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J Am Soc Nephrol 13:1-9, 2002
© 2002 American Society of Nephrology

Peripheral Benzodiazepine Receptor Mapping in Rat Kidney. Effects of Angiotensin II-Induced Hypertension

Estelle Bribes*, Pierre Casellas*, Hubert Vidal*, Danielle Dussossoy* and Daniel Casellas{dagger}

*Department of Immunology and Oncology, Sanofi Synthélabo Montpellier, France; and {dagger}Groupe Rein et Hypertension, IURC, Montpellier, France.

Correspondence to Dr. Daniel Casellas, Groupe Rein et Hypertension, Institut Universitaire de Recherche Clinique, 641 Avenue du Doyen Gaston Giraud, 34093 Montpellier Cedex 5, France. Phone: 33-467-415-927; Fax: 33-467-542-731; E-mail: casellas{at}iurc1.iurc.montp.inserm.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
ABSTRACT. Intrarenal distribution and function(s) of the peripheral benzodiazepine receptor (PBR) remain uncertain. The goals of this study were to (1) develop a specific anti-rat PBR antibody and (2) map intrarenal immunoreactive PBR (irPBR) in untreated rats and in rats that received chronic angiotensin II infusion (200 ng/kg per min, subcutaneously, 17 d). A polyclonal rabbit antibody was raised against the C-terminal end of rat PBR (aa 159 to 169). The antibody specifically recognized a single 18-kD protein in whole kidney extracts, and confocal microscopy showed exclusive mitochondrial localization of irPBR in cultured rat glial C6 cells. In control rats, irPBR was found along thick ascending limbs of Henle’s loops, including the macula densa area, along distal tubules, and along collecting ducts. Vascular smooth-muscle cells were PBR-positive. General irPBR distribution was unaffected by angiotensin II treatment (systolic BP, 205 ± 9 mmHg). However, irPBR appeared in parietal glomerular epithelial cells, atrophic proximal tubules, and infiltrating mononuclear cells. In conclusion, the results suggest previously unsuspected roles of PBR in the control of glomerular dynamics and in proximal tubular injury/repair processes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The peripheral benzodiazepine receptor (PBR) is expressed in relatively great quantities in the kidney and other peripheral organs such as the adrenals (1). Although its specific renal roles remain elusive, covariations have been demonstrated between renal PBR density, acute changes in angiotensin II (AngII) levels (2), and arterial hypertension in rat models associated or not to environmental stress (1,3). To the best of our knowledge, however, changes in renal PBR expression in response to chronic AngII exposure, or associated with renal tissue injury that occurs in this hypertensive model (4–6) are not known.

For assessing tissue distribution and modulation of PBR density, most studies have relied on the characteristically high affinity of PBR to exogenous ligands such as the benzodiazepine Ro 5-4864 or to the isoquinoline carboxamide derivative PK 11195 (for recent review, see reference (1)).

In the rat kidney, and by means of autoradiography (7), PBR was detected along the thick ascending limb of the loop of Henle (TAL) and distal convoluted tubules (DCT). Broader tissue distribution was obtained by binding studies performed on microdissected tubular segments and isolated glomeruli (8). In this study (8), specific binding was found in glomeruli and from the medullary TAL all the way down to the medullary collecting duct (MCD).

In this context, assessment of PBR distribution, or modulation of its density, has been limited by the inherently poor spatial resolution of radioligand studies and by potential competition with endogenous ligands (1). Designed specifically to overcome these difficulties, a recent immunohistochemical study was based on a polyclonal antibody raised against rat mitochondrial PBR (9). It revealed immunoreactive PBR (irPBR) along distal convoluted tubules only (9). In view of the differing patterns of tubular distribution obtained by binding or immunohistochemical studies (7–9), uncertainty thus persists as to the extent and precise topology of PBR expression. Furthermore, PBR expression along the renal vasculature remains unexplored.

