【作者】 查冬青；

【导师】 丁国华；

【作者基本信息】 武汉大学 ， 内科学， 2013， 博士

【摘要】 近年来,对蛋白尿产生及发展机制的深入研究发现,足细胞结构和功能的异常可能是导致蛋白尿的主要原因。Nephrin是定位于足突裂孔膜的结构分子和信号分子,在维持裂孔膜结构完整性和足细胞功能方面起关键作用。Nephrin异常及其磷酸化改变与蛋白尿形成、足突融合和细胞丢失密切相关,是足细胞损伤的重要机制之一。但Nephrin异常是否影响足细胞骨架改变、足细胞与基底膜的粘附以及两者间是否存在某种信号机制相互作用,目前尚不清楚。PINCH-1-ILK-α-parvin (PIP)复合物是由PINCH-1、ILK、α-parvin三种分子所组成的异聚体,是哺乳动物黏着斑的主要成份。PIP复合物是连接细胞与细胞外基质的重要结构。它也可连接整合素和肌动蛋白骨架,参与细胞骨架定位,调节细胞与细胞外基质粘附的信号转导,从而在细胞粘附、伸展、存活等方面起着重要作用,尤其是在肾小球足细胞结构改变以及与基底膜粘附中尤为重要。但是,PIP复合物受哪些病理生理信号的调节,引起足细胞骨架改变、足突融合和粘附异常,目前知之甚少。本文旨在探讨nephrin表达改变对足细胞骨架、足细胞伸展及粘附能力的影响及其分子机制。我们首先建立嘌呤霉素氨基核苷肾病大鼠模型,检测在蛋白尿动物模型中nephrin/磷酸化nephrin、PIP复合物、F-actin的表达变化。结果发现,大鼠嘌呤霉素氨基核苷肾病模型类似于人类微小病变型肾病。在大鼠嘌呤霉素氨基核苷肾病模型中,PIP复合物在肾小球中表达异常,其改变与尿蛋白的改变一致,PIP复合物表达减少可能与足突融合、细胞骨架改变及蛋白尿产生相关,PIP复合物相互作用的改变可能与nephrin磷酸化调节有关。为证实PIP复合物在足细胞骨架改变、足细胞粘附中的作用,我们在体外培养的条件永生性小鼠足细胞中,采用PINCH-1siRNA下调PIP复合物的表达,观察PIP复合物改变对足细胞骨架、足细胞伸展及粘附的影响,结果发现PIP复合物改变可导致足细胞骨架紊乱重组、足细胞伸展及粘附性降低。但PIP复合物改变不影响nephrin及磷酸化nephrin表达。为进一步证实nephrin表达在PIP复合物介导足细胞粘附改变中的作用及机制,我们在体外培养小鼠足细胞中采用nephrin siRNA以及Src激酶抑制剂(PP2)抑制nephrin及其磷酸化的表达,观察nephrin及其磷酸化nephrin改变对PIP复合物、足细胞骨架、足细胞粘附的影响。结果显示抑制nephrin或磷酸化nephrin可降低PIP复合物表达、足细胞骨架发生紊乱重组、足细胞伸展及粘附性降低。综上所述,nephrin表达异常可导致足细胞骨架改变、足细胞伸展及粘附功能降低,从而影响足突与基底膜的粘附。且nephrin在足细胞骨架、足细胞伸展及粘附中的作用,至少部分与nephrin磷酸化调节PIP复合物有关。第一部分Nephrin、磷酸化nephrin、PINCH-1-ILK-α-parvin(PIP)复合物在大鼠嘌呤霉素氨基核苷肾病模型中的表达目的研究嘌呤霉素氨基核苷肾病(puromycin aminonucleoside nephropathy; PAN)大鼠模型中nephrin、磷酸化nephrin、PIP复合物的表达,评价其表达与蛋白尿及足细胞损伤的关系。方法36只SPF级雄性SD大鼠,体重120-140g,随机分为对照组(n=6)和模型组(n=30。14,7,14,28d五个时间点,每组6只)。模型组一次性腹腔注射嘌呤霉素氨基核苷(puromycin aminonucleoside, PA,15mg/100g),分别于注射后第1,4,7,14,28天处死大鼠。对照组予等体积生理盐水腹腔注射。各组大鼠处死前一天代谢笼收集24h尿液,免疫透射比浊法测24h尿蛋白定量。硫酸钠-聚丙烯酰胺凝胶电泳行尿蛋白考马斯亮蓝染色,普通光镜和电镜观察肾脏形态学改变,免疫共沉淀法检测PINCH-1、ILK、α-parvin三者间相互作用及PIP复合物在肾脏的表达,免疫荧光检测肾小球nephrin的表达,免疫印迹法检测肾小球nephrin、磷酸化nephrin及F-actin的表达。结果(1)注射PA后第4天,大鼠24h尿蛋白定量较对照组升高(P<0.05),第7天达高峰(P<0.01),第14天开始减少,第28天降至正常。(2)PAS染色显示注射PA后第7天、14天肾小管内可见大量蛋白管型,肾小球未见明显病理病变。注射PA后第1天、第4天及第28天大鼠肾小球及肾小管未见明显病理改变。(3)透射电镜显示注射PA后第4天大鼠足突部分融合,第7、14天足突广泛融合,第28天足突形态有所恢复。(4)免疫印迹显示nephrin于注射PA后第1天开始降低,第7天时降至最低,第14天时逐渐升高,第28天时升高至正常。磷酸化nephrin表达在第14天时仍保持低水平,第28天时恢复正常。(5)免疫沉淀及免疫印迹法显示注射PA后第4天PIP复合物的表达与对照组相比有所降低(P<0.05)。第7天PIP复合物表达明显低于对照组(P<0.01)。第28天PIP复合物表达已恢复正常。PIP复合物改变与尿蛋白改变一致。(6)F-actin于注射PA后第1天降至最低,后逐渐恢复至正常。结论大鼠PAN模型类似于人类微小病变型肾病；PIP复合物的表达减少可能引起足突融合、细胞骨架改变,导致蛋白尿的产生；PIP复合物的表达减少可能与nephrin磷酸化调节相关。第二部分PINCH-1-ILK-α-parvin (PIP)复合物对足细胞骨架及粘附的影响目的通过培养条件永生小鼠足细胞,探讨PIP复合物在足细胞中的表达、PIP复合物在足细胞粘附改变中的作用以及可能的调节机制。方法培养条件永生性小鼠足细胞,应用PINCH-1siRNA转染,实验共分为三组：正常组、PINCH-1siRNA转染组、scrambled siRNA组。采用免疫印迹及免疫荧光检测nephrin、磷酸化nephrin在足细胞中的表达,采用免疫沉淀检测PINCH-1、 ILK、α-parvin的相互作用,免疫荧光检测细胞骨架F-actin的改变,采用倒置显微镜观察细胞伸展性并计数。通过细胞粘附实验检测足细胞粘附能力。结果(1)PIP复合物在足细胞有表达。(2)转染PINCH-1siRNA明显抑制PINCH-1、ILK、α-parvin三者的相互作用,足细胞细胞骨架发生明显紊乱、重组,足细胞伸展性及粘附性明显降低。(3)抑制PIP复合物表达后nephrin及磷酸化nephrin蛋白水平表达无明显改变。结论PIP复合物表达改变可导致足细胞骨架紊乱重组,足细胞伸展性及粘附能力降低,且PIP复合物介导的信号并不影响nephrin及磷酸化nephrin的表达。第三部分Nephrin表达对足细胞骨架、伸展及粘附的影响目的通过培养条件永生小鼠足细胞,探讨nephrin表达改变对足细胞骨架、足细胞伸展性及粘附能力的影响及其可能机制。方法体外培养永生性小鼠足细胞,转染nephrin siRNA,实验共分三组：正常对照组、nephrin siRNA组、scrambled siRNA组。采用免疫印迹法观察nephrin和磷酸化nephrin的表达和分布,采用免疫荧光法观察足细胞细胞骨架F-actin的改变。采用倒置显微镜观察细胞伸展性并计数。