Vol. 274, Issue 3, F556-F563, March 1998
Extrarenal resistance to atrial natriuretic peptide in rats
with experimental nephrotic syndrome
Jean-Pierre
Valentin1,
Wei-Zhong
Ying1,
William G.
Couser2, and
Michael H.
Humphreys1
1 Divisions of Nephrology, San
Francisco General Hospital, University of California San Francisco, San
Francisco, California 94143; and
2 University of Washington,
Seattle, Washington 98195
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ABSTRACT |
Nephrotic syndrome is associated with resistance to the renal
actions of atrial natriuretic peptide (ANP). We performed experiments in anesthetized, acutely nephrectomized rats 21-28 days after injection of adriamycin (7-8 mg/kg iv) or 9-14 days after
injection of anti-Fx1A antiserum (5 ml/kg ip) (passive Heymann
nephritis; PHN) to test whether extrarenal resistance also occurred.
Proteinuria was significantly elevated in both models compared with
controls before study. ANP infusion (1 µg · kg
1 · min1)
caused arterial pressure to decrease similarly in control rats, adriamycin-treated rats, and rats with PHN (by 8.2 ± 1.0, 9.4 ± 2.3, and 9.0 ± 2.0%, respectively; all
P < 0.05 vs. both baseline and
vehicle-infused control rats). In control rats, hematocrit increased
progressively to a maximal value 9.5 ± 0.9% over baseline as a
result of the infusion, an increase corresponding to a reduction in
plasma volume of 16.1 ± 0.9%. The ANP-induced increase in
hematocrit was preserved in adriamycin-treated rats (9.2 ± 1.3%)
but was markedly blunted in rats with PHN (2.4 ± 1.3%;
P < 0.0001 vs. ANP infusion in
control rats). ANP infusion increased plasma ANP levels to the same
extent in the three groups, whereas plasma guanosine
3',5'-cyclic monophosphate was significantly lower in rats
with PHN compared with both control and adriamycin-treated rats.
Infusion of a subpressor dose of angiotensin II (ANG II, 2.5 ng · kg1 · min1)
fully restored the ANP-induced increase in hematocrit in rats with PHN.
This study demonstrates that 1) the
hemoconcentrating and hypotensive actions of ANP are preserved in
adriamycin-treated rats, 2) the
effect of ANP on hematocrit and fluid distribution is blunted in rats
with PHN while its hypotensive action is preserved, and
3) low-level ANG II infusion
normalizes the hemoconcentrating effect of exogenously infused ANP in
rats with PHN. Thus deficient ANG II generation in rats with PHN, but
not adriamycin nephrosis, may contribute to extrarenal ANP resistance.
angiotensin II; arterial pressure; guanosine
3',5'-cyclic monophosphate; nephrectomized animals; nephrotic edema; plasma volume; proteinuria; renin-angiotensin system; vascular permeability
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INTRODUCTION |
IN ADDITION to its potent diuretic, natriuretic, and
vasodilator properties, atrial natriuretic peptide (ANP) also causes an
increase in hematocrit not fully accounted for by urinary fluid loss
(4). This rise in hematocrit is due to a decrease in plasma volume
resulting from a transfer of plasma fluid and proteins from the
intravascular to the interstitial compartment (1, 6, 27, 30-32,
35, 40). Such an effect of ANP may result from an increase in
filtration pressure, filtration surface area, and/or
permeability at the capillary level. Although the mechanism of this
effect is still not fully understood, it requires the presence of
angiotensin II (ANG II) (27). Moreover, we recently reported that the
hemoconcentrating but not the hypotensive activity of ANP was blunted
in rats with experimental diabetes mellitus (32), a model associated
with resistance to the renal actions of ANP (24) as well as with
alterations in the renin-angiotensin system (12).
