Interleukin 6 mediated activation of the mineralocorticoid receptor in the aldosterone-sensitive distal nephron.

Hypertension is characterized by increased sodium (Na+) reabsorption along the aldosterone-sensitive distal nephron (ASDN) as well as chronic systemic inflammation. Interleukin-6 (IL-6) is thought to be a mediator of this inflammatory process. Interestingly, increased Na+ reabsorption within the ASDN does not always correlate with increases in aldosterone (Aldo), the primary hormone that modulates Na+ reabsorption via the mineralocorticoid receptor (MR). Thus, understanding how increased ASDN Na+ reabsorption may occur independent of Aldo stimulation is critical. Here, we show that IL-6 can activate the MR by activating Rac1 and stimulating the generation of reactive oxygen species (ROS) with a consequent increase in thiazide-sensitive Na+ uptake. Using an in vitro model of the distal convoluted tubule (DCT2), mDCT15 cells, we observed nuclear translocation of eGFP-tagged MR after IL-6 treatment. To confirm the activation of downstream transcription factors, mDCT15 cells were transfected with mineralocorticoid response element (MRE)-luciferase reporter constructs; then treated with vehicle, Aldo, or IL-6. Aldosterone or IL-6 treatment increased luciferase activity that was reversed with MR antagonist cotreatment, but IL-6 treatment was reversed by Rac1 inhibition or ROS reduction. In both mDCT15 and mpkCCD cells, IL-6 increased amiloride-sensitive transepithelial Na+ current. ROS and IL-6 increased 22Na+ uptake via the thiazide-sensitive sodium chloride cotransporter (NCC). These results are the first to demonstrate that IL-6 can activate the MR resulting in MRE activation and that IL-6 increases NCC-mediated Na+ reabsorption, providing evidence for an alternative mechanism for stimulating ASDN Na+ uptake during conditions where Aldo-mediated MR stimulation may not occur.

Myristoylated alanine-rich C kinase substrate-like protein-1 regulates epithelial sodium channel activity in renal distal convoluted tubule cells.

The epithelial sodium channel (ENaC) regulates blood pressure by fine-tuning distal nephron sodium reabsorption. Our previous work has shown that ENaC gating is regulated by anionic phospholipid phosphates, including phosphatidylinositol 4,5-bisphosphate (PIP2). The PIP2-dependent regulation of ENaC is mediated by the myristoylated alanine-rich protein kinase C substrate-like protein-1 (MLP-1). MLP-1 binds to and is a reversible source of PIP2 at the plasma membrane. We examined MLP-1 regulation of ENaC in distal convoluted tubule clonal cell line DCT-15 cells. Wild-type MLP-1 runs at an apparent molecular mass of 52 kDa despite having a predicted molecular mass of 21 kDa. Native MLP-1 consists of several distinct structural elements: an effector domain that is highly positively charged, sequesters PIP2, contains serines that are the target of PKC, and controls MLP-1 association with the membrane; a myristoylation domain that promotes association with the membrane; and a multiple homology 2 domain of previously unknown function. To further examine MLP-1 in DCT-15 cells, we constructed several MLP-1 mutants: WT, a full-length wild-type protein; S3A, three substitutions in the effector domain to prevent phosphorylation; S3D mimicked constitutive phosphorylation by replacing three serines with aspartates; and GA replaced the myristoylation site glycine with alanine, so GA could not be myristoylated. Each mutant was tagged with either NH2-terminal 3XFLAG or COOH-terminal mCherry or V5. Transfection with MLP mutants modified ENaC activity in DCT-15 cells: activity was highest in S3A and lowest in S3D, and the activity after transfection with either construct was significantly different from WT. In Western blots, when transfected with 3XFLAG-tagged MLP-1 mutants, the expression of the full length of MLP-1 at 52 kDa increased in mutant S3A-MLP-1-transfected DCT-15 cells and decreased in S3D-MLP-1-transfected DCT-15 cells. Several lower molecular mass bands were also detected that correspond to potential presumptive calpain cleavage products. Confocal imaging shows that the different mutants localize in different subcellular compartments consistent with their preferred location in the membrane or in the cytosol. Activation of protein kinase C increases phosphorylation of endogenous MLP-1 and reduces ENaC activity. Our results suggest a complicated role for proteolytic processing in MLP-1 regulation of ENaC.

