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We recently reported that skin vasoconstriction to suppress transepidermal water loss (TEWL) leads to hypertension in renal injury model rats with impaired urine concentration ability. In this study, we investigated the pathogenesis of hypertension in spontaneously hypertensive rats (SHRs) from the perspective of renal water loss and skin water conservation. We compared the urinary concentration ability, body sodium and water balance, blood pressure, and TEWL in SHRs and control normotensive Wistar-Kyoto rats (WKYs). SHRs showed significantly higher urine volume and lower urinary osmolality than those of WKYs, while there were no significant differences in water intake, urinary osmolyte excretion, and plasma osmolarity between the groups. SHRs exhibited significantly higher blood pressure, skin sodium content, and lower TEWL compared with those is WKYs. Skin vasodilation, induced by elevating body temperature, increased TEWL in both SHRs and WKYs, and significantly reduced blood pressure in SHRs but not WKYs. These findings suggest that physiological adaptation can reduce dermal water loss in SHRs to compensate for renal water loss. Vasoconstriction required for successful cutaneous water conservation explains SHR hypertension. Renal concentration ability and skin barrier function for water conservation may become a novel therapeutic target for essential hypertension.

We have recently reported that the urea osmolyte-associated water conservation system is activated in fluid loss models such as high salt-induced natriuresis, renal injury-induced impaired renal concentrating ability, or skin barrier dysfunction-induced transepidermal water loss. The system consists of the interaction of multiple organs including renal urea recycling, hepato-muscular ureagenesis, and suppression of cardiovascular energy expenditure. Here, we determined the effect of pharmacological fluid loss induced by tolvaptan, a selective vasopressin V

Background Tissue sodium (Na+) content in patients on maintenance hemodialysis (MHD) and peritoneal dialysis (PD) was previously explored using 23Na+ magnetic resonance imaging (23NaMRI). Larger studies would provide a better understanding of Na+ stores in patients on dialysis as well as the factors influencing this Na+ accumulation. Methods In this cross-sectional study, we quantified the calf muscle and skin Na+ content in 162 subjects (10 PD, 33 MHD patients and 119 controls) using 23NaMRI. Plasma levels of interleukin-6 (IL-6) and high-sensitivity C-reactive protein (hsCRP) were measured to assess systemic inflammation. Sixty-four subjects had repeat 23NaMRI scans that were analyzed to assess the repeatability of the 23NaMRI measurements. Results Patients on MHD and PD exhibited significantly higher muscle and skin Na+ accumulation compared with controls. African American patients on dialysis exhibited greater muscle and skin Na+ content compared with non–African Americans. Multivariable analysis showed that older age was associated with both higher muscle and skin Na+ and male sex was associated with increased skin Na+ deposition. Greater ultrafiltration was associated with lower skin Na+ in patients on PD (Spearman’s ρ = −0.68, P = 0.035). Higher plasma IL-6 and hsCRP levels correlated with increased muscle and skin Na+ content in the overall study population. Patients with higher baseline tissue Na+ content exhibited greater variability in tissue Na+ stores on repeat measurements. Conclusions Our findings highlight greater muscle and skin Na+ content in dialysis patients compared with controls without kidney disease. Tissue Na+ deposition and systemic inflammation seen in dialysis patients might influence one another bidirectionally.

Therapeutic inhibition of the sodium–glucose co-transporter 2 (SGLT2) leads to substantial loss of energy (in the form of glucose) and additional solutes (in the form of Na+ and its accompanying anions) in urine. However, despite the continuously elevated solute excretion, long-term osmotic diuresis does not occur in humans with SGLT2 inhibition. Rather, patients on SGLT2 inhibitor therapy adjust to the reduction in energy availability and conserve water. The metabolic adaptations that are induced by SGLT2 inhibition are similar to those observed in aestivation — an evolutionarily conserved survival strategy that enables physiological adaptation to energy and water shortage. Aestivators exploit amino acids from muscle to produce glucose and fatty acid fuels. This endogenous energy supply chain is coupled with nitrogen transfer for organic osmolyte production, which allows parallel water conservation. Moreover, this process is often accompanied by a reduction in metabolic rate. By comparing aestivation metabolism with the fuel switches that occur during therapeutic SGLT2 inhibition, we suggest that SGLT2 inhibitors induce aestivation-like metabolic patterns, which may contribute to the improvements in cardiac and renal function observed with this class of therapeutics. SGLT2 inhibitors induce a number of metabolic adaptations in response to increased glucose and Na+ excretion. This Perspective article describes how these adaptations suggest that SGLT2 inhibition triggers a body water-conserving mechanism, and discusses how these metabolic adjustments may contribute to the favourable cardiovascular and renal outcomes of this class of therapeutics.

OBJECTIVE Lipedema is characterized by pain, fatigue, and excessive adipose tissue and sodium accumulation of the lower extremities. This case-control study aims to determine whether sodium or vascular dysfunction is present in the central nervous system. METHODS Brain magnetic resonance imaging was performed at 3 T in patients with lipedema (n = 15) and control (n = 18) participants matched for sex, age, race, and BMI. Standard anatomical imaging and intracranial angiography were applied to evaluate brain volume and vasculopathy, respectively; arterial spin labeling and sodium magnetic resonance imaging were applied to quantify cerebral blood flow (CBF) (milliliters per 100 grams of tissue/minute) and brain tissue sodium content (millimoles per liter), respectively. A Mann-Whitney U test (significance criteria P

Immune checkpoint inhibitor (ICI) therapy has revolutionized the breast cancer treatment landscape. However, ICI-induced systemic inflammatory immune-related adverse events (irAE) remain a major clinical challenge. Previous studies in our laboratory and others have demonstrated that a high-salt (HS) diet induces inflammatory activation of CD4+T cells leading to anti-tumor responses. In our current communication, we analyzed the impact of dietary salt modification on therapeutic and systemic outcomes in breast-tumor-bearing mice following anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA4) monoclonal antibody (mAb) based ICI therapy. As HS diet and anti-CTLA4 mAb both exert pro-inflammatory activation of CD4+T cells, we hypothesized that a combination of these would lead to enhanced irAE response, while low-salt (LS) diet through blunting peripheral inflammatory action of CD4+T cells would reduce irAE response. We utilized an orthotopic murine breast tumor model by injecting Py230 murine breast cancer cells into syngeneic C57Bl/6 mice. In an LS diet cohort, anti-CTLA4 mAb treatment significantly reduced tumor progression (day 35, 339 ± 121 mm3), as compared to isotype mAb (639 ± 163 mm3, p < 0.05). In an HS diet cohort, treatment with anti-CTLA4 reduced the survival rate (day 80, 2/15) compared to respective normal/regular salt (NS) diet cohort (8/15, p < 0.05). Further, HS plus anti-CTLA4 mAb caused an increased expression of inflammatory cytokines (IFNγ and IL-1β) in lung infiltrating and peripheral circulating CD4+T cells. This inflammatory activation of CD4+T cells in the HS plus anti-CTLA4 cohort was associated with the upregulation of inflammasome complex activity. However, an LS diet did not induce any significant irAE response in breast-tumor-bearing mice upon treatment with anti-CTLA4 mAb, thus suggesting the role of high-salt diet in irAE response. Importantly, CD4-specific knock out of osmosensitive transcription factor NFAT5 using CD4cre/creNFAT5flox/flox transgenic mice caused a downregulation of high-salt-mediated inflammatory activation of CD4+T cells and irAE response. Taken together, our data suggest that LS diet inhibits the anti-CTLA4 mAb-induced irAE response while retaining its anti-tumor efficacy.