This rat study was undertaken with the following goals: (1) to develop and characterize a specific rabbit polyclonal antibody directed against the C-terminal region of the rat PBR; (2) to assess, via immunohistochemistry, the tubulovascular topology of irPBR in adult rats; and (3) to assess potential changes in irPBR distribution brought on by chronic AngII-induced hypertension.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and Protocols
These animal experiments complied with the European and French laws and with the guiding principles for experimental procedures as set forth in the Declaration of Helsinki. Studies were conducted in 13 adult male Sprague-Dawley rats (Iffa Credo, Lyon, France). Three of these rats had their kidneys removed under pentobarbital anesthesia (50 mg/kg, intraperitoneally) for Western blot analyses. Four rats were used as controls. In six rats, systemic hypertension was induced by continuous subcutaneous delivery of AngII (Sigma, St. Louis, MO; 200 ng/kg per min for 17 d) via osmotic minipumps (Alzet model 2002; Alza Corp., Palo Alto, CA), as described elsewhere (10). Systolic BP (SBP) was assessed in conscious animals by tail-cuff manometry (Narco BioSystems, Houston, TX) 1 d before AngII treatment, after 17 d of AngII treatment, and at similar periods in time in control rats. Increased albuminuria was taken as indicative of glomerular barrier/proximal tubular injury and determined on 24-h urine collections by laser immunonephelometry (11). Under pentobarbital anesthesia, the left kidney was flushed of its blood with albuminated Krebbs-ringer bicarbonate buffer via a catheter inserted into the abdominal aorta. The kidney was then perfusion-fixed with 10% buffered formalin (Accustain; Sigma), hemisectioned, and kept overnight in the fixative before histology.

Preparation and Characterization of Anti-Rat PBR Antibody
A polyclonal antibody was produced by aiming at the C-terminal region of rat PBR (aa 159 to 169). Rat PBR C-terminal peptide (SGRRGGSRLTE) and bovine serum albumin-conjugated peptide were obtained from Neosystem S.A. (Strasbourg, France). Immunosera were raised in rabbits by use of bovine serum albumin as carrier protein. Antibodies were purified by affinity chromatography against the synthetic peptide coupled to a sepharose matrix. Purified antibodies (concentration, 1 µg/ml) were stored at -80°C.

Western Blot Analyses
Whole kidney homogenates were made in a buffer that contained 2 mM Hepes (pH 7.4), 70 mM sucrose, 200 mM mannitol, 1 mM ethylenediaminetetraacetate, and a protein inhibitor cocktail (Boehringer, Mannheim, Germany) and centrifuged at 100,000 x g (20 min, 4°C). Pellets were suspended in 20 mM Tris-HCl buffer (pH 7.4). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (4% to 20% polyacrylamide) and electroblotted onto nitrocellulose. Membranes were soaked for 60 min in Tween-phosphate-buffered saline buffer (10 mM sodium phosphate [pH 7.6], 150 mM NaCl, and 0.1% Tween 20) that contained 5% dried milk. Blots were then incubated for 3 h in Tween-phosphate-buffered saline buffer that contained 1 µg/ml anti-PBR antibody. Blots were incubated with anti-rabbit IgG-horseradish perodidase-conjugated antibody (Dako; 60 min), and proteins were visualized by use of an enhanced chemiluminescent detection system (BCA Protein Assay Reagent kit; Pierce Chemical Company, Rockford, IL) and a Curix 60 developer (AGFA). Analyses were performed in triplicate.

Confocal Microscopy
Specificity of anti-PBR antibody was assessed at the subcellular level in C6 cell line developed from rat glial tumor (American Type Culture Collection, Manassas, VA) and characterized by high, exclusively mitochondrial PBR expression (12,18). Cells were grown until confluence (3 d, 37°C) on Ham’s F10 medium (82.5%) that contained 15% horse serum and 2.5% fetal bovine serum (Life Technologies, Grand Island, NY). Cells were incubated with a FITC-conjugated mitochondrial probe (MitoTracker, Molecular Probes Inc; Eugene, OR) as specified by the manufacturer, fixed with 1% paraformaldehyde (Sigma), and permeabilized with 0.1% saponin. After blockade with normal goat serum, cells were exposed to anti-PBR antibody (1:400, 60 min, room temperature), washed in phosphate-buffered saline buffer, and exposed to indocicarbocyanine-5-conjugated goat anti-rabbit IgG (1:200; Jackson Immunoresearch/Interchim, Paris, France). Analysis of cell distribution and degree of colocalization of irPBR and mitochondrial probe was performed with 650/670 and 492/520 nm filter sets, respectively. Experiments were performed with a laser scanning confocal microscope (LSM 410; Zeiss, Oberkochen, Germany), equipped with a 63x Zeiss planapo oil immersion objective (numerical aperture 1.4) by use of LSM 410 image analysis software, as described elsewhere (13). Specificity controls were carried out by preincubation of anti-PBR antibody overnight (1:400, 4°C) with the immunizing peptide (10 µg/ml).

