NaCl Intake Research Articles

Angiotensinergic and cholinergic receptors of the subfornical organ mediate sodium intake induced by GABAergic activation of the lateral parabrachial nucleus

Bilateral injections of the GABAA agonist muscimol into the lateral parabrachial nucleus (LPBN) induce 0.3M NaCl and water intake in satiated and normovolemic rats, a response reduced by intracerebroventricular (icv) administration of losartan or atropine (angiotensinergic type 1 (AT1) and cholinergic muscarinic receptor antagonists, respectively). In the present study, we investigated the effects of the injections of losartan or atropine into the subfornical organ (SFO) on 0.3M NaCl and water intake induced by injections of muscimol into the LPBN. In addition, using intracellular calcium measurement, we also tested the sensitivity of SFO-cultured cells to angiotensin II (ANG II) and carbachol (cholinergic agonist). In male Holtzman rats with cannulas implanted bilaterally into the LPBN and into the SFO, injections of losartan (1μg/0.1μl) or atropine (2nmol/0.1μl) into the SFO almost abolished 0.3M NaCl and water intake induced by muscimol (0.5nmol/0.2μl) injected into the LPBN. In about 30% of the cultured cells of the SFO, carbachol and ANG II increased intracellular calcium concentration ([Ca2+]i). Three distinct cell populations were found in the SFO, i.e., cells activated by either ANG II (25%) or carbachol (2.6%) or by both stimuli (2.3%). The results suggest that the activation of angiotensinergic and cholinergic mechanisms in the SFO is important for NaCl and water intake induced by the deactivation of LPBN inhibitory mechanisms with muscimol injections. They also show that there are cells in the SFO activated by both angiotensinergic and cholinergic stimuli, perhaps those involved in the responses to muscimol into the LPBN.

Prostaglandin E2 induces chloride secretion through crosstalk between cAMP and calcium signaling in mouse inner medullary collecting duct cells

Under conditions of high dietary salt intake, prostaglandin E2 (PGE2) production is increased in the collecting duct and promotes urinary sodium chloride (NaCl) excretion; however, the molecular mechanisms by which PGE2 increases NaCl excretion in this context have not been clearly defined. We used the mouse inner medullary collecting duct (mIMCD)-K2 cell line to characterize mechanisms underlying PGE2-regulated NaCl transport. When epithelial Na(+) channels were inhibited, PGE2 exclusively stimulated basolateral EP4 receptors to increase short-circuit current (Isc(PGE2)). We found that Isc(PGE2) was sensitive to inhibition by H-89 and CFTR-172, indicating that EP4 receptors signal through protein kinase A to induce Cl(-) secretion via cystic fibrosis transmembrane conductance regulator (CFTR). Unexpectedly, we also found that Isc(PGE2) was sensitive to inhibition by BAPTA-AM (Ca(2+) chelator), 2-aminoethoxydiphenyl borate (2-APB) (inositol triphosphate receptor blocker), and flufenamic acid (FFA) [Ca(2+)-activated Cl(-) channel (CACC) inhibitor], suggesting that EP4 receptors also signal through Ca(2+) to induce Cl(-) secretion via CACC. Additionally, we observed that PGE2 stimulated an increase in Isc through crosstalk between cAMP and Ca(2+) signaling; BAPTA-AM or 2-APB inhibited a component of Isc(PGE2) that was sensitive to CFTR-172 inhibition; H-89 inhibited a component of Isc(PGE2) that was sensitive to FFA inhibition. Together, our findings indicate that PGE2 activates basolateral EP4 receptors and signals through both cAMP and Ca(2+) to stimulate Cl(-) secretion in IMCD-K2 cells. We propose that these signaling pathways, and the crosstalk between them, may provide a concerted mechanism for enhancing urinary NaCl excretion under conditions of high dietary NaCl intake.

Transforming Growth Factor-β Regulates Endothelial Function During High Salt Intake in Rats

Previous studies have demonstrated that an increase in dietary NaCl (salt) intake stimulated endothelial cells to produce transforming growth factor-β (TGF-β). The intent of the present study was to determine the functional significance of increased TGF-β on endothelial cell function. Young Sprague-Dawley rats were fed diets containing 0.3 or 8.0% NaCl for 2 days before treatment with a specific inhibitor of the TGF-β receptor I/activin receptor-like kinase 5 kinase, or vehicle for another 2 days. At day 4 of study, endothelial phosphorylated Smad2 (S465/467) increased and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) levels decreased in the high-salt–treated rats. In addition, phosphorylated Akt (S473) and phosphorylation of the endothelial isoform of NO synthase (NOS3) at S1177 increased. Treatment with the TGF-β receptor I/activin receptor-like kinase 5 inhibitor reduced Smad2 phosphorylation to levels observed in rats on the low-salt diet and prevented the downstream signaling events induced by the high-salt diet. In human umbilical vein endothelial cells, reduction in PTEN levels increased phosphorylated Akt and NOS3. Treatment of macrovascular endothelial cells with TGF-β1 increased phosphorylated NOS3 and the concentration of NO metabolites in the medium but had no effect on either of these variables in cells pretreated with small interfering RNA directed against PTEN. Thus, during high salt intake, an increase in TGF-β directly promoted a reduction in endothelial PTEN levels, which in turn regulated Akt activation and NOS3 phosphorylation. This effect closes a feedback loop that potentially mitigates the effect of TGF-β on the vasculature.

