laser scanning confocal microscope. Nephrin protein expression and its phosphorylation level were examined by immunofluorescence and Western blot.
Results The relative cell adhesion and cell area of CD2AP siRNA transfected podocytes were apparently less than that of control group(P<0.05). The apoptotic rate in CD2AP siRNA transfected podocytes was significantly higher than control podocytes(P<0.05). 100μg/ml PAN could markedly induce podocytes to apoptosis and impaired cell adhesion ability(P<0.05). Nevertheless, no obvious difference was founed to cell body spreading(P>0.05). The distribution of F-actin in CD2AP depletion podocytes was apparently altered. The expression of nephrin protein and its phosphorylation level were conspicuous descended to some degree(P<0.05).
Conclusion CD2AP depletion facilitated podocytes to apoptosis and impaired the cell adhesion function. Cytoskeleton confusion and nephrin signaling weakness that caused by CD2AP depletion might be responsible for the decline of cell adhesion and spreading.
Objective To observe the proteinuria endocytosis of podocytes and the part the CD2AP plays in it.
Methods In this study, conditionally immortalized murine podocytes cell line, which was established from transgenic H-2Kb-tsA58 mice, was cultured in RPMI 1640 medium at 33℃permissive conditions. FITC conjugated bovine serum albumin (BSA) was prepared to mimic the podocytes floating in Bowman's capsule under proteinuria circumstance in vivo. The endocytosis of BSA in podocytes was investigated by flow cytometer and laser scanning confocal microscope (LSCM). The co-localization of CD2AP and FITC-BSA was detected during endocytosis. CD2AP expression profile was measured by Western blot.
Results The results demonstrated that podocytes had endocytosis ability to albumin even under physiological threshold (0.05mg/ml). Endocytosis curve of FITC-BSA with time was made at large concentration (1mg/ml) and exhibited an“S”shape. A dynamic equilibrium of endocytosis was observed at 2h or so. Co-localization of CD2AP and FITC-BSA was conformed by LSCM in podocytes. CD2AP protein expression was markedly up-regulated 24h later. Apoptosis podocytes and ultrastructure changes induced by BSA were observed.
Conclusion Beyond acting as the filtration barrier component, podocytes also exert its endocytosis to proteinuria in microalbuminuria and macroalbuminuria individual. CD2AP is involved in the urine protein endocytosis of podocytes.
引文
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24 Winder SJ, Ayscough KR. Actin-binding proteins. J Cell Sci. 2005; 118:651-654.
25 Lehtonen S, Zhao F, Lehtonen E. CD2-associated protein directly interacts with the actin cytoskeleton. Am J Physiol Renal Physiol. 2002; 283:F734-743.
26 Saleem MA, Ni L, Witherden I, et al. Co-localization of nephrin, podocin, and the actin cytoskeleton: evidence for a role in podocyte foot process formation.Am J Pathol. 2002;161:1459-1466.
27 Welsch T, Endlich N, G?kce G, et al. Association of CD2AP with dynamic actin on vesicles in podocytes. Am J Physiol Renal Physiol. 2005; 289:F1134-1143.
28 Kirsch KH, Georgescu MM, Shishido T, et al. The adapter type protein CMS/CD2AP binds to the proto-oncogenic protein c-Cbl through a tyrosine phosphorylation-regulated Src homology 3 domain interaction. J Biol Chem. 2001; 276:4957-4963.
29 Lynch DK, Winata SC, Lyons RJ, et al. A Cortactin-CD2-associated protein (CD2AP) complex provides a novel link between epidermal growth factor receptor endocytosis and the actin cytoskeleton. J Biol Chem. 2003; 278:21805-21813.
30 Rothschild BL, Shim AH, Ammer AG, et al. Cortactin overexpression regulates actin-related protein 2/3 complex activity, motility, and invasion in carcinomas with chromosome 11q13 amplification. Cancer Res. 2006; 66:8017-8025.
31 SchifferM, Mundel P, Shaw AS, et al. A novel role for the adaptor molecule CD2-associated protein in transforming growth factor-beta induced apoptosis. J Biol Chem. 2004; 279: 37004-37012.
32 Huber TB, Hartleben B, Kim J, et al. Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKT-dependent signaling. Mol Cell Biol. 2003; 23: 4917-4928.
33 Monzo P, Gauthier NC, Keslair F, et al. Clues to CD2-associated protein involvement in cytokinesis. Mol Biol Cell. 2005; 16: 2891-2902.
2 Asanuma K, Mundel P. The role of podocytes in glomerular pathobiology. Clin Exp Nephrol. 2003; 7:255-259.
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13 Li C, Ruotsalainen V, Tryggvason K, et al. CD2AP is expressed with nephrin in developing podocytes and is found widely in mature kidney and elsewhere. Am J Physiol Renal Physiol. 2000; 279:785-792.
14 Kim JM, Wu H, Green G, et al. CD2-associated protein haploinsufficiency is linked to glomerular disease susceptibility. Science. 2003; 300(5623):1298-1300.
15 Kirsch KH, Georgescu MM, Ishimaru S, et al. CMS: an adapter molecule involved in cytoskeletal rearrangements. Proc Natl Acad Sci U S A. 1999; 96:6211-6216.
16 Patrakka J, Tryggvason K. Nephrin--a unique structural and signaling protein of the kidney filter.Trends Mol Med. 2007; 13:396-403.
17 Roselli S, Gribouval O, Boute N, et al. Podocin localizes in the kidney to the slit diaphragm area. AmJ Pathol. 2002; 160:131-139.
18 Schwarz K, Simons M, Reiser J, et al. Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin. J Clin Invest. 2001; 108:1621-1629.
