ANG II AT1 and AT2 receptors in
developing kidney of normal microswine
Susan P.
Bagby1,
Linda
S.
LeBard1,
Zaiming
Luo1,
Bryan
E.
Ogden2,
Christopher
Corless3,
Elizabeth D.
McPherson1, and
Robert C.
Speth4
Departments of 1 Medicine,
2 Comparative Medicine, and 3 Pathology,
Oregon Health and Science University, and Portland Veterans Affairs
Medical Center, Portland, Oregon 97201-2940; and
4 Departments of Veterinary and Comparative Anatomy and
of Pharmacology and Physiology, School of Veterinary Medicine,
Washington State University, Pullman, Washington 99164-6520
 |
ABSTRACT |
To identify an appropriate model of human
renin-angiotensin system (RAS) involvement in fetal origins of adult
disease, we quantitated renal ANG II AT1 and
AT2 receptors (AT1R and AT2R, respectively) in fetal
(90-day gestation, n = 14), neonatal (3-wk, n = 5), and adult (6-mo, n = 8)
microswine by autoradiography (125I-labeled
[Sar1Ile8]ANG II+cold CGP-42112 for AT1R,
125I-CGP-42112 for AT2R) and by whole kidney radioligand
binding. The developmental pattern of renal AT1R in microswine, like
many species, exhibited a 10-fold increase postnatally
(P < 0.001), with maximal postnatal density in
glomeruli and lower density AT1R in extraglomerular cortical and outer
medullary sites. With aging, postnatal AT1R glomerular profiles
increased in size (P < 0.001) and fractional area
occupied (P < 0.04), with no change in the number per
unit area. Cortical levels of AT2R by autoradiography fell with age
from
5,000 fmol/g in fetal kidneys to
60 and 20% of fetal levels
in neonatal and adult cortex, respectively (P < 0.0001). The pattern of AT2R binding in postnatal pig kidney mimicked
that described in human and simian, but not rodent, species: dense AT2R
confined to discrete cortical structures, including pre- and
juxtaglomerular, but not intraglomerular, vasculature. Our results
provide a quantitative assessment of ANG II receptors in developing pig
kidney and document the concordance of pigs and primates in
developmental regulation of renal AT1R and AT2R.
developing kidney; AT1; AT2; quantitative
autoradiography; swine
 |
INTRODUCTION |
THE RENIN-ANGIOTENSIN
SYSTEM (RAS) plays an important role in the development of renal
vascular and tubular structures (1, 27). Although ureteral
bud induction during early nephrogenesis occurs in in vitro organ
culture despite ANG II AT1 receptor (AT1R) blockade
(1), the in vivo role of ANG II in determining nephron number remains controversial. Emerging evidence also implicates the ANG
II AT2 receptor (AT2R) in renal development
(11), both in morphogenesis of the urogenital system
(6) and in fetal vasculogenesis and vascular
differentiation (32).
Renal AT1R and AT2R also play critical roles in postnatal renal
hemodynamics and renal tubular function in both physiological and
pathological states. Thus AT1R stimulation promotes vasoconstriction and Na retention in the normal kidney and activates cellular growth and
fibrosis in inflammatory disease states (27). New evidence supports an active role of AT2R in attenuation of renal AT1R responses (16) via AT2R activation of nitric oxide- and
bradykinin-mediated vasodilation (4, 8, 23), inhibition of
pressure-natriuresis (20), growth
inhibition/apoptosis (33), and antifibrotic
actions (24). These actions underscore the importance of a
balance between AT1R and AT2R activities in determining net in vivo
renovascular response to ANG II and thus vulnerability to hypertension
when AT1R actions are unopposed (23).
Much of our information about AT1R and AT2R derives from rodent models.
However, studies suggest potentially important differences between
humans and rodents in the distribution of intrarenal AT2R (12). Autoradiographic studies of renal ANG II receptors
in rodents have demonstrated the absence of vascular AT2R (28, 30), whereas similar human and simian studies reveal the
consistent presence of AT2R in preglomerular arterioles (12, 13,
26, 34). To identify a nonprimate animal model that is optimal
for the study of human RAS involvement in intrauterine growth
retardation and associated adult hypertension, we have examined ANG II
receptors in microswine, a species with cardiovascular, renal, and
immune systems uniquely similar to those of humans (7,
19). We have quantitatively assessed renal AT1R and AT2R in
normal fetal, neonatal, and adult microswine by autoradiography and
membrane radioligand binding. Results indicate striking developmental
differences in renal AT1R and AT2R density and/or distribution,
document the histological structures responsible for AT1R/AT2R binding,
and emphasize the similar prominence of AT2R in postnatal preglomerular vasculature of porcine and human kidneys.
 |
METHODS |
Animal/Tissue Preparation
Time-bred normal microswine were obtained from Charles River
Laboratories and maintained in the Oregon Health and Science University
Animal Care Facility until study, according to institutionally approved
protocols (A439). For fetal studies, sows at 90 days' gestation
underwent sterile caesarian section, sequential fetal removal, and then
euthanasia with 85 mg/kg Beuthanasia-D (Schering; iv). Each piglet was
immediately killed, and the kidneys were removed, their length and
weight were recorded, and the tissue was immediately immersed in
ice-cold PBS. Under isofluorane anesthesia, 3-wk-old and 6-mo-old pigs
underwent a midline thoracoabdominal incision for kidney removal.
Coronal slices from the mid-right kidney were kept in ice-cold PBS for
3-4 h before being processed for frozen sections. Tissue from each
left kidney was placed in ice-cold PBS for membrane binding or
snap-frozen in liquid nitrogen for RNA extraction. Additional renal
tissue sections were quickly distributed to zinc-formalin (10%),
Carnoy's solution, or fresh 4% paraformaldehyde for histology.
