Megumi Fujita

Sympathoexcitation by Oxidative Stress in the Brain Mediates Arterial Pressure Elevation in Obesity-Induced Hypertension

Background— Obesity is one of the major risk factors for cardiovascular disease and is often associated with increased oxidative stress and sympathoexcitation. We have already suggested that increased oxidative stress in the brain modulates the sympathetic regulation of arterial pressure in salt-sensitive hypertension, which is often associated with obesity. The present study was performed to determine whether oxidative stress could mediate central sympathoexcitation in the initial stage of obesity-induced hypertension. Methods and Results— Four-week-old male Sprague-Dawley rats were fed a high-fat (45% kcal as fat) or low-fat (10% kcal as fat) diet for 6 weeks. Fat loading elicited hypertension and sympathoexcitation, along with visceral obesity. In urethane-anesthetized and artificially ventilated rats, arterial pressure and renal sympathetic nerve activity decreased in a dose-dependent fashion when 53 or 105 &mgr;mol/kg tempol, a membrane-permeable superoxide dismutase mimetic, was infused into the lateral cerebral ventricle. Central tempol reduced arterial pressure and renal sympathetic nerve activity to a significantly greater extent in high-fat diet–fed hypertensive rats than in low-fat diet–fed normotensive rats. Intracerebroventricular apocynin or diphenyleneiodonium, a reduced NADPH oxidase inhibitor, also elicited markedly greater reductions in arterial pressure and renal sympathetic nerve activity in the high-fat diet–fed rats. In addition, fat loading increased NADPH oxidase activity and NADPH oxidase subunit p22phox, p47phox, and gp91phox mRNA expression in the hypothalamus. Conclusions— In obesity-induced hypertension, increased oxidative stress in the brain, possibly via activation of NADPH oxidase, may contribute to the progression of hypertension through central sympathoexcitation.

Sympathoexcitation by Oxidative Stress in the Brain Mediates Arterial Pressure Elevation in Salt-Sensitive Hypertension

Central sympathoexcitation is involved in the pathogenesis of salt-sensitive hypertension. We have suggested that oxidative stress in the brain modulates the sympathetic regulation of arterial pressure. Thus, we investigated whether oxidative stress could mediate central sympathoexcitation in salt-sensitive hypertension. Five- to 6-week-old male Dahl salt-sensitive rats and salt-resistant rats were fed with a normal (0.3%) or high- (8%) salt diet for 4 weeks. In urethane-anesthetized and artificially ventilated rats, arterial pressure, renal sympathetic nerve activity, and heart rate decreased in a dose-dependent fashion, when 20 or 40 &mgr;mol of tempol, a membrane-permeable superoxide dismutase mimetic, was infused into the lateral cerebral ventricle. The same degree of reduction was noted in salt-sensitive and salt-resistant rats without salt loading. Salt loading significantly increased central tempol-induced reductions in arterial pressure (−29.1±4.8% versus −10.6±3.3% at 40 &mgr;mol; P<0.01), sympathetic nerve activity (−18.7±2.0% versus −7.1±1.8%; P<0.01), and heart rate (−10.7±2.8% versus −2.0±0.7%; P<0.05) in salt-sensitive rats but not in salt-resistant rats. Intracerebroventricular diphenyleneiodonium, a reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor, also elicited significantly greater reduction in each parameter in salt-loaded salt-sensitive rats. Moreover, salt loading increased reduced nicotinamide-adenine dinucleotide phosphate–dependent superoxide production in the hypothalamus in salt-sensitive rats but not in salt-resistant rats. In addition, reduced nicotinamide-adenine dinucleotide phosphate oxidase subunits p22phox, p47phox, and gp91phox mRNA expression significantly increased in the hypothalamus of salt-loaded salt-sensitive rats. In conclusion, in salt-sensitive hypertension, increased oxidative stress in the brain, possibly via activation of reduced nicotinamide-adenine dinucleotide phosphate oxidase, may elevate arterial pressure through central sympathoexcitation.

