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Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease. Besides glycemic and blood pressure control, environmental factors such as cigarette smoking (CS) adversely affect the progression of DN. The effects of CS on DN progression have been attributed to combustion-generated molecules without consideration to the role of nicotine (NIC), responsible for the addictive properties of both CS and electronic cigarettes (ECs). Podocytes are essential to preserve the structure and function of the glomerular filtration barrier, and strong evidence indicates that early podocyte loss promotes DN progression. We performed experiments in human podocytes and in a mouse model of diabetes that develops nephropathy resembling human DN. We determined that NIC binding to podocytes in concentrations achieved with CS and ECs activated NADPH oxidase, which sets in motion a dysfunctional molecular network integrated by cyclooxygenase 2, known to induce podocyte injury; downregulation of AMP-activated protein kinase, important for maintaining cellular energy stores and antioxidation; and upregulation of CD36, which increased lipid uptake and promoted apoptosis. In diabetic mice, NIC increased proteinuria, a recognized marker of chronic kidney disease progression, accompanied by reduced glomerular podocyte synaptopodin, a crucial stabilizer of the podocyte cytoskeleton, and increased fibronectin expression. This novel study critically implicates NIC itself as a contributor to DN progression in CS and EC users. NEW & NOTEWORTHY In this study, we demonstrate that nicotine increases the production of reactive oxygen species, increases cyclooxygenase-2 expression, and upregulates Cd36 while inducing downregulation of AMP-activated protein kinase. In vivo nicotine increases proteinuria and fibronectin expression in diabetic mice. This study demonstrates that effects of nicotine on podocytes are responsible, at least in part, for the deleterious effects of smoking in the progression of chronic kidney disease, including diabetic nephropathy.

It has been shown that the inflammatory cytokine tumor necrosis factor α (TNFα) plays a role in the development of hypertension and end-stage renal diseases. We hypothesize that TNFα contributes to endothelial dysfunction and cardiac and vascular injury in deoxycorticosterone acetate (DOCA)/salt-hypertensive mice. The wild-type or TNFα-deficient mice were uninephrectomized and implanted with DOCA pellet treatment for 5 weeks; the mice were given either tap water or 1% NaCl drinking water. DOCA mice developed hypertension (systolic blood pressure (SBP): 167±5 vs. 110±4 mmHg in control group, p<0.05), cardiac and vascular hypertrophy, and the impairment of endothelium-dependent relaxation to acetylcholine (EDR). TNFα deficiency improved EDR and lowered cardiac and vascular hypertrophy with a mild reduction in SBP (152±4 vs. 167±5 mmHg in DOCA group, p<0.05) in DOCA mice. The mRNA expressions of the inflammatory cytokines, including TNFα, interleukin 1β (IL1β), monocyte chemotactic protein 1 (MCP1), and monocyte/macrophage marker F4/80 were significantly increased in the aorta of DOCA-hypertensive mice; TNFα deficiency reduced these inflammatory gene expressions. DOCA-hypertensive mice also exhibited an increase in the vascular oxidative fluorescence intensities, the protein expressions of gp91phox and p22phox, and the fibrotic factors transforming growth factor β and fibronectin. TNFα deficiency reduced oxidative stress and fibrotic protein expressions. The DOCA mice also showed a decrease in the protein expression of eNOS associated with increased miR155 expression; TNFα deficiency prevented a decrease in eNOS expression and an increase in miR155 expression in DOCA mice. These results support the idea that TNFα significantly contributes to vascular inflammation, vascular dysfunction, and injury in hypertension.

Monocyte/macrophage recruitment is closely associated with the degree of hypertensive renal injury. Here we investigated the direct role of macrophage using Liposome encapsulated clodronate (LEC) to deplete monocyte/macrophage in Ang II-induced hypertensive renal injury. C57BL/6 mice were treated with a pressor dose of angiotensin II (Ang, 1.4 mg/kg/day by mini-pump) plus LEC or the PBS-liposome for two weeks. Ang II mice developed hypertension (186 ± 5 vs. 110 ± 4 mmHg in control, p+ cells) and the protein expression of renal tumor necrosis factor α and interleukin β1, which were significantly reduced in Ang II mice treated with LEC. Ang II increased renal oxidative stress (as demonstrated by increased oxidative fluorescence densities, NADPH oxidative activity and the protein expression of NADPH oxidase subunits gp91phox and p22phox) and the expression of profibrotic factors transform growth factor β1 and fibronectin. Ang II also inhibited the phosphorylation of endothelial nitric oxide synthesis (eNOS, ser1177). LEC revised most of the changes in the Ang II-induced molecules as mentioned above. Our results suggest that renal macrophage is a main mediator to contribute to Ang II-induced hypertensive renal injury and fibrosis, the underlying mechanisms may involve reduction in macrophage-drove renal inflammation and restoration of the balance between renal oxidative stress and eNOS. These findings may lead us to develop a novel therapy directed at targeting the infiltration of macrophages or the macrophages-derived cytokines for treatment of hypertensive renal diseases.

