Concerted action of dopamine on renal and
intestinal Na+-K+-ATPase in the rat remnant
kidney
M. A.
Vieira-Coelho,
P.
Serrão,
J. T.
Guimarães,
M.
Pestana, and
P.
Soares-Da-Silva
Institute of Pharmacology and Therapeutics, Faculty of Medicine,
4200 Porto, Portugal
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ABSTRACT |
The present study evaluated renal and intestinal adaptations in
sodium handling in uninephrectomized (Unx) rats and the role of
dopamine. Two weeks after uninephrectomy, the remnant kidney in Unx
rats weighed 33 ± 2% more than the corresponding kidney in
sham-operated (Sham) animals. This was accompanied by increases in
urinary levels of dopamine and major metabolites
[3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid] and
increases in maximal velocity values (169 vs. 115 nmol · mg
protein
1 · 15 min
1) for renal
aromatic L-amino acid decarboxylase, the enzyme responsible for the synthesis of renal dopamine. High salt (HS) intake increased (P < 0.05) the urinary excretion of dopamine and DOPAC
in Unx and Sham rats. However, the urinary levels of
L-3,4-dihydroxyphenylalanine, dopamine, and DOPAC in Sham
rats during HS intake were lower than in Unx rats. Blockade of dopamine
D1 receptors (Sch-23390, 2 × 30 µg/kg) reduced the
urinary excretion of sodium in Unx (31% decrease) more pronouncedly
than in Sham (19% decrease) rats. However, inhibition of renal
Na+-K+-ATPase activity by dopamine was of
similar magnitude in Unx and Sham rats. In parallel, it was observed
that uninephrectomy resulted in a significant reduction in jejunal
sodium absorption and Na+-K+-ATPase activity in
jejunal epithelial cells. In jejunal epithelial cells from Sham rats,
dopamine (1 µM) failed to inhibit
Na+-K+-ATPase activity, whereas in Unx rats it
produced a significant reduction. It is concluded that uninephrectomy
results in increased renal dopaminergic activity and dopamine-sensitive
enhanced natriuresis. Furthermore, it is suggested that decreased
jejunal absorption of sodium may take place in response to partial
renal ablation, as an example of renal-intestinal cross talk.
intestine; sodium-potassium-adenosine-5'-triphosphatase
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INTRODUCTION |
DOPAMINE OF RENAL ORIGIN
EXERTS natriuretic and diuretic effects by activating
D1-like receptors located at various regions in the nephron
(23). At the level of the proximal tubule, the overall
increase in sodium excretion produced by dopamine and D1-receptor agonists results from inhibition of main sodium
transport mechanisms at the basolateral and apical membranes,
respectively, Na+-K+-ATPase (1,
36) and the Na+/H+ exchanger
(7). The physiological importance of the renal actions of
dopamine mainly depends on the sources of the amine in the kidney and
on the availability of this dopamine to activate the amine-specific
receptors. The proximal tubules, but not distal segments of the
nephron, are endowed with a high aromatic L-amino acid
decarboxylase (AADC) activity, and epithelial cells of proximal tubules
have been demonstrated to synthesize dopamine from circulating or
filtered L-3,4-dihydroxyphenylalanine (L-dopa)
(17, 36, 41). This nonneuronal renal dopaminergic system
appears to be highly dynamic, and the basic mechanisms for the
regulation of this system are thought to depend mainly on the
availability of L-dopa, its fast decarboxylation into
dopamine, and on precise and accurate cell-outward amine transfer
mechanisms (40). Although one of the most important
factors determining the synthesis of dopamine is the amount of sodium
delivered to the kidney (17, 36, 41), high levels of
metabolic enzymes, such as type A and B monoamine oxidases (MAO-A and
MAO-B, respectively) and catechol-O-methyltransferase (COMT), may also determine overall availability of renal dopamine (5, 8-10, 49).
