1 Department of Nephrology, Friedrich-Alexander-University Erlangen-Nürnberg, D-91054 Erlangen; 2 Department of Chemistry and Physics, Federal Center for Meat Research, D-95326 Kulmbach; and Departments of 3 Biochemistry and 4 Physiology, Free University of Berlin, D-14195 Berlin, Germany
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent evidence suggested that Na can be stored in an osmotically inactive form. We investigated whether osmotically inactive Na storage is reduced in a rat model of salt-sensitive (SS) hypertension. SS and salt-resistant (SR) Dahl-Rapp rats as well as Sprague-Dawley (SD) rats were fed a high (8%)- or low (0.1%)-NaCl diet for 4 wk (n = 10/group). Mean arterial pressure (MAP) was measured at the end of the experiment. Wet and dry weights, water content, total body Na (TBS), and bone Na content were measured by dessication and dry ashing. MAP was higher in both Dahl strains than in SD rats. In SS rats, 8% NaCl led to Na accumulation, water retention, and hypertension due to impaired renal Na excretion. There was no dietary-induced Na retention in SR and SD rats. TBS was variable; nevertheless, TBS was significantly correlated with body water and MAP in all strains. However, the extent of Na-associated volume and MAP increases was strain specific. Osmotically inactive Na in SD rats was threefold higher than in SS and SR rats. Both SS and SR Dahl rat strains displayed reduced osmotically inactive Na storage capacity compared with SD controls. A predisposition to fluid accumulation and high blood pressure results from this alteration. Additional factors, including impaired renal Na excretion, probably contribute to hypertension in SS rats. Our results draw attention to the role of osmotically inactive Na storage.
salt sensitive; bone sodium; Dahl rats
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE RELATIONSHIP BETWEEN SALT intake and blood pressure has been extensively studied in humans and provides evidence that subpopulations of humans are sensitive to alterations in salt intake. A well-established animal model for salt-sensitive (SS) hypertension is the Dahl rat. SS rats develop hypertension when fed a high-NaCl diet (6). Salt-resistant (SR) Dahl rats developed increased blood pressure after they received a transplanted kidney from an SS donor animal. Conversely, transplantation of SR donor kidneys into SS rats lowers blood pressure (5, 10). Despite the fact that the glomerular filtration rate is not different between both strains (2, 14), SS rats have an impaired ability to excrete Na (1, 2). However, this feature does not fully explain the development of SS hypertension (11). Thus the role of Na retention in the development of hypertension is unclear.
Recent studies on long-term Na balance in humans showed that high dietary Na consumption with Na retention does not necessarily lead to expansion of the extracellular volume (8). This finding suggests that Na might be stored in an osmotically inactive form. We studied the relationship between osmotically inactive Na storage, total body Na (TBS), total body water (TBW), and hypertension in Dahl SS rats and control animals. Our initial primary hypothesis was that Dahl SS rats exhibit a reduced capacity for osmotically inactive Na storage that would predispose these animals to volume retention and thus lead to SS hypertension. The secondary hypothesis was that osmotically inactive Na storage is also deficient in Dahl SR rats, which are genetically distinct from the Sprague-Dawley (SD) founder strain (12) and exhibit higher blood pressure than the founder strain. To determine Na and water distribution under high-dietary Na consumption, we investigated SS and SR Dahl rats (and SD rats as an additional control).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
Animals and diets. Male Dahl rats [20 SS and 20 SR; Dahl JR strain (inbred strain), M&B, Ry, Denmark] were fed regular rat chow (0.61% NaCl) for 1 wk after arrival at our animal care facility. Then, the animals were divided into four subgroups. Group 1 (10 SS, 264 ± 5.9 g) and group 2 (10 SR, 263.1 ± 10.2 g) were were fed a practically NaCl-free diet (<0.1% NaCl). Group 3 (10 SS, 264 ± 10 g) and group 4 (10 SR, 263.1 ± 7.24 g) were fed a high-NaCl diet (8% NaCl) for 4 wk. Both diets contained 0.95% calcium and 0.70% potassium.
Cages and metabolic measurements. Five animals of each subgroup were kept in metabolic cages. The animals were weighed daily, and daily food and water intake as well as urine output were monitored. The remaining animals were kept in regular cages. All rats received water and food ad libitum. The cages were located in a room with constant temperature (22 ± 2°C) and humidity (60 ± 5%) and a 12:12-h light-dark cycle.
Controls. Ten male SD rats (control group 1, 248.8 ± 1.9 g) were fed a 0.1% NaCl diet. Ten SD rats (control group 2, 250.3 ± 1.6 g) were fed an 8% NaCl diet. The controls were kept in regular cages for 4 wk.
