Role of TGF-β in chronic kidney disease: an integration of tubular,glomerular and vascular effects

Transforming growth factor beta (TGF-β) has been recognized as an important mediator in the genesis of chronic kidney diseases (CKD), which are characterized by the accumulation of extracellular matrix (ECM) components in the glomeruli (glomerular fibrosis, glomerulosclerosis) and the tubular interstitium (tubulointerstitial fibrosis). Glomerulosclerosis is a major cause of glomerular filtration rate reduction in CKD and all three major glomerular cell types (podocytes or visceral epithelial cells, mesangial cells and endothelial cells) participate in the fibrotic process. TGF-β induces (1) podocytopenia caused by podocyte apoptosis and detachment from the glomerular basement membrane; (2) mesangial expansion caused by mesangial cell hypertrophy, proliferation (and eventually apoptosis) and ECM synthesis; (3) endothelial to mesenchymal transition giving rise to glomerular myofibroblasts, a major source of ECM. TGF-β has been shown to mediate several key tubular pathological events during CKD progression, namely fibroblast proliferation, epithelial to mesenchymal transition, tubular and fibroblast ECM production and epithelial cell death leading to tubular cell deletion and interstitial fibrosis. In this review, we re-examine the mechanisms involved in glomerulosclerosis and tubulointerstitial fibrosis and the way that TGF-β participates in renal fibrosis, renal parenchyma degeneration and loss of function associated with CKD.     

Effect of sarpogrelate on altered STZ-diabetes induced cardiovascular responses to 5-hydroxytryptamine in rats

Sarpogrelate, a specific 5-HT_2A receptor antagonist is reported to produce a number of beneficial cardiovascular effects in diabetes mellitus. In the present investigation we have studied the effects of sarpogrelate on 5-HT receptors in heart and platelets in streptozotocin (STZ)-diabetic rats. Diabetes was induced by a single tail vein injection of STZ (45 mg/kg) and sarpogrelate (1 mg/kg, i.p.) was administered daily for 6 weeks. Injection of STZ produced significant loss of body weight, polyphagia, polydypsia, hyperglycemia, hypoinsulinemia, hypertension and bradycardia. Treatment with sarpogrelate significantly lowered fasting glucose levels with corresponding increase in insulin levels. It also significantly prevented STZ-induced polydypsia, hyperphagia, hypertension, and bradycardia but not the loss of body weight. 5-HT produced dose-dependent positive inotropic effect that was found to be decreased significantly in STZ-diabetic rats. Hearts obtained from sarpogrelate treated diabetic rats did not show any decrease in responsiveness to 5-HT. Relative platelet aggregation per se was found to be higher in STZ-diabetic rats as compared to control and this was significantly prevented by sarpogrelate treatment. 5-HT produced a dose-dependent increase in platelet aggregation in non-diabetic and sarpogrelate treated diabetic rats. However, 5-HT failed to produce any increase in platelet aggregation in untreated diabetic rats. Our data suggest that STZ-induced diabetes may produce down-regulation of cardiac 5-HT_2A receptors and increased platelet aggregation. Treatment with sarpogrelate seems to prevent STZ-induced down-regulation of 5-HT receptors and increase in platelet activity in diabetic rats.     

Transforming Growth Factor β1 (TGF-β1) Enhances Expression of Profibrotic Genes through a Novel Signaling Cascade and MicroRNAs in Renal Mesangial Cells

