Articles

Expression of the Subtype 2 Angiotensin (AT2) Receptor Protein in Rat Kidney

Ryoji Ozono, Zhi-Qin Wang, Allan F. Moore, Tadashi Inagami, Helmy M. Siragy, Robert M. Carey
https://doi.org/10.1161/01.HYP.30.5.1238
Hypertension. 1997;30:1238-1246
Originally published November 1, 1997

Jump to

Abstract

Abstract In situ hybridization studies have suggested that the subtype 2 angiotensin (AT2) receptor gene is expressed in fetal and newborn rat kidney but is undetectable in the adult animals. In the present study, we investigated the expression of AT2 receptor protein in the fetal (days 14 and 19 of fetal life), newborn (day 1 postpartum), and adult (4-week-old and 3-month-old) rat kidney. Polyclonal anti-peptide antiserum was raised against the amino terminus of the native AT2 receptor. The selectivity of the antiserum was validated by recognition of the AT2 receptor in a stably transfected COS-7 cell line by Western blot and immunocytochemical analysis. As a positive control, the AT2 receptor signal was detected strongly in the adrenal gland. Positive immunohistochemical staining was observed in the mesenchymal cells and ureteric buds of the 14-day fetal kidney and in the glomeruli, tubules, and vessels in the 19-day fetal and newborn kidney. Glomeruli expressing the AT2 receptor were localized mainly in the outer layer of the renal cortex. In the young (4-week-old) and mature (3-month-old) adult rat on normal sodium intake, renal AT2 receptor immunoreactivity was present in glomeruli but substantially diminished compared with that of newborn rats. In both young and mature adult rats, dietary sodium depletion increased the renal AT2 receptor signal, mainly in the glomeruli and interstitial cells. Preimmune and preadsorption controls were negative. Western blot analysis detected a single 44-kD band in the fetal and newborn rat kidney and in the young and mature adult rat kidney. Dietary sodium depletion increased the density of the AT2 receptor band in mature adult rat kidneys. These data provide evidence that the AT2 receptor protein is expressed in the fetal and newborn rat kidney, diminishes in adult life, and is reexpressed in the adult in response to sodium depletion.

Angiotensin II (Ang II) plays a major role in the regulation of blood pressure and fluid and electrolyte balance by binding to Ang II receptors. After the discovery of Ang II receptor subtype–specific antagonists, two pharmacologically distinct subclasses of Ang II receptors (AT1 and AT2) were identified and characterized.1 Subsequently, the cDNAs that encode the AT1 (AT1A and AT1B) and AT2 receptors were cloned, and the cell-signaling pathways that transduce their biological actions were investigated.2 3 4 5 6 7 Both subclasses of Ang II receptors have seven transmembrane domains typical of G protein–coupled receptors. However, AT1 and AT2 receptors share only 30% sequence homology and have distinctive functional properties and cell-signaling mechanisms on stimulation by Ang II.2

The AT1 receptor is localized to the brain, peripheral vasculature, heart, kidney, and adrenal gland and mediates most of the known functions of Ang II.1 In the kidney, all of the actions of Ang II on hemodynamic and tubular function are thought to be mediated via the AT1 receptor, including afferent and efferent arteriolar vasoconstriction, decreased glomerular filtration rate and renal plasma flow, and stimulation of sodium and fluid reabsorption in the proximal tubules.8 9 10 11 AT1 receptors are thought also to mediate growth and differentiation in the kidney.12

The biological function of the AT2 receptor is largely unknown.1 11 13 14 15 Recent studies have suggested that AT2 receptors mediate cell-signaling pathways related to inhibition of cell growth and differentiation.16 17 18 During fetal life, the AT2 receptor gene is expressed predominantly in areas of active mesenchymal differentiation, but the mRNA expression level decreases rapidly and disappears within a few days after birth.19 20 21 22 23 24 In the adult, AT2 receptor mRNA has been detected in the adrenal gland, heart, and brain.1 20 25 AT2 receptor mRNA is expressed in the fetal and neonatal rat kidney but disappears after the neonatal period and is not expressed in the normal adult.20 21 22 23

Recently, we have demonstrated that renal AT2 receptors may be activated during sodium depletion or Ang II administration in the conscious adult rat. We found that the AT2 receptor antagonist PD 123319 abolished the increase in renal interstitial fluid cGMP engendered by sodium depletion or Ang II infusion.26 AT2 receptor blockade with PD 123319 augmented AT1-receptor–mediated increases in renal interstitial fluid prostaglandin E2 levels.26 These observations suggested that the renal AT2 receptor may be reexpressed in the adult kidney in response to Ang II and may be physiologically important in the control of renal function.

