High glucose and angiotensin II increase β1 integrin and integrin-linked kinase synthesis in cultured mouse podocytes

Alterations of integrin α3β1 may play a role in the development of diabetic nephropathy. We have investigated the effects of high glucose and angiotensin II on the expression of integrin α3 and β1, and whether these changes are associated with integrin-linked kinase (ILK) in cultured mouse podocytes. Integrin β1 and ILK mRNA expression and protein production were rapidly up-regulated in a dose-dependent manner by high glucose and angiotensin II stimulation. ILK mRNA levels in the mouse podocytes exposed to 30 mmol/l glucose were 1.66, 1.89, and 1.28 times higher than those in control cells at 6, 24, and 72 h exposure, respectively. ILK mRNA levels in mouse podocytes exposed to 1 nM, 10 nM, and 100 nM angiotensin II for 6 h were 1.38, 1.55, and 1.93 times higher, respectively, than those in control cells. Angiotensin-II-induced integrin β1 and ILK mRNA expression was significantly inhibited by treatment with losartan (100 μM). In addition, the up-regulation of ILK synthesis induced by these stimuli was related to β1 integrin synthesis and increased ILK kinase activity. Cell adhesion assay displayed inhibitory effects when podocytes were exposed to high concentrations of angiotensin II. Interestingly, glucose and angiotensin II stimulation induced shrinkage of the cell body and elongation of the podocyte processes, a phenotype similar to that of immature podocytes. In addition, β1 integrin showed higher levels of staining on both the cell membranes and the cell-cell contact areas. Thus, high glucose and angiotensin II may affect the regulation of the integrin-ILK system in podocytes; this system may therefore play a role in the pathogenesis of diabetic nephropathy and other renal diseases affecting podocytes. The results presented in this paper have not been published previously in whole or part, except in abstract form. This work was supported by grant R01–2002–000–00139–0 from the Basic Research Program of the Korea Science & Engineering Foundation.     

Calcitriol regulates angiotensin-converting enzyme and angiotensin converting-enzyme 2 in diabetic kidney disease

To investigate the effects of calcitriol on angiotensin-converting enzyme (ACE) and ACE2 in diabetic nephropathy. Streptozotocin (STZ) induced diabetic rats were treated with calcitriol for 16 weeks. ACE/ACE2 and mitogen activated protein kinase (MAPK) enzymes were measured in the kidneys of diabetic rats and rat renal tubular epithelial cells exposed to high glucose. Calcitriol reduced proteinuria in diabetic rats without affecting calcium-phosphorus metabolism. ACE and ACE2 levels were significantly elevated in diabetic rats compared to those in control rats. The increase in ACE levels was greater than that of ACE2, leading to an elevated ACE/ACE2 ratio. Calcitriol reduced ACE levels and ACE/ACE2 ratio and increased ACE2 levels in diabetic rats. Similarly, high glucose up-regulated ACE expression in NRK-52E cells, which was blocked by the p38 MAPK inhibitor SB203580, but not the extracellular signal-regulated kinase (ERK) inhibitor FR180204 or the c-Jun N-terminal kinase (JNK) inhibitor SP600125. High glucose down-regulated ACE2 expression, which was blocked by FR180204, but not SB203580 or SP600125. Incubation of cells with calcitriol significantly inhibited p38 MAPK and ERK phosphorylation, but not JNK phosphorylation, and effectively attenuated ACE up-regulation and ACE2 down-regulation in high glucose conditions. The renoprotective effects of calcitriol in diabetic nephropathy were related to the regulation of tubular levels of ACE and ACE2, possibly by p38 MAPK or ERK, but not JNK pathways.     