The number of cancer survivors with cardiovascular disease is increasing. However, the effect of cancer on body fluid regulation remains to be clarified. In this study, we evaluated body osmolyte and water imbalance in rats with hepatocellular carcinoma.Wistar rats were administered diethylnitrosamine, a carcinogenic drug, to establish liver cancer. We analyzed tissue osmolyte and water content, and their associations with aldosterone secretion.Hepatocellular carcinoma rats had significantly reduced body mass and the amount of total body sodium, potassium, and water. However, these rats had significantly increased relative tissue sodium, potassium, and water content per tissue dry weight. Furthermore, these changes in sodium and water balance in hepatocellular carcinoma rats were significantly associated with increased 24-h urinary aldosterone excretion. Supplementation with 0.25% salt in drinking water improved body weight reduction associated with sodium and water retention in hepatocellular carcinoma rats, which was suppressed by treatment with spironolactone, a mineralocorticoid receptor antagonist. Additionally, the urea-driven water conservation system was activated in hepatocellular carcinoma rats.These findings suggest that hepatocellular carcinoma induces body mass loss in parallel with activation of the water conservation system including aldosterone secretion and urea accumulation to retain osmolyte and water. The osmolyte and water retention at the tissue level may be a causative factor for ascites and edema formation in liver failure rats.

We recently reported that a 4% high-salt diet + saline for drinking (HS + saline) leads to a catabolic state, reduced heart rate, and suppression of cardiovascular energy expenditure in mice. We suggested that HS + saline reduces heart rate via the suppression of the sympathetic nervous system to compensate for the high salt intake-induced catabolic state. To test this hypothesis, we directly measured renal sympathetic nerve activity (RSNA) in conscious Sprague-Dawley (SD) rats using a radiotelemetry system. We confirmed that HS + saline induced a catabolic state. HS + saline decreased heart rate, while also reducing RSNA in SD rats. In contrast, Dahl salt-sensitive (DSS) rats exhibited no change in heart rate and increased RSNA during high salt intake. Renal denervation significantly decreased heart rate and attenuated the catabolic state independent of blood pressure in DSS rats fed HS + saline, suggesting that salt-sensitive animals were unable to decrease cardiovascular energy consumption due to abnormal renal sympathetic nerve activation during high salt intake. These findings support the hypothesis that RSNA mediates heart rate during high salt intake in SD rats. However, the insensitivity of heart rate and enhanced RSNA observed in DSS rats may be additional critical diagnostic factors for salt-sensitive hypertension. Renal denervation may benefit salt-sensitive hypertension by reducing its effects on catabolism and cardiovascular energy expenditure.

ObjectivePatients with SLE frequently have debilitating fatigue and reduced physical activity. Intermuscular adipose tissue (IMAT) accumulation is associated with reduced physical exercise capacity. We hypothesised that IMAT is increased in patients with SLE and associated with increased fatigue, reduced physical activity and increased inflammation.MethodsIn a cross-sectional study, 23 patients with SLE and 28 control participants were evaluated. IMAT was measured in the calf muscles using sequentialT1-weighted MRI. Patient-reported physical activity and fatigue were measured and a multiplex proteomic assay was used to measure markers and mediators of inflammation.ResultsIMAT accumulation (percentage of IMAT area to muscle area) was significantly higher in SLE versus control participants (7.92%, 4.51%–13.39% vs 2.65%, 1.15%–4.61%, median, IQR, pConclusionPatients with SLE have greater IMAT accumulation than controls in the calf muscles. Increased IMAT is associated with greater fatigue and lower physical activity. Future studies should evaluate the effectiveness of interventions that improve muscle quality to alleviate fatigue in patients with SLE.

Hyperosmolality is common in critically ill patients during body fluid volume reduction. It is unknown whether this is only a result of decreased total body water or an active osmole-producing mechanism similar to that found in aestivating animals, where muscle degradation increases urea levels to preserve water. We hypothesized that fluid volume reduction in critically ill patients contributes to a shift from ionic to organic osmolytes similar to mechanisms of aestivation. We performed a post-hoc analysis on data from a multicenter observational study in adult intensive care unit (ICU) patients in the postresuscitative phase. Fluid, electrolyte, energy and nitrogen intake, fluid loss, estimated glomerular filtration rate (eGFR), and estimated plasma osmolality (eOSM) were registered. Contributions of osmolytes Na+, K+, urea, and glucose to eOSM expressed as proportions of eOSM were calculated. A total of 241 patients were included. eOSM increased (median change 7.4 mOsm/kg [IQR−1.9–18]) during the study. Sodium's and potassium's proportions of eOSM decreased (P ClinicalTrials.org Identifier: NCT03972475.

Therapeutic inhibition of the sodium-glucose co-transporter 2 (SGLT2) leads to substantial loss of energy (in the form of glucose) and additional solutes (in the form of Na

Objective— A commonly accepted pivotal mechanism in fluid volume and blood pressure regulation is the parallel relationship between body Na + and extracellular fluid content. Several recent studies have, however, shown that a considerable amount of Na + can be retained in skin without commensurate water retention. Here, we asked whether a salt accumulation shown to result in VEGF (vascular endothelial growth factor)-C secretion and lymphangiogenesis had any influence on lymphatic function. Approach and Results— By optical imaging of macromolecular tracer washout in skin, we found that salt accumulation resulted in an increase in lymph flow of 26% that was noticeable only after including an overnight recording period. Surprisingly, lymph flow in skeletal muscle recorded with a new positron emission tomography/computed tomography method was also increased after salt exposure. The transcapillary filtration was unaffected by the high-salt diet and deoxycorticosterone-salt treatment, suggesting that the capillary barrier was not influenced by the salt accumulation. A significant reduction in lymph flow after depletion of macrophages/monocytes by clodronate suggests these cells are involved in the observed lymph flow response, together with collecting vessels shown here to enhance their contraction frequency as a response to extracellular Na + . Conclusions— The observed changes in lymph flow suggest that the lymphatics may influence long-term regulation of tissue fluid balance during salt accumulation by contributing to fluid homeostasis in skin and muscle. Our studies identify lymph clearance as a potential disease-modifying factor that might be targeted in conditions characterized by salt accumulation like chronic kidney disease and salt-sensitive hypertension.

