Combination of aspirin with telmisartan suppresses the augmented TGFβ/smad signaling during the development of streptozotocin-induced type I diabetic nephropathy

Diabetic nephropathy (DN) is the most common indication for the development of end stage renal diseases. Inflammation is increasingly seen as the core process in the development of diabetes. Inflammatory markers e.g. NFκB (p65 levels), TNFα, COX-2 and TGFβ–smad signaling are the key elements in the development of DN. Renin–angiotensin system suppressors like telmisartan have been used to treat DN, but they are not able to prevent completely because of development of resistance against them. Anti-inflammatory agents like, aspirin acts through both COX dependent and COX independent pathways. Hence, we thought that combining aspirin with telmisartan will be better therapeutic option in preventing the progression of nephropathy in diabetes. In the present study we studied the effect of this combination on inflammatory markers [COX-2, NFκB (p65 levels), TNFα], TGFβ–smad expression in preventing the progression of streptozotocin-induced type I diabetic nephropathy. Treatment of aspirin significantly prevented the progression of nephropathy and inhibited the augmented COX-2, NFκB (p65 levels), TNFα, and TGFβ–smad expression. Combination of aspirin with telmisartan resulted in a further decrease in the development of nephropathy and inflammatory markers in comparison to aspirin alone treatment. This is the first report which shows that aspirin in combination with telmisartan is more proficient in the treatment of diabetic nephropathy than any single drug therapy and involves the change in expression of inflammatory markers and TGFβ–smad signaling.     

Nimbolide ameliorates the streptozotocin-induced diabetic retinopathy in rats through the inhibition of TLR4/NF-κB signaling pathway

BackgroundDiabetic retinopathy (DR) is a common problem in the diabetic patients due to the high blood glucose level. DR affects more number of diabetic patients worldwide with irreversible vision loss.ObjectiveThe current investigation was focused to reveal the therapeutic actions of nimbolide against the streptozotocin (STZ)-provoked DR in rats through inhibition of TLR4/NF-κB pathway.MethodologyDR was provoked to the rats through administering a single dose of STZ (60 mg/kg) intraperitoneally. The DR rats were then supplemented with the 50 mg/kg of nimbolide for 60 days. The bodyweight and blood glucose level was measured using standard methods. The lipid profiles (cholesterol, TG, LDL, and HDL), inflammatory markers, and antioxidants level was detected using respective kits. The level of MCP-1, VEGF, and MMP-9 was quantified using kits. The morphometric analysis of retinal tissues were done. The mRNA expressions of target genes were studied using RT-PCR assay.ResultsNimbolide treatment effective decreased the food intake and blood glucose, and improved the bodyweight of STZ-provoked animals. The levels of pro-inflammatory mediators, cholesterol, TG, LDL, and HDL, MCP-1, VEGF, and MMP-9 was remarkably suppressed by the nimbolide treatment. Nimbolide also improved the antioxidants, retinal thickness and cell numbers. The TLR4/NF-κB pathway was appreciably inhibited by the nimbolide.ConclusionOverall, our findings demonstrated that the nimbolide attenuated the STZ-provoked DR in rats through inhibiting the TLR4/NF-κB pathway.     

Curcumin reverses diabetic nephropathy in streptozotocin-induced diabetes in rats by inhibition of PKCβ/p66Shc axis and activation of FOXO-3a

This study investigated if the nephroprotective effect of Curcumin in streptozotocin-induced type 1 diabetes mellitus (DM) in rats involves downregulation/inhibition of p⁶⁶Shc and examined the underlying mechanisms. Rats were divided into 4 groups (n = 12/group) as control, control + Curcumin (100 mg/kg), T1DM, and T1DM + Curcumin. Curcumin was administered orally to control or diabetic rats for 12 weeks daily. As compared to diabetic rats, Curcumin didn't affect either plasma glucose or insulin levels but significantly reduced serum levels of urea, blood urea nitrogen, and creatinine, and concurrently reduced albumin/protein urea and increased creatinine clearance. It also prevented the damage in renal tubules and mitochondria, mesangial cell expansion, the thickness of the basement membrane. Mechanistically, Curcumin reduced mRNA and protein levels of collagen I/III and transforming growth factor- β-1 (TGF-β1), reduced inflammatory cytokines levels, improved markers of mitochondrial function, and suppressed the release of cytochrome-c and the activation of caspase-3. In the kidneys of both control and diabetic rats, Curcumin reduced the levels of reactive oxygen species (ROS), increased mRNA levels of manganese superoxide dismutase (MnSOD) and gamma-glutamyl ligase, increased glutathione (GSH) and protein levels of Bcl-2 and MnSOD, and increased the nuclear levels of nuclear factor2 (Nrf2) and FOXO-3a. Besides, Curcumin reduced the nuclear activity of the nuclear factor-kappa B (NF-κB), downregulated protein kinase CβII (PKCβII), NADPH oxidase, and p⁶⁶Shc, and decreased the activation of p⁶⁶Shc. In conclusion, Curcumin prevents kidney damage in diabetic rats by activating Nrf2, inhibiting Nf-κB, suppressing NADPH oxidase, and downregulating/inhibiting PKCβII/p⁶⁶Shc axis.     