High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway

Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in the development and progression of diabetic nephropathy (DN). Cyclin-dependent kinase 5 (Cdk5), who is an atypical but essential member of the Cdk family of proline-directed serine/threonine kinases, has been shown as a key regulator of podocyte differentiation, proliferation and morphology. Our previous studies demonstrated that the expression of Cdk5 was significantly increased in podocytes of diabetic rats, and was closely related with podocyte injury of DN. However, the mechanisms of how expression and activity of Cdk5 are regulated under the high glucose environment have not yet been fully elucidated. In this study, we showed that high glucose up-regulated the expression of Cdk5 and its co-activator p35 with a concomitant increase in Cdk5 kinase activity in conditionally immortalized mouse podocytes in vitro. When exposed to 30 mM glucose, transforming growth factor-β1 (TGF-β1) was activated. Most importantly, we found that SB431542, the Tgfbr1 inhibitor, significantly decreased the expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-treated podocytes. Moreover, high glucose increased the expression of early growth response-1 (Egr-1) via TGF-β1-ERK1/2 pathway in podocytes and inhibition of Egr-1 by siRNA decreased p35 expression and Cdk5 kinase activity. Furthermore, inhibition of Cdk5 kinase activity effectively alleviated podocyte apoptosis induced by high glucose or TGF-β1. Thus, the TGF-β1-ERK1/2-Egr-1 signaling pathway may regulate the p35 expression and Cdk5 kinase activity in high glucose-treated podocytes, which contributes to podocyte injury of DN.     

Reactive oxygen species, PKC-beta1, and PKC-zeta mediate high-glucose-induced vascular endothelial growth factor expression in mesangial cells

Vascular endothelial growth factor (VEGF) is implicated in the development of proteinuria in diabetic nephropathy. High ambient glucose present in diabetes stimulates VEGF expression in several cell types, but the molecular mechanisms are incompletely understood. Here primary cultured rat mesangial cells served as a model to investigate the signal transduction pathways involved in high-glucose-induced VEGF expression. Exposure to high glucose (25 mM) significantly increased VEGF mRNA evaluated by real-time PCR by 3 h, VEGF cellular protein content assessed by immunoblotting or immunofluorescence within 24 h, and VEGF secretion by 24 h. High-glucose-induced VEGF expression was blocked by an antioxidant, Tempol, and antisense oligonucleotides directed against p22(phox), a NADPH oxidase subunit. Inhibition of protein kinase C (PKC)-beta(1) with the specific pharmacological inhibitor LY-333531 or inhibition of PKC-zeta with a cell permeable specific pseudosubstrate peptide also prevented enhanced VEGF expression in high glucose. Enhanced VEGF secretion in high glucose was prevented by Tempol, PKC-beta(1), or PKC-zeta inhibition. In normal glucose (5.6 mM), overexpression of p22(phox) or constitutively active PKC-zeta enhanced VEGF expression. Hypoxia inducible factor-1alpha protein was significantly increased in high glucose only by 24 h, suggesting a possible contribution to high-glucose-stimulated VEGF expression at later time points. Thus reactive oxygen species generated by NADPH oxidase, and both PKC-beta(1) and -zeta, play important roles in high-glucose-stimulated VEGF expression and secretion by mesangial cells.     

Rapid change of glucose concentration promotes mesangial cell proliferation via VEGF: inhibitory effects of thiazolidinedione

Diabetic nephropathy is a common complication in diabetes mellitus (DM). Thiazolidinedione (TZD) is thought to ameliorate diabetic nephropathy, however, the mechanism has not been elucidated. We hypothesized that VEGF participates in the pathogenesis of diabetic nephropathy and that TZD may be beneficial for the treatment of diabetic nephropathy through its effect on VEGF. Increased VEGF expression was demonstrated in the glomeruli of DM rats and rat mesangial cells (RMC) incubated with high medium glucose. It was also demonstrated that VEGF promoted mesangial cell proliferation, which was inhibited by TZD. It was shown that a rapid fall and rise of ambient glucose concentration induces more VEGF production and cell proliferation in RMC than in cells with continuously high glucose medium, which was also inhibited by TZD. Prostaglandin J2 and protein C kinase inhibitors significantly inhibited [3H]thymidine incorporation in RMC incubated with VEGF, which was inhibited by TZD. These findings indicate that a rapid change of glucose concentration promotes RMC proliferation by the increased production of VEGF. TZD has an inhibitory action through, at least in part, PPAR-gamma.     

