Protein kinase C (PKC) ␤ isoform activity is increased in myocardium of diabetic rodents and heart failure patients. Transgenic mice overexpressing PKC␤2 (PKC␤2Tg) in the myocardium exhibit cardiomyopathy and cardiac fibrosis. In this study, we characterized the expression of connective tissue growth factor (CTGF) and transforming growth factor ␤ (TGF␤) with the development of fibrosis in heart from PKC␤2Tg mice at 4 -16 weeks of age. Heart-to-body weight ratios of transgenic mice increased at 8 and 12 weeks, indicating hypertrophy, and ratios did not differ at 16 weeks. Collagen VI and fibronectin mRNA expression increased in PKC␤2Tg hearts at 4 -12 weeks. Histological examination revealed myocyte hypertrophy and fibrosis in 4-to 16-week PKC␤2Tg hearts. CTGF expression increased in PKC␤2Tg hearts at all ages, whereas TGF␤ increased only at 8 and 12 weeks. In 8-week diabetic mouse heart, CTGF and TGF␤ expression increased two-and fourfold, respectively. Similarly, CTGF expression increased in rat hearts at 2-8 weeks of diabetes. This is the first report of increased CTGF expression in myocardium of diabetic rodents suggesting that cardiac injury associated with PKC␤2 activation, diabetes, or heart failure is marked by increased CTGF expression. CTGF could act independently or together with other cytokines to induce cardiac fibrosis and dysfunction. Diabetes 51:2709 -2718, 2002 C ardiomyopathy associated with diabetes can occur regardless of coronary artery disease and is characterized by myocyte hypertrophy and fibrosis (1,2). Ventricles from diabetic patients show accumulation of PAS-positive glycoproteins, collagen, and active fibroblasts (2). Similarly, ventricle from diabetic rodents show increased fibrosis and expression of the extracellular matrix (ECM) components fibronectin, collagen IV, and collagen VI (3-5). Ventricular remodeling along with changes such as defective calcium transport or fatty acid metabolism can impair contractility. Animals and patients with diabetes show reduced stroke volume and increased end-diastolic pressure (6 -9), which collectively could precipitate heart failure.Activation of the protein kinase C (PKC)/diacylglycerol (DAG) signaling pathway is one mechanism by which hyperglycemia may exert adverse cardiovascular effects. Inoguchi et al. (10) reported increased membranous PKC activity and total DAG in diabetic rat heart. PKC␤2 isoform was preferentially increased in the membranous fraction of heart and aorta, suggesting that this isoform contributes to diabetic vascular complications. Indeed, administration of the PKC␤ selective inhibitor LY333531 to diabetic rats ameliorated increased glomerular filtration, albumin excretion, and retinal circulation rates (11). Ventricles from patients with end-stage heart failure show increased expression of PKC␤ and increased membranous PKC activity within cardiac myocytes (12). Furthermore, targeted overexpression of PKC␤2 in mouse myocardium resulted in left ventricular hypertrophy, fibrosis, and decreased left ventricular performance, s...

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Diabetic nephropathy (DN) is a major cause of end-stage renal disease (ESRD) worldwide. Glycemic and blood pressure (BP) control are important but not sufficient to attenuate the incidence and progression of DN. Sodium–glucose cotransporter (SGLT) 2 inhibitors are a new class of glucose-lowering agent suggested to exert renoprotective effects in glucose lowering-dependent and independent fashions. Experimental studies have shown that SGLT2 inhibitors attenuate DN in animal models of both type 1 diabetes (T1D) and type 2 diabetes (T2D), indicating a potential renoprotective effect beyond glucose reduction. Renoprotection by SGLT2 inhibitors has been demonstrated in T2D patients with a high cardiovascular risk in randomized controlled trials (RCTs). These favorable effects of SGLT2 inhibitors are explained by several potential mechanisms, including the attenuation of glomerular hyperfiltration, inflammation and oxidative stress. In this review article, we discuss the renoprotective effects of SGLT2 inhibitors by integrating experimental findings with the available clinical data.