Dopamine is also relatively abundant in the intestine mucosal cell
layer (4, 6), and studies on the formation of dopamine from exogenous L-dopa along the rat digestive tract showed
that the highest AADC activity is located in the jejunum
(48). A high salt (HS) intake has been found to constitute
an important stimulus for the production of dopamine in rat jejunal
epithelial cells, and this is accompanied, in 20-day-old animals, by a
decrease in intestinal sodium absorption (12). This effect
is accompanied, at the cellular level, by inhibition of
Na+-K+-ATPase activity (46). The
relative importance of this system in controlling sodium absorption
assumes particular relevance in view of the findings that 40-day-old
rats subjected to a HS intake have a fault in intestinal dopamine
production during salt loading, in contrast to that occurring in
20-day-old animals. The lack of changes in the jejunal function in
response to HS intake coincides with the period in which renal function
has reached maturation (34), suggesting the occurrence of
complementary functions between the intestine and the kidney during development.
Reduction in total renal mass leads to an increase in the filtration
rates of the remaining nephrons and increased excretion of sodium per
nephron (18). Although changes in solute reabsorption after reductions in renal mass may be related to altered expression of
sodium transporters (24), it is not known whether this is accompanied by enhanced activity of natriuretic endogenous substances. The hypothesis we have explored in the present study concerns the
occurrence of adaptations in the activity of the renal dopaminergic system in uninephrectomized (Unx) rats and their putative role in
tubular sodium absorption. For this purpose, we measured in Unx and
sham-operated (Sham) rats, during normal salt (NS) and HS intake, the
urinary excretion of water and electrolytes, dopamine, its immediate
precursor L-dopa, and the metabolites
3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine
(3-MT), and homovanillic acid (HVA). In addition, the activities
of enzymes involved in the synthesis and degradation of dopamine,
namely, AADC, MAO, and COMT, were measured in the same animals to
assess the state of activation of the renal dopaminergic system. The
effect of dopamine on proximal tubular
Na+-K+-ATPase activity in these different
groups of rats was also evaluated. In parallel, we have also determined
in Unx and Sham rats the state of activation of the intestinal
dopaminergic system, Na+-K+-ATPase activity,
and its sensitivity to inhibition by dopamine.
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MATERIALS AND METHODS |
In Vivo Studies
Normotensive male Wistar rats (Biotério do Instituto
Gulbenkian de Ciência, Oeiras, Portugal), 40-45 days old and
weighing 200-220 g, were selected after a 7-day period of
stabilization and after adaptation to blood pressure measurements.
Animals were kept under controlled environmental conditions (12:12-h
light-dark cycle and room temperature 22 ± 2°C); fluid intake
and food consumption were monitored daily throughout the study. All
animals were fed ad libitum throughout the study with ordinary rat chow
containing 0.4% sodium (Letica, Barcelona, Spain). Blood pressure
(systolic and diastolic) and heart rate were measured in conscious
restrained animals between 7:00 and 10:00 AM, using a photoelectric
tail-cuff pulse detector (LE 5000, Letica).
Uninephrectomy.
Rats were anesthetized with pentobarbital sodium (60 mg/kg ip), and the
left kidney was exposed under sterile conditions through an incision in
the abdominal wall. Thereafter, the renal pedicle was isolated and the
left kidney was removed. Control animals were Sham rats and submitted
to the same procedures as Unx rats. After surgery, the animals were
returned to their cages, where they had free access to food and water.
HS intake.
In some experiments, Unx and Sham animals were subdivided into two
groups according to their daily sodium intake. In each group (Unx and
Sham) there were subgroups of rats on NS and HS intake. Animals on NS
intake received tap water, and their daily sodium intake averaged 0.5 mmol/100 g body wt. Rats on HS intake had 1.0% (wt/vol) NaCl in their
drinking water, and daily sodium intake averaged 5.0 mmol/100 g body
wt. All four groups of rats were maintained in metabolic cages for the
duration of the study (24 h). The vials collecting 24-h urine contained
1 ml of 6 M HCl to prevent spontaneous decomposition of monoamines and
amine metabolites. After completion of this protocol, rats were
anesthetized with pentobarbital sodium (60 mg/kg ip). Blood from the
vena cava was then collected, and the kidneys and the jejunum were
rapidly removed through an abdominal midline incision. The blood was
collected in tubes containing heparin for later determination of plasma catecholamines and biochemical parameters. The kidneys were rinsed free
from blood with saline (0.9% NaCl), decapsulated, cut in half, and
placed in ice-cold saline. Thereafter, the outer cortex was cut out
with fine scissors, and fragments weighing ~100 mg were placed in
vials containing 500 µl of 0.2 M perchloric acid. Segments of jejunum
~10 cm in length were opened longitudinally with fine scissors,
rinsed free from blood and intestinal contents with cold saline (0.9%
NaCl), and the jejunal mucosa was removed with a scalpel. The mucosae
thus removed were blotted onto filter paper, weighed, and placed in 0.5 ml 0.2 M perchloric acid. The samples were stored at
80°C until
quantification of catecholamines and metabolites by HPLC with
electrochemical detection.