Renal Tissue Preparation and PBR Immunohistochemistry
Perfusion-fixed kidneys were paraffin-embedded and cut at 2- to 4-µm thickness. After deparaffinization, sections from control and AngII-treated rats were stained with Masson’s trichrome (Accustain trichrome stain Masson, HT15; Sigma). All other sections were microwaved for antigen retrieval (800 watts, 3 x 5 min, in citrate buffer [pH 6]). After H2O2/methanol blockade of endogenous peroxidases, sections were subjected to an avidin-biotin immunoperoxidase procedure, with the use of H2O2/diaminobenzidine as chromogenic substrate (brown label, Vectastain ABC and DAB substrate kits; Vector Laboratories, Burlingame, CA), or to an indirect streptavidin-biotin method that used H2O2/3-amino-9-ethylcarbazole as chromogenic substrate (red label, Dako ChemMate Detection kit, peroxidase/AEC, Rabbit/mouse; Dako A/S, Glostrup, Denmark). Sections were counterstained with hematoxylin.

Sections were exposed to anti-PBR antibody (dilution 1:300) for 30 min at room temperature. For AEC staining, optimal chromogenic development in tubules was achieved in 2 min; development was extended to 3 min for optimal vascular staining. For DAB staining, the optimal development time was 3 min.

Specificity was demonstrated by absence of staining after preadsorbing anti-PBR antibody overnight at 4°C with the immunizing peptide. Negative controls were also obtained by absence of staining after omission of the primary antibody. Photographs were taken through a Laborlux D-FS microscope (Ernst Leitz GMBH, Wetzlar, Germany) equipped with 4x, 10x, 25x, and 40x dry objectives and Fujichrome films (Provia, RDP II 100, daylight). Identification of vascular and tubular segments in sections was made on the basis of previously well-documented anatomical characteristics (14–16).

Identification of PBR-Positive Cell Type along Collecting Ducts
Collecting ducts consist of a heterogeneous population of principal and intercalated cells (16). We took advantage of the fact that the vasopressin-regulated water channel, aquaporin 2 (AQP2), is expressed exclusively in principal cells (17) to identify PBR-positive cell type. Double labeling of deparaffinized/microwaved sections was performed in control and AngII-treated rats with our anti-PBR and a previously characterized (17) specific rabbit polyclonal antibody raised against the amino acid residues 254 to 271 of mouse and rat AQP2 (reference 178612, Calbiochem-Novabiochem Corporation, San Diego, CA). The procedure was performed according to the manufacturer’s instructions, by use of a double labeling kit (Dako EnVision Double stain System, Dako A/S, Glostrup, Denmark). Sections were first exposed to anti-PBR antibody (dilution 1:300; 30 min) and to DAB substrate (brown label). Sections were then exposed to anti-AQP2 antibody (dilution 1:600, 30 min) and to Fast Red substrate (red label). No nuclear counterstain was applied. Stained sections were mounted in aqueous medium (Dako Glycergel mounting medium). In double-stained sections, Fast Red signal could be viewed separately from DAB stain under epifluorescence (Leitz Ploemopak, filter cube N2.1, 515 to 560/580 nm). Specificity was demonstrated by absence of staining after preadsorbing each antibody overnight at 4°C with its corresponding immunizing peptide. Negative controls were also obtained by absence of staining after omission of the primary antibodies.

Statistical Analyses
Within-group analyses were carried out with Wilcoxon’s rank sum test for paired data (albuminuria) or paired t test. P < 0.05 was considered statistically significant. Values are given as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Characterization of Anti-PBR Antibody
As shown in Figure 1, the anti-PBR antibody was specific for the immunizing peptide and recognized a single 18-kD band in whole-kidney extracts, in complete agreement with PBR molecular weight predicted from its complete DNA sequence or obtained experimentally (9). Subcellular specificity of anti-PBR antibody was demonstrated, as illustrated in Figure 2. PBR-associated immunofluorescence was confined to mitochondria, as demonstrated by complete overlap with mitochondrial probe.



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  		Figure 1. Western blot analysis for peripheral benzodiazepine receptor (PBR) in whole-kidney extract. A single 18-kD band is indicated by an arrow in the left lane. No band is detectable in the right lane (PBR + peptide), because the anti-PBR antibody was preadsorbed with the immunizing peptide.

 


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  		Figure 2. Assessment by confocal microscopy of the subcellular distribution of immunoreactive PBR (irPBR) in a cultured rat glial C6 cell (N, nucleus). (a) Fluorescence distribution of irPBR. (b) Fluorescence distribution of the mitochondrial probe. (c) Preadsorbing anti-PBR antibody with immunizing peptide eliminates irPBR signal. (d) Merges panels a and b and demonstrates almost complete colocalization of both probes. Scale bar applies to all panels.

 
SBP, Body Weight, and Albuminuria
In AngII-treated rats, SBP averaged 121 ± 2 and 205 ± 9 mmHg (P < 0.05, paired t test, n = 6) before and at the end of AngII treatment, respectively. In untreated control rats, SBP averaged, respectively, 119 ± 5 and 134 ± 3 mmHg (n = 4). In control rats, mean body weight increased from 288 ± 7 to 380 ± 12 g (n = 4) during the 18-d period, whereas AngII-treated rats increased their mean body weight from 280 ± 6 to 333 ± 15 g (n = 6). AngII treatment induced a significant, 20-fold increase of mean albuminuria from a baseline of 142 ± 23 to 3073 ± 1196 µg/24 h (P < 0.05, Wilcoxon’s rank sum test, n = 6), whereas mean albuminuria of control rats was stable and averaged 142 ± 23 and 57 ± 34 µg/24 h (n = 4) at corresponding periods of time.

Intrarenal Topology of irPBR in Control Rats
Corticopapillary distribution of irPBR and specificity of immunostaining are illustrated in Figure 3, a through e and f, respectively. In the cortex and outer stripe of the outer medulla, TAL, DCT, and cortical collecting ducts exhibited similar levels of irPBR, whereas all proximal tubular profiles were PBR negative (for overview, see Figure 3, a and b; for details, see Figure 4, a through d). Thick ascending limbs and MCD were PBR positive along the entire inner stripe of the outer medulla, whereas vasa recta bundles were negative (Figure 3, c and d). Thin descending limbs identified at the outer/inner stripe boundary as originating from late proximal tubular loops, were occasionally PBR-positive (data not shown). Of importance, the TAL segments located in the inner stripe exhibited lesser irPBR than their counterparts located in the outer stripe and cortex (Figure 3, a versus c). Collecting ducts exhibited a decrease in irPBR in their medullary segments via a decrease in the density of PBR-positive cells (Figure 3, a versus c). Rarefaction of PBR-positive cells occurred along MCD, near the inner stripe/inner medulla boundary (Figure 3d) and continued toward papilla, where no positive cells were found (Figure 3e). Double staining with anti-PBR and anti-AQP2 antibodies allowed us to identify PBR-positive cells located along MCD segments near the inner stripe/inner medulla boundary as the intercalated (i.e., AQP2-negative) cell minority (Figure 4, d and e). Deep cells of the transitional epithelium of the pelvic mucosa (and ureter) were PBR-positive (data not shown). Elongated, perinuclear deposits of irPBR were found in smooth-muscle cells of all segments of the preglomerular vasculature, including main arcuate arteries, arcuate arterial branches, interlobular arteries, and afferent arterioles (Figure 4c). Endothelial cells exhibited weaker perinuclear staining than adjacent smooth-muscle cells in control rats (data not shown).



