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Endocrinology Vol. 140, No. 4 1622-1629
Copyright © 1999 by The Endocrine Society

ARTICLES

Functional Receptors in the Avian Kidney for C-Type Natriuretic Peptide

Daniela Brenner and Rüdiger Gerstberger

Max-Planck-Institute for Physiological and Clinical Research, W. G. Kerckhoff-Institute, Parkstrasse 1, D-61231 Bad Nauheim, Germany

Address all correspondence and requests for reprints to: Rüdiger Gerstberger, Ph.D., W.G. Kerckhoff-Institute, MPI, Parkstrasse 1 D-61231 Bad Nauheim, Germany. E-mail: rgerst{at}kerckhoff.mpg.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Renal actions of avian-specific C-type natriuretic peptide (chCNP) were investigated in the conscious Pekin duck. Under conditions of steady-state renal water and salt elimination, systemic chCNP administration (6 and 30 pmol/min·kg BW for 20 min) dose dependently induced transient natriuresis and diuresis. Mean arterial pressure and heart rate remained constant throughout the experiment. Employing receptor autoradiography, binding sites specific for [125I]BH-chCNP could be localized at high density in glomeruli of both reptilian- and mammalian-type nephrons, and arterioles of the avian kidney. The distal tubular zone revealed [125I]BH-chCNP binding sites at medium, the medullary cone area at low density. Using an enriched kidney membrane fraction, competitive displacement studies with [125I]BH-chCNP as radioligand and various unlabeled peptide analogs (chANP, chCNP, rANP, rBNP, frANP, rANP(4–23)) allowed the discrimination of high-affinity (IC50 values 10-10–10-9 M) and low-affinity (IC50 values 10-8–10-7 M) binding sites different from typical mammalian receptor subtypes. Intracellular cyclic GMP formation could be demonstrated immunocytochemically for both types of glomeruli and cells of the distal tubular zone in fixed tissue sections after in vivo application of chCNP (0.8 nmol/min·kg BW; 5 min). The results obtained by combination of physiological in vivo studies and in vitro receptor analysis indicate an important role for chCNP in the modulation of avian kidney function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
C-TYPE NATRIURETIC PEPTIDE (CNP), first isolated from the porcine and rat brain (1, 2) represents a rather new member of the natriuretic peptide family (ANP, BNP, CNP, VNP) with a 17-amino acid ring structure in common (3). The extraction of CNP-like factors from the brain tissue of chicken (chCNP) (4), amphibians, and some elasmobranchs (3) indicates a wide distribution of homologous peptides among vertebrates. According to radioimmunological and immunocytochemical studies, CNP is distinctly expressed in hypothalamic nuclei of the rat brain, and high CNP levels were found in human cerebrospinal fluid (5, 6), suggesting a neuromodulatory rather than endocrine function, in line with contradictory reports concerning the presence of CNP as a circulating peptide in humans, rabbits or rats (5, 6, 7, 8). On the other hand, the detection of significant CNP expression in peripheral tissues like the kidney, intestine, lung, and testis, but also endothelial cells of major arteries supports a tissue-specific autocrine or paracrine function, with special emphasis placed on intrarenally formed CNP (9).

Common to the natriuretic peptides in mammals is their affinity to two types of cell membrane receptors, of which the first one possesses intrinsic guanylyl cyclase activity (10, 11) and may be subdivided (GC-A, GC-B) according to different affinites for ANP, BNP, and CNP (12, 13), whereas the second one does not reveal intrinsic guanylyl cyclase activity and possibly acts as a "clearance receptor" internalizing the ligand (14, 15).

In all classes of vertebrates investigated, circulating ANP/BNP-like peptides of cardiac origin are primarily involved in body fluid homeostasis and cardiovascular control under conditions of extracellular hypervolemia, with the kidneys and other ion transporting epithelia, the adrenals and vascular smooth muscle cells representing important target tissues (3, 16, 17, 18). Information concerning the endocrine or paracrine functions for CNP, however, remains sparse with studies on renal functions limited to mammalian species. Under conditions of anesthesia, systemically administered CNP was shown to possess only modest or even no diuretic and natriuretic activity but proved to be a potent vasodilator (2, 7, 19). On the other hand, experiments performed in unanesthetized animals or human subjects revealed natriuretic and diuretic CNP actions comparable to those of ANP for the sheep (20, 21) but weaker than those of ANP for humans and monkeys (22, 23). In elasmobranchs, CNP acted as a strong secretagogue for rectal gland chloride secretion (24).

