Glomerulosclerosis is one of the complications of diabetes that occurs after many years of uncontrolled hyperglycemia. Mesangial cells (MCs) exposed to high glucose (HG) for short periods have shown that transforming growth factor-beta (TGF-beta) and activated diacylglycerol-dependent protein kinase C (PKC) mediate increased collagen formation. Our study examined collagen formation by MCs exposed to HG for 8 weeks. Exposure to HG in overnight culture resulted in the activation of all PKC isoforms. In contrast, 8-week exposure to HG resulted in the persistent activation of PKC-delta, did not change PKC-alpha or -beta activity, and decreased PKC-epsilon activity while increasing collagen I and IV gene and protein expression. Collagen IV accumulation was reversed by specific PKC-delta inhibition. Collagen IV gene expression was completely normalized by TGF-beta neutralization; however, this was associated with plasminogen activator inhibitor-1 (PAI-1) overexpression and a modest reduction in collagen protein. Our studies suggest that prolonged exposure to HG results in PKC-delta-driven collagen accumulation by MCs mediated by PAI-1 but independent of TGF-beta.

Other than stimulation of cell contractility, little is known about the potential metabolic effects induced by sulfonylureas, independently of insulin action. Previous studies from our laboratory demonstrated complete abrogation of glomerulosclerosis in an experimental model of type 1 diabetes chronically (9 mo) treated with low-dose sulfonylureas (Biederman JI, Vera E, Pankhaniya R, Hassett C, Giannico G, Yee J, Cortes P. Kidney Int 67: 554-565, 2005). Therefore, the effects of glibenclamide (Glib) on net collagen I, collagen IV, and fibronectin medium net secretion and cell layer collagen I deposition were investigated in mesangial cells continuously exposed to 25 mM glucose for 8 wk and treated with predetermined increasing concentrations of Glib for the same period. Clinically relevant concentrations (0.01 microM) of Glib fully suppressed the high glucose-enhanced accumulation of collagen I, collagen IV, and fibronectin in the medium and inhibited collagen I deposition in the cell layer. These effects occurred while transforming growth factor (TGF)-beta1 medium concentration remained elevated and glucose uptake was increased to levels above those in 25 mM glucose-incubated cultures. The decreased collagen I accumulation occurred simultaneously with enhanced collagen I mRNA expression in concert with marked suppression of plasminogen inhibitor type-1 (PAI-1) mRNA and protein expression. This strongly suggests an accelerated matrix turnover favoring breakdown. Glib-induced effects demonstrated a biphasic pattern, being absent or reversed in cells treated with higher Glib concentrations (0.1 or 1 microM). Therefore, chronic Glib treatment at low concentrations markedly diminishes the high glucose-induced enhanced accumulation of extracellular matrix components by suppression of steady-state PAI-1 transcriptional activity. These results and those previously reported in vivo suggest that long-term Glib treatment may prevent glomerulosclerosis in insulin-deficient diabetes.

BackgroundOur laboratory reported that male mice with cardiomyocyte-selective knockout of the prostaglandin E2 EP4 receptor sub-type (EP4 KO) exhibit reduced cardiac function. Gene array on left ventricles (LV) showed increased fractalkine, a chemokine implicated in heart failure. We therefore hypothesized that fractalkine is regulated by PGE2 and contributes to depressed contractility via alterations in intracellular calcium.MethodsFractalkine was measured in LV of 28–32 week old male EP4 KO and wild type controls (WT) by ELISA and the effect of PGE2 on fractalkine secretion was measured in cultured neonatal cardiomyocytes and fibroblasts. The effect of fractalkine on contractility and intracellular calcium was determined in Fura-2 AM-loaded, electrical field-paced cardiomyocytes. Cardiomyocytes (AVM) from male C57Bl/6 mice were treated with fractalkine and responses measured under basal conditions and after isoproterenol (Iso) stimulation.ResultsLV fractalkine was increased in EP4 KO mice but surprisingly, PGE2 regulated fractalkine secretion only in fibroblasts. Fractalkine treatment of AVM decreased both the speed of contraction and relaxation under basal conditions and after Iso stimulation. Despite reducing contractility after Iso stimulation, fractalkine increased the Ca2+ transient amplitude but decreased phosphorylation of cardiac troponin I, suggesting direct effects on the contractile machinery.ConclusionsFractalkine depresses myocyte contractility by mechanisms downstream of intracellular calcium.

Human and bovine platelets contain a 58,000-dalton vimentinlike protein that cross-reacts with antivimentin antibody. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blots indicate that this protein is present in whole platelet lysates and triton insoluble cytoskeletons. Transmission electron microscopy of platelets reveals an isotropic network of individual intermediate filaments distributed throughout the platelets. High salt, triton extracted, glutaraldehyde and tannic acid fixed platelets reveal 10-nm filaments that can be seen to form a peripheral ring, as well as an isotropic network in the body of the cells. Indirect immunofluorescence of resting and spread platelets demonstrates a circumferential staining pattern close to the cell membrane, with additional fibrillar staining throughout the platelets. Our data suggest that the 58,000-dalton vimentinlike protein may be associated with the microtubule coil and the plasma membrane, and may thus help to maintain the resting platelet's discoid shape.