Facilitation of sodium intake by combining noradrenaline into the lateral parabrachial nucleus with prazosin peripherally

Injections of noradrenaline into the lateral parabrachial nucleus (LPBN) increase arterial pressure and 1.8% NaCl intake and decrease water intake in rats treated with the diuretic furosemide (FURO) combined with a low dose of the angiotensin converting enzyme inhibitor captopril (CAP). In the present study, we investigated the influence of the pressor response elicited by noradrenaline injected into the LPBN on FURO+CAP-induced water and 1.8% NaCl intake. Male Holtzman rats with bilateral stainless steel guide-cannulas implanted into LPBN were used. Bilateral injections of noradrenaline (40nmol/0.2μl) into the LPBN increased FURO+CAP-induced 1.8% NaCl intake (12.2±3.5, vs., saline: 4.2±0.8ml/180min), reduced water intake and strongly increased arterial pressure (50±7, vs. saline: 1±1mmHg). The blockade of the α1 adrenoceptors with the prazosin injected intraperitoneally abolished the pressor response and increased 1.8% NaCl and water intake in rats treated with FURO+CAP combined with noradrenaline injected into the LPBN. The deactivation of baro and perhaps volume receptors due to the cardiovascular effects of prazosin is a mechanism that may facilitate water and NaCl intake in rats treated with FURO+CAP combined with noradrenaline injected into the LPBN. Therefore, the activation of α2 adrenoceptors with noradrenaline injected into the LPBN, at least in dose tested, may not completely remove the inhibitory signals produced by the activation of the cardiovascular receptors, particularly the signals that result from the extra activation of these receptors with the increase of arterial pressure.

Disorder of cholesterol and electrolytes metabolism in hypertension

Hypertension is an asymptomatic, important disease regarded as a multifactorial disorder of modern life. The frequency of hypertension range from 6-32%. People with hypertension have lipid abnormality and electrolyte abnormalities when compared to individual with normotensive. There is positive association between sodium chloride (Nacl) intake and blood pressure. In present study we are going to evaluate serum cholesterol and electrolyte level in hypertensive and in normotensive. The present study was carried out on total 200 subjects, which were divided into two groups. First group of experimental group consists of 100 subjects with known hypertensive patient (B.P>140/90mm of Hg). Another group is control group consists of 100 subjects who were healthy normotensive (B.P<140/90 mm of Hg) with no past or present history of hypertension.12 hours overnight fasting venous blood sample was collected from both the subject group in plane vacutainer and the sample was centrifuged for the estimation of fasting cholesterol level and electrolytes i.e. sodium (Na + ), Potassium (K + ). The study was carried out on two group of population, one group was subject group i.e. hypertensive group, consisting of 65 male and 35 females (total population =100) and another group is the control group i.e normotensive, consisting of 60 male and 40 females (total population = 100). The total cholesterol level significantly elevated in subject group compared to control group (p<0.001). The sodium level is statistically elevated in subject group when compared to control group (P<0.001). The potassium level significantly decreased in subject group when compared to control group (P<0.001). It has been noticed that the serum cholesterol level, serum sodium level were stastically elevated in hypertensive subjects, while serum potassium level is significantly reduced in hypertensive subject when compared to normotensive subjects.

