Na In Urine Research Articles

Abstract P227: Prostaglandin EP 4 Receptor Mediate Angiotensin II-induced Hypertension via Activation of PRR/ENaC Pathway

Activation of COX-2/EP 4 pathway is shown to upregulate renal (pro) renin receptor (PRR) contributing angiotensin II (Ang II)-induced hypertension but the underlying mechanism remains unclear. Here, we tested the hypothesis that activation of EP 4 receptors increases ENaC expression/activity through PRR in the setting of Ang II treatment. Sprague-Dawley rats were infused for 2 weeks with vehicle, Ang II alone (100 ng/kg/min), or in combination with an EP 4 antagonist ONO-AE3-208 (ONO) (0.2 mg/kg/d). Following 14-d AngII treatment, 24-h urine volume and 24-h urinary Na + excretion both exhibited a significant increase which was completely blocked by ONO. Ang II infusion selectively elevated α-ENaC protein abundance (1.9-fold) in the renal medulla but not in the renal cortex, which was completely blocked by ONO, as assessed by immunoblotting. Interestingly, Ang II infusion elevated the cleaved γ-ENaC protein in both medulla and cortex, which was similarly abolished by ONO. In response to Ang II infusion, the increases in renal medullary mRNA were 1.7-fold for α-ENaC, 1.3-fold for β-ENaC, 2.4-fold for γ-ENaC as assessed by qRT-PCR; in contrast, none of the ENaC subunits showed obvious changes in the renal cortex. Ang II-induced increases in α-ENaC mRNA were reduced 41% by ONO with a modest effect on the response of β-ENaC and γ-ENaC. In cultured mpkCCD cells, Ang II treatment at 500 nM for 24 h induced a 2.5-fold increase of ENaC activity as assessed by epithelial volt-ohmmeter, which was completely blocked by ONO (10 μM) or a specific PRR inhibitor PRO20 (1.0 μM). To further determine the role of the EP4 receptor in the regulation of ENaC activity, the mpkCCD cells were treated with vehicle, CAY10580 alone (agonist of the EP 4 receptor, 10 μM), or in combination with PRO20 for 24 h. The ENaC activity showed a 2.0-fold increase in response to CAY10580, which was nearly completely abolished by PRO20 (n = 6-8, p<0.05). CAY10580 induced a 2.1-fold increase in PRR protein abundance. Overall, these results suggest that activation of EP 4 induces PRR, leading enhancement of ENaC expression/activity, eventually contributing to increased blood pressure in response to Ang II treatment.

Abstract 103: Decreased Ability to Excrete a Na Load and Hypertension in the ALMS1 (Alstrom Syndrome 1) Knockout Rat

Few genes involved in obesity are known to be involved in hypertension. The Alstrom Syndrome 1 protein (ALMS1) is involved in obesity in humans. We found it is expressed in the thick ascending limb (TAL) where it interacts with the Na/K/2Cl cotransporter (NKCC2). ALMS1 is also expressed in other nephron segments such as the proximal tubule and collecting ducts. We hypothesized that ALMS1 deletion leads to hypertension which is likely due to a decrease ability to excrete a salt load secondary to enhanced NaCl transport by the TAL and other nephron segments. To study the role of ALMS1 in renal function we generated ALMS1 knockout (KO) rats in a Dahl-salt sensitive genetic background via zinc-finger nuclease targeting by MCW gene targeting core. Using radio-telemetry we found the KO rats to have a higher baseline systolic blood pressure on normal Na chow compared to the Wild-type salt sensitive (WT) rats (KO:146±2 and WT:136±1 mmHg, *p=0.0012). To explore whether hypertension is related to increase renal Na reabsorption in KO rats we performed metabolic cage protocols to measure the acute excretion of a Na load (1% of body weight) in conscious rats. Our data show that it took longer for KO rats to excrete the Na load; cumulative or at individual time points (6, 9, 24 h) (cumulative Na, KO:4251±590 μmols/24h and WT:8788±994 μmols/24h, *p=0.0028). To test the role of different nephron segments in higher Na reabsorption we used a single dose of diuretics and measured urine Na and volume excretion. The NKCC2 inhibitor bumetanide (5mg/kg), induced a higher natriuretic response in KO rats (KO:485.2±37.1 μmols/8h, WT:221.8±32 μmols/8h, *p<0.05). The NCC inhibitor hydrochlorothiazide (HCTZ) 200mg/kg, caused a similar natriuretic effect in KO rats (KO:206.8±16 μmols/8h and WT:155.6±20 μmols/8h, p=0.0717). The ENaC inhibitor benzamil (10mg/kg), caused a similar natriuretic response in KO rats (KO:214.5±33 μmols/8h and WT:253.4±22 μmols/8h, p=0.3477). We conclude that ALMS1 KO have a decreased ability to excrete a salt load and this is primarily mediated by enhanced TAL-mediated Na reabsorption. Higher TAL Na absorption is likely involved in hypertension in ALMS1 KO rats. These data show that ALMS1 is important for blood pressure control and renal function.