Long-term elevated blood sugar levels result in tissue matrix compositional changes in patients with diabetes mellitus type 2 (T2DM). We hypothesized that hemodialysis patients with T2DM might accumulate more tissue sodium than control hemodialysis patients. To test this, 23Na magnetic resonance imaging (23Na MRI) was used to estimate sodium in skin and muscle tissue in hemodialysis patients with or without T2DM. Muscle fat content was estimated by 1H MRI and tissue sodium content by 23Na MRI pre- and post-hemodialysis in ten hemodialysis patients with T2DM and in 30 matched control hemodialysis patients. We also assessed body fluid distribution with the Body Composition Monitor. 1H MRI indicated a tendency to higher muscle fat content in hemodialysis patients with T2DM compared to non-diabetic hemodialysis patients. 23Na MRI indicated increased sodium content in muscle and skin tissue of hemodialysis patients with T2DM compared to control hemodialysis patients. Multi-frequency bioimpedance was used to estimate extracellular water (ECW), and excess ECW in T2DM hemodialysis patients correlated with HbA1c levels. Sodium mobilization during hemodialysis lowered muscle sodium content post-dialysis to a greater degree in T2DM hemodialysis patients than in control hemodialysis patients. Thus, our findings provide evidence that increased sodium accumulation occurs in hemodialysis patients with T2DM and that impaired serum glucose metabolism is associated with disturbances in tissue sodium and water content.

Impairment in the ability of the skin to properly store Na+ nonosmotically (without water) has recently been hypothesized as contributing to salt-sensitive hypertension. Our laboratory has shown that endothelial production of endothelin-1 (ET-1) is crucial to skin Na+ handling. Furthermore, it is well established that loss of endothelin type B receptor (ETB) receptor function impairs Na+ excretion by the kidney. Thus we hypothesized that rats lacking functional ETB receptors (ETB-def) will have a reduced capacity of the skin to store Na+ during chronic high-salt (HS) intake. We observed that ETB-def rats exhibited salt-sensitive hypertension with an approximate doubling in the diurnal amplitude of mean arterial pressure compared with genetic control rats on a HS diet. Two weeks of HS diet significantly increased skin Na+ content relative to water; however, there was no significant difference between control and ETB-def rats. Interestingly, HS intake led to a 19% increase in skin Na+ and 16% increase in water content (relative to dry wt.) during the active phase (zeitgeber time 16) versus inactive phase (zeitgeber time 4, P < 0.05) in ETB-def rats. There was no significant circadian variation in total skin Na+ or water content of control rats fed normal or HS. These data indicate that ETB receptors have little influence on the ability to store Na+ nonosmotically in the skin during long-term HS intake but, rather, appear to regulate diurnal rhythms in skin Na+ content and circadian blood pressure rhythms associated with a HS diet.

OBJECTIVE To test the hypothesis that tissue sodium and adipose content are elevated in patients with lipedema; if confirmed, this could establish precedence for tissue sodium and adipose content representing a discriminatory biomarker for lipedema. METHODS Participants with lipedema (n = 10) and control (n = 11) volunteers matched for biological sex, age, BMI, and calf circumference were scanned with 3.0-T sodium and conventional proton magnetic resonance imaging (MRI). Standardized tissue sodium content was quantified in the calf skin, subcutaneous adipose tissue (SAT), and muscle. Dixon MRI was employed to quantify tissue fat and water volumes of the calf. Nonparametric statistical tests were applied to compare regional sodium content and fat-to-water volume between groups (significance: two-sided P ≤ 0.05). RESULTS Skin (P = 0.01) and SAT (P = 0.04) sodium content were elevated in lipedema (skin: 14.9 ± 2.9 mmol/L; SAT: 11.9 ± 3.1 mmol/L) relative to control participants (skin: 11.9 ± 2.0 mmol/L; SAT: 9.4 ± 1.6 mmol/L). Relative fat-to-water volume in the calf was elevated in lipedema (1.2 ± 0.48 ratio) relative to control participants (0.63 ± 0.26 ratio; P

SUMMARY Sodium accumulates in the interstitium and promotes inflammation through poorly defined mechanisms. We describe a pathway by which sodium enters dendritic cells (DCs) through amiloride-sensitive channels including the alpha and gamma subunits of the epithelial sodium channel and the sodium hydrogen exchanger 1. This leads to calcium influx via the sodium calcium exchanger, activation of protein kinase C (PKC), phosphorylation of p47phox, and association of p47phox with gp91phox. The assembled NADPH oxidase produces superoxide with subsequent formation of immunogenic isolevuglandin (IsoLG)-protein adducts. DCs activated by excess sodium produce increased interleukin-1β (IL-1β) and promote T cell production of cytokines IL-17A and interferon gamma (IFN-γ). When adoptively transferred into naive mice, these DCs prime hypertension in response to a sub-pressor dose of angiotensin II. These findings provide a mechanistic link between salt, inflammation, and hypertension involving increased oxidative stress and IsoLG production in DCs., In Brief Barbaro et al. describe a pathway by which increased extracellular sodium activates dendritic cells. This pathway potentially explains the link between excessive salt intake, inflammation, and high blood pressure.

Rationale and Objectives Sodium and proton magnetic resonance imaging (23Na/1H-MRI) have shown that muscle and skin can store Na+ without water. In chronic renal failure and in heart failure, Na+ mobilization occurs, but is variable depending on age, dialysis vintage, and other features. Na+ storage depots have not been studied in patients with acute kidney injury (AKI). Materials and Methods We studied 7 patients with AKI (mean age: 51.7 years; range: 25–84) and 14 age-matched and gender-matched healthy controls. All underwent 23Na/1H-MRI at the calf. Patients were studied before and after acute hemodialysis therapy within 5–6 days. The 23Na-MRI produced grayscale images containing Na+ phantoms, which served to quantify Na+ contents. A fat-suppressed inversion recovery sequence was used to quantify H2O content. Results Plasma Na+ levels did not change. Mean Na+ contents in muscle and skin did not significantly change following four to five cycles of hemodialysis treatment (before therapy: 32.7 ± 6.9 and 44.2 ± 13.5 mmol/L, respectively; after dialysis: 31.7 ± 10.2 and 42.8 ± 11.8 mmol/L, respectively; P > .05). Water content measurements did not differ significantly before and after hemodialysis in muscle and skin (P > .05). Na+ contents in calf muscle and skin of patients before hemodialysis were significantly higher than in healthy subjects (16.6 ± 2.1 and 17.9 ± 3.2) and remained significantly elevated after hemodialysis. Conclusions Na+ in muscle and skin accumulates in patients with AKI and, in contrast to patients receiving chronic hemodialysis and those with acute heart failure, is not mobilized with hemodialysis within 5–6 days.