Increased expression of transforming growth factor-β1 (TGF-β1) in glomerular mesangial cells (MC) augments extracellular matrix accumulation and hypertrophy during the progression of diabetic nephropathy (DN), a debilitating renal complication of diabetes. MicroRNAs (miRNAs) play key roles in the pathogenesis of DN by modulating the actions of TGF-β1 to enhance the expression of profibrotic genes like collagen. In this study, we found a significant decrease in the expression of miR-130b in mouse MC treated with TGF-β1. In parallel, there was a down-regulation in miR-130b host gene 2610318N02RIK (RIK), suggesting host gene-dependent expression of this miRNA. TGF-β receptor 1 (TGF-βR1) was identified as a target of miR-130b. Interestingly, the RIK promoter contains three NF-Y binding sites and was regulated by NF-YC. Furthermore, NF-YC expression was inhibited by TGF-β1, suggesting that a signaling cascade, involving TGF-β1-induced decreases in NF-YC, RIK, and miR-130b, may up-regulate TGF-βR1 to augment expression of TGF-β1 target fibrotic genes. miR-130b was down-regulated, whereas TGF-βR1, as well as the profibrotic genes collagen type IV α 1 (Col4a1), Col12a1, CTGF, and PAI-1 were up-regulated not only in mouse MC treated with TGF-β1 but also in the glomeruli of streptozotocin-injected diabetic mice, supporting in vivo relevance. Together, these results demonstrate a novel miRNA- and host gene-mediated amplifying cascade initiated by TGF-β1 that results in the up-regulation of profibrotic factors, such as TGF-βR1 and collagens associated with the progression of DN.     

Effects of sarpogrelate on serotonin-induced increases in cytosolic Ca2+ in cultured rat mesangial cells

The effects of the new 5-HT_2A receptor antagonist sarpogrelate on the cellular action of serotonin were examined in cultured rat mesangial cells by measuring cytosolic free calcium concentration ([Ca²⁺]i). Sarpogrelate inhibited serotonin-induced increases in [Ca²⁺]i in a concentration-dependent manner. M1, a major metabolite of sarpogrelate, also exhibited an inhibitory effect exceeding that of sarpogrelate. The inhibitory effects of sarpogrelate and M1 were abolished by washing out these compounds. In contrast, the increase in [Ca²⁺]i induced by angiotensin II or arginine vasopressin was not affected by pretreatment of the cells with sarpogrelate or M1. These results suggest that sarpogrelate and its major metabolite (M1) act as reversible and specific 5-HT_2A receptor antagonists against the contractile action of platelet-derived serotonin in mesangial cells.     

FOG2 Protein Down-regulation by Transforming Growth Factor-β1-induced MicroRNA-200b/c Leads to Akt Kinase Activation and Glomerular Mesangial Hypertrophy Related to Diabetic Nephropathy

Glomerular hypertrophy is a hallmark of diabetic nephropathy. Akt kinase activated by transforming growth factor-β1 (TGF-β) plays an important role in glomerular mesangial hypertrophy. However, the mechanisms of Akt activation by TGF-β are not fully understood. Recently, miR-200 and its target FOG2 were reported to regulate the activity of phosphatidylinositol 3-kinase (the upstream activator of Akt) in insulin signaling. Here, we show that TGF-β activates Akt in glomerular mesangial cells by inducing miR-200b and miR-200c, both of which target FOG2, an inhibitor of phosphatidylinositol 3-kinase activation. FOG2 expression was reduced in the glomeruli of diabetic mice as well as TGF-β-treated mouse mesangial cells (MMC). FOG2 knockdown by siRNAs in MMC activated Akt and increased the protein content/cell ratio suggesting hypertrophy. A significant increase of miR-200b/c levels was detected in diabetic mouse glomeruli and TGF-β-treated MMC. Transfection of MMC with miR-200b/c mimics significantly decreased the expression of FOG2. Conversely, miR-200b/c inhibitors attenuated TGF-β-induced decrease in FOG2 expression. Furthermore, miR-200b/c mimics increased the protein content/cell ratio, whereas miR-200b/c inhibitors abrogated the TGF-β-induced increase in protein content/cell. In addition, down-regulation of FOG2 by miR-200b/c could activate not only Akt but also ERK, which was also through PI3K activation. These data suggest a new mechanism for TGF-β-induced Akt activation through FOG2 down-regulation by miR-200b/c, which can lead to glomerular mesangial hypertrophy in the progression of diabetic nephropathy.     