Monocyte/macrophage recruitment is closely associated with the degree of hypertensive renal injury. We investigated the direct role of macrophages using liposome-encapsulated clodronate (LEC) to deplete monocytes/macrophages in hypertensive renal injury. C57BL/6 mice were treated with a pressor dose of angiotensin (Ang, 1.4 mg/kg/day) II plus LEC or the PBS-liposome for 2 weeks. Ang II mice developed hypertension, albuminuria, glomerulosclerosis, and renal fibrosis. LEC treatment reduced systolic blood pressure (SBP), albuminuria, and protected against renal structural injury in Ang II mice. Ang II significantly increased renal macrophage infiltration (MOMA2+ cells) and the expression of renal tumor necrosis factor α and interleukin β1, which were significantly reduced in Ang II/LEC mice. Ang II increased renal oxidative stress and the expression of profibrotic factors transforming growth factor (TGF) β1 and fibronectin. Ang II also inhibited the phosphorylation of endothelial nitric oxide synthase [phospho-endothelial nitric oxide synthesis (eNOS), ser1177]. LEC treatment reduced renal oxidative stress and TGFβ1 and fibronectin expressions, and increased phospho-eNOS expression in the Ang II mice. In Dahl rats of salt-sensitive hypertension, LEC treatment for 4 weeks significantly attenuated the elevation of SBP induced by high salt intake and protected against renal injury and fibrosis. Our results demonstrate that renal macrophages play a critical role in the development of hypertension and hypertensive renal injury and fibrosis; the underlying mechanisms may be involved in the reduction in macrophage-driven renal inflammation and restoration of the balance between renal oxidative stress and eNOS. Therefore, macrophages should be considered as a potential therapeutic target to reduce the adverse consequences of hypertensive renal diseases.

Obesity and cigarette smoke are major cardiovascular (CV) risk factors and, when coexisting in the same individuals, have additive/synergistic effects upon CVD. We studied the mechanisms involved in nicotine enhancement of CVD in Sprague Dawley rats with diet-induced obesity. The rats were fed either a high fat (HFD) or normal rat chow diet with or without nicotine (100 mg/L in drinking water) for 20 weeks. HFD rats developed central obesity, increased systolic blood pressure (SBP), aortic superoxide (O2-) production, and impaired endothelial nitric oxide synthase (eNOS) and endothelium-dependent relaxation to acetylcholine (EDR). Nicotine further increased SBP, O2- and impaired eNOS and EDR in obese rats. In the peritoneal macrophages from obese rats, tumor necrosis factor (TNF) α, interleukin 1β and CD36 were increased, and were further increased in nicotine-treated obese rats. Using PCR array we found that 3 of 84 target proinflammatory genes were increased by 2-4 fold in the aorta of obese rats, 11 of the target genes were further increased in nicotine-treated obese rats. HUVECs, incubated with conditioned medium from the peritoneal macrophages of nicotine treated-obese rats, exhibited reduced eNOS and increased NADPH oxidase subunits gp91phox and p22phox expression. Those effects were partially prevented by adding anti-TNFα antibody to the conditioned medium. Our results suggest that nicotine aggravates the CV effects of diet-induced obesity including the oxidative stress, vascular inflammation and endothelial dysfunction. The underlying mechanisms may involve in targeting endothelium by enhancement of macrophage-derived TNFα.

Cigarette smoking is a major risk factor for atherosclerosis and cardiovascular disease. CD36 mediates oxidized LDL (oxLDL) uptake and contributes to macrophage foam cell formation. We investigated a role for the CD36 pathway in nicotine-induced activation of macrophages and foam cell formation in vitro and in vivo. Nicotine in the same plasma concentration range found in smokers increased the CD36+/CD14+cell population in human peripheral blood mononuclear cells, increased CD36 expression of human THP1 macrophages, and increased macrophage production of reactive oxygen species, PKCδ phosphorylation, and peroxisome proliferator-activated receptor-γ (PPARγ) expression. Nicotine-induced CD36 expression was suppressed by antioxidants and by specific PKCδ and PPARγ inhibitors, implicating mechanistic roles for these intermediates. Nicotine synergized with oxLDL to increase macrophage expression of CD36 and cytokines TNF-α, monocyte chemoattractant protein-1, IL-6, and CXCL9, all of which were prevented by CD36 small interfering (si)RNA. Incubation with oxLDL (50 μg/ml) for 72 h resulted in lipid deposition in macrophages and foam cell formation. Preincubation with nicotine further increased oxLDL-induced lipid accumulation and foam cell formation, which was also prevented by CD36 siRNA. Treatment of apoE−/−mice with nicotine markedly exacerbated inflammatory monocyte levels and atherosclerotic plaque accumulation, effects that were not seen in CD36−/−apoE−/−mice. Our results show that physiological levels of nicotine increase CD36 expression in macrophages, a pathway that may account at least in part for the known proinflammatory and proatherogenic properties of nicotine. These results identify such enhanced CD36 expression as a novel nicotine-mediated pathway that may constitute an independent risk factor for atherosclerosis in smokers. The results also suggest that exacerbated atherogenesis by this pathway may be an adverse side effect of extended use of high concentrations of nicotine independent of their mode of administration.

The physiologic actions of insulin in the vasculature serve to couple regulation of metabolic and hemodynamic homeostasis. Insulin activation of the phosphatidylinositol-3-kinase (PI3K) pathway promotes glucose uptake in insulin-responsive tissues and nitric oxide (NO) production in the endothelium. NO induces vasodilation and inhibits platelet aggregation and vascular smooth muscle cell growth. In contrast, insulin activation of the mitogen-activated protein kinase (MAPK) leads to vasoconstriction and pathologic vascular cellular growth. In states of insulin resistance, insulin activation of PI3K is selectively impaired, whereas the MAPK pathway is spared and activated normally. In the endothelium, selective impairment of insulin-mediated NO production may contribute to the development of hypertension, endothelial dysfunction, atherogenesis, and insulin resistance. This article reviews experimental and clinical data elucidating the physiologic and pathophysiologic role of insulin in the vasculature and the mechanisms contributing to the development of vascular and metabolic diseases.