The Ras GTPase-activating-like protein IQGAP1 is downregulated in human diabetic nephropathy and associated with ERK1/2 pathway activation

Podocyte injury may contribute to the pathogenesis of diabetic nephropathy (DN), but the underlying mechanism of hyperglycemia induced podocyte damage is not fully understood. The Ras GTPase-activating-like protein IQGAP1 is associated to the slit diaphragm proteins and the actin cytoskeleton in podocyte. Here, we studied IQGAP1 expression alterations in human DN biopsies and extracellular signal-regulated kinase (ERK)-dependent pathways of IQGAP1 expression in podocyte under high glucose (HG) media. In vivo, analysis of renal biopsies from patients with DN revealed a significant reduction in IQGAP1 expression compared to controls. In vitro, IQGAP1 mRNA and protein expression were observed to decline under HG media at 48 h. But phosphorylation of ERK1/2 was activated under HG media at 24 h and 48 h. However, HG-induced downregulation of IQGAP1 protein was attenuated by specific ERK1/2 activation inhibitor PD98059. Taken together, these results highlight the importance of IQGAP1 in DN, and suggest that IQGAP1 expression in podocyte under HG media is modulated by the ERK1/2 pathway, which may lead to the future development of therapies targeting IQGAP1 dysfunction in podocytes in DN.     

The roles of connective tissue growth factor and integrin-linked kinase in high glucose-induced phenotypic alterations of podocytes

Emerging evidence has suggested that podocytes undergo epithelial-mesenchymal transition (EMT) in diabetic nephropathy (DN). Connective tissue growth factor (CTGF) and integrin-linked kinase (ILK) are involved in the progression of DN. However, the underlying mechanisms of EMT are not well understood. The study aimed to investigate the roles of CTGF and ILK in high glucose-induced phenotypic alterations of podocytes and determine whether ILK signaling is downstream of CTGF. The epithelial marker of nephrin and the mesenchymal marker of desmin were investigated by real-time RT-PCR and Western blotting. The results demonstrated that podocytes displayed a spreading, arborized morphology in normal glucose, whereas they had a cobblestone morphology in high glucose conditions, accompanied by decreased nephrin expression and increased desmin expression, suggesting podocytes underwent EMT. In response to high glucose, CTGF and ILK expression in podocytes were increased in a dose- and time-dependent manner, whereas the increase did not occur in the osmotic control. Furthermore, the inhibition of CTGF with anti-CTGF antibody prevented the phenotypic transition, as demonstrated by the preservation of epithelial morphology, the suppression of high glucose-induced desmin overexpression and the restoration of nephrin. Of note, the upregulation of ILK induced by high glucose was partially blocked by the inhibition of CTGF. In summary, these findings suggested that CTGF and ILK were involved in high glucose-induced phenotypic alterations of podocytes. ILK acted as a downstream kinase of CTGF and high glucose-induced ILK expression might occur through CTGF-dependent and -independent pathways.     

Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells

Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor beta1 (TGF-beta1), and addition of TGF-beta1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-beta1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFbeta1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFbeta1-driven matrix production within a diabetic milieu.     

Beyond proteinuria: VDR activation reduces renal inflammation in experimental diabetic nephropathy

