High glucose alters actin assembly in glomerular mesangial and epithelial cells

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.

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.

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.

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.