Salt-fed Rats Research Articles

Input from afferent renal nerves is involved in progression of hypertensive heart failure through central vasopressin-mediated sympathoexcitation

Introduction: Paraventricular nucleus of hypothalamus (PVN), a control center of sympathetic outflow, receives the input from afferent renal nerves. Although acute stimulation of afferent renal nerves increases central sympathetic outflow, it remains unclear if the renal afferents contribute to progression of heart failure. Acute stimulation of afferent renal nerves was reported to increase plasma vasopressin concentration. In addition, central dendritic release of vasopressin by activation of vasopressin-expressing neurons located in the PVN may cause sympathoexcitation via stimulation of neighboring presympathetic neurons in the PVN. Therefore, we hypothesized that the chronic input from afferent renal nerves contributes to sympathoexcitation and hypertensive cardiac dysfunction along with activation of vasopressin-expressing neurons and presympathetic neurons in the PVN. Methods: Male Dahl salt-sensitive rats were fed low salt diet (LS) or 8% high salt diet from 6 weeks of age, and the high salt-fed rats were allocated to selective afferent renal denervation (HS-ARDN) or sham surgery (HS-Sham) at 9 weeks. The effect of ARDN on central sympathetic regulation was investigated at 12 weeks (pre-heart failure phase). In another cohort, its effect on cardiac phenotypes was evaluated at 16 weeks (heart failure phase). Results: At 12 weeks, plasma norepinephrine level was increased in HS-Sham compared to LS and was attenuated by ARDN (LS 188.7±16.5 pg/ml, HS-Sham 563.8±82.7 pg/ml, HS-ARDN 327.4±50.2 pg/ml; n=15, 12, 14; p<0.05). The c-Fos expression, a marker of neuronal activity, in presympathetic neurons in the PVN was also increased in HS-Sham and attenuated in HS-ARDN, which was in parallel with the c-Fos expression in vasopressin-expressing neurons in the PVN. At 16 weeks, the decrease of left ventricular (LV) fractional shortening (HS-Sham 25.9±1.7 % vs LS 46.7±0.6 %, n=9, 9; p<0.01) was improved in HS-ARDN (42.2±1.2 %, n=7; p<0.01 vs HS-Sham). LV weight and lung weight were significantly increased in HS-Sham compared to LS (LV weight/body weight: 4.39±0.19 mg/g vs 2.53±0.03 mg/g; lung weight/body weight: 5.29±0.37 mg/g vs 3.55±0.05 mg/g; p<0.05), and these changes were attenuated in HS-ARDN (LV weight/body weight: 3.73±0.14 mg/g; lung weight/body weight: 4.17±0.11 mg/g; p<0.05 vs HS-Sham). Histological LV fibrosis and fibrosis marker expression, such as connective tissue growth factor, in LV were significantly attenuated in HS-ARDN compared to HS-Sham. ARDN did not significantly impact the high salt-induced blood pressure elevation in both cohorts. Conclusion: The input from afferent renal nerves contributes to cardiac dysfunction, probably via the central vasopressin-mediated sympathoexcitation even without blood pressure changes in hypertensive heart failure model. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract P174: Sex Differences In The Pro-hypertensive Effect Of Carnitine, The Precursor Of Gut-derived Metabolite TMAO

Recent evidence reports sexually divergent mechanisms that differentially drive the severity of hypertension. Our data show that female Dahl Salt-Sensitive (SS) rats are significantly protected from salt-induced hypertension and renal injury and have stark differences in gut microbiota composition compared to males. Gut-derived metabolites are increasingly being recognized as mechanistic links between the gut microbiota and hypertension. One such metabolite is trimethylamine N-oxide (TMAO), which is derived from the bacterial metabolism of carnitine and is gaining notoriety for its role in cardiovascular disease. Metabolomics analysis in high salt-fed SS rats revealed a trend for increased TMAO (1.3-fold, p=0.11) in the serum of males compared to females (n=6). TMAO appears to be specifically derived from gut bacteria since oral antibiotic treatment nearly eliminated circulating TMAO levels in both males and females (99.3% and 88.9% reduction, respectively; p<0.001). Interestingly, antibiotic treatment reduced salt-sensitive hypertension in males but not females. There was also a corresponding increase in the TMAO precursor carnitine (1.9-fold, p<0.01) in the serum of males versus females. Thus, we hypothesized that administration of carnitine (400 mg/kg/day) in the drinking water would exacerbate salt-sensitive hypertension, renal damage, and gut inflammation in male and female SS rats challenged with high salt (4% NaCl). There was a trend for carnitine treatment to exacerbate mean arterial pressure in both males (160±9 vs 146±2 mmHg, n=4-6, p=0.22) and females (155±6 vs 139±2 mmHg, n=2, p=0.14) compared to vehicle. Despite elevated pressure in both sexes, carnitine-treated males exhibited greater increases in albuminuria (340±136 vs 194±29 mg/day, carnitine vs vehicle, p=0.28) than females (55±33 vs 26±5 mg/day). Carnitine treatment also significantly increased the number of CD3+ T cells in the colonic lamina propria (24.3±6.0 vs 2.4±0.5 x 10 6 cells/g tissue, n=5, p<0.05) of male rats compared to vehicle. Together, these data identify gut microbiota-mediated carnitine/TMAO metabolism as a potentially detrimental pathway that promotes greater salt-sensitivity, renal damage, and gut inflammation in males versus females.