Clinical and experimental studies have provided evidence suggesting that statins exert renoprotective effects. To investigate the mechanisms by which statins may exert renoprotection, we utilized the hypertensive Dahl salt-sensitive (DS) rat model, which manifests cardiovascular and renal injury linked to increased angiotensin II-dependent activation of NADPH oxidase and decreased nitric oxide (NO) bioavailability. DS rats given high salt diet (4% NaCl) for 10 wk exhibited hypertension [systolic blood pressure (SBP) 200 ± 8 vs. 150 ± 2 mmHg in normal salt diet (0.5% NaCl), P < 0.05], glomerulosclerosis, and proteinuria (158%). This was associated with increased renal oxidative stress demonstrated by urinary 8-F2α-isoprostane excretion and NADPH oxidase activity, increased protein expression of transforming growth factor (TGF)-β (63%) and fibronectin (181%), increased mRNA expression of the proinflammatory molecules monocyte chemoattractant protein-1 (MCP-1) and lectin-like oxidized LDL receptor-1 (LOX-1), as well as downregulation of endothelial NO synthase (eNOS) activity (−44%) and protein expression. Return to normal salt had no effect on SBP or any of the measured parameters. Atorvastatin (30 mg·kg−1·day−1) significantly attenuated proteinuria and glomerulosclerosis and normalized renal oxidative stress, TGF-β1, fibronectin, MCP-1 and LOX-1 expression, and eNOS activity and expression. Atorvastatin-treated rats showed a modest reduction in SBP that remained in the hypertensive range (174 ± 8 mmHg). Atorvastatin combined with removal of high salt normalized SBP and proteinuria. These findings suggest that statins mitigate hypertensive renal injury by restoring the balance among NO, TGF-β1, and oxidative stress and explain the added renoprotective effects observed in clinical studies using statins in addition to inhibitors of the renin-angiotensin system.

Background We have previously shown that in hypertensive Dahl salt-sensitive (DS) rats, impaired endothelium-dependent relaxation to acetylcholine and to insulin is mechanistically linked to up-regulation of angiotensin (Ang) II actions and the production of reactive oxygen species (ROS) and to activation of the proinflammatory transcription factor (NF)κB. Here we investigated whether Ang II activation of NFκB contributed to insulin resistance in the skeletal muscle of this animal model. Methods DS rats were fed either a normal (NS, 0.5% NaCl) or high (HS, 4% NaCl) salt diet for 6 weeks. In addition, 3 separate groups of HS rats were given angiotensin receptor 1 blocker candesartan (ARB, 10 mg/kg/day in drinking water), antioxidant tempol (1 mmol/L in drinking water) or NFκB inhibitor PDTC (150 mg/kg in drinking water). Results DS rats manifested an increase in soleus muscle Ang II content, ROS production and phosopho-IκBα/IκBα ratio, ARB or tempol reduced ROS and phospho-IκBα/IκBα ratio. Hypertensive DS rats also manifested a reduction in glucose infusion rate, impaired insulin-induced Akt phosphorylation and Glut-4 translocation in the soleus muscle, which were prevented with treatment of either ARB, tempol, or PDTC. Data from the rat diabetes signaling pathway PCR array showed that 8 genes among 84 target genes were altered in the muscle of hypertensive rats with the increase in gene expression of ACE1 and 5 proinflammatory genes, and decrease of 2 glucose metabolic genes. Incubation of the muscle with NFκB SN50 (a specific peptide inhibitor of NFκB) ex vivo reversed changes in hypertension-induced gene expression. Conclusion The current findings strongly suggest that the activation of NFκB inflammatory pathway by Ang II play a critical role in skeletal muscle insulin resistance in salt-sensitive hypertension.

Background Although there is overwhelming evidence that hypertension promotes atherosclerosis, the relative contribution and/or interaction of vasoactive and hemodynamic factors remain undefined. Endothelial dysfunction complicates hypertension and is a precursor of atherosclerosis. It is characterized by a reduction in the bioavailability of vasodilators, particularly nitric oxide, and an increase in the activity of vasoconstrictors, including angiotensin (Ang) II and reactive oxygen species (ROS). Nitric oxide antagonizes the vasoconstrictive and pro-atherosclerotic effects of Ang II, whereas Ang II decreases nitric oxide bioavailability by promoting oxidative stress. Objectives The present review will focus on the interaction among nitric oxide, Ang II, and ROS in the endothelium and will examine their role in vascular tone and atherogenesis. In this context, studies from our laboratory will be reviewed demonstrating that salt-sensitive hypertension is a vascular diathesis characterized by a local activation of Ang II and NAD(P)H oxidase-derived ROS in the setting of insufficient nitric oxide. In hypertensive Dahl salt-sensitive rats, a paradigm of human salt-sensitive hypertension, inhibition of Ang II type 1 receptor or NAD(P)H oxidase-derived ROS prevented the development of endothelial dysfunction, upregulation of pro-atherogenic molecules, and vascular ROS generation, independently of blood pressure. Conclusions Salt sensitivity, an independent risk factor for increased cardiovascular morbidity and mortality, affects approximately 50% of hypertensives. Our studies suggest that, in salt-sensitive hypertension, atherogenesis is more closely linked to oxidative stress than to the hemodynamic stress of hypertension. To prevent or arrest atherosclerosis, antihypertensive therapy should aim at restoring the homeostatic balance between vasoactive factors in the vascular wall.

