transforming-growth-factor-beta and Hyperglycemia

Post-surgical adhesions are a major complication leading to organ dysfunctions, pain, intestinal obstruction, and infertility. The incidence of post-surgical adhesion is really high. The factors involved in the pathogenesis of post-surgical fibrosis, are largely unknown, for example why two patients with similar abdominal operation have a different risks of adhesion severity? High secretion of pro-inflammatory cytokines and growth factors, includes tumour necrosis factor α (TNF-α), interleukin 6 (IL6), and transforming growth factor β (TGF-β) by persistent recruitment of immune cells and the inappropriate proliferated fibroblast/mesothelial cells can stimulate signalling pathways particularly TGF-β leads to the up-regulation of some pro-fibrotic genes that impair fibrinolytic activity and promote extracellular matrix (ECM) accumulation. In this review, we focus on the role of diabetes and hyperglycaemia on post-surgical fibrosis, including the molecular mechanisms affected by hyperglycaemia that cause inflammation, oxidative stress, and increase the expression of pro-fibrotic molecules.

Topics: Cytokines; Fibrosis; Humans; Hyperglycemia; Interleukin-6; Tissue Adhesions; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Islet dysfunction is a hallmark of type 2 diabetes mellitus (T2DM). Compelling evidence suggests that accumulation of islet amyloid in the islets of Langerhans significantly contribute to β-cell dysfunction and diabetes. Emerging evidence implicates a role for cystic fibrosis transmembrane-conductance regulator in the regulation of insulin secretion from pancreatic islets. Impaired first-phase insulin responses and glucose homeostasis have also been reported in cystic fibrosis patients. The transforming growth factor-β protein superfamily is central regulators of pancreatic cell function, and has a key role in pancreas development and pancreatic disease, including diabetes and islet dysfunction. It is also becoming clear that islet inflammation plays a key role in the development of islet dysfunction. Inflammatory changes, including accumulation of macrophages, have been documented in type 2 diabetic islets. Islet dysfunction leads to hyperglycemia and ultimately the development of diabetes. In this review, we describe these risk factors and their associations with islet dysfunction.

Topics: Amyloid; Animals; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Diabetes Mellitus, Type 2; Disease Progression; Glucose; Homeostasis; Humans; Hyperglycemia; Inflammation; Insulin; Insulin Secretion; Islets of Langerhans; Macrophages; Pancreas; Risk Factors; Transforming Growth Factor beta

The Notch signaling cascade is an evolutionarily ancient system that allows cells to interact with their microenvironmental neighbors through direct cell-cell interactions, thereby directing a variety of developmental processes. Recent research is discovering that Notch signaling is also responsive to a broad variety of stimuli beyond cell-cell interactions, including: ECM composition, crosstalk with other signaling systems, shear stress, hypoxia, and hyperglycemia. Given this emerging understanding of Notch responsiveness to microenvironmental conditions, it appears that the classical view of Notch as a mechanism enabling cell-cell interactions, is only a part of a broader function to integrate microenvironmental cues. In this review, we summarize and discuss published data supporting the idea that the full function of Notch signaling is to serve as an integrator of microenvironmental signals thus allowing cells to sense and respond to a multitude of conditions around them.

Topics: Animals; Cellular Microenvironment; Extracellular Matrix; Humans; Hyperglycemia; Hypoxia; Integrins; Models, Biological; Receptor Cross-Talk; Receptors, Notch; Signal Transduction; Stress, Physiological; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Wnt Signaling Pathway

Nephropathy is one of the major complications of diabetes which further directs to end stage renal disease. Extensive work has been done to find out the mechanisms involved in pathogenesis of the DN. Now, many researchers have been convinced that almost all of the molecular mediators and intracellular signaling pathways involved in progression of diabetic nephropathy have involvement in transforming growth factor beta (TGF- β) at some stage. In DN, hyperglycemia causes increase in the expression of TGF- β genes, TGF- β proteins and their receptors. Increased glucose level mediates these effects through activation of polyol pathway, protein kinase C pathway, hexosamine pathway, increases advanced glycation end products (AGE) and increases oxidative stress. Hyperglycemia also activates the TGF- β via activation of glucose transporters (GLUT), angiotensine II and platelet derived growth factor (PDGF). Activated TGF-β further leads to glomerular basement membrane (GBM) thickening and glomerulosclerosis through activation of connective tissue growth factor (CDGF) and vascular endothelial growth factor (VEGF). We have discussed the progression of hyperglycemia to DN via TGF- β, whose schematic presentation may serve as an effective way to understand the mechanisms and to find out an effective way for the management of diabetic nephropathy.

Topics: Diabetic Nephropathies; Disease Progression; Glucose Transporter Type 5; Glycation End Products, Advanced; Humans; Hyperglycemia; Intercellular Signaling Peptides and Proteins; Kidney Failure, Chronic; Oxidative Stress; Protein Kinase C; Receptors, Platelet-Derived Growth Factor; Signal Transduction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Oxidative stress has been linked to the pathogenesis of the major complications of diabetes in the kidney, the heart, the eye or the vasculature. NADPH oxidases of the Nox family are a major source of ROS (reactive oxygen species) and are critical mediators of redox signalling in cells from different organs afflicted by the diabetic milieu. In the present review, we provide an overview of the current knowledge related to the understanding of the role of Nox in the processes that control cell injury induced by hyperglycaemia and other predominant factors enhanced in diabetes, including the renin-angiotensin system, TGF-β (transforming growth factor-β) and AGEs (advanced glycation end-products). These observations support a critical role for Nox homologues in diabetic complications and indicate that NADPH oxidases are an important therapeutic target. Therefore the design and development of small-molecule inhibitors that selectively block Nox oxidases appears to be a reasonable approach to prevent or retard the complications of diabetes in target organs. The bioefficacy of these agents in experimental animal models is also discussed in the present review.

Topics: Diabetes Complications; Enzyme Inhibitors; Glycation End Products, Advanced; Humans; Hyperglycemia; NADPH Oxidase 4; NADPH Oxidases; Reactive Oxygen Species; Renin-Angiotensin System; Transforming Growth Factor beta

Diabetic glomerulosclerosis is characterized by accumulation of extracellular matrix proteins, mesangial expansion, and tubulointerstitial fibrosis. Hyperglycemia accelerates development of the disease, a direct result of increased intracellular glucose availability. The facilitative glucose transporter GLUT1 mediates mesangial cell glucose flux which leads to activation of signaling cascades favoring glomerulosclerosis, including pathways mediated by angiotensin II (Ang II), transforming growth factor β (TGF-β), connective tissue growth factor (CTGF), and vascular endothelial growth factor (VEGF). Ang II has both hemodynamic and metabolic effects directly inducing GLUT1 and/or matrix protein synthesis through diacyl glycerol (DAG) or protein kinase C (PKC) induction, mesangial cell stretch, and/or through transactivation of the epidermal growth factor receptor, the platelet-derived growth factor receptor, and the insulin-like growth factor-1 receptor, all of which may stimulate GLUT1 synthesis via an ERK-mediated pathway. Conversely, inhibition of Ang II effects suppresses GLUT1 and cellular glucose uptake. GLUT1-mediated glucose flux leads to metabolism of glucose via glycolysis, with induction of DAG, PKC, TGF-β1, CTGF and VEGF. VEGF in turn triggers both GLUT1 and matrix synthesis. New roles for GLUT1-mTOR and GLUT1-mechano-growth factor interactions in diabetic glomerulosclerosis have also recently been suggested. Recent mouse models confirmed roles for GLUT1 in vivo in stimulating glomerular growth factor expression, growth factor receptors and development of glomerulosclerosis. GLUT1 may therefore act in concert with cytokines and growth factors to induce diabetic glomerulosclerosis. Further clarification of the pathways involved may prove useful for the therapy of diabetic nephropathy. New directions for investigation are discussed.

Topics: Angiotensin II; Animals; Connective Tissue Growth Factor; Diabetic Nephropathies; Glucose; Glucose Transporter Type 1; Humans; Hyperglycemia; Signal Transduction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Human fibroblasts in culture have been employed as an in vitro system to investigate some pathophysiological mechanisms of diabetes mellitus also associated with the development of diabetic nephropathy. In fact, there is increasing evidence that genetic factors either convey the risk of, or protect from, diabetic nephropathy and that the expression profiles and/or the behaviour of the cultured skin fibroblasts from type 1 diabetic patients could reflect these genetic influences. On the other hand, alterations could be attributable not only to changes in DNA sequence, but also to epigenetic factors. Our aim is to make a critical overview of the studies involving primary cultures of skin fibroblasts as tools to investigate the pathophysiology of diabetic nephropathy performed until now in this area. Cultured skin fibroblasts could be useful not only for the identification of patients at risk of developing diabetic renal disease, but also for a better understanding of the complex multifactorial mechanisms leading to the long-term complications in diabetes.

Topics: Cell Proliferation; Cells, Cultured; Collagen; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Fibroblasts; Humans; Hyperglycemia; Protein Kinase C; Proteomics; Risk Assessment; Skin; Sodium-Hydrogen Exchangers; Transforming Growth Factor beta

Podocyte loss is a common feature in human diabetes as well as in experimental diabetes in rodents. Almost all components of the diabetic milieu lead to serious podocyte stress, driving the cells towards cell cycle arrest and hypertrophy, detachment and apoptosis. Common pathway components induced by high glucose and advanced glycation end-products are reactive oxygen species, cyclin-dependent kinases (p27(Kip1)) and transforming growth factor-beta. In addition, mechanical stresses by stretch or shear forces, insulin deficiency or insulin resistance are independent components resulting in podocyte apoptosis and detachment. In this review, we discuss the common pathways leading to podocyte death as well as novel pathways and concepts of podocyte dedifferentiation and detachment that influence the progression of diabetic glomerulopathy.

Topics: Actins; Apoptosis; Chemokines; Cyclin-Dependent Kinase Inhibitor p27; Cytokines; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Glucose; Glycation End Products, Advanced; Humans; Hyperglycemia; Insulin; Insulin Resistance; Podocytes; Stress, Mechanical; Transforming Growth Factor beta

Diabetes mellitus is characterized by a lack of insulin causing elevated blood glucose, often with associated insulin resistance. Over time, especially in genetically susceptible individuals, such chronic hyperglycemia can cause tissue injury. One pathological response to tissue injury is the development of fibrosis, which involves predominant extracellular matrix (ECM) accumulation. The main factors that regulate ECM in diabetes are thought to be pro-sclerotic cytokines and protease/anti-protease systems. This review will examine the key markers and regulators of tissue fibrosis in diabetes and whether their levels in biological fluids may have clinical utility.

Topics: Animals; Basement Membrane; Biomarkers; Cardiomyopathies; Connective Tissue Growth Factor; Diabetes Complications; Diabetic Angiopathies; Diabetic Nephropathies; Diabetic Retinopathy; Endothelium, Vascular; Extracellular Matrix; Fatty Liver; Fibrosis; Glycation End Products, Advanced; Heart Diseases; Heart Failure; Humans; Hyperglycemia; Immediate-Early Proteins; Insulin Resistance; Intercellular Signaling Peptides and Proteins; Liver Cirrhosis; Metalloproteases; Peptide Fragments; Procollagen; Renin-Angiotensin System; Transforming Growth Factor beta; Tunica Intima; Up-Regulation

Diabetic nephropathy characterized by proteinuria and sclerosis is the leading cause of renal failure, but its mechanisms are not well understood. Zucker Obese (ZO) rat model of obesity, insulin resistance, and hypertension has been used to study nephropathy. We hypothesize that chronically elevated intrarenal angiotensin II (ANG II) down-regulates nephrin, a key slit-pore protein and up-regulates fibrogenic molecule transforming growth factor (TGFbeta1) and thus result in progression of nephropathy in type 2 diabetes. Untreated or angiotensin converting enzyme (ACE) inhibitor, captopril, treated ZO and control Lean (ZL) rats were used to measure intrarenal levels of ANG II, glomerular nephrin, TGFbeta1, collagen and fibronectin with age using radioimmunoassay, RT-PCR and immunoblot techniques. Progression of nephropathy was established by measuring proteinuria and sclerosis. ZO rats developed obesity, hyperglycemia, hyperinsulinimia, increase in intrarenal ANG II and proteinuria. Expression of glomerular nephrin decreased while expression of TGFbeta1 and matrix components increased in ZO rats. Captopril treatment prevented increase in intrarenal ANG II, and reversed expression of nephrin, TGFbeta1, collagen and fibronectin. We conclude that in this model of type 2 diabetic nephropathy, chronically elevated intrarenal ANG II causes proteinuria via decrease in nephrin and glomerulosclerosis via TGFbeta1 mediated increase in matrix component.

Topics: Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Animals; Animals, Genetically Modified; Blood Pressure; Chromatography; Collagen; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Disease Models, Animal; Fibronectins; Glucose; Hyperglycemia; Immunoblotting; Insulin; Insulin Resistance; Kidney; Male; Models, Statistical; Obesity; Proteinuria; Radioimmunoassay; Rats; Rats, Zucker; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Topics: Animals; Combined Modality Therapy; Diabetic Nephropathies; Disease Progression; Glycation End Products, Advanced; Humans; Hyperglycemia; Hypertension, Renal; Kidney Glomerulus; Peptidyl-Dipeptidase A; Polymers; Protein Kinase C; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Transforming Growth Factor beta

Diabetic retinopathy is a micro-angiopathy affecting predominantly small vessels of the retina. Proliferative diabetic retinopathy is characterised by preretinal neovascularisation and fibrosis leading to vitreous heamorrhage and tractional retinal detachment. Chronic hyperglicemia may cause growth factor alterations that are likely to participate in tissue remodeling typical for this late complication. Numerous angiogenic and mitogenic factors have been demonstrated to be present in the eye, including transforming growth factor-beta (TGF-beta), insulin-like growth factors, fibroblast growth factor, tumor necrosis factor and vascular endothelial growth factor. TGF-beta is involved in the control of endothelial cell proliferation, adhesion and deposition of extracellular matrix, thus TGF-beta may play a role in the control of endothelial cell proliferation seen in the disease. The role of TGF-beta in diabetic retinopathy enables better understanding, and thus in the future better intensive antidiabetic therapy in aspect of ophthalmic complications.

Topics: Chronic Disease; Diabetic Retinopathy; Humans; Hyperglycemia; Receptors, Transforming Growth Factor beta; Retina; Transforming Growth Factor beta

Topics: Diabetic Nephropathies; Extracellular Matrix; Humans; Hyperglycemia; Kidney Glomerulus; Signal Transduction; Transforming Growth Factor beta

Hyperglycemia, a well recognized pathogenetic factor of long-term complications in diabetes mellitus, not only generates more reactive oxygen species but also attenuates antioxidative mechanisms through glycation of the scavenging enzymes. Therefore, oxidative stress has been considered to be a common pathogenetic factor of the diabetic complications including nephropathy. A causal relationship between oxidative stress and diabetic nephropathy has been established by observations that (1) lipid peroxides and 8-hydroxydeoxyguanosine, indices of oxidative tissue injury, were increased in the kidneys of diabetic rats with albuminuria; (2) high glucose directly increases oxidative stress in glomerular mesangial cells, a target cell of diabetic nephropathy; (3) oxidative stress induces mRNA expression of TGF-beta1 and fibronectin which are the genes implicated in diabetic glomerular injury, and (4) inhibition of oxidative stress ameliorates all the manifestations associated with diabetic nephropathy. Proposed mechanisms involved in oxidative stress associated with hyperglycemia are glucose autooxidation, the formation of advanced glycosylation end products, and metabolic stress resulting from hyperglycemia. Since the inhibition of protein kinase C (PKC) effectively blocks not only phorbol ester-induced but also high glucose- and H2O2-induced fibronectin production, the activation of PKC under diabetic conditions may also have a modulatory role in oxidative stress-induced renal injury in diabetes mellitus.

