The internal environment and its regulation have drawn rapt attention since the 19th Century [1–3]. The basic tenet of electrolyte balance has been that extracellular bodily fluids readily equilibrate, allowing the kidney to achieve strict constancy in body sodium content, which is the key to blood pressure (BP) control and thus has a central standing in cardiovascular medicine [4–7]. Early evidence from physiological experiments in humans suggested that salt retention in the body might be always ‘isotonic’, meaning that a 140–150 mmol salt accumulation in the extracellular space inevitably leads to a 1 l fluid accumulation . The strict coupling between sodium and water balance is part of the widely accepted rules in salt and water homeostasis: first, sodium accumulation in the body seems to be primarily extracellular; second, sodium accumulated in the extracellular space will result in fluid retention until initial differences in extracellular sodium concentrations have equilibrated; and third, extracellular sodium content is maintained steady within very narrow limits to prevent fluid overload and perhaps blood pressure increase. Multiple physiological regulatory systems are operative to prevent surplus or loss of body sodium and thereby control the extracellular fluid matrix . One of the main predictions of the infinite gain theory of blood pressure control by the kidney is that long-term arterial blood pressure regulation is ‘principally a function of the body's fluid balance system and the ability of the kidney to excrete sodium’ . This theory stands on the assumption that ‘the close relationships between sodium and body fluid volume are already well established’ and that ‘essentially all of the factors … that alter fluid volume, especially extracellular fluid volume, cause parallel and almost proportional changes in body sodium at the same time’ . This review focuses on the recent evidence which suggests that the relationship between sodium and body fluid volume is not as close and as predictable as believed, because large amounts of sodium are stored in the body. Readers who would believe that the resulting expanded model of sodium balance is novel are advised to study the first description of skin chloride storage in dogs by Padtberg  more than 100 years ago, the experiments in rats by Ivanova et al. , and Cannon's  textbook comment on skin sodium homeostasis.
SODIUM BALANCE THEORY TODAY COMES FROM SHORT-TERM STUDIES WHICH WERE DESIGNED IN THE 19TH CENTURY
Clinicians’ thinking on sodium balance derives from experiments introduced by Carl Ludwig  who studied renal salt excretion in individuals exposed to extremes (very high versus very low) in salt intake. When urinary output again approached input, steady-state regulation of sodium balance was generally assumed in these studies [8,9]. In these classic experiments, steady state is achieved after transient accumulation or loss of body sodium, which leads to parallel changes in extracellular fluid volume (Fig. 1a): ‘If dietary intake is abruptly increased from a low-sodium diet, only about one-half is excreted on the first day. The remainder is retained augmenting sodium stores. This state of affairs elevates the plasma osmolality, stimulating both thirst and secretion of antidiuretic hormone. The increments in water intake and renal water reabsorption produce water retention, resulting in increases in effective circulating volume and weight. After 3–4 days, a new steady state is achieved in which renal sodium excretion matches intake. The same sequence occurs in reverse if sodium intake is reduced’ .
