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(Hypertension. 1999;33:96-101.)
© 1999 American Heart Association, Inc.

Scientific Contributions

Differential Regulation of Renal Angiotensin Subtype AT_1A and AT₂ Receptor Protein in Rats With Angiotensin-Dependent Hypertension

Zhi-Qin Wang; Lesley J. Millatt; Nicholas T. Heiderstadt; Helmy M. Siragy; Roger A. Johns; Robert M. Carey

From the Departments of Medicine (Z.Q.W., N.T.H., H.M.S., R.M.C.) and Anesthesiology (L.J.M., R.A.J.), University of Virginia Health Sciences Center, Charlottesville.

Correspondence to Robert M. Carey, MD, Box 395, University of Virginia Health Sciences Center, Charlottesville, VA 22908. E-mail RMC4C{at}virginia.edu

	Abstract

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Abstract—This study was designed to investigate distribution and regulation of the renal AT_1A and AT₂ subtype receptors in rats with either systemic angiotensin II (Ang II)–induced hypertension or acute phase renal hypertension (2-kidney, 1-clip [2K1C] or 2-kidney, 1-figure-of-8-wrap [2K1W]). In normal rat kidneys, positive immunostaining for the AT_1A receptor was observed in the intrarenal vasculature, glomeruli, proximal and distal tubules, and collecting ducts. The AT₂ receptor was localized mainly to the glomeruli. The AT_1A but not AT₂ receptor protein expression was significantly reduced in rats with 10-day systemic Ang II–induced hypertension. In both 7-day 2K1C and 3-day 2K1W rats, the AT_1A receptor was significantly reduced in ischemic and contralateral kidneys compared with sham-operated control rats. Reduction in AT₂ receptor expression was observed only in the ischemic kidneys in 2K1C and 2K1W renal hypertensive rats. These results demonstrate that the AT_1A receptor is widely distributed in the glomerulus and all other nephron segments of the rat kidney. Renal AT_1A but not AT₂ receptor protein is downregulated in rats with Ang II–induced hypertension. In renal hypertensive rats, the AT_1A receptor is bilaterally downregulated and the AT₂ receptor is downregulated only in the ischemic kidney.

Key Words: receptors, angiotensin II • immunohistochemistry • kidney • hypertension, renal

	Introduction

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The kidney is an important target organ for angiotensin II (Ang II), which plays a critical role in the regulation of blood flow, glomerular filtration, tubular sodium reabsorption, and renin secretion. The 2 subtypes of the AT₁ receptor (AT_1A and AT_1B) are encoded by separate genes on different chromosomes.¹ Their cDNAs have 91% identity in their nucleotide sequences within the coding regions but exhibit only 60% homology in the 5' and 3' untranslated regions.¹ Both AT_1A and AT_1B receptor mRNAs are present in the kidney, with the AT_1A receptor as the predominant isoform,² and their mRNA expression is differentially regulated.³ However, the intrarenal localization of the AT₁ receptor subtype protein remains unknown largely because of the lack of specific ligand for different AT₁ receptor isoforms. The AT₂ receptor is expressed in the adult kidney at a much lower level and is localized mainly to the glomerular mesangial cells.^{4 5} The majority of studies suggest that the renal actions of Ang II are mediated primarily by the AT₁ receptor. However, activation of renal AT₂ receptors recently has been reported to regulate pressure-natriuresis,⁶ to cause vasodilation in the preglomerular afferent arteriole,⁷ and to stimulate renal nitric oxide production.⁸

Both 2-kidney, 1-clip (2K1C) (Goldblatt) and 2-kidney, 1-figure-of-8-wrap (2K1W) (Grollman) renal hypertension are renin dependent.^{9 10} Differential regulation of renal AT₁ subtype receptor mRNA in renal hypertension has been reported in 2K1C rats.¹¹ However, regulation of the AT₂ receptor expression in renal hypertension is unknown. The present study is designed to investigate the distribution and regulation of renal subtype AT_1A and AT₂ receptor protein in Ang II–dependent hypertension in rats induced by systemic Ang II infusion and rats in the acute phase of renal hypertension (either 7-day 2K1C or 3-day 2K1W hypertension).

	Methods

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All animal procedures were conducted with the approval of the Animal Research Committee of the University of Virginia School of Medicine.

