In salt-sensitive hypertension, the accumulation of Na(+) in tissue has been presumed to be accompanied by a commensurate retention of water to maintain the isotonicity of body fluids. We show here that a high-salt diet (HSD) in rats leads to interstitial hypertonic Na(+) accumulation in skin, resulting in increased density and hyperplasia of the lymphcapillary network. The mechanisms underlying these effects on lymphatics involve activation of tonicity-responsive enhancer binding protein (TonEBP) in mononuclear phagocyte system (MPS) cells infiltrating the interstitium of the skin. TonEBP binds the promoter of the gene encoding vascular endothelial growth factor-C (VEGF-C, encoded by Vegfc) and causes VEGF-C secretion by macrophages. MPS cell depletion or VEGF-C trapping by soluble VEGF receptor-3 blocks VEGF-C signaling, augments interstitial hypertonic volume retention, decreases endothelial nitric oxide synthase expression and elevates blood pressure in response to HSD. Our data show that TonEBP-VEGF-C signaling in MPS cells is a major determinant of extracellular volume and blood pressure homeostasis and identify VEGFC as an osmosensitive, hypertonicity-driven gene intimately involved in salt-induced hypertension.

Dietary salt intake has been investigated in relation to hypertension since the early 20th century.1 Mendelian forms of hypertension have underscored the role of salt intake in the development of hypertension.2 Clinicians have advised patients in terms of their dietary intake and have relied on 24 hour urine collections to verify dietary compliance. 3 Nonetheless, the results have been suboptimal. Clinicians have been limited to measuring Na + in plasma and urine, and the relationship to these sources for determining salt-sensitivity has been disappointing. The issue is important, as salt-sensitivity portends an earlier death. 4 Na + is bound to negatively charged proteoglycans that are very abundant in the skin, the body's largest organ. 5 We showed recently that signaling mechanisms exist in skin that control skin electrolyte storage. 6 When these mechanisms are perturbed, salt-sensitive hypertension results. Translating such findings to humans has been challenging. For that reason, we implemented quantitative 23 Na magnetic resonance imaging ( 23 Na-MRI) to visualize Na + in skin and soft tissues. 7 We reported on rodents measured with 23 Na-MRI and with MR spectroscopy, a small number of normal subjects, and 5 patients with primary aldosteronism, who were studied before and after definitive treatment. We have now extended our observations to larger numbers of normal men and women, as well as to patients with essential hypertension. We believe that 23 Na-MRI shows promise to be of clinical utility in further defining the relationship between salt and hypertension. MethodsWe implemented 23 Na-MRI for quantitative analysis in men; the methods were recently published. 7 We measured Na + content in lower leg muscle and skin with a 23 Na knee-coil (Stark-Contrast, Erlangen, Germany) at 3.0 T with a MRI scanner (Magnetom-Trio, Siemens Healthcare, Erlangen, Germany) using a 2D-FLASH sequence (total acquisition time, TA=13.7 minutes; echo time, TE=2.07 ms; repetition time, TR=100 ms; flip angle, FA=90°; 128 averages, resolution:Abstract-High dietary salt intake is associated with hypertension; the prevalence of salt-sensitive hypertension increases with age. We hypothesized that tissue Na + might accumulate in hypertensive patients and that aging might be accompanied by Na + deposition in tissue. We implemented 23 Na magnetic resonance imaging to measure Na + content of soft tissues in vivo earlier, but had not studied essential hypertension. We report on a cohort of 56 healthy control men and women, and 57 men and women with essential hypertension. The ages ranged from 22 to 90 years.23 Na magnetic resonance imaging measurements were made at the level of the calf. We observed age-dependent increases in Na + content in muscle in men, whereas muscle Na + content did not change with age in women. We estimated water content with conventional MRI and found no age-related increases in muscle water in men, despite remarkable Na + accumulation, indicating water-free Na + storage in muscle. With increasing age, there was ...

The skin interstitium sequesters excess Na + and Cl -in salt-sensitive hypertension. Mononuclear phagocyte system (MPS) cells are recruited to the skin, sense the hypertonic electrolyte accumulation in skin, and activate the tonicity-responsive enhancer-binding protein (TONEBP, also known as NFAT5) to initiate expression and secretion of VEGFC, which enhances electrolyte clearance via cutaneous lymph vessels and increases eNOS expression in blood vessels. It is unclear whether this local MPS response to osmotic stress is important to systemic blood pressure control. Herein, we show that deletion of TonEBP in mouse MPS cells prevents the VEGFC response to a high-salt diet (HSD) and increases blood pressure. Additionally, an antibody that blocks the lymph-endothelial VEGFC receptor, VEGFR3, selectively inhibited MPS-driven increases in cutaneous lymphatic capillary density, led to skin Cl -accumulation, and induced salt-sensitive hypertension. Mice overexpressing soluble VEGFR3 in epidermal keratinocytes exhibited hypoplastic cutaneous lymph capillaries and increased Na + , Cl -, and water retention in skin and salt-sensitive hypertension. Further, we found that HSD elevated skin osmolality above plasma levels. These results suggest that the skin contains a hypertonic interstitial fluid compartment in which MPS cells exert homeostatic and blood pressure-regulatory control by local organization of interstitial electrolyte clearance via TONEBP and VEGFC/VEGFR3-mediated modification of cutaneous lymphatic capillary function.

The steady-state concept of Na(+) homeostasis, based on short-term investigations of responses to high salt intake, maintains that dietary Na(+) is rapidly eliminated into urine, thereby achieving constant total-body Na(+) and water content. We introduced the reverse experimental approach by fixing salt intake of men participating in space flight simulations at 12 g, 9 g, and 6 g/day for months and tested for the predicted constancy in urinary excretion and total-body Na(+) content. At constant salt intake, daily Na(+) excretion exhibited aldosterone-dependent, weekly (circaseptan) rhythms, resulting in periodic Na(+) storage. Changes in total-body Na(+) (±200-400 mmol) exhibited longer infradian rhythm periods (about monthly and longer period lengths) without parallel changes in body weight and extracellular water and were directly related to urinary aldosterone excretion and inversely to urinary cortisol, suggesting rhythmic hormonal control. Our findings define rhythmic Na(+) excretory and retention patterns independent of blood pressure or body water, which occur independent of salt intake.

Hypertension is linked to disturbed total-body sodium (Na(+)) regulation; however, measuring Na(+) disposition in the body is difficult. We implemented (23)Na magnetic resonance spectroscopy ((23)Na-MR) and imaging technique ((23)Na-MRI) at 9.4T for animals and 3T for humans to quantify Na(+) content in skeletal muscle and skin. We compared (23)Na-MRI data with actual tissue Na(+) content measured by chemical analysis in animal and human tissue. We then quantified tissue Na(+) content in normal humans and in patients with primary aldosteronism. We found a 29% increase in muscle Na(+) content in patients with aldosteronism compared with normal women and men. This tissue Na(+) was mobilized after successful treatment without accompanying weight loss. We suggest that, after further refinements, this tool could facilitate understanding the relationships between Na(+) accumulation and hypertension. Furthermore, with additional technical advances, a future clinical use may be possible.