Abstract P220: High Salt Intake Induces Catabolism Accompanied by Hepatic Urea Osmolyte Production and Decreases Renal Sympathetic Nerve Activity

Introduction: We have recently reported that high salt intake induces the energy-intensive nature of hepatic urea osmolyte production that accompanied the decrease in heart rate (HR). These findings suggest that high salt intake decreases sympathetic nerve activity to reduce cardiovascular energy expenditure for the hepatic urea production. Therefore, we examined the effects of high salt intake on renal sympathetic nerve activity (RSNA) by using a radiotelemetry system in rats. Methods: We inserted the radiotelemetry system into male Sprague Dawley (SD) rats for recording RSNA and HR. After 2 weeks recovery, we fed the rats 0.3% NaCl diet (NS) with tap water to drink (NS + tap), 4% NaCl diet with tap water (HS + tap), and 4% NaCl diet with saline (HS + saline) for 4 days in this order, and kept monitoring their HR and RSNA. In the separate experiment, we also fed male SD rats NS + tap or HS + saline for 6 consecutive weeks to confirm a catabolic state in HS + saline rats. Results: Rats exhibited marked reductions in RSNA and HR during HS + saline phase compared with other phases. HS + saline rats significantly decreased body weight (369±35 g) compared with NS + tap rats (405±48 g) despite of the similar daily food intake (18.5±1.3 vs. 19.2±1.6 g/day). And HS + saline rats increased liver arginase, a urea producing enzyme, activity (1561±121 units/L/mg) compared with NS + tap rats (1337±131 units/L/mg) at 6 weeks after the feeding. Conclusion: High salt intake decreased RSNA accompanied by the catabolic urea production in rats. These findings suggest that the sympathetic nervous system is one of the key regulators to reduce cardiovascular energy expenditure, which could support the energy-intensive nature of hepatic urea production in high salt-fed rats.

Moderate (20%) fructose-enriched diet stimulates salt-sensitive hypertension with increased salt retention and decreased renal nitric oxide.

Previously, we reported that 20% fructose diet causes salt‐sensitive hypertension. In this study, we hypothesized that a high salt diet supplemented with 20% fructose (in drinking water) stimulates salt‐sensitive hypertension by increasing salt retention through decreasing renal nitric oxide. Rats in metabolic cages consumed normal rat chow for 5 days (baseline), then either: (1) normal salt for 2 weeks, (2) 20% fructose in drinking water for 2 weeks, (3) 20% fructose for 1 week, then fructose + high salt (4% NaCl) for 1 week, (4) normal chow for 1 week, then high salt for 1 week, (5) 20% glucose for 1 week, then glucose + high salt for 1 week. Blood pressure, sodium excretion, and cumulative sodium balance were measured. Systolic blood pressure was unchanged by 20% fructose or high salt diet. 20% fructose + high salt increased systolic blood pressure from 125 ± 1 to 140 ± 2 mmHg (P < 0.001). Cumulative sodium balance was greater in rats consuming fructose + high salt than either high salt, or glucose + high salt (114.2 ± 4.4 vs. 103.6 ± 2.2 and 98.6 ± 5.6 mEq/Day19; P < 0.05). Sodium excretion was lower in fructose + high salt group compared to high salt only: 5.33 ± 0.21 versus 7.67 ± 0.31 mmol/24 h; P < 0.001). Nitric oxide excretion was 2935 ± 256 μmol/24 h in high salt‐fed rats, but reduced by 40% in the 20% fructose + high salt group (2139 ± 178 μmol /24 hrs P < 0.01). Our results suggest that fructose predisposes rats to salt‐sensitivity and, combined with a high salt diet, leads to sodium retention, increased blood pressure, and impaired renal nitric oxide availability.