Mesenteric arteries from stroke-prone spontaneously hypertensive rats exhibit an increase in nitric-oxide-dependent vasorelaxation.

The endothelium is crucial for the maintenance of vascular tone by releasing several vasoactive substances, including nitric oxide (NO). Systemic mean arterial pressure is primarily regulated by the resistance vasculature, which has been shown to exhibit increased vascular reactivity, and decreased vasorelaxation during hypertension. Here, we aimed to determine the mechanism for mesenteric artery vasorelaxation of the stroke-prone spontaneously hypertensive rat (SHRSP). We hypothesized that endothelial NO synthase (eNOS) is upregulated in SHRSP vessels, increasing NO production to compensate for the endothelial dysfunction. Concentration-response curves to acetylcholine (ACh) were performed in second-order mesenteric arteries; we observed decreased relaxation responses to ACh (maximum effect elicited by the agonist) as compared with Wistar-Kyoto (WKY) controls. Vessels from SHRSP incubated with Nω-nitro-l-arginine methyl ester and (or) indomethacin exhibited decreased ACh-mediated relaxation, suggesting a primary role for NO-dependent relaxation. Vessels from SHRSP exhibited a significantly decreased relaxation response with inducible NO synthase (iNOS) inhibition, as compared with WKY vessels. Western blot analysis showed increased total phosphorylated NF-κB, and phosphorylated and total eNOS in SHRSP vessels. Overall, these data suggest a compensatory role for NO by increased eNOS activation. Moreover, we believe that iNOS, although increasing NO bioavailability to compensate for decreased relaxation, leads to a cycle of further endothelial dysfunction in SHRSP mesenteric arteries.

The sodium chloride cotransporter (NCC) and epithelial sodium channel (ENaC) associate.

The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α- and γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.

Pendrin localizes to the adrenal medulla and modulates catecholamine release.

Pendrin (Slc26a4) is a Cl(-)/HCO3 (-) exchanger expressed in renal intercalated cells and mediates renal Cl(-) absorption. With pendrin gene ablation, blood pressure and vascular volume fall, which increases plasma renin concentration. However, serum aldosterone does not significantly increase in pendrin-null mice, suggesting that pendrin regulates adrenal zona glomerulosa aldosterone production. Therefore, we examined pendrin expression in the adrenal gland using PCR, immunoblots, and immunohistochemistry. Pendrin protein was detected in adrenal lysates from wild-type but not pendrin-null mice. However, immunohistochemistry and qPCR of microdissected adrenal zones showed that pendrin was expressed in the adrenal medulla, rather than in cortex. Within the adrenal medulla, pendrin localizes to both epinephrine- and norepinephrine-producing chromaffin cells. Therefore, we examined plasma catecholamine concentration and blood pressure in wild-type and pendrin-null mice under basal conditions and then after 5 and 20 min of immobilization stress. Under basal conditions, blood pressure was lower in the mutant than in the wild-type mice, although epinephrine and norepinephrine concentrations were similar. Catecholamine concentration and blood pressure increased markedly in both groups with stress. With 20 min of immobilization stress, epinephrine and norepinephrine concentrations increased more in pendrin-null than in wild-type mice, although stress produced a similar increase in blood pressure in both groups. We conclude that pendrin is expressed in the adrenal medulla, where it blunts stress-induced catecholamine release.

Compensatory renal hypertrophy following uninephrectomy is calcineurin-independent.

Calcineurin is a calcium-dependent phosphatase that is involved in many cellular processes including hypertrophy. Inhibition or genetic loss of calcineurin blocks pathological cardiac hypertrophy and diabetic renal hypertrophy. However, calcineurin does not appear to be involved in physiological cardiac hypertrophy induced by exercise. The role of calcineurin in a compensatory, non-pathological model of renal hypertrophy has not been tested. Therefore, in this study, we examined activation of calcineurin and the effect of calcineurin inhibition or knockout on compensatory hypertrophy following uninephrectomy (UNX). UNX induces ~15% increase in the size of the remaining kidney; the data show no change in the generation of reactive oxygen species (ROS), Nox4 or transforming growth factor-ß expression confirming the model as one of compensatory hypertrophy. Next, analyses of the remaining kidney reveal that total calcineurin activity is increased, and, to a lesser extent, transcriptional activity of the calcineurin substrate nuclear factor of activated T cell is up-regulated following UNX. However, inhibition of calcineurin with cyclosporine failed to prevent compensatory renal hypertrophy. Likewise, hypertrophy was comparable to WT in mice lacking either isoform of the catalytic subunit of calcineurin (CnAα-/- or CnAß-/-). In conclusion, similar to its role in the heart, calcineurin is required for pathological but not compensatory renal hypertrophy. This separation of signalling pathways could therefore help further define key factors necessary for pathological hypertrophy including diabetic nephropathy.