Objective Angiotensin (Ang) II increases reactive oxygen species (ROS), decreases nitric oxide (NO) bioavailability and promotes cardiovascular remodeling. ROS have been identified as critical second messengers of the trophic responses by Ang II. In rats with Ang II-induced hypertension, we investigated the role of ROS in cardiac hypertrophy as well as the remodeling of aortas and mesenteric (resistance) arteries. Methods Sprague-Dawley rats received Ang II (0.7 mg/kg per day by mini-pump, n = 7) or vehicle (n = 7) for 5 days. Endothelium-dependent relaxation to acetylcholine (EDR) in aortas was determined in organ baths and in mesenteric resistance vessels in a pressurized myograph. Superoxide (O 2 - ) production was measured by lucigenin chemiluminescence, laser-confocal fluorescence microscopy (LCM) and NADPH oxidase assay. Results Ang II-treated rats developed hypertension (183 ± 3 versus 138 ± 4 mmHg, P

To investigate the effects of environment on tissue norepinephrine (tNE) levels of fish, we examined tNE concentrations in the adult chum salmon (Oncorhynchus keta) from fresh water (FW) and from sea water (SW). Plasma norepinephrine (pNE) and tNE of 15 organs, including the gill, heart, liver, aorta, spleen, stomach, jejunum, colon, brain stem, cerebrum, muscle, bone, sexual organ and kidney (head and body), were determined in river and sea salmon by high performance pressure liquid chromatography with electrochemical detection. tNE concentrations of all organs except the gill of sea salmon were significantly lower in comparison with those in river salmon. The pNE concentration was higher in sea salmon than in river salmon of male but not female. In river salmon, tNE concentrations of 12 organs of females were higher than those of male, whereas in sea salmon, there was no significant difference in tNE concentrations of all organs except the liver between male and female. Plasma osmolality and sodium concentration were higher in sea salmon than in river salmon. These results indicate that tNE concentrations in most organs of salmon in a SW environment are significantly decreased compared with those in a FW environment, and that tNE concentrations are higher in female salmon than in male salmon in a FW environment but not in a SW environment. J. Exp. Zool. 284:107–111, 1999. © 1999 Wiley-Liss, Inc.

To determine organ and development related differences in tissue norepinephrine concentration (tNE) in Dahl salt-sensitive (S) and -resistant (R) rats, we measured the tNE of 16 organs, including the heart (left ventricle), kidney, cerebrum, brain stem, stomach, jejunum, ileum, colon, spleen, pancreas, liver, aorta, lung, bone, salivary gland, and muscle, at 1, 3, 5, 7, 9, 11 weeks old. Large differences were found in tNE among the organs of both S and R rats, ranging from 4.0±1.1 ng/g tissue (the bone of S rats) to 1234.8±32.5 ng/g tissue (the salivary gland of R rats). tNE in R rats increased development-dependently in 12 of 16 organs, but did not significantly change in the other three organs, and decreased in the bone. On the other hand, the development-dependent increase in tNE was suppressed in S rats, and the tNE values of S rats were significantly lower than those of R rats in 14 of 16 organs. To eliminate the baroreflexive effects on tNE, another group of 5-week-old S and R rats were subjected to sinoaortic denervation (SAD) or the sham operation. The tNE was measured in 10 organs in these animals at 9 weeks old. SAD did not alter the tNE in most of the organs in both S and R rats. There was no significant differences in mean arterial pressure (MAP) between S and R rats with baroreceptor intact at 9 weeks old. SAD slightly but significantly increased MAP in S rats, whereas not in R rats. There was no significant differences in plasma NE concentration (pNE) between S and R rats with the baroreceptor intact. SAD did not alter pNE in S or R rats. These results demonstrate that variations of the tNE were dependent on the organ and development. Many organs of S rats had lower tNE than those of R rats. The developmental-dependent increases in tNE in S rats were suppressed, compared with those in R rats. These tNE behaviors in S rats may not be related to blood pressure or baroreflex sensitivity, but might be involved in an abnormal sympathetic nerve activity.

The incidence of metabolic syndrome is rapidly increasing in the United States and worldwide. The metabolic syndrome is a complex metabolic and vascular disorder that is associated with inappropriate activation of the renin–angiotensin–aldosterone system (RAAS) in the cardiovascular (CV) system and increased CV morbidity and mortality. Insulin activation of the phosphatidylinositol-3-kinase (PI3K) pathway promotes nitric oxide (NO) production in the endothelium and glucose uptake in insulin-sensitive tissues. Angiotensin (Ang) II inhibits insulin-mediated PI3K pathway activation, thereby impairing endothelial NO production and Glut-4 translocation in insulin-sensitive tissues, which results in vascular and systemic insulin resistance, respectively. On the other hand, Ang II enhances insulin-mediated activation of the mitogen-activated protein kinase (MAPK) pathway, which leads to vasoconstriction and pathologic vascular cellular growth. Therefore, the interaction of Ang II with insulin signaling is fully operative not only in insulin-sensitive tissues but also in CV tissues, thereby linking insulin resistance and CV disease. This notion is further supported by an increasing number of experimental and clinical studies indicating that pharmacological blockade of RAAS improves insulin sensitivity and endothelial function, as well as reduces the incidence of new-onset diabetes in high-risk patients with CV disease. This article reviews experimental and clinical data elucidating the physiological and pathophysiological role of the interaction between insulin and RAAS in the development of insulin resistance as well as CV disease.