In another set of experiments, Unx and Sham animals were orally given
20 ml/kg 1.0% (wt/vol) NaCl and placed in metabolic cages for 6 h. The two groups (Unx and Sham) were subdivided into two subgroups of
rats receiving (ip) vehicle (water) or the dopamine D1-receptor antagonist Sch-23390 (30 µg/kg, every 3 h). Urine and plasma samples were collected as described above for the
assay of electrolytes and creatinine.
Volume expansion.
The animals were anesthetized with an intraperitoneal injection of
pentobarbitone sodium (60 mg/kg) and placed on a servo-controlled heating pad to maintain the rectal temperature at 37°C. Polyethylene catheters were inserted into the right jugular vein and carotid artery,
the former for infusion of [3H]inulin (2-µCi bolus,
followed by 1 µCi · 100 g body
wt
1 · h
1) and isotonic saline
(volume expansion), and the latter for continuous blood pressure
measurements and blood sampling. The urinary bladder was catheterized
through a suprapubic incision for urine sampling.
After completion of surgical procedures, isotonic saline was infused
continuously at 0.5 ml · h
1 · 100 g body
wt
1. After 60 min of equilibration (from
t = 0-60 min), the volume expansion was started by
infusing the isotonic saline solution 5 ml · h
1 · 100 g body wt
1
(t = 60-90 min). After volume expansion was
completed, the rats were given intraduodenally (at t = 90 min) 0.1 mg/kg L-dopa or vehicle (0.9% saline, 0.4 ml/kg), and isotonic saline solution was again infused at 0.5 ml · h
1 · 100 g body wt
1
(t = 90-240 min). Urine sampling was performed
every 30 min until the end of the experiment (t = 240 min). The animals were killed by an intravenous injection of
pentobarbitone sodium (100 mg/kg) before excision of kidneys, which
were then bled out and weighed.
Jejunal sodium absorption.
An in vivo perfusion technique was used to determine net jejunal fluid
and sodium transport (11, 31). In brief, in anesthetized (pentobarbitone sodium; 60 mg/kg ip) rats a polyethylene catheter was
inserted into the right jugular vein for fluid replacement with
isotonic saline, and the jejunum was cannulated below the suspensory
muscle of the duodenum and again 10 cm below the proximal cannula. This
segment was rinsed and flushed with 10 ml warmed air. The proximal
cannula was connected by a polyvinyl tube to an infusion pump. The
jejunal segment was perfused at a rate of 10 ml · h
1 · 100 g body wt
1
with the following isotonic perfusion fluid: 155 mM Na+, 5 mM K+, 25 mM HCO3
, 115 mM
Cl
, [14C]polyethylene glycol (PEG;
molecular weight 4,000), 2.5 µCi/100 ml, and unlabeled PEG (5g/l), as
a nonabsorbable water marker. The pH was fixed at 7.4 by gassing with
95% O2-5% CO2. After 50 min of equilibration,
effluents from two periods of 20 min each (t = 0-20 and
t = 20-40 min) were collected.
In Vitro Studies
In vitro studies included mainly the assay of enzymes involved
in the synthesis (AADC) and metabolism (MAO and COMT) of renal dopamine. For these studies, renal tissues were obtained from the same
two experimental groups of rats mentioned above: Sham animals
(n = 5) and Unx animals (n = 5).