Not all investigators, however, have corroborated this interpretation. Heer et al.  performed a detailed balance study in which normal men were given sodium intakes ranging from 50 to 550 mmol/day. Heer et al. found that plasma volume dose dependently increased with increasing salt intake and was increased by about 300 ml with the 550 mmol/day sodium intake. However, total body water did not increase and there was no change in body weight, even though total body sodium content was increased by 1700 mmol. The authors concluded from the results of their two independently conducted studies that in contrast to present opinion, the sodium accumulation did not result in commensurate body water storage, but instead induced a relative fluid shift from the interstitial into the intravascular space . These findings were corroborated in a study in which sodium intake was not fixed, but could be monitored in three men for 135 days . The studies suggested that healthy humans may store large amounts of sodium without fluid retention, a hypothesis that was not in line with textbook explanations . A simulated space flight to Mars has recently allowed re-inspection of this theory [15▪▪]. Healthy individuals were confined to an enclosed, restricted environment in two independent studies for 105 and 520 days, which allowed two ultra-long-term Na+ balance studies in humans. Practically all balance studies, except for one , which were used for modelling the kinetics of sodium homeostasis, had studied the body's short-term adaptation to extremes in salt intake . Now, for the first time, the reverse was studied during this simulated trip to Mars, namely sodium homeostasis in response to ultra-long-term constancy in salt intake [15▪▪]. The patients received three fixed levels of sodium intake; 12 g salt/day, 9 g salt/day, and 6 g salt/day. Microgravity was not simulated. Actually, the mock spacemen did not upset the steady-state theory. They ate almost 15 kg of salt during the studies and excreted 90–95% of the dietary salt in their urine. However, the way that this steady state was achieved was startling and has been reviewed in detail previously . When salt intake was fixed for weeks and months, there was considerable day-to-day variability in 24 h Na+ excretion, accompanied with fluctuations of aldosterone, cortisol, and cortisone that peaked with a periodicity of about 1 week. Regular fluctuations of total body Na+ content were also observed, but with a longer periodicity of approximately 1 month. The patients thereby rhythmically accumulated and released body Na+ independent of daily salt intake, presumably regulated by (neuro)-endocrine rhythmical clocks (Fig. 1b). This rhythmical accumulation and release of body sodium was not paralleled by changes in body weight, indicating that sodium was stored in the body. These first long-term balance studies did not support the accepted notion that Na+ retention inevitably leads to volume retention (assumption 1), and that body Na+ therefore is to maintained constant within very narrow limits (assumption 2). In contrast, the rather ‘spooky’ Na+ balance studies initiated by the European Space program suggested that Na+ is stored in the body [13,14,15▪▪]. To transfer these findings into the clinical arena, our next question was where is the salt?
SEEING AND QUANTIFYING SODIUM STORES WITH 23NAMRI
To address this question, we implemented 23NaMRI for quantitative detection of tissue sodium stores in the muscle and the skin. Figure 2a shows an example of tissue sodium content in a patient with primary hyperaldosteronism before and after removal of an aldosterone-producing tumor . High aldosterone levels were paralleled by marked sodium storage in muscle. Surgical adenoma removal reduced tissue stores by 30% in five patients with Conn's syndrome. Similarly, spironolactone treatment reduced sodium stores in a patient with bilateral adrenal hyperplasia (Fig. 2b). The dramatic reduction in muscle sodium content was not paralleled by a reduction in body weight, indicating that the sodium had been stored without commensurate water retention. The reduction in body sodium content and blood pressure in this example of (salt-sensitive) secondary hypertension had taken place without a detectable correction of body water content. Experimental mineralocorticoid receptor activation in deoxycorticosterone acetate (DOCA) salt-treated rats has confirmed these findings, showing that skeletal muscle is a relevant sodium reservoir in this experimental model of secondary hypertension . Muscle sodium storage in the DOCA-salt model is predominately intracellular, with a parallel loss of intracellular potassium . The molecular mechanisms by which mineralocorticoid receptor activation leads to intracellular sodium storage in muscle are unclear. It is also unclear to which extent this sodium accumulation is primarily regulated by muscle uptake, or whether this phenomenon is secondary to reduced renal sodium excretion, and extracellular sodium and volume overload. Experiments in mice with muscle-specific mineralocorticoid receptor deletion may provide more specific and mechanistic insights in the future.
