Sodium-, potassium-, chloride-, and bicarbonate-related effects on blood pressure and electrolyte homeostasis in deoxycorticosterone acetate-treated rats

Agata Ziomber, Agnes Machnik, Anke Dahlmann, Peter Dietsch, Franz-Xaver Beck, Hubertus Wagner, Karl F. Hilgers, Friedrich C. Luft, Kai-Uwe Eckardt, Jens Titze

Abstract

Na+ loading without Cl fails to increase blood pressure in the DOCA model. We compared the changes in the total body (TB) effective Na+, K+, Cl, and water (TBW) content as well as in intracellular (ICV) or extracellular (ECV) volume in rats receiving DOCA-NaCl, DOCA-NaHCO3, or DOCA-KHCO3. We divided 42 male rats into 5 groups. Group 1 was untreated, group 2 received 1% NaCl, and groups 3, 4, and 5 were treated with DOCA and received 1% NaCl, 1.44% NaHCO3, or 1.7% KHCO3 to drink. We measured mean arterial blood pressure (MAP) directly after 3 wk. Tissue electrolyte and water content was measured by chemical analysis. Compared with control rats, DOCA-NaCl increased MAP while DOCA-NaHCO3 and DOCA-KHCO3 did not. DOCA-NaCl increased TBNa+ 26% but only moderately increased TBW. DOCA-NaHCO3 led to similar TBNa+ excess, while TBW and ICV, but not ECV, were increased more than in DOCA-NaCl rats. DOCA-KHCO3 did not affect TBNa+ or volume. At a given TB(Na++K+) and TBW, MAP in DOCA-NaCl rats was higher than in control, DOCA-NaHCO3, and DOCA-KHCO3 rats, indicating that hypertension in DOCA-NaCl rats was not dependent on TB(Na++K+) and water mass balance. Skin volume retention was hypertonic compared with serum and paralleled hypertension in DOCA-NaCl rats. These rats had higher TB(Na++K+)-to-TBW ratio in accumulated fluid than DOCA-NaHCO3 rats. DOCA-NaCl rats also had increased intracellular Cl concentrations in skeletal muscle. We conclude that excessive cellular electrolyte redistribution and/or intracellular Na+ or Cl accumulation may play an important role in the pathogenesis of salt-sensitive hypertension.

  • DOCA-salt
  • salt-induced hypertension
  • autoregulation
  • osmotically inactive sodium

a close relationship between extracellular volume (ECV) and blood pressure is believed to play a pivotal role in the pathogenesis of hypertension. Guyton et al. (13, 14) suggested that the predominant integral system important for the control of arterial blood pressure is the renal-body fluid pressure control system. They argued that the body fluid integral pressure control system in the long term completely overrides other proportional control systems such as baroreceptor systems, endocrine, or neurally mediated vasoconstriction, by its infinite gain. The kidney therefore acts as a servo-controller of arterial blood pressure by rapidly eliminating excess fluid and Na+ to prevent ECV increases that could increase blood pressure. When renal function is reduced and the ECV increases, elevated blood pressure promotes a pressure natriuresis that corrects the rate of body fluid volume change toward zero. Hence, ECV homeostasis is maintained at the expense of hypertension. This overriding hypothesis on the pathogenesis of hypertension, particularly salt-sensitive hypertension, links a causal interrelationship between Na+ and fluid balance, the kidney, and blood pressure. The concept receives support from the fact that many genetically determined (22) and acquired (7, 12, 32) forms of hypertension “go with the kidneys” when the organs are transplanted. In the systems analysis approach, the factor controlled with infinite gain is the rate of change in body fluid volume. The concept relies on an “already established relationship between body Na+ and body fluid volume” (13). This relationship between Na+ and body fluid volume is based on a two-compartment model in which ≈95% of osmotically active cations that hold water to maintain isosmolality between the extra- and the intracellular space are Na+ or K+ (31, 33, 41). These cations serve as “effective osmolytes” since they determine shifts in water (tonicity changes) from one compartment to another, as opposed to noneffective osmolytes such as urea that engender no volume shifts.

Mineralocorticoid excess models further supported the renal-body fluid blood pressure concept, since positive Na+ balance and therefore positive water balance goes along with a blood pressure increase, which promotes a pressure natriuresis. Thus a new steady state in Na+ and water balance is achieved. This mineralocorticoid action escape is at the expense of elevated blood pressure (15), returning the rate of change of body volume precisely to zero. However, recent experimental evidence in deoxycorticosterone acetate (DOCA)-treated rats suggests that changes in the relationship between body electrolytes and body fluid may have a stronger effect on the generation of blood pressure increases than changes in body volume. O'Donaughy et al. (27, 28) have suggested that hypertonicity of the accumulated fluid may lead to sustained sympathoexcitation and hypertension, driven by increased NaCl levels relative to water. However, whether an increased Na+ concentration solely determines the instantaneous blood pressure level or actually impacts long-term blood pressure regulation is unclear.

