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Am J Physiol Renal Physiol 283: F755-F764, 2002. First published May 7, 2002; doi:10.1152/ajprenal.00313.2001
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Vol. 283, Issue 4, F755-F764, October 2002

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 congruent 5,000 fmol/g in fetal kidneys to congruent 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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  		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.



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  		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 (congruent 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.


                              
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  		Table 1.   ANG II receptor binding in fetal kidney membranes



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  		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).]


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  		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.



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  		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.



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  		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.



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  		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 congruent 15% that of fetal cortex and congruent 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).


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  		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).



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  		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.


                              
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  		Table 2.   ANG II receptors in neonatal vs. adult kidney cortex by quantitative autoradiography

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 congruent 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.


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  		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 congruent 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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  		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 alpha -actin immunochemistry in parallel with AT2R autoradiography, it has been found that AT2R-positive cells, while alpha -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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