Salt Excess Causes Left Ventricular Diastolic Dysfunction in Rats With Metabolic Disorder

Metabolic syndrome is a highly predisposing condition for cardiovascular disease and could be a cause of excess salt–induced organ damage. Recently, several investigators have demonstrated that salt loading causes left ventricular diastolic dysfunction associated with increased oxidative stress and mineralocorticoid receptor activation. We, therefore, investigated whether excess salt induces cardiac diastolic dysfunction in metabolic syndrome via increased oxidative stress and upregulation of mineralocorticoid receptor signals. Thirteen-week-old spontaneously hypertensive rats and SHR/NDmcr-cps, the genetic model of metabolic syndrome, were fed a normal salt (0.5% NaCl) or high-salt (8% NaCl) diet for 4 weeks. In SHR/NDmcr-cps, salt loading induced severe hypertension, abnormal left ventricular relaxation, and perivascular fibrosis. Salt-loaded SHR/NDmcr-cps also exhibited overproduction of reactive oxygen species and upregulation of mineralocorticoid receptor–dependent gene expression, such as Na+/H+ exchanger-1 and serum- and glucocorticoid-inducible kinase-1 in the cardiac tissue. However, in spontaneously hypertensive rats, salt loading did not cause these cardiac abnormalities despite a similar increase in blood pressure. An antioxidant, tempol, prevented salt-induced diastolic dysfunction, perivascular fibrosis, and upregulation of mineralocorticoid receptor signals in SHR/NDmcr-cps. Moreover, a selective mineralocorticoid receptor antagonist, eplerenone, prevented not only diastolic dysfunction but also overproduction of reactive oxygen species in salt-loaded SHR/NDmcr-cps. These results suggest that metabolic syndrome is a predisposed condition for salt-induced left ventricular diastolic dysfunction, possibly via increased oxidative stress and enhanced mineralocorticoid receptor signals.

Mice Lacking Hypertension Candidate Gene ATP2B1 in Vascular Smooth Muscle Cells Show Significant Blood Pressure Elevation

We reported previously that ATP2B1 was one of the genes for hypertension receptivity in a large-scale Japanese population, which has been replicated recently in Europeans and Koreans. ATP2B1 encodes the plasma membrane calcium ATPase isoform 1, which plays a critical role in intracellular calcium homeostasis. In addition, it is suggested that ATP2B1 plays a major role in vascular smooth muscle contraction. Because the ATP2B1 knockout (KO) mouse is embryo-lethal, we generated mice with vascular smooth muscle cell-specific KO of ATP2B1 using the Cre-loxP system to clarify the relationship between ATP2B1 and hypertension. The KO mice expressed significantly lower levels of ATP2B1 mRNA and protein in the aorta compared with control mice. KO mice showed significantly higher systolic blood pressure as measured by tail-cuff method and radiotelemetric method. Similar to ATP2B1, the expression of the Na+-Ca2+ exchanger isoform 1 mRNA was decreased in vascular smooth muscle cells of KO mice. However, ATP2B4 expression was increased in KO mice. The cultured vascular smooth muscle cells of KO mice showed increased intracellular calcium concentration not only in basal condition but also in phenylephrine-stimulated condition. Furthermore, phenylephrine-induced vasoconstriction was significantly increased in vascular rings of the femoral artery of KO mice. These results suggest that ATP2B1 plays important roles in the regulation of blood pressure through alteration of calcium handling and vasoconstriction in vascular smooth muscle cells.

Sympatho-Inhibitory Action of Endogenous Adrenomedullin Through Inhibition of Oxidative Stress in the Brain

Central sympathetic activation is one of the possible mechanisms underlying hypertension, in which reactive oxygen species may play a role. Thus, we examined whether adrenomedullin, an antioxidant peptide, is involved in the central regulation of arterial pressure through sympatho-modulatory action. Adrenomedullin knockout mice were fed with high-salt diet for 4 weeks to stimulate adrenomedullin production. In the wild-type littermates, brain adrenomedullin content was significantly increased with salt loading, but not in the knockout mice. Intracerebroventricular hyperosmotic saline increased arterial pressure and sympathetic nerve activity in a dose-dependent fashion. With the normal salt diet, the hyperosmotic saline-induced response did not significantly differ between the knockout and wild-type mice; with the high-salt diet, however, the response was significantly greater in the knockout mice than in wild-type littermates (arterial pressure: 35.3±5.7% versus 20.1±2.1%, P<0.05; sympathetic nerve activity: 30.3±4.8% versus 15.9±1.5%, P<0.05; respectively). Moreover, pretreatment with 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (tempol), a membrane-permeable superoxide dismutase mimetic, inhibited the augmented response to central hyperosmotic saline in salt-loaded knockout mice. Consistently, the hyperosmotic saline-induced production of reactive oxygen species, measured by the lucigenin chemiluminescence method, was significantly greater in the isolated hypothalamus of salt-loaded knockout mice than in that of salt-loaded wild-type ones. In conclusion, endogenous adrenomedullin in the brain may inhibit sympathetic activation through its antioxidant action.

Protective Effect of Dietary Potassium against Cardiovascular Damage in Salt-Sensitive Hypertension: Possible Role of its Antioxidant Action

It is well known that high salt intake induces hypertension and cardiovascular damage, while dietary potassium supplementation counteracts these harmful effects. Actually, the protective effect of potassium is strengthened with excess salt as compared with salt depletion. Although the precise mechanisms have not been fully elucidated, in our previous reports, the antihypertensive effect of dietary potassium was accompanied by sympathetic nerve inhibition in salt-sensitive hypertension. Also, potassium supplement suppressed salt-induced insulin resistance. These effects of dietary potassium can explain its cardio- and vasculo-protective action in addition to the potassium supplementation induced decreased salt-induced rise in blood pressure. On the other hand, salt-sensitive hypertension is associated with reactive oxygen species (ROS) overproduction. Moreover, sympathoexcitation can be induced by central ROS upregulation and insulin resistance can be caused by ROS excess in the target organs of insulin, such as skeletal muscle. Conversely, the seemingly different actions of potassium can be explained by the antioxidant effect of dietary potassium; in our recent studies, potassium supplementation inhibits salt-induced progress of cardiac diastolic dysfunction and vascular neointima formation by cuff placement around arteries, associated with the inhibition of regional ROS overproduction, in salt-sensitive hypertension. Thus, it is possible that dietary potassium protects against salt-induced cardiovascular damage by the reduction of ROS generation and by central sympatholytic action and amelioration of insulin resistance induced through its antioxidant effect.

Sympathoexcitation by brain oxidative stress mediates arterial pressure elevation in salt-induced chronic kidney disease.

Hypertension is very prevalent in chronic kidney disease and critical for its prognosis. Sympathoexcitation and oxidative stress have been demonstrated to be involved in chronic kidney disease. We have shown previously that sympathoexcitation by brain oxidative stress mediates arterial pressure elevation in the salt-sensitive hypertension model, Dahl salt-sensitive rats. Thus, we investigated whether sympathoexcitation by excessive brain oxidative stress could contribute to arterial pressure elevation in salt-induced chronic kidney disease model rats. Young (3-week–old) male Sprague-Dawley rats were randomly assigned to a uninephrectomy or sham operation and then subjected to either a normal salt (0.5%) or high-salt (8.0%) diet for 4 weeks. The young salt-loaded uninephrectomized rats exhibited sympathoexcitation, hypertension, and renal injury, proteinuria and global glomerulosclerosis together with tubulointerstitial damage. Under urethane anesthesia and artificial ventilation, renal sympathetic nerve activity, arterial pressure, and heart rate decreased to a greater degree in the salt-loaded uninephrectomized rats than in the nonsalt-loaded uninephrectomized rats and the salt-loaded or nonsalt-loaded sham-operated rats, when Tempol, a membrane-permeable superoxide dismutase mimetic, was infused acutely into the lateral cerebral ventricle. Oxidative stress in the hypothalamus, measured by lucigenin chemiluminescence, was also significantly greater. Furthermore, in the salt-loaded uninephrectomized rats, antioxidant treatment with chronic intracerebroventricular Tempol decreased sympathetic nerve activity and arterial pressure, which, in turn, led to a decrease in renal damage. Similar effects were elicited by treatment with oral moxonidine, the central sympatholytic agent. In conclusion, sympathoexcitation by brain oxidative stress may mediate arterial pressure elevation in salt-induced chronic kidney disease.

Mineralocorticoid receptor activation: a major contributor to salt-induced renal injury and hypertension in young rats

Excessive salt intake is known to preferentially increase blood pressure (BP) and promote kidney damage in young, salt-sensitive hypertensive human and animal models. We have suggested that mineralocorticoid receptor (MR) activation plays a major role in kidney injury in young rats. BP and urinary protein were compared in young (3-wk-old) and adult (10-wk-old) uninephrectomized (UNx) Sprague-Dawley rats fed a high (8.0%)-salt diet for 4 wk. The effects of the MR blocker eplerenone on BP and renal injury were examined in the high-salt diet-fed young UNx rats. Renal expression of renin-angiotensin-aldosterone (RAA) system components and of inflammatory and oxidative stress markers was also measured. The effects of the angiotensin receptor blocker olmesartan with or without low-dose aldosterone infusion, the aldosterone synthase inhibitor FAD286, and the antioxidant tempol were also studied. Excessive salt intake induced greater hypertension and proteinuria in young rats than in adult rats. The kidneys of young salt-loaded rats showed marked histological injury, overexpression of RAA system components, and an increase in inflammatory and oxidative stress markers. These changes were markedly ameliorated by eplerenone treatment. Olmesartan also ameliorated salt-induced renal injury but failed to do so when combined with low-dose aldosterone infusion. FAD286 and tempol also markedly reduced urinary protein. UNx rats exposed to excessive salt at a young age showed severe hypertension and renal injury, likely primarily due to MR activation and secondarily due to angiotensin receptor activation, which may be mediated by inflammation and oxidative stress.

Mineralocorticoid receptor--Rac1 activation and oxidative stress play major roles in salt-induced hypertension and kidney injury in prepubertal rats.

Objectives: To elucidate the roles that renal mineralocorticoid receptor–Rac1 interactions and oxidative stress play in salt-induced hypertension and renal injury in prepubertal rats. Methods: Three-week-old male Sprague Dawley rats were uninephrectomized (UNx) and fed a high-salt (8% NaCl) diet for 4 weeks. Five were left untreated, whereas the remaining rats were administered an mineralocorticoid receptor blocker (n = 5), a Rac1 inhibitor (n = 5), a Rho-kinase inhibitor (n = 5), or the superoxide dismutase mimetic tempol (n = 5). A control group of young UNx rats (n = 5) was fed a normal-salt (0.5% NaCl) diet. The rats were sacrificed after a 4-week experimental period. Blood pressure, urinary protein, histological morphology, and renal serum-regulated and glucocorticoid-regulated kinase (Sgk) 1 and Rac1 expression were evaluated. The effect of adrenalectomy with dexamethasone supplementation in young salt-loaded UNx rats (n = 5) was also evaluated. Results: Excessive salt intake induced hypertension and proteinuria in the young UNx rats, whose kidneys showed marked histological injury, Sgk1 overexpression and Rac1 activation. Both mineralocorticoid receptor blockade and Rac1 inhibition markedly prevented these abnormalities associated with a reduction in renal Rac1 expression. Adrenalectomy, but not Rho-kinase inhibition, also prevented salt-induced renal injury. Interestingly, tempol inhibited renal Rac1 activation and renal injury. Conclusions: These findings suggest that Rac1-related mineralocorticoid receptor activation contributed to salt-induced hypertension and kidney injury in young UNx rats. Furthermore, as adrenalectomy abrogated salt-induced proteinuria, Rac1 may be an enhancer of aldosterone-induced mineralocorticoid receptor activation. Oxidative stress may also modify the interaction between Rac1 and mineralocorticoid receptor.

Enhanced Angiotensin Receptor-Associated Protein in Renal Tubule Suppresses Angiotensin-Dependent Hypertension

We have previously shown that angiotensin II type 1 receptor-associated protein (ATRAP/Agtrap) interacts with the angiotensin II type 1 receptor and promotes constitutive internalization of the receptor so as to inhibit the pathological activation of its downstream signaling but preserve baseline physiological signaling activity. The present study was designed to investigate the role of renal ATRAP in angiotensin II–dependent hypertension. We generated transgenic mice dominantly expressing ATRAP in the renal tubules, including renal distal tubules. The renal ATRAP transgenic mice exhibited no significant change in blood pressure at baseline on normal salt diet. However, in the renal ATRAP transgenic mice compared with wild-type mice, the following took place: (1) the development of high blood pressure in response to angiotensin II infusion was significantly suppressed based on radiotelemetry, (2) the extent of daily positive sodium balance was significantly reduced during angiotensin II infusion in metabolic cage analysis, and (3) the renal Na+-Cl− cotransporter activation and &agr;-subunit of the epithelial sodium channel induction by angiotensin II infusion were inhibited. Furthermore, adenoviral overexpression of ATRAP suppressed the angiotensin II–mediated increase in the expression of &agr;-subunit of the epithelial sodium channel in mouse distal convoluted tubule cells. These results indicate that renal tubule–dominant ATRAP activation provokes no evident effects on blood pressure at baseline but exerts an inhibitory effect on the pathological elevation of blood pressure in response to angiotensin II stimulation, thereby suggesting that ATRAP is a potential target of interest in blood pressure modulation under pathological conditions.