Am J Physiol Regul Integr Comp Physiol 285: R782-R790, 2003.
First published June 12, 2003; doi:10.1152/ajpregu.00133.2003
0363-6119/03 $5.00
REGULATION IN GENETICALLY MODIFIED ANIMALS
Renal and blood pressure phenotype in 18-mo-old bradykinin B2R(-/-)CRD mice
Lisa M. Harrison-Bernard,1
Susana Dipp,2 and
Samir S. El-Dahr2
Departments of 1Physiology and
2Pediatrics, Section of Pediatric Nephrology, Tulane
University Health Sciences Center, New Orleans, Louisiana 70112
Submitted 12 March 2003
; accepted in final form 12 June 2003
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ABSTRACT
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Aberrant gene-environment interactions are implicated in the pathogenesis
of congenital renal dysgenesis (CRD), a leading cause of renal failure in
infants and children. We have recently developed an animal model of CRD that
is caused by gestational salt stress (5% NaCl diet; HS) of bradykinin
B2R null mice [B2R(-/-)CRD; El-Dahr SS,
Harrison-Bernard LM, Dipp S, Yosipiv IV, and Meleg-Smith S. Physiol
Genomics 3: 121-131, 2000.]. Developing B2R(-/-)CRD
mice exhibit tubular and glomerular cysts, stromal expansion, and loss of
corticomedullary differentiation. In addition,
B2R(-/-)CRD mice exhibit transient hypertension from 2
to 4 mo of age. The present study was designed to determine the long-term
consequences of CRD on renal morphology and salt sensitivity of blood pressure
in B2R(-/-)CRD mice. One-year- and 18-mo-old
B2R(-/-)CRD mice exhibited stunted renal growth,
glomerular cystic abnormalities, and collecting duct ectasia. Moreover, tumors
of mesenchymal cell origin emerged in the dysplastic kidneys of 90% of
1-yr-old and 100% of 18-mo-old B2R(-/-)CRD mice but not
in age-matched B2R(-/-) or wild-type mice. When challenged with an
HS diet, 18-mo-old B2R(-/-)CRD exhibited a significant
rise in systolic and diastolic blood pressures and more pronounced natriuresis
and diuresis compared with salt-loaded 18-mo-old wild-type mice. Kidney
aquaporin-2 expression was decreased by 50%, whereas renin, ANG type 1
receptor, and Na+-K+-ATPase levels were not different in
B2R(-/-)CRD mice compared with controls. In conclusion,
this study demonstrates that B2R(-/-)CRD mice exhibit
permanent phenotypic and functional abnormalities in renal growth and
differentiation. This novel model of human disease links gene-environment
interactions with renal development and blood pressure homeostasis.
kidney development; salt sensitivity; kallikrein-kinin; aquaporin
CONGENITAL RENAL DYSGENESIS (CRD) accounts for 
30% of
chronic renal failure cases in infants and children
(12). We recently developed a
model of CRD caused by gestational salt stress of mice with targeted
disruption of the bradykinin B2 receptor gene (gene:
Bdkr2; protein: B2R; see Ref.
10). The developing kidney
expresses all of the components of the tissue kallikreinkinin system
(8,
9). The kidney abnormality in
B2R(-/-) progeny is evident histologically on embryonic day
16 and consists of distorted renal architecture, foci of tubular
dysgenesis, and cyst formation
(10). Moreover, the abnormality
is intrinsic to the embryo, because B2R homozygous offspring from
heterozygous parents exhibit the same phenotype as offspring from homozygous
null parents (10). In contrast,
B2R mutant mice maintained on a normal salt intake or salt-loaded
wild-type mice do not develop renal abnormalities
(10). Thus this model of CRD
depends on the cooperation of both a defined genetic defect and a specific
environmental stressor heretofore termed
B2R(-/-)CRD.
Several lines of evidence suggest that the aberrant renal phenotype of
B2R(-/-)CRD mice is the result of impaired terminal
epithelial differentiation. First, the renal phenotype appears relatively late
in fetal development in concert with morphological and functional tubular
differentiation. Second, histomorphometric analysis indicates that the early
inductive events of nephrogenesis proceed normally in the B2R null
mice (10). In addition to renal
dysplasia, B2R(-/-)CRD mice develop early onset
hypertension (4). The elevated
blood pressure (BP) can be measured as early as 6-8 wk of age and persists
until 12-14 wk of age, when it gradually declines toward normal values.