Local inflammation is thought to contribute to the progression of diabetic nephropathy. The vitamin D receptor (VDR) activator paricalcitol has an antiproteinuric effect in human diabetic nephropathy at high doses. We have explored potential anti-inflammatory effects of VDR activator doses that do not modulate proteinuria in an experimental model of diabetic nephropathy to gain insights into potential benefits of VDR activators in those patients whose proteinuria is not decreased by this therapy. The effect of calcitriol and paricalcitol on renal function, albuminuria, and renal inflammation was explored in a rat experimental model of diabetes induced by streptozotocin. Modulation of the expression of mediators of inflammation by these drugs was explored in cultured podocytes. At the doses used, neither calcitriol nor paricalcitol significantly modified renal function or reduced albuminuria in experimental diabetes. However, both drugs reduced the total kidney mRNA expression of IL-6, monocyte chemoattractant protein (MCP)-1, and IL-18. Immunohistochemistry showed that calcitriol and paricalcitol reduced MCP-1 and IL-6 in podocytes and tubular cells as well as glomerular infiltration by macrophages, glomerular cell NF-κB activation, apoptosis, and extracellular matrix deposition. In cultured podocytes, paricalcitol and calcitriol at concentrations in the physiological and clinically significant range prevented the increase in MCP-1, IL-6, renin, and fibronectin mRNA expression and the secretion of MCP-1 to the culture media induced by high glucose. In conclusion, in experimental diabetic nephropathy VDR activation has local renal anti-inflammatory effects that can be observed even when proteinuria is not decreased. This may be ascribed to decreased inflammatory responses of intrinsic renal cells, including podocytes, to high glucose.     

Amelioration of diabetic nephropathy by oral administration of d-α-tocopherol and its mechanisms

Diabetic nephropathy (DN) is a diabetic vascular complication, and abnormal protein kinase C (PKC) activation from increased diacylglycerol (DG) production in diabetic hyperglycemia is one of the causes of DN. Diacylglycerol kinase (DGK) converts DG into phosphatidic acid. In other words, DGK can attenuate PKC activity by reducing the amount of DG. Recently, we reported that intraperitoneally administered d-α-tocopherol (vitamin E, αToc) induces an amelioration of DN in vivo through the activation of DGKα and the prevention of podocyte loss. However, the effect of the oral administration of αToc on DN in mice remains unknown. Here, we evaluated the effect of oral administration of αToc on DN and its molecular mechanism using streptozocin-induced diabetic mice. Consequently, the oral administration of αToc significantly ameliorated the symptoms of DN by preventing the loss of podocytes, and it was revealed that the inhibition of PKC?activity was involved in this amelioration.     

PKC alpha mediates beta-arrestin2-dependent nephrin endocytosis in hyperglycemia

Nephrin, the key molecule of the glomerular slit diaphragm, is expressed on the surface of podocytes and is critical in preventing albuminuria. In diabetes, hyperglycemia leads to the loss of surface expression of nephrin and causes albuminuria. Here, we report a mechanism that can explain this phenomenon: hyperglycemia directly enhances the rate of nephrin endocytosis via regulation of the β-arrestin2-nephrin interaction by PKCα. We identified PKCα and protein interacting with c kinase-1 (PICK1) as nephrin-binding proteins. Hyperglycemia induced up-regulation of PKCα and led to the formation of a complex of nephrin, PKCα, PICK1, and β-arrestin2 in vitro and in vivo. Binding of β-arrestin2 to the nephrin intracellular domain depended on phosphorylation of nephrin threonine residues 1120 and 1125 by PKCα. Further, cellular knockdown of PKCα and/or PICK1 attenuated the nephrin-β-arrestin2 interaction and abrogated the amplifying effect of high blood glucose on nephrin endocytosis. In C57BL/6 mice, hyperglycemia over 24 h caused a significant increase in urinary albumin excretion, supporting the concept of the rapid impact of hyperglycemia on glomerular permselectivity. In summary, we have provided a molecular model of hyperglycemia-induced nephrin endocytosis and subsequent proteinuria and highlighted PKCα and PICK1 as promising therapeutic targets for diabetic nephropathy.     