Topics: Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Humans; Hyperglycemia; Kidney Glomerulus; Oxidative Stress; Protein Kinase C; Rats; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta

The important role of hyperglycemia in the genesis of diabetic renal disease has been strengthened by tissue culture studies, experimental animal models, and clinical trials. A mechanistic understanding of the cellular and biochemical processes that link hyperglycemia with the development of diabetic nephropathy is indispensable for directing the most optimal therapeutic interventions. Likely mediators of the effects of high ambient glucose include activation of the polyol pathway, increased protein kinase C activity, nonenzymatic glycation of circulating or matrix proteins, and/or aberrant synthesis or actions of cytokines and vasomodulatory agents. The latter include angiotensin II, thromboxane, platelet-derived growth factor, endothelins, insulin-like growth factor-1, and transforming growth factor-beta. The studies we review here argue strongly in support of the hypothesis that elevated production and/or activity of transforming growth factor-beta in the kidney is a final common mediator of diabetic renal hypertrophy and mesangial matrix expansion.

Topics: Animals; Cytokines; Diabetic Nephropathies; Glucose; Humans; Hyperglycemia; Kidney; Transforming Growth Factor beta

Renal cells are a rich source of transforming growth factor (TGF)-beta, and they serve as targets for its actions. Our hypothesis that activation of the TGF-beta system in the kidney is implicated in the development of diabetic renal disease stems from the close similarity of actions of TGF-beta and high ambient glucose on renal cell growth and extracellular matrix metabolism. Proximal tubule cells and glomerular mesangial cells cultured in high glucose concentration express increased TGF-beta 1 mRNA and protein levels, and treatment with anti-TGF-beta antibodies results in prevention of the effects of high glucose to induce cellular hypertrophy and stimulate collagen biosynthesis. Several in vivo studies by different groups of investigators have reported overexpression of TGF-beta in the glomeruli in human and experimental diabetes. We have also observed that the development of renal hypertrophy in the insulin-dependent diabetic BB rat and NOD mouse is associated with increased expression of TGF-beta 1 in the kidney and that short-term administration of antibodies capable of neutralizing the activity of TGF-beta in the streptozotocin mouse model of diabetes results in attenuation of whole kidney and glomerular hypertrophy and overexpression of mRNAs encoding matrix components. Together, these findings are consistent with the hypothesis that the diabetic state stimulates TGF-beta expression in the kidney and that in turn this growth factor may mediate, in an autocrine/paracrine manner, some of the principal early manifestations of diabetic renal disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Cells, Cultured; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Extracellular Matrix; Gene Expression; Glomerular Mesangium; Humans; Hyperglycemia; Kidney; Kidney Tubules, Proximal; Mice; Mice, Inbred NOD; Rats; Rats, Inbred BB; Transforming Growth Factor beta

Diabetes mellitus is associated with typical patterns of long term vascular complications which vary with the organ involved. The microvascular kidney disease (Olgemoller and Schleicher, 1993) is characterized by thickening of the capillary basement membranes and increased deposition of extracellular matrix components (ECM), while loss of microvessels with subsequent neovascularisation is predominant in the eye and peripheral nerves. On the other hand macrovascular disease is characterized by accelerated atherosclerosis. These complications are dependent on long term hyperglycemia. Specific biochemical pathways linking hyperglycaemia to microvascular changes were proposed: the polyol pathway (Greene et al., 1987), non-enzymatic glycation of proteins (Brownlee et al., 1988), glucose autooxidation and oxidative stress (Hunt et al., 1990), hyperglycemic pseudohypoxia (Williamson et al., 1993) enhanced activation of protein kinase C by de novo-synthesis of diacyl glycerol (Lee et al., 1989; DeRubertis and Craven 1994) and others. These pathways are not mutually exclusive (Larkins and Dunlop, 1992; Pfeiffer and Schatz, 1992). They may be linked to alterations in the synthesis of growth factors particularly since atherosclerosis and angioneogenesis are associated with increased proliferation of endothelial and smooth muscle cells. Increased synthesis of ECM components is stimulated by growth factors like transforming growth factor beta (TGF beta) (Derynck et al., 1984) and insulin-like growth factor I (IGF-I) (Moran et al., 1991). This review will summarize some of the recent evidence for an involvement of growth factors in diabetic vascular complications and will attempt to assign their emergence in the sequence of events leading to vascular complications.

Topics: Animals; Arteriosclerosis; Diabetic Angiopathies; Diabetic Nephropathies; Diabetic Retinopathy; Epidermal Growth Factor; Fibroblast Growth Factor 2; Growth Hormone; Growth Substances; Humans; Hyperglycemia; Insulin Resistance; Insulin-Like Growth Factor I; Receptors, Somatotropin; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

It has been suggested that transforming growth factor beta (TGFb) mediates liver fibrosis, which can be monitored by the serum determination of the N-terminal peptide of type III procollagen (PIIIP) and laminin. Fibrosis is also an important phenomenon in patients with chronic pancreatitis (CP). In 23 patients with CP, 38 with liver cirrhosis (LC) and 20 healthy controls we compared the serum patterns of PIIIP, laminin and TGFb and assessed whether in CP these markers are correlated with exocrine and endocrine function. In patients with LC, PIIIP and laminin levels were significantly higher, whereas TGFb levels were significantly lower than those of controls. In CP patients, no significant variations were found for PIIIP and laminin, although levels were high in 7/23 and in 5/23 patients, respectively. TGFb levels in CP patients were higher than those in LC patients, levels being raised in 6/23 patients. In LC patients an inverse correlation was found between PIIIP and TGFb, whereas in CP patients, a direct correlation was found between TGFb and PIIIP. Moreover, in CP patients, there was also a positive correlation between TGFb and fasting serum glucose levels, while laminin was correlated with PABA test results.. serum biochemical markers of liver fibrosis can be considered of limited value in assessing pancreatic fibrosis; in liver cirrhosis there may be a negative feed-back regulation between TGFb production and the fibrogenetic process; and in chronic pancreatitis TGFb appears to favor fibrosis on the one hand and the development of hyperglycemia on the other.

Topics: Adult; Aged; Bilirubin; Biomarkers; Blood Glucose; Chronic Disease; Female; Fibrosis; Follow-Up Studies; Humans; Hyperglycemia; Laminin; Liver Cirrhosis; Male; Middle Aged; Pancreatitis; Procollagen; Transforming Growth Factor beta

Chronic non healing wounds in diabetic patients still impose a major problem in modern medicine. Especially additional peripheral vascular disease complicates treatment success in these patients. Thus, we analyzed the effects of 11,12 epoxyeicosatrienoic acid (EET) in a combined model of hyperglycemia and ischemia in mice. Hyperglycemia was induced by Streptozotozin 2 weeks prior to wounding. 3 days before wound creation 2 of the 3 suppling vessels of the moue ear were cautherized for ischemia. Either 11,12 EET or solvent for control was applied. Wound closure as well as TNF-α, TGF-β, SDF-1α, VEGF, CD31, and Ki67 were measured. The wounds closed on day 14.4 ± 0.4 standard deviation (SD). 11,12 EET treatment enhanced healing to 9.8 ± 0.6 SD. TNF-α level was augmented on day 9 compared to control and receded on day 18. TGF-β seemed to be elevated all days observed after 11,12 EET treatment. SDF-1α was enhanced on day 6 and 9 by 11,12 EET, and VEGF on day 6 and 18 as well as CD13 on day 3, 6, and 18. 11,12 EET did not alter Ki67. 11,12 EET are able to rescue deteriorated wound healing in a combined model of hyperglycamia and ischemia by resolution of inflammation, augmentation of neovascularization and increasing expression of TGF-β as well as SDF-1α.

Topics: Animals; Chemokine CXCL12; Diabetes Mellitus; Hyperglycemia; Ischemia; Ki-67 Antigen; Mice; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Vascular Endothelial Growth Factor A; Wound Healing

Inflammation drives the growth of tumors and is an important predictor of cancer aggressiveness. CD68, a marker of tumor-associated macrophages (TAM), is routinely used to aid in prognosis and treatment choices for breast cancer patients. We report that thrombospondin-4 (TSP-4) mediates breast cancer inflammation and growth in mouse models in response to hyperglycemia and TGF-beta by increasing TAM infiltration and production of inflammatory signals in tumors. Analysis of breast cancers and noncancerous tissue specimens from hyperglycemic patients revealed that levels of TSP-4 and of macrophage marker CD68 are upregulated in diabetic tissues. TSP-4 was colocalized with macrophages in cancer tissues. Bone-marrow-derived macrophages (BMDM) responded to high glucose and TGF-beta by upregulating TSP-4 production and expression, as well as the expression of inflammatory markers. We report a novel function for TSP-4 in breast cancer: regulation of TAM infiltration and inflammation. The results of our study provide new insights into regulation of cancer growth by hyperglycemia and TGF-beta and suggest TSP-4 as a potential therapeutic target.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Cell Line, Tumor; Disease Models, Animal; Female; Gene Expression Regulation, Neoplastic; Humans; Hyperglycemia; Inflammation; Macrophages; Male; Mammary Neoplasms, Experimental; Mice, Inbred C57BL; Mice, Knockout; Thrombospondins; Transforming Growth Factor beta

To investigate the role of LncRNA PVT1 (plasmacytoma variant translocation 1) in hyperglycemia-triggered cartilage damage using the diabetic osteoarthritis (OA) mice model.. Streptozotocin (STZ) was used to induce mouse diabetes. Knee OA model was induced through transection of anterior cruciate ligament (ACLT). Severity of arthritis was assessed histologically by Safranin O-Fast Green Staining using Mankin Scores. LncRNA PVT1 and miR-146a were detected by real-time polymerase chain reaction (PCR) in cartilage tissue. Moreover, the interaction among PVT1, miR-146a, and SMAD4 was examined by luciferase reporter assays. Mice were injected intra-articularly with ad-siRNA-PVT1 and ad-siRNA scramble control. Articular concentrations of TNF-α, IL-1, IL-6 and TGF-β1 were determined using enzyme-linked immunosorbent assay. Levels of type II Collagen (COL2A1), TGF-β1, p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4 and nuclear SMAD4 were detected by western blot analysis.. PVT1 expression was significantly increased, whereas miR-146a was markedly decreased in diabetic OA mice than in non-diabetic OA and control. Increased PVT1 expression in diabetic OA mice was significantly associated with Mankin score and reduced miR-146a as well as Collagen alpha-1(II) (COL2A1) expressions. In vivo, intra-articular injection of ad-siRNA-PVT1 efficiently increased miR-146a and COL2A1 expressions, alleviated joint inflammation, decreased the expression of pro-inflammatory mediators, and suppressed TGF-β/SMAD4 pathway in diabetic OA mice.. Our results demonstrate LncRNA PVT1 is involved in cartilage degradation in diabetic OA and correlated with disease severity. Efficiency of ad-siRNA-PVT1 in controlling joint inflammation in diabetic OA mice is associated with the suppression of the expression of miR-146a, pro-inflammatory cytokines and activation of TGF-β/SMAD4 pathway.

Topics: Adenoviridae; Animals; Base Sequence; Cartilage, Articular; Collagen Type II; Diabetes Mellitus, Experimental; Down-Regulation; HEK293 Cells; Humans; Hyperglycemia; Matrix Metalloproteinase 13; Matrix Metalloproteinase 3; Mice, Inbred C57BL; MicroRNAs; Osteoarthritis; RNA, Long Noncoding; RNA, Small Interfering; Severity of Illness Index; Signal Transduction; Smad4 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Growing evidence indicates that prorenin receptor (PRR) is upregulated in collecting duct (CD) of diabetic kidney. Prorenin is secreted by the principal CD cells, and is the natural ligand of the PRR. PRR activation stimulates fibrotic factors, including fibronectin, collagen, and transforming growth factor-β (TGF-β) contributing to tubular fibrosis. However, whether high glucose (HG) contributes to this effect is unknown. We tested the hypothesis that HG increases the abundance of PRR at the plasma membrane of the CD cells, thus contributing to the stimulation of downstream fibrotic factors, including TGF-β, collagen I, and fibronectin. We used streptozotocin (STZ) male Sprague-Dawley rats to induce hyperglycemia for 7 days. At the end of the study, STZ-induced rats showed increased prorenin, renin, and angiotensin (Ang) II in the renal inner medulla and urine, along with augmented downstream fibrotic factors TGF-β, collagen I, and fibronectin. STZ rats showed upregulation of PRR in the renal medulla and preferential distribution of PRR on the apical aspect of the CD cells. Cultured CD M-1 cells treated with HG (25 mM for 1 h) showed increased PRR in plasma membrane fractions compared to cells treated with normal glucose (5 mM). Increased apical PRR was accompanied by upregulation of TGF-β, collagen I, and fibronectin, while PRR knockdown prevented these effects. Fluorescence resonance energy transfer experiments in M-1 cells demonstrated augmented prorenin activity during HG conditions. The data indicate HG stimulates profibrotic factors by inducing PRR translocation to the plasma membrane in CD cells, which in perspective, might be a novel mechanism underlying the development of tubulointerstitial fibrosis in diabetes mellitus.

Topics: Animals; Collagen; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Disease Models, Animal; Fibronectins; Gene Expression Regulation; Glucose; Humans; Hyperglycemia; Kidney Tubules, Collecting; Prorenin Receptor; Rats; Receptors, Cell Surface; Transforming Growth Factor beta

Topics: Animals; Antioxidants; Cell Line; Cell Proliferation; Glutathione; Heme Oxygenase-1; Hydrogen Peroxide; Hyperglycemia; Oxidative Stress; Rats; Reactive Oxygen Species; Retinal Ganglion Cells; Signal Transduction; Transforming Growth Factor beta; Transforming Growth Factors

Topics: Animals; Antihypertensive Agents; Blood Glucose; Blood Urea Nitrogen; Captopril; Creatinine; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Diuretics; Drug Combinations; Drug Repositioning; Gene Expression Regulation; Hyperglycemia; Hypoglycemic Agents; Male; MicroRNAs; Rats; Rats, Wistar; Spironolactone; Streptozocin; Transforming Growth Factor beta; Treatment Outcome

Topics: AMP-Activated Protein Kinases; Animals; Antimicrobial Cationic Peptides; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Cardiomyopathies; Echocardiography; Endothelial Cells; Endothelium; Epithelial-Mesenchymal Transition; Fibrosis; Glucose; Hemodynamics; Hyperglycemia; Male; Mice; Mice, Inbred C57BL; Pyrazoles; Pyrroles; Signal Transduction; Transforming Growth Factor beta

Metformin was found to protect against hyperglycemia-induced injury in osteoblasts, but the cellular mechanisms involved remain unclear. Therefore, the aim of this study was to determine the effect of metformin on hyperglycemia-induced apoptosis and differentiation suppression in osteoblasts and to explore its relationships with the TLR4 signaling pathway.. A mouse osteoblast cell line, MC3T3-E1, and a diabetic rat model were used to survey the protective effects of metformin on hyperglycemia-induced injury. TLR4 expression was altered using small interfering (si)RNA and lentivirus-mediated TLR4 overexpression. LPS was used as a specific TLR4 activator, and CLI-095 was used as a TLR4 inhibitor.. Metformin improved osteoblast differentiation, reduced apoptosis in hyperglycemic osteoblasts, and inhibited TLR4, MyD88 and NF-κB expression in a dose-dependent manner. Down-regulating the expression or inhibiting the activity of TLR4 enhanced these protective effects of metformin on osteoblast differentiation, cell viability and cell apoptosis in hyperglycemic conditions, whereas up-regulating the expression or activating the activity of TLR4 had the opposite effects. Activating NF-κB suppressed the protective effects of metformin, while inhibiting NF-κB activity had the opposite effects. Metformin increased ALP and OCN secretion, enhanced BMP-2 expression, improved bone mineral density (BMD), and decreased TLR4, MyD88 and NF-κB levels in the femur tissues of diabetic rats.. Taken together our experimentation support the hypothesis that metformin may alleviate hyperglycemia-induced apoptosis and differentiation suppression in osteoblasts by inhibiting the TLR4/MyD88/NF-κB signaling pathway.

