Acute Sodium Loading Research Articles

Renal Medullary Overexpression of Sphingosine-1-Phosphate Receptor 1 Transgene Attenuates Deoxycorticosterone Acetate (DOCA)-Salt Hypertension.

Our previous studies showed that renal medullary sphingosine-1-phosphate receptor 1 (S1PR1) mediated sodium excretion, high salt intake increased S1PR1 level, deoxycorticosterone acetate (DOCA) blocked high salt-induced S1PR1 in the renal medulla, and that conditional knockout of S1PR1 in the collecting duct aggravated DOCA-salt hypertension. The present study tested the hypothesis that overexpression of S1PR1 transgene in the renal medulla attenuates the sodium retention and hypertension in DOCA-salt mouse model. Male C57BL/6J mice received renal medullary transfection of control or S1PR1-expressing plasmids and then DOCA-salt treatment. Renal sodium excretion and arterial pressure were compared between control and S1PR1-overexpressed mice in response to high salt loading or pressure natriuresis. S1PR1-transfected mice showed significantly enhanced urinary sodium excretion in response to acute sodium loading (0.93 ± 0.27 in control vs. 4.72 ± 1.12 µmol/min/gKW in S1PR1-overexpressed mice, P < 0.05) and the pressure natriuresis (3.58 ± 1.77 vs. 9.52 ± 1.38, P < 0.05), less positive sodium balance in response to chronic high-salt intake (3.05 ± 0.39 vs. 1.65 ± 0.39 mmol/72 hr, P < 0.05), and consequently, the attenuation of DOCA-salt hypertension (134.2 ± 6.79 vs. 109.8 ± 3.54 mm Hg, P < 0.05). The αENaC protein amount in the renal medulla was not changed, however, the βENaC was significantly decreased and the γENaC was significantly increased in S1PR1-overexpressed mice. The immunostaining showed apical membrane translocation of γENaC, while no change of αENaC and βENaC in control mice, and that the apical membrane translocation of γENaC was blocked in S1PR1-treasffected mice. These results suggested that activation of S1PR1 in the renal medulla attenuates DOCA-induced sodium retention and salt-sensitive hypertension associated with inhibition of ENaC.

Mid-Point of the Active Phase Is Better to Achieve the Natriuretic Effect of Acute Salt Load in Mice

Excess sodium intake and insufficient potassium intake are a prominent global issue because of their influence on high blood pressure. Supplementation of potassium induces kaliuresis and natriuresis, which partially explains its antihypertensive effect. Balancing of minerals takes place in the kidney and is controlled by the circadian clock; in fact, various renal functions exhibit circadian rhythms. In our previous research, higher intake of potassium at lunch time was negatively associated with blood pressure, suggesting the importance of timing for sodium and potassium intake. However, the effects of intake timing on urinary excretion remain unclear. In this study, we investigated the effect of 24 h urinary sodium and potassium excretion after acute sodium and potassium load with different timings in mice. Compared to other timings, the middle of the active phase resulted in higher urinary sodium and potassium excretion. In Clock mutant mice, in which the circadian clock is genetically disrupted, urinary excretion differences from intake timings were not observed. Restricted feeding during the inactive phase reversed the excretion timing difference, suggesting that a feeding-induced signal may cause this timing difference. Our results indicate that salt intake timing is important for urinary sodium and potassium excretion and provide new perspectives regarding hypertension prevention.

Glucocorticoids Promote Na+ Excretion in the Renal Epithelia of Heart Failure Rats by Suppressing Transporter Proteins Involved in Acute Sodium Loading.

Glucocorticoid receptors are essential for normal development and stress responses. Their role in H 2 O and Na + metabolism, especially in chronic heart failure (CHF), is not well defined. In a previous study, we found that glucocorticoids potentiate urination in CHF and promote H 2 O excretion by inhibiting the vasopressin receptor 2 pathway. The present study examines the effect of glucocorticoids on renal Na + excretion and the underlying mechanisms in CHF rats with acute sodium loading. CHF was induced by left coronary artery ligation for 8 weeks. Rats were randomly assigned to 5 groups: control, CHF, dexamethasone (DEX)-administered CHF, DEX-administered CHF treated with RU486 (mifepristone, a glucocorticoid receptor antagonist), and RU486-treated CHF. An acute sodium loading test was performed 6 hours after DEX administration. Blood and urine samples were collected, and hemodynamics were measured. The expression and localization of Na + transporter proteins were determined by immunoblotting and immunohistochemistry. DEX increased the urine volume and urinary sodium and improved cardiac function and the estimated glomerular filtration rate in CHF rats. The upregulation of the epithelial sodium channel β and γ subunits, Na-K-2Cl cotransporter, serum glucocorticoid-regulated kinase 1 (SGK1), and Na + /K + -ATPase in the renal epithelium of CHF rats was downregulated by DEX. These beneficial effects were abolished by RU486. The expression of natriuretic peptide receptor A was opposite that of the above proteins. Glucocorticoids might induce profound natriuresis in CHF rats during acute sodium loading, which is associated with downregulating some Na + transporter proteins in the renal epithelium and improving intrarenal hemodynamics.

