HIGH GLUCOSE ALTERS ACTIN ASSEMBLY IN GLOMERULAR MESANGIAL AND EPITHELIAL-CELLS

BACKGROUND: Glomerular mesangial and epithelial cell structure and fun ction are maintained by cytoskeletal protein organization and function To determine whether the diabetic milieu alters filamentous (F-) acti n assembly, the spatial distributions and content of F- and monomeric (G-) actin were analyzed in rat mesangial and glomerular epithelial ce lls (10 to 15 passages) cultured for 5 days in high (22.4 mM) or norma l (5.2 mM) glucose and in cells of whole glomeruli isolated from strep tozotocin-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 s canning microscopy (N = 60 cells per group). Total and G-actin were me asured in mesangial cells by a spectrophotometric-based DNase-1 inhibi tion assay. RESULTS: In response to endothelin-1, 0.1 mu M, vasopressi n 1.0 mu M, or angiotensin II 1.0 mu M mesangial cells cultured in nor mal 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 (mu g/mg cellular protein) was reduced significantly with no c hange in absolute G-actin compared with normal glucose exposure. The e ffect of high glucose on mesangial cell actin was reversed by returnin g the cells to normal glucose for 2 days, stimulation with insulin 2 m u g/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 mu M) or vasopressin (1.0 mu M measured by videomicroscopy. High glucose-induced F-actin disassembly, possibly due to activated protein kinase C, could account for smaller cell siz e and lack of response to vasopressor agents. Glomerular epithelial ce lls cultured in normal glucose demonstrated F-actin disassembly and in creased G-actin fluorescence intensity in response to A23187 (5 mu M) 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 remai ned unresponsive to agonists. The cells of glomeruli isolated from the diabetic rat displayed the same increase in G-actin, no change in F-a ctin fluorescence, and loss of response to agonist stimulation as obse rved 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 ce llular dysfunction in early diabetes.