The circulating renin-angiotensin system is not activated in
B2R(-/-) mutants on the BL6 genetic background
(4), yet these mice are highly
sensitive to the chronic hypertensive effect of exogenous ANG II
(5). The long-term relation of
renal dysplasia with hypertension is not clear because tubular dysgenesis is
usually associated with salt wasting
(17a). Therefore, the
objectives of the current study were to determine the long-term consequences
of CRD on renal morphology and salt sensitivity of BP in
B2R(-/-)CRD mice.
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METHODS
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Animals and experimental protocols. Breeding pairs of
B2R(-/-) mice (C57/BL6) were placed on a high-salt diet of
isocaloric chow (HS; 5% NaCl, TD no. 92102; Harlan Teklad, Madison, WI) 1 day
before mating and for the duration of gestation to induce CRD in the progeny,
as described previously (4,
10). The maternal diet was
switched to normal salt (NS; 0.3% NaCl) 1 day postpartum. After weaning
(days 21-25), the B2R(-/-)CRD progeny were
continued on NS. B2R(-/-) and B2R(+/+) mice were
maintained on life-long NS and served as controls for the effects of age on
renal structure and BP. To determine the long-term effects of CRD on BP,
systolic BP (SBP) was measured in the same animals at 12 and 17 mo of age in
B2R(-/-)CRD and B2R(+/+) mice on a life-long
NS diet. To determine the long-term consequences of CRD on salt handling,
17-mo-old B2R(-/-)CRD and B2R(+/+) mice were
placed on HS for 4 wk. BP was measured during the 4-wk HS period, and plasma
electrolytes and urinary sodium excretion were analyzed at the end of the 4-wk
HS diet, as outlined in Fig. 1
and described below.
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Fig. 1. Experimental design. Congenital renal dysgenesis (CRD) B2
receptor (B2R)-deficient [B2R(-/-)CRD] and
B2R wild-type [B2R(+/+)] mice were exposed to a
gestational high-salt diet (HS) or a normal salt diet (NS), respectively. All
mothers and suckling pups were placed on the NS diet on postnatal day
1. Systolic blood pressures (SBP) were measured by the tail-cuff method
in the same animals at 12, 17, and 18 mo of age. Twenty-four-hour urine
samples were collected at 17 and 18 mo of age on NS and HS diets,
respectively. Acute blood pressures (BP) and blood samples were obtained under
pentobarbital sodium anesthesia at the end of the protocol (18 mo only).
Kidneys were obtained at postnatal day 2 and 12 and 18 mo of age for
histological investigation of renal morphology and protein extraction.
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Conscious SBP measurements. Serial measurements of conscious SBP
were performed on male and female B2R(-/-)CRD
(n = 20) and male B2R(+/+) (n = 6) mice at 12,
17, and 18 mo of age using a computer-automated tail-cuff system (Visitech
BP-2000 Blood Pressure Analysis System; Visitech Systems; Apex, NC). Animals
were placed on a heated platform and underwent 10 preliminary cycles. The
average of three 10-cycle measurements, which each have a minimum of 6 out of
10 successful measurement cycles, was used for data analysis.
Anesthetized BP measurements. Before anesthesia, 18-mo-old male
and female B2R(-/-)CRD (n = 18) and male
B2R(+/+) (n = 4; originally 6 mice, 2 died between 12 and
18 mo of age) mice treated with an HS diet for 4 wk were administered 3 mg/100
g body wt bromodeoxyuridine (BrDU; Zymed Laboratories) intraperitoneally to
measure the proliferative index in the kidneys. Animals were anesthetized with
50 mg/kg ip pentobarbital sodium and were placed on a heated surgical table.
