Expression of natriuretic peptides, nitric oxide synthase, and guanylate cyclase activity in frog...

GENERAL AND COMPARATIVE

ENDOCRINOLOGY

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General and Comparative Endocrinology 137 (2004) 166–176

Expression of natriuretic peptides, nitric oxide synthase,and guanylate cyclase activity in frog mesonephros during

the annual cycle

Carla Fenoglio,a,* Livia Visai,b Concetta Addario,a Giuseppe Gerzeli,a Gloria Milanesi,a

Rita Vaccarone,a and Sergio Barnia

a Dipartimento di Biologia Animale, Universit�a di Pavia, Piazza Botta 10, 27100 Pavia, Italyb Dipartimento di Biochimica—Sezione di Medicina, Universit�a di Pavia, Via Taramelli, 3/B, 27100 Pavia, Italy

Received 28 February 2003; revised 17 February 2004; accepted 10 March 2004

Available online 24 April 2004

Abstract

Natriuretic peptides (NPs), a family of structurally related hormones and nitric oxide (NO), generated by nitric oxide synthase

(NOS), are believed to be involved in the regulation of fluid balance and sodium homeostasis. Differential expression and regulation

of these factors depend on both physiological and pathological conditions. Both NPs and NO act in target organs through the

activation of guanylate cyclase (GC) and the generation of guanosine 30,50-cyclic monophosphate (cGMP), which is considered a

common messenger for the action of these factors. The present study was designed to investigate—by histochemical methods—the

expression of some NPs (proANP and ANP) and isoforms of NOS (neuronal NOS, nNOS, and inducible NOS, iNOS) in the

mesonephros of Rana esculenta in different periods of the year including hibernation, to evaluate possible seasonal changes in their

expression. We also studied the enzyme activity of NOS-related nicotinamide adenine dinucleotide phosphate diaphorase (NAD-

PHd) and of GC. The experiments were performed on pieces of kidney of R. esculenta collected in their natural environment during

active and hibernating life. The study was carried out using immunohistochemical techniques to demonstrate proANP, ANP, and

some NOS isoforms. Antigen capture by enzyme linked immunosorbent assay (ELISA) was also performed to determine the

presence of NPs in the frog kidney extract. Enzyme histochemistry was used to demonstrate the NOS-related NADPHd activity at

light microscopy; GC activity was visualized at the electron microscope, using cerium as capture agent. The application of the

immunohistochemical techniques demonstrated that frog mesonephros tubules express different patterns of distribution and/or

expression of ANP and NOS during the annual cycle. Comparing the results obtained on active and hibernating frogs has provided

interesting data; the NOS/NADPHd and GC activities showed some variations as well. Furthermore, the presence of NPs in the frog

kidney extract was evidenced by dose-dependent response in the ELISA. The data suggest that both ANP and NO are intra-renal

paracrine and/or autocrine factors which may modulate the adaptations of frog renal functions to seasonal changes through the

action of the cGMP generated from GC activity.

� 2004 Elsevier Inc. All rights reserved.

Keywords: Natriuretic peptides; Nitric oxide; Amphibians; Mesonephros; Hibernation

1. Introduction

The regulation of ions and water balance in different

vertebrate organs and principally in the kidney is greatly

influenced by hormones and hormone-like substances.

Among these, molecules like natriuretic peptides (NPs)

* Corresponding author. Fax: +39-0382-506406.

E-mail address: [email protected] (C. Fenoglio).

0016-6480/$ - see front matter � 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.ygcen.2004.03.008

and nitric oxide (NO) are attracting an increasing

interest.

NPs constitute, in different species of each vertebrate

class, a hormonal system that includes three types of

peptides: atrial NP (ANP), brain NP (BNP), and C-type

NP (CNP). Both ANP and BNP are synthesized mainly

in the heart and in the brain, and stored in granule formfor release into the bloodstream, whereas CNP is

expressed principally in the brain (for reviews see

mail to: [email protected]