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  		Figure 3. Overlapping microscopic fields showing corticomedullary distribution of irPBR in a control rat, appearing as brown peroxidase deposits (a through e). Specificity of primary antibody is demonstrated on a contiguous section by lack of irPBR after preabsorbtion with corresponding peptide (f). From the cortex (a) down to the outer stripe/inner stripe boundary (dotted line in b), no proximal tubular profiles are labeled. (a and b) Thick ascending limbs (single arrow), collecting ducts (double arrows), and distal convoluted tubules (triple arrows) are strongly positive. (c) Spans most of inner stripe of outer medulla (characterized by the absence of proximal tubular profiles); note the negative vasa recta bundle (*) and positive thick ascending limbs (single arrow) and medullary collecting ducts (double arrows). Note decreased irPBR in thick ascending limbs (c, single arrow), compared with their cortical counterparts (a, single arrow), and decreased density of positive cells along collecting ducts (c [double arrows] versus a [double arrows]). (d) Transition from outer to inner medulla (inner stripe/inner medulla boundary indicated by dotted line and characterized by disappearance of thick ascending limbs); note the ensuing rarefaction of positive cells in collecting ducts (double arrows). Rarefaction of positive cells (double arrows) is complete in papilla (p, e). Scale bar, 500 µm, applies to all panels.

 


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  		Figure 4. Detailed topology of irPBR in control rats. (a through c) irPBR appears as red (AEC substrate and hematoxylin counterstain) or brown deposits (DAB substrate and no counterstain). (a) irPBR in macula densa plaque (triple arrows) and in directly connected distal tubule (double arrow: transition of straight part into convoluted part of distal tubule). (b) Distal convoluted tubular profile (single arrow), connecting tubule (double arrows), and directly connected cortical collecting duct (triple arrows) exhibit similar levels of irPBR. (c) Tangential section of an arcuate arterial branch; it gives rise to a cortical radial artery (middle right vessel) and an afferent arteriole ({star}). Note the clearly perinuclear irPBR (small arrows) following the smooth-muscle cell orientation. (d) Double staining for aquaporin 2 (AQP2; red Fast Red deposit) and PBR (brown DAB deposit) along collecting duct profiles (*) in the inner medulla (i.e., the area that corresponds to lower part of Figure 3d). PBR-positive cells (arrows) appear to be AQP2 negative. (e) Fast Red fluorescence view of the same microscopic field confirms that PBR-positive cells (arrows) are AQP2 negative. (f) double stain specificity shown by absence of staining after preadsorbtion of both primary antibodies with corresponding peptides. Scale bars: 50 µm in a and c; 100 µm in b; and 25 µm in d through f.

 
Intrarenal Topology of irPBR in AngII-Treated Rats
In renal zones devoid of injury, irPBR was found along TAL, DCT, cortical collecting ducts, MCD, and preglomerular vessels, following a general pattern (overview not shown) similar to that of control rats (Figure 3). Studies elsewhere (4–6) have demonstrated that chronic AngII-induced hypertension is associated with focal cortical zones of tubulointerstitial injury, collapsed/atrophic proximal tubules or dilated tubules with casts, interstitial collagen deposition, and monocyte/macrophage infiltration. Similar injuries were found in this study, as illustrated in Figure 5a. Within these injured zones, profiles of collapsed/atrophic proximal tubules, starting occasionally from Bowmans capsule (Figure 5b), exhibited irPBR, in association with PBR-positive mononuclear cells infiltering renal interstitium. In these injured cortical areas, parietal epithelial cells of Bowman capsules were also frequently PBR-positive (Figure 5, b and c). Of note, a few PBR-positive cells occasionally clustered along an otherwise "normal" proximal tubular profile in association with PBR-positive mononuclear cells in nearby interstitium (Figure 5d). In addition to forming inflammatory infiltrates, PBR-positive mononuclear cells attached to venous endothelium or were present within glomerular capillary loops. In contrast, no irPBR was found in hypertrophied glomeruli and proximal tubules. Furthermore, distended TAL and DCT with correspondingly thinned walls had reduced PBR immunostaining as compared to nearby "normal" structures (Figure 5b). Vascular injuries, identified by their characteristic focal media thickening (4,6), also exhibited strong PBR expression in smooth-muscle as well as endothelial cells (Figure 5e).