The right carotid artery was cannulated with a short PE-10 catheter connected
to a PE-50 catheter, as previously described
(4). Direct arterial BP was
measured using a P23XL transducer (Astro-Med) and was converted to a digital
signal by using a Pentium 200-MHz computer and an analog-to-digital
data-acquisition system (model MP100; Biopac Systems, Santa Barbara, CA) after
a 30- to 45-min equilibration period. Average SBP, mean arterial pressure
(MAP), diastolic BP (DBP), and heart rate (HR) were obtained over a 10- to
15-min period. Sampling rate was set at 400 samples/s. Blood samples were
taken from the carotid artery cannula at the end of the data collection period
(5 mM ETDA). Hematocrit and plasma protein concentration (AO Refractometer;
American Optical, Buffalo, NY) were measured. Plasma sodium and potassium
concentrations were measured as described below. Kidneys were removed, blotted
dry, and weighed. One kidney was immersed in liquid nitrogen and stored at
-80°C until the time of protein extraction. The other kidney was processed
for histology and BrDU staining. SBP and urine excretion were measured in the
same mice at 17 mo of age on the NS diet and during the 4-wk period of the HS
diet. However, anesthetized BPs, blood samples, and kidney samples were only
obtained at the end of the HS diet.
Urine collections. Male B2R(+/+) (n = 5) and
B2R(-/-)CRD (n = 7) animals were placed in
metabolic cages for a period of 24 h while on the NS diet at 17 mo of age and
again at 18 mo of age after 4 wk on the HS diet. Urine was collected and
analyzed for volume, sodium concentration, potassium concentration, and
osmolality. Water intake was measured on the HS diet. Sodium and potassium
were determined with a flame photometer (model 943; Instrumentation
Laboratory, Lexington, MA). Osmolality was measured with a vapor pressure
osmometer (model 5500; Wescor, Logan, UT).
Western blot analysis of kidney protein. Western blot analysis was
performed on kidneys obtained from 18-mo-old B2R(-/-)CRD
(n = 6) and B2R(+/+) (n = 4) mice on HS, as
previously described (18). The following primary antibodies were used:
anti-rat sheep polyclonal angiotensinogen antibody (1:6,000; see Ref.
7), anti-human rabbit
polyclonal ANG type 1 receptor (AT1) antibody (1:200; N-10,
sc-1173; Santa Cruz), anti-rat rabbit polyclonal aquaporin-2 (AQP-2) antibody
(1:200; AB3066; Chemicon), anti-rabbit mouse monoclonal
Na+-K+-ATPase 
1 (1:1,500; 05-369;
Upstate Biotechnology), and anti-human rabbit polyclonal renin antibody
(1:2,000; see Ref. 3).
Membranes were reprobed with 
-actin antibody (monoclonal
anti--actin antibody, 1:4,000; A5441; Sigma). Signals were detected
using enhanced chemiluminescence (Amersham), and protein expression was
analyzed densitometrically using the Digital Imaging and Analysis Systems
(Apha Innotech).
Immunohistochemical analysis of kidney tissue sections. Kidneys
from 12- and 18-mo-old mice were fixed in 10% buffered formalin, dehydrated in
graded solutions of alcohol, and embedded in paraffin blocks, and 5-µm
sections were made and mounted on slides with Vectabond (Vector Laboratories,
Burlingame, CA). Immunostaining was performed by the immunoperoxidase
technique using the Vectastain Elite kit (Vector Laboratories, as previously
described (10). Primary
antibodies used include anti-human mouse monoclonal -smooth muscle
actin (1:100; NCL-SMA; Nova Castra Laboratories), AQP-2 (1:100; Chemicon
International), anti-BrDU mouse monoclonal antibody (1:50; ZBU30; Zymed),
anti-human rabbit polyclonal -catenin (1:200; H-102, sc-7199; Santa Cruz
Biotechnology), and E-cadherin (1:200; H-108, Santa Cruz Biotechnology).
Controls consisted of tissue sections in which the primary antibodies were
substituted with PBS or nonimmune serum.
Lectin histochemistry. Tissue sections were incubated with
Dolichos biflorus agglutinin (0.0125 mg/ml; Sigma), as previously
described (10).
Data analysis. Statistical analyses were performed using SigmaStat
Statistical Software on the raw data by two-way ANOVA, followed by Tukey's
test or by unpaired t-test, as appropriate. A P value
<0.05 was considered statistically significant. All data are presented as
means ± SE.