In mammals, a major confounding factor in the experimental analysis of renal actions of natriuretic peptides, as of osmoregulatory peptides in general, is their interference with cardiovascular control. In contrast, circulatory side effects are minimal or absent in birds. Thus, birds are particularly suited as animal models, as demonstrated especially for the domestic duck, in which detailed analysis of renal and extrarenal actions of angiotensin II (ANGII), the antidiuretic hormone and chANP were carried out (17, 25, 26). Accordingly, the present study has investigated the putative modulatory role for renal sodium and water excretion of bird-specific CNP (chCNP) in the duck by combining physiological experiments in unanesthetized animals with the pharmacological and histochemical characterization of the duck’s renal natriuretic peptide receptor system.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
The studies were performed using adult female Pekin ducks (Anas platyrhynchos) (W. G. Kerckhoff-Institute, Bad Nauheim, Germany) with body weights (BW) of 2.0–2.5 kg, housed in flocks of approximately 10 animals at 20 ± 1 C ambient temperature under a natural day-night cycle. For immunohistochemical detection of cyclic GMP (cGMP) in kidney sections, female ducks 8 weeks of age were used. The animals were maintained on tap water ad libitum and fed commercial dry chicken food enriched with vitamins and minerals. All experiments were carried out according to the highest standards of human animal care and were approved by the Hessian Ethics Committee in Germany.

Experimental procedure
All physiological experiments were performed with conscious birds accustomed to the experimental setup and procedures. Twenty-four hours before experimentation, the animal was kept single with drinking water available ad libitum, whereas food was withdrawn to prevent fecal contamination of the urine. At the beginning of the experiment, the duck was placed in a cotton sling allowing free movement of legs and neck. A catheter (Vasocan Braunula 20G; Braun, Melsungen, Germany) was placed in a leg vein by percutaneous venipuncture for iv infusions. To avoid disturbances, the animal was screened from the adjacent laboratory, where experimental manipulations were carried out. Steady-state renal salt and water elimination was induced by continuous iv infusion of a 200 mosmol/kg D-glucose and isotonic NaCl solution at a 7:3 ratio and total flow rate of 0.7 ml/min (Perfusor secura; Braun). A perforated bulb was inserted into the cloaca (at the level of the ureteral orifice), and urine was continuously aspirated by mild suction into graded cylinders. Urine volume was determined at intervals of 5 min (experimental periods) or 10 min (control periods) at an accuracy of ± 0.1 ml.

The effects of bird-specific CNP (chCNP) on renal excretion were examined after 2–3 h when steady-state conditions of kidney excretion had been established. Synthetic chCNP (Bachem AG, Bubendorf, Switzerland) was added to the systemic infusion at concentrations of 6 and 30 pmol/min·kg BW. Each dose of chCNP was administered for 20 min followed by a recovery period of at least 2 h before further peptide infusions. For comparison, chANP was infused at 30 pmol/min·kg BW. Two to three infusion periods were carried out in randomized sequence per experimental day and animal (series 1). Urine osmolality was measured after centrifugation (10,000 x g, 2 min, 20 C) by vapor pressure osmometry (5100 C; Wescor, Logan, UT). Sodium and potassium concentrations in the urine were determined by flame photometry (IL 943; Instrumentation Laboratories, München, Germany). For analysis of extracellular fluid status before and after the single experiments, small aliquots (500 µl) of blood were obtained via the iv catheter, the hematocrit was determined in microcapillary tubes (Hawksley, Sussex, UK) and plasma osmolality was measured by vapor pressure osmometry after centrifugation (4,000 x g, 2 C, 10 min).

To monitor possible cardiovascular and stressful actions of the highest chCNP dose applied, animals studied in a second series of experiments under identical conditions were implanted under local anesthesia (Xylestin; Espe, Seefeld, Germany) with a heparinized (Vetren; Promonta, Hamburg, Germany) brachial arterial catheter (PP 60; Portex, Hythe, UK) one day before the experiment (series 2). During a first infusion period of the peptide, mean arterial pressure (MAP) and heart rate (HR) were continuously recorded with an Endevco pressure transducer (N 8510; San Juan Capistrano, CA) connected to a blood pressure measuring unit (Servomed SMS 302; Hellige, Freiburg, Germany). HR was determined by a storage oscilloscope (Tektronix 7623; Köln, Germany). MAP and HR were measured at 2-min intervals before, during and after the hormone application period (20 min). During a second, comparable chCNP infusion period, arterial blood samples were collected for subsequent radioimmunological determination of plasma concentrations for [Arg8]vasotocin (AVT) and corticosterone. Blood samples were taken 30 and 10 min before and 5, 20, 30, and 50 min after start of the peptide infusion. To keep the catheter patent throughout the experiment, heparinized isotonic saline (4,000 i.e. Vetren/liter) was constantly infused at low flow rate (0.1 ml/min), in addition compensating for respiratory evaporative water loss (27).