Neonatal and adult, but not fetal, animals included in this report
represent partial commonality with control pigs used in studies of
intrauterine growth retardation (3-wk-old pigs: 2745,
2746, 2748, 2749, and 2750;
6-mo-old adult pigs: 1285, 1286, 1153,
and 1155).
Film Autoradiography for ANG II Receptors
Autoradiography for both AT1R and AT2R was performed within
24 h of tissue harvest on the basis of preliminary observations that AT1R, but not AT2R, binding declines in storage (Bagby S, unpublished observations; Ref. 25). Tissue blocks were
snap-frozen in optimal cutting tissue compound using a pentane-dry-ice
slurry; on the day of harvest, sequential 20-µm frozen sections were
cut, placed on gelatin-coated slides, and kept at
80°C until study. On the following day, sections were thawed and preincubated for 30 min
at room temperature in isotonic buffer [150 mM NaCl, 50 mM Tris, 50 µM Plummer's inhibitor (carboxypeptidase inhibitor), 20 µM
bestatin (aminopeptidase inhibitor), 5 mM EDTA, 1.5 mM
1,10-phenanthroline, and 0.1% heat-treated protease-free BSA, pH
7.4]. Consecutive sections were incubated for 25 min in one of four
isotonic-buffer solutions: 0.4 nM 125I-labeled
[Sar1Ile8]ANG II (*SIAII) for total ANG II
receptor binding; 0.4 nM *SIAII plus 10
6 M
[Sar1]ANG II (nonselective analog) for nonspecific
binding; 0.4 nM *SIAII plus 10
6 M valsartan (Val; an
AT1-selective antagonist) to demonstrate AT2R; and 0.4 nM
*SIAII plus 10
6 M CGP-42112 (an AT2R-selective compound)
to demonstrate AT1R. To independently confirm AT2R binding, we exposed
a second set of four consecutive sections to 0.4 nM
125I-CGP-42112 (*CGP) for total binding;
*CGP+[Sar1]ANG II for nonspecific binding; *CGP+Val for
the absence of AT1R binding; and *CGP+PD-123319 (a chemically distinct
AT2R-selective compound) for AT2R specificity of CGP-42112 in
microswine. Radioligands were purchased from the Peptide
Radioiodination Service Center (Washington State University, Pullman
WA). Sections were rinsed, dried, placed on Kodak Biomax MR1 X-ray
film, and maintained at
80°C for 24 h. A standard slide with
calibrated concentrations ranging from 1 to 600 nCi/mg 125I
(Microscales, Amersham-Pharmacia Biotech, Piscataway, NJ) was included
in each cassette. The film was developed using an automated Kodak film processor.
Image Analysis
Images were captured using an AIS image-analysis system (Imaging
Research, St. Catharines, ONT) via an analog camera. The average
density of ANG II receptors was determined by reference to the
125I standards for each film. Because AT1R binding in fetal
kidneys was low and diffusely distributed, the average density was
assessed across the whole kidney area. In postnatal kidneys, using a
threshold setting, AT1R binding was analyzed for size, number per area, and fractional area of the cortex occupied by the distinctly denser glomerular profiles (identity confirmed; see below). AT1R density was
assessed both within glomeruli and separately in the extraglomerular tissue. Fetal AT2R density was individually assessed in the pelvic wall
("pelvis"), papilla, outer medulla, main cortex (excluding the
outer rim), and subcapsular cortex. In postnatal cortical areas, where
the AT2R occupied only a portion of the cross-sectional area, a
threshold setting was used to assess the average AT2R density, size,
and number per unit area of AT2R-rich "hotspots" and also to
estimate the fraction (%) of the cortical cross-sectional area
occupied by AT2R. Specific AT2R binding in intervening cortical tissue
was undetectable in both neonatal and adult kidneys. Results for AT2R
using *SIAII+Val were qualitatively similar but of lower resolution
than those using *CGP-42112. Thus formal analysis was restricted to
*SIAII+CGP-42112 for quantitating AT1R and *CGP-42112 for AT2R, each
with subtraction of the relevant nonspecific binding density.
Image-analysis data for each receptor subtype were analyzed by ANOVA,
either one-way with age as the experimental factor or, if data
permitted, two-way with age and region of kidney as factors. Sex-related differences were evaluated only in fetal kidney.
To identify histological sites of ATR binding, adjacent frozen sections
were processed for autoradiography or immunostained by an avidin-biotin
protocol previously detailed (5) for factor VIII-related
antigen (1:4,000 dilution, Dako). Slide images were digitized,
identically sized, and electronically overlaid.
ANG II Receptor Binding in Fresh Kidney Membranes
Freshly harvested tissue from 14 fetal and 8 adult animals was
processed according to standard methods, including homogenization, preparation of a crude membrane fraction via 100,000-g
ultracentrifigation, protein assay (Bio-Rad), and incubation of
20-100 µg protein/well at 37°C with appropriate radioligands
in an assay volume of 150 µl. Each binding assay included *SIAII and
*CGP saturation curves (each in triplicate) and *SIAII displacement
curves using CGP-42112 (displacement), Val (AT2R displacement), or ANG
II (AT1R and AT2R displacement), each done in duplicate with a
nonspecific binding well. Average specific binding was calculated in
Excel (Microsoft, Redmond WA) as femtomoles radioligand per milligram
lysate protein. Curves were analyzed in PRISM (GraphPad Software, San
Diego, CA). Statistical analysis was carried out using SigmaStat
(Jandel Scientific Software, San Rafael, CA) via ANOVA for effects of
age (fetal vs. adult) or sex (fetal data only) on 1) maximum
binding (Bmax) for *SIAII (AT1R+AT2R), *CGP (AT2R), and
AT1R by difference; 2) Kd for each
radioligand; 3) %AT1R using Val displacement curves; %AT2R
using CGP-42112 displacement curves; and log IC50 for each displacing ANG II analog by standard PRISM formulas.
 |
RESULTS |
AT2R
Fetal kidney.