C. Fenoglio et al. / General and Comparative Endocrinology 137 (2004) 166–176 167

Beltowski and Wojcicka, 2002; Forssmann et al., 1998;Takei, 2000, 2001). Moreover, a peptide closely related

to ANP—urodilatin (URO)—has been localized within

the distal tubules of human kidney and urine (Beltowski

and Wojcicka, 2002; Forssmann et al., 1998; Kone,

2001). URO and ANP derive from the same gene and a

common peptide but, unlike ANP, urodilatin is syn-

thesized, processed, and secreted by the kidney, and

does not circulate in the blood.Nitric oxide (NO) is a highly diffusible gas generated

from LL-arginine by nitric oxide synthase (NOS, EC

1.14.13.39) exerting a variety of functions in different

tissues and organs, including the kidney (for reviews see

Aoki et al., 1995; Moncada and Higgs, 1993). In mam-

mals, multiple isoforms of NOS have been identified,

i.e., neuronal NOS (nNOS), inducible NOS (iNOS) and

endothelial NOS (eNOS)—also termed NOS1, NOS2and NOS3, respectively—which vary considerably in

their intracellular location, structure, and function.

Moreover, variants of these isoforms are now beginning

to be identified (Alderton et al., 2001).

Regarding the kidney, compelling evidence indicates

the presence of nNOS in rat kidney macula densa,

whereas existing data concerning the presence of iNOS

in rat renal tubules are controversial (Bachmann et al.,1995; Morrissey et al., 1994; Terada et al., 1992); eNOS

has been detected mainly in kidney vascular components

(Bachmann et al., 1995; Kone, 2001). All three isoforms

possess NADPH diaphorase activity which, unlike other

NADPH diaphorases, survive fixation. Therefore,

NADPHd activity can be specifically used as a marker

for NOS in different paraformaldehyde-fixed tissues

(Nakos and Gossrau, 1994).A number of reports conducted both in vivo and in

vitro support the assumption that both ANP and NO

play an important role in fluid and electrolyte homeo-

stasis by modulating ion transport pathways and blood

pressure. It is generally believed that they inhibit

transepithelial Naþ and water resorption in tubule cells

mainly through the action of Naþ, Kþ-ATPase (for re-

views see Beltowski and Wojcicka, 2002; Kourie andRive, 1999).

In previous immunohistochemical studies, antibodies

against both mammalian ANP and NOS have been

successfully employed in different tissues of non-mam-

malian vertebrates, including amphibians (Bodegas

et al., 1995; Chapeau et al., 1985; De Falco et al., 2002;

Feuilloley et al., 1993; Netchitailo et al., 1988; Reinecke

et al., 1989). In particular, specific research has dem-onstrated that the structure of frog proANP and ANP

shows a structural homology with the mammalian

peptides (Gilles et al., 1990; Lazure et al., 1988; Sakata

et al., 1988).

The biological effects of NPs and NO on the target

cells are mediated by the second messenger guanosine

30,50-cyclic monophosphate (cGMP) through the action

of guanylate cyclase (GC, EC 4.6.1.2). Numerous stud-ies have documented the presence of GC-coupled na-

triuretic peptide receptors (NPR) in different segments

of the rat renal tubule (Grandclement and Morel, 1998;

Hirsch et al., 2001; Ritter et al., 1995; Terada et al.,

1991). Moreover, studies have shown the presence of

ANP binding sites also in the kidney of some species of

amphibians (Rana temporaria, Kloas and Hanke, 1992a;

Xenopus laevis Kloas and Hanke, 1992b; Ambystoma

mexicanus, Kloas and Hanke, 1993; Bufo marinus, Meier

et al., 1999).

It is a well known fact that, in most amphibians,

environmental conditions induce a series of complex

physiological adaptations. Moreover, during hiberna-

tion many amphibians drastically reduce most of their

metabolic processes, including osmotic and ionic regu-

latory processes. Therefore, amphibians are of particu-lar interest with respect to osmotic regulation and the

endocrine mechanisms they utilize for this process

(Bentley, 1998).

In a previous study performed on the mesonephros of

active and hibernating frogs, we evidenced some struc-

tural and cytochemical modifications of this organ, in-

cluding a variation of Naþ, Kþ-ATPase activity

(Fenoglio et al., 1996).To extend our knowledge on the different mecha-

nisms behind body fluid homeostasis, we studied the

distribution of ANP and NOS in the frog kidney

throughout active life and hibernation. The ELISA was

also used to demonstrate immunoreactive NPs in kidney

extracts. We also tested the expression of the ANP-

propeptide (proANP) to verify the synthetic activity of

the organ. For this purpose, immunoreactions for pro-ANP, ANP, and NOS and enzyme cytochemical studies

for NOS/NADPHd and GC were performed in the

mesonephros of the Rana esculenta during the different

periods of the natural annual cycle.

