Osmotically inactive skin Na storage in rats - 《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 13:49 |只看该作者 |倒序浏览 |打印

作者：Jens Titze, Rainer Lang, Christoph Ilies, Karl H. Schwind, Karl A. Kirsch, Peter Dietsch, Friedrich C. Luft,  Karl F. Hilgers作者单位：1 Department of Nephrology, Friedrich-Alexander University Erlangen-Nürnberg, D-91054 Erlangen; Department of Chemistry and Physics, Federal Center for Meat Research, D-95326 Kulmbach; Departments of Physiology and Biochemistry, Free University of Berlin, D-14195 Berlin; and Franz Volhard Clini
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      【摘要】! t! a2 u5 r' k/ w- [
   Compared with age-matched men, women are resistant to the hypertensive effects of dietary NaCl; however, after menopause, the incidence of salt-sensitive hypertension is similar in women and men. We recently suggested that osmotically inactive Na storage contributes to the development of salt-sensitive hypertension. The connective tissues, including those immediately below the skin that may serve as a reservoir for osmotically inactive Na storage, are affected by menopause. We tested the hypothesis that ovariectomy (OVX) might reduce osmotically inactive Na storage capacity in the body, particularly in the skin. Male, female-fertile, and female OVX Sprague-Dawley (SD) rats were fed a high (8%)- or low (0.1%)-NaCl diet. The groups received the diet for 4 or 8 wk. At the end of the experiment, subgroups received 0.9% saline infusion and urinary Na and K excretion was measured. Wet and dry weight (DW), water content in the body and skin, total body Na (rTBNa ) and skin Na (rSKNa ) content were measured relative to DW by desiccation and dry ashing. There were no gender differences in osmotically inactive Na storage in SD rats. All SD rats accumulated Na if fed 8% NaCl, but rTBNa was lower in OVX rats than in fertile rats on a low ( P P OVX decreased rSKNa ( P diet led to Na accumulation ( SKNa ) in the skin in all SD rats. Osmotically inactive skin Na accumulation was 66% of SKNa in female and 82% in male-fertile rats, but there was no osmotically inactive Na accumulation in OVX rats fed 8% NaCl. We conclude that skin is an osmotically inactive Na reservoir that accumulates Na when dietary NaCl is excessive. OVX leads to an acquired reduction of osmotically inactive Na storage in SD rats that predisposes the rats to volume excess despite a reduced Na content relative to body weight. $ @( R1 E6 u8 u% Q& a
      【关键词】 ovariectomy sodium reservoir total body sodium Dahl rats hypertension% v( B; O# ~" `( E% R% C8 |
   AFTER A high-NaCl diet, part of the Na that accumulates in the body does not equilibrate with body water but appears to be stored in an osmotically inactive form. We recently reported that reduced osmotically inactive Na storage capacity might contribute to the development of hypertension in Dahl rats ( 27 ). However, we could not precisely localize the compartment responsible for osmotically inactive Na storage. Potential osmotically inactive Na reservoirs in mammals may include bone, cartilage, and connective tissue ( 8 ). In our previous investigation, we observed that bone Na content was not responsible for the difference in osmotically inactive Na storage between Sprague-Dawley (SD) rats and both Dahl strains. These results drew attention to other potential Na reservoirs, for example, the skin and subcutaneous tissue. We therefore tested the hypothesis that the skin tissue Na concentration increases with high-NaCl consumption and/or increased total body Na  .. |! `) k$ a: `: X# x9 [: B