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RESULTS
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Renal morphology. Histological studies were performed on kidney
sections of B2R(+/+) and B2R(-/-)CRD mice
(Fig. 2). Normal renal
morphology is observed on postnatal day 2 of B2R(+/+) mice
(Fig. 2A), 12 mo of
age (Fig. 2D), and 18
mo of age (data not shown). Newborn B2R(-/-)CRD mice
exhibit developmental renal abnormalities similar to what has previously been
described (10), consisting of
tubular cysts, a disorganized cortex, and poorly developed medullary rays
(Fig. 2, B and
C). Dysgenic tubules in 18-mo-old
B2R(-/-)CRD mouse kidneys stain positively for D.
biflorus lectin, indicating collecting duct origin
(Fig. 2E). The
dysplastic tubules stained positively but faintly for AQP-2
(Fig. 2F), consistent
with downregulated expression of distal nephron differentiation markers (see
results of AQP-2 Western blot below). Hyperplastic islands or
"tumorlets" are observed in the dysplastic kidneys of 20/22
1-yr-old and 18/18 18-mo-old B2R(-/-)CRD mice. These
tumorlets were not observed at birth or at 4 or 6 mo of age (not shown). By
immunohistochemistry, proliferating cells
(Fig. 2, G-I) are
frequently seen in the tumorlets. An average of three to six tumorlets are
observed per section and are located in the inner cortex closely associated
with renal arteries, veins, and glomeruli
(Fig. 2G).
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Fig. 2. Renal morphology in B2R(+/+) (A and D) and
B2R(-/-)CRD (B, C, and E-I) mice at
birth and 12 and 18 mo of age. Normal renal morphology is seen in
B2R(+/+) mice on postnatal day 2 (A) and at 1
yr(D). Nephrogenic zone (NZ) is demarcated in A, and normal
glomerular morphology (arrow) is shown in D.
B2R(-/-)CRD mice exposed to gestational HS demonstrate
tubular cysts (*) at postnatal day 2 (B and at higher
magnification in C) and at 18 mo of age (E). High-power
magnification in E shows that the epithelial cells (arrows) lining
the renal cysts are positive for the collecting duct marker Dolichos
biflorus (DB; E). Aquaporin-2 (AQP-2) expression is
downregulated in dysplastic collecting ducts as shown by the presence of a
faint immunoreactivity (arrows in F). Renal tumors are found at 18 mo
of age in B2R(-/-)CRD mice (G-I). These tumors
are positive for bromodeoxyuridine (BrDU; G and H and higher
magnification in I; arrows). PT, proximal convoluted tubule; CD,
collecting duct; a, artery; v, vein. Original magnification x20 (A,
B, and H), x40 (C, D, G, and I), and
x100 (E and F).
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Figure 3A and
higher-magnification Fig.
3B show that smooth muscle -actin positively
stained blood vessels are seen at the center of a renal tumorlet, indicating
high vascularization. The tumorlets are composed predominantly of mesenchymal,
myofibroblastic cells (smooth muscle -actin-positive;
Fig. 3B) and, to a
lesser extent, vimentin-positive cells (data not shown). Although the
periphery of the tumorlet is positive for CD-45, a marker of hematopoietic
cells, the tumorlet's core is negative
(Fig. 3C). Moreover,
the tumorlets are positive for proliferating cell nuclear antigen
(Fig. 3D) but negative
for tubular epithelial markers, including -catenin
(Fig. 3, E and
F), AT1 receptor
(Fig. 4, A-C),
angiotensinogen (Fig. 4, D and
E), AQP-2, Na+-K+-ATPase, and
E-cadherin (data not shown). In the case of epithelial cell markers, no
specific staining was observed in the tumorlets in spite of using
intentionally high concentrations of antibodies. Importantly, 1-yr-old
B2R(-/-) mice that were maintained on an NS diet during
embryogenesis and post-natally (n = 5) have normal renal architecture
and no evidence of renal tumorigenesis
(Fig. 3, G and
H).
