Kidney function in mice lacking aldosterone.
Natalia Makhanova*§, Gene Lee*#, Nobuyuki Takahashi*+, Maria L. Sequeira Lopez**, R.
Ariel Gomez**, Hyung-Suk Kim*, and Oliver Smithies*
*Department of Pathology & Laboratory Medicine, +Department of Cellular and Molecular
Physiology, University of North Carolina, Chapel Hill, NC, USA
**Department of Pediatrics, University of Virginia, Charlottesville, VA, USA
§ Institute of Cytology and Genetics, Siberian Division, Russian Academy of Sciences,
Running head: Abnormal kidney function in mice lacking aldosterone
Oliver Smithies, D.Phil.
Department of Pathology & Laboratory Medicine
University of North Carolina at Chapel Hill
701 Brinkhous-Bullitt Building
Chapel Hill, NC 27599-7525 USA
Articles in PresS. Am J Physiol Renal Physiol (August 23, 2005). doi:10.1152/ajprenal.00257.2005
Copyright © 2005 by the American Physiological Society.
To explore the effects of decreased amounts or absence of aldosterone, we have disrupted
the gene coding for aldosterone synthase (AS) in mice and investigated blood pressure and
kidney function in AS+/+, AS+/- and AS-/- mice. AS+/- mice have normal blood pressures and
show no abnormalities in electrolytes or kidney gene expression but they have significantly
higher than normal urine volume and lower urine osmolality. In contrast, the AS-/- mice have
low blood pressure, abnormal electrolyte homeostasis (increased plasma concentrations of K+,
Ca2+ and Mg2+ and decreased concentrations of HCO-3and Cl-but no difference in the plasma
Na+level), and disturbances in water metabolism (higher urine output, decreased urine
osmolality, and impaired urine concentrating and diluting ability). Absence of aldosterone in the
AS-/- mice induced several compensatory changes: an increased food intake/body weight ratio,
an elevated plasma concentration of glucocorticoids, and strong activation of the renin-
angiotensin system. Parallel with the markedly increased synthesis and release of renin, the AS-/-
mice showed increased expression of COX-2 in macula densa. On salt supplementation, plasma
electrolyte concentrations and kidney renin and COX-2 levels became similar to those of wild
type mice, but the lower blood pressure of the AS-/- mice was not corrected. Thus, absence of
aldosterone in AS-/- mice results in impairment of sodium reabsorption in the distal nephron,
decreased blood pressure, and strong RAS activation. Our data show the substantial correction
of these abnormalities, except the low blood pressure, by high dietary salt does not depend on
Keywords: aldosterone synthase, blood pressure, electrolytes, renin, COX-2
The most important function of aldosterone is in control of fluid and electrolyte
homeostasis. In the kidney, aldosterone-dependent regulation of sodium reabsorption and
potassium secretion takes place in the aldosterone-sensitive distal nephron, which includes the
second half of the distal convoluted tubule (DCT), the connecting tubule (CNT), and the cortical
and outer and inner medullary collecting ducts (22). The effectors of the response to aldosterone
are the amiloride– sensitive epithelial Na+channel (ENaC), the K+channel (ROMK) and the
serosal Na+/K+-ATPase, with the Na+/K+-ATPase pump providing the electrochemical driving
force necessary for the luminal entry of sodium and exit of potassium. The limiting step in
sodium reabsorption appears to be the amiloride– sensitive epithelial Na+channel (ENaC).
However, the luminal thiazide-sensitive Na+/Cl-cotransporter (NCC) is also an important
effector (14). ENaC, the mineralocorticoid receptor (MR), and 11β-hydroxysteroid
dehydrogenase type2 (11 β -HSD2) - a key enzyme for aldosterone selectivity of MR - are
localized in the terminal part of the DCT, the CNT and the collecting duct in mice. The thiazide-
sensitive Na+/Cl-cotransporter is localized in the proximal and distal segments of the DCT.
Several aldosterone-sensitive genes have recently been identified, including a serum and
glucocorticoid-inducible kinase (Sgk), the small monomeric Kirsten RAS GTP-binding protein
(ki-ras) and the corticosteroid hormone-induced factor (CHIF). Sgk has received the most
attention (36); it appears to phosphorylate other proteins involved in trafficking of ENaC into
and retrieval from the cell membrane. Recently binding of ubiquitin to specific membrane
proteins has been shown to lead to their internalization and degradation by proteosomes. For
example, ubiquitin ligase Nedd4-2 binds specifically to the “ PY domain” at the COOH-terminal
region of all three ENaC subunits, and Snyder et al. have shown that Sgk binds to and
phosphorylates Nedd4-2, which reduces its activity (34).