The present study was directed toward the identification and site-specific distribution of the AT2 receptor protein in the rat kidney, to our knowledge for the first time. We employed a combination of immunohistochemistry and Western blot analysis to localize the AT2 receptor protein in the fetal, neonatal, and young (4-week-old) and mature (3-month-old) adult rat kidney and to determine whether the AT2 receptor is recruited in the adult in response to sodium depletion.

Methods

Animals

Timed pregnant Sprague-Dawley rats, as well as adult female Sprague-Dawley rats at an age of 4 weeks (80 g) and 3 months (about 250 g), were obtained from Hilltop Inc (Scottsdale, Pa). The fetus was removed from the uterus of pregnant rats at gestation days 14 (F14) and 19 (F19). Neonatal rats from the pregnant mothers were subjected to study at the age of 1 day (D1). For the sodium-depletion study, the young (4-week-old) and mature (3-month-old) adult rats were placed on sodium-deficient rat chow (containing <0.05% sodium) for 1 week. Sodium depletion was validated by measurement of 24-hour urine sodium excretion with the rats in metabolic cages before study. All animal procedures were in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals.

AT2 Receptor Antiserum

Polyclonal antibody was raised against a synthetic peptide sequence (MKDNFSFAATSRNITSS) derived from the amino terminus of predicted AT2 receptor amino acid sequence (Fig 1⇓). This sequence was selected because of its uniqueness to the AT2 receptor, with absence of significant homology to any other known protein. The antiserum was IgG affinity purified before use as described previously.27 Selectivity of the antiserum was validated by recognition of the AT2 receptor expressed in a stably transfected COS-7 cell line (kindly provided by Dr Tadashi Inagami, Vanderbilt University) by immunohistochemistry and Western blot analysis.

Figure 1.
Figure 1.

Rat AT2 receptor amino acid sequence depicting the intracellular and extracellular domains and the seven transmembrane regions. The 17–amino acid sequence used as antigenic peptide in this study is denoted by an asterisk next to the extracellular NH3-terminal tail of the receptor.

Immunohistochemistry of the AT2 Receptor

Immunohistochemistry was performed as described previously.27 The AT2 receptor–transfected and nontransfected COS-7 cells were grown on plastic slides with solution chamber (Nunc, chamber slide) and fixed in 2% paraformaldehyde in PBS. For the fetal kidney, the fetus was removed from uterus of timed pregnant Sprague-Dawley rats on F14 or F19, and the tissue, including the premature kidney and urogenital area, dissected under a low-magnification microscope. Neonatal and adult (young or mature) rat kidneys and adult adrenal glands were removed from Sprague-Dawley rats at ages D1, 4 weeks, or 3 months, respectively. These tissues were immediately immersion fixed in 2% paraformaldehyde in PBS for 1 hour, cryoprotected overnight at 4°C in 30% sucrose in PBS, and frozen sections (8 to 12 μm) were cut. The endogenous peroxidase and nonspecific binding sites of secondary goat antibody were blocked with 0.3% hydrogen peroxide in methanol, 3% normal goat serum, and 1% nonfat dry milk in PBS, respectively. The cells then were incubated for 24 to 36 hours at 4°C with one of the following, diluted in 1.5% normal goat serum and 0.5% nonfat dry milk in PBS: (1) antiserum diluted at 1: 250 to 1: 500, (2) preimmune serum, or (3) antiserum preadsorbed against a 10-fold molar excess of the pure peptide immunogen. For the study of AT2 receptor–transfected cells, the nontransfected COS-7 cells were used as a negative control. AT2 receptor–positive staining was visualized with the avidin-biotin immunoperoxidase reaction (Vectastain ABC kit) using diaminobenzidine (Fast DAB tablets, Sigma).

Western Blot Analysis of the AT2 Receptor Protein

AT2 receptor–transfected or nontransfected COS-7 cells were extracted with lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.02% sodium azide, 100 μg/mL PMSF, 1 μg/mL aprotinin, and 1% NP-40) and then centrifuged at 12 000g for 5 minutes. The supernatant was used for the analysis. The rat kidneys, adrenal glands, and whole brains were dissected, minced, and homogenized in buffer A (10% glycerol, 20 mmol/L Tris-HCl, 100 mmol/L NaCl, 2 mmol/L PMSF, 2 mmol/L EDTA, 2 mmol/L EGTA, 10 mmol/L sodium orthovanadate, 10 μg/L leupeptin, and 10 μg/L aprotinin). The homogenate was centrifuged at 30 000g for 30 minutes at 4°C. The pellet was resuspended in buffer B (buffer A with 1% NP-40), stirred for 2 hours at 4°C, and centrifuged again at 30 000g for 30 minutes at 4°C. The supernatant was used for the analysis.27