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Fig. 3. Immunohistochemistry of tumorlets in 12-mo-old
B2R(-/-)CRD dysplastic kidneys. Control section without
primary antibody (A) and consecutive section immunostained for smooth
muscle -actin (B) showing the localization of a blood vessel
at the center of the tumor (arrow). Tumorlets are negative for CD45, a marker
of hematopoietic cell lineage (C). The dotted line outlines the core
of the tumorlet, which is CD45 negative. The tumorlet is positive for
proliferating cell nuclear antigen (PCNA; arrow), a proliferation marker
(D). Anti--catenin antibody staining (E and
F) shows positive immunoreactivity in the tubules (arrow), whereas
the tumorlets are -catenin negative (arrowhead). Kidney sections from
12-mo-old NS/B2R(-/-) mice (maintained on an NS diet since the time
of conception) show normal morphology and an absence of dysplastic tubules and
tumors (G and higher magnification in H). Original
magnification x20 (A, C, D, and G) and x40
(B, E, F, and H).
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Fig. 4. Immunohistochemistry of tumorlets in 12-mo-old
B2R(-/-)CRD dysplastic kidneys. A-C: ANG II
type 1 (AT1) receptor immunoreactivity is present in renal tubules
(arrowhead in B) and blood vessels (C) but not in the
tumorlet or the dysplastic primitive duct (arrow in B). D
and E: angiotensinogen (AGT) is expressed in tubules (arrows) but not
in the tumorlet. The background nonspecific staining is the result of the use
of intentionally high concentrations of AGT antibody. A glomerular cyst (glom
cyst) is seen in D. Original magnification x20 (A) and
x40 (B-E).
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Excretory function of
B2R(-/-)CRD and
B2R(+/+) mice on NS and HS
diets. A 24-h urine collection was obtained from 17-mo-old male
B2R(+/+) (n = 5) and B2R(-/-)CRD
(n = 7) mice while on the NS diet. Body weights were significantly
higher in NS/B2R(-/-)CRD than NS/B2R(+/+)
mice (Table 1). No significant
weight change occurred in either group after 4 wk of HS diets
(Table 1). Twenty-four-hour
urine volume, urinary Na+ and K+ excretion, factored for
body weight, and urine osmolality, are not different between 17-mo-old
NS/B2R(-/-)CRD and NS/B2R(+/+) mice
(Table 1). A second 24-h urine
collection and 24-h water intake were measured after placing these two groups
of mice on an HS diet for 4 wk. HS/B2R(+/+) mice showed a fivefold
increase in urinary sodium excretion (P = 0.08) and a significant
increase in urine volume compared with NS (P < 0.05;
Table 1). The degree of
natriuresis in HS/B2R(+/+) mice is similar to that reported for
wild-type mice on a 1-wk HS diet by Oliverio et al.
(17).
HS/B2R(-/-)CRD mice had statistically significant
increases in urine volume (3-fold), sodium excretion (10-fold), and potassium
excretion (40%) compared with values on the NS diet
(Table 1). Importantly, urine
volume (2-fold), sodium excretion (3-fold), and potassium excretion (3-fold)
of HS/B2R(-/-)CRD mice, factored for body weight, were
significantly greater than HS/B2R(+/+)
(Table 1). Twenty-four-hour
water intake, factored for body weight, was similar in
HS/B2R(-/-)CRD and HS/B2R(+/+) mice
(Table 1).
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Table 1. Urinary excretory parameters before (17 mo of age) and after (18 mo)
salt loading of male B2R(+/+) and B2R(-/-)CRD
mice
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SBP profile in
B2R(-/-)CRD and
B2R(+/+) mice on the NS
diet. We have previously reported that B2R(-/-)CRD
mice exposed to gestational HS and switched to an NS diet postnatally exhibit
significantly higher SBP at 2 and 3 mo of age with a gradual return of SBP to
normal values by 4 mo of age
(4). SBP of
B2R(-/-)CRD mice measured at 12 and 17 mo of age
averaged 116 ± 2(n = 12) and 114 ± 2(n = 20)
mmHg, respectively. SBP of B2R(+/+) mice measured at 12 and 17 mo
of age averaged 112 ± 2 (n = 8) and 115 ± 5 (n
= 5) mmHg, respectively. There were no significant differences in SBP at 12
and 17 mo of age in B2R(-/-)CRD mice compared with
B2R(+/+) mice maintained on the NS diet throughout postnatal life
(Fig. 5).
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Fig. 5. SBP at 2, 3, 4, 12, and 17 mo of age in
NS/B2R(-/-)CRD (solid bars; n = 10-20) and
NS/B2R(+/+) (hatched bars; n = 5-8) mice. Data at 2, 3,
and 4 mo adapted from Ref. 4.
