Internal sodium balance in DOCA-salt rats: a body composition study - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:04 |只看该作者 |倒序浏览 |打印

Department of Nephrology and Hypertension, Friedrich-Alexander-University, Erlangen-Nürnberg, Germany

Institute of Biochemistry, Charité Campus Benjamin Franklin, Berlin, Germany
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Federal Center for Meat Research, Kulmbach, Germany
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Charité Campus Buch, Franz Volhard Clinic, HELIOS Klinikum-Berlin and Max Delbrück Center for Molecular Medicine, Berlin, Germany0 R- E- u/ n5 O! @4 s
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ABSTRACT5 o6 n; J& x1 {. b# o1 b" @3 ~
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The idea that Na  retention inevitably leads to water retention is compelling; however, were Na  accumulation in part osmotically inactive, regulatory alternatives would be available. We speculated that in DOCA-salt rats Na  accumulation is excessive relative to water. Forty female Sprague-Dawley rats were divided into four subgroups. Groups 1 and 2 (controls) received tap water or 1% saline (salt) for 5 wk. Groups 3 and 4 received subcutaneous DOCA pellets and tap water or salt. Na , K , and water were measured in skin, bone, muscle, and total body by desiccation and consecutive dry ashing. DOCA-salt led to total body Na  excess (0.255 ± 0.022 vs. 0.170 ± 0.010 mmol/g dry wt; P
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hypertension; sodium storage; hypertensive rats; mineralocorticoid escape) z  i8 I; u: C# v# l4 I

SODIUM AND ACCOMPANYING ANIONS make up the effective osmoles of the extracellular fluid volume. K  salts account for most of the intracellular osmoles. The cells are permeable to both cations; however, Na  and K  are able to function as effective osmolytes because they are restricted to their respective compartments by the activity of the Na -K -ATPase pump in the cell membrane. Sodium balance is generally assumed to function as a two-compartment model. Were 140 mmol Na  retained as the Cl– salt, the extracellular space would be expected to increase by 1 liter. However, balance studies have been performed in which Na , as the Cl– salt, did not invariably lead to volume retention (8, 17, 21). Furthermore, negative Na  balance has been observed that was not paralleled by volume losses (6). Farber and Soberman (4) noticed a "high ratio of body sodium to water" in patients with cardiac edema over 50 years ago. They speculated that Na  might be stored in "bone, cartilage, or connective tissue." Their startling conclusions imply that Na  may be exchanged for some other intracellular cation or might be stored in an osmotically inactive form. We have found that osmotically inactive Na  storage in rats takes place largely in the skin (20, 23). We also observed that salt-sensitive Dahl rats have a reduced osmotically inactive storage capacity for Na , compared with controls (19, 22). DOCA and a high-salt intake provide a favorite mineralocorticoid-induced model of hypertension. When salt intake is increased, the animals quickly achieve Na  balance without further Na  retention, a phenomenon termed "mineralocorticoid escape." In this case, the escape is external because Na  excretory mechanisms are operative despite mineralocorticoid. We speculated that an additional "internal Na " escape might exist in DOCA rats in the form of osmotically inactive Na  storage. Were such a mechanism regulated, it might be of relevance to salt-induced hypertension.7 R' ?: u0 |) f+ I

METHODS0 M' H. S0 r7 q% K& A6 H
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Experiment 1. Twenty female Sprague-Dawley rats were randomly assigned to four groups. Groups 1 and 2 were untreated control groups, whereas groups 3 and 4 received DOCA pellets implanted subcutaneously under methohexital anesthesia. New pellets were placed in these rats after 3 wk. All rats were fed a
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Experiment 2. Twenty female Sprague-Dawley rats were assigned to four groups. Group 1 (180.7 ± 5.4 g), group 2 (173.4 ± 6.8 g), group 3 (179.0 ± 3.5 g), and group 4 (186.6 ± 2.9 g). Each group received the same dietary regimen and DOCA treatment protocol as in experiment 1. Blood sampling and carcass preparation for dry ashing was similar to experiment 1, with the exception that the quadriceps muscles were additionally removed and ashed separately. The local animal care committee approved the protocols. The requirements correspond to those outlined by the American Physiological Society., o: [( k' i' a  G