AADC activity.
AADC activity was determined in homogenates of renal tissues and
jejunal mucosa, using L-dopa (100-10,000 µM) as the
substrate (42). The assay of dopamine was performed by
HPLC with electrochemical detection.
MAO activity.
MAO activity was determined in renal tissues, as previously described
(10). MAO activity was determined with
[3H]5-hydroxytryptamine (5-HT; 75-3,000 µM) as a
preferential substrate for MAO-A and
[14C]
-phenylethylamine (
-PEA; 10-500 µM) as
a preferential substrate for MAO-B. The deaminated products were
measured by liquid scintillation counting.
COMT activity.
COMT activity was evaluated by the ability of tissue homogenates to
methylate epinephrine (1-2,000 µM) to metanephrines, as previously described (49). The assay of metanephrine was
performed by HPLC with electrochemical detection.
Na+-K+-ATPase activity.
Na+-K+-ATPase activity was measured by the
method of Quigley and Gotterer (33) and adapted in our
laboratory with slight modifications (45). Isolated rat
renal proximal tubules and jejunal epithelial cells were obtained as
described (30, 46). Na+-K+-ATPase
activity is expressed as nanomoles Pi per milligram protein per minute and determined as the difference between total and ouabain-sensitive ATPase. The protein content in cell suspension (~2
mg/ml), as determined by the method described by Bradford (3) with human serum albumin as a standard, was similar in all samples.
Alkaline phosphatase and 3-glutamyl transferase activities.
Alkaline phosphatase (ALKP) and
-glutamyl transferase (
-GT)
activities in homogenates of renal cortex were measured, as previously
described (45).
Assay of Catecholamines
The assay of catecholamines and its metabolites in urine
[dopamine, norepinephrine, epinephrine, 5-HT, DOPAC, HVA, and
5-hydroxyindolacetic acid (5-HIAA)]; plasma samples
(L-dopa, norepinephrine, epinephrine, dopamine and DOPAC);
renal tissues and jejunal mucosa (L-dopa, norepinephrine,
dopamine, and DOPAC); and in samples from AADC (dopamine) and COMT
(metanephrine) studies was performed by HPLC with electrochemical
detection, as previously described (30, 45). The lower
limit of detection of L-dopa, dopamine, norepinephrine, epinephrine, metanephrine, DOPAC, 3-MT, HVA, 5-HT, and 5-HIAA ranged
from 350 to 1,000 fmol.
Plasma and Urine Ionogram and Biochemistry
Urinary sodium and potassium were measured by flame photometry
and urine and plasma osmolality by means of an osmometer
(30). Urinary and plasma creatinine and plasma urea were
measured by a wavelength photometer (30).
Drugs
The compounds used were
S-adenosyl-L-methionine; DOPAC;
L-dopa; dopamine hydrochloride; L-epinephrine
bitartrate; HVA; 5-HT; 5-HIAA; DL-metanephrine
hydrochloride; 3-MT; norepinephrine bitartrate; and ouabain and
pargyline hydrochloride and were obtained from Sigma (St. Louis, MO).
Sch-23390, SKF-83566, and (S)-sulpiride were obtained from
Research Biochemicals International (Natick, MA).
[3H]5-hydroxytryptamine creatinine sulfate (23.6 Ci/mmol), [14C]
-phenylethylamine hydrochloride (50 Ci/mmol) were obtained from NEN Chemicals. [3H]inulin
(2.6 Ci/mmol) and [14C]polyethylene glycol (11.2 mCi/g)
were obtained from Nycomed Amersham. Tolcapone was kindly donated by
the late Professor Mosé Da Prada (Hoffman La Roche, Basel, Switzerland).
Statistics
Results are means ± SE of values for the indicated number
of determinations. Maximal velocity (Vmax) and
Michaelis-Menten coeffecient (Km) values for the
decarboxylation of L-dopa, O-methylation of
epinephrine, or deamination of [3H]5-HT and
[14C]
-PEA were calculated from nonlinear regression
analysis by using the GraphPad Prism statistics software package
(25). Geometric means are given with 95% confidence
limits, and arithmetic means are given with SE. Statistical analysis
was performed by one-way ANOVA followed by Student's t-test
for unpaired comparisons. P < 0.05 was assumed to
denote a significant difference.