通过细胞粘附实验检测足细胞粘附能力。采用免疫共沉淀法检测PINCH-1、ILK、α-parvin三者之间的相互作用。结果(1)转染nephrin siRNA,可明显抑制nephrin、磷酸化nephrin表达。(2)抑制nephrin表达,可使足细胞骨架明显紊乱重组,足细胞伸展性及粘附能力明显降低。(3)抑制nephrin表达后,PINCH-1、ILK、α-parvin三者之间的相互作用明显减少。结论Nephrin表达异常可使足细胞骨架紊乱重组,足细胞伸展性及粘附能力降低；Nephrin异常可调节PINCH-1-ILK-α-parvin (PIP)复合物的形成。第四部分Nephrin影响足细胞骨架、伸展及粘附的分子机制目的Nephrin磷酸化改变是足细胞损伤的重要机制之一,Src家族激酶抑制剂(PP2)可抑制nephrin磷酸化。Nephrin磷酸化异常是否影响足细胞与基底膜的粘附,尚需进一步证实。而PIP复合物在肾小球足细胞结构改变、存活以及与基底膜粘附中起着重要作用,己被证实可维持足细胞骨架稳定、介导足细胞伸展及粘附。本部分实验通过对条件永生性小鼠足细胞的研究,探讨nephrin磷酸化水平改变对PIP复合物介导足细胞粘附改变的影响。方法体外培养永生性小鼠足细胞,使用Src家族激酶抑制剂(PP2)干预刺激,实验共分二组：正常对照组、PP2组。采用免疫印迹法观察磷酸化nephrin的表达,采用免疫荧光法观察足细胞骨架F-actin改变。采用倒置显微镜观察细胞伸展性并计数。通过细胞粘附实验检测足细胞粘附能力。采用免疫共沉淀法检测PINCH-1、ILK、a-parvin三者之间的相互作用。结果抑制nephrin磷酸化,可使PINCH-1-ILK-a-parvin (PIP)复合物表达减少,足细胞骨架紊乱重组,足细胞伸展性和粘附性下降。结论Nephrin磷酸化通过调节PINCH-1-ILK-a-parvin (PIP)复合物,介导足细胞骨架以及粘附信号,从而改变足突与基底膜的粘附能力。更多还原

【Abstract】 Nephrin, a key structural component of the slit diaphragm, is also a signaling molecule, which transduces signal after phosphorylation. Inhibition of nephrin or nephrin phosphorylation is correlated with podocyte injury, such as foot process effacement and detachment from the basement membrane. The PINCH-1-ILK-α-parvin (PIP) complex, an important molecule in the process of focal adhesion, plays a crucial role in cell adhesion and cytoskeleton formation. In this study, we hypothesized that nephrin could influence cytoskeleton and cell adhesion in podocytes by regulating the PINCH-1-ILK-a-parvin complex. Wistar rats were received either puromycin aminonucleoside (15mg/100g) or normal saline via one-time intraperitoneal injection.24-hour urinary protein excretion and serum creatinine were measured on0,1,4,7,14,28days after the injection. The light microscopy was used to observe the morphology of glomeruli. The transmission election microscopy was used to observe the ultrastructure of podocytes. Nephrin and phosphorylation of nephrin expression were analyzed by Western blot and immuno fluorescence. PIP complex was evaluated by co-immunoprecipitation. F-actin was analyzed by Western blot and immunofluorescence. Nephrin expression, PIP complex formation, and F-actin in Wistar rats intraperitoneally injected with puromycin aminonucleoside were gradually decreased but increased with time, coinciding with the recovery from glomerular/podocyte injury and proteinuria. In cultured podocytes, PINCH-1-ILK-a-parvin complex knockdown using siRNA resulted in cytoskeleton reorganization and decreased cell spreading and adhesion. Western blot and immunofluorescence showed that nephrin and its phosphorylation were unaffected after PINCH-1-ILK-a-parvin complex knockdown. Furthermore, inhibition of nephrin or nephrin phosphorylation reduced PINCH-1-ILK-a-parvin complex expression, disorganized podocyte cytoskeleton, and decreased cell adhesion and spreading. These findings indicate that alterations in nephrin expression can result in the cytoskeleton disorganization and decrease cell adhesion through a PINCH-1-ILK-a-parvin complex-dependent mechanism. Part1. Expression of nephrin, phosphorylated nephrin, PIP complex and F-actin