Abstract P188: Nephron-wide Deletion of Nos3 Impairs Salt Excretion and Causes Hypertension

In vitro studies have suggested that nephron nitric oxide synthase 3 (NOS3) plays a role in the regulation of urinary Na + excretion (UNaV) and potentially contributes to blood pressure (BP) control. To assess whether NOS3 is indeed involved in the physiological regulation of UNaV and BP, mice with doxycycline-inducible nephron-wide deletion of NOS3 were generated. These mice were homozygous for loxP-flanked exons 9-12 of the NOS3 gene (contains the calmodulin binding site) and hemizygous for Pax8-rtTA and LC-1 transgenes (Pax8 promoter-rtTA confers nephron-specific targeting and LC-1 transgene contains doxycycline/rtTA-inducible Cre recombinase). Mice were treated with either vehicle (controls) or doxycycline (knockouts, KO) at 1 month of age for 12 days and studied at ~3 months of age. Nephron-specific NOS3 KO mice had renal-specific NOS3 gene recombination and reduced mRNA levels in microdissected nephron segments. Renal vascular NOS3 mRNA levels were markedly increased in KO mice, presumably as a compensatory response to nephron NOS3 KO. KO mice had modestly increased mean arterial pressure (100 ± 2 mmHg in controls and 103 ± 1 mmHg in KO, N=6, p<0.05) on a normal (0.3% Na + ) salt diet that increased further during high (3.2% Na + ) salt intake (103 ± 2 mmHg in controls and 118 ± 3 mmHg in KO, N=6, p<0.05). KO mice had reduced daily UNaV and greater cumulative Na + retention compared to controls over the 7 days of high salt feeding. PRC tended to be lower in KO mice on a high salt diet compared to controls (85.8 ± 13.5 ng/ml in controls and 60.1 ± 10.3 ng/ml in KO, N=12, p<0.07). Control mice excreted most of an acute I.P. salt load by 4 hr post-load, while KO mice excreted the acute salt load between 5-7 hr after loading. Furosemide, but not hydrochlorothiazide, abolished the differences in UNaV between control and control mice following an acute salt load. No sex-dependent differences in UNaV or BP were observed. In summary, these findings indicate that nephron NOS3 is involved in acute and chronic control of renal Na + excretion and BP during high salt intake. Taken together with previous studies showing that collecting duct-specific KO of NOS3 does not alter UNaV or BP, our data suggest that thick ascending limb is at least partly involved – further mechanistic studies are ongoing.

Diuretic activity of the aqueous crude extract and hot tea infusion of Moringa stenopetala (Baker f.) Cufod. leaves in rats.

Moringa stenopetala (Baker f.) Cufod. is a medicinal plant that has been used for the treatment of different ailments such as hypertension and diabetes in Ethiopia. This study aims to assess the diuretic activity of the aqueous crude extract and hot tea infusion of M. stenopetala leaves in saline-loaded rats. Male Wistar rats were divided into ten groups (n = 5). The control group received distilled water (5 mL/kg), whereas the reference group received Furosemide (10 mg/kg). Groups III–X orally received different doses of aqueous crude extract (62.5, 125, 250, and 500 mg/kg) and hot tea infusion (1, 2, 4, and 6 teaspoons [Tsp]) based on community use. Urine volume was recorded every hour until the end of the 5th hour, and total urine volume of each animal was calculated. The diuretic activity and diuretic action were determined based on the urine output. Additionally, concentration of urinary sodium, chloride, and potassium ions was determined. The urinary Na+/K+ ratio and carbonyl anhydrase activity (Cl−/(Na+/K+)) were also assessed. The findings verified that the aqueous crude extract as well as the hot tea infusion of the leaves of M. stenopetala possesses significant (P < 0.01) diuretic, natriuretic, and kaliuretic effects. The aqueous crude extract (125 mg/kg) and hot tea infusion (2 Tsp) displayed the highest diuretic activity (101% and 96%, respectively) comparable to the reference drug, Furosemide (10 mg/kg). They also displayed a good natriuretic activity. The aqueous crude extract and hot tea infusion revealed a significant Na+ urinary excretion (P < 0.001) and Na+/K+ ratio (P < 0.05) at all test doses. There was also a significant (P < 0.01) Cl− urinary excretion at all test doses of aqueous crude extract except 62.5 mg/kg and all test doses of hot tea infusion except higher doses (4 and 6 Tsp). Thus, the aqueous crude extract as well as the hot tea infusion of the leaves of M. stenopetala causes a plausible increase in the urine volume and concentration of urinary electrolytes in rats.

Trial of Amiloride in Type 2 Diabetes With Proteinuria

IntroductionRenal sodium (Na+) retention and extracellular fluid volume expansion are hallmarks of nephrotic syndrome, which occurs even in the absence of activation of hormones that stimulate renal Na+ transporters. Plasmin-dependent activation of the epithelial Na+ channel has been proposed to have a role in renal Na+ retention in the setting of nephrotic syndrome. We hypothesized that the epithelial Na+ channel inhibitor amiloride would be an effective therapeutic agent in inducing a natriuresis and lowering blood pressure in individuals with macroscopic proteinuria.MethodsWe conducted a pilot double-blind randomized cross-over study comparing the effects of daily administration of either oral amiloride or hydrochlorothiazide to patients with type 2 diabetes and macroscopic proteinuria. Safety and efficacy were assessed by monitoring systolic blood pressure, kidney function, adherence, weight, urinary Na+ excretion, and serum electrolytes. Nine subjects were enrolled in the trial.ResultsNo significant difference in systolic blood pressure or weight was seen between subjects receiving hydrochlorothiazide and those receiving amiloride (P ≥ 0.15). Amiloride induced differences in serum potassium (P < 0.001), with a 0.88 ± 0.30 mmol/l greater acute increase observed. Two subjects developed acute kidney injury and hyperkalemia when treated with amiloride. Four subjects had readily detectable levels of urinary plasminogen plus plasmin, and 5 did not. Changes in systolic blood pressure in response to amiloride did not differ between individuals with versus those without detectable urinary plasminogen plus plasmin.DiscussionIn summary, among patients with type 2 diabetes, normal renal function, and proteinuria, there were reductions in systolic blood pressure in groups treated with hydrochlorothiazide or amiloride. Acute kidney injury and severe hyperkalemia were safety concerns with amiloride.