2. Materials and methods

Adult samples of edible Rana esculenta L. of both

sexes were caught in their natural environment near

Pavia for two consecutive years. Active frogs were col-

lected from rice-field drains in June and September

(environmental temperature ranging from 16 to 25 �C);frogs hibernating underground were collected in Janu-

ary (environmental temperature about 0 �C). Five ani-

mals for each period were used immediately aftercapture. To avoid circadian variations, all samplings

were conducted between 15.00 and 16.00 h. The experi-

ments were performed in compliance with the Italian

law. The animals were stunned and killed by decapita-

tion and the kidneys were removed, excised, and pro-

cessed for the following studies: (a) recognition of ANP

in frog kidney extract by ELISA, (b) proANP and ANP

168 C. Fenoglio et al. / General and Comparative Endocrinology 137 (2004) 166–176

immunohistochemistry, (c) NOS immunohistochemis-try, (d) NOS/NADPH-diaphorase histochemistry, and

(e) guanylate cyclase cytochemistry.

We compared the relative intensity of both immu-

nostaining and enzyme reactions between hibernating

and active samples on the same slide to avoid possible

discrepancies resulting from different processing times.

The relative expression levels of immunodeposits and

the enzyme reaction product of different specimens wereevaluated by two independent observers. Different val-

ues of positivity were scored as weak (+), moderate

(++), and intense (+++), respectively. Sections lacking

reactivity were graded as negative ()).

2.1. Frog kidney extract

Fresh frog renal tissue samples were homogenizedunder liquid nitrogen using mortar and pestle and

suspended in 0.1M hydrochloric acid and 0.6% Triton

X-100. After sonication (6� 20 s) the material was cen-

trifuged at 60,000g for 20min and the supernatant was

filtered and kept at )20 �C. The protein contents of the

extracts were determined by the bicinchoninic acid assay

(BCA) following the manufacturer’s specifications

(Pierce).

2.2. Frog NPs ELISA

ELISAs were performed as previously described (Visai

et al., 2000). Briefly, microtitre wells were coated with

100 ll of 2 lg/ml of commercial available human ANP

(HANP), human urodilatin (HURO) or frog ANP

(FANP) (Sigma) in coating buffer (50mM sodiumcarbonate, pH 9.5) overnight at 4 �C. To control for non-

specific binding, some of the wells were coated with

bovine serum albumin (BSA), laminin, fibronectin, and

fibrinogen as negative controls. After three washes with

PBS (pH 7.4) containing 0.1% (v/v) Tween 20 (PBST), the

wells were blocked by incubating them with 200 ll PBScontaining 2% (w/v) BSA for 1 h at 22 �C. Diluted (1:600)

rabbit anti-HANP (Biogenesis) in PBST containing 1%BSA was added to each well and incubated for 2 h at

37 �C. The wells were subsequently washed with PBST

and incubated for 1 h at 22 �C with 100 ll horseradishperoxidase (HRP)-conjugated goat anti-rabbit IgG

(1:500 dilution in 1% BSA). The wells were finally

incubated with 100 ll of development solution (phos-

phate–citrate buffer containing o-phenylenediamine

dihydrochloride substrate, as prepared according tomanufacturer’s specifications). The reaction was stopped

with 100 ll of 0.5M H2SO4 and absorbance values

(490 nm) were measured using a microplate reader

(Bio-Rad).

Similarly, microtitre wells were also coated with

100 ll of increasing concentrations of the frog kidney

extract prepared as previously described and diluted in

coating buffer overnight at 4 �C. After washing, blockedwells were incubated with rabbit anti-HANP or rabbit

pre-immune antibodies (dilution 1:600 in 1% BSA for

both antibodies) for 2 h at 37 �C. The pre-immune an-

tibodies used did not recognize wells coated with

commercial HANP or FANP, indicating the absence of

anti-ANP antibodies. The wells were washed and then

incubated with for 1 h at 22 �C with HRP-conjugated

goat anti-rabbit IgG (1:500 dilution in 1% BSA). Thecolor development reaction was performed and mea-

sured as previously described.