Reduced numbers and activity of circulating progenitor cells are associated with aging and have been linked with coronary artery disease. To determine the impact of aging and atherosclerotic disease on the chemotaxic activity of bone marrow derived cells (BMCs), we examined CXCR4 surface expression on BMCs from aged and atherosclerotic mice.CXCR4 expression and cellular mobility were compared between BMCs of young (6-week old) ApoE null mice (ApoE(-/-)) and aged ApoE(-/-) mice that had been fed with a high-fat, high-cholesterol diet for 6-months.Age and atherosclerosis correlated with significantly lower surface expression of CXCR4 that was less inducible by calcium. The impaired calcium response was associated with defective calcium influx and was partially recovered by treatment with the calcium ionophore ionomycin. ApoE(-/-) mice fed high fat diet for 6-months had defective CXCR4 expression and SDF-1 regulation that is equivalent to that of 24-month old wild type mice. BMCs from aged, atherogenic ApoE(-/-) mice also displayed defective homing to SDF-1, and the animals had lower serum and bone marrow levels of SDF-1.Evolution of atherosclerosis in ApoE(-/-) mice is paralleled by progressive loss of mobility of BMCs with reductions of CXCR4 expression, and reduced levels of SDF-1 in both serum and bone marrow. These changes mute the homing capability of BMCs and may contribute to the progression of atherosclerosis in this model.

Activation of the nuclear factor kappa B (NF kappaB) inflammatory pathway by angiotensin II and reactive oxygen species may play an important role in the development of insulin resistance and cardiovascular injury in hypertensive and metabolic diseases. We have shown that in hypertensive Dahl salt-sensitive rats, upregulation of angiotensin II and reactive oxygen species contributed to increased vascular inflammatory gene expression, endothelial dysfunction, and insulin resistance. Herein, we investigated whether activation of NF kappaB contributes to the development of endothelial dysfunction, vascular injury, and vascular and peripheral insulin resistance in salt-sensitive hypertension.Dahl salt-sensitive rats were fed a normal (0.5% NaCl) or high-salt diet (4% NaCl), or high-salt diet and pyrrolidine dithiocarbamat (150 mg/kg in drinking water), an inhibitor of NF kappaB activation, for 6 weeks.Hypertensive Dahl salt-sensitive rats manifested impaired endothelium-dependent relaxation to acetylcholine, aortic hypertrophy (35%), increased plasma C-reactive protein (25%), vascular superoxide (O2) production (148%), and expression of monocyte chemoattractant protein-1, tumor necrosis factor alpha, phospho-I kappaB alpha, and phospho-(Ser536)-p65NF kappaB. Pyrrolidine dithiocarbamat significantly improved endothelium-dependent relaxation, reduced vascular O2, and normalized aortic hypertrophy and systemic and local inflammation, despite only mildly reducing blood pressure. Hypertensive Dahl salt-sensitive rats also manifested impaired insulin-mediated vasorelaxation and Akt/endothelial nitric oxide synthase phosphorylation and decreased insulin sensitivity by hyperinsulinemic-euglycemic clamp (glucose infusion rate, -32%). Pyrrolidine dithiocarbamat significantly improved insulin-mediated vascular relaxation and Akt/endothelial nitric oxide synthase phosphorylation as well as insulin sensitivity.The current findings strongly suggest that activation of the NF kappaB inflammatory pathway by angiotensin II-induced reactive oxygen species generation may importantly contribute to vascular injury, systemic inflammation, as well as vascular and peripheral insulin resistance in salt-sensitive hypertension.

Insulin activation of the phosphatidylinositol 3-kinase (PI3K) pathway stimulates glucose uptake in peripheral tissues and synthesis of nitric oxide (NO) in the endothelium. Insulin resistance (IR) and hypertension frequently coexist, particularly among individuals with salt-sensitive hypertension. The mechanisms underlying this association are poorly understood. We investigated these mechanisms in a model of salt-sensitive hypertension in which we have previously shown that endothelial dysfunction is mediated by superoxide anion (O2−) linked to local ANG II. Dahl salt-sensitive rats were fed, for 6 wk, a normal salt diet (NS; 0.5% NaCl), high-salt diet (HS; 4% NaCl), HS plus the ANG II type 1 receptor (AT1R) blocker (ARB) candesartan (10 mg·kg−1·day−1), or HS plus the antioxidant tempol (172 mg/l in drinking water). Hypertensive (mean arterial pressure: 145 ± 4 vs. 102 ± 5 mmHg in NS, P < 0.05) rats manifested increased aortic AT1R mRNA (210%) and protein (101%) expression and O2− production (104%) and impaired endothelium-dependent relaxation (EDR) to acetylcholine [maximal response ( Emax): 68 ± 9 vs. 91 ± 8% in NS, P < 0.05]. ARB or tempol normalized O2− and EDR despite that they did not normalize mean arterial pressure, which was reduced only 25%. Hypertensive rats manifested metabolic IR (36% reduction in the glucose infusion rate by insulin clamp), impaired NO-mediated insulin-induced EDR ( Emax: 12 ± 5 vs. 32 ± 4% in NS, P < 0.05), and impaired insulin activation of PI3K/endothelial NO synthase. ARB or tempol improved insulin-mediated EDR, PI3K, Akt/ endothelial NO synthase phosphorylation, and metabolic IR (all P < 0.05). This study provides insight into the mechanisms that underlie the association between metabolic and hypertensive cardiovascular diseases and support the notion that O2− overproduction linked to tissue ANG II interferes with shared insulin signaling pathways in metabolic and cardiovascular tissues.