Abstract 357: Role of Blood Pressure in Aldosterone-Mediated Cardiac Injury

Excess aldosterone (ALDO) causes hypertension (HTN), and cardiac hypertrophy, injury and dysfunction. The role of HTN in ALDO-mediated cardiac injury remains controversial. Whether cardiac injury is blood pressure (BP)-dependent, -independent or exacerbated by HTN is not fully understood. We aim to study the role of BP in ALDO-mediated cardiac hypertrophy, inflammation and fibrosis. Eight-week old uninephrectomized male Sprague Dawley rats (N=6/grp) were randomly assigned to the following 3 groups: Control, ALDO/SALT, ALDO/SALT+3-AHT (Triple Anti-Hypertensive Therapy). ALDO (0.75 μg/h) or vehicle (PEG 300) was administered by osmotic minipumps. SALT (1.0 % NaCl + 0.3 % KCl) was administered in tap water. 3-AHT (240 mg/kg hydralazine, 75 mg/kg hydrochlorothiazide, 15 mg/kg reserpine) was administered in the food. Another set of animals (N=6/grp) was implanted with radiotelemetry probes for BP monitoring. Left ventricle (LV) gene expression (GE) was quantified by qRT-PCR. 3-AHT reduced MAP (126 ± 4 vs. 178 ± 3 mm Hg, p&lt;0.05 vs. ALDO, controls: 108 ± 1). 3-AHT attenuated cardiac (3.8 ± 0.1 vs. 4.0 ± 0.1 HW/BW, p&lt;0.05 vs. ALDO; controls: 3.5 ± 0.1) and LV (2.8 ± 0.1 vs. 3.0 ± 0.1 LVW/BW, p&lt;0.05 vs. ALDO; controls: 2.4 ± 0.1) hypertrophy. 3-AHT abolished ALDO-mediated LV GE upregulation of fibrosis (collagen I, collagen III, fibronectin, Timp1, periostin) and inflammation (osteopontin, MCP-1, TGFβ, Hmox1) markers. A similar effect was observed with novel LV ALDO-target genes such as Reg3b, collagen VIII, Prss35, Ltbp2 or Pcp4. 3-AHT drug administration in the food caused no change in NaCl intake. However, to avoid the confounding effect of the natriuretic drug hydrochlorothiazide, a group of animals was treated with hydralazine and reserpine alone (2-AHT). 2-AHT treatment attenuated HTN (135 ± 4 vs. 178 ± 3 mm Hg, p&lt;0.05 vs. ALDO) but did not modify ALDO-mediated cardiac or LV hypertrophy. However, 2-AHT also abolished ALDO-mediated GE upregulation of fibrosis and inflammation markers, as well as novel LV ALDO-target genes. These results suggest that ALDO-mediated LV GE regulation in vivo is BP-dependent without excluding that ALDO may exacerbate the HTN-mediated cardiac injury. A stricter BP management may benefit patients with elevated ALDO levels.

Abstract 553: Prostaglandin E2 EP3 Receptor Downregulates COX2 Expression in the Medullary Thick Ascending Limb (mTAL) Induced by Hypertonic NaCl Intake

We recently showed that a novel negative feedback mechanism involving PGE 2 acting on EP3 receptors regulates cyclooxygenase-2 (COX-2) expression in the thick ascending limb (TAL) induced by the selective COX-2 inhibitors, celecoxib and rofecoxib. In the present study we tested the hypothesis that inhibition of EP3 facilitates COX-2 expression in the TAL induced by ingestion of hypertonic NaCl or exposure of mTAL cells to hypertonic media. COX-2 protein expression in the outer medulla (OM) increased 2.2±0.3 fold in mice given free access to 1% NaCl in the drinking water for 3 days compared with tap water; p&lt;0.05. The increase was associated with a 4-fold elevation in COX-2 mRNA accumulation (tap water: 0.5±0.1 vs 1% NaCl: 1.9±0.4; p&lt;0.05), and higher PGE 2 production by freshly isolated mTAL tubules (tap water: 87.6±9.4 vs 1% NaCl: 203.3±19.3 pg/mg protein; p&lt;0.05). EP3 mRNA levels also increased approximately 2-fold in OM of mice ingesting 1% NaCl (tap water: 0.7±0.2 vs 1% NaCl: 1.3±0.2). Administration of a selective EP3 receptor antagonist (L-798106: 20μg/kg/day) for 2 days increased COX-2 mRNA accumulation in mTAL tubules (L-798106: 2.1±0.1 fold change vs control; p&lt;0.05), and the elevation in COX-2 protein expression induced by 1% NaCl was increased an additional 50% in mice given L-798106. COX-2 mRNA accumulation in primary cultures of mTAL cells increased 2-fold in response to media made hypertonic by addition of NaCl (400 mosmol/kg H 2 O), compared with cells incubated in isotonic media. L-798106 increased COX-2 mRNA 2-fold in isotonic media and 4-fold in cells exposed to 400 mosmol/kg H 2 O. PGE 2 production by mTAL cells increased approximately 4-fold in response to challenge with 400 mosmol/kg H 2 O for 9 hr, and was inhibited in cells transiently transfected with a lentivirus shRNA construct (EGFP-C2-ex5) to silence COX-2 (286.1±14.8 to 64.3±5.2 pg/mg protein; p&lt;0.05). Collectively, the data suggest that local hypertonicity in the mTAL is associated with an increase in COX-2 expression concomitant with elevated EP3 receptor expression, which attenuates COX-2 activity in this segment of the nephron. Moreover, PGE 2 signaling via EP3 receptors in the TAL may be part of a mechanism that regulates mTAL COX-2 expression and function in response to diverse stimuli.