Lepidium sativum alleviates diabetic nephropathy in a rat model by attenuating glucose levels,oxidative stress,and inflammation with concomitant suppression of TGF-β1

In this research, the treatment of diabetic nephropathy in rats induced by streptozotocin with L. sativium whole-plant aqueous extract was examined, and the mechanism of action was proposed. Adult male rats were grouped into: control, L. sativum, T1DM, and T1DM + L. sativum-treated groups. For 8 weeks, L. sativum S was given to rats at a final dose of 250 mg/kg. Treatment with L. sativum reduced the amount of fasting glucose, increased the amount of fasting insulin, and diminished the increase in hepatic and serum cholesterol, free fatty acid, and triglyceride levels. The level of serum LDL-c was reduced. At the level of the kidney, L. sativum reduced urine volume and albumin excretion and spiked creatinine excretion. It also attenuated the tubular damage in the rats’ kidneys and reduced the amounts of major inflammatory markers, including nuclear factor-kappaα (NF-κB), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6). Interestingly, L. sativium reduced the amount of mRNA transforming growth factor-β1 (TGF-β1), stimulated mRNA superoxide dismutase (SOD) and catalase (CAT), reduced lipid peroxide levels (MDA), and increased the glutathione (GSH), SOD, and CAT in the rat kidneys of the control and T1DM-treated group. In conclusion, L. sativum is a novel therapy against DN owing to its hypoglycemic effect, insulin-releasing, and antioxidant potential.     

Nephroprotective potential of Quercus infectoria galls against experimentally induced diabetic nephropathy in rats through inhibition of renal oxidative stress and TGF-β

Diabetic nephropathy (DN) is the leading cause of death in diabetic patients and the current treatment options available have a limited significance. The insect galls of Quercus infectoria are traditionally important in the treatment of numerous diseases including diabetes. Hence, the present study was undertaken to evaluate the effect of Q. infectoria gall extract (QIGE) against experimental DN. Type 2 diabetes was induced by feeding rats with a high-fat diet (HFD) initially for 5 weeks, followed by a single intraperitoneal injection of streptozotocin (STZ, 35?mg/kg?bw/day). QIGE was administered to the rats orally at doses of 100 and 200?mg/kg?bw/day, respectively. At the end of the experimental period, various glycemic and renal function parameters were evaluated in the serum, urine and kidney tissues. The QIGE treatment significantly (p?p?via the inhibition of hyperglycemia-induced oxidative stress and renal TGF-β expression and is, therefore, a potential therapeutic agent in the treatment of diabetic complications, especially DN.     

Obesity reduces bone density associated with activation of PPARγ and suppression of Wnt/β-catenin in rapidly growing male rats

Background

It is well established that excessive consumption of a high fat diet (HFD) results in obesity; however, the consequences of obesity on postnatal skeletal development have not been well studied.

Methodology and Principal Findings

Total enteral nutrition (TEN) was used to feed postnatal day 27 male rats intragastrically with a high 45% fat diet (HFD) for four weeks to induce obesity. Fat mass was increased compared to rats fed TEN diets containing 25% fat (medium fat diet, MFD) or a chow diet (low fat diet, LFD) fed ad libitum with matched body weight gains. Serum leptin and total non-esterified fatty acids (NEFA) were elevated in HFD rats, which also had reduced bone mass compared to LFD-fed animals. This was accompanied by decreases in bone formation, but increases in the bone resorption. Bone marrow adiposity and expression of adipogenic genes, PPARγ and aP2 were increased, whereas osteoblastogenic markers osteocalcin and Runx2 were decreased, in bone in HFD rats compared to LFD controls. The diversion of stromal cell differentiation in response to HFD stemmed from down-regulation of the key canonical Wnt signaling molecule β-catenin protein and reciprocal up-regulation of nuclear PPARγ expression in bone. In a set of in vitro studies using pluripotent ST2 bone marrow mesenchymal stromal cells treated with serum from rats on the different diets or using the free fatty acid composition of NEFA quantified in rat serum from HFD-fed animals by GC-MS, we were able to recapitulate our in vivo findings.