In both males and females, hypertension is an important cardiovascular risk factor; however, there are gender differences in the incidence of hypertension ( 22, 23 ). Whether there are gender differences in osmotically inactive Na storage in rats is not known. Compared with men, women develop high blood pressure, particularly systolic hypertension, at an increased rate as they age. Menopause exaggerates this age-related blood pressure increase. Compared with age-matched men, women are resistant to the hypertensive effects of dietary NaCl before menopause. However, thereafter the incidence of salt-sensitive hypertension is similar in women and men ( 17 ), underscoring the blood pressure-lowering effects of female sex hormones.
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Female sex hormones not only prevent bone loss ( 12 ), but they also play an important role in the generation of proteoglycans that are essential in the skin and subcutaneous tissue ( 15, 21, 25, 28 ). These proteoglycans may play a role in osmotically inactive Na storage. We speculated that ovariectomy (OVX) might lead to an aquired reduction in osmotically inactive Na storage capacity in the skin. This reduced osmotically inactive Na storage capacity would lead to a relative predominance of osmotically active Na in the body, with a consecutive volume and blood pressure increase. We therefore investigated the relationship between Na and water accumulation in the body and skin and its effect on blood pressure in fertile female, OVX rats and male SD rats to investigate gender differences in osmotically inactive Na storage. Our hypothesis was that OVX might lead to an aquired osmotically inactive Na storage incapacity and thus would predispose the rats to a volume-dependent blood pressure increase. Additionally, we reanalyzed the role of skin Na storage in male Dahl rats, presuming that a reduced skin Na storage capacity accounted for reduced osmotically inactive Na storage capacity in the rats.
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MATERIALS AND METHODS, i" ?' R0 s6 }( L/ ~$ ~

All animal experiments were done according to American Physiological Society guidelines and were duly approved by local government authorities.+ C, r9 a$ J/ \8 d6 _4 v) b
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Animals and Diets
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Female rats. Forty female SD rats (Charles River) were divided into four groups with similar body weight (BW) ( group 1 : 193.3 ± 3.6 g; group 2 : 198.5 ± 2.1 g; group 3 : 198.0 ± 4.0 g; group 4 : 197.1 ± 3.5 g) and fed a regular rat chow (0.61% NaCl) for 1 wk after arrival at our animal care facility. During week 1, the animals from groups 2 and 4 were ovariectomized (OVX). From week 2, the rats were fed different diets. Ten fertile ( group 1 ) and 10 OVX ( group 2 ) rats were fed a virtually Na -free diet ( wk. The other 10 fertile ( group 3 ) and 10 OVX ( group 4 ) rats were fed a high-Na diet (8% NaCl) for 8 wk. Both Na diets contained 0.95% Ca 2  and 0.70% K  .5 M7 w& y& Z. S7 I1 ^9 ~
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Male rats. Six male SD rats ( group 5, weight 246.8 ± 1.7 g) were fed a 0.1% NaCl diet and six male SD rats ( group 6, weight 244.3 ± 1.9 g) were fed an 8% NaCl diet for 4 consecutive wk. Additionally, we reanalyzed the role of skin Na content in 20 salt-sensitive (SS) and 20 salt-resistant (SR) male Dahl rats (Dahl-JR-Strain, M&B, Ry, Denmark), which had been killed in a previously reported experiment ( 27 ). Ten SS (264.0 ± 5.9 g) and 10 SR (263.5 ± 10.2 g) rats had been given 0.1% NaCl. The remaining 10 SS (BW 264.0 ± 10.0 g) and 10 SR (263.1 ± 7.24 g) rats had been fed a high-NaCl diet (8% NaCl) for 4 wk.8 V5 s( _3 K& k

Blood Pressure Measurement2 o2 j6 d" u  ~
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At the end of the experiment, the animals received intraperitoneal anesthesia with 100 mg/kg BW methohexital and the left femoral arteries were catheterized. Arterial lines were connected to Statham transducers and a Gould polygraph, and mean arterial blood pressure (MAP) was measured in the completely conscious animals kept in a restrainer.

Volume Expansion Protocol  j. r9 V. w" o. u# p7 ]. C$ \

Together with the arterial line, the left femoral vein was catheterized and a catheter was implanted in the urinary bladder in five rats per subgroup. The rats were placed in a restrainer and received an intravenous background infusion of 3.75 ml/h 0.9% NaCl. Four hours after the operation, urine was sampled for a 30-min control period in completely conscious animals. Then, the rats received a volume expansion (VE) with intravenous saline (0.9% NaCl, 5% of BW) within 30 min. Urine was sampled during the VE period and for an additional 90 min thereafter (post-VE diuresis).