We next analyzed sodium stores in a cross-sectional 23NaMRI study in 113 European Caucasian patients with essential hypertension. We found that sodium storage in muscle and in skin mimics the development of arterial hypertension. In line with the general population characteristics, SBP in the 23NaMRI cohort increased with age in both sexes, and men had lower blood pressure than women. In the same patients, we found that sodium storage in muscle and skin increased with age, was more pronounced in men than in women, and was associated with the elevated SBP [22▪▪]. Despite substantial increases in the sodium content, muscle water remained constant at all ages. Skin sodium storage with age was paralleled by small but detectable increases in skin water content. This state of affairs leads us to the hypothesis that tissue sodium storage characterizes a disruption of internal environment composition, which may be causally linked to primary hypertension. The age-related increase in tissue sodium content we observed is similar to the age-dependent increase in blood pressure and pulse wave velocity reported by Vaitkevicius et al.  20 years ago.
We speculate that sodium storage might represent a previously undetected cardiovascular risk factor. This hypothesis could be tested in prospective studies only if sodium storage was reversible in response to therapeutic intervention. We therefore tested the hypothesis that drug intervention may reduce sodium stores in the body. In line with our initial observation in a patient with hyperaldosteronism , our cross-sectional analysis of tissue sodium content in patients with refractory essential hypertension showed that patients with spironolactone treatment had significantly reduced muscle sodium content [22▪▪]. We were also surprised to find that correction of hypernatremia in a patient with diabetes insipidus was not due to correction of a total body water deficit, but presumably due to correction of secondary hyperaldosteronism and mobilization of massive sodium storage in the muscle . Most recent evidence from a first 23NaMRI study in dialysis patients has shown that 4–5 h of dialysis treatment can reduce sodium and water stored in the muscle and in the skin; however, skin sodium storage can be excessive in dialysis patients, especially with advancing age . The removal of tissue sodium was primarily dependent on the ultrafiltration rate. In younger patients with end-stage renal disease, a single 4–5 h hemodialysis treatment without vigorous ultrafiltration was sufficient to reduce elevated tissue sodium content to the levels observed in age-matched, healthy controls. However, excess skin sodium storage which occurred in older dialysis patients or in patients with an antilymphangiogenic serum profile was difficult to correct with the same hemodialysis treatment regime. Whether sodium stores could be mobilized in these patients with more vigorous ultrafiltration during the treatment session is unclear.
LYMPHATIC CLEARANCE OF SKIN ELECTROLYTES BY HOMEOSTATIC IMMUNE CELLS
Approximately 70–80% of the extracellular fluids are interstitial and thereby not immediately controlled by renal salt and water excretion. We have established the concept that local Na+ homeostasis in the gel-like interstitium cannot be maintained by renal function alone and relies on additional extrarenal regulatory mechanisms. Significant amounts of Na+ are stored in the skin in animals [20,21,25–31,32▪▪,33▪▪] and humans [17,19,22▪▪], and lymph vessels and pro-lymphangiogenic growth factors play a key role in regulating this depot [17,29,30,33▪▪,34▪▪]. Skin sodium is actively concentrated in the keratinocyte/blood vascular counter-current system of the skin ; other fractions of the skin sodium reservoir could be bound in the interstitial gel by electrostatic interaction with the negatively charged extracellular matrix [11,27,28], and significant amounts of sodium can be located in the intracellular space . This unconventional view of the principles of electrolyte homeostasis resulted in elucidation of unexpected regulatory events. Cutaneous lymph capillaries are apparently part of a local ‘kidney-like’ clearance system, in which immune cells act as mobile Na+ sensors and as regulators of lymphatic mobilization of skin sodium stores [29,30,33▪▪]. We have found that macrophages sense Na+ overload in skin sodium stores. We found that the osmoprotective transcription factor tonicity enhancer binding protein (TonEBP, gene name Nfat5) is a master regulator of homeostatic function in cells of the innate immune system. Macrophages enter areas of high Na+ concentrations in the skin interstitium and express TonEBP in the interstitial microenvironment. This salt-driven osmotic stress reaction releases vascular endothelial growth factor C (VEGF-C), which leads to hyperplasia of the cutaneous lymph-capillary system and facilitates sodium and chloride clearance from the skin interstitium. Failure of this macrophage-driven clearance mechanism leads to skin electrolyte overload and increases blood pressure [29,30]. We have more recently shed additional light on the specificity of this regulatory process [33▪▪]. First, genetic deletion of TonEBP in cells of the mononuclear phagocyte system (MPS cells) in LysMcre TonEBPflox/flox mice prevented the lymph capillary response, increased the cutaneous electrolyte content, and resulted in salt-sensitive hypertension. Second, selective pharmacological blockade of the VEGFR3 receptor prevented the MPS-driven modification of cutaneous lymph capillaries, increased skin electrolyte content, and resulted in salt-sensitive hypertension. Third, skin-specific trapping of VEGF-C in K14-FLT4 mice with overexpression of soluble VEGFR3 in the skin resulted in hypoplastic cutaneous lymph capillaries, skin electrolyte accumulation, and increased blood pressure. The findings support the idea that MPS cells exert homeostatic immune function in the skin, where they regulate skin electrolyte clearance via cutaneous lymph capillaries and thereby control SBP.