Characteristics of Polyclonal Antisera
The peptide (²²⁵AYEIQKNKPRNDD²³⁷), a part of the third cytoplasmic domain of the rat AT_1A receptor,¹² was cross-linked to thyroglobulin and used to generate a specific anti–AT_1A receptor polyclonal antiserum in the rabbit.^{13 14} This antiserum recognized a specific protein with an estimated molecular mass of 60 kd, consistent with the predicted molecular weight of the glycosylated form of the AT_1A receptor.¹³ The specificity of the antiserum was further supported by¹ a single band of 60 kd detected from the AT_1A receptor–rich adult (12-week old) rat liver but not from the AT_1B receptor–rich adult (12-week old) rat pituitary gland (Pel-Freez; Rogers, Ariz) by immunoblot analysis, performed as described in the following section (Figure 1, left)² ; a significant decrease in the enzyme-linked immunosorbent assay response (10 ng of the peptide used as antigen target) after preadsorption challenge of the antiserum with the peptide at 120 µg/mL diluted antiserum, performed as described¹⁵ (Figure 1, right). The polyclonal antiserum to the AT₂ receptor was raised in the rabbit against a synthetic peptide sequence (¹MKDNFSFAATSRNITSS¹⁷ ) derived from the rat AT₂ receptor, and its specificity has been documented.^{4 16 17}

View larger version (24K): [in this window] [in a new window]   Figure 1. Characteristics of polyclonal antiserum against rat AT1A subtype receptor. Left, Representative immunoblot of AT1A receptor protein from liver (lane 1) and pituitary gland (lane 2) of adult (12-week-old) rat (40 µg of total protein per lane). Right, Summary of the enzyme-linked immunosorbent assay response after preadsorption challenge of AT1A receptor antiserum with peptide immunogen. n=3, **P<0.01 vs antiserum.

Animal Preparations
Female Sprague-Dawley rats (200 to 220 g body weight, Hilltop Laboratory Animals; Scottsdale, Pa) were randomly allocated to the following groups¹ : 2K1C hypertension and a sham-operated control group² ; 2K1W hypertension and a sham-operated control group; and Ang II–induced hypertension and a vehicle-infused control group.³ After rats were anesthetized with pentobarbital sodium (50 mg/kg body wt intraperitoneally), the left kidneys were exposed by a midline laparotomy in the first 2 groups. In the first group, the left renal artery was clipped by placement of a solid U-shaped silver clip. The effective clipping of the left renal artery was confirmed by a 70% to 75% reduction in renal cortical blood flow as determined by a laser Doppler probe (superficial probe, type C, Advance Co, Ltd) coupled to a laser Doppler flowmeter (ALF 21D Dual Channel Flowmeter, Advance Co, Ltd). In the second group, the left kidney was tautly wrapped in a figure-of-8 pattern around the kidney to compress the poles and body with 2.0 silk thread (Ethicon Inc). All rats in the sham control groups received identical surgical treatment apart from clip placement or renal wrap. In the third group, Ang II (25 ng/min in lactated Ringer's solution) or vehicle was infused into the jugular vein through mini-osmotic pumps (model 2002, Alza). After 3 days (for 2K1W and sham-operated control rats), 7 days (for 2K1C and sham-operated control rats), or 10 days (for Ang II–infused or vehicle-infused rats), systolic arterial blood pressure was measured by a tail-cuff photosensor (Rat Tail Monometer-Tachometer system, Natsume model KN-210, Peninsula Laboratories). The kidneys were quickly perfused and then harvested for immunohistochemical studies (n=3/group) and Western blot analysis (n=7/group) from rats deeply anesthetized with pentobarbital sodium (80 mg/kg body wt intraperitoneally).

Light Microscopic Immunohistochemistry
Indirect immunoperoxidase immunohistochemistry was performed as previously described.^{4 16} Frozen sections (10 to 12 µm) were incubated with one of the following, diluted 1:500 in 1.5% normal goat serum and 0.5% nonfat dry milk in phosphate-buffered saline¹ : AT_1A (7.7 protein mg/mL) or AT₂ (3.1 mg protein/mL) receptor primary antiserum² ; AT_1A or AT₂ receptor primary antiserum preabsorbed against its respective synthetic peptide antigen. For preadsorption, antiserum was incubated overnight at 4°C with its respective peptide immunogen at 120 µg/mL (for AT_1A) or 50 µg/mL (for AT₂) diluted antiserum. Immunoreactive signal was detected with an avidin-biotin immunoperoxidase reaction (Vectastain ABC kit, Vector Laboratory) and visualized by exposure to diaminobenzidine for 40 seconds.

