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. Author manuscript; available in PMC: 2011 Mar 29.
Published in final edited form as: Kidney Int. 2010 May 26;78(4):363–373. doi: 10.1038/ki.2010.137

Figure 4. Ectopic expression of ILK induces podocyte EMT, migration, and Snail expression.

Figure 4

(a) Immunofluorescence staining showed that exogenous ILK expression resulted in suppression of ZO-1 and induction of mesenchymal marker desmin, α-SMA, fibronectin, and MMP-9 in podocytes. Podocytes were infected with adenovirus harboring either Flag-ILK (Ad.Flag-ILK) or β-galactosidase gene (Ad.LacZ). Overexpression of exogenous ILK (Flag-ILK) and induction of desmin, α-SMA and fibronectin in podocytes were also confirmed by western blot analysis (b). Of note, infection with control adenovirus (Ad.LacZ) did not significantly affect the basal expression of ILK, desmin, α-SMA and fibronectin. (c) Real-time RT-PCR showed that ectopic expression of exogenous ILK inhibited nephrin mRNA expression. Relative mRNA level over the Ad.LacZ controls (value = 1.0) is presented. *P < 0.05 (n = 6). (d, e) Forced expression of ILK promoted podocyte migration. Representative micrographs show podocyte migration in a Boyden chamber motility assay (d). Quantitative determination of the migrated podocytes per field in different groups is presented (e). *P < 0.05 (n = 3). (f) Ectopic expression of ILK induced Snail expression. Cell lysates were prepared at 48 h after infection with Ad.Flag-ILK or Ad.LacZ adenovirus, and immunoblotted with antibodies against Snail and α-tubulin, respectively. Cells without infection with adenovirus were denoted as control. (g) Overexpression of ILK impaired the filtration barrier function of podocytes. Podocyte monolayer on collagen-coated Transwell filters was infected with Ad.LacZ or Ad.Flag-ILK adenovirus, and 24 h later albumin permeability across podocyte monolayer was determined. Data are presented as means ± s.e.m. (n = 6). *P < 0.01 versus Ad.LacZ control. α-SMA, α-smooth muscle actin; EMT, epithelial–mesenchymal transition, GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ILK, integrin-linked kinase; MMP-9, matrix metalloproteinase-9.