ARTICLE |
Correspondence to: Angelo Parini, INSERM U388, Pharmacologie Moléculaire et Physiopathologie Rénale, Institut Louis Bugnard, Bat. L3, CHU Rangeuil, 31403 Toulouse Cedex 4, France. E-mail: parini@toulouse.inserm.fr
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Summary |
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Dopamine D4 receptors mediate inhibition of vasopressin-dependent sodium reabsorption by dopamine in collecting tubules. At present, the distribution of D4 receptors in other renal districts remains an open issue. The renal distribution of D4 receptor was assessed in normally innervated and denervated male SpragueDawley rats by quantitative immunohistochemistry using an anti-dopamine D4 receptor rabbit polyclonal antibody. D4 receptor protein immunoreactivity was observed perivascularly in the adventitia and the adventitiamedia border. The density of perivascular dopamine D4 receptor was higher in afferent and efferent arterioles than in other segments of the renal vascular tree. Renal denervation abolished perivascular dopamine D4 receptor protein immunoreactivity. In renal tubules, the epithelium of collecting tubules showed the highest dopamine D4 receptor protein immunoreactivity, followed by the epithelium of proximal and distal tubules. No dopamine D4 receptor protein immunoreactivity was observed in the epithelium of the loop of Henle. Denervation did not change dopamine D4 receptor protein immunoreactivity in renal tubules. These results indicate that rat kidney expresses dopamine D4 receptors located both prejunctionally and nonprejunctionally in collecting, proximal, and distal tubules. This suggests that the dopamine D4 receptor may be involved in the control of neurotransmitter release and in renal hemodynamic and tubule function.
(J Histochem Cytochem 50:10911096, 2002)
Key Words: D4 receptors, kidney, rat, immunohistochemistry
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Introduction |
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Increasing evidence has emphasized the role of dopamine in the regulation of renal function (
The D3 receptor is the renal dopamine D2-like receptor subtype most investigated. It was characterized by reverse transcription-polymerase chain reaction (RT-PCR) (
To define the post- and prejunctional localization of renal D4 receptors, we investigated their microanatomic localization in kidneys of sham-operated and denervated rats.
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Materials and Methods |
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Animals and Tissue Treatment
Male SpragueDawley rats (n=20; bw 290 g) were used. Ten animals were anesthetized with an IP injection of pentobarbital sodium (60 mg/kg) and laparotomized. The right renal artery was exposed and denervated by stripping the artery surgically and swabbing it with a 10% phenol solution dissolved in ethanol according to the procedure detailed elsewhere (
The remaining rats were perfused through the abdominal aorta with a 0.9% NaCl solution containing heparin (20 IU/100 ml). This solution was replaced by a fixative solution of a 4% freshly prepared paraformaldehyde and 0.4% picric acid in phosphate buffer (pH 6.9). At the end of perfusion, kidneys were removed, cut into slices of 0.20.5 cm, and put in the same perfusion for an additional 72 hr. At the end of fixation, slices were washed, dehydrated in ethanol, and embedded in a semi-synthetic paraffin. Paraffin blocks were cut serially using a motorized microtome and mounted on gelatin-coated microtome slides. Each slide contained two 10-µm-thick sections of kidney.
NE Assay
The upper halves of the right and left kidneys were homogenized in ice-cold 0.1 M HClO4 (5 ml/g tissue). The homogenate was centrifuged at 2000 x g for 20 min at 4C and the resulting supernatant was used for NE assay. NE was assayed by high-pressure liquid chromatography (HPLC) with electrochemical detection (
Western Blotting Analysis
The upper halves of the right and left kidneys were homogenized in 0.32 M sucrose. The homogenate was centrifuged at 2000 x g for 20 min at 4C to remove nuclei and cell debris, and the resulting supernatant was centrifuged at 14,000 x g for 30 min. The pellet representing kidney membranes was re-suspended in a buffer containing phenylmethyl sulfonylfluoride, aprotinin, and leupeptin. Equivalent amounts of proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose. Anti-dopamine D4 receptor protein antibody was dissolved in 0.1 M PBS containing BSA (1%) and Tween-20 (0.05%) at a dilution of 1:400. This antibody concentration was established in a series of preliminary experiments. The specificity of the immune reaction was assessed using an antibody pre-adsorbed with the peptide used for generating it. A 0.05% 3-3'-diaminobenzidine tetrahydrochloride (DAB) solution was used as a chromogen for the peroxidase reaction.
Dopamine D4 Receptor Protein Immunohistochemistry
Groups of six consecutive slides were used. The first section was stained with hematoxylineosin to verify microanatomic details. The second and fourth sections were exposed to the primary anti-dopamine receptor antibody in phosphate buffer containing 0.2% (w/v) BSA, 0.03% Triton X-100, and 0.1% (w/v) sodium azide, diluted 1:2500. The third and fifth sections were incubated as above with the primary antibody pre-adsorbed with its peptide antigen at a dilution of 10 µg/ml. The sixth section was exposed to a non-immune serum instead of the primary antiserum. Incubation with antibody was accomplished in a humid chamber at 4C for 1218 hr. Optimal antisera dilutions and incubation times were assessed in a series of preliminary experiments.