Solubilized samples were subjected to SDS-polyacrylamide gel electrophoresis (5% acrylamide stacking gel and 8% running gel). Proteins were transferred onto a nitrocellulose membrane (0.2 μm, Schleicher & Schuell) by semidry electroblotting (Trans Blot SD DNA, Bio-Rad) as previously described.27 The nitrocellulose membrane was soaked in Tris-buffered saline (10 mmol/L Tris-HCl, 250 mmol/L NaCl) containing 5% nonfat powdered milk and 0.1% Tween 20 for 2 hours to block nonspecific sites and then incubated with the AT2 receptor antiserum (3.1 mg/mL, 1:1000 dilution in Tris-buffered saline with 5% nonfat milk and 0.1% Tween 20) for 2 hours at room temperature. Blots were washed and incubated with peroxidase-conjugated donkey anti-rabbit secondary antibody (1:2500 dilution, Amersham) for 2 hours. Immunoreactivity was visualized with the ECL Western blotting detection kit (Amersham). Densitometry was quantitated using a Phosphor Imager (Molecular Dynamics), and data were expressed as mean±SE. Results were analyzed by the Student’s two-tailed t test for paired samples.

Results

Validation of the Specificity of the Antiserum for the AT2 Receptor

The AT2 receptor–transfected COS-7 cells were positively stained by AT2 receptor antiserum diluted at 1:500 (Fig 2A⇓), whereas the following controls were all negative: (1) substitution of preimmune serum at the same dilution (data not shown), (2) substitution of the preadsorbed antiserum (Fig 2B⇓), and (3) incubation of antiserum with a nontransfected COS-7 cell line (Fig 2C⇓). A single 44-kD band was detected in the Western blot analysis of the AT2 receptor–transfected COS-7 cells (Fig 3⇓). This band was not observed when the antiserum directed against the AT2 receptor was substituted with an antiserum that had been preadsorbed with the pure peptide immunogen (data not shown) or when the nontransfected COS-7 cells were used instead of the transfected cells (Fig 3⇓).

Figure 2.
Figure 2.

Light photomicrograph of AT2-transfected COS-7 cells. A, Cells demonstrating positive staining with antiserum directed toward the amino terminus of the AT2 receptor (1:500 dilution). B, Preadsorption control. C, Nontransfected COS-7 cells. Cells were lightly counterstained with hematoxylin. Magnification ×400.

Figure 3.
Figure 3.

Western blot analysis of the rat AT2 receptor in AT2 receptor–transfected and nontransfected COS-7 cells and various rat tissues. A representative immunoblot from three experiments showed a single 44-kD band of AT2 receptor protein (10 μg protein per lane, except that 30 μg protein was loaded in lanes 7 and 8). Lane 1, adrenal gland; lane 2, whole rat brain; lane 3, nontransfected COS-7 cells; lane 4, COS-7 cells transfected with the rat AT2 receptor; lane 5, fetal kidney (F 14); lane 6, newborn kidney (day 1); lane 7, young (4-week-old) adult rat kidney; and lane 8, mature (3-month-old) adult rat kidney. MW indicates molecular weight.

Immunohistochemistry of the AT2 Receptor in the Rat Adrenal Gland and Kidney

In the mature adult (3-month-old) rat adrenal gland, employed as a positive control, the AT2 receptor immunohistochemical signal was detected strongly in the zona glomerulosa (Fig 4A⇓). AT2 receptor signal also was detected in the adrenal zona fasciculata (Fig 4A⇓) and the adrenal medulla (data not shown) but was absent from the cortical zona reticularis. Preimmune (data not shown) and preadsorption (Fig 4B⇓) controls were negative.

Figure 4.
Figure 4.

Photomicrographs of frozen sections of the adult rat adrenal gland. A, AT2 receptor antiserum (1:500 dilution) in a section of the adrenal cortex showing heavy staining in the zona glomerulosa and less intense signal in the zona fasciculata. B, Preadsorption control. Magnification ×200.

In the F14 fetus, heavy immunohistochemical staining for the AT2 receptor was observed in the undifferentiated mesenchymal cells surrounding the epithelial structures (Fig 5A⇓) and in the ureteric buds (data not shown). Preadsorption controls showed no staining (Fig 5B⇓). In the F19 fetus, AT2 receptor signal was detected in the S-shaped glomeruli, primitive tubules, and mesenchymal tissue (Fig 6A⇓ and 6C⇓). Preadsorption controls (Fig 6B⇓ and 6D⇓) were negative.

Figure 5.
Figure 5.

Photomicrographs of frozen sections of the rat kidney at fetal day 14 (F14) demonstrating positive immunohistochemical staining for the AT2 receptor. A, AT2 receptor antiserum (1:500 dilution). B, Preadsorption control. Sections were counterstained with hematoxylin. Positive signal was observed in mesenchymal cells. Magnification ×400.