We previously performed body composition analysis of Na+, K+, and water in DOCA-salt rats, suggesting that large amounts of the body Na+ escaped isosmolality apart from the serum Na+ concentration (37, 39). Our data supported the notion that local fluid tonicity per se may play an important role in long-term blood pressure regulation. Hypertension in the DOCA-salt model coincides with both increased total body water and an increased total body (Na++K+)-to-water relationship. Thus discriminating between an increase in absolute body Na+ and total body water (external Na+ and water balance) or an increase in the total body (Na++K+)-to-total body water relationship (internal Na+, K+, and water balance) as a cause for hypertension has not been possible. Selective Na+ loading without a concomitant Cl increase fails to induce hypertension (1921). We hypothesized that changes in internal (Na++K+)-to-water relationship are a stronger predictor of blood pressure increases than the absolute Na+ and water content of the body. To test this hypothesis, we investigated the changes in Na+, K+, and water content and blood pressure in DOCA-treated rats with high-NaCl, -NaHCO3, or -KHCO3 diets. We included Cl measurements in these studies and used them to estimate the ECV.

METHODS

Animal experiments.

We used male Sprague-Dawley rats as outlined elsewhere (3739). The rats were fed a <0.1% NaCl diet and received tap water (control) or salt (1% NaCl water ad libitum) or were DOCA-salt treated (1% NaCl water ad libitum), DOCA-NaHCO3 treated (1.44% NaHCO3 water ad libitum), or DOCA-KHCO3 treated (1.7% KHCO3 water ad libitum). The rats were not uninephrectomized. Rats were randomly assigned to five groups: 1) control (n = 10; 292.7 ± 13.0 g); 2) salt (n = 11; 295.8 ± 19.6 g); 3) DOCA-NaCl (n = 12; 308.0 ± 23.0 g); 4) DOCA-NaHCO3 (n = 11; 311.4 ± 21.3 g); 5) DOCA-KHCO3 (n = 9; 309.9 ± 18.1 g). Body weights were not different among the groups. At the end of the experiment, the rats were anesthetized with 1.5–2% isoflurane anesthesia and the right femoral artery was catheterized for direct mean arterial blood pressure (MAP) determinations (37, 39). Thereafter, blood samples were taken before the animals were killed. We analyzed arterial blood gases with a clinical blood gas analyzer (Radiometer, Copenhagen, Denmark), including Na+, K+, and Cl measurements by ion-selective electrodes. Total serum Na+ and K+ concentrations were measured by a flame photometer (model EFIX, Eppendorf, Hamburg, Germany). All animal experiments were done in accordance with the guidelines of the American Physiological Society and were approved by the animal care and use committee of local government authorities (AZ 54-2531.31-5/07; Regierung von Mittelfranken, Ansbach, Germany).

Ashing procedure.

The skins, carcasses, and muscles were weighed [wet weight (WW)] and then desiccated at 90°C for 72 h [dry weight (DW)] (3739). Because weights were unchanged with further drying, the difference between WW and DW was considered as tissue water content. The tissues were then ashed at 190° and 450°C for 24 h at each temperature level, and the bones were sieved from the carcass ashes. The separated tissues were further ashed at 600°C for 48 h and then dissolved in 5% or 10% HNO3. Na+ and K+ concentrations were measured by atomic adsorption spectrometry (model 3100, Perkin Elmer, Rodgau, Germany). Cl concentration ([Cl]) in the ashes was measured by titration with 0.1 N silver nitrate (model Titrando, German Metrohm, Filderstadt, Germany). Bone mineral Cl contents were not included for total body Cl space calculations. Cl space as a measure of the ECV (6, 25) was then calculated from the tissue Cl content and the serum [Cl]: Math(1)

Electron microprobe analysis.

Tissues from skeletal muscle were shock frozen in an isopentane-propane mixture (1:4) at −196°C. One-micrometer sections were cut at −90°C and freeze-dried. Element concentrations were determined with a scanning transmission electron microscope fitted to an X-ray detector system as described previously (2).

Data analysis.