Ashing procedure. The skin, the carcasses and the muscles were weighed (wet weight) and then desiccated at 90°C for 72 h (dry weight). Because further drying left weights unchanged, the difference between wet weight and dry weight was considered as tissue water content. The dried skin, carcasses, and muscles were then ashed at 190 and 450°C for 24 h at each temperature level. Thereafter, we removed the bones from the carcasses by sieving the ashes with a stainless steel sieve. Bones, skin, muscles, and rest ashes from the completely skinned and bone-removed rest carcasses were then completely ashed at 600°C for an additional 48 h. The ashes were then dissolved in 10% HNO3.
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Electrolyte measurements. Na  and K  concentrations in the blood samples were measured with a flame photometer (model EFIX 5055; Eppendorf, Hamburg, Germany). Na  and K  concentrations ([Na ] and [K ], respectively) in the dissolved ashes were measured with an atomic absorption spectrometer in the flame photometry modus (model 3100; Perkin Elmer, Rodgau, Germany).
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Data analysis. Data are expressed as average ± SD. Data from the various tissue Na , K , and water contents were analyzed by multivariate analysis (general linear model). Post hoc tests were performed with the Bonferroni algorithm. All comparisons of means were performed with the SPSS software (version 12.0). Curve fitting in scatterplots was done with ORIGIN software (version 6.0). The terms "increased" or "decreased" are used solely when the results are statistically significant (P
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Na  abundance relative to water. Water "free" sodium accumulation is characterized by increases in the Na -to-water ratio (RNa /water) in the tissues. We investigated the relationship between changes of Na  content and alterations of water content in total body, skin, bone, muscle, and rest carcass without normalizing the data for tissue weight by calculating the ratio between total tissue Na  (in mmol) and total tissue water (in ml) content in each rat:! d+ m. h! [8 v8 A

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The absolute amount of excess Na  accumulation relative to water [Na(i) , mmol] was calculated from the difference between tissue RNa /water on a low- and on a high-NaCl diet tissue (RNa /water; in mmol/ml) and tissue water content (ml):
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From the assumption that Na  is the major extracellular and K  the major intracellular cation where both cations exert osmotic activity, increases in the tissue RNa /water were compared with its (Na K )-to-water ratio [R(Na  K )/water]. Parallel increases in RNa /water and R(Na  K )/water indicated Na  and cation abundance relative to water and hence osmotically inactive Na  storage in the tissue. In case the RNa /water was increased in the tissue while its R(Na  K )/water was unchanged, Na  excess relative to water had been balanced by K  loss relative to water, indicating an osmotically neutral Na /K  exchange in the tissue.* U; F0 z- t* i9 p. O! g$ p5 o

Osmotically active Na  accumulation. Based on the assumption that water accumulation (W; ml) was due to osmotically active Na  and K  accumulation, osmotically active Na K  retention [(Na  K )(a); mmol] was estimated from the difference between the relative tissue water content on a low- and a high-NaCl diet (rW; ml/g wet weight), the tissue wet weight (WW; g), and the serum Na K  concentration ([Na  K ]serum; mmol/ml):

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Taking into consideration the difference between serum [Na ] and serum [K ], (Na  K )(a) roughly matched Na (a) in the rats.- @6 w1 y9 }: u1 z' y