*P < 0.05 vs. B2R(+/+) mice at a given time
point.
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Effect of HS diet on SBP profile in
B2R(-/-)CRD and
B2R(+/+) mice. In response
to 4 wk of elevated dietary salt, 18-mo-old
HS/B2R(-/-)CRD exhibited a significant rise in SBP,
whereas SBP in HS/B2R(+/+) was not altered
(Fig. 6). SBP of
HS/B2R(-/-)CRD increased significantly from 114 ±
2 to 127 ± 3 mmHg after 18 days compared with the NS diet. SBP
continued to increase over the following week in
HS/B2R(-/-)CRD and was significantly higher on days
24 and 28 compared with HS/B2R(+/+) mice. SBP
averaged 133 ± 3 in HS/B2R(-/-)CRD and 118
± 8 mmHg in HS/B2R(+/+) (P < 0.05) at the end of
4 wk of the HS diet. The impact of salt loading on SBP is evidenced by the
increase of 18 vs. 4 mmHg in HS/B2R(-/-)CRD and
HS/B2R(+/+) mice, respectively
(Fig. 6, inset).
BP profile in anesthetized
B2R(-/-)CRD and
B2R(+/+) mice on the HS
diet. Direct measurements of arterial BP were collected in anesthetized
mice to confirm the observation of salt sensitivity in
B2R(-/-)CRD mice made by tail-cuff SBP measurement.
HS/B2R(-/-)CRD mice exhibited higher DBP compared with
HS/B2R(+/+) mice when studied under pentobarbital sodium anesthesia
[66 ± 3 and 51 ± 3 mmHg, respectively (P < 0.05);
Fig. 7C]. MAP
(Fig. 7A) and SBP
(Fig. 7B) tended to be
higher in HS/B2R(-/-)CRD than HS/B2R(+/+)
mice, averaging 79 ± 4 and 62 ± 3 mmHg, respectively (P
= 0.06). HR was not different between HS/B2R(-/-)CRD and
HS/B2R(+/+) mice (449 ± 24 vs. 444 ± 61 beats/min;
Fig. 7D).
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Fig. 7. Hemodynamic profile of anesthetized B2R(-/-)CRD
(solid bars; n = 14) and B2R(+/+) (hatched bars;
n = 4) mice on the HS diet. Mean arterial (A) and systolic
blood (B) pressures of B2R(-/-)CRD mice tended
to be higher than B2R(+/+) mice. Diastolic BP (C) was
significantly elevated in B2R(-/-)CRD compared with
B2R(+/+) mice. Heart rate (D) was not different between
the two groups. *P < 0.05 vs. B2R(+/+) mice.
bpm, beats/min.
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Body weight was not different in male and female 18-mo-old
HS/B2R(-/-)CRD and male HS/B2R(+/+) mice
(Table 2). Plasma sodium and
potassium concentrations were not different between the groups. Plasma protein
concentration was significantly higher (P < 0.05); however,
hematocrit values were not different in HS/B2R(-/-)CRD
compared with HS/B2R(+/+) mice. Consistent with the postnatal
phenotype of renal dysgenesis, total kidney weight and kidney-to-body weight
ratios were reduced significantly in adult
HS/B2R(-/-)CRD compared with HS/B2R(+/+) mice
(Table 2).
Kidney protein expression in
B2R(-/-)CRD and
B2R(+/+) mice on the HS
diet. To assess whether the urinary excretory abnormalities in
HS/B2R(-/-)CRD are associated with changes in tubular
sodium and water transport mechanisms, the abundance of the renin-angiotensin
system components, Na+-K+-ATPase, and AQP-2 protein
expressions were determined in kidneys obtained from 18-mo-old
HS/B2R(-/-)CRD and HS/B2R(+/+) mice by
Western blot analysis. AQP-2 protein expression was significantly lower by 52%
in kidneys of HS/B2R(-/-)CRD compared with
HS/B2R(+/+) mice (P < 0.05;
Fig. 8).
Na+-K+-ATPase, renin, and AT1 receptor
protein expressions were not different between the two groups, but there was a
50% elevation in angiotensinogen protein in
HS/B2R(-/-)CRD (P < 0.05; data not
shown).