Plasminogen Activator Inhibitor-1 Is a Transcriptional Target of the Canonical Pathway of Wnt/β-Catenin Signaling

Plasminogen activator inhibitor-1 (PAI-1) is a multifunctional glycoprotein that plays a critical role in the pathogenesis of chronic kidney and cardiovascular diseases. Although transforming growth factor (TGF)-β1 is a known inducer of PAI-1, how it controls PAI-1 expression remains enigmatic. Here we investigated the mechanism underlying TGF-β1 regulation of PAI-1 in kidney tubular epithelial cells (HKC-8). Surprisingly, overexpression of Smad2 or Smad3 in HKC-8 cells blocked PAI-1 induction by TGF-β1, whereas knockdown of them sensitized the cells to TGF-β1 stimulation, suggesting that Smad signaling is not responsible for PAI-1 induction. Blockade of several TGF-β1 downstream pathways such as p38 MAPK or JNK, but not phosphatidylinositol 3-kinase/Akt and ERK1/2, only partially inhibited PAI-1 expression. TGF-β1 stimulated β-catenin activation in tubular epithelial cells, and ectopic expression of β-catenin induced PAI-1 expression, whereas inhibition of β-catenin abolished its induction. A functional T cell factor/lymphoid enhancer-binding factor-binding site was identified in the promoter region of the PAI-1 gene, which interacted with T cell factor upon β-catenin activation. Deletion or site-directed mutation of this site abolished PAI-1 response to β-catenin or TGF-β1 stimulation. Similarly, ectopic expression of Wnt1 also activated PAI-1 expression and promoter activity. In vivo, PAI-1 was induced in kidney tubular epithelia in obstructive nephropathy. Delivery of Wnt1 gene activated β-catenin and promoted PAI-1 expression after obstructive injury, whereas blockade of Wnt/β-catenin signaling by Dickkopf-1 gene inhibited PAI-1 induction. Collectively, these studies identify PAI-1 as a direct downstream target of Wnt/β-catenin signaling and demonstrate that PAI-1 induction could play a role in mediating the fibrogenic action of this signaling.     

Inhibition of Plasminogen Activator Inhibitor-1 Attenuates Transforming Growth Factor-β-Dependent Epithelial Mesenchymal Transition and Differentiation of Fibroblasts to Myofibroblasts

Transforming growth factor-β (TGF-β) is central during the pathogenesis of pulmonary fibrosis, in which the plasminogen activator inhibitor-1 (PAI-1) also has an established role. TGF-β is also known to be the strongest inducer of PAI-1. To investigate the link between PAI-1 and TGF-β in fibrotic processes, we evaluated the effect of SK-216, a PAI-1-specific inhibitor, in TGF-β-dependent epithelial-mesenchymal transition (EMT) and fibroblast to myofibroblast differentiation. In human alveolar epithelial A549 cells, treatment with TGF-β induced EMT, whereas co-treatment with SK-216 attenuated the occurrence of EMT. The inhibition of TGF-β-induced EMT by SK-216 was also confirmed in the experiment using murine epithelial LA-4 cells. Blocking EMT by SK-216 inhibited TGF-β-induced endogenous production of PAI-1 and TGF-β in A549 cells as well. These effects of SK-216 were not likely mediated by suppressing either Smad or ERK pathways. Using human lung fibroblast MRC-5 cells, we demonstrated that SK-216 inhibited TGF-β-dependent differentiation of fibroblasts to myofibroblasts. We also observed this inhibition by SK-216 in human primary lung fibroblasts. Following these in vitro results, we tested oral administration of SK-216 into mice injected intratracheally with bleomycin. We found that SK-216 reduced the degree of bleomycin-induced pulmonary fibrosis in mice. Although the precise mechanisms underlying the link between TGF-β and PAI-1 regarding fibrotic process were not determined, PAI-1 seems to act as a potent downstream effector on the pro-fibrotic property of TGF-β. In addition, inhibition of PAI-1 activity by a PAI-1 inhibitor exerts an antifibrotic effect even in vivo. These data suggest that targeting PAI-1 as a downstream effector of TGF-β could be a promising therapeutic strategy for pulmonary fibrosis.     