Bone and Carcass Ashing( Q% I  [$ ~( t, b4 z# j

The intestines were completely removed from all carcasses to exclude remains of chow. Small slices of femur cartilage were cut and shock-frozen in liquid nitrogen. We removed both femurs from all animals for histological and radiological investigations. The carcasses were dry ashed according to a protocol described in detail previously ( 27 ). Water content was determined by desiccation of the carcasses and skin 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, all cervical, thoracic, lumbar and sacral vertebrae, 12 caudal vertebrae, the forelimb bones (without forefeet), the tibias, and the pelvis were removed and ashed seperately at 700°C for an additional 24 h. Then, each sample was dissolved 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 g 5% HNO 3.$ W6 H% {- y( d. ^5 L9 ?4 q
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Skin Ashing

Female rats. Six of 10 carcasses from groups 1 - 4 were skinned completely to determine total skin Na and skin water content. In the remaining four carcasses per group, we removed a piece of back skin to investigate Na and water content relative to skin weight.6 C/ c. k0 d  ~5 q4 c
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Male rats. All carcasses from male SD rats ( groups 5 and 6 ) were skinned completely to determine total skin Na and skin water content in the animals. In Dahl rats, we removed a piece of skin from the back (average WW 5.82 ± 0.37 g) for isolated water and Na determination.6 n/ B" y  e( Z  r/ q  m* B

All skin samples were dessicated at 90°C for 72 h. Skin water content was calculated from the difference between WW and DW. After dry ashing at 190°C for 24 h and 600°C for an additional 48 h, the skin ashes were dissolved in 20 ml 10% HNO 3.

Electrolyte Measurements
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Na concentrations in blood samples were measured with a flame photometer (model EFIX, Eppendorf, Hamburg, Germany). Na concentrations in the dissolved ashes were measured with flame photometry (model 3100, PerkinElmer, Rodgau, Germany).6 U0 N7 l* _# G
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Data Analysis
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Data are expressed as means ± SE. MAP, weights, water contents, and electrolyte concentrations were analyzed with multivariate analysis (GLM, SPSS, version 10.0). Post hoc tests were performed with the Bonferroni algorithm. Scatter plots and curve fitting were done with Origin software (version 6.0)./ j, o& A: i, h
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Na and water relationship. In contrast to female-fertile rats, increased body weight and redistribution of carcass composition with decreases of fat-free DW and increase of carcass fat are well-known effects of OVX in rats ( 16 ). Our previous osmotically inactive Na calculations in male rats were based on the relationship between Na and water content relative to WW ( 27 ), assuming a rather constant fat content in male rats. Assuming that Na and water balance takes place in fat-free body compartments predominately, increased carcass fat in OVX rats would lead to erroneous osmotically active Na estimations after correction for BW in OVX rats, if compared with fertile rats. Thus we investigated the relationship between changes in Na content and alterations of water content in skin without normalizing the data for skin weight by calculating the ratio between total skin Na (SKNa  ; mmol) and total skin water (SKW; ml) content in each rat

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Increased R (SKNa /SKW) indicated osmotically inactive Na storage in skin./ W9 r- |( z; M2 `9 n' t& `

We then estimated osmotically inactive skin Na storage without normalizing the data to body weight. On the basis of the assumption that water accumulation in skin ( SKW; ml) was due to osmotically active extracellular Na accumulation, the fraction of osmotically active was estimated from the relative skin water accumulation ( rSKW; ml/g), skin wet weight (SWW; g), and the serum concentration
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Osmotically inactive Na accumulation in skin was characterized by an increasing ratio between SKNa and SKW ( Eq. 1 ) on a high-NaCl diet compared with a low-NaCl diet [ R (SKNa /SKW); mmol/ml]. Thus the fraction of osmotically inactive Na accumulation was estimated from R (SKNa /SKW) with the help of the skin water content) g3 {1 i; }/ k
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( 3 )

Internal Na balance. In addition to the extracellular volume (ECV), skin and bone are other Na compartments in the body that contain Na in an osmotically inactive form. To determine changes in internal Na balance in the rats, we calculated the ratio between bone Na (mmol) in the removed bones and total body Na (TBNa  ; mmol) in each animal. Accordingly, the ratio between total skin Na (SKNa  ; mmol) and TBNa in the completely skinned carcass was calculated
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( 4 )