Objectives Increased endothelial production of reactive oxygen species and decreased nitric oxide bioactivity, associated with the upregulation of monocyte chemoattractant protein (MCP)-1 and lectin-like oxidized low-density lipoprotein receptor (LOX)-1, link hypertension with atherogenesis. We investigated whether the beneficial effects of thiazide diuretics are exclusively related to a reduction in the biomechanical stress of hypertension or are also endowed with pleiotropic vasculoprotective effects that are independent of their effect upon blood pressure. Methods Dahl salt-sensitive (DSS) rats, a paradigm of human salt-sensitive hypertension, were given a diet with normal salt (0.5% NaCI), high salt (4% NaCI), or a high salt diet plus either hydrochlorothiazide 75 mg/l, chlorthalidone 37 or 75 mg/l in their drinking water for 6 weeks. We determined systolic blood pressure (SBP), left ventricular hypertrophy (LVH), proteinuria, aortic superoxide anion (O 2 - ) production, endothelium-dependent relaxation (EDR) to acetylcholine, and aortic angiotensin II type 1 (AT 1 ) receptor, LOX-1, and MCP-1 messenger RNA expression (by real-time polymerase chain reaction). Results DSS rats on a high salt diet developed hypertension, LVH, proteinuria, increased production of aortic O 2 - (106%), impaired EDR, and aortic upregulation of AT 1 receptor (198%), LOX-1 (135%), and MCP-1 (145%). Hydrochlorothiazide as well as the high and low dose of chlorthalidone reduced SBP, LVH, and proteinuria, but did not reduce O 2 - production, AT 1 receptor, LOX-1, or MCP-1 expression, or improved EDR. Conclusions This study demonstrates that thiazide diuretics do not reduce oxidative stress, improve endothelial function, or prevent the expression of proatherogenic molecules. We conclude that thiazide diuretics may not fully provide long-term global cardiovascular protection beyond lowering blood pressure..

Endothelial dysfunction (ED) complicates hypertension and is a precursor of atherosclerosis. Reduced NO bioactivity, because of increased reduced NAD(P)H oxidase–derived reactive oxygen species (ROS), plays a critical role in ED. gp91 phox , predominantly expressed in the endothelium and adventitia, is a subunit of NAD(P)H oxidase important for its activation in response to angiotensin (Ang) II. Human atherosclerotic plaques are heavy laden with gp91 phox . We have shown that in Dahl salt-sensitive (DS) rats, a paradigm of low renin salt-sensitive (SS) hypertension in humans, Ang II receptor blockade normalizes ROS production and endothelium-dependent relaxation (EDR) without significantly affecting systolic blood pressure (SBP). To additionally elucidate the mechanisms involved in the functional association of Ang II in SS hypertension, we administered a cell-permeable inhibitor of the assembly of p47 phox with gp91 phox in NAD(P)H oxidase, gp91ds-tat (10 mg/kg body weight, 3 weeks by minipump), to DS rats fed a 4% salt diet. Control rats received either vehicle or an inactive scramb-tat peptide. Vehicle-treated DS developed hypertension (SBP 168±5 mm Hg), left ventricular hypertrophy (LVH), proteinuria, impaired EDR, and increased aortic ROS production (superoxide 115% and peroxynitrite 157%) and expression of the proatherogenic molecules LOX-1 (130%) and MCP-1 (166%). gp91ds-tat, but not scramb-tat, normalized ROS and EDR, as well as LOX-1 and MCP-1, despite nonsignificant effects on SBP (159±5 mm Hg; P >0.05), left ventricular hypertrophy, and proteinuria. Our findings support the notion that in SS hypertension, activation of NAD(P)H oxidase promotes ED and atherogenesis via decreased nitric oxide bioactivity and increased LOX-1 and MCP-1, independent of blood pressure.

A balance among nitric oxide (NO), angiotensin II (Ang II), and reactive oxygen species (ROS) in the endothelium is necessary for maintaining the homeostasis of the vascular wall. Oxidative stress has been shown to play a critical role in the development of hypertension and atherosclerosis. Although there is overwhelming evidence that hypertension promotes atherosclerosis, the relative contribution and/or interaction of hemodynamic and oxidative stress remains undefined. NO is synthesized in the endothelium by NO synthase and antagonizes the vasoconstrictive and proatherosclerotic effects of Ang II. On the other hand, Ang II decreases NO bioavailability by promoting oxidative stress. A better understanding of the pathophysiologic mechanisms involved in the link between hypertension and atherosclerosis may aid in developing therapeutic interventions. We propose that those antihypertensive agents that lower blood pressure and concomitantly restore the homeostatic balance of vasoactive agents in the endothelium would be more effective in preventing or arresting atherosclerosis.

Although initially adaptive, the changes that accompany hypertension, namely, cell growth, endothelial dysfunction, and extracellular matrix deposition, eventually can become maladaptive and lead to end-organ disease such as heart failure, coronary artery disease, and renal failure. A functional imbalance between angiotensin II (Ang II) and nitric oxide (NO) plays an important pathogenetic role in hypertensive end-organ injury. NO, an endogenous vasodilator, inhibitor of vascular smooth muscle and mesangial cell growth, and natriuretic agent, is synthesized in the endothelium by a constitutive NO synthase. NO antagonizes the effects of Ang II on vascular tone, cell growth, and renal sodium excretion, and also down-regulates the synthesis of angiotensin-converting enzyme (ACE) and Ang II type 1 receptors. On the other hand, Ang II decreases NO bioavailability by promoting oxidative stress. A better understanding of the pathophysiologic mechanisms involved in hypertensive end-organ damage may aid in identifying markers of cardiovascular susceptibility to injury and in developing therapeutic interventions. We propose that those antihypertensive agents that lower blood pressure and concomitantly restore the homeostatic balance of vasoactive agents such as Ang II and NO within the vessel wall would be more effective in preventing or arresting end-organ disease.