The actions of aldosterone are mediated through the MR, which is a nuclear receptor.
MR-knockout mice die between 8 and 13 days after birth with reduced weight and severe
dehydration due to failure to reabsorb sodium. At day 8 they show hyponatremia, hyperkalemia,
high renal salt wasting and a strongly activated renin-angiotensin system (RAS) (2), thus
demonstrating the central role of MR in aldosterone action.
Aldosterone synthase deficiency in humans is a rare autosomal recessively inherited
disorder caused by mutations in the aldosterone synthase gene (CYP11B2). Aldosterone
synthase (AS) catalyzes the last step in the synthesis of aldosterone, which involves 11β-
hydroxylation of 11-deoxycorticosterone to form corticosterone, 18-hydroxylation of
corticosterone to form 18-hydroxycorticosterone and oxidation at position C18 to give
aldosterone (18-alcorticosterone). There are two forms of aldosterone synthase deficiency:
corticosterone methyloxidase (aldosterone synthase) deficiency types I and II (CMO I and CMO
II) (39). CMO I is characterized by decreased levels of 18-hydroxycorticosterone, whereas CMO
II is characterized by increased levels of this steroid. In both syndromes aldosterone
biosynthesis is impaired and may results in sodium loss, but the symptoms decline with age (29,
40). Patients with CMO I and CMO II deficiency are most often diagnosed because of
hyperkalemia, metabolic acidosis and markedly elevated plasma renin activity (29).
To study the effects of decreased amounts or absence of aldosterone, we have disrupted
the gene coding for AS in mice. In a previous paper (21), we described the generation of mice
lacking AS and the homeostatic responses that resulted in their adrenal glands. Here we describe
the regulation of blood pressure, electrolyte homeostasis and kidney function in AS+/+, AS+/-
and AS-/- mice.
Materials and Methods.
Mice. The AS-null mice were generated by gene targeting in strain 129-derived ES cells as
described previously (21). Male chimeras were mated with 129/ SvEv females to derive
coisogenic offspring. Wild type mice (AS+/+), heterozygous (AS+/-) and homozygous (AS-/-)
littermates were used in this study. Mice were fed normal mouse chow (0.8% NaCl, Harlan
Teklad) or a high salt diet (8% NaCl). All animals had free access to food and water. All
experiments were conducted with female and male mice about 3-4 months old and were
approved by the Institutional Animal Care and Use Committee of the University of North
Carolina, Chapel Hill.
Blood pressure measurement. Blood pressures were measured in conscious mice with a
computerized a tail-cuff system (Visitech Systems, Cary, NC) (19).
Blood analysis. Blood was drawn under anesthesia from the retro-orbital sinus, and plasma
electrolyte concentrations were measured using a VT250 Chemical Analyzer (Orthodiagnostic
Clinical Inc.). Plasma aldosterone was measured with the Coat-A-Count RIA procedure
(Diagnostic Products, Los Angeles, CA). Plasma corticosterone was measured with an RIA kit
(ICN Biomedicals, Palo Alto,CA). Hematologic analyses were with the Animal Blood Counter
(Roche Diagnostic). Plasma renin concentration was measured as described (15) by RIA for
Angiotensin I (Perkin Elmer, USA). The concentration of Angiotensin II (ANG II) was measured
with an RIA kit (Peninsula Laboratory, Inc, CA).
Histological Analysis. Organs were fixed in 4% buffered paraformaldehyde overnight,
embedded in paraffin, sectioned and stained with hematoxylin and eosin, periodic acid-Schiff, or
Masson-Trichrome for light microscopy.
Renin and COX-2 immunostaining in kidney. The immunohistochemical detection of renin
was performed as described previously (17) by incubating deparaffinized kidney sections with a
polyclonal renin antibody (1:10,000, gift from Dr. Tadashi Inagami, Vanderbilt University,
Nashville, TN). For COX-2 immunostaining, frozen sections (5µm) of kidney were incubated
with primary antibody (COX-2, Cayman Chemicals) at 4ºC overnight. Sections were then
washed, and biotinylated secondary antibody was applied, followed by ABC-complex (Vector
Laboratories) with diaminobenzidine as the chromogen.