Angiotensin-(1-7) Selectively Induces Relaxation and Modulates Endothelium-Dependent Dilation in Mesenteric Arteries of Salt-Fed Rats

This study investigated the acute effects of angiotensin-(1-7) and AVE0991 on active tone and vasodilator responses to bradykinin and acetylcholine in isolated mesenteric arteries from Sprague-Dawley rats fed a high-salt (HS; 4% NaCl) versus a normal salt (NS; 0.4% NaCl) diet. Angiotensin-(1-7) and AVE0991 elicited relaxation, and angiotensin-(1-7) unmasked vasodilator responses to bradykinin in arteries from HS-fed rats. These effects of angiotensin-(1-7) and AVE0991 were inhibited by endothelium removal, A779, PD123319, HOE140 and <smlcap>L</smlcap>-NAME. Angiotensin-(1-7) also restored the acetylcholine-induced relaxation that was suppressed by the HS diet. Vasodilator responses to bradykinin and acetylcholine in the presence of angiotensin-(1-7) were mimicked by captopril and the AT₂ receptor agonist CGP42112 in arteries from HS-fed rats. Thus, in contrast to salt-induced impairment of vascular relaxation in response to vasodilator stimuli, angiotensin-(1-7) induces endothelium-dependent and NO-mediated relaxation, unmasks bradykinin responses via activation of mas and AT₂ receptors, and restores acetylcholine-induced vasodilation in HS-fed rats. AT₂ receptor activation and angiotensin-converting enzyme (ACE) inhibition shared the ability of angiotensin-(1-7) to enhance bradykinin and acetylcholine responses in HS-fed rats. These findings suggest a therapeutic potential for mas and/or AT₂ receptor activation and ACE inhibition in restoring endothelial function impaired by elevated dietary salt intake or other pathological conditions.

Altered potassium ATP channel signaling in mesenteric arteries of old high salt-fed rats.

[Purpose]Both aging and the consumption of a high salt diet are associated with clear changes in the vascular system that can lead to the development of cardiovascular disease; however the mechanisms are not clearly understood. Therefore, we examined whether aging and the consumption of excess salt alters the function of potassium ATP-dependent channel signaling in mesenteric arteries[Methods]Young (7 months) and old (29 months) Fischer 344 x Brown Norway rats were fed a control or a high salt diet (8% NaCl) for 12 days and mesenteric arteries were utilized for vascular reactivity measurements.[Results]Acetylcholine-induced endothelium relaxation was significantly reduced in old arteries (81 ± 4%) when compared with young arteries (92 ± 2%). Pretreatment with the potassium-ATP channel blocker glibenclamide reduced relaxation to acetylcholine in young arteries but did not alter dilation in old arteries. On a high salt diet, endothelium dilation to acetylcholine was significantly reduced in old salt arteries (60 ± 3%) when compared with old control arteries (81 ± 4%). Glibenclamide reduced acetylcholine-induced dilation in young salt arteries but had no effect on old salt arteries. Dilation to cromakalim, a potassium-ATP channel opener, was reduced in old salt arteries when compared with old control arteries.[Conclusion]These findings demonstrate that aging impairs endothelium-dependent relaxation in mesenteric arteries. Furthermore, a high salt diet alters the function of potassium-ATP-dependent channel signaling in old isolated mesenteric arteries and affects the mediation of relaxation stimuli.

Endogenous hydrogen peroxide in the hypothalamic paraventricular nucleus regulates neurohormonal excitation in high salt-induced hypertension

Reactive oxygen species (ROS) in the brain plays an important role in the progression of hypertension and hydrogen peroxide (H2O2) is a major component of ROS. The aim of this study is to explore whether endogenous H2O2 changed by polyethylene glycol-catalase (PEG-CAT) and aminotriazole (ATZ) in the hypothalamic paraventricular nucleus (PVN) regulates neurotransmitters, renin-angiotensin system (RAS), and cytokines, and whether subsequently affects the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) in high salt-induced hypertension. Male Sprague–Dawley rats received a high-salt diet (HS, 8% NaCl) or a normal-salt diet (NS, 0.3% NaCl) for 10 weeks. Then rats were treated with bilateral PVN microinjection of PEG-CAT (0.2 i.u./50nl), an analog of endogenous catalase, the catalase inhibitor ATZ (10nmol/50nl) or vehicle. High salt-fed rats had significantly increased MAP, RSNA, plasma norepinephrine (NE) and pro-inflammatory cytokines (PICs). In addition, rats with high-salt diet had higher levels of NOX-2, NOX-4 (subunits of NAD(P)H oxidase), angiotensin-converting enzyme (ACE), interleukin-1beta (IL-1β), glutamate and NE, and lower levels of gamma-aminobutyric acid (GABA) and interleukin-10 (IL-10) in the PVN than normal diet rats. Bilateral PVN microinjection of PEG-CAT attenuated the levels of RAS and restored the balance of neurotransmitters and cytokines, while microinjection of ATZ into the PVN augmented those changes occurring in hypertensive rats. Our findings demonstrate that ROS component H2O2 in the PVN regulating MAP and RSNA are partly due to modulate neurotransmitters, renin-angiotensin system, and cytokines within the PVN in salt-induced hypertension.

