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The current study investigated the potential of green tea (GT) to improve uncoupling of endothelial nitric oxide synthase (eNOS) in diabetic conditions. In rats with streptozotocin-induced diabetes, nitric oxide (NO) bioavailability was reduced by uncoupling eNOS, characterized by a reduction in tetrahydrobiopterin (BH(4)) levels and a decrease in the eNOS dimer-to-monomer ratio. GT treatment ameliorated these abnormalities. Moreover, immortalized human mesangial cells (ihMCs) exposed to high glucose (HG) levels exhibited a rise in reactive oxygen species (ROS) and a decline in NO levels, which were reversed with GT. BH(4) and the activity of guanosine triphosphate cyclohydrolase I decreased in ihMCs exposed to HG and was normalized by GT. Exogenous administration of BH(4) in ihMCs reversed the HG-induced rise in ROS and the decline in NO production. However, coadministration of GT with BH(4) did not result in a further reduction in ROS production, suggesting that reduced ROS with GT was indeed secondary to uncoupled eNOS. In summary, GT reversed the diabetes-induced reduction of BH(4) levels, ameliorating uncoupling eNOS, and thus increasing NO bioavailability and reducing oxidative stress, two abnormalities that are involved in the pathogenesis of diabetic nephropathy. [ABSTRACT FROM AUTHOR]

Background: The authors compared the levels of HIF1-α, VEGF, TNF-α, and IL-10 in peri-implant crevicular fluid between patients with or without peri-implantitis. HIF-1α levels were significantly high in the peri-implantitis possibly due to hypoxia triggered by persistent inflammation., Objective: This study aimed to compare the levels of HIF1-α, VEGF, TNF-α, and IL-10 in the peri-implant crevicular fluid of patients with and without peri-implantitis., Methods: Forty patients, comprising 16 with and 24 without peri-implantitis were selected., Results: Patients with peri-implantitis exhibited significantly higher HIF-1α levels than those without peri-implantitis (p=0.0005). TNF-α revealed significant positive correlations with IL-10 (p=0.0008) and VEGF (p=0.0246), whereas HIF-1α and IL-10 levels (p=0.0041) demonstrated a negative and significative correlation in the peri-implantitis group., Conclusion: This study, for the first time demonstrates the balance of HIF-1α, TNFα, IL-10, and VEGF in peri-implantitis. It shows an elevated HIF-1α levels in patients with peri-implantitis, which could have stemmed from persistent inflammation- triggered hypoxia. Furthermore, the positive correlation between TNF-α and VEGF suggests intensified proinflammatory activity in peri-implantitis. Nevertheless, further studies are essential to understand these immune dynamics in peri-implantitis., Background: Higher levels of HIF-1α in patients with peri-implantitis occurred possibly due to persistent hypoxia triggered by inflammation., Background: Tissue hypoxia in peri-implantitis induced increase in HIF-1α consequently increased VEGF and angiogenesis, contributing to the persistence of inflammation.

In patients with diabetes, it has been suggested that physical exercise may reduce albuminuria and the progression of renal disease. However, the molecular mechanism by which physical exercise protects the kidney in diabetes remains poorly understood. The aim of the present study was to determine the contribution of muscle irisin secretion induced by aerobic physical exercise with the subsequent activation of AMPK for kidney protection under diabetic conditions. Aerobic physical exercise in rats protected the kidney in streptozotocin-induced diabetes. It reduced albuminuria, glomerular hypertrophy, and glomerular expression of collagen IV and fibronectin, as well as markers of kidney inflammation, when compared to sedentary diabetic rats. These effects were associated with elevation in muscle FNDC5/irisin and activity of AMPK in the diabetic kidney. However, the beneficial effects of exercise were lost when the diabetic rats were treated with CycloRGDyK, that in the bone it has been described as an irisin receptor blocker. In cultured human tubular (HK-2) cells, treatment with recombinant irisin counteracted the effect of high glucose in a dose-dependent manner. Irisin, per se, also activated AMPK in HK-2 cells. It is concluded that in diabetes, the renal protective effect of exercise may be mediated by the irisin/AMPK pathway., (© 2022. The Author(s).)

Aim: To evaluate the initial adhesion and formation of Streptococcus mutans biofilm in vitro in the presence of saliva, human colostrum and 3'-sialyllactose., Methods: Human colostrum and salivas were collected from 30 mothers and newborn postpartum. Eighteen hours culture of S. mutans was treated with colostrum or 3'-sialyllactose in three different moments: before, during, and after 24 h from the microbial inoculation. Salivas were also tested in conjunction with colostrum. The assays were realized in sterile 96-well flat-bottom microtiter plates for 24 h. The biofilms were fixed, washed, stained with crystal violet, and extracted. Absorbance was measured to evaluate biofilm growth mass., Results: Colostrum applied after and during the inoculation decreased biofilm formation when compared with the control ( p  < .05). The presence of saliva increased the biofilm biomass ( p  < .05). The application of 3'-sialyllactose reduced biofilm formation independently of moments of application ( p  < .05)., Conclusion: Saliva contributed to the proliferation of biofilm and colostrum did not prevent the initial adhesion, but interfered in the accumulation and development of microorganisms in biofilms. 3'-sialyllactose significantly decreased biofilm formation. This information expands the importance of colostrum as a potent oral antimicrobial biofluid.

Metformin, an AMP-activated protein kinase (AMPK) activator, has been shown in previous studies to reduce kidney fibrosis in different models of experimental chronic kidney disease (CKD). However, in all of these studies, the administration of metformin was initiated before the establishment of renal disease, which is a condition that does not typically occur in clinical settings. The aim of the present study was to investigate whether the administration of metformin could arrest the progression of established renal disease in a well-recognized model of CKD, the subtotal kidney nephrectomy (Nx) model. Adult male Munich-Wistar rats underwent either Nx or sham operations. After the surgery (30 days), Nx rats that had systolic blood pressures of >170 mmHg and albuminuria levels of >40 mg/24 h were randomized to a no-treatment condition or to a treatment condition with metformin (300 mg·kg -1 ·day -1 ) for a period of either 60 or 120 days. After 60 days of treatment, we did not observe any differences in kidney disease parameters between Nx metformin-treated and untreated rats. However, after 120 days, Nx rats that had been treated with metformin displayed significant reductions in albuminuria levels and in markers of renal fibrosis. These effects were independent of any other effects on blood pressure or glycemia. In addition, treatment with metformin was also able to activate kidney AMPK and therefore improve mitochondrial biogenesis. It was concluded that metformin can arrest the progression of established kidney disease in the Nx model, likely via the activation of AMPK.

Objective: To evaluate the density of anti-galectin-3-immunostained cells, collagen percentage, mast cell density and presence of pathological processes in intestinal muscle biopsies of patients., Methods: Thirty-five patients who underwent intestinal biopsy were selected from 1997 to 2015. Patients were divided into three groups: chagasic patients with mucosal lesion (n=13), chagasic patients with intact mucosa (n=12) and non-chagasic patients with no mucosal lesion (n=10). Histological processing of the biopsied fragments and immunohistochemistry for galectin-3 were performed. Additional sections were stained with hematoxylin and eosin to evaluate the general pathological processes, picrosirius for evaluation of collagen and toluidine blue to evaluate the mast cell density., Results: Patients of mucosal lesion group had a significantly higher frequency of ganglionitis and myositis when compared to the chagasic patients with intact mucosa and non-chagasic group. The density of anti-galectin-3-immunostained cells was significantly higher in the chagasic patients with intact mucosa group when compared to the non-chagasic group. The group of chagasic patients with intact mucosa presented a higher percentage of collagen in relation to the patients with mucosal lesion and to the non-chagasic group, with a significant difference. There was no significant difference in mast cell density among the three groups., Conclusion: The higher density of anti-galectin-3-immunostained cells in patients in the chagasic patients with intact mucosa group suggested the need for greater attention in clinical evaluation of these patients, since this protein is associated with neoplastic transformation and progression.