Salt-Sensitive Hypertension and the Kidney.

Salt-sensitive hypertension (SS-HT) is characterized by blood pressure elevation in response to high dietary salt intake and is considered to increase the risk of cardiovascular and renal morbidity. Although the mechanisms responsible for SS-HT are complex, the kidneys are known to play a central role in the development of SS-HT and the salt sensitivity of blood pressure (SSBP). Moreover, several factors influence renal function and SSBP, including the renin-angiotensin-aldosterone system, sympathetic nervous system, obesity, and aging. A phenotypic characteristic of SSBP is aberrant activation of the renin-angiotensin system and sympathetic nervous system in response to excessive salt intake. SSBP is also accompanied by a blunted increase in renal blood flow after salt loading, resulting in sodium retention and SS-HT. Obesity is associated with inappropriate activation of the aldosterone mineralocorticoid receptor pathway and renal sympathetic nervous system in response to excessive salt, and mineralocorticoid receptor antagonists and renal denervation attenuate sodium retention and inhibit salt-induced blood pressure elevation in obese dogs and humans. SSBP increases with age, which has been attributed to impaired renal sodium handling and a decline in renal function, even in the absence of kidney disease. Aging-associated changes in renal hemodynamics are accompanied by significant alterations in renal hormone levels and renal sodium handling, resulting in SS-HT. In this review, we focus mainly on the contribution of renal function to the development of SS-HT.

Evaluation of renal sodium handling in heart failure with preserved ejection fraction: A pilot study.

The pathophysiology behind sodium retention in heart failure with preserved ejection fraction (HFpEF) remains poorly understood. We hypothesized that patients with HFpEF have impaired natriuresis and diuresis in response to volume expansion and diuretic challenge, which is associated with renal hypo-responsiveness to endogenous natriuretic peptides. Nine HFpEF patients and five controls received saline infusion (0.25 mL/kg/min for 60 min) followed by intravenous furosemide (20 mg or home dose) 2 h after the infusion. Blood and urine samples were collected at baseline, 2 h after saline infusion, and 2 h after furosemide administration; urinary volumes were recorded. The urinary cyclic guanosine monophosphate (ucGMP)/plasma B-type NP (BNP) ratio was calculated as a measure of renal response to endogenous BNP. Wilcoxon rank-sum test was used to compare the groups. Compared to controls, HFpEF patients had reduced urine output (2480 vs.3541 mL; p = 0.028), lower urinary sodium excretion over 2 h after saline infusion (the percentage of infused sodium excreted 12% vs. 47%; p = 0.003), and a lower baseline ucGMP/plasma BNP ratio (0.7 vs. 7.3 (pmol/mL)/(mg/dL)/(pg/mL); p = 0.014). Patients with HFpEF had impaired natriuretic response to intravenous saline and furosemide administration and lower baseline ucGMP/plasma BNP ratios indicating renal hypo-responsiveness to NPs.

Aldosterone acutely stimulates NCC activity via a SPAK-mediated pathway.

Hypertension is a leading cause of morbidity and mortality worldwide, and disordered sodium balance has long been implicated in its pathogenesis. Aldosterone is perhaps the key regulator of sodium balance and thus blood pressure. The sodium chloride cotransporter (NCC) in the distal convoluted tubule of the kidney is a major site of sodium reabsorption and plays a key role in blood pressure regulation. Chronic exposure to aldosterone increases NCC protein expression and function. However, more acute effects of aldosterone on NCC are unknown. In our salt-abundant modern society where chronic salt deprivation is rare, understanding the acute effects of aldosterone is critical. Here, we examined the acute effects (12-36 h) of aldosterone on NCC in the rodent kidney and in a mouse distal convoluted tubule cell line. Studies demonstrated that aldosterone acutely stimulated NCC activity and phosphorylation without affecting total NCC abundance or surface expression. This effect was dependent upon the presence of the mineralocorticoid receptor and serum- and glucocorticoid-regulated kinase 1 (SGK1). Furthermore, STE20/SPS-1-related proline/alanine-rich kinase (SPAK) phosphorylation also increased, and gene silencing of SPAK eliminated the effect of aldosterone on NCC activity. Aldosterone administration via a minipump in adrenalectomized rodents confirmed an increase in NCC phosphorylation without a change in NCC total protein. These data indicate that acute aldosterone-induced SPAK-dependent phosphorylation of NCC increases individual transporter activity.

