Search

Background and Purpose: The purpose of the study was to investigate renal endothelium-dependent vasodilatation in a model of severe hypertension associated with kidney injury.Experimental Approach: Changes in perfusion pressure were measured in isolated, perfused kidneys taken from 18-week-old Wistar-Kyoto rat (WKY), spontaneously hypertensive rats (SHR) and SHR treated for 2 weeks with N(ω) -nitro-L-arginine methyl ester in the drinking water (L-NAME-treated SHR, 6 mg·kg(-1) ·day(-1) ).Key Results: Acetylcholine caused similar dose-dependent renal dilatation in the three groups. In vitro administration of indomethacin did not alter the vasodilatation, while the addition of N(w) -nitro-L-arginine (L-NA) produced a differential inhibition of the vasodilatation, (inhibition in WKY > SHR > L-NAME-treated SHR). Further addition of ODQ, an inhibitor of soluble guanylyl cyclase, abolished the responses to sodium nitroprusside but did not affect the vasodilatation to acetylcholine. However, the addition of TRAM-34 (or charybdotoxin) inhibitors of Ca(2+) -activated K(+) channels of intermediate conductance (K(Ca) 3.1), blocked the vasodilatation to acetylcholine, while apamin, an inhibitor of Ca(2+) -activated K(+) channels of small conductance (K(Ca) 2.3), was ineffective. Dilatation induced by an opener of K(Ca) 3.1/K(Ca) 2.3 channels, NS-309, was also blocked by TRAM-34, but not by apamin. The magnitude and duration of NS-309-induced vasodilatation and the renal expression of mRNA for K(Ca) 3.1, but not K(Ca) 2.3, channels followed the same ranking order (WKY < SHR < L-NAME-treated SHR).Conclusions and Implications: In SHR kidneys, an EDHF-mediated response, involving activation of K(Ca) 3.1 channels, contributed to the mechanism of endothelium-dependent vasodilatation. In kidneys from L-NAME-treated SHR, up-regulation of this pathway fully compensated for the decrease in NO availability. [ABSTRACT FROM AUTHOR]

BACKGROUND AND PURPOSE The purpose of the study was to investigate renal endothelium-dependent vasodilatation in a model of severe hypertension associated with kidney injury. EXPERIMENTAL APPROACH Changes in perfusion pressure were measured in isolated, perfused kidneys taken from 18-week-old Wistar-Kyoto rat (WKY), spontaneously hypertensive rats (SHR) and SHR treated for 2 weeks with Nω-nitro-L-arginine methyl ester in the drinking water (L-NAME-treated SHR, 6 mg·kg−1·day−1). KEY RESULTS Acetylcholine caused similar dose-dependent renal dilatation in the three groups. In vitro administration of indomethacin did not alter the vasodilatation, while the addition of Nw-nitro-L-arginine (L-NA) produced a differential inhibition of the vasodilatation, (inhibition in WKY > SHR > L-NAME-treated SHR). Further addition of ODQ, an inhibitor of soluble guanylyl cyclase, abolished the responses to sodium nitroprusside but did not affect the vasodilatation to acetylcholine. However, the addition of TRAM-34 (or charybdotoxin) inhibitors of Ca2+-activated K+ channels of intermediate conductance (KCa3.1), blocked the vasodilatation to acetylcholine, while apamin, an inhibitor of Ca2+-activated K+ channels of small conductance (KCa2.3), was ineffective. Dilatation induced by an opener of KCa3.1/KCa2.3 channels, NS-309, was also blocked by TRAM-34, but not by apamin. The magnitude and duration of NS-309-induced vasodilatation and the renal expression of mRNA for KCa3.1, but not KCa2.3, channels followed the same ranking order (WKY < SHR < L-NAME-treated SHR). CONCLUSIONS AND IMPLICATIONS In SHR kidneys, an EDHF-mediated response, involving activation of KCa3.1 channels, contributed to the mechanism of endothelium-dependent vasodilatation. In kidneys from L-NAME-treated SHR, up-regulation of this pathway fully compensated for the decrease in NO availability. [ABSTRACT FROM AUTHOR]