in the glomeruli of rats with puromycin aminonucleoside nephrosisObjective:To investigate the expression alteration of nephrin, phosphorylated nephrin, PIP complex and F-actin in the development of proteinuria in rats with puromycin aminonucleoside nephrosis (PAN).Methods:PAN model was established by a single injection of puromycin aminonucleoside.24hour urinary protein excretion was detected by immunity transmission turbidity assay. The glomerular morphology and podocyte ultrastructure were observed under the light and transmission election microscopies respectively. The expression of nephrin, phosphorylated nephrin and F-actin were detected by Western blot. The expression of nephrin was also detected by immunofluorescence. The interaction of PINCH、ILK、α-parvin was detected by co-immunoprecipitation.Results:(1) The rats injected with puromycin aminonucleoside showed a significant increase in24h urinary protein on day4,7, and14compared with the normal rats.24h urinary protein was gradually increased but decreased with time. Coomassie brilliant blue staining of urinary protein exhibited no bands in the normal rats and70kD bands in the rats on day1,4, and28after the injection of puromycin aminonucleoside. And extensive bands were detected on day7and14.(2) Light microscopy showed a large number of protein casts in renal tubules, and no significant pathological changes in glomeruli on day7and14after the injection of puromycin aminonucleoside were observed. No significant pathological changes were observed both in glomeruli and renal tubuli on day0,1,4, and28after the injection of puromycin aminonucleoside.(3) Electron microscopy showed a portion of foot process effacement on day4and diffuse foot process fusion or effacement on day7and14. However, the effaced and fused foot processes recovered on day28.(4) The expression of nephrin and phosphorylated nephrin were decreased significantly on day1,4and7after the injection of puromycin aminonucleoside compared with that of control rats on day0. Furthermore, nephrin expression was recovered on day14and resumed to the normal control on day28. Phosphorylated nephrin was low on day14and recovered on day28.(5) The PIP complex was decreased on day4after the injection, reached its lowest level on day7, and then restored to control levels on day28.(6) F-actin was reduced on day1after the injection of PA and then started to recover on day4. Conclusions:The rat model injected with puromycin aminonucleoside was similar to human minimal change nephrotic syndrome. A dramatic alteration of PIP complex, nephrin, phosphorylated nephrin, F-actin expression in PAN suggests the potential relationship of PIP complex with the foot process fusion, cytoskeletal changes and the development of proteinuria and nephrin expression alterations may modulate the PINCH-1-ILK-a-parvin complex formation. Part2. Effects of PINCH-1-ILK-a-parvin complex on cytoskeleton, cell spreading and adhesion in PodocytesObjective To investigate the expression of PINCH-1-ILK-a-parvin (PIP) com plex in podocytes and the effect of PIP complex on pococyte