FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2060122043> ?p ?o ?g. }

• W2060122043 endingPage "1600" @default.
• W2060122043 startingPage "1593" @default.
• W2060122043 abstract "Although previous studies have demonstrated that diabetic nephropathy is attributable to early extracellular matrix accumulation in glomerular mesangial cells, the molecular mechanism by which high glucose induces matrix protein deposition remains not fully elucidated. Rat mesangial cells pretreated with or without inhibitors were cultured in high-glucose or advanced glycation end product (AGE) conditions. Streptozotocin-induced diabetic rats were given superoxide dismutase (SOD)-conjugated propylene glycol to scavenge superoxide. Transforming growth factor (TGF)-β1, fibronectin expression, Ras, ERK, p38, and c-Jun activation of glomerular mesangial cells or urinary albumin secretion were assessed. Superoxide, not nitric oxide or hydrogen peroxide, mediated high glucose- and AGE-induced TGF-β1 and fibronectin expression. Pretreatment with diphenyliodonium, not allopurinol or rotenone, reduced high-glucose and AGE augmentation of superoxide synthesis and fibronection expression. High glucose and AGEs rapidly enhanced Ras activation and progressively increased cytosolic ERK and nuclear c-Jun activation. Inhibiting Ras by manumycin A reduced the stimulatory effects of high glucose and AGEs on superoxide and fibronectin expression. SOD or PD98059 pretreatment reduced high-glucose and AGE promotion of ERK and c-Jun activation. Exogenous SOD treatment in diabetic rats significantly attenuated diabetes induction of superoxide, urinary albumin excretion, 8-hydroxy-2′-deoxyguanosine, TGF-β1, and fibronectin immunoreactivities in renal glomerular mesangial cells. Ras induction of superoxide activated ERK-dependent fibrosis-stimulatory factor and extracellular matrix gene transcription of mesangial cells. Reduction of oxidative stress by scavenging superoxide may provide an alternative strategy for controlling diabetes-induced early renal injury. Although previous studies have demonstrated that diabetic nephropathy is attributable to early extracellular matrix accumulation in glomerular mesangial cells, the molecular mechanism by which high glucose induces matrix protein deposition remains not fully elucidated. Rat mesangial cells pretreated with or without inhibitors were cultured in high-glucose or advanced glycation end product (AGE) conditions. Streptozotocin-induced diabetic rats were given superoxide dismutase (SOD)-conjugated propylene glycol to scavenge superoxide. Transforming growth factor (TGF)-β1, fibronectin expression, Ras, ERK, p38, and c-Jun activation of glomerular mesangial cells or urinary albumin secretion were assessed. Superoxide, not nitric oxide or hydrogen peroxide, mediated high glucose- and AGE-induced TGF-β1 and fibronectin expression. Pretreatment with diphenyliodonium, not allopurinol or rotenone, reduced high-glucose and AGE augmentation of superoxide synthesis and fibronection expression. High glucose and AGEs rapidly enhanced Ras activation and progressively increased cytosolic ERK and nuclear c-Jun activation. Inhibiting Ras by manumycin A reduced the stimulatory effects of high glucose and AGEs on superoxide and fibronectin expression. SOD or PD98059 pretreatment reduced high-glucose and AGE promotion of ERK and c-Jun activation. Exogenous SOD treatment in diabetic rats significantly attenuated diabetes induction of superoxide, urinary albumin excretion, 8-hydroxy-2′-deoxyguanosine, TGF-β1, and fibronectin immunoreactivities in renal glomerular mesangial cells. Ras induction of superoxide activated ERK-dependent fibrosis-stimulatory factor and extracellular matrix gene transcription of mesangial cells. Reduction of oxidative stress by scavenging superoxide may provide an alternative strategy for controlling diabetes-induced early renal injury. Diabetic nephropathy is characterized by excessive deposition of extracellular matrix in the kidney, causing glomerular mesangial expansion and fibrosis.1Ha H. Lee H.B. Reactive oxygen species and matrix remodeling in diabetic kidney.J Am Soc Nephrol. 2003; 14: S246-S249Google Scholar Previous studies have attributed diabetic nephropathy to hyperglycemia-induced deposition of extracellular matrix, glycosylation of matrix proteins, mesangial cell dysfunction, and global glomerular sclerosis, leading to promotion of endothelial albumin permeability.2Harris R.D. Steffes M.W. Bilous R.W. et al.Global glomerular sclerosis and glomerular arteriolar hyalinosis in insulin dependent diabetes.Kidney Int. 1991; 40: 107-114Google Scholar, 3Heilig C.W. Concepcion L.A. Riser B.L. et al.Overexpression of glucose transporters in rat mesangial cells cultured in a normal glucose milieu mimics the diabetic phenotype.J Clin Invest. 1995; 96: 1802-1814Google Scholar, 4Carraro M. Mancini W. Artero M. et al.Albumin permeability in isolated glomeruli in incipient experimental diabetes mellitus.Diabetologia. 2000; 43: 235-241Google Scholar High glucose is reported to alter renal remodeling-stimulatory or -inhibitory factor activities by increasing intercellular thrombospondin I5Wang S. Skorczewski J. Feng X. et al.Glucose up-regulates thrombospondin I gene transcription and transforming growth factor-beta activity through antagonism of cGMP-dependent protein kinase repression via upstream stimulatory factor 2.J Biol Chem. 2004; 279: 34311-34322Google Scholar or downregulating pigment epithelium-derived factor expression6Wang J.J. Zhang S.X. Lu K. et al.Decreased expression of pigment epithelium-derived factor is involved in the pathogenesis of diabetic nephropathy.Diabetes. 