Medullary overexpression of sphingosine‐1‐phosphate receptor 1 transgene attenuates sodium retention and hypertension in deoxycorticosterone acetate (DOCA) salt‐treated mice

Our previous studies have showed that sphingosine‐1‐phosphate (S1P) agonist stimulates urinary sodium excretion and the S1P type 1 receptor (S1PR1) knockout in the collecting duct led to increased sodium retention and enhanced salt‐sensitive hypertension under the treatment of deoxycorticosterone (DOCA) and high salt. We hypothesized that the increase in medullary S1PR1 would attenuate sodium retention by promoting sodium excretion in such model. Male C57BL/6J mice around 25g bodyweight received intramedullary transfection of S1PR1 plasmids to achieve renal medullary S1PR1 overexpression (OE). The mice were then implanted with the silastic DOCA sheet subcuntaneously and 1% NaCl drinking water to induce salt‐sensitive hypertension (SS‐HTN). Thereafter, the mice underwent either acute or chronic assessments on blood pressure and urinary sodium excretion. Under DOCA‐salt challenge, mice with S1PR1‐OE showed increased urine volume (UV) and urinary sodium excretion (UNAV) in responses to the acute sodium loading (IV of normal saline at 5% body weight in 30 min); the UV and UNAV were 12.51±2.31 vs. 28.50±4.23mL/min/gKW and 0.93±0.27 vs. 4.72±1.12mmole/min/gKW (p&lt;0.05) in control and S1PR1‐OE mice, respectively. The mice with S1PR1‐OE also showed enhanced pressure natriuretic responses (UV, 18.73±7.57 vs. 40.61±6.43mL/min/gKW, p&lt;0.05; UNAV, 3.58±1.77 vs. 9.52±1.38nmole/min/gKW, p&lt;0.05). In the long‐term DOCA‐salt treatment, S1PR1‐OE mice showed less daily sodium balance with the greater sodium excretion (2.72±0.56 vs. 5.60±0.72mmole/day, p&lt;0.05) and ameliorated sodium retention (3.05±0.39 vs. 1.65±0.39mmole/72hr, p&lt;0.05), which was accompanied with the attenuation of SS‐HTN (SBP 134.2±6.79 vs. 109.8±3.54mmHg, p&lt;0.05). The level of alpha subunit of epithelial sodium channel (αENaC) was not changed. However, the beta subunit (βENaC) was significantly decreased (55%, p&lt;0.01). Although ENaCγ was significantly increased (350%) in the S1PR1‐OE group, immunostaining showed that the DOCA‐stimulated translocation of γENaC onto the apical membrane was inhibited. These results suggested that the overexpression of S1PR1 in the renal medulla attenuates sodium retention by the inhibition of ENaC function in the DOCA‐induced SS‐HTN.

Sequential Afferent‐Targeted and Total Renal Denervation and Impact on Regulation of Arterial Pressure, Glomerular Filtration, and Sodium Handling in Sprague Dawley Male Rats

Renal denervation (RDNx) is often reported to chronically decrease mean arterial pressure (MAP) in conscious, unchallenged Sprague Dawley (SD) rats, but has no effect of salt-sensitivity of MAP. Although these findings indicate an important role of renal nerves in long-term regulation of MAP under unchallenged conditions, the individual contributions of sympathetic and afferent renal nerves in MAP and renal homeostasis remain unclear. The present study was conducted to test the hypothesis that efferent (sympathetic) renal nerves are the primary contributors both MAP and renal function responses to RDNx in normotensive Sprague Dawley rats. To test this hypothesis, 12 adult male Sprague Dawley rats (300-325g) were subjected to sequential denervation procedures to ablate afferent and efferent nerves separately by subjecting rats to the following surgical sequence: Sham, then afferent-targeted RDNx (A-RDNx), then complete (T)-RDNx. A-RDNx was achieved by periaxonal application of 33mM capsaicin, and T-RDNx was achieved by surgical dissection followed by perivascular application of 10%(v/v) phenol in ethanol. One week was permitted for recovery from surgery before cardiovascular and renal measurements were collected. 24-hour mean arterial pressure (MAP) was recorded by implanted telemetry. Glomerular filtration rate (GFR) was assessed in unrestrained, conscious rats by acute FITC-sinistrin clearance. Renal excretory function was assessed by measuring the urinary sodium excretion response to an acute saline challenge (150mM PBS, 10% bodyweight, i.p.) over four hours. Data was analyzed by one-way repeated-measures ANOVA with Bonferroni post-hoc test (α=0.05). Data presented as mean±SEM. Sham surgery had no significant effect on MAP, GFR or Na Clearance. Following A-RDN treatment, MAP increased ∆5.4±0.9mmHg; (p<0.01); however, no significant changes in GFR or the excretory response to acute sodium load were detected. In contrast, following T-RDN, MAP was decreased 4.9±0.9mmHg (p<.01) following T-RDN vs. compared to A-RDN. Regarding renal function, GFR increased (1.20±0.05 vs. 1.06±0.04 mL/min/100gBWT; p<.01) as did the sodium excretory response to a saline load (28.2±2.3 vs. 18.6±1.0% Na load excreted per 4 hours; p<.01). Together, these data partly support our hypothesis and solidify the role for renal efferent (sympathetic) nerves contribute to long-term regulation of MAP, GFR, and sodium handling in the conscious SD rat. Contrary to our hypothesis, A-RDNx resulted in a small, yet significant, increase in MAP; however, no impact on renal function was observed. We speculate that the increased GFR and decrease in MAP are likely due to a decrease in renal vascular resistance and dilation of the renal afferent arterioles. Moreover, renal afferent nerves appear to contribute to maintenance of MAP in Sprague Dawley rats, which we speculate is through a tonic sympatho-inhibition due to the increase MAP following A-RDNx. These data highlight the important, yet differential roles of efferent and afferent renal nerves, and further studies are underway to further test this hypothesis.