Data are expressed as averages ±± SD. Data from the various tissue Na++, K++, and water contents were analyzed by multivariate analysis [general linear model (GLM)]. The amount of osmotically active, osmotically inactive, and osmotically neutral Na++ accumulation was investigated from the relationship between changes of Na++ content and alterations of water content in total body, skin, bone, and rest carcass. We used SPSS software for statistical analysis (version 12.0). A detailed list of abbreviations used is given in the supplemental data.1

RESULTS

Both DOCA-NaCl and DOCA-NaHCO3 treatment caused hypochloremic metabolic alkalosis and hypokalemia (Table 1). Serum Na+ concentrations did not differ between the groups. Compared with the control rats, DOCA-NaCl rats had an increased anion gap. The anion gap was not significantly different between DOCA-NaCl and DOCA-NaHCO3 rats. DOCA-KHCO3-treated rats were investigated to control for the effect of HCO3 supplementation in the absence of dietary Na+. DOCA-KHCO3 treatment led to mild hypokalemia and less pronounced hypochloremic metabolic alkalosis compared with DOCA treatment in the presence of Na+. Compared with the control rats, DOCA-NaHCO3 decreased DW at the total body level, in skinned and bone-removed rest carcass, skin, and bone (Table 2). To adjust for the differences in body weights across the groups, we calculated the “relative” total Na+ and K+ content in the total body (rTBNa+ and rTBK+; mmol/g DW), in the skinned and bone-removed rest carcass (rCarNa+ and rCarK+; mmol/g DW), in the skin (rSKNa+ and rSKK+; mmol/g DW), and in the bone (rBoneNa+ and rBoneK+; mmol/g bone ash) in all rats (Fig. 1). Compared with the control rats, rTBNa+ was increased by ≈30% in DOCA-NaCl and DOCA-NaHCO3 rats, but no significant changes in rTBK+ were found (Fig. 1A). A similar change pattern was found in the rest carcasses (Fig. 1B) and in the skin (Fig. 1C). In the bone, DOCA-NaCl or DOCA-NaHCO3 treatment increased the Na+ content relative to bone ash by ≈12–13%. This Na+ retention went along with a corresponding bone K+ loss, indicating osmotically neutral Na+/K+ exchange. NaCl without DOCA increased skin Na+ content slightly, which was not detected at the total body level. DOCA-KHCO3 treatment did not lead to body Na+ retention but increased the skin K+ content.

Fig. 1.

Na+ and K+ content in control rats fed low-salt and tap water, given 1% saline to drink (NaCl), and in deoxycorticosterone acetate (DOCA)-NaCl-, DOCA-NaHCO3-, and DOCA-KHCO3-treated rats in total body [rTBNa+ and rTBK+, mmol/g dry weight (DW); A], in completely skinned and bone-removed rest carcasses (rCarNa+ and rCarK+, mmol/g DW; B), in skin (rSKNa+ and rSKK+, mmol/g DW; C), and in bone (rBoneNa+ and rBoneK+, mmol/g ash; D). DOCA-NaCl and DOCA-NaHCO3 treatment significantly increased rTBNa+ by ≈30%, while no significant changes in rTBK+ were found. No significant Na+ retention in DOCA-KHCO3 rats was found. *P < 0.05 vs. control; †P < 0.05 vs. DOCA-KHCO3.

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Table 1.

Arterial blood pH and electrolyte and HCO3 concentrations in control and DOCA-NaCl-, DOCA-NaHCO3-, and DOCA-KHCO3-treated rats

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Table 2.

Absolute wet weight and dry weight, electrolyte content, and water content in various tissues of control, NaCl-treated, DOCA-NaCl-treated, DOCA-NaHCO3-treated, and DOCA-KHCO3-treated rats

Na+ retention in DOCA-NaCl- and DOCA-NaHCO3-treated rats was paralleled by water accumulation at the total body level in the completely skinned and bone-removed rest carcasses and in skin (Fig. 2). However, despite similar Na+ retention, fluid accumulation was more pronounced in DOCA-NaHCO3 rats compared with DOCA-NaCl rats at the total body level and in the rest carcasses (Fig. 2A), but not in the skin (Fig. 3C). Compared with the control rats, NaCl alone and DOCA-KHCO3 treatment did not significantly increase body water retention.

Fig. 2.