By autoradiography, AT2R were overwhelmingly predominant in fetal
kidney (Figs. 1 and
2), maximally dense in primordial
papillary regions (P < 0.025 vs. other areas) and
extending in spokelike cords to a distinct outer nephrogenic region.
Despite trends toward higher AT2R density in several regions of female
kidneys (Fig. 2), there were no significant sex differences in
autoradiographic AT2R density. By radioligand binding (Table
1, Fig.
3), fetal kidney again exhibited high
levels of total ANG II receptors (combined average: 352 ± 276 fmol/mg protein, n = 14) that were 96 ± 3% AT2R
by competition binding and by a comparison of *SIAII Bmax (total ATR) vs. *CGP Bmax (AT2R only) (Table 1). As in
autoradiographic AT2R binding, there were no significant sex
differences in receptor number (Bmax) or subtype
distribution. However, Kd values for both *SIAII
and *CGP-42112 were significantly higher in females than in males
(Table 1), indicating a lower AT2R affinity in the fetal female kidney.
(Because of the predominance of AT2R, *SIAII binding affinity in this
context also reflects primarily that of the AT2R.) Since differing
Kd values could bias results of autoradiographic
receptor density, we reexamined the effect of sex on AT2R density after
adjusting for sex-dependent Kd differences. However, even after this adjustment, AT2R density values did not differ
significantly between fetal males and females.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 1.
ANG II receptors in fetal kidney. Top:
representative autoradiographic images of ANG II AT2
receptors (AT2R) by 0.4 nM 125I-CGP-42112 (*CGP) binding
that reflect (left to right) total binding
[AT2R+nonspecific (Nonspec)]; nonspecific binding {*CGP+cold
[Sar1]ANG II ([S1]AII)}; AT2R
[*CGP+valsartan (Val), an ANG II AT1-selective analog];
and ANG II AT1 receptors (AT1R) [*CGP + PD-123319
(PD), an unrelated AT2-selective compound used to confirm
AT2R selectivity of CGP]. Bottom: 0.4 nM
125I-labeled [Sar1Ile8]ANG II
(*SIAII) autoradiographic images reflect (left to
right) total ATR (AT1R+AT2R+nonspecific); nonspecific
binding (*SIAII+cold [S1]AII); AT2R (*SIAII+Val); and
AT1R (*SIAII+PD). Each row represents a matched set of adjacent tissue
sections. Hot, relevant radioligand; cold, nonradioactive analog.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 2.
Regional ANG II receptor densities in male vs. female
fetal kidneys by quantitative image analysis. Receptor densities are
presented according to sex for indicated kidney regions and are shown
for AT2R (left set of 4 paired bars) and AT1R
(right paired bars). AT2R density was high in all regions;
the primordial medullary region significantly exceeded other regions
(P < 0.001), which did not differ from each other.
AT1R density was uniformly low ( 1/40th of AT2R density) and
diffusely distributed. Note differing y-axis scale for AT1R
(right) compared with AT2R (left). There were no
significant sex differences despite a trend toward higher AT2R and
lower AT1R numbers in females.
|
|

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
ANG II radioligand binding in fetal kidney membranes.
Representative binding curves indicate specific AT2R saturation binding
using AT2-selective radioligand *CGP (A),
specific total (AT1R+AT2R) saturation binding via nonselective
radioligand *SIAII (B), and competition binding curves using
0.55 nM *SIAII displaced by increasing concentrations of valsartan
(AT1 selective), CGP-42112 (AT2 selective), or
ANG II (nonselective) (C). D: AT1R binding.
Direct confirmation of AT1R was made using *SIAII saturation binding in
the presence of 10 5 M cold CGP-42112. Collectively,
results show overwhelming dominance of AT2R in 90-day fetal kidney.
Results shown are representative of 14 experiments summarized in Table
1.
|
|
Neonatal kidney.
By comparison with fetal kidney, 3-wk-old neonatal kidney (Fig.
4) exhibited a lower density of cortical
AT2R by autoradiography (Fig. 5 vs. Fig.
2) (P = 0.002 vs. fetal)
and a substantially altered distribution pattern (Fig. 4 vs. Fig.
1). Thus AT2R binding in neonatal cortex was confined to
discrete AT2R-rich clusters (AT2R hotspots) with no detectable specific
binding in the intervening cortical tissue (Fig. 5). Average AT2R
density within neonatal hotspots in both the subcapsular and main
cortex significantly exceeded the density of the linear outer medullary
and the low-diffuse papillary AT2R (Fig. 5) (regional difference
P < 0.001). Intense pelvic wall AT2R binding was
similar to cortical hotspot levels. AT2R hotspots in the subcapsular
cortex occupied a higher fractional area (P < 0.025)
and were more numerous per unit area (P < 0.01) than
those of the main cortex (Fig. 6). AT2R
hotspot size did not differ between the two cortical regions. The
AT2R-dense structures were often linear and sometimes branching. On the
basis of overlays of autoradiographic and adjacent histological images
immunostained for factor VIII-related antigen (Fig. 7,
left), AT2R hotspots included
some, but not all, vessels (specifically, small intracortical arterioles and microvessels of the outer medulla); juxtaglomerular, but
not intraglomerular, structures; and large neural trunks accompanying penetrating pelvic vessels. [Note that attempted radioligand
binding using both *SIAII and *CGP in neonatal whole kidney membrane
homogenates yielded barely detectable binding in only 2 of 8 neonatal
kidneys tested (data not shown).]

View larger version (65K):
[in this window]
[in a new window]
|
Fig. 4.
ANG II receptors in neonatal kidney by autoradiography. Images are
labeled as described for Fig. 1. AT2R [top, first and third
panels (left to right)] are prominent in cortex
as discrete AT2R-dense structures ("hotspots") with variable
shapes; there are no detectable AT2R in the intervening cortical tissue
(see also Fig. 7). AT2R were also present in outer medulla, papilla,
and pelvic wall. AT1R (bottom,
far-right) cortical binding shows discrete, intensely
AT1R-positive "hotdots" (glomerular profiles; see Fig. 7)
superimposed on less dense, homogenous extraglomerular and medullary
AT1R binding. AT1R was not visible in the pelvic wall.
|
|

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 5.