The (pro)renin receptor (PRR) has recently been demonstrated to bind equally well renin and its precursor, prorenin, leading to a similar intracellular signaling independent of angiotensin II. In this study, we report that human embryonic kidney cells (HEK) exposed to renin or prorenin for 24 h in the presence of a blocking concentration of the angtiotensin-converting enzyme inhibitor perindoprilate increased superoxide anion production as measured by luminescence (lucigenin) and electron spin resonance spectroscopy (hydroxylamine radical transition). Also, both renin and prorenin increased Nox4 expression while Nox2, p47phox, and p67phox remained unchanged. In an investigation of the effects of renin and prorenin on fibrosis genes, it appeared that both proteins stimulated transforming growth factor-β (TGF-β), fibronectin, and plasm inogen activator inhibitor type 1 (PAl-1) expression and therefore participated to an overall switch toward a profibrotic state of the kidney cells. When the cells were transfected with a siRNA targeting the PRR, Nox4 expression was efficiently prevented as well as the increase in superoxide production, TGF-13, fibronectin, and PAl- 1. Finally, we demonstrated that transfection of the cells with a Nox4- specific small interfering (si) RNA also prevented fibrosis gene expression following treatment with renin or prorenin. The results demonstrate that renin and prorenin, through their specific membrane receptor and independently of angiotensin II, promote fibrosis gene expression via a Nox4-dependent mechanism. [ABSTRACT FROM AUTHOR]

Arachidonic acid metabolites, some of which may activate thromboxane A[sub 2] receptors (TPr) and contribute to the development of diabetes complications, including nephropathy, are elevated in diabetes. This study determined the effect of blocking TPr with $18886 or inhibiting cyclooxygenase with aspirin on oxidative stress and the early stages of nephropathy in streptozotocin-induced diabetic apolipoprotein E-/- mice. Diabetic mice were treated with S18886 (5 mg ⋅ kg-1 ⋅ day-1) or aspirin (30 mg ⋅ kg-1 ⋅ day-1) for 6 weeks. Neither $18886 nor aspirin affected hyperglycemia or hypercholesterolemia. There was intense immunohistochemical staining for nitrotyrosine in diabetic mouse kidney. In addition, a decrease in manganese super-oxide dismutase (MnSOD) activity was associated with an increase in MnSOD tyrosine-34 nitration. Tyrosine nitration was significantly reduced by $18886 but not by aspirin. Staining for the NADPH oxidase subunit p47phox, inducible nitric oxide synthase, and 12-lipoxygenase was increased in diabetic mouse kidney, as were urine levels of 12-hydroxyeicosatetraenoic acid and 8-iso-prostaglandin F2α. $18886 attenuated all of these markers of oxidant stress and inflammation. Furthermore, $18886 significantly attenuated microalbuminuria in diabetic mice and ameliorated histological evidence of diabetic nephropathy, including transforming growth factor-β and extracellular matrix expression. Thus, in contrast to inhibiting cyclooxygenase, blockade of TPr may have therapeutic potential in diabetic nephropathy, in part by attenuating oxidative stress. [ABSTRACT FROM AUTHOR]

Objective: Anoxia followed by reoxygenation (A/R) increases endothelial cell superoxide (O2-) generation which is implicated in E-selectin overexpression. The mechanisms which govern these processes are not fully understood and therefore the goal of our study was to determine the functional importance of NADPH oxidase in the regulation of E-selectin expression in human umbilical veins endothelial cells (HUVECs) submitted to A/R. Methods: O2- production was estimated using lucigenin chemiluminescence and formazan accumulation. NADPH oxidase expression in HUVECs was studied by RT-PCR and Western blot and E-selectin by Northern blot analysis. NFκB activation was assessed by electrophoretic mobility shift assay. Results: A/R caused an increased O2- production which was inhibited by the superoxide dismutase mimetic M40403 (50 μmol/l), the protein kinase C inhibitor chelerythrine (10 μmol/l), the NADPH oxidase inhibitor diphenyleneiodonium (DPI, 10 μmol/l) and the NADPH oxidase assembly blocker apocynin (600 μmol/l). At the end of the anoxic period, the mRNA expression and the protein p47phox was increased as compared to normoxic HUVECs. NFκB activation of anoxic HUVECs was maximal after 1 h of reoxygenation and returned to basal normoxic levels after 2 h of reoxygenation. Apocynin reduced the NFκB activation at 1 h of reoxygenation. E-selectin mRNA expression was increased after 3 h of reoxygenation of anoxic HUVECs and the SOD mimetic M40403 as well as apocynin prevented this overexpression. Conclusions: Activated NADPH oxidase is a critical enzyme in E-selectin overexpression after A/R of HUVECs. Moreover, A/R increased expression of membranous and cytosolic NADPH oxidase subunits as well as the protein p47phox. Strategies aimed at preventing endothelial NADPH oxidase activation and/or activity may be useful in controlling leukocyte adhesion during ischemia/reperfusion. [Copyright &y& Elsevier]

Studies the direct effects of lidoflazine on arterial and venous smooth muscle. Materials and methods; Concentration-dependent depression of the amplitude of the spontaneous contractions; Effect of increasing concentrations of lidoflazine; Influence of the increasing concentrations of lidoflazine on the response to electrical stimulation.