Although the salivary glands present several functions, there are few studies evaluating these glands in Chagas disease (CD). This study aimed to compare the percentage of collagen, the presence of inflammation, the density of chimase and tryptase mast cells, the area and density of lingual salivary gland acini in autopsied individuals with and without (CD). We analyzed 400 autopsy reports performed in a tertiary public hospital from 1999 to 2015 and selected all the cases in which tongue fragments were collected (27 cases), 12 with chronic CD without megaesophagus (CH) and 15 without CD (non-chagasic - NC). The histological sections of the tongue were stained by Picrosirius red for collagen evaluation and Hematoxylin-eosin for morphometric evaluation of salivary gland acini and inflammation. Anti-chimase and anti-tryptase antibodies were used for the immunohistochemical evaluation of mast cells. The chagasic patients presented higher volume and lower density of salivary glands acini. There was no difference in the collagen percentage, inflammation and density of mast cell chymase and tryptase between the groups. Although we did not observe a significant difference between the groups regarding the collagen percentage, inflammatory process and mast cell density, our results suggest that even without megaesophagus, chagasic patients present hypertrophy of the lingual salivary glands and lower acinar density probably due to mechanisms independent of the esophagus-glandular stimulus.

Purpose: Outer blood retinal barrier breakdown is a neglected feature of diabetic retinopathy (DR). We demonstrated that the agonism of the δ opioid receptor (DOR) by epicatechin preserves the tight junction proteins in ARPE-19 cells under diabetic conditions. Presently, we aimed to evaluate the possible role of the DOR on the maintenance of tight junction of RPE layer and on the early markers of experimental DR., Methods: DR markers and external retinal tight junction proteins were evaluated in CL57B diabetic mice submitted to intravitreous injection of short hairpin RNA (shRNA)-DOR (108 transducing units [TU]/mL) treated or not with DOR agonist (0.05 g/animal/d of epicatechin in drinking water) for 16 weeks. The presence of DOR in human retina from postmortem eyes from diabetic and nondiabetic donors were also performed., Results: DOR is present in RPE layer and in neuro retina. The treatment with DOR agonist prevented the upregulation of the early markers of retinopathy (glial fibrillary acidic protein, VEGF) and the downregulation of pigment epithelium-derived factor, occludin, claudin-1, and zonula occludens-1 tight junction expressions. The silencing of DOR in retina of diabetic mice partially abolished the protective effects of epicatechin. In human retina specimens, DOR is present throughout the retina, similarly in nondiabetic and diabetic donors., Conclusions: This set of experiments strongly indicates that the DOR agonism preserves RPE tight junctions and reduces the early markers of retinopathy in model of diabetes. These novel findings designate DOR as a potential therapeutic tool to treat DR with preservation of the RPE tight junction proteins.

Purpose: Müller cells (MCs) are a major source of VEGF in diabetic retinopathy (DR). Vascular endothelial growth factor is the main therapeutic target for treating DR. This study aimed to investigate whether autophagy is involved in MC response under high glucose (HG)., Methods: Rat retinal Müller cells (rMCs) were exposed to normal or high glucose in and out of presence of pharmacologic inhibitors and activators and small interfering RNA (siRNA) for p62/SQTSM1 for 24 hours., Results: High glucose induces increase of early and late autophagic markers, accumulation of p62/SQTSM1 and endoplasmic reticulum (ER) stress response associated with apoptosis augmentation (P < 0.01). The inhibition of autophagy in HG leads to higher rMC apoptotic rate (P < 0.001). By silencing the p62/SQTSM1, ER stress is ameliorated (p<0.0001), preventing apoptosis. Retinal MCs in HG treated with rapamycin (mTOR inhibitor) show autophagy machinery activation and reestablishment of cargo degradation, protecting cells from apoptosis (P < 0.0001). Rapamycin improves lysosomal proteolytic activity by improving cathepsin L activity restoring autophagic cargo degradation, and preventing increased VEGF release (P < 0.0001). In experimental model of diabetes, Beclin-1 and p62/SQTSM-1 were found to be marked increased in retinas from diabetic Wystar Kyoto rats compared with control group (P < 0.003) with reduction of cathepsin L activity., Conclusions: High glucose upregulates autophagy but accumulates p62/SQTSM1 cargo due to lysosomal dysfunction, leading to massive VEGF release and cell death of rMCs. Lysosomal impairment and autophagic dysfunction are early events present in the pathogenesis of diabetic retinopathy (DR). This might be valuable for developing a novel therapeutic strategy to treat DR.

Prior research has shown that in experimental diabetes mellitus, green tea reduces albuminuria by decreasing podocyte apoptosis through activation of the WNT pathway. We investigated the effect of green tea polyphenols (GTP) on residual albuminuria of diabetic subjects with nephropathy. We conducted a randomised, double-blind study in 42 diabetic subjects with a urinary albumin-creatinine ratio (UACR) >30 mg/g, despite administration of the maximum recommended dose of renin-angiotensin (RAS) inhibition. Patients were randomly assigned to two equal groups to receive either GTP (containing 800 mg of epigallocatechin gallate, 17 with type 2 diabetes and 4 with type 1 diabetes) or placebo (21 with type 2 diabetes) for 12 weeks. Treatment with GTP reduced UACR by 41%, while the placebo group saw a 2% increase in UACR (p = 0.019). Podocyte apoptosis (p = 0.001) and in vitro albumin permeability (p < 0.001) were higher in immortalized human podocytes exposed to plasma from diabetic subjects compared to podocytes treated with plasma from normal individuals. In conclusion, GTP administration reduces albuminuria in diabetic patients receiving the maximum recommended dose of RAS. Reduction in podocyte apoptosis by activation of the WNT pathway may have contributed to this effect.

Bone marrow-derived cells were demonstrated to improve organ function, but the lack of cell retention within injured organs suggests that the protective effects are due to factors released by the cells. Herein, we tested cell therapy using early outgrowth cells (EOCs) or their conditioned media (CM) to protect the retina of diabetic animal models (type 1 and type 2) and assessed the mechanisms by in vitro study. Control and diabetic (db/db) mice (8 weeks of age) were randomized to receive a unique intravenous injection of 5×105EOCs or 0.25 ml thrice weekly tail-vein injections of 10x concentrated CM and Wystar Kyoto rats rendered diabetic were randomized to receive 0.50 ml thrice weekly tail-vein injections of 10x concentrated CM. Four weeks later, the animals were euthanized and the eyes were enucleated. Rat retinal Müller cells (rMCs) were exposed for 24 h to high glucose (HG), combined or not with EOC-conditioned medium (EOC-CM) from db/m EOC cultures. Diabetic animals showed increase in diabetic retinopathy (DR) and oxidative damage markers; the treatment with EOCs or CM infusions significantly reduced this damage and re-established the retinal function. In rMCs exposed to diabetic milieu conditions (HG), the presence of EOC-CM reduced reactive oxygen species production by modulating the NADPH-oxidase 4 system, thus upregulating SIRT1 activity and deacetylating Lys-310-p65-NFκB, decreasing GFAP and VEGF expressions. The antioxidant capacity of EOC-CM led to the prevention of carbonylation and nitrosylation posttranslational modifications on the SIRT1 molecule, preserving its activity. The pivotal role of SIRT1 on the mode of action of EOCs or their CM was also demonstrated on diabetic retina. These findings suggest that EOCs are effective as a form of systemic delivery for preventing the early molecular markers of DR and its conditioned medium is equally protective revealing a novel possibility for cell-free therapy for the treatment of DR.