Abstract 480: NaCl-Independent Dietary Effects are Associated with Altered DNA Methylation in Dahl Salt-Sensitive (SS) Hypertension

Salt-sensitive hypertension and renal damage were compared in colonies of SS rats fed casein-and grain-based diets. When 6 week old rats were fed a low NaCl diet (n=7-11/group), mean arterial pressure (MAP) and urinary albumin (UAlb) excretion were not significantly different in grain-fed rats compared to values in casein-fed rats (107±2 mmHg and 6±2 mg/day). Increasing dietary NaCl intake to 4.0% for three weeks led to a significantly greater increase in MAP (171±7 vs 119±7 mmHg), UAlb (164±19 vs 24±6 mg/day) and renal histological damage in SS rats fed the casein diet (p&lt;.05). To assess mechanisms of this NaCl independent effect, an RNA-Seq analysis of renal outer medullary tissue of rats (n=4/group) fed the grain- or casein-based diets was performed. Over 1500 known genes were differentially expressed between the casein- and grain-fed rats on low salt; over 2100 were different between the groups fed the 4.0% chow (FDR&lt;0.05); and 897 genes were common to these two data sets. The differentially expressed genes were significantly enriched for genes related to hematopoietic cell lineage, the complement and coagulation cascade, B-cell signaling, NK cell-mediated cytotoxicity, and primary immunodeficiency. To understand potential mechanisms of the regulation of gene expression, a genome-wide DNA methylation analysis of the renal outer medulla at single-base resolution was performed. Of nearly 3,000 methylated CpG islands identified, 534 and 650 were differentially methylated between the casein and grain fed groups when comparing effects of low to high salt diets, respectively (FDR&lt;0.05). Approximately 200 differentially methylated CpG islands were located in the proximity of transcriptional start sites. We first focused our attention on several genes which exhibited a reciprocal pattern of DNA methylation and RNA abundance. These include Klotho (an aging-related gene), and Cyp39a1 (a cytochrome P450) which exhibited significantly more 5-methylcytosine modifications in the transcription start site and reduced mRNA expression in rats fed the casein-based diet with low salt. These data provide evidence that epigenetic modifications due to NaCl-independent dietary effects may alter salt-sensitive blood pressure and renal disease phenotypes.

Would right atrial stretch inhibit sodium intake following GABAA receptor activation in the lateral parabrachial nucleus?

The knowledge of the mechanisms underlying circulating volume control may be achieved by stretching a balloon placed at the junction of the superior vena cava–right atrial junction (SVC–RAJ). We investigated whether the inflation of a balloon at the SVC–RAJ inhibits the intake of 0.3M NaCl induced by GABAA receptor activation in the lateral parabrachial nucleus (LPBN) in euhydrated and satiated rats. Male Wistar rats (280–300g) with bilateral stainless steel LPBN cannulae and balloons implanted at the SVC–RAJ were used. Bilateral injections of the GABAA receptor agonist muscimol (0.5ηmol/0.2l) in the LPBN with deflated balloons increased intake of 0.3M NaCl (30.1±3.9 vs. saline: 2.2±0.7)ml/210min, n=8) and water (17.7±1.9 vs. saline: 2.9±0.5ml/210min). Conversely, 0.3M NaCl (27.8±2.1ml/210min) and water (22.8±2.3ml/210min) intake were not affected in rats with inflated balloons at the SVC–RAJ. The results show that sodium and water intake induced by muscimol injected into the LPBN was not affected by balloon inflation at the SVC–RAJ. We suggest that the blockade of LPBN neuronal activity with muscimol injections impairs inhibitory mechanisms activated by signals from cardiopulmonary volume receptors determined by balloon inflation.

Interaction between dietary content of protein and sodium chloride on milk urea concentration, urinary urea excretion, renal recycling of urea, and urea transfer to the gastrointestinal tract in dairy cows