RIAs
For plasma hormone analysis of AVT and corticosterone, arterial blood samples were rapidly collected on ice, the plasma separated by centrifugation (4,000 x g, 2 C, 10 min) and stored at -24 C. AVT was extracted from plasma with chilled (-20 C) acetone and petroleum benzine. The aqueous phase was lyophilized in a Speed-Vac concentrator (Lyovac GT-2; Leybold-Heraeus, Hanau, Germany). The AVT RIA was carried out according to Gray and Simon (28) with the highly AVT-specific polyclonal H5 antiserum, [125I]AVT as radioligand and synthetic AVT as standard for the RIA. Recovery of the peptide amounted to 90% on average, and intra as well as interassay variabilities proved to be lower than 7% each. Corticosterone was extracted with dichloromethane and the lipid phase was lyophilized. [1,2,6,7-3H]corticosterone was used as radioligand (Amersham Life Science, Braunschweig, Germany) and synthetic corticosterone as standard. The polyclonal antiserum was obtained commercially (ICN Biomedicals, Inc., Eschwege, Germany), recovery of the steroid amounted to 75% and assay variabilities of <8% were calculated.

Radioiodination of chCNP
To obtain a radioligand of high specific activity for receptor binding studies, chCNP lacking a tyrosine residue was radioiodinated according to the indirect method of Bolton and Hunter (29) employing [125I]Bolton-Hunter reagent ([125I]BH; Amersham). One millicurie (0.5 nmol) of the reagent was dried under N2 in a lead-shielded safety system and incubated for 7 h at 2 C with 10 µg (4 nmol) chCNP in 10 µl of 0.2 M phosphate buffer, pH 8.5. Radioiodinated [125I]BH-chCNP was separated from free125I, unreacted [125I]BH reagent, unlabeled chCNP, and biologically inactive radioiodinated peptides by reversed-phase HPLC (NovaPak C-18; Waters Associates/Millipore Corp., Eschborn, Germany). Elution was performed with HPLC-grade acetonitrile (Baker B.V., Deventer, The Netherlands), using a gradient of 26–35% containing 0.1% trifluoroacetic acid (Sigma Chemical Co., München, Germany) at a flow rate of 1.2 ml/min. Aliquots of the radioligand, diluted with phosphate buffer containing 0.1% BSA, were stored at -30 C. The specific activity of the radioligand was determined according to Bürgisser (30) via modified Scatchard analysis using duck kidney membranes as 700–800 Ci/mmol, comparable with the value obtained for [125I]BH-chANP (16).

Characterization of chCNP-specific renal binding sites
Kidneys quickly obtained from animals killed by decapitation were minced, and the tissue homogenized with an ultra-turrax (Janke & Kunkel, Staufen, Germany) and a Teflon-fitted Elvehjem glass homogenizer (Wheaton, Chicago, IL) in ice-cold 30 mM Tris-HCl buffer, pH 6.5, containing 25 mM NaCl, 90 mM sucrose, 10 mM MgCl2, 1 mM EGTA, and 0.1 mM phenylmethylsulfonylfluoride (Sigma Chemical Co.) (= RRA-buffer). The supernatant of an initial low speed centrifugation (500 x g, 2 C, 8 min) was subjected to a second centrifugation (45,000 x g, 2 C, 20 min). The fluffy top layer of the resulting pellet was resuspended in cold buffer and centrifuged under identical conditions. The final membrane-enriched top layer of the pellet was dispersed in ice-cold RRA-buffer containing Trasylol (100 i.e./ml; Bayer, Leverkusen, Germany) and 0.125% phenanthroline as enzyme inhibitors. The protein concentrations were determined by Bradford assay (Bio-Rad Laboratories, Inc., München, Germany).

Competitive displacement studies were performed using the enriched membrane fraction of duck kidney (12–20 µg protein) and 60 pM [125I]BH-chCNP as radioligand according to modifications established for [125I]BH-chANP binding (17). Incubations were carried out for 40 min at 4 C in the presence of logarithmically increasing concentrations (1.88 x 10-12 M to 10-6 M) of unlabeled ANP/CNP peptide analogs such as avian-specific CNP (chCNP) and ANP (chANP), rat-specific ANP (rANP) and BNP (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) (rBNP), frog-specific ANP (frANP) and rANP (4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) (cANP) binding with high affinity to the clearance receptor. Except rANP (Peninsula, Heidelberg, Germany), all peptides were purchased from Bachem AG. The incubation was stopped by rapid filtration through Whatman GF/C filters (Waters Associates/Millipore Corp.) presoaked in 0.1% polyethylenimine (Sigma Chemical Co.) using a cell harvester (M-24R; Brandel, Gaithersburg, MD), including four subsequent washes with ice-cold PBS, pH 7.4, containing 10 mM Na2HPO4, 1.8 mM KH2PO4, 136 mM NaCl, 2.7 mM KCl and 0.02% BSA. Filter-bound radioactivity was counted in a {gamma}-spectrometer (LB 951 G; Berthold, Wildbad, Germany), and data were analyzed by a PC-fitted version of LIGAND (GraphPad 2.0 Software, Inc.; ISI Software).