A: water content in control rats fed low-salt and tap water, given 1% saline to drink (NaCl), and in DOCA-NaCl-, DOCA-NaHCO3-, and DOCA-KHCO3-treated rats in total body [rTBW; ml/g wet weight (WW)] and in completely skinned and bone-removed rest carcasses (rCarW; ml/g WW). B: skin water content (SKW; ml/g WW) and mean arterial blood pressure (MAP; mmHg). DOCA-NaCl treatment increased rTBW (≈3%) and rCarW (≈2.5%), and DOCA-NaHCO3 increased rTBW (≈4.5%) and rCarW (≈3.8%) further. DOCA-salt treatment led to salt-sensitive hypertension, which coincided with Na+ (Fig. 1) and volume retention. DOCA-NaHCO3 treatment did not increase blood pressure from control levels despite similar Na+ retention and even more pronounced water retention; DOCA-KHCO3 did not increase blood pressure in the absence of significant Na+ and water retention compared with control rats. *P < 0.05 vs. control; †P < 0.05 vs. DOCA-NaHCO3; ‡P < 0.05 vs. DOCA-KHCO3.

Fig. 3.

Relationship between total body (Na++K+) accumulation [rTB(Na++K+), mmol/g DW] and total body water retention (rTBW, ml/g DW) (A), body Cl space [rTBClSp, as an estimate for extracellular volume (ECV) retention, ml/g DW; B], and body Cl-free water space [rTBW-ClSp, as an estimate for intracellular volume (ICV) retention, ml/g DW; C]. Increasing rTB(Na++K+) was paralleled by increasing rTBW and rTB(W-ClSp), while the relationship between rTB(Na++K+) and rTBClSp was blunted. Group-specific scatter plots from A are presented in Fig. 4.

Cl space measurements for ECV and tissue water − Cl space (W − ClSp) measurements for intracellular volume (ICV) estimation in DOCA-treated rats compared with control rats are shown in Table 3. Compared with the control rats, DOCA-NaCl and DOCA-NaHCO3 treatment did not increase the total body Cl content despite massive Na+ retention (Fig. 1), resulting in increased Na+-to-Cl ratio. In the completely skinned and bone-removed rest carcasses, rCarW − rCarClSp expansion was found in DOCA-NaCl rats, which was increased even further in DOCA-NaHCO3-treated rats, suggesting ICV expansion. No rCarClSp expansion was found in DOCA-NaCl- or DOCA-NaHCO3-treated rats. In contrast, both DOCA-NaCl and DOCA-NaHCO3 treatments led to increased rSKClSp, suggesting ECV expansion, while rSKW − rSKClSp was unchanged. DOCA-KHCO3 treatment slightly increased rSKW − rSKClSp in the skinned and bone-removed rest carcass and ECV in skin. Fluid intake, and thereby Na+ intake, was higher in DOCA-NaCl rats than in DOCA-NaHCO3 rats (Table 3).

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Table 3.

Tissue Cl content and its derived ECV and ICV calculations and water and electrolyte intake

DOCA-NaCl treatment led to ≈10 mmol/kg WW intracellular Cl accumulation, while DOCA-NaHCO3 treatment did not (Table 4). Both DOCA-NaCl and DOCA-NaHCO3 rats increased intracellular muscle Na+ by 30–40 mmol/kg WW and decreased intracellular muscle K+ content, indicating osmotically neutral Na+/K+ exchange. However, muscle K+ loss was more pronounced in DOCA-NaHCO3 rats, and thereby their muscle (Na++K+) content was lower, compared with DOCA-NaCl rats. These cellular changes in K+ content were not detected at the total body electrolyte content level (Fig. 1); however, similar changes were visible when investigating the relationship between Na+, K+, and water accumulation in the body (Table 5). At the total body level, Na+ accumulation in DOCA-NaCl rats was 0.035 mmol/g DW and was paralleled with 0.189 ml/g DW water retention, resulting in a (Na++K+) concentration in the accumulated fluid of 0.185 mmol/ml. Therefore, the (Na++K+) concentration in the accumulated fluid exceeded the serum (Na++K+) concentration, indicating that ≈30% of the (Na++K+) accumulated with DOCA-NaCl treatment escaped isosmolality. Similarly, ≈30% of the (Na++K+) accumulated in the completely skinned and bone-removed rest carcasses escaped isosmolality, and ≈50% of the (Na++K+) accumulated in the skin was stored as osmotically inactive in DOCA-NaCl rats. In contrast, total body (Na++K+) and water accumulation in DOCA-NaHCO3 rats was isosmolal to their serum (Na++K+) concentration (Table 1), although their osmotically inactive skin Na+ storage capacity was maintained and both rSKClSp and rSKW − rSKClSp volumes were not different compared with DOCA-NaCl rats (Table 3). In contrast to DOCA-NaCl rats, the Δ(Na++K+)-to-Δwater ratio in DOCA-NaHCO3 rats indicated that the fluid accumulated in the completely skinned and bone-removed rest carcasses was hypotonic compared with the serum (Na++K+) concentration (Table 1).

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Table 4.

Muscle electrolyte and water content in control and DOCA-NaCl-, DOCA-NaHCO3-, and DOCA-KHCO3-treated rats

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Table 5.

(Na++K+) and water content in control and DOCA-NaCl- and DOCA-NaHCO3-treated rats

We next investigated the relationship between increasing (Na++K+) content and water content (Fig. 3). Body (Na++K+) accumulation increased body water content in the rats (Fig. 3A); however, the relationship between (Na++K+) and water accumulation in the rats was group specific. First, only control and DOCA-NaCl rats showed the expected close direct relationship between body (Na++K+) and water accumulation, while this relationship was lost in DOCA-NaHCO3 rats and blunted in DOCA-KHCO3 rats (Fig. 4 and Table 5). Second, the slope between increasing (Na++K+) and water content was steep in control rats and right-shifted in DOCA-NaCl treated rats, indicating augmented osmotically inactive Na+ storage with DOCA-NaCl treatment. The relationship between changes in body (Na++K+) content and rTBClSp and rTB(W − rClSp) volumes as estimates for ECV or ICV changes is given in Fig. 3, B and C. No significant direct relationship between increasing body (Na++K+) and rTBClSp was found in the rats (Fig. 3B, Table 5), while a robust direct relationship between increasing body (Na++K+) and rTBW − rTBClSp was found in control and DOCA-NaCl rats. This finding suggests that increasing body (Na++K+) led to substantial intracellular electrolyte and volume accumulation.

Fig. 4.

Relationship between rTB(Na++K+) and rTBW in control rats with low salt and in DOCA-NaCl rats (A), DOCA-NaHCO3 rats (B), and DOCA-KHCO3 rats (C). The close relationship between “effective osmolyte” retention and body fluid accumulation was blunted in DOCA-NaHCO3 and DOCA-KHCO3 rats.

To investigate the relationship between tissue (Na++K+) retention in excess of water and changes in fluid compartments in an extracellular tissue, we next analyzed (Na++K+)-to-W ratio and ClSp or (W − rClSp) volumes in the skin. Analyzing all rats, increases in SK(Na++K+)/SKW were paralleled by increases in rSKClSp (Fig. 5A, Table 6), suggesting that ECV retention was paralleled by augmented Na+ retention relative to water. However, this effect was restricted to DOCA-NaCl rats (Fig. 5B), while the relationship between SK(Na++K+)/SKW and rSKClSp was blunted in the other groups. In contrast, the relationship between SK(Na++K+)/SKW and rSK(W − ClSp) was negative in the rats (Fig. 5C, Table 6), suggesting that accumulation of Na+ in excess over water was paralleled by cellular shrinkage in the skin. We conclude that increases in SK(Na++K+)/SKW were not exclusively “osmotically inactive,” but osmotic stress was a critical feature of increasing Na+ content in the skin. This inverse correlation was not only restricted to DOCA-NaCl rats but also occurred in control and DOCA-NaHCO3 rats (Fig. 5D).

Fig. 5.

Relationship between skin (Na++K+) in abundance over water [SK(Na++K+)/SKW, mmol/ml] and the skin Cl space volume (rSKClSp, ml/g DW) as an estimate of ECV retention (A and B) and relationship between SK(Na++K+)/SKW and Cl-free water space volume [rSK(W-ClSp), ml/g DW] as an estimate of ICV changes (C and D). ECV retention in the skin was paralleled by increasing SK(Na++K+)/SKW, suggesting that ECV retention was characterized by Na+ accumulation in excess over water, especially in DOCA-NaCl rats (B). In parallel, skin Na+ accumulation in excess over water was paralleled by reduced rSK(W-ClSp) in the skin (C), indicating cell shrinkage with accumulation of Na+ in excess over water. This effect was present in control, DOCA-NaCl, and DOCA-NaHCO3 rats (D).

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Table 6.

Relationship between electrolyte content, body fluid volumes, and blood pressure

Finally, we investigated the relationship between body water, volume retention, and MAP. We found no relationship between body (Na++K+) content and MAP (Fig. 6A; Table 6). The variability in rTB(Na++K+) allowed us to investigate MAP at similar rTB(Na++K+) levels in all four treatment groups. At a given rTB(Na++K+), MAP was higher in DOCA-NaCl than in control, DOCA-NaHCO3, and DOCA-KHCO3 rats, indicating that hypertension in DOCA-salt rats was not directly linked with (Na++K+) mass balance changes. Because (Na++K+) accounts for >95% of the effective osmolytes, similar results were seen when investigating the relationship between body water content and blood pressure (Fig. 6B). At a given rTBW, MAP was higher in DOCA-NaCl rats than in other treatment groups or control rats. However, increases in rSKClSp were paralleled by MAP increases in DOCA-NaCl but not DOCA-NaHCO3 or DOCA-KHCO3 rats (Fig. 6C; Table 6). Given that fluid volumes accumulated in the skin were hypertonic compared with serum (Na++K+) concentrations in DOCA-NaCl and DOCA-NaHCO3 rats (Tables 1 and 5), increases in skin (Na++K+) content were not only paralleled by increases in rSKClSp volume but also were accompanied by decreases in rSK(W − ClSp) volume (Fig. 5C), suggesting osmotic stress in the skin. The corresponding decreases in rSK(W − ClSp) volume were paralleled by increasing blood pressure in both DOCA-NaCl and DOCA-NaHCO3 rats (Fig. 6D; Table 6).