Metformin treatment improves erectile function in an angiotensin II model of erectile dysfunction.

INTRODUCTION: Increased angiotensin II (AngII) levels cause hypertension, which is a major risk factor for erectile dysfunction (ED). Studies have demonstrated that increased AngII levels in penile tissue are associated with ED. A recent study showed that metformin treatment restored nitric oxide synthase (NOS) protein expression in penile tissue in obese rats; however, whether metformin treatment can be beneficial and restore erectile function in a model of ED has not yet been established. AIM: The goal of this study was to test the hypothesis that AngII induces ED by means of increased corpus cavernosum contraction, and that metformin treatment will reverse ED in AngII-treated rats. METHODS: Male Sprague-Dawley rats were implanted with mini-osmotic pumps containing saline or AngII (70 ng/minute, 28 days). Animals were then treated with metformin or vehicle during the last week of AngII infusion. MAIN OUTCOME MEASURES: Intracavernosal pressure; corpus cavernosum contraction and relaxation; nNOS protein expression; extracellular signal-regulated kinase (ERK1/2), AMP-activated protein kinase (AMPK), and eNOS protein expression and phosphorylation. RESULTS: AngII-induced ED was accompanied with an increase in corpus cavernosum contractility, decreased nitrergic relaxation, and increased ERK1/2 phosphorylation. Metformin treatment improved erectile function in the AngII-treated rats by reversing the increased contraction and decreased relaxation. Metformin treatment also resulted in an increase in eNOS phosphorylation at ser1177. CONCLUSIONS: Metformin treatment increased eNOS phosphorylation and improved erectile function in AngII hypertensive rats by reestablishing normal cavernosal smooth muscle tone.

Epithelial N-methyl-D-aspartate (NMDA) receptors mediate renal vasodilation by affecting kidney autoregulation.

Background: N-methyl-D-aspartate receptor (NMDAR) are amino acid receptors that are well studied in brain physiology; however, their role in kidney is poorly understood. Nonetheless, NMDAR inhibitors can increase serum K+ and reduce GFR, which suggests they have an important physiological role in the kidney. We hypothesized that NMDARs in the distal nephron induce afferent-arteriole vasodilation through the vasodilator mechanism connecting-tubule-glomerular feedback (CNTGF) that involves ENaC activation. Methods and results: Using a tubule-specific transcriptome database combined with molecular biology and microscopy techniques, we showed kidney expression of NMDAR subunits along the nephron and specifically in ENaC-positive cells. This receptor is expressed in both male and female mice, with higher abundance in females (p=0.02). Microperfusing NMDAR agonists into the connecting tubule induced afferent-arteriole vasodilation (EC50 10.7 vs. 24.5 mM; p<0.001) that was blunted or eliminated with the use of NMDAR blocker MK-801 or with the ENaC inhibitor Benzamil, indicating a dependence on CNTGF of the NMDAR-induced vasodilation. In vivo, we confirmed this CNTGF-associated vasodilation using kidney micropuncture (Stop-flow pressure 37.9±2.6 vs. 28.6±1.9 mmHg, NMDAR agonist vs vehicle; p<0.01). We explored NMDAR and ENaC channel interaction by using mpkCCD cells and split-open connecting tubules. We observed increased amiloride-sensitive current following NMDAR activation that was prevented by MK-801 (1.14 vs. 0.4 µAmp; p=0.03). In split-open tubules, NMDAR activation increased ENaC activity (Npo Vehicle vs. NMDA; p=0.04). Conclusion: NMDARs are expressed along the nephron, including ENaC-positive cells, with higher expression in females. Epithelial NMDAR mediates renal vasodilation through the connecting-tubule-glomerular feedback, by increasing ENaC activity.