An increased R (TSKNa /TBNa ) indicated TBNa redistribution in favor of the skin.+ l: ?, d" v; [6 d7 K- Z$ N
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Skin Na Storage in Dahl Rats$ D% n3 W. f/ K& v  y
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Initially focusing on the role of bone in osmotically inactive Na storage, we only removed a piece of back skin in Dahl rats. Thus the role of skin in internal Na balance could not be measured in Dahl rats by separating total skin Na and total body Na as indicated in equation 4. We thus investigated internal Na balance in Dahl rats by normalizing total body Na (rTBNa  ; mmol/g DW) and skin Na content (rSKNa  ; mmol/g DW) relative to tissue DW
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( 5 )

An increased R (rTSKNa /rTBNa ) would thus indicate TBNa redistribution in favor of the skin." h1 ?8 j, p7 b. H) [! R0 y' ^" d. O
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RESULTS

Body Weight, Bone Ash Mass, and Bone Na

Gender differences. Body weight ( Table 1 ) was higher in male than in female-fertile rats, whereas bone ash mass and bone Na content relative to DW ( Table 1 ) were lower in male than in female SD rats. Relative bone Na content was unchanged in female rats fed 8% NaCl. In male rats, NaCl consumption increased relative bone Na content not because of inorganic Na storage in bone, but due to decreased carcass DW and stable bone mass in rats fed 8% NaCl.7 d4 ]% ~: b/ o  R6 O0 ^

Table 1. Descriptive statistics on body weight, bone mass, and bone Na in fertile and OVX Sprague-Dawley rats& N% _: E- |! {

OVX. OVX led to increased WW, DW, and bone ash mass in female rats ( Table 1 ), whereas relative bone ash mass was decreased. OVX rats fed 8% NaCl increased bone ash mass, whereas carcass weight was decreased. Thus bone Na content relative to BW was increased in OVX rats fed 8% NaCl. The ratio between bone Na and TBNa was unchanged in fertile and OVX rats fed 8% NaCl, indicating that Na distribution between bone and the other Na compartments of the body was not altered on different diets.% }3 J/ g+ v9 A8 C5 G# V0 }

Total Body Na  , Skin Na  , and Tissue Water Content( [8 ]! [' H. m
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Gender differences. Both genders increased total body Na relative to body mass (rTBNa  ; mmol/g DW) if fed a high-NaCl diet ( Fig. 1 A ). In contrast to female rats, male rats did not accumulate water on a high-NaCl diet ( Fig. 1 C ). Corresponding gender differences were apparent in relative skin Na (rSKNa  ; mmol/g DW) in the rats ( Fig. 1, B and D ). Relative skin water content (rSKW; ml/g WW) was increased in female rats fed 8% NaCl ( Fig. 1 D ) and was also higher in male rats fed a high-salt diet [GLM: P (diet) = 0.06; nonparametric Mann-Whitney test: P (diet)2 C; I1 I# _3 U. Q
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Fig. 1. A : total body Na [rTBNa  ; mmol/g dry weight (DW)] relative to DW. B : relative skin Na (rSKNa  ; mmol/g DW). C : total body water [rTBW; ml/g wet weight (WW)] relative to WW. D : relative skin water (rSKW; ml/g WW) in fertile female and male or ovariectomized (OVX) Sprague-Dawley (SD) rats fed 0.1 or 8% NaCl. * P (diet) P (diet) P (OVX) P (OVX) P (OVX)6 Q, @1 ^4 c. ^

OVX. rTBNa and rTBW were lower in OVX rats than in fertile rats [ P (OVX) rats increased relative body Na and water on a high-salt diet. rSKNa and rSKW were lower in OVX rats than in fertile rats [ P (OVX) diet increased Na and water content in the skin of OVX rats.