NADPH Oxidase p67phox Subunit Contributes to the Maintenance of Diurnal Blood Pressure Rhythms in Dahl Salt‐Sensitive Rats

Loss of diurnal blood pressure (BP) rhythm, such as absence of dipping during the inactive phase is a known risk factor for cardiovascular disease. Salt‐sensitive individuals have higher susceptibility for dysfunctional diurnal BP rhythms as well as for developing hypertension. In this study, we hypothesized that NADPH oxidase, specifically the p67phox subunit, impairs diurnal BP rhythm and circadian clock gene mRNA expression in the Dahl salt‐sensitive (SS) rats. We utilized SS and p67phox knockout SS (SSp67phox−/−) rats (n=8 per group) to determine 24‐hr BP, heart rate and locomotor activity by telemetry while on normal (0.4% NaCl), low (0.04% NaCl) and high (4% NaCl) salt diets (Dyets, AIN‐76A). Our results show that SSp67phox−/− rats and SS rats have similar diurnal BP rhythms (average amplitude 3.43 vs. 3.13 mmHg, respectively) when on a normal salt diet. Cosinor analysis was used to calculate the amplitude, which is defined as the difference between peak and mean value of a 24‐hr oscillation. 24‐hr mean arterial pressure (MAP) was lower in SSp67phox−/− than SS (116 ± 1 vs. 129 ± 1 mmHg, respectively, p&lt;0.0001). Heart rate groups and locomotor activity did not differ across genotypes or diets. On one week of high salt diet, the increase in 24‐hr MAP from normal salt diet was larger in SS rats compared to SSp67phox−/− (+11.6 ± 0.8 vs. +7.0 ± 1.1 mmHg, respectively, p=0.017). The diurnal BP rhythm (day vs. night) in SS and SSp67phox−/− on high salt diet became more pronounced than on normal salt diet (average amplitude 7.99 and 6.11 mmHg, respectively). Low salt diet did not change 24‐hr MAP in either genotype, however diurnal BP rhythm was abolished in SS rats and maintained in the SSp67phox−/− rats (average amplitude 2.35 and 3.30 mmHg, respectively). We also measured 12‐hr urinary sodium (Na+) excretion via metabolic cages under normal, low and high salt diets. During the dark/active phase on high salt diet, SSp67phox−/− had less water intake and urine output compared to SS rats, while light/inactive phase measurements were similar. Urinary Na+ excretion was also lower in SSp67phox−/− compared to SS controls on high salt diet during the light/active phase, while Na+ excretion was similar during the light/inactive phase. Food intake was similar between genotypes, regardless of time of day. On normal and low salt diets, SSp67phox−/− and SS rats had similar urine output, food and water intake, and urinary Na+ excretion. To assess if core clock gene, Bmal1, was altered with salt diets, we examined Bmal1 mRNA expression in renal vessels at 12‐hr intervals under either low or high salt feeding. Bmal1 mRNA expression was higher at zeitgeber time 0 (ZT0) compared to ZT12 in renal vessel and buccal cells. In contrast to our prior findings using renal inner medullary tissue, neither salt diet or genotype changed the magnitude of Bmal1 mRNA expression. Taken together, these data suggest that NADPH oxidase p67phox expression contributes to the maintenance of diurnal BP rhythms in SS rats on low salt diet.Support or Funding InformationThis work was supported by the American Heart Association Strategically Focused Research Network grant (15SFRN2390002)

Adaptive changes in GFR, tubular morphology, and transport in subtotal nephrectomized kidneys: modeling and analysis.

Removal of renal mass stimulates anatomical and functional adaptations in the surviving nephrons, including elevations in single-nephron glomerular filtration rate (SNGFR) and tubular hypertrophy. A goal of this study is to assess the extent to which the concomitant increases in filtered load and tubular transport capacity preserve homeostasis of water and salt. To accomplish that goal, we developed computational models to simulate solute transport and metabolism along nephron populations in a uninephrectomized (UNX) rat and a 5/6-nephrectomized (5/6-NX) rat. Model simulations indicate that nephrectomy-induced SNGFR increase and tubular hypertrophy go a long way to normalize excretion, but alone are insufficient to fully maintain salt balance. We then identified increases in the protein density of Na+-K+-ATPase, Na+-K+-2Cl- cotransporter, Na+-Cl- cotransporter, and epithelial Na+ channel, such that the UNX and 5/6-NX models predict urine flow and urinary Na+ and K+ excretions that are similar to sham levels. The models predict that, in the UNX and 5/6-NX kidneys, fractional water and salt reabsorption is similar to sham along the initial nephron segments (i.e., from the proximal tubule to the distal convoluted tubule), with a need to further reduce Na+ reabsorption and increase K+ secretion primarily along the connecting tubules and collecting ducts to achieve balance. Additionally, the models predict that, given the substantially elevated filtered and thus transport load among each of the surviving nephrons, oxygen consumption per nephron segment in a UNX or 5/6-NX kidney increases substantially. But due to the reduced nephron population, whole animal renal oxygen consumption is lower. The efficiency of tubular Na+ transport in the UNX and 5/6-NX kidneys is predicted to be similar to sham.