Effects of high fructose and salt feeding on systematic metabonome probed via (1) H NMR spectroscopy.

Diets rich in high fructose and salt are increasingly popular in our daily life. A combination consumption of excessive fructose and salt can induce insulin resistance (IR) and hypertension (HT), which are major public health problems around the world. However, the effects of high fructose and salt on systematic metabonome remain unknown, which is very important for revealing the molecular mechanism of IR and HT induced by this dietary pattern. The metabolic profiling in urine, plasma, and fecal extracts from high fructose and salt-fed rats was investigated by use of (1) H nuclear magnetic resonance (NMR)-based metabonomics approach in this study. Multivariate analysis of NMR data showed the effects of high fructose and salt on the global metabonome. The metabolite analysis in urine and fecal extracts showed the time-dependent metabolic changes, which displayed metabonomic progression axes from normal to IR and HT status. The changes of 2-oxoglutarate, creatine and creatinine, citrate, hippurate, trimethylamine N-oxide (TMAO), and betaine in urine, together with gut microbiota disorder in feces, were observed at the preliminary formation stage of IR and HT (fourth week). At the severe stage (eighth week), the previously mentioned metabolic changes were aggravated, and the changes of lipid and choline metabolism in plasma suggested the increased risk of cardiovascular diseases. These findings provide an overview of biochemistry consequences of high fructose and salt feeding and comprehensive insights into the progression of systematic metabonome for IR and HT induced by this dietary pattern.

Response to “Does Angiotensin-Dependent Superoxide Production Help to Prevent Salt-Induced Endothelial Dysfunction in 2 Kidney–1 Clip Hypertensive Rats?”

To the Editor: We recently reported the surprising finding that the salt-induced endothelial dysfunction and downregulation of copper (Cu)/zinc (Zn) superoxide dismutase (SOD) that occurred in cerebral arteries of sham-operated animals were absent in 2 kidney–1 clip (2K1C) hypertensive rats, which exhibit prolonged elevations of plasma angiotensin II (ANG II) levels, and that AT1 receptor blockade with losartan eliminated endothelium-dependent vasodilation in 2K1C rats.1 We concluded that the elevated ANG II levels in the 2K1C rats played a key role in protecting against salt-induced vascular dysfunction and suppression of Cu/Zn SOD by upregulating antioxidant defense mechanisms in the arteries. In a letter to the editor entitled “Could AT1 Receptor Activation Increase Antioxidant Defense to Prevent Salt-Induced Vascular Dysfunction of 2 Kidney-1 Clip Hypertensive Rats?”, Dr Valdir A. Braga stated that it is difficult to draw conclusions about the vascular actions of ANG II without looking at oxidant enzymes and downstream production of reactive oxygen species. Braga cites a list of well-known and indisputable findings showing that ANG II, acting via AT1 receptors, activates NADPH oxidase to produce superoxide (O2 −) radicals and that ANG II has been linked to oxidative stress in the context of vascular dysfunction and hypertension. Based on those conclusions, Braga suggests that the upregulation of Cu/Zn SOD occurs in response to increases in O2 − rather than a possible antioxidant effect of ANG II. In our estimation, Braga’s conclusion that O2 − production in response to the elevated ANG II levels in the 2K1C rats may play an important role in preventing salt-induced endothelial dysfunction through upregulation of Cu/Zn SOD is quite plausible and could provide an explanation for the surprising findings in our study (i.e., the “antioxidant effect” of ANG II to upregulate Cu/Zn SOD in 2K1C rats). In support of that hypothesis, Papaiahgari et al.2 reported that O2 − produced by NADPH oxidase was essential to activate the ERK 1/2 pathway in C10 alveolar epithelial cells exposed to hyperoxia and that this led to upregulation of the nuclear transcription factor Nrf2, which upregulates multiple antioxidant and cell protective enzymes, including Cu/Zn SOD. Consistent with those findings, McEwen et al.3 showed that the protective effect of low-dose ANG II infusion to restore vascular relaxation mechanisms in cerebral arteries of salt-fed Sprague-Dawley rats is eliminated by AT1 receptor blockade and by inhibiting the ERK 1/2 pathway. Further emphasizing the importance of physiological levels of ANG II in maintaining normal vascular function is our recent study,4 which showed that diet-induced obesity, which is known to elevate plasma ANG II levels, restores normal endothelial function in a model of chronically low levels of plasma ANG II, the Dahl salt-sensitive rat.The precise mechanisms by which O2 − radicals formed by NADPH oxidase in response to elevated ANG II levels contribute to maintenance of antioxidant defense mechanisms (including Cu/Zn SOD) in 2K1C hypertensive rats remain to be determined. However, investigation of the relationship between ANG II levels, vascular O2 −, and activation of the Nrf2 antioxidant defense system appears to be a promising avenue of investigation.