Conclusions/Significance

These observations strongly suggest that increased NEFA in serum from rats made obese by HFD-feeding impaired bone formation due to stimulation of bone marrow adipogenesis. These effects of obesity on bone in early life may result in impaired attainment of peak bone mass and therefore increase the prevalence of osteoporosis later on in life.     

Nrf2 participates in the anti-apoptotic role of zinc in Type 2 diabetic nephropathy through Wnt/β-catenin signaling pathway

Zinc (Zn), as an essential trace element, has been approved to serve many roles in diabetic studies. Also Zn deficiency will aggravate renal damage in diabetes through suppression of nuclear factor-erythroid 2-related factor 2 (Nrf2) expression and function. The purpose of this study was to illustrate the role of Zn in renal apoptosis in diabetes and whether Nrf2 participated in the process. Type 2 diabetes mice model was induced by a single dose of streptozotocin (STZ) injection after high-fat diet (HFD) feeding for 3 months, then the mice were given diets supplemented with different concentrations of Zn (control, 30 ppm; low-concentration, 0.85 ppm). After 12-week treatment, morphology and associated protein expressions were examined. The results showed that low Zn diet significantly aggravated the level of renal apoptosis during diabetes, performed as the upregulation of caspase-3 expression. In addition, either low Zn diet or diabetes or both dramatically decreased the expression of Nrf2 and P-AKT in kidney. Moreover, the expression of β-catenin in kidney was increased markedly in diabetic groups. Mechanistic study applying human renal tubular epithelial cells (HK11) confirmed the role of Nrf2, as silencing Nrf2 expression abolished Zn supplementation protection against high sugar + high fat + low Zn-induced apoptosis and downregulation of β-catenin expression. All these results suggest that Nrf2 plays a key role in Zn protection against Type 2 diabetes induced renal apoptosis, which might be through Wnt/β-catenin signaling pathway.     

The role of TGF-β and epithelial-to mesenchymal transition in diabetic nephropathy

Transforming Growth Factor-beta (TGF-β) is a pro-sclerotic cytokine widely associated with the development of fibrosis in diabetic nephropathy. Central to the underlying pathology of tubulointerstitial fibrosis is epithelial-to-mesenchymal transition (EMT), or the trans-differentiation of tubular epithelial cells into myofibroblasts. This process is accompanied by a number of key morphological and phenotypic changes culminating in detachment of cells from the tubular basement membrane and migration into the interstitium. Ultimately these cells reside as activated myofibroblasts and further exacerbate the state of fibrosis. A large body of evidence supports a role for TGF-β and downstream Smad signalling in the development and progression of renal fibrosis. Here we discuss a role for TGF-β as the principle effector in the development of renal fibrosis in diabetic nephropathy, focusing on the role of the TGF-β1 isoform and its downstream signalling intermediates, the Smad proteins. Specifically we review evidence for TGF-β1 induced EMT in both the proximal and distal regions of the nephron and describe potential therapeutic strategies that may target TGF-β1 activity.     

Acetylshikonin from Zicao ameliorates renal dysfunction and fibrosis in diabetic mice by inhibiting TGF-β1/Smad pathway

Diabetic nephropathy (DN) is the major cause of end-stage renal disease in diabetic patients. Zicao, a well-known Chinese traditional medicine, has attracted much attention due to its beneficial effects in various medical fields. In this study, we attempted to investigate the effects and mechanisms of action of acetylshikonin, the main ingredient of Zicao, on renal dysfunction in DN. Our results showed that administration with acetylshikonin not only decreased blood urea nitrogen, urine creatinine and the mean kidney-to-body weight ratio in streptozotocin-induced diabetic mice, but also restored the loss of body weight, whereas the blood glucose was not changed. Masson’s trichrome staining showed that acetylshikonin treatment resulted in a marked decrease in kidney fibrosis from diabetic mice. The increased expression of fibrosis proteins, such as plasminogen activator inhibitor type 1 (PAI-1), connective tissue growth factor, and collagen III and IV, were reduced after acetylshikonin administration. In addition, the expressions of interleukin-1β, interleukin-6, monocyte chemoattractant protein-1, intercellular adhesion molecule 1 and infiltration of macrophages in kidney tissues were decreased in acetylshikonin-treated diabetic mice. Acetylshikonin led to a reduction of transforming growth factor-β1 (TGF-β1) expression and Smad-2/3 phosphorylation, as accompanied by increased Smad7 expression. Furthermore, in vitro treatment with acetylshikonin markedly attenuated TGF-β1-induced the PAI-1, collagen III and IV, and Smad-2/3 phosphorylation in HK2 immortalized human proximal tubule epithelial cells. Acetylshikonin also prevented epithelial-to-mesenchymal transition induced by TGF-β1. Collectively, our study provides evidences that acetylshikonin attenuates renal fibrosis though inhibiting TGF-β1/Smad signaling pathway, suggesting that acetylshikonin may be a novel therapeutic agent for the treatment of DN.     

Urotensin-2 promotes collagen synthesis via ERK1/2-dependent and ERK1/2-independent TGF-β1 in neonatal cardiac fibroblasts

U2 (urotensin-2) is the most potent vasoconstrictor in mammals which is involved in cardiac remodelling, including cardiac hypertrophy and cardiac fibrosis. Although the cellular mechanisms of the U2-induced vasoconstriction have been extensively studied, the signalling pathways involved in U2-induced TGF-β1 (transforming growth factor-β1) expression and collagen synthesis remain unclear. In this study, we show that U2 promoted collagen synthesis and ERK1/2 (extracellular signal-regulated kinase 1/2) activation in neonatal cardiac fibroblasts. The U2-induced collagen synthesis and TGF-β1 production were significantly but not completely inhibited by blocking ERK1/2. Both ERK1/2 inhibitor and TGF-β1 antibody could separately inhibit U2-induced collagen synthesis, and the synergistic inhibition effect was observed by blocking ERK1/2 and TGF-β1 simultaneously. These data suggest that U2 promotes collagen synthesis via ERK1/2-dependent and independent TGF-β1 pathway in neonatal cardiac fibroblasts.     

Curcumin decreases renal triglyceride accumulation through AMPK–SREBP signaling pathway in streptozotocin-induced type 1 diabetic rats

Diabetic kidney disease has been associated with the presence of lipid deposits. We assumed that curcumin, a polyphenol, would attenuate the tissue dyslipidemic condition through activation of 5' adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation and suppression of sterol regulatory element-binding protein (SREBP)-1c in the kidney and would prevent renal progression in experimental type 1 diabetic rats. Diabetes was induced with streptozotocin (STZ) (55 mg/kg) by intraperitoneal injection in male Sprague–Dawley rats. Three weeks after STZ injection, rats were divided into three groups, namely, control, diabetic and diabetic treated with curcumin (100 mg/kg/day) by gavage for 8 weeks. We found that curcumin decreased plasma triglyceride and the amount of renal triglyceride significantly. Furthermore, treatment of diabetic rats with curcumin increased the phosphorylation of AMPK and prevented the increased renal expression of SREBP-1c and, as a result, decreased the expression of acetyl CoA carboxylase and fatty acid synthase as well as adipose differentiation-related protein, a marker of cytoplasmic droplets. We also demonstrate that curcumin significantly suppressed the increased expression of transforming growth factor β, vascular endothelial growth factor and extracellular matrix proteins such as type IV collagen and fibronectin. In addition, curcumin treatment increased nephrin expression to near-normal levels in diabetic rats. These results demonstrated that curcumin protects against the development of diabetic nephropathy through the AMPK–SREBP pathway and the reduction of renal triglyceride accumulation which could be a possible mechanism by which curcumin preserves renal function in diabetes.     

TGF-β1 mediates sirolimus and cyclosporine A-induced alteration of barrier function in renal epithelial cells via a noncanonical ERK1/2 signaling pathway

The immunosuppressant drugs cyclosporine A (CsA) and sirolimus (SRL) used in combination demonstrated beneficial effects in organ transplantation, but this combination can also result in increased adverse effects. We previously showed that not only CsA treatment but also its combination with SRL decreased paracellular permeability in renal proximal tubular cells by modification of the tight junction proteins, claudins, through ERK1/2 signaling pathway. In this present study, evidence is presented that not only CsA but also the combination of CsA/SRL may have adverse effects on the barrier function of renal proximal cells, at least in part, through the expression of the cytokine transforming growth factor (TGF)-β(1). CsA treatment upregulated TGF-β(1) gene expression and this upregulation was enhanced when CsA and SRL were applied together. Addition of TGF-β(1) (5 ng/ml) altered the barrier function with increased transepithelial electrical resistance (TER) and claudin-1 expression. Use of a TGF-β(1)-blocking antibody or blockage of TGF-β(1) receptor kinase activity with SD208 prevented the CsA- and CsA/SRL-induced increase in TER. No evidence was found in the present studies to indicate that CsA or CsA/SRL treatment activated the TGF-β(1) Smad canonical signaling pathway, whereas addition of TGF-β(1) (5 ng/ml) did activate the Smad pathway. Addition of the ERK1/2 signaling inhibitor U0126 was able to prevent the TGF-β(1)-mediated increase in TER and claudin expression. It is most likely that the CsA- and CsA/SRL-induced increases in TGF-β(1) expression may not be sufficient to trigger the Smad pathway but however may trigger other TGF-β(1) receptor-mediated signaling including the ERK1/2 signaling pathway.     

TGF-β1 expression is associated with invasion and metastasis of intrahepatic cholangiocarcinoma

Background

Transforming growth factor (TGF)-β is involved in many physiologic processes, it often promotes metastasis, and its high expression is correlated with poor prognosis. In the present study, we analyzed the correlation between transforming growth factor beta 1 (TGF-β1) expression and prognosis in intrahepatic cholangiocarcinoma.

Results

We examined the expression of TGF-β1 in 78 intrahepatic cholangiocarcinomas by immunohistochemistry and correlated the expression with clinicopathological parameters. TGF-β1 was expressed in 37 of 78 (47.4 %) intrahepatic cholangiocarcinomas. The expression of TGF-β1 was significantly correlated with lymph node metastasis, distant metastasis, and tumour recurrence. Patients with TGF-β1-positive tumours had significantly shorter survival time. In a multivariant analysis, the expression of TGF-β1 and the tumour stage were independent prognostic factors.

Conclusions

Our data suggest that expression of TGF-β1 is a novel prognostic marker for intrahepatic cholangiocarcinoma.     

TRAF6 mediates IL-1β/LPS-induced suppression of TGF-β signaling through its interaction with the type III TGF-β receptor

Transforming growth factor-β1 (TGF-β1) is an important anti-inflammatory cytokine that modulates and resolves inflammatory responses. Recent studies have demonstrated that inflammation enhances neoplastic risk and potentiates tumor progression. In the evolution of cancer, pro-inflammatory cytokines such as IL-1β must overcome the anti-inflammatory effects of TGF-β to boost pro-inflammatory responses in epithelial cells. Here we show that IL-1β or Lipopolysaccharide (LPS) suppresses TGF-β-induced anti-inflammatory signaling in a NF-κB-independent manner. TRAF6, a key molecule in IL-1β signaling, mediates this suppressive effect through interaction with the type III TGF-β receptor (TβRIII), which is TGF-β-dependent and requires type I TGF-β receptor (TβRI) kinase activity. TβRI phosphorylates TβRIII at residue S829, which promotes the TRAF6/TβRIII interaction and consequent sequestration of TβRIII from the TβRII/TβRI complex. Our data indicate that IL-1β enhances the pro-inflammatory response by suppressing TGF-β signaling through TRAF6-mediated sequestration of TβRIII, which may be an important contributor to the early stages of tumor progression.     

MiR-130b increases fibrosis of HMC cells by regulating the TGF-β1 pathway in diabetic nephropathy

Basement membrane thickening, glomerular hypertrophy, and deposition of multiple extracellular matrix characterize the pathological basis of diabetic nephropathy (DN), a condition which ultimately leads to glomerular and renal interstitial fibrosis. Here, we identified a novel microRNA, miR-130b, and investigated its role and therapeutic efficacy in alleviating DN. Introduction of miR-130b dramatically increased cell growth and fibrosis in DN cells. We found that transforming growth factor (TGF)-β1 was a functional target of miR-130b in human glomerular mesangial cells (HMCs) and overexpression of miR-130b increased expressions of the downstream signaling molecules of TGF-β1, t-Smad2/3, p-Smad2/3, and SMAD4. An ectopic application of miR-130b increased messenger RNA and protein expressions of collagen type I (colI), colIV, and fibronectin, whose expression levels were correlated with the expression of miR-130b. Taken together, the findings of this study reveal that miR-130b in HMC cells plays an important role in fibrosis regulation and may thus be involved with the pathogenesis of DN. Therefore, miR-130b may serve as a novel therapeutic target for the prevention and the treatment of DN.