TGF-β1 Protects against Mesangial Cell Apoptosis via Induction of Autophagy

Autophagy can lead to cell death in response to stress, but it can also act as a protective mechanism for cell survival. We show that TGF-β1 induces autophagy and protects glomerular mesangial cells from undergoing apoptosis during serum deprivation. Serum withdrawal rapidly induced autophagy within 1 h in mouse mesangial cells (MMC) as determined by increased microtubule-associated protein 1 light chain 3 (LC3) levels and punctate distribution of the autophagic vesicle-associated-form LC3-II. We demonstrate that after 1 h there was a time-dependent decrease in LC3 levels that was accompanied by induction of apoptosis, evidenced by increases in cleaved caspase 3. However, treatment with TGF-β1 resulted in induction of the autophagy protein LC3 while suppressing caspase 3 activation. TGF-β1 failed to rescue MMC from serum deprivation-induced apoptosis upon knockdown of LC3 by siRNA and in MMC from LC3 null (LC3^−/−) mice. We show that TGF-β1 induced autophagy through TAK1 and Akt activation, and inhibition of PI3K-Akt pathway by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or dominant-negative Akt suppressed LC3 levels and enhanced caspase 3 activation. TGF-β1 also up-regulated cyclin D1 and E protein levels while down-regulating p27, thus stimulating cell cycle progression. Bafilomycin A1, but not MG132, blocked TGF-β1 down-regulation of p27, suggesting that p27 levels were regulated through autophagy. Taken together, our data indicate that TGF-β1 rescues MMC from serum deprivation-induced apoptosis via induction of autophagy through activation of the Akt pathway. The autophagic process may constitute an adaptive mechanism to glomerular injury by inhibiting apoptosis and promoting mesangial cell survival.     

Hydrogen Peroxide–Inducible Clone-5 Regulates Mesangial Cell Proliferation in Proliferative Glomerulonephritis in Mice

Hydrogen peroxide-inducible clone-5 (Hic-5) is a transforming growth factor (TGF)-β1-inducible focal adhesion protein. We previously demonstrated that Hic-5 was localized in mesangial cells and its expression was associated with glomerular cell proliferation and matrix expansion in human and rat glomerulonephritis (GN). In the present study, we first assessed the role of Hic-5 in mesangioproliferative GN by injecting Habu venom into heminephrectomized wild type (Hic-5+/+) and Hic-5-deficient (Hic-5-/-) mice. Hic-5+/+ GN mice exhibited glomerular cell proliferation on day 7. Surprisingly, glomerular cell number and Ki-67-positive cells in Hic-5-/- GN mice were significantly greater than those in Hic-5+/+ GN mice on day 7, although the number of glomerular apoptotic cells and the expression of growth factors (platelet-derived growth factor-BB and TGF-β1) and their receptors were similarly increased in both Hic-5+/+ and Hic-5-/- GN mice. In culture experiments, proliferation assays showed that platelet-derived growth factor-BB and TGF-β1 enhanced the proliferation of Hic-5-/- mesangial cells compared with Hic-5+/+ mesangial cells. In addition, mitogenic regulation by Hic-5 was associated with altered and coordinated expression of cell cycle-related proteins including cyclin D1 and p21. The present results suggest that Hic-5 might regulate mesangial cell proliferation in proliferative GN in mice. In conclusion, modulation of Hic-5 expression might have a potential to prevent mesangial cell proliferation in the acute mitogenic phase of glomerulonephritis.     

Role of MicroRNA 1207-5P and Its Host Gene,the Long Non-Coding RNA Pvt1, as Mediators of Extracellular Matrix Accumulation in the Kidney: Implications for Diabetic Nephropathy

Diabetic nephropathy is the most common cause of chronic kidney failure and end-stage renal disease in the Western World. One of the major characteristics of this disease is the excessive accumulation of extracellular matrix (ECM) in the kidney glomeruli. While both environmental and genetic determinants are recognized for their role in the development of diabetic nephropathy, epigenetic factors, such as DNA methylation, long non-coding RNAs, and microRNAs, have also recently been found to underlie some of the biological mechanisms, including ECM accumulation, leading to the disease. We previously found that a long non-coding RNA, the plasmacytoma variant translocation 1 (PVT1), increases plasminogen activator inhibitor 1 (PAI-1) and transforming growth factor beta 1 (TGF-β1) in mesangial cells, the two main contributors to ECM accumulation in the glomeruli under hyperglycemic conditions, as well as fibronectin 1 (FN1), a major ECM component. Here, we report that miR-1207-5p, a PVT1-derived microRNA, is abundantly expressed in kidney cells, and is upregulated by glucose and TGF-β1. We also found that like PVT1, miR-1207-5p increases expression of TGF-β1, PAI-1, and FN1 but in a manner that is independent of its host gene. In addition, regulation of miR-1207-5p expression by glucose and TGFβ1 is independent of PVT1. These results provide evidence supporting important roles for miR-1207-5p and its host gene in the complex pathogenesis of diabetic nephropathy.     

