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摘要:
背景 肾小球滤过屏障由足细胞及足突裂隙膜构成，病理状态下足细胞损伤破坏足突裂隙膜结构，造成肾小球滤过功能改变而产生蛋白尿。钠钙交换蛋白1(sodium-calcium exchanger 1，NCX1)作为Ca2+通道，具有调节钙稳态、维持细胞结构的重要作用。 目的 本研究拟证实NCX1在足细胞损伤中的作用，解析蛋白尿发生的新机制。 方法 建立被动型海曼肾炎(passive Heymann nephritis，PHN)大鼠模型，观察模型的NCX1表达变化；体外培养小鼠足细胞，建立补体激活足细胞损伤模型；Ca2+荧光染色探针Fluo-3检测足细胞胞内Ca2+水平变化；免疫荧光检测足细胞标志物及细胞骨架蛋白表达与结构的变化；Western blot 检测Ca2+下游信号通路蛋白表达的变化；应用NCX1反向模式(reverse mode)抑制剂KB-R7943处理补体激活足细胞损伤模型，观察其是否可有效减轻足细胞损伤。 结果 PHN大鼠模型NCX1表达随造模时间逐渐降低，在21 d达到最低；补体激活足细胞损伤模型NCX1表达亦降低；胞内Ca2+浓度明显升高，活化型RhoA、ROCK1和ROCK2蛋白的表达水平明显增高；NCX1反向模式抑制剂KB-R7943可明显降低胞内Ca2+浓度及下游信号通路蛋白活性及表达水平；补体激活足细胞损伤模型中足细胞标志物Nephrin及Synaptopodin表达减少，细胞骨架蛋白F-actin表达异常，而KB-R7943可显著改善上述异常蛋白表达从而减轻足细胞损伤。 结论 NCX1可通过调控钙离子内流激活下游RhoA/ROCK信号通路改变足细胞骨架结构，损伤足细胞，导致蛋白尿。 Abstract:Background The glomerular filtration barrier is composed of podocytes and slit diaphragm. Under pathological condition, the structure of slit diaphragm has been damaged during podocytes injury, resulting in changes in glomerular filtration function and proteinuria. As a Ca2+ channel, sodium-calcium exchanger 1 (NCX1) plays an important role in regulating calcium homeostasis and maintaining cell structure. Objective To confirm the role of NCX1 in podocyte injury and analyze a new mechanism of proteinuria. Methods A Passive Hyman Nephritis (PHN) rat model was established and the expression of NCX1 was detected in the animal model. Mouse podocytes were cultured and a complement injured podocyte model was established. The intracellular Ca2+ level of podocytes was detected with a Ca2+ fluorescence probe fluo-3. Immunofluorescence was used to confirm the changes of podocyte injury and cytoskeleton structure rearrangement and the downstream signaling pathway proteins were analyzed by Western blot. NCX1 reverse mode inhibitor KB-R7943 was applied to the complement injured podocyte model to observe whether the podocytes injury was effectively reduced. Results NCX1 expression in PHN rat model decreased gradually with time and reached the minimum level at 21 d. Meanwhile, in complement injured podocytes we also found the decreased expression of NCX1. The concentration of Ca2+ increased significantly, and the protein expression of RhoA, ROCK1 and ROCK2 also increased. NCX1 reverse mode was activated to promote Ca2+ influx and RhoA/ROCK signaling pathway was activated. The expression of podocyte markers Nephrin and Synaptopodin decreased with F-actin rearragement. The podocytes injury could be significantly reduced by KB-R7943. Conclusion NCX1 regulates calcium influx to active RhoA/ROCK signaling pathway mediated podocyte injury with cytoskeleton structure rearragement, thus leading to proteinuria. -
Key words:
- sodium-calcium exchanger 1 /
- calcium /
- podocyte /
- cytoskeleton /
- signaling pathway /
- complement
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图 1 免疫荧光染色检测PHN大鼠模型中NCX1的表达(Con组：对照组；PHN模型组7 d、14 d、21 d、28 d；蓝色：细胞核)
Figure 1. Expression of NCX1 in PHN rat model detected by immunofluorescence staining (Con: control group; PHN model groups for 7 days, 14 days, 21 days, 28 days; Blue: nuclei)
图 2 补体激活足细胞损伤模型中NCX1及Ca2+水平的变化(蓝色：细胞核)
A：足细胞免疫荧光染色NCX1；B和C：足细胞Ca2+荧光染色图像及荧光数据分析；Con：对照组；C5b-9：补体激活足细胞损伤模型组；KB-R7943：NCX1反向模式抑制剂KB-R7943处理组(n=10，C5b-9 vs Con，KB-R7943 vs C5b-9，aP<0.05)
Figure 2. Immunofluorescence staining of NCX1 and intracellular Ca2+ in podocytes (Blue: nuclei)
A: Immunofluorescence staining of NCX1; B and C: Fluorescence imaging and quantitative analysis of Ca2+; Con: Control group; C5b-9: Complement injured podocyte model group; KB-R7943: KB-R7943 treatment group (n=10, C5b-9 vs Con, KB-R7943 vs C5b-9, aP<0.05)
图 3 Western blot检测RhoA/ROCK信号通路蛋白活性
Con：对照组；C5b-9：补体激活足细胞损伤模型组；KB-R7943：NCX1反向模式抑制剂KB-R7943处理组(n=5，C5b-9 vs Con，KB-R7943 vs C5b-9，aP<0.05)
Figure 3. RhoA/ROCK signaling pathway proteins activities detected by Western blot
Con: Control group; C5b-9: Complement injured podocyte model group; KB-R7943: KB-R7943 treatment group (n=5, C5b-9 vs Con, KB-R7943 vs C5b-9, aP<0.05)
图 4 足细胞F-actin、Nephrin和Synaptopodin的表达与结构改变(蓝色：细胞核 )
A：免疫荧光染色检测F-actin、Nephrin和Synaptopodin；B：荧光数据分析；Con：对照组；C5b-9：补体激活足细胞损伤模型组；KB-R7943组：NCX1反向模式抑制剂KB-R7943处理组(n=12，C5b-9 vs Con，KB-R7943 vs C5b-9，aP<0.05)
Figure 4. Immunofluorescence staining of F-actin, Nephrin and Synaptopodin in podocytes (Blue: nuclei)
A: Immunofluorescence imaging of F-actin, Nephrin and Synaptopodin; B: Quantitative analysis of immunofluorescence images; Con: Control group; C5b-9:Complement injured podocyte model group; KB-R7943: KB-R7943 treatment group (n=12, C5b-9 vs Con, KB-R7943 vs C5b-9, aP<0.05)
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