| Abstract |
Glomerular permeability for macromolecules depends partially on proper attachment of the glomerular epithelial cells (GEC) to the glomerular basement membrane (GBM). The latter requires integrity of the actin cytoskeleton, which in turn is regulated by specific actin associated proteins. Since several glomerulopathies characterized by heavy proteinouria are associated with increased glomerular tumor necrosis factor α (TNF-α) expression, we studied the interaction of TNF-α with the actin cytoskeleton of cultured rat GEC. Incubation of GEC with 10 ng/ml TNF-α for variable time periods runding from 15 min to 24 hr demonstrated a marked accentuation and redistribution of actin microfilaments, as shown by direct fluorescence analysis and confocal laser scanning microscopy. Quantitative biochemical determination of the G/total actin ratio confirmed the above observations. Indeed, this ratio was significantly reduced, indicating substantial polymerization of G-actin and formation of F-actin. Concurrently, TNF-α rapidly induced tyrosine phosphorylation of both paxillin and focal adhesion kinase, without affecting the expression levels of these two proteins. In addition, tyrosine phosphorylation of vinculin became evident, indicating involvement of this focal adhesion marker in the observed actin reorganization. Inhibition of tyrosine phosphorylation by genistein prevented the reorganization of the actin cytoskeleton by TNF-α. We conclude that TNF-α induces substantial reorganizatoin of actin cytoskeleton and focal adhesion. These effects occur simultaneously, with a promet TNF-α induced tyrosine phosphorylation of paxillin and focal adhesion kinase, indicating that these proteins, shown to regulate actin polyemrization and formation of focal adhesions, may be directly involved in the mechanism controllign the observed actin redistribution. We studied effects of TNFα upon the proliferation, cell volume and apoptosis glomerular epithelial cells. Incubation of GEC evidence with TNF-α (10 ng/ml) for 15 min, 2h, 4h and 24h increased of cell volume, while there is not evidence proliferation and apoptosis. These findings suggest that the observed TNF-α-actin cytoskeleton interactions may relate to the pathogenesis of glomerulopathies with heavy proteinuria, in which increased glomerular expression of TNF-α is associated with disturbances in the attachement of podocytes to the GBM.
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In Vitro Μελέτη της Επίδρασης του TNFa στον κυτταροσκελετό σπειραματικών επιθηλιακών κυττάρων (ποδοκυττάρων)