Real-Time RT-PCR. Expression of genes in the kidney was determined by quantitative real-
time PCR with an Applied Biosystems 7700 Sequence Detection System (Perkin-Elmer), as
described (16). RNA was isolated from tissue with ABI Prism 6700 automated nucleic acid work
station. Relative levels of gene expression are expressed as percentage of wild type.
Analyses of urine and kidney function. Mice were maintained on a 12 hour light-dark cycle
with water and food ad libitum. To estimate 24 hour water and food intake, urine volume and
excretion of electrolyte, mice were housed in metabolic cages for 3 days. Urine osmolality was
determined by freezing point depression. Urine electrolytes and creatinine were measured using
VT250 Chemical Analyzer. To determine the ability of mice to concentrate urine, they were
deprived of water for 20 hours. To test the ability of the kidney to dilute urine, mice were given
water (4% body weight) by gavage (25). Creatinine clearance was used as a measure of
glomerular filtration rate (13). To detect ketones in urine, we used Boehringer Mannheim
Chemstrips (Boehringer Mannhem Diagnostics, Indianapolis, IN).
Statistical analysis. All statistical analyses were performed using JMP statistical Software (SAS
Institute, Cary, NC), and are presented as the mean±SEM. Statistical significances were
assessed with ANOVA and post hoc analyses were performed using the unpaired t-test.
Body and kidney weights, blood data in AS+/+, AS+/-, AS-/- mice. The body weights of the
AS-/- homozygous mice were significantly lower than their heterozygous and wild type
littermates (Table 1). The gross weights of the kidneys were significantly reduced in the AS-/-
mice compared to AS+/+ and AS+/- mice, but the ratio of kidney (also heart, liver, spleen) to
body weight was not significantly different in mice of the three genotypes, indicating that the
absence of aldosterone causes a general rather than organ-related impairment of growth.
Table 1. Body and kidney weights, blood data in AS+/+, AS+/-, AS-/- mice on normal salt
AS+/+ AS+/- AS-/-
BW, g 25.1±0.7 (10) 24.6±1.1 (10) 20.3±1.1 (10)**, #
Kidney weight, g 0.35±0.03 (10) 0.32±0.02 (10) 0.25±0.02 (10)*
Kidney weight, g/20g BW 0.27±0.01 (10) 0.26±0.01 (10) 0.25±0.02 (10)
Food intake, g/ 20g BW 3.01±0.14 (14) 3.43±0.12 (15)* 3.90±0.19 (16)***, #
53.0±2.2 (10) 51.5±3.8 (10) 54.9±1.9 (10)
5.8±0.1 (16) 6.0±0.1 (15) 6.4±0.2 (15)**, #
212±9 (15) 214±12 (9) 223±8 (18)
Aldosterone, pg/ ml
604±130(7) 515±70 (6)
51±11(11) 67±16 (7) 79±6 (11)*
Values are means ±SE; parentheses (number of animals); BW-body weight; ND-not detectable.
* p<0.05 vs AS+/+ mice, ** p<0.01 vs AS+/+ mice, *** p<0.001 vs AS+/+ mice, #p<0.05 vs
AS+/- mice, + p<0.05 vs AS+/+ mice.
Food intake per 20g BW was increased in the homozygous AS null mice compared with wild
type mice (p<0.001) and heterozygous (p<0.05) mice. However, the heterozygous mice also
consumed more food per 20 g body weight than wild type mice (Table 1).
Blood data showed that the heterozygotes and the homozygous AS null mice did not
differ significantly from wild type in their hematocrits, but the homozygous null animals had
increased total plasma protein concentrations (Table 1). There were no differences in the plasma
glucose concentration among the three genotypes. Plasma aldosterone was not detectable in the
AS-/- mice. The concentration of aldosterone in the plasma of the heterozygotes was not
significantly different from wild type. Absence of aldosterone in the AS-/- mice resulted in an
increased level of corticosterone compared with wild type (p<0.05) (Table 1). The concentration
of corticosterone in the heterozygous mice was intermediate between those of the wild type and
homozygous null mice, although the difference did not reach significance.
Abnormal electrolyte levels in AS -/- mice. To determine the effects of absence of aldosterone
on electrolyte homeostasis, we measured plasma and urine electrolytes in the AS+/+, AS+/- and
AS-/- mice. There were no differences in the plasma Na+concentration and urinary excretion of
sodium among the three genotypes on normal salt diet (Table 2). However, there were significant
differences in the concentrations of K+, Ca2+, Mg2+ and Cl-in plasma in the homozygous null
mice compared with wild type and heterozygous mice. The heterozygous mice showed no
significant differences from wild type.