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RESULTS |
Uninephrectomy had no adverse effects on body growth, as Unx rats
attained the same weight at 2 wk as did Sham rats. Kidney growth,
however, was significantly altered, and the remnant kidney weighed
33 ± 2% more than the corresponding kidney in Sham animals (Table 1). Uninephrectomy led to small
but statistically significant increases in blood urea nitrogen and
plasma creatinine at 2 wk after surgery, this being accompanied by a
40% reduction in creatinine clearance. Plasma levels of electrolytes
(sodium, potassium, and chloride) and plasma osmolality were, however,
similar in Sham and Unx rats (Table 1). The fractional excretion of
both Na+ and K+ was markedly (P < 0.05) increased in the hypertrophied kidney, which might explain why
the urinary excretion of these electrolytes was similar in renal-intact
and Unx rats. Systolic and diastolic blood pressure and heart rate in
Unx rats did not differ from that in Sham rats (Table 1).
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Table 1.
Body and kidney weight, metabolic balance, renal function, blood
pressure, and heart rate in sham-operated and uninephrectomized
rats 14 days after uninephrectomy
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When the urinary dopamine and amine metabolites are expressed in
nanomoles per gram kidney per kilogram body weight per day, it became
evident that the urinary excretion of dopamine, DOPAC, and HVA, but not
3-MT, in Unx rats was greater than in Sham rats (Table
2). The urinary excretion of
norepinephrine did not differ between Sham and Unx rats. Taken
together, these data suggest that Unx rats may have an enhanced
capability to synthesize dopamine, which is then converted to
deaminated (DOPAC) and deaminated plus methylated (HVA) metabolites. In
agreement with this view is the result of an increase in AADC in the
remnant kidney. AADC activity was determined in homogenates of renal
cortex with L-dopa (100-10,000 µM), which resulted
in a concentration-dependent formation of dopamine (Fig.
1). The Vmax
values for renal AADC using L-dopa as the substrate in Unx
rats were found to be significantly (P < 0.01) higher
than those observed in Sham rats (Table
3). The decarboxylation reaction was a
saturable process, with Km values of the same
magnitude. By contrast, Vmax values for
intestinal AADC in Unx rats were similar to those observed in Sham rats
(Table 3). This difference between renal and intestinal AADC in
response to uninephrectomy suggests that the increase in AADC in renal tissues may correspond to a local response. On the other hand, to gain
confidence for the interpretation that increases in AADC were unrelated
to increases in total protein, we also measured the activities of ALKP
and 3-GT in the renal cortex from Unx and Sham and found no differences
between the two groups (ALKP: Sham, 395 ± 46; Unx, 366 ± 36 mU/mg protein;
-GT: Sham, 1811 ± 52; Unx, 1,258 ± 94 mU/mg protein).
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Table 2.
Urinary excretion of L-dopa, dopamine, DOPAC, 3-MT, HVA,
norepinephrine, 5-HT, and 5-HIAA in sham-operated and
uninephrectomized rats
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Fig. 1.
Aromatic L-amino acid decarboxylase (AADC)
activity in homogenates of renal cortex (A) and jejunal
mucosa (B) obtained from uninephrectomized (Unx;
) and sham-operated (Sham; ) rats. AADC
activity is expressed as the rate of formation of dopamine (in
nmol · mg protein 1 · 15 min 1) vs. concentration of
L-3,4-dihydroxyphenylalanine (L-dopa; µM).
Symbols represent means of 5 experiments/group, and error bars
represent SE.
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Table 3.
