Mesangial cell volume increases and proliferation correlate with the development of diabetic nephropathy. Metabolic alterations associated with diabetes, principally chronic hyperglycemia, are believed to produce cell inositol depletion which in turn is expected to influence cell phosphoinositide turnover, a signalling mechanism of proposed importance in cell function and proliferation, these effects may also be pertinent to mesangial cell changes associated with diabetes mellitus. More distal than signals in the chain of events leading to these changes may be the actin cytoskeletal system, believed important for cell shape, cell movement, cell volume and possibly cell proliferation. Recent data implicates phosphatidylinositol-4, 5-bisphosphate in the regulation of two key actin-modulating proteins, profilin and gelsolin, that latter also being influenced by micromolar calcium concentrations; calcium is also believed to activate cytoplasmic myosin by phosphorylation it though the medium of calmodulin and myosin kinases. Methods have also recently been developed for examining high affinity interactions between these regulatory proteins and actin, for quantifying the state of assembly of actin biochemically and for evaluating the three-dimensional architecture of actin in the cell. All of these assays can be done on small samples of cells or tissue. The investigators propose to examine effects of diabetes-associated metabolic perturbations such as hyperglycemia, inositol depletion, and aldose reductase inhibition on the state of actin assembly, associations between profilin and actin and gelsolin and actin, and the extent of myosin phosphorylation, first in blood cells in which the actin system has been more completely studied, and then in cultured mesangial cells. The three-dimensional architecture of actin filaments in control and experimentally diabetic mesangial cells will also be determined.
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