Fig. 6.

A: relationship between total body rTB(Na++K+) and MAP. No significant correlation was found. Furthermore, at a given rTB(Na++K+), MAP was higher in DOCA-NaCl rats than in individual rats from other treatment groups. This finding suggests that MAP increases were not dependent on the absolute body Na+ content. B: relationship between rTBW and MAP. No significant correlation was found. Furthermore, at a given rTBW, MAP was higher in DOCA-NaCl rats than in individual rats from other treatment groups. This finding suggests that MAP increases were not dependent on the absolute body volume content. C: relationship between skin Cl space volume retention (rSKClSp, as an estimate of ECV retention, ml/g DW) and MAP. A positive relationship between skin rSKClSp volume retention and MAP was only found in DOCA-NaCl rats, suggesting that ECV retention was paralleled by hypertension in DOCA-NaCl rats. D: relationship between skin Cl-free volume retention (rSKW-rSKClSp, as an estimate of changes in ICV, ml/g DW) and MAP. An inverse relationship between skin Cl-free volume retention and MAP was found in both DOCA-NaCl and DOCA-NaHCO3 rats.

DISCUSSION

Our chemical analysis of DOCA-NaCl hypertension provides some novel findings. First, different MAP levels occurred at similar Na+ contents, suggesting that blood pressure increases, especially in DOCA-NaCl rats, were not predictable by mass balance changes in body Na+ content and corresponding volume changes. Second, we found skin Na+ retention in excess over water without parallel K+ losses, indicating interstitial hypertonicity. Increased MAP paralleled hypertonicity in both DOCA-NaCl- and DOCA-NaHCO3-treated groups. Third, electrolyte redistribution, namely, increased intracellular Na+ and Cl contents, were found, which may play an additional relevant role in DOCA-NaCl hypertension. These three findings are not in line with the widely accepted view that Na+ retention almost exclusively takes place in the ECV and inevitably is paralleled by volume retention leading to hypertension. We reiterate these findings point by point.

Blood pressure was not primarily dependent on (Na++K+) mass balance and therefore not dependent on volume-mass balance changes.

This statement is evidenced not only by the finding that average Na+ retention was similar in DOCA-NaCl and DOCA-NaHCO3 rats, while MAP levels were different (Figs. 1 and 2), but also by the fact that at a given rTB(Na++K+) of 0.35–0.40 mmol/g DW MAP was higher in DOCA-NaCl rats than in control, DOCA-NaHCO3, and DOCA-KHCO3 rats (Fig. 6A). Because (Na++K+) are believed to account for >95% of the body's effective osmolytes, we were not surprised to find that at a given TBW content of 0.67–0.69 ml/g WW, MAP was highest in DOCA-NaCl rats and lower in control, DOCA-NaHCO3, and DOCA-KHCO3 rats (Fig. 6B). We conclude that the subsequent mass changes in body (Na++K+) and body water content that accompany DOCA-induced Na+ retention with high salt are not an isolated causal event for the development of DOCA salt-sensitive hypertension.

Skin Na+ retention in excess over water was paralleled by interstitial hypertonicity and blood pressure increases.