2.3. ANP immunohistochemistry

Pieces of kidney were fixed in 2% (w/v) paraformal-

dehyde in 0.1M phosphate buffer, embedded in parap-

last-wax and sectioned at 6 lm. Sections were dewaxedin xylene, rehydrated, and treated with 3% H2O2 in 10%

methanol for 15min to block endogenous peroxidase

activity. They were then briefly (15min) incubated with

0.5% Triton X, rinsed and then treated with 10% bovine

serum albumin (BSA) in PBS for 45min to prevent non-

specific staining. For immunohistochemical staining the

following primary antisera were applied: anti-human

proANP 26–92, anti-human or anti-rat aANP, pur-chased from Peninsula. All antisera were raised in rab-

bits and diluted 1:200. The anti-HANP antibody from

Biogenesis was also tested, diluted 1:100. The sections

were incubated overnight in a moist chamber at 4 �C.After rinsing, the sections were treated with the sec-

ondary peroxidase-conjugated swine anti-rabbit anti-

body (Dako), diluted 1:100 for 45min, and afterwards

incubated for 5min for peroxidase reaction, in a me-dium containing 0.05% 3,30-diaminobenzidine tetrahy-

drochloride (DAB) and 0.01% H2O2 in 0.05M Tris–HCl

buffer. Slides were dehydrated and mounted.

The specificity of the immunoreaction was controlled

by replacing the primary antisera with PBS or normal

serum; no staining was observed under these conditions.

2.4. NOS immunohistochemistry

Paraformaldehyde-fixed, paraplast-embedded sec-

tions (6 lm thick) were dewaxed and rehydrated in

graded ethanol. After washing in PBS, endogenous

peroxidase activity was blocked by incubation in 3%

H2O2 in 10% methanol for 15min. Sections were incu-

bated in 0.5% Triton X and then treated with 10%

normal swine serum in PBS (45min) to block non-spe-cific staining. For immunohistochemical staining the

sections were incubated overnight at 4 �C in a humid

chamber with the respective rabbit anti-rat nNOS, 1:500

dilution (Chemicon) or rabbit anti-mouse iNOS, 1:100

dilution (Serotec). After washing in PBS the sections

were incubated for 45min at room temperature with

peroxidase-conjugated swine anti-rabbit antibody

Fig. 1. Binding of anti-HANP antibody to HANP, FANP, and HURO

in the ELISA. Human and frog atrial natriuretic peptides, human

urodilatin, and BSA were immobilized (2lg/well) to microtitre wells,

incubated with rabbit anti-human ANP antibody (1:600), and the

bound antibody was detected with HRP-conjugated goat anti-rabbit

IgG. Data represent means�SD of triplicate measurements.


(Dako), diluted 1:100. After rinsing off the secondaryantibody with PBS the peroxidase reaction was per-

formed with 0.05% DAB in 0.05M Tris–HCl buffer, pH

7.6, containing 0.01% H2O2, for 5min. Finally, the

slides were dehydrated and mounted for examination.

For the negative controls, primary antibodies were

replaced by normal rabbit serum or PBS; they did not

show any immunoreactivity.

2.5. NOS/NADPH-diaphorase histochemistry

Kidneys were frozen in liquid nitrogen in closed vials.

Cryostat sections, 8 lm thick, were cut on a cryostat at a

cabinet temperature of )21 �C. Sections from both ac-

tive and hibernating samples were picked up on poly-LL-

lysine coated slides; several slides were stored in the

cryostat cabinet until used.To demonstrate the catalytic activity of NOS specif-

ically, cryostat sections were prefixed in 4% parafor-

maldehyde, buffered with 0.1M phosphate (pH 7.4) for

15min at 4 �C, washed in phosphate buffer for 10min,

and then incubated in a medium according to Nakos

and Gossrau (1994), with our modifications (Fenoglio

et al., 1997). The standard medium consisted of poly-

vinyl alcohol (PVA) 15% in 100mM phosphate buffer,pH 7.4, 5mM levamisole, 2mM bNADPH (Sigma),

3mM NBT (Sigma), 0.2% Triton X, and 0.2% para-

formaldehyde. Incubation was performed for 50min at

37 �C in the dark.

Controls were carried out with incubation media: (a)

free of bNADPDH and (b) with NADH instead of

NADPH. In both cases no reaction product was

observed in the tissue sections.