SALT-DRIVEN TH17 POLARIZATION OF T CELLS
Th17 cells are now considered the most potent effector T cell for inducing tissue inflammation in experimental autoimmune disease models, as well as in humans, and play an important role in the pathogenesis of arterial hypertension . Recent evidence suggests that T cells exposed to high salt concentrations, which are comparable to microenvironment changes in the salt overloaded skin reservoir and its draining lymphatic system, polarize into a pro-inflammatory autoimmune Th17 phenotype, and worsen experimental autoimmune encephalitis [32▪▪,38]. A similar response in blood vessels embedded into the salt-rich interstitium in sodium reservoir tissue could result in vascular inflammation, blood pressure increase, and cardiovascular disease. This hypothesis may provide novel mechanistic insights into how salt storage can increase blood pressure without parallel volume expansion [22▪▪]. First, increasing interstitial sodium and chloride concentrations may result in increased intracellular sodium or calcium levels, or both, and increased vascular tone. A second hypothesis is that chronic interstitial activation of immune cells in the sodium overloaded interstitium may lead to a chronic pro-inflammatory state, which in turn may lead to vascular target organ damage, increased vascular stiffness, and elevated blood pressure levels. A chronic inflammatory response in sodium reservoirs could provide the missing pathophysiologic link between increasing tissue sodium stores, chronic pro-inflammation, and cardiovascular disease, especially in ageing populations .
Conventional teaching maintains that extracellular bodily fluids readily equilibrate, electrolyte concentrations in the various compartments are constant, and the kidney is solely responsible for controlling body Na+ content. Recent counter-intuitive findings in sodium stores question this paradigm. Surprisingly, although the hairpin-like lymphatic and blood capillary structures are well described, physiological studies on electrolyte concentration by counter-currents in skin have never been performed. Such ‘kidney-like’ lymphatic and blood vessel counter-current systems in the skin, intestines, bone, and elsewhere may locally contribute to reabsorption and extrarenal clearance of tissue electrolytes. Novel ideas that the immune system acts a homeostatic regulator of interstitial electrolyte homeostasis and that salt leads to pro-inflammatory immune cell polarization may open an entirely new perspective on immune function that extends immunity's ancient invasion defense to physiological adaptation, interstitial fluid matrix regulation, blood pressure control, and cardiovascular disease in the ageing organism. Novel 23NaMRI methodology has allowed rapid transfer of new research concepts from preclinical animal experiments into the clinical arena.
Financial support and sponsorship
The author is supported by grants from the German Federal Ministry for Economics and Technology/DLR Forschung unter Weltraumbedingungen (50WB0920), the Interdisciplinary Centre for Clinical Research (IZKF Junior Research Group 2), the NIH (RO1 HL118579–01), the AHA (14SFRN20770008), and a Clinical Translational Science Award 1UL-1RR024975 from the National Center for Research Resources.
Conflicts of interest
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
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