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Table 1 shows the body compositions of all four groups. Ten rats were studied for each measurement with the exception of muscle, where the quadriceps was excised from five rats from each treatment group. DOCA-salt treatment resulted in slightly lower body (wet) weight than the other three groups. After desiccation (dry weight), this difference was more pronounced. DOCA alone and DOCA-salt reduced dry weight. The same was true for the skin, while muscle wet and dry weights were not perturbed. Bone ash weight was lower with DOCA-salt treatment than in all other groups. Rest carcass dry weight was lower in the DOCA groups compared with the control groups, whereas there were no differences in wet weights between the groups. This finding indicates that a substantial mass loss in the DOCA rest carcasses was "hidden" by water retention. Total body Na  was higher in DOCA-salt and DOCA compared with the control groups, whereas total body K  was lower in the DOCA-salt group but not in the DOCA-alone group compared with the controls. Total body water seemed not to be influenced by salt, DOCA-alone, or DOCA-salt, compared with controls. Skin Na  increased with salt, DOCA, or DOCA-salt, compared with controls. Skin K  was reduced in the DOCA-salt group, compared with DOCA alone or controls. Bone Na  was unchanged across the groups. However, when bone Na  was expressed as millimoles per gram ash, increases were seen in the DOCA-alone and DOCA-salt groups, compared with controls. Thus DOCA rats maintained bone Na  despite substantial bone mineral (bone ash) loss by changing their bone mineral composition. In contrast, bone K  (expressed as mmol/g ash) was unchanged in DOCA rats, indicating that the observed decreases in bone K  were due to bone mineral loss and not due to alterations in the bone mineral composition. Rest carcass Na  increased with DOCA alone and DOCA-salt compared with controls, whereas rest carcass K  decreased in DOCA-salt rats. Rest carcass water was unchanged. Serum Na  increased slightly with DOCA alone and DOCA-salt, whereas serum K  decreased with salt, DOCA alone, and DOCA-salt compared with controls.
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View this table:

To adjust for the differences in body weights across the groups, we calculated the "relative" total body water (all ml/g wet wt), rest carcass water, and skin water (Fig. 1). DOCA increased relative total body water (rTBW), relative carcass water, and relative skin water compared with both controls. Muscle values were not expressed in relative terms because muscle (quadriceps) weights were not different in the groups. Muscle water (MW) was unperturbed, with the exception that a slightly higher value was found in DOCA-salt compared with DOCA alone. Thus, with the possible exception of muscle, DOCA treatment with or without salt appears to slightly increase water in the tissues (per g body wt).
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DOCA increased relative total body Na  (rTBNa ; all mmol/g dry wt), whereas DOCA-salt increased this value further (Fig. 2). The relative total body K  (rTBK ; mmol/g dry wt) increased with DOCA alone but decreased with DOCA-salt to the control level. There was no dietary effect on rTBNa  and relative carcass Na  (rCarNa ) in the control rats, whereas salt increased relative skin Na  (rSKNa ) in both the DOCA groups and controls given salt. Thus rCarNa  becomes an additional Na  reservoir with DOCA treatment. In the skin, a site that we have already identified as a Na  reservoir, salt resulted in increased rSKNa  values, whereas DOCA alone and DOCA-salt increased these values further, and the relative K  values with salt or DOCA, alone or with salt, were unchanged. In muscle, similar patterns were seen. DOCA increased muscle Na  (mmol), and DOCA-salt increased this value further. DOCA decreased muscle K , whereas this value was not altered by salt.