2.6. Guanylate cyclase cytochemistry

Small sections from kidneys were prefixed in 2%

paraformaldehyde and 0.25% glutaraldehyde in 0.05M

cacodylate buffer (pH 7.4) for 1 h at 4 �C. After fixation,

they were washed overnight in cacodylate buffer at 4 �C.GC activity was localized ultracytochemically by us-

ing cerium as capture agent. The incubation medium

consisted of 0.05M Tris–maleate buffer, pH 7.4, with

5% sucrose, 1mM guanylyl-imidodiphosphate sodium

salt (Gpp(NH)p, Sigma) 10mM theophylline (as inhib-

itor of phosphodiesterase), 10mM levamisole (as in-

hibitor of alkaline phosphatase), 6mM MnCl2, and

2mM CeCl3. The incubation lasted for 45min at 37 �C;the original medium was replaced with a freshly pre-pared medium after 20min. Some specimens were in-

cubated in a standard medium plus URO 1 lM (Sigma)

as enzyme activator. Control samples were incubated in

a substrate-free medium.

After incubation, the samples were washed several

times in Tris–maleate buffer and in cacodylate buffer,

refixed for 60min at 4 �C in 1.5% glutaraldehyde, and

washed again before postfixation with OsO4 for 2 h at4 �C. Samples were dehydrated in a graded ethanol se-

ries and embedded in Epon 812. Some ultrathin sections

were left unstained while others were stained with uranyl

acetate and lead citrate, and observed in a Zeiss EM 900.

Controls incubated in the absence of substrate did

not show any specific deposits of the enzyme reaction

product.

3. Results

3.1. Morphological aspect

Sections of frog mesonephros stained with conven-

tional H&E showed that the frog nephron consists of the

following parts: glomerulus, proximal tubule, neck andintermediate segment, distal tubule, collecting tubule,

and duct. No macula densa was observed in any of the

samples.

The distal tubules occupy the ventromedial region of

the mesonephros while the rest are located in the dor-

solateral region. The glomeruli are present between the

two regions.

3.2. Recognition of frog kidney NPs through ELISA

The ELISA—a sensitive biochemical technique—was

performed to determine the presence of natriuretic

peptides in the frog kidney extract before proceeding to

Fig. 2. Percentage of binding of anti-ANP antibody to frog kidney

extract by ELISA. Increasing concentrations (from 50 ng to 5lg) offrog kidney extract prepared as described in ‘‘Section 2’’ were immo-

bilized to microtitre wells, incubated with rabbit anti-HANP (1:600) or

rabbit pre-immune antibody (1:600), and the bound antibody was

detected with HRP-conjugated goat anti-rabbit IgG. Data represent

means� SD of triplicate measurements.


other immunohistochemical analyses. With this same

technique, the immunoreactivity of the rabbit anti-

HANP was then tested on HANP, FANP, and HURO.

Fig. 1 shows that the antibody preparation used reactsstrongly not only with immobilized HANP as expected

but also with FANP and HURO. As can be seen, rabbit

anti-HANP (the negative control) did not bind to BSA.

The ELISA test further indicated that the anti-HANP

antibody did not bind to other immobilized proteins

such as ovalbumin, laminin, fibronectin, and fibrinogen

Fig. 3. Immunohistochemical stainings for ANP in active (A) and hibernati

most distal and proximal tubules. (B) Dense immunodeposits of ANP can be

labelled. gm, glomeruli; dt, distal tubules; pt, proximal tubules, 130�.

(data not shown), a fact which underscores the speci-ficity of the antibody. Fig. 2 shows the percentage of

binding of the rabbit anti-HANP antibody to frog kid-

ney extract. The anti-HANP antibody reactivity to in-

creasing concentrations of immobilized frog kidney

extract indicates the presence of NPs in the frog kidney

extract. The rabbit pre-immune antibody showed no

binding whatsoever to the frog kidney extract.

3.3. ANP immunohistochemistry

The immunohistochemical experiment with the anti-

sera against NPs resulted in a generally positive labelling

in several tubule segments. Essentially, similar staining

patterns were obtained with different antisera. No sig-

nificant differences in the distribution of immunoreac-

tive deposits were noted in any samples using eitherhuman or rat ANP antiserum.

Interestingly, some differences in immunostaining

were observed when comparing active and hibernating

frogs. In active frogs, moderate to intense ANP-like

immunostaining was detected in some tracts of proxi-

mal, distal, and collecting tubules (Fig. 3A). Moreover,

within the tubule segments some cells showed different

degrees of positivity. In hibernating specimens ANP-likeimmunoreactivity was mainly observed as dense depos-

its in some distal tubules, whereas other segments of

renal tubule—mainly proximal ones—showed only weak

immunostaining (Fig. 3B). Some cells slightly positive to

ANP-like immunostaining were observed within the

glomeruli of both active and hibernating frogs (Figs. 3A

and B).

