Distribution and Expression of Podocin in Puromycin Aminonucleoside Injured Mouse Podocyte Cell Line

Abstract

Objective To explore the podocin mRNA expression and distribution in a time series scale in the couse of podocyte injury in vitro. Methods Mouse podocyte cell line (MPC5) were regenerated at 33℃ for proliferation in the presence of γ-inferon, and then subcultured at 37℃ without γ-inferon for differentiation about 8~10 days, which would be subsequently subject to puromycin aminonucleoside (PAN) treatment. The podocyte morphology was observed by the phase-contrast microscope. The podocin mRNA expression level and distribution were examined by indirect immunocytofluorescence, semi-quantitative RT-PCR at 8 h, 24 h, and 48 h, respectively. Results ①The well-developed podocyte arborization was formed after the in vitro induction, the PAN treatment led to the podocyte foot processe retraction and effacement together with the MPC5 shrinkage and the loss of cell contact. ② Podocin was evenly distributed in a filamentous pattern with the small dots across the well-differentiated MPC5, which was shifted to an uneven, discontinuous coarse puncta limited in the nuclei immediately after PAN treatment. ③ Semi-quantitative RT-PCR found that mRNA level had no significant changes after PAN treatment (P>0.05); however, immunocytochemistry suggested podocin disappeared in the cellular membrane and cytoplasm significantly (P<0.05). Conclusion Because of the intimate concomitance, the changed distribution pattern of podocin was the prominent effect of the podocyte injury, which might be related to the disease mechanism. Key words: podocyte cell line; podocin; puromycin aminonucleoside(PAN)