Dietary protein and salt affect the concentration of milk urea nitrogen (MUN; mg of N/dL) and the relationship between MUN and excretion of urea nitrogen in urine (UUN; g of N/d) of dairy cattle. The aim of the present study was to examine the effects of dietary protein and sodium chloride (NaCl) intake separately, and their interaction, on MUN and UUN, on the relationship between UUN and MUN, on renal recycling of urea, and on urea transfer to the gastrointestinal tract. Twelve second-parity cows (body weight of 645±37kg, 146±29d in milk, and a milk production of 34.0±3.28kg/d), of which 8 were previously fitted with a rumen cannula, were fitted with catheters in the urine bladder and jugular vein. The experiment had a split-plot arrangement with dietary crude protein (CP) content as the main plot factor [116 and 154g of CP/kg of dry matter (DM)] and dietary NaCl content as the subplot factor (3.1 and 13.5g of Na/kg of DM). Cows were fed at 95% of the average ad libitum feed intake of cows receiving the low protein diets. Average MUN and UUN were, respectively, 3.90mg of N/dL and 45g of N/d higher for the high protein diets compared with the low protein diets. Compared with the low NaCl diets, MUN was, on average, 1.74mg of N/dL lower for the high NaCl diets, whereas UUN was unaffected. We found no interaction between dietary content of protein and NaCl on performance characteristics or on MUN, UUN, urine production, and renal clearance characteristics. The creatinine clearance rate was not affected by dietary content of protein and NaCl. Urea transfer to the gastrointestinal tract, expressed as a fraction of plasma urea entry rate, was negatively related to dietary protein, whereas it was not affected by dietary NaCl content. We found no interaction between dietary protein and NaCl content on plasma urea entry rate and gastrointestinal urea entry rate or their ratio. The relationship between MUN and UUN was significantly affected by the class variable dietary NaCl content: UUN=−17.7±7.24 + 10.09±1.016 × MUN + 2.26±0.729 × MUN (for high NaCl); R2=0.85. Removal of the MUN × NaCl interaction term lowered the coefficient of determination from 0.85 to 0.77. In conclusion, dietary protein content is positively related to MUN and UUN, whereas dietary NaCl content is negatively correlated to MUN but NaCl content is not related to UUN. We found no interaction between dietary protein and NaCl content on performance, MUN, UUN, or renal urea recycling, nor on plasma urea entry rate and urea transfer to the gastrointestinal tract. For a proper interpretation of the relationship between MUN and UUN, the effect of dietary NaCl should be taken into account, but we found no evidence that the effect of dietary NaCl on MUN is dependent on dietary protein content.

Periodontal disease reduces water and sodium intake induced by injection of muscimol into the lateral parabrachial nucleus

ObjectiveGamma-aminobutyric acid A (GABAA) receptor activation with muscimol in the lateral parabrachial nucleus (LPBN) induces water and 0.3M NaCl intake. The purpose of this study was to investigate whether a local inflammatory event, such as periodontal disease (PD), is able to alter the effects of muscimol on water and 0.3M NaCl intake in fluid-replete rats and in rats treated with furosemide (FURO) combined with captopril (CAP) injected subcutaneously. DesignMale Wistar rats were divided into two groups: with PD and those without PD (control condition). Fifteen days after PD, both groups had cannulas implanted bilaterally into the LPBN. ResultsIn fluid-replete rats without PD, injections of muscimol (0.5nmol/0.2μl) into the LPBN induced 0.3M NaCl and water intake and a pressor response. In fluid-replete rats with PD, a decrease was observed in water intake and pressor response but not in 0.3M NaCl intake. In control rats with FURO+CAP treatment, injections of muscimol into the LPBN increased 0.3M NaCl and water intake. In PD rats with FURO+CAP treatment, a decrease was observed in 0.3M NaCl and water intake after muscimol in the LPBN. Alveolar bone loss and interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) plasmatic concentration were higher in PD rats in comparison with controls. ConclusionThese results suggest that PD is able to reduce the pressor response and the dipsogenic and natriorexigenic effects induced by the activation of GABAA receptors in the LPBN, probably due to the elevation of the plasmatic concentration of pro-inflammatory cytokines IL-6 and TNF-α.

Baclofen into the lateral parabrachial nucleus induces hypertonic sodium chloride intake during cell dehydration

BackgroundActivation of GABAB receptors with baclofen into the lateral parabrachial nucleus (LPBN) induces ingestion of water and 0.3 M NaCl in fluid replete rats. However, up to now, no study has investigated the effects of baclofen injected alone or combined with GABAB receptor antagonist into the LPBN on water and 0.3 M NaCl intake in rats with increased plasma osmolarity (rats treated with an intragastric load of 2 M NaCl). Male Wistar rats with stainless steel cannulas implanted bilaterally into the LPBN were used.ResultsIn fluid replete rats, baclofen (0.5 nmol/0.2 μl), bilaterally injected into the LPBN, induced ingestion of 0.3 M NaCl (14.3 ± 4.1 vs. saline: 0.2 ± 0.2 ml/210 min) and water (7.1 ± 2.9 vs. saline: 0.6 ± 0.5 ml/210 min). In cell-dehydrated rats, bilateral injections of baclofen (0.5 and 1.0 nmol/0.2 μl) into the LPBN induced an increase of 0.3 M NaCl intake (15.6 ± 5.7 and 21.5 ± 3.5 ml/210 min, respectively, vs. saline: 1.7 ± 0.8 ml/210 min) and an early inhibition of water intake (3.5 ± 1.4 and 6.7 ± 2.1 ml/150 min, respectively, vs. saline: 9.2 ± 1.4 ml/150 min). The pretreatment of the LPBN with 2-hydroxysaclofen (GABAB antagonist, 5 nmol/0.2 μl) potentiated the effect of baclofen on 0.3 M NaCl intake in the first 90 min of test and did not modify the inhibition of water intake induced by baclofen in cell-dehydrated rats. Baclofen injected into the LPBN did not affect blood pressure and heart rate.ConclusionsThus, injection of baclofen into the LPBN in cell-dehydrated rats induced ingestion of 0.3 M NaCl and inhibition of water intake, suggesting that even in a hyperosmotic situation, the blockade of LPBN inhibitory mechanisms with baclofen is enough to drive rats to drink hypertonic NaCl, an effect independent of changes in blood pressure.