The AS-/- mice had a significantly higher plasma K+ concentration than the heterozygous
(p<0.05) and wild type mice (p<0.01). However, urinary excretion of potassium was similar in
three genotypes (Table 2). The concentration of chloride in plasma in the AS-/- mice was lower
than in AS+/+ and AS+/- mice (p<0.05) (Table 2). The AS-/- mice had also higher
concentrations of calcium and magnesium in plasma than AS+/+ and AS+/- mice. There were no
differences in the plasma lactate and phosphorus concentrations among the three genotypes on
Table 2. Plasma electrolytes, urinary excretion of electrolytes and creatinine clearance in
AS+/+, AS+/- and AS-/-mice
AS+/+ AS+/- AS-/-
136.8 ±1.5 (12) 135.7±1.2 (11) 137.2 ±1.29 (9)
5.9 ±0.1 (18) 6.2±0.2 (24) 6.7±0.2 (20) **, #
10.1 ±0.2 (11) 9.8±0.2 (9) 10.9±0.3 (10)*, ##
2.0 ±0.01 (14) 1.9±0.1 (13) 2.2±0.1 (10) *, ##
126.9±0.8 (20) 126.8±1.0 (19) 123.6±1.2 (16) *, #
Phosphorus , mg/dl
5.5±0.2 (6) 6.2±0.5 (5) 5.3±0.3 (6)
6.3±0.3 (6) 6.2±0.6 (5) 6.5±0.6 (6)
24.1 ±0.6 (6)
253±12 (11) 285±12 (11) 278±11 (13)
386±17 (10) 428± 16 (11) 387±15 (13)
Ketones ND (6) ND (6) ND (6)
Cr clearence, ml/min 0.38±0.09 (8) 0.51±0.12 (7) 0.49±0.07 (9)
Values are means ±SE, parentheses (number of animals); Cr – creatinine; U NaV –urinary sodium
excretion; U KV -urinary potassium excretion; ND-not detectable.
*p<0.05 vs AS+/+ mice, ** p<0.01 vs AS+/+ mice, #p<0.05 vs AS+/- mice, ## p<0.01 vs AS+/-
normal salt diet. The pH of plasma and plasma concentration of HCO-3were both decreased in
AS null mice compared with AS+/+ mice (Table 2). Tests for ketones in urine were negative in
all genotypes. (Table 2)
In comparison, plasma electrolytes of AS+/- mice were not different from wild type mice.
Blood pressure. The blood pressure of the AS -/- mice (96±2 mmHg) was significantly lower
than that of wild type (110±2 mmHg, p<0.0001) and AS+/- (107±2mmHg) mice (Fig 1A).
Fig 1. Blood pressure
(A); plasma renin
concentration, PRC (B);
plasma angiotensin II
concentration (C) in
AS+/+, AS+/- and AS-/-
mice on normal salt diet.
Parentheses (number of
RAS. The AS-null mice had extremely strong activation of the RAS, as judged by their ~40
times normal plasma renin concentration (1293±241 Ang I ng/ml/h compared with AS+/+ mice,
33±8 Ang I ng/ml/h, p<0.001) (Fig 1B). The concentration of ANG II in the plasma of the AS-
null mice (135±37 pg/ml) was ~4 times higher than in wild type mice (38±13 pg/ml, p<0.05)
(Fig 1C). AS+/- heterozygous, on the other hand, had normal plasma concentrations of renin
(40±8 Ang I ng/ml/h) and ANG II (40±11 pg/ml) (Fig 1 B, C).
Kidney function. To further characterize the impact of absence of aldosterone on kidney
function, we used creatinine clearance as a measure of glomerular filtration rate, and found no
significant differences in creatinine clearance between the genotypes (Table 2). However, as
shown in Fig 2A, the 24hour urine volume per 20g body weight of the AS-/- mice was ~2 times
more that of wild type mice (p<0.0001), with the heterozygotes being intermediate at ~ 1.3 times
wild type (p<0.001). Correspondingly, the AS-null mice consumed significantly more water per
20g body weight than the wild type mice (p<0.0001, Fig. 2B). Urine osmolality in the
homozygous null mice on a normal salt intake was significantly less than wild type mice
(p<0.0001), with the heterozygotes again being intermediate although not significant different
from wild type (p= 0.057) (Fig.2C).
Fig 2. Metabolic study in
AS+/+, AS+/- and AS-/-
mice: A. Urine volume . B.
Water intake in AS+/+,
AS+/-, AS-/- mice. C.
Urine osmolality before
and after water deprivation.