19 Welsch T, Endlich N, Kriz W, et al. CD2AP and p130Cas localize to different F-actin structures in podocytes. Am J Physiol Renal Physiol. 2001; 281:F769-777.
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5 Rops AL, van der Vlag J, Jacobs CW, et al. Isolation and characterization of conditionally immortalized mouse glomerular endothelial cell lines.Kidney Int. 2004; 66:2193-2201.
6 Monzo P, Gauthier NC, Keslair F, et al. Clues to CD2-associated protein involvement in cytokinesis. Mol Biol Cell. 2005; 16: 2891-2902.
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1 Asanuma K, Mundel P. The role of podocytes in gomerular pathobiology. Clin Exp Nephrol. 2003; 7:255-259.
2 Kriz W. Podocyte is the major culprit accounting for the progression of chronic renal disease. Microsc Res Tech. 2002; 57:189-195.
3 Shih NY, Li J, Karpitskii V, et al. Congenital nephritic syndrome in mice lacking CD2-associated protein. Science. 1999; 286: 312-315.
4 Kim JM, Wu H, Green G, et al. CD2-associated protein haploinsufficiency is linked to glomerular disease susceptibility. Science. 2003; 300:1298-1300.
5 Benzing T. Signaling at the slit diaphragm. J Am Soc Nephrol. 2004; 15:1382-1391.
6 Dustin ML, Olszowy MW, Holdorf AD, et al. A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts. Cell. 1998; 94: 667-677.
7 Welsch T, Endlich N, Kriz W, Endlich K. CD2AP and p130Cas localize to different F-actin structures in podocytes. Am J Physiol Renal Physiol. 2001; 281:F769-777.
8 Li C, Ruotsalainen V, Tryggvason K, et al. CD2AP is expressed with nephrin in developing podocytes and is found widely in mature kidney and elsewhere. Am J Physiol Renal Physiol. 2000; 279:F785-792.
9 Kirsch KH, Georgescu MM, Ishimaru S, et al. CMS: an adapter molecule involved in cytoskeletal rearrangements. Proc Natl Acad Sci U S A. 1999; 96:6211-6216.
10 Grunkemeyer JA, Kwoh C, Huber TB, et al. CD2-associated protein (CD2AP) expression in podocytes rescues lethality of CD2AP deficiency. J Biol Chem. 2005; 280: 29677-29681.
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14 Patrakka J, Tryggvason K. Nephrin--a unique structural and signaling protein of the kidney filter.Trends Mol Med. 2007; 13:396-403.
15 Ruotsalainen V, Ljungberg P, Wartiovaara J, et al . Nephrin is specifically located at the slit diaphragm of glomerular podocyte. Proc Natl Acad Sci USA. 1999; 96: 7962-7967.
16 Shih NY, Li J, Cotran R, et al. CD2AP localizes to the slit diaphragm and binds to nephrin via a novel C-terminal domain. Am J Pathol. 2001; 159:2303-2308.
17 Roselli S, Gribouval O, Boute N, et al. Podocin localizes in the kidney to the slit diaphragm area. AmJ Pathol. 2002; 160:131-139.
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22 Kestila M, Lenkkeri U, Mannikko M, et al. Positionally cloned gene for a novel glomerular protein nephrin is mutated in congenital nephritic syndrome. Mol Cell. 1998; 1:572-578.
23 Gubler MC. Podocyte differentiation and hereditary proteinuria/nephrotic syndromes. J Am Soc Nephrol. 2003; 14 Suppl 1:S22-26.
24 Winder SJ, Ayscough KR. Actin-binding proteins. J Cell Sci. 2005; 118:651-654.
25 Lehtonen S, Zhao F, Lehtonen E. CD2-associated protein directly interacts with the actin cytoskeleton. Am J Physiol Renal Physiol. 2002; 283:F734-743.
26 Saleem MA, Ni L, Witherden I, et al. Co-localization of nephrin, podocin, and the actin cytoskeleton: evidence for a role in podocyte foot process formation.Am J Pathol. 2002;161:1459-1466.
27 Welsch T, Endlich N, G?kce G, et al. Association of CD2AP with dynamic actin on vesicles in podocytes. Am J Physiol Renal Physiol. 2005; 289:F1134-1143.
28 Kirsch KH, Georgescu MM, Shishido T, et al. The adapter type protein CMS/CD2AP binds to the proto-oncogenic protein c-Cbl through a tyrosine phosphorylation-regulated Src homology 3 domain interaction. J Biol Chem. 2001; 276:4957-4963.
29 Lynch DK, Winata SC, Lyons RJ, et al. A Cortactin-CD2-associated protein (CD2AP) complex provides a novel link between epidermal growth factor receptor endocytosis and the actin cytoskeleton. J Biol Chem. 2003; 278:21805-21813.
30 Rothschild BL, Shim AH, Ammer AG, et al. Cortactin overexpression regulates actin-related protein 2/3 complex activity, motility, and invasion in carcinomas with chromosome 11q13 amplification. Cancer Res. 2006; 66:8017-8025.
31 SchifferM, Mundel P, Shaw AS, et al. A novel role for the adaptor molecule CD2-associated protein in transforming growth factor-beta induced apoptosis. J Biol Chem. 2004; 279: 37004-37012.
32 Huber TB, Hartleben B, Kim J, et al. Nephrin and CD2AP associate with phosphoinositide 3-OH kinase and stimulate AKT-dependent signaling. Mol Cell Biol. 2003; 23: 4917-4928.
33 Monzo P, Gauthier NC, Keslair F, et al. Clues to CD2-associated protein involvement in cytokinesis. Mol Biol Cell. 2005; 16: 2891-2902.
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