Mineralocorticoid receptor in NTS stimulates salt intake during 4th ventricular infusions of aldosterone

The purpose of this study was to determine if neurons within the NTS that possess the mineralocorticoid receptor (MR) play a role in aldosterone stimulation of salt intake. Adult WKY rats received microinjections into the NTS of a small, hairpin RNA for the MR (shRNA; n=9) or a scrambled RNA (scRNA; n=8). After the viral construct injections, aldosterone‐filled osmotic mini‐pumps were implanted subcutaneously and connected to tubing within the 4th ventricle to infuse aldosterone at a rate of 20ng/h. Prior to and after surgeries, rats had ad libitum access to food and two graduated drinking bottles filled with distilled water and 0.3M NaCl and salt intake expressed as 100 × the ratio of 0.3M NaCl intake to total fluid intake. Prior to surgeries, basal salt intake was 4.7% ± 2.9% in the scRNA group and 3.91% ± 2.6% in the shRNA group. 24 days post‐surgery salt intake was 20.6% ± 2.9% in the scRNA group and 4.3% ± 2.8% in the shRNA group. Post‐mortem immunohistochemistry revealed a significant reduction in the number of NTS neurons exhibiting immunoreactivity for the MR (scRNA 33 ± 2 cells/section; shRNA 23 ± 1 cells/section; p=.008). These results indicate 4th ventricular infusions of aldosterone stimulate salt intake and that at least part of the stimulation of salt intake during 4th ventricular infusions of aldosterone is mediated by hindbrain NTS neurons that possess the MR.

Expressions of epithelial Na+ channel subunits in the supraoptic nucleus of the hypothalamus in the male rat in response to dietary NaCl intake

All three epithelial Na+ channel (ENaC) subunits (α, β, and γ) were demonstrated in vasopressin (VP) synthesizing magnocellular neurons (MNCs) in the hypothalamic supraoptic (SON) and paraventricular nuclei. Our recent findings showed that ENaC mediates a Na+ leak current that changes the frequency of action potentials in MNCs. In the present study, we assessed the effect of dietary NaCl intake on ENaC subunits in the SON. Male rats were fed either a 8% NaCl diet, a NaCl deficient, or a control diet for two weeks. Then, relative changes in the subunit mRNAs in the SON and kidney were measured by real‐time RT‐PCR. The 8% NaCl diet caused significant increase in β‐ and γ‐ENaC mRNAs in the SON, while expression of α‐ENaC did not change. In contrast, the 8% NaCl diet did not cause any marked changes in expressions of the subunits in the kidney, while a deficiency in dietary NaCl caused a significant rise in α‐ENaC mRNA. Change in the subcellular distribution of α‐ENaC immunoreactivety in MNCs was also observed using confocal microscopy. The labeling in the MNCs from the 8% NaCl diet group was distinctively concentrated towards the plasma membrane in all 5 rats tested, while those from the NaCl deficient and control groups were mainly dispersed in the cytoplasm with no distinct labeling at the plasma membrane. These findings imply that dietary NaCl affects ENaC activity in VP MNCs, which, in turn, affects VP secretion.NIH grant: R21 HL093728

Hypertonic NaCl intake induces renal TNF production by a pathway involving NKCC2A and NFAT5