Topics: Animals; Apoptosis; Bone Density; Bone Morphogenetic Protein 2; Cell Differentiation; Cell Line; Diabetes Mellitus, Experimental; Down-Regulation; Hyperglycemia; Hypoglycemic Agents; Male; Metformin; Mice; NF-kappa B; Osteoblasts; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Signal Transduction; Toll-Like Receptor 4; Transforming Growth Factor beta; Up-Regulation

The occurrence of biochemical alterations that last for a long period of time in diabetic individuals even after adequate handling of glycemia is an intriguing phenomenon named metabolic memory. In this study, we show that a kidney pathway is gradually altered during the course of diabetes and remains persistently changed after late glycemic control in streptozotocin-induced diabetic rats. This pathway comprises an early decline of uric acid clearance and pAMPK expression followed by fumarate accumulation, increased TGF-β expression, reduced PGC-1α expression, and downregulation of methylation and hydroxymethylation of mitochondrial DNA. The sustained decrease of uric acid clearance in treated diabetes may support the prolonged kidney biochemical alterations observed after tight glycemic control, and this regulation is likely mediated by the sustained decrease of AMPK activity and the induction of inflammation. This manuscript proposes the first consideration of the possible role of hyperuricemia and the underlying biochemical changes as part of metabolic memory in diabetic nephropathy development after glycemic control.

Topics: Adenylate Kinase; Animals; Blood Glucose; Diabetes Mellitus, Experimental; DNA, Mitochondrial; Fasting; Fumarates; Hyperglycemia; Kidney; Male; Malondialdehyde; Mitochondria; Models, Biological; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Phosphorylation; Rats, Wistar; Transforming Growth Factor beta

Atherosclerosis, a major macrovascular complication associated with diabetes, poses a tremendous burden on national health care expenditure. Despite extensive efforts, cost-effective remedies are unknown. Therapies for atherosclerosis are challenged by a lack of targeted drug delivery approaches. Toward this goal, we turn to a biology-derived drug delivery system utilizing nanoparticles formed by the plant virus, Cowpea mosaic virus (CPMV). The aim herein is to investigate the anti-atherogenic potential of the beneficial mineral nutrient, trivalent chromium, loaded CPMV nanoparticles in human aortic smooth muscle cells (HASMC) under hyperglycemic conditions. A non-covalent loading protocol is established yielding CrCl3-loaded CPMV (CPMV-Cr) carrying 2000 drug molecules per particle. Using immunofluorescence microscopy, we show that CPMV-Cr is readily taken up by HASMC in vitro. In glucose (25 mM)-stimulated cells, 100 nM CPMV-Cr inhibits HASMC proliferation concomitant to attenuated proliferating cell nuclear antigen (PCNA, proliferation marker) expression. This is accompanied by attenuation in high glucose-induced phospho-p38 and pAkt expression. Moreover, CPMV-Cr inhibits the expression of pro-inflammatory cytokines, transforming growth factor-β (TGF-β) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), in glucose-stimulated HASMCs. Finally glucose-stimulated lipid uptake is remarkably abrogated by CPMV-Cr, revealed by Oil Red O staining. Together, these data provide key cellular evidence for an atheroprotective effect of CPMV-Cr in vascular smooth muscle cells (VSMC) under hyperglycemic conditions that may promote novel therapeutic ventures for diabetic atherosclerosis.

Topics: Aorta; Atherosclerosis; Azo Compounds; Cell Proliferation; Cells, Cultured; Chlorides; Chromium Compounds; Comovirus; Cytokines; Drug Delivery Systems; Glucose; Humans; Hyperglycemia; Lipids; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Myocytes, Smooth Muscle; Nanoparticles; NF-kappa B; Proliferating Cell Nuclear Antigen; Spectrophotometry, Ultraviolet; Transforming Growth Factor beta

The generation of reactive oxygen species (ROS), particularly superoxide, by damaged or dysfunctional mitochondria has been postulated to be an initiating event in the development of diabetes complications. The glomerulus is a primary site of diabetic injury, and podocyte injury is a classic hallmark of diabetic glomerular lesions. In streptozotocin-induced type 1 diabetes, podocyte-specific EGF receptor (EGFR) knockout mice (EGFR(podKO)) and their wild-type (WT) littermates had similar levels of hyperglycemia and polyuria, but EGFR(podKO) mice had significantly less albuminuria and less podocyte loss compared with WT diabetic mice. Furthermore, EGFR(podKO) diabetic mice had less TGF-β1 expression, Smad2/3 phosphorylation, and glomerular fibronectin deposition. Immunoblotting of isolated glomerular lysates revealed that the upregulation of cleaved caspase 3 and downregulation of Bcl2 in WT diabetic mice were attenuated in EGFR(podKO) diabetic mice. Administration of the SOD mimetic mito-tempol or the NADPH oxidase inhibitor apocynin attenuated the upregulation of p-c-Src, p-EGFR, p-ERK1/2, p-Smad2/3, and TGF-β1 expression and prevented the alteration of cleaved caspase 3 and Bcl2 expression in glomeruli of WT diabetic mice. High-glucose treatment of cultured mouse podocytes induced similar alterations in the production of ROS; phosphorylation of c-Src, EGFR, and Smad2/3; and expression of TGF-β1, cleaved caspase 3, and Bcl2. These alterations were inhibited by treatment with mito-tempol or apocynin or by inhibiting EGFR expression or activity. Thus, results of our studies utilizing mice with podocyte-specific EGFR deletion demonstrate that EGFR activation has a major role in activating pathways that mediate podocyte injury and loss in diabetic nephropathy.

Topics: Albuminuria; Animals; Caspase 3; Diabetic Nephropathies; ErbB Receptors; Hyperglycemia; Male; Mice, Knockout; Podocytes; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Smad Proteins, Receptor-Regulated; src-Family Kinases; Transforming Growth Factor beta

Mesenchymal stromal/stem cells (MSCs) are multipotent cells that have the ability to express and secrete a wide range of immunomodulatory molecules, cytokines, growth factors and antiapoptotic proteins. MSCs modulate both innate and adaptive immune responses making them potential candidates for the treatment of patients with type 1 diabetes mellitus (T1D). However, one problem frequently associated with the systemic MSCs administration is the entrapment of the cells mainly in the lungs. In this sense, trying to avoid the lung barrier, the purpose of this study was to evaluate the long-term therapeutic efficacy and biodistribution of allogeneic adipose tissue-derived MSCs (ADMSCs) injected via two different delivery routes (intrasplenic/I.Sp and intrapancreatic/I.Pc) in a murine model of diabetes induced by streptozotocin (STZ).. Experimental diabetes was induced in C57BL/6 male mice by multiple low-doses of STZ. MSCs were isolated from adipose tissue (ADMSCs) of Balb/c mice. A single dose of 1x10(6) ADMSCs was microinjected into the spleen or into the pancreas of diabetic mice. Control group received injection of PBS by I.Sp or I.Pc delivery routes. Glycemia, peripheral glucose response, insulin-producing β cell mass, regulatory T cell population, cytokine profile and cell biodistribution were evaluated after ADMSCs/PBS administration.. ADMSCs injected by both delivery routes were able to decrease blood glucose levels and improve glucose tolerance in diabetic mice. ADMSCs injected by I.Sp route reverted hyperglycemia in 70% of diabetic treated mice, stimulating insulin production by pancreatic β cells. Using the I.Pc delivery route, 42% of ADMSCs-treated mice responded to the therapy. Regulatory T cell population remained unchanged after ADMSCs administration but pancreatic TGF-β levels were increased in ADMSCs/I.Sp-treated mice. ADMSCs administrated by I.Sp route were retained in the spleen and in the liver and ADMSCs injected by I.Pc route remained in the pancreas. However, ADMSCs injected by these delivery routes remained only few days in the recipients.. Considering the potential role of MSCs in the treatment of several disorders, this study reports alternative delivery routes that circumvent cell entrapment into the lungs promoting beneficial therapeutic responses in ADMSCs-treated diabetic mice.

Topics: Adipose Tissue; Animals; Blood Glucose; Cell Movement; Cells, Cultured; Diabetes Mellitus, Experimental; Hyperglycemia; Insulin; Insulin-Secreting Cells; Lung; Lymphocyte Count; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Transgenic; Spleen; Streptozocin; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Esculetin (6, 7- dihydroxycoumarin) was found to be protective against hepatic and renal damage associated with Streptozotocin (STZ) induced type 1 diabetes, because of its radical scavenging property. However, there are no reports regarding its effect on vascular dysfunction under hyperinsulinemic and hyperglycemic conditions. Hence, the present study aimed to investigate the effect of esculetin on vascular dysfunction under these conditions. Non-genetic model of hyperinsulinemia and hyperglycemia were developed by high fat diet (HFD) feeding and HFD + Streptozotocin (STZ, 35 mg/kg, I.P) treatment in Wistar rats, respectively. Esculetin was administered at 50 and 100 mg/kg/day (P.O, 2 weeks) doses and biochemical, vascular reactivity and immunohistochemical experiments were performed to assess the effect of esculetin on vascular dysfunctions. Esculetin treatment significantly attenuates metabolic perturbations, alleviates insulin levels in hyperinsulinemic condition. Thoracic aorta of hyperinsulinemic and hyperglycemic rats showed hyper-responsiveness to Ang II mediated contraction and impaired acetylcholine mediated relaxation, and esculetin attenuates alterations in vascular reactivity to Ang II and acetylcholine challenges. In addition, immunohistochemical evaluations revealed that esculetin prevents increase in AT1R, AT2R, Keap1, TGF-β, and decrease in ACE2 expression in aorta of hyperinsulinemic and hyperglycemic rats.

Topics: Acetylcholine; Angiotensin II; Angiotensin-Converting Enzyme 2; Animals; Antioxidants; Aorta; Blood Pressure; Diabetes Mellitus, Experimental; Hyperglycemia; Hyperinsulinism; Hypertension; Male; Peptidyl-Dipeptidase A; Rats, Wistar; Transforming Growth Factor beta; Umbelliferones

1-[4-[2-(4-Bromobenzene-sulfonamino)ethyl]phenylsulfonyl]-3-(trans-4-methylcyclohexyl) urea (G004, CAS865483-06-3) is a synthetic sulfonylurea, incorporating the hypoglycemic active structure of glimepiride (CAS 93479-97-1) and anti-TXA2 receptor (TP) active structure of BM-531(CAS 284464-46-6). In this study, we evaluated the effect of G004 on hyperglycemia and dyslipidemia as well as diabetic nephropathy (DN) in db/db mice by gavage over 90 consecutive days of treatment. The fasting blood glucose (FBG), glucose, and insulin tolerance as well as dyslipidemia were effectively ameliorated in db/db mice treated with G004. Interestingly, renal histological results of db/db mice revealed that G004 markedly reversed the expansion of mesangial extracellular matrix (ECM), the early hallmark of DN. Indeed, G004 treatment downregulated the renal expressions of type 4 collagen (Col IV) and transforming growth factor-β1 (TGF-β1) in db/db mice. In addition, imbalance in expressions of matrix metalloproteinase-9 (MMP-9) and its tissue inhibitor-1 (TIMP-1) in db/db mice kidneys was observed. However, G004 increased and decreased the expressions of MMP-9 and TIMP-1, respectively. It is well known that TGF-β pathway signaling plays an essential role in hyperglycemia-induced cell protein synthesis. On the other hand, MMP/TIMP system is responsible for the breakdown and turnover of ECM. Thus, we speculate that G004 possibly attenuated ECM accumulation via remodeling the synthesis and degradation of ECM component Col IV through modulation in TGF-β1 and MMP-9/TIMP-1 expressions in kidneys of db/db mice. Results from this study provide a strong rationale for G004 to be an efficient glucose-controlling agent with significant reno-protective properties.

Topics: Animals; Blood Glucose; Cholesterol; Collagen Type IV; Diabetes Mellitus; Diabetic Nephropathies; Disease Models, Animal; Dyslipidemias; Hyperglycemia; Hypoglycemic Agents; Kidney; Liver; Male; Matrix Metalloproteinase 9; Mice; Organ Size; Protective Agents; Sulfonylurea Compounds; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Triglycerides

The anti-inflammatory cytokine IL-35 is produced by regulatory T (Treg) cells to suppress autoimmune and inflammatory responses. The role of IL-35 in type 1 diabetes (T1D) remains to be answered. To elucidate this, we investigated the kinetics of Treg cell response in the multiple low dose streptozotocin induced (MLDSTZ) T1D model and measured the levels of IL-35 in human T1D patients. We found that Treg cells were increased in MLDSTZ mice. However, the Treg cells showed a decreased production of anti-inflammatory (IL-10, IL-35, TGF-β) and increased pro-inflammatory (IFN-γ, IL-2, IL-17) cytokines, indicating a phenotypic shift of Treg cells under T1D condition. IL-35 administration effectively both prevented development of, and counteracted established MLDSTZ T1D, seemingly by induction of Eos expression and IL-35 production in Treg cells, thus reversing the phenotypic shift of the Treg cells. IL-35 administration reversed established hyperglycemia in NOD mouse model of T1D. Moreover, circulating IL-35 levels were decreased in human T1D patients compared to healthy controls. These findings suggest that insufficient IL-35 levels play a pivotal role in the development of T1D and that treatment with IL-35 should be investigated in treatment of T1D and other autoimmune diseases.

