Theobromine increases NAD⁺/Sirt-1 activity and protects the kidney under diabetic conditions.

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)-ß1 signaling. It is suggested that Sirt-1 activation by theobromine may have therapeutic potential for diabetic nephropathy.

Polyphenol-enriched cocoa protects the diabetic retina from glial reaction through the sirtuin pathway.

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.

Inactivation of AMPK mediates high phosphate-induced extracellular matrix accumulation via NOX4/TGFß-1 signaling in human mesangial cells.

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.

Spatial distribution of theobromine--a low MW drug--in tissues via matrix-free NALDI-MS imaging.

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.

Increase in AMPK brought about by cocoa is renoprotective in experimental diabetes mellitus by reducing NOX4/TGFß-1 signaling.

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 TGFß-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 TGFß-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 TGFß-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 TGFß-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/TGFß-1 signaling may have a therapeutic potential in diabetic nephropathy.

S-nitrosoglutathione inhibits inducible nitric oxide synthase upregulation by redox posttranslational modification in experimental diabetic retinopathy.

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.

Green tea is neuroprotective in diabetic retinopathy.

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.

The concomitance of hypertension and diabetes exacerbating retinopathy: the role of inflammation and oxidative stress.

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.

Uncoupling endothelial nitric oxide synthase is ameliorated by green tea in experimental diabetes by re-establishing tetrahydrobiopterin levels.

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.

Reduction of inducible nitric oxide synthase via angiotensin receptor blocker prevents the oxidative retinal damage in diabetic hypertensive 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.

Exogenous SOD mimetic tempol ameliorates the early retinal changes reestablishing the redox status in diabetic hypertensive rats.

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.

Diabetic retinal neurodegeneration is associated with mitochondrial oxidative stress and is improved by an angiotensin receptor blocker in a model combining hypertension and diabetes.

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.

Arterial hypertension exacerbates oxidative stress in early diabetic retinopathy.

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.

Hypertension increases retinal inflammation in experimental diabetes: a possible mechanism for aggravation of diabetic retinopathy by hypertension.

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.

Prevention of hypertension abrogates early inflammatory events in the retina of diabetic hypertensive rats.

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.