Receptor autoradiography
For receptor autoradiography, freshly dissected kidney slices were immediately frozen in dry ice and stored at -24 C until further use. Sections of 20 µm were cut in a cryostat at -25 C, thaw-mounted on poly-L-lysine coated slides, dehydrated (freeze-dried) at 4 C for 12 h, and stored in sealed boxes at -24 C. The binding assay was performed according to the method established by H. Schütz (16, 17) for [125I]BH-chANP. After a 20-min preincubation at 4 C in RRA-buffer containing 0.2% BSA, sections were incubated with 0.3 nM [125I]BH-chCNP for 40 min in the absence (TO) or presence (NSB) of 10-6 M unlabeled chCNP. After three 2-min washes in BSA-free buffer, the sections were dried under cold air and exposed to AgfaScopix XR3 film (Bender, Frankfurt, Germany) for 14 days.

Immunocytochemistry
To localize cGMP as putative second messenger of chCNP in the duck kidney, three 8-week-old female ducks were deeply anesthetized with 60 mg sodium-pentobarbitone (Nembutal; Ceva, Düsseldorf, Germany). To stimulate intracellular de novo synthesis of cGMP, two animals received an iv infusion of chCNP (0.8 nmol/min·kg BW) dissolved in 0.1 M phosphate buffer, pH 7.4, containing 10-3 M isobutylmethylxanthin (IBMX; Sigma Chemical Co.) as phosphodiesterase inhibitor at a flow rate of 1.5 ml/min for 5 min. The third duck received a systemic infusion of chCNP-free, IBMX-containing buffer (control). Immediate transcardial perfusion with 0.1 M phosphate buffer, pH 7.4, at a flow rate of 100 ml/min under constant pressure of 105 mmHg for 2–3 min was followed by perfusion with 4% freshly prepared paraformaldehyde in perfusion buffer (fixative) for 4–5 min at the same flow rate. The kidneys were dissected immediately after perfusion, cut into smaller pieces, postfixed in the same fixative for 2–4 h at 4 C and infiltrated with 20% sucrose for 24 h at 4 C. Cryostat sections (20 µm) of duck kidney were mounted onto poly-L-lysine coated slides and air-dried.

Indirect immunocytochemical detection of cGMP was performed using a primary antiserum against cGMP raised in sheep (31) at a final dilution of 1:4,000 and a rhodamine-conjugated donkey antisheep secondary antibody (1:200 dilution) (Rockland, Gilbertsville, PA). The air dried tissue sections were rehydrated for antibody permeation in immuno-buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 136 mM NaCl, 2.7 mM KCl and 0.3% Triton X-100, pH 7.4) for 1.5 h at RT. Sections were subsequently preincubated for 1 h with 5% FCS (Boehringer Mannheim, Mannheim, Germany) in immuno-buffer. The incubation with the primary antiserum was carried out for 48 h at 4 C in a humidified chamber, followed by three 10-min washes in immuno-buffer. Sections were then incubated for 90 min at RT with the rhodamine-coupled secondary antibody in immuno-buffer, washed three times, embedded (Cityfluor, London, UK) and examined under a Nikon fluorescence microscope (Nikon, Düsseldorf, Germany).

For interpreting the results obtained by autoradiography and immunohistochemistry, the kidney sections were subjected to a modified Trichrom-Masson-Goldner counter-staining. Except those mentioned above, all other chemicals were purchased from Merck & Co., Inc. (Darmstadt, Germany).

Statistics
The experimental results are presented as means with SEM. All comparisons within one series were performed by one-way ANOVA. For multiple comparisons, the posttest according to Tukey was applied, and differences were considered as significant when the null hypothesis was rejected at P <= 0.05 (*) and P <= 0.01(**), respectively (GraphPad Software, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Renal, cardiovascular, and hormone releasing actions of chCNP
The continuous iv infusion of a 200 mosmol/kg D-glucose and isotonic NaCl solution (at a ratio of 7:3) caused a reduction in plasma osmolality from 295.1 ± 1.8 to 285.1 ± 2.1 mosmol/kg (ANOVA: P <= 0.05). Hematocrit was not significantly reduced from 39.2 ± 1.5 to 38.5 ± 1.4%, indicative of only marginal intravascular volume expansion. Steady-state diuresis and natriuresis were established after 2–3 h, with the kidneys eliminating some 84% of the water and 70% of the sodium administered.

