肾脏缺血再灌注中足细胞损伤机制的研究

【作者】 黄轶嵘；

【导师】 李荣山； Saleem；

【作者基本信息】 山西医科大学 ， 内科学， 2008， 博士

【摘要】 目的通过观察肾脏缺血再灌注中血管紧张素Ⅱ(AngⅡ)、足细胞形态学及其相关蛋白分子nephrin、podocin表达变化,探讨AngⅡ在肾脏缺血再灌注足细胞损伤中的作用及其可能机制,为其预防和治疗提供新的靶点。方法(1)取健康雄性SD大鼠,通过夹闭双侧肾动脉制作肾脏缺血模型,60分钟后恢复肾脏血流,并于再灌注30分钟,1小时,3小时,6小时,12小时,24小时后分别处死动物,苦味酸法检测血清肌酐、ELISA法检测血清及肾组织中AngⅡ含量,并行光镜及电镜观察肾组织形态学损伤。(2)根据电镜结果在足细胞损伤最严重时间点加用AngⅡ的1型受体阻断剂(ARB)氯沙坦灌胃治疗,检测肾功能、电镜观察肾组织足细胞损伤、免疫荧光及Westernblot检测肾组织nephrin、podocin表达,同假手术组,缺血组,缺血再灌注组比较,观察上述指标变化。(3)体外培养人肾小球足细胞,观察其生长特点并经间接免疫荧光法检测细胞上nephrin、podocin的表达。通过在培养箱中通入高浓度氮气制作细胞缺氧复氧模型,ELISA法检测细胞上清液中AngⅡ含量变化。(4)模拟缺血再灌注模型体内环境变化,直接在足细胞培养基中加入AngⅡ刺激,并分别给予ARB及蛋白激酶C抑制剂(PKCI)干预,观察各组细胞形态学改变,Western blot检测足细胞PKC活性变化,流式细胞仪检测细胞nephrin、podocin表达。结果(1)缺血组大鼠血清肌酐上升,同假手术组相比有统计学意义。再灌注后血清肌酐进一步升高,1小时达高峰,6小时后开始逐渐下降,24小时恢复至正常水平。缺血后血清及肾组织中AngⅡ含量也升高,同假手术组相比有统计学意义。再灌注后血清中AngⅡ即开始下降,但组织中AngⅡ含量仍维持在高水平并可持续至再灌注后24小时。光镜下缺血再灌注组织主要表现为小管间质损伤,肾小球无明显改变。电镜下再灌注组可看到足细胞足突融合,部分脱落,以1小时及3小时最为严重,6小时后逐渐恢复。免疫荧光及Westernblot检测缺血再灌注组织中nephrin、podocin表达明显减少,且存在分布异常。(2)氯沙坦治疗组血清肌酐明显下降,且足细胞损伤减轻,nephrin、podocin表达有所恢复,同缺血再灌注组相比有统计学意义。(3)体外培养的人肾小球足细胞呈现两种状态,在33℃条件下细胞胞体较小,鹅卵石状,生长速度快,呈现增殖状态,具有双核的特点;从33℃转移到37℃后,足细胞增殖能力明显减弱,胞体逐渐增大,细胞开始长出细小足突,之后这些足突会逐渐长长,细胞呈现星形。随时间延长,细胞完全失去增殖能力,胞体继续变大,足突进一步伸长呈树枝状。在两周左右细胞完全分化成熟,不同细胞之间的足突会相互接触。经间接免疫荧光检测可见到细胞表面有nephrin、podocin表达。在培养箱中通入高浓度氮气制作细胞缺氧模型,在复氧后1小时及3小时检测,细胞上清液中AngⅡ含量有所上升,但同对照组相比,无明显统计学意义。(4)体外培养人肾小球足细胞对AngⅡ呈现剂量依赖毒性,随AngⅡ浓度升高,足细胞存活率逐渐降低,台盼蓝及LDL释放实验显示,AngⅡ浓度为100nmol/L时细胞死亡率小于5%,因此我们选择100nmol/L作为AngⅡ的刺激浓度。在AngⅡ刺激组,HE及Gimsa染色可见到细胞失去原有形态,变小变圆,核浓缩,Western blot结果显示足细胞胞膜PKC与胞浆PKC比值升高,活性增强,流式细胞仪检测细胞nephrin、podocin荧光强度明显减弱。给予ARB及PKCI干预抑制了细胞中PKC的激活,异常形态的细胞减少,nephrin、podocin的表达也有所恢复。结论肾脏缺血再灌注过程中存在有足细胞的损伤。AngⅡ参与了这一过程,给予其1型受体阻断剂(ARB)可减轻缺血再灌注导致的足细胞损伤。AngⅡ对足细胞的损伤作用部分由PKC信号转导通路介导,给予ARB及PKCI可抑制PKC的活性,使足细胞相关的功能性蛋白nephrin、podocin表达有所恢复,起到一定的足细胞保护作用。更多还原

【Abstract】 ObjectiveTo investigate the effects of AngⅡon podocyte after ischemia/ reperfusion and to discuss the possible mechanism.Methods（1）The rat models induced by ischemia and reperfusion of kidney were established.These rats were sacrified at 0.5h,1h,3h,6h,12h and 24h after reperfusion. The blood samples were obtained for the measurement of serum creatinine, the level of AngⅡin plasma and kidney tissue was tested by ELISA. The change of renal histology was observed by electron microscope.（2）The ischemia/reperfusion rats were treated with angiotensin receptor blockers（ARB）-Losartan.Tested the serum creatinine and the histology change of podocyte.The expression of the nephrin and podocin in renal tissue were examined by indirect immunofluorescence staining and Western-blot.（3）The conditionally immortalized human podocyte cell lines were cultured.The histology change of the cell was observed,and the expression of nephrin and podocin on the differentiated podocytes was tested by indirect immunofluorescence.Then the cells were cultured in 95%N₂ incubater for 1h,3h and 6h to estabilish model.The level of AngⅡin supernatant was measured by ELISA.（4）Human podocyte cell lines were stimulated with angiotensinⅡat different concentrations,and then ARB and PKCI were added.Western-blot was applied to assess protein expression of PKC,and the change of nephrin、podocin was analyzed by flow cytometry.Results（1）Following the I/R injury,the serum creatinine was increased,and the level of AngⅡin serum and kidney tissue were also increased.The podocyte injury was observed,and the expression of nephrin and podocin was decreased,as compared to the control group.（2）The ARB can prevent the change of podocyte.（3）The conditionally immortalized human podocyte cell lines proliferate at a temperature of 33℃,and transform into a quiescent,differentiated phenotype after transfer to 37℃.Podocyte markers,nephrin and podocin,were expressed when the cells were kept at 37℃for 14 days.The AngⅡsecretion of podocytes did not significantly change after the cells were cultured in high concentration of N₂.（4）The survival rate of podocyte stimulated with AngⅡwas in dose dependent manner.In cultured podocyte cells stimulated with angiotensinⅡ,the activity of PKC was increased and the expression of nephrin,podocin was decreased.In cultured podocyte cells treated with ARB or PKCI,the change was reversed partly.Conclusion:1.The injury was observed in podocyte after I/R.2.AngⅡmediated this change,and ARB can protect podocyte from ischemia/reperfusion injury on renal histology and function,3.The effect of AngⅡon podocyte may be,at least in part,due to its activation of PKC signaling pathway.PKCI can reversed the effect partly.更多还原