The application of the proANP resulted in a weak tomoderate immunoreactive labelling of some proximal

ng frog mesonephros (B). (A) Anti-ANP immunolabeling is intense in

observed mainly in distal tubules (arrows); proximal tubules are faintly


and distal tubule segments both during the active andhibernating periods, being more intense in active frogs

(data not shown).

3.4. NOS immunohistochemistry and histochemistry

In active frogs, iNOS immunoreactivity was weak to

moderate and it was mainly localized in cells consti-

tuting the proximal segments of the mesonephros tu-bules (Fig. 4A). In hibernating samples, moderate

immunohistochemical staining for iNOS was mainly

found in most proximal tubules. A dense rim of im-

munodeposits labelled the apical portion of these tu-

bules (Fig. 4B).

Fig. 4. Immunolocalization of iNOS (A and B). (A). Light to moderate imm

sonephros. (B). Moderate to intense immunostaining (arrows) can be obs

NADPHd activity. (C) Most proximal tubules of the active frog mesonephro

proximal tubule cells during hibernation. pt, proximal tubules, 200�.

In all samples considered, no immunostaining couldbe detected by using an antiserum against nNOS (data

not shown).

Following NOS/NADPHd histochemistry, the epi-

thelial cells lining the proximal tracts of the renal tubules

showed a different intensity of reaction, depending on

the seasonal periods we considered. In particular, during

the active life, the cells constituting proximal tubules

were particularly positive to the NOS/NADPHd reac-tion (Fig. 4C), whereas distal and collecting tubules

showed little, if any, NOS/NADPHd staining. Hiber-

nating specimens displayed a decrease in reaction in-

tensity at the same sites (Fig. 4D). Glomeruli were not

stained in any of the samples.

unopositivity is noted in proximal tubules (arrow) in active frog me-

erved in proximal tubules of the hibernating frog. (C and D) NOS/

s show moderate to intense reactivity. (D). Low staining is observed in


3.5. Guanylate cyclase cytochemistry

In all samples, most epithelial cells constituting the

proximal tubules showed granules of the reaction

product on the apical plasma membranes; fine reaction

product granules were detected also on the lateral

membranes of these cells (Fig. 5A). Fine precipitates

were also evidenced on the cell membrane of the col-

lecting tubule cells (Fig. 5B). Granules of the reactionproduct were also detected within the cytoplasm of the

distal tubule cells (Fig. 5C). The localization of the re-

action product was the same in both active and hiber-

nating frogs, although the granules of the reaction

product were less numerous in the latter (Fig. 5D) than

in the former (Fig. 5A). In the glomeruli, mainly of the

active frogs, the podocytes and their foot processes ex-

hibited some reactivity. The reaction product was de-tected within the cytoplasm or lining some podocyte

processes (Figs. 6A and B).

The addition of URO in the basal medium caused an

increase of the granules mainly on the apical side of the

plasma membrane of the cells constituting the proximal

tubules (data not shown).

Fig. 5. Ultracytochemical demonstration of GC activity in active frogs (A–C

brane and brush border (bb) of proximal tubule cells (arrows), 9000�. (B). R

cells of the collecting tubule (arrow), 12,000�. (C). Cytoplasmic localization

8500�. (D). Hibernating frog. Granules of the reaction product are localize

detected on the brush border (bb) in comparison with active sample (A), 12

All the results are summarized in Tables 1 and 2.

4. Discussion

The present data show that the mesonephros of R.

esculenta is a site of synthesis and/or storage of ANP.

From the comparative point of view, and taking into

account the existing differences between amphibianmesonephros and mammalian metanephros, the distri-

bution of the immunodeposits in the frog kidney showed

some interesting similarities with mammalian species.

Immunoreactive sites were visualized by using anti-

sera against mammalian molecules (proANP, ANP). In

fact, literature data indicate that antigenic determinants

of frog ANP are related structurally to rat or human

ANP. Moreover, as concerns ANP the results we ob-tained with the ELISA performed on the frog extract

indicates the presence of NPs in the frog kidney. With

the ELISA technique we cannot distinguish between

proANP, mature ANP or URO but we may have evi-

dences of their effective presence in frog kidney extract.

It is of note that, in a study recently conducted on the

). (A) Abundant reaction product is detected on apical plasma mem-

eaction product granules can be observed on the plasma membranes of

of reaction product granules can be observed within distal tubule cells,

d on the luminal side of the proximal tubule cells; fewer granules are

,000�.