Collecting duct-specific knockout of nitric oxide synthase 3 impairs water excretion in a sex-dependent manner.

Nitric oxide (NO) inhibits collecting duct (CD) Na+ and water reabsorption. Mice with CD-specific knockout (KO) of NO synthase 1 (NOS1) have salt-sensitive hypertension. In contrast, the role of NOS3 in CD salt and water reabsorption is unknown. Mice with CD NOS3 KO were generated with loxP-flanked exons 9-12 (encodes the calmodulin binding site) of the NOS3 gene and the aquaporin-2 promoter-Cre transgene. There were no differences between control and CD NOS3 KO mice, irrespective of sex, in food intake, water intake, urine volume, urinary Na+ or K+ excretion, plasma renin concentration, blood pressure, or pulse during 7 days of normal (0.3%), high (3.17%), or low (0.03%) Na+ intake. Blood pressure was similar between genotypes during DOCA-high salt. CD NOS3 KO did not alter urine volume or urine osmolality after water deprivation. In contrast, CD NOS3 KO male, but not female, mice had lower urine volume and higher urine osmolality over the course of 7 days of water loading compared with control mice. Male, but not female, CD NOS3 KO mice had reduced urinary nitrite+nitrate excretion compared with controls after 7 days of water loading. Urine AVP and AVP-stimulated cAMP accumulation in isolated inner medullary CD were similar between genotypes. Western analysis did not reveal a significant effect of CD NOS3 KO on renal aquaporin expression. In summary, these data suggest that CD NOS3 may be involved in the diuretic response to a water load in a sex-specific manner; the mechanism of this effect remains to be determined.

Dietary sodium and potassium intake and their association with blood pressure in a non-hypertensive Iranian adult population: Isfahan salt study.

The association of sodium (Na) and potassium (K) intake with blood pressure (BP) is an ongoing debate, especially in central Iran. We aimed to examine the mean Na and K intake, major sources of Na and the relationship between BP and dietary and urinary Na and K. This cross-sectional study was performed in central Iran in 2013-2014. A total of 796 non-hypertensive adults aged >18 years were randomly recruited. The semi-quantitative food frequency questionnaire was used to assess dietary Na and K intake. Moreover, 24-hour urine samples were collected to measure 24-hour urinary Na (UNa) and K (UK) as biomarkers. BP was measured twice on each arm using a standard protocol. The mean Na and K intake were 4309.6 ± 1344.4 and 2732.7 ± 1050.5 mg/day, respectively. Table and cooking salt were the main sources of Na. Odds ratio (OR) (95% confidence interval (CI)) of the crude model in the highest quartile of UNa indicated a significant association with the higher risk of prehypertension (OR (95% CI): 2.09 (1.09-4.05); P for trend = 0.007). After adjustment for potential confounders, prehypertension was significantly associated with increasing dietary Na/K ratio (OR (95% CI): 1.28 (1.01-1.57); P for trend = 0.046) and UNa/UK ratio (OR (95% CI): 2.15(1.08-4.55); P for trend = 0.029). Increasing dietary and urinary Na/K ratios and UNa were associated with elevated BP and prehypertension occurrence. These findings support the necessity of developing a salt reduction programme in our country.

ISH NIA PS 02-04 Urinary Na and K excretion values in outpatients with hypertension compared to patients with other diseases

Objective: Restricting salt intake is considered important in the treatment of hypertension, and knowledge of the profile of salt and potassium (K) intakes is required in outpatients with hypertension. We collected spot urine from outpatients to determine urinary sodium (Na) and K excretion values and urinary creatinine to calculate daily salt and K intakes. Design and Method: Spot urine from outpatients with hypertension (n = 377) or other diseases (n = 282) was collected when they presented to our hospital to receive medical examination from morning to noon. Urinary Na and K levels were determined, and daily excretion values were calculated based on the urinary Na (mEq/L) or K (mEq/L) values, body weight (BW, kg), height (cm), urinary creatinine value (mg/dl), age (years) and sex, according to previously reported method. Results: Urinary Na and K excretion values were significantly lower in patients with hypertension (Na, 188.9 ± 3.9 mEq/day; K, 46.7 ± 0.7 mEq/day) than in patients with other diseases (Na, 204.5 ± 4.7 mEq/day; K, 50.1 ± 0.9 mEq/day). Body mass index was significantly higher in patients with hypertension (23.8 ± 0.2 kg/m2) than in patients with other disease (22.8 ± 0.2 kg/m2). Age did not differ significantly between groups (patients with hypertension, 72.0 ± 0.6 years; patients with others, 71.9 ± 0.8 years). Urinary Na and K excretion values correlated negatively with age and positively with body weight and BMI in both groups. Conclusions: Lower Na excretion values in patients with hypertension might be attributable to effective education about the importance of lowering salt intake. On the other hand, lower K excretion suggests lower K intake, which might act to increase blood pressure. Education on this issue may help to increase K ingestion and body weight might be decreased. A simple method to determine 24-h urinary Na and K excretion in outpatients is useful for clinical application.

Abstract P206: Increased Short-term Dietary Sodium Intake Does Not Increase Blood Pressure or Measures of Aortic Stiffness in Young Healthy Adults

Background: Sodium (Na) sensitivity is defined as the short-term increase in blood pressure (BP) after increased Na intake. The impact of Na sensitivity on measures of aortic stiffness has not been previously characterized in non-hypertensives. We tested the feasibility of an oral Na loading protocol to characterize the effects of increased dietary Na on blood pressure and measures of aortic stiffness in health young adults. Methods: In an open-label repeated measures pilot study, we enrolled 40 healthy adults age 21-30 years (18 females). Subjects were evaluated before and after 7 days of oral Na loading (12g (205 mmol) of salt tablets/day). BP (mmHg, cuff method) was measured in supine, seated and standing positions. Radial artery tonometry (Sphygmacor) was used to assess measures of aortic stiffness: aortic BP (mmHg), aortic pulse pressure (mmHg), aortic augmentation index (%) and adjusted Tr, time of return of the reflected pressure wave as an index of aortic pulse wave velocity (m/s). Serum aldosterone, urine Na and potassium (K) were measured. Results: Nine subjects dropped out due to GI intolerance of the salt pills. In the remaining 31 subjects, Na loading did not change BP in any position, aortic BP, aortic pulse pressure, or Tr (Table: mean±SD for seated R arm BP and measures of aortic stiffness). There was no measurable aortic augmentation in the cohort. Na loading decreased serum aldosterone (9.1±1.5 to 5.2±0.8 ng/dl, p=0.02), and increased urine Na:K ratio (1.8±0.2 to 3.5±0.5, p=0.002). Conclusion: Oral Na loading was associated with suppression of the renin-angiotensin aldosterone system, did not increase BP, or measures of aortic stiffness in healthy young adults.

Abstract 095: Ovariectomy Restores Purinergic Receptor Activation of Endothelin-dependent Natriuresis

We have recently reported interplay between renal medullary endothelin-1 (ET-1) and purinergic (P2) systems, which play central roles in controlling Na + homeostasis in male rats. Evidence suggests that sex hormones regulate ET-1 and P2 systems. To test whether activation of the renal medullary P2 receptors promotes ET-dependent natriuresis in females and whether ovariectomy (OVX) modulates this potential interaction, we studied the effect of medullary NaCl loading on Na + excretion in adult intact female and OVX SD rats in the presence and absence of P2 or ET receptor antagonism. Isosmotic saline (284 mOsmol/kg H 2 O) was infused into the renal medullary interstitium during a baseline urine collection period, followed by isosmotic or hyperosmotic saline (1800 mOsmol/kg H 2 O) infusion. Blood pressure, renal blood flow, urine Na + , K + and osmolality were measured. Medullary NaCl loading significantly enhanced Na + excretion in intact females and OVX (from 0.8±0.2 to 6.2±1.6 and from 0.7±0.1 to 5.6±0.8 μmol/min, respectively, n=6-8, p&lt;0.05). The natriuretic effect of NaCl loading in intact females was not attenuated by P2 or ET receptor blockade. Whereas, intramedullary infusion of the P2 receptor antagonist, suramin, inhibited the natriuresis induced by medullary NaCl loading in OVX (from 0.4±0.2 to 0.9±0.4 μmol/min, n=6). Additionally, combined ET A/B receptor blockade (ABT-627 + A-192621) abolished the natriuretic response to medullary NaCl load in OVX rats (from 0.2±0.1 to 1.4±0.7 μmol/min, n=4). Activation of medullary purinergic (P2Y 2/4 ) receptors by UTP infusion had no significant effect in intact females, but enhanced Na + excretion in OVX rats (from 0.5±0.1 to 2.3±0.8 μmol/min, n=5, p&lt;0.05). Combined ET A/B receptor blockade significantly inhibited the natriuretic response to UTP observed in OVX rats. These data suggest that increased medullary NaCl loading induces ET-independent and P2-independent natriuresis in intact females. In OVX, activation of medullary P2 receptors promotes ET-dependent natriuresis, similar to our previous findings in male rats and suggests that OVX restores the interplay between the renal ET-1 and purinergic (P2) signaling systems to facilitate Na + excretion. Funded by AHA 15POST25090329 to EYG and P01 HL95499 to DMP

Abstract P330: Role of Na-H Exchanger Regulatory Factor 1 (NHERF1) In Salt-sensitive Hypertension and Inflammation in Aging

Aging animals develop salt-sensitive hypertension due, in part, to desensitization of dopamine receptors and sensitization of AT1R in renal proximal tubules. We demonstrated old FBN rats develop salt-dependent hypertension. Preliminary data showed loss of NHERF1 expression in 22m old F344 rats. We hypothesized that loss of NHERF1 may contribute to salt reabsorption in aging. To address this hypothesis, Fischer Brown Norway (FBN) rats (1m, 4m, 12m, and 24m old) were fed 1% or 8% NaCl diet for one week and, urinary volume, sodium, potassium, and chloride and expression of Na-K ATPase α1 subunit (NKA), NHERF1, and D1R were measured. Feeding 8% NaCl increased 24 h urine volume compared to rats fed 1% NaCl diet in 1m (13.6±0.98 vs 29.2±3.8 mL) and 4m (15.3±1.5 vs 39.2±2.4) but not in 24 m rats (20.7±4.7 vs 15.9±2.4). 24 h urine Na increased 7 fold in 4 m old rats fed an 8% NaCl diet but only 3 fold in 24m old rats. To our surprise, NHERF1 expression increased with age in FBN rats (0.21±.03 (4m) vs 0.71±0.07 (24m) AU ratio NHERF1:actin). In F344 rats NHERF1 expression decreased with age (0.86±0.14 vs 0.39±0.05). Increased NHERF1 expression with age in FBN rats was associated with decreased D1R expression (0.8±0.16 vs 0.4±0.04) while in F344 rats D1R expression increased with age (0.6±0.1 vs 0.9±0.1). FBN rats are salt-sensitive while F344 are salt-resistant. F344 rats develop kidney inflammation with aging. We determined TNFα in kidney homogenates from FBN and F344 rats. TNFα increased with age in both F344 (4.04±0.15 vs 6.9±0.42) and FBN rats (2.1±0.3 vs 4.05±0.31 ng/mg protein). To determine if lack of NHERF1 is responsible for salt wasting, we measured urine volume and Na in 18 m old WT and NHERF1 KO mice. The urine volume in WT mice (2.7±0.8 (1% NaCl) vs 5.6±1.2 mL/24 h (8% NaCl)) were significantly lower than KO mice (3.6±0.57(1% NaCl) vs 7.5±0.88 mL/24 h (8% NaCl)). Similar results were observed in Na excretion. D1R expression increased in NHERF1 KO mice as compared to WT mice. TNFα increased significantly in NHERF1 KO mice kidneys as compared to WT mice. We conclude that increased NHERF1 expression as seen in FBN rats results in salt retention while lack of NHERF1 as seen F344 rats and NHERF1 KO mice increases renal inflammation. Further studies are required to delineate the two roles of NHERF1.

Abstract 069: β-Catenin Signaling Contributes to Angiotensin II-Induced Hypertension via Activation of Sodium Transporters in the Distal Nephron

β-Catenin signaling plays an important role in regulation of development as well as tissue homeostasis. Recently, we have shown that this pathway is involved in regulation of collecting duct (CD) function. Here, we examined the role of β-catenin signaling in AngII-induced hypertension. C57/BL6 mice were administered for 7 d with vehicle, AngII alone (300 ng/kg/min) or in combination with an inhibitor of β-catenin pathway, ICG-001 (50 mg/kg/d) both via an osmotic mini-pump. Radiotelemetry demonstrated that ICG-001 effectively attenuated AngII-induced hypertension (MAP on day 7: 112.5 ± 2.02 in Control group vs. 137±2.07 in AngII group vs. 119.2±2.26 mmHg in AngII/ICG-001 group, n = 7-9, p&lt;0.05) accompanied with a 34% (34 of 100) increase in 24-h urine volume and a 35% (35 of 100) increase in 24-h urinary Na+ excretion. Following AngII treatment, urinary renin activity and renin content both exhibited a more than 10-fold increase which was completely blocked by ICG-001. AngII infusion selectively elevated α-ENaC protein abundance in the renal medulla but not in the renal cortex; the renal medullary upregulation of α-ENaC protein expression was attenuated by ICG-001. Similarly, renal cortical NCC and NKCC2 protein expression was elevated by AngII infusion; this upregulation of NCC and NKCC2 protein expression was again abolished by ICG-001. In contrast, NHE3 protein abundance remained constant. In cultured mpkCCD cells transfected with a β-catenin-driven luciferase construct, AngII treatment at 500 nM for 24 h induced a 10-fold increase in the reporter activity which was sensitive to ICG-001 (1 μM). In these cells, the AngII treatment induced amiloride-sensitive Na + transport as assessed by epithelial volt-ohmmeter, which was completely blocked by ICG-001. Protein expression of α-ENaC exhibited the similar pattern of changes as ENaC activity whereas the expression of β- or γ-ENaC remained constant. ICG-001 was not associated with noticeable toxicity in vivo or in vitro. In summary, these results suggest that β-catenin signaling mediates AngII-induced hypertension at least in part through regulation of intrarenal renin response and the expression of Na+ transporters in the distal nephron.

Abstract P608: Obesity Induced Hypertension Exacerbated Through Upregulation Of Renal Naci Cotransporter, Reversed By Increasing Pgc-1α-ho-1 Gene Expression To Restore Mitochondrial Function

Introduction: Hypertension caused by chronic obesity as a result of high calorie food intake or in leptin receptor deficient db/db mice may be linked to mitochondrial dysfunction. Previously we and others have shown that an epoxyeicosatrienoic acid agonist (EET-A), reduced adiposity and ROS resulting in normalization of BP by unknown mechanisms. We hypothesize that EET-A will attenuate BP by restoring mitochondrial function through increasing the PGC-1α-HO-1 axis and increasing urinary sodium excretion by downregulating NCC channels. Methods: Db/db mice at 16-wks of age were divided into 3 treatment groups and for an additional 16-wks received: A) control, B) EET-A 1.5mg/100g BW i.p. 2x/week and C) EET-A and lentiviral (Ln)- PGC-1α shRNA (to suppress PGC-1α protein). Oxygen consumption (VO 2 ), visceral fat and blood glucose were determined. Additionally, renal tissues were harvested to measure the type 2 Na-K-Cl cotransporters (NKCC2), epithelial Na channels- (ENaC), NaCl cotransporters (NCC), PGC-1α, HO-1, insulin receptors, and mitochondrial biogenesis markers. Results: At the conclusion of 32 weeks: Group A, developed hypertension and presented with decreased urinary Na excretion, decreased VO 2 , decreased downstream PGC-1α signaling, and mitochondrial dysfunction. There were increased levels of NCCs but not of NKCC2s or ENaCs. Renal PGC-1α, HO-1, pAMPK, and mitochondrial fusion protein Mfn 1/2, and Opa1 were decreased, p&lt;0.05. Group B, exhibited restoration of renal levels of PGC-1α, HO-1, pAMPK, and mitochondrial biogenesis proteins Mfn 1/2 and Opa1. NCC expression was reduced and was associated with an increase in urinary Na excretion; (p&lt;0.05). The beneficial effect of EET-A observed in group B was suppressed in group C using Ln- PGC-1α shRNA which suppressed PGC-1α expression in renal tissue &gt; 50% and was accompanied by the onset of even more severe suppression of urinary Na excretion than in Group A. Conclusion: Treatment of obese mice with EET-agonists leads to the recruitment of PGC-1α-HO-1 which enhances mitochondrial function and induces the downregulation of NCC channels and increased sodium excretion. EET may serve as a powerful therapeutic agent for the treatment of obesity induced hypertension.

Abstract 011: An Orally Absorbable Potent NHE3 Inhibitor Attenuates Angiotensin II-induced Hypertension Primarily by Inhibiting NHE3 in the Proximal Tubule of the Kidney

We have recently shown that angiotensin (ANG II)-induced hypertension was attenuated in mice with global ( Nhe3 -/- ) and Nhe3 -/- mice with transgenic rescue of the NHE3 gene selectively in small intestines (tg Nhe3 -/- ), suggesting an important role of NHE3 in the development of ANG II-dependent hypertension. In this study, we specifically tested whether the pharmacological inhibition of NHE3 mainly in the proximal tubules of the kidney attenuates ANG II-dependent hypertension induced by a low and slow pressor dose of ANG II supplemented with a high salt diet. Overall, 9 groups (n=5-12) of adult male C57BL/6J mice were infused with or without ANG II (500 μg/kg/day, i.p. via minipump) and supplemented with or without a 2% NaCl diet to slowly and moderately increase systolic blood pressure (SBP) in 2 weeks. ANG II alone increased SBP from 116 ± 2 mmHg to 140 ± 2 mmHg ( p &lt;0.01), and supplement of ANG II with a 2% NaCl diet further increased SBP to 147 ± 4 mmHg ( p &lt;0.05). Concurrent treatment with an orally active, absorbable NHE3 inhibitor AVE0657 (Sanofi-Aventis; 20 mg/kg/day, p.o.) significantly decreased SBP to 125 ± 4 mmHg in ANG II-infused mice ( p &lt;0.01), and to 134 ± 6 mmHg in ANG II-infused mice supplemented with 2% NaCl ( p &lt;0.01), respectively. Further treatment with AVE0657 and losartan, an AT 1 receptor blocker (20 mg/kg/day, p.o.), completely normalize SBP in mice treated with ANG II and 2% NaCl to control (115 ± 5 mmHg, p &lt;0.01). In the kidney, AVE0657 significantly increased 24h urinary Na + excretion from 157.1 ± 6.7 to 207.7 ± 8.1 μmol/24h ( p &lt;0.01) without altering 24h urine excretion or SBP. Furthermore, AVE0657 did not significantly alter 24 h fecal Na + excretion in non ANG II-infused (4.99 ± 0.37 μmol/24h, n.s.) or ANG II-infused mice (4.19 ± 0.67 μmol/24h, n.s.), compared with control (4.02 ± 0.20 μmol/24h, n.s. ) or global Nhe3 -/- mice (50.8 ± 0.8 μmol/24h, p &lt;0.01). Since small intestines in the gut and the proximal tubules of the kidney express the vast majority of NHE3 in the body, these results provide preclinical evidence and perspectives that orally absorbable NHE3 inhibitors may be pharmacologically beneficial to prevent and treat hypertension induced by ANG II and a high salt, mainly by inhibiting NHE3 in the proximal tubule of the kidney.

Abstract 098: A Novel Mechanism for Buffering Dietary Salt in Humans: Effects of Salt Loading on Skin Sodium, VEGF-C and Blood Pressure

Introduction: Dietary sodium is an important trigger for hypertension. Animal studies show that the skin buffers dietary salt and salt-loading induces lymphangiogenesis mediated by VEGF-C, maintaining normal BP. Our primary objective was to determine whether increased skin Na + on salt loading attenuates the expected rise in blood pressure. Methods: We assessed skin and urine electrolytes, systemic haemodynamics, ambulatory BP and plasma VEGF-C in 48 healthy participants. Participants were placed on a low salt diet (70mmol sodium/day) and received placebo and slow sodium (200mmol daily for 7 days) in a randomised order. Skin Na + and K + concentrations were measured in mg/g tissue by inductively coupled plasma optical emission spectroscopy. Results were expressed as the ratio of Na + :K + to correct for variability in sample hydration. Plasma VEGF-C was analysed by ELISA. Results: Mean age was 30 ± 8 years (24 male). 24-hr urine Na + was higher following salt than placebo in males (221.9 ± 16.5 vs. 86.3 ± 10.3mmol, p &lt; 0.001) and females (227.1 ± 19.9 vs. 60.0 ± 6.8mmol, p &lt; 0.001). In males, skin Na + :K + was higher after salt loading (2.87 ± 0.12 vs. 2.59 ± 0.09 mg/g , p = 0.008), but not significantly different in females (3.36 ± 0.12 vs. 3.23 ± 0.10 mg/g, p = 0.49). Females showed a significant increase in 24-hr MAP with salt loading (88 ± 1 vs. 85 ± 1 mmHg, p=0.004) but not males (90 ± 2 vs. 90 ± 2 mm Hg, p = 0.91). In males only, skin Na + :K + correlated with supine MAP post-placebo (r = 0.52, p = 0.004) and post-salt (r = 0.51, p = 0.01). No significant change was noted in plasma VEGF-C. Conclusions: Salt loading causes an increase in BP in healthy females but not in males, whilst skin sodium rises in males. This indicates that the skin may buffer the haemodynamic consequences of increased salt intake in a gender-specific manner.

Effect of Dapagliflozin Treatment on Fluid and Electrolyte Balance in Diabetic Rats

AimThis study evaluates the effect of dapagliflozin, a SGLT2 inhibitor, on fluid or electrolyte balance and its effect on urea transporter-A1 (UT-A1), aquaporin-2 (AQP2) and Na-K-2Cl cotransporter (NKCC2) protein abundance in diabetic rats. MethodsDiabetes mellitus (DM) was induced by injection of streptozotocin into the tail vein. Serum Na+, K+, Cl− concentration, urine Na+, K+, Cl− excretion, blood glucose, urine glucose excretion, urine volume, urine osmolality and urine urea excretion were analyzed after the administration of dapagliflozin. UT-A1, AQP2 and NKCC2 proteins were detected by western blot. ResultsDapagliflozin treatment decreased blood glucose concentration by 38% at day 7 and by 47% at day 14 and increased the urinary glucose excretion rate compared with the untreated diabetic animals. Increased 24-hour urine volume, decreased urine osmolality and hyponatremia, hypokalemia and hypochloremia observed in diabetic rats were attenuated by dapagliflozin treatment. Western blot analysis showed that UT-A1, AQP2 and NKCC2 proteins are upregulated in DM rats over control rats; dapagliflozin treatment results in a further increase in inner medulla tip UT-A1 protein abundance by 42% at day 7 and by 46% at day 14, but it did not affect the DM-induced upregulation of AQP2 and NKCC2 proteins. ConclusionDapagliflozin treatment augmented the compensatory changes in medullary transport proteins in DM. These changes would tend to conserve solute and water even with persistent glycosuria. Therefore, diabetic rats treated with dapagliflozin have a mild osmotic diuresis compared to nondiabetic animals, but this does not result in an electrolyte disorder or significant volume depletion.

Prenatal Testosterone Exposure Decreases Aldosterone Production but Maintains Normal Plasma Volume and Increases Blood Pressure in Adult Female Rats

Plasma testosterone levels are elevated in pregnant women with preeclampsia and polycystic ovaries; their offspring are at increased risk for hypertension during adult life. We tested the hypothesis that prenatal testosterone exposure induces dysregulation of the renin-angiotensin-aldosterone system, which is known to play an important role in water and electrolyte balance and blood pressure regulation. Female rats (6 mo old) prenatally exposed to testosterone were examined for adrenal expression of steroidogenic genes, telemetric blood pressure, blood volume and Na+ and K+ levels, plasma aldosterone, angiotensin II and vasopressin levels, and vascular responses to angiotensin II and arg8-vasopressin. The levels of Cyp11b2 (aldosterone synthase), but not the other adrenal steroidogenic genes, were decreased in testosterone females. Accordingly, plasma aldosterone levels were lower in testosterone females. Plasma volume and serum and urine Na+ and K+ levels were not significantly different between control and testosterone females; however, prenatal testosterone exposure significantly increased plasma vasopressin and angiotensin II levels and arterial pressure in adult females. In testosterone females, mesenteric artery contractile responses to angiotensin II were significantly greater, while contractile responses to vasopressin were unaffected. Angiotensin II type-1 receptor expression was increased, while angiotensin II type-2 receptor was decreased in testosterone arteries. These results suggest that prenatal testosterone exposure downregulates adrenal Cyp11b2 expression, leading to decreased plasma aldosterone levels. Elevated angiotensin II and vasopressin levels along with enhanced vascular responsiveness to angiotensin II may serve as an underlying mechanism to maintain plasma volume and Na+ and K+ levels and mediate hypertension in adult testosterone females.

2 Renin-angiotensin-aldosterone profiles predictive of superimposed preeclampsia

Introduction Women with preexisting, or chronic hypertension (CHT) in pregnancy are at heightened risk for significant morbidity and death, primarily due to further elevations in blood pressure (BP) and development of superimposed preeclampsia (SPE). The pathophysiology for the elevations in BP remains unresolved. Objectives We investigated the hypothesis that renin–angiotensin aldosterone profiles (RAAS) are associated with BP and SPE. Methods We performed a prospective, longitudinal trial of 108 women with CHT (RO1 HL 48846. PI: P. August). We measured plasma renin activity (PRA), 24h urine Na, K and aldosterone (UA) at 12, 20, 28, and 36weeks gestation and post-martum to investigate the association between these potentially pathophysiologic parameters and elevations in BP and SPE. Results Mean Arterial Pressure (MAP) was inversely related to PRA ( r =−0.23, P r =−0.11, P =0.047). PRA and UA were significantly and positively associated with each other ( r =0.5327, P P =0.005) and UA ( P =0.039) were significantly lower at 28weeks in those who developed SPE compared to those who did not develop SPE. Interestingly, PRA was significantly lower in black women compared to other racial groups ( p =0.026 at 36weeks and P P =0.0018) were significant predictors of PRA, while gestational age ( P P =0.0001), urine K ( P P =0.0005) were predictors of UA. Conclusions We conclude that renin–angiotensin–aldosterone profiles, ascertained in the third trimester of pregnancy, foretell the development of SPE in pregnant women with CHT. The RAAS profiles characterized in our investigation, in addition to serving as prognostic biomarkers, advance sodium retention as the primary mechanism for the elevation in BP in women with SPE.