Figure 6.
Figure 6.

Photomicrographs of frozen sections of rat kidney at fetal day 19 (F19) demonstrating positive immunohistochemical staining for the AT2 receptor. A, Renal cortex with AT2 receptor antiserum (1:250 dilution). B, Preadsorption control for A. C, Renal medulla with AT2 receptor antiserum (1:250). D, Preadsorption control for C. Sections were counterstained with hematoxylin. Positive signal was observed in S-shaped glomeruli, tubules, and mesenchymal cells. Magnification ×400.

In the newborn rat kidney at D1, positive staining was observed in glomeruli, proximal and distal renal tubule cells, and blood vessels (Fig 7A⇓). Nephron maturation proceeds from the outer cortical zone inward. The positive glomeruli were mainly in the outer layer of the cortex, where the more undifferentiated glomeruli were localized. In the inner medullary region, immunoreactive signal was localized to collecting tubules (Fig 7C⇓). Preadsorption controls (Fig 7B⇓ and 7D⇓) were negative.

Figure 7.
Figure 7.

Photomicrographs of frozen sections of the newborn rat kidney at postnatal day 1 (D1) demonstrating positive immunohistochemical staining for the AT2 receptor. A, Outer cortex demonstrating positive signal in glomeruli, tubules, blood vessels, and interstitial space. B, Preadsorption control for A. C, Inner medulla showing positive signal in collecting tubules. D, Preadsorption control for C. Sections were counterstained with hematoxylin. Magnification ×400.

In young adult (4-week-old) rats, dietary sodium restriction decreased urinary sodium excretion from 1049±75 to 41±30 μEq/24 h (P<.001). In the young adult rat on normal sodium intake, renal staining for the AT2 receptor was markedly reduced but remained barely detectable in the glomeruli and tubules (Fig 8A⇓). In contrast, in the kidneys of sodium-depleted young adult rats, glomerular, tubular, and mesenchymal staining was reexpressed (Fig 8B⇓). The glomerular staining was localized to mesangial cells and also was present in the interstitial cells of the outer cortex (Fig 8C⇓).

Figure 8.
Figure 8.

Photomicrographs of frozen sections of the young adult (4-week-old) rat kidney. A, Kidney from a rat on normal sodium intake showing barely detectable signal in the glomeruli. Magnification ×400. B, Kidney from a sodium-depleted rat showing marked augmentation of signal in the glomeruli and interstitial cells of the outer cortex. Magnification ×400. C, High-power view of a glomerulus in a sodium-depleted rat. Magnification ×800 D, Preadsorption control for B.

In mature adult (3-month-old) rats, dietary sodium depletion resulted in a decrease in urinary sodium excretion from 1100±79 to 226±49 μEq/24 h (P<.001). In the mature adult rat on normal sodium intake, there was some glomerular and tubule expression of AT2 receptor protein signal (Fig 9A⇓). However, in mature adult rats during sodium depletion, renal AT2 receptor staining was enhanced predominantly in the glomeruli. Signal also was reexpressed in the interstitial cells of the outer cortex in response to sodium depletion (Fig 9B⇓ and 9C⇓).

Figure 9.
Figure 9.

Photomicrographs of frozen sections of the mature (3-month-old) rat kidney. A, Kidney from a rat on normal sodium intake showing low level of signal. B, Kidney from a sodium-depleted rat showing reexpression of signal in the glomeruli. Magnification ×400. C, Kidney from a sodium-depleted rat showing reexpression of signal in the glomerulus and interstitial cells of the outer cortex. Magnification ×800. D, Preadsorption control for B.

Western Blot Analysis

In agreement with the findings of immunohistochemistry, single bands of the predicted molecular mass for AT2 receptor (44 kD) were detected in the fetal and newborn kidney (Fig 3⇑). In kidneys of mature adult rats on normal sodium intake, a single 44-kD band was observed that was significantly enhanced in the kidneys of rats subjected to dietary sodium depletion (Fig 10⇓).

Figure 10.
Figure 10.

Western blot analysis for AT2 receptor protein (33 μg protein per lane) in the non–sodium-depleted (NS, lane 1) or sodium-depleted (LS, lane 2) mature adult (3-month-old) rat kidney. A representative Western blot and densitometric analysis of six experiments are depicted. *P<.05 vs NS. MW indicates molecular weight.

Discussion

In the present study using a specific affinity-purified anti-AT2 receptor peptide antiserum, we provide evidence for the expression of AT2 receptor protein in the fetal and newborn rat kidney and its regression in the adult kidney. We also found that AT2 receptor protein was reexpressed (upregulated) during sodium depletion in the adult kidney.

