Mobilization of osmotically inactive Na by growth and by dietary salt restrict - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-22 09:40 |只看该作者 |倒序浏览 |打印

作者：Markus Schafflhuber, Nicola Volpi, Anke Dahlmann, Karl F. Hilgers, Francesca Maccari, Peter Dietsch, Hubertus Wagner, Friedrich C. Luft, Kai-Uwe Eckardt, and Jens Titze作者单位：1 Department of Nephrology and Hypertension, Friedrich-Alexander-University, Erlangen-Nürnberg; 2 Department of Animal Biology, University of Modena and Reggio Emilia, Modena and Reggio Emilia, Italy; 3 Institute of Biochemistry, Charité Campus Benjamin Franklin, Berlin; 4 Federal Resear ; ^, I8 f4 }! H9 A/ s



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      【摘要】
   The idea that an osmotically inactive Na storage pool exists that can be varied to accommodate states of Na retention and/or Na loss is controversial. We speculated that considerable amounts of osmotically inactive Na are lost with growth and that additional dietary salt excess or salt deficit alters the polyanionic character of extracellular glycosaminoglycans in osmotically inactive Na reservoirs. Six-week-old Sprague-Dawley rats were fed low-salt (0.1%; LS) or high-salt (8%; HS) diets for 1 or 4 wk. At their death, we separated the tissues and determined their Na  , K  , and water content. Three weeks of growth reduced the total body Na content relative to dry weight (rTBNa ) by 23%. This "growth-programmed" Na loss originated from the bone and the completely skinned and bone-removed carcasses. The Na loss was osmotically inactive (45-50%) or osmotically active (50-55%). In rats aged 10 wk, compared with HS, 4 wk of LS reduced rTBNa by 9%. This dietary-induced Na loss was osmotically inactive ( 50%) and originated largely from the skin, while 50% was osmotically active. LS for 1 wk did not reduce skin Na content. The mobilization of osmotically inactive skin Na with long-term salt deprivation was associated with decreased negatively charged skin glycosaminoglycan content and thereby a decreased water-free Na binding capacity in the extracellular matrix. Our data not only serve to explain discrepant results in salt balance studies but also show that glycosaminoglycans may provide an actively regulated interstitial cation exchange mechanism that participates in volume and blood pressure homeostasis. 9 f+ p/ I( f* S0 W; K" X; m3 Y
      【关键词】 extrarenal sodium balance hypertension volume sodium reservoirs glycosaminoglycans& P& B4 N8 j7 T
   OSMOTICALLY INACTIVE Na reservoirs undoubtedly exist. However, earlier ( 10, 13 ) and recent data from human ( 12, 17, 26 ) and animal studies (22-24, 27) suggest that osmotically inactive Na storage pools may be variable during states of Na retention or Na loss. Thus the contribution of osmotically inactive Na reservoirs to extracellular volume and blood pressure control may be variable under differing circumstance. The currently favored hypothesis of Na or K retention and/or loss is based on a two-compartment model, where 90-95% of osmotically active cations that act to hold water in the extracellular or intracellular space are Na or K ( 19, 28 ). Accordingly, Na is restricted mainly to the extracellular fluid and K to the intracellular space. The energy-requiring Na -K -ATPase maintains the disequilibrium across the cellular membrane. Na concentrations are not equal in the serum and the interstitial fluid due to large, negatively charged proteins in the intravascular space that attract cations and repel anions according to Gibbs-Donnan forces ( 18 ).

We argue that Na and K homeostasis is more complex and have suggested a more open system with respect to Na and K handling. In cartilage, the negative charge density of glycosaminoglycans (GAGs) may increase the local interstitial Na concentration to 450 mmol/l ( 14 ). This interstitial concentration far exceeds the serum Na concentration. Interstitial matrix GAGs and their negative charge density hence provide attractive molecules to test the hypothesis of whether changes in the polyanionic character of our internal environment are important in osmotically inactive Na storage ( 5, 6, 27 ). Conceivably, isotope dilution studies ( 2, 3, 8, 9 ) could shed light on these issues. However, these gold-standard techniques are limited. Forbes and Lewis ( 7 ) pointed out that dilution techniques may well underestimate true total body content for Na  , Cl -, and K  . They concluded that "the discrepancy is greatest in the case of Na  ." Na reservoir tissues could work slowly by changing their polyanionic character and could also be affected by growth. Equilibration between the osmotically active and inactive Na pool could occur with changes in body composition and thereby take place slowly. As milestone isotope dilution studies on the relationship among the exchangeable body Na  , K  , water, and the serum Na concentration were performed with equilibration times that did not exceed 48 h ( 3, 4 ), osmotically inactive Na metabolism could thereby have escaped the experimental evidence. Furthermore, the total body Na and water content relative to body weight is higher in children than in adults ( 9 ). Growth leads to a redistribution of body fluids and electrolytes, and cell growth reduces the extracellular space in favor of intracellular volumes ( 1 ). Na loss in the growing organism is presumably osmotically active and thus paralleled by a corresponding water loss. However, the relative predominance of the extracellular space not only alters body fluid distribution but also changes the internal environment's polyanionic character. Given the large numbers of negatively charged GAGs that are a major compound of the extracellular matrix and growing bone, we speculated that growth could lead to a loss of osmotically inactive Na  . Furthermore, we speculated that additional long-term dietary salt loading increases the negative charge density and thereby the Na pool in terms of osmotically inactive Na reservoirs.