TNF is produced by the medullary thick ascending limb (mTAL), however, the effect of hypertonicity on production of this cytokine has not been determined. Mice given 1% NaCl in the drinking water for 3 days exhibited a two‐fold increase in urinary, but not plasma, TNF levels compared with mice given tap water. A concomitant 3‐fold increase in NKCC2A and 2‐fold increase in NFAT5 mRNA accumulation was observed in mTAL tubules from mice given 1% NaCl. Increases in urinary TNF levels were abolished after treatment with furosemide or intrarenal injection of a lentivirus shRNA construct that knocked down NKCC2A (EGFP‐N2A‐ex4); plasma levels of TNF did not change. NFAT5 mRNA abundance also decreased after injection of EGFP‐N2A‐ex4. TNF production by primary cultures of mTAL cells increased approximately 6‐fold in response to high NaCl concentration (400 mosmol/kg H2O) and was inhibited in cells treated with bumetanide or transiently transfected with a dnNFAT5 construct (3.0 ± 0.9 to 0.9±0.2 pg/μg protein; p&lt;0.05). Transduction of mTAL cells with EGFP‐N2A‐ex4 prevented increases in TNF production in response to high NaCl concentration and reduced transcriptional activity of an NFAT5 promoter construct by more than 60%. Since NKCC2A expression is restricted to the TAL segment of the nephron, NKCC2A‐dependent activation of NFAT5 is part of a pathway contributing to TNF production by the TAL in response to hypertonic NaCl intake.

Deletion of cyclooxygenase-2 in the mouse increases arterial blood pressure with no impairment in renal NO production in response to chronic high salt intake

Experiments were designed to test the hypothesis that cyclooxygenase-2 (COX-2) activity attenuates the blood pressure increase during high NaCl intake by stimulation of endothelial nitric oxide synthase (eNOS)-mediated NO synthesis in the kidney medulla. COX-2(-/-) (C57BL6) an COX-2(+/+) mice were fed a diet with 0.004% (low salt, LS) or 4% (high salt, HS) NaCl for 18 days. Arterial blood pressure was recorded continuously using indwelling catheters. Food and water intake and diuresis were measured in metabolic cages. Urine osmolality and excretion of electrolytes, cGMP, cAMP, and NOx were determined, as well as plasma NOx and cGMP. There was a significant dependence of blood pressure on salt intake and genotype: COX-2(-/-) exhibited higher blood pressure than COX-2(+/+) both on HS and LS intake. COX-2(+/+) littermates displayed an increase in blood pressure on HS versus LS (102.3 ± 1.1 mmHg vs. 91.9 ± 0.9 mmHg) day and night. The mice exhibited significant blood pressure increases during the awake phase (night) that were larger in COX-2(-/-) on HS diet compared with COX-2(+/+). Water intake, diuresis, Na(+), and osmolyte excretions and NOx and cGMP excretions were significantly and similarly elevated with HS in COX-2(-/-) and COX-2(+/+). In summary, C57BL6 mice exhibit a salt intake-dependent increase in arterial blood pressure with increased renal NO production. COX-2 activity has a general lowering effect on arterial blood pressure. COX-2 dampens NaCl-induced increases in arterial blood pressure in the awake phase. In conclusion, COX-2 activity attenuates the changes in nocturnal blood pressure during high salt intake, and COX-2 activity is not necessary for increased renal nitric oxide formation during elevated NaCl intake.

Effects of high dietary sodium chloride content on performance and sodium and potassium balance in growing pigs

Thirty castrated male Moo Lath pigs (6-8 weeks of age) were used in a 15-week growth trial to study the effect of high dietary sodium chloride (NaCl) content on feed and water intake, performance, sodium (Na) and potassium (K) balance, and plasma aldosterone concentration. The pigs were randomly allocated (ten per treatment) to diets containing 0.24 % Na (Na0.24), 0.28 % Na (Na0.28), and 0.32 % Na (Na0.32) per kg diet. Feed and water was provided ad libitum, and water consumption, feed offered, and feed residues were recorded daily. Every third week, the pigs were weighed, blood samples were collected, and a 3-day total collection of urine and feces was performed. Water intake was higher (P = 0.001) in pigs fed with diets Na0.28 (3.7 L/day) and Na0.32 (3.9 L/day) than in pigs fed with diet Na0.24 (3.4 L/day), and dry matter (DM) intake was higher on diet Na0.32 (P = 0.041) than on the other diets. The average daily body weight (BW) gain was higher on diet Na0.32 than on the other diets (P = 0.031). The feed conversion ratio (in kilogram feed DM to kilogram BW gain) was 4.6, 4.6, and 4.1 on treatments Na0.24, Na0.28, and Na0.32, respectively (P = 0.14). The highest Na balance was observed on diet Na0.32 followed by diets Na0.28 and Na0.24 (P < 0.001), while there was no treatment-related pattern for the K balance. The Na/K ratio in feces and urine increased (P < 0.001), and the K/Na ratio in feces (P < 0.001) decreased with increasing Na content in the diet. Plasma aldosterone concentration decreased (P < 0.001) with increasing dietary content of Na. These results indicate that high NaCl intake and free access to water will increase Na balance but do not negatively influence feed intake and performance of growing local pigs.

Variable effects of parabrachial nucleus lesions on salt appetite in rats depending upon experimental paradigm and saline concentration.