The specificity of the antiserum directed toward AT2 was validated by (1) the uniqueness of the amino acid sequence that is not homologous with the product of any known gene, including those of both the AT1A and AT1B receptor genes; (2) detection of a single band of the appropriate molecular weight in AT2 receptor–transfected COS-7 cells and its absence in nontransfected COS-7 cells and in preadsorption controls; (3) positive immunostaining of AT2 receptor–transfected COS-7 cells and absence of staining in nontransfected cells and preadsorption controls (these AT2 receptor–transfected COS-7 cells have been previously documented to have strong expression of AT2 receptor gene and exhibit characteristic ligand binding and functional features28 ); and (4) detection of AT2 receptor immunoreactivity in the adrenal zona glomerulosa and medulla, in keeping with previously described distribution of AT2 receptor mRNA within the adrenal gland.14 20

Regarding the site-specific localization of AT2 receptor protein within the kidney, our present observations are consistent with previous reports of AT2 receptor mRNA, in that the AT2 receptor was mainly observed in the mesenchyme of the fetal kidney and receptor expression decreased markedly after birth.20 22 23 However, we detected a strong signal for AT2 receptor protein in the ureteric buds in the fetus, and our results show a very different expression pattern for the AT2 receptor protein compared with its mRNA distribution in the newborn kidney. Whereas AT2 receptor mRNA was found only in subcapsular cortical nephrogenic tissue and undifferentiated interstitial tissue lying between medullary rays20 21 and AT2 receptor binding sites are associated with advancing tubules and ampullae of the ureteric bud and within the metanephric mass in the cortex,24 we detected AT2 receptor protein distributed throughout the renal cortex of the newborn rat. In the present study, a strong signal for AT2 receptor protein was detected in cortical glomeruli and tubular structures, as well as in undifferentiated mesenchymal cells. The reasons for the discrepancy in the cellular localization of AT2 protein and mRNA are unclear. It is possible that the glomerular expression of receptor protein reflects mesangial localization, as glomerular mesangial cells originate from mesenchyme. In the young and mature adult rat on normal sodium intake, the renal AT2 receptor protein was present predominantly in glomeruli but was substantially diminished compared with that of newborn rats. This distribution pattern of AT2 receptor protein is substantially different from the previous studies reporting immunolocalization of renal AT1 receptor in adult rats, which detected strong expression of AT1 receptor protein in all small arteries and arterioles and glomeruli, as well as in proximal tubules and thick ascending limb epithelium.29 Ernsberger et al30 have identified AT2 receptors by classic pharmacological binding techniques in cultured rat mesangial cells. Jacobs and Douglas31 also have shown that Ang II mediates a phospholipase A2–dependent signaling pathway through an AT2 receptor that was localized to the apical cell membrane of cultured rabbit proximal tubule cells. These latter studies are consistent with the results of the current study with respect to the cellular distribution of the AT2 receptor in the kidney. However, the specific glomerular cellular and glomerular and tubular subcellular distribution of AT2 receptor protein awaits clarification with electron microscopic immunocytochemistry.

The concept of reexpression (upregulation) of several components of the renin-angiotensin system has been established from previous studies in which physiological manipulations or pathological situations increase the expression of a protein that was present in the fetus but not in adult life. Our group has shown that renin is expressed throughout the renal vasculature of the fetal rat, but only in the juxtaglomerular cells of the afferent arteriole in the adult.32 However, renin is reexpressed in the renal vasculature during angiotensin-converting enzyme inhibition or AT1 receptor blockade with losartan.33 34 Interestingly, sodium depletion significantly increases the gene expression of the AT1A receptor and decreases AT1B receptor mRNA levels in the rat kidney.35 AT2 receptor mRNA has been demonstrated to be reexpressed in pathological situations involving tissue remodeling or repair, such as vascular neointima formation36 and wound healing.37 AT2 receptor mRNA expression also is upregulated in rat ovary granulosa cells undergoing apoptosis.38 Whether the renal AT2 receptor is reexpressed/upregulated under certain (patho)-physiological conditions has not been explored previously.