D. Diluting ability.
Parentheses (number of
To determine the ability of mice to concentrate urine, they were deprived of water for 20
hours. After this water deprivation, urine osmolality was increased in all three genotypes,
although osmolalities in the AS-/- mice (p versus wild type <0.0001) and the AS+/- mice (p
versus wild type <0.05) were still less than in wild type. The ability of AS-/- mice to dilute
water was also impaired (p versus wild type <0.05) (Fig.2D).
RNA studies in the kidney. To characterize the kidney molecular phenotype in the AS+/+,
AS+/- and AS-/- mice, we used quantitative RT-PCR to determine gene expression. In agreement
with the strong RAS activation detected in plasma, the renin mRNA level in the AS-/- kidney
was ~ 12 times the level in wild type mice (p<0.0001) and ~ 7.5 times the level in heterozygotes
(p<0.0001). Renin mRNA in the heterozygotes was ~ 1.5 times that in wild type mice, although
the difference did not reach significance (p=0.12) (Table 3). Kidney MR mRNA in the AS-/-
Table 3. mRNA levels in the kidney of AS+/+, AS+/-, AS-/- mice on normal salt diet
AS +/+ AS+/- AS-/-
100±7 (19) 157±36 (18) 1171±109 (20)****, ####
100±7 (19) 95±6 (18) 83±7 (20)
GR 100±8 (19)
66±5 (20)**, #
100±7 (19) 98±8 (18) 82±12 (20)
100±8 (19) 120±12 (18) 145±6 (20)*
100±8 (19) 114±16 (18) 97±10 (20)
100±9 (19) 99±4 (17) 111±8 (20)
β ENaC 100±6 (19) 108±11 (18) 113±9 (20)
100±7 (19) 93±8 (18) 117±8 (20)
NCC 100±5 (19) 91±8 (18) 76±5 (20) ***
100±7 (20) 105±7 (17) 104±6(20)
100±6 (20) 98±7 (17) 95±7(20)
100±15 (9) 82±13 (9) 59±10 (8)*
100±16(9) 102±12 (9) 105±16 (9)
100±15(9) 122±19 (9) 539±91 (9)***, ###
Values (wild type as 100%) are means ±SE, parentheses (number of animals); * p<0.05 vs AS+/+
mice; ** p<0.01 vs AS+/+ mice; *** p<0.001 vs AS+/+ mice; **** p<0.0001 vs AS+/+ mice;
#p<0.05 vs AS+/- mice, ### p<0.001 vs AS+/- mice; #### p<0.0001 vs AS+/- mice.
mice on normal salt diet was not different from AS+/+ mice. However, the increased level of
glucocorticoids observed in the AS-/- (Table 1) was accompanied by a modest decrease in the
mRNA of glucocorticoid receptors (GR) to ~ 2/3 of that in AS+/+ and AS+/- mice.
Glucocorticoids are selectively inactivated by 11β-HSD2 in aldosterone-sensitive tissues, and we
found that the level of 11β-HSD2 mRNA in the AS-/- mice was increased on normal salt diet to
~1.5 that of wild type. The level of 11β-HSD1 mRNA, which converts inactivate
glucocorticoids to their active form, was not significantly decreased in the AS-/- and AS+/- mice,
although there was a downward trend.
The abundance of mRNA for the aldosterone-sensitive gene Sgk, the α, β, and γ subunits
of ENaC, Na+/K+/2Cl-cotransporter (NKCC2), and Na+/H+exchanger type 3 (NHE3) in the
kidney were unaffected by the absence of aldosterone. However, the mRNA level of NCC was
decreased in AS-null mice to about 75% of wild type. In agreement with the observations by
Wald et al. (41) that cortical ROMK expression is regulated by aldosterone and K+, the level of
ROMK mRNA in the kidney cortex of AS-null mice was 59 % of wild type (p<0.05, Table 3).
Cyclooxygenase-2 (COX-2) derived prostaglandins in the renal cortex have recently
been shown to play a role in regulating synthesis and/or secretion of renin (7, 9, 31). We
therefore investigated the mRNA level of COX-2 and COX-1 in our mice. The COX-1 mRNA
levels in the renal cortex did not differ between the genotypes, but the COX-2 mRNA level in
the renal cortex in the AS-null mice was ~ 5 times that of wild type mice (Table 3).
Expression of genes in the kidneys of AS+/- heterozygotes was not significantly different
from wild type mice.