Both clinical and experimental nephrotic syndromes are associated with
altered fluid balance and blood volume homeostasis (9). Two models of
experimental nephrosis have been particularly useful in exploring
nephrotic sodium metabolism. They result from the intravenous injection
of the anthracyclin, adriamycin, which induces a glomerular lesion
similar to minimal change nephropathy in humans (3), and from the
intraperitoneal administration of anti-glomerular epithelial cell
(anti-Fx1A) antibodies (23) to induce passive Heymann nephritis (PHN),
a model of membranous nephropathy in humans. Both models are
characterized by no change or an increase in the plasma concentration
of ANP (9, 28, 33) and blunted renal responsiveness to exogenously
administered ANP (8, 14, 19) and/or to endogenously secreted
ANP elicited by volume expansion (2, 5, 28, 33). In addition, patients with nephrotic syndrome have altered renal responses to changes in
plasma ANP induced by either water immersion or isotonic volume expansion (11, 20, 21). However, to date no information is available
regarding the extrarenal effect of ANP on transcapillary shift of
plasma fluid toward the interstitium in the nephrotic state.
In the present studies, we found that rats with nephrotic syndrome
resulting from PHN exhibited blunted hemoconcentration in response to
ANP infusion despite a normal hypotensive response. The blunted
extrarenal response to ANP was restored by the simultaneous infusion of
a subpressor dose of ANG II. In contrast, rats with adriamycin
nephrosis had both hypotensive and hemoconcentrating responses to
infused ANP that did not differ from those in control rats.
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METHODS |
Animal model.
We carried out studies in male Sprague-Dawley rats (Bantin-Kingman,
Fremont, CA) housed in climate-controlled conditions and provided
standard rat laboratory diet and water ad libitum. Two models of
nephrotic syndrome were studied. Adriamycin nephrosis was induced with
a single intravenous injection of adriamycin (doxorubicin, Sigma
Chemical, St. Louis, MO; 7-8 mg/kg at a concentration of 10 mg/ml)
dissolved in normal saline to seven rats weighing 140-160 g (3,
28). PHN was induced by the intraperitoneal injection of anti-Fx1A
antiserum (5 ml/kg body wt) to 17 rats weighing 140-200 g (13, 23,
33). Appropriate control rats, matched for age and weight at the time
of the adriamycin or anti-Fx1A administration, received an equal volume
of normal saline (n = 31). Rats were
studied 21-28 and 9-14 days after the injection of adriamycin
or the anti-Fx1A antiserum, respectively. To determine 24-h water
intake, urine volume, and daily urinary excretion of sodium, potassium,
and proteins, rats were placed in metabolic cages on the last day of
the experiment.
General procedure.
On the day of the acute experiment, animals were anesthetized with an
intraperitoneal injection of 100 mg/kg of Inactin (Andrew Lockwood and
Associates, Sturtevant, WI) and placed on a heated table to maintain
rectal temperature at 37 ± 0.5°C. Animals underwent tracheostomy and breathed spontaneously; they were prepared for acute
experimentation as previously described (27, 29-32). Briefly, catheters were inserted into a femoral artery and vein and the right
carotid artery for sampling blood, infusing fluids and drugs, and
continuous measurement of arterial pressure via a Statham pressure
transducer (model P23 ID, Gould Instruments, Oxnard, CA) connected to a
Grass polygraph (model 7D, Grass Instrument, Quincy, MA). Both kidneys
were then removed through retroperitoneal flank incisions. During the
surgical preparation, rats received a constant intravenous infusion of
plasma substitute (Hespan, 6% hetastarch in 0.9% sodium chloride, Du
Pont Pharmaceuticals, Wilmington, DE) at a rate of 40 µl/min via a
syringe pump (Harvard Apparatus, S. Natick, MA) until a total volume of
0.5% body weight was administered, to replace estimated fluid losses.
Thereafter, the infusion rate was reduced to 10 µl/min for the
duration of the studies. Experiments were started 30-45 min after
completion of surgical procedures. After a 45-min control period,
control rats received either rat ANP-(1
28) (Peninsula Laboratories,
Belmont, CA; n = 15) at a dose of 1 µg · kg1 · min1
in normal saline or vehicle alone (n = 10). Nephrotic rats received the ANP infusion. The infusion of vehicle
or ANP was at a rate of 10 µl/min for 45 min. In additional
experiments, the influence of ANG II on the responses to ANP was
evaluated in six control rats and eight rats with PHN. For this
purpose, a subpressor dose of ANG II (2.5 ng · kg1 · min1;
Sigma) was infused throughout the experiment, starting at the beginning
of the control period as previously described (27). These animals
received the ANP infusion, and the total infusion rate was limited to
10 µl/min to maintain the administered volume at a constant level.
Three 50-µl samples of blood were taken 15, 30, and 45 min after the
start of the control and experimental periods for determination of
hematocrit and plasma protein concentration. Hematocrit was measured in
duplicate on each blood sample by spinning blood at 12,000 rpm in a
Microfuge (Clay Adams, Parsippany, NJ) for 3 min. Plasma protein
concentration was estimated in duplicate by refractometry (National
Instrument, Baltimore, MD). Plasma volume was measured by the dilution
principle using the Evans blue dye technique (32). For this purpose, 50 µl of Evans blue (Sigma; 5 mg/ml dissolved in normal saline) were
injected intravenously, and the tubing was flushed with 200 µl of
normal saline 5 min before the end of the 45-min infusion period. At
the end of the experimental period, 5 ml of arterial blood were
withdrawn and rapidly transferred into an ice-cold tube containing the
following protease inhibitors: 1 mg of EDTA, 500 kallikrein inhibitor
units of aprotinin, 10 µg of pepstatin A, and 100 µg of
phenylmethylsulfonyl fluoride per milliliter of blood. Blood was
immediately centrifuged (4,000 rpm) at 4°C for 15 min, and plasma
was kept frozen at 
70°C until subsequently analyzed for
Evans blue dye concentration and immunoreactive ANP and guanosine
3',5'-cyclic monophosphate (cGMP).
Analytical techniques, calculations, and statistical evaluation.
Analytical methods used in the laboratory have previously been
described (28-33). Briefly, urine sodium and potassium
concentrations were measured by flame photometry (model 943, Instrument
Laboratories, Lexington, MA) and plasma Evans blue dye concentration in
a spectrophotometer at 620 nm (Titertek Multiskan Plus, Labsystem and
Flow Laboratories, Finland). Urinary protein concentration was
determined by the Coomassie blue method.
Plasma concentration of immunoreactive ANP was determined after
extraction on a Sep-Pak C18 column
(Waters Chromatography Div., Millipore, Milford, MA) preequilibrated
with 0.1% trifluoracetic acid. After elution with 75% methanol in
0.1% trifluoracetic acid and evaporation to dryness under a stream of
nitrogen, the residue was reconstituted in assay buffer and measured
for ANP immunoreactivity with a commercially available kit (Peninsula).
Plasma concentration of cGMP was determined after extraction with
water-saturated ethyl ether. After evaporation to dryness under a
stream of air, the residue was reconstituted in assay buffer and
measured for cGMP immunoreactivity with a commercially available kit
(Du Pont).
Estimated changes in plasma volume were calculated according to the
following formula: dV = (100/100 Hi) × [100 × (Hi Hf)/Hf],
where dV is the percent change in plasma volume, and Hi and
Hf are the initial and final
hematocrits, respectively. Data are expressed as means ± SE.
Analysis of variance with or without repeated measures followed by
Dunnett's test was used to assess significance among and between
groups, respectively (Statview, Brain Power, Calabasas, CA).
Relationships between variables were assessed by logarithmic regression
analysis. P = 0.05 was considered the
minimal level of significance.
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RESULTS |
Group characteristics.
Results from the metabolic cage study indicated that nephrotic rats had
similar water intake, urine volume, and urinary excretion of sodium and
potassium compared with controls (Table 1).
Urinary protein excretion was significantly elevated in both
adriamycin-treated rats and rats with PHN compared with controls and
was greater in adriamycin-treated rats than in rats with PHN (Table 1).
Effect of ANP infusion on systemic hemodynamics, hematocrit, plasma
protein concentration, and plasma volume in control rats.
Infusion of ANP induced a decrease in mean arterial pressure (MAP) of
8.2 ± 1.0% (P < 0.05 vs. both
baseline and vehicle-infused control rats) from a basal value of 120 ± 3 mmHg (Fig. 1). Heart rate was not
affected by ANP (356 ± 11 vs. 359 ± 11 beats/min; NS). As
depicted in Fig.
2A, ANP
infusion led to a progressive rise in hematocrit of 5.6 ± 0.6, 8.3 ± 0.8, and 9.5 ± 0.6% at 15, 30, and 45 min, respectively,
from a basal value of 46.0 ± 0.9% (all
P < 0.05 vs. both baseline and
vehicle-infused control rats). The maximal increase in plasma protein
concentration (PPC) resulting from the 45-min ANP infusion (3.9 ± 0.7%, from a basal value of 4.97 ± 0.05 g/dl;
P < 0.05 vs. both baseline and
vehicle-infused control rats) was of smaller magnitude than the
corresponding change in hematocrit (Fig.
2B). The decrease in plasma volume calculated from the change in hematocrit amounted to 16.1 ± 0.9% for ANP. This calculated change was confirmed by the
direct measurement of plasma volume at the end of the experiment using
Evans blue dye. As shown in Fig.
3A, plasma
volume was significantly lower in ANP-infused compared with
vehicle-infused control rats (21.3 ± 1.4 vs. 25.2 ± 1.1 ml/kg;
P < 0.05 between groups). Such a
decrease in plasma volume should have increased PPC by ~20%, much
greater than the observed increase of only 3.9%, indicating that some loss of plasma protein had occurred in response to ANP infusion. In the
vehicle group, no significant change in MAP (Fig. 1) or heart rate (318 ± 10 vs. 312 ± 11 beats/min) was noted over the course of the
experiment, whereas hematocrit and PPC decreased slightly (1.4 ± 0.6 and 0.8 ± 0.5%, respectively, both
P < 0.05 vs. baseline; Fig. 2).
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Fig. 1.
Response of mean arterial pressure to a 45-min infusion of atrial
natriuretic peptide (ANP) in control rats (CTL;  ;
n = 15), adriamycin-treated rats (ADR;
 ; n = 7), and rats with passive
Heymann nephritis (PHN;  ; n = 9) or
vehicle (Veh) in control rats ( ; n = 10). ANP was also administered in control rats ( ;
n = 6) and rats with PHN ( ;
n = 8) acutely infused with ANG II
(2.5 ng · kg1 · min1)
for duration of experiment (i.e., 90 min). Values are means ± SE.
§ P < 0.05 vs. baseline by
repeated-measures analysis of variance (ANOVA).
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Fig. 2.
Time course of relative changes in hematocrit
(A) and plasma protein concentration
(B) during 45-min infusion of ANP in
control (), adriamycin-treated rats (), and rats with PHN ()
or vehicle in control rats (). Values are means ± SE.
* P < 0.05 vs. baseline by
repeated-measures ANOVA; § P < 0.05 comparison between ANP-infused control or adriamycin-treated
rats and both vehicle-infused control rats and rats with PHN.
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Fig. 3.
Effect of a 45-min infusion of vehicle or ANP on plasma volume
(A) and plasma concentrations of
immunoreactive (ir) ANP (B) and
ircGMP (C) in control rats,
adriamycin-treated rats, and in rats with PHN. Plasma concentrations of
ANP and cGMP as well as plasma volume were determined at end of acute
experiment. § P < 0.05 vs.
control rats infused with vehicle;
* P < 0.05 comparison between
remaining groups.
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Effect of ANP infusion in nephrotic rats.
Baseline values for MAP and hematocrit were significantly lower in
nephrotic rats compared with control animals, whereas neither heart
rate nor PPC differed significantly between groups (Table 2). The decrease in MAP observed in both
adriamycin-treated animals and rats with PHN (9.4 ± 2.3 and 9.0 ± 2.0%; both P < 0.05 vs. baseline; Fig. 1) was similar in magnitude to that observed in control
animals infused with ANP. As shown in Fig. 2, the ANP-induced increase
in hematocrit was markedly blunted in rats with PHN (1.9 ± 0.8, 2.8 ± 1.2, and 2.4 ± 1.3% at 15, 30, and 45 min, respectively; all
P < 0.05 vs. ANP infusion in control
animals), and the ANP-induced increase in PPC did not occur. These
responses to ANP infusion were preserved in adriamycin-treated rats
(Fig. 2), with the values being no different than the changes observed
in control animals infused with ANP. The decrease in plasma volume
calculated from the change in hematocrit amounted to 3.9 ± 2.3 and
13.9 ± 1.6%, respectively, in rats with PHN and adriamycin-treated
rats at the end of the infusion period. As shown in Fig.
3A, rats with PHN had a significantly
higher plasma volume than both ANP- and vehicle-infused control rats.
Adriamycin-treated rats infused with ANP had plasma volume similar to
that measured in ANP-infused control animals.
Effect of ANP infusion on plasma concentrations of ANP and cGMP.
Infusion of ANP for 45 min elevated plasma immunoreactive ANP
concentration to the same extent in control, adriamycin-treated rats,
and rats with PHN (Fig. 3B). In
contrast, plasma immunoreactive cGMP concentration, measured at the
conclusion of the experiment, was significantly lower in rats with PHN
(141 ± 25 pmol/ml; P < 0.05)
compared with control (281 ± 18 pmol/ml) and adriamycin-treated rats (290 ± 49 pmol/ml; Fig.
3C). When values from the four
groups of animals were pooled together, a positive correlation existed between the maximal change in hematocrit and plasma concentration of
cGMP (Fig. 4;
r = 0.79, P < 0.0001), whereas no correlation was observed between changes in hematocrit and plasma concentration of
ANP.
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Fig. 4.
Correlations between relative changes in hematocrit and plasma ircGMP
concentration induced by a 45-min infusion of ANP in control rats
(), adriamycin-treated rats (), and rats with PHN () or
vehicle in control rats (). Plasma concentration of cGMP was
measured at end of acute experiment.
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Influence of ANG II infusion on responses to ANP in rats with PHN.
Baseline MAP did not differ significantly in animals infused with ANG
II (2.5 ng · kg1 · min1)
compared with their respective controls (Table 2). MAP in response to
ANP infusion decreased similarly in control rats and rats with PHN
infused with ANG II (by 8.8 ± 2.8 and 10.0 ± 3.3%,
respectively; both P < 0.05 vs.
baseline and vs. vehicle-infused control rats) and to the same extent
as in controls not infused with ANG II (Fig. 1). Acute infusion with
ANG II did not affect the hemoconcentrating response to ANP in control
rats but restored to normal the ANP-induced increase in hematocrit and
PPC in rats with PHN (Fig. 5).
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Fig. 5.
Maximal relative changes in hematocrit and plasma protein concentration
observed during 45-min infusion of ANP in control rats (solid bars) and
rats with PHN (hatched bars) or of vehicle in control rats (open bars).
ANP was also infused in control rats (checkered bars) and rats with PHN
(cross-hatched bars) receiving an infusion of a subpressor dose of ANG
II (2.5 ng · kg1 · min1)
for duration of experiment (i.e., 90 min). Values are means ± SE.
§ P < 0.05 vs.
vehicle-infused control rats;
* P < 0.05 comparison between
remaining groups; NS, not significant.
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DISCUSSION |
The present studies, performed in bilaterally nephrectomized rats, have
demonstrated that the extrarenal effects of ANP are preserved in
adriamycin-treated nephrotic rats. In contrast, the ANP-induced
hemoconcentration resulting from a reduction in plasma volume is
blunted in rats with PHN, but its hypotensive action is preserved.
Acute infusion of a subpressor dose of ANG II in rats with PHN restored
the ANP-mediated increase in hematocrit to normal. Although the
reported changes in some cases were small and not outside the likely
variability in measurement, they nevertheless describe a cogent picture
of extrarenal ANP action in these models of experimental nephrosis.
In control rats, infusion of ANP at a pharmacological but mildly
hypotensive dose (1 µg · kg1 · min1)
resulted in a roughly 8-10% increase in hematocrit and a decrease in plasma volume of around 14-16% as measured by the Evans blue dye method. These results are similar to those that we (27, 30-32)
and others (1, 6, 35, 40) have previously reported. Because renal fluid
losses were absent in these bilaterally nephrectomized rats, the
decrease in plasma volume resulted from a shift of fluid from the
vascular space to the interstitial compartment. A similar increase in
hematocrit occurs at lower doses of infused ANP (33, 35, 40) as well as
when a more modest increase in plasma concentration of ANP (4- to
6-fold) is induced by endothelin infusion (29). Thus, although the
plasma concentration of ANP achieved by our infusion is well out of the
physiological range, the differences that we have observed may also
take place at more physiologically relevant concentrations. In this
regard, we have observed that ANP-mediated effects on hematocrit and
plasma volume are most pronounced at mild to moderate plasma
concentrations of ANP, with values greater than fivefold over baseline
having little further effect to increase hematocrit (29).
Although the mechanism or mechanisms by which ANP alters fluid
distribution are not known, the accumulation of plasma proteins in
susceptible vascular beds (30, 35, 40) suggests that increased
capillary permeability may be an important component. The abnormal
response of rats with PHN suggests that this component is reversibly
altered in this experimental model of nephrotic syndrome, whereas it
remains intact in rats with adriamycin nephrosis. The blunted
hemoconcentration that we observed in rats with PHN after ANP infusion
was accompanied by an increased plasma volume compared with control
animals infused with either vehicle or ANP. There are several possible
explanations for this abnormal response to ANP. Enhanced catabolism of
the infused peptide seems unlikely in view of the nearly identical
plasma concentrations measured in the three experimental groups.
Moreover, as discussed above, the concentrations were in any case much
higher than those required to elicit a maximal increase in vascular
permeability in control rats based on our previous studies (29). Rats
with PHN had lower resting arterial pressure than normal rats, and it
is possible that this lower pressure in some way rendered them
resistant to the infused ANP. The impact of this lower blood pressure
on ANP-induced fluid partition is probably minimal if any. Trippodo and
Barbee (26) found that spinal cord transection, a maneuver which
produced a more substantial lowering of arterial pressure, did not
influence the effect of ANP on fluid distribution. Moreover,
adriamycin-treated rats had a preserved hemoconcentrating response to
ANP despite significantly lower baseline blood pressure compared with
control rats. Furthermore, the hemoconcentrating activity of ANP was
restored in rats with PHN during simultaneous infusion of a subpressor dose of ANG II. In addition, despite lower baseline blood pressure, nephrotic animals had a quantitatively similar hypotensive response to
infused ANP as seen in the control animals, indicating that this action
of ANP is preserved in nephrosis. Interestingly, a blunted hypotensive
response to a bolus injection of brain natriuretic peptide has recently
been reported in adriamycin nephrosis (38), suggesting the possibility
of differential responsiveness to the vascular actions of members of
the natriuretic peptide family.
Clinical and experimental nephrotic syndromes have been associated with
either normal or elevated resting plasma ANP concentration (9, 28, 33).
One consequence of chronic elevation of plasma ANP concentration could
be a downregulation of biologically active ANP receptors. However, we
could not find any differences in either density or affinity of ANP
renal receptors in both adriamycin-treated rats (28) and rats with PHN
(33). These observations are in agreement with those of Perico et al.
(18), reporting that the binding of ANP to inner medullary collecting
duct cells did not differ between controls and nephrotics. Thus renal
resistance to ANP in vivo is unlikely to result from reduced binding of
the peptide to its receptors. Whether these findings regarding normal renal ANP receptor characteristics in experimental nephrosis can be
extended to extrarenal ANP receptors is not known at the present time.
Our data indicate that responsiveness of the vasculature with respect
to blood pressure is preserved in the nephrotic state, implying that
the receptors mediating vasorelaxation are functionally intact. These
observations make it likely that the characteristics of the natriuretic
peptide receptors responsible for ANP-related increases in
permeability, presumably on capillary endothelial cells, are also
normal in rats with PHN, particularly since the low-dose ANG II
infusion was able to restore responsiveness to normal.
A fourth possible explanation for this abnormal responsiveness to
infused ANP that we observed is impaired cell signaling in response to
the binding of ANP to its biologically active receptors. We observed
that the plasma concentration of cGMP in rats with PHN after ANP
infusion was only half the value measured in both control and
adriamycin-treated rats. Although the source of circulating cGMP is not
known with certainty, it probably originates in vascular tissue
including endothelial cells (4, 39). The lower plasma cGMP level in
rats with PHN could result from impaired generation or enhanced
degradation of intracellular cGMP, currently the only recognized
intracellular second messenger of ANP (4, 36). We recently reported
that enhanced cGMP phosphodiesterase activity could contribute to
abnormal cGMP metabolism and account for the resistance to the
natriuretic actions of ANP observed in both adriamycin nephrosis and
PHN (28, 33). Moreover, because the hypotensive action of ANP was
preserved in rats with PHN, the defect may be a selective one, found in
the kidney (33) and possibly the endothelium, but not in vascular
smooth muscle. A reduced plasma cGMP concentration could result from
diminished ANP binding to its receptors, impaired cell signaling after
binding, or enhanced phosphodiesterase activity in these selective
tissues manifesting ANP resistance, and it is not possible to
distinguish among these possibilities at present.
The hemoconcentrating activity of ANP in rats with PHN was restored by
the simultaneous infusion of ANG II at a low, subpressor dose. We
previously demonstrated the importance of ANG II in ANP-induced hemoconcentration: chronic pretreatment of rats with the
angiotensin-converting enzyme (ACE) inhibitor, captopril, or acute
pretreatment with the ACE inhibitor, enalaprilat, or the ANG II type 1 receptor antagonist, losartan, abolished the ANP-induced increase in
hematocrit (27). Furthermore, the effect of ANP on hematocrit was
restored in ACE inhibitor-treated rats by the simultaneous infusion of a subpressor dose of ANG II (i.e., 2.5 ng · kg1 · min1).
In the context of the present studies, rats with PHN have depressed 1) plasma renin activity and
concentration (37), 2) plasma ANG II
concentration (25), and 3) renal
renin mRNA (10). Human membranous nephropathy has also been
characterized as a low-renin, plasma volume-expanded state (9, 16). In
contrast, adriamycin-treated rats have high levels of both tissue and
blood ACE (34) and similar glomerular renin staining (22) compared with
control animals. Taken together, these results suggest that the
renin-angiotensin system is either normal or enhanced in adriamycin
nephrosis but is depressed in rats with PHN. Although we did not
measure plasma ANG II levels in our experiments, it is conceivable that
they were insufficient in rats with PHN to allow ANP to increase
hematocrit. The mechanism of such an effect has previously been
discussed (27) and could be explained by changes in local hydrostatic forces. By analogy with the renal microcirculation, it is conceivable that ANP-induced precapillary vasodilatation is detectable only in
vessels previously partially constricted with ANG II. A reduction in
the preferential postcapillary constrictor effect of ANG II, as
expected in rats with PHN, would then prevent the increase in the
transcapillary pressure gradient and thus fluid movement toward the
interstitial compartment induced by ANP. Alternatively, ANP and ANG II
may interact at the endothelial level, where ANP receptors have been
located (15) and contrasting effects of ANP and ANG II on electrolyte
transport have been observed (7, 17). Because bilateral nephrectomy
would have removed the major source of circulating renin in both
groups, the preserved response to infused ANP in rats with adriamycin
nephrosis indicates that sufficient residual ANG II, whether generated
from circulating renin or from other tissue sources, was present in
this model but not in rats with PHN.
In summary, adriamycin nephrosis in the rat is characterized by normal
responsiveness to the hypotensive and hemoconcentrating actions of
exogenous ANP, whereas rats with PHN, a form of experimental nephrotic
syndrome resembling human membranous nephropathy, exhibit a blunted
response to ANP-induced increases in hematocrit and protein
extravasation while showing a normal decrease in blood pressure. PHN
induces a selective impaired responsiveness to ANP that may relate to
an interaction with ANG II at target cells governing vascular
permeability, since simultaneous infusion of a subpressor dose of ANG
II restored the responsiveness to infused ANP.
Perspectives
Analysis of the mechanisms involved in nephrotic sodium retention has
led to two competing hypotheses (9). In one, the underfill model,
sodium retention is initiated by a diminished plasma volume brought on
by a reduced plasma oncotic pressure from low albumin concentration,
whereas in the other, the overflow model, sodium retention occurs
despite an expanded plasma volume. Renal resistance to ANP is a regular
occurrence in both experimental and clinical nephrotic syndrome (9, 19)
and has been linked to sodium retention. The present experiments
demonstrate that selective resistance to the extrarenal actions of ANP
also occurs in an immunologically mediated form of experimental
nephrotic syndrome (PHN) but not in a toxic model (adriamycin
nephrosis). In each case, the hypotensive action of infused ANP was
preserved, whereas the hemoconcentrating effect, which is due to a
reduction in plasma volume, was normal in adriamycin nephrosis but
markedly blunted in rats with PHN. These rats also exhibited expanded
plasma volume, suggesting that this extrarenal resistance to ANP is a determinant of the expanded plasma volume. PHN is known to have suppression of the renin-angiotensin system, and infusion of a low dose
of ANG II restored the hemoconcentrating effect of infused ANP to
normal. These observations will allow analysis of the relationship between plasma volume and sodium retention in nephrotic syndrome to
take place in a new context and suggest that interactions between ANG
II and ANP may provide a basis to account for divergent measurements of
plasma volume in this condition, helping to reconcile the controversy between underfill and overflow mechanisms of nephrotic edema.
| |
ACKNOWLEDGEMENTS |
This work was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grants DK-31623 and DK-34198 and American
Heart Association Grants-in-Aid 891124 and 941229. J. P. Valentin was
supported by research fellowships awards from ICI-Pharma (Zeneca) and
the American Heart Association, California Affiliate.
| |
FOOTNOTES |
Current addresses: J. P. Valentin, Div. of Cardiovascular Diseases,
Centre de Recherche Pierre Fabre, Castres, France; W.-Z. Ying, Div. of
Nephrology, University of Alabama, Birmingham, AL 35294.
Address for reprint requests: M. H. Humphreys, Div. of Nephrology,
University of California, San Francisco, Bldg. 100, Rm. 350, Box 1341, Renal Center/San Francisco General Hospital, San Francisco, CA
94143-1341.
Received 11 August 1997; accepted in final form 16 November 1997.
| |
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