Western Blot Analysis
Western blot analysis was performed as previously described.^{4 16} The nitrocellulose membrane was incubated with the AT_1A or AT₂ receptor antibody (both 1:1000 dilution in Tris-buffered saline with 5% nonfat dry milk and 0.1% Tween 20) in random order. Blots were then incubated with peroxidase-conjugated donkey anti-rabbit secondary antibody (1:5000 dilution, Amersham, Arlington Heights, Ill). The immunoreactivity was visualized with an enhanced chemiluminescence Western blotting detection kit (Amersham). Membranes were stripped between incubations with different antibodies in a Tris-buffered solution containing 2% sodium dodecyl sulfate and 100 mmol/L ß-mercaptoethanol at 50°C.

Quantitative assessment of band densities was performed by scanning densitometry (ImageQuant, Molecular Dynamics). Statistical analysis was performed with a Macintosh StatView program (Abacus Concepts). All data were expressed as mean±SEM. Comparisons were made with either the unpaired t test or 1-way ANOVA followed by Fisher protected least significant difference test for multiple comparisons when appropriate. A value of P<0.05 was considered statistically significant.

	Results

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The 2K1C and 2K1W animals exhibited significantly increased systolic blood pressure compared with their respective sham-operated controls (166±6 vs 95±4 mm Hg in 2K1C, P<0.01; 148±2 vs 104±3 mm Hg in 2K1W, P<0.01). Ang II–infused rats also had elevated blood pressure compared with vehicle-infused controls (159±9 vs 100±7 mm Hg, P<0.01).

Intrarenal Distribution of AT_1A and AT₂ Subtype Receptors
In the frozen sections of normal rat kidney, intense immunoreactivity for the AT_1A receptor was observed in glomeruli, proximal and distal tubules, and collecting ducts (Figure 2, A and B). The macula densa cells were strongly stained (Figure 2, A). Positive immunostaining also was observed in the intrarenal vasculature (Figure 2, A), including juxtaglomerular cells (Figure 2, C) and interlobular artery (Figure 2, D). No specific staining was observed in consecutive sections incubated with preabsorbed antiserum (Figure 2, E). Notably, less staining than normal control rats was observed in the kidney of the Ang II–infused rat (Figure 2, F) and both ischemic and contralateral kidneys of the 2K1C (Figure 2, G and H) and 2K1W (Figure 2, I and J) rats, respectively.

View larger version (151K): [in this window] [in a new window]   Figure 2. Light photomicrographs of frozen sections of rat kidneys immunostained for AT1A receptor expression. In normal rat kidney, positive immunoreactivity was observed in glomeruli, proximal and distal tubules, and collecting ducts (A, B). Macula densa cells were strongly stained (A, arrow). Positive immunostaining was also detected in intrarenal vasculature (A), including afferent arteriole (C, arrow) and interlobar artery (D). No immunoreactive signal was observed in consecutive sections incubated with preabsorbed antiserum (E). Systemic Ang II infusion apparently decreased the intensity of immunostaining (F). AT1A receptor immunoreactivity also was notably decreased in clipped (G) and unclipped (H) kidneys of 2K1C and in wrapped (I) and unwrapped (J) kidneys of 2K1W rats. Magnification x250 except C, x500.

The immunoreactive signal for the AT₂ receptor was present mainly in the glomeruli and also lightly in the proximal tubules of the normal rat kidney (Figure 3, A). No specific staining was observed in consecutive sections incubated with preabsorbed antiserum (Figure 3, B). The AT₂ receptor staining was absent in the clipped kidney of the 2K1C rat (Figure 3, C) and apparently decreased in both the glomeruli and tubules in the wrapped kidney of the 2K1W rat (Figure 3, D). However, both the glomeruli and tubular epithelium were positively stained in the contralateral (nonischemic) kidneys of the 2K1C (Figure 3, E) and 2K1W rats (Figure 3, F). No apparent change from the normal rat in the renal AT₂ receptor staining was observed in the Ang II–infused rat (Figure 3, G).