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DISCUSSION
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Genetic and environmental factors are implicated in the pathogenesis of
CRD. The environmental component includes the fetal-maternal milieu. As such,
we have developed a model of CRD resulting from the combination of an
environmental stressor (gestational HS) combined with genetic deletion of the
bradykinin B2 receptor. This animal model exhibits congenital
collecting duct dysgenesis (Ref.
10 and this study). Long-term
follow-up in the present study demonstrates that the collecting duct
dysgenesis is permanent and irreversible. In addition, microscopic tumors of
mesenchymal cells emerge in the dysplastic kidneys. Functional studies
performed at 17-18 mo of age revealed that CRD mice show abnormalities in salt
and water handling and a propensity to develop salt-induced hypertension.
Importantly, 1-yr-old B2R(-/-) maintained on an NS diet since the
time of conception revealed no abnormalities in renal development and had no
renal tumors. Collectively, these findings present direct genetic evidence
linking aberrant intrauterine gene-environment interactions with abnormal
renal development and salt-sensitive hypertension.
A surprising finding of this study is the emergence of tumor growths in
kidneys of 1-yr- and 18-mo-old B2R(-/-)CRD mice.
Interestingly, studies in humans have described the emergence of nodular
blastema tumors within dysplastic kidneys
(16). Immunohistochemical
staining of 1-yr-old and 18-mo-old B2R(-/-)CRD kidneys
identified the cellular masses as proliferative (BrDU positive) and of
mesenchymal cell origin (smooth muscle -actin and vimentin positive).
In comparison, the tumorlets were negative for tubular epithelial cell markers
such as angiotensinogen, Na+-K+-ATPase, AQP-2,
E-cadherin, and -catenin. Given the proximity of the tumorlets to
glomeruli, the possibility that these cells originated from the
extraglomerular mesangium was considered. Unfortunately, there are no specific
markers for mesangial cells in the mouse. Finally, the core of the tumorlet
was CD45 negative, indicating that the cells are not of hematopoeitic origin.
We hypothesize that the tumorlets originated from a population of renal
stromal stem cells that maintained uncontrolled proliferation in the aberrant
microenvironment of the dysplastic kidney. Additional studies are necessary to
elucidate the cell biology of abnormal growth regulation in this model,
particularly in light of the fact that hypertensive patients are pre-disposed
to renal cancer (6,
13).
Alfie et al. (1) reported
that SBP and MAP were higher in adult B2R(-/-) mice maintained on
the HS diet for 8 wk compared with B2R(-/-) on the NS diet. Madeddu
et al. (15) have shown that
SBP and MAP were higher in B2R(-/-) than B2R(+/+) mice
on the NS diet. It is possible that the difference between the two studies is
related to the genetic background of the mice under study (129Sv vs. mixed
129/BL6). We have previously shown that B2R null mice exposed to a
"postnatal" HS diet for 4 mo develop hypertension yet do not show
signs of renal dysplasia (4).
Only the combination of gestational HS diet and lack of B2R
receptors leads to renal dysplasia. We have capitalized on this unique model
of renal dysplasia to investigate the long-term effects of salt and water
handling on BP, and renal morphology. The results of the present study show
that, under conditions of normal salt intake,
B2R(-/-)CRD mice remain normotensive up to 17 mo of age.
However, B2R(-/-)CRD mice have a propensity to develop
hypertension when challenged with a chronic dietary salt load. Loss of
bradykinin's natriuretic actions cannot be the sole reason for salt
sensitivity in HS/B2R(-/-)CRD, since bradykinin's
actions via the B2R are natriuresis and diuresis, and these mice
exhibited substantial increases in both salt and water excretion in response
to chronic salt loading. Therefore, additional studies are warranted to
elucidate the mechanisms of hypertension in 18-mo-old
HS/B2R(-/-)CRD mice.