Regional ANG II receptor densities in neonatal kidney by
quantitative image analysis. Autoradiographic receptor densities for
indicated regions of the kidney are shown for AT2R (left set
of 6 bars) and AT1R (right set of 6 bars). AT2R-rich
cortical hotspots were of similar density in subcapsular (Subcaps Ctx)
vs. main cortex and significantly greater than in the intervening
("background"; "Bkgd") cortical tissue (undetectable) and in
the outer medullary region and papillary areas (P < 0.001 overall region effect). Pelvic wall AT2R density was high and
similar to that of cortical hotspots. AT1R density in glomerular
profiles was similar in subcapsular vs. main cortical regions and was
significantly higher than that in extraglomerular (background)
cortical, medullary, or papillary regions. AT1R was undetectable in the
pelvic wall.
|
|

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 6.
ANG II receptor distribution patterns in neonatal kidney
cortex by image analysis. Data are shown for AT2R (left) and
AT1R (right) and plotted separately for subcapsular cortex
(hatched bars) vs. main cortex (open bars). See also Table 2. AT2R in
cortex were present only within hotspots. The visual appearance of more
confluent AT2R in the subcapsular vs. main cortical region (e.g., Fig.
4, top left) was corroborated by significant increases in
AT2R fractional area and number of hotspots/unit area without a size
difference compared with main cortex. The AT1R distribution pattern was
notable for a 6-fold higher fractional area occupied by glomerular
profiles in subcapsular (nephrogenic) vs. main cortex. However, the
apparent increase in size and fractional area of glomerular profiles is
overestimated, whereas glomerular density is underestimated because the
high density of normally sized glomerular profiles led to merging of
some outlines during image analysis. #/kpxl, No. of receptor-rich
structures (glomerular profiles for AT1R, hotspots for AT2R)/kilopixel
of cross-sectional area.
|
|

View larger version (147K):
[in this window]
[in a new window]
|
Fig. 7.
Microscopic ANG II binding sites in neonatal kidney. Adjacent
kidney sections were autoradiographically processed for AT2R
(middle left) or AT1R (middle right) binding or
were immunochemically stained for factor VIII-related antigen [brown
stain in endothelial cells of glomerular tufts (Gl) and vasculature
(top)]. The most intense (but not all) ATR-positive sites
in each autoradiogram were captured by setting a density threshold,
then overlaid on the matched, identically sized immunostained image
(bottom). AT2R binding sites (left) were
associated with intracortical and medullary microvessels (Mv),
juxtaglomerular (but not intraglomerular) structures, and with nerves
(Nv) accompanying pelvic arteries/veins. AT1R binding sites
(right) were maximally dense within glomerular tufts. All
extraglomerular structures were homogenously positive for AT1R at a
lower density, including tubules and microvessels.
|
|
Adult kidney.
In adult kidney cortex, AT2R similarly exhibited a "starry-sky"
distribution pattern: discrete clusters of intense AT2R binding with no
detectable binding in the intervening tissue (Figs.
8 and
9). Unlike in neonates, there was no
distinct subcapsular region in *CGP autoradiographs of adult kidneys.
Average AT2R density of adult cortical hotspots (Fig. 9) was
15%
that of fetal cortex and
25% that of neonatal hotspots (Table
2; age effect: P < 0.0001). As in neonates, selected adult autoradiographs demonstrated AT2R binding in larger renal vessels (e.g., Fig. 8). Compared with
neonatal hotspots, adult AT2R hotspots were larger (P = 0.05) but less numerous per unit area (P < 0.01), thus
occupying a comparable fractional area of the cortex (Table 2).

View larger version (62K):
[in this window]
[in a new window]
|
Fig. 8.
ANG II receptors in adult kidney by autoradiography. Images are
labeled as described for Fig 1. AT2R (top) in cortex were
distributed in a magnified version of the neonatal pattern, discrete
hotspots with no detectable specific binding in intervening tissue.
Dense binding in a large preglomerular penetrating artery is visible at
the corticomedullary junction. However, unlike neonates, there was no
AT2R binding in the outer medulla or papilla of the adult kidney. AT1R
(bottom right) again exhibits a pattern of maximally dense
glomerular profiles superimposed on diffuse extraglomerular AT1R
binding of lower density, prominently including the outer medullary
stripe. There was no distinguishable subcapsular cortical region in
adult kidneys. When present in autoradiographs, the pelvic wall was
consistently AT2R positive (not shown).
|
|

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 9.
Regional ANG II receptor densities in adult kidney by
quantitative image analysis. AT2R density in shown in the
left set of bars, AT1R in the right set. AT2R
binding was high in cortical hotspots and undetectable in intervening
cortical tissue and medulla. AT1R density of adult glomerular profiles
was statistically similar to neonatal levels and exceeded both the
diffuse extraglomerular cortical and the outer medullary AT1R densities
in the adult kidney.
|
|
With the use of autoradiographic vs. histological images from adult
kidney sections as described for neonates, AT2R-positive structures
were again found to include small intracortical arterioles and
juxtaglomerular sites, whereas the main glomerular tufts were consistently negative for AT2R binding (not shown). Although not present with enough consistency in autoradiographic sections for statistical analysis, the adult pelvic wall typically exhibited intense
AT2R binding. In contrast, unlike in the neonatal kidney, there were no
detectable AT2R in adult medulla (Fig. 8).
By radioligand binding in adult kidney membranes, the total number of
ANG II receptors was low, detectable in only four of eight kidneys
studied and averaging
1% of fetal total ANG II receptor levels.