View larger version (106K): [in this window] [in a new window]   Figure 3. Light photomicrographs of frozen sections of rat kidneys immunostained for AT2 receptor expression. Positive immunoreactivity was observed mainly in glomeruli of normal rat kidney (A). No specific signal was observed in consecutive sections incubated with preabsorbed antiserum (B). AT2 receptor staining was absent in clipped kidney of 2K1C rat (C) and apparently decreased in wrapped kidney of 2K1W rat (D). However, both the glomeruli and tubular epithelium were positively stained in contralateral (nonischemic) kidneys of 2K1C (E) and 2K1W (F) rats. No apparent change from normal rat in renal AT2 receptor signal was detected in Ang II–infused rats (G). Magnification x250.

Western Blot Analysis of Renal AT_1A and AT₂ Subtype Receptors
In both 2K1C and 2K1W renal hypertensive rats, the AT_1A receptor protein was significantly reduced in the ischemic and contralateral kidneys compared with sham-operated control rats (n=7/group, P<0.01 in 2K1C and 2K1W, Figure 4). However, the reduction in the AT₂ receptor protein expression was observed only in the ischemic kidneys in both 2K1C (n=7/group, P<0.01) and 2K1W (n=7/group, P<0.05) rats (Figure 5). Renal AT_1A receptor protein expression was significantly decreased in rats with Ang II–induced hypertension compared with vehicle-infused controls (n=7/group, P<0.05, Figure 4). No significant difference in renal AT₂ receptor protein was observed between Ang II–infused and vehicle-infused rats (n=7/group, P>0.05, Figure 5).

View larger version (39K): [in this window] [in a new window]   Figure 4. Representative immunoblot (left) and summary of densitometry quantitation (right) of AT1A receptor protein in 3 groups of hypertensive rats (40 µg of total kidney protein per lane, n=7/group). Top, AT1A receptor from both contralateral (lanes 4, 5, and 6, CTL) and clipped (lanes 7, 8, and 9, CLP) kidneys of 2K1C rats was significantly less than that of sham-operated control rats (lanes 1, 2, and 3, C). Middle, AT1A receptor from both contralateral (lanes 4, 5, and 6, CTL) and wrapped (lanes 7, 8, and 9, WRP) kidneys of 2K1W rats is significantly less than sham-operated control rats (lanes 1, 2, and 3, C). Bottom, Renal AT1A receptor from Ang II–infused hypertensive rats (lanes 4, 5, and 6, Ang II) was significantly decreased when compared with vehicle-infused control rats (lanes 1, 2, and 3, C). *P<0.05, **P<0.01 vs C. Migration and size of molecular weight markers (MW, values x103) are at left. Approximate molecular mass of AT1A receptor is 60 kd.

View larger version (39K): [in this window] [in a new window]   Figure 5. Representative immunoblot (left) and summary of densitometry quantitation (right) of AT2 receptor protein in 3 groups of hypertensive rats (40 µg of total kidney protein per lane, n=7/group). Top, Expression of AT2 receptor in clipped (lanes 7, 8, and 9, CLP) kidneys of 2K1C rats was almost undetectable, whereas its expression in contralateral (lanes 4, 5, and 6, CTL) kidneys of 2K1C rats is not significantly different from sham-operated control rats (lanes 1, 2, and 3, C). Middle, Expression of AT2 receptor in wrapped (lanes 7, 8, and 9, WRP) but not in contralateral (lanes 4, 5, and 6, CTL) kidneys of 2K1W rats was significantly reduced when compared with sham-operated control rats (lanes 1, 2, and 3, C). Bottom, Renal AT2 receptor protein from Ang II–infused hypertensive rats (lanes 4, 5, and 6, Ang II) was not significantly different from vehicle-infused control rats (lanes 1, 2, and 3, C). **P<0.01 vs C. Migration and size of molecular weight markers (MW, values x103) are at left. Approximate molecular mass of AT2 receptor is 44 kd.