Na and Water Distribution in the Body
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Gender differences. Na and water distribution in the body were not gender specific. In both fertile female and male SD rats, dietary NaCl loading predominantly increased the skin Na content ( Fig. 2 A ) as indicated by an increased ratio between SKNa (mmol) and TBNa (mmol). Although body Na had been preferably distributed into skin in fertile rats of both genders, the relationship between total SKW and total water content in the completely skinned rest carcasses [R (SKW/TBW); Fig. 2 B ] was unchanged, indicating that Na had been stored in skin in an osmotically inactive form.
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Fig. 2. A : ratio (R) between total skin Na (SKNa  ; mmol) and total body Na (TBNa  ; mmol). B : ratio between total skin water (SKW; ml) and total body water (TBW; ml). C : ratio between skinned carcass Na (CNa  ; mmol) and water content (CW; ml). D : ratio between SKNa (mmol) and SKW (ml). Completely skinned female-fertile, OVX, and male-fertile SD rats fed 0.1 or 8% NaCl. * P (diet) P (diet) P (diet) P (OVX)0 Z: y5 \, F1 g2 ]/ E6 y  U2 V
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OVX. R (SKNa /TBNa ) was higher in OVX rats than in fertile rats on a low-salt diet. In contrast to fertile rats, R (SKNa /TBNa ) was unchanged in OVX rats fed 8% NaCl ( Fig. 2 A ). Similar to fertile rats, R (SKW/TBW) was unchanged in OVX rats fed 8% NaCl ( Fig. 2 B ).
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Carcass and Skin Na Content and Its Relationship to Water Content

Gender differences. As judged from the relationship between Na and water in the carcasses, both genders fed 8% NaCl identically accumulated skin Na ( Fig. 2 D ) in an osmotically inactive form, as indicated by an increased ratio between absolute Na (mmol) and water (ml) content. There was no gender difference in Na -induced water accumulation between the rats, neither in the skinned carcasses [ P (gender) 0.1; Fig. 2 C ] nor in the skin [ P (gender) 0.1; Fig. 2 D ].# ]% h8 }  ?2 U2 ^, \" A1 y
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OVX. The relationship between carcass Na (CNa  ; mmol) and carcass water (CW; ml) was lower in OVX than in female-fertile rats, indicating reduced osmotically inactive Na storage in OVX skinned carcasses irrespective from the diet ( Fig. 2 C ). In contrast to fertile rats, high-NaCl consumption was not associated with osmotically inactive Na storage in OVX rats, as indicated by an unchanged R (SKNa /SKW) in OVX rats fed 8% NaCl ( Fig. 2 D ).8 X: J6 P4 ~! P  u5 u" W  h
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Osmotically Inactive Na Storage in Skin1 S! u. w8 m3 v6 Q- T

In female-fertile rats fed 8% NaCl, the fraction of osmotically active skin Na accumulation, according to Eq. 2 ( Fig. 1 D, Table 2 ), was 0.053 ml/g x 44.5 g x 0.152 mmol/ml = 0.36 mmol in the average rat skin. According to Eq. 3 ( Fig. 2 D, Table 2 ), the fraction of osmotically inactive Na accumulation was 0.027 mmol/ml x 25.6 ml = 0.69 mmol in the average rat skin. In male-fertile rats, was 0.020 ml/g x 66.0 g x 0.156 mmol/ml = 0.21 mmol in the average rat skin. was 0.024 mmol/ml x 39.1 ml = 0.94 mmol in the average rat skin. In OVX rats, where no osmotically inactive Na accumulation was identified in skin [as indicated by an unchanged R (SKNa ) on a high-salt diet], was 0.077 ml/g x 58.5 g x 0.155 mmol/ml = 0.70 mmol in rats fed 8% NaCl in the average rat skin. In summary, osmotically inactive skin Na accumulation was 66% in female- and 82% in male-fertile rats, whereas there was no osmotically inactive Na accumulation in OVX rats fed 8% NaCl ( Fig. 3 ).
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Table 2. SWW, SKW, and serum Na concentration, R( K3 k' g, {6 d' Y
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Fig. 3. Osmotically inactive and osmotically active Na accumulation in skin ( SKNa  ; mmol/total skin) in fertile female, OVX, and fertile male SD rats fed 8% NaCl.6 ^9 @6 d. Z' D- Y( `  O
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Total Body Na and Water and Its Relationship to Blood Pressure