The Effects of Short-Term Changes in Sodium Intake on Plasma Marinobufagenin Levels in Patients with Primary Salt-Sensitive and Salt-Insensitive Hypertension.

Increased marinobufagenin (MBG) synthesis has been suggested in response to high dietary salt intake. The aim of this study was to determine the effects of short-term changes in sodium intake on plasma MBG levels in patients with primary salt-sensitive and salt-insensitive hypertension. In total, 51 patients with primary hypertension were evaluated during acute sodium restriction and sodium loading. Plasma or serum concentrations of MBG, natriuretic pro-peptides, aldosterone, sodium, potassium, as well as hematocrit (Hct) value, plasma renin activity (PRA) and urinary sodium and potassium excretion were measured. Ambulatory blood pressure monitoring (ABPM) and echocardiography were performed at baseline. In salt-sensitive patients with primary hypertension plasma MBG correlated positively with diastolic blood pressure (ABPM) and serum NT-proANP concentration at baseline and with serum NT-proANP concentration after dietary sodium restriction. In this subgroup plasma MBG concentration decreased during sodium restriction, and a parallel increase of PRA was observed. Acute salt loading further decreased plasma MBG concentration in salt-sensitive subjects in contrast to salt insensitive patients. No correlation was found between plasma MBG concentration and left ventricular mass index. In conclusion, in salt-sensitive hypertensive patients plasma MBG concentration correlates with 24-h diastolic blood pressure and dietary sodium restriction reduces plasma MBG levels. Decreased MBG secretion in response to acute salt loading may play an important role in the pathogenesis of salt sensitivity.

Distinct osmoregulatory responses to sodium loading in patients with altered glycosaminoglycan structure: a randomized cross-over trial

BackgroundBy binding to negatively charged polysaccharides called glycosaminoglycans, sodium can be stored in the body—particularly in the skin—without concurrent water retention. Concordantly, individuals with changed glycosaminoglycan structure (e.g. type 1 diabetes (DM1) and hereditary multiple exostosis (HME) patients) may have altered sodium and water homeostasis.MethodsWe investigated responses to acute (30-min infusion) and chronic (1-week diet) sodium loading in 8 DM1 patients and 7 HME patients in comparison to 12 healthy controls. Blood samples, urine samples, and skin biopsies were taken to investigate glycosaminoglycan sulfation patterns and both systemic and cellular osmoregulatory responses.ResultsHypertonic sodium infusion increased plasma sodium in all groups, but more in DM1 patients than in controls. High sodium diet increased expression of nuclear factor of activated t-cells 5 (NFAT5)—a transcription factor responsive to changes in osmolarity—and moderately sulfated heparan sulfate in skin of healthy controls. In HME patients, skin dermatan sulfate, rather than heparan sulfate, increased in response to high sodium diet, while in DM1 patients, no changes were observed.ConclusionDM1 and HME patients show distinct osmoregulatory responses to sodium loading when comparing to controls with indications for reduced sodium storage capacity in DM1 patients, suggesting that intact glycosaminoglycan biosynthesis is important in sodium and water homeostasis.Trial registration These trials were registered with the Netherlands trial register with registration numbers: NTR4095 (https://www.trialregister.nl/trial/3933 at 2013-07-29) and NTR4788 (https://www.trialregister.nl/trial/4645 at 2014-09-12).

Sports Dietitians Australia Position Statement: Nutrition for Exercise in Hot Environments.