Topics: Animals; Cytokines; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Forkhead Transcription Factors; Humans; Hyperglycemia; Interleukin-10; Interleukin-2; Interleukins; Leukocytes, Mononuclear; Male; Mice; Mice, Inbred NOD; Minor Histocompatibility Antigens; Phenotype; Receptors, Cytokine; Recombinant Proteins; Spleen; Streptozocin; T-Lymphocytes, Helper-Inducer; T-Lymphocytes, Regulatory; Thymus Gland; Transforming Growth Factor beta

Increase in matrix protein content in the kidney is a cardinal feature of diabetic kidney disease. While renal matrix protein content is increased by chronic hyperglycemia, whether it is regulated by acute elevation of glucose and insulin has not been addressed. In this study, we aimed to evaluate whether short duration of combined hyperglycemia and hyperinsulinemia, mimicking the metabolic environment of prediabetes and early type 2 diabetes, induces kidney injury. Normal rats were subjected to either saline infusion (control, n = 4) or 7 h of combined hyperglycemic-hyperinsulinemic clamp (HG+HI clamp; n = 6). During the clamp, plasma glucose and plasma insulin were maintained at about 350 mg/dl and 16 ng/ml, respectively. HG+HI clamp increased the expression of renal cortical transforming growth factor-β (TGF-β) and renal matrix proteins, laminin and fibronectin. This was associated with the activation of SMAD3, Akt, mammalian target of rapamycin (mTOR) complexes, and ERK signaling pathways and their downstream target events in the initiation and elongation phases of mRNA translation, an important step in protein synthesis. Additionally, HG+HI clamp provoked renal inflammation as shown by the activation of Toll-like receptor 4 (TLR4) and infiltration of CD68-positive monocytes. Urinary F2t isoprostane excretion, an index of renal oxidant stress, was increased in the HG+HI clamp rats. We conclude that even a short duration of hyperglycemia and hyperinsulinemia contributes to activation of pathways that regulate matrix protein synthesis, inflammation, and oxidative stress in the kidney. This finding could have implications for the control of short-term rises in blood glucose in diabetic individuals at risk of developing kidney disease.

Topics: Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Blood Glucose; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Enzyme Activation; Fibronectins; Fibrosis; Hyperglycemia; Hyperinsulinism; Inflammation; Kidney; Laminin; Male; MAP Kinase Signaling System; NF-kappa B; Oxidative Stress; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; RNA, Messenger; Smad3 Protein; Toll-Like Receptor 4; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

Diabetes mellitus in pregnancy causes defects in infant heart, including the outflow tracts (OFTs). Development of the aorta and pulmonary artery, which are derived from the common OFT in the embryo, is regulated by the transforming growth factor β (TGFβ) and Wnt families, and can be perturbed by hyperglycemia-generated intracellular stress conditions. However, the underlying cellular and molecular mechanisms remain to be delineated.. Female mice were induced diabetic with streptozotocin. Embryonic and fetal OFTs were examined morphologically and histologically. Cell proliferation was assessed using 5'-bromo-2'-deoxyuridine incorporation assay. Oxidative and endoplasmic reticulum (ER) stress markers and TGFβ factors were detected using immunohistochemistry. The expression of genes in the Wnt-signaling system was assessed using real-time reverse transcription polymerase chain reaction array. The role of activin-A in cell proliferation was addressed by treating embryos cultured in high glucose with activin-A.. Maternal diabetes caused complex abnormalities in the OFTs, including aortic and pulmonary stenosis and persistent truncus arteriosus. The development of the endocardial cushions was suppressed, manifested with insufficient cellularization of the tissues. Cell proliferation was significantly decreased under oxidative and ER stress conditions. The expression of genes in the Wnt signaling was significantly altered. Activin-A and Smad3 were found to be expressed in the OFT. Treatment with activin-A rescued cell proliferation in the endocardial cushions.. Maternal diabetes generates oxidative and ER stress conditions, suppresses TGFβ and Wnt signaling, inhibits cell proliferation and cellularization of the endocardial cushions, leading to OFT septal defects. Activin-A plays a role in hyperglycemia-suppressed proliferation of the endocardial cells.

Topics: Activins; Animals; Aorta; Aortic Valve Stenosis; Cardiac Output; Cell Proliferation; Diabetes Mellitus, Experimental; Diabetes, Gestational; Embryo Culture Techniques; Embryo, Mammalian; Endocardial Cushions; Endoplasmic Reticulum Stress; Female; Gene Expression Regulation, Developmental; Glucose; Heart Defects, Congenital; Hyperglycemia; Mice; Mice, Inbred C57BL; Neural Crest; Oxidative Stress; Pregnancy; Pulmonary Artery; Pulmonary Valve Stenosis; Smad3 Protein; Streptozocin; Transforming Growth Factor beta; Truncus Arteriosus, Persistent; Wnt Proteins; Wnt Signaling Pathway

Diabetes is a risk factor for respiratory infection, and hyperglycaemia is associated with increased glucose in airway surface liquid and risk of Staphylococcus aureus infection.. To investigate whether elevation of basolateral/blood glucose concentration promotes airway Staphylococcus aureus growth and whether pretreatment with the antidiabetic drug metformin affects this relationship.. Human airway epithelial cells grown at air-liquid interface (±18 h pre-treatment, 30 μM-1 mM metformin) were inoculated with 5×10(5) colony-forming units (CFU)/cm(2) S aureus 8325-4 or JE2 or Pseudomonas aeruginosa PA01 on the apical surface and incubated for 7 h. Wild-type C57BL/6 or db/db (leptin receptor-deficient) mice, 6-10 weeks old, were treated with intraperitoneal phosphate-buffered saline or 40 mg/kg metformin for 2 days before intranasal inoculation with 1×10(7) CFU S aureus. Mice were culled 24 h after infection and bronchoalveolar lavage fluid collected.. Apical S aureus growth increased with basolateral glucose concentration in an in vitro airway epithelia-bacteria co-culture model. S aureus reduced transepithelial electrical resistance (RT) and increased paracellular glucose flux. Metformin inhibited the glucose-induced growth of S aureus, increased RT and decreased glucose flux. Diabetic (db/db) mice infected with S aureus exhibited a higher bacterial load in their airways than control mice after 2 days and metformin treatment reversed this effect. Metformin did not decrease blood glucose but reduced paracellular flux across ex vivo murine tracheas.. Hyperglycaemia promotes respiratory S aureus infection, and metformin modifies glucose flux across the airway epithelium to limit hyperglycaemia-induced bacterial growth. Metformin might, therefore, be of additional benefit in the prevention and treatment of respiratory infection.

Topics: Animals; Bacterial Load; Blood Glucose; Bronchoalveolar Lavage Fluid; Cells, Cultured; Chemokine CXCL9; Epithelial Cells; Granulocyte-Macrophage Colony-Stimulating Factor; Hyperglycemia; Interleukin-1alpha; Interleukin-6; Metformin; Mice; Mice, Inbred C57BL; Permeability; Pseudomonas aeruginosa; Receptors, Leptin; Respiratory System; Staphylococcus aureus; Transforming Growth Factor beta

Hyperglycemia plays a pivotal role in the development of diabetic nephropathy (DN) and may be related to epigenetic metabolic memory. One of the most crucial epigenetic mechanisms is histone modification, which is associated with the expression of a fibrosis factor in vascular injury. Aim .In this study, we investigated the ubiquitination of histones H2A and H2B to explore the epigenetic mechanisms of DN.. The GMCs were cultured as follows: normal group, high glucose group, mannitol group, and intervention group. After 12 hr, 24 hr, and 48 hr, histones ubiquitination, transforming growth factor-β (TGF-β), and fibronectin (FN) were measured using WB, RT-PCR, and IF.. High glucose can induce the upregulation of FN. H2A ubiquitination in GMCs increased in high glucose group (P < 0.01), whereas it decreased significantly in intervention group (P < 0.05). In contrast, H2B ubiquitination decreased with an increasing concentration of glucose, but it was recovered in the intervention group (P < 0.05). Expression of TGF-β changed in response to abnormal histone ubiquitination.. The high glucose may induce H2A ubiquitination and reduce H2B ubiquitination in GMCs. The changes of histone ubiquitination may be due in part to DN by activating TGF-β signaling pathway.

Topics: Animals; Cells, Cultured; Cysteine Proteinase Inhibitors; Fibronectins; Glucose; Histones; Hyperglycemia; Leupeptins; Mesangial Cells; Rats; Transforming Growth Factor beta; Ubiquitination; Up-Regulation

In diabetes the endothelium is either chronically or transiently exposed to hyperglycemic conditions. In addition, endothelial dysfunction in diabetes is related to changes in the inflammatory response and the turnover of extracellular matrix. This study was undertaken to study the effects of inflammatory stimuli on one particular matrix component, the heparan sulfate (HS) proteoglycans (PGs) synthesized by primary human umbilical cord vein endothelial cells (HUVEC). Such cells were cultured in vitro in 5 mM and 25 mM glucose. The latter concentration was used to mimic hyperglycemic conditions in short-term experiments. HUVEC were also cultured in the presence of the inflammatory agents tumor necrosis factor α (TNF-α), interleukin 1α (IL-1α), interleukin 1β (IL-1β) and transforming growth factor β (TGF-β). The cells were labeled with (35)S-sulfate and (35)S-PGs were recovered for further analyses. The major part of the (35)S-PGs was secreted to the medium, irrespective of type of stimuli. Secreted (35)S-PGs were therefore isolated and subjected to further analyses. TNF-α and IL-1α slightly increased the release of (35)S-PGs to the culture medium, whereas IL-1β treatment gave a significant increase. The different treatments neither changed the ratio of (35)S-HS and (35)S-chondroitin sulfate (CS) nor the macromolecular properties of the (35)S-PGs. However, the (35)S-HS chains were slightly increased in size after TNF-α treatment, and slightly decreased after TGF-β treatment, but not affected by the other treatments. Compositional analysis of labeled disaccharides showed changes in the amount of 6-O-sulfated glucosamine residues after treatment with TNF-α, IL-1α and IL-1β. Western immunoblotting showed that major HSPGs recovered from these cells were collagen XVIII, perlecan and agrin, and that secretion of these distinct PGs was increased after IL-1β stimulation. Hence, short term inflammatory stimuli increased the release of HSPGs in HUVEC and affected both the size and sulfation pattern of HS, depending on type of stimuli.

Topics: Agrin; Cells, Cultured; Chondroitin Sulfates; Collagen Type XVIII; Cytokines; Diabetes Mellitus; Endothelium; Extracellular Matrix; Glucosamine; Glucose; Heparan Sulfate Proteoglycans; Human Umbilical Vein Endothelial Cells; Humans; Hyperglycemia; Interleukin-1alpha; Interleukin-1beta; Sulfur Radioisotopes; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Diabetic nephropathy is the most common cause of chronic renal failure in industrialized countries. Depletion of podocytes plays an important role in the progression of diabetic glomerulopathy. Various factors in the diabetic milieu lead to serious podocyte stress driving the cells toward cell cycle arrest (p27(Kip1)), hypertrophy, detachment, and apoptosis. Mitochondria are responsible for oxidative phosphorylation and energy supply in podocytes. Recent studies indicated that mitochondrial dysfunction is a key factor in diabetic nephropathy. In the present study, we investigated metabolic profiles of podocytes under diabetic conditions. We examined oxygen consumption rates (OCRs) and oxidative phosphorylation complex activities in murine podocytes. Cells were exposed to high glucose for 48 hours, cultured for 10 passages under high-glucose conditions (30 mmol/L), or incubated with transforming growth factor-β (5 ng/mL) for 24 hours. After prolonged exposure to high glucose, podocytes showed a significantly increased OCR at baseline and also a higher OCR after addition of oligomycin, indicating significant changes in mitochondrial energy metabolism. Higher OCRs after inhibition of respiration by rotenone also indicated changes in nonmitochondrial respiration. Podocytes stimulated with a proapoptotic concentration of transforming growth factor-β displayed similar bioenergetic profiles, even with decreased citrate synthase activity. In all tested conditions, we found a higher cellular nicotinamide adenine dinucleotide content and changes in activities of respiratory chain complexes. In summary, we provide for the first time evidence that key factors of the diabetic milieu induce changes in glucose metabolism and mitochondrial function in podocytes.

Topics: Animals; Blood Glucose; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Fluorometry; Glomerular Basement Membrane; Glomerular Filtration Barrier; Glucose Transporter Type 1; Hyperglycemia; Immunohistochemistry; Indicators and Reagents; Kidney Glomerulus; Male; Mice; Mice, Inbred Strains; Mitochondria; Oxazines; Oxidation-Reduction; Oxygen Consumption; Phosphorylation; Podocytes; Reactive Oxygen Species; Succinate Cytochrome c Oxidoreductase; Succinate Dehydrogenase; Transforming Growth Factor beta; Xanthenes

Topics: Animals; Blood Glucose; Diabetic Nephropathies; Hyperglycemia; Kidney Glomerulus; Male; Mitochondria; Oxygen Consumption; Podocytes; Transforming Growth Factor beta

In sugar cataract formation in rats, aldose reductase (AR) activity is not only linked to lenticular sorbitol (diabetic) or galactitol (galactosemic) formation but also to signal transduction changes, cytotoxic signals and activation of apoptosis. Using both in vitro and in vivo techniques, the interrelationship between AR activity, polyol (sorbitol and galactitol) formation, osmotic stress, growth factor induction, and cell signaling changes have been investigated. For in vitro studies, lenses from Sprague Dawley rats were cultured for up to 48 h in TC-199-bicarbonate media containing either 30 mM fructose (control), or 30 mM glucose or galactose with/without the aldose reductase inhibitors AL1576 or tolrestat, the sorbitol dehydrogenase inhibitor (SDI) CP-470,711, or 15 mM mannitol (osmotic-compensated media). For in vivo studies, lenses were obtained from streptozotocin-induced diabetic Sprague Dawley rats fed diet with/without the ARIs AL1576 or tolrestat for 10 weeks. As expected, lenses cultured in high glucose/galactose media or from untreated diabetic rats all showed a decrease in the GSH pool that was lessened by ARI treatment. Lenses either from diabetic rats or from glucose/galactose culture conditions showed increased expression of basic-FGF, TGF-β, and increased signaling through P-Akt, P-ERK1/2 and P-SAPK/JNK which were also normalized by ARIs to the expression levels observed in non-diabetic controls. Culturing rat lenses in osmotically compensated media containing 30 mM glucose or galactose did not lead to increased growth factor expression or altered signaling. These studies indicate that it is the biophysical response of the lens to osmotic stress that results in an increased intralenticular production of basic-FGF and TGF-β and the altered cytotoxic signaling that is observed during sugar cataract formation.

Topics: Aldehyde Reductase; Animals; Blotting, Western; Cataract; Diabetes Mellitus, Experimental; Electrophoresis, Polyacrylamide Gel; Fibroblast Growth Factor 2; Galactose; Glucose; Glutathione; Hyperglycemia; Lens, Crystalline; Male; MAP Kinase Signaling System; Organ Culture Techniques; Osmotic Pressure; Rats; Rats, Sprague-Dawley; Sorbitol; Stress, Physiological; Transforming Growth Factor beta

Diabetes is a proinflammatory state. The pattern recognition receptors, Toll-like receptors (TLRs), are increased in diabetic patients and have been suggested to play a role in diabetic nephropathy (DN). Progression of DN involves altered mesangial cell (MC) function with an expansion of the mesangial matrix. There is a paucity of data examining the role of TLR and its expression in MC. We hypothesize the expression of TLRs in the mesangium might be an important factor contributing to mesangium expansion and nephropathy. Thus we evaluated the effect of high glucose on TLR2 and TLR4 expression in mouse mesangial cells (MMC) in vitro. Exposure of MMC to 25 mM glucose for 24 h resulted in increased TLR4 mRNA and cell surface receptor expression compared with 5.5 mM glucose (P < 0.05). Interestingly, we were not able to detect expression of TLR2 in MMC. Furthermore, expression of a TLR4 downstream signaling cascade including myeloid differentiation factor 88 (MyD88), interferon regulatory factor 3 (IRF3), and Toll interleukin receptor domain containing adaptor inducing interferon-β (TRIF)-related adaptor molecule (TRAM) were significantly increased in cells exposed to 25 mM glucose (P < 0.05). There was also a significant increase in NF-κB activation along with increased secretion of inflammatory cytokines IL-6 and monocyte chemotactic protein-1. Levels of transforming growth factor-β were also significantly increased in the presence of 25 mM glucose (P < 0.05). Collectively, these data suggest that hyperglycemia activates TLR4 expression and activity in MC and could contribute to DN.

Topics: Animals; Cell Line; Cells, Cultured; Chemokine CCL2; Diabetic Nephropathies; Glucose; Hyperglycemia; Interleukin-6; Mesangial Cells; Mice; Signal Transduction; Toll-Like Receptor 4; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Diabetic tendinopathy is characterized by increased stiffness, thickness, and excess calcification of affected tendons. We investigated the hypothesis that proteoglycans (PGs), as key regulators of tendon structure and calcification, are altered in diabetic tendons.. Adult porcine patellar tendons were incubated in iso-osmolar media with high or normal glucose levels for 2 weeks. The PG fraction was isolated and analyzed. Protein and mRNA levels of five PGs were measured. PG production was assessed in primary tenocyte monolayers by (35)S-sulfate labeling in high and normal glucose conditions with and without exposure to advanced glycation end-products (AGEs). Levels of transforming growth factor β, which commonly mediates some effects of hyperglycemia, were also measured and the effects of free radical scavengers on (35)S-sulfate incorporation were determined.. PG levels were significantly decreased in tendons exposed to high glucose media compared with tendons in iso-osmolar control media. Relative quantities of individual PGs were unchanged by exposure to hyperglycemia and mRNAs for PGs were variably affected. High glucose media decreased PG production by tenocytes as measured by (35)S-sulfate labeling, whereas AGE-modified type I collagen and free radical scavengers had no effects. Hyperglycemic conditions increased levels of transforming growth factor β1 in an AGE-independent manner.. Hyperglycemia produces a reduction in PG levels related to decreased synthesis or sulfation of glycosaminoglycans, which may contribute to the tendon pathology observed clinically in diabetes.

Topics: Animals; Collagen Type I; Diabetes Complications; Glycation End Products, Advanced; Hyperglycemia; Organ Culture Techniques; Peptidoglycan; Swine; Tendinopathy; Tendons; Transforming Growth Factor beta

Acrolein has been implicated in retinal pigment epithelium (RPE) cell death, and has been associated with diabetic retinopathy. Our purpose was to investigate the potential effect of high glucose in influencing acrolein-mediated RPE cytokine production and cell death. We investigated the influence of the acrolein effect on ARPE-19 cells in high glucose conditions and quantified the release of transforming growth factor β (TGFβ1 and 2) and vascular endothelial growth factor (VEGF). We assessed the ability of N-benzylhydroxylamine(NBHA) as well as TGFβ pathway inhibitors SIS3 and SB431542 to prevent this effect of acrolein on ARPE-19 cells.. Confluent ARPE-19 cells were treated with acrolein and/or NBHA in both 5.5 and 18.8 mM glucose conditions. Cells were also pretreated with SIS3, a specific inhibitor of the SMAD3 pathway, and SB431542, a specific inhibitor of TGFβ signaling pathway, before treating them with acrolein. Viable cells were counted and ELISAs were performed to measure the cytokines TGFβ1 and 2, and VEGF released into the conditioned media.. In ARPE-19 cells exposed to acrolein and hyperglycemia there was reduced cell viability and an increase in the cell media of VEGF, TGFβ1, and TGFβ2, which was reversed by NBHA. Acrolein/hyperglycemia-induced cell viability reduction and cytokine overproduction was also reduced by TGFβ pathway blockade.. We conclude that the effect of acrolein on the reduction of viability and VEGF increase by ARPE-19 cells in hyperglycemic media is conducted through the TGFβ signaling pathway. Our results suggest that benefits of sequestering acrolein by NBHA and the blockage of the TGFβ pathway by SB431542 and SIS3 offer suggestions as to potential useful pharmacological drug candidates for the prevention of diabetes-induced complications in the eye.

Topics: Acrolein; Cell Line; Cell Survival; Diabetic Retinopathy; Enzyme-Linked Immunosorbent Assay; Humans; Hyperglycemia; Retinal Pigment Epithelium; Signal Transduction; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

The predominant transcription factors regulating key genes in diabetic kidney disease have not been established. The transcription factor upstream stimulatory factor 1 (USF1) is an important regulator of glucose-mediated transforming growth factor (TGF)-β1 expression in mesangial cells; however, its role in the development of diabetic kidney disease has not been evaluated. In the present study, wild-type (WT; USF1 +/+), heterozygous (USF1 +/-), and homozygous (USF1 -/-) knockout mice were intercrossed with Akita mice (Ins2/Akita) to induce type 1 diabetes. Mice were studied up to 36 wk of age. The degree of hyperglycemia and kidney hypertrophy were similar in all groups of diabetic mice; however, the USF1 -/- diabetic mice had significantly less albuminuria and mesangial matrix expansion than the WT diabetic mice. TGF-β1 and renin gene expression and protein were substantially increased in the WT diabetic mice but not in USF1 -/- diabetic mice. The underlying pathway by which USF1 is regulated by high glucose was investigated in mesangial cell culture. High glucose inhibited AMP-activated protein kinase (AMPK) activity and increased USF1 nuclear translocation. Activation of AMPK with AICAR stimulated AMPK activity and reduced nuclear accumulation of USF1. We thus conclude that USF1 is a critical transcription factor regulating diabetic kidney disease and plays a critical role in albuminuria, mesangial matrix accumulation, and TGF-β1 and renin stimulation in diabetic kidney disease. AMPK activity may play a key role in high glucose-induced regulation of USF1.

Topics: Albuminuria; Alleles; AMP-Activated Protein Kinases; Animals; Cell Line; Cell Nucleus; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Disease Progression; Extracellular Matrix; Female; Hyperglycemia; Hypertrophy; Kidney; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Renin-Angiotensin System; RNA, Messenger; Transforming Growth Factor beta; Upstream Stimulatory Factors

Extracts of Andrographis paniculata Nees are used for various ethnomedical conditions including hyperglycemia and hypertension complications.. The purpose of this study is to evaluate the anti-diabetic nephropathy effect of diterpene lactones andrographolide (AP1) and 14-deoxy-11,12-didehydroandrographolide (AP2) from Andrographis paniculata.. MES-13, a SV40-transformed murine glomerular mesangial cell line, was cultured in high concentration of glucose to induce diabetic nephropathy phenotypes, which include secretion of extracellular matrix protein fibronectin, cytokine TGF-β, states of oxidative stress, and apoptosis marker caspase-3.. Our data suggest that addition of compounds AP1 or AP2 reduces the phenotypes indicating diabetic nephropathy in MES-13 cells. The compound AP2 showed potent activity than AP1 in the reduction of apoptosis marker caspase-3, fibrosis marker TGF-β, and PAI-1. Furthermore, AP1 and AP2 do not have antioxidant ability in acellular environment; however, addition of AP1 and AP2 reduced intracellular oxidative states in high glucose cultured MES-13 cells.. This is the first report on anti-diabetic nephropathy effect of AP1 and AP2 in part due to the regulation of intracellular signaling transduction, not mere clearance of reactive oxygen species. Thus, this study may be useful for drug development or food supplement for diabetes and nephropathy from Andrographis paniculata.

Topics: Andrographis; Animals; Apoptosis; Cell Line, Transformed; Diabetic Nephropathies; Diterpenes; Fibronectins; Fibrosis; Hyperglycemia; Mesangial Cells; Mice; Oxidation-Reduction; Oxidative Stress; Plant Components, Aerial; Plant Extracts; Plasminogen Activator Inhibitor 1; Reactive Oxygen Species; Transforming Growth Factor beta

Nutrient overabundance is known to promote cellular hypertrophy, a significant pathological event in diseases like diabetes and cancer, although mechanisms have remained unclear. In this issue of Developmental Cell, Wu and Derynck provide a new model that links metabolism and cell growth by demonstrating that hyperglycemia can increase TGF-beta-dependent activation of the mTOR pathway to promote cellular hyperplasia.

Topics: Animals; Glucose; Hyperglycemia; Hypertrophy; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

In multicellular organisms, cell size is tightly controlled by nutrients and growth factors. Increasing ambient glucose induces enhanced protein synthesis and cell size. Continued exposure of cells to high glucose in vivo, as apparent under pathological conditions, results in cell hypertrophy and tissue damage. We demonstrate that activation of TGF-beta signaling has a central role in glucose-induced cell hypertrophy in fibroblasts and epithelial cells. Blocking the kinase activity of the TbetaRI receptor or loss of its expression prevented the effects of high glucose on protein synthesis and cell size. Exposure of cells to high glucose induced a rapid increase in cell surface levels of the TbetaRI and TbetaRII receptors and a rapid activation of TGF-beta ligand by matrix metalloproteinases, including MMP-2 and MMP-9. The consequent autocrine TGF-beta signaling in response to glucose led to Akt-TOR pathway activation. Accordingly, preventing MMP-2/MMP-9 or TGF-beta-induced TOR activation inhibited high glucose-induced cell hypertrophy.

Topics: Active Transport, Cell Nucleus; Animals; Cell Line; Cell Size; Diabetes Mellitus; Epithelial Cells; Fibroblasts; Glucose; Hyperglycemia; Hypertrophy; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Mice; Mice, Knockout; Neoplasms; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Rats; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; RNA Interference; Signal Transduction; Smad3 Protein; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

Magnolol, a natural product isolated from Magnolia officinalis, has various pharmacological effects, such inhibition of effect on inflammation and tumor metastasis, protection against cerebral ischaemic injury, and potent antioxidant activity. In this present study, we evaluated the inhibitory effects of magnolol on transforming growth factor-beta1 (TGF-beta1) and fibronectin expression induced by high concentrations of glucose or S100b (a specific receptor of advance glycation end products ligand) in human retinal pigment epithelial cells (human RPE cells). No effect on cell growth was found with magnolol (up to 20 microg/ml) using a colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltertrazolium bromide (MTT) assay. High glucose (25 mM) or S100b (5 microg/ml) induced increases in expression of TGF-beta1 and fibronectin. The increases in TGF-beta1 and fibronectin expression with high glucose or S100b were prevented by magnolol in a dose-dependent manner. Also, magnolol inhibited extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)/Akt activation. The present study demonstrates that high glucose- or S100b-induced TGF-beta1 and fibronectin expression, but this increased expression is inhibited by magnolol via the ERK/MAPK/Akt signaling pathway in human RPE cells.

Topics: Biphenyl Compounds; Blotting, Western; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Fibronectins; Gene Expression; Glucose; Humans; Hyperglycemia; Lignans; Lipid Peroxidation; Magnolia; Molecular Structure; Nerve Growth Factors; Phosphorylation; Pigment Epithelium of Eye; Proto-Oncogene Proteins c-akt; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Transforming Growth Factor beta

Diabetic nephropathy is a life-threatening disease associated with diabetes mellitus. Longstanding hyperglycemia induces pathological reactions of glomerular mesangial cells, such as overproduction of extracellular matrix, which finally lead to nephropathy. However, the mechanisms underlying its pathogenesis have not been completely elucidated. Using the Streptozotocin-induced model of diabetes, we report that mice deficient in the growth factor midkine (Mdk-/-) exhibited strikingly milder nephropathy than Mdk+/+ mice, even though both mice showed similar extents of hyperglycemia after Streptozotocin injection. Midkine expression was induced in the glomerular mesangium of Mdk+/+ mice with diabetic nephropathy and in primary cultured mesangial cells exposed to high glucose. Mdk-/- mesangial cells exhibited reduced phosphorylation of protein kinase C and extracellular signal-regulated kinase as well as reduced production of transforming growth factor-beta(1) on high glucose loading. Addition of exogenous midkine restored extracellular signal-regulated kinase phosphorylation in Mdk-/- cells under high glucose conditions, whereas a midkine antisense oligodeoxynucleotide suppressed midkine in Mdk+/+ cells. Therefore, this study identifies midkine as a key molecule in diabetic nephropathy and suggests that midkine accelerates the intracellular signaling network evoked by hyperglycemia in nephropathy.

Topics: Animals; Blotting, Western; Cells, Cultured; Cytokines; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Enzyme-Linked Immunosorbent Assay; Extracellular Signal-Regulated MAP Kinases; Female; Glomerular Mesangium; Hyperglycemia; Immunohistochemistry; Mice; Mice, Mutant Strains; Midkine; Protein Kinase C; Transforming Growth Factor beta; Transforming Growth Factor beta1

GLUT1 upregulation and increased glucose transport activity may contribute to extracellullar matrix (ECM) accumulation characterizing diabetic nephropathy (DN). Rats of the Milan hypertensive strain (MHS) are resistant to both hypertensive and diabetic renal disease, due to a haemodynamic protection. On the contrary, those of the Milan normotensive strain (MNS) develop spontaneous glomerulosclerosis, and when rendered diabetic, show typical morphological and haemodynamic changes.. To assess whether susceptibility to diabetic glomerulopathy in MNS rats is associated with higher glucose transporter 1 (GLUT1) expression (and glucose transport activity) vs MHS rats, diabetic and nondiabetic MNS and MHS rats were followed for 6 months and mesangial cells derived from these animals were exposed to high glucose (HG) vs normal glucose (NG) conditions.. Glomerular expression of GLUT1 protein and ECM and transforming growth factor-beta (TGF-beta) mRNA was significantly upregulated in diabetic vs nondiabetic MNS, but not MHS rats. Upon exposure to HG and/or TGF-beta, mesangial cells from 1- and 8-month-old MNS rats showed higher glucose transport activity and GLUT1 membrane expression than those from age-matched MHS rats. Likewise, ECM and TGF-beta production increased more markedly in response to HG and/or TGF-beta in MNS vs MHS mesangial cells.. These data indicate that susceptibility to diabetic glomerulopathy in MNS rats is associated with increased GLUT1-dependent glucose transport activity in response to hyperglycaemia and/or TGF-beta, which may amplify ECM overproduction. Conversely, the haemodynamic protection from glomerulosclerosis in MHS rats is associated with lack of upregulation of TGF-beta/GLUT1 axis, thus supporting the concept that this axis may represent the link between haemodynamic and metabolic mechanisms of injury.

Topics: Animals; Diabetes Complications; Diabetes Mellitus, Experimental; Disease Susceptibility; Extracellular Matrix; Glomerulonephritis; Glucose; Glucose Transporter Type 1; Hyperglycemia; Hypertension; Immunity, Innate; Rats; Rats, Mutant Strains; Transforming Growth Factor beta; Up-Regulation

Diabetic nephropathy is characterized early in its course by glomerular hypertrophy and, importantly, mesangial hypertrophy, which correlate with eventual glomerulosclerosis. The mechanism of hypertrophy, however, is not known. Gene disruption of the tumor suppressor PTEN, a negative regulator of the phosphatidylinositol 3-kinase/Akt pathway, in fruit flies and mice demonstrated its role in size control in a cell-specific manner. Here, we investigated the mechanism of mesangial hypertrophy in response to high extracellular glucose. We link early renal hypertrophy with significant reduction in PTEN expression in the streptozotocin-induced diabetic kidney cortex and glomeruli, concomitant with activation of Akt. Similarly, exposure of mesangial cells to high concentrations of glucose also decreased PTEN expression and its phosphatase activity, resulting in increased Akt activity. Expression of PTEN inhibited high-glucose-induced mesangial cell hypertrophy, and expression of dominant-negative PTEN was sufficient to induce hypertrophy. In diabetic nephropathy, the hypertrophic effect of hyperglycemia is thought to be mediated by transforming growth factor-beta (TGF-beta). TGF-beta significantly reduced PTEN expression in mesangial cells, with a reduction in its phosphatase activity and an increase in Akt activation. PTEN and dominant-negative Akt attenuated TGF-beta-induced hypertrophy of mesangial cells. Finally, we show that inhibition of TGF-beta signal transduction blocks the effect of high glucose on PTEN downregulation. These data identify a novel mechanism placing PTEN as a key regulator of diabetic mesangial hypertrophy involving TGF-beta signaling.

Topics: Animals; Blood Glucose; Diabetes Mellitus, Experimental; Gene Expression Regulation, Enzymologic; Glomerular Mesangium; Hyperglycemia; Hypertrophy; Kidney Cortex; PTEN Phosphohydrolase; Rats; Signal Transduction; Transforming Growth Factor beta

TGF-beta and oxidant stress have been considered to play key roles in the pathogenesis of diabetic vascular complications; however, the stimulus for these factors in humans is not clear. The purpose of this in vivo study was to determine whether transient hyperglycemia in humans is sufficient to increase renal production of TGF-beta1 and urinary isoprostanes in normal humans. A hyperglycemic clamp procedure was performed on 13 healthy volunteers. An infusion of glucose was delivered to maintain the plasma glucose between 200 and 250 mg/dl for 120 min. Timed urine samples, collected on an overnight period before the study, at each void on completion of the procedure, and the following overnight, were assayed for TGF-beta1, F2-isoprostanes, and creatinine. Plasma samples were assayed for TGF-beta1 before and at timed intervals throughout hyperglycemia. Mean baseline TGF-beta1 in plasma was 4.57 +/- 0.22 ng/ml, and no change in plasma TGF-beta1 levels was detected throughout the hyperglycemia period. Baseline urine TGF-beta1 was 4.14 +/- 1.16 pg/mg creatinine. The fractional urine samples showed a sharp increase in TGF-beta1 excretion in the 12-h period after exposure to hyperglycemia, with a mean peak TGF-beta1 of 30.43 +/- 8.05 pg/mg (P = 0.002). TGF-beta1 excretion in the subsequent overnight urine sample was not different from baseline (4.62 +/- 1.21 pg/mg). Urinary isoprostanes increased from a baseline of 4.92 +/- 0.74 to 13.8 +/- 3.37 ng/mg creatinine. It is concluded that 120 min of hyperglycemia in normal humans is sufficient to induce an increase in renal TGF-beta1 and isoprostane production.

Topics: Adult; Female; Humans; Hyperglycemia; Isoprostanes; Male; Transforming Growth Factor beta

We generated the homozygous transgenic mice with expression of the active form of TGF-beta1 by the glucagon promoter (homozygous NOD-TGF-beta1). The homozygous NOD-TGF-beta1 showed severe diabetes in 84.6%, impaired glucose tolerance, and low serum insulin levels. The final size of endocrine and whole pancreas decreased, respectively, to 6 and 34%, compared to wild-type mice. The homozygous N(2) backcross to C57BL/6 (B6-TGF-beta1) showed no diabetes, but impaired glucose tolerance and low serum insulin levels. In homozygous NOD-TGF-beta1, the expression of p15(INK4b) was induced by 3.4-fold in pancreatic islets than that in wild-type mice. Based on these, we conclude first that excessive paracrine TGF-beta1 signaling in islets results in endocrine and exocrine pancreatic hypoplasia, second that TGF-beta1decrease the final size of endocrine and exocrine pancreas presumably through regulating cell cycle via p15(INK4b) at least in endocrine pancreas, and third that hypoplastic action of TGF-beta1 of pancreatic islets is independent of the genetic background.

Topics: Animals; Cell Cycle; Cell Cycle Proteins; Crosses, Genetic; Cyclin-Dependent Kinase Inhibitor p15; Diabetes Mellitus, Type 1; Female; Glucagon; Glucose Tolerance Test; Homozygote; Hyperglycemia; Insulin; Islets of Langerhans; Male; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Transgenic; Pancreas, Exocrine; Promoter Regions, Genetic; Swine; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Suppressor Proteins

Peroxisome proliferator-activated receptor-gamma (PPARgamma) are ligand-activated transcription factors that regulate cell growth, inflammation, lipid metabolism, and insulin sensitivity. We recently demonstrated that PPARgamma agonists limit high glucose-induced inflammation in a model of proximal tubular cells (PTC; Panchapakesan U, Pollock CA, and Chen XM. Am J Physiol Renal Physiol 287: F528-F534, 2004). However, the role of PPARgamma in the excess extracellular matrix production is largely unknown. We evaluated the effect of 24- to 48-h 8 microM l-805645 or 10 microM pioglitazone on 25 mM D-glucose-induced markers of fibrosis in HK-2 cells. High D-glucose induced nuclear binding of activator protein-1 (AP-1) to 140.8 +/- 10.9% (P < 0.05), which was attenuated with L-805645 and pioglitazone to 82.3 +/- 14.4 (P < 0.01 vs. high D-glucose) and 99.3 +/- 12.2% (P < 0.05 vs. high D-glucose), respectively. High D-glucose increased total production of transforming growth factor (TGF)-beta(1) 139.6 +/- 6.5% (P < 0.05), which was reversed with L-805645 and pioglitazone to 68.73 +/- 5.7 (P < 0.01 vs. high D-glucose) and 112 +/- 13.6% (P < 0.05 vs. high D-glucose). L-805645 and pioglitazone reduced high d-glucose-induced fibronectin from 156.0 +/- 24.9 (P < 0.05) to 81.9 +/- 16.0 and 57.4 +/- 12.7%, respectively (both P < 0.01 vs. high D-glucose). Collagen IV was not induced by high d-glucose. L-805645 and pioglitazone suppressed collagen IV to 68.0 +/- 14.5 (P < 0.05) and 46.5 +/- 11.6% (P < 0.01) vs. high D-glucose, respectively. High D-glucose increased the nuclear binding of NF-kappaB to 167 +/- 22.4% (P < 0.05), which was not modified with PPARgamma agonists. In conclusion, PPARgamma agonists exert antifibrotic effects in human PTC in high glucose by attenuating the increase in AP-1, TGF-beta(1), and the downstream production of the extracellular matrix protein fibronectin.

Topics: Cell Culture Techniques; Cell Proliferation; Fibrosis; Glucose; Humans; Hyperglycemia; Hypoglycemic Agents; Inflammation; Insulin Resistance; Kidney; Kidney Tubules, Proximal; Lipid Metabolism; Pioglitazone; PPAR gamma; Thiazolidinediones; Transcription Factor AP-1; Transforming Growth Factor beta; Transforming Growth Factor beta1

Besides the classical cardiovascular diseases, high levels of blood glucose directly interfere with cardiomyocytes. The mechanisms responsible for this have not yet been explored in detail. This study aims to determine if hyperglycaemia has any impact on prominent signalling molecules and on the contractile function of cardiomyocytes. Freshly isolated cardiomyocytes from adult rats were treated with various concentrations of glucose. Formed free radicals were measured by DCF-fluorescence. TGFbeta expression and p38 MAP-kinase (MAPK) activation were measured by Western blotting. The contractile efficiency was determined by measurement of the maximal amount of cell shortening. Glucose (30 mM) caused an increase in formation of radicals, phosphorylation of p38 MAPK, and TGFbeta expression. Under conditions of low viscosity (1 cp), contractile responses to hyperglycaemia (15 mM) were not altered in contrast to control. However, enhancement of viscosity (400 cp) effected a limitation of contractile function. The responsiveness to beta-adrenoceptor stimulation did not change. Neither inhibition of p38 MAPK with SB 202190 (1 microM) nor inhibition of reactive oxygen species with vitamin C did alter these measured functional parameters. Diabetes mellitus directly influences the activation degree of prominent signalling molecules and the contractile function of adult ventricular cardiomyocytes, which results in facilitating in the development of diabetic cardiomyopathy.

Topics: Animals; Antioxidants; Ascorbic Acid; Cells, Cultured; Diabetes Mellitus; Electrophysiology; Glucose; Hyperglycemia; Imidazoles; Isoproterenol; Male; Muscle Contraction; Myocytes, Cardiac; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Kinase Inhibitors; Pyridines; Rats; Rats, Wistar; Reactive Oxygen Species; Receptors, Adrenergic, beta; Signal Transduction; Transforming Growth Factor beta

Diabetic nephropathy is one of the major microvascular complications in diabetes and is the leading cause of end-stage renal disease worldwide. Among various factors, angiogenesis-associated factors such as vascular endothelial growth factor (VEGF)-A and angiopoietin (Ang)-2 are involved in the development of diabetic nephropathy. We previously reported the therapeutic efficacy of antiangiogenic tumstatin peptide in the early diabetic nephropathy model. Here, we examine the effect of endostatin peptide, a potent inhibitor of angiogenesis derived from type XVIII collagen, in preventing progression in the type 1 diabetic nephropathy mouse model. Endostatin peptide did not affect hyperglycemia induced by streptozotocin (STZ). Glomerular hypertrophy, hyperfiltration, and albuminuria were significantly suppressed by endostatin peptide (5 mg/kg) in STZ-induced diabetic mice. Glomerular mesangial matrix expansion, the increase of glomerular type IV collagen, endothelial area (CD31(+)), and F4/80(+) monocyte/macrophage accumulation were significantly inhibited by endostatin peptide. Increase in the renal expression of VEGF-A, flk-1, Ang-2, an antagonist of angiopoietin-1, transforming growth factor-beta1, interleukin-6, and monocyte chemoattractant protein-1 was inhibited by endostatin peptide in diabetic mice. Decrease of nephrin mRNA and protein in diabetic mice was suppressed by treatment with endostatin peptide. The level of endostatin in the renal cortex and sera was increased in diabetic mice. Endogenous renal levels of endostatin were decreased in endostatin peptide-treated groups in parallel with VEGF-A. Although serum levels of endostatin were decreased in the low-dose endostatin-peptide group, high-dose administration resulted in elevated serum levels of endostatin. These results demonstrate the potential use of antiangiogenic endostatin peptide as a novel therapeutic agent in diabetic nephropathy.

Topics: Albuminuria; Amino Acid Sequence; Animals; Blood Glucose; Body Weight; Collagen Type IV; Creatinine; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Endostatins; Female; Hyperglycemia; Hypertrophy; Immunohistochemistry; Integrin alpha5beta1; Kidney; Kidney Glomerulus; Membrane Proteins; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Organ Size; Peptide Fragments; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

We quantified the glomerular expression of thrombospondin-1 (THBS1, also known as TSP-1), transforming growth factor beta 1 (TGFB1, also known as TGF-beta1) and connective tissue growth factor (CTGF) at each stage of diabetic nephropathy. We also examined the roles of THBS1 and CTGF in mediating high-glucose- and glycated-albumin-induced synthesis of the matrix protein, fibronectin, by mesangial cells.. THBS1, latent and active TGFB1, and CTGF, were detected by immunohistochemistry and in situ hybridisation in biopsies from 19 insulin-dependent diabetic patients with incipient, manifest and advanced diabetic nephropathy, and in 11 control kidneys. Findings were quantified by image analysis. Human mesangial cells were cultured with normal or high glucose, albumin or glycated albumin (Amadori product), +/-THBS1 or CTGF antisense oligonucleotides, or with peptide W, an inhibitor of TGFB1 bioactivation by THBS1. Proteins were measured by western blot analysis or ELISA.. In glomeruli of normal kidneys, mRNA and protein levels for THBS1, latent-TGFB1 and CTGF were low. They were increased in the incipient stage of diabetic nephropathy, predominantly in mesangial areas, with further increases at later stages of the disease. Little or no active TGFB1 immunostaining was detected prior to manifest diabetic nephropathy. In contrast to high-glucose conditions, increases in fibronectin synthesis that were stimulated by glycated albumin were not dependent on THBS1 activation of latent TGFB1. However, increased fibronectin synthesis in both conditions required CTGF.. Increased glomerular expression of all three factors occurs from the earliest stage of diabetic nephropathy. In contrast to THBS1, CTGF is required for mesangial synthesis of fibronectin stimulated by high glucose or glycated albumin, and is thus a potential therapeutic target.

Topics: Adult; Aged; Blotting, Western; Case-Control Studies; Connective Tissue Growth Factor; Diabetes Mellitus, Type 1; Diabetic Nephropathies; Extracellular Matrix Proteins; Female; Fibronectins; Gene Expression Regulation; Glomerular Mesangium; Glucose; Glycated Serum Albumin; Glycation End Products, Advanced; Humans; Hyperglycemia; Immediate-Early Proteins; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; RNA, Messenger; Serum Albumin; Thrombospondin 1; Transforming Growth Factor beta

Cardiovascular defects are common in diabetic offspring, but their etiology and pathogenesis are poorly understood. Extracellular matrix accumulates in adult tissues in response to hyperglycemia, and transforming growth factor-beta1 (TGF beta1) likely mediates this effect. The objective of this study was to characterize TGF beta expression in the organogenesis-stage mouse heart and to evaluate TGF beta and fibronectin expression in embryonic mouse heart exposed to hyperglycemia. Prominent TGF beta1, and minimal TGF beta2 or TGF beta 3, protein expression was demonstrated in embryonic day (E) 9.5-E13.5 hearts. Hyperglycemia for 24 hr produced significantly increased fibronectin, slightly increased TGF beta1, and unchanged TGF beta2 or TGF beta 3, by immunohistochemistry. Increased TGF beta1 was demonstrated by enzyme-linked immunosorbent assay in embryonic fluid and isolated hearts after hyperglycemia for 24 hr, but not 48 hr. Hyperglycemia increased fibronectin protein and mRNA expression in embryonic hearts after 24 hr, and pericardial injection of TGF beta1 also increased fibronectin mRNA in the embryonic heart. It is proposed that TGF beta1 and fibronectin may play a role in diabetes-induced cardiac dysmorphogenesis.

Topics: Animals; Embryo, Mammalian; Fibronectins; Hyperglycemia; Mice; Myocardium; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Transforming Growth Factor beta3

Reduction of renal mass is frequently associated with progressive loss of kidney function. We examined the effects of hyperlipidemia on renal pathology and mediators of tissue damage in B6.ROP Os/+ mice, a model of reduced renal mass.. C57BL/6 control mice and B6.ROP Os/+ mice were fed normal rodent chow or a high fat, high cholesterol (HFHC) diet for 12 weeks. Kidney function and renal pathology were assessed.. Hyperlipidemia led to a decline in kidney function in C57BL/6 mice. Renal pathology was characterized by an increase in glomerular matrix and cellularity, glomerular and tubulointerstitial macrophage influx, and increased tubular epithelial cell turnover. Chow-fed B6.ROP Os/+ animals demonstrated glomerular hypertrophy with an increase in mesangial matrix and cellularity that was characterized by macrophage influx and increased proliferation. The tubulointerstitium showed increased macrophages as well as tubular atrophy and dilation. Renal pathology was accompanied by an increase in blood urea nitrogen (BUN) and proteinuria. Hyperlipidemia in B6.ROP Os/+ mice resulted in increased plasma BUN compared to chow-fed B6.ROP Os/+ animals and aggravated renal pathology by further increasing glomerular matrix and glomerular hypercellularity. Glomerular hypercellularity was associated with increased expression of platelet-derived growth factor-B (PDGF B) and its receptor beta. Glomerular transforming growth factor-beta (TGF-beta) mRNA expression was increased in B6.ROP Os/+ mice, hyperlipidemic C57BL/6 mice and hyperlipidemic B6.ROP Os/+ animals compared to controls and correlated with the amount of mesangial matrix.. This study demonstrates that hyperlipidemia worsens renal pathology in B6.ROP Os/+ mice with a decline in renal function mediated at least in part through increased renal expression of the cytokines PDGF B and TGF-beta.

Topics: Animals; Chemokine CCL2; Extracellular Matrix; Female; Glomerular Mesangium; Hyperglycemia; Hyperlipidemias; Hypertrophy; Insulin Resistance; Kidney Diseases; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Microscopy, Electron; Obesity; Proto-Oncogene Proteins c-sis; RNA, Messenger; Transforming Growth Factor beta

Cyclin kinase inhibitor p27(Kip(1)) (p27) has been shown to be upregulated in glomeruli of diabetic animals and mesangial cells cultured under high glucose. This study was an investigation of the role of p27 in the progression of diabetic nephropathy. Mice deficient in p27 (p27 -/-) and wild-type mice (p27 +/+) were studied 12 wk after diabetes induction by streptozotocin. Blood glucose and BP were comparable between diabetic p27 +/+ and p27 -/- mice. The kidney weight to body weight ratio and glomerular volume increased in diabetic p27 +/+ mice. In contrast, these parameters did not change in diabetic p27 -/- mice. Similarly, albuminuria developed in diabetic p27 +/+ mice but not in diabetic p27 -/- mice. The mesangial expansion was significantly milder in diabetic p27 -/- mice than that in diabetic p27 +/+ mice. These changes were associated with a similar increase in glomerular TGF-beta expression in diabetic p27 +/+ and p27 -/- mice. However, glomerular protein expression of fibronectin, a target of TGF-beta, increased only in diabetic p27 +/+ mice. In mesangial cells cultured from p27 +/+ mice, exposure to high glucose caused significant increases in total protein content and [(3)H]-leucine incorporation. On the other hand, high glucose caused a significant reduction in these parameters in cells from p27 -/- mice. Phosphorylation of 4E-BP1, the translation inhibitor, increased after exposure to high glucose in p27 +/+ cells. In p27 -/- cells, the level of phosphorylated 4E-BP1 was higher than that in control p27 +/+ cells and decreased under high glucose conditions. In conclusion, renal hypertrophy, glomerular hypertrophy, and albuminuria did not develop, and mesangial expansion was milder in diabetic p27 -/- mice despite glomerular TGF-beta upregulation. These results suggest that controlling p27 function may ameliorate diabetic nephropathy.

Topics: Adaptor Proteins, Signal Transducing; Albuminuria; Animals; Blood Group Antigens; Carrier Proteins; Cell Cycle Proteins; Cyclin-Dependent Kinase Inhibitor p27; Diabetic Nephropathies; Disease Progression; Eukaryotic Initiation Factors; Fibronectins; Glomerular Mesangium; Hyperglycemia; Leucine; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Phosphoproteins; Phosphorylation; Proliferating Cell Nuclear Antigen; Thymidine; Transforming Growth Factor beta; Tritium; Tumor Suppressor Proteins

Various renal cell types have been shown to contribute to the excessive matrix deposition observed in diabetic nephropathy. The present study examined the effect of high ambient glucose and transforming growth factor-beta1 (TGF-beta1) on matrix production by human renal fibroblasts.. Human renal fibroblasts (TK173) were used to examine the effects of high glucose and TGF-beta1 on fibronectin and collagen type III expression. Stable transfectants were generated of TK173 cells expressing a dominant negative TGF-beta type II receptor. Matrix components were measured in enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR).. Fibronectin secretion by renal fibroblasts was increased upon exposure to high glucose, but with delayed kinetics compared to TGF-beta1-induced fibronectin. Exposure to high glucose resulted in an increased secretion of latent TGF-beta1. However, treatment with neutralizing pan-specific anti-TGF-beta antibodies could not attenuate the effects of glucose. Furthermore, collagen type III was up-regulated by high glucose, but not by TGF-beta1. Importantly, fibroblasts expressing a dominant negative TGF-beta type II receptor were defective in TGF-beta1-induced fibronectin production, whereas glucose-induced fibronectin and collagen type III were unaffected.. These data show that in renal fibroblasts exposure to high glucose can increase matrix production independent of endogenous TGF-beta1. Although glucose activation is accompanied by an increased production of latent TGF-beta1, which can have an important role in vivo, the data suggest involvement of alternative growth factors in the mechanism by which hyperglycemic conditions can modulate matrix accumulation in diabetic nephropathy.

Topics: Antibodies; Cell Line, Transformed; Collagen Type III; Extracellular Matrix Proteins; Fibroblasts; Fibronectins; Gene Expression; Glucose; Humans; Hyperglycemia; Kidney; Kinetics; Receptors, Transforming Growth Factor beta; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

SPARC (Secreted Protein, Acidic and Rich in Cysteine) is a matricellular protein that inhibits mesangial cell proliferation and also affects production of extracellular matrix (ECM) by regulating transforming growth factor-beta1 (TGF-beta1) and type I collagen in mesangial cells. This study is an investigation of the role of SPARC in streptozotocin (STZ)-induced diabetic nephropathy (DN) of 6-mo duration in wild type (WT) and SPARC-null mice. SPARC expression was evaluated by immunohistochemistry (IHC) and by in situ hybridization (ISH). Deposition of type I and IV collagen and laminin was evaluated by IHC, and TGF-beta 1 mRNA was assessed by ISH. Renal function studies revealed no significant difference in BUN between diabetic SPARC-null mice and diabetic WT mice, whereas a significant increase in albumin excretion was detected in diabetic WT relative to diabetic SPARC-null mice. Diabetic WT animals exhibited increased levels of SPARC mRNA and protein in glomerular epithelial cells and in interstitial cells, in comparison with nondiabetic WT mice. Neither SPARC mRNA nor protein was detected in SPARC-null mice. Morphometry revealed a significant increase in the percentage of the glomerular tufts occupied by ECM in diabetic WT compared with nondiabetic WT mice, although there was no difference in the mean glomerular tuft area among groups. In contrast, diabetic SPARC-null mice did not show a significant difference in the percentage of the glomerular tufts occupied by ECM relative to nondiabetic null mice. Tubulointerstitial fibrosis was ameliorated in diabetic SPARC-null mice compared with diabetic WT animals. Further characterization of diabetic SPARC-null mice revealed diminished glomerular deposition of type IV collagen and laminin, and diminished interstitial deposition of type I and type IV collagen correlated with decreases in TGF-beta 1 mRNA compared with WT diabetic mice. These observations suggest that SPARC contributes to glomerulosclerosis and tubulointerstitial damage in response to hyperglycemia through increasing TGF-beta 1 expression in this model of chronic DN.

Topics: Animals; Collagen; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Extracellular Matrix; Hyperglycemia; Laminin; Male; Mice; Models, Animal; Osteonectin; Transforming Growth Factor beta; Transforming Growth Factor beta1

The peritoneal membrane of long-term peritoneal dialysis patients is characterized by a loss of ultrafiltration capacity, associated morphologically with submesothelial fibrosis and neoangiogenesis. Exposure to high glucose concentrations in peritoneal dialysate and the resultant advanced glycation end-products (AGE) accumulation have been implicated in the development of these changes, but their exact pathophysiological role is unknown. We examined the effect of the interaction of AGE with one of their receptors (i.e., RAGE) on the function and structure of the peritoneum exposed to high ambient glucose concentrations. Streptozotocin-induced diabetic rats and control rats were treated during 6 wk with either neutralizing monoclonal anti-RAGE antibodies or control antibodies. The expression of RAGE was strongly enhanced in the peritoneal membrane of the diabetic animals. The diabetic peritonea were characterized by an elevated transport of small solutes, lower ultrafiltration rates, a higher vascular density, and an upregulation of endothelial nitric oxide synthase expression. These parameters were unaffected by treatment with anti-RAGE antibodies. In contrast, anti-RAGE but not control antibodies prevented upregulation of TGF-beta, development of submesothelial fibrosis, and fibronectin accumulation in the peritoneum of diabetic animals. In conclusion, binding of AGE to RAGE increases the expression of TGF-beta and contributes to the development of submesothelial fibrosis. Neoangiogenesis and the resultant loss of ultrafiltration capacity are mediated by different pathogenetic pathways.

Topics: Albumins; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Biological Transport; Diabetes Mellitus, Experimental; Epithelium; Female; Fibronectins; Fibrosis; Glucose; Glycation End Products, Advanced; Hyperglycemia; Immunoblotting; Immunohistochemistry; Neovascularization, Pathologic; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Peritoneum; Rats; Rats, Wistar; Receptor for Advanced Glycation End Products; Receptors, Immunologic; Transforming Growth Factor beta; Up-Regulation; Urea

Hyperglycemia is a crucial factor in the development of diabetic nephropathy. We previously showed that high glucose upregulates thrombospondin 1 (TSP1)-dependent transforming growth factor (TGF)-beta activation by altering cGMP-dependent protein kinase (PKG) activity as a result of decreased nitric oxide signaling. In the present study, we showed that high glucose concentrations significantly reduced endogenous PKG activity. To further examine the mechanisms by which PKG regulates TSP1 expression and TSP1-dependent TGF-beta activation, we generated stably transfected rat mesangial cells (RMCs) with inducible expression tetracycline-induced gene expression of the catalytic domain of PKG. After tetracycline induction, the catalytic domain of PKG is expressed as a cGMP-independent active kinase. Expression of the catalytic domain prevented high glucose-mediated increases in transcription of the TSP1 gene with no alteration in TSP1 mRNA stability. Glucose stimulation of TSP1 protein expression and TGF-beta bioactivity were also downregulated. TGF-beta-dependent fibronectin and type IV collagen expression under high glucose conditions were significantly reduced upon catalytic domain expression in transfected RMCs. These results show that constitutively active PKG inhibits the fibrogenic potential of high glucose through repression of TSP1-dependent TGF-beta bioactivity, suggesting that gene transfer of the catalytic domain of PKG might provide a new strategy for treatment of diabetic renal fibrosis.

Topics: Animals; Anti-Bacterial Agents; Catalytic Domain; Cell Line; Collagen; Cyclic GMP-Dependent Protein Kinases; Diabetic Nephropathies; Enzyme Activation; Extracellular Matrix; Fibronectins; Gene Expression Regulation, Enzymologic; Glomerular Mesangium; Glucose; Humans; Hyperglycemia; Rats; Tetracycline; Thrombospondin 1; Transfection; Transforming Growth Factor beta

Chronic elevated glucose levels and activation of the renal renin-angiotensin system have been implicated in the pathogenesis of diabetic nephropathy. We tested the ability of lisofylline (LSF), a novel antiinflammatory compound, to prevent extracellular matrix (ECM) accumulation and growth factor production by human mesangial cells (HMCs) cultured in chronic elevated glucose (HG) or angiotensin II (AngII). HMCs were cultured in normal glucose (NG) (5.5 mm) and in HG (25 mm) for 7 d or with 10-7 m AngII for 4 h with or without LSF. Levels of the ECM protein fibronectin and TGF-beta in media were shown to increase in HG compared with NG. LSF decreased HG-induced fibronectin and TGF-beta production to control levels. Increased expression of collagen type IV and laminin was observed in AngII-cultured HMCs. LSF protected HMCs from the AngII induction of these key matrix proteins. cAMP-responsive binding element phosphorylation was significantly higher in both HG and AngII-cultured HMCs. LSF reduced phosphorylation of both cAMP-responsive binding element and p38 MAPK compared with control. These data demonstrate that LSF protects HMCs from HG- and AngII-mediated ECM deposition by the reduction of matrix protein secretion possibly through regulation of TGF-beta production and modulation of the p38 MAPK pathway. These results suggest that LSF may provide therapeutic benefit for prevention or treatment of diabetic nephropathy.

Topics: Angiotensin II; Anti-Inflammatory Agents, Non-Steroidal; Cells, Cultured; Connective Tissue Growth Factor; Cyclic AMP Response Element-Binding Protein; Extracellular Matrix; Extracellular Matrix Proteins; Fibronectins; Gene Expression; Glomerular Mesangium; Glucose; Humans; Hyperglycemia; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Pentoxifylline; Phosphorylation; Transforming Growth Factor beta; Vasoconstrictor Agents

Previous studies demonstrated that 2 mo of dietary supplementation with alpha-lipoic acid (LA) prevented early glomerular injury in non-insulin-treated streptozotocin diabetic rats (D). The present study examined the effects of chronic LA supplementation (30 mg/kg body wt per d) on nephropathy in D after 7 mo of diabetes. Compared with control rats, D developed increased urinary excretion of albumin and transforming growth factor beta, renal insufficiency, glomerular mesangial matrix expansion, and glomerulosclerosis in association with depletion of glutathione and accumulation of malondialdehyde in renal cortex. LA prevented or ameliorated all of these changes in D. Because chronic LA supplementation also attenuated hyperglycemia in D after 3 mo, its effects on renal injury were compared with treatment of rats with sufficient insulin to maintain a level of glycemic control for the entire 7-mo period (D-INS) equivalent to that observed with LA during the final 4 mo. Despite superior longitudinal glycemic control in D-INS, urinary excretion of albumin and transforming growth factor beta, glomerular mesangial matrix expansion, the extent of glomerulosclerosis, and renal cortical malondialdehyde content were all significantly greater, whereas cortical glutathione content was lower than corresponding values in D given LA. Thus, the renoprotective effects of LA in D were not attributable to improved glycemic control alone but also likely reflected its antioxidant activity. The combined antioxidant and hypoglycemic actions of LA both may contribute to its utility in preventing renal injury and other complications of diabetes.

Topics: Albuminuria; Animals; Antioxidants; Blood Glucose; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Female; Glomerular Mesangium; Glutathione; Hyperglycemia; Hypoglycemic Agents; Kidney Cortex; Malondialdehyde; Rats; Renal Insufficiency; Thioctic Acid; Transforming Growth Factor beta

Activation of the renal renin-angiotensin system has been implicated in the pathogenesis of diabetic nephropathy. Because previous in vitro studies demonstrated the angiotensin II (ang II)-mediated up-regulation of the prosclerotic transforming growth factor beta 1 (TGF) we studied the molecular mechanism of ang II-induced TGF-beta 1 gene activation.. Mesangial cells were stimulated with 100 nmol/l ang II with or without inhibitors of protein kinase C (PKC) and p38 MAPK and the TGF-beta 1 promoter activity was determined by promoter-reporter assays. The effect of ang II on the binding of nuclear proteins to the regulatory AP-1 site B, previously shown to mediate the high glucose-response of the TGF-beta 1 promoter, was studied by electrophoretic mobility shift assays.. Ang II enhanced the activity of the TGF-beta1 promoter fragment -453/+11 approximately 1.6-fold. Mutation of each of two AP-1 binding sites or inhibition of the PKC- and p38 MAPK-dependent pathways blocked the ang II-stimulated activity completely. Furthermore, ang II activated the binding of nuclear proteins to the AP-1 box B of the TGF-beta 1 promoter. These effects were similar to those previously observed with high glucose. Co-incubation with ang II and high glucose had no additive effect on TGF-beta 1 promoter activity, protein binding to the AP-1 box B or activation of p38 MAPK.. The findings indicate that ang II and hyperglycaemia stimulate the TGF-beta 1 gene activation through the same PKC- and p38 MAPK-dependent pathways by the same regulatory elements of the TGF-beta 1 promoter. Our data could also be relevant for e.g. hypertension-induced glomerulosclerosis.

Topics: Angiotensin II; Animals; Base Sequence; beta-Galactosidase; Binding Sites; Cell Line; Gene Expression Regulation; Genes, Reporter; Glomerular Mesangium; Glucose; Humans; Hyperglycemia; Mitogen-Activated Protein Kinases; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Recombinant Proteins; Swine; Transcription Factors; Transcriptional Activation; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Biological Transport; Blood Glucose; Diabetic Nephropathies; Humans; Hyperglycemia; Immediate-Early Proteins; Nuclear Proteins; Protein Serine-Threonine Kinases; Renal Insufficiency; Sodium; Transforming Growth Factor beta

Topics: Diabetic Nephropathies; Glucose Transporter Type 1; Humans; Hyperglycemia; Monosaccharide Transport Proteins; Transforming Growth Factor beta

The accumulation of extracellular matrices and integrins by high glucose has been reported in relation to diabetic complications. We previously reported that PDL cells expressed a higher amount of VLA-5 when cultured in high-glucose (4500 mg/L) medium than those cultured in low-glucose (1100 mg/L) medium. In this study, we aimed to address (1) whether this effect was mediated by the transcriptional level of the gene or the degradative level of the protein, and (2) whether this effect was mediated by TGF-beta. The results indicated that the level of mRNA encoding alpha5 integrin did not change in PDL cells regardless of the concentration of glucose. Alternatively, high glucose suppressed cathepsin B+L activity. Additionally, the level of mRNA encoding TGF-beta was not affected by high glucose, nor did an anti-TGF-beta neutralizing antibody have an effect on the expression of beta5 gene or cathepsin activity. Therefore, the effects of high glucose appeared to be mediated by impaired protein degradation, but not by autocrine TGF-beta.

Topics: Autocrine Communication; Cathepsins; Cell Adhesion; Cells, Cultured; Culture Media; Down-Regulation; Fibroblasts; Glucose; Humans; Hyperglycemia; Periodontal Ligament; Receptors, Fibronectin; Statistics, Nonparametric; Transcription, Genetic; Transforming Growth Factor beta

The hexosamine pathway has been implicated in the pathogenesis of diabetic complications. We determined first that hyperglycemia induced a decrease in glyceraldehyde-3-phosphate dehydrogenase activity in bovine aortic endothelial cells via increased production of mitochondrial superoxide and a concomitant 2.4-fold increase in hexosamine pathway activity. Both decreased glyceraldehyde-3-phosphate dehydrogenase activity and increased hexosamine pathway activity were prevented completely by an inhibitor of electron transport complex II (thenoyltrifluoroacetone), an uncoupler of oxidative phosphorylation (carbonyl cyanide m-chlorophenylhydrazone), a superoxide dismutase mimetic [manganese (III) tetrakis(4-benzoic acid) porphyrin], overexpression of either uncoupling protein 1 or manganese superoxide dismutase, and azaserine, an inhibitor of the rate-limiting enzyme in the hexosamine pathway (glutamine:fructose-6-phosphate amidotransferase). Immunoprecipitation of Sp1 followed by Western blotting with antibodies to O-linked GlcNAc, phosphoserine, and phosphothreonine showed that hyperglycemia increased GlcNAc by 1.7-fold, decreased phosphoserine by 80%, and decreased phosphothreonine by 70%. The same inhibitors prevented all these changes. Hyperglycemia increased expression from a transforming growth factor-beta(1) promoter luciferase reporter by 2-fold and increased expression from a (-740 to +44) plasminogen activator inhibitor-1 promoter luciferase reporter gene by nearly 3-fold. Inhibition of mitochondrial superoxide production or the glucosamine pathway prevented all these changes. Hyperglycemia increased expression from an 85-bp truncated plasminogen activator inhibitor-1 (PAI-1) promoter luciferase reporter containing two Sp1 sites in a similar fashion (3.8-fold). In contrast, hyperglycemia had no effect when the two Sp1 sites were mutated. Thus, hyperglycemia-induced mitochondrial superoxide overproduction increases hexosamine synthesis and O-glycosylation of Sp1, which activates expression of genes that contribute to the pathogenesis of diabetic complications.

Topics: Acetylglucosamine; Animals; Cattle; Cells, Cultured; Glycosylation; Hexosamines; Hyperglycemia; Mitochondria; Phosphoserine; Phosphothreonine; Plasminogen Activator Inhibitor 1; Promoter Regions, Genetic; Sp1 Transcription Factor; Superoxides; Transforming Growth Factor beta

Hyperglycemia-induced overproduction of the prosclerotic cytokine transforming growth factor-beta1 (TGF-beta1) has been implicated in the pathogenesis of diabetic nephropathy. Because high glucose and phorbol esters (PMA) increase TGF-beta1 mRNA levels in mesangial cells, this study was designed to characterize these effects on the human TGF-beta1 promoter activity. With the use of luciferase reporter gene constructs containing TGF-beta1 5'-flanking sequence (from -453 to +11 bp) transfected into mesangial cells, it was found that 30 mM glucose induced a nearly twofold increase in TGF-beta1 promoter activity after 24 h of incubation in human and porcine mesangial cells. Stimulation by PMA was more effective (2.3-fold). Mutagenesis in either one of the two or both activating protein-1 (AP-1) binding sites abolished the high glucose and the PMA effect. Furthermore, addition of the AP-1 inhibitor curcumin obliterated the glucose response. Corresponding experiments revealed that the transcription factor stimulating protein 1 was not involved in mediating the glucose effect. The high glucose-induced TGF-beta1 promoter activation was also prevented by inhibitors of protein kinase C and p38 mitogen-activated proteinkinase. Electrophoretic mobility shift assays with oligonucleotides containing one of the two AP-1 binding sites showed that glucose treatment markedly enhanced the binding activity of nuclear proteins of mesangial cells, particularly to box B. Supershift assays demonstrated that JunD and c-Fos were present in the protein-DNA complexes under control and hyperglycemic conditions. The functional and structural results show that glucose regulates human TGF-beta1 gene expression through two adjacent AP-1 binding sites and gives rise to the involvement of protein kinase C and p38 mitogen-activated protein kinase in hyperglycemia-induced TGF-beta1 gene expression.

Topics: Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Gene Expression Regulation; Glomerular Mesangium; Glucose; Humans; Hyperglycemia; Imidazoles; Indoles; Maleimides; Mitogen-Activated Protein Kinases; Nuclear Proteins; p38 Mitogen-Activated Protein Kinases; Podophyllin; Podophyllotoxin; Promoter Regions, Genetic; Pyridines; RNA, Messenger; Sp1 Transcription Factor; Tetradecanoylphorbol Acetate; Tissue Distribution; Transcription Factor AP-1; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Animals; Gene Expression Regulation; Glomerular Mesangium; Glycosaminoglycans; Hyperglycemia; Swine; Transforming Growth Factor beta

Recent experimental work implicates transforming growth factor-beta (TGF-beta) as an aetiologic mediator of diabetic nephropathy and the ubiquitous glucose transporter GLUT1 as an important permissive factor for the tissue injury caused by hyperglycaemia. High ambient glucose increases GLUT1 expression and glucose transport activity when compared with physiologic glucose concentrations. Treatment of rat mesangial cells with TGF-beta up-regulates GLUT1 mRNA and protein levels and significantly increases glucose uptake. Addition of neutralizing anti-TGF-beta antibody prevents the stimulatory effects of high glucose on GLUT1 expression. Cultured rat mesangial cells transduced with the human GLUT1 gene and thus overexpressing the GLUT1 protein show marked increase in glucose uptake and the synthesis of extracellular matrix molecules, even when grown in normal ambient glucose concentrations. Thus, TGF-beta and GLUT1, two proteins that are up-regulated in glomerular mesangial cells in a hyperglycaemic milieu, can influence the expression of one another. It is therefore fair to conclude that, with successful interruption of the TGF-beta-GLUT1 axis, the beneficial effects of strict glucose control on the development of diabetic nephropathy could likely be augmented.

Topics: Animals; Diabetic Nephropathies; Glucose Transporter Type 1; Humans; Hyperglycemia; Monosaccharide Transport Proteins; Rats; Transforming Growth Factor beta

The primary etiologic factor in diabetic glomerulosclerosis appears to be an overproduction of transforming growth factor-beta by mesangial cells, which in turn reflects a hyperglycemically mediated overactivation of protein kinase C (PKC) throughout the glomerulus. Membrane-active antioxidants, fish oil, and angiotensin-converting enzyme inhibitors can act to down-regulate glomerular PKC activity, via a variety of mechanisms that may include activation of diacylglycerol kinase and suppression of phosphatidate phosphohydrolase, support of endothelial nitric oxide and heparan sulfate production, inhibition of thromboxane and angiotensin synthesis/activity, and correction of glomerular hypertension. The beneficial impact of these measures on vascular endothelial function may be of more general utility in the prevention of diabetic complications such as retinopathy, neuropathy, and atherosclerosis. Adjunctive use of gamma-linolenic acid is indicated for prevention of neuropathy, and it is conceivable that bioactive chromium will have protective activity not solely attributable to improved glycemic control. Re-establishing euglycemia must clearly remain the core strategy for preventing diabetic complications, but when glycemic control remains suboptimal, practical, safe measures are at hand for decreasing risk.

Topics: Angiotensin II; Angiotensin-Converting Enzyme Inhibitors; Antioxidants; Diabetic Angiopathies; Diabetic Nephropathies; Enzyme Activation; Fish Oils; Heparitin Sulfate; Humans; Hyperglycemia; Kidney Glomerulus; Lipid Peroxidation; Models, Biological; Nitric Oxide; Protein Kinase C; Thromboxane A2; Transforming Growth Factor beta

Induction of protein kinase C (PKC) pathway in the vascular tissues by hyperglycemia has been associated with many of the cellular changes observed in the complications of diabetes. Recently, we have reported that the use of a novel, orally effective specific inhibitor of PKC beta isoform (LY333531) normalized many of the early retinal and renal hemodynamics in rat models of diabetes. In the present study, we have characterized a spectrum of biochemical and molecular abnormalities associated with chronic changes induced by glucose or diabetes in the cultured mesangial cells and renal glomeruli that can be prevented by LY333531. Hyperglycemia increased diacylglycerol (DAG) level in cultured mesangial cells exposed to high concentrations of glucose and activated PKC alpha and beta1 isoforms in the renal glomeruli of diabetic rats. The addition of PKC beta selective inhibitor (LY333531) to cultured mesangial cells inhibited activated PKC activities by high glucose without lowering DAG levels and LY333531 given orally in diabetic rats specifically inhibited the activation of PKC beta1 isoform without decreasing PKC alpha isoform activation. Glucose-induced increases in arachidonic acid release, prostaglandin E2 production, and inhibition of Na+-K+ ATPase activities in the cultured mesangial cells were completely prevented by the addition of LY333531. Oral feeding of LY333531 prevented the increased mRNA expression of TGF-beta1 and extracellular matrix components such as fibronectin and alpha1(IV) collagen in the glomeruli of diabetic rats in parallel with inhibition of glomerular PKC activity. These results suggest that the activation of PKC, predominately the beta isoform by hyperglycemia in the mesangial cells and glomeruli can partly contribute to early renal dysfunctions by alteration of prostaglandin production and Na+-K+ ATPase activity as well as the chronic pathological changes by the overexpression of TGF-beta1 and extracellular matrix components genes.

Topics: Administration, Oral; Animals; Arachidonic Acid; Cells, Cultured; Collagen; Diabetes Mellitus, Experimental; Diglycerides; Dinoprostone; Enzyme Activation; Enzyme Inhibitors; Extracellular Matrix Proteins; Fibronectins; Glomerular Mesangium; Glucose; Hyperglycemia; Indoles; Kidney Glomerulus; Male; Maleimides; Prostaglandins; Protein Kinase C; Protein Kinase C beta; Rats; Rats, Sprague-Dawley; Reference Values; RNA, Messenger; Sodium-Potassium-Exchanging ATPase; Transcription, Genetic; Transforming Growth Factor beta

Post-mitotic cultures of human mesangial cells were maintained in media containing 4-30 mM D-glucose for up to 28 days. Changes in mRNA and protein levels for specific macromolecules occurred between 7 and 14 days after initiating hyperglycaemic conditions. Slot blot analysis showed 2-3-fold increases in mRNAs for collagen type I, fibronectin, versican and perlecan, whereas mRNA for decorin was increased by up to 20-fold. Levels of mRNAs for biglycan and syndecan were unaffected by hyperglycaemic culture. Reverse transcriptase PCR (RT-PCR) confirmed that decorin mRNA levels are greatly elevated and also showed increased transcription of the TGF-beta 1 gene in hyperglycaemic cultures. Western analysis and ELISA indicated accumulations of collagen types I and III, laminin and fibronectin in the cell layers and media of hyperglycaemic cultures with increasing time. Type IV collagen did not accumulate in either compartment of hyperglycaemic mesangial cell cultures. Collagen types I, III, and fibronectin did not accumulate in the cell layers of hyperglycaemic human dermal fibroblasts, indicating a cell-specific response in mesangial cultures. Decorin and versican, but not biglycan, were increased in the hyperglycaemic mesangial cell culture media. There were no apparent changes in core proteins for decorin and biglycan in fibroblast media. Transforming growth factor beta 1 (TGF-beta 1) in hyperglycaemic mesangial cell cultures increased 5-fold after 7 days, but decreased thereafter to only approx. 2-fold after 28 days. The changes in TGF-beta 1 mRNA, as detected by RT-PCR, and protein followed one another closely.

Topics: Base Sequence; Cells, Cultured; DNA Primers; Extracellular Matrix Proteins; Female; Fibroblasts; Glomerular Mesangium; Glucose; Humans; Hyperglycemia; Kidney Cortex; Kinetics; Middle Aged; Mitosis; Molecular Sequence Data; Polymerase Chain Reaction; RNA, Messenger; Skin; Time Factors; Transcription, Genetic; Transforming Growth Factor beta

We investigated the intrarenal distribution of transforming growth factor-beta 1 (TGF-beta 1) protein and the TGF-beta 1 mRNA levels in the glomeruli and renal cortex of Wistar rats with streptozotocin-induced diabetes before and after the onset of diabetic nephropathy. Monthly urinary albumin excretion, glomerular filtration rate, glomerular volume, renal histology and immunohistochemical reaction for type-I collagen were also studied. The results showed progressively higher glomerular immunohistochemical TGF-beta 1 staining in rats with a diabetes duration of 24 and 40 weeks which was correlated with albuminuria (r = 0.905, p < 0.01) and was temporally associated with the appearance of glomerular deposition of total and type-I collagen. The glomerular content of TGF-beta 1 mRNA was higher in rats diabetic for 20 weeks while lower cortical RNA-TGF-beta 1 levels were found in rats with a diabetes duration of 1-40 weeks. These data suggest that this polypeptide may be an important mediator of diabetic glomerulosclerosis.

Topics: Animals; Blotting, Northern; Diabetic Nephropathies; Female; Hyperglycemia; Immunohistochemistry; Insulin; Kidney Cortex; Kidney Function Tests; Rats; Rats, Wistar; RNA, Messenger; Sclerosis; Transforming Growth Factor beta