High glucose alters actin assembly in glomerular mesangial and epithelial cells.

BACKGROUND: Glomerular mesangial and epithelial cell structure and function are maintained by cytoskeletal protein organization and function. To determine whether the diabetic milieu alters filamentous (F-) actin assembly, the spatial distributions and content of F- and monomeric (G-) actin were analyzed in rat mesangial and glomerular epithelial cells (10 to 15 passages) cultured for 5 days in high (22.4 mM) or normal (5.2 mM) glucose and in cells of whole glomeruli isolated from streptozotocin-treated diabetic or normal rats. EXPERIMENTAL
DESIGN: Cells were labeled with the fluorescent probes rhodamine-phalloidin and FITC-DNase-1 specific for F- and G-actin, respectively. The average pixel intensities per cell were measured using dual channel confocal laser scanning microscopy (N = 60 cells per group). Total and G-actin were measured in mesangial cells by a spectrophotometric-based DNase-1 inhibition assay.
RESULTS: In response to endothelin-1, 0.1 microM, vasopressin 1.0 microM, or angiotensin II 1.0 microM, mesangial cells cultured in normal glucose displayed partial disassembly of F-actin characterized by decreased fluorescence intensity (microfilament bundle pattern changed to network) with no change in G-actin fluorescence. In high glucose, but not mannitol (22.4 mM), partial disassembly of F-actin and loss of response to the agonists were observed. In high glucose, the F-actin content (micrograms/mg cellular protein) was reduced significantly with no change in absolute G-actin compared with normal glucose exposure. The effect of high glucose on mesangial cell actin was reversed by returning the cells to normal glucose for 2 days, stimulation with insulin 2 micrograms/ml, or with a protein kinase C inhibitor. Mesangial cells in high glucose were smaller in planar area and exhibited loss of contractile response to endothelin-1 (0.1 microM) or vasopressin (1.0 microM) measured by videomicroscopy. High glucose-induced F-actin disassembly, possibly due to activated protein kinase C, could account for smaller cell size and lack of response to vasopressor agents. Glomerular epithelial cells cultured in normal glucose demonstrated F-actin disassembly and increased G-actin fluorescence intensity in response to A23187 (5 microM) or bradykinin (10 nM). When cultured in high glucose, but not mannitol, increased epithelial G-actin fluorescence and loss of F- and G-actin response to agonists were observed. Although stimulation with insulin reversed the high glucose effect on epithelial G-actin, F-actin remained unresponsive to agonists. The cells of glomeruli isolated from the diabetic rat displayed the same increase in G-actin, no change in F-actin fluorescence, and loss of response to agonist stimulation as observed in cultured epithelial cells.
CONCLUSIONS: These findings suggest that high glucose alters actin assembly in both glomerular mesangial and epithelial cells in vitro and in vivo, possibly contributing to cellular dysfunction in early diabetes.