Short-term systemic infusion of chCNP in two concentrations led to a clearly dose-dependent rapid but transient increase in urine flow rate and renal sodium excretion (Fig. 1Go) (series 1). At the higher dose of 30 pmol/min·kg BW, chCNP caused a pronounced elevation in renal water elimination from 0.58 ± 0.05 (control) to 1.02 ± 0.13 and 1.05 ± 0.08 ml/min during the first and second 5-min periods of hormone application, respectively (Fig. 1AGo). This marked diuresis was accompanied by a significant elevation in renal sodium excretion from 25.5 ± 3.6 (control) to 45.7 ± 7.2 and 50.0 ± 5.8 µEq/min 5 and 10 min, respectively after the beginning of hormone administration (Fig. 1CGo). Systemic infusion of chANP under identical experimental conditions and at equimolar concentration (30 pmol/min·kg BW) stimulated urine flow and renal sodium elimination only from 0.59 ± 0.04 ml/min and 22.9 ± 4.0 µEq/min to 0.77 ± 0.05 ml/min and 33.9 ± 5.5 µEq/min, respectively (n = 8), and thus significantly less.



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  		Figure 1. Time course of dose-dependent chCNP action on renal water and sodium excretion. Urine flow rate [ml/min] (A, B) and sodium excretion [µEq/min] (C, D) were determined before, during and after systemic application of chCNP (20 min) at 30 (A, C) and 6 (B, D) pmol/min·kg BW. To induce steady-state diuresis and natriuresis, conscious freshwater-acclimated Pekin ducks received a continuous iv infusion of 200 mosmol/kg D-glucose and isotonic saline solution (7:3 ratio) at 0.7 ml/min. Statistical significance of differences was tested by one-way ANOVA with subsequent posthoc testing according to Tukey (*, P <= 0.05; **, P <= 0.01). Values represent means ± SEM (number of animals = N, number of experiments = n).

 
While both urine flow rate and sodium excretion were initially enhanced during chCNP application with partial decline toward control values already during the second half of the hormone infusion period, sodium concentration of the urine only gradually increased during and even after chCNP infusion (30 pmol/min·kg BW). Maximal values were reached 20 min after termination of chCNP infusion (63.8 ± 5.8 compared with 43.0 ± 4.6 mEq/liter; ANOVA: P <= 0.05). Urine osmolality and sodium concentration were tightly correlated throughout the experiments (r2 = 0.93).

The higher chCNP dose (30 pmol/min·kg BW) did not significantly alter mean arterial pressure nor heart rate, which remained essentially constant throughout the entire observation time of 80 min at basal values of about 130 to 135 mmHg and 90–100 beats/min, respectively (n = 6). Plasma corticosterone levels also remained in the normal range of about 6–10 ng/ml throughout the experiment (n = 6). AVT plasma concentration varied between 12 and 15 pg/ml and, although ANOVA indicated a significant treatment effect, it could not be decided whether this small change was a chCNP effect or secondary to the transient diuresis (series 2).

Characterization of renal chCNP-specific binding sites
To pharmacologically characterize putative membrane-intrinsic binding sites for chCNP in the duck kidney, competitive displacement experiments were performed using an enriched kidney membrane fraction and [125I]BH-chCNP as radioligand. Computer-assisted data analysis indicated functional expression of both a low- and high-affinity binding site (Fig. 2Go). The avian-specific analogs of the radioligand as well as the natriuretic peptide family, chANP and chCNP, revealed binding to the high-affinity site with mean IC50 values of 3.0 and 9.8·10-10 M, respectively, and to the low-affinity site at 100-fold reduced potency (Table 1Go). For frANP, both binding affinities were comparable to those of chCNP. Mammalian ANP (rANP) as the classical representative of the A-type natriuretic peptides, preferentially binding to GC-A receptors, competed for the high-affinity binding site at four times lower affinity compared with chCNP. Rat-specific BNP (rBNP), as representative of the B-type natriuretic peptides with high structural homology to chANP [which should better be classified as B-type-specific (3)] competed with the highest potency to displace [125I]BH-chCNP at the high- and low-affinity site, with average IC50 values of 1.9·10-10 M and 2.6·10-8 M, respectively (Fig. 2Go; Table 1Go). The rANP fragment rANP(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) (=cANP), specifically binding to the clearance receptors in mammalian tissues, showed significant competition with [125I]BH-chCNP preferentially at the high-affinity binding site, and only weak potency to displace the radioligand from its low-affinity site (Table 1Go).



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  		Figure 2. Receptor analysis: competitive displacement studies. Radioiodinated [125I]BH-chCNP (60 pM) bound to an enriched membrane fraction of the duck kidney was competitively displaced by increasing concentrations (2·10-12–10-6 M) of the unlabeled peptide analogs chCNP, chANP, and rANP4–23 (cANP). Points represent the means of triplicate measurements ± SEM. Number of experiments = n.

 

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  		Table 1. Competitive displacement of [125I]BH-chCNP

 
The following sequences demonstrate declining potency of the various ANP analogs to compete with the radioligand for the high-affinity (Hi) and low-affinity (Lo) binding site:

(Hi) rBNP > chANP > cANP > chCNP = frANP > rANP.

(Lo) rBNP > rANP > chANP > chCNP = frANP > cANP.

As delineated from computer-assisted analysis, percentage of the high-affinity binding sites amounted to some 40% on average of the total receptor population.