Fig. 6. Ultrastructural localization of GC activity in active frogs (A and B). Details of two glomeruli showing fine precipitates of the reaction product

in the cytoplasm of a podocyte (po) and also within and around some podocyte processes (arrows). (A) 14,000� and (B) 18,000�.

Table 1

Evaluation of immunohistochemical expression of natriuretic peptides

and NOS in active and hibernating frogs

Samples proANP ANP nNOS iNOS

Active frogs ++ +++ ) +/++

Hibernating

Frogs

+ ++ ) ++

+++, intense staining; ++, moderate staining; +, weak staining;

and ), no staining.

Table 2

Evaluation of enzyme activities in active and hibernating frogs

Samples NOS/NADPHd GC

Active frogs ++/+++ +++

Hibernating frogs +/++ ++

+++, intense staining; ++, moderate staining; and +, weak stain-

ing.


kidney of two species of anurans—R. esculenta and Rana

italica—De Falco et al. (2002) have demonstrated the

presence of ANP by using a mammalian antibody.

However, the authors found that the immunoreaction

was mainly present in the kidney of the R. esculenta, a

species living in an aquatic habitat. The authors suggest

that different patterns of ANP positivity could be related

to the different physiologies of the two frog species.In the two conditions we considered—activity and

hibernation—some interesting differences in the ANP-

like immunostaining distribution along the renal tubule

were evidenced. In both conditions the distal tubules

were the most reactive sites, being sometimes more

intense in hibernating animals.

These dense ANP immunodeposits in the hibernating

samples could represent a storage form of natriuretic

peptide, probably released in response to appropriate

stimuli when frogs awake. Consistent with this hypoth-

esis, Lenz et al. (1999) have demonstrated that HEK 293

cells (which display several characteristics of distal tu-

bule cells)—unless stimulated—are able to store a certain

amount of natriuretic peptide in the cytoplasm.As concerns NOS, in this study, no immunoreaction

resulted after incubation of kidney samples with nNOS

antibody probably due to the absence of a structure

similar to macula densa. It is of interest that in a review

of Sokabe and Ogawa (1974) have questioned the

presence of such a structure in different amphibian

species, suggesting that a macula densa is absent in

amphibian kidney.Apart from macula densa, the presence of NOS in

other nephron segments is still a controversial subject.

However, some authors have identified iNOS expression

in some segments of the rat renal tubule, including the

proximal (Liang and Knox, 2000; Mohaupt et al., 1994;

Morrissey et al., 1994; Shin et al., 1999; Yu, 1997). It has

also been demonstrated that iNOS mRNA is constitu-

tively expressed in rat proximal-tubule cells (Liang andKnox, 2000) and that the proximal convoluted tubule is

the primary site in mammal kidney to synthesize argi-

nine from citrulline (Levillain et al., 1993; Morel et al.,

1996). Moreover, the proximal tubule is able to produce

large quantities of NO upon a variety of stimuli (Liang

and Knox, 2000; McLay et al., 1994).

In frog mesonephros, the immunoreaction for iNOS

and the histochemical reaction for NOS/NADPHd


activity were both expressed mainly in the proximaltubules. Although the localization for the two reactions

was similar in both active and inactive frogs, their in-

tensity varied during the annual cycle and demonstrated

opposite results. In particular, active frog specimens

showed an intense NOS/NADPHd activity as compared

to the hibernating animals. The seasonal differences in

the NOS/NADPHd-staining intensity observed in the

proximal tubules could reflect different iNOS activation.In fact, some studies have shown that in mammals the

proximal tubule is able to produce large quantities of

NO after appropriate stimuli (i.e., LPS, cytokines, and

hypoxia) even after a few minutes (McLay et al., 1994;

Yaqoob et al., 1996; Yu et al., 1994). Consistent with

these findings, upregulation of iNOS could occur inde-

pendently of a de novo synthesis of the enzyme. In this

regard, some tyrosine kinases and phosphatases andANP are believed to modulate the functional activity of

this enzyme (Kiemer and Vollmar, 1998; Pan et al.,

1996).