Kinetic parameters (Vmax and Km) of AADC,
MAO-A, MAO-B, and COMT activities in homogenates of renal cortex and
jejunum from sham-operated and uninephrectomized rats
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Because the increase in the urinary excretion of dopamine metabolites
(DOPAC and HVA) was slightly greater than the increase in urinary
dopamine, we felt it was worthwhile to examine the activities of the
enzymes responsible for these metabolic transformations (MAO-A, MAO-B,
and COMT). Homogenates of renal cortex from both Unx and Sham rats were
found to deaminate quite actively both [3H]5-HT and
[14C]
-PEA. Deamination of [14C]
-PEA
was a high-affinity process when compared with that for [3H]5-HT, as expected for specific substrates of MAO-B
and MAO-A, respectively. As shown in Table 3,
Vmax and Km values were
similar in both experimental groups, suggesting that increases in the urinary excretion of DOPAC in Unx rats was not related to increases in
deamination of renal dopamine. Similarly, Vmax
and Km values for COMT in Unx rats did not
differ from those for Sham rats, suggesting that increases in the
urinary excretion of HVA in Unx rats was not related to increases in
the O-methylation of renal dopamine or DOPAC.
Considering the high rate of output of renal dopamine, tissue levels of
the amine may not reflect the rate of synthesis (40). As
shown in Table 4, tissue levels of
L-dopa, dopamine, DOPAC, and norepinephrine in fragments
from renal cortex of Unx rats were slightly lower than those in Sham
rats, although the difference did not achieve statistical significance.
This contrasts with the urinary excretion of dopamine and DOPAC in Unx
rats and reinforces the view that renal dopamine may not be accumulated
in storage structures (40, 43). Levels of
L-dopa, dopamine, and DOPAC in the jejunal mucosa were
similar to those occurring in the renal cortex and did not differ
between Unx and Sham rats. Higher levels of norepinephrine in the
kidney than those occurring in the jejunal mucosa reflect the intensity
of sympathetic innervation of the former.
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Table 4.
Absolute and relative tissue levels of L-dopa, dopamine and
norepinephrine in the renal cortex and jejunal mucosa from
sham-operated and uninephrectomized rats
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Effect of HS Intake
HS intake for 24 h did not affect body weight in Sham and Unx
rats. Liquid and food intake in both Sham and Unx rats did not differ
during either NS or HS intake (Table 5).
The urine volume in Unx rats on NS intake did not differ from that in
Sham rats, and both groups of rats responded with an increase in urine
volume during HS intake (Table 5). The urinary excretion of creatinine between Unx and Sham rats on NS and HS intake was not significantly different, but creatinine clearance was significantly lower in Unx rats
(Table 5). Fractional excretion of sodium during NS intake in Unx was
significantly greater than in Sham rats (Fig. 2). As expected, fractional excretion of
sodium during HS intake increased significantly, and this increase was
more pronounced in Unx than in Sham rats (Fig. 2). Urinary excretion of
potassium was similar in Sham and Unx rats and did not change during HS intake (Table 5). Urine osmolality in Unx rats on a NS intake was
slightly lower than that in Sham rats. During HS intake, Unx and Sham
rats responded with a slight decrease in urine osmolality (Table 5).
Plasma levels of electrolytes (sodium, potassium, and chloride) and
plasma osmolality were similar in Sham and Unx rats and were not
affected by HS intake (data not shown).
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Table 5.
Body weight, renal mass, liquid intake, food intake,
Na+ and K+
intake, urine volume, urine creatinine, urinary
Na+ and K+,
urine osmolality, and creatinine clearance in sham-operated and
uninephrectomized rats on NS or HS intake
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Fig. 2.
Effect of high salt intake on the fractional excretion of
sodium in Unx and Sham rats. Bars represent means of 5 determinations/group, and error bars represent SE.
#Significantly different from corresponding values for rats
on normal salt (NS) intake within the group (P < 0.05;
Student's t-test). *Significantly different from
corresponding values for Sham rats (P < 0.05;
Student's t-test).