Localization of chCNP-specific binding sites and signal transduction in the duck kidney
Receptor autoradiography with [125I]BH-chCNP as radioligand was employed to localize chCNP-specific binding sites in the Pekin duck kidney. Marked specific labeling (as proven by complete displacement of the radioligand in the presence of 10-6 M unlabeled chCNP) could be demonstrated for nearly all glomeruli of both the reptilian- and mammalian-type nephrons (Fig. 3Go). Reptilian-type nephrons lacking the loop of Henle and comprising some 80% of all nephrons in the duck kidney (Gerstberger, R., unpublished observation) possess small glomeruli of 35–40 µm diameter, which are arranged in a circular pattern of each kidney lobule around the central vein (vena intralobularis) (Fig. 3Go, a and c, f, g). The larger glomeruli of mammalian-type nephrons (80–105 µm diameter) bearing binding sites for [125I]BH-chCNP (Fig. 3Go, c–e) are located in close vicinity to medullary cone areas (32, 33, 34). Autoradiograms with high resolution and counter-staining of serial kidney sections according to a modified Masson-Goldner method indicated a high density of [125I]BH-chCNP-specific binding sites especially in the subcapsular region and capillaries of the glomeruli.



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  		Figure 3. Receptor analysis: localization of [125I]BH-chCNP-specific renal binding sites. Receptor autoradiography was performed on cryostat sections (20 µm) of the duck kidney employing [125I]BH-chCNP (0.3 nM) as radioligand. (a, b) Specificity of [125I]BH-chCNP binding to reptilian-type glomeruli and distal tubules proven by ligand incubation in the absence (a) or presence (b) of unlabeled chCNP (10-6 M). c–e, Specific labeling of both reptilian- and mammalian-type glomeruli, the distal tubular zone, and arterioles in the medullary cone area. (f, g) ring-like arrangement of [125I]BH-chCNP labeled reptilian-type glomeruli surrounding the central vena intralobularis. A, Arteriole; DT, distal tubules; MG, mammalian-type glomerulus; RG, reptilian-type glomerulus; Vin, vena intralobularis. The bars represent 200 µm.

 
Specific binding of the radioligand at medium density could additionally be observed in the distal tubular zone around the central vein (Fig. 3Go, f and g), and at moderate (optical) density in medullary cone areas containing cortical and medullary collecting ducts and some loops of Henle. Small and medium-size renal arterioles (50–200 µm in diameter; Fig. 3Go, c–e) in close vicinity to mammalian type glomeruli were also labeled with [125I]BH-chCNP, whereas venules showed no binding.

cGMP represents the classical second messenger for natriuretic peptides in mammalian tissues, and its formation could also be demonstrated in the duck kidney during systemic stimulation with chCNP (8 nmol/min·kg BW; 5 min) using indirect immunocytochemistry (Fig. 4Go). In congruency to the localization of [125I]BH-chCNP-specific binding sites, intracellular cGMP formation could be demonstrated for both reptilian- (Fig. 4Go, b–e) and mammalian-type glomeruli (Fig. 4Go, e and f). Intense immunofluorescence was found to be concentrated in the subcapsular and pericapillary region for both classes of glomeruli. Direct comparison to counter-stained kidney sections (Masson-Goldner) showed that almost all glomeruli were immunopositive for cGMP, and a physiological control experiment in the absence of circulating chCNP did not reveal any immunocytochemical staining for the second messenger (not shown).



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  		Figure 4. Immunohistochemical localization of cGMP in the duck kidney. Renal cGMP formation induced by systemic infusion of chCNP (0.8 nmol/min·kg BW; 5 min) under anesthesia and followed immediately by transcardial fixation is demonstrated in 20 µm cryostat kidney sections. a, Immunofluorescence specific for cGMP in individual epithelial cells of the distal tubular zone. b–d, Immunofluorescence specific for cGMP in reptilian-type glomeruli (b–d) arranged in linear and circular patterns as well as in individual cells of the distal tubular zone (d). e and f, Both reptilian- and mammalian-type glomeruli proved to be immunopositive for cGMP. Proximal tubules showed slight autofluorescence. DT, Distal tubules; MG, mammalian-type glomerulus; PT, proximal tubules; RG, reptilian-type glomerulus. The bar represents 100 µm.