*SIAII Bmax in the four adult kidneys averaged 5 ± 3 fmol/mg protein, of which 0-30% were AT2R. Representative binding
curves are shown in Fig. 10. However,
AT2R as assessed by *CGP saturation binding were undetectable in all
eight adult kidneys. Of potential importance, the insensitivity of *CGP
despite its high affinity for AT2R may be due to the unusually high
nonspecific binding in renal membranes, this despite the remarkably low
nonspecific binding in renal autoradiographs. Separating cortex vs.
medulla in binding assays did not improve the sensitivity of
radioligand binding in adult pig kidneys (data not shown). Thus, as in
neonates, radioligand binding in adult porcine whole kidney membranes
was insufficiently sensitive to consistently quantitate ANG II receptor status.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 10.
ANG II radioligand binding adult kidney membranes.
A: representative *SIAII saturation binding curve indicates
total ANG II receptors in adult kidneys. Maximum binding is 100-fold
lower than that of fetal kidneys in Fig 3. *CGP binding was
undetectable in 8 of 8 adult kidneys (data not shown), indicating AT2R
are below assay sensitivity in the whole kidney preparation.
B: competition binding curves using 0.55 nM *SIAII displaced
by analogs valsartan (AT1 selective), CGP-42112
(AT2 selective), or ANG II (nonselective) show virtually
100% AT1R present; AT2R ranged from 0 to 30%.
|
|
AT1R
Fetal kidney.
By autoradiography, AT1R binding in fetal kidney was very low but
consistently detectable, distributed diffusely throughout the cortex
while being absent in primordial papilla (Fig. 1, bottom right). AT1R density in fetal kidney was ~1/40th that of fetal AT2R (Fig. 2) (note separate y-axis scale for AT1R vs.
AT2R). There were no AT1R-dense structures in the fetal cortex
comparable to the glomerular profiles present in postnatal kidneys.
Although AT1Rs were difficult to directly detect by competition binding due to the large excess of AT2R, comparison of *SIAII (total) vs. *CGP
(AT2R only) binding in kidney membranes yielded an estimate of 4 ± 3% AT1Rs, which were also detectable by *SIAII saturation binding
carried out in the presence of AT2R blockade with 1 µM *CGP (Table 1;
Fig. 3D).
Neonatal kidney.
The pattern of AT1R distribution in 3-wk-old neonatal cortex tissue
(Fig. 4, bottom right) was one of the AT1R-dense glomerular profiles, seen as uniformly round "dots," superimposed on diffuse, less-dense AT1R binding in the intervening cortical tissue. AT1R density of neonatal glomerular profiles was 10-fold higher than that
for the fetal cortex, comparable in the glomerular profiles of
subcapsular and main cortical regions, and significantly higher than
that in the extraglomerular cortex or medullary tissue (Fig. 5).
However, neonatal subcapsular vs. main cortical areas exhibited significant differences in binding patterns: AT1R-dense glomerular profiles occupied a >10-fold larger fraction of the subcapsular area
than of the main cortical area (P < 0.001; Fig. 6).
Also, the average size of the glomerular profiles by image analysis was
4-fold increased in the subcapsular vs. main cortex. However, this
apparently increased size of neonatal subcapsular > main cortical
glomeruli was not supported by histological assessment (Fig. 7,
right). Instead, normal-sized, factor VIII-antigen-positive glomeruli in the subcapsular cortex were densely packed along with less
mature glomeruli, which lacked factor VIII-antigen immunoreactivity but
did exhibit AT1R binding (Fig. 7). This high density of subcapsular glomeruli caused overlapping outlines of adjacent glomerular profiles during use of the threshold setting for image analysis, resulting in an
overestimation of glomerular size while the number per unit area was
underestimated. Thus it is the increased number of glomeruli per unit area and the concomitant increase in fractional area occupied
by glomerular profiles (but not the size of glomeruli) that
distinguishes the subcapsular (nephrogenic) from the main cortex.
(Overlapping of glomerular profiles was not an issue in the main cortex
of neonates nor in adult kidney cortex.)
In adult kidney, there was, as with AT2R, no distinguishable
subcapsular cortical region in AT1R autoradiographs (Fig. 8). Concordance of AT1R-rich "hotdots" and glomerular profiles in adult
kidney was confirmed as described for neonatal kidney (not shown).
Average AT1R density of adult glomerular profiles was significantly
higher than that of extraglomerular cortical and outer medullary areas
(Fig. 9; P < 0.025) and was comparable to that of
neonatal glomeruli (P = NS) (Table 2). The average size (cross-sectional area) of AT1R-dense adult glomerular profiles was more
than threefold larger than that of the glomerular profiles in the main
cortex of neonates (P = 0.001). Adult glomerular
profiles occupied 10 ± 5% of cortical area, compared with 3 ± 2% of the main cortical area in neonates (P = 0.03)
(Table 2). The number of glomerular profiles per unit area did not
differ between adult cortex and neonatal main cortex (Table 2).
 |
DISCUSSION |
We have examined the distribution and densities of AT1R and AT2R
binding in the normal kidneys of developing microswine, a species with
renal anatomy and physiology uniquely similar to that of the human
kidney (19). Our major findings include 1) reciprocal developmental changes in AT1R (increasing) and AT2R (decreasing) density in microswine kidney from gestation day
90 (of 115) to postpubertal adulthood; 2)
identification and quantitation of distinct postnatal regional
distribution patterns for AT1R (maximally dense in glomeruli with
diffuse extraglomerular cortical and outer medullary binding) vs. AT2R
(confined to pre- and juxtaglomerular arterioles, outer medullary
microvessels, and some neural elements) in swine renal tissue;
3) sex differences in AT2R binding affinity in fetal kidney;
and 4) similarity of porcine and human kidneys in the
prominent presence and distribution pattern of AT2R (13, 34).