As indicated in Table 1, there was no dietary or OVX effect on average blood pressure in the rats. However, multivariate comparison of means of dietary or OVX effects on blood pressure does not consider the individual impact of increasing body Na content on total body water and blood pressure in the rats. Figure 4 A shows the relationship between rTBNa and MAP (mmHg) in OVX and fertile rats. Increased rTBNa correlated directly with MAP in OVX rats, but not in fertile rats. Additionally, there was a direct correlation between rTBW and MAP in OVX rats ( Fig. 4 B ), whereas there was no relationship between water retention and MAP in fertile rats. Similar to fertile female rats, there was no direct correlation between rTBNa or rTBW and MAP in male SD rats (data not shown)." @( s* x- O" M: I: ?7 ^
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Fig. 4. A : relationship between rTBNa (mmol/g DW) and mean arterial blood pressure (MAP; mmHg). B : relationship between rTBW (ml/g WW) and MAP in fertile or OVX SD rats fed 0.1 or 8% NaCl.& i. E: j! r3 i* b1 t) s
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Skin Na Content and Natriuresis During Saline Infusion
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Table 3 shows natriuresis (U Na V; mmol/g WW) and Na accumulation in OVX and fertile female and male rats during saline infusion. The rats received a continuous 3.75-ml/h saline background infusion and a 30-min VE with 5% BW 0.9% NaCl. U Na V was measured 30 min previous to VE (control), during 30 min VE, and 90 min after VE had been stopped (post). U Na V in response to mild saline background infusion previous to VE was affected by the previous dietary regime in female- and male-fertile rats ( Table 3 ), but not in OVX rats. Fertile rats fed a low-NaCl diet tended to accumulate Na  , whereas Na balance in fertile rats fed 8% NaCl was negative during background infusion ( rTBNa control). Within 90 min after acute VE had been stopped, there was no significant Na accumulation in all groups of rats.

Table 3. Natriuresis, Na accumulation, urinary Na concentration, and urinary K concentration during 3.75 ml/h 0.9% saline background infusion, acute VE, and sampled for 90 min after VE

In fertile rats of both genders, osmotically inactive skin Na content [as indicated by an increased R (SKNa /SKW) ] correlated positively with urinary Na concentration (U Na; mmol/l; Fig. 5 A ) and negatively with urinary K concentration (U K; mmol/l; Fig. 5 C ) during mild saline infusion previous to VE. In contrast, there was no correlation between R (SKNa /SKW) and U Na ( Fig. 5 B ) in OVX rats, whereas a negative correlation between R (SKNa /SKW) and U K was still evident in OVX rats ( Fig. 5 D ). Correspondingly, during acute VE, R (SKNa /SKW) and U Na correlated directly in both female ( P P whereas there was no correlation between R (SKNa /SKW) and U Na in OVX rats ( P 0.1; data not shown).$ B3 o% P, t! t6 n- |/ O

Fig. 5. Relationship between R (SKNa /SKW) (mmol/ml) and urinary Na concentration (U Na; mmol/l) in fertile female and male SD rats ( A ) and OVX SD rats ( B ). Relationship between R (SKNa /SKW) and urinary K concentration (U K; mmol/l) in fertile female and male SD rats ( C ) and OVX SD rats ( D ). U Na and U K measured during 30-min mild saline infusion (3.75 ml/h). The scatter points in brackets have not been taken into consideration for curve fitting.

Skin Na Storage in Dahl Rats
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Figure 6 A shows the skin Na content (rSKNa ) relative to skin DW in SS and SR Dahl rats fed a high- or a low-NaCl diet. rSKNa was unchanged in SS and SR rats fed 8% NaCl. The relationship between rSKNa and total body Na [R (rSKNa /rTBNa ); mmol/g skin DW and mmol/g total body DW] was decreased in SS rats and unchanged in SR rats fed 8% NaCl ( Fig. 6 B ). In contrast to fertile SD rats ( Fig. 2 D ), there was no significant osmotically inactive Na storage in skin in Dahl rats, as indicated by an unchanged R (SKNa /SKW) ( Fig. 6 C ). In contrast to fertile SD rats, there were neither positive correlations between R (SKNa /SKW) and U Na ( P 0.1) nor negative correlations between R (SKNa /SKW) and U K ( P 0.1) in Dahl rats (data not shown).
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Fig. 6. A : rSKNa (mmol/g DW). B : ratio between rSKNa (mmol/g skin DW) and rTBNa (mmol/g total body DW). C : ratio between SKNa (mmol) and SKW (ml) in salt-resistant (SR) and salt-sensitive (SS) Dahl rats fed 0.1 or 8% NaCl. * P (diet)2 |* S" R7 B# d2 k3 K+ H8 ]+ U