Renal denervation (RDN) has been introduced for reducing blood pressure (BP) in treatment-resistant hypertension (TRH). The precise mechanism how RDN exerts its BP-lowering effects are not yet fully understood. It is widely accepted that sodium (Na+) plays a crucial role in the pathogenesis of hypertensive disease. However, there is increasing evidence of osmotically inactive Na+ storage. We investigated the impact of RDN on Na+ homeostasis using estimation of salt intake, and measurement of tissue Na+ content. In a study 41 patients with TRH (office BP ≥140/90 mmHg and diagnosis confirmed by 24-h ambulatory BP monitoring) underwent RDN. Tissue Na+ content was assessed non-invasively with 3.0 T magnetic resonance imaging before and 6 months after RDN. In addition, 24-h urinary Na+ excretion as an estimate of salt intake and spot urine Na+/K+ excretion were assessed. The study was registered at http://www.clinicaltrials.gov (ID: NCT01687725). There was a significant fall in BP (office: −17 ± 20/−10 ± 12 mmHg; 24-h: −11 ± 13/−6 ± 9 mmHg, all p

Blood pressure salt sensitivity and salt resistance are mechanistically imperfectly explained. A prescient systems medicine approach by Guyton and colleagues—more than 50 years ago—suggested how salt intake might influence blood pressure. They proposed that a high-salt diet engenders sodium accumulation, volume expansion, cardiac output adjustments, and then autoregulation for flow maintenance. The autoregulation in all vascular beds increases systemic vascular resistance, causing the kidneys to excrete more salt and water, thus reducing systems to normal and minimizing any changes in blood pressure. This schema, which is remarkably all encompassing, included all regulatory mechanisms Guyton could identify at the time. Guyton introduced the idea that the kidney is central, particularly concerning the regulation of renal pressure natriuresis. Numerous criticisms have been subsequently raised, particularly recently. Kurtz and colleagues argue that the ability of individuals to respond with an appropriate vasodilatory response to increased salt intake is pivotal. Data exist to address that issue. Salt-resistant hypertensive models provide additional information. We identified a mendelian form of hypertension not related to sodium reabsorption in the distal nephron. The hypertension develops because of increased systemic vascular resistance. In addition, we rediscovered a third salt-storage glycose-aminoglycan–related compartment, largely in the skin. This compartment operates independently of renal function, and when perturbed, is associated with salt sensitivity. More recently, we found novel molecular mechanisms demonstrating how large salt quantities are excreted by the kidneys with minimal water losses. We introduce novel interpretations as to how the kidneys excrete salt when the intake is high. The findings could have relevance as to how blood pressure may be regulated at varying salt intakes. Our purposes are to provide the readership with a banquet of thoughts to digest, to pursue Guyton's ideas, and to adjust them accordingly.

Purpose 23Na MRI demonstrated increased tissue sodium concentrations in a number of pathologies. Acute atrophy results in muscle fibre volume shrinking that may result in a relative increase of extracellular volume and might affect sodium concentration. Thus, we hypothesized that local unloading of the calf muscles would lead to a decrease in muscle volume and an increase in muscle tissue sodium concentration. Method One lower leg of 12 healthy male subjects was submitted to a 60 day long period of unloading using the Hephaistos orthosis, while the other leg served as control. 23Na MRI and 2D PD-weighted Dixon turbo spin echo were obtained from the control and orthosis leg using a 3T scanner. For quantification, a sodium reference phantom was used with 10, 20, 30, and 40 mmol/L NaCl solution. Result Tissue sodium concentration (TSC) increased as an effect of unloading in the orthosis leg. Relative increases were 17.4 ± 16.8% (P = 0.005) in gastrocnemius medialis muscle, 11.1 ± 12.5 (P = 0.037) in gastrocnemius lateralis muscle, 16.2 ± 4.7% (P < 0.001) in soleus muscle, 10.0 ± 10.5% (P = 0.009) in the ventral muscle group, and 10.7 ± 10.0% (P = 0.003) in the central muscle group, respectively. TSC in the control leg did not significantly change. In the orthosis leg, muscle volume decreased as follows: medial gastrocnemius muscle: −5.4 ± 8.3% (P = 0.043) and soleus muscle: −7.8 ± 15.0% (P = 0.043). Conclusion Unloading atrophy is associated with an increase in muscle sodium concentration. 23Na MRI is capable of detecting these rather small changes.

The common notion is that the body Na + is maintained within narrow limits for fluid and blood pressure homeostasis. Several studies have, however, shown that considerable amounts of Na + can be retained or removed from the body without commensurate water loss and that the skin can serve as a major salt reservoir. Our own data from rats have suggested that the skin is hypertonic compared with plasma on salt storage and that this also applies to skin interstitial fluid. Even small electrolyte gradients between plasma and interstitial fluid would represent strong edema-generating forces. Because the water accumulation has been shown to be modest, we decided to reexamine with alternative methods in rats whether interstitial fluid is hypertonic during salt accumulation induced by high-salt diet (8% NaCl and 1% saline to drink) or deoxycorticosterone pellet implantation. These treatments resulted both in increased systemic blood pressure, skin salt, and water accumulation and in skin hyperosmolality. Interstitial fluid isolated from implanted wicks and lymph draining the skin was, however, isosmotic, and Na + concentration in fluid isolated by centrifugation and in lymph was not different from plasma. Interestingly, by eluting layers of the skin, we could show that there was an osmolality and urea gradient from epidermis to dermis. Collectively, our data suggest that fluid leaving the skin as lymph is isosmotic to plasma but also that the skin can differentially control its own electrolyte microenvironment by creating local gradients that may be functionally important.

Chronic renal failure leads to muscle mass loss and hypertension, which according to textbook teaching occur secondary to an inability of the kidneys to excrete solutes and water. We found instead that rats with experimental chronic renal failure constantly lost body water, because their kidneys could not sufficiently concentrate the urine. Physiological adaptation to body water loss, termed aestivation, is an evolutionary conserved survival strategy that relies on complex physiologic-metabolic adjustment across multiple organs to prevent otherwise lethal dehydration. We show that rats with chronic renal failure utilize these ancient water conservation motifs to successfully stabilize their body water. Metabolic aestivation responses to chronic renal failure require nitrogen-rich organic osmolyte production. Continuous endogenous energy and nitrogen supply from skeletal muscle in support of this metabolic requirement explains “renal” muscle mass loss. Accompanying circulatory aestivation responses are designed to limit skin water loss. This process requires vasoconstriction, which explains “renal” hypertension.