Addressing global disparities in blood pressure control: perspectives of the International Society of Hypertension.

ABSTRACT: Raised blood pressure (BP) is the leading cause of preventable death in the world. Yet, its global prevalence is increasing, and it remains poorly detected, treated, and controlled in both high- and low-resource settings. From the perspective of members of the International Society of Hypertension based in all regions, we reflect on the past, present, and future of hypertension care, highlighting key challenges and opportunities, which are often region-specific. We report that most countries failed to show sufficient improvements in BP control rates over the past three decades, with greater improvements mainly seen in some high-income countries, also reflected in substantial reductions in the burden of cardiovascular disease and deaths. Globally, there are significant inequities and disparities based on resources, sociodemographic environment, and race with subsequent disproportionate hypertension-related outcomes. Additional unique challenges in specific regions include conflict, wars, migration, unemployment, rapid urbanization, extremely limited funding, pollution, COVID-19-related restrictions and inequalities, obesity, and excessive salt and alcohol intake. Immediate action is needed to address suboptimal hypertension care and related disparities on a global scale. We propose a Global Hypertension Care Taskforce including multiple stakeholders and societies to identify and implement actions in reducing inequities, addressing social, commercial, and environmental determinants, and strengthening health systems implement a well-designed customized quality-of-care improvement framework.

Aorta from angiotensin II hypertensive mice exhibit preserved nitroxyl anion mediated relaxation responses.

Hypertension is a disorder affecting millions worldwide, and is a leading cause of death and debilitation in the United States. It is widely accepted that during hypertension and other cardiovascular diseases the vasculature exhibits endothelial dysfunction; a deficit in the relaxatory ability of the vessel, attributed to a lack of nitric oxide (NO) bioavailability. Recently, the one electron redox variant of NO, nitroxyl anion (NO(-)) has emerged as an endothelium-derived relaxing factor (EDRF) and a candidate for endothelium-derived hyperpolarizing factor (EDRF). NO(-) is thought to exist protonated (HNO) in vivo, which would make this species more resistant to scavenging. However, no studies have investigated the role of this redox species during hypertension, and whether the vasculature loses the ability to relax to HNO. Thus, we hypothesize that aorta from angiotensin II (AngII)-hypertensive mice will exhibit a preserved relaxation response to Angeli's Salt, an HNO donor. Male C57Bl6 mice, aged 12-14 weeks were implanted with mini-osmotic pumps containing AngII (90ng/min, 14 days plus high salt chow) or sham surgery. Aorta were excised, cleaned and used to perform functional studies in a myograph. We found that aorta from AngII-hypertensive mice exhibited a significant endothelial dysfunction as demonstrated by a decrease in acetylcholine (ACh)-mediated relaxation. However, vessels from hypertensive mice exhibited a preserved response to Angeli's Salt (AS), the HNO donor. To confirm that relaxation responses to HNO were maintained, concentration response curves (CRCs) to ACh were performed in the presence of scavengers to both NO and HNO (carboxy-PTIO and L-cys, resp.). We found that ACh-mediated relaxation responses were significantly decreased in aorta from sham and almost completely abolished in aorta from AngII-treated mice. Vessels incubated with l-cys exhibited a modest decrease in ACh-mediated relaxations responses. These data demonstrate that aorta from AngII-treated hypertensive mice exhibit a preserved relaxation response to AS, an HNO donor, regardless of a significant endothelial dysfunction.

Highlights from the International Society of Hypertension's New Investigators Network during 2019.

: The New Investigators Committee (NIC) of the International Society of Hypertension (ISH) is a dynamic group of junior doctors and scientists, actively involved in various society activities. This report highlights the events (scientific meetings and summer schools) and activities (social media, mentorship and networking) during 2019 including May Measurement Month and collaborative efforts with the ISH Women in Hypertension Research Committee (WiHRC). The ISH NIC is proud to sponsor awards for outstanding work by junior and emerging researchers at hypertension conferences and also provides opportunities to showcase their work on our social media features such as 'Our Fellows Work' and the New Investigator Spotlight of the month. In 2020, the ISH NIC aims to promote women in leadership roles and to foster strong collaborations with and between society committees and other scientific organizations.

TNF-alpha knockout mice have increased corpora cavernosa relaxation.