We are unable to identify prior reports of renal ANG II receptors in
swine. Grone et al. (14) reported that virtually 100% of
ANG II receptors in human fetal kidney are AT2R, in agreement with our
findings in fetal pig kidney. ATR binding in the postnatal human kidney
by autoradiography has been described in four studies: three of four
reported that AT2R predominated in preglomerular arteries in an
adventitial location (12-14, 34); glomeruli and outer
medullary vascular bundles exhibited dense AT1R, and tubules showed
less dense AT1R. Similarly, in simian kidney, AT2R were dominant in
arteries, whereas glomeruli contained only AT1R and the juxtaglomerular
apparatus exhibited both subtypes (12). One report that
failed to find AT2R in human intrarenal vasculature (29)
was based on limited human material. An immunochemical study in human
kidneys reported mild-to-moderate immunoreactive AT2R protein in blood
vessels, together with weak staining in glomeruli (21).
Overall, our autoradiographic findings in fetal and postnatal porcine
kidney closely match these prior reports in primate kidneys. Our
finding that AT1R-rich hotdots represent glomerular profiles in pigs is
in conformance with results in other species using emulsion
autoradiography (12-14, 34). Overlays of our
autoradiographic and histological images of pig kidney indicate that
postnatal porcine glomeruli exhibit little if any AT2R, whereas
cortical arterioles and outer medullary microvessels show dense AT2R
binding. Our results do not indicate whether small-arteriole-associated AT2R reflects binding by medial wall, adventitia, and/or associated microneural elements. Comparison of extracortical neonatal vs. adult
pig kidneys demonstrates loss or marked reduction of AT2R binding in
medullary microvessels with aging, whereas the pelvic wall retains
dense AT2R binding in adults. Finally, on the basis of the diffuse and
homogenous specific AT1R binding in extraglomerular cortical tissue,
our results indicate that the diverse tubular and vascular elements
within this compartment contain AT1R at comparable densities.
The primate-porcine pattern of AT2R is in distinct contrast to reports
of AT1R/AT2R distribution in the rodent kidney. In a paired
autoradiographic study of adult human and rat kidney, Gibson et al.
(12) found AT2R in human renal vasculature but not in rat
kidney. Song et al. (30) also found only AT1R in adult rat
renal structures. Immunochemical studies of rat kidney by Ozono et al.
(28), using a well-validated anti-AT2R antibody, demonstrated the virtual disappearance of AT2R protein by 4 wk of age,
whereas a low-Na diet induced the reappearance of AT2R protein. This
was localized to glomerular and interstitial sites, not to vasculature
sites (28). In disagreement with these reports, Miyata et
al. (22) found immunoreactive AT2R protein throughout the
rat kidney except in the glomerulus and thick ascending limb. Finally,
Cao et al. (3) applied radioiodinated CGP-42112
autoradiography to rat kidney sections and found extensive AT2R
binding, most prominently in the inner cortex. However, this did not
reproduce the pattern reported in primate and porcine kidney (as
described above in this section). Studies of AT2R mRNA in rat kidney
are also discrepant. Norwood et al. (26) found no AT2R
mRNA in 1-mo-old rat kidney, and Kakuchi et al. (17) noted
the disappearance of rat renal AT2R mRNA at birth. However, Miyata et
al. (22) reported positive AT2R mRNA in all rat renal
compartments except glomeruli and medullary thick ascending limb.
Because we were unable to reconcile these differences in reported
studies of AT2R binding and mRNA in postnatal rodent kidney, we
examined AT1R and AT2R in three normal adult rat kidneys by autoradiographic techniques identical to those used in our adult microswine. Confirming the results of Gibson et al. (12),
no specific AT2R binding was detectable in adult rat kidney after 24-h
exposure (Fig. 11). This does not
exclude AT2R in rat kidney; 3-4 wk of exposure are required for
the detection of macrovascular AT2R in pigs. However, the differences
noted demonstrate a substantially lower AT2R density in adult rat
kidney compared with adult porcine kidney.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 11.
ANG II receptors in normal adult rat kidney by
autoradiography. Because of discrepancy among published reports, we
examined 3 adult rat kidneys for AT1R (left) and AT2R
(right) by autoradiographic methods identical to those
applied to microswine kidneys reported here. After 24-h exposure, AT2R
binding by *CGP autoradiography was undetectable, whereas AT1R binding
using *SIAII+CGP autoradiography was abundant.
|
|
Previous reports of vascular AT2R in primate kidney localize the
receptors to the adventitial layer (12, 34). We have recently examined localization of ANG II receptors in conduit arteries
(including the main renal artery) in normal microswine (2). We found that AT2R localize to a discrete
circumferential cell layer along the medial-adventitial border in what
would typically be described as adventitia. However, using smooth
muscle-specific
-actin immunochemistry in parallel with AT2R
autoradiography, it has been found that AT2R-positive cells, while
-actin negative, in fact extend into the outermost medial layer,
occupying what Stenmark and colleagues (9, 10) have
described as "intersitital spaces" between longitudinal muscle
bundles. These medial-adventitial boundary cells, which formed a
continuous layer extending from the abdominal aorta into its branch
artery walls, are optimally positioned to mediate AT2R-dependent
effects of ANG II generated locally in vascular interstitium (15,
31). Recent studies support an active role for AT2R in both
modulating renovascular responses to ANG II and regulating systemic
blood pressure. Thus in vivo infusion of AT2R-antisense oligonucleotide
into the renal interstitial space in normal rats induced hypertension
and enhanced pressor sensitivity to exogenous ANG II (23),
indicating a tonically active role for AT2R in the renal vasculature
even in the rodent. Accordingly, the abundance of preglomerular
arterial AT2R may well determine not only the renal hemodynamic
response to ANG II but also the set point of renal homeostatic
regulation of blood pressure.