We recently reported that a 4% high-salt diet + saline for drinking (HS + saline) leads to a catabolic state, reduced heart rate, and suppression of cardiovascular energy expenditure in mice. We suggested that HS + saline reduces heart rate via the suppression of the sympathetic nervous system to compensate for the high salt intake-induced catabolic state. To test this hypothesis, we directly measured renal sympathetic nerve activity (RSNA) in conscious Sprague-Dawley (SD) rats using a radiotelemetry system. We confirmed that HS + saline induced a catabolic state. HS + saline decreased heart rate, while also reducing RSNA in SD rats. In contrast, Dahl salt-sensitive (DSS) rats exhibited no change in heart rate and increased RSNA during high salt intake. Renal denervation significantly decreased heart rate and attenuated the catabolic state independent of blood pressure in DSS rats fed HS + saline, suggesting that salt-sensitive animals were unable to decrease cardiovascular energy consumption due to abnormal renal sympathetic nerve activation during high salt intake. These findings support the hypothesis that RSNA mediates heart rate during high salt intake in SD rats. However, the insensitivity of heart rate and enhanced RSNA observed in DSS rats may be additional critical diagnostic factors for salt-sensitive hypertension. Renal denervation may benefit salt-sensitive hypertension by reducing its effects on catabolism and cardiovascular energy expenditure.

Dietary salt consumption leads to cutaneous Na(+) storage and is associated with various disorders, including osteopenia. Here, we explore the impact of Na(+) and the osmoprotective transcription factor nuclear factor of activated T cell 5 (NFAT5) on bone density and osteoclastogenesis. Compared with treatment of mice with high-salt diet, low-salt diet (LSD) increased bone density, decreased osteoclast numbers, and elevated Na(+) content and Nfat5 levels in the BM. This response to LSD was dependent on NFAT5 expressed in myeloid cells. Simulating in vivo findings, we exposed osteoclast precursors and osteoblasts to elevated Na(+) content (high-salt conditions; HS¢), resulting in increased NFAT5 binding to the promotor region of RANKL decoy receptor osteoprotegerin (OPG). These data not only demonstrate that NFAT5 in myeloid cells determines the Na(+) content in BM, but that NFAT5 is able to govern the expression of the osteoprotective gene OPG. This provides insights into mechanisms of Na(+)-induced cessation of osteoclastogenesis and offers potentially new targets for treating salt-induced osteopenia.

Introduction: We recently reported that kidneys accumulate urea as an alternative osmolyte in the renal medulla to concentrate sodium into the urine during high salt intake. This urea-driven water conservation process is coupled with the energy-intensive nature of hepatic urea production, which leads to a catabolic state in mice fed high salt. In this study, we examined the effects of renal denervation on the salt-driven catabolic state since previous studies found that the renal sympathetic nervous system regulates urinary sodium excretion and energy metabolism of some organs such as liver and muscle. Methods: Sham-operated (sham) or renal denervated (RDX) male C57/B6J mice were fed on 0.3% NaCl diet with tap water (NS) or 4% NaCl high salt diet + 0.9% NaCl water to drink (HS) for 4 consecutive weeks (ad libitum), followed by 2 weeks of pair-feeding to match energy intake in all groups. We measured daily food intake and body weight, 24 hours urinary sodium excretion, body sodium content, and activity of liver arginase, a urea producing enzyme. Results: NS + sham and HS + sham mice showed similar body weight gain (NS + sham: +3.6 ± 0.2, HS + sham: +3.4 ± 0.2 g) during ad libitum feeding although HS significantly increased food intake (NS + sham: 3.1 ± 0.04, HS + sham: 3.3 ± 0.04 g/day). After the pair-feeding, HS mice significantly decreased their body weight (NS + sham: +0.9 ± 0.07, HS + sham: -0.6 ± 0.05 g) and increased liver arginase activity (NS + sham: 3792 ± 322, HS + sham: 5412 ± 499 units/L/tissue), confirming the high salt-induced catabolic hepatic urea production. On the other hand, HS did not increase food intake (NS + RDX: 3.2 ± 0.1, HS + RDX: 3.1 ± 0.01 g/day) and liver arginase activity (NS + RDX: 3599 ± 316, HS + RDX: 3363 ± 369 units/L/tissue) in RDX group. In addition, HS + RDX mice did not show pair feeding-induced body weight loss (NS + RDX: +0.6 ± 0.3, HS + RDX: +0.3 ± 0.3 g). HS + RDX mice did not alter 24 hours urinary sodium excretion (HS + sham: 135 ± 6.3, HS + RDX: 142 ± 13 mmol/d/kg) and body sodium content (HS + sham: 0.15 ± 0.003, HS + RDX: 0.15 ± 0.006 mmol/d/kg) compared with HS + sham mice. Conclusion: These findings suggest that renal denervation attenuates the HS-induced body weight loss and catabolic urea production independently of urinary sodium excretion.

Simple Summary Cell based immunotherapy is rapidly emerging as a promising cancer treatment. Salt (sodium chloride) treatment to immune cell cultures is known to induce inflammatory activation. In our current study, we analyzed the anti-cancer ability of salt treatment on immune cells outside the host followed by reinfusion into the host. Using a pre-clinical breast cancer model, we demonstrated that external salt treatment on T-cell subset of immune cells produced a viable anti-cancer response, which may have future human clinical application. Abstract Cell based immunotherapy is rapidly emerging as a promising cancer treatment. A modest increase in salt (sodium chloride) concentration in immune cell cultures is known to induce inflammatory phenotypic differentiation. In our current study, we analyzed the ability of salt treatment to induce ex vivo expansion of tumor-primed CD4 (cluster of differentiation 4)+T cells to an effector phenotype. CD4+T cells were isolated using immunomagnetic beads from draining lymph nodes and spleens from tumor bearing C57Bl/6 mice, 28 days post-injection of Py230 syngeneic breast cancer cells. CD4+T cells from non-tumor bearing mice were isolated from splenocytes of 12-week-old C57Bl/6 mice. These CD4+T cells were expanded ex vivo with five stimulation cycles, and each cycle comprised of treatment with high salt (Δ0.035 M NaCl) or equimolar mannitol controls along with anti-CD3/CD28 monoclonal antibodies for the first 3 days, followed by the addition of interleukin (IL)-2/IL-7 cytokines and heat killed Py230 for 4 days. Ex vivo high salt treatment induced a two-fold higher Th1 (T helper type 1) expansion and four-fold higher Th17 expansion compared to equimolar mannitol treatment. Importantly, the high salt expanded CD4+T cells retained tumor-specificity, as demonstrated by higher in vitro cytotoxicity against Py230 breast cancer cells and reduced in vivo syngeneic tumor growth. Metabolic studies revealed that high salt treatment enhanced the glycolytic reserve and basal mitochondrial oxidation of CD4+T cells, suggesting a role of high salt in enhanced pro-growth anabolic metabolism needed for inflammatory differentiation. Mechanistic studies demonstrated that the high salt induced switch to the effector phenotype was mediated by tonicity-dependent transcription factor, TonEBP/NFAT5. Using a transgenic murine model, we demonstrated that CD4 specific TonEBP/NFAT5 knock out (CD4cre/creNFAT5flox/flox) abrogated the induction of the effector phenotype and anti-tumor efficiency of CD4+T cells following high salt treatment. Taken together, our data suggest that high salt-mediated ex vivo expansion of tumor-primed CD4+T cells could induce effective tumor specific anti-cancer responses, which may have a novel cell-based cancer immunotherapeutic application.

Background Recent data suggest that sodium (Na+) is stored in the muscle and skin without commensurate water retention in maintenance hemodialysis (MHD) patients. In this study, we hypothesized that excessive Na+ accumulation would be associated with abnormalities in peripheral insulin action. Methods Eleven MHD patients and eight controls underwent hyperinsulinemic–euglycemic–euaminoacidemic clamp studies to measure glucose (GDR) and leucine disposal rates (LDR), as well as lower left leg 23Na magnetic resonance imaging to measure Na+ concentration in the muscle and skin tissue. Results The median GDR and LDR levels were lower, and the median muscle Na+ concentration was higher in MHD patients compared with controls. No significant difference was found regarding skin Na+ concentration between group comparisons. Linear regression revealed inverse relationships between muscle Na+ concentration and GDR and LDR in MHD patients, whereas no relationship was observed in controls. There was no association between skin Na+ content and GDR or LDR in either MHD patients or controls. Conclusions These data suggest that excessive muscle Na+ content might be a determinant of IR in MHD patients, although the causality and mechanisms remain to be proven.

The aim of this work was to investigate possible sex differences in the patterns of sodium deposition between muscle and skin using sodium MRI. A total of 38 subjects were examined for comparisons: 20 males, aged 2579 years with a median age of 51; 18 females, aged 38-66 years, median age 53. All subjects underwent sodium MRI scans of the calf muscles together with cross sections through four calibration standards containing known sodium contents (10 mM, 20 mM, 30 mM, and 40 mM). Tissue sodium concentrations (TSC) in muscle and skin were then calculated by comparing signal intensities between tissues and reference standards using a linear analysis. A Wilcoxon rank sum test was applied to the Delta TSC (=TSCmuscle - TSCskin) series of males and females to examine if they were significantly different. Finally, a multiple linear regression was utilized to account for the effects from two potential confounders, age and body mass index (BMI). We found that sodium content appears to be higher in skin than in muscle for men, however women tend to have higher muscle sodium than skin sodium. This sex-relevant sodium deposition is statistically significant (P = 3.10 x 10(-5)) by the Wilcoxon rank sum test, and this difference in distribution seems to be more reliable with increasing age. In the multiple linear regression, gender still has a statistically significant effect (P < 1.0 x 10(-4)) on the difference between sodium deposition in muscle and skin, while taking the effects of age and BMI into account. (C) 2016 Elsevier Inc. All rights reserved.

Objective. Skin fibrosis is the predominant feature of SSc and arises from excessive extracellular matrix deposition. Glycosaminoglycans are macromolecules of the extracellular matrix, which facilitate Na(+) accumulation in the skin. We used (23)Na-MRI to quantify Na(+) in skin. We hypothesized that skin Na(+) might accumulate in SSc and might be a biomarker for skin fibrosis. Methods. In this observational case–control study, skin Na(+) was determined by (23)Na-MRI using a Na(+) volume coil in 12 patients with diffuse cutaneous SSc and in 21 control subjects. We assessed skin fibrosis by the modified Rodnan skin score prior to (23)Na-MRI and on follow-up 12 months later. Results. (23)Na-MRI demonstrated increased Na(+) in the fibrotic skin of SSc patients compared with skin from controls [mean (s.d.): 27.2 (5.6) vs 21.4 (5.3) mmol/l, P < 0.01]. Na(+) content was higher in fibrotic than in non-fibrotic SSc skin [26.2 (4.8) vs 19.2 (3.4) mmol/l, P < 0.01]. Furthermore, skin Na(+) amount was correlated with changes in follow-up modified Rodnan skin score (R(2) = 0.68). Conclusions. (23)Na-MRI detected increased Na(+) in the fibrotic SSc skin; high Na(+) content was associated with progressive skin disease. Our findings provide the first evidence that (23)Na-MRI might be a promising tool to assess skin Na(+) and thereby predict progression of skin fibrosis in SSc.

Humans have had a long and complex relationship with salt. Although highly valued in many societies, dietary salt has long been associated with high blood pressure1–3 and, more recently, with other diseases.4–6 Some individuals with hypertension often display salt-sensitive blood pressure changes, which is a condition more prevalent among blacks, older people, and individuals with renal insufficiency or diabetes mellitus.7–9 In general, for those with salt-sensitive hypertension, excess sodium intake is associated with higher blood pressure, whereas a low-salt diet decreases blood pressure.3 In spite of this well-known association, the basic molecular and cellular mechanisms underlying the effects of salt on blood pressure regulation are still not well understood. Furthermore, individuals with high blood pressure are at increased risk for multiple diseases (ie, coronary artery disease, heart failure, stroke, and renal disease) although at present whether or not a high dietary salt intake can directly lead to these diseases (ie, in the absence of hypertension) is not known. Our understanding of the effect of salt on health has grown even more complex recently. Researchers have reported a new connection between salt and autoimmunity: a high-salt diet was shown to accelerate autoimmune activity in a mouse model of multiple sclerosis.10,11 In addition, a close connection between hypertension and the immune system has been revealed.12–16 However, the causal relationships between salt, immunity, and hypertension (eg, how salt could mediate interactions between the immune system and the vasculature, brain, or kidney to increase blood pressure) are not well understood. The National Heart, Lung, and Blood Institute convened a Working Group (WG) in 2014 to discuss this new emerging scientific area in hypertension research. The WG brought together experts from diverse backgrounds including hypertension, epidemiology, preeclampsia, cardiovascular disease, …

Sodium and potassium intake are modifiable determinants of hypertension in the general population but have not been studied in patients with systemic lupus erythematosus (SLE). We examined the relationship between urinary excretion of sodium and potassium, as an estimate of intake, and blood pressure in patients with SLE. We studied 178 SLE patients and 86 controls, matched for age, sex, and race. Urine sodium (Na+) and potassium (K+) were measured by flame photometry. Blood pressure was the average of two resting measurements. The associations between systolic (SBP) and diastolic blood pressures (DBP) and estimated 24-hour urinary Na+, K+, and Na+:K+ ratio were tested. The estimated mean 24-hour urinary K+ excretion was lower, and the Na+:K+ ratio was higher in patients with SLE than controls. There were no significant differences in the estimated 24-hour urinary Na+. In patients with SLE, a higher urinary Na+:K+ ratio was associated with higher SBP (β coefficient = 4.01, p = 0.023) and DBP (β coefficient = 4.41, p = 0.002) after adjusting for age, sex, and race. SLE patients had significantly lower estimated 24-hour urinary K+ and higher estimated 24-hour urinary Na+: K+ ratio than controls. The urinary Na+:K+ ratio was significantly associated with SBP and DBP.