	Discussion

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Because all known AT₁ receptor ligands bind with equivalent affinities to AT_1A and AT_1B receptor subtypes, the distribution of the AT₁ subtype receptor in the kidney has been difficult to evaluate with ligand binding and autoradiographic techniques. Moreover, previous efforts to localize the renal AT₁ receptor protein failed to distinguish AT_1A from AT_1B receptor subtypes because the anti-peptide antibodies used were raised against shared amino acid sequences between AT_1A and AT_1B receptor subtypes.¹⁸ In this study, AT_1A subtype receptor protein was localized to the glomeruli and all other nephron segments of rat kidney by immunohistochemistry with the use of a specific anti–AT_1A receptor antibody. Our results are in good agreement with the expression pattern of renal AT_1A receptor mRNA in that the AT_1A receptor subtype is the major (>80%) isoform of AT₁ receptor mRNA detected in all nephron segments except the glomerulus, where AT_1A and AT_1B mRNA are expressed in similar proportions.² Renal AT_1A receptor expression in the afferent and efferent arteriolar smooth muscle and in the glomerular mesangial cells was recently demonstrated indirectly by immunohistochemical localization of ß-galactosidase in the heterozygous AT_1A receptor of mutant mice, generated by a targeted replacement of the AT_1A receptor loci by the lacZ gene.¹⁹ However, whether there is any tubular localization of the AT_1A receptor was not reported. Ang II influences single-nephron glomerular filtration rate by modifying glomerular vascular tone, inducing contraction of mesangial cells, and reducing glomerular ultrafiltration coefficient.²⁰ Ang II regulates sodium and bicarbonate transport in proximal tubules^{21 22} ; modulates sodium, chloride, and bicarbonate transport in distal tubules^{23 24} ; and modifies H⁺/ATPase activity and amiloride-sensitive sodium transport in the cortical collecting ducts.^{23 25} Our findings provide structural evidence for the possible role of the AT_1A receptor in these Ang II–mediated renal effects.

Renal hypertension produced by unilateral renal artery clipping or renal compression is associated with increased renin release and Ang II production from the ischemic kidney²⁶ (also unpublished data). The current findings provide direct evidence for the bilateral downregulation of the renal AT_1A subtype receptor protein in rats with acute-phase 2K1C and 2K1W renal hypertension. Renal AT_1A receptor mRNA expression is decreased after systemic administration of a nonpressor dose of Ang II²⁷ and in the clipped kidney 2 days after unilateral renal artery clipping.¹¹ These results are in agreement with the notion that increased Ang II exerts a negative regulatory effect on the renal expression of the AT_1A receptor.

This study demonstrated differential regulation of the renal AT₂ receptor in renal hypertension. The AT₂ receptor was downregulated only in the ischemic kidneys of 2K1C and 2K1W hypertensive rats, whereas renal AT₂ receptor expression was not significantly affected in Ang II–induced hypertensive rats. These results suggest that increased Ang II itself may not be directly responsible for this downregulation. Hypoperfusion and/or other local changes secondary to reduced blood supply to the clipped/wrapped kidney are possible causes of suppressed AT₂ receptor expression in the ischemic kidneys of renal hypertensive rats. A lack of inducible AT₂ receptor expression in cultured rat glomerular mesangial cells from stroke-prone spontaneously hypertensive rats and aortic vascular smooth muscle cells from spontaneously hypertensive rats has been described.^{5 28} The AT₂ receptor not only causes vasodilation in the preglomerular afferent arteriole⁷ but also exerts an antiproliferative effect²⁹ and induces apoptosis.³⁰ Recently, accelerated renal interstitial fibrosis and collagen deposition has been observed in adult AT₂ receptor null mutant mice during unilateral ureteral obstruction.³¹ Thus, decreased AT₂ receptor expression in the ischemic kidney may aggravate renal ischemia and promote cell proliferation/fibrosis, possibly augmenting kidney damage in renal hypertension.

In summary, we localized the AT_1A subtype receptor protein to glomeruli, proximal and distal tubules (including macula densa), and collecting ducts, as well as the intrarenal vasculature. Renal AT_1A but not AT₂ receptor protein was downregulated in Ang II–induced hypertension. Expression of AT_1A and AT₂ receptor protein in the kidney was differentially regulated in renal vascular hypertensive rats: The AT_1A receptor was bilaterally downregulated, whereas the AT₂ receptor was downregulated only in the ischemic kidney. Differential regulation of angiotensin receptor subtypes may play a role in the pathophysiology of renal hypertension.

	Acknowledgments

 
The authors wish to thank Dr M. Ian Phillips of the University of Florida for providing the AT_1A receptor antiserum and Nancy L. Howell and Dawn M. Baramki for their excellent technical assistance. This work was supported in part by grants RO1-HL-49575 and RO1-HL-59948 (R.M.C.), RO1-HL-47669 (H.M.S.), and RO1-HL-39706 and RO1-GM-49111 (R.A.J.) from the National Institutes of Health and by American Heart Association Fellowship VA-96-F-10 (L.J.M.).

Received January 30, 1998; first decision March 4, 1998; accepted August 28, 1998.

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