2005; 54: 243-250Google Scholar to control the action of transforming growth factor (TGF)-β1 on matrix protein synthesis in rodent and human mesangial cells. Advanced glycation end products (AGEs) synthesized by Amadori rearrangement of excess glucose and free amino acid in chronic hyperglycemia have been implicated as secondary irreversible metabolic products of hyperglycemia.7Singh A.K. Mo W. Dunea G. et al.Effect of glycated proteins on the matrix of glomerular epithelial cells.J Am Soc Nephrol. 1998; 9: 802-810Google Scholar We have shown that AGEs are coincided with increased inflammatory cytokine secretion in patients with chronic renal failure.8Lin C.L. Huang C.C. Yu C.C. et al.Reduction of advanced glycation end product levels by on-line hemodiafiltration in long-term hemodialysis patients.Am J Kidney Dis. 2003; 42: 524-531Google Scholar AGEs have been found to promote cell apoptosis, vascular dysfunction, and tubular epithelial-myofibroblast transient, resulting in renal failure.9Brownlee M. Biochemistry and molecular cell biology of diabetic complications.Nature. 2001; 414: 813-820Google Scholar, 10Li J.H. Wang W. Huang X.R. et al.Advanced glycation end products induce tubular epithelial-myofibroblast transient through RAGE-ERK1/2 MAP kinas signaling pathway.Am J Pathol. 2004; 164: 1389-1397Google Scholar, 11Yamagishi S. Inagaki Y. Okamoto T. et al.Advanced glycation end products inhibit de novo protein synthesis and induce TGF-beta overexpression in proximal tubular cells.Kidney Int. 2003; 63: 464-473Google Scholar The administration of AGE inhibitor has been reported to inhibit progression of extracellular matrix accumulation and glomerular injury in mice.12Ohashi S. Abe H. Takahashi T. et al.Advanced glycation end products increase collage-specific chaperone protein in mouse diabetic nephropathy.J Biol Chem. 2004; 279: 19816-19823Google Scholar Alteration of reactive radicals has been shown to regulate diabetes-induced renal injury. Increased oxidative stress is reported to coincide with vascular dysfunction, lipid peroxidation, and glyco-oxidative molecule production in diabetes.13Takeuchi M. Makita Z. Yanagisawa K. et al.Detection of noncarboxymethyllysine and carboxymethyllysine advanced glycation end products (AGE) in serum of diabetic patients.Mol Med. 1999; 5: 393-405Google Scholar, 14Liu B.F. Miyata S. Hirota Y. et al.Methylglyoxal induces apoptosis through activation of p38 mitogen-activated protein kinase in rat mesangial cells.Kidney Int. 2003; 63: 947-957Google Scholar Patients with diabetic nephropathy are associated with decreased antioxidant enzyme expression.15Hodgkinson A.D. Barltlett T. Oates P.J. et al.The response of antioxidant genes to hyperglycemia is abnormal in patients with type 1 diabetes and diabetic nephropathy.Diabetes. 2003; 52: 846-851Google Scholar Increased hydrogen peroxide inhibits Na+/glucose transporter activity in renal proximal tubular cells exposed to high-glucose conditions.16Han H.J. Lee Y.J. Park S.H. et al.High glucose-induced oxidative stress inhibits Na+/glucose cotransporter activity in renal proximal tubule cells.Am J Physiol Renal Physiol. 2005; 288: 988-996Google Scholar Decreased nitric oxide bioavailability or formation of cytotoxic peroxynitrite seems to be associated with overproduction of superoxide in high glucose, leading to human glomerular endothelial cell dysfunction and patients with diabetic nephropathy.17Hoshiyama M. Li B. Yao J. et al.Effect of high glucose on nitric oxide and endothelial nitric oxide synthase protein expression in human glomerular endothelial cells.Nephron Exp Nephron. 2003; 95: e62-e68Google Scholar Upregulated reactive oxygen radicals and TGF-β1 synthesis have been found in glomerular mesangial cells cultured in high glucose.18Ha H. Lee H.B. Reactive oxygen species as glucose signaling molecules in mesangial cells cultured under high glucose.Kidney Int. 2000; 77: S19-S25Google Scholar, 19Ziyadeh F.N. Sharma K. Ericksen M. et al.Stimulation of collagen gene expression and protein synthesis in murine mesangial cells by high glucose is mediated by autocrine activation of transforming growth factor-beta.J Clin Invest. 1994; 93: 536-542Google Scholar Reduction of oxidative stress reportedly alleviates high glucose-induced apoptosis of endothelial cells and mesangial cells,20Weidig P. McMaster D. Bayraktutan U. High glucose mediates pro-oxidant and antioxidant enzyme activities in coronary endothelial cells.Diabetes Obes Metab. 2004; 6: 432-441Google Scholar, 21Catherwood M.A. Powell L.A. Anderson P. et al.Glucose-induced oxidative stress in mesangial cells.Kidney Int. 2002; 61: 599-608Google Scholar implying that reactive radicals act as potent mediators controlling high-glucose induction of early glomerular mesangial injury. However, the molecular mechanisms and signaling transduction by which high glucose induces early mesangial cell dysfunction are not clearly understood. We hypothesize that oxidative radicals mediate high-glucose and glyco-oxidative molecule (AGEs) activation of intracellular signaling pathway to induce excess extracelluar matrix accumulation in mesangial cells. Reduction of oxidative stress by cell-permeable superoxide dismutase (SOD)-conjugated propylethyl glycol (SOD-PEG) may ameliorate diabetes promotion of fibrosis-stimulatory factor and matrix protein expression in mesangial cells. The purposes of this study are as follows: investigate which species of oxidative radicals are involved in regulating high-glucose and AGE induction of excess extracellular matrix synthesis, what the signal transduction pathways are responsible for these biological reactions, and determine whether antioxidant treatment by SOD-PEG alleviates oxidative damage, excess TGF-β1, and fibronectin synthesis of renal