The TNF-α serum level increases after rhabdomyolysis and is involved in the subsequent cardiorenal injury. In the present study, we investigated the TNF-α-dependent cell signaling pathways implicated in cellular injury in these organs. Rhabdomyolysis was induced by intramuscular glycerol injection in rats. Renal function, cardiac and renal pathology, and activation of caspases were evaluated during the first 24 h after glycerol injection. TNF-α blockade with infliximab reduced tubular necrosis and cardiorenal apoptosis. Cellular Fas-associated protein with death domain-like IL-1β-converting enzyme inhibitory protein (cFLIP), an inhibitor of caspase-8, was overexpressed in the kidney but not in the heart. The inhibitory effect of cFLIP blunted caspase-8 activation in the kidney. In this condition, the cellular response to the TNF-α stimulus was driven to receptor-interacting protein-1 (RIP1)-mediated necroptosis. Treatment with RIP1 inhibitor (necrostatin-1) isolated or in combination with infliximab showed a similar reduction in tubular necrosis, underscoring the importance of TNF-α-mediated tubular necroptosis in this model. TNF-α played a positive regulatory role in the transcription of proapoptotic Bax and p53-upregulated modulator of apoptosis (PUMA) proteins. Infliximab treatment reduced caspase-9-mediated apoptosis in both organs. Treatment with a caspase-8 inhibitor showed that caspase-8 participated in the process of apoptosis only in the heart, upstream of caspase-9 activation. TNF-α-mediated necroptosis is the predominant form of tubular injury observed in the glycerol model. TNF-α up regulates Bax and PUMA proapoptotic proteins, resulting in activation of the intrinsic pathway of apoptosis in the kidney and heart., (Copyright © 2015 the American Physiological Society.)

In diabetes mellitus (DM), podocyte apoptosis leads to albuminuria and nephropathy progression. Low-density lipoprotein receptor-related protein 6 (LRP6) is WNT pathway receptor that is involved in podocyte death, adhesion and motility. Glycogen synthase kinase 3 (GSK3) interaction with p53 (GSK3-p53) promotes apoptosis in carcinoma cells. It is unknown if GSK3-p53 contributes to podocyte apoptosis in DM. In experimental DM, green tea (GT) reduces albuminuria by an unknown mechanism. In the present study, we assessed the role of the GSK3β-p53 in podocyte apoptosis and the effects of GT on these abnormalities. In diabetic spontaneously hypertensive rats (SHRs), GT prevents podocyte's p-LRP6 expression reduction, increased GSK3β-p53 and high p53 levels. In diabetic SHR rats, GT reduces podocyte apoptosis, foot process effacement and albuminuria. In immortalized mouse podocytes (iMPs), high glucose (HG), silencing RNA (siRNA) or blocking LRP6 (DKK-1) reduced p-LRP6 expression, leading to high GSK3β-p53, p53 expression, apoptosis and increased albumin influx. GSK3β blockade by BIO reduced GSK3β-p53 and podocyte apoptosis. In iMPs under HG, GT reduced apoptosis and the albumin influx by blocking GSK3β-p53 following the rise in p-LRP6 expression. These effects of GT were prevented by LRP6 siRNA or DKK-1. In conclusion, in DM, WNT inhibition, via LRP6, increases GSK3β-p53 and podocyte apoptosis. Maneuvers that inactivate GSK3β-p53, such as GT, may be renoprotective in DM., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Reduction in sirtuin 1 (Sirt-1) is associated with extracellular matrix (ECM) accumulation in the diabetic kidney. Theobromine may reduce kidney ECM accumulation in diabetic rats. In the current study, we aimed to unravel, under diabetic conditions, the mechanism of kidney ECM accumulation induced by a reduction in Sirt-1 and the effect of theobromine in these events. In vitro, we used immortalized human mesangial cells (iHMCs) exposed to high glucose (HG; 30 mM), with or without small interfering RNA for NOX4 and Sirt-1. In vivo, spontaneously hypertensive rats (SHR) were rendered diabetic by means of streptozotocin and studied after 12 wk. The effects of treatment with theobromine were investigated under both conditions. HG leads to a decrease in Sirt-1 activity and NAD(+) levels in iHMCs. Sirt-1 activity could be reestablished by treatment with NAD(+), silencing NOX4, and poly (ADP-ribose) polymerase-1 (PARP-1) blockade, or with theobromine. HG also leads to a low AMP/ATP ratio, acetylation of SMAD3, and increased collagen IV, which is prevented by theobromine. Sirt-1 or AMPK blockade abolished these effects of theobromine. In diabetic SHR, theobromine prevented increases in albuminuria and kidney collagen IV, reduced AMPK, elevated NADPH oxidase activity and PARP-1, and reduced NAD(+) levels and Sirt-1 activity. These results suggest that in diabetes mellitus, Sirt-1 activity is reduced by PARP-1 activation and NAD(+) depletion due to low AMPK, which increases NOX4 expression, leading to ECM accumulation mediated by transforming growth factor (TGF)-β1 signaling. It is suggested that Sirt-1 activation by theobromine may have therapeutic potential for diabetic nephropathy., (Copyright © 2015 the American Physiological Society.)

Cocoa is rich in flavonoids, which are potent antioxidants with established benefits for cardiovascular health but unproven effects on neurodegeneration. Sirtuins (SIRTs), which make up a family of deacetylases, are thought to be sensitive to oxidation. In this study, the possible protective effects of cocoa in the diabetic retina were assessed. Rat Müller cells (rMCs) exposed to normal or high glucose (HG) or H2O2 were submitted to cocoa treatment in the presence or absence of SIRT-1 inhibitor and small interfering RNA The experimental animal study was conducted in streptozotocin-induced diabetic rats randomized to receive low-, intermediate-, or high-polyphenol cocoa treatments via daily gavage for 16 weeks (i.e., 0.12, 2.9 or 22.9 mg/kg/day of polyphenols). The rMCs exposed to HG or H2O2 exhibited increased glial fibrillary acidic protein (GFAP) and acetyl-RelA/p65 and decreased SIRT1 activity/expression. These effects were cancelled out by cocoa, which decreased reactive oxygen species production and PARP-1 activity, augmented the intracellular pool of NAD(+), and improved SIRT1 activity. The rat diabetic retinas displayed the early markers of retinopathy accompanied by markedly impaired electroretinogram. The presence of diabetes activated PARP-1 and lowered NAD(+) levels, resulting in SIRT1 impairment. This augmented acetyl RelA/p65 had the effect of up-regulated GFAP. Oral administration of polyphenol cocoa restored the above alterations in a dose-dependent manner. This study reveals that cocoa enriched with polyphenol improves the retinal SIRT-1 pathway, thereby protecting the retina from diabetic milieu insult., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: Retinal pigment epithelium cells, along with tight junction (TJ) proteins, constitute the outer blood retinal barrier (BRB). Contradictory findings suggest a role for the outer BRB in the pathogenesis of diabetic retinopathy (DR). The aim of this study was to investigate whether the mechanisms involved in these alterations are sensitive to nitrosative stress, and if cocoa or epicatechin (EC) protects from this damage under diabetic (DM) milieu conditions., Methods: Cells of a human RPE line (ARPE-19) were exposed to high-glucose (HG) conditions for 24 hours in the presence or absence of cocoa powder containing 0.5% or 60.5% polyphenol (low-polyphenol cocoa [LPC] and high-polyphenol cocoa [HPC], respectively)., Results: Exposure to HG decreased claudin-1 and occludin TJ expressions and increased extracellular matrix accumulation (ECM), whereas levels of TNF-α and inducible nitric oxide synthase (iNOS) were upregulated, accompanied by increased nitric oxide levels. This nitrosative stress resulted in S-nitrosylation of caveolin-1 (CAV-1), which in turn increased CAV-1 traffic and its interactions with claudin-1 and occludin. This cascade was inhibited by treatment with HPC or EC through δ-opioid receptor (DOR) binding and stimulation, thereby decreasing TNF-α-induced iNOS upregulation and CAV-1 endocytosis. The TJ functions were restored, leading to prevention of paracellular permeability, restoration of resistance of the ARPE-19 monolayer, and decreased ECM accumulation., Conclusions: The detrimental effects on TJs in ARPE-19 cells exposed to DM milieu occur through a CAV-1 S-nitrosylation-dependent endocytosis mechanism. High-polyphenol cocoa or EC exerts protective effects through DOR stimulation., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

The ability of nano-assisted laser desorption-ionization mass spectrometry imaging (NALDI-IMS) to provide selective chemical monitoring with appropriate spatial distribution of a low molecular drug in a biological tissue was investigated. NALDI-IMS is a matrix-free laser desorption ionization (LDI) protocol based on imprinting of tissue constituents on a nanostructured surface. Using the accumulation of theobromine in rat kidney as a model, NALDI-IMS was found to provide well-resolved images of the special distribution of this low molecular weight (MW) drug in tissue., (Copyright © 2014 John Wiley & Sons, Ltd.)