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Table 2 gives the shifts in "internal balance" in terms of cation movements across the regimens. Carcass Na  to total body Na  decreased slightly with salt but increased with DOCA alone and increased further with DOCA-salt. In skin, the skin Na  per total body Na  increased with salt. DOCA alone and DOCA-salt had no influence. In bone, the ratio of bone Na  to total body Na  actually decreased with DOCA alone and decreased further with DOCA salt compared with the control groups. Compared with Na , the ratio of carcass K  to total body K , skin K  to total body K , and bone K  to total body K  were relatively constant. However, irrespective of diet, DOCA increased the carcass K -to-total body K  ratio (0.746 ± 0.020 vs. 0.726 ± 0.022), whereas the skin K -to-total body K  ratio was decreased (0.114 ± 0.011 vs. 0.124 ± 0.011). Carcass water to total body water was decreased by DOCA alone compared with controls, but this was increased by DOCA-salt compared with DOCA alone. Skin water-to-total body water ratio was increased by DOCA alone compared with control but decreased by DOCA-salt compared with diet control or DOCA alone. We interpret these data as indicating that skin and carcass were the sites of Na  storage, whereas the K  losses stemmed primarily from the skin. The rest carcass was better able to defend its K .
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View this table:  Y9 @6 G  t) ]2 G7 U0 k
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Table 3 shows the relationships (ratios) between Na , K , and the sum of the two cations (total effective osmolytes) with total body water. Expressing the data in this fashion provides more insight into where the Na  is going and from whence the K  is leaving. Na  is the primary extracellular cation, whereas K  is the primary intracellular cation. The sum of the two respective primary (>98%) extracellular and intracellular cations represents the effective osmolyte concentration, provided that they are free osmoles. In the controls, the ratio of total body Na  per total body water was not increased by salt. However, with DOCA alone, an increase was observed that increased further with DOCA-salt. This relationship in rest carcass Na  per carcass water was different. Here, salt in drinking water actually decreased carcass Na  per carcass water slightly in control rats. However, DOCA alone and DOCA-salt increased the relationship in a stepwise fashion. In skin, the ratio skin Na  per skin water was increased with salt but not with DOCA alone. However, with DOCA-salt, the highest ratio of skin Na  to skin water was observed. The relationship changed little in the Na  bone per total body water across the regimens. In muscle, the ratio muscle Na  per MW was increased with DOCA, whereas salt did not significantly alter this value. The ratio of K  to water was generally decreased by DOCA-salt and little influenced by salt alone or DOCA alone in the various compartments. Salt alone did not affect the ratio of total body Na K  to water. DOCA alone and DOCA-salt increased the ratio. Similar changes in the ratio occurred in the completely skin- and bone-removed rest carcass. In skin, the ratio of skin Na K  to skin water increased with DOCA-salt, reflecting the movement of Na  to skin. Across bone and muscle, the Na K -to-water ratio in these structures did not change.
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We next (Fig. 3A) examined the correlation between rTBNa  (mmol/g dry wt) and rTBW (ml/g wet wt). In control rats receiving tap water or salt, a steep relationship was found, whereby a range of rTBNa  (0.15–0.20 mmol/g dry wt) encompassed a range of rTBW (0.63–0.67 ml/g wet wt). In rats receiving DOCA, this relationship exhibited a far less steep slope so that a range in rTBNa  from 0.20 to 0.30 mmol/g dry wt covered a range of rTBW from 0.65 to 0.70 ml/g wet wt. This relationship implies that the presence of DOCA was associated with a lesser span in water accumulation for given total body sodium accumulation. We calculated similar data for K  (Fig. 3B). The rTBK  was plotted against rTBW for control animals given tap water or salt. A linear relationship was found. In animals receiving DOCA alone or DOCA-salt, no relationship was found. These data imply that, normally, rTBW increases with increases in rTBK . However, in the presence of DOCA, with or without salt, this relationship is perturbed, presumably because of K  losses in the presence of water retention. We also calculated the same relationship examining the total effective osmolyte concentration (rTBNa rTBK  as mmol/g dry wt) against relative total body water (Fig. 3C). A robust linear relationship for all groups irrespective of salt intake or DOCA administration was found. However, in rats receiving DOCA, the relationship was moved rightward and upward. This right shift reflected the significant increase in the total body Na K -to-water relationship (0.208 ± 0.009 vs. 0.196 ± 0.006 mmol/ml) that was observed in DOCA rats, indicating an abundance of Na  and K  over water. This finding implies that DOCA increased the total Na  concentration per dry weight.# X% Q, J. o6 L6 ~5 e# d

We investigated the same relationships for rCarNa  (mmol/g dry wt) and relative carcass water (rCarW; ml/g wet wt), relative carcass K  vs. rCarW, and rCar for Na K  vs. rCarW (Fig. 4). The relationships were mostly similar in kind to those shown for relative Na  shown above. The same relationships were also plotted for skin (Fig. 5). In skin, the relationship between rSKNa  and relative skin water (rSKW) was linear in tap water controls and salt controls. However, in DOCA rats, a curvilinear relationship was generated. The relationship indicates that the amount of Na  per the amount of water in the skin increased markedly with DOCA treatment. The relative skin K  per rSKW was a linear relationship; DOCA treatment moved the slope upward, so that a greater amount of water was present per a given amount of K . The relationship between relative skin Na K  and rSKW was linear in the controls and in DOCA rats on a low-salt diet and became curvilinear only in DOCA rats with skin Na  excess. There was no relationship between muscle Na  and MW (Fig. 6) either with or without DOCA. Muscle K  and MW were correlated in the control groups but not in the DOCA groups. Although there was no correlation between muscle Na  and MW, and although muscle K  and MW only correlated in control rats, the relationship between muscle Na muscle K  and MW was highly significant across the groups.3 M, K% P2 E" I0 E

The Na K -to-water relationship in skin was higher than in muscle (0.208 ± 0.015 vs. 0.172 ± 0.004 mmol/ml in control rats; 0.209 ± 0.024 vs. 0.174 ± 0.010 mmol/ml in DOCA rats), indicating cation abundance relative to water in skin compared with muscle. In DOCA rats, skin acted as an osmotically inactive Na  reservoir: dietary salt loading increased the skin Na  content not only per dry weight (Fig. 2) but also relative to water (Table 3). This abundance of Na  relative to water was not balanced by a corresponding K  loss and thus resulted in excess tissue Na K  relative to water. This excess of Na  and K  relative to water can only be explained by water-free accumulation of the cation load, which is osmotically inactive Na  storage. The reaction of the muscle to the Na  challenge in DOCA rats was different. DOCA induced muscle Na  retention (Fig. 2) was balanced by K  loss. Thus increases in muscle Na  were compensated by decreases in muscle K  and the Na K -to-water ratio remained constant. Whether this osmotically neutral Na /K  exchange was achieved by the exchange of osmotically active or inactive cations remains unclear. As judged from the serum electrolyte concentrations (Table 1), the differences in the Na K -to-water ratio between the extracellular fluid and the muscle (0.150 mmol/ml vs. 0.174 mmol/ml) indicated that 14% of the muscle Na  or K  were present in an osmotically inactive form. This amount corresponds well to the actual muscle K  loss observed in DOCA rats (Fig. 2).2 |9 f1 O+ @) B' E7 Z4 s+ F% B

Finally, we quantified the absolute effect of DOCA-salt treatment on Na  gain (Fig. 7).We calculated the contribution of osmotically active Na  retention (active; Eq. 3), osmotically inactive Na  retention (inactive; Eq. 2), and osmotically neutral Na  retention (calculated as the difference between water free Na  gain and water free K  loss). Rest carcass included all skeletal muscle. The gain in bone Na  relative to water was small (0.29 mmol) and was completely balanced by K  loss. Water-free skin Na  gain was 0.89 mmol of which 0.34 mmol were balanced by skin K  losses, relative to water. This relationship resulted in 0.55 mmol osmotically inactive skin Na  storage. Rest carcass Na  accumulation in excess of water was 2.54 mmol, of which 1.09 mmol were balanced as osmotically neutral by corresponding K  losses. The remaining Na , 1.45 mmol, was accumulated as osmotically inactive. The fraction of osmotically active Na  accumulation in DOCA-salt rats was 1.16 mmol. The latter was calculated from their water retention and serum Na  and K  concentrations (Eq. 3). Thus 75–80% of the Na  gained by the DOCA-salt rats was either inactive or neutral (balanced by K  losses) and could not have contributed to volume expansion.

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This study is the first to examine Na  metabolism in all body compartments in the DOCA-salt rat model of hypertension. The important findings were that with DOCA-salt, the rats retained 4.75 mmol Na . This degree of Na  accumulation should lead to a 32 ml volume retention. However, we detected only water accumulation of 7.75 ml that corresponded to 1.16 mmol Na  as an active extracellular osmolyte. The remaining 75–80% Na  was retained in abundance over water, a finding quite similar to that reported by Farber and Soberman (4) in their patients. The observation implies that "internal Na  escape" occurs with DOCA treatment and was achieved by osmotically inactive storage as shown by the concomitant increases in tissue Na -to-water ratio and the Na K -to-water ratio. Furthermore, we found an osmotically neutral Na /K  exchange, as evidenced by an increased Na  to water balance with concomitant decreases in the tissue K -to-water ratio. As a result, the Na K -to-water ratio remained almost unchanged despite massive Na  accumulation. The presence of both mechanisms challenges the traditional view of Na  balance (25). Osmotically inactive Na  storage questions the notion that extracellular Na  accumulation inevitably leads to water retention. Moreover, osmotically neutral Na /K  exchange challenges the view that Na  retention is restricted to the extracellular fluid volume. Strauss et al. (18) developed a kinetic concept of Na  excretion that has been the traditional view. However, Strauss et al. also commented that equating 140 mmol Na  retained with 1 liter of water retained in humans was an oversimplification (18).2 \1 O/ u% J2 A$ H" I
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Our idea of "internal Na  escape" receives support from earlier studies. Brands and Hall (3) found a moderate 1.2-liter increase in the sodium iothalamate space when dogs were given DOCA-salt despite a 450 mmol Na  retention. In rats, one of the most careful balance studies performed by Mhring and Mhring (14, 15) showed a circadian rhythm of DOCA escape. They found that after 4 days of DOCA-salt "sodium balance returned to control values from 7 PM to 7 AM and remained normal during the following days." That finding indicates Na  balance with DOCA escape during the locomotor period. However, the investigators reported further, "from 7 AM to 7 PM, sodium retention persisted" in their rats. The data reported by the Mhrings confirmed still earlier observations by Green et al. (7). These investigators reported persistent small amounts of Na  retention throughout a 4-wk DOCA treatment in rats. K  excretion is thought not to escape from DOCA-related effects (7). However, the question from where the K  originated or how water balance could be achieved during continuous DOCA activity, were K  losses not compensated for by Na  retention, remained unanswered. We believe that our data shed light on this question.

We did not perform balance studies in our rats. We doubt that we could top the meticulous experiments done by the Mhrings (14, 15). However, in our study, we abandoned the "black box" approach necessarily resulting from balance studies. Instead, we looked inside the box. We confirmed our recent observations on skin as a Na  storage depot. Skin accounted for 25% of the depot, whereas K  was lost from skin. However, skin was not the major site in our study. In muscle, DOCA-salt resulted in Na  accumulation that was balanced by K  losses. Increases in intracellular muscle Na  concentrations in DOCA rats have been reported earlier (5, 9, 10, 24). We were not able to separate the complete muscle mass in our studies and therefore could not assess the total muscle contribution. We furthermore did not determine the extra- and intracellular muscle volume in this experiment and hence cannot provide experimental data on the intracellular Na  concentration in the muscle in DOCA-treated rats. However, we nonetheless suggest that muscle provides an osmotically neutral Na  storage site by means of Na /K  exchange. Bone as a storage site has been studied much earlier (1, 2). We found that water-free bone Na  storage was a relatively minor component. Bone Na  gain was accompanied by bone K  loss.
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We observed that DOCA-salt rats exhibited growth retardation. The treatment resulted in decreased dry weight of the skinned carcasses and decreased bone ash to 90% of control values. Skin mass was reduced to 79% of controls. The reduced skin mass was responsible for the small skin K -to-total body K  ratio exhibited by the DOCA-salt group, despite an unchanged skin K  content relative to skin mass. Conceivably, the continuous K  losses reported for DOCA-salt rats coincide with a reduced cellular mass; however, this hypothesis remains to be tested. Also unanswered by our study is what the fate of Cl– might be. Kurtz and Morris (12, 13) showed earlier that only Na  as the Cl– salt causes hypertension in the DOCA-salt model, whereas equimolar amounts of NaHCO3 do not. That DOCA-salt results in hypertension is well known. Focusing on salt and water balance, we did not measure blood pressure in this experiment. The relationship between Na  (as the Cl– or the HCO3– salt), body fluids, and hypertension in DOCA rats remains to be investigated.
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Although the kinetics of Na  and K  balance in rats and humans may be different, we believe that our data could have clinical implications. They address the discrepancies observed in earlier balance studies (6, 8, 11, 17, 21), and they may explain the insightful speculations raised by Farber and Soberman regarding Na  storage in edematous states (4). Furthermore, we believe that water-free Na  accumulation should be taken into consideration in the treatment of dysnatremias (16). Finally, our data may provide an additional avenue in the study of Na -related effects on blood pressure.
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GRANTS
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This work was supported by grants from the Else Krner-Fresenius-Stiftung and the Erlanger Leistungsbezogene Anschubfinanzierung und Nachwuchsfrderung fonds to J. Titze.
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ACKNOWLEDGMENTS

The authors thank B. Weigel for help with the animal experiments and E. Prell for help with the ashing procedures.

FOOTNOTES

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.

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