Abnormalities in kidney structure. Histological examinations demonstrated no notable
differences between the kidneys of the AS+/+ and AS+/- mice. In contrast, the kidneys of the
AS-/- mice exhibited several abnormalities. In five out of nine AS null mice the kidneys showed
hydronephrosis, varying in severity from mild to moderate. Fig 3B shows the appearance of a
kidney from an AS null mouse with moderate hydronephrosis.
Fig3. Histological and
of kidney of AS-null mice.
A. Kidney of AS+/+ mice.
B. AS-null mice had
hydronephrosis of varying
C. Immunostainning for renin
in wild type showed classic
adult pattern with renin
staining conferred to
D. AS-null mice had marked
increase in number of renin
expressing cells in
Juxtaglomerular areas and
along the preglomerular
E. Macula densa in kidney in
F. Extensive enlargement of
the macula densa in AS-null
mice. G. Absence of COX-2
immunostaining in AS+/+
mice. H. COX-2
immunostainig in the macula
densa in AS-null mice.
Immunostaining for renin in wild type (Fig. 3C) and heterozygous mice (data not shown)
showed the classic adult pattern with renin staining confined to the juxtaglomerular apparatus
and with the glomeruli showing no renin staining. However, the AS null mice showed a marked
increase in the number of renin expressing cells in the juxtaglomerular areas and along the
preglomerular arterioles (Fig. 3D). In addition, there was an increase in size of individual renin-
producing cells in the AS-/- mice. The AS-null mice also showed hypertrophy of the
juxtaglomerular apparatus and extensive enlargement of the macula densa (Fig. 3F) compared
with wild type mice (Fig. 3E).
COX-2 is normally expressed at low levels in the tubular cells of the thick ascending
limb of Henle included the macula densa region. No immunoreactive COX-2 was detectable in
the macula densa region of the wild type (Fig. 3G) and heterozygous mice (data not shown).
However, a strongly positive immunohistochemical reaction for COX-2 was observed in the
macula densa cells of the homozygous null mice (Fig. 3H), in agreement with the increased
COX-2 mRNA in these mice (Table 3).
Salt supplement in the AS-/- mice. To investigate whether dietary salt supplement could
correct any of the abnormalities in the AS null mice, wild type and AS-/- mice were fed a high
salt diet. Surprisingly, as shown in Fig. 4A, the high salt diet had no effect on the blood pressure
of the AS-/- mice (96 mmHg in the normal salt diet and 95mmHg in the high salt diet).
Nevertheless, there was clear recovery in the plasma concentration of K+in the AS-/- mice (5.8
in +/+ and -/- mice) (Fig 4B) and of other electrolytes (Ca2+, Mg2+ and Cl-) (data not shown).
However, the most striking effect of the high salt was an almost complete return to normal of the
synthesis and secretion of renin and of COX-2 expression in the AS-null mice (Fig. 4 C, D, E).
Thus PRC was not significantly different between the AS+/+ and AS-/- mice on the high salt
diet, and the level of renin mRNA level in the kidney decreased from ~ 40 times wild type to ~ 2
times on the high salt diet (Fig 4C).
Fig 4. Blood pressure (A), plasma K+(B); plasma renin concentration, PRC (C); level renin
mRNA in kidney (D); level COX-2 mRNA in cortex of kidney (E) in AS+/+, AS+/-, AS-/- mice
on normal (NS) and high (HS) salt diets.
Parallel with the renin decrease, the mRNA level of COX-2 in the kidney cortex decreased from
about 6 times wild type to only ~1.3 times (Fig 4E). Immunohistochemical signals for COX-2
were no longer detectable in the macula densa cells of AS-/- kidneys on the high salt diet (data
We have previously reported the use of gene targeting to generate mice homozygous
(AS-/-) for a disrupted allele of the gene coding for aldosterone synthase, and have described
their general characteristics and the homeostatic changes that occur in their adrenal glands in
response to their inability to synthesize aldosterone (21). The present study focuses on changes
that occur in the kidney function of the AS-/- and AS+/- mice, and on how these changes are
modified by feeding a high salt diet. Our data demonstrate that adult AS-/- mice have low blood
pressure, abnormal electrolyte homeostasis and water handling. The water handling ability of the
AS+/- heterozygotes is also affected.