cytoskeleton, podocyte spreading and adhesion.Methods Cultured murine podocytes were pretreated with or without PINCH-1siRNA. F-actin was analyzed by immunofluorescence with FITC-phalloidin. The expression of nephrin, phosphorylated nephrin were detected by Western blot. The interaction of PIP complex was detected by co-immunoprecipitation. Cell spreading was analyzed under inverted microscope. Cell adhesion was measured with spectropho-tometry.Results (1) Transfection of podocytes with PINCH-1siRNA significantly inhibited the interaction of PINCH-1、ILK、α-parvin compared with normal podocytes and cells transfected with scrambled siRNA.(2) After inhibition of PIP complex, the cytoskeleton was disorganized.(3) PIP complex knockdown decreased podocyte spreading and adhesion.(4) Inhibition of PIP complex did not affect the expression of nephrin and phosphorylated nephrin.Conclusions PIP complex plays an important role in the cytoskeleton, cell spreading and adhesion in podocytes. Part3. Effect of nephrin expression on cytoskeleton, cell spreading and adhesion in PodocytesObjective To investigate the effect of nephrin expression on pococyte cytoskeleton, podocyte spreading and adhesion.Methods Cultured murine podocytes were pretreated with or without nephrin siRNA. The expression of nephrin, phosphorylated nephrin were analyzed by Western blot. The interaction of PIP complex was evaluated by co-immunoprecipitation. F-actin was analyzed by immunofluorescence with FITC-phalloidin. Cell spreading was evaluated under inverted microscope. Cell adhesion was measured with spectrophot-ometry.Results (1) Transfection of nephrin siRNA significantly inhibited the expression of nephrin and phosphorylated nephrin.(2) Knockdown of nephrin disorganized the podocyte cytoskeleton.(3) Inhibition of nephrin decreased cell spreading and adhesion in podocytes.(4) Knockdown of nephrin suppressed the interaction of PINCH-1、ILK、 α-parvin.Conclusions Alterations in nephrin expression disorganize podocyte cytoskeleton and decrease cell spreading and adhesion. Part4. Molecular mechanism of nephrin on cytoskeleton, cell spreading and adhesion in PodocytesObjective To investigate the molecular mechanism of nephrin on pococyte cytoskeleton, spreading and adhesion.Methods Cultured murine podocytes were treated with the Src family kinase inhibitor (PP2;10μM). Phosphorylation of nephrin was analyzed by Western blot. The interaction of PINCH-1、ILK、α-parvin was evaluated by co-immunoprecipitation. F-actin was analyzed by immunofluorescence with FITC-phalloidin. Cell adhesion was measured with spectrophotometry. Cell spreading was evaluated under inverted micro-scope. Results (1) Reduction of phosphorylated nephrin inhibited the interaction of PINCH-1、ILK、α-parvin.(2) Inhibition of phosphorylated nephrin resulted in the podocyte cytoskeleton rearrangement.(3) Inhibition of phosphorylated nephrin decreased cell spreading and adhesion in podocytes.Conclusion Abnormalities of nephrin phosphorylation disorganize podocyte cytoskeleton and decrease cell adhesion through a PIP complex-dependent mechanism.更多还原