After incubation, slides were rinsed twice in phosphate buffer and exposed for 30 min at 25C to a biotinylated anti-rabbit secondary antibody at a dilution of 1:200. The product of the immune reaction was then revealed using a biotinstreptavidin immunostaining kit with 0.05% DAB in 0.1% H2O2 as a chromogen. Sections were then washed, dehydrated in ethanol, and mounted in a synthetic mounting medium.
Microdensitometry
The intensity of immunoreactivity developed in the epithelia of the proximal tubule, loop of Henle, and distal and collecting tubules was assessed microdensitometrically. For microdensitometry, six sections of right and left kidneys in five denervated and sham-operated rats were examined at a final x400 magnification. Fifty proximal, distal, and collecting tubules or loop of Henle epithelial cells per section were identified and delineated by a measuring diaphragm whose size was adapted to cover one cell at a time. In parallel sections, proximal tubules were distinguished from distal tubules by alkaline phosphatase histochemistry. Microdensitometry was performed using an IAS 2000 image analyzer (Delta Sistemi; Rome, Italy) connected via a TV camera to a Leica 500 AC microscope. Microdensitometry system was calibrated taking as zero the background obtained in sections exposed to non-immune serum. The intensity of immune staining was then measured using a microdensitometry program of the image analyzer. Microdensitometry technique was validated in a series of preliminary experiments in which the primary antibody was used in a range of dilutions from 1:10,000 to 1:500. The antibody dilution causing the lowest background (1:2500) was considered optimal and was used in subsequent microdensitometry experiments.
Antibodies and Chemicals
Rabbit anti-dopamine D4 receptor protein antibody (cat. no. 324405, lot no. B17850) was purchased from CalbiochemNovabiochem (San Diego, CA). Peptide immunogen corresponding to that used for generating antibody was synthesized by the Department of Medicinal Chemistry of Camerino University. It corresponded to amino acids 175186 for the human D4 receptor. The biotinstreptavidin immunostaining kit was from CalbiochemNovabiochem. Other chemicals were purchased from Sigma Chemical (St Louis, MO).
Statistics
Data for microdensitometry of dopamine receptor protein immunostaining in the different types of tubule cells were analyzed statistically by ANOVA followed by Duncan's multiple-range test as a post-hoc test.
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Results |
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The renal distribution of D4 receptors was investigated by immunohistochemistry using an anti-dopamine D4 receptor antibody raised against the amino acid fragment 176185 of the human D4 receptor protein. The use of an antibody directed against the same peptide allowed the demonstration of D4 receptor expression in rat cortical collecting duct by immunohistochemistry (
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Immunohistochemistry was performed in kidneys of sham-operated and denervated rats. The efficacy of renal denervation was evaluated by the measure of renal NE concentrations. In right kidneys of sham-operated rats, the NE concentration averaged 40 + 2.1 ng/g tissue. Denervation caused a decrease of NE concentration to 3.5 + 0.1 ng/g tissue (p<0.01 vs sham-operated). NE concentrations in left kidneys were not different from those assayed in right kidneys of sham-operated rats (data not shown). Sections of sham-operated kidneys exposed to antibodies against dopamine D4 receptor protein developed a sparse dark-brown staining in the adventitiamedia of renal arteries (Fig 2A) and in an appreciable percentage (approximately 40%) of nerve fibers present in nerve trunks visible in the hilum (data not shown). Perivascular dopamine D4 receptor protein immunoreactivity was located in large segmental arteries and in arcuate, interlobar, and interlobular arteries (data not shown). This immunoreactivity disappeared in denervated kidneys (Fig 2C). Sections exposed to pre-immune serum or to antibodies pre-adsorbed with the corresponding receptor protein peptide developed background only (Fig 2E). Dopamine D4 receptor protein immunoreactivity was also located perivascularly in locations corresponding to afferent (Fig 2B) and, to a lesser extent, efferent (Fig 2B) glomerular arterioles. This immunoreactivity was no longer observed in denervated kidneys (Fig 2D) or after pre-exposure to antibodies pre-adsorbed with the corresponding receptor protein peptide (Fig 2F).
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No dopamine D4 receptor protein immunoreactivity was observed in the glomerular tuft (Fig 2A and Fig 2D) or in the loop of Henle epithelium (Table 1). Slight D4 receptor protein immunoreactivity was found in proximal and distal tubules (Table 1) and moderate immunoreactivity in collecting tubule epithelium (Fig 3A and Fig 3C; Table 1). Reaction was specific for D4 receptor protein because it did not occur when an antibody pre-adsorbed with blocking peptide was used (Fig 3B) and was unaffected by renal denervation (Fig 3C; Table 1).
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Discussion |
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During the past decade, several efforts have been made to determine the molecular, pharmacological, and functional properties of dopamine D4 receptors. Most of the studies concerned the role of these receptors in the regulation of brain function and their abnormalities in neurological disorders (
In this study, extending the indirect results of other investigations (
In conclusion, we have demonstrated that dopamine D4 receptors are distributed in different segments of the nephron and, in the vascular distribution, they also have a prejunctional localization. These results may represent a starting point for the further characterization of the functional properties of D4 receptors in kidney and for comprehension of the mechanisms of renal function regulation by dopamine.
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Acknowledgments |
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Supported in part by an INSERM/CNR collaborative grant and by a financial grant from Servier Laboratories.
Received for publication August 6, 2001; accepted March 13, 2002.
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