It is the position of Sports Dietitians Australia (SDA) that exercise in hot and/or humid environments, or with significant clothing and/or equipment that prevents body heat loss (i.e.,exertional heat stress), provides significant challenges to an athlete's nutritional status, health, and performance. Exertional heat stress, especially when prolonged, can perturb thermoregulatory, cardiovascular, and gastrointestinal systems. Heat acclimation or acclimatization provides beneficial adaptations and should be undertaken where possible. Athletes should aim to begin exercise euhydrated. Furthermore, preexercise hyperhydration may be desirable in some scenarios and can be achieved through acute sodium or glycerol loading protocols. The assessment of fluid balance during exercise, together with gastrointestinal tolerance to fluid intake, and the appropriateness of thirst responses provide valuable information to inform fluid replacement strategies that should be integrated with event fuel requirements. Such strategies should also consider fluid availability and opportunities to drink, to prevent significant under- or overconsumption during exercise. Postexercise beverage choices can be influenced by the required timeframe for return to euhydration and co-ingestion of meals and snacks. Ingested beverage temperature can influence core temperature, with cold/icy beverages of potential use before and during exertional heat stress, while use of menthol can alter thermal sensation. Practical challenges in supporting athletes in teams and traveling for competition require careful planning. Finally, specific athletic population groups have unique nutritional needs in the context of exertional heat stress (i.e.,youth, endurance/ultra-endurance athletes, and para-sport athletes), and specific adjustments to nutrition strategies should be made for these population groups.

Blunted diuretic and natriuretic responses to acute sodium loading early after catheter-based renal denervation in normotensive sheep.

Catheter-based renal denervation (RDN) was introduced as a treatment for resistant hypertension. There remain critical questions regarding the physiological mechanisms underlying the hypotensive effects of catheter-based RDN. Previous studies indicate that surgical denervation reduces renin and the natriuretic response to saline loading; however, the effects on these variables of catheter-based RDN, which does not yield complete denervation, are largely unknown. The aim of this study was to investigate the effects of catheter-based RDN on glomerular-associated renin and regulation of fluid and sodium homeostasis in response to physiological challenges. First, immunohistochemical staining for renin was performed in normotensive sheep (n = 6) and sheep at 1 wk (n = 6), 5.5 mo (n = 5), and 11 mo (n = 5) after unilateral RDN using the same catheter used in patients (Symplicity). Following catheter-based RDN (1 wk), renin-positive glomeruli were significantly reduced compared with sham animals (P < 0.005). This was sustained until 5.5 mo postdenervation. To determine whether the reduction in renin after 1 wk had physiological effects, in a separate cohort, Merino ewes were administered high and low saline loads before and 1 wk after bilateral RDN (n = 9) or sham procedure (n = 8). After RDN (1 wk), the diuretic response to a low saline load was significantly reduced (P < 0.05), and both the diuretic and natriuretic responses to a high saline load were significantly attenuated (P < 0.05). In conclusion, these findings indicate that catheter-based RDN acutely alters the ability of the kidney to regulate fluid and electrolyte balance. Further studies are required to determine the long-term effects of catheter-based RDN on renal sodium and water homeostasis.

Sodium loading, treadmill walking, and the acute redistribution of bone mineral content on dual energy X-ray absorptiometry scans.

The purpose of this study was to assess relationships between plasma sodium concentration ([Na+]) and bone mineral content (BMC) after an acute sodium load plus treadmill walking and then quantify the amount of sodium the dual energy X-ray absorptiometry (DXA) scan could detect. The primary study was a single-blind randomized control crossover trial under two conditions: ingestion of six flour tablets (placebo trial) or six 1-g NaCl tablets (salt intervention trial). The tablets were ingested after baseline blood and urine collection followed immediately by the DXA scan. After 60 min of rest, a 45-min treadmill walk was conducted. Immediately postexercise, blood and urine were collected and the DXA scan was repeated. Main outcomes included changes (∆: post minus pre) in plasma [Na+] and BMC. Additionally, six 1-g NaCl tablets were superimposed over a DXA spine phantom for separate quantification of sodium as BMC. Fourteen subjects completed the primary study. Two-way repeated measures ANOVA tests revealed significant interaction ( F = 13.06; P = 0.0007), condition ( F = 21.88; P < 0.001), and time ( F = 6.51; P = 0.014) effects in plasma [Na+]. A significant condition ( F = 6.46; P = 0.014) effect was also noted in urine [Na+]. Total body BMC∆ was negatively correlated with plasma [Na+]∆ ( r = -0.43; P = 0.02) and urine [Na+]∆ ( r = -0.47; P = 0.01). Total body BMC∆ in the salt intervention trial [-5.5 (27) g] closely approximated the amount of NaCl ingested and subsequently absorbed into the bloodstream. The DXA scan quantified 67% of NaCl tablets as BMC in spine phantom analyses. Total body BMC∆ was negatively related to plasma and urine [Na+]∆ after treadmill walking. Reductions in total body BMC closely approximated the amount of NaCl ingested (~6 g). The DXA scan quantified NaCl as BMC.