Low-Dose Angiotensin II Infusion Restores Vascular Function in Cerebral Arteries of High Salt-Fed Rats by Increasing Copper/Zinc Superoxide Dimutase Expression

This study examined the vasoprotective role of circulating angiotensin II (ANG II) levels in the cerebral circulation of high salt (HS)-fed (SS.BN-(D13hmgc41-13hmgc23)/Mcwi) (Ren1-BN) congenic rats, which carry a normally functioning renin allele from the Brown Norway (BN) rat on the Dahl salt-sensitive genetic background. Ren1-BN rats were placed on an HS (4.0% NaCl) diet for 3 days. The vasodilator response to acetylcholine (ACh; 10(-10) - 10(-6) mol/L) was assessed in isolated middle cerebral arteries (MCAs), and Western blots were performed to assess the expression of the antioxidant enzymes copper (Cu)/zinc (Zn) superoxide dismutase (SOD) and manganese (Mn) SOD in cerebral resistance vessels. A separate group of HS-fed animals were infused with either a subpressor dose of ANG II (100ng/kg/min) or saline vehicle via osmotic minipump for 3 days. HS diet eliminated acetylcholine (ACh)-induced dilation in the MCAs of the congenic rats. Western blot analysis of antioxidant enzymes showed that Cu/Zn SOD and Mn SOD expression were significantly reduced in the cerebral resistance arteries of the HS-fed rats compared with control animals fed a normal salt diet. Infusion of ANG II restored the vasodilator response to ACh in the MCAs and increased Cu/Zn SOD (but not Mn SOD) expression compared with saline-infused animals. These results indicate that prevention of salt-induced ANG II suppression prevents vascular dysfunction in the cerebral circulation by preventing the downregulation of Cu/Zn SOD and vascular oxidant stress that normally occurs with HS diet.

Sympathetically Mediated Changes in Capacitance

“To be accepted as a paradigm, a theory must seem better than its competitors, but it need not, and in fact never does, explain all the facts with which it can be confronted.” Thomas Kuhn, The Structure of Scientific Revolutions ### Heard on Rounds “A 57-year-old patient with a history of heart failure states that she began experiencing worsening shortness of breath 4 days prior to admission, with lower extremity edema developing 1 day prior to admission. She is compliant with her medications, though has had similar admissions previously. She states that her weight at home increased by “a couple of pounds” 2 days ago and that she increased her diuretic dose but her symptoms worsened. She says that she generally adheres to her low salt diet, but admits to eating pretzels 5 days ago. The physical examination showed BP 136/84, HR 94, weight 3 lbs above baseline, elevated JVP, basilar rales, and 1+ pedal edema.” As the intern finishes the presentation, the Senior expresses surprise that the patient's weight had increased only 3 pounds, and comments about salt and volume overload leading to acute decompensated heart failure (ADHF). The Attending adds that recent data reveal that many patients presenting with ADHF have minimal or no weight gain, but that there is no clear mechanism to explain this phenomenon. They set in place plans for diuresis, increasing vasodilators, and further patient education. On the surface, there is nothing remarkable about this case, and the usual approaches to treat this entity have remained remarkably consistent since the advent of loop diuretics over 50 years ago: salt restriction, patient education, uptitration of medications if not already at optimal doses, and diuresis. However, approximately 1 in 4 such patients are readmitted to the hospital within 30 days,1 and the disease continues to progress. It is clear …

Acute and chronic angiotensin-(1–7) restores vasodilation and reduces oxidative stress in mesenteric arteries of salt-fed rats

This study determined the effect of ANG-(1-7) on salt-induced suppression of endothelium-dependent vasodilatation in the mesenteric arteries of male Sprague-Dawley rats. Chronic intravenous infusion of ANG-(1-7), oral administration of the nonpeptide mas receptor agonist AVE-0991, and acute preincubation of the arteries with ANG-(1-7) and AVE-0991 all restored vasodilator responses to both ACh and histamine that were absent in the arteries of rats fed a high-salt (4% NaCl) diet. The protective effects of ANG-(1-7) and AVE-0991 were inhibited by acute or chronic administration of the mas receptor antagonist A-779, the ANG II type 2 (AT(2)) receptor blocker PD-123319, or N-nitro-l-arginine methyl ester, but not the ANG II type 1 receptor antagonist losartan. Preincubation with the antioxidant tempol or the nitric oxide (NO) donor diethylenetriamine NONOate and acute and chronic administration of the AT(2) receptor agonist CGP-42112 mimicked the protective effect of ANG-(1-7) to restore vascular relaxation. Acute preincubation with ANG-(1-7) and chronic infusion of ANG-(1-7) ameliorated the elevated superoxide levels in rats fed a high-salt diet, but the expression of Cu/Zn SOD and Mn SOD enzyme proteins in the vessel wall was unaffected by ANG-(1-7) infusion. These results indicate that both acute and chronic systemic administration of ANG-(1-7) or AVE-0991 restore endothelium-dependent vascular relaxation in salt-fed Sprague-Dawley rats by reducing vascular oxidant stress and enhancing NO availability via mas and AT(2) receptors. These findings suggest a therapeutic potential for mas/AT(2) receptor activation in preventing the vascular oxidant stress and endothelial dysfunction associated with elevated dietary salt intake.