METHODS+ G7 N: B1 y: e8 @' {# O( {

Animal studies. In experiment 1, 30 male Sprague-Dawley rats (6 wk of age, Charles River) were divided into 4 groups. Group 1 (127.9 ± 3.0 g) was fed 0.1% NaCl (low-salt diet; LS), and group 2 (130.4 ± 5.9 g) received 8% NaCl chow (high-salt diet; HS) for 1 wk. Group 3 (125.4 ± 5.4 g) received LS for four consecutive weeks. Group 4 (126.0 ± 5.0 g) was fed HS for 4 wk. The rats received tap water ad libitum. After 1 or 4 wk on their specified diets, the rats received intravenous saline infusion and blood samples were taken. Thereafter, the rats were killed. In experiment 2, 18 male Sprague-Dawley rats (6 wk of age) were divided into two groups and fed LS or HS for four consecutive weeks. At the end of the experiment, blood samples were taken and the rats were killed. 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 621-2531-31-28/00; Regierung von Mittelfranken, Ansbach, Germany).
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Volume expansion procedure. At the end of the experiment, the rats were anesthetized with 100 mg/kg body wt ketamine and 45 mg/kg xylazine and the right femoral artery was catheterized. All rats were placed into restrainers. Together with the arterial line, the right femoral vein was catheterized and a cannula was implanted in the urinary bladder. The rats received an intravenous background infusion of 0.9% NaCl at 3.75 ml/h. Four hours after the operation, urine was sampled for a 30-min control period in completely conscious animals. The rats then underwent volume expansion with intravenous saline (0.9% NaCl, 5% of body weight) within 30 min. Urine was sampled during the period of volume expansion and for a further 90 min thereafter (post-volume diuresis)./ Y# ?+ w4 }8 D
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Dry ashing procedure. In all carcasses from experiment 1, the intestines were completely removed to exclude remains of chow. The carcasses were skinned completely to determine total skin Na  , K  , and water content in the animals. Skin water content (SKW; ml) was determined by desiccation at 90°C for 72 h from the difference between skin wet weight (g) and skin dry weight (g). After dry ashing at 190°C for 24 h and 600°C for a further 48 h, the skin ashes were dissolved in 20 ml 10% HNO 3. Water content in the skinned carcasses was determined by desiccation at 90°C for 72 h from the difference between wet weight (WW) and dry weight (DW). After dry ashing at 190 and 450°C for 24 h at each temperature level, we removed the skull, all cervical, thoracic, lumbar, and sacral vertebrae, 10 caudal vertebrae, the forelimb bones (without forefeet), the tibias, and the pelvis and ashed these bones separately at 700°C for a further 24 h. Then, the bone samples were dissolved separately in 20 ml 10% HNO 3. End ashing of the carcasses after bone removal was done at 600°C for 48 h. Carcass ashes were dissolved in 100 ml 5% HNO 3. Na and K concentrations in the dissolved ashes were measured with flame photometry (model 3100, PerkinElmer, Rodgau, Germany). Total body Na and K content was calculated from the Na and K content in skin, bone, and the rest carcass. A list of the derived parameters and their abbreviations is given in the online supplement (all supplementary material is available in the online version of this article).
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Assessment of osmotically active and water-free Na and K loss. Growth and/or dietary salt loading led to changes in the total body Na and K relative to tissue mass [ rTB(Na    K ); mmol/g DW] content and to concomitant changes in the body water content ( rTBW; ml/g WW). Total body Na and K accumulation or loss relative to water was assessed from the relationship between TB(Na    K ) and TBW. To compare TB(Na    K ) and TBW in rats at 7 and 10 wk of age, electrolyte and water contents were adjusted for body DW. Adjusting for DW (g) at 10 wk of age, adjusted WW (g) in rats 7 wk of age was2 B) X; b4 Q. i$ k$ T
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( 1 )

TBW (g) in rats 7 wk of age adjusted for DW in rats 10 wk of age was
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( 2 )/ I. [) W. H4 [' f! N9 g

Total body Na    K content [TB(Na    K ); mmol] in rats 7 wk of age adjusted for DW in rats 10 wk of age was calculated from the total body Na    K content relative to DW [rTB(Na    K ); mmol/g DW] in rats 7 wk of age and DW (g) in rats 10 wk of age

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Given that Na or K 90% of the extracellular and intracellular body osmotically active cations and that the osmotically active intracellular Na    K concentration ([Na    K ] (a) intra; mmol/ml) and the osmotically active extracellular Na    K concentration ([Na    K ] (a)extra; mmol/ml) are in equilibrium, osmotically active Na    K accumulation [ (Na    K ) a; mmol] in the body leads to water retention ( W, ml) to maintain isosmolality between the intracellular (intra) and extracellular (extra) body fluid volumes9 Z+ G6 m- q( i. `% _6 _$ N