Genetic Variation in SLC8A1 Gene Involved in Blood Pressure Responses to Acute Salt Loading

Salt sensitivity of blood pressure (SSBP) increases the risk of cardiovascular complications, and the heritability of SSBP is about 50% in Chinese population. However, studies identifying genes involved in BP responses to acute sodium loading and diuresis shrinkage are still limited. A total of 342 essential hypertensives from Beijing were recruited in our study. A modified Sullivan's acute oral saline load and diuresis shrinkage test was conducted to each individual. Medical history and lifestyle risk factors were obtained by questionnaire. Generalized linear model was used to examine the associations of 29 single-nucleotide polymorphisms (SNPs) with SSBP and false discovery rate (FDR) was used to correct P values for multiple testing. In the process of acute sodium loading, after adjusting for age and 24-hour urinary sodium concentration, SNPs in CYP11B2, PRKG1, SLC8A1 genes were significantly associated with systolic BP (SBP) rising in the additive and recessive model; SNPs in CYP4A11, PRKG1, SLC8A1, and ADRB2 genes were significantly associated with diastolic BP (DBP) rising. In the process of diuresis shrinkage, SNPs of CLCNKA, eNOS, PRKG1 gene were associated with SBP and DBP decreasing. After FDR correction, rs434082 in SLC8A1 gene was still significantly associated with blood pressure rising during salt load. In the additive model, A allele increased DBP of 2.8 mm Hg (FDR_q = 0.029) and MAP of 3.1 mm Hg (FDR_q = 0.029) after adjusting for age and 24-hour urinary sodium concentration. SLC8A1 gene may contribute to BP change in the process of acute sodium loading in a Han Chinese population.

Infusion of Valproic Acid Into the Renal Medulla Activates Stem Cell Population and Attenuates Salt-Sensitive Hypertension in Dahl S Rats

Background: Our previous study has detected a stem cell deficiency in the renal medulla in Dahl salt-sensitive (S) rats. This study determined whether infusion of valproic acid (VA), an agent known to stimulate the stem cell function, attenuated salt-sensitive hypertension in Dahl S rats. Methods: Uninephrectomized Dahl S rats were infused with vehicle or VA (50mg/kg/d) into the renal medulla and fed with a low (LS) or high salt diet (HS). Stem cell marker and number were analyzed by immunohistochemistry, Real-time RT-PCR and Western blot. Sodium excretion and blood pressure were measured. Results: VA significantly increased the mRNA and protein levels of FGF2, a stem cell niche factor, and CD133, a stem cell marker. The number of CD133+ cells was significantly increased in the renal medulla in VA-treated rats. Meanwhile, high salt-induced increases in the mRNA level of proinflammatory factors interleukin-1β and interleukin-6 were blocked in VA-treated rats. Functionally, sodium excretion in response to the blood pressure increase and acute sodium loading was significantly enhanced, sodium retention attenuated, high salt-induced increase of blood pressure reduced in VA-treated rats. Conclusion: Activation of stem cell function by VA inhibits the activation of proinflammatory factors and attenuates salt-sensitive hypertension in Dahl S rats.

Abstract P324: Reduction in Excreted Sodium Load in Obese Sheep: Role of Antenatal Betamethasone and Sex

Our previous studies have demonstrated the programmed impairment of sodium excretion in lean antenatal betamethasone exposed sheep whereby the males exhibited blunted excretion of a sodium load when compared to control males or control or antenatal betamethasone exposed females. Obesity has been recognized as a risk factor for developing early stage kidney failure in individuals exposed to unfavorable intrauterine conditions by virtue of fetal programming. We hypothesize that obesity (second hit) can accentuate the impairment of sodium excretion in antenatal betamethasone exposed offspring in a sex specific manner. Pregnant ewes received either betamethasone or vehicle at 80-81 days gestation. The male (n=6 per group) or female (n=5 per group) offspring were overfed to become obese (40% weight gain over a course of 3 months) at age of 1.5 years. Acute sodium load (sodium chloride: 0.13g/kg) was administrated intravenously. Sodium excretion (excreted sodium load), blood pressure (BP), renal plasma flow (RPF), and glomerular filtration rate (GFR) were measured. Data for sodium excretion are expressed as percentage of excreted sodium load. ANOVA and Student’s t test were used for data analysis. The excreted sodium load was lower in obese male antenatal betamethasone exposed offspring (OMB) than in obese male vehicle exposed offspring (OMV) (13.5±3.1% vs. 33.2±9.9% p=0.04). However, the excreted sodium load was not significantly different between obese female antenatal betamethasone exposed offspring (OFB) and obese female vehicle exposed offspring (OFV) (60.4±6.2% vs. 72.0±8.2%, p=0.31). Compared male offspring with female offspring, the excreted sodium load was lower in male offspring (33.2±9.9% vs. 72.0±8.2%, p=0.001, OMV vs. OFV; 13.5±3.2% vs. 60.4±6.2%, p&lt;0.0001, OMB vs. OFB). During the experiments BP, RPF and GFR did not change significantly. . These data suggest that obesity, acting as a second hit, accentuates impairment of sodium excretion in antenatal betamethasone exposed male but not female offspring. Thus the sex specific effect of antenatal betamethasone on sodium excretion persists in in animals experiencing a secondary insult of obesity in adulthood.