The finding that the (Na++K+) concentration of the fluid accumulated in skin in both DOCA-NaCl and DOCA-NaHCO3 rats was remarkably higher than the serum (Na++K+) concentration in the same rats (Table 1 and Table 5) underscores this statement. Previous experiments support the notion of “osmotically inactive Na+ storage” (35, 3740), “a situation in which it would be exchangeable but would not exert the anticipated osmotic effect for sodium in these biological fluids,” as suggested by Farber et al. (810). This wording was based on the idea that plasma and interstitial fluid should be in equilibrium according to Gibbs-Donnan equilibrium (see Ref. 31), even suggesting lower Na+ concentrations in the interstitium than in plasma because of higher plasma albumin concentrations. The skin is the largest organ of the body. The predominant extracellular, and hence interstitial, compartment of this organ warrants careful consideration. First, the skin contains large amounts of interstitial glycosaminoglycans (GAGs), which are the predominant polyanions that attract Na+ and repel Cl by their negative charge density. We showed previously (35, 40) that skin Na+ retention is paralleled by increases in the negative GAG charge density. GAGs may be viewed as negatively charged interstitial condensers. Electrolyte redistribution around GAGs hence might include local hypertonicity, as evidenced in cartilage interstitium (23). Second, the anatomic organization of subcutaneous blood capillary very much resembles the countercurrent organization of blood vessels in the renal medulla, which also could contribute to the generation of hypertonicity in the skin interstitium compared with blood. Hence osmotic stress may be a critical event of interstitial Na+ accumulation. In contrast to our data, the inulin dilution studies by Passmore et al. (30) and Zicha and Kunes (43) showed that DOCA-treated rats had more pronounced ECV expansion with dietary NaCl than with dietary Na+ loading without Cl, while we found no differences in the skin Cl space between DOCA-NaCl and DOCA-NaHCO3 rats. However, the accumulation of Na+ in the skin included skin volume accumulation, with (Na++K+)/water concentrations that were significantly higher than in serum (Table 1, Table 5). In parallel, we found an increase in the skin Cl space, suggesting ECV retention (Table 3, Fig. 5, A and B) of hypertonic fluid compared with serum. Our concept receives additional support from the finding that the accumulation of fluid hypertonic to serum into skin was paralleled by a reduction in the skin Cl-free space. This finding suggests cell shrinkage in response to interstitial hypertonicity (Fig. 5, C and D), suggesting that osmotic stress might be a critical feature of interstitial Na+ accumulation. However, the accuracy of Cl space measurements of ECV may be limited, especially when acid-base disturbances are present. In metabolic alkalosis, cellular Cl/HCO3 exchange counteracts intracellular alkalosis (5). For instance, intracellular Cl content in skeletal muscle was increased in alkalotic DOCA-NaCl rats. Possibly, intracellular Cl escape resulted in overestimation of ECV and underestimation of ICV measurements with the Cl space method. Overestimation of ECV and parallel underestimation of ICV by Cl space measurements is relevant to our DOCA-NaCl treated rats, where muscle Cl content doubled from 12 mmol/kg WW to 24 mmol/kg WW (Table 4). Cell shrinkage in DOCA-NaCl rats hence may have been overestimated, while ECV expansion might have been underestimated. In contrast, despite similar extracellular alkaloses, we did not find relevant intracellular Cl escape in control, DOCA-NaHCO3, and DOCA-KHCO3 rats (Table 4). This result suggests that Cl space measurements may provide a more accurate estimation of ECV and ICV in these groups, compared with DOCA-NaCl rats. The finding that increasing (Na++K+)-to-water ratios were paralleled by decreasing ICV supports the notion that interstitial Na+ accumulation without commensurate water retention resulted in osmotic stress in the skin (Fig. 5D). This finding is in line with vapor pressure osmometry measurements in lymphatic tissues providing evidence that hypertonicity is a critical feature of the lymphatic, and therefore interstitial, microenvironment (11). Our data also suggest that interstitial osmotic stress might be a critical feature of blood pressure regulation, because the accumulation of hypertonic fluid in the skin went along with increases in the skin Cl space that were paralleled by MAP increases in DOCA-NaCl rats (Fig. 6C). However, reduction in water-free skin Cl space volumes, as a surrogate marker for osmotic stress with interstitial Na+ accumulation, were paralleled by MAP increases in control, DOCA-NaCl, and DOCA-NaHCO3 rats (Fig. 6D). This finding suggests that not only the absolute volume accumulated but also the tonicity of the fluid accumulated might play a decisive role in blood pressure regulation. The mechanisms involved remain unclear; however, the recent data from Oberleithner et al. (29) suggest that increasing extracellular Na+ concentrations led to vascular endothelial stiffening and reduced nitric oxide release. Similarly, hypertonic interstitial Na+ overload in the skin, which harbors most of the body's resistance vessels, may lead to increased peripheral resistance, especially in DOCA-NaCl rats. These dramatic changes in interstitial Na+ and water distribution occurred without any changes in the serum Na+ concentration (Table 1). Similar change patterns in humans would therefore inevitably escape the physician's clinical notice. Our data are therefore not in accord with the currently favored two-compartment model of interstitial electrolyte and body fluid homeostasis (31).

Electrolyte redistribution, namely, increased intracellular Na+ and Cl contents, paralleled DOCA-NaCl hypertension.