DISCUSSION
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Our data indicate that skin Na storage plays a central role in osmotically inactive Na metabolism in rats. Fertile SD rats of both genders reacted to dietary Na loading with osmotically inactive Na storage in skin ( Fig. 1 ). Total body Na was distributed in favor of the skin ( Fig. 2 A ), whereas total body water distribution was unchanged ( Fig. 2 B ) in SD rats fed 8%, indicating a shift of internal Na balance in favor of skin as an osmotically inactive Na reservoir. The data support the notion that, in NaCl excess, extracellular volume homeostasis is not only maintained by osmotically active Na excretion through the kidneys but also by osmotically inactive Na storage in skin and internal Na balance redistribution in favor of osmotically inactive Na reservoirs.
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We additionally investigated inherited and acquired osmotically inactive Na storage incapacity in rats. As a model of inherited Na storage incapacity, we recently reported that male Dahl rats display a reduced osmotically inactive Na storage capacity ( 27 ), but we could not precisely localize the compartment responsible for osmotically inactive Na storage. Reanalyzing our data, we thus focused on skin Na storage in Dahl rats. Na storage in skin was deficient in Dahl SS and SR rats compared with SD rats. In both Dahl strains, high NaCl did not increase skin Na content ( Fig. 6 A ) and there was no osmotically inactive Na storage in reaction to a high-salt diet in Dahl rats ( Fig. 6 C ). Thus Na excess in Dahl SS rats led to Na redistribution in favor of osmotically active Na compartments (the details have been outlined earlier; 27 ), and internal Na balance was redistributed in favor of Na compartments other than skin ( Fig. 6 B ). This reduced osmotically inactive Na storage capacity predisposed the rats to a volume-sensitive blood pressure increase.: a7 k  B6 Y' n: V! B" p& J& s* b; A0 b
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As a model of acquired osmotically inactive skin Na storage capacity, we ovariectomized female SD rats. In contrast to Dahl rats, both fertile and OVX rats accumulated Na in their skin if fed 8% NaCl ( Fig. 1 ). However, as indicated by an unchanged SKNa /SKW ratio in rats fed a high-salt diet ( Fig. 2 D ), there was less osmotically inactive skin Na accumulation on a high-NaCl diet in OVX rats compared with fertile rats. Correspondingly, the relationship between Na and water in the skinned carcasses was lower in OVX rats than in fertile rats ( Fig. 2 C ). The reduced osmotically inactive Na storage capacity in OVX rats' skin was associated with an unchanged SKNa /TBNa ratio in the body, whereas intact osmotically inactive skin Na storage in fertile rats fed a high-salt diet was associated with an internal Na balance shift into skin as indicated by an increased SKNa /TBNa ratio ( Fig. 2 A ).; S( M5 p2 f6 g7 t8 v( Y+ E2 w

Increased natriuresis and decreased kaliuresis characterize a suppression of the renin-angiotensin-aldosterone system. In fertile SD rats, increased osmotically inactive Na storage in skin correlated directly with urinary Na concentration ( Fig. 5 A ) and inversely with urinary K concentration ( Fig. 5 C ) during controlled saline infusion experiments. We conclude that not only the circulating volume but also the Na content in osmotically inactive Na reservoirs may be operative in the regulation of body volume and total body Na  . Furthermore, the Na content in osmotically inactive Na reservoirs influenced natriuresis and not only the volume of the isotonic saline infused. Thus natriuresis (as the effector of Na homeostasis) is not only regulated by the circulating volume but also by the Na content in osmotically inactive Na reservoirs. Na homeostasis is regarded to be regulated by changes in the extracellular fluid volume. In contrast to osmoregulation, where the system has a set point at a plasma osmolality, and the effector is vasopressin-mediated water excretion within minutes or hours, Na -regulatory mechanisms operate with a remarkable sluggishness ( 14, 29 ). The very existence of a set point for total body Na has been a matter of debate ( 1, 10 ). This debate was based on the assumption that changes in total body Na are always changes in extracellular volume. Our data indicate a dissociation of Na homeostasis and volume homeostasis. Na and volume excess in rats is characterized by renal Na and water excretion and water-free Na accumulation in the skin. Thus Na homeostasis is only partially characterized by volume homeostasis. We conclude that discussions on the existence of a set point for total body Na should include the potential role of osmotically inactive Na reservoirs and discriminate between volume regulation and Na homeostasis.