The aims of the present study were to investigate, in diabetes mellitus (DM), the mechanism of NOX4 up-regulation, its link with 5' adenosine monophosphate-activated protein kinase (AMPK) inactivation and transforming growth factor (TGF) ß-1 signaling in determining the accumulation of kidney extracellular matrix (ECM), and the possible action of cocoa enriched with polyphenols (CH) in these events. After 16 weeks of DM, spontaneously hypertensive rats showed increased kidney TGFβ-1 levels and expression of phosphorylated smad2, collagen IV and fibronectin in parallel with elevated NOX4 expression and reduced phosphorylated AMPK. CH treatment in diabetic rats prevented all of these abnormalities. In immortalized human mesangial cells exposed to high glucose (HG), or TGFβ-1, CH, nicotinamide adenine dinucleotide phosphate blocker, or silencing NOX4 ameliorated enhanced phosphorylated smad2 and collagen IV. Reduction in phosphorylated AMPK induced by HG or TGFβ-1 was ameliorated by CH or activation of AMPK, which reduced phosphorylation of smad2 and collagen IV via reduction in NOX4 expression. The effects of CH were abolished by AMPK blockade. These results suggest that inactivation in AMPK leads to NOX4 up-regulation, activation of TGFβ-1 signaling and increased ECM accumulation. Additionally, increased TGF-ß1 per se leads to the amplification of ECM production by reducing AMPK and promoting the activation of NOX4. It is suggested that the activation of AMPK by CH followed by reduction in NOX4/TGFβ-1 signaling may have a therapeutic potential in diabetic nephropathy., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Purpose: Diabetic retinopathy (DR) is associated with nitrosative stress. The purpose of this study was to evaluate the beneficial effects of S-nitrosoglutathione (GSNO) eye drop treatment on an experimental model of DR., Methods: Diabetes (DM) was induced in spontaneously hypertensive rats (SHR). Treated animals received GSNO eye drop (900 nM or 10 μM) twice daily in both eyes for 20 days. The mechanisms of GSNO effects were evaluated in human RPE cell line (ARPE-19)., Results: In animals with DM, GSNO decreased inducible nitric oxide synthase (iNOS) expression and prevented tyrosine nitration formation, ameliorating glial dysfunction measured with glial fibrillary acidic protein, resulting in improved retinal function. In contrast, in nondiabetic animals, GSNO induced oxidative/nitrosative stress in tissue resulting in impaired retinal function. Nitrosative stress was present markedly in the RPE layer accompanied by c-wave dysfunction. In vitro study showed that treatment with GSNO under high glucose condition counteracted nitrosative stress due to iNOS downregulation by S-glutathionylation, and not by prevention of decreased GSNO and reduced glutathione levels. This posttranslational modification probably was promoted by the release of oxidized glutathione through GSNO denitrosylation via GSNO-R. In contrast, in the normal glucose condition, GSNO treatment promoted nitrosative stress by NO formation., Conclusions: In this study, a new therapeutic modality (GSNO eye drop) targeting nitrosative stress by redox posttranslational modification of iNOS was efficient against early damage in the retina due to experimental DR. The present work showed the potential clinical implications of balancing the S-nitrosoglutathione/glutathione system in treating DR.

Background/aims: High phosphate (Pi) levels and extracellular matrix (ECM) accumulation are associated with chronic kidney disease progression. However, how high Pi levels contribute to ECM accumulation in mesangial cells is unknown. The present study investigated the role and mechanism of high Pi levels in ECM accumulation in immortalized human mesangial cells (iHMCs)., Methods: iHMCs were exposed to normal (0.9 mM) or increasing Pi concentrations (2.5 and 5 mM) with or without diferent blockers or activators. NOX4, phosphorylated AMPK (p-AMPK), phosphorylated SMAD3 (p-SMAD3), fibronectin (F/N), collagen IV (C-IV) and alpha-smooth muscle actin (α-SMA) expression was assessed via western blot and immunofluorescence. Lucigenin-enhanced chemiluminescence, and dihydroethidium (DHE) assessed NADPH oxidase activity and superoxide (SO), respectively., Results: In iHMCs, a Pi transporter blocker (PFA) abrogated high Pi-induced AMPK inactivation, increase in NADPH oxidase-induced reactive oxygen species (ROS) levels, NOX4, p-SMAD3, α-SMA and C-IV expression. AMPK activation by AICAR, NOX4 silencing or NADPH oxidase blocker prevented high Pi-induced DHE levels, p-SMAD3, F/N, C-IV and α-SMA expression., Conclusion: AMPK inactivation with NOX4-induced ROS formation and transforming growth factor ß-1 (TGFß-1) signaling activation mediates high Pi-induced ECM accumulation in iHMCs. Maneuvers increasing AMPK or reducing NOX4 activity may contribute to renal protection under hyperphosphatemia., (© 2014 S. Karger AG, Basel.)

Diabetes and hypertension frequently coexist and constitute the most notorious combination for the pathogenesis of DR. Large clinical trials have clearly demonstrated that tight control of glycaemia and/or blood pressure significantly reduces the incidence and progression of DR. The mechanism by which hypertension interacts with diabetes to exacerbate the retinal disease is not completely understood. From experimental studies, increasing evidence demonstrates that chronic inflammation and oxidative stress are involved. In the present review, we summarize data obtained from our research along with those from other groups to better understand the role of hypertension in the pathogenesis of DR. It is suggested that oxidative stress and inflammation may be common denominators of retinal damage in the presence of hypertension in diabetic patients.

Background: Loss of β-cell function hastens deterioration of metabolic control in type 2 diabetes patients. Besides amyloid deposit and glucolipotoxicity, advanced glycation end products (AGEs) acting through their receptors (RAGE) seem to contribute to this process by promoting islet apoptosis. In order to investigate the role of AGEs in β-cell deterioration, we evaluated the temporal and dose effects of AGE compounds on apoptosis rate, reactive oxygen species generation and expression of pro-apoptotic and anti-apoptotic genes in cultured islets., Methods: Rat pancreatic islets were exposed or not for 24, 48, 72 and 96 h to albumin modified by glycoaldehyde. Apoptosis, reactive oxygen species and superoxide content and NADPH oxidase activity were evaluated as well as RNA expression of the genes Ager (codes for RAGE), Bax, Bcl2 and Nfkb1., Results: In 24 and 48 h, glycoaldehyde elicited a decrease in apoptosis rate in comparison with the control condition concomitantly with a reduction in Bax/Bcl2 RNA ratio and in Nfkb1 RNA expression. In contrast, after 72 and 96 h, glycoaldehyde promoted an increase in apoptosis rate concomitantly with an increase in Bax/Bcl2 RNA ratio and in Nfkb1 RNA expression. In 24 h, glycoaldehyde elicited a decrease in the islet content of reactive oxygen species, whereas after 48 and 72 h, it promoted an opposite effect, increasing superoxide generation. The NADPH oxidase inhibitor VAS2870 attenuated superoxide production, implicating NADPH oxidase as an important source of reactive oxygen species in islets exposed to AGEs., Conclusions: Albumin modified by glycoaldehyde exerted a dual effect in cultured pancreatic islets, being protective against apoptosis after short exposure but pro-apoptotic after prolonged exposure., (Copyright © 2013 John Wiley & Sons, Ltd.)

Purpose: Green tea (GT), widely studied for its beneficial properties in protecting against brain ischemia, is a rich source of polyphenols, particularly (-)-epigallocatechin gallate (EGCG). The results presented here demonstrate the beneficial effects of GT in diabetic retinas and in retinal cells under diabetic conditions., Methods: Diabetes was induced in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats. Treatment animals received GT orally for 12 weeks. A vehicle was administered orally to the control animals. The protective effects of GT were also evaluated in Müller and in ARPE-19 cells., Results: In diabetic rats, there was an increase in the expression of glial fibrillary acidic protein (GFAP), oxidative retinal markers, and glutamine synthetase levels. In addition, there was a decrease in occludin and glutamate transporter and receptor. Diabetic SHR also demonstrated blood-retinal barrier breakdown and impaired electroretinography results. Müller cells exposed to high-glucose medium produced higher levels of reactive oxygen species (ROS) and glutamine synthetase but reduced levels of glutathione, glutamate transporter, and glutamate receptor. Similarly, ARPE-19 cells exhibited increased ROS production accompanied by decreased expression of claudin-1 and glutamate transporter. Treatment with GT fully restored all the above-mentioned alterations in diabetic animals as well as in retinal cells., Conclusions: GT protected the retina against glutamate toxicity via an antioxidant mechanism. These findings reveal a novel mechanism by which GT protects the retina against neurodegeneration in disorders such as diabetic retinopathy.