Our studies raise questions about potential male-female differences in
the AT2R, which is located on the X chromosome (18). In
radioligand binding studies of fetal kidneys, membranes from females
exhibited a significantly lower affinity (higher
Kd) for both *CGP and *SIAII radioligands. In
autoradiographic studies, fetal female kidneys showed a higher average
density of AT2R in cortical and outer medullary regions, although this
was not significant, even after an adjustment for observed
Kd differences. Unfortunately, our small numbers
and serendipitous male/female distribution did not permit a formal
assessment of sex differences in either neonatal or adult animals.
In summary, we have demonstrated that developmental regulation of ANG
II receptors in microswine follows directional changes similar to those
reported in other species: increasing AT1R and decreasing AT2R
densities with aging. Furthermore, AT1R density and distribution over
the course of postnatal development are similar to those reported in
both rodent and primate kidneys. In contrast, AT2R in postnatal pig
kidney localize predominantly to cortical preglomerular and
juxtaglomerular vasculature (and, in neonates, to outer medullary
vasculature), thus following a primate rather than a rodent pattern of
renal AT2R distribution. Importantly, our results do not differentiate
which vascular wall components exhibit AT2R binding. Our findings
provide new information on developmental AT1R and AT2R regulation in
the pig kidney, support potentially important species differences in
AT2R abundance and distribution, and validate the swine model for study
of the renal RAS in fetal origins of human disease.
 |
ACKNOWLEDGEMENTS |
The authors acknowledge the contributions of Vicki Feldmann, Oregon
Health and Science University (OHSU) Laboratory Animal Technician; the
support and technical contributions of Carolyn Gendron and Linda
Jauron-Mills of OHSU Heart Research Center Imaging Core Laboratory; the
skills of photographer Michael Moody of the Portland VAMC Medical Media
Service; and the administrative assistance of Kathleen Beebe in
preparation of the manuscript.
 |
FOOTNOTES |
This work was supported by National Institute of Child Health and Human
Development Grant PO1-HD-34430.
Address for reprint requests and other correspondence:
S. P. Bagby, Div. of Nephrology, Oregon Health and Sciences
Univ., Suite 262, 3314 SW US Veterans Hospital Rd., Portland, OR
97201-2940 (E-mail: bagbys{at}ohsu.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.
May 22, 2002;10.1152/ajprenal.00313.2001
Received 11 October 2001; accepted in final form 15 April 2002.
 |
REFERENCES |
1.
Alcorn, D,
McCausland JE,
and
Maric C.
Angiotensin receptors and development: the kidney.
Clin Exp Pharmacol Physiol Suppl
3:
S88-S92,
1996[Medline].
2.
Bagby, SP,
LeBard LS,
Luo Z,
Speth RC,
Ogden BE,
and
Corless CL.
Angiotensin II Type 1 and 2 receptors in conduit arteries of normal developing microswine.
Arterioscler Thromb Vasc Biol
22:
1113-1121,
2002[Abstract/Free Full Text].
3.
Cao, Z,
Kelly DJ,
Cox A,
Casley D,
Forbes JM,
Martinello P,
Dean R,
Gilbert RE,
and
Cooper ME.
Angiotensin type 2 receptor is expressed in the adult rat kidney and promotes cellular proliferation and apoptosis.
Kidney Int
58:
2437-2451,
2000[ISI][Medline].
4.
Carey, RM,
Jin X,
Wang Z,
and
Siragy HM.
Nitric oxide: a physiological mediator of the type 2 (AT2) angiotensin receptor.
Acta Physiol Scand
168:
65-71,
2000[ISI][Medline].
5.
Corless, CL,
Kibel AS,
Iliopoulos O,
and
Kaelin WG.
Immunostaining of the von Hippel-Lindau gene product in normal and neoplastic human tissues.
Hum Pathol
28:
459-464,
1997[ISI][Medline].
6.
Curhan, GC,
Chertow GM,
Willett WC,
Spiegelman D,
Colditz GA,
Manson JE,
Speizer FE,
and
Stampfer MJ.
Birth weight and adult hypertension and obesity in women.
Circulation
94:
1310-1315,
1996[Abstract/Free Full Text].
7.
Danser, AH,
van Kats JP,
Admiraal PJ,
Derkx FH,
Lamers JM,
Verdouw PD,
Saxena PR,
and
Schalekamp MA.
Cardiac renin and angiotensins. Uptake from plasma versus in situ synthesis.
Hypertension
24:
37-48,
1994[Abstract].
8.
Dimitropoulou, C,
White RE,
Fuchs L,
Zhang H,
Catravas JD,
and
Carrier GO.
Angiotensin II relaxes microvessels via the AT(2) receptor and Ca2+-activated K+ BKCa channels.
Hypertension
37:
301-307,
2001[Abstract/Free Full Text].
9.
Frid, MG,
Aldashev AA,
Dempsey EC,
and
Stenmark KR.
Smooth muscle cells isolated from discrete compartments of the mature vascular media exhibit unique phenotypes and distinct growth capabilities.
Circ Res
81:
940-952,
1997[Abstract/Free Full Text].
10.
Frid, MG,
Dempsey EC,
Durmowicz AG,
and
Stenmark KR.
Smooth muscle cell heterogeneity in pulmonary and systemic vessels. Importance in vascular disease.
Arterioscler Thromb Vasc Biol
17:
1203-1209,
1997[Abstract/Free Full Text].
11.
Gallinat, S,
Busche S,
Raizada MK,
and
Sumners C.
The angiotensin II type 2 receptor: an enigma with multiple variations.
Am J Physiol Endocrinol Metab
278:
E357-E374,
2000[Abstract/Free Full Text].
12.
Gibson, RE,
Thorpe HH,
Cartwright ME,
Frank JD,
Schorn TW,
Bunting PB,
and
Siegl PK.
Angiotensin II receptor subtypes in renal cortex of rats and rhesus monkeys.
Am J Physiol Renal Fluid Electrolyte Physiol
261:
F512-F518,
1991[Abstract/Free Full Text].
13.
Goldfarb, DA,
Diz DI,
Tubbs RR,
Ferrario CM,
and
Novick AC.