There is an assumption based on experimental

findings that ANP and NO can interact in the control

of renal functions (Chatterjee et al., 1999; Eitle et al.,

1998; Kone, 2001; McLay et al., 1995). These studies,

using different methods and experimental models,consider cGMP the common messenger for both the

diuretic and natriuretic actions of ANP and NO on the

renal tubule. In particular, the modulation of tubular

sodium transport by ANP is mediated through acti-

vation of GC and generation of cGMP after they bind

to specific natriuretic peptide receptors, notably NPR-

A for both ANP and urodilatin. Therefore, the

presence of receptors for natriuretic peptides may beindirectly demonstrated through the cytochemical lo-

calization of ANP-stimulated GC activity.

As concerns NPRs Sekiguchi et al. (2001) have per-

formed a comparative study by cloning the NPR-A re-

ceptor subtype from the brain of the bullfrog Rana

catesbeiana. Interestingly, they found that the bullfrog

receptor is related to its mammalian counterpart,

showing a very high similarity (�92%) in the catalyticdomain where GC region resides. They also demon-

strated a dose-dependent stimulatory effect by ANP on

specific receptors in eliciting the cGMP production.

In mammal renal tubules ANP receptors or ANPR-

mRNA have been found in different segments of rat

nephron (Grandclement and Morel, 1998; Ritter et al.,

1995; Terada et al., 1991). Although the proximal tubule

is not considered the main target of ANP, there is evi-dence that ANP receptors also in the cells of this seg-

ment mainly occur in response to an increase in the

expression of ANP (Mistry et al., 2001; Terada et al.,

1991). In amphibians, some studies have found ANP-

binding sites on the glomeruli and renal tubules by au-

toradiography (Kloas and Hanke, 1993; Meier et al.,

1999).

In the present paper we evidenced the presence of GCultrastructurally, in particular in proximal and distal

tubules, its activity being more relevant in samples of

active frogs. Some reactivity was observed within some

podocytes and their processes of the glomeruli, mainly

in the active frogs. Discrepancies concerning the detec-

tion of ANP binding sites in the kidney of amphibians

between previous researches and the present study may

be due to differences in methodological approaches and/or amphibian species studied.

However, the seasonal differences we observed in the

GC activity can be reasonably associated to variations

in diuresis and natriuresis. In fact, NPs are believed to

act via cGMP to control different mechanisms involved

in ion and water balance, including the modulation of

the Naþ, Kþ ATPase activity (Beltowski and Wojcicka,

2002; Kourie and Rive, 1999; McLay et al., 1995;Scavone et al., 1995).

Naþ/Kþ ATPase plays a major role for sodium re-

absorption in different tubule segments. Evidence exists

that some hormones, including ANP, regulate the cat-

alytic activity of the renal Naþ/Kþ ATPase through

phosphorylation and dephosphorylation by second

messengers and protein kinases (Bertorello et al., 1991;

Ewart and Klip, 1995; F�eraille et al., 1999; Ominatoet al., 1996).

Our previous research provided some information

regarding the changes in the activity of the Naþ/Kþ

ATPase-pump in frog kidney during the annual cycle

(Fenoglio et al., 1996), whereas immunostaining for the

subunit a of the Naþ/Kþ ATPase remained practically

unchanged (Fenoglio et al., 2000). Therefore, we believe

that, in our experimental model, changes in enzymeactivity could be related to different ANP and also NO

production which, acting as paracrine and/or autocrine

factors, may modulate frog nephron functions directly.

In addition to the mesonephros, the skin and the

urinary bladder of R. esculenta both seem to be involved

in osmoregulatory processes, as we had previously de-

duced from cytochemical studies of the Naþ/Kþ ATPase

activity (De Piceis Polver et al., 1988, 1999). Some in-teresting studies have also investigated the possible in-

fluence of ANP on both skin and urinary bladder of

some amphibians (Coviello et al., 1989; Meier and

Donald, 2002; Uchiyama et al., 1998; Vagnetti et al.,

2001).

Lastly, we observed some seasonal variations in ANP

production in the heart of R. esculenta (Addario et al.,

2001). In this respect, we would like to mention amorphometrical study conducted by Aoki et al. (1989)

on the heart of Bufo arenarum showing seasonal changes

in this species regarding the production of ANP granules

by myocardiocytes.

In conclusion, it is possible to assume that integrated

stimuli of ANP and NO acting locally within the me-

sonephros or on different anatomical structures in


combination with other hormonal factors, play a sub-stantial role in controlling osmoregulation in frog.

Acknowledgments

The authors wish to express their thanks to Prof.

M.A. Masini for her suggestions. This research was

supported by a FAR grant from the Italian Ministry of

the University and Scientific Research.

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