In the current study, we found that the AT2 receptor protein was reexpressed (upregulated) during sodium depletion in the adult kidney. The predominant site of AT2 receptor reexpression in the adult rat was the glomerulus, although the receptor also was reexpressed to a lesser extent in the tubules, renal vasculature, and interstitial cells. The reexpression of the AT2 receptor in the adult rat is consistent with our recent observations26 that dietary sodium depletion in the adult rat led to a stepwise increase in renal interstitial fluid cGMP levels, an effect totally abolished by administration of the selective nonpeptide AT2 receptor antagonist PD 123319. Renal interstitial fluid cGMP was unchanged in response to PD 123319 during normal sodium intake.26 Previous studies have failed to demonstrate a function for the renal AT2 receptor in the adult,39 40 probably because these studies were conducted during normal sodium intake. We also demonstrated in the sodium-depleted conscious dog that Ang II stimulated renal bradykinin production and cGMP formation by a non-AT1 receptor.41 Clark et al42 demonstrated an action of Ang II at AT2 receptors in sodium-depleted dogs, but they used relatively high infusion rates of PD 123319, which would be predicted to interact with AT1 receptors. Taken together with the available physiological studies, our present observations indicate that the AT2 receptor protein is expressed only at a low level in the normal adult kidney but is reexpressed in response to sodium depletion. Glomerular and tubular AT2 receptor reexpression as demonstrated in the present study may also provide an explanation for recent findings that renal AT2 receptors modulate pressure natriuresis in the rat.43

A question that remains to be answered is how dietary sodium restriction induces reexpression of the AT2 receptor during sodium depletion. Renin release and Ang II formation are relatively high in the fetal and early newborn period but decline significantly after birth.44 45 Compensatory mechanisms, such as circulating and/or local renal Ang II and release of aldosterone, are activated in response to sodium depletion and may play a role in AT2 receptor reexpression in the adult rat. Although the precise function of the reexpressed AT2 receptors remains to be elucidated, on the basis of previous studies,26 41 43 it is reasonable to infer that reexpression of the AT2 receptor in response to Ang II may mediate renal vasodilation and/or inhibition of tubular sodium reabsorption by stimulating bradykinin, nitric oxide, and/or eicosanoid production.

In summary, we demonstrated the site-specific localization of the AT2 receptor protein in the fetal and newborn rat kidney. In the fetal kidney, the AT2 receptor is highly expressed in mesenchymal tissue and the ureteral bud. In the newborn kidney, the AT2 receptor is present in cortical glomeruli and tubules, as well as in cortical and medullary mesenchyme. In the adult kidney, the expression level of AT2 receptor protein is markedly diminished compared with that in the fetal and neonatal rat but is significantly upregulated in the glomeruli and interstitium in response to sodium depletion. The mechanisms and function of renal AT2 receptor reexpression during dietary sodium restriction in the adult rat deserve additional investigation.

Acknowledgments

This study was supported by National Institutes of Health grants R01-HL-47669 and K04-HL-03006 (Dr Siragy) and R01-HL-49575 (Dr Carey). The authors are indebted to Dr Tadashi Inagami of Vanderbilt University for provision of the AT2 receptor–transfected COS-7 cells, to Suzanne J. Botkin for technical assistance, and to Cynthia Allen for secretarial help.

Footnotes

  • Reprint requests to Dr Robert M. Carey, Box 395, University of Virginia Health Sciences Center, Charlottesville, VA 22908.

  • R. Ozono and Z.Q. Wang contributed to this work equally.

  • Received March 6, 1997.
  • Revision received April 1, 1997.
  • Accepted April 29, 1997.