Spironolactone (SPR), a mineralocorticoid receptor blocker, diminishes hyperglycemia-induced reduction in glucose-6-phosphate dehydrogenase (G6PD) activity, improving oxidative stress damage. This study investigated whether SPR ameliorates nephropathy by increasing G6PD activity and reducing oxidative stress in spontaneously hypertensive diabetic rats (SHRs). The streptozotocin-induced diabetic rats received or not SPR 50 mg/kg per day, for eight weeks. A human mesangial cell line was cultured in normal or high glucose conditions, with or without SPR, for 24 h. Plasma glucose levels and systolic blood pressure were unaltered by diabetes or by SPR treatment. Albuminuria, fibronectin expression, 8-OHdG urinary levels, lipid peroxidation and p47phox expression were higher in the diabetic rats compared with the control and were reduced by SPR. The antioxidant GSH/GSSG ratio was reduced in the diabetic rats and the treatment reestablished it. Diabetes-induced SGK1 up-regulation was inhibited by SPR. Reactive oxygen species (ROS) and superoxide production induced by NADPH oxidase were increased by hyperglycemia and high glucose, in vivo and in vitro, respectively, and were reduced with SPR. Hyperglycemia and high glucose decreased G6PD activity, which was restored with SPR. These results suggest that SPR ameliorates nephropathy in diabetic SHRs by restoring G6PD activity and diminishes oxidative stress without affecting glycaemia and blood pressure.

Background/aims: In diabetic hypertensive rats, tempol reduces albuminuria by restoring the redox imbalance. Increased formation of reactive oxygen species leading to activation of poly(ADP-ribose) polymerase (PARP)-1 and podocyte loss by apoptosis contribute to albuminuria in diabetes mellitus (DM). In the present study, we investigated the hypothesis that in DM tempol reduces albuminuria by inhibition of PARP-induced podocyte apoptosis., Methods: DM was induced in 4-week-old spontaneously hypertensive rats by streptozotocin. Mouse and human podocyte cell lines were cultured in normal or high-glucose conditions, with or without tempol and/or a PARP-1 inhibitor, PJ34., Results: In diabetic rats, tempol treatment did not affect plasma glucose levels or systolic blood pressure. Albuminuria was higher in diabetic rats, and it was reduced by tempol. DM leads to an elevation of glomerular apoptotic cells and to podocyte loss; both were prevented by tempol treatment. DM increases the expression of poly(ADP-ribose)-modified proteins in isolated glomeruli, and it was reduced by tempol. In vitro, high glucose increased caspase-3 activity and led to a higher number of apoptotic cells that were prevented by tempol and the PARP-1 inhibitor., Conclusion: In DM, tempol reduces albuminuria associated with reduction of podocyte apoptosis and decreasing oxidative stress via PARP signaling., (Copyright © 2012 S. Karger AG, Basel.)

This study identified the pattern of alcohol consumption among high school students in Passos, Minas Gerais State, and its association with socio-demographic factors. Study participants included 1,967 adolescents from public and private educational institutions. The AUDIT questionnaire was used. For data analysis, descriptive statistics, the chi-square test and multiple logistic regression were applied. The average age of participants was 15.84±1.23 years. The first contact with alcohol occurred at 13.37±1.92 years. Among the adolescents, 30.96% were teetotalers, 45.76% had moderate drinking behavior, 16.47%, were risk drinkers, 3.51% were high-risk drinkers, and 3.31% possible addicts. There was a significant association between the risk of drinking and sex, the relationship of adolescents with their mothers, work, economic bracket and education of the head of the family. In multivariate analysis, there was a higher risk for adolescent males, as well as workers with a poor relationship with their mothers. It was found that teenagers have early contact with alcohol and high prevalence of risk-related drinking behavior.

Diabetes and hypertension frequently coexist and constitute the most notorious combination for the pathogenesis of diabetic nephropathy and retinopathy. Large clinical trials have clearly demonstrated that tight control of glycemia and/or blood pressure significantly reduces the incidence and progression of diabetic retinopathy (DR) and nephropathy. However, the mechanism by which hypertension interacts with diabetes to induce and/or exacerbate nephropathy and retinopathy is very unclear. Substantial evidence implicates the involvement of chronic inflammation and oxidative stress in the pathogenesis of DR and nephropathy. In addition, hypertension causes oxidative stress and inflammation in the kidney and retina. In the present review, we summarized data obtained from our research along with those from other groups to better understand the role of hypertension in the pathogenesis of diabetic nephropathy and retinopathy. It is suggested that oxidative stress and inflammation may be common denominators of kidney and retinal damage in the concomitant presence of diabetes and hypertension.

Purpose: The purpose of this study was to investigate the efficacy of tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), a superoxide dismutase mimetic, in preventing early retinal molecular changes in a model that combines hypertension and diabetes., Methods: Four-week-old spontaneously hypertensive rats (SHR) were rendered diabetic by streptozotocin. Diabetic SHR rats (DM-SHR) were randomized to receive or not receive tempol treatment. After 20 days of induction of diabetes, the rats were euthanatized, and their retinas were collected., Results: The early molecular markers of diabetic retinopathy (DR), glial fibrillary acidic protein, and fibronectin were evaluated by Western blot assays and showed an increase in DM-SHR compared with the SHR group. The oxidative balance, evaluated by superoxide production and nitric oxide end product levels estimated by a nitric oxide analyzer, and the counterpart antioxidative defense revealed an accentuated imbalance in DM-SHR compared with the SHR group. As a result, the product peroxynitrite, which was detected by immunohistochemistry for nitrotyrosine, was higher in the DM-SHR group. The retinal poly-ADP-ribose (PAR)-modified proteins, which reflect the activation of PAR polymerase (PARP), and the inducible nitric oxide synthase (iNOS) expressions were found to have increased in this group. Treatment with tempol reestablished the oxidative parameters and decreased the PAR-modified proteins, thus preventing extracellular matrix accumulation and glial reaction., Conclusions: The administration of tempol prevented oxidative damage, decreased iNOS levels, and ameliorated the activation of PARP in the retinas of diabetic hypertensive rats. Consequently, the early molecular markers of DR, such as glial reaction (glial fibrillary acidic protein [GFAP]) and extracellular matrix accumulation (fibronectin), were prevented in tempol-treated rats.

Purpose: To investigate if nitric oxide (NO) system contributes to the beneficial effect of angiotensin II type 1 receptor (AT(1)) blocker losartan in the retina of diabetic spontaneously hypertensive rats (SHR)., Methods: Diabetic SHR were randomized to receive oral treatment with losartan (DM-SHRLos). After 20 days, the rats were euthanized and the retinas collected., Results: Diabetic SHR rats exhibited a significant increase in glial fibrillary acidic protein (GFAP) and decrease in occludin, markers of early diabetic retinopathy (DR). The oxidative status, evaluated by NO end-products (NO(x)(-)) levels along with the antioxidative system superoxide dismutase, revealed an accentuated imbalance in favor to oxidants in DM-SHR leading to a higher tyrosine nitration and DNA damage. The inducible NO synthase (iNOS) was also elevated in DM-SHR rats. The treatment with losartan ameliorated all of the above alterations., Conclusions: Oral treatment with losartan reduces iNOS expression and reestablishes the redox status, thus ameliorating the early markers of DR in a model of diabetes and hypertension.

