Effect of the knockdown of podocin mRNA on nephrin and alpha-actinin in mouse podocyte.

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, semiquantitative 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 beta-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.