glomerular mesangial cell in streptozotocin-induced diabetic rats. Mesangial cells were cultured in 35 mM D-glucose or 100 μg/ml AGEs for 8 days. Real-time polymerase chain reaction (PCR) results showed that D-glucose or AGEs significantly increased TGF-β1 (Figure 1a ) and fibronectin gene expression in 1 day (Figure 1b). High glucose and AGEs elicited the highest promoting effects on TGF-β1 and fibronectin gene expression at 2 days after treatment, respectively (Figure 1). We further investigated the action mechanisms of high-glucose and AGE promotion of fibronectin expression. Pretreatments with 100 μMN-nitro-L-arginine methyl ester or 500 U/ml catalase did not significantly affect high-glucose and AGE promotion of TGF-β1 (Figure 2a ) and fibronectin mRNA expression (Figure 2b). Pretreatment with 500 U/ml SOD significantly reduced the enhancing effect of high glucose and AGEs on TGF-β1 (Figure 2a) and fibronectin expression (Figure 2b), indicating that superoxide mediates TGF-β1 and fibronectin synthesis in mesangial cells.Figure 2Superoxide, but not hydrogen peroxide or nitric oxide, mediated high glucose- or AGE-enhanced TGF-β1 and fibronectin mRNA expression of mesangial cells. Cell cultures (1 × 106 cell/well, in six-well plate) were pretreated with 500 U/ml SOD-PEG or 500 U/ml catalase-PEG or 100 μMN-nitro-L-arginine methyl ester (L-NAME) and subjected to 35 mM D-glucose or 100 μg/ml AGE treatment for 2 days. The experimental results graphed represent the relative abundance of the TGF-β1 and fibronectin gene by real-time PCR normalized to housekeeping gene β-actin. Experimental results are presented as means±s.e.'s errors calculated from six-paired triplicate experiments. *, #, and + show differences (P<0.05) from the vehicle, high glucose-, and AGE-treated groups, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Results of SOD-inhibitable cytochrome c reduction assays showed that high glucose and AGEs (Figure 3a ) rapidly increased superoxide synthesis in 1 h. Pretreatment with rotenone or allopurinol did not significantly affect high-glucose and AGE promotion of superoxide (Figure 3b), TGF-β1 (Figure 3c), and fibronectin synthesis (Figure 3d). Pretreatment with diphenyloniodium (DPI) significantly reduced the enhancing effect of high glucose and AGEs on superoxide (Figure 3b), TGF-β1 (Figure 3c), and fibronectin expression (Figure 3d), indicating that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is responsible for regulating superoxide induction. Immunoblotting results showed that AGE and high-glucose treatments promoted cytosolic extracellular signal-regulated protein kinase (ERK) (Figure 4a ) and nuclear c-Jun activation in 6 h (Figure 4c). AGE and high-glucose treatments did not evidently alter p38 activation throughout the study period (Figure 4b). Moreover, high glucose and AGEs rapidly increased Ras activation in 1 h (Figure 5a ). Pretreatment of DPI or SOD did not evidently affect high-glucose or AGE promotion of Ras activation (Figure 5b). Inhibiting Ras activity by manumycin A significantly reduced the promoting effect of high glucose and AGEs on superoxide synthesis in 1 h (Figure 5c).Figure 5High glucose and AGEs rapidly increased Ras activation. (a) Inhibition of Ras activity by manumycin A reduced the promoting effect of high glucose and AGEs on (b) Ras activation and (c) superoxide synthesis in 1 h. Cell cultures (1 × 106 cells/well, in six-well plate) were pretreated with 10 μM manumycin A and subjected to high glucose or AGE treatment for 2 days. Experimental results are presented as means values±s.e.'s calculated from six-paired triplicate experiments. *, #, and + show differences (P<0.05) from the vehicle, high glucose-, and AGE-treated groups, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Inhibition of superoxide synthesis by SOD or DPI or suppression of ERK activity by PD98059 or inhibition of Ras activity by manumycin A significantly reduced the enhancing effect of high glucose and AGEs on cytosolic ERK (Figure 6a ), and nuclear c-Jun activation (Figure 6b), TGF-β1 (Figure 6c), and fibronectin mRNA expression (Figure 6d). These findings suggest that high glucose and AGEs through Ras and NADPH oxidase induce superoxide to activate cytosolic ERK and nuclear c-Jun. In comparison with the normal control group, diabetes significantly increased systemic superoxide (Figure 7a ), which coincided with urinary albumin excretion (Figure 7b). Exogenous 50 U/kg/day SOD-PEG treatments significantly reduced the promoting effect of diabetes on systemic superoxide levels (Figure 7a) and urinary albumin excretion (Figure 7b). Immunoblotting showed that diabetes increased TGF-β1 and fibronectin levels in kidney tissue (Figure 7c). Superoxide easily reacts with nucleotide, resulting in DNA damage to form 8-hydroxy-2′-deoxyguanosine (a marker of DNA oxidative damage). We found that diabetes promoted 8-hydroxy-2′-deoxyguanosine expression in kidney tissue. SOD treatment alleviated diabetes promotion of TGF-β1, fibronectin, and 8-hydroxy-2′-deoxyguanosine expression (Figure 7c). In the absence of primary antibodies, no immunostaining of TGF-β1, fibronectin, or 8-hydroxy-2′-deoxyguanosine was visible (Figure 8 ). Mesangial cells in glomeruli showing positive for immuno-expression exhibited brown color in the cell peripheri and cytoplasm. In the control group, cells located at the glomeruli in renal cortex expressed weak fibronectin and TGF-β1 expression. In the diabetes group, mesangial cells displayed strong fibronectin and TGF-β1 expression (Figure 8). Cells positive for 8-hydroxy-2′-deoxyguanosine displayed brown staining in nuclei. Diabetes apparently induced 8-hydroxy-2′-deoxyguanosine expression of mesangial cells in glomeruli (Figure 8). Mesangial cells expressed slight 8-hydroxy-2′-deoxyguanosine, TGF-β1, and fibronectin in diabetic renal cortex after SOD treatment (Figure 8). Immunohistomorphometry results showed that diabetes significantly increased TGF-β1, fibronectin, and 8-hydroxy-2′-deoxyguanosine expression of glomerular mesangial cells compared with those in control group (Table 1 ). Exogenous SOD treatment significantly downregulated oxidative DNA damages, which coincided with reduced TGF-β1 and fibronectin in the renal glomeruli of diabetic rats (Table 1).Table 1Expression of TGF-β1, fibronectin, and 8-hydroxy-2′-deoxyguanosine in renal glomerular mesangial cells of diabetic rats with or without SOD treatmentDMExpressionNormalVehicleSOD-PEGTGF-β146±9246±25*88±19#Fibronectin35±8187±29*53±11#8-Hydroxy-2′-deoxyguanosine24±6197±31*94±12#DM, diabetes mellitus; SOD-PEG, superoxide dismutase-conjugated propylethyl glycol; TGF, transforming growth factor.Data are means±standard errors calculated from number of positive immunostained cells in glomeruli per high-power field (0.75 mm2) in five areas from three sections of eight rats.* and #, respectively, show difference from normal and DM-vehicle group, P<0.05. Open table in a new tab DM, diabetes mellitus; SOD-PEG, superoxide dismutase-conjugated propylethyl glycol; TGF, transforming growth factor. Data are means±standard errors calculated from number of positive immunostained cells in glomeruli per high-power field (0.75 mm2) in five areas from three sections of eight rats. * and #, respectively, show difference from normal and DM-vehicle group, P<0.05. In this study, elevations in TGF-β1 and fibronectin levels in mesangial cell cultures followed high-glucose and AGE stresses, which induced membrane-bound Ras- and NADPH oxidase-dependent superoxide. This reactive oxygen radical progressively activated cytosolic ERK and nuclear c-Jun. Although several previous studies have demonstrated that excess extracellular matrix expansion and increased TGF-β1 levels in mesangial cells impair glomerular function leading to diabetic nephropathy,22Tsilibary E.C. Microvascular basement membranes in diabetes mellitus.J Pathol. 2003; 200: 537-546Google Scholar, 23Lodha S. Dani D. Mehta R. et al.Angiotensin II-induced mesangial cell apoptosis: role of oxidative stress.Mol Med. 2002; 8: 830-840Google Scholar, 24Hayashida T. Schnaper H.W. High ambient glucose enhances sensitivity to TGF-beta1 via extracellular signal-regulated kinase and protein kinase Cdelta activities in human mesangial cells.J Am Soc Nephrol. 2004; 15: 2032-2041Google Scholar mechanisms underlying high glucose-induced extracellular matrix remodeling of renal mesangial cells are not well understood. Our current findings provide evidence that renal mesangial cells respond to high glucose and AGEs by raising superoxide mediation of intracellular signal transduction to promote fibrosis-stimulatory factor and extracellular matrix gene transcription. We propose that it is the increased oxidative stress and fibrosis response of mesangial cells that brings about the pathogenesis of diabetes-induced early renal injury. High glucose and AGEs have been found to disturb cell homeostasis and bioactive molecule influx,9Brownlee M. Biochemistry and molecular cell biology of diabetic complications.Nature. 2001; 414: 813-820Google Scholar possibly indicating that bioactive molecules may initialize intracellular signal. In this study, superoxide, but not hydrogen peroxide or nitric oxide, mediated TGF-β1 and fibronectin expression of mesangial cells following high-glucose and AGE stresses. In contrast to previous studies, which demonstrated high glucose-induced mitochondria-dependent superoxide synthesis in human mesangial cells,25Kiritishi S. Nishikawa T. Sonoda K. et al.Reactive oxygen species from mitochondria induces cyclooxygenase-2 gene expression in human mesangial cells: potential role in diabetic nephropathy.Diabetes. 2003; 52: 2570-2577Google Scholar we showed that NADPH oxidase was responsible for high glucose- and AGE-induced superoxide production. These findings are based on the facts that superoxide synthesis, TGF-β1, and fibronectin expression were inhibited by DPI, but not by rotenone or allopurinol. We speculate that the regulatory effect of high glucose and AGEs on superoxide synthesis is dependent on the model system and cell type used. Our findings are in agreement with those in previous studies, which have demonstrated that NADPH-dependent oxidoreductases are active in non-phagocytic cells and act as generators of redox signal in response to stresses.26Li J.M. Shah A.M. ROS generation by nonphagocytic NADPH oxidase: potential relevance in diabetic nephropathy.J Am Soc Nephrol. 2003; 14: S221-S226Google Scholar Inhibiting Ras activity by manumycin A reduced the stimulatory effect of high glucose and AGEs on Ras activation and subsequently reduced superoxide, ERK phosphorylation, and fibronectin levels. We propose that membrane-bound Ras acts as a crucial regulator to transmit extracellular stress by increasing redox reaction and progressively activating intracellular signal transduction pathways. Previous studies have demonstrated that ERK is involved in regulating reactive oxygen radical-induced apoptosis of proximal tubular cells.27Hannken T. Schroeder R. Zahner G. et al.Reactive oxygen species stimulate p44/42 mitogen-activated protein kinase and induce p27 (Kip1): role in angiotensin II-mediated hypertrophy of proximal tubular cells.J Am Soc Nephrol. 2000; 11: 1387-1397Google Scholar Mutation of the alkaline phosphatase-1-binding site or alkaline phosphatase-1 inhibitor abrogated high glucose-induced TGF-β promoter activity.28Hiramatsu N. Kasai A. Yao J. et al.AP-1-independent sensitization to oxidative stress-induced apoptosis by proteasome inhibitors.Biochem Biophys Res Commun. 