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As mentioned before, HS intake has been shown to increase the synthesis
of renal dopamine (17, 36, 41), this being accompanied by
increases in the urinary excretion of dopamine and DOPAC (2, 14,
15, 45). As shown in Fig. 3, when
Unx rats were placed on a HS intake the urinary excretion of
L-dopa, dopamine, and DOPAC was greater than that during NS
intake. In Sham rats, HS intake produced no increase in the urinary
excretion of L-dopa but significantly increased urinary
levels of dopamine and DOPAC that were, however, less marked than those
in Unx rats. The response of urinary dopamine to HS intake varies
greatly with the strain used. In our laboratory, the increase in
urinary dopamine after HS intake in Wistar rats [31% increase in the
present study; 16% increase in a previous study (47)]
was markedly lower than that observed in Fischer rats [~100%
increase; (45)]. The enhanced urinary excretion of
dopamine, DOPAC, and HVA by Unx rats in response to HS intake may
reflect their enhanced ability to synthesize dopamine, as previously
indicated by increases in AADC. It is interesting to note, however,
that the urinary excretion of norepinephrine in Unx rats on NS
intake (11.4 ± 1.3 nmol · g
kidney
1 · kg body
wt
1 · day
1) was similar to that in
Sham rats (9.3 ± 1.2 nmol · g
kidney
1 · kg body
wt
1 · day
1) and that HS intake
produced no significant change in the urinary excretion of this amine
in both groups of animals (Sham, 12.5 ± 1.4; Unx, 14.4 ± 1.2 nmol · g kidney
1 · kg body
wt
1 · day
1).

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Fig. 3.
Effect of high salt intake on urinary levels of L-dopa
(A), dopamine (B), 3,4-dihydroxyphenylacetic acid
(DOPAC; C), 3-methoxytyramine (3-MT; D),
and homovanillic acid (HVA; E) in Unx and Sham rats. Bars
represent means of 5 determinations/group, and error bars represent SE.
#Significantly different from values for rats on NS intake
within the group (P < 0.05; Student's
t-test). *Significantly different from corresponding values
for Sham rats (P < 0.05; Student's
t-test).
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Because we were interested to know whether the enhanced urinary
excretion of sodium in Unx rats during HS intake might be related to
their enhanced ability to produce renal dopamine, we decided to test
the effect of a D1 dopamine-receptor antagonist. Because
Sch-23390, one of the most used D1-receptor antagonists in
in vivo experimental studies, is endowed with a short half-life, it was
decided to submit Unx and Sham rats to acute oral HS intake and measure
urinary sodium excretion during a 6-h period. During this period, two
intraperitoneal injections of Sch-23390 (30 µg/kg) were given every
3 h to secure an effective D1 receptor blockade. The
experiment started with the oral administration of 20 ml/kg 0.1%
saline, and the rats were then placed in metabolic cages for the
collection of urine; during this 6-h period, animals had free access to
food and 1.0% saline in their drinking water. As shown in Fig.
4, the urinary excretion of sodium was
markedly greater in Unx than in Sham rats. The effect of Sch-23390 was a marked (P < 0.05) decrease in the urinary excretion
of sodium in Unx (31% reduction), whereas in Sham rats the decrease in
urinary sodium (19% reduction) did not attain a significant
difference. Liquid intake during the 6-h period in Sch-23390-treated
Unx rats (93.1 ± 10.3 ml/kg) was similar to that in
Sch-23390-treated Sham rats (80.6 ± 8.3 ml/kg).

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Fig. 4.
Effect of Sch-23390 (60 µg/kg) on the urinary excretion
of sodium in Unx and Sham rats during HS intake. Bars represent means
of 5 determinations/group, and error bars represent SE.
#Significantly different from corresponding control values
(P < 0.05; Student's t-test).
*Significantly different from corresponding values for Sham rats
(P < 0.05; Student's t-test).
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Isotonic Saline Volume Expansion
The urinary sodium excretion during (0-60 min) and after
(90-240 min) isotonic saline volume expansion (2.5% of body wt)
in Sham and Unx rats is depicted in Fig.
5A. L-Dopa (0.1 mg/kg) or vehicle (0.9% saline, 0.4 ml/kg) was administered
intraduodenally immediately after volume expansion was completed. The
accumulated sodium excretion during the first 90 min of the experiment
(t = 0-90 min; i.e., just after completion of
volume expansion and before L-dopa administration) was
24.3 ± 5.1 µmol in Unx and 23.1 ± 6.6 µmol in Sham rats
(P = