ANF IN RENOVASCULAR HYPERTENSION
ATRIAL NATRIURETIC FACTOR IN TWO KIDNEY - TWO CLIP RENOVASCULAR
HYPERTENSION IN THE RAT
ANA M. PUYO1, GUSTAVO W.
VEGA2, AMANDA PELLEGRINO de IRALDI3, LILIANA E. ALBORNOZ2, MARIA I.
ROSON2, PAULA SCAGLIA2, MARIA M. CELENTANO2, JUAN P. CORAZZA3, ERNESTO
L. PALUMBO2, BELISARIO E. FERNANDEZ1, IGNACIO J. de la RIVA2
1Departamento de Ciencias
Biológicas, Facultad de Farmacia y Bioquímica, UBA; 2Departamento de
Ciencias Fisiológicas, 3Instituto de Biología Celular y
Neurociencias Profesor Eduardo de Robertis, Facultad de Medicina,
Universidad de Buenos Aires
Key words: atrial natriuretic factor, hypertension,
renovascular hypertension, vascular reactivity
levels of circulating atrial natriuretic factor (ANF) have been
reported in several physiopathologic conditions like hypertension,
heart and renal failure, pregnancy and high sodium intake.
Nevertheless, neither relationships with water-sodium space regulation
nor the role of an ANF vascular relaxant effect have been yet defined.
The aim of present experiments was to characterize the contribution of
circulating ANF and its vascular relaxing effects in the two
kidney-two clip (2K2C) experimental model of renovascular
hypertension. Complementary, plasma metabolites nitrite/nitrate of
nitric oxide (NO) was examined because of mediation for both (NO an
ANF) through cGMP. The results showed (two-four weeks after surgery):
indirect sistolic blood pressure (mmHg), 186 ± 4 in HT and 122 ± 1
in SH (p < 0.001); a significant increase of plasma ANF (fmol/ml)
in HT (n = 7, 1221 ± 253) vs. SH (n = 9, 476 ± 82; p < 0.02).
Nitrate/nitrite plasma concentrations (µmol/l) were mpt different
between SH and. The relaxant effect of ANF (10-9, 10-8 and 10-7 M) on
phenylephrine (3,5 x 10-6 M) contracted rings from HT rats was smaller
than SH rats (10-8 M, p < 0.05). Contractions to phorbol 12,
13-dibutyrate (seven weeks after surgery) were significantly higher in
rings from HT rats (p < 0.001). We conclude: 1) in addition to
decreased granularity in atrial myocardiocytes, high circulating
values of ANF here described suggest an increased turnover of the
peptide in 2K2C hypertensive rats; 2) lower significant vascular
relaxant effects in HT rats would indicate down regulation of ANF
receptors in this model; the latter would derive from high plasma ANF
concentration and, tentatively, because of greater activity of protein
kinase C in the vascular wall; 39 similar values of plasma
nitrite/nitrate in SH and HT rats would indicate a comparable NO
circulating availability in both groups.
natriurético atrial en la hipertensión dos riñones-dos clips en la
rata. Niveles circulantes elevados del factor natriurétrico atrial
(ANF) han sido referidos en varias condiciones fisiopatológicas tales
como la hipertensión arterial, la insuficiencia cardíaca y renal, el
embarazo y la ingesta de sodio elevada. Sin embargo, aún no está
claramente establecida su participación en la regulación del espacio
sodio-agua ni su importancia como relajante vascular. El objetivo del
presente trabajo ha sido caracterizar la contribución del ANF
circulante y sus efectos sobre el músculo liso vascular en el modelo
experimental de hipertensión renovascular dos riñones dos clip
(2R2C). Complementariamente, se examinó la concentración plasmática
de metabolitos del óxido nítrico (NO, nitrito/nitrato), dado que
para ambas sustancias (NO y ANF) los efectos son mediados por GMPc.
Los resultados mostraron (dos-cuatro semanas después de la cirugía):
presión arterial sistólica (indirecta, mmHg), 186 ± 4 en HT y 122
± 1 en SH (p < 0.001); significando aumento del ANF plasmático
(fmol/ml) en las ratas HT n = 7), 1221 ± 253 con respecto a las SH (n
= 9), 476 ± 82 (p < 0.02); las concentraciones de nitrito/nitrato
en plasma (µm/l) no fueron diferentes entre HT y SH. El efecto
relajante del ANF (10-9, 10-8 y 10-7 M) fue menor en los anillos de
aorta de ratas HT (p < 0.001). En conclusión: 1) las altas
concentraciones de ANF circulantes, acompañadas por una
degranulación de los miocitos atriales, sugieren un recambio
aumentado del mismo en HT 2R2C; 2) el menor efecto relajante en
anillos de aorta de ratas HT pre-contraídos con Phe, indicaría una
desensibilización de los receptores para ANF en este modelo,
atribuíble a las altas concentraciones de ANF circulante y,
tentativamente, a una mayor actividad de proteína kinasa C en la
pared vascular; 3) la similar concentración plasmática de
nitrito/nitrato en SH e HT indicaría una disponibilidad de NO
circulante comparable en ambos grupos
Postal address: Dr. Ignacio J. de la Riva, Departamento de
Ciencias Fisiológicas, Facultad de Medicina, Universidad de Buenos
Aires, Paraguay 2155, 1121 Buenos Aires, Argentina. Fax: 54-1-963-6287
Received: 4-II-1998 Accepted: 18-II-1998
The presence of secretory like granules linked to striated cardiac
muscle cells was observed by electron microscopy in guinea pig atrial
as far as in 19561. Many years later, in 1981, de Bold et al2
demonstrated that intravenous administration of rat atrial homogenates
enriched in such granules resulted in a fast and short but impressive
diuresis, natriuresis and consistent decrease in blood presure. Later
on, the peptidic nature of the natriuretic factor localed in the
granules was established3 and the fact was confirmed by
immunohistochemical studies4. In addition to the atrial natriuretic
factor (ANF) two other natriuretic peptides, B and C types (BNP and
CNP), with a high degree of sequence homology to ANF, each derived
from a separate gene and with some similar biological effects, were
identified during the past deca-de5, 6.
We have previously reported that ANF specific granules as observed by
electron microscopy, decrease in 2K2C rats with subacute (two weeks)
and chronic (six weeks) hypertension7. It was suggested that vanishing
of granularity in hypertensive rats 72 hs after clipping and further
on, would indicate the release of the natriuretic peptide as a
cooperative system which favour sodium-water equilibrium in
renovascular hypertension. At least two opposite conditions could
account for the results: 1) a decrease in synthesis and 2) an
increased delivery of the peptide. In order to distinguish between the
two possibilities, plasma ANF concentration analysis were performed in
present experiments after two-three weeks of clipping both renal
arteries. We speculated that, if plasma concentration were elevated,
the decreased atrial granularity previously described could express
the increased release of the peptide. Moreover, since renal clearance
of ANF by kidneys with artery stenosis has been reported to be
normal8, 9, it would suggest an enhanced turnover of the peptide
during the subacute period of 2K2C renovascular hypertension.
Concerning the hypotensive action of ANF, some authors10 suggested
that it could specially derive from a decrease in cardiac output.
However, a vascular relaxant effect of ANF through cGMP has been
clearly defined on aorta and renal rabbit strips11, 12. In this
regard, the endothelium-derived relaxing factor, which is nitric oxide
(NO) or a nitroso compound, also yields vasodilation by cGMP in both
conductance and resistance vessels13-16. Consequently, in present
experiments ANF and NO metabolites (nitrite/nitrate) circulating
levels and vascular “in vitro” relaxation to ANF were additionally
determined to characterize their relationships in the experimental
2K2C type of renovascular hypertension; as far as we know, this
information has not been reported before. This model allows to
particularly analize primary Goldblatt ischemic mechanisms since it
excludes the involvement of an untouched contralateral kidney (as in
2K1C rats) or the simultaneous reduction in kidney mass (as in 1K1C
rats), which is accompanied by a significant increase in sodium
Material and Methods
Male Wistar rats (250-270 g) were used. Rats were maintained on
commercial standard food (Asociación Cooperativa Argentina) and tap
water “ad libitum”. Room temperature was maintained at 22 ± 1°C
and the air was adequately recycled. Hypertension was elicited by
applying a solid silver clip (0.29 mm lumen) to each renal artery18
under ether anesthesia (HT rats); in control Sham rats (SH) all
surgical procedures were performed except to apply the clips. The day
before sacrificing the animals, indirect systolic blood pressure (BP)
was determined by means of a photoelectric tail-cuff connected to an
amplififer (II TC model 47, Woodland Hills, California, USA) in series
with an oscilloscope (type 532, TEKTRONIC inc., Portland, Oregon,
Two-three weeks after surgery, the animals were anesthetized by
intraperitoneal injection of 3.5% Chloral Hydrate (0.8 ml/100 g).
Blood samples for ANF analysis were obtained from the jugular vein and
immediately placed in ice-chilled plastic tubes with EDTA and then
centrifuged at 2,000g at 4°C for 30 min. Plasma samples were kept at
-70°C until ANF assay19. Briefly, samples were acidified by adding
100 µl/ml of 1 M HCI and passed three times through Sep-Pak C-18
cartridges previously activated with 5 ml of acetonitrile containing
0.1% trifluoroacetic acid (TFA) followed by 5 ml of 0.1% TFA. The
cartridges with the adsorbed peptide were washed with 20 ml of 0.1%
TFA and then eluted with 3 ml of 80% acetonitrile containing 0.1% TFA.
Samples were dried and then stored at -20°C until assayed.
Lyophilized dried samples were reconstituted in 1 ml phosphate buffer
(pH 7.4) containing 0.1% bovine serum albumin, 0.01% sodium azide,
0.05 M NaCl, and 0.1% Triton and supernatants were assayed for ANF by
radioimmunoassay. Anti-rat ANF (99-126) antibody was purchased from
Peninsula Lab. Inc. (Belmont, CA) and labeled human ANF (99-126) from
New England Nuclear (Boston, MA). ANF concentration was expressed as
fmol/ml of plasma.
Nitrite-nitrate in plasma
Nitrite/nitrate plasma levels were measured in SH and HT rats after
four weeks of surgery by the fluorometric assay described by Misko et
al.20 Briefly, plasma samples were filtered through 5.000 cutoff
microcentrifuge filters (Sigma Chemical Co St. Louis, MO) for 45 min
at 7.500g. Nitrate was converted to nitrite by the action of 20 mU
Nitrate Reductase from Aspergillus species (Boehringer Mannheim
Biochemical, Mannheim, Germany) in presence of 40 µM NADPH and 20 mM
TRIS pH 7.6. The reaction was stopped after 5 minutes at 20°C, by
dilution with equal volume of destilled water followed by the addition
of 2, 3-diaminonaphthalene (DAN, 0.05 mg/ml in 0.62 M HCI) for
determination of nitrite. After a 10 min incubation at 20°C, the
reaction was stopped with NaOH 2.8 N. DAN reacted with nitrite to form
1-(H)-naphthotriazole, a fluorescent product. Fluorescense was
measured in a JASCO FP-770 fluorometer. Nitrite/nitrate plasma
concentration was expressed as µmol/l.
Contractility of aorta rings
To study vascular contractility, the same animals in which blood
samples for ANF were obtained were decapitated and the abdominal aorta
was removed and placed in cold Krebs-bicarbonate solution: (mM) NaCl,
120; KCl, 4.8; KH2PO4, 1.2; MgSO4.7H2O, 1.3; CaCl2, 1.6; NaHCO3, 25;
Dextrose, 10; CaNa2.EDTA, 0.03. The excess of adventitia was excised
and rings of the arteries were cut (3 mm wide) to be suspended in
tissue baths with Krebs solution conveniently gassed by 95% O2 and 5%
CO2. Tension development was registered by isometric force transducers
(GRASS FT03) connected to an amplifier in series with a PC with a
special computer program for registration of vascular smooth muscle
contraction. After one hour of equilibration at 2 g of basal tension
(readjusted every fifteen min), contractions were induced in abdominal
aorta rings of SH and HT by 3.5 x 10-6 M phenylephrine (Phe, SIGMA)
for three min; then, relaxations to three different doses of ANF
(10-9, 10-8 and 10-7 M) were tested on the same rings and results were
expressed as percent relaxation of Phe contraction. In another group
of rats (seven weeks after clipping), 10-5 M phorbol 12, 13-dibutyrate
(PDBu, SIGMA) was used to stimulate protein kinase C in order to
induce contraction as an indirect index of protein kinase C activity.
Results were expressed as mg of tension development.
Results were expressed as means ± SEM. The unpaired Student’s
t-test were used and differences at a level of p < 0.05 were
Values (mean ± SEM) in HT rats were significan- tly higher than in
SH rats (p < 0.001): 186 ± 4 vs 122 ± 1 mmHg.
Striking similarities were observed in kidney mass (expressed in g)
of HT and SH rats in all groups. The fact indicates that fairly
comparable mass of functional kidney tissue was present in SH and
clipped rats. Group of rats in which ANF and vascular contractility
were determined: left kidney: HT (n = 7) 1.36 ± 0.04 vs SH (n = 9)
1.35 ± 0.04; right kidney: HT (n = 7) 1.41 ± 0.05 vs SH (n = 9) 1.40
± 0.04. Group of rats in which nitrite/nitrate were determined, left
kidney: HT (n = 8) 1.31 ± 0.06 vs SH (n = 9) 1.25 ± 0.04; right
kidney: HT (n = 8) 1.33 ± 0.04 vs SH (n = 9) 1.28 ± 0.03.
ANF and nitrite/nitrate in plasma
Plasma ANF value (fmol/ml)) (Fig. 1, upper panel) were higher in HT
(n = 7, 1.221 ± 253) than in SH rats (n = 9, 476 ± 82, p < 0.02).
On the other hand, nitrite/nitrate plasma concentration (µmol/l) did
not differ (Fig. 1, lower panel) between HT and SH rats: HT (n = 8)
25.9 ± 2.0 vs SH (n = 9) 22.4 ± 3.3, NS.
Contractility of aorta rings
A significant lower contractile response to 3.5 x 10-6 M Phe was
observed in aorta rings obtained from HT rats (p < 0.05; Fig. 2
upper panel) as compare with rings from SH rats. On the contrary, PDBu
induced higher contractions (Fig. 2, lower panel) on aorta rings from
HT rats vs rings from SH rats (p < 0.001). Relaxation responses to
ANF on Phe precontracted aorta rings were smaller in rings from HT
rats as compared with SH rats, but results were significant only for
one of the three concentrations used (Fig. 3).
The study of ANF contribution in renovascular hypertension is
relevant in view of its recognized interaction with the
renin-angiotensin system (RAS). The latter is a powerful
vasoconstrictor (specially on renal vessels), increases water and
sodium reabsorption and stimulates aldosterone release. On the
contrary, ANF is a direct endogenous antagonist to the
renin-angiotensin-aldosterone system21, 22 and may induce peripheral
vasodilation. Furthermore, the RAS and the ANF (in addition to BNP and
CNP of the same peptide family) are both present in central neurons.
The actions of ANF and BNP in this location appear also to be opposite
to the RAS since ANF inhibits thirst associated with dehydration and
hemorrhage23 and suppresses vasopressin and ACTH release24.
The information in the literature has clearly related ANF circulating
levels to water-sodium balance25-28. Nevertheless, distinction between
the stimulus originated in total sodium space or particularized to the
interstitial or intravascular fluid compartment has been poorly
defined. With this respect, in the 1K1C experimental model or
renovascular hypertension, in which a characteristic increase in total
exchangeable sodium was reported17, particularly high ANF circulating
values were observed28. Furthermore, elevated plasma ANF were also
found in humans without edema with both unilateral and bilateral renal
artery stenosis or with essential hypertension29. On the contrary, in
patients with cirrhosis and edema (in the advanced period, when blood
volume is contracted in spite of high total extracelular fluid),
circulating ANF was reported to be within normal ranges30.
Up to date, little information is available about ANF in experimental
unilateral renal artery stenosis31, 32 and no data was found in the
literature concerning the experimental 2K2C model. In these latter
2K2C hypertensive rats we have previously observed the increase in
22Na space four weeks after clipping when hypertension was moderate
(BP < 170 mmHg), but no difference with controls was found in
severe hypertension (BP > 170 mmHg)33. In present experiments the
mean BP of hypertensive rats reached 186 ± 4 mmHg after three weeks;
thus this group of severe 2K2C hypertensive rats should be devoided of
water-sodium expansion. Consequently, high ANF plasma levels in
present experiments might not be necessary ascribed to an increased
total water-sodium space. This assertion is in agreement with the high
circulating ANF levels described in 2K1C rats34 in which hypertension
is not either accompanied by water-sodium retention as long as the
contralateral untouched kidney remains undamaged. Furthermore, our
results would support that the primary specific stimulus to ANF
release is cardiac muscle stretch35 which might derive from high BP
and/or the primary tendency of increasing intravascular fluid volume
space (blood volume), the latter observed in situations like heart
failure36, renal failure37, pregnancy38 and high sodium intake30.
Concerning vascular responses, in 1984 García et al.12 provided for
the first time convincing evidence that the fall in blood pressure
resulting from the infusion of crude atrial extracts might involve a
direct relaxant effect of a purified natriuretic factor on blood
vessels. Nevertheless, dilation of resistance-sized arteries has been
questioned39 and some studies have demonstrated that the hypotensive
action of ANF is mainly due to a decrease in cardiac output10;
actually, regional and total resistance might increase10, 40. With
this respect, Lappe et al.10 suggested that the rapid fall in cardiac
output observed by the infusion of the synthetic natriuretic peptide
atriopeptin II (AP II) would indicate a preferential venodilation
effect with a marked reduction in venous return. As a consequence, the
progressive fall in cardiac output and arterial pressure stimulates
baroreflex mechanisms increasing sympathetic vasoconstrictor tone
which could mask any moderate ANF peripheral vasodilation on the
arterial side. It is worth to note, the same authors10 recognize that
low concentrations of AP II (avoiding a significant effect on cardiac
output) cause measurable vasodilatory responses. In summary, vascular
actions of natriuretic peptides generate conflicting results in
relation to the use of different doses and/or whether the effect is
observed on isolated blood vessels or in the entire animal. In our
2K2C experimental model, neither the diuretic-natriuretic effect, the
possible depressor effect on cardiac output and/or peripheral
vasodilation via ANF, nor its well known opposite action on RAS, were
effective for high circulating ANF to inhibit the increase of BP. It
could be speculated that any intent to decrease BP in these 2K2C rats
would derive in renal ischemia and thus in renin secretion,
counteracting cardiovascular depressor effects of ANF.
Our results showed that rings of abdominal aorta of 2K2C rats
stimulated by 3.5 x 10-6 M Phe (a submaximal dose) contracted less
than rings from SH rats (Fig. 2). Accordingly, we have previously
reported that strips of the same vessel (abdominal aorta) and
experimental model (2K2C), contracted less than controls to
norepinephrine, indicating that vessels from this type of renovascular
hypertensive animals would be less responsive to catecholamines41.
With regard to relaxation, a lower relaxant effect to ANF on rings
from hypertensive rats was observed in present experiments
(significant for the medium dose used, 10-8 M). García et al.27 and
others33 have reported down-regulation of ANF receptors in presence of
high plasma values of ANF in 2K1C and 1K1C hypertensive rats. Thus the
high plasma values observed in our 2K2C rats could be responsable, in
part, for lower peripheral vasodilation mediated by down-regulation of
ANF receptors. With this respect, protein kinase C has been postulated
to be a regulator of ANF-B receptors42; to further approach this
possibility, responses to PDBu (a protein kinase C stimulant) on aorta
rings were examined and tension development was found to be
significantly increased in rings from HT rats (Fig. 2, lower panel).
This fact suggests that greater activity of protein kinase C in the
vessel wall may contribute to down-regulation of ANF-B receptors in
On the other hand, it is a well known fact that vascular endothelium
plays a major role in controlling vascular tone by releasing relaxing
and contracting factors43. Among the former, NO or a closely related
compound mediates its vascular relaxant effect by the same second
messenger (cGMP) as ANF. Nevertheless, ANF activates membrane
particulate guanylate cyclase while NO activates intracellular soluble
guanylate cyclase; accordingly, ANF stimulation correlates with plasma
cGMP concentration while NO stimulation correlates with cGMP content
in the vascular wall44. Consequently, we would like to speculate that
the NO effect might be particularly ascribed to local paracrine
vasodilation and elevated plasma ANF would specially account for
humoral modulation of peripheral vascular resistance in our 2K2C rats.
It can be concluded that high circulating values of ANF, along with
the previously described decrease in granularity of atrial
myocardiocytes, suggest an increased turnover of the peptide in 2K2C
hypertensive rats. We further speculate that the primary stimulus for
releasing ANF peptide is cardiac muscle stretch by high BP and/or the
increase (or the tendency to increase) of the intravascular fluid
volume; high total sodium space, in the presence of simulatneous
normal or decreased blood volume and without hypertension (like in the
advanced period of cirrhosis), would be denied as responsable for
elevated circulating ANF. Moreover, high levels of ANF here reported
might be particularly involved in humoral modulation of peripheral
vascular resistance in renovascular hypertension. According to the
literature, NO and CNP (which belongs to the peptide natriuretic
family) are better candidates for local paracrine regulation of blood
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Fig. 1.- ANF (upper panel) and nitrite/nitrate (lower panel)
concentration in plasma.
Fig. 3.- Percent relaxation to atrial natriuretic factor in abdominal
aorta rings precontracted by 3.5 x 10-6 M phenylephrine.
Fig. 2.- Contraction of abdominal aorta rings to phenylephrine 3.5 x
10-6 M (upper panel); contraction of aorta rings to phorbol 12,
13-dibutyrate 10-5 M (lower panel).