Study on molecular effect and interaction among podocyte molecules by gene knockdown
- VernacularTitle:通过基因敲低探讨多个足细胞分子作用及分子间反应
- Author:
Qingfeng FAN
;
Jie DING
;
Yan XING
;
Na GUAN
;
Jingjing ZHANG
- Publication Type:Journal Article
- Keywords:
RNA interference;
Actinin;
Knockdown;
Podocyte molecule
- From:
Chinese Journal of Nephrology
2005;0(10):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the molecular effect and interaction among nephrin, podocin, CD2AP and ?-actinin-4. Methods Firstly, the recombinant RNA interference (RNAi) plasmid-psiRNA-hH1GFPzeo, specifically targeting to the mRNA of nephrin, podocin, CD2AP or ?-actinin-4, was respectively tansfected into the mouse podocyte clone (MPC5) to each knockdown (KD) the expression of nephrin, podocin, CD2AP or ?-actinin-4. Molecular distributions were revealed by confocal microscopy, and the mRNA and protein expressions were detected with semi-quantitative RT-PCR and Western blotting. Results (1)In podocin KD group (siPod966 and siPod54), the mRNAs of podocin and nephrin were not detected, their protein decreased 92% and 79%, 82% and 67%, respectively. The mRNA and protein level of CD2AP increased 62% and 42%, 71% and 46%, respectively, whereas ?-actinin-4 did not change. In nephrin KD group (siNep492), the mRNA expression and protein level of nephrin were not detected, CD2AP increased 35% and 48%, respectively; and whereas podocin and ?-actinin-4 did not change. In CD2AP KD group (siCda744 and siCda21), the mRNA of expression CD2AP was not detected, and its protein level decreased 92% and 83%, the mRNA and protein of nephrin decreased 60% and 48%, 76% and 72%, respectively; podocin increased 38% and 22%, 56% and 44%, respectively; whereas ?-actinin-4 did not change. In ?-actinin-4 KD group (siAct1790 and siAct319), the mRNAs expression of ?-actinin-4 and nephrin decreased 69% and 58%, 64% and 49%, respectively; their protein level decreased 81% and 55%, 71% and 64%, respectively. However, the mRNAs of podocin and CD2AP increased 50% and 34%, 45% and 28%, respectively; and their protein level increased 64% and 46%, 65% and 42%, respectively. (2) With their expression change, the distributions of nephrin, podocin and CD2AP shifted evidently from the cell membrane surface to the nucleus circumference, whereas ?-actinin-4 showed no change, which was still localized in the cytoplasm and further extended to foot processes. Conclusion (1) Nephrin might more independently play a crucial role in the slit diaphragm complex. (2) Alpha-actinin-4 might interact direcdy or indirectly with nephrin, podocin and CD2AP. (3) The relationship among these podocyte molecules might not be spontaneous, either a single-directional or bi-directional reaction. (4) The normal localization of these podocyte molecules might depend on their normal expression quantity.