Altering expression of alpha3beta1 integrin on podocytes of human and rats with diabetes

The adhesion molecule integrin alpha3beta1 is the major receptor of podocyte to the glomerular capillary basement membrane (GBM). Since progressive alteration of the glomerular extracellular matrix (ECM) compartment leading to GBM thickening is common in diabetic nephropathy, we investigated the cellular distribution of alpha3beta1 integrin in podocytes of patients with diabetic nephropathy and streptozotocin-induced diabetic rats, and we evaluated the effects of high glucose on the cultured rat podocytes. Both human and rat kidneys were stained using the immunoelectron microscopy and immunoperoxidase technique with mouse monoclonal antibodies to human integrin alpha3 subunit. The results showed that both the number of immunogold particles and the staining of integrin alpha3 subunit on podocytes were weaker in patients with diabetic nephropathy than those of control kidneys. The staining of alpha3 on podocytes in the poorly-controlled diabetic rats was also weaker after one and three months of hyperglycemia. However, the staining was identical to controls in rats with only one week of hyperglycemia. High glucose (25 mM) but not streptozotocin in vitro suppressed the alpha3 expression of cultured rat podocytes. Our results demonstrated that the expression of integrin alpha3beta1 on podocytes was suppressed in both human and rats with diabetes, possibly due to the effects of hyperglycemia, and the suppression became more severe with the duration of diabetes.     

High glucose stimulates synthesis of fibronectin via a novel protein kinase C,Rap1b,and B-Raf signaling pathway

The molecular mechanism(s) by which high glucose induces fibronectin expression via G-protein activation in the kidney are largely unknown. This investigation describes the effect of high glucose (HG) on a small GTP-binding protein, Rap1b, expression and activation, and the relevance of protein kinase C (PKC) and Raf pathways in fibronectin synthesis in cultured renal glomerular mesangial cells (MCs). In vivo experiments revealed a dose-dependent increase in Rap1b expression in glomeruli of diabetic rat kidneys. Similarly, in vitro exposure of MCs to HG led to an up-regulation of Rap1b with concomitant increase in fibronectin (FN) mRNA and protein expression. The up-regulation of Rap1b mRNA was mitigated by the PKC inhibitors, calphostin C, and bisindolymaleimide, while also reducing HG- induced FN expression in non-transfected MCs. Overexpression of Rap1b by transfection with pcDNA 3.1/Rap1b in MCs resulted in the stimulation of FN synthesis; however, the PKC inhibitors had no significant effect in reducing FN expression in Rap1b-transfected MCs. Transfection of Rap1b mutants S17N (Ser --> Asn) or T61R (Thr --> Arg) in MCs inhibited the HG-induced increased FN synthesis. B-Raf and Raf-1 expression was investigated to assess whether Rap1b effects are mediated via the Raf pathway. B-Raf, and not Raf-1, expression was increased in MCs transfected with Rap1b. HG also caused activation of Rap1b, which was largely unaffected by anti-platelet-derived growth factor (PDGF) antibodies. HG-induced activation of Rap1b was specific, since Rap2b activation and expression of Rap2a and Rap2b were unaffected by HG. These findings indicate that hyperglycemia and HG cause an activation and up-regulation of Rap1b in renal glomeruli and in cultured MCs, which then stimulates FN synthesis. This effect appears to be PKC-dependent and PDGF-independent, but involves B-Raf, suggesting a novel PKC-Rap1b-B-Raf pathway responsible for HG-induced increased mesangial matrix synthesis, a hallmark of diabetic nephropathy.     

Stretch-induced retinal vascular endothelial growth factor expression is mediated by phosphatidylinositol 3-kinase and protein kinase C (PKC)-zeta but not by stretch-induced ERK1/2, Akt, Ras, or classical/novel PKC pathways.