Angiotensin II receptor subtypes in the human renal cortex and renal cell carcinoma.
J Urol
151:
208-213,
1994[ISI][Medline].
14.
Grone, HJ,
Simon M,
and
Fuchs E.
Autoradiographic characterization of angiotensin receptor subtypes in fetal and adult human kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
262:
F326-F331,
1992[Abstract/Free Full Text].
15.
Hollenberg, NK,
Fisher ND,
and
Price DA.
Pathways for angiotensin II generation in intact human tissue: evidence from comparative pharmacological interruption of the renin system.
Hypertension
32:
387-392,
1998[Abstract/Free Full Text].
16.
Ichiki, T,
Labosky PA,
Shiota C,
Okuyama S,
Imagawa Y,
Fogo A,
Niimura F,
Ichikawa I,
Hogan BL,
and
Inagami T.
Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor.
Nature
377:
748-750,
1995[ISI][Medline].
17.
Kakuchi, J,
Ichiki T,
Kiyama S,
Hogan BL,
Fogo A,
Inagami T,
and
Ichikawa I.
Developmental expression of renal angiotensin II receptor genes in the mouse.
Kidney Int
47:
140-147,
1995[ISI][Medline].
18.
Lazard, D,
Briend-Sutren MM,
Villageois P,
Mattei MG,
and
Strosberg AD.
Molecular characterization and chromosome localization of a human angiotensin AT2 receptor gene highly expressed in tissues.
Receptors Channels
2:
271-280,
1994[ISI][Medline].
19.
Lee, KT.
Swine as animal models in cardiovascular research.
In: Swine in Biomedical Research, edited by Tumbleson ME.. New York: Plenum, 1986, p. 1481-1496.
20.
Liu, KL,
Lo M,
Grouzmann E,
Mutter M,
and
Sassard J.
The subtype 2 of angiotensin II receptors and pressure-natriuresis in adult rat kidneys.
Br J Pharmacol
126:
826-832,
1999[Abstract/Free Full Text].
21.
Mifune, M,
Sasamura H,
Nakazato Y,
Yamaji Y,
Oshima N,
and
Saruta T.
Examination of angiotensin II type 1 and type 2 receptor expression in human kidneys by immunohistochemistry.
Clin Exp Hypertens
23:
257-266,
2001[ISI][Medline].
22.
Miyata, N,
Park F,
Li XF,
and
Cowley AW.
Distribution of angiotensin AT1 and AT2 receptor subtypes in the rat kidney.
Am J Physiol Renal Physiol
277:
F437-F446,
1999[Abstract/Free Full Text].
23.
Moore, AF,
Heiderstadt NT,
Huang E,
Howell NL,
Wang ZQ,
Siragy HM,
and
Carey RM.
Selective inhibition of the renal angiotensin type 2 receptor increases blood pressure in conscious rats.
Hypertension
37:
1285-1291,
2001[Abstract/Free Full Text].
24.
Morrissey, JJ,
and
Klahr S.
Effect of AT2 receptor blockade on the pathogenesis of renal fibrosis.
Am J Physiol Renal Physiol
276:
F39-F45,
1999[Abstract/Free Full Text].
25.
Moulik, S,
Speth RC,
and
Rowe BP.
Differential loss in function of angiotensin II receptor subtypes during tissue storage.
Life Sci
66:
L233-L237,
2000.
26.
Norwood, VF,
Craig MR,
Harris JM,
and
Gomez RA.
Differential expression of angiotensin II receptors during early renal morphogenesis.
Am J Physiol Regul Integr Comp Physiol
272:
R662-R668,
1997[Abstract/Free Full Text].
27.
Oliverio, MI,
and
Coffman TM.
Angiotensin II receptor physiology using gene targeting.
News Physiol Sci
15:
171-175,
2000[Abstract/Free Full Text].
28.
Ozono, R,
Wang ZQ,
Moore AF,
Inagami T,
Siragy HM,
and
Carey RM.
Expression of the subtype 2 angiotensin (AT2) receptor protein in rat kidney.
Hypertension
30:
1238-1246,
1997[Abstract/Free Full Text].
29.
Sechi, LA,
Grady EF,
Griffin CA,
Kalinyak JE,
and
Schambelan M.
Distribution of angiotensin II receptor subtypes in rat and human kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
262:
F236-F240,
1992[Abstract/Free Full Text].
30.
Song, K,
Zhuo J,
Allen AM,
Paxinos G,
and
Mendelsohn FA.
Angiotensin II receptor subtypes in rat brain and peripheral tissues.
Cardiology 79 Suppl
1:
45-54,
1991.
31.
Voors, AA,
Pinto YM,
Buikema H,
Urata H,
Oosterga M,
Rooks G,
Grandjean JG,
Ganten D,
and
van Gilst WH.
Dual pathway for angiotensin II formation in human internal mammary arteries.
Br J Pharmacol
125:
1028-1032,
1998[Abstract].
32.
Yamada, H,
Akishita M,
Ito M,
Tamura K,
Daviet L,
Lehtonen JY,
Dzau VJ,
and
Horiuchi M.
AT2 receptor and vascular smooth muscle cell differentiation in vascular development.
Hypertension
33:
1414-1419,
1999[Abstract/Free Full Text].
33.
Yamada, T,
Horiuchi M,
and
Dzau VJ.
Angiotensin II type 2 receptor mediates programmed cell death.
Proc Natl Acad Sci USA
93:
156-160,
1996[Abstract/Free Full Text].
34.
Zhuo, J,
Dean R,
MacGregor D,
Alcorn D,
and
Mendelsohn FA.
Presence of angiotensin II AT2 receptor binding sites in the adventitia of human kidney vasculature.
Clin Exp Pharmacol Physiol Suppl
3:
S147-S154,
1996[Medline].
Am J Physiol Renal Fluid Electrolyte Physiol 283(4):F755-F764