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  		Figure 5. Tubulointerstitial and vascular injury in angiotensin II-treated rats. (a) Low-power view (trichrome stain) shows focal tubulointerstitial injury surrounded by unaffected tissue (*). Two glomeruli (G), the upper one with a "normal" appearance (note macula densa facing vascular pole) and the lower, smaller one with a thickened arteriole (double arrows) and collapsed, injured proximal tubule directly connected to Bowman’s capsule (single arrow); {star}, distal, dilated tubular profiles. In subsequent views, irPBR appears as dense red deposits (AEC label). (b) G with positive Bowman capsule (single arrow) giving rise to a collapsed, PBR-positive proximal tubule (double arrows) and another PBR-positive proximal loop (triple arrows); positive mononuclear cells (arrowhead) within expanded interstitium, among which dilated, distal convoluted tubular loops (*) have reduced irPBR deposits, compared with nearby positive profiles with thicker, "normal" walls. (c) G with positive Bowman’s capsule surrounded by expanded interstitium with numerous, positive mononuclear cells (arrowheads). Several profiles of collapsed proximal tubules (*) exhibit irPBR deposits; compare with PBR-positive thick ascending limbs ({star}). (d) Occurrence of clustered PBR-positive cells (between arrows) in proximal tubules with no sign of collapse, associated with positive mononuclear cells (arrowhead), {star}, PBR-positive thick ascending limb. (e) Focal injury (between large arrows, characterized by thickened media) along a cortical radial artery (*), with positive smooth-muscle and endothelial cells (small arrows); {star}, positive nonproximal tubular profiles. (f) Specificity PBR staining indicated by lack of AEC label after preadsorption of anti-PBR antibody with its immunizing peptide; nuclear counterstain. Scale bar: 100 µm in a; 50 µm in b through f.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We report the production and characterization of a novel polyclonal anti-rat PBR antibody. Producing an antibody that recognizes the C-terminal region of the rat PBR molecule is a proved valid strategy, because this part of the molecule is likely the most immunogenic (13). Our antibody specifically recognized a single 18-kD protein in whole-kidney extracts. This molecular weight fits with the PBR weight that could be predicted from its complete sequence. Finally, our antibody demonstrated adequate subcellular specificity when applied to a rat cultured cell model in which PBR is exclusively present in the mitochondrial membrane (12,18,19).

Use of this novel anti-PBR antibody in normotensive rats revealed a broad distribution of specific irPBR in renal epithelia, including the pelvic and ureteral mucosa. This finding contrasts with a rat study elsewhere in which irPBR was detected in DCT only (9). The reason for such a difference is not clear. It may relate to our selective targeting of the hydrophilic PBR C-terminus, which faces cytosol (13), and may thus be more accessible to the antibody, whereas whole rat PBR extracted from adrenal mitochondria was used elsewhere (9). Nevertheless, our results are in good agreement with PBR distribution obtained in binding studies performed on microdissected rat tubules (8).

Studies elsewhere on renal PBR showed an almost exclusively epithelial, nonproximal distribution, which focused attention on relationships between PBR and anion transport or vasopressin and mineralocorticoid action on PBR-bearing tubular segments (8,20,21). This immunohistochemical study provides, for the first time, detailed topology of renal PBR and reveals some as yet unknown distribution patterns of potentially new functional significance.

In the renal cortex, irPBR was equally present along TAL, macula densa, DCT, and cortical collecting ducts. Bowman’s capsules and proximal tubules were all PBR-negative. At the level of the inner stripe of outer medulla, TAL had reduced levels of irPBR. It is intriguing to note that the corticomedullary gradient of irPBR along TAL is opposite to that of mitochondrial density in this segment (16). MCD also showed a reduction of irPBR levels, particularly beyond the inner stripe/inner medulla boundary. This occurred through a distinct pattern of rarefaction of PBR-positive cells. When AQP2 was used as a specific marker of principal cells (17), we found that, in the inner stripe/inner medulla region, PBR-positive cells represented the intercalated, vasopressin-insensitive cell minority. Of interest, a similarly abrupt decrease in cell density was demonstrated at the outer/inner medulla boundary for medullary type A intercalated cells in rat kidneys (22).