Stretch-induced expression of vascular endothelial growth factor (VEGF) is thought to be important in mediating the exacerbation of diabetic retinopathy by systemic hypertension. However, the mechanisms underlying stretch-induced VEGF expression are not fully understood. We present novel findings demonstrating that stretch-induced VEGF expression in retinal capillary pericytes is mediated by phosphatidylinositol (PI) 3-kinase and protein kinase C (PKC)-zeta but is not mediated by ERK1/2, classical/novel isoforms of PKC, Akt, or Ras despite their activation by stretch. Cardiac profile cyclic stretch at 60 cpm increased VEGF mRNA expression in a time- and magnitude-dependent manner without altering mRNA stability. Stretch increased ERK1/2 phosphorylation, PI 3-kinase activity, Akt phosphorylation, and PKC-zeta activity. Signaling pathways were explored using inhibitors of PKC, MEK1/2, and PI 3-kinase; adenovirus-mediated overexpression of ERK, PKC-alpha, PKC-delta, PKC-zeta, and Akt; and dominant negative (DN) mutants of ERK, PKC-zeta, Ras, PI 3-kinase and Akt. Although stretch activated ERK1/2 through a Ras- and PKC classical/novel isoform-dependent pathway, these pathways were not responsible for stretch-induced VEGF expression. Overexpression of DN ERK and Ras had no effect on VEGF expression in these cells. In contrast, DN PI 3-kinase as well as pharmacologic inhibitors of PI 3-kinase blocked stretch-induced VEGF expression. Although stretch-induced PI 3-kinase activation increased both Akt phosphorylation and activity of PKC-zeta, VEGF expression was dependent on PKC-zeta but not Akt. In addition, PKC-zeta did not mediate stretch-induced ERK1/2 activation. These results suggest that stretch-induced expression of VEGF involves a novel mechanism dependent upon PI 3-kinase-mediated activation of PKC-zeta that is independent of stretch-induced activation of ERK1/2, classical/novel PKC isoforms, Ras, or Akt. This mechanism may play a role in the well documented association of concomitant hypertension with clinical exacerbation of neovascularization and vascular permeability.     

Knockdown of thioredoxin-interacting protein ameliorates high glucose-induced epithelial to mesenchymal transition in renal tubular epithelial cells

Epithelial to mesenchymal transition (EMT) of tubular cells contributes to the renal accumulation of matrix protein that is associated with diabetic nephropathy. Both high glucose and transforming growth factor-β (TGF-β) are able to induce EMT in cell culture. In this study, we examined the role of the thioredoxin-interacting protein (TXNIP) on EMT induced by high glucose or TGF-β1 in HK-2 cells. EMT was assessed by the expression of α-smooth muscle actin (α-SMA) and E-cadherin and the induction of a myofibroblastic phenotype. High glucose (30 mM) was shown to induce EMT at 72 h. This was blocked by knockdown of TXNIP or antioxidant NAC. Meanwhile, we also found that knockdown of TXNIP or antioxidant NAC inhibited high glucose-induced generation of reactive oxygen species (ROS), phosphorylation of p38 MAPK and ERK1/2 and expression of TGF-β1. HK-2 cells that were exposed to TGF-β1 (4 ng/ml) also underwent EMT. The expression of TXNIP gene and protein was increased in HK-2 cells treated with TGF-β1. Transfection with TXNIP shRNA was able to attenuate TGF-β1 induced-EMT. These results suggested that knockdown of TXNIP antagonized high glucose-induced EMT by inhibiting ROS production, activation of p38 MAPK and ERK1/2, and expression of TGF-β1, highlighting TXNIP as a potential therapy target for diabetic nephropathy.     

O-GlcNAcylation involvement in high glucose-induced cardiac hypertrophy via ERK1/2 and cyclin D2