TDAG51 induces renal interstitial fibrosis through modulation of TGF-β receptor 1 in chronic kidney disease

Chronic kidney disease (CKD) is characterized by the gradual loss of renal function and is a major public health concern. Risk factors for CKD include hypertension and proteinuria, both of which are associated with endoplasmic reticulum (ER) stress. ER stress-induced TDAG51 protein expression is increased at an early time point in mice with CKD. Based on these findings, wild-type and TDAG51 knock-out (TDKO) mice were used in an angiotensin II/deoxycorticosterone acetate/salt model of CKD. Both wild-type and TDKO mice developed hypertension, increased proteinuria and albuminuria, glomerular injury, and tubular damage. However, TDKO mice were protected from apoptosis and renal interstitial fibrosis. Human proximal tubular cells were used to demonstrate that TDAG51 expression induces apoptosis through a CHOP-dependent mechanism. Further, a mouse model of intrinsic acute kidney injury demonstrated that CHOP is required for ER stress-mediated apoptosis. Renal fibroblasts were used to demonstrate that TGF-β induces collagen production through an IRE1-dependent mechanism; cells treated with a TGF-β receptor 1 inhibitor prevented XBP1 splicing, a downstream consequence of IRE1 activation. Interestingly, TDKO mice express significantly less TGF-β receptor 1, thus, preventing TGF-β-mediated XBP1 splicing. In conclusion, TDAG51 induces apoptosis in the kidney through a CHOP-dependent mechanism, while contributing to renal interstitial fibrosis through a TGF-β-IRE1-XBP1 pathway.Subject terms: Endoplasmic reticulum, Apoptosis, End-stage renal disease, Preclinical research, Chronic inflammation     

Asiatic Acid Isolated From Centella Asiatica Inhibits TGF-β1-induced Collagen Expression in Human Keloid Fibroblasts via PPAR-γ Activation

Keloids are fibroproliferative disorders characterized by exuberant extracellular matrix deposition and transforming growth factor (TGF)-β/Smad pathway plays a pivotal role in keloid pathogenesis. Centella asiatica extract has been applied in scar management for ages. As one of its major components, asiatic acid (AA) has been recently reported to inhibit liver fibrosis by blocking TGF-β/Smad pathway. However, its effect on keloid remains unknown. In order to investigate the effects of AA on cell proliferation, invasion and collagen synthesis, normal and keloid fibroblasts were exposed to TGF-β1 with or without AA. Relevant experiments including 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2-deoxyuridine (EdU) incorporation assay, Transwell invasion assay, enzyme-linked immunosorbent assay, Western blot, quantitative polymerase chain reaction and RNA interference assay were conducted. As a result, keloid fibroblasts showed higher responsiveness to TGF-β1 stimulation than normal fibroblasts in terms of invasion and collagen synthesis. AA could suppress TGF-β1-induced expression of collagen type I, inhibit Smad 2/3 phosphorylation and plasminogen activator inhibitor-1 (PAI-1) expression, while elevate Smad 7 protein level. Noteworthy, the effects of AA on keloid fibroblasts could be abrogated by PPAR-γ antagonist GW9662 and by silencing of PPAR-γ. The present study demonstrated that AA inhibited TGF-β1-induced collagen and PAI-1 expression in keloid fibroblasts through PPAR-γ activation, which suggested that AA was one of the active constituents of C. asiatica responsible for keloid management, and could be included in the arsenal for combating against keloid.     

A Novel Role for SIRT-1 in L-Arginine Protection against STZ Induced Myocardial Fibrosis in Rats

Background

L-arginine (L-ARG) effectively protects against diabetic impediments. In addition, silent information regulator (SIRT-1) activators are emerging as a new clinical concept in treating diabetic complications. Accordingly, this study aimed at delineating a role for SIRT-1 in mediating L-ARG protection against streptozotocin (STZ) induced myocardial fibrosis.