One important new finding is that PBR is expressed along the entire preglomerular vasculature, including main arcuate arteries, arcuate arterial branches, cortical radial arteries, and afferent arterioles. However, no irPBR was detected in glomerular capillaries and vasa recta bundles. This may simply reflect differing cell compositions of the various vascular beds. In preglomerular vessels, irPBR was mostly confined to the smooth-muscle cell population, and only weak perinuclear expression was detected in endothelial cells. Glomerular capillaries and vasa rectae, in contrast, do not possess smooth-muscle cells but consist of an endothelial tube surrounded by podocytes and pericytes, respectively (16). Studies elsewhere have pointed to a vasomotor role of PBR, because it may, for instance, participate in the control of airway smooth-muscle tone (23). Furthermore, it has been proposed that PBR controls the production of cytochrome P-450-derived eicosanoids, in particular that of 20-hydroxyeicosatetraenoic acid (1). This compound is of particular relevance in the context of renal circulation, given that it has been recently identified as a mediator of pressure-induced autoregulatory responses of preglomerular vessels (24). Therefore, the presence of irPBR both at the macula densa and along the vasculature provides the anatomic ground for a role of PBR in the control of glomerular and renal hemodynamics.

In this study, we explored renal PBR distribution in rats made hypertensive by chronic administration of AngII. Chronic AngII-induced hypertension is associated with an array of well-documented renal alterations (4–6) and thereby affords the opportunity to pinpoint in vivo modulation of irPBR in relation to pathologic cell processes.

The tubulovascular patterns of irPBR currently observed in intact renal areas of AngII-treated rats was essentially the same as that in normotensive rats. Our results thus suggest robust tubulovascular distribution of PBR, insensitive to chronic AngII treatment and/or to the associated systemic hypertension. Decreases in renal PBR binding have been reported elsewhere after acute AngII infusion (2) or in association with systemic hypertension in several hypertensive rat models (3,25). Because immunohistochemistry is nonquantitative, our results do not allow us to make conclusions on this point.

Our observations on irPBR distribution in renal areas with overt AngII-induced alterations deserve specific comments. Focal injuries develop along preglomerular vessels during chronic AngII hypertension (4,6). They were easily detected in this study by thickened media and were associated with increased or maintained irPBR levels within smooth-muscle and endothelial cells. A variety of tubular alterations were present in AngII hypertensive rats, as has been documented elsewhere (5,6). Dilated Bowman’s capsules with cast, leading to dilated proximal loops, were observed and remained PBR negative. Dilated/stretched nonproximal loops exhibited reduced irPBR levels. By contrast, Bowman’s capsules connected to or located nearby collapsed/atrophic proximal tubules were PBR positive. In collapsed/atrophic proximal tubules, irPBR also accumulated. Furthermore, clusters of PBR-positive cells were occasionally found in the wall of proximal tubular profiles with patent lumina and a "normal" appearance. This apparently de novo epithelial synthesis of PBR was associated with PBR-positive mononuclear cell infiltrates. PBR-positive mononuclear cells likely represent the monocyte/macrophage population that infiltrates renal interstitium during AngII-induced hypertension (5,6), and monocytes are known to express PBR (26). In addition to its known spacial proximity to antiapoptotic proteins such as Bcl-2 (27), PBR is a survival factor in hematopoietic cells in vitro (28). Further studies are thus warranted to assess the potential involvement of PBR in tubular injury/repair processes associated with inflammation.

In conclusion, a novel polyclonal anti-rat PBR antibody was developed that allowed detailed mapping of renal PBR by immunohistochemistry. A similar pattern of tubulovascular expression was obtained in normotensive and in AngII hypertensive rats in renal zones devoid of tubulointerstitial injury. The presence of irPBR in smooth-muscle cells of preglomerular vessels and at the macula densa suggest a role of PBR in glomerular dynamics. The appearance of irPBR along collapsed/atrophic proximal tubules in AngII hypertensive rats suggests a role of PBR in epithelial injury/repair processes.


   Acknowledgments
 
Studies were supported by a research grant from Sanofi Synthélabo (AED 0199C156) and by the Ministère de l’Education Nationale de l’Enseignement Supérieur et de la Recherche. We acknowledge Dr Joëlle Simony-Lafontaine (CRLC, Montpellier, France) for allowing access to her histologic facilities. Annie Artuso, Danielle Briand, and Nadine Lequeux provided expert technical assistance. Micrographs were printed by Patrick Schuman (ISIS 24, Montpellier, France). Requests for antibody should be sent to the corresponding author.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication March 29, 2001. Accepted for publication July 10, 2001.




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