Effect of the knockdown of podocin mRNA on nephrin and α-actinin in mouse podocyte
Effect of the knockdown of podocin mRNA on nephrin and α-actinin in mouse podocyte
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DOI：
10.1177/153537020422900914
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发表时间：
2004-10-01
影响因子：
3.2
通讯作者：
Deng, JH
中科院分区：
文献类型：
--
作者：
Fan, QF;Ding, J;Deng, JH
最近，新的足细胞蛋白 podocin、nephrin 和 alpha-actinin-4 已分别在三种先天性/家族性肾病综合征中被鉴定出来。进一步的研究表明，这些足细胞蛋白参与了一些获得性肾病综合征和各种蛋白尿实验模型。然而，这些足细胞蛋白之间的分子相互作用仍不清楚。在本研究中，为了研究podocin、nephrin和α-actinin-4之间的分子相互作用，我们重建了专门针对podocin mRNA的RNA干扰（RNAi）表达载体pSilencer 2.1-U6，并将其转染到小鼠足细胞克隆（MPC5）中。采用免疫荧光染色、免疫双标记、共聚焦显微镜、半定量逆转录聚合酶链式反应（RT-PCR）和Western blotting检测podocin、nephrin、α-actinin-4和glyserogenic-3-磷酸脱氢酶（GAPDH）/β-actin的分布和表达。足细胞内podocin和nephrin的荧光强度明显减弱，从细胞膜表面到细胞核周围的分布也发生明显变化。与 GAPDH 相关，观察到 podocin 和 nephrin 的 mRNA 分别减少了约 65% 和 70%。干扰组中podocin蛋白表达量过低，无法检测到。与 P-肌动蛋白相比，去氧肾上腺素的蛋白质水平降低了约 78%。 α-辅肌动蛋白的分布以及mRNA和蛋白水平没有显示出明显的变化。 α-辅肌动蛋白主要定位于细胞质，也延伸至突起。因此，通过敲除podocin mRNA，检测到nephrin的表达显着降低以及重新分布，而α-actinin-4的表达和分布没有变化。这些结果表明，podocin 可能与去氧肾上腺素直接相互作用，但不与 α-辅肌动蛋白相互作用。
Recently, the novel podocyte proteins podocin, nephrin, and alpha-actinin-4 have been identified in three congenital/family nephrotic syndromes, respectively. Further studies showed that these podocyte proteins were involved in some acquired nephrotic syndromes and various experimental models of proteinuria. However, the molecular interactions among these podocyte proteins remain unclear. In this study, to investigate the molecular interactions among podocin, nephrin, and alpha-actinin-4, we reconstructed the RNA interference (RNAi) expression vector, pSilencer 2.1-U6, specifically targeting podocin mRNA, and it was transfected into the mouse podocyte clone (MPC5). Immunofluorescence staining, double-immunolabeling, confocal microscopy, serniquantitative reverse transcription polymerase chain reaction (RT-PCR), and Western blotting were used to detect the distribution and expression of podocin, nephrin, alpha-actinin-4, and glyseraldehyde-3-phosphate dehydrogenase (GAPDH)/beta-actin. The fluorescence intensity of podocin and nephrin decreased obviously, along with the evident distribution change from the cell membrane surface to the nucleus circumference in podocyte. In relation to GAPDH, the mRNA reductions of podocin and nephrin were observed by about 65% and 70%, respectively. The expression of podocin protein was too low to be detected in the interference group. In relation to P-actin, the protein level of nephrin decreased by about 78%. The distribution and the mRNA and protein level of alpha-actinin showed no appreciable change. Alpha-actinin localized mainly in the cytoplasm and also extended to the processes. Thus, the significant decreased expression of nephrin along with the redistribution were detected with the knockdown of podocin mRNA, whereas the expression and distribution of alpha-actinin-4 showed no change. These results suggest that podocin may interact directly with nephrin, but not with alpha-actinin.