Objective: Diabetic retinopathy displays the features of a neurodegenerative disease. Oxidative stress is involved in the pathogenesis of diabetic retinopathy. This investigation sought to determine whether hypertension exacerbates the oxidative stress, neurodegeneration, and mitochondrial dysfunction that exists in diabetic retinopathy and whether these changes could be minimized by the angiotensin II type 1 (AT(1)) receptor blocker (ARB) losartan., Research Design and Methods: Diabetes was induced in spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats. The diabetic SHRs were assigned to receive or not receive losartan., Results: The level of apoptosis in the retina was higher in diabetic WKY rats than in the control group, and higher levels were found in diabetic SHRs. The apoptotic cells expressed neural and glial markers. The retinal glial reaction was more evident in diabetic WKY rats and was markedly accentuated in diabetic SHRs. Superoxide production in retinal tissue increased in diabetic WKY rats, and a greater increase occurred in diabetic SHRs. Glutathione levels decreased only in diabetic SHRs. As a consequence, the levels of nitrotyrosine and 8-hydroxy 2'-deoxyguanosine, markers of oxidative stress, were elevated in diabetic groups, mainly in diabetic SHRs. Mitochondrial integrity was dramatically affected in the diabetic groups. The ARB treatment reestablished all of the above-mentioned parameters., Conclusions: These findings suggest that concomitance of hypertension and diabetes exacerbates oxidative stress, neurodegeneration, and mitochondrial dysfunction in the retinal cells. These data provide the first evidence of AT(1)blockage as a neuroprotective treatment of diabetic retinopathy by reestablishing oxidative redox and the mitochondrial function.

Green tea (GT), through its antioxidant properties, may be useful to treat or prevent human diseases. Because several lines of evidence suggest that oxidative stress contributes to the pathogenesis of diabetic nephropathy, we tested the hypothesis that GT prevents diabetes and hypertension-related renal oxidative stress, attenuating renal injury. Spontaneously hypertensive rats (SHR) with streptozotocin-induced diabetes and nondiabetic SHR were treated daily with tap water or freshly prepared GT (13.3 g/L). After 12 wk, the systolic blood pressure did not differ between treated and untreated nondiabetic or diabetic rats. However, body weight was less (P < 0.05) and glycemia was greater in diabetic SHR rats than in nondiabetic rats. Renal oxidative stress variables such as 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nitrotyrosine expression, NADPH oxidase-dependent superoxide generation, and the expression of renal cortex Nox4 were greater (P < 0.05) in diabetic rats that received water (DW) than in nondiabetic rats that received water (CW). The 8-OHdG and NADPH oxidase-dependent superoxide generation were significantly less in rats treated with GT. Nitrotyrosine and Nox4 expression were significantly less in diabetic rats that received GT (DGT) than in DW. Likewise, the indices of renal injury, albuminuria, and renal expression of collagen IV were significantly greater in DW than in CW. These differences were significantly less in DGT than in DW. GT reestablished the redox state and reduced the indicators of nephropathy without altering glycemia and blood pressure levels in diabetic SHR. These findings suggest that the consumption of GT may ameliorate nephropathy in diabetic hypertensive patients.

Aims: The presence of hypertension increases renal oxidative stress by increasing NADPH oxidase-dependent superoxide production and by decreasing antioxidant defense in the early stage of experimental diabetes mellitus (DM). In the present study, we investigated whether the administration of an antioxidant mimetic of the superoxide dismutase (SOD) (tempol) corrects the oxidative imbalance and oxidative stress-induced renal injury in the presence of DM and hypertension., Methods: DM was induced in spontaneously hypertensive rats (SHR) by streptozotocin at 4 weeks of age. The diabetic rats either did or did not receive tempol for 20 days. Oxidative-stress parameters and indices of renal injury were evaluated., Results: Tempol reestablished the imbalance in redox status induced by DM. It elevated the expression of renal antioxidant extracellular SOD, p < 0.0001; decreased (p = 0.049) the production of renal NADPH-dependent superoxide production, and diminished (p = 0.016) a marker of oxidative stress-induced DNA damage, 8-hydroxy-2'-deoxyguanosine. Reduction of oxidative stress markers was associated with reduction in renal damage parameters associated with DN. DM-induced albuminuria and elevation in renal expression of collagen IV were reduced to the level observed in control rats., Conclusion: We conclude that an imbalance in renal redox status is associated with markers of renal injury in the early stage of DM and hypertension. Antioxidant treatment reestablished the redox status and prevented oxidative stress-induced renal damage.

Purpose: Hyperglycemia and hypertension contribute to the development of diabetic retinopathy, and this may involve alterations in the normal retinal cell cycle. In this work, we examined the influence of diabetes and hypertension on retinal cell replication in vivo and the relationship between these changes and several early markers of diabetic retinopathy., Methods: Diabetes was induced with streptozotocin in 4- and 12-week-old spontaneously hypertensive rats (SHR) and their Wistar Kyoto (WKY) controls. The rats were killed 15 days later. Retinal cells stained with bromodeoxyuridine (BrdU) were seen in rats of both ages., Results: In 12-week-old rats, the number of BrdU-positive retinal cells was higher in SHR than in WKY rats. After 15 days of diabetes mellitus, there was a marked reduction in cell replication only in diabetic SHR (p=0.007). The BrdU-positive cells expressed neural, glial, or vascular progenitor markers. There was greater expression of p27(Kip1) in the ganglion cell layer of both diabetic groups (p=0.05), whereas in the inner nuclear layer there was enhanced expression only in diabetic SHR (p=0.02). There was a marked increase in the retinal expression of fibronectin (p=0.04) and vascular endothelial growth factor (p=0.02) in diabetic SHR that was accompanied by blood-retinal barrier breakdown (p=0.01)., Discussion: Concomitant diabetes and hypertension attenuated the proliferation of retinal cells, and it is associated with an increase in p27(Kip1) expression, fibronectin accumulation, and blood-retinal barrier breakdown. The replicative retinal cells displayed characteristics of progenitor cells.

Aims: The combination of hypertension and diabetes exacerbates renal oxidative stress. The aim of the present study was therefore to evaluate the pro-oxidant and antioxidant mechanisms responsible for the induction of renal oxidative stress in the presence of hypertension and diabetes mellitus., Methods: Diabetes was induced in spontaneously hypertensive rats (SHR) and their genetically normotensive control Wistar-Kyoto (WKY) rats by streptozotocin at 12 weeks of age. After 10 days, pro-oxidant, antioxidant and oxidative stress parameters were evaluated in the renal tissue., Results: NADPH oxidase-dependent superoxide generation in the renal cortex was significantly elevated in WKY and SHR diabetic (D) groups compared to the respective control (C) groups (p < 0.005, n = 5). However, the highest level of superoxide generation was observed in the SHR-D group compared to all other groups. The expression of the gp91phox subunit of NADPH oxidase was significantly elevated in the SHR-D (p < 0.05, n = 5), but not in the WKY-D group, compared to the respective control groups. The renal cortical extracellular-superoxide dismutase level was found to be markedly decreased in the SHR groups compared to the WKY groups (p < 0.05, n = 5). The antioxidant glutathione level was found to be lower in the SHR-D (p = 0.03, n = 15), but not in the WKY-D group, compared to the respective control groups. Finally, nitrotyrosine and 8-hydroxy-2'-deoxyguanosine, markers of oxidative stress, were found to be similar in the kidneys of WKY-C and WKY-D, but were elevated in the SHR-D compared to the SHR-C group., Conclusion: We therefore conclude that hypertension increases pro-oxidant generation and decreases antioxidant defense, and thereby induces renal oxidative stress in early diabetes., ((c) 2007 S. Karger AG, Basel.)

The present study was undertaken to investigate the redox status in the retina of an experimental model that combines hypertension and diabetes. Spontaneously hypertensive rats (SHR) and their control Wystar Kyoto (WKY) rats were rendered diabetic and, after 20 days, the rats were sacrificed and the retinas collected. The superoxide production was higher in diabetic than in control WKY (p < 0.03) and SHR rats showed elevated superoxide production compared with WKY groups (p < 0.009). The glutathione antioxidant system was diminished only in diabetic SHR (p < 0.04). Tirosyne nitration was higher in diabetic WKY and control SHR compared with control WKY (p < 0.03), and further increment was observed in diabetic SHR (p < 0.02). The DNA damage estimated by immunohystochemistry for 8-OHdG was higher in control SHR than in WKY, mainly in diabetic SHR (p < 0.0001). Hypertension aggravates oxidative-induced cytotoxicity in diabetic retina due to increasing of superoxide production and impairment of antioxidative system.