References

  1. ↵
    Goodfriend TL, Elliott ME, Catt KJ. Angiotensin receptors and their antagonists. N Engl J Med. 1996;334:1649-1654.
    OpenUrlCrossRefPubMed
  2. ↵
    Yamada T, Horiuchi M, Dzau VJ. Angiotensin II type 2 receptor mediates programmed cell death. Proc Natl Acad Sci U S A. 1996;93:156-160.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Murphy TJ, Alexander RW, Griendling KK, Runge MS, Bernstein KE. Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor. Nature. 1991;351:233-236.
    OpenUrlCrossRefPubMed
  4. ↵
    Sasaki K, Inagami T. Cloning of a complimentary DNA encoding a bovine adrenal angiotensin II type I receptor. Nature. 1991;351:230-233.
    OpenUrlCrossRefPubMed
  5. ↵
    Kambayashi Y, Bardhan S, Takahashi K, Tsuzuki S, Inui H, Hamakubo T, Inagami T. Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition. J Biol Chem. 1993;268:24543-24546.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Mukoyama M, Nakajima M, Horiuchi M, Sasamara H, Pratt RE, Dzau VJ. Expression cloning of the type-2 angiotensin receptor reveals a unique class of seven transmembranes receptor. J Biol Chem. 1993;268:24539-24542.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Murphy TJ, Takeuchi K, Alexander RW. Molecular cloning of AT1 angiotensin receptors. Am J Hypertens. 1992;5:236S-242S.
    OpenUrlPubMed
  8. ↵
    Navar LG, Rosivall L. Contribution of the renin-angiotensin system to the control of intrarenal hemodynamics. Kidney Int. 1984;25:857-868.
    OpenUrlPubMed
  9. ↵
    Harris PJ, Navar LG. Tubular transport responses to angiotensin. Am J Physiol. 1985;248:F621-F630.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Ichikawa I, Harris RC. Angiotensin actions in the kidney: renewed insight into the old hormone. Kidney Int. 1991;40:583-596.
    OpenUrlPubMed
  11. ↵
    Douglas JG. The curtain rises on the renin-angiotensin system: AT2 receptors are in the spotlight. J Clin Invest. 1996;97:1787.
    OpenUrlPubMed
  12. ↵
    Tufro-McReddie A, Johns DW, Geary KM, Dagli H, Everett AD, Chevalier RL, Carey RM, Gomez RA. Angiotensin II type 1 receptor: role in renal growth and gene expression during normal development. Am J Physiol. 1994;266:F911-F918.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    Smith RD, Chiu AT, Wong PC, Timmermans PB. Pharmacology of nonpeptide angiotensin II receptor antagonists. Annu Rev Pharmacol Toxicol. 1992;32:135-165.
    OpenUrlPubMed
  14. ↵
    Timmermans PB, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini D. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev. 1993;45:205-251.
    OpenUrlPubMed
  15. ↵
    deGasparo M, Levens NR. Pharmacology of angiotensin II receptors in the kidney. Kidney Int. 1994;46:1486-1491.
    OpenUrlPubMed
  16. ↵
    Tufro-McReddie A, Romano LM, Harris JM, Ferder L, Gomez RA. Angiotensin II regulates nephrogenesis and renal vascular development. Am J Physiol. 1995;269:F110-F115.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Levy BI, Benessiano J, Henrion D, Caputo L, Heymes C, Duriez M, Poltevin P, Samuel JL. Chronic blockade of AT-2-subtype receptors prevents the effect of angiotensin II on rat vascular structure. J Clin Invest. 1996;98:418-425.
    OpenUrlPubMed
  18. ↵
    Stoll M, Steckelings UM, Paul M, Bottari SP, Metzger R, Unger T. The angiotensin AT2 receptor mediates inhibition of cell proliferation in coronary endothelial cells. J Clin Invest. 1995;95:651-657.
  19. ↵
    Grady EF, Sechi LA, Griffin CA, Schambelan M, Kalinyak JE. Expression of AT2 receptors in the developing rat fetus. J Clin Invest. 1991;88:921-933.
  20. ↵
    Shanmugam S, Llorens-Cortes C, Clauser E, Corvol P, Gase JM. Expression of angiotensin AT2 receptor mRNA during development of the rat kidney and adrenal gland. Am J Physiol. 1995;268:F922-F930.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Shanmugam S, Corvol P, Gase JM. Angiotensin II type 2 receptor mRNA expression in the developing cardiopulmonary system of the rat. Hypertension. 1996;28:91-97.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Shanmugam S, Lenkei ZG, Gase JM, Corvol P. Ontogeny of angiotensin II type 2 (AT2) receptor mRNA in the rat. Kidney Int. 1995;47:1095-1100.
    OpenUrlPubMed
  23. ↵
    Kakuchi J, Ichiki T, Kiyama S, Hogan BL, Fogo A, Inagami T, Ichikawa I. Developmental expression of renal angiotensin II receptor genes in the mouse. Kidney Int. 1995;47:140-147.
    OpenUrlPubMed
  24. ↵
    Aguilera G, Kapur S, Feuilan P, Sunar-Akbasak B, Bethia AJ. Developmental changes in angiotensin II receptor subtypes and AT1 receptor mRNA in rat kidney. Kidney Int. 1994;46:973-979.
    OpenUrlPubMed
  25. ↵
    Gehlert DR, Grackenheimer SL, Reel JK, Lin H-S, Steinberg MI. Non-peptide angiotensin II receptor antagonists discriminate subtypes of 125 I-angiotensin II binding sites in the rat brain. Eur J Pharmacol. 1990;187:123-126.