Sustained oliguria during fluid resuscitation represents a perplexing problem in patients undergoing therapy for septic acute kidney injury. Here, we tested whether lipopolysaccharide induces filtrate leakage from the proximal tubular lumen into the interstitium, thus disturbing the recovery of urine output during therapy, such as fluid resuscitation, aiming to restore the glomerular filtration rate. Intravital imaging of the tubular flow rate in the proximal tubules in mice showed that lipopolysaccharide did not change the inflow rate of proximal tubule filtrate, reflecting an unchanged glomerular filtration rate, but significantly reduced the outflow rate, resulting in oliguria. Lipopolysaccharide disrupted tight junctions in proximal tubules and induced both paracellular leakage of filtered molecules and interstitial accumulation of extracellular fluid. These changes were diminished by conditional knockout of Toll-like receptor 4 in the proximal tubules. Importantly, these conditional knockout mice showed increased sensitivity to fluid resuscitation and attenuated acute kidney injury. Thus, lipopolysaccharide induced paracellular leakage of filtrate into the interstitium via a Toll-like receptor 4-dependent mechanism in the proximal tubules of endotoxemic mice. Hence, this leakage might diminish the efficacy of fluid resuscitation aiming to maintain renal hemodynamics and glomerular filtration rate. publishedVersion

Infection and inflammation are able to induce diet-independent Na+-accumulation without commensurate water retention in afflicted tissues, which favors the pro-inflammatory activation of mouse macrophages and augments their antibacterial and antiparasitic activity. While Na+-boosted host defense against the protozoan parasite Leishmania major is mediated by increased expression of the leishmanicidal NOS2 (nitric oxide synthase 2, inducible), the molecular mechanisms underpinning this enhanced antibacterial defense of mouse macrophages with high Na+ (HS) exposure are unknown. Here, we provide evidence that HS-increased antibacterial activity against E. coli was neither dependent on NOS2 nor on the phagocyte oxidase. In contrast, HS-augmented antibacterial defense hinged on HIF1A (hypoxia inducible factor 1, alpha subunit)-dependent increased autophagy, and NFAT5 (nuclear factor of activated T cells 5)-dependent targeting of intracellular E. coli to acidic autolysosomal compartments. Overall, these findings suggest that the autolysosomal compartment is a novel target of Na+-modulated cell autonomous innate immunity. Abbreviations: ACT: actins; AKT: AKT serine/threonine kinase 1; ATG2A: autophagy related 2A; ATG4C: autophagy related 4C, cysteine peptidase; ATG7: autophagy related 7; ATG12: autophagy related 12; BECN1: beclin 1; BMDM: bone marrow-derived macrophages; BNIP3: BCL2/adenovirus E1B interacting protein 3; CFU: colony forming units; CM-H2DCFDA: 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester; CTSB: cathepsin B; CYBB: cytochrome b-245 beta chain; DAPI: 4,6-diamidino-2-phenylindole; DMOG: dimethyloxallyl glycine; DPI: diphenyleneiodonium chloride; E. coli: Escherichia coli; FDR: false discovery rate; GFP: green fluorescent protein; GSEA: gene set enrichment analysis; GO: gene ontology; HIF1A: hypoxia inducible factor 1, alpha subunit; HUGO: human genome organization; HS: high salt (+ 40 mM of NaCl to standard cell culture conditions); HSP90: heat shock 90 kDa proteins; LDH: lactate dehydrogenase; LPS: lipopolysaccharide; Lyz2/LysM: lysozyme 2; NFAT5/TonEBP: nuclear factor of activated T cells 5; MΦ: macrophages; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence intensity; MIC: minimum inhibitory concentration; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; NaCl: sodium chloride; NES: normalized enrichment score; n.s.: not significant; NO: nitric oxide; NOS2/iNOS: nitric oxide synthase 2, inducible; NS: normal salt; PCR: polymerase chain reaction; PGK1: phosphoglycerate kinase 1; PHOX: phagocyte oxidase; RFP: red fluorescent protein; RNA: ribonucleic acid; ROS: reactive oxygen species; sCFP3A: super cyan fluorescent protein 3A; SBFI: sodium-binding benzofuran isophthalate; SLC2A1/GLUT1: solute carrier family 2 (facilitated glucose transporter), member 1; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like kinase 1; v-ATPase: vacuolar-type H+-ATPase; WT: wild type

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

Sodium can be buffered in the skin, which mechanism is altered during aging and in certain diseases such as hypertension. High salt environment can promote autoimmunity by expanding pathogenic IL-17 producing T helper (Th17) cells. Psoriasis is a relapsing and remitting inflammatory autoimmune disease affecting the skin and joints and involves proinflammatory Th17 cells. Here we tested the hypothesis if psoriatic skin has a higher sodium content in mice and humans. We used two psoriasis mouse models; the K14-IL-17A ind/+ mice overexpressing IL-17A in K14-positive keratinocytes and the imiquimod (IMQ) mouse model by applying 62.5 mg IMQ cream (5%) on the shaved back and ears of FVB/N mice for 5 days daily. End of the study skins of mice were collected, weighted, dried and ashed to measure water and sodium content. Additionally, skin sodium and water content were measured in psoriasis patients and aged matched healthy controls by non-invasive 23 Na-MRI on non-affected flexor site of the lower leg and by 23 Na-spectroscopy to compare affected and non-affected sites of the leg. K14-IL-17A ind/+ mice had significantly higher sodium content compared to control IL-17A ind/+ mice (0.191±0.021 vs. 0.137±0.023 mg/g dry weight) together with an elevated water content. IMQ-treated back skin had significantly higher sodium content compared to untreated ventral skin of the same mice (0.175±0.023 vs. 0.143±0.014 mg/g dry weight), whereas sham mice had a significantly lower content in both regions (0.116±0.010 vs. 0.107±0.005 mg/g dry weight). IMQ treatment led to significant expansion of IL-17 producing γδT cells in the skin, regional lymph nodes and in the spleen with typical skin lesions. Patients with psoriasis area and severity index (PASI) >5 had significantly higher sodium content in the skin compared to those with lower PASI or with healthy controls (17.73±1.52 vs. 14.32±1.54 vs. 14.30±2.59 AU, respectively); this elevation was water coupled. PASI significantly correlated with skin sodium content (Pearson’s r=0.598, P5 has higher sodium content in the affected skin compared to non-affected skin of the same patient. Data from animal models and humans argue for higher sodium accumulation in the inflamed skin.