Viscum album (mistletoe) extract prevents changes in levels of red blood cells, PCV, Hb, serum proteins and ESR in high salt-fed rats

Aim of the study The present study was aimed to determine the effects of Viscum album (mistletoe) on red blood cells, packed cell volume, Hb content, absolute haematological values {mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), colour index (CI)}, plasma proteins and ESR in high salt-fed rats. Materials and methods 24 male albino Wistar rats were divided into 4 groups of 6 rats each. Group 1 received normal rat pellets + drinking water. Group 2 took same as group 1 + mistletoe extract (150 mg/kg body weight, orally once daily). Group 3 took high salt (8% NaCl) diet + 1% NaCl drinking water. Group 4 took same as group 3 + mistletoe extract (150 mg/kg body weight, orally once daily). The feeding regimens lasted for 6 weeks. Results We observed that the mean RBC, PCV and Hb in the control group were 5.21 ± 0.09 × 10 6 cells/mm 3, 43.50 ± 1.61%, and 10.88 ± 0.21 g/dl respectively. The extract significantly ( P < 0.05) reduced the RBC (5.72 ± 0.08 × 10 6 cells/mm 3), PCV (54.50 ± 2.64%) and Hb (14.33 ± 5.78 g/dl) in high salt-fed rats to near control levels. The extract also brought the elevated total plasma protein levels and reduced ESR in the high salt-fed rats (86.77 ± 1.08 g/L and 1.83 ± 0.31 mm/h respectively) to near control levels (82.23 ± 0.91 g/L and 2.83 ± 0.31 mm/h respectively), indicating the ability of the extract to prevent marked changes in the blood viscosity. The MCV, MCH, MCHC, and CI were not significantly altered by either extract or salt loading. Conclusion Crude mistletoe extract prevents changes in RBC, PCV, plasma protein levels, and ESR, and indication that the extract prevents changes in blood viscosity a major determinant of arterial blood pressure.

Angiotensin II maintains cerebral vascular relaxation via EGF receptor transactivation and ERK1/2

This study identified, on the integrative level, two components of the ANG II signaling pathway that lay downstream from the ANG II type 1 (AT(1)) receptor and are critically involved in maintaining vascular relaxation in cerebral resistance arteries. In these experiments, the relaxation of isolated middle cerebral arteries (MCA) in response to ACh (10(-9)-10(-5) M), iloprost (10(-16)-10(-11) g/ml), and reduced PO(2) was lost and the ratio of phospho-ERK/ERK1/2 was significantly reduced in aortas of male Sprague-Dawley rats fed a high-salt (HS; 4% NaCl) diet to suppress plasma ANG II levels. In salt-fed rats, relaxation of MCA in response to these vasodilator stimuli was restored by chronic (3 days) intravenous infusion of either ANG II (5 ngxkg(-1)xmin(-1)) or epidermal growth factor (EGF; 2 microg/h). The protective effect of ANG II infusion to restore vascular relaxation was eliminated by coinfusion of either the EGF receptor kinase inhibitor AG-1478 (20 microg/h), the ERK1/2 inhibitor PD-98059 (10 microg/h), or the protein synthesis inhibitor cycloheximide (5 microg/h). In rats fed a low-salt (0.4% NaCl) diet, MCA relaxation in response to ACh, reduced PO(2), and iloprost was eliminated by intravenous infusion of AG-1478, PD-98059, or cycloheximide. In ANG II-infused rats fed HS diet, and in rats fed LS diet, vasodilator responses to reduced PO(2) and iloprost were unaffected by the p38 MAP kinase inhibitor SB-203580 and the phosphatidylinositol 3-kinase inhibitor wortmannin. These findings indicate that maintenance of normal vascular relaxation mechanisms by ANG II in rat MCA requires activation of the EGF receptor kinase and ERK1/2.