2004; 316: 545-552Google Scholar The PD98059 inhibition of ERK activity and SOD scavenging of superoxide reduced the enhancing effect of high glucose and AGEs on nuclear c-Jun activation and TGF-β1 and fibronectin expression, suggesting that ERK is an important oxidant-sensitive regulator able to transmit signal imparted by high glucose into the nuclei to activate cascades of fibrosis-stimulatory factor and extracellular matrix gene transcription. We cannot exclude the possibilities that AGEs and high-glucose activation of ERK and fibrosis factor gene transcription may be directly or indirectly regulated by redox reactions or other membrane-bound signaling molecules. We suggest that Ras pathway is, at least in part, responsible for superoxide, TGF-β1, and fibronectin production of renal mesangial cells after high-glucose or AGE stress. Few previous studies have focused on the influence of high glucose-induced oxidative stress on the kidney microenvironment in vivo. Redox control of TGF-β1 and fibronectin expression in mesangial cells of the diabetic kidney have not, to our knowledge, been previously reported. This study provides the immunohistochemical evidence that mesangial cells in diabetic kidneys displayed intensive 8-hydroxy-2′-deoxyguanosine associated with TGF-β1 and fibronectin immunostaining. This phenomenon in the experimental animal model is in line with that in the cell culture model. In response to oxygen radical, DNA increases 8-hydroxy-2′-deoxyguanosine and impairs cellular metabolism and biological activities.29Kakimoto S. Inoguchi T. Sonta T. et al.Accumulation of 8-hydroxy-2′-deoxyguanosine and mitochondrial DNA deletion in kidney diabetic rats.Diabetes. 2002; 51: 1588-1595Google Scholar In our studies, suppression of superoxide levels by exogenous SOD evidently alleviated DNA oxidative damage, TGF-β1, and fibronectin levels of renal glomerular mesangial cells in diabetic rats. We suggest that renal mesangial cells actively respond to high-glucose stress by increasing TGF-β1 and fibronectin expression via superoxide mediation of DNA oxidation and subsequently impair mesangial cell function. Moreover, reduced urinary albumin secretion in diabetic rats with exogenous SOD treatment also provided a physiological explanation for oxidative stress mediation of diabetic nephropathy. The effect of antioxidants on controlling diabetic nephropathy remains controversial. Previous studies have demonstrated that increased antioxidant levels were observed in streptozotocin-induced diabetic rats.30Sechi L.A. Ceriello A. Griffin C.A. et al.Renal antioxidant enzyme mRNA levels are increased in rats with experimental diabetes mellitus.Diabetologia. 1997; 40: 23-29Google Scholar High glucose likely promoted SOD gene expression, but did not alter SOD activity. Diabetes-induced lipid oxidation and glyco-oxidation can impair SOD antioxidant activity.31Sharpe P.C. Yue K.K. Catherwood M.A. et al.The effects of glucose-induced oxidative stress on growth and extracellular matrix gene expression of vascular smooth muscle cells.Diabetologia. 1998; 41: 1210-1219Google Scholar Recent studies have demonstrated that vitamins C, lipoic acid, N-acetylcysteine, and nordihydroguairetic acid are potent antioxidants that alleviate diabetes-induced peripheral neuropathy, microangiopathy, and glomerular proteinuria in patients and experimental animals.32Anjaneyulu M. Chopra K. Nordihydroguairetic acid, a lignin, prevents oxidative stress and the development of diabetic nephropathy in rats.Pharmacology. 2004; 72: 42-50Google Scholar, 33Haak E. Usadel K.H. Kusterer K. et al.Effects of alpha-lipoic acid on microcirculation in patients with peripheral diabetic neuropathy.Exp Clin Endocrinol Diabetes. 2000; 108: 168-174Google Scholar, 34Montero A. Munger K.A. Khan R.Z. et al.F (2)-isoprostanes mediate high glucose-induced TGF-beta synthesis and glomerular proteinuria in experimental type I diabetes.Kidney Int. 2000; 58: 1963-1972Google Scholar, 35Chiarelli F. Santilli F. Sabatino G. et al.Effects of vitamin E supplementation on intracellular antioxidant enzyme production in adolescents with type 1 diabetes and early microangiopathy.Pediatr Res. 2004; 56: 720-725Google Scholar In this experimental animal model, exogenous cell-permeable SOD reduced diabetes-induced systemic superoxide levels and early renal injury, as demonstrated by intensive fibrosis-stimulatory factor and extracellular matrix accumulation. Increase in antioxidant activity by SOD gene overexpression or SOD-mimetic agents protected experimental animals against diabetes-induced glomerular injury and chronic renal failure.36Craven P.A. Melhem M.F. Phillips S.L. et al.Overexpression of Cu2+/Zn2+ superoxide dismutase protects against early diabetic glomerular injury in transgenic mice.Diabetes. 