Methods

Male Wistar rats were allocated into five groups; (i) normal control rats received 0.1 M sodium citrate buffer (pH 4.5); (ii) STZ at the dose of 60 mg/kg dissolved in 0.1 M sodium citrate buffer (pH 4.5); (iii) STZ + sirtinol (Stnl; specific inhibitor of SIRT-1; 2 mg/Kg, i.p.); (iv) STZ + L-ARG given in drinking water (2.25%) or (v) STZ + L-ARG + Stnl.

Results

L-ARG increased myocardial SIRT-1 expression as well as its protein content. The former finding was paralleled by L-ARG induced reduction in myocardial fibrotic area compared to STZ animals evidenced histopathologically. The reduction in the fibrotic area was accompanied by a decline in fibrotic markers as evident by a decrease in expression of collagen-1 along with reductions in myocardial TGF-β, fibronectin, CTGF and BNP expression together with a decrease in TGF-β and hydroxyproline contents. Moreover, L-ARG increased MMP-2 expression in addition to its protein content while decreasing expression of PAI-1. Finally, L-ARG protected against myocardial cellular death by reduction in NFκ-B mRNA as well as TNF-α level in association with decline in Casp-3 and FAS expressions andCasp-3protein content in addition to reduction of FAS positive cells. However, co-administration of L-ARG and Stnl diminished the protective effect of L-ARG against STZ induced myocardial fibrosis.

Conclusion

Collectively, these findings associate a role for SIRT-1 in L-ARG defense against diabetic cardiac fibrosis via equilibrating the balance between profibrotic and antifibrotic mediators.     

Latent TGF-β1 protects against diabetic kidney disease via Arkadia/Smad7 signaling

TGF-β1 has long been considered as a key mediator in diabetic kidney disease (DKD) but anti-TGF-β1 treatment fails clinically, suggesting a diverse role for TGF-β1 in DKD. In the present study, we examined a novel hypothesis that latent TGF-β1 may be protective in DKD mice overexpressing human latent TGF-β1. Streptozotocin-induced Type 1 diabetes was induced in latent TGF-β1 transgenic (Tg) and wild-type (WT) mice. Surprisingly, compared to WT diabetic mice, mice overexpressing latent TGF-β1 were protected from the development of DKD as demonstrated by lowing microalbuminuria and inhibiting renal fibrosis and inflammation, although blood glucose levels were not altered. Mechanistically, the renal protective effects of latent TGF-β1 on DKD were associated with inactivation of both TGF-β/Smad and nuclear factor-κB (NF-κB) signaling pathways. These protective effects were associated with the prevention of renal Smad7 from the Arkadia-induced ubiquitin proteasomal degradation in the diabetic kidney, suggesting protection of renal Smad7 from Arkadia-mediated degradation may be a key mechanism through which latent TGF-β1 inhibits DKD. This was further confirmed in vitro in mesangial cells that knockdown of Arkadia failed but overexpression of Arkadia reversed the protective effects of latent TGF-β1 on high glucose-treated mesangial cells. Latent TGF-β1 may protect kidneys from TGF-β1/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation in diabetes through inhibiting Arkadia-mediated Smad7 ubiquitin degradation.     

The effects of sarpogrelate on cardiomyocyte hypertrophy

Sarpogrelate was developed as an antiplatelet agent antagonizing 5-hydroxytryptamine (5-HT) receptors. It had been reported that 5-HT receptors were expressed in cardiovascular system, and that sarpogrelate had antihypertrophic effects in vascular smooth muscle cells. Cardiac hypertrophy is a major problem in cardiac diseases, so the present study was designed to elucidate the effects of sarpogrelate on cardiac hypertrophy. Cultured rat cardiomyocytes (MCs) and cardiac nonmyocytes (NMCs) were prepared by Percoll gradient and adhesion method and MCs were incubated with (MCs/NMCs) or without NMCs. As an index of protein synthesis of MCs, [3H]-leucine uptake into MCs and MCs/NMCs was measured. Sarpogrelate decreased [3H]-leucine uptake into MCs (maximum 62.6+/-20.6% of control at 10(-4)M, p<0.05 vs. control). Sarpogrelate also significantly attenuated angiotensin-II- and endothelin-1-induced [3H]-leucine uptake. These results indicated that sarpogrelate might have antihypertrophic effects and could be a useful aid for cardiovascular disease.     