INTRODUCTION: Erectile dysfunction is considered an early clinical manifestation of vascular disease and an independent risk factor for cardiovascular events associated with endothelial dysfunction and increased levels of pro-inflammatory cytokines. Tumor necrosis factor-alpha (TNF-alpha), a pro-inflammatory cytokine, suppresses endothelial nitric oxide synthase (eNOS) expression. AIM: Considering that nitric oxide (NO) is of critical importance in penile erection, we hypothesized that blockade of TNF-alpha actions would increase cavernosal smooth muscle relaxation. METHODS: In vitro organ bath studies were used to measure cavernosal reactivity in wild type and TNF-alpha knockout (TNF-alpha KO) mice and NOS expression was evaluated by western blot. In addition, spontaneous erections (in vivo) were evaluated by videomonitoring the animals (30 minutes). Collagen and elastin expression were evaluated by Masson trichrome and Verhoff-van Gieson stain reaction, respectively. MAIN OUTCOME MEASURES: Corpora cavernosa from TNF-alpha KO mice exhibited increased NO-dependent relaxation, which was associated with increased eNOS and neuronal NOS (nNOS) cavernosal expression. RESULTS: Cavernosal strips from TNF-alpha KO mice displayed increased endothelium-dependent (97.4 +/- 5.3 vs. CONTROL: 76.3 +/- 6.3, %) and nonadrenergic-noncholinergic (93.3 +/- 3.0 vs. CONTROL: 67.5 +/- 16.0; 16 Hz) relaxation compared to control animals. These responses were associated with increased protein expression of eNOS and nNOS (P < 0.05). Sympathetic-mediated (0.69 +/- 0.16 vs. CONTROL: 1.22 +/- 0.22; 16 Hz) as well as phenylephrine-induced contractile responses (1.6 +/- 0.1 vs. CONTROL: 2.5 +/- 0.1, mN) were attenuated in cavernosal strips from TNF-alpha KO mice. Additionally, corpora cavernosa from TNF-alpha KO mice displayed increased collagen and elastin expression. In vivo experiments demonstrated that TNF-alpha KO mice display increased number of spontaneous erections. CONCLUSION: Corpora cavernosa from TNF-alpha KO mice display alterations that favor penile tumescence, indicating that TNF-alpha plays a detrimental role in erectile function. A key role for TNF-alpha in mediating endothelial dysfunction in ED is markedly relevant since we now have access to anti-TNF-alpha therapies.

Moving the Needle on Hypertension: What Knowledge Is Needed?

This review highlights the gaps in knowledge and methodological challenges discussed during the Experimental Biology 2019 expert panel session titled "Moving the Needle on Hypertension: What Knowledge Is Needed?" Hypertension is a critical public health burden. Despite a demonstrated benefit of blood pressure reduction on measures of hypertension-related morbidity and mortality, rates for successful blood pressure control remain low. Dietary sodium reduction has been shown to reduce both systolic blood pressure by approximately 3.2 mm Hg and diastolic blood pressure by 2.3 mm Hg, depending on baseline blood pressure and degree of sodium reduction. The updated Dietary Reference Intakes for adults released by the National Academies of Sciences, Engineering, and Medicine include a Chronic Disease Risk Reduction sodium intake level of 2300 mg/d, highlighting the importance of dietary sodium intake in reducing elevated blood pressure and indicating that reducing intakes to this level is expected to reduce blood pressure and risk of cardiovascular disease. The average US daily sodium intake of 3400 mg/d is well above the Chronic Disease Risk Reduction of 2300 mg/d, suggesting that dietary sodium reduction has the potential to significantly improve public health. Although the National Academies of Sciences, Engineering, and Medicine report presents intake recommendations based on a systematic, comprehensive, and thorough evaluation of the evidence, several challenges to moving the needle on hypertension remain. Success will require a more advanced understanding of sodium and potassium physiology, as well as development of the tools needed to effectively address existing research gaps and reduce barriers to sodium intake reduction.

Blood collection in unstressed, conscious, and freely moving mice through implantation of catheters in the jugular vein: a new simplified protocol.