2001; 50: 2114-2125Google Scholar, 37Vaziri N.D. Dicus M. Ho N.D. et al.Oxidative stress and dysregulation of superoxide dismutase and NADPH oxidase in renal insufficiency.Kidney Int. 2003; 63: 179-185Google Scholar The in vitro and in vivo findings in this study indicated that reduction of oxidative stress by controlling superoxide synthesis alleviates early diabetic nephropathy. Moreover, modulating Ras/ERK signaling transduction may be an alterative strategy in the future for controlling diabetes induction of oxidative stress and early renal mesangial cell dysfunction. Taken together, this study provided evidence that renal mesangial cells respond to high-glucose and AGE stresses by increasing Ras-dependent superoxide production and subsequently inducing cytosolic ERK and nuclear c-Jun activation, leading to excess fibrosis-stimulatory factor and extracellular matrix accumulation. This study also provides further evidence that regulation of redox reactions or modulation of high-glucose responsive fibrosis-stimulatory signaling transduction can be an alternative strategy in the future for preventing diabetes-induced nephropathy. Rat mesangial cells (American Type Culture Center, CRL-2573™) were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) in a 5% CO2, 37°C incubator for 6 days. Cells were harvested by trypsinization and re-suspended in medium for further studies. Cell viability was determined using trypan blue exclusion. Cells (1 × 106 cells/well, in six-well plate) were cultured in Dulbecco's modified Eagle's medium, 10% fetal bovine serum without or with 35 mM D-glucose or 100 μg/ml AGEs (Calbiochem, La Jolla, CA, USA) for 48 h.38Chang P.C. Chen T.H. Chang C.J. et al.Advanced glycosylation end products induce inducible nitric oxide synthase (iNOS) expression via a p38 MAPK-dependent pathway.Kidney Int. 2004; 65: 1664-1675Google Scholar, 39Xu D. Kyriakis J.M. Phosphatidylinositol 3′-kinase-dependent activation of renal mesangial cell Ki-Ras and ERK by advanced glycation end products.J Biol Chem. 2003; 278: 39349-39355Google Scholar To investigate the role of superoxide, hydrogen peroxide, and nitric oxide in high-glucose and AGE promotion of TGF-β1 and fibronectin expression, subconfluent cell cultures were pretreated with or without the following inhibitors: 500 U/ml bovine erythrocyte SOD-PEG, catalase-PEG, 100 μMN-nitro-L-arginine methyl ester, 20 μM DPI, 30 μM allopurinol, or 30 μM rotenone (Sigma-Aldrich, St Louis, MO, USA). In some experiments, subconfluent cell cultures were pretreated with 10 μM PD98059 (Calbiochem, La Jolla, CA, USA) or manumycin A (Sigma-Aldrich, St Louis, MO, USA) to inhibit ERK or Ras activity for 4 h before high-glucose and AGE treatment, respectively. Superoxide production of mesangial cell cultures (1 × 105 cells/well, in 96-well plate) with or without high-glucose or AGE treatment or mononuclear cells (1 × 106 cells/well, in 96-well plate) of peripheral blood from diabetic rats with or without SOD treatment were determined using horse heart cytochrome c reduction assay in the absence and presence of SOD, and calculated from the molar extinction coefficient of 0.0282 μM −1 cm−1 as described previously.40Wang F.S. Wang C.J. Sheen-Chen S.M. et al.Superoxide mediates shock wave induction of ERK-dependent osteogenic transcription factor (CBFA1) and mesenchymal cell differentiation toward osteoprogenitors.J Biol Chem. 2002; 277: 10931-10937Google Scholar Total RNA was extracted and purified from 106 cells with and without high glucose or AGE treatment using Tri reagent (Sigma Chemical Inc., St Louis, MI, USA). In all, 1 μg of total RNA was reversely transcribed (RT) into complementary DNA, followed by PCR amplification using rat gene-specific primers: TGF-β1 (forward) (5′-TGA GTG GCT GTC TTT TGA CG-3′), (reverse) (5′-TGG GAC TGA TCC CAT TGA TT-3′) (154-base pair expected); fibronectin (forward) (5′-GTG GCT GCC TTC AAC TTC TC-3′), (reverse) (5′-AGT CCT TTA GGG CGG TCA AT-3′) (231-base pair expected); β-actin (forward) (5′-CGC CAA CCG CGA GAA GAT-3′), β-actin (reverse) (5′-CGT CAC CGG AGT CCA TCA-3′) (168-base pair expected). The parameters for reverse transcriptase-PCR cycling were set as described previously.41Wang F.S. Lin C.L. Chen Y.J. et al.Secreted frizzled-related protein 1 modulates glucocorticoid attenuation of osteogenic activities and bone mass.Endocrinology. 2005; 146: 2415-2423Google Scholar A volume of 25 μl of PCR mixture containing complementary DNA template equivalent to 20 ng total RNA, 2.5 μM each of forward, reverse primer, and 2 × iQ™ SYBR Green Supermix was amplified using the iCycler iQ® Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) with an initial melt at 95°C for 5 min followed by 40 cycles at 94°C for 15 s, 52°C for 20 s, and 72°C for 30 s. Rat gene-specific primer oligonucleotide sequences were used as follows: TGF-β1 (forward) (5′-TGA GTG GCT GTC TTT TGA CG-3′), (reverse) (5′-TGG GAC TGA TCC CAT TGA TT-3′) (154-base pair expected); fibronectin (forward) (5′-GTG GCT GCC TTC AAC TTC TC-3′), (reverse) (5′-AGT CCT TTA GGG CGG TCA AT-3′) (231-base pair expected); β-actin (forward) (5′-CGC CAA CCG CGA GAA GAT-3′), β-actin (reverse) (5′-CGT CAC CGG AGT CCA TCA-3′) (168-base pair expected). The number of amplification steps required to reach an arbitrary intensity threshold (Ct) was computed. The relative gene expression levels were presented as 2(−Δct ), where ΔΔCt = Cttarget - Ctβ - actin. Fold change for the treatment was defined as the relative expression, compared with the vehicle, and was calculated as 2−ΔΔCt, where ΔΔCt = ΔCttreatment - ΔCtvehicle.41Wang F.S. Lin C.L. Chen Y.J. et al.Secreted frizzled-related protein 1 modulates glucocorticoid attenuation of osteogenic activities and bone mass.Endocrinology. 