Sarpogrelate inhibits the expression of ICAM-1 and monocyte–endothelial adhesion induced by high glucose in human endothelial cells

Hyperglycemia is the major cause of diabetic angiopathy. Sarpogrelate hydrochloride is an antiplatelet drug, and expected to be useful in the treatment of chronic arterial occlusive diseases. The aim of our study was to evaluate the possible effects of sarpogrelate hydrochloride on adhesion molecule expression and its underlying mechanism in the prevention and treatment of cardiovascular disorders. Intercellular adhesion molecule-1 (ICAM-1) expression and superoxide dismutase (SOD) activity were determined after endothelial cells were exposed to high glucose in the absence and presence of sarpogrelate hydrochloride. Coincubation of endothelial cells with high glucose for 24 h resulted in a significant increase of monocyte–endothelial cell adhesion and the expression of ICAM-1 (P < 0.01). These effects were abolished by sarpogrelate hydrochloride and sarpogrelate hydrochloride significantly increased SOD activities (40 ± 8 vs. 47 ± 7, n = 8, P < 0.01). The low dose sarpogrelate group (0.1 μM) had significantly higher monocyte–endothelial cell adhesion and the expression of ICAM-1 than medium dose sarpogrelate group (1.0 μM) and high dose sarpogrelate group (10.0 μM) (P < 0.05 for comparison among three groups and P < 0.01 for difference between low and high dose sarpogrelate groups). These findings suggested that sarpogrelate hydrochloride was able to protect vascular endothelium from dysfunction induced by high glucose.     

Antidiabetic Effects of Chamomile Flowers Extract in Obese Mice through Transcriptional Stimulation of Nutrient Sensors of the Peroxisome Proliferator-Activated Receptor (PPAR) Family

Given the significant increases in the incidence of metabolic diseases, efficient strategies for preventing and treating of these common disorders are urgently needed. This includes the development of phytopharmaceutical products or functional foods to prevent or cure metabolic diseases. Plant extracts from edible biomaterial provide a potential resource of structurally diverse molecules that can synergistically interfere with complex disorders. In this study we describe the safe application of ethanolic chamomile (Matricaria recutita) flowers extract (CFE) for the treatment and prevention of type 2 diabetes and associated disorders. We show in vitro that this extract activates in particular nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) and its isotypes. In a cellular context, in human primary adipocytes CFE administration (300 µg/ml) led to specific expression of target genes of PPARγ, whereas in human hepatocytes CFE-induced we detected expression changes of genes that were regulated by PPARα. In vivo treatment of insulin-resistant high-fat diet (HFD)-fed C57BL/6 mice with CFE (200 mg/kg/d) for 6 weeks considerably reduced insulin resistance, glucose intolerance, plasma triacylglycerol, non-esterified fatty acids (NEFA) and LDL/VLDL cholesterol. Co-feeding of lean C57BL/6 mice a HFD with 200 mg/kg/d CFE for 20 weeks showed effective prevention of fatty liver formation and hepatic inflammation, indicating additionally hepatoprotective effects of the extract. Moreover, CFE treatment did not reveal side effects, which have otherwise been associated with strong synthetic PPAR-targeting molecules, such as weight gain, liver disorders, hemodilution or bone cell turnover. Taken together, modulation of PPARs and other factors by chamomile flowers extract has the potential to prevent or treat type 2 diabetes and related disorders.     

Transforming growth factor-β1 regulation of laminin γ1 and fibronectin expression and survival of mouse mesangial cells