Statins, inhibitors of cholesterol biosynthesis, are endowed with pleiotropic effects that may contribute to their favorable clinical results. Hypertensive Dahl salt-sensitive (DS) rats have endothelial dysfunction and cardiorenal injury associated with decreased NO bioavailability and increased superoxide (O 2 − ) production linked to a functional upregulation of angiotensin II. We investigated whether atorvastatin (30 mg/kg per day; by gavage) would prevent endothelial nitric oxide (eNOS) downregulation and the increase in O 2 − in DS rats, thereby reducing end-organ injury. DS rats given a high-salt diet (4% NaCl) for 10 weeks developed hypertension (systolic blood pressure [SBP] 200±8 versus 150±2 mm Hg in DS rats fed 0.5% NaCl diet [NS]; P P 2 − (2632±316 versus 1176±112 counts/min per milligram in NS; P 2 α isoprostanes. Atorvastatin prevented the decrease in eNOS activity (1.5±0.3 nmol/min per gram protein) as well as the increase in O 2 − (1192±243 counts/min per milligram) and plasma 8-F 2 α isoprostanes, reduced LVH and proteinuria, and normalized endothelial function and vascular response to endothelin-1, although reduction in SBP was modest (174±8 mm Hg). Atorvastatin combined with removal of high salt normalized aortic eNOS activity, SBP, LVH, and proteinuria. These findings strongly suggest that concomitant prevention of vascular eNOS downregulation and inhibition of oxidative stress may contribute to the protection against end-organ injury afforded by this statin in salt-sensitive hypertension.

Calcium channel blockers such as amlodipine are effective antihypertensive agents. In this study we investigated the effects of amlodipine on vascular oxidative stress, expression of the lectin-like oxidized low-density lipoprotein receptor (LOX-1), and endothelial function in angiotensin (Ang) II-infused rats.Sprague-Dawley rats were treated with Ang II (0.7 mg/kg/day subcutaneously injected by mini-pump), with or without amlodipine (10 mg/kg/day by gavage), for 5 days and compared with control rats. Levels of aortic ring superoxide (O(2)(-)) and peroxynitrite (ONOO(-)) were determined, and systolic blood pressure (SBP) and endothelium-dependent relaxation were evaluated.Compared with control rats, Ang II-infused rats developed hypertension (175 +/- 3 v 135 +/- 2 mm Hg, P.05), aortic hypertrophy (16.9 +/- 1.3 v 13.2 +/- 0.3 mg/cm, P.05), left ventricular hypertrophy (0.236 +/- 0.003 v 0.204 +/- 0.004 g/100 g body weight, P.05), and impaired endothelium-dependent relaxation (ED(50): 6.6 +/- 0.2 v 8.0 +/- 0.2 -log mol/L acetylcholine concentration, P.05). Compared with control rats, Ang II-infused rats also had higher aortic levels of LOX-1 mRNA expression, O(2)(-)production (1005 +/- 140 v 608 +/- 159 counts/min/mg, P.05), ONOO(-) production (1875 +/- 295 v 782 +/- 115 counts/min/mg, P.05), and plasma free 8-F(2)alpha-isoprostanes (67.4 +/- 19.1 v 27.2 +/- 6.1 pg/mL, P.05). In Ang II-infused rats SBP, aortic hypertrophy, endothelial dysfunction, LOX-1 expression, aortic O(2)(-) and ONOO(-) production, and plasma free 8-F(2)alpha-isoprostane levels were significantly reduced by amlodipine treatment.Amlodipine has antihypertensive and antioxidant activity in vivo, which effectively inhibits many of the oxidative stress-dependent mechanisms involved in Ang II-mediated cardiovascular injury.

The balance between endothelial nitric oxide (NO) and angiotensin II (Ang II) maintains the homeostasis of the cardiovascular and renal systems. We tested the hypothesis that increased oxidant stress linked to a functional imbalance between NO and Ang II might play a central pathogenetic role in salt-sensitive (SS) hypertension. We studied Dahl SS (DS) rats during the prehypertensive (5 days) and hypertensive (12 weeks) phases of a high-salt (4% NaCl) diet. Control rats received a normal-salt (0.5% NaCl, [NS]) diet. Prehypertensive DS rats (systolic blood pressure [SBP] 138±2 mm Hg) manifested a 35% increase ( P 2 − ) production without evidence of end-organ damage. Hypertensive DS rats (SBP 214±11 mm Hg) had impaired endothelium-dependent relaxation (EDR) and increased aortic O 2 − production (320%), urinary isoprostane excretion (83%), aortic (20%) and left ventricular (LVH, 21%) hypertrophy, and proteinuria (124%). In prehypertensive DS rats, candesartan (10 mg · kg −1 · d −1 ) an Ang II type 1 receptor blocker (ARB), normalized O 2 − production. In hypertensive DS rats, the ARB decreased aortic O 2 − production by 71% and normalized EDR without affecting SBP (212±8 mm Hg), aortic hypertrophy, LVH, or proteinuria. Switching hypertensive DS rats to an NS diet did not affect SBP (208±8 mm Hg), LVH, aortic hypertrophy, or proteinuria and had minimal effects on O 2 − and EDR. Concomitant ARB administration plus a switch to an NS diet normalized SBP (138±8 mm Hg) as well as end-organ damage. Dahl salt-resistant rats fed an HS diet for 12 weeks did not show hypertension or increased O 2 − production. Thus, SS hypertension might represent a specific vascular diathesis linked to functional upregulation of Ang II action (increased O 2 − synthesis) accompanied by insufficient NO bioavailability, which promotes severe endothelial dysfunction.