2005; 146: 2415-2423Google Scholar Membrane, cytosolic, and nuclear extracts of cell cultures were prepared as described previously.42Wang F.S. Wang C.J. Chen Y.J. et al.Ras induction of superoxide activates ERK-dependent angiogenic transcription factor HIF-1alpha and VEGF-A expression in shock wave-stimulated osteoblasts.J Biol Chem. 2004; 279: 10331-10337Google Scholar Aliquots of cytosolic and nuclear extracts (50 μg) were subjected to Western blot assay. Total cytosolic ERK, p38, and nuclear c-Jun on the blots were recognized by respective antibodies against ERK, p38, and c-Jun antibodies (Cell Signaling Technology, Beverly, MA, USA), followed by horseradish peroxidase-conjugated immunoglobulin G, and visualized with chemiluminescence agents. Phosphorylated ERK, p38, and nuclear c-Jun on the blots were recognized by anti-phospho-ERK, anti-phospho-p38, and anti-phospho-c-Jun antibodies (Cell Signaling Technology, Beverly, MA, USA). Ras activation was assessed using the Ras activation assay kit (Upstate Biotechnology, Charlottesville, VA, USA) according to the manufacturer's instructions. Briefly, cell lysates were precleared with glutathione-agarose, followed by incubation with specific Raf Ras-binding domain conjugate. The immunoprecipitates were subjected to immunoblotting. Activated Ras proteins on the blots were recognized by using Ras antibodies as the primary antibody and horseradish peroxidase-immunoglobulin G as the secondary antibody, and visualized using chemiluminescence agents.42Wang F.S. Wang C.J. Chen Y.J. et al.Ras induction of superoxide activates ERK-dependent angiogenic transcription factor HIF-1alpha and VEGF-A expression in shock wave-stimulated osteoblasts.J Biol Chem. 2004; 279: 10331-10337Google Scholar Male Wistar rats, 4-month old (National Experimental Animals Production Center, Taiwan), were caged in pairs and maintained on rodent chow and water ad libitum. Rats with diabetes were induced by a single intraperitoneal injection of streptozotocin (50 mg/kg; Sigma-Aldrich, St Louis, MO, USA).43Fujikawa M. Yamazaki K. Hamazaki T. et al.Effect of eicosapentaenoic acid ethyl ester on albuminuria in streptozotocin-induced diabetic rats.J Nutr Sci Vitaminol. 1994; 40: 49-61Google Scholar At 7 days after injection, blood sugar was measured in tail blood samples. Rats with blood sugar>300 mg/dl were defined as successful induction of diabetes and then used for succeeding experiments. To equalize blood sugar level at 200 mg/dl, diabetic rats were subcutaneously administered intermittent-acting insulin (1–2 unit/kg; Montard® Novo Nordisk A/S) once a day until the animals were killed with an overdose of sodium pentobarbital. All studies were approved by the Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital. A total of 16 rats with diabetes were randomly divided into two groups. Rats in each group received intraperitoneal injection with 50 U/kg/day SOD-PEG (n=8) or normal saline (n=8) for 28 consecutive days. Eight rats without streptozotocin injection were used as normal control. Blood was harvested from each group of rats via an intracardiac needle and processed to collect mononuclear cell fraction using Ficoll-Paque gradient density separation as described previously.40Wang F.S. Wang C.J. Sheen-Chen S.M. et al.Superoxide mediates shock wave induction of ERK-dependent osteogenic transcription factor (CBFA1) and mesenchymal cell differentiation toward osteoprogenitors.J Biol Chem. 2002; 277: 10931-10937Google Scholar At day 28, urinary albumin and creatinine levels were measured using respective assay kits (Sigma-Aldrich, St Louis, MO, USA) according to the manufacturer's instructions. In a pilot study, diabetic rats received intraperitoneal injections of 0, 10, 50, and 100 U/kg/day SOD–PEG for 28 days, respectively. Both 50 and 100 U/kg/day SOD-PEG treatment evidently reduced the promoting effect of diabetes on systemic superoxide levels and urinary albumin excretion compared with injections of 0 and 10 U/kg/day SOD-PEG. Therefore, 50 U/kg/day SOD-PEG treatments were used for the succeeding experiments. Normal and diabetic rats with or without SOD-PEG treatment gained body weight throughout the study period. Rats were killed with an overdose of pentobarbital sodium and their kidneys were dissected. After perfusion with phosphate-buffered saline, fresh kidney tissues were ground with a mortar and pestle under liquid nitrogen, lysed with ice-cold phosphate-buffered saline containing 1% NP-40, 0.1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 100 μg/ml phenylmethylsulfonyl fluoride and 30 μg/ml aprotinin, and homogenized by ultrasonication. Aliquots of kidney tissue homogenate (50 μg) were subjected to assessment of TGF-β1, fibronectin, and 8-hydroxy-2′-deoxyguanosine expression using immunoblotting. Kidneys were fixed in 4% phosphate-buffered saline-buffered formaldehyde, embedded in paraffin, and then sliced longitudinally into 5-μm thick sections. Monoclonal antibodies against TGF-β1, fibronectin, and 8-hydroxy-2′-deoxyguanosine (Chemicon International Inc., Temecula, CA, USA) were used for immunohistochemistry. Immunoreactivity in sections was demonstrated using a horseradish peroxidase-3′-, 3′-diaminobenzidine kit (R & D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions, followed by counterstaining with hematoxylin, dehydration, and mounting. Sections without primary antibodies were enrolled as negative controls for immunostaining. Six regions within renal glomeruli from three sections obtained from eight rats were studied. Five areas (3 mm2) containing positive immunostained cells were microscopically and quantitatively analyzed (Carl Zeiss, Gottingen, Germany). Three random images of 0.75 mm2 from each selected region were then taken, captured, and analyzed under × 400 magnifications using a cool CCD camera and image-analysis software (Media Cybernetics, Sliver Spring, MD, USA). The number of positive immunolabeled cells in each area was counted. Renal mesangial cells were identified morphologically. All values were expressed as means±s.e.'s. The Wilcoxon test was used to evaluate differences between each sample of interest and its respective control. For analysis of time course, a multiple range of analysis of variance and Bonferroni post hoc tests were used. A P-value of <0.05 was considered statistically significant. This work was supported in part by grants from the National Science Council, Taiwan (NSC93-2314-B-182A-116), and Chang Gung Memorial Hospital (CMRPG63004), Taiwan." @default.