Increasing evidence suggests that Na+/K+ redistribution in smooth muscle cells plays a pivotal role in mediating vascular smooth muscle contractility and smooth muscle differentiation via Ca2+-dependent (4, 16, 17) and Ca2+-independent (36, 42) pathways, thereby translating body Na+ retention and ECV expansion into salt-sensitive hypertension by increases in intracellular Na+ content. However, if ECV expansion and/or elevated serum Na+ concentrations with subsequent increases in intracellular Na+ content invariably lead to vascular smooth muscle cell contraction, we would have expected MAP increases in both DOCA-NaCl- and DOCA-NaHCO3-treated rats, because Na+ concentrations in skeletal muscle were not different between these groups. In line with our data, Motoyota et al. (24) compared the effect of a 7% NaCl diet and an equimolar Na+ but Cl-free diet in DOCA-treated rats. They found increased intracellular Na+ contents in muscle and aorta with DOCA treatment. The authors additionally showed further increased intracellular Na+ concentrations in the high-Na+/low-Cl group compared with the NaCl group. However, in the absence of simultaneous K+ measurements, changes in intracellular tonicity could not be estimated in their study. In addition to these data, our chemical analysis of intracellular electrolyte contents corroborates the notion that osmotically inactive Na+ storage and/or local hypertonicity with DOCA-NaCl was not restricted to the skin but may have also occurred inside the muscle cell (Table 4). As shown previously, muscle Na+ retention with DOCA-NaCl is not paralleled by muscle water retention (18, 37). Intracellular muscle water content was ≈0.75 × WW. The estimated intracellular (Na++K+) concentration was (47+93) mmol/kg WW/0.75 l/kg = 185 mmol/l in DOCA-NaCl rats and only (39+85) mmol/kg WW/0.75 l/kg = 165 mmol/l in DOCA-NaHCO3 rats (Table 3). Therefore, our chemical ashing data (Tables 2 and 4) at the tissue level and our electron microprobe analysis (Table 3) both indicate that the internal environment of cells was different between the groups, suggesting either hypertonic electrolyte accumulation or osmotically inactive Na+ storage inside the cells with DOCA-NaCl treatment but not with DOCA-NaHCO3 treatment. Whether acute experimental induction of extracellular hypertonicity (2628) causes an intracellular electrolyte and volume challenge similar to that in our DOCA-NaCl rats, or instead leads to acute water efflux out of the cell and concomitant volume shifts in favor of the ECV, remains to be investigated. Finally, the differences in intracellular skeletal muscle Cl content between DOCA-NaCl and DOCA-NaHCO3 rats did not escape our notice (Table 4). Compared with all other groups, the hypertensive DOCA-NaCl group was the only group with an increased intracellular Cl content in muscle (Table 3). Increased vascular smooth muscle Cl content was found in mice with mutations of the K+-Cl cotransporter and was paralleled by increased myogenic tone and hypertension (34). Similarly, increased contractility of vascular smooth muscle cells has been associated with increased intracellular Cl content (1). Whether or not similar increases in intracellular vascular smooth muscle Cl content in vascular smooth muscle cells are present in DOCA-NaCl rats as well remains to be investigated.

In summary, our data suggest that the redistribution of body electrolytes and water may play a pivotal role in the pathogenesis of hypertension in the DOCA-salt model. Local hypertonic fluid accumulation compared with serum could lead to osmotic stress as well as redistribution of body electrolytes. Such effects could play an important role in the development of hypertension. Na+-induced extra- and intracellular hypertonicity, as well as intracellular Cl entry, might be of particular relevance for the development of hypertension in DOCA-NaCl rats. In contrast to the currently favored two-compartment model of interstitial electrolyte and body fluid homeostasis (31), we suggest that not only the absolute amount of ECV or ICV but also the relationship between interstitial electrolyte and volume content, namely fluid tonicity, and/or the redistribution of Cl may be a critical component of interstitial volume and blood pressure homeostasis in the DOCA-salt model. This different view on basic regulatory concepts of the “milieu interieur” (3) may open new research avenues for understanding cellular function in the interstitial space and its impact on blood pressure regulation.

GRANTS

This study was supported by grants to J. Titze from the Interdisziplinäres Zentrum für klinische Forschung (IZKF) Erlangen, the Bundesministerium für Bildung und Forschung-Forschung unter Weltraumbedingungen (DLR/BMBF), and a Fresenius Nephro-Core Stipend to A. Ziomber.

Acknowledgments

We thank Elke Prell and Jennifer Goss for their technical assistance.

Footnotes

  • 1 The online version of this article contains supplemental material.

  • The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

REFERENCES

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