We have previously reported that (+/-)-1-(2.5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), a 5-HT2 receptor agonist, induced renal vasodilation in anesthetized dogs. The present study was designed to investigate whether DOI-induced renal vasodilation might be mediated by increased nitric oxide (NO) release/production in renal tissue. The experiments were performed in anesthetized dogs. A 23-gauge needle was inserted into the left renal artery for infusion of drug solutions. Renal blood flow was measured with an electromagnetic flowmeter. The microdialysis probes were implanted into the renal cortex to collect the dialysate for measurement of guanosine 3',5'-cyclic monophosphate (cGMP) and nitrite/nitrate (NO2/NO3) concentration. Intrarenal infusion of DOI at a rate of 5 microg/kg/min resulted in a significant increase, by 30 +/- 4%, in renal blood flow, indicating renal vasodilation. The renal interstitial concentrations of NO2/NO3 and cGMP also increased by 70 +/- 6% and 60 +/- 6%, respectively. These changes induced by DOI were completely abolished by the intrarenal pretreatment with N(w)-nitro-L-arginine methyl ester (L-NAME, a NO synthase inhibitor, 100 microg/kg/min) or sarpogrelate (100 microg/kg/min, a highly selective 5-HT2 receptor antagonist). DOI infusion increased urine volume and urinary excretion of Na+, which were also blocked by L-NAME or sarpogrelate. These results suggest that DOI caused renal vasodilation due to increased NO release/production by stimulation of 5-HT2 receptors in the kidney. The natriuretic effect of DOI might also be related to increased intrarenal NO production.

Objectives To clarify whether endothelium-derived contracting factor (EDCF) is developed in renal artery of hypertensive Dahl rats and whether prolonged oral L-arginine treatments prevent development of EDCF and hypertension. Design The effect of prolonged salt treatment with or without L-arginine on the renal artery was examined. Methods and Results Dahl salt-sensitive and -resistant rats were fed a 0.4 or an 8% NaCl diet for 4 weeks. High sodium intake increased arterial pressure in Dahl salt-sensitive rats. The rings of renal arteries were suspended for isometric tension recording. Only in the hypertensive rats, more than 1 μmol/l acetylcholine induced an endothelium-dependent contraction response. The contraction was completely inhibited by indomethacin or ONO-3708 [prostaglandin H 2 (PGH 2 )/thromboxane A 2 (TXA 2 ) receptor antagonist], and partially inhibited by OKY-046 (TXA 2 synthetase inhibitor). Acetylcholine-induced relaxation was significantly depressed in hypertensive rats, which was partially improved by SQ29548 (PGH 2 /TXA 2 receptor antagonist). Oral L-arginine, but not ONO-8809 (orally active PGH 2 /TXA 2 receptor antagonist) treatment, inhibited the contraction and amended the relaxation. The endothelium-independent contraction to TXA 2 receptor agonist U46619 and relaxation to nitroprusside were not altered by L-arginine treatment The L-Arginine treatment reduced blood pressure and sodium retention with increases in urinary NO - 2 /NO - 3 and cGMP excretion. Hydralazine treatment also inhibited development of EDCF. Conclusions The present results suggest that impaired endothelium-dependent relaxation to acetylcholine is caused in part by induction of EDCF synthesis/release in renal arteries of hypertensive Dahl rats. L-arginine can attenuate sodium retention and development of hypertension, which lead to a decrease in EDCF synthesis in renal arteries.

The present study was designed to determine whether and how potassium supplementation improves the endothelial function of carotid arteries of hypertensive Dahl rats. Dahl salt-sensitive rats were fed a high sodium diet, a high sodium plus potassium-supplemented diet, a normal rat chow, or a potassium-supplemented diet for 4 weeks. High sodium intake significantly increased the blood pressure, which was attenuated by potassium supplementation. The isometric tension of rat-isolated carotid rings was measured. In norepinephrine-precontracted rings, the relaxation in response to acetylcholine, adenosine 5'-diphosphate (ADP), and isoproterenol were significantly attenuated in hypertensive Dahl rats, which was improved by potassium supplementation. Pretreatment with N(G)-nitro-L-arginine methyl ester blocked the responses to acetylcholine and ADP, and eliminated the difference in relaxation in response to isoproterenol. The endothelium-independent relaxation in response to forskolin, S-nitroso-N-acetyl-DL-penicillamine, and sodium nitroprusside was significantly attenuated in hypertensive Dahl rats, which was not affected by potassium supplementation. The results indicated that salt-induced hypertension was associated with marked alterations in the endothelial and vascular smooth muscle functions of the carotid arteries of Dahl rats. Potassium supplementation ameliorated the endothelial but not the smooth muscle function. The protective effect of potassium appeared to be achieved through increased endothelial nitric oxide production. The current studies, in conjunction with our recent studies on nitric oxide synthase activity in the kidney, strongly suggest that potassium attenuates development of hypertension by increasing nitric oxide production in Dahl rats.