S17834 is a synthetic polyphenol (hydroxy triallyl farnisine) that attenuates atherogenesis in type 1 diabetic LDLr[sup -/-] mice. The objective of this study was to examine its role in influencing atherogenesis and the expression of NF-κB-inducible genes, vascular cell adhesion molecule (VCAM)-1 and inducible nitric oxide synthase (iNOS) in insulin resistant LDLr[sup -/-] mice fed a high fat high sucrose diet (HFHS, 30% fat and 30% sucrose) for 6 wks. HFHS LDLr[sup -/-] mice developed significantly more atherosclerotic lesions in the aortic root than did LDLr[sup -/-] mice on normal chow [25±1.9 vs. 6±0.4 x 1000 µm² (n=8), respectively, P<0.001]. S17834 (130 mg/kg/day, in HFHS diet) significantly attenuated lesions in HFHS LDLr[sup -/-] mice [10±l.l xl000 µm² (n=8), P<0.001]. Immunohistochemical staining showed that HFHS diet increased VCAM-1, but decreased iNOS expression in aortic lesions compared to LDLr[sup -/-]mice on normal chow, and that this was reversed by S17834. Mechanistic studies in cultured rat vascular smooth muscle cells show that S17834 reduced IL-1β-induced VCAM-1 expression, but enhanced IL-1β-induced iNOS expression and NO production. Although S17834 alone did not induce iNOS expression, it enhanced IL-1β-induced ERK-activation which is responsible for prolonged NF-κB activity and iNOS expression. Furthermore, EGF-induced DNA synthesis ([³H]-thymidine incorporation) was reduced 62% by IL-1β alone (P<0.01) and 70% by IL-1β plus S17834 (P<0.01), which was partially reversed by L-NMMA. S17834 alone inhibited 52% of EGF-induced [³H]-thymidine incorporation (P<0.01), suggesting that both NO-dependent and independent mechanisms were involved in the anti-proliferative action. In conclusion, the anti-atherogenic effect of S17834 may relate to regulating the inflammatory process by reducing VCAM-1 expression, enhancing iNOS expression, and inhibiting smooth muscle cell proliferation. [ABSTRACT FROM AUTHOR]

Sterol regulatory element binding protein (SREBP) is a key lipogenic transcription factor that has been implicated in the pathogenesis of dyslipidemia. We previously demonstrated that high glucose and streptozotocin (STZ)-induced type 1 diabetes decrease AMPK activity and cause lipid accumulation both in HepG2 hepatocytes in vitro, and in mouse liver in vivo, associated with hyperlipidemia. These changes were prevented by metformin or a synthetic polyphenol, S17834. We have further investigated the role of S 17834 in regulating SREBP and lipid metabolism. In STZ-induced diabetic mice, the nuclear active form (∼68 kDa) of liver SREBP-1 was increased 5-fold, and the increase was significantly prevented by S17834 (130 mg/kg/day), suggesting that the lipid-lowering effect of S17834 is associated with suppression of SREBP-1. Exposure of HepG2 cells to high glucose in culture resulted in increased expression of the precursor and nuclear active form of SREBP-1, as well as enhanced nuclear translocation of SREBP- 1 as detected by immunofluorescent staining. An increase in the SREBP-1 target enzyme, fatty acid synthase (FAS), was associated with an increase in hepatocellular triglyceride accumulation. In contrast, S17834 (l0 micro;M) or metformin (2 mM) significantly increased AMPK activity and inhibited activation of SREBP and induction of FAS, and thereby lowered hepatocellular triglyceride levels. Moreover, the ability of S17834 to downregulate SREBP-1 and FAS is mimicked by overexpression of the constitutively active AMPK, and largely prevented by a dominant-negative AMPK. These results indicate that polyphenols attenuate hepatic lipid accumulation associated with high glucose via inhibition of de novo fatty acid synthesis via activation of AMPK and suppression of SREBP-1, and point to new approaches using small molecules for treating dyslipidemia associated with diabetes. [ABSTRACT FROM AUTHOR]

