Figure 3.
Effect of Opn treatment on cultured murine podocytes. (A) Western blot analysis of cultured murine podocytes treated with recombinant murine Opn (50 ng/ml) and harvested after the indicated time points. Membranes were probed as indicated with phospho-IκBα antibodies, total IκBα, and β-actin serving as loading controls. When indicated, cells were preincubated with 5 μM Bay11-7082 (Bay) or 100 μM PDTC. The data are a representative of three independent experiments. (B) Transfactor NF-κB p50 DNA binding assay of control and Opn-treated cells in the presence or absence of indicated inhibitors. (C) Cultured podocytes were treated with 50 ng/ml mouse recombinant Opn for 1, 4, 6 or 24 h. uPA and MMP-2 and -9 mRNA levels were determined by QRT-PCR. (D) Cells were incubated with Bay of PDTC followed by treatment with recombinant Opn (for 6 h). Significance was determined by one-way ANOVA. Scrape wound assay was performed on podocytes in the presence and absence of recombinant Opn and various inhibitors of Bay (5 μM) or PDTC (100 μM). The number of migrated cells was counted (by DAPI staining) 16 h after Opn incubation. (E) Representative images of control (sham), Opn, Bay, or PDTC plus Opn-treated podocytes are shown. (F) Quantification of three independent experiments. *Statistically significant difference (P < 0.05) compared with control. (G) The proposed model of Opn action in podocytes mediating albuminuria.