The present study was undertaken to identify whether the age at induction of experimental diabetes modifies macrophage infiltration in the kidney. Renal macrophage infiltration was studied 10 days after the induction of experimental diabetes in 4-week-old pre-pubertal and 12-week-old adult male rats of normotensive [Wistar-Kyoto (WKY) rats] and hypertensive (spontaneously hypertensive rats, SHRs) background. Renal macrophage infiltration was evaluated by immunohistochemistry for ED1. Plasma glucose levels were similar in all diabetic groups. Adult SHRs were hypertensive, and induction of diabetes did not alter blood pressure (BP) in any group. Induction of diabetes in pre-pubertal rats did not induce macrophage infiltration in the kidney. However, in adult rats, tubulointerstitial macrophage infiltration was increased in both WKY (22.86 +/- 3.93 vs 7.86 +/- 2.16 per high-power field, P < 0.001) and SHR (26.41 +/- 5.91 vs 11.48 +/- 1.23, P < 0.001) groups after induction of diabetes. Glomerular macrophage infiltration was also increased after induction of diabetes in the adult WKY group (1.83 +/- 0.50 vs 1.16 +/- 0.26 per glomerular cross section, P = 0.029), which was not significant in the adult SHRs (2.52 +/- 0.34 vs 1.95 +/- 0.35). We conclude that the pre-pubertal induction of diabetes apparently protects against early renal macrophage infiltration, while the induction of diabetes in adults induces exaggerated macrophage infiltration in the kidney.

Hypertension is an important risk factor associated with development and progression of diabetic retinopathy (DR). The mechanisms by which hypertension increases the risk for DR are poorly understood. As the inflammatory mechanisms play a pivotal role in the pathogenesis of DR, in the present study, we investigated the effects of diabetes, hypertension, and combination of diabetes and hypertension on early inflammatory phenomena in the retina, and the effects of blood pressure control on retinal inflammation. Four-week-old spontaneously hypertensive rats (SHR) and their normotensive counterpart Wistar Kyoto (WKY) rats were rendered diabetic by intravenous injection of streptozotocin. Diabetic SHR rats were randomized to receive no antihypertensive drug (Sd), an antihypertensive drug that acts on renin-angiotensin system (losartan, Sd+Los), or antihypertensive drug that do not affect renin-angiotensin system (triple therapy, Sd+Tri). After 20 days, rats were sacrificed and the retinas were collected. The number of immunohistochemically detected ED1/microglial positive cells and the expression of ICAM-1 in the retina were significantly higher in diabetic SHR than in control SHR (p=0.003). The NF-kappaB p65 levels were higher in SHR compared with WKY groups (p=0.001) and its increment in diabetic SHR was not significant. These abnormalities in diabetic SHR rats were completely prevented by both types of antihypertensive drugs. The concomitance of diabetes and hypertension leads to exuberant inflammatory response in the retina, and the prevention of hypertension abrogates these abnormalities. It is suggested that the inflammatory events may be involved in the mechanism by which hypertension exacerbates retinopathy in patients with diabetes.

Inflammation is pivotal to the pathogenesis of diabetic retinopathy (DR). Hypertension is the main secondary risk factor associated with DR. The mechanisms by which hypertension increases the risk for DR are poorly understood. The aim of the current study was to investigate the contribution of genetic hypertension to early retinal inflammation in experimental diabetes. Diabetes was induced in 4-week-old (developing hypertension) and 12-week-old (fully hypertensive) spontaneously hypertensive rats (SHR) and age-matched control normotensive Wistar Kyoto (WKY) rats by administration of streptozotocin (50 mg/kg, i.v); after 20 days the rats were sacrificed and the retinas were collected. ED1 positive cells, ICAM-1 and VEGF levels were significantly higher in diabetic SHR in both prehypertensive and hypertensive ages (p < 0.005). NF-kappaB p65 levels were higher in prehypertensive SHR and in hypertensive diabetic SHR (p < 0.05). Induction of diabetes in normotensive WKY rats did not show any alteration in retinal expression of inflammatory parameters. Therefore, we conclude that the developing hypertension and also the fully developed hypertension lead to earlier development of inflammation in diabetic retina. Aggravation of the inflammatory process may be involved in the mechanism by which essential hypertension exacerbates retinopathy in the presence of diabetes.

The present study was undertaken to identify whether inflammation or oxidative stress is the primary abnormality in the kidney in spontaneously hypertensive rats (SHR). Renal inflammation and oxidative stress were evaluated in 2- and 3-week-old prehypertensive SHR and age-matched genetically normotensive control Wistar-Kyoto (WKY) rats. Blood pressure was similar in WKY and SHR rats at 2 and 3 weeks, of age. Renal inflammation (macrophage and nuclear factor-kappaB) was elevated in SHR at 3 weeks, but not at 2 weeks, of age compared with age-matched WKY rats. Renal oxidative stress (nitrotyrosine, 8-hydroxy-2'-deoxyguanosine and p47phox) was also clearly elevated in 3-week-old SHR compared with age-matched WKY rats. Additionally, NADPH oxidase subunit p47phox was found elevated in 2-week-old SHR compared to age-matched WKY rats. Moreover, antioxidant (N-acetyl-L-cysteine and Tempol) treatment reduced renal inflammation in prehypertensive SHR. We therefore conclude that the oxidative stress appears before inflammation as a primary abnormality in the kidney in prehypertensive SHR.

Background and Aims: Several lines of clinical evidence support the concept that the reduction of blood pressure may be useful in the prevention of diabetic kidney disease. In young diabetic spontaneously hypertensive rats (SHR), prevention of hypertension reduces several early renal abnormalities including albuminuria. However, the contribution of nephrin loss to albuminuria in this early stage of experimental diabetes is unknown. Therefore, we investigated whether elevation of albuminuria in young diabetic SHR is associated with nephrin loss, and if prevention of hypertension, with or without inhibition of the renin-angiotensin system, precludes these abnormalities., Methods: Diabetes was induced by streptozotocin injection in 4-week-old still normotensive SHR and their genetically normotensive control, Wistar-Kyoto rats. Diabetic SHR were randomized for no treatment, or treatment with captopril, losartan, or triple therapy (hydrochlorothiazide, reserpine and hydralazine) for 20 days., Results: The increase in systolic blood pressure was equally prevented by all treatments. Albuminuria was higher in diabetic SHR and similarly reduced (p < 0.05) by captopril, losartan, and triple therapy. Glomerular expression of nephrin was significantly reduced in diabetic SHR in comparison with non-diabetic controls. The antihypertensive treatment prevented the reduction in glomerular expression of nephrin., Conclusions: These results demonstrate that the loss of nephrin is associated with albuminuria in a model of genetic hypertension and diabetes, and that the prevention of development of hypertension restores nephrin and prevents albuminuria. This finding suggests a crucial role of blood pressure in diabetes as determinant of nephrin expression and albuminuria., ((c) 2007 S. Karger AG, Basel.)

Background/aims: Previous partial hepatectomy (HPTX) can attenuate glycerol-induced acute kidney injury (Gly-AKI). The aim of this study was to explore the pathophysiological mechanisms and the role of hepatocyte growth factor (HGF) in kidney protection., Methods: Rats were subjected to HPTX 24 h before glycerol administration. Renal function, acute tubular necrosis, apoptosis, leukocyte infiltration, and the expression of HGF, c-met, monocyte chemoattractant protein-1, interleukin-1beta, and heme oxygenase-1 were evaluated 24 h after glycerol injection. The regenerative response was analyzed from 6 to 72 h after glycerol injection (BrdU incorporation). In a separate series of experiments, Gly-AKI+HPTX rats were treated with anti-HGF antibody., Results: Gly-AKI+HPTX rats showed an increased expression of renal HGF and c-met as well as an improved creatinine clearance and reduced acute tubular necrosis and apoptosis, cytokine expression, and leukocyte infiltration. The regenerative response was less intense 24 and 72 h after glycerol administration in this group. The anti-HGF treatment disclosed an important role of HGF in the reduction of tubular injury, particularly apoptosis. Overexpression of heme oxygenase-1 was observed in Gly-AKI+HPTX rats, but was not associated with HPTX-induced renal protection., Conclusion: We conclude that Gly-AKI+HPTX rats have a reduced susceptibility to renal injury instead of an increased regenerative response and that endogenous HGF overexpression is responsible for suppression of tubular apoptosis., (Copyright 2007 S. Karger AG, Basel.)