Our recent studies show that the synthetic polyphenol, S 17834, stimulates AMP-activated protein kinase (AMPK) more potently than resveratrol or metformin. Stimulation of AMPK by S17834 was shown to attenuate high glucose-induced lipid accumulation in HepG2 cells, as well as to be associated with a reduction in hepatic and serum lipids, and atherosclerosis in type 1 diabetic LDL receptor deficient (LDLR-/-) mice. To determine the effect of S17834 in obesity-induced insulin resistance and type 2 diabetes, LDLR-/- mice were fed a high fat, high sucrose (HFHS) diet for 16 weeks. Compared with normal chow, body weight gain increased ∼3-fold, and insulin resistance index, as determined by the homeostasis model assessment for insulin resistance (HOMAIR), was increased ∼2.5-fold. Hepatic steatosis caused by the HFHS diet was accompanied in the liver by decreased phosphorylation of AMPK and the AMPK downstream effector, acetyl CoA carboxylase (ACC) compared with chow fed mice. The HFHS diet caused a ∼2-fold increase in plasma cholesterol levels as well as a 4-fold increase in oil red O stained atherosclerotic lesions in aortic mot cross sections. S17834 administration (130 mg/kg/day, 16 weeks) significantly protected mice against diet-induced obesity and systemic insulin resistance. Treatment with S 17834 was accompanied by increased AMPK and ACC phosphorylation in the liver and improvement in hepatic steatosis. S17834 also significantly decreased plasma cholesterol levels and attenuated the development of aortic atherosclerotic lesions. These data show that protection afforded by S17834 from the exaggerated hyperlipidemia and vascular disease caused by type 1 diabetes extends to type 2 diabetes in LDLR-/- mice. Improvements in body weight, insulin resistance, hepatic steatosis, hyperlipidemia, and atherosclerosis may be attributed to the ability of the polyphenol to stimulate AMPK as demonstrated here to occur in the mouse liver. Activation of AMPK by polyphenols, like S17834, represents a major new avenue for treatment of hyperlipidemia and vascular disease in both type 1 and type 2 diabetes. [ABSTRACT FROM AUTHOR]

Sirt1 is one of seven mammalian homologs of Sir2, an NAD-dependent deacetylase that functions as a novel metabolic sensor connecting aging and metabolism. We recently discovered that polyphenols stimulate AMPK activity and lower hepatic lipids, contributing to their beneficial effects on accelerated hyperlipidemia and atherosclerosis in diabetes. Here we examined if Sirt1 mediates the effect of polyphenols on AMPK and its lipid metabolic effects. The in vitro Fluor de Lys deacetylation assay showed that Sirt1 catalytic activity was significantly increased by polyphenols including resveratrol and a synthetic polyphenol, S17834, and decreased by Sirt1 inhibitors, nicotinamide and suramin, respectively. The two polyphenolic Sirt1 activators substantially stimulated phosphorylation of AMPK and its downstream effector, acetyl CoA carboxylase, in cultured human HepG2 hepatocytes and HEK293 cells, but not in HeLa cells deficient in LKB1. These responses were largely blocked by inhibition of Sirt1 deacetylation with nicotinamide or splitomicin. Importantly, downregulation of Sirt1 using lentivirus-mediated Sirt1 shRNA completely abolished the basal and polyphenol-induced AMPK activation. Furthermore, studies with a dominant negative Sirt1 (Sirt1H355A) and Sirt1 inhibitors revealed that the effects of polyphenols on hepatocellular AMPK signaling and lipids depend on Sirt1 activity in a HepG2 cell model of high glucose-induced lipid accumulation. Overexpression of wild type Sirt1 also stimulated AMPK, which was inhibited by high glucose, as well as reduced triglyceride levels that were enhanced by high glucose. The effects of wild type Sirtl were completely blocked by a dominant negative AMPK (AMP-Ka1phaK45R). These results implicate Sirt1 stimulation of AMPK as a key regulator of lipid metabolism in bepatocytes. Because of the associated improvements seen in hyperlipidemia and atherosclerosis caused by polyphenols in type 1 and type 2 mouse models, these data suggest that Sirt1 and AMPK may be attractive therapeutic targets for diabetes. [ABSTRACT FROM AUTHOR]