Blood pressure measurement. After 4 wk on their specified diets, the animals received intraperitoneal anesthesia with 100 mg/kg body wt methohexital, the left femoral arteries were catheterized, and blood samples were taken. Intra-arterial lines were connected to Statham transducers and a Gould polygraph, and blood pressure was measured 50-65 min after anesthesia, when oscillating signals occured before complete consciousness, and then 150 min later in completely conscious animals to validate the initial measurements.
Ashing procedures.
Although subtle protocols for Na balance studies in rats exist for
animals fed regular chow (9), cumulative Na balance calculations are not precise enough to investigate changes in TBS
during our long-term experiment. When fed a high-NaCl diet (8% NaCl),
rats very often suffer diarrhea. In this case, a mixture of chow,
stool, and urine with high-Na content sticks in the cages and accounts
for large sampling errors. Therefore, we developed a dry ashing
protocol to investigate TBS in rats. We removed both femurs and tibias
and a piece of back fur from all animals for future histological and
computertomographic investigations. Intestines were completely removed
to exclude remains of chow. The carcasses were weighed [wet weight
(WW)] and then dessicated at 90°C for 72 h [dry weight (DW)].
Because further drying left weights unchanged, we considered the
difference between WW and DW as TBW
![]() |
(1) |
Electrolyte measurements. Na concentrations ([Na]) in urine and blood samples were measured with a flame photometer (model EFIX 5055; Eppendorf, Hamburg, Germany). [Na] in dissolved ashes was measured with flame photometry (model 3100, PerkinElmer, Rodgau, Germany).
Data analysis. Data are expressed as means ± SE. Mean arterial blood pressures (MAP), weights, water contents, and electrolyte concentrations were analyzed with multivariate analysis (general linear model). Post hoc tests were performed with the Bonferroni algorithm. All comparisons of means were analyzed with SPSS software (version 10.0). Curve fitting in scatterplots was done with ORIGIN software (version 6.0).
Osmotically inactive Na determination.
Figure 1 gives an example of Na
content and water content in rats fed a 0.1 or 8% NaCl chow. Na
content [TBS(dry)] is given as millimoles per gram DW,
and water content [TBW(%)] is given as the percentage of
WW. Dietary-induced Na accumulation (TBS) is associated with
increased body water content (
TBW).
|
![]() |
(2) |
![]() |
(3) |
![]() |
(4) |
![]() |
(5) |
![]() |
(6) |
![]() |
(7) |
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Electrolyte and water contents.
Weights and calculated water contents are shown in Fig.
2. High-NaCl consumption led to stunted
growth in Dahl SS (78.7 ± 5.39 vs. 128.1 ± 4.42 g DW,
P < 0.001) and SD rats (113.2 ± 4.17 vs.
123.63 ± 2.46 g DW, P < 0.05) but not in
Dahl SR rats.
|
Electrolyte balance studies.
Figure 3 shows average daily natriuresis
(µmol/g DW) and Na intake (µmol/g DW). On a 0.1% NaCl diet, there
was no difference in natriuresis between SS and SR rats. On an 8% NaCl
diet, SR rats ingested 5.6% more NaCl than did SS rats (109.8 ± 1.43 vs. 104.0 ± 1.57 µmol/g, P < 0.01).
Natriuresis was 15.3% higher in SR rats (107.3 ± 1.06 vs.
93.1 ± 1.79 µmol/g, P < 0.001).
|
Arterial blood pressure. Figure 2D shows MAP measured 50-65 min after anesthesia, when oscillating blood pressure signals appeared. In all strains, average MAP was not different when measured 150 min later in completely conscious animals.
Fed 0.1% NaCl, SD rats had the lowest MAP (112.2 ± 4.06 vs. 133.0 ± 3.74 mmHg in SR rats, P < 0.01, and 165.00 ± 11.95 mmHg in SS rats, P < 0.01). On 0.1% NaCl, MAP in SR rats was lower than in SS rats (P < 0.05). Fed 8% NaCl, all strains again had a different MAP. SD rats had the lowest MAP (122.8 ± 4.29 vs. 139.4 ± 4.06 mmHg in SR rats, P < 0.05, and 229.5 ± 7.69 mmHg in SS rats, P < 0.001). On 8% NaCl, MAP in SR rats was lower than in SS rats (P < 0.001). A high-NaCl diet increased MAP in the SS rats (229.5 ± 7.69 vs. 165.0 ± 11.95 mmHg, P < 0.001) but not in the SR or SD strains.Na storage.