The aim of this study was to evaluate the effect of prevention of hypertension on glomerular hypertrophy, renal cell replication and accumulation of glomerular fibronectin in a model of genetic hypertension and experimental diabetes. Four-week-old streptozotocin induced spontaneously hypertensive rats (SHR) were randomized for no treatment, or for treatment with captopril, losartan or triple therapy (hydrochlorothiazide, reserpine and hydralazine) for 20 days. Increase in systolic blood pressure was equally prevented by captopril (118+/-15 mmHg), losartan (111+/-9) and triple therapy (112+/-14, p<0.0001). Glomerular size was higher (p<0.005) in diabetic SHR (27,300+/-2130 microm(2)) compared with non-diabetic SHR (23,800+/-307). The antihypertensive therapy with captopril (23,900+/-175), losartan (23,800+/-120), and triple therapy (23,400+/-210) prevented the glomerular enlargement in diabetic SHR. Glomerular expression of fibronectin was increased in diabetic SHR (7.61+/-1.22 densitometric unit) as compared to the controls (2.27+/-2.15, p<0.0001), and was decreased (p<0.0001 vs diabetic SHR) with captopril (2.49+/-1.42), losartan (1.57+/-1.1) and triple therapy (2.04+/-1.42). The number of replicating glomerular cell significantly decreased in diabetic SHR and it was restored by all three antihypertensive regimes. The glomerular expression of p27(Kip1) was increased in diabetic SHR but it was not modified by antihypertensive treatment. Strict blood pressure control, in diabetic SHR independently of the class of antihypertensive agent, restores glomerular hypertrophy and renal cellular replication, and prevents the increment in glomerular fibronectin.

In this work we defined experimental conditions for site-directed gene replacement of the Xanthomonas axonopodis pv. citri (Xac), an economically relevant pathogen of citrus plants. The procedure involved, first, optimizing the electrotransformation conditions of the Xac 306 strain and, second, constructing non-replicative suicide vectors carrying knockout copies of the target gene. Using specific experimental conditions, transformation efficiencies of Xac were at least 100 fold higher than those achieved with electroporation protocols previously designed for X. campestris transformation. Successful gene replacement events were achieved with a suicide vector derived from R6K plasmid (pWR-SS) but not with those with ColE1 replication origin. We have chosen the oppA as a target gene, encoding the binding component (OppA) of the major oligopeptide uptake system found in the genome of the Xac 306 strain, although not in X. campestris pv. campestris (Xcc). Defining the experimental conditions, which allow for the specific mutagenesis of the Xac 306 strain, represents a step in the understanding of both genetics and physiology of this economically important bacterial species.

Caspases are the main executioners of apoptosis as well as interleukin (IL)-1beta and IL-18 conversion to active forms. They are activated after acute kidney injuries. In this study, we evaluated the importance of the caspase family in the pathogenesis and recovery of glycerol-induced acute renal failure in rats (Gly-ARF). Rats were treated with pan-caspase or selective caspase 1 and 3 inhibitors at the moment we injected glycerol. Renal function, renal histology (HE), transferase-mediated deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling staining for apoptosis, leukocytes infiltration (immunohistochemistry), renal expression of IL-1beta and IL-18 (immunohistochemistry and Western blot), tubular regeneration (5-bromo-2'-deoxyuridine (BrdU) incorporation), and P27(Kip) expression (Western blot) were evaluated at appropriate times. All inhibitors reduced the renal function impairment. Pan-caspase and caspase-3 inhibitors reduced cellular death (necrosis and apoptosis) 24 h after Gly-ARF. All caspases inhibitors reduced macrophages infiltration. The expression of total IL-1beta was enhanced in Gly-ARF, but the active IL-1beta and IL-18 forms were abolished in pan-caspase treated rats. Caspase-1 inhibitor attenuated Gly-ARF but not tubular injury suggesting glomerular hemodynamic improvement. There was striking regenerative response 48 h after Gly-ARF characterized by enhanced BrdU incorporation and reduced expression of p27(Kip). This response was not blunted by caspases inhibition. Our findings demonstrate that caspases participate in important pathogenic mechanisms in Gly-ARF such as inflammation, apoptosis, vasoconstriction, and tubular necrosis. The early inhibition of caspases attenuates these mechanisms and reduces the renal function impairment in Gly-ARF.

Background: The combination of diabetes and hypertension increases the incidence and severity of kidney disease in an additive manner. Inflammatory and oxidative stress mechanisms contribute to renal damage in both diabetes and hypertension. Therefore, we investigated whether renal macrophage infiltration and oxidative stress events are additive from the beginning in diabetic animals with coexisting hypertension., Methods: Diabetes was induced in spontaneously hypertensive rats (SHRs) and their genetically normotensive control Wistar Kyoto (WKY) rats by streptozotocin injection at 12 weeks of age for 10 days, and the effects of hyperglycemia on renal macrophage infiltration and oxidative stress were evaluated., Results: Blood pressure was higher in SHR than in WKY groups. Markers of oxidative stress-induced DNA and protein modification, 8-hydroxy- 2'-deoxyguanosine (8-OHdG) and nitrotyrosine, respectively, and the antioxidant glutathione levels were found to be similar in WKY-control and WKY-diabetic groups. However, 8-OHdG was significantly elevated (p = 0.014), the nitrotyrosine level tended to be elevated (p = 0.068) and the glutathione level was significantly reduced (p = 0.034) in the SHR-diabetic group compared to the SHR-control group. On the other hand, glomerular and tubulointerstitial macrophage infiltration was significantly higher in both WKY-diabetic and SHR-diabetic groups than the respective control groups., Conclusions: A short duration of diabetes mellitus induces renal oxidative stress in the presence of hypertension; however, renal macrophage infiltration becomes evident in early diabetes regardless of the presence or absence of hypertension. We conclude that the combination of diabetes and hypertension adversely affects oxidative stress in the kidney, but the combination has no additive effect on renal macrophage infiltration, at least in early diabetes., (Copyright (c) 2006 S. Karger AG, Basel.)

Elevated arterial pressure is a major risk factor for progression to ESRD in diabetic nephropathy. However, the component of arterial pressure and level of BP control for optimal renal outcomes are disputed. Data from 1590 hypertensive patients with type 2 diabetes in the Irbesartan Diabetic Nephropathy Trial (IDNT), a randomized, double-blind, placebo-controlled trial performed in 209 clinics worldwide, were examined, and the effects of baseline and mean follow-up systolic BP (SBP) and diastolic BP and the interaction of assigned study medications (irbesartan, amlodipine, and placebo) on progressive renal failure and all-cause mortality were assessed. Other antihypertensive agents were added to achieve predetermined BP goals. Entry criteria included elevated baseline serum creatinine concentration up to 266 micromol/L (3.0 mg/dl) and urine protein excretion >900 mg/d. Baseline BP averaged 159/87 +/- 20/11 mmHg. Median patient follow-up was 2.6 yr. Follow-up achieved SBP most strongly predicted renal outcomes. SBP >149 mmHg was associated with a 2.2-fold increase in the risk for doubling serum creatinine or ESRD compared with SBP <134 mmHg. Progressive lowering of SBP to 120 mmHg was associated with improved renal and patient survival, an effect independent of baseline renal function. Below this threshold, all-cause mortality increased. An additional renoprotective effect of irbesartan, independent of achieved SBP, was observed down to 120 mmHg. There was no correlation between diastolic BP and renal outcomes. We recommend a SBP target between 120 and 130 mmHg, in conjunction with blockade of the renin-angiotensin system, in patients with type 2 diabetic nephropathy.