 
Apart from slight autofluorescence in proximal tubules and blood cells (Fig. 4cGo), individual epithelial cells of the distal tubules showed significant and specific synthesis of cGMP upon systemic chCNP stimulation (Fig. 4aGo). In contrast, cGMP generation could not be detected for medullary cones containing the collecting ducts as well as for arterioles. Methodological control experiments without the specific primary antiserum or use of a nonspecific secondary antiserum led to complete suppression of the cGMP-specific, immunocytochemical staining.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Employing the Pekin duck as an experimental model to elucidate efferent hormonal control mechanisms involved in body fluid homeostasis (35), this study reports dose-dependent diuretic and natriuretic actions of avian C-type natriuretic peptide (chCNP) via interaction with renal membrane-intrinsic receptor proteins. With a RIA for chCNP not available, equimolar infusions of chANP (30 pmol/min·kg BW) resulted in a rise in chANP plasma concentration from 100 to 230 pg/ml, well within the physiological range (36). The absence of changes in arterial pressure and heart rate in response to chCNP infusion in the duck corresponds to what is known for other osmoregulatory peptides and excludes major effects secondary to circulatory actions in this experimental model. The stable plasma levels of corticosterone excluded stress-induced alterations of renal function and contrasted with the CNP-induced significant elevation of plasma cortisol in the conscious sheep (21). The avian mineralocorticoid aldosterone was not measured in this study, but increases in plasma concentration were unlikely in the condition of NaCl loading (32 µEq/min) (37) and, moreover, would not have interfered with the rather rapid diuretic/natriuretic chCNP effect.

Most studies investigating the renal and vascular effects of C-type natriuretic peptide in mammals (CNP) were performed in anesthetized animals. In rats, bolus applications of CNP (0.4–32 nmol) during anesthesia induced mild natriuresis and diuresis at 100-fold lower potency when compared with ANP and BNP. Anesthetized dogs responded to systemic infusions of CNP (4 and 40 pmol/min·kg BW) with a distinct drop in arterial blood pressure at unchanged renal sodium and water elimination, whereas equimolar ANP caused a 5-fold increase in sodium excretion (7, 19). On the other hand, only a limited number of experiments have been reported for conscious animals. In the awake sheep, iv infusion of CNP (10 pmol/min·kg BW) caused a marked natriuresis comparable to that seen during ANP stimulation (20, 21). After systemic application of either analog, cGMP plasma concentrations proved to be significantly augmented at unaltered levels of cAMP (21). Also in conscious monkeys, systemically administered CNP (10 nmol/kg·BW) in combination with a neutral endopeptidase inhibitor led to an elevation in renal sodium excretion from 400 to 1040 µEq within 2 h, accompanied by increased renal cGMP excretion (22). ANP and BNP, however, induced comparable effects at ten times lower concentrations (38). Recent studies in humans finally indicated a significant dose-dependent increase in both complete and fractional sodium excretion at unchanged urine flow rate after systemic application of CNP (2, 4 or 20 pmol/min·kg BW) (23).

In accordance with those results obtained from conscious sheep, monkeys, and humans, bird-specific chCNP induced a significant, dose-dependent augmentation in renal sodium and water excretion in the conscious Pekin duck at hormone concentrations comparable to the studies quoted. Whereas the CNP-induced diuresis and natriuresis in all the mammalian species tested proved to be maximal after one hour of peptide application, systemic application of chCNP in the duck caused a rapid and transient increase in renal sodium and water elimination with subsequent compensatory mild antidiuresis. The distinct diversity with regard to effect and time course of CNP-intrinsic natriuretic and/or diuretic activity reported in the literature might therefore have been due to species-specific variations, the way of hormone administration (bolus or constant infusion) and especially the state of animal consciousness. Not least, effects secondary to circulatory actions of the peptide cannot be excluded in mammals.

When compared with avian-specific chANP in the present study, chCNP at equimolar concentration proved to be of higher diuretic and natriuretic potency at a comparable time course of action under the experimental condition of hypotonic saline loading. For the Pekin duck, it could be shown that the degree of natriuresis/diuresis elicited by chANP increased with the degree of salt loading (39). Diuresis and natriuresis proved to be even more enhanced in chicken (40) and in saltwater-acclimated ducks with elevated plasma sodium concentration (17) under conditions of isotonic saline loading at 145 and 120 µEq/min, respectively. In dogs, ANP-induced natriuretic and diuretic actions were enhanced under conditions of hypertonic hydration (41), and augmented sodium intake in rats also resulted in a markedly stimulated, ANP-mediated diuresis (38).

The transient diuresis and natriuresis during systemic chCNP application was followed by mild antidiuresis and antinatriuresis at elevated urinary sodium concentration and plasma AVT concentration, indicative of AVT-mediated action (42). The slight but significant stimulation of neurohypophyseal AVT release seen in the present study could not be identified as a direct chCNP effect and was not demonstrated during chANP application in the duck (39). Intravascular adsorption of circulating chANP and possibly also chCNP to iv-injected chANP-specific antibodies even led to augmented AVT concentration in the plasma and an enhanced sensitivity of the central AVT system to osmotic stimulation (43).

The general hypotensive properties of exogenously administered mammalian CNP in experiments with anesthetized dogs and monkeys could not be repeated in conscious sheep and humans (7, 20, 21, 22, 23, 44). Different from the unanesthetized Pekin duck, in which neither chCNP nor chANP affected arterial pressure and heart rate (17), anesthetized chicken responded to systemic chANP with hypotension (45).