Inhibition of Angiogenesis by High Salt Diet is Associated with Impaired Muscle Performance Following Chronic Muscle Stimulation

High dietary salt has been demonstrated to inhibit angiogenesis in skeletal muscle. The purpose of this study was to determine whether high salt impairs steady state muscle performance following a chronic stimulation protocol. Sprague-Dawley rats were placed on a control diet (CD, 0.4% NaCl) or high salt diet (HSD, 4.0% NaCl) prior to implantation of an electrical muscle stimulator. In chronically stimulated animals, hind limb muscles were stimulated to contract eight hours daily for seven days. Sham animals received a stimulator that was never activated. Following chronic stimulation, tibialis anterior (TA) muscles of animals on CD demonstrated an 84.6% increase in force of contraction at the end of an acute stimulation bout relative to sham animals fed CD. Decreased muscle fatigue was associated with an increase in capillaries per TA fiber (C:F). Chronic stimulation in HSD rats induced a smaller improvement (52.2%) in final force compared to HSD sham rats. This impairment of muscle performance in high salt-fed rats correlated with inhibited angiogenesis. Infusion of angiotensin II in HSD animals restored angiogenesis and muscle fatigue to CD levels. This study suggests that angiogenic inhibition by high salt is associated with impaired skeletal muscle performance following chronic stimulation.

Long-Term Inhibition of the Central α 2B-Adrenergic Receptor Gene Via Recombinant AAV-Delivered Antisense in Hypertensive Rats

Salt-induced hypertension is mediated via the alpha(2B)-adrenergic receptor (AR) subtype. In alpha(2B)-AR gene knockout mice, blood pressure (BP) does not rise with salt loading, and in rats with salt-induced hypertension, BP decreases transiently with antisense (AS) treatment targeting the alpha(2B)-AR gene. The present experiments were designed to explore the possibility of gene transfection in the brain by intracerebroventricular (ICV) delivery of AS-DNA via adeno-associated virus (AAV) to prolong alpha(2B)-AR inhibition and hence reversal of salt-dependent hypertension. A recombinant AAV (rAAV) vector preparation encoding the alpha(2B)-AS fragment (previously tested in vitro for inhibition of alpha(2B)-AR protein production in cells) and containing green fluorescence protein (GFP) for visualization was injected ICV into subtotally nephrectomized, salt-fed rats. Control rats received rAAV-GFP (n = 8 per group). We observed that BP rose from a baseline of 120 +/- 10 to 184 +/- 12 mm Hg. Injection of rAAV-alpha(2B)-AS produced a 35 +/- 12 mm Hg fall in BP, lasting without evidence of diminishing for at least 16 days, whereas rAAV-GFP-injected rats showed a continued rise in BP. Rats treated with rAAV-alpha(2B)-AS treated had a 45% to 65% decrease in alpha(2B)-AR protein levels in key regulatory regions of the brain. Neither group had signs of immunologic response to the virus injection. These results indicate that our construct, when given by ICV means, could reach multiple sites of the central nervous system relevant to BP regulation and could safely inhibit the central alpha(2B)-adrenergic receptor, thereby achieving prolonged reversal of salt-induced hypertension.

Angiotensin II infusion restores stimulated angiogenesis in the skeletal muscle of rats on a high-salt diet

Elevated dietary salt intake has previously been demonstrated to have dramatic effects on microvascular structure and function. The purpose of this study was to determine whether a high-salt diet modulates physiological angiogenesis in skeletal muscle. Male Sprague-Dawley rats were placed on a control diet (0.4% NaCl by weight) or a high-salt diet (4.0% NaCl) before implantation of a chronic electrical stimulator. After seven consecutive days of unilateral hindlimb muscle stimulation, animals on control diets demonstrated a significant increase in microvessel density in the tibialis anterior muscle of the stimulated hindlimb relative to the contralateral control leg. High salt-fed rats demonstrated a complete inhibition of this angiogenic response, as well as a significant reduction in plasma ANG II levels compared with those of control animals. To investigate the role of ANG II suppression on the inhibitory effect of high-salt diets, a group of rats that were fed high salt were chronically infused with ANG II at a low dose. Maintenance of ANG II levels restored stimulated angiogenesis to control levels in animals fed a high-salt diet. Western blot analysis indicated that inhibition of angiogenesis in high salt-fed rats was not due to changes in VEGF or VEGF receptor type 1 protein expression in response to stimulation; however, the degree to which VEGF receptor 2 protein increased with stimulation was significantly lower in high salt-fed animals. This study demonstrates an inhibitory effect of high salt intake on stimulated angiogenesis and suggests a critical role for ANG II suppression in mediating this antiangiogenic effect.