( 4 )
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However, in tissues where polyvalent anion concentrations are substantial (e.g., cartilage and skin), the Na and K concentrations are considerably higher in interstitial fluid than in plasma ( 11, 14 ), because of the extracellular matrix negative charge density that prevents interstitial Na and K to readily equilibrate with serum Na and K  . The resulting disequilibrium can be viewed as osmotically inactive cation storage. On the assumption that the serum Na    K concentration (serum [Na    K ]; mmol/ml) roughly matches osmotically active [Na    K ] (a) extra, the amount of accumulated osmotically inactive Na and K ions that escapes isosomolality [ (Na    K ) i ] can be estimated relative to water by

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At the total body (TB) level, we then estimated the amount of osmotically inactive Na    K accumulation relative to body mass [ rTB(Na    K ) i; mmol/g DW] by" @9 P! }0 U5 z6 S

( 6 )
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The amount of osmotically active Na    K accumulation relative to body mass [ rTB(Na    K ) a; mmol/g DW] was calculated by
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Similar to the changes at the total body level, the relationship among Na  , K  , and water accumulation was calculated at the tissue levels in the completely skinned and bone-removed carcasses and in the skin. As our ashing protocol does not provide information on the bone water content, changes in bone Na and K content could only be expressed relative to bone ash mass in the rats.
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Skin GAG analysis. Approximately 1 g of rat skin was cut into small slices and digested with 7 mg proteinase K (Sigma) in 10 ml 20 mM Tris-buffer (pH 7) at 55°C for 12 h. The homogenate was then filtered to a centrifugation tube, 180 ml acetone were added, and the tube was stored at -20°C for 12 h. After precipitation, the tubes were centrifuged at 4,000 rpm for 5 min. After centrifugation, the fluid was poured out of the tube and the precipitate was dried at 50°C overnight. The dried material was dissolved with 10 ml distilled water and brought to glass columns containing 9 ml of an anion exchange resin (Sephadex QAE, Amersham Biosciences). After the resin had been washed with 2 x 10 ml 100 mM NaCl, pH 4, the GAGs were eluted from the column with 15 ml 2.5 M NaCl, pH 4. The eluate was transferred to centrifugation tubes and again precipitated with 180 ml acetone at -20°C for 12 h. After precipitation, the tubes were centrifuged at 4,000 rpm for 5 min, and then the fluids were removed and the precipitate was dried at 50°C for a further 12 h. The dried material with the purified GAGs was then dissolved with 10 ml water, transferred to a dialysis bag (Spectra/Por 3 Membrane, Spectrum), and dialyzed against distilled water for 24 h. After lyophilization, the purified skin GAGs were ready for agarose gel and HPLC-disaccharide analysis.- B- l1 x$ N% r! G
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Agarose gel electrophoresis. GAGs were separated by discontinuous agarose gel electrophoresis. Agarose gels were prepared at a concentration of 0.5% in 0.04 M barium acetate buffer, pH 5.8. Fifteen microliters of samples were layered by micropipettes. The run was in 0.05 M HCl for 180 min at 200 mA and in 0.04 M barium acetate (buffered at pH 5.8 with acetic acid) for 60 min at 100 mA. After migration, the plate was soaked in a solution of 0.2% cetylpyridinium chloride and then stained with toluidine blue always freshly prepared (0.2% in ethanol-water-acetic acid 50:49:1) for 30 min, and destained with ethanol-water-acetic acid 50:49:1. Plates were further stained with Stains-All (25 mg in 500 ml ethanol-water 50:50 overnight in the dark and destained with water) to reveal hyaluronan. Quantitative analysis of hyaluronan and dermatan sulfate was performed with densitometry. Specific calibration curves were performed by pure standard of hyaluronan (Sigma) and dermatan sulfate (Sigma) from 0.1 to 5 µg.

Disaccharide-HPLC analysis and GAG charge density measurement. Ten microliters of samples (10 mg/ml) were treated with 5 mU of chondroitin ABC lyase (E.C. 4.2.2.4  ) in 50 µl of 100 mM Tris/150 mM sodium acetate buffer, pH 8.0, at 37°C for 12 h. Boiling the solutions for 1 min blocked the reaction. The unsaturated disaccharides were analyzed by strong anion-exchange (SAX)-HPLC separation using a 150 x 4.6-mm spherisorb 5-SAX stainless steel column and detection at 232 nm. Isocratic separation was from 0 to 5 min with 0.05 M NaCl, pH 4.00, and linear gradient separation was from 5 to 90 min with 100% 0.05 M NaCl, pH 4.00, to 100% 1.2 M NaCl, pH 4.00. The flow rate was 1.2 ml/min. The amount of each identified disaccharide was determined by purified standards and reported as weight percentage. The charge density of the various skin samples was calculated considering the amount of sulfate groups per disaccharide. HPLC equipment was from Jasco (pump model PU-1580, UV detector model UV-1570, Rheodyne injector equipped with a 100-µl loop, software Jasco-Borwin rel. 1.5).0 T: A' T! N7 @: w# @3 O4 r
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Determination of the GAG Na binding capacity. Five milligrams of purified dermatan sulfate (Sigma C3788) and 5 mg of hyaluronan (Sigma H5388) were brought separately to a stirred ultrafiltration cell (8AMC, Amicon) and dialyzed with 0.01 M HCl for 6 h. After protonation, the GAGs were washed with distilled water for 6 h and then transferred to a stirred tiration cell. The protonated GAGs then were titrated with 0.1 M NaOH, resulting in a sigmoidal pH curve. After Na was exchanged with H  , the GAG Na binding capacity corresponded to the amount of base titrated at the occurrence of a rapidly increasing pH.8 C" Z: x! L$ p5 _/ W
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Data analysis. The data were tested by multivariate analysis (general linear model; GLM) for significant average differences. We tested the parameters for the dietary effect P (diet), growth effect P (growth), and interaction between diet and growth P (diet * growth). Post hoc tests were performed with the Bonferroni algorithm. All data are presented as average ± SD. The terms increased or decreased are used only if the results were significant at P + s1 j) `0 |$ S5 t

RESULTS2 f* Y9 J! V; h0 ]3 S6 A
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Compared with rats 7 wk of age, rats 10 wk of age increased total body WW by 73% and total body DW by 86% ( Table 1 ). Similar weight changes were found in the completely skinned and bone-removed rest carcasses, skin, and bone ash. Compared with LS (0.1% NaCl), HS (8% NaCl) reduced body and tissue weights, especially in rats fed HS for 4 wk (supplementary Table I ). As the differences in body and tissue mass led to concomitant changes in absolute electrolyte and water content in the rats, we adjusted Na  , K  , and water contents relative to body weight.

Table 1. Wet wt, dry wt, and Na  , K  , and water content in rats 7 or 10 wk of age at the total body level, skinned and bone-removed carcasses, skin, and bone+ K1 u2 k, S( M$ X0 b
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Growth was the major determinant of the total body Na content relative to body mass (rTBNa  ; mmol/g DW, Fig. 1 A ). Growth reduced rTBNa  , while total body K content relative to body weight (rTBK  ; mmol/g DW) was not significantly affected by age. A similar pattern was found in the completely skinned and bone-removed rest carcasses (rCarNa and rCarK  , respectively; mmol/g DW, Fig. 1 B ). In bone, both Na and K content were reduced with growth ( Fig. 1 D ). In contrast to these tissues, skin electrolyte content was less altered by growth but changed with diet. One week of LS vs. HS did not lead to significant differences in skin Na content (rSKNa  ; mmol/g DW) or skin K content (rSKK  ; mmol/g DW), while long-term dietary intervention for 4 wk led to significant differences in rSKNa ( Fig. 1 C ). These differences in rSKNa were not attributable to skin Na accumulation with HS but to skin Na loss in rats during 4-wk salt deprivation. The dietary effects on rSKNa came through at the total body Na level ( Fig. 1 A ). Growth and/or diet had no effect on rSKK in the rats. In summary, growth increased the fraction of total body Na present in the skin ( Table 2 ). In contrast to Na balance, body K balance was redistributed with growth in favor of the bone-removed and skinned rest carcass, while the fraction of total body K located in the skin and in the bone was reduced with growth. Irrespective of the diet, the pronounced tissue Na and K loss with growth was not paralleled by changes in the serum Na concentration ( week 7 : 147.5 ± 1.5 vs. week 10 : 148.2 ± 1.2 mmol/l; P 0.1) or in the serum K concentration ( week 7 : 3.75 ± 0.25 vs. week 10 : 3.89 ± 0.31 mmol/l; P 0.1). Dietary salt for 1 wk increased serum K in rats 7 wk of age, while long-term HS had no effect on serum Na or K in the rats ( Table 2 ). Salt decreased serum aldosterone levels, and long-term salt loading in rats 10 wk of age further suppressed serum aldosterone. Compared with rats 7 wk of age fed LS for 1 wk, long-term salt restriction in rats 10 wk of age did not further increase serum aldosterone levels.
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Fig. 1. Na and K contents relative (r) to dry weight (DW) in 6-wk-old male Sprague-Dawley rats that were fed a 0.1 or 8% NaCl for 1 or 4 wk. A : total body Na (rTBNa ) and K contents (rTBK ). B : bone-removed and completely skinned rest carcass Na (rCarNa ) and K (rCarK ) contents. C : skin Na (rSKNa ) and K contents (rSKK ). Bone Na and K content was normalized to bone ash mass ( D ). * P (diet)
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Table 2. Internal Na  , K  , and water distribution and serum Na  , K  , and aldosterone concentrations in rats 7 or 10 wk of age fed a low-salt or high-salt diet

Some of the growth- and diet-related differences in electrolyte content led to parallel changes in the water content of the tissues ( Fig. 2 ). Growth reduced the water content relative to WW not only at the total body level (rTBW; Fig. 2 A ) and in the skinned and bone-removed rest carcasses (rCarW; Fig. 2 B) but also in the skin (rSKW; Fig. 2 C). LS for 4 wk, but not for 1 wk, reduced rTBW and rCarW in the rats, while skin Na loss ( Fig. 1 C ) after a 4-wk LS diet compared with 4-wk HS was not accompanied by concomitant skin water loss.
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Fig. 2. Water content relative to wet weight (WW) in total body (rTBW; A ), bone-removed and completely skinned rest carcass (rCarW; B ), and skin (rSKW; C ) in 6-wk-old male Sprague-Dawley rats that were fed a 0.1 or 8% NaCl for 1 or 4 wk. * P (diet) 9 v" f2 ~4 _$ Q6 s7 U/ U: @- a
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To compare the relationship between tissue electrolyte and water content in rats 7 wk of age and rats 10 wk of age ( Table 1 ), we adjusted electrolyte and water contents for DW and then calculated the differences in tissue Na or K content ( Na  , K  ; mmol) and water content ( water; ml). At the total body level, DW-adjusted TB(Na    K ) was -6.5 mmol, and TBW was -23 ml, resulting in a TB(Na    K )/ TBW ratio of 0.283 mmol/ml. On the assumption that osmotically active TB(Na    K )/ TBW ratio and serum (Na    K ) were in equilibrium ( Eq. 4 ), 45-50% of the TB(Na    K ) lost with growth escaped isosmolality. This finding suggests that a large portion of Na and K lost from the body with growth were osmotically inactive, while 50-55% of the (Na    K ) lost with growth was osmotically active. As summarized in Table 1 and Fig. 3 A, a greater proportion of Na and K lost from the completely skinned and bone-removed rest carcass was osmotically active (65-70%). The small amount of Na and K mobilized by growth from skin went along with a corresponding water loss, indicating the mobilization of osmotically active Na from the skin. Figure 3 A, right, summarizes the (Na    K ) from the skinned and bone-removed rest carcass tissues, the skin, and the bone and their contribution to total body (Na    K ) loss in rats 10 wk of age compared with rats 7 wk of age after body weight adjustment. The loss of body (Na    K ) associated with the growth originated primarily from the skinned and bone-removed rest carcasses. The growth-related internal environmental changes in bone and skin played a less predominant role in terms of (Na    K ) mobilization in the rats.! p. E1 y+ r% k% \% Y( i. r) m( s
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Fig. 3. A, left : effect of growth on Na    K content relative to tissue DW (mmol/g DW) in terms of osmotically active or osmotically inactive cation loss in 10-wk-old rats compared with 7-wk-old rats. Right : summary of contribution of absolute (Na    K ) loss from the skinned and bone-removed rest carcass, skin, and bone to total body (Na    K ) loss in 10-wk-old rats compared with 7-wk-old rats (adjusted for body weight). B, left : effect of 4 wk 0.1% NaCl (LS) vs. 8% NaCl (HS) in 10-wk-old rats on Na    K content relative to tissue DW (mmol/g DW) in terms of osmotically active or osmotically inactive cation loss. Right : contribution of skin (Na    K ) loss to total body (Na    K ) loss in rats 10 wk of age fed LS vs. HS for 4 consecutive weeks. Data are derived from Tables 1 and 3.

We next investigated the effect of the diet on the relationships among Na  , K  , and water in rats fed LS vs. HS. One week LS or HS in rats 7 wk of age did not lead to significant differences in the tissue Na or K content ( Fig. 1 ). Long-term salt restriction for 4 wk led to body Na and water loss, while no changes in body K content were observed, as shown in Table 3. Adjusted for body weight, TB(Na    K ) with LS was -2.8 mmol, and TBW was -9.4 ml, resulting in a TB(Na    K )/ TBW ratio of 0.298 mmol/ml. On the assumption that osmotically active TB(Na    K )/ TBW ratio and serum (Na    K ) are in equilibrium ( Eq. 4 ), 50% of the TB(Na    K ) lost with long-term salt restriction was osmotically inactive, while the remainder (Na    K ) loss with LS was osmotically active. As summarized in Table 3 and Fig. 3 B, the skin was the predominant osmotically inactive Na reservoir from which Na was mobilized during long-term dietary salt restriction. Our measurements were not sensitive enough to detect significant changes in rest carcass or bone Na and/or K content with long-term salt restriction. However, as indicated by the difference between TB(Na    K ) and SK(Na    K ) in Fig. 3 B, right, undetectably small Na  , K  , and water losses from these tissues had also been operative in Na  , K  , and water mobilization during dietary salt scarcity.
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Table 3. Wet weight, dry weight, and Na  , K  , and water content at the total body level and skin in rats 10 wk of age fed 0.1 or 8% NaCl for 4 wk

We next investigated in saline infusion experiments whether differences in the tissue Na content were paralleled by similar changes in the renal excretion of Na relative to water ( Fig. 4 ). LS for 1 or 4 wk reduced urinary Na excretion. Long-term salt restriction not only further reduced skin Na content ( Fig. 1 C ) but also decreased urinary Na concentration () and increased urinary K concentration () after 4-wk LS. The resulting decrease in urinary / suggests that salt scarcity for 4 wk led to maximum renal Na conservation.  in rats fed LS for 1 wk was significantly higher than in rats with long-term salt restriction for 4 wk, reflecting the kidney's effort to restore the pronounced Na loss relative to water at the tissue level ( Fig. 1C ), while the serum Na concentration was unchanged ( Table 2 ). During brisk intravenous volume loading with 5% body weight saline within 30 min ( Fig. 4 B ), and also 90 min after VE when the volume load was excreted ( Fig. 4C ),  was still higher in rats fed HS than in rats fed LS; however, the effect of skin Na loss induced by 4 wk LS was not reflected by a further reduction in  during volume expansion. The decrease in  and the increase in  especially in rats with LS for 4 wk were paralleled by decreased natriuresis () and increased kaliuresis (). Furthermore, compared with the other groups, rats fed LS for 4 wk showed marked Na retention and K loss during 0.9% saline infusion experiments (Supplementary Table II ). These findings suggest that renal Na conservation and mobilization of osmotically inactive Na from reservoir tissues both contribute to Na -water homeostasis during long-term salt deprivation." C$ O7 B/ y! {0 t3 h

Fig. 4. Urinary Na and K concentration and urinary Na /K ratio during 0.9% saline infusion in 7- or 10-wk-old rats fed a 0.1 or 8% NaCl for 1 or 4 wk. Dietary salt increased urinary Na concentration and urinary Na /K ratio during background infusion ( A ), volume expansion (VE; B ), and after VE ( C ). Low dietary salt for 4 wk resulted in reduced urinary Na concentration and increased K concentration, suggesting increased mineralocorticoid action in the rats on LS for 4 wk. This effect was lost during and after 0.9% saline excess. * P (diet)

Having shown that the loss of skin Na during long-term salt restriction was mobilization of osmotically inactive Na from a reservoir tissue located in the skin, we finally addressed the question in an additional experiment of whether LS for 4 wk might change the polyanionic character of skin GAGs in the osmotically inactive Na reservoir. A representative-HPLC disaccharide analysis of skin GAGs from rats fed LS vs. HS for 4 wk is shown in Fig. 5, A and B. LS for 4 wk decreased the degree of GAG sulfatation and hence the negative GAG charge density of the reservoir tissue. Respectively, 43% of the skin GAGs were sulfated in rats fed LS for 4 wk, while 59% of skin GAGs were sulfated in rats fed HS for 4 wk ( Table 4 ). Additional titration experiments indicated an increased GAG Na binding capacity as a result of increased GAG charge density. At physiological pH, the Na binding capacity of dermatan sulfate (DS) was 13 µmol Na /5 mg DS and only 7.5 µmol Na /5 mg hyaluronan (titration curve; see supplementary Fig. A). Agarose gel electrophoresis of GAGs purified from the skin of rats fed LS or HS confirmed the HPLC data ( Fig. 5 C, Table 4 ). Figure 6 summarizes the relationship between dietary salt restriction and/or salt excess, skin Na storage, and skin GAG charge density in the rats. LS for 4 wk increased serum aldosterone and led to mobilization of osmotically inactive Na from the skin, as indicated by an decreased skin (Na    K )-to-water ratio ( Fig. 6 A ). A similar relationship was found between skin Na -to-water ratio and serum aldosterone concentration, while we found no correlation between skin K -to-water ratio and serum aldosterone in the rats ( R 2 = 0.06, P = 0.22). The reduction in the polyanionic character of the reservoir tissue, as indicated by the decreased skin GAG charge density, was paralleled by increases in the serum aldosterone in rats fed LS for 4 wk ( Fig. 6 B ). HS for 4 wk suppressed serum aldosterone levels and resulted in osmotically inactive Na storage [increased skin (Na    K )-to-water ratio], which went along with increases in the polyanionic character of the reservoir tissue (increased skin GAG charge density).5 s$ h- t5 {* w" R4 ?8 P

Fig. 5. Representative glycosaminoglycan (GAG)-disaccharide [Di-hyaluronic acid (HA)]-HPLC analysis from skin GAGs in rats 10 wk of age fed 0.1 ( A ) or 8% NaCl ( B ) for 4 consecutive weeks. Compared with rats on HS, rats fed LS for 4 wk displayed a higher amount of unsulfated GAG disaccharides (Di-HA) relative to sulfated disaccharides (Di-4s; Di-6s) in their skin. This finding was confirmed by agarose gel electrophoresis ( C ), where we found increased HA content relative to dermatan sulfate (DS) present in skin of rats fed LS. Respectively, 57% of the skin GAGs in rats fed LS were unsulfated, while only 41% of the skin GAGs were unsulfated with HS ( Table 4 ). This finding indicates a reduction in the negative GAG charge density in rats with long-term salt restriction, which coincided with a reduction in osmotically inactive Na in the skin reservoir.7 ^# e7 y0 [3 W

Table 4. Glycosaminoglycan agarose gel electrophoresis HPLC-disaccharide analysis, and serum aldosterone concentration in skin of rats fed a low- or a high-salt diet for 4 consecutive wk
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Fig. 6. Relationship between serum aldosterone level and SK(Na    K )/SKW ratio ( experiment 1 in A ) and between serum aldosterone and skin GAG charge density ( experiment 2 in B ) in rats fed 0.1 or 8% NaCl. LS increased serum aldosterone, reduced the SK(Na    K )/SKW ratio (indicating Na mobilization from the osmotically inactive Na reservoir), and reduced the negative skin GAG charge density in reservoir tissue.
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DISCUSSION

A novel finding in this study is that long-term salt deprivation was associated with decreased negative charge density of skin GAGs. This decrease in the polyanionic character was associated with the mobilization of osmotically inactive Na from reservoir tissue ( Fig. 6 ). This finding suggests that besides the negatively charged, intravascular proteins, other molecules with polyanionic character localized in the interstitium are operative in body Na distribution. Furthermore, the findings suggest that Gibbs-Donnan calculations based on oversimplified and closed two-compartment models may not adequately reflect the tissue heterogeneity in the maintenance of internal body Na balance. Compared with rats 7 wk of age, rats 10 wk of age with a total body water content of 193.1 ml lost 6.5 mmol Na    K ( 6 mmol Na ). In case this electrolyte loss had originated as osmotically active from the water space, one would expect a -6.5 mmol/193.1 ml = -34 mmol/l decrease in serum Na concentration in rats at 10 wk of age. However, we found no decreases in serum Na or K concentrations between rats 7 and 10 wk of age ( Table 1 ). Our experimental animal data suggest that, in contrast to the data leading to the Edelman equation ( 4 ) and its derivations ( 15, 19 ), the exchangeable total body Na pool can be variable without concomitant changes in the serum Na concentration. This finding sheds new light on the term "exchangeable" as coined by Edelman et al. ( 4 ). As originally indicated by Forbes and Perley ( 8 ), "the isotopic dilution method measures that portion of the total body sodium which is most intimately concerned with the body fluid framework" ( 8 ). Evidence based on this method ( 4, 9 ) hence primarily addresses osmotically active electrolyte metabolism. In addition, our chemical analysis indicates that beyond such osmotically active electrolytes, an even greater portion of exchangeable Na  , namely, the exchangeable osmotically inactive Na pool, may be operative in total body Na homeostasis in the rat. While variations in exchangeable osmotically active Na or K are essential in body fluid homeostasis, the physiological and pathophysiological role of osmotically inactive Na metabolism is unclear.1 V+ [/ S' T; Z8 l* q0 }7 e
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The mobilization of osmotically inactive Na by growth or dietary salt restriction was tissue specific. Growth led to mobilization of osmotically inactive Na from the completely skinned and bone-removed rest carcasses, and the bone, but not from the skin. Dietary salt restriction led to mobilization of osmotically inactive Na from the skin, while the sodium content in the bone and the completely skinned and bone-removed rest carcass was not affected by diet. With growth, there are internal environmental changes characterized by a loss of extracellular space/matrix ( 1 ). The data suggest that Na loss attributable to growth and its associated changes in the internal environment were insensitive to changes in dietary salt intake and originated primarily from the completely skinned and bone-removed rest carcass (muscle, internal organs) or from bone, while only a small amount of Na from the skin was mobilized with growth. In contrast, the skin served as a predominant osmotically inactive Na reservoir that reacted to dietary salt loading by changing its polyanionic character. These differential changes in the internal environment are relevant to notions regarding Na balance, extracellular volume regulation, and possibly to the putative relationship between salt intake and arterial hypertension.4 P* l5 S, E3 E* @3 P5 R5 @& S. }
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These findings make two important points. First, they underscore the important role of the kidneys in maintaining rTBNa in rats, as dietary salt excess did not lead to total body Na excess. Growing rats achieved a positive Na balance despite dietary LS (supplementary Table I ). Second, the observations indicate the relevance of an extrarenal regulation to maintain total body Na balance, as "growth-programmed" reduction in rTBNa was tremendous ( Fig. 3 ), but nonetheless resistant to dietary extremes. Our data also suggest that renal and extrarenal regulation of total body Na and total body K are coordinated within the organism. The depletion of the osmotically inactive skin Na pool, which occurred in rats with long-term salt deficit but not in rats fed LS for 1 wk ( Fig. 1 C ), was consistent with further decreases in urinary Na excretion relative to water and with marked Na retention during infusion of 0.9% saline ( Fig. 4 A ).- D5 Y1 b0 e& E3 ~9 ~" v0 p7 }2 |: T5 \

The redistribution of the total body Na with growth led to pronounced tissue Na loss, but only moderate water loss. The Na loss in excess over water was not balanced by a corresponding K gain, suggesting the mobilization of osmotically inactive Na  . Compared with internal Na distribution in rats 7 wk of age, rats 10 wk of age lost 6.5 mmol Na and K ( Table 1, Fig. 3 ). If such a Na and K loss had been extracellular and osmotically active, this amount would be predicted to decrease extracellular volume by 6.5 mmol/0.152 mmol/ml = 43 ml and would decrease extracellular volume 60-70% in a 300-g animal. Instead, only a 23-ml decrease in volume was recorded ( Table 1 ). As 45-50% of the Na load escaped isosmolality ( Fig. 3 A ), we conclude that 3 mmol Na and K was lost from osmotically inactive Na pools and saved the animals from an additional 20-ml volume loss. Compared with growth, the effects of dietary salt restriction on osmotically inactive body Na loss were moderate. LS for 4 consecutive wk mobilized 2.8 mmol Na and K  . If such Na loss had been extracellular and osmotically active, this amount would be predicted to decrease extracellular volume 2.8 mmol/0.152 mmol/ml = 18.4 ml and would decrease extracellular volume 30% in a 300-g animal. Instead, only a 9.4-ml decrease in volume was recorded ( Table 3 ). Even at the tissue level, we found no significant mobilization of osmotically neutral Na in exchange with K in rats with LS ( Table 3 ).; t, T4 f8 J7 o6 }

In an earlier study ( 27 ), we suggested that osmotically inactive skin Na storage in rats might be an active process characterized by an increased skin GAG content. However, we could not provide data on the GAG charge density. Thus the polyanionic character of the skin matrix and its association with the osmotically inactive skin Na pool in these experiments were not defined. The new data in this report indicate that long-term LS features mobilization of osmotically inactive Na from the skin that coincides with a decreased GAG sulfatation and charge density. As a result, the polyanionic character of the extracellular matrix decreases. The data suggest that an interstitial cation exchange mechanism may be important in osmotically inactive Na storage and/or the mobilization of osmotically inactive Na from a reservoir tissue. Osmotically inactive Na storage appears to be an active process that includes the modulation of the polyanionic character of our internal environment by GAG polymerization and/or GAG sulfatation.  x) H4 T; E. X3 s3 m
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What the signals might be that regulate GAG chain elongation and/or sulfatation with HS intake is unknown. In these studies, we measured aldosterone concentrations; however, we presented no evidence that this hormone is a mediator. Our hyperbolic correlations ( Fig. 6 ) have no causal implications. Furthermore, the mobilization of osmotically inactive Na in rats with long-term salt deprivation fed LS for 4 wk ( Fig. 1, A and C ) was not paralleled by further increases in the serum aldosterone concentration in this subgroup ( Table 1 ). Gene expression studies might provide some insight into candidates that regulate osmotically inactive Na reservoirs. Although the loss of osmotically inactive Na with growth most probably was attributable to a reduction of extracellular space leading to a reduction in GAG-rich, polyanionic interstitial matrix molecules (such as bone cartilage and interstitial matrix in internal organs and muscle), we cannot provide direct evidence for such growth-associated changes and their relationship to total body electrolyte balance. Growth-related changes in the polyanionic character of the extracellular interstitial space await experimental clarification.

On the one hand, our studies address the discrepancies in Na balance studies in humans, suggesting that a HS diet does not necessarily induce the expected amount of volume retention ( 12, 13, 17, 26 ). The notion of water-free Na retention also receives support from a recent study in marathon runners ( 16 ). On the other hand, Seeliger et al. ( 20, 21 ) performed Na  , K  , and water balance studies of 4 days' duration in freely moving dogs and concluded that "the notion of osmotically inactive Na storage during Na accumulation appears to be invalid" ( 21 ), as the actual experimental evidence from humans ( 12, 26 ) and preliminary data from animal experiments ( 23, 24, 27 ) failed to account for K balance. Seeliger et al. ( 20 ) argued that changes in total body Na frequently include osmotically active Na /K redistributions among the extracellular and the intracellular fluid compartments and that Na accumulation in abundance over water could hence be balanced by corresponding K loss. They concluded that we had misinterpreted our own data because we had not measured concomitant changes in total body K ( 21 ).# a; E8 c) j9 y1 Z( V2 |3 J  l& b
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Our present experiments have addressed this criticism. We have eliminated the possibility that the alterations in skin Na content relative to water with LS vs. HS are balanced by opposite redistribution of K to maintain isosmolality. Our data thus support the notion that the skin is an osmotically inactive Na reservoir and that its osmotically inactive Na pool in the male Sprague-Dawley rat varies with the diet. However, we must attribute changes in the osmotically inactive Na content after dietary intervention in this and previous experiments ( 23, 24, 27 ) rather to Na loss with LS than to Na accumulation with HS. In contrast, balance experiments in humans suggested that much more moderate salt diets may increase total body Na by 40% within 1-4 wk ( 12, 17 ). We were able to achieve similar excess Na accumulation in rats only by adding mineralocorticoid treatment ( 22, 25 ). Those studies suggested that not only the absolute amount of Na accumulated (external Na balance) but also the long-term redistribution of Na within the organism (internal Na balance) are important in body fluid and blood pressure regulation ( 25 ). Possibly, the rat is not a perfect model to experimentally simulate water-free Na retention in humans. However, besides these quantitative aspects, our model provides a robust tool for investigating the mechanisms of osmotically inactive Na storage, for instance in the skin.  I6 S/ R$ P9 |0 ^7 K3 p& l8 S
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The Else Kröner-Fresenius-Stiftung and the " Interdisziplinäres Zentrum für klinische Forschung" (IZKF) Erlangen supported the study.

ACKNOWLEDGMENTS

Elke Prell helped with the ashing procedures.3 _, D6 l$ V/ Y4 v
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