Recent evidence from our laboratory has demonstrated that high salt (Δ0.05 M NaCl) induced inflammatory response and cancer cell proliferation through salt inducible kinase-3 (SIK3) upregulation. As calcium influx is known to effect inflammatory response and drug resistance, we examined the impact of high salt on calcium influx in breast cancer cells. Treatment of MCF-7 and MDA-MB-231 cells with high salt induced an enhanced intracellular calcium intensity, which was significantly decreased by store operated calcium entry (SOCE) inhibitor co-treatment. Further, high salt induced P-glycoprotein (P-gp) mediated paclitaxel drug resistance in breast cancer cells. Murine tumor studies demonstrated that injection of MCF-7 cells cultured in high salt, exerted higher tumorigenicity compared to the basal cultured counterpart. Knock down of SIK3 by specific shRNA inhibited tumorigenicty, expression of SOCE regulators and P-gp activity, suggesting SIK3 is an upstream mediator of SOCE induced calcium influx. Furthermore, small molecule inhibitor, prostratin, exerted anti-tumor effect in murine models through SIK3 inhibition. Taken together, we conclude that SIK3 is an upstream regulator of store operated calcium entry proteins, Orai1 and STIM1, and mediates high salt induced inflammatory cytokine responses and P-gp mediated drug resistance. Therefore, small molecule inhibitors, such as prostratin, could offer novel anti-cancer approaches.

Purpose of review Textbook theory holds that blood pressure (BP) is regulated by the brain, by blood vessels, or by the kidney. Recent evidence suggests that BP could be regulated in the skin. Recent findings The skin holds a complex capillary counter current system, which controls body temperature, skin perfusion, and apparently systemic BP. Epidemiological data suggest that sunlight exposure plays a role in controlling BP. Ultraviolet A radiation produces vasodilation and a fall in BP. Keratinocytes and immune cells control blood flow in the extensive countercurrent loop system of the skin by producing nitric oxide, a key regulator of vascular tone. The balance between hypoxia-inducible factor-1α and hypoxia-inducible factor-2α activity in keratinocytes controls skin perfusion, systemic thermoregulation, and systemic BP by nitric oxide-dependent mechanisms. Furthermore, the skin accumulates Na which generates a barrier to promote immunological host defense. Immune cells control skin Na metabolism and the clearance of Na via the lymphatic system. Reduced lymphatic clearance increases BP. Summary Apart from the well-known role of the brain, blood vessels, and the kidney, the skin is important for systemic BP control in humans and in experimental animals.

Accurately collected 24-hour urine collections are presumed to be valid for estimating salt intake in individuals. We performed 2 independent ultralong-term salt balance studies lasting 105 (4 men) and 205 (6 men) days in 10 men simulating a flight to Mars. We controlled dietary intake of all constituents for months at salt intakes of 12, 9, and 6 g/d and collected all urine. The subjects’ daily menus consisted of 27 279 individual servings, of which 83.0% were completely consumed, 16.5% completely rejected, and 0.5% incompletely consumed. Urinary recovery of dietary salt was 92% of recorded intake, indicating long-term steady-state sodium balance in both studies. Even at fixed salt intake, 24-hour urine collection for sodium excretion (UNaV) showed infradian rhythmicity. We defined a ±25 mmol deviation from the average difference between recorded sodium intake and UNaV as the prediction interval to accurately classify a 3-g difference in salt intake. Because of the biological variability in UNaV, only every other daily urine sample correctly classified a 3-g difference in salt intake (49%). By increasing the observations to 3 consecutive 24-hour collections and sodium intakes, classification accuracy improved to 75%. Collecting seven 24-hour urines and sodium intake samples improved classification accuracy to 92%. We conclude that single 24-hour urine collections at intakes ranging from 6 to 12 g salt per day were not suitable to detect a 3-g difference in individual salt intake. Repeated measurements of 24-hour UNaV improve precision. This knowledge could be relevant to patient care and the conduct of intervention trials.

Sodium balance is achieved within a matter of days and everything that enters should come out; sodium stores are of questionable relevance and sodium accumulation is accompanied by weight gain. Careful balance studies oftentimes conflicted with this view, and long-term studies suggested that total body sodium (TBNa) fluctuates independent of intake or body weight. We recently performed the opposite experiment in that we fixed sodium intake for weeks at three levels of sodium intake and collected all urine made. We found weekly (circaseptan) patterns in sodium excretion that were inversely related to aldosterone and directly related to cortisol. TBNa was not dependent on sodium intake, but instead exhibited far longer (greater than or equal to monthly) infradian rhythms independent of extracellular water, body weight or blood pressure. To discern the mechanisms further, we delved into sodium magnetic resonance imaging (Na-MRI) to identify sodium storage clinically. We found that sodium stores are greater in men than in women, increase with age and are higher in hypertensive than normotensive persons. We have suggestive evidence that these sodium stores can be mobilized, also in dialysis patients. The observations are in accordance with our findings that immune cells regulate a hypertonic interface in the skin interstitium that could serve as a protective barrier. Returning to our balance studies, we found that due to biological variability in 24-h sodium excretion, collecting urine for a day could not separate 12, 9 or 6 g/day sodium intakes with the precision of tossing a coin. Every other daily urine sampling correctly classified a 3-g difference in salt intake less than half the time, making the gold standard 24-h urine collection of little value in predicting salt intake. We suggest that wobbles in expected outcomes can lead to novel clinical insights even with respect to banal salt questions.

The current study was designed to determine whether vascular endothelial-derived endothelin-1 (ET-1) is important for skin Na+ buffering. In control mice (C57BL/6J), plasma Na+ and osmolarity were significantly elevated in animals on high- vs. low-salt (HS and LS, respectively) intake. The increased plasma Na+ and osmolarity were associated with increased ET-1 mRNA in vascular tissue. There was no detectable difference in skin Na+:H2O in HS fed mice (0.119 ± 0.005 mM vs. 0.127 ± 0.007 mM; LS vs. HS); however, skin Na+:H2O was significantly increased by blockade of the endothelin type A receptor with ABT-627 (0.116 ± 0.006 mM vs. 0.137 ± 0.007 mM; LS vs. HS; half-maximal inhibitory concentration, 0.055 nM). ET-1 peptide content in skin tissue was increased in floxed control animals on HS (85.9 ± 0.9 pg/mg vs. 106.4 ± 6.8 pg/mg; P < 0.05), but not in vascular endothelial cell endothelin-1 knockout (VEET KO) mice (76.4 ± 5.7 pg/mg vs. 65.7 ± 7.9 pg/mg; LS vs. HS). VEET KO mice also had a significantly elevated skin Na+:H2O (0.113 ± 0.007 mM vs. 0.137 ± 0.005 mM; LS vs. HS; P < 0.05). Finally, ET-1 production was elevated in response to increasing extracellular osmolarity in cultured human endothelial cells. These data support the hypothesis that increased extrarenal vascular ET-1 production in response to HS intake is mediated by increased extracellular osmolarity and plays a critical role in regulating skin storage of Na+.—Speed, J. S., Heimlich, J. B., Hyndman, K. A., Fox, B. M., Patel, V., Yanagisawa, M., Pollock, J. S., Titze, J. M., Pollock, D. M. Endothelin-1 as a master regulator of whole-body Na+ homeostasis.