The mouse has become the most common mammalian animal model used in biomedical research. However, laboratory techniques used previously in rats and other larger animals to sample blood had to be adapted in mice due to their lower mouse plasma volume. Sampling is further confounded by the variability in plasma hormone and metabolite concentrations that can occur from the stress or the anesthesia that accompanies the collection. In this article, we describe in detail a protocol we developed for blood sampling in conscious, unrestrained mice. Our protocol implements the use of chronic indwelling catheters in the right external jugular vein, allowing the mice to recover fully in their home cages, untethered until the time of blood sampling. This protocol employs catheters that remain patent for days and does not require the purchase of expensive equipment. We validated this protocol by measuring the time course of plasma norepinephrine (NE) concentration during and after the relief of acute immobilization stress in wild type (WT) and pendrin knockout (KO) mice and compared these results with our previously published values. We found that following relief from immobilization stress, it takes longer for plasma NE concentration to return to basal levels in the pendrin KO than in the wild type mice. These results highlight the potential utility of this protocol and the potential role of pendrin in the neuroendocrine response to acute stress.

Protein kinase Cα deletion causes hypotension and decreased vascular contractility.

AIM: Protein kinase Cα (PKCα) is a critical regulator of multiple cell signaling pathways including gene transcription, posttranslation modifications and activation/inhibition of many signaling kinases. In regards to the control of blood pressure, PKCα causes increased vascular smooth muscle contractility, while reducing cardiac contractility. In addition, PKCα has been shown to modulate nephron ion transport. However, the role of PKCα in modulating mean arterial pressure (MAP) has not been investigated. In this study, we used a whole animal PKCα knock out (PKC KO) to test the hypothesis that global PKCα deficiency would reduce MAP, by a reduction in vascular contractility. METHODS: Radiotelemetry measurements of ambulatory blood pressure (day/night) were obtained for 18 h/day during both normal chow and high-salt (4%) diet feedings. PKCα mice had a reduced MAP, as compared with control, which was not normalized with high-salt diet (14 days). Metabolic cage studies were performed to determine urinary sodium excretion. RESULTS: PKC KO mice had a significantly lower diastolic, systolic and MAP as compared with control. No significant differences in urinary sodium excretion were observed between the PKC KO and control mice, whether fed normal chow or high-salt diet. Western blot analysis showed a compensatory increase in renal sodium chloride cotransporter expression. Both aorta and mesenteric vessels were removed for vascular reactivity studies. Aorta and mesenteric arteries from PKC KO mice had a reduced receptor-independent relaxation response, as compared with vessels from control. Vessels from PKC KO mice exhibited a decrease in maximal contraction, compared with controls. CONCLUSION: Together, these data suggest that global deletion of PKCα results in reduced MAP due to decreased vascular contractility.

Nitroxyl Anion Mediates Relaxation in Mesenteric Arteries from Angiotensin II Hypertensive Mice.

AIMS: Nitroxyl anion (HNO) has recently become an emerging candidate in vascular regulation. NO- is a potent vasodilator of both conduit and small resistance vessels and mediates relaxation in a soluble guanylate cyclase-dependent manner. Interestingly, HNO activates voltage-dependent K+ (K+ V) channels, whereas Nitric Oxide (NO) activates calcium-activated K+ Ca channels. To date, there are few studies investigating the role of HNO in hypertension, and the possible mechanisms, which may be altered during this condition. We hypothesized that mesenteric arteries from angiotensin II-induced (AngII) hypertensive mice would exhibit an increased dependence upon NO- for relaxation, which may be mediated through K+ V channels. Methods and Key Results: C57/Bl6 mice, aged 12-14 weeks were implanted with mini-pumps containing angiotensin II (AngII, 3600ng/kg/min) for 14 days. For this study, we proposed to investigate the role of HNO in the resistance vasculature, and so first order mesenteric arteries were isolated and used in functional studies, or were frozen for Western blot analysis. We observed that mesenteric arteries from AngII mice (AngII) exhibited a decrease in HNO-mediated relaxation, which was endotheliumindependent. With HNO scavenging by L-cysteine [3mM], the maximal acetylcholine (ACh)-mediated relaxation response was decreased in sham, whereas mesenteric arteries from AngII exhibited a decrease in sensitivity. Incubation with the K+ V channel inhibitor, 4-aminopyridine [1mM], decreased AChmediated relaxation responses in sham, but almost completely abolished relaxation in AngII. CONCLUSION: We reveal that exogenous HNO-mediated relaxation, via Angeli's Salt, is impaired in mesenteric arteries from AngII-treated mice, yet endogenous HNO-mediated relaxation may be more important during hypertension.