Serum [Na] was 144.3 ± 0.39 mmol/l in all pooled rats (no
differences between strains and diets, P > 0.1).
Increased body Na content went along with increased body water in all
strains (Fig. 4A). However,
the extent of Na-associated fluid accumulation showed immense
variations.
|
Na and water content and their association with blood pressure. Body Na content and blood pressure in individual rats were correlated significantly in all strains (Fig. 4C). Again, the slope of the Na content/blood pressure curve differed between strains. In SS rats, Na accumulation from 0.139 to 0.240 mmol/g increased MAP by 64.5 mmHg, from 165 to 229.5 mmHg (P < 0.001). In SR rats, increased TBS from 0.147 to 0.179 mmol/g increased MAP from 130 to 143.1 mmHg (P = 0.01). In SD rats, TBS elevation from 0.143 to 0.180 mmol/g increased MAP from 111.2 to 124.1 mmHg (P < 0.05). Respectively, a 0.01 mmol/g TBS increase elevated MAP by 6.4 mmHg in SS rats, 4.1 mmHg in SR rats, and 3.5 mmHg in the SD strain.
Increased body water content was associated with increased blood pressure in all strains (Fig. 4D). The extent of volume-associated hypertension showed different strain specificity. In SS rats, increased water content from 60.7 to 69.7% increased MAP from 165 to 229.5 mmHg. In SR rats, increased water content from 62.2 to 65.4% increased MAP from 130 to 143.1 mmHg. In SD rats, TBW elevation from 64.9 to 66.6% increased MAP from 111.2 to 124.1 mmHg. Respectively, TBW expansion by 1% was associated with 7.2 mmHg blood pressure increase in SS rats and 7.6 mmHg in SD rats. In SR rats, the effect of elevated water content was small. One percent increases in TBW were associated with only a 4.1-mmHg blood pressure increase.Bone Na contents. Bone Na can be regarded as a potent osmotically inactive Na compartment. To investigate its contribution to TBS(i), we determined the bone Na content in the rats.
With regard to low-salt diets, bone Na content per gram of rat DW was similar in both strains of Dahl rats but higher than in SD rats (P < 0.05 and < 0.001, respectively; Fig. 5A). In rats fed 8% NaCl, SR and SD bone Na did not differ significantly, whereas bone Na content in SS was higher (P < 0.001). Within groups of rats, high dietary NaCl consumption coincided with elevated skeletal Na contents in SS (39.9 ± 1.90 vs. 27.8 ± 1.55 µmol/g, P < 0.001) and also in SD rats (27.7 ± 1.61 vs. 24.8 ± 1.38 µmol/g, P < 0.001) but not in SR rats. Similar to TBS, in SR rats bone Na content showed large variations that were independent from the diet. Increased TBS increased bone Na in all rats (Fig. 5C).
|
Na distribution and its association with fluid volume and blood
pressure.
A high-NaCl diet led to Na redistribution in SS, but not in SR and SD,
rats. In SS rats, 8% NaCl reduced the bone Na fraction of TBS (Fig.
5B). Such Na-altered distribution in the SS strain was
associated with parallel changes in fluid volume and blood pressure
(Fig. 6). Decreased relative bone Na
content observed in individual SS rats under different dietary regimes
was associated with body fluid expansion and increased MAP. In
SR and SD rats, there was no correlation between relative bone Na
content and body fluids or blood pressure (data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
High dietary NaCl consumption led to Na retention in SS rats but not in SR and SD strains. These findings confirm the data from Tsunooka and Morita (15) in Iwai Dahl inbred strains, whereas Schackow and Dahl (13) originally reported that a high-salt diet had no effect on TBS in SS rats but did affect TBS in SR rats. Our data also confirm the finding that differences between Dahl strains are characterized by a reduced ability to excrete Na loads in SS rats (1, 2), with subsequent Na accumulation and hypertension. Although Na accumulation increased bone Na by ~43% (Fig. 3A), skeletal Na storage capacity was inadequate to cope with Na overload in SS rats. This state of affairs led to an altered Na distribution between bone and extracellular space in these rats. Furthermore, salt-induced volume expansion led to elevated blood pressure in SS rats compared with the SR strain. This observation points to the previously reported (7, 11) notion that, in addition to altered salt-induced volume expansion, volume-independent aspects contribute to the development of Dahl SS hypertension.
Irrespective of NaCl consumption, TBS in individual SR and SD rats showed large variations. The reason for these different Na contents is not clear. The elevated Na contents were correlated with increased TBW in each strain. The relationship between Na and water was strain specific. The fraction of osmotically inactive Na in SD rats was threefold higher than in Dahl SS and SR rats, as judged by the relationship between body Na and body water. We conclude that compared with SD rats, both SS and SR Dahl strains are characterized by impaired storage of osmotically inactive Na. Presumably, the Dahl rats react to increased TBS with expansion of the extracellular space. Fluid volumes consecutively rose to levels higher than those in SD rats.
Bone could be viewed as a potential osmotically inactive Na reservoir (3, 4). In all strains, increased TBS led to increased bone Na content. As indicated by an inverse correlation between TBS and the bone Na/TBS ratio (Fig. 5, B and D), in both Dahl strains (with their reduced osmotically inactive Na storage capacitiy), increased TBS not only increased bone Na but also changed Na distribution in favor of Na compartments other than bone. Dahl SS rats additionally had a reduced ability to excrete Na that led to Na and water excess in these animals if they were fed a high-NaCl diet. This Na and water excess in SS rats was characterized by bone Na excess. Dahl SS rats fed 8% NaCl suffered a 0.1 mmol/g TBS increase (Fig. 2C) and a 12.1 µmol/g bone Na increase (Fig. 5A), indicating that 12% of the Na accumulated had been stored in this osmotically inactive Na compartment. Because the fraction of osmotically inactive Na accumulation was 28.2% (Fig. 4B), we conclude that bone is not the only osmotically inactive Na reservoir in SS rats. Such osmotically inactive Na reservoirs might function as a buffer that receives Na from a Na-overloaded extracellular space. We speculate that Na storage in an osmotically inactive compartment could be an alternative pathway for Na clearance from the extracellular space when Na loads are excessive or renal Na excretion is inadequate. Such a mechanism could reduce volume expansion and hypertension in Na-overloaded organisms. However, the mechanism is apparently inadequate to cope with the impaired renal Na excretion. In SD rats, the role of bone as a Na reservoir that osmotically inactivates extracellular Na is less readily apparent. Although these rats demonstrated the highest capacity to store osmotically inactive Na, the bone Na was lowest in SD rats. These findings suggest that other Na reservoirs in addition to bone, such as cartilage and mixed connective tissue, may be operative.
In the Dahl model, SR rats are considered the normotensive strain compared with SS rats. Apparently, renal Na handling abnormality led to Na and blood pressure excess in SS rats. Presumably, impaired renal Na excretion is associated with increased sympathetic nerve activity (16) in SS rats. Thus, besides Na and water retention, increased sympathetic nerve activity and its impact on the cardiovascular system may be an important factor in Dahl SS hypertension.
However, compared with SD rats, from which both Dahl strains were originally inbred, both SS and SR strains exhibited higher blood pressure. Even if animals were fed an NaCl-free diet, MAP in SS and SR strains was higher than in SD rats (Fig. 2). Although MAP was different, TBS in Dahl SR and SD rats, whether they were fed 0.1 or 8% NaCl, was comparable to TBS in SS rats fed 0.1% NaCl. Thus factors other than Na retention must explain the somewhat higher blood pressure in SS and SR rats compared with SD rats.
In contrast to our initial hypothesis, salt resistance in Dahl SR rats
could not be explained by a higher capacity than in SS to store Na as
osmotically inactive. Similar to SS rats, high-TBS levels led to
exaggerated water content in the SR strain. Consequently, SR rats
appear to be "water resistant" or "volume resistant." The
relationship between MAP and
TBW was quite similar in SS and SD
rats. In contrast, increased
TBW from 58 to 68% left blood pressure
almost unchanged in SR rats (Fig. 4D). To the extent that
TBW reflects changes in extracellular volume and cardiac output, the
lack of an increase of blood pressure points to lower peripheral
resistance in SR. In the absence of cardiac output measurements, we can
only speculate that lower peripheral resistance might be an inbred,
advantageous adaptation in SR rats rather than another disadvantage in
the SS strain.
Similar to SS rats, increased water content correlated with blood pressure in SD rats (Fig. 4D), indicating a body water-blood pressure relationship comparable to that in Dahl SS rats. However, because of osmotically inactive Na storage, water content increases did not exceed 6% of WW in SD rats. We conclude that osmotically inactive Na storage is an important mechanism to buffer volume and blood pressure in SD rats. The contribution of osmotically inactive Na to Na balance has been the focus of recent studies in humans (8). The variation and amount of osmotically inactive Na in SD rats fit well with similar findings in healthy men (in humans during a terrestrial space station simulation study; Titze J, Lang R, and Kirsch KA, unpublished observations). The role of osmotically inactive Na storage should be taken into account in the pathophysiological approach to volume expansion and hypertension.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors thank B. Weigel and S. Böhm for help with the animal experiments and E. Prell, H. Mohs, and A. Ziegler for help with the anorganic analyses. K. H. Schwind was mainly involved in the development of the ashing procedures. G. Weidemann helped with the analysis of urine electrolytes, and B. Johannes helped with the data analysis. We thank H. D. Rupprecht for support and F. C. Luft for very helpful comments on the manuscript.
![]() |
FOOTNOTES |
---|
This work was made possible by grant support of the Else Kröner-Fresenius-Stiftung to J. Titze (project "Langsame Natriumspeicher").
Address for reprint requests and other correspondence:
K. F. Hilgers, Nephrologische Forschungslaboratorien
Medizinische Klinik IV, Loschgestrae 8, D-91054 Erlangen, Germany
(E-mail:
karl.hilgers{at}rzmail.uni-erlangen.de).
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.
First published January 29, 2002;10.1152/ajprenal.00323.2001
Received 26 October 2001; accepted in final form 17 January 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Ben-Ishay, D,
and
Dahl LK.
Absence of an exaggerated renal response to acute salt loading in salt-hypertensive rats.
Proc Soc Exp Biol Med
123:
304-309,
1966.
2.
Ben-Ishay, D,
Knudsen KD,
and
Dahl LK.
Renal function studies in the early stage of salt hypertension in rats.
Proc Soc Exp Biol Med
125:
515-518,
1967.
3.
Bergstrom, WH,
and
Wallace WM.
Bone as a sodium and potassium reservoir.
J Clin Invest
33:
867-873,
1954[ISI].
4.
Bergstrom, WH.
The participation of bone in total body sodium metabolism in the rat.
J Clin Invest
34:
997-1004,
1955[ISI].
5.
Dahl, LK,
Heine M,
and
Thompson K.
Genetic influence of the kidneys on blood pressure. Evidence from chronic renal homografts in rats with opposite predispositions to hypertension.
Circ Res
40:
94-101,
1974[Medline].
6.
Dahl, LK.
Salt and blood pressure.
Lancet
1:
622-623,
1969.
7.
Dobesova, Z,
Kunes J,
and
Zicha J.
Body fluid alterations and organ hypertrophy in age-dependent salt hypertension of Dahl rats.
Physiol Res
44:
377-387,
1995[ISI][Medline].
8.
Heer, M,
Baisch F,
Kropp J,
Gerzer R,
and
Drummer C.
High dietary sodium chloride consumption may not induce body fluid retention in humans.
Am J Physiol Renal Physiol
278:
F585-F595,
2000
9.
Möhring, J,
and
Möhring B.
Evaluation of sodium and potassium balance in rats.
J Appl Physiol
33:
688-692,
1972
10.
Morgan, DA,
DiBona GF,
and
Mark AL.
Effects of interstrain renal transplantation on NaCl-induced hypertension in Dahl rats.
Hypertension
15:
436-442,
1990[Abstract].
11.
Qi, N,
Rapp JP,
Brand PH,
Metting PJ,
and
Britton SL.
Body fluid expansion is not essential for salt-induced hypertension in SS/Jr rats.
Am J Physiol Regulatory Integrative Comp Physiol
277:
R1392-R1400,
1999
12.
Rapp, JP.
Genetic analysis of inherited hypertension in the rat.
Physiol Rev
80:
135-172,
2000
13.
Schackow, E,
and
Dahl LK.
Effects of chronic excess salt ingestion: lack of gross salt retention in salt-hypertension.
Proc Soc Exp Biol Med
122:
952-957,
1966.
14.
Sterzel, RB,
Luft FC,
Gao Y,
Schnermann J,
Briggs JP,
Ganten D,
Waldherr R,
Schnabel E,
and
Kriz W.
Renal disease and the development of hypertension in salt-sensitive Dahl rats.
Kidney Int
33:
1119-1129,
1988[ISI][Medline].
15.
Tsunooka, K,
and
Morita H.
Effect of a chronic high-salt diet on whole-body and organ sodium contents of Dahl rats.
J Hypertens
15:
851-856,
1997[ISI][Medline].
16.
Wu, X,
Vieth R,
Milojevic S,
Sonnenberg H,
and
Melo LG.
Regulation of sodium, calcium and vitamin D metabolism in Dahl rats on a high-salt/low-potassium diet: genetic and neural influences.
Clin Exp Pharmacol Physiol
27:
378-383,
2000[ISI][Medline].