Generally in birds, glomerular filtration represents a prime target for circulating osmoregulatory factors (32, 46). Indeed, in the presence of circulating chCNP, renal sodium excretion in the duck proved to be significantly elevated at constant urinary sodium concentration, indicative of a primarily glomerular action of the peptide. Comparable experiments performed in saltwater-acclimated ducks revealed a pronounced enhancement of glomerular filtration (GFR) and effective renal plasma flow during the systemic infusion of chANP (17). ANP-mediated increase in GFR via elevation of glomerular hydraulic pressure caused by dilation of the afferent arterioles was also demonstrated in several studies for mammals (47, 48, 49).

In accordance with the physiological data, receptor autoradiography employing radioiodinated [125I]BH-chCNP and [125I]BH-chANP as radioligands revealed specific labeling of both reptilian-type and mammalian-type glomeruli in the duck kidney at high density and identical patterns for both radioligands. Functionality of these glomerular binding sites was deduced from the up-regulation (increased Bmax) of chANP-specific receptors under conditions of extracellular volume depletion with concomitantly diminished concentrations of circulating natriuretic peptides in the duck (50). Additional binding of [125I]BH-chCNP to arterioles, distal tubules, and also to collecting ducts in the medullary cones suggests that intrarenal hemodynamic and/or tubular adjustments participate in the overall natriuretic and diuretic response.

Competitive displacement studies with either [125I]BH-chCNP (present study) or [125I]BH-chANP (50) as radioligands for the duck renal natriuretic peptide receptor(s) did not demonstrate a sequence of binding affinities characterizing GC-A and GC-B receptors like in mammals. However, functional expression of both a high and a low affinity binding site could be verified in the duck with IC50 values in the upper picomolar to low nanomolar range, and upper nanomolar range, respectively (50). All peptide analogs revealed comparable patterns of radioligand displacement, supporting the notion of two yet unidentified receptor subtypes for natriuretic peptides in the bird without preferential ligand specificity. Receptor autoradiograms of consecutive tissue sections and alternate application of [125I]BH-chCNP and [125I]BH-chANP as radioligands showed identical patterns of labeling, with glomeruli of both reptilian- and mammlian-type nephrons and the distal tubular zone marked preferentially (17), again suggestive of identical binding sites for chANP and chCNP in the Pekin duck kidney.

Coupling of at least one of the two receptor subtypes to intracellular guanylate cyclase activity could be shown for the duck kidney by significant cGMP formation in response to systemic chCNP administration in both types of glomeruli and in individual cells of the distal tubules. Demonstration of a chCNP-specific signal transduction pathway via cGMP clearly emphasizes that the glomerular as well as distal tubular binding sites labeled by [125I]BH-chCNP represent functional membrane-spanning receptor proteins. CNP-induced formation of cGMP in the glomeruli of the mammalian kidney is still debated. While transcripts of the guanylyl cyclase coupled B-type receptor (GC-B) could be detected in the hamster renal cortex, the same authors failed to stimulate glomerular production of cGMP with CNP (51). With rather large amounts of GC-B receptor PCR products also detected in glomeruli of the rat kidney, cGMP accumulation in the glomeruli after CNP stimulation could, on the other hand, be verified by Terada and co-workers (52), whereas Brown and Zuo (53) solely described a reduction in glomerular cAMP formation. In cultured human mesangial cells, both binding of radiolabeled CNP and dose-dependent formation of cGMP could be demonstrated (54).

Specific binding of radiolabeled chCNP was also detected in arterioles and to a weaker extent in the medullary cone of the duck kidney containing loops of Henle of mammalian-type nephrons and collecting ducts. Different from studies performed with cells of the human collecting duct (55), however, cGMP formation could not be observed in corresponding avian cells. High binding affinity of cANP(4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23) to whole kidney membranes and the presence of two receptor subtypes might indicate expression of a clearance receptor (C-receptor) in the collecting duct zone of the duck kidney.

In the present study, renal diuretic and natriuretic actions of CNP comparable to those of ANP and the characteristics of the receptors involved were demonstrated for the first time in an avian species. Histochemically, the largely congruent distribution of binding sites and of chCNP activated cGMP production shows that functional receptors of yet unknown subtype-specificity are primarily located at the level of the glomeruli.


   Acknowledgments
 
The authors very much appreciate the excellent technical assistance of Mrs. Roswitha Bender, Diana Fuchs, and Irene Küchenmeister in animal surgery, ion analysis, RIA determinations, and figure preparations. The cGMP-specific antiserum was a generous gift by Dr. Jan de Vente from the Department of Psychiatry and Neuropsychology, University of Limburg, Maastricht, The Netherlands. The authors thank Prof. Dr. Eckhart Simon and Dr. Richard Paley for critical proofreading of the manuscript.

Received July 23, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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