Department of Pediatrics (J.T., T.M., H.K., Y.M., I.I.), Department of Medicine (T.M., I.I.), and Department of Biochemistry (M.T.), Vanderbilt University School of Medicine, Nashville, Tennessee 37232; and Institute of Medical Science, Molecular and Cellular Nephrology (T.M.), Department of Pediatrics (T.H., I.I.), Tokai University, Isehara, Kanagawa 259-1193, Japan
Address all correspondence and requests for reprints to: Taiji Matsusaka, M.D., Ph.D., Institute of Medical Science, Molecular and Cellular Nephrology, Tokai University, Bohseidai, Isehara City, Kanagawa 259-1193, Japan. E-mail: taijim{at}is.icc.u-tokai.ac.jp
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ABSTRACT |
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To study the effect of Ang II on glomerulosa cells that may operate independently of plasma potassium in situ, we used chimeric mice made of cells with or without the intact AT1A gene (Agtr1a). When animals were fed a normal diet or chronically infused with Ang II, the aldosterone synthase mRNA was detectable only in Agtr1a+/+ but not Agtr1a-/- zona glomerulosa cells. After 2 wk of sodium restriction, plasma aldosterone increased (1.51 ± 0.27 ng/ml) and potassium remained on average at 4.5 ± 0.2 mEq/liter, with aldosterone synthase mRNA expressed intensively in Agtr1a+/+, but not detectable in Agtr1a-/- cells. Simultaneous sodium restriction and losartan treatment caused increases in plasma potassium (5.5 ± 0.1 mEq/liter) and aldosterone (1.84 ± 0.38 ng/ml), with both Agtr1a-/- and Agtr1a+/+ cells intensively expressing aldosterone synthase mRNA. Thus, aldosterone production is regulated by Ang II in the adrenal gland during chronic alterations in extracellular fluid volume when plasma potassium is maintained within the normal range. In the light of a previous observation that dietary potassium restriction superimposed on sodium restriction abolished secondary hyperaldosteronism in angiotensinogen null-mutant mice, the present findings demonstrate that when the renin-Ang system is compromised, plasma potassium acts as an effective alternative mechanism for the volume homeostasis through its capacity to induce hyperaldosteronism.
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INTRODUCTION |
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However, it has been documented repeatedly that administration of Ang I converting enzyme (ACE) inhibitors or AT1 antagonists in humans and animals fails to suppress plasma aldosterone levels (6, 7, 8, 9, 10, 11, 12, 13, 14, 15). In this regard, we previously showed that null-mutant mice for the angiotensinogen gene (Agt), which can generate no Ang II, were perfectly capable of achieving secondary hyperaldosteronism when they were placed on dietary sodium restriction (16). In these mice, plasma potassium was markedly elevated, and plasma aldosterone concentration was proportional to that of potassium. Superimposition of potassium restriction on sodium restriction to normalize plasma potassium levels led to an abolition of hyperaldosteronism in Agt-/- mice. These findings unequivocally indicate that the establishment of secondary hyperaldosteronism does not require an intact renin-Ang system and suggest that in the absence of Ang II, potassium plays an intermediary role in secondary hyperaldosteronism. Thus, despite the experimental observations suggesting otherwise, it now becomes conceivable that Ang II is not of vital importance to achieve secondary hyperaldosteronism, at least for animals that lack Ang II from the earliest embryonic stage.
In the present study, we first studied whether the above Ang-independent hyperaldosteronism can operate in wild-type mice carrying an intact renin-Ang system. In addition, we undertook a new experimental approach that allows us to study the direct effect of Ang II on glomerulosa cells operating independently of the systemic milieu including plasma potassium. The approach involved the use of chimeric mice made up with two genetically different cell types, one with and the other without the intact AT1A (the major subtype of AT1 receptor in mice) gene (Agtr1a). Within a given chimeric mouse, both types of cells are exposed to the same systemic milieu, and the different behavior between the two types of cells represents the local and direct action of Ang II in the adrenal gland. With this chimeric mouse system, we demonstrated in situ the direct action of Ang II for the first time in the chronic setting of ECF volume depletion. In addition, the chimeric mice served to unveil the existence of a separate mechanism for aldosterone production that plays a vitally important role in ECF volume homeostasis.
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RESULTS |
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Generation of AT1A Intact-Deficient Chimeric Mice
To study the direct action of Ang II on glomerulosa cells that may
operate independently of the systemic milieu, we generated chimeric
mice that were made up with AT1A-deficient (Agtr1a-/-) and
intact cells. We used ROSA26 transgenic (ROSA) mice as a
source of AT1A-intact cells. Since ROSA mice ubiquitously
express the lacZ transgene and have an intact
Agtr1a gene, staining for ß-galactosidase should allow us
to distinguish Agtr1a-/- and intact cells.
ß-Galactosidase staining confirmed that all cells in the adrenal
gland in ROSA mice, but not in Agtr1a-/- mice,
intensely express the lacZ gene (Fig. 2, a and c). To study the expression of
AT1 receptor in the adrenal gland of ROSA and
Agtr1a-/- mice, we performed binding autoradiography using
[125I-Sar1-Ile8]Ang
in the presence of an AT2 antagonist, PD123319. The study confirmed
that zona glomerulosa and zona fasciculata of ROSA mice
intensely express AT1 receptor (Fig. 2b
). Although
Agtr1a-/- mice have an intact gene for AT1B receptor, the
second subtype of AT1 receptor in mice, no binding signal was
detectable in Agtr1a-/- mice (Fig. 2d
).
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To correlate the cellular expression pattern of the AT1 receptor and
the lacZ gene in the adrenal gland of Agtr1a-/-
ROSA mice, we performed Ang binding autoradiography and lacZ
staining in the adjacent sections. The pattern of Ang binding
completely overlapped that of lacZ-positive cells, with the most
intense signal localized to the zona glomerulosa (Fig. 2
, e and f; g
and h). No binding signal was observed in lacZ-negative cells. These
results established that the positive and negative lacZ staining
faithfully reflects the presence and absence of AT1 receptor in the
adrenal gland of Agtr1a-/-
ROSA mice.
The Expression of Aldosterone Synthase mRNA in
Agtr1a-/- ROSA Mice
To examine the role of Ang II in aldosterone production, we
performed in situ hybridization for aldosterone synthase
mRNA in the adrenal gland of Agtr1a-/- ROSA
mice placed under various conditions. The genotype of adrenal cells was
determined by lacZ staining in the adjacent sections.
In Agtr1a-/- ROSA mice fed a normal sodium
diet, aldosterone synthase mRNA was observed only in ROSA
cells, but not in Agtr1a-/- cells, within the zona
glomerulosa (Fig. 3
, a and b). This
demonstrates that aldosterone synthesis is dependent on Ang II in this
condition.
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We next examined the response to exogenous Ang II. Ang II (1 ng/g body
wt/min) was infused for 2 wk into Agtr1a-/-
ROSA mice using an osmotic minipump. A significant
expression of aldosterone synthase mRNA was found in ROSA
glomerulosa cells, whereas no hybridization signal was observed in
Agtr1a-/- glomerulosa cells (Fig. 3
, c and d).
We then placed Agtr1a-/- ROSA mice on a
low-sodium diet. After 2 wk of sodium restriction, plasma potassium
remained within a normal range (4.5 ± 0.2 mEq/liter). Aldosterone
synthase mRNA was expressed selectively in ROSA glomerulosa
cells, whereas no hybridization signal was observed in
Agtr1a-/- glomerulosa cells (Fig. 3
, e and f; g and h).
This indicates that in the presence of an intact renin-Ang system, the
expression of aldosterone synthase mRNA during sodium restriction is
also dependent on Ang II, and the plasma potassium remains within
normal range.
Finally, we placed Agtr1a-/- ROSA mice on
sodium restriction and simultaneously treated them with losartan for 2
wk. In these mice, the expression of aldosterone synthase mRNA was not
confined to ROSA cells in the zona glomerulosa, but was also
found in Agtr1a-/- cells (Fig. 3
, i and j; k and l). Of
note, in these chimeric mice, plasma potassium levels were
significantly elevated to the range of 5.25.8 mEq/liter. These
findings demonstrate that an increase in plasma potassium acts as an
alternative mechanism for the secondary hyperaldosteronism
independently of the renin-Ang system when the latter is
compromised.
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DISCUSSION |
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Unlike humans, mice have two subtypes of AT1 receptor, AT1A and
AT1B, which are encoded by Agtr1a and Agtr1b,
respectively. RT-PCR analysis of total RNA extracted from a whole mouse
adrenal gland showed that only 10% of AT1 mRNA is of Agtr1b
origin (20, 21). Since the expression of AT1B is
restricted to the zona glomerulosa (22), whereas AT1A is
expressed in both zonae glomerulosa and fasciculata, it is conceivable
that the relative contribution of AT1B in the zona glomerulosa may be
more than 10% of total AT1 mRNA. In the current study, however, the
Agtr1a-/- cells in the adrenal gland of
Agtr1a-/- ROSA chimeric mice showed no
detectable Ang binding and aldosterone synthase mRNA, indicating that
the amount of AT1B protein and its function are negligible in the
adrenal gland of mice fed normal sodium diet. We chronically infused
Ang II into Agtr1a-/-
ROSA chimeric mice. In
this condition, aldosterone synthase mRNA was expressed selectively in
ROSA cells in the zona glomerulosa, but
Agtr1a-/- cells expressed no detectable aldosterone
synthase mRNA. We concluded that the uniform expression of aldosterone
synthase mRNA in Agtr1a-/-
ROSA mice fed
low-sodium diet and treated with AT1 antagonist is ascribed not to the
intact AT1B, but to a non-Ang II mechanism.
In the Agtr1a-/- ROSA chimeric mice placed
on normal or low-sodium diet, hybridization signals for aldosterone
synthase mRNA were strictly confined to lacZ-positive, AT1A-intact
cells. In these mice, plasma potassium was within the normal range.
Clearly, therefore, aldosterone production is dependent on Ang II when
plasma potassium is within the normal range. This is an unequivocal
demonstration that the hyperaldosteronism secondary to chronic ECF
volume depletion is dependent on Ang II.
In the present study, plasma potassium was elevated in both wild-type mice and chimeric mice placed on dietary sodium restriction with administration of AT1 antagonist. In our previous study, simultaneous restriction of potassium during sodium restriction nullified hyperaldosteronism in Agt-/- mice (16). Early studies have indicated that plasma potassium is also an important modulator of aldosterone synthesis (26). However, the effect of potassium on aldosterone synthesis has largely been attributed to the activation of the adrenal renin-angiotensin system (4). In this regard, our present findings provided evidence that the potassium-mediated pathway for aldosterone synthesis operates independently of angiotensin. We also demonstrated that blockade of angiotensin in sodium-restricted mice resulted in a greater increase in plasma aldosterone, suggesting that the potassium-mediated pathway is more potent than angiotensin-dependent stimulation of aldosterone synthesis in this setting.
Collectively, these data indicate that an increase in plasma potassium becomes the intermediary regulator of aldosterone synthesis, allowing successful achievement of secondary hyperaldosteronism during ECF volume depletion when the renin-Ang system is compromised.
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MATERIALS AND METHODS |
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Chimeric mice were generated by an aggregation method as previously
described (23, 24). The mice used as embryo donors had a
mixed genetic background of C57BL/6, 129/Ola, and ICR. Chimeric mice
generated from Agtr1a-/- and ROSA26 transgenic
(ROSA) embryos were designated as Agtr1a-/-
ROSA, and those from wild-type and ROSA embryos
as Agtr1a+/+
ROSA. Chimeric mice thus produced
had varying degrees of ROSA-derived cells ranging between 0
and 100%, and the contribution of ROSA cells in the adrenal
gland was similar to that in the skin. Chimeric mice (7 to 26 wk of
age) in which 4090% of the skin was of ROSA origin were
used in this study. The number of Agtr1a-/-
ROSA mice analyzed was 5 (normal sodium diet), 5 (Ang II
infusion), 5 (low-sodium diet), and 4 (low-sodium diet plus losartan).
Three Agtr1a+/+
ROSA mice were fed normal
sodium diet and analyzed.
All animals were handled in accordance with the institutional animal care policy of the Vanderbilt University Medical School.
Dietary Sodium Restriction
Mice were fed either a low-sodium diet (0.02% Na, Ralston Purina Co., Battle Creek, MI) or normal chow (0.46% Na,
0.72% K) for 2 wk. Some mice fed a low-sodium diet were also treated
with an AT1 antagonist, losartan (100 mg/liter in drinking water)
(Merck & Co., Inc., West Point, PA), which was started
2 d before dietary sodium restriction. All mice had free access to
food and tap water.
Chronic Ang II Infusion
Ang II (1.0 ng/g wt/min) was infused sc by osmotic minipump
(model 2002; Alza Corp., Palo Alto, CA) for 2 wk.
Analysis of the Adrenal Gland
Adrenal glands were embedded in OCT compound and frozen at -70
C. Serial sections (12 µm) were taken alternatively for lacZ staining
and Ang II binding or in situ hybridization for aldosterone
synthase mRNA. For ß-galactosidase staining, sections were fixed in
2% glutaraldehyde for 5 min, rinsed in PBS, and incubated in a
staining solution consisting of 0.01% sodium deoxycholate, 0.02%
IGAEPAL-CA630, 1 mM MgCl2,
2 mM
K3Fe(CN)6, 2
mM
K4Fe(CN)6, 1 mg/ml X-Gal
(Life Technologies, Inc., Gaithersburg, MD) in PBS at 37 C
for 10 h. Ang binding autoradiography was performed using
[125I-Sar1-Ile8]Ang
in the presence of AT2 antagonist, PD123319, as described previously
(23). In situ hybridization was performed as
described previously (23), using a specific probe for
mouse aldosterone synthase (a gift from Dr. Keith L. Parker, Duke
University, Durham, NC) (25).
Measurement of Plasma Aldosterone
Trunk blood was collected into ice-cold tubes in the presence of
EDTA (10 mM) and centrifuged, and the plasma was kept
frozen at -70 C until measurement. Plasma aldosterone concentration
was determined by two methods. One method was RIA utilizing a
commercially available kit (Coat-A-Count, Diagnostic Products, Los Angeles, CA). The second method used
HPLC-atmospheric pressure chemical ionization-tandem mass spectrometry.
Chromatography was performed on a Vydac (Hesperia, CA) C18 reverse
phase column (2.0 x 150 mm, 5 µm). The mobile phase consisted
of methanol/water (80:20, vol/vol) delivered at a flow rate of 200
µl/min. A 100-µl aliquot of extracted sample was injected onto the
column. A Finnigan TSQ7000 mass spectrometer (Thermo Quest, Manchester,
UK) was used for quantitative mass spectrometric detection. An
atmospheric pressure chemical ionization source was used in the
negative-ionization mode. The corona needle current, heated capillary,
and vaporizer temperature were set at 5.0 µA, 230 C, and 400 C,
respectively.
Measurement of Plasma Potassium
Blood was collected from the tail artery into heparinized
hematocrit capillary tubes to avoid hemolysis. Potassium concentration
was measured with a flame photometer (Instrumentation Laboratory,
Lexington, MA).
Statistical Analysis
Data were expressed as the mean ± SE.
Statistical significance was assessed by using the unpaired
t test and regression analysis. A value of P
< 0.05 was considered significant.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: Ang, Angiotensin; AT1, Ang II type 1; ECF, extracellular fluid
Received for publication April 5, 2001. Accepted for publication August 16, 2001.
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REFERENCES |
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