Because aldosterone is a key factor in the regulation of electrolytes, abnormalities in
electrolyte homeostasis were expected in the AS null mice. Thus they exhibited modest but
significant increases in plasma concentration of K+ and decreases in plasma concentration of Cl-,
HCO3-and pH. These abnormalities agree with the presumption that sodium reabsorption in the
aldosterone-sensitive distal nephron is impaired, as the diminished activity of Na+channels
results in a lumen negative voltage, which in turn decreased Cl-reabsorbtion and K+and H+
secretion (30). Also in agreement with previous clinical and experimental data indicating
profound interaction between Na+, Ca2+ and Mg2+ transport within the distal convoluted tubule,
the AS null mice have increased Ca2+ and Mg2+ in their plasma and decreased urinary excretion
of these electrolytes.
Absence of aldosterone in the AS-/- mice can cause metabolic acidosis by at least two
mechanisms. The impaired reabsorption of Na+in the CNT and the collecting duct will make
the tubular lumen less negative and cause a decrease in proton secretion (30). Additionally, since
aldosterone directly increases the activity of the H+-ATPase in the collecting duct (38), its
absence will result in decreased proton secretion.”
Since aldosterone normally stimulates sodium reabsorption by activating ENaC and NCC
in the aldosterone-sensitive distal nephron, its absence was expected to lead to Na loss.
However, the AS-/- mice have a concentration of sodium in plasma not significantly different
from wild type mice. Evidently, compensation of some type occurs in the AS-/- mice.
Compensatory enhancement of Na+reabsorption in proximal tubule can be stimulated by
glucocorticoids (32, 44) and/or by angiotensin II (4, 6, 27). Since AS-/- mice on normal chow
diet have ~ 1.6 times the wild type concentration of plasma glucocorticoids and ~ 4 times the
wild type plasma level of ANG II, it is probable that the compensation is at least in part in the
Although previous studies have shown that chronic administration of aldosterone
increases the abundance of α-ENaC at both the protein (23) and mRNA levels (37), the levels of
mRNA of all subunits (α, β and γ) of the amiloride-sensitive channels-ENaC did not
significantly differ in the AS-/-, heterozygous AS+/- and wild type mice. Similarly, Berger et al.
(2) found no changes in mRNA levels for the ENaC subunits in MR knockout mice. The absence
of changes in the mRNA of the ENaC subunits in the AS-/- and MR-/- mice suggests that either
compensatory changes induced in these animals are not at transcriptional levels or that
compensation is essentially completes possibly as a result of increased levels of glucocorticoids
and of ANG II. The latter may be the case, since glucocorticoid treatment in MR knockout mice
increases the abundance of α-ENaC mRNA (33), and Beutler et al. (3) have also demonstrated
that angiotensin II administration increases the expression of α-ENaC at both the mRNA and
protein levels in AT1R+/-heterozygous mice. In addition, the absence of aldosterone in AS -/-
mice could activate RAS in the central nervous system leading to a stimulation of thirst and to
vasopressin secretion. Studies by Ecelbarger et al (5) have shown that chronic exposure to
vasopressin in rats results in a marked increase in abundances of β and γ-ENaC in whole kidney.
The most significant changes in the expression of genes in the AS-/- kidneys were
increases in renin mRNA to ~12 times wild type and in COX-2 (renal cortex) to ~5 times wild
type. Both of these increases were readily documented by histochemistry. They were
accompanied with ~40 times wild type PRC and ~4 times plasma ANGII concentration.
Significant although less marked changes include an increase in the expression of 11-β HSD2,
and a decrease in NCC and ROMK (renal cortex). The AS -/- mice also showed histological
abnormalities in renal structure, including hypertrophy of the juxtaglomerular apparatus and in
some animals hydronephrosis. Several factors may be involved in the very high expression and
release of renin in the AS null mice. Previous studies have shown that alterations in luminal
chloride change the rate of Na+/K+/2Cl-cotransport in the macula densa (26, 31), which
stimulates COX-2, the enzyme responsible for prostaglandins synthesis (1). Although the role
of COX-2 in the macula densa is not fully understood, it has been suggested that COX-2 derived
prostaglandins from macula densa may regulate renin expression and release (7, 12). Several
studies have also demonstrated that COX-2 expression in macula densa/ cTALH increases in
high-renin states (salt restriction, angiotensin-converting enzyme inhibition, renovascular
hypertension) at both mRNA and immunoreactive protein levels (8, 10, 11, 42). Furthermore,
Zhang MZ et al. (45) demonstrated that COX-2 expression in the renal cortex of mature rats is
negatively regulated by aldosterone and to some extend by glucocorticoids. Our finding that the
levels of COX-2 mRNA in the renal cortex of the AS-/- and AS+/- mice were increased to ~ 5
times and ~1.3 times wild type is consistent with their observations.
Our study of the AS-/- and AS+/-mice demonstrates a role of aldosterne in regulation of
water balance, as might be expected from previous work of others (20, 24, 28). On normal
chow both have higher urine output, decreased urine osmolality and an impaired the ability to
concentrate and dilute urine. The absence of aldosterone impairs sodium reabsorption in the CNT
and collecting duct, which leads to less water reabsorption in the collecting ducts. Changes in the
expression of aquaporins have also been described in adrenalectomized rats maintained with
glucocorticoids but without aldosterone ( 20, 24). This could at least partly explain the impaired
ability of AS-/- and AS+/- mice to concentrate urine. Aldosterone deficiency alters also fluid
balance through effects on hemodynamic. Klar et al. (18) have shown that renal afferent
arterioles, including their juxtaglomerular portion, express MR. There is also evidence that
aldosterone is required for normal function of thick ascending limb (TAL). Thus in vivo TAL
microperfusion studies by Stanton (35) showed a 33% reduction in sodium reabsorbtion in
adrenalectomized rats, which was restored by aldosterone, but not by glucocorticoids.
Experiments by Work and Jamison (43) have confirmed these findings in vitro. Absence of
aldosterone is therefore likely to decrease salt reabsorption in TAL and consequently the
maintenance of an axial osmolar gradient, thereby impairing the ability of the kidney to
Although hydronephrosis is present in about half of the AS-null adults, it is unlikely to be
the cause of the abnormalities in water handling for several reasons: because we observed
impaired ability to concentrate urine in animals without hydronephrosis, because it is never more
than moderate, and because the kidney/body weights of the AS-/- mice are not different from
wild type mice. In agreement with this reasoning, urine volumes in the heterozygotes on water
ad lib and osmolalities achieved during water deprivation are both significantly different from
wild type mice even though none of the heterozygotes have hydronephrosis. The death of 30% of
the AS-/- pups prior to weaning, apparently from dehydration, and the occurrence of
hydronephrosis in half the surviving adults suggests that the balance in neonatal mice is
precarious between a failure to compete with siblings for milk, leading to dehydration, and
salt/water wasting leading to hydronephrosis. The normal hematocrit and creatinine clearance of
the AS-/- mice that survive to adulthood indicates that they can achieve an almost normal
balance between water intake and water excretion.
Perhaps the most important finding of the current study is that high dietary salt is able to
normalize the plasma electrolyte concentrations in AS-/- mice, even though they remain
hypotensive and that increased expression of renin and COX-2 in AS null mice are minimized by
the high salt. This suggests that electrolyte disturbances in the AS-/- mice may change the
activity of the macula densa Na+-K+-2Cl-cotransporter leading to an increase in COX-2 activity.
COX-2 derived prostaglandins may then increase the synthesis and release of renin. Because the
AS null mice have reduced blood pressure, it is likely that renin expression and release are also
stimulated by an increase in renal sympathetic nerve activity. However, despite the restoration of
electrolyte homeostasis, and the near normalization of renin and СOX-2 synthesis their blood
pressure remained low.
Finally, we comment on the phenotype of AS+/- heterozygotes. It is a common
experience with mice in which genes have been disrupted that the effects of homozygous
absence of gene function are easy to demonstrate while the effects of heterozygosity may be
detectable but not reach significance. Yet the heterozygous effects are important indicators of
the likelihood that small differences in expression of gene of interest will have effects in humans.
In present case, in 16 out of 20 variables for which homozygous null animals significantly
differed from wild type, the value for the heterozygote differed from wild type in the same
direction as did the homozygote even though not reach significance. In 3 additional variables,
related to water handling the heterozygous mice differed significantly from wild type. Thus it is
likely that polymorphisms that affect the levels of AS gene expression in human population, will
have effects, albeit small, on water balance and possibly blood pressure.
In summary, our data show that absence of aldosterone results in impairment of sodium
reabsorption in the distal nephron and low blood pressure, which activates several compensatory
mechanisms, including increased sodium uptake, a huge stimulation of the RAS and elevated
level of glucocorticoids. They also show that a high intake of dietary sodium chloride largely
normalizes these impairments with the exception of the low blood pressure.
Grants. This work was supported by National Institutes of Health Grant HL49277.
Acknowledgments. We thank Dr Nobuyo Maeda, Emily Riggs and Gang Cui for their help and
Current address of Gene Lee: Department of Oral Biochemistry College of Dentistry, Seoul
National University, Seoul, South Korea.
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