In contrast to fertile rats, there was no direct correlation between skin Na storage and urinary Na excretion in OVX, although an increased SKNa /SKW ratio and urinary K concentration still correlated negatively ( Fig. 5 ). We conclude that the close functional relationship between volume regulation and skin Na storage was disturbed in OVX rats. The reduced osmotically inactive Na storage capacity in OVX rats was characterized by a TBNa -associated total body water increase. This total body water increase correlated directly with blood pressure in OVX rats ( Fig. 4 ). Blood pressure is sensitive to alterations of female sex hormones ( 23 ). Estradiol substitution tends to lower blood pressure in postmenopausal women ( 7 ), whereas the extent of postmenopausal osteoporosis coincides with hypertension in women ( 2 ). OVX deteriorates or induces salt-sensitive hypertension in Dahl SS and spontaneously hypertensive rats ( 9, 20 ), and estradiol substitution was reported to attenuate hypertension in OVX SS Dahl rats ( 24 ). The pathophysiological approach toward the effect of estradiol on blood pressure has focused on the effects of the sex hormone on vascular resistance ( 7 ), vascular tone ( 3 - 5 ), vascular growth ( 6, 13 ), the sympathetic nerve system ( 9, 11, 26 ), and estradiol-induced renoprotection ( 18, 19 ). Our data suggest that a reduced osmotically inactive Na storage capacity in skin was present in OVX rats, raising still another possible estradiol-related effect. Although there was no significant dietary effect on blood pressure in OVX rats ( Table 1 ), we speculate that the occurrence of a volume-sensitive blood pressure reaction might be one factor contributing to the multifactorial pathophysiological puzzle leading to hypertension. Of interest, the direct correlation between total body water and blood pressure occurred in OVX SD rats with the lowest rTBNa and thus lowest relative total body water. We conclude that the ability to partially osmotically inactivate TBNa  , and not the absolute TBNa value itself, might play an important role in the relationship between fluid volume and blood pressure.1 Y5 h* M, a! k2 E2 t0 s! U% |! q
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In summary, our data demonstrate an inherited incapacity in (male) Dahl rats to transfer Na into skin in salt excess and an acquired incapacity to osmotically inactivate Na in OVX rats. Osmotically inactive Na storage incapacity in skin predisposed the rats to a volume-sensitive blood pressure increase. Whether this concept is transferable to OVX experiments in female salt-sensitive models (such as Dahl or spontaneously hypertensive rats) remains to be investigated. In conclusion, we suggest that impaired capacity for osmotically inactive Na storage may be an alternative or additional factor contributing to the salt-sensitive increase in blood pressure after menopause.! w, W# w# j2 F+ q" j

DISCLOSURES1 E2 p9 ^" c' p- G( P; e
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This work was made possible by grant support of the Else Kröner-Fresenius-Stiftung (project Langsame Natriumspeicher ) and the Deutsche Nierenstiftung (supported from Ortho Biotech Janssen-Cilag; project Auswirkung von OVX auf osmotisch inaktive Na-Speicher ) to J. Titze." h1 q( q6 I5 o. d

ACKNOWLEDGMENTS
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The authors thank B. Weigel and S. Böhm for help with the animal experiments and E. Prell and H. Mohs for help with the inorganic analyses. H. Hecht was mainly involved in the development of the ashing procedures.0 j/ V( X/ k7 u
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