• W2060122043 created "2016-06-24" @default.
• W2060122043 creator A5014676064 @default.
• W2060122043 creator A5018725286 @default.
• W2060122043 creator A5046130292 @default.
• W2060122043 creator A5058841234 @default.
• W2060122043 creator A5061234672 @default.
• W2060122043 creator A5080313060 @default.
• W2060122043 creator A5084198356 @default.
• W2060122043 date "2006-05-01" @default.
• W2060122043 modified "2023-10-16" @default.
• W2060122043 title "Ras modulation of superoxide activates ERK-dependent fibronectin expression in diabetes-induced renal injuries" @default.
• W2060122043 cites W1588997471 @default.
• W2060122043 cites W1964094408 @default.
• W2060122043 cites W1978657102 @default.
• W2060122043 cites W1982371349 @default.
• W2060122043 cites W1983678974 @default.
• W2060122043 cites W1996976658 @default.
• W2060122043 cites W1997339787 @default.
• W2060122043 cites W2005454124 @default.
• W2060122043 cites W2006382243 @default.
• W2060122043 cites W2006721690 @default.
• W2060122043 cites W2008010521 @default.
• W2060122043 cites W2011035462 @default.
• W2060122043 cites W2014281402 @default.
• W2060122043 cites W2023102331 @default.
• W2060122043 cites W2024038126 @default.
• W2060122043 cites W2028886206 @default.
• W2060122043 cites W2029864685 @default.
• W2060122043 cites W2036457406 @default.
• W2060122043 cites W2044437167 @default.
• W2060122043 cites W2046401692 @default.
• W2060122043 cites W2048275130 @default.
• W2060122043 cites W2048393160 @default.
• W2060122043 cites W2049564409 @default.
• W2060122043 cites W2050154568 @default.
• W2060122043 cites W2051370085 @default.
• W2060122043 cites W2053988280 @default.
• W2060122043 cites W2074382350 @default.
• W2060122043 cites W2075578721 @default.
• W2060122043 cites W2094766335 @default.
• W2060122043 cites W2106447864 @default.
• W2060122043 cites W2106752458 @default.
• W2060122043 cites W2123503495 @default.
• W2060122043 cites W2129150771 @default.
• W2060122043 cites W2136785786 @default.
• W2060122043 cites W2137348715 @default.
• W2060122043 cites W2144721274 @default.
• W2060122043 cites W2145356697 @default.
• W2060122043 cites W2145843617 @default.
• W2060122043 cites W2159932011 @default.
• W2060122043 cites W2160201851 @default.
• W2060122043 cites W2161356142 @default.
• W2060122043 cites W2468150319 @default.
• W2060122043 cites W82935974 @default.
• W2060122043 doi "https://doi.org/10.1038/sj.ki.5000329" @default.
• W2060122043 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16572112" @default.
• W2060122043 hasPublicationYear "2006" @default.
• W2060122043 type Work @default.
• W2060122043 sameAs 2060122043 @default.
• W2060122043 citedByCount "86" @default.
• W2060122043 countsByYear W20601220432012 @default.
• W2060122043 countsByYear W20601220432013 @default.
• W2060122043 countsByYear W20601220432014 @default.
• W2060122043 countsByYear W20601220432015 @default.
• W2060122043 countsByYear W20601220432016 @default.
• W2060122043 countsByYear W20601220432018 @default.
• W2060122043 countsByYear W20601220432019 @default.
• W2060122043 countsByYear W20601220432020 @default.
• W2060122043 countsByYear W20601220432021 @default.
• W2060122043 countsByYear W20601220432022 @default.
• W2060122043 countsByYear W20601220432023 @default.
• W2060122043 crossrefType "journal-article" @default.
• W2060122043 hasAuthorship W2060122043A5014676064 @default.
• W2060122043 hasAuthorship W2060122043A5018725286 @default.
• W2060122043 hasAuthorship W2060122043A5046130292 @default.
• W2060122043 hasAuthorship W2060122043A5058841234 @default.
• W2060122043 hasAuthorship W2060122043A5061234672 @default.
• W2060122043 hasAuthorship W2060122043A5080313060 @default.
• W2060122043 hasAuthorship W2060122043A5084198356 @default.
• W2060122043 hasBestOaLocation W20601220431 @default.
• W2060122043 hasConcept C107038049 @default.
• W2060122043 hasConcept C123079801 @default.
• W2060122043 hasConcept C126322002 @default.
• W2060122043 hasConcept C134018914 @default.
• W2060122043 hasConcept C142362112 @default.
• W2060122043 hasConcept C181199279 @default.
• W2060122043 hasConcept C185592680 @default.
• W2060122043 hasConcept C189165786 @default.
• W2060122043 hasConcept C2780091579 @default.
• W2060122043 hasConcept C2780795997 @default.
• W2060122043 hasConcept C2991844318 @default.
• W2060122043 hasConcept C502942594 @default.
• W2060122043 hasConcept C55493867 @default.
• W2060122043 hasConcept C555293320 @default.
• W2060122043 hasConcept C57074206 @default.
• W2060122043 hasConcept C62478195 @default.
• W2060122043 hasConcept C71924100 @default.

• next