Previous studies have demonstrated that bilateral lesions of the gustatory (medial) zone of the parabrachial nucleus (PBN) in the pons eliminate the salt (sodium chloride; NaCl) appetite induced in rats by treatment with the diuretic drug, furosemide. The present studies reexamined NaCl intake of rats with PBN lesions induced by ibotenic acid, using multiple models of salt appetite. The impairment of a conditioned taste aversion, an established consequence of PBN damage, was used as an initial screen with which to assess the effectiveness of the lesions. Rats with PBN lesions did not drink either 0.3 of a molar (M) solution of NaCl or 0.5 M NaCl in response to daily treatment with desoxycorticosterone acetate. These findings suggest that the excitatory stimulus of salt appetite mediated by mineralocorticoids is abolished by PBN lesions. In contrast, rats with PBN lesions drank some 0.5 M NaCl and more 0.3 M NaCl, in addition to water, in response to hypovolemia induced by subcutaneous injection of 30% polyethylene glycol solution. Those findings suggest that an excitatory stimulus of salt appetite, presumably mediated by Angiotensin II, is not abolished by PBN lesions. These and other observations indicate that lesions of the gustatory PBN in rats may or may not eliminate salt appetite, depending on which model is used and which concentration of NaCl solution is available.

NKCC2A and NFAT5 regulate renal TNF production induced by hypertonic NaCl intake

Pathways that contribute to TNF production by the kidney are not well defined. Mice given 1% NaCl in the drinking water for 3 days exhibited a 2.5-fold increase in urinary, but not plasma, TNF levels compared with mice given tap water. Since furosemide attenuated the increase in TNF levels, we hypothesized that hypertonic NaCl intake increases renal TNF production by a pathway involving the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). A 2.5-fold increase in NKCC2A mRNA accumulation was observed in medullary thick ascending limb (mTAL) tubules from mice given 1% NaCl; a concomitant 2-fold increase in nuclear factor of activated T cells 5 (NFAT5) mRNA and protein expression was observed in the outer medulla. Urinary TNF levels were reduced in mice given 1% NaCl after an intrarenal injection of a lentivirus construct designed to specifically knockdown NKCC2A (EGFP-N2A-ex4); plasma levels of TNF did not change after injection of EGFP-N2A-ex4. Intrarenal injection of EGFP-N2A-ex4 also inhibited the increase of NFAT5 mRNA abundance in the outer medulla of mice given 1% NaCl. TNF production by primary cultures of mTAL cells increased approximately sixfold in response to an increase in osmolality to 400 mosmol/kgH2O produced with NaCl and was inhibited in cells transiently transfected with a dnNFAT5 construct. Transduction of cells with EGFP-N2A-ex4 also prevented increases in TNF mRNA and protein production in response to high NaCl concentration and reduced transcriptional activity of a NFAT5 promoter construct. Since NKCC2A expression is restricted to the TAL, NKCC2A-dependent activation of NFAT5 is part of a pathway by which the TAL produces TNF in response to hypertonic NaCl intake.

Activation of μ-opioid receptors in the central nucleus of the amygdala induces hypertonic sodium intake

Opioid mechanisms are involved in the control of water and NaCl intake and opioid receptors (ORs) are present in the central nucleus of the amygdala (CeA), a site of important facilitatory mechanisms related to the control of sodium appetite. Therefore, in the present study we investigated the effects of the activation of μ-ORs in the CeA on 0.3M NaCl and water intake in rats. Male Sprague–Dawley rats with stainless steel cannulas implanted bilaterally in the CeA were used. In rats submitted to water deprivation–partial rehydration, bilateral injections of the selective μ-OR agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) in the doses of 1, 2, and 4nmol into the CeA induced a dose-related increase of 0.3M NaCl intake and water intake, and bilateral injections of the selective μ-OR antagonist D-Phe-Cys-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) in the doses of 0.5, 1, and 2nmol into the CeA produced a dose-related decrease of 0.3M NaCl and water intake induced by DAMGO 2nmol into the same site. In rats treated with the diuretic furosemide (10mg/kgb.w.) combined with the angiotensin-converting enzyme inhibitor captopril (5mg/kgb.w.) injected subcutaneously, bilateral injections of DAMGO 2nmol into the CeA increased 0.3M NaCl intake and water intake and the blockade of μ-ORs with CTAP 1nmol injected into the CeA reduced the increase in 0.3M NaCl intake and water intake induced by DAMGO 2nmol into the same site. Bilateral injections of DAMGO into the CeA did not change urinary volume, sodium urinary excretion and mean arterial pressure, but increased activity. Thus stimulating μ-ORs in the CeA increases hypertonic sodium intake, whereas antagonizing these sites inhibits hypertonic sodium intake. Together, our results implicate μ-ORs in the CeA in a positive regulation of sodium intake.