Continuous hyperglycemia is considered to be the most significant pathogenesis of diabetic cardiomyopathy, which manifests as cardiac hypertrophy and subsequent heart failure. O-GlcNAcylation has attracted attention as a post-translational protein modification in the past decade. The role of O-GlcNAcylation in high glucose-induced cardiomyocyte hypertrophy remains unclear. We studied the effect of O-GlcNAcylation on neonatal rat cardiomyocytes that were exposed to high glucose and myocardium in diabetic rats induced by streptozocin. High glucose (30 mM) incubation induced a greater than twofold increase in cell size and increased hypertrophy marker gene expression accompanied by elevated O-GlcNAcylation protein levels. High glucose increased ERK1/2 but not p38 MAPK or JNK activity, and cyclin D2 expression was also increased. PUGNAc, an inhibitor of β-N-acetylglucosaminidase, enhanced O-GlcNAcylation and imitated the effects of high glucose. OGT siRNA and ERK1/2 inhibition with PD98059 treatment blunted the hypertrophic response and cyclin D2 upregulation. OGT inhibition also prevented ERK1/2 activation. We also observed concentric hypertrophy and similar changes of O-GlcNAcylation level, ERK1/2 activation and cyclin D2 expression in myocardium of diabetic rats induced by streptozocin. In conclusion, O-GlcNAcylation plays a role in high glucose-induced cardiac hypertrophy via ERK1/2 and cyclin D2.     

Glucose upregulates plasminogen activator inhibitor-1 gene expression in vascular smooth muscle cells

We investigated the effects of high concentrations of glucose on plasminogen activator inhibitor-1 (PAI-1) gene expression in cultured rat vascular smooth muscle cells (VSMC). In response to a high glucose concentration (27.5 mM), PAI-1 mRNA increased within 2 h, peaked at 4 h, remained elevated for another 4 h, then decreased to basal levels at 24 h. On the other hand, mannose at the same concentration (22.5 mM mannose plus 5.5 mM glucose) as an osmotic control had little effect on PAI-1 mRNA expression. The expression of PAI-1 mRNA that was also increased by H(2)O(2), angiotensin II, or phorbol myristate acetate, was reversed by the MAPK kinase (MEK) inhibitor PD98059 or the specific protein kinase C (PKC) inhibitor GF109203X. High glucose appeared to activate MAPK and PKC in VSMC judging from Elk-1 and AP-1 activation, respectively. PD98059 inhibited and GF109203X prevented subsequent PAI-1 induction by glucose. These results suggest that glucose at high concentrations induces PAI-1 gene expression in VSMC at least partially via MAPK and PKC activation. This direct effect of glucose might have important implications for the increased plasma concentrations of PAI-1 and possibly atherosclerosis that are associated with diabetes.     

NADPH oxidase NOX1 is involved in activation of protein kinase C and premature senescence in early stage diabetic kidney

Increased oxidative stress and activation of protein kinase C (PKC) under hyperglycemia have been implicated in the development of diabetic nephropathy. Because reactive oxygen species derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, NOX1 accelerate the translocation of PKC isoforms, NOX1 is postulated to play a causative role in the development of diabetic nephropathy. Hyperglycemia was induced in wild-type and Nox1-deficient mice (KO) by two doses of streptozotocin injection. At 3 weeks after the induction of hyperglycemia, glomeruli and cortical tubules were isolated from kidneys. The mRNA level of Nox1 was significantly upregulated in the renal cortex at 3 weeks of hyperglycemia. Urinary albumin and expression of inflammatory or fibrotic mediators were similarly elevated in diabetic wild-type and KO; however, increases in glomerular volume and mesangial matrix area were attenuated in diabetic KO. Nox1 deficiency significantly reduced the levels of renal thiobarbituric acid-reacting substances and 8-hydroxydeoxyguanosine, membranous translocation of PKCα/β, activity of PKC, and phosphorylation of p38 mitogen-activated protein kinase in the diabetic kidney. Furthermore, increased staining of senescence-associated β-galactosidase in glomeruli and cortical tubules of diabetic mice was significantly suppressed in KO. Whereas the levels of cyclin-dependent kinase inhibitors, p16^INK4A and p21^Cip1, were equivalent between the genotypes, increased levels of p27^Kip1 and γ-H2AX, a biomarker for DNA double-strand breaks, were significantly attenuated in isolated glomeruli and cortical tubules of diabetic KO. Taken together, NOX1 modulates the p38/p27^Kip1 signaling pathway by activating PKC and promotes premature senescence in early stage diabetic nephropathy.