Reduced Arteriolar Responses to Skeletal Muscle Contraction after Ingestion of a High Salt Diet

We previously reported that in skeletal muscle arterioles of rats fed a very high salt (HS; 7%) diet, the bioavailability of endothelium-derived nitric oxide (NO) is reduced through scavenging by reactive oxygen species. Because arteriolar NO can play an important role in local blood flow control, we investigated whether arteriolar responses to increased tissue metabolism become compromised in skeletal muscle of salt-fed rats. Consumption of a HS (4%) diet for 4 weeks had no effect on arteriolar diameters, volume flow or shear stress in resting spinotrapezius muscle. Arteriolar responses to a modest elevation in metabolic demand (0.5 Hz contraction) were not different from those in rats fed a normal diet, but diameter responses to a greater elevation in metabolic demand (4 Hz contraction) were significantly less in HS rats than in rats fed a normal diet. In both groups, the NO synthase inhibitor N^G-monomethyl-L-arginine reduced resting arteriolar diameters and flow by a similar amount and had little or no effect on arteriolar diameter or flow responses to muscle contraction. Arterioles in HS rats exhibited an increase in overall oxidant activity (tetranitroblue tetrazolium reduction) but not in superoxide activity (dihydroethidine oxidation). Reactive oxygen species scavengers (2,2,6,6-tetramethylpiperidine-N-oxyl and catalase) did not normalize the reduced arteriolar responses to muscle contraction in HS rats. These findings suggest that increased oxidant activity in the arteriolar network of salt-fed rats is not due to accumulation of superoxide anion and that neither this oxidant activity nor reduced NO availability can account for the blunted active arteriolar dilation in rats fed a 4% salt diet.

Exaggerated response to adenosine in kidneys from high salt-fed rats: role of epoxyeicosatrienoic acids

Cytochrome P-450 (CYP)-dependent epoxyeicosatrienoic acids (EETs) dilate rat preglomerular microvessels when adenosine(2A) receptors (A(2A)R) are stimulated. As high salt (HS) intake increases epoxygenase activity and adenosine levels, we hypothesized that renal adenosine responses would be greater in HS-fed rats. Male Sprague-Dawley rats were fed either HS (4.0% NaCl) or normal salt (NS; 0.4% NaCl) diet. On day 8, isolated kidneys were perfused with Krebs' buffer containing indomethacin (10 microM) and L-NAME (200 microM) and preconstricted to approximately 150 mmHg with infusion of phenylephrine (10(-7) M). Renal effluents were extracted for analysis of eicosanoids by gas chromatography-mass spectrometry. Bolus injections of the stable adenosine analog 2-chloroadenosine (2-CA; 0.1-10 microg) resulted in dose-dependent dilation; at 10 microg, perfusion pressure (PP) was lowered to a greater extent in the kidneys of HS rats compared with NS rats (-60 +/- 4 vs. -31 +/- 8 mmHg; P < 0.05) and the area of response was increased (27 +/- 6 vs. 9 +/- 4 mm(2); P < 0.05), as was EET release (132 +/- 23 vs. 38 +/- 18 ng; P < 0.05). HS treatment increased A(2A)R and CYP2C23 protein expression. A selective epoxygenase inhibitor, MS-PPOH (12 microM), significantly reduced the response to 2-CA in HS rats; PP, area of response, and EET release decreased by 40, 70, and 81%, respectively, whereas lesser changes were evident in NS kidneys. Thus the greater vasodilator response to 2-CA seen in kidneys obtained from HS-fed rats was mediated by increased EET release. As EETs are renal vasodilator and natriuretic eicosanoids, interactions between adenosine and EETs may contribute to the adaptive response to HS intake.

Development of structural vascular changes in salt-fed rats

The hypothesis that long-term administration of a physiologically relevant high salt diet to rats leads to the development of structural vascular changes that predispose to hypertension was tested. Adult male Sprague-Dawley rats were fed 2% NaCl diet for 3 or 6 months; rats fed 0.7% NaCl diet were controls. Then, the systemic circulation of the rats was perfusion-fixed at 100 mm Hg. The junction of the mesentery and small intestine, the renal cortex, and segments of left carotid artery, thoracic aorta, and second order mesenteric arteries were embedded in paraffin or epoxy for morphometric measurements. The average monthly tail systolic blood pressure (BP) of salt-fed rats at 3 and 6 months were unchanged. The following morphometric changes in salt-fed rats were observed: 1) dilatation of the carotid artery at 3 months ( P < .05); 2) dilatation and reduced wall-to-lumen ratio of the second order mesenteric artery ( P < .01); 3) increased wall-to-lumen ratio of small mesenteric resistance arteries ( P < .01); 4) reduced wall-to-lumen ratio of renal cortical resistance arteries at 6 months ( P <. 05); and 5) unchanged structure of aorta. The long-term administration of a high salt diet leads to structural vascular changes in normotensive rats. There are important regional and segmental variations in the long-term adaptation of arteries to a high salt diet.