Impaired sodium-evoked paraventricular nucleus neuronal activation and blood pressure regulation in conscious Sprague-Dawley rats lacking central Gαi2 proteins.

AimWe determined the role of brain Gαi2 proteins in mediating the neural and humoral responses of conscious male Sprague–Dawley rats to acute peripheral sodium challenge.MethodsRats pre‐treated (24‐h) intracerebroventricularly with a targeted oligodeoxynucleotide (ODN) (25 μg per 5 μL) to downregulate brain Gαi2 protein expression or a scrambled (SCR) control ODN were challenged with an acute sodium load (intravenous bolus 3 m NaCl; 0.14 mL per 100 g), and cardiovascular parameters were monitored for 120 min. In additional groups, hypothalamic paraventricular nucleus (PVN) Fos immunoreactivity was examined at baseline, 40, and 100 min post‐sodium challenge.ResultsIn response to intravenous hypertonic saline (HS), no difference was observed in peak change in mean arterial pressure between groups. In SCR ODN pre‐treated rats, arterial pressure returned to baseline by 100 min, while it remained elevated in Gαi2 ODN pre‐treated rats (P < 0.05). No difference between groups was observed in sodium‐evoked increases in Fos‐positive magnocellular neurons or vasopressin release. V1a receptor antagonism failed to block the prolonged elevation of arterial pressure in Gαi2 ODN pre‐treated rats. A significantly greater number of Fos‐positive ventrolateral parvocellular, lateral parvocellular, and medial parvocellular neurons were observed in SCR vs. Gαi2 ODN pre‐treated rats at 40 and 100 min post‐HS challenge (P < 0.05). In SCR, but not Gαi2 ODN pre‐treated rats, HS evoked suppression of plasma norepinephrine (P < 0.05).ConclusionThis highlights Gαi2 protein signal transduction as a novel central mechanism acting to differentially influence PVN parvocellular neuronal activation, sympathetic outflow, and arterial pressure in response to acute HS, independently of actions on magnocellular neurons and vasopressin release.

Abstract 275: The Effect of High Salt-diet on Pioglitazone Treated-db/db-/- mice

The objective of this study was to evaluate the impact of high-salt-diet on pioglitazone treated db/db mice. Groups of 6 weeks-old db/db mice obtained from Jackson Laboratory were given either pioglitazone (0.02%) in diet or pioglitazone (0.02%) plus high salt-diet (8% NaCl) for 6 to 12 weeks. Control groups received either pioglitazone or normal salt diet. During the course of treatment, urine volume, urine sodium, creatinine and blood glucose were measured in the animals. For urine collection, mice were placed in metabolic cages. In order to evaluate whether differences exist between diabetic animals that received pioglitazone or not in the handling of a sodium load and to characterize the transport mechanisms involved, at the end of experimental period the animals were given an acute sodium load (physiological saline), 1.25 ml/100 g body weight by intraperitoneal route. The sodium load was repeated in mice that was treated 15 minutes earlier with hydrochlorothiazide (40 mg/kg i.p.) or furosemide (8 mg/kg i.p.), or amiloride (1.65 mg/kg). The animals were placed in metabolic cages and urine voided was collected over 5 hr. for determination of urine volume and sodium. Sodium was determined by flame photometer. No significant changes were observed in mean arterial blood pressure in all the groups (at p 0.05). There was significant increase in UNav with all the diuretics in db/db mice that received high salt diet at 6 weeks. The blood pressure did not increase with the sodium diet. This might be due to natriuresis resulting from polyurea in the diabetic condition of the mice.

PP.41.01

Objective: The endothelial surface layer (ESL) consists of negatively charged glycosaminoglycans that can osmotically inactivate sodium. The ESL may therefore affect sodium homeostasis and blood pressure (BP). In mice, we investigated the consequences of combined heterozygous loss of EXT-1 and EXT-2 on BP after acute and chronic sodium loading. Both EXT genes are involved in polymerization of heparan sulfate, the predominant ESL glycosaminoglycan. Design and method: After a two-week normal sodium (0.3%) diet, 11-week old EXT1+/-EXT2+/- and wildtype mice were fed a normal or high sodium (8.0%) diet and water ad libitum for one week while monitoring BP with tail cuff measurements. We trained all mice one week before performing tail cuff measurements. At the end of the experiment, mice on normal sodium diet were subjected to intravenous saline loading (1.8%, 8 μl/g) while measuring BP invasively in the carotid artery. After exsanguination, we prepared mesenteric arteries for wire myography to study methacholine-induced (10 nmol/l-10 μmol/l) endothelium-dependent vasorelaxation after initial vasoconstriction using U46116. All statistical analyses were performed using non-parametric tests. Results: Baseline mean arterial pressure (MAP) was comparable between wildtype (89.2 (8.0) mmHg, n = 11) and EXT1+/-EXT2+/- mice (88.3 (8.4) mmHg, n = 16; p = 0.79). After a 7-day high sodium diet, MAP change in wildtype (+0.6 (8.4) mmHg, n = 6; p = 0.88) and EXT1+/-EXT2+/- mice (-5.2 (10.5) mmHg, n = 10, p = 0.15) was not significantly different (p = 0.18). Infusion of 220 (16) μl saline in 100 (20) seconds resulted in a significant 15.4 (10.8)% (p = 0.018) MAP decrease in wildtype mice while EXT1+/-EXT2+/- mice showed a non-significant 2.9 (13.4)% increase (p = 0.65).This significant variation in BP response (p = 0.030) was not accompanied by any change in heart rate in wildtype (-4.0 (8.0)%, p = 0.28) or EXT1+/-EXT2+/- mice (-0.7 (1.1)%, p = 0.26). Methacholine dose-response curves showed impaired vasodilation of mesenteric arteries of EXT1+/EXT2+/- mice (41 (10)%) when compared to wildtype mice (65 (22)%; p = 0.030). Conclusions: A defect in heparan sulfate polymerization in mice alters BP response to acute sodium loading, possibly due to impaired vasodilatory capacity. This indicates that heparan sulfates may be pivotal in vascular responses to acute sodium loading.

Abstract 535: Impaired Natriuresis and Increased Salt Sensitivity in Response to Acute Salt Load During Western Diet-intake: Role of Trpv1

Western diet (WD) intake increases morbidity of obesity, diabetes, and salt sensitive hypertension albeit mechanisms are largely unknown. We tested the hypothesis that the transient receptor potential vanilloid type 1 (TRPV1) channels are activated during WD intake to protect against salt-induced increases in blood pressure possibly via enhancing natriuresis. Wild-type (WT, C57BL/6) or gene-targeted TRPV1-null mutant (TRPV1 -/- ) mice were fed a normal (CON) or Western (WD) diet for 16-18 weeks. Mean arterial pressure (MAP), determined by radiotelemetry after acute sodium loading (NS, 0.9% NaCl) or vehicle (C) by gavage with or without treatment with Nω-nitro-l-arginine methyl ester ( L - NAME, 10mg/kg, ip. ) or a TRPV1 agonist, N-oleoyldopamine ( OLDA, 0.1mg/kg, ip ), was not different between two strains fed a CON diet. WT or TRPV1-/- mice fed a WD diet had increased MAP after NS, with a greater magnitude in TRPV1-/- mice (p&lt;0.05). OLDA decreased while L-NAME increased MAP in WT-WD-NS but not TRPV1-/-WD-NS mice (p&lt;0.05). The urinary nitrates plus nitrites excretion (UNOx, umol*4h urine/kg BW), an indicator of renal nitric oxide (NO) production, was increased in both strains fed a CON diet after NS (p&lt;0.05). OLDA further increased while L-NAME prevented NS-induced increases in UNOx in WT-CON-NS only or in both strains, respectively. TRPV1 ablation with WD intake decreased UNOx levels before NS and abolished NS-induced increment in UNOx (WT-WD-C: 0.3 ±0.06 vs. TRPV1-/-WD-C: 0.2 ±0.05; and WT-WD-NS: 0.7 ±0.1 vs. TRPV1-/-WD-NS: 0.3 ±0.1; p&lt;0.05). OLDA further increased while L-NAME prevented NS-induced increment in UNOx in WT-WD-NS mice only (p&lt;0.05). Urinary sodium excretion was increased in both strains fed a CON diet after NS (p&lt;0.05), which was unaffected by OLDA or L-NAME. UNa was increased in WT-WD-NS but not TRPV1-/-WD-NS after NS. OLDA further increased while L-NAME prevented NS-induced increases in UNa in WT-WD-NS only. Thus, TRPV1 ablation increases salt sensitivity during WD intake possibly via impaired NS-induced renal NO production and sodium excretion. Activation of TRPV1 with OLDA enhances renal NO production and sodium excretion, resulting in prevention of increased salt sensitivity during WD intake.

Feedback inhibition of the epithelial Na+ channel (ENaC) during acute sodium loading in vivo

The in vivo role of feedback inhibition of ENaC by intracellular [Na+] ([Na+]i) was studied by investigating the effect of amiloride during an acute sodium load to Na+‐deprived rats in the presence of an aldosterone mini‐pump. Amiloride blocks sodium entry into principal cells of the cortical collecting ducts (CCD) and presumably decreases [Na+]i. ENaC activity was evaluated by measuring amiloride‐sensitive currents (INa) in split‐open CCDs using whole‐cell patch clamp. ENaC surface expression was assessed by an in vivo biotinylation protocol. A four‐hour acute sodium load decreased INa by 70%. The presence of amiloride did not significantly change INa after an acute sodium load. Whole‐kidney expression of cleaved α, β, and cleaved γ ENaC decreased with an acute sodium load by 2.6, 3.4, and 6.2 fold, respectively. In addition, cell‐surface expression of β and cleaved γ ENaC decreased with an acute sodium load by 2.5 and 4.7 fold, respectively. Amiloride increased whole‐kidney expression of cleaved α and cleaved γ ENaC after an acute sodium load by 2.1 and 3.0 fold, respectively, and cell‐surface expression of β and cleaved γ ENaC by 2.2 and 5.2 fold, respectively. These results suggest that [Na+]i can regulate ENaC expression in vivo but changes in factors other than [Na+]i and aldosterone are responsible for down regulating ENaC activity in response to an acute sodium load.NIH RO1‐DK59659 (LGP) and T32‐GM07739 (ABP).