    OpenUrlCrossRefPubMed
  26. ↵
    Siragy HM, Carey RM. The subtype-2 (AT2) angiotensin receptor regulates renal cyclic guanosine 3′,5′-monophosphate and AT1 receptor-mediated prostaglandin E2 production in conscious rats. J Clin Invest. 1996;97:1978-1982.
    OpenUrlPubMed
  27. ↵
    Ozono R, O’Connell DP, Vaughan CJ, Botkin SJ, Walk SF, Felder RA, Carey RM. Expression of the subtype 1A dopamine receptor in the rat heart. Hypertension. 1996;27:693-703.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Inagami T, Guo DF, Kitami Y. Molecular biology of angiotensin II receptors: an overview. J Hypertens. 1994;12(suppl 10):S83-S94.
  29. ↵
    Paxton WG, Runge M, Horaist C, Cohen C, Alexander RW, Bernstein KE. Immunohistochemical localization of rat angiotensin II AT1 receptor. Am J Physiol. 1993;264:F989-F995.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Ernsberger P, Zhou J, Douglas JG. Angiotensin II receptor subtypes in cultured rat mesangial cells. Am J Physiol. 1992;263:F411-F416.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Jacobs LS, Douglas JG. Angiotensin II type 2 receptor subtype mediates phospholipase A2–dependent signalling in rabbit proximal tubular epithelial cells. Hypertension. 1996;28:663-668.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Gomez RA, Lynch KR, Sturgill BC, Elwood JP, Chevalier RL, Carey RM, Peach MJ. Distribution of renin mRNA and its protein in the developing kidney. Am J Physiol. 1989;257:F850-F858.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Gomez RA, Chevalier RL, Everett AD, Elwood JP, Peach MJ, Lynch KR, Carey RM. Recruitment of renin gene expressing cells in adult rat kidneys. Am J Physiol. 1990;259:F660-F665.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    Gomez RA, Lynch KR, Chevalier RL, Everett AD, Johns DW, Wilfong N, Peach MJ, Carey RM. Renin and angiotensinogen gene expression and intrarenal renin distribution during ACE inhibition. Am J Physiol. 1988;254:F900-F906.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Du Y, Yao A, Guo DF, Inagami T, Wang DH. Differential regulation of angiotensin II receptor subtypes in rat kidney by low dietary sodium. Hypertension. 1995;25:872-877.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Janiak P, Pillon A, Prost JF, Vilaine JP. Role of angiotensin subtype 2 receptor in neointima formation after vascular injury. Hypertension. 1992;20:737-745.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Viswanathan M, Saavedra JM. Expression of angiotensin II AT2 receptors in the rat skin during experimental wound healing. Peptides. 1992;13:783-786.
    OpenUrlCrossRefPubMed
  38. ↵
    Tanaka M, Onishi J, Ozawa Y, Sugimoto M, Usuki S, Naruse M, Murakami K, Miyazaki H. Characterization of angiotensin II receptor subtype II during differentiation and apoptosis of rat ovarian cultured granulosa cells. Biochem Biophys Res Commun. 1995;207:593-598.
    OpenUrlCrossRefPubMed
  39. ↵
    Kaiser JA, Bjork FA, Hodges JC, Taylor DG Jr. Renal hemodynamic and excretory responses to PD 123319 and losartan nonpeptide AT1 and AT2 subtype-specific angiotensin II ligands. J Pharmacol Exp Ther. 1992;262:1154-1160.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Macari D, Whitebread S, Cumin F, deGasparo M, Levens NR. Renal actions of the angiotensin AT2 receptor ligands CGP 42112 and PD 123319 after blockade of the renin-angiotensin system. Eur J Pharmacol. 1994;259:27-36.
    OpenUrlCrossRefPubMed
  41. ↵
    Siragy HM, Jaffa AA, Margolius HS, Carey RM. Renin-angiotensin system modulates renal bradykinin production. Am J Physiol. 1996;40:R1090-R1095.
    OpenUrl
  42. ↵
    Clark KL, Robertson MJ, Drew GM. Role of angiotensin AT1 and AT2 receptors in mediating the renal effects of angiotensin II in the anaesthetized dog. Br J Pharmacol. 1993;109:148-156.
    OpenUrlPubMed
  43. ↵
    Lo M, Liu K, Lantelme P, Sassard J. Subtype 2 of angiotensin II receptors controls pressure natriuresis in rats. J Clin Invest. 1995;95:1394-1397.
  44. ↵
    Jelinek J, Hackenthal R, Hilgenfelt TW, Schaechtelin G, Hackenthal E. The renin-angiotensin system in the perinatal period in rats. J Dev Physiol. 1986;8:33-41.
    OpenUrlPubMed
  45. ↵
    Pipkin FB. The fetal renin-angiotensin system. In: Robertson JIS, Nicholls MG, eds. The Renin-Angiotensin System. London, UK: Gower Medical Publishing; 1993:51.1-51.10.
View Abstract

This Issue

Jump to

Article Tools

Share this Article

  • Email
  • Expression of the Subtype 2 Angiotensin (AT2) Receptor Protein in Rat Kidney
    Ryoji Ozono, Zhi-Qin Wang, Allan F. Moore, Tadashi Inagami, Helmy M. Siragy and Robert M. Carey
    Hypertension. 1997;30:1238-1246, originally published November 1, 1997
    https://doi.org/10.1161/01.HYP.30.5.1238
    Permalink:
    del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo