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Objective: Accumulation of visceral fat is one of the major risk factors for the development of cardiovascular disease in peritoneal dialysis (PD) patients. Adiponectin, an adipokine commonly regarded as a negative indicator of metabolic disease, is reported to be downregulated in its gene level in end-stage renal disease patients. Since excessive fat deposit is involved in increased reactive oxygen species (ROS), PD solution (PDS) may contribute to ROS production, resulting in dysregulation of adiponectin. In this study, we tested our hypothesis that oxidative stress induced by PDS may play a role in the regulation of adiponectin., Methods: Commercial PDS containing 3.86% glucose (20 - 30 mL) was administered to SD rats for 12 weeks with and without N-acetylcysteine (NAC; 10 mmol/L). ELISA was used to quantify adiponectin in plasma and spent dialysate. For in vitro studies, fully differentiated 3T3-L1 adipocytes and adipocytes isolated from abdominal fat were treated with a high glucose solution, PDS, and H(2)O(2). Adiponectin levels in the conditioned media were measured by ELISA and immunoblot assays. The mRNA levels of adiponectin in mature adipocytes were examined using real-time RT-PCR., Results: The levels of adiponectin in plasma and spent dialysate were significantly downregulated by PDS and this effect was suppressed by NAC. In 3T3-L1 adipocytes, adiponectin secretion was inhibited by 50 mmol/L glucose, PDS diluted 2-fold, and H(2)O(2) (200 μmol/L). In addition, H(2)O(2) downregulated expression of adiponectin mRNA and secretion of adiponectin oligomer complexes., Conclusions: Our data suggest that ROS induced by conventional glucose-based PDS may contribute to pathophysiological changes in abdominal fat and downregulate adiponectin secreted from adipocytes during long-term PD.

Diabetic nephropathy is characterized by excessive accumulation of extracellular matrix (ECM) in the kidney. Reactive oxygen species (ROS) play a central role in the ECM synthesis and degradation in the glomeruli and tubulointerstitium leading to renal fibrosis. High glucose (HG) induces cellular ROS through protein kinase C (PKC)-dependent activation of NADPH oxidase and through mitochondrial metabolism. ROS thus generated activate signal transduction cascade (PKC, mitogen-activated protein kinases, and janus kinase/signal transducers and activators of transcription) and transcription factors (nuclear factor-kappaB, activated protein-1, and specificity protein-1), up-regulate transforming growth factor-beta1 (TGF-beta1), angiotensin II (Ang II), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) gene and protein expression, and promote formation of advanced glycation end-products (AGE). PKC, TGF-beta1, Ang II, and AGE also induce cellular ROS and signal through ROS leading to enhanced ECM synthesis. NF-kappaB-MCP-1 pathway is activated by ROS and promotes monocyte recruitment and profibrotic process in the kidney. HG- and TGF-beta1-induced PAI-1 up-regulation is mediated by ROS and contribute to ECM accumulation via suppression of plasmin ativity. TGF-beta1-induced myofibroblast transformation of renal tubular epithelial cells (epithelial-mesenchymal transition) is also mediated by ROS and contribute to tubulointerstitial fibrosis. In summary, ROS transduce and amplify glucose signalling in renal cells under high glucose environment and play a critical role in excessive ECM deposition in the diabetic kidney. A better understanding of ROS production and removal will allow more effective therapeutic strategies in diabetic renal and other vascular complications.

Background: Plasminogen activator inhibitor-1 (PAI-1) plays an important role in remodeling of extracellular matrix (ECM) in the glomeruli. PAI-1 is up-regulated by high glucose and is overexpressed in diabetic kidney. Since reactive oxygen species (ROS) mediate ECM accumulation in diabetic glomeruli and was recently found to mediate transforming growth factor-beta1 (TGF-beta1)-induced PAI-1 up-regulation in glomerular mesangial cells, we examined the role of ROS in high glucose-induced PAI-1 expression in cultured glomerular mesangial cells and in streptozotocin-induced diabetic rat glomeruli., Methods: Growth arrested and synchronized primary rat mesangial cells were treated with different concentrations of glucose in the presence or absence of N-acetylcysteine (NAC) or trolox, or after cellular reduced form of glutathione (GSH) depleted with DL-buthionine-(S,R)-sulfoximine (BSO). Taurine was administered to diabetic rats from 2 days to 4 weeks after streptozotocin injection. Urinary protein excretion, glomerular volume, and fractional mesangial area were measured as markers of renal injury and lipid peroxide (LPO) as an oxidative stress marker. PAI-1 mRNA expression was measured by Northern blot analysis in mesangial cells and reverse transcription-polymerase chain reaction (RT-PCR) in glomeruli, PAI-1 protein by Western blot analysis and enzyme-linked immunosorbent assay (ELISA), and plasmin activity by fluorometry., Results: High glucose significantly increased PAI-1 mRNA and protein expression and decreased plasmin activity in mesangial cells. Equimolar concentrations of l-glucose or mannitol did not affect PAI-1 expression. BSO pretreatment significantly increased basal PAI-1 expression and amplified the response to high glucose. NAC effectively inhibited high glucose-induced, but not basal, PAI-1 expression. Reduced plasmin activity in mesangial cells by high glucose was rescued by antioxidants. Anti-TGF-beta antibody inhibited both high glucose- and H(2)O(2)-induced PAI-1 up-regulation. Taurine significantly reduced plasma LPO, glomerular PAI-1 expression, glomerular volume, fractional mesangial area, and proteinuria in streptozotocin-induced diabetic rats., Conclusion: These results demonstrate that ROS mediate high glucose-induced up-regulation of PAI-1 expression in cultured mesangial cells and in diabetic glomeruli. Since both high glucose and TGF-beta1 induce cellular ROS and ROS mediate both high glucose- and TGF-beta1-induced PAI-1, ROS appear to amplify TGF-beta1 signaling in high glucose-induced PAI-1 up-regulation. Antioxidants can prevent accumulation of ECM protein in diabetic glomeruli partly by abrogating up-regulation of PAI-1 and suppression of plasmin activity.

Background: We previously demonstrated that high glucose up-regulates fibronectin mRNA and protein expression by human peritoneal mesothelial cells (HPMC) through activation of protein kinase C (PKC). PKC is known to induce cellular reactive oxygen species (ROS) and PKC-dependent activation of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has recently been shown to be responsible, in part, for increased oxidative stress in diabetes. On the other hand, high glucose-induced mitochondrial overproduction of superoxide anion was found to activate PKC. We, therefore, hypothesized that high glucose-induced activation of PKC in HPMC may increase cellular ROS and ROS, in turn, may activate PKC and thus provide signal amplification in high glucose-induced fibronectin up-regulation in HPMC., Methods: The role of ROS in high glucose- and PKC-induced fibronectin expression was examined by quantification of cellular ROS after stimulation with high glucose and phorbol 12-myristate 13-acetate (PMA), by the effect of hydrogen peroxide (H(2)O(2)) and PMA on fibronectin expression, and finally by inhibition of ROS and PKC. The source of cellular ROS was further examined by inhibition of NADPH oxidase and mitochondrial metabolism., Results: D-glucose increased dichlorofluorescein (DCF)-sensitive cellular ROS in HPMC in a dose-dependent manner. l-glucose did not induce ROS generation and cytochalasin B completely blocked high glucose-induced ROS generation, suggesting that glucose uptake, but not media hyperosmolality, is required in ROS generation in HPMC. PMA increased cellular ROS and fibronectin secretion. A single dose of H(2)O(2) or H(2)O(2) continuously generated by glucose oxidase up-regulated fibronectin expression [corrected]. Antioxidants trolox and catalase inhibited high glucose- and PMA-induced fibronectin mRNA and protein expression. Inhibition of PKC inhibited high glucose-and H(2)O(2)-induced fibronectin secretion. NADPH oxidase inhibitors (diphenyleneiodinium and apocynin) and an inhibitor of mitochondrial electron transport chain subunit I (rotenone) all effectively inhibited high glucose-induced cellular ROS generation and fibronectin secretion., Conclusion: The present data demonstrate that high glucose increases cellular ROS in HPMC through activation of PKC, NADPH oxidase, and mitochondrial metabolism and that ROS, thus generated, up-regulate fibronectin expression by HPMC. ROS are not only downstream but also upstream signaling molecules to PKC and provide signal amplification in high glucose-induced fibronectin expression by HPMC. The present data imply that cellular ROS may be potential therapeutic targets in progressive accumulation of extracellular matrix in the peritoneal tissue of long-term peritoneal dialysis patients using high glucose-containing peritoneal dialysis solutions.

Agmatine, an amine and organic cation, reduced H(2)O(2) that was generated by hyperglycemia, and transcription factors such as NF-kappaB and AP-1 activity in the mesangial cells that were exposed to high glucose. However, spermine which shares a strong nucleophilic structure with agmatine decreased the H(2)O(2) levels and AP-1, but not the NF-kappaB activity. Possible roles for agmatine and spermine in decreasing fibronectin are discussed, and the signaling pathway for agmatine-reduced fibronectin accumulation is presented.

Although high glucose (HG) has been shown to induce nuclear factor-kappaB (NF-kappaB) activation in vascular cells, the upstream regulation and the biologic significance of NF-kappaB activation in diabetic renal injury are not clear. It was, therefore, examined if HG-induced generation of reactive oxygen species (ROS) and protein kinase C (PKC) activation are involved in NF-kappaB activation in mesangial cells (MC), and the role of NF-kappaB activation in HG-induced monocyte chemoattractant protein-1 (MCP-1) expression by MC was further investigated. Recent observations suggest that MCP-1 may play a role in the development and progression of diabetic nephropathy. HG rapidly induced NF-kappaB activation in MC as estimated by electrophoretic mobility shift assay. Supershift assay suggests that most of the binding activity arose from p50/p50 and p50/p65 dimers. Antioxidants, pyrrolidine dithiocarbamate, N-acetyl-L-cystein, and trolox effectively inhibited HG-induced NF-kappaB activation in MC. HG rapidly generated dichlorofluorescin-sensitive intracellular ROS in MC as measured by laser-scanning confocal microscopy. HG also activated PKC rapidly in MC. Inhibition of PKC effectively blocked HG-induced intracellular ROS generation and NF-kappaB activation in MC. HG increased MCP-1 mRNA expression by 1.9-fold and protein secretion by 1.6-fold that of control glucose in MC transfected with control vector but not in MC transfected with dominant negative mutant inhibitor of NF-kappaB (IkappaBalphaM). Inhibition of either PKC or ROS effectively blocked HG-induced, but not basal, MCP-1 protein secretion by MC transfected with control vector. Thus this study demonstrates that HG rapidly activates NF-kappaB in MC through PKC and ROS and suggests that HG-induced NF-kappaB activation in MC may play a role in diabetic renal injury through upregulation of MCP-1 mRNA and protein expression.

Background/aim: Increased nitric oxide (NO) generation and action have been suggested to be associated with glomerular hyperfiltration and increased vascular permeability early in diabetes. However, previous studies have primarily focused on the constitutive nitric oxide synthase (cNOS) pathway present in endothelial cells, and the role of the inducible NOS (iNOS) pathway in diabetic nephropathy has remained unclear. This study examined whether high glucose modulates NO synthesis by the iNOS pathway in rat mesangial cells. In addition, the effect of inhibition of the iNOS pathway on fibronectin production was determined to examine the role of the iNOS pathway in high glucose-induced extracellular expansion by mesangial cells., Methods: NO synthesis by the iNOS pathway was evaluated by nitrite and iNOS mRNA and protein productions. The effects of protein kinase C (PKC) inhibitor and aldose reductase inhibitor on the iNOS mRNA expression and aminoguanidine, a relatively specific inhibitor of the iNOS on fibronectin protein production were examined., Results: High 30 mM glucose concentration led to significant increases in nitrite production of rat mesangial cells upon stimulation with lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) compared with control 5.6 mM glucose concentration. Mesangial iNOS mRNA expression and protein production also increased significantly in response to high glucose. The addition of calphostin C, a PKC inhibitor, and 6-bromo-1,3-dioxo-1H-benz[d,e]isoquinoline-2(3H)-acetic acid, an aldose reductase inhibitor, significantly suppressed the enhancement of iNOS mRNA expression in high glucose concentration. High glucose also significantly increased fibronectin protein production of mesangial cells upon stimulation with LPS plus IFN-gamma compared to control glucose. Aminoguanidine reversed this high glucose-induced fibronectin production at dose inhibiting iNOS mRNA expression., Conclusions: These results indicate that high glucose enhances cytokine-induced NO production by rat mesangial cells, and that the activation of PKC and aldose reductase pathway may play a role in this enhancement. In addition, high glucose-induced NO production by the iNOS pathway may promote extracellular matrix accumulation by mesangial cells under certain condition., (Copyright 2002 S. Karger AG, Basel)

Sequential effects of high glucose on mesangial cell transforming growth factor-β1 and fibronectin synthesis. Background. Transforming growth factor (TGF)-β is recognized as the final common mediator of the principal lesions of diabetic nephropathy such as renal hypertrophy and mesangial expansion. To gain better understanding of the temporal relationships between high glucose (HG) and mesangial cell (MC) TGF-β1 synthesis and between TGF-β1 and extracellular matrix (ECM) synthesis, the present study examined early and sequential effects of HG on TGF-β1 and fibronectin (FN) mRNA expression and protein synthesis. Methods. Confluent primary rat MC was stimulated with 5.6 (control) or 30 (high) mM glucose after synchronizing the growth by incubation with serum-free media for 48 hours. Results. Mesangial cell TGF-β1 mRNA expression increased significantly in six hours and continued to increase until 48 hours in response to HG. The level of TGF-β1 mRNA was 1.5-fold higher than that of control glucose at six hours and 1.8-fold at 48 hours. TGF-β activity in heat-activated conditioned media under HG increased 1.5- and 1.6-fold at 24 and 48 hours, respectively, compared to control glucose. FN mRNA increased significantly at 24 and 48 hours and 1.4-fold that of control glucose at both time points. FN protein also increased 1.5-fold that of control glucose at 48 hours. Anti-TGF-β antibody completely abolished HG-induced FN synthesis. Conclusions. The present findings demonstrate that HG stimulates TGF-β1 very early and prior to FN production and that HG-induced FN production is mediated by TGF-β. This finding is consistent with the view that TGF-β mediates increased ECM accumulation by MC under high glucose conditions. [ABSTRACT FROM AUTHOR]

Reactive oxygen species as glucose signaling molecules in mesangial cells cultured under high glucose. Background Oxidative stress is one of the important mediators of vascular complications in diabetes including nephropathy. High glucose (HG) generates reactive oxygen species (ROS) as a result of glucose auto-oxidation, metabolism, and formation of advanced glycosylation end products. The concept of ROS-induced tissue injury has recently been revised with the appreciation of new roles for ROS in signaling pathways and gene expression. Methods and Results. High glucose rapidly generated dichlorofluorescein-sensitive cytosolic ROS in rat and mouse mesangial cells. Neither L -glucose nor 3-O-methyl- D -glucose increased cytosolic ROS and cytochalasin B, an inhibitor of glucose transporter, effectively inhibited HG-induced ROS generation, suggesting that glucose uptake and subsequent metabolism are required in HG-induced cytosolic ROS generation. H 2 O 2 up-regulated fibronectin mRNA expression and protein synthesis; this up-regulation was effectively inhibited by protein kinase C (PKC) inhibitor or by depletion of PKC. The HG-induced generation of ROS was, in turn, related to activation of PKC and transcription factors nuclear factor-κ;B (NF-κ;B) and activator protein-1 (AP-1) as well as to the up-regulation of transforming growth factor-β1 (TGF-β1), fibronectin mRNA expression and protein synthesis, because antioxidants effectively inhibited HG-induced PKC, NF-κ;B, AP-1 activation, and TGF-β1 and fibronectin expression in mesangial cells cultured under HG. Conclusions Although signal transduction pathways linking HG, ROS, PKC, transcription factors, and extracellular matrix (ECM) protein synthesis in mesangial cells have not been fully elucidated, the current data provide evidence that ROS generated by glucose metabolism may act as integral signaling molecules under HG as in other membrane receptor signaling. [ABSTRACT FROM AUTHOR]

Reactive oxygen species amplify protein kinase C signaling in high glucose-induced fibronectin expression by human peritoneal mesothelial cells. Background. We previously demonstrated that high glucose up-regulates fibronectin mRNA and protein expression by human peritoneal mesothelial cells (HPMC) through activation of protein kinase C (PKC). PKC is known to induce cellular reactive oxygen species (ROS) and PKC-dependent activation of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase has recently been shown to be responsible, in part, for increased oxidative stress in diabetes. On the other hand, high glucose-induced mitochondrial overproduction of superoxide anion was found to activate PKC. We, therefore, hypothesized that high glucose-induced activation of PKC in HPMC may increase cellular ROS and ROS, in turn, may activate PKC and thus provide signal amplification in high glucose-induced fibronectin up-regulation in HPMC. Methods. The role of ROS in high glucose- and PKC-induced fibronectin expression was examined by quantification of cellular ROS after stimulation with high glucose and phorbol 12-myristate 13-acetate (PMA), by the effect of hydrogen peroxide (H2O2) and PMA on fibronectin expression, and finally by inhibition of ROS and PKC. The source of cellular ROS was further examined by inhibition of NADPH oxidase and mitochondrial metabolism. Results. d-glucose increased dichlorofluorescein (DCF)-sensitive cellular ROS in HPMC in a dose-dependent manner. l-glucose did not induce ROS generation and cytochalasin B completely blocked high glucose-induced ROS generation, suggesting that glucose uptake, but not media hyperosmolality, is required in ROS generation in HPMC. PMA increased cellular ROS and fibronectin secretion. A single dose of H2O2 or H2O2 continuously generated by glucose oxidase fibronectin expression. Antioxidants trolox and catalase inhibited high glucose- and PMA-induced fibronectin mRNA and protein expression. Inhibition of PKC inhibited high glucose- and H2O2-induced fibronectin secretion. NADPH oxidase inhibitors (diphenyleneiodinium and apocynin) and an inhibitor of mitochondrial electron transport chain subunit I (rotenone) all effectively inhibited high glucose-induced cellular ROS generation and fibronectin secretion. Conclusion. The present data demonstrate that high glucose increases cellular ROS in HPMC through activation of PKC, NADPH oxidase, and mitochondrial metabolism and that ROS, thus generated, up-regulate fibronectin expression by HPMC. ROS are not only downstream but also upstream signaling molecules to PKC and provide signal amplification in high glucose-induced fibronectin expression by HPMC. The present data imply that cellular ROS may be potential therapeutic targets in progressive accumulation of extracellular matrix in the peritoneal tissue of long-term peritoneal dialysis patients using high glucose-containing peritoneal dialysis solutions. [ABSTRACT FROM AUTHOR]