Primary aldosteronism and low-renin hypertension: a continuum?

Primary aldosteronism (PA) was first described by Jerome Conn almost 60 years ago, and is thus sometimes called Conn’s syndrome. Although Conn believed that PA may constitute 20% of all hypertension, within a decade of his initial findings, it was thought (and taught) that PA was relatively benign, accounted for <1% of hypertension, with hypokalaemia required for diagnosis, none of which we now know to be the case. PA is now recognized as causing ∼10% of hypertension, frequently occurring with normokalaemia, and to carry a much higher level of cardiovascular risk—atrial fibrillation, non-fatal myocardial infarction and stroke—than age, sex and blood pressure (BP)-matched essential hypertensives [1]. The radically revised figure for the prevalence of PA reflects the currently widely accepted use of the plasma aldosterone to renin ratio (ARR) for potential case detection. If a patient has low levels of plasma renin activity (PRA: reflecting generation of angiotensin II from endogenous angiotensinogen) or of plasma renin concentration per se, coupled with a plasma aldosterone concentration (PAC) in the upper levels of the normal range or higher, they are candidates for the diagnosis of PA. PA is thus defined as inappropriate aldosterone secretion despite suppressed levels of renin plus normal or low levels of plasma [K + ], the two major stimuli of aldosterone secretion. Although the definition of a cut-off for normal versus elevated ARR varies between laboratories, most find ∼20% of hypertensives to have a raised ARR, of whom approximately half have PA as confirmed by subsequent testing, commonly involving PAC being less than normally responsive to acute sodium loading, or other tests (captopril challenge, fludrocortisone suppression). While levels of aldosterone in PA rarely equal those found in profound chronic sodium deficiency, the clinical picture differs markedly between the two. In sodium deficiency, the elevated PAC is homeostatic, to maximize epithelial sodium retention, and BP is low normal; in PA, the elevated PAC causes inappropriately high epithelial sodium retention and volume expansion. The vascular wall is a physiological target tissue for aldosterone, with the high PAC in sodium deficiency serving to maintain the vascular tone in the face of a reduced circulating volume; in PA, the elevated PAC is inappropriately vasoconstrictor, in the face of the expanded plasma volume. Taken together, the epithelial and vascular actions of inappropriately elevated aldosterone levels in PA provide a cogent explanation of the resultant BP elevation. This contrasts with low-renin hypertension (LRH), in which renin is suppressed (as is the case for PA), but PAC levels are apparently normal. This appears to be the case for ∼30% of hypertensives; given that renin is secreted in response to sympathetic stimulation, the elevated BP prima facie appears not to reflect increased systemic sympathetic overactivity; low renin rules out direct effects of angiotensin on sodium retention or vascular tone, as similarly do the apparently normal levels of PAC. In contrast with PA, then, LRH is a description of rather than a mechanism-based diagnosis for the observed BP elevation.

Feedback inhibition of ENaC during acute sodium loading in vivo

The epithelial Na(+) channel (ENaC) is tightly regulated by sodium intake to maintain whole body sodium homeostasis. In addition, ENaC is inhibited by high levels of intracellular Na(+) [Na(+)](i), presumably to prevent cell Na(+) overload and swelling. However, it is not clear if this regulation is relevant in vivo. We show here that in rats, an acute (4 h) oral sodium load decreases whole-cell amiloride-sensitive currents (I(Na)) in the cortical collecting duct (CCD) even when plasma aldosterone levels are maintained high by infusing the hormone. This was accompanied by decreases in whole-kidney cleaved α-ENaC (2.6 fold), total β-ENaC (1.7 fold), and cleaved γ-ENaC (6.2 fold). In addition, cell-surface β- and γ-ENaC expression was measured using in situ biotinylation. There was a decrease in cell-surface core-glycosylated (2.2 fold) and maturely glycosylated (4.9 fold) β-ENaC and cleaved γ-ENaC (4.7 fold). There were no significant changes for other apical sodium transporters. To investigate the role of increases in Na(+) entry and presumably [Na(+)](i) on ENaC, animals were infused with amiloride prior to and during sodium loading. Blocking Na(+) entry did not inhibit the effect of resalting on I(Na). However, amiloride did prevent decreases in ENaC expression, an effect that was not mimicked by hydrochlorothiazide administration. Na(+) entry and presumably [Na(+)](i) can regulate ENaC expression but does not fully account for the aldosterone-independent decrease in I(Na) during an acute sodium load.