The small GTPase Rho and its effector Rho-kinase are involved in the pathogenesis of diabetic nephropathy. Accumulating evidence shows that hypoxia-inducible factor-1α (HIF-1α) is a key regulator of renal sclerosis under diabetic conditions. However, the interactions of Rho-kinase and HIF-1α in the development of renal dysfunction have not been defined. Here, we assessed whether Rho-kinase blockade attenuates HIF-1α induction and the subsequent fibrotic response using type 2 diabetic mice and cultured mesangial cells. Fasudil, a Rho-kinase inhibitor, reduced urinary albumin excretion, mesangial matrix expansion, and the expression of fibrotic mediators in db/db mice. Mechanistically, HIF-1α accumulation and the expression of its target genes that contribute to diabetic glomerulosclerosis were also prevented by fasudil in the renal cortex. In mesangial cells, Rho/Rho-kinase signaling was activated under hypoxic conditions. Further in vitro studies showed that pharmacological and genetic inhibition of Rho-kinase promoted proteasomal HIF-1α degradation, which subsequently suppressed HIF-1-dependent profibrotic gene expression by upregulation of prolyl hydroxylase 2. Thus, we found a previously unrecognized renoprotective mechanism for the effects of Rho-kinase inhibition and this could be a potential therapeutic target for the treatment of diabetic nephropathy.

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Activation of the diacylglycerol-protein kinase C (DAG-PKC) cascade by excess glucose has been implicated in vascular complications of diabetes. Its involvement in diabetic embryopathy has not been established. We examined DAG production and PKC activities in embryos and decidua of streptozotocin (STZ)-diabetic or transiently hyperglycemic mice during neural tube formation. STZ diabetes significantly increased DAG and total PKC activity in decidua (1.5-and 1.4-fold, respectively) and embryos (1.7-and 1.3-fold, respectively) on day 9.5. Membrane-associated PKC ␣, ␤II, ␦, and were increased in decidua by 1.25-to 2.8-fold. Maternal hyperglycemia induced by glucose injection on day 7.5, the day before the onset of neural tube formation, also increased DAG, PKC activity, and PKC isoforms (1.1-, 1.6-, and 1.5-fold, respectively) in the embryo on day 9.5. Notably, membrane-associated PKC activity was increased 24-fold in embryos of diabetic mice with structural defects. These data indicate that hyperglycemia just before organogenesis activates the DAG-PKC cascade and is correlated with congenital defects.

The small GTPase Rho and its downstream effector, Rho-associated coiled-coil containing protein kinase (Rho-kinase), regulate a number of cellular processes, including organization of the actin cytoskeleton, cell adhesion, and migration. While pharmacological inhibitors of Rhokinase signaling are known to block renal inflammation, the molecular basis for this effect is unclear. Here, we provide evidence that proinflammatory TNF-␣ promotes mesangial expression of macrophage colony-stimulating factor (M-CSF), a key regulator for the growth and differentiation of mononuclear phagocytes, in a Rhokinase-dependent manner. Consistent with this observation, TNF-␣-mediated renal expression of M-CSF in insulin-resistant db/db mice was downregulated by Rho-kinase inhibition. Small interfering RNAfacilitated knockdown of Rho-kinase isoforms ROCK1 and ROCK2 indicated that both isoforms make comparable contributions to regulation of M-CSF expression in mesangial cells. From a mechanistic standpoint, Western blotting and EMSA showed that Rho-kinase and its downstream target p38 MAPK regulate nuclear translocation of NF-B RelA/p65 and subsequent DNA binding activity, with no significant effects on IB␣ degradation and RelA/p65 phosphorylation. Moreover, we showed that Rho-kinase-mediated cytoskeletal organization is required for the nuclear uptake of RelA/p65. Collectively, these findings identify Rho-kinase as a critical regulator of chemokine expression and macrophage proliferation.