In the present study, we observed decreased kidney/collecting duct AQP-2
expression in HS/B2R(-/-)CRD compared with age- and
sex-matched wild-type mice, which may contribute, at least partly, to the
diuresis. Interpretation of the exaggerated natriuresis in
HS/B2R(-/-)CRD mice is more complex, because estimates
of dietary salt intake are not available. It is important to note that higher
body weights were observed in B2R(-/-)CRD compared with
B2R(+/+) mice in both the mice on the NS and HS diets
(Table 1), yet exaggerated
natriuresis was only observed in the HS/B2R(-/-)CRD
group. Also, differences in appetite cannot account for the natriuresis
because a primary increase in salt appetite, resulting in a threefold increase
in steady-state urinary sodium excretion, would be expected to cause a
significant weight gain as a result of higher caloric intake. However, this
did not occur because NS/B2R(-/-)CRD and
HS/B2R(-/-)CRD had similar body weights on NS and HS
diets (Table 1). The precise
mechanisms of hypertension in HS/B2R(-/-)CRD remain to
be defined. From a "clinical" standpoint, patients born with CRD
initially present with complex electrolyte disturbances secondary to renal
tubular salt wasting and/or concentrating defects, renal tubular acidosis, and
varying degrees of renal functional impairment. Treatment involves careful
replacement of fluid and electrolyte losses to maintain euvolemia
(12). It is unknown whether
euvolemic CRD patients are susceptible to salt-induced hypertension, similar
to the B2R(-/-)CRD mice.
In summary, 1-yr-old and 18-mo-old B2R(-/-)CRD mice
with CRD exhibit persistent structural abnormalities such as renal tubular
ectasia and glomerular cysts. An unexpected new finding was the development of
mesenchymal-type tumor growth in the kidneys of
B2R(-/-)CRD mice. BP, urine flow, and sodium excretion
are normal at 17 mo of age in B2R(-/-)CRD mice
maintained on an NS diet throughout postnatal life. However, when challenged
with the HS diet for 4 wk, 18-mo-old B2R(-/-)CRD mice
exhibit a rise in SBP and greater natriuretic and diuretic responses than
salt-loaded B2R(+/+) mice. The diuresis is consistent with the
downregulation of kidney AQP-2 expression. The development of salt-induced
hypertension in the face of enhanced urinary salt/water excretion in 18-mo-old
B2R(-/-)CRD is puzzling. To our knowledge, there are no
published studies that have addressed renal handling of a salt load in adult
humans born with renal dysgenesis and whether these individuals have increased
susceptibility to salt-induced hypertension. Our study suggests such
possibilities. In conclusion, the B2R(-/-)CRD mouse is a
novel model of human disease that links gene-environment interactions with
renal development and BP homeostasis.
|
DISCLOSURES
|
|---|
This work was supported by National Institutes of Diabetes and Digestive
and Kidney Diseases Grant DK-56264 (to S. S. El-Dahr). L. M. Harrison-Bernard
was supported by American Heart Association Scientist Development Grant
9930120N and NIDDK Grant DK-62003-01. Digital images were obtained at the
Tulane Hypertension and Renal Center of Excellence Imaging Core Facility made
possible by the HEF2001-06-07 Louisiana Board of Regents through the
Millennium Trust Health Excellence Fund.
|
ACKNOWLEDGMENTS
|
|---|
We thank Drs. Conrad Sernia and Dan Catanzaro for generously providing the
angiotensinogen and renin antibodies and Dr. Kenneth D. Mitchell for critical
review of the manuscript.
|
FOOTNOTES
|
|---|
Address for reprint requests and other correspondence: S. S. El-Dahr, Dept. of
Pediatrics, SL37, Tulane Univ. Health Sciences Center, 1430 Tulane Ave., New
Orleans, LA 70112-2699 (E-mail:
seldahr{at}tulane.edu).
The costs of publication of this article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
|
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Copyright © 2003 by the American Physiological Society.
TOP
ABSTRACT
METHODS
; n =
13-20) and B2R(+/+) (
; n = 4-5) mice beginning at 17
mo of age. Bars indicate time points for NS and HS diets. SBP was not
different between the groups on NS, remained constant in B2R(+/+)
on the HS diet, but increased significantly in
B2R(-/-)CRD mice on the HS diet. The change in SBP from
the last day of NS to the last day of HS is shown in inset [solid
bar, B2R(-/-)CRD; hatched bar, B2R(+/+)].
*P < 0.05 vs. NS. 
P < 0.05 vs.
B2R(+/+) mice.