The transforming growth factor-beta (TGF-β) 1 is a mediator of extracellular matrix (ECM) gene expression in mesangial cells and the development of diabetic glomerulopathy. Here, we investigate the effects of TGF-β1 on laminin γ1 and fibronectin polypeptide expression and cell survival in mouse mesangial cells (MES-13). TGF-β1 (10 ng/ml) stimulates laminin-γ1 and fibronectin expression ~two-fold in a time-dependent manner (0–48 h). TGF-β1 treatment also retards laminin-γ1 mobility on SDS-gels, and tunicamycin, an inhibitor of the N-linked glycosylation, blocks the mobility shift. TGF-β1 increases the binding of laminin γ1 to WGA-agarose and the binding is abolished by tunicamycin suggesting that laminin γ1 is modified by N-linked glycosylation. TGF-β1 also elevates fibronectin glycosylation but its mobility is not altered. The degradation of laminin γ1 and fibronectin proteins is reduced by their glycosylation. In addition, TGF-β1 enhances mesangial cell viability and metabolic activities initially (0–24 h); however, eventually leads to cell death (24–48 h). TGF-β1 elevates pro-apoptotic caspase-3 activity and decrease cell cycle progression factor cyclin D1 expression, which parallels cell death. These results indicate that TGF-β1 plays an important role in ECM expression, protein glycosylation and demise of mesangial cells in the diabetic glomerular mesangium. (Mol Cell Biochem 278: 165–175, 2005)     

Dimethylfumarate Attenuates Renal Fibrosis via NF-E2-Related Factor 2-Mediated Inhibition of Transforming Growth Factor-β/Smad Signaling

TGF-β plays a key role in the development of renal fibrosis. Suppressing the TGF-β signaling pathway is a possible therapeutic approach for preventing this disease, and reports have suggested that Nrf2 protects against renal fibrosis by inhibiting TGF-β signaling. This study examines whether dimethylfumarate (DMF), which stimulates Nrf2, prevents renal fibrosis via the Nrf2-mediated suppression of TGF-β signaling. Results showed that DMF increased nuclear levels of Nrf2, and both DMF and adenovirus-mediated overexpression of Nrf2 (Ad-Nrf2) decreased PAI-1, alpha-smooth muscle actin (α-SMA), fibronectin and type 1 collagen expression in TGF-β-treated rat mesangial cells (RMCs) and renal fibroblast cells (NRK-49F). Additionally, DMF and Ad-Nrf2 repressed TGF-β-stimulated Smad3 activity by inhibiting Smad3 phosphorylation, which was restored by siRNA-mediated knockdown of Nrf2 expression. However, downregulation of the antioxidant response element (ARE)-driven Nrf2 target genes such as NQO1, HO-1 and glutathione S-transferase (GST) did not reverse the inhibitory effect of DMF on TGF-β-induced upregulation of profibrotic genes or extracellular matrix proteins, suggesting an ARE-independent anti-fibrotic activity of DMF. Finally, DMF suppressed unilateral ureteral obstruction (UUO)-induced renal fibrosis and α-SMA, fibronectin and type 1 collagen expression in the obstructed kidneys from UUO mice, along with increased and decreased expression of Nrf2 and phospho-Smad3, respectively. In summary, DMF attenuated renal fibrosis via the Nrf2-mediated inhibition of TGF-β/Smad3 signaling in an ARE-independent manner, suggesting that DMF could be used to treat renal fibrosis.     

High-Fat Diet-Induced Adiposity,Adipose Inflammation,Hepatic Steatosis and Hyperinsulinemia in Outbred CD-1 Mice

High-fat diet (HFD) has been applied to a variety of inbred mouse strains to induce obesity and obesity related metabolic complications. In this study, we determined HFD induced development of metabolic disorders on outbred female CD-1 mice in a time dependent manner. Compared to mice on regular chow, HFD-fed CD-1 mice gradually gained more fat mass and consequently exhibited accelerated body weight gain, which was associated with adipocyte hypertrophy and up-regulated expression of adipose inflammatory chemokines and cytokines such as Mcp-1 and Tnf-α. Increased fat accumulation in white adipose tissue subsequently led to ectopic fat deposition in brown adipose tissue, giving rise to whitening of brown adipose tissue without altering plasma level of triglyceride. Ectopic fat deposition was also observed in the liver, which was associated with elevated expression of key genes involved in hepatic lipid sequestration, including Ppar-γ2, Cd36 and Mgat1. Notably, adipose chronic inflammation and ectopic lipid deposition in the liver and brown fat were accompanied by glucose intolerance and insulin resistance, which was correlated with hyperinsulinemia and pancreatic islet hypertrophy. Collectively, these results demonstrate sequentially the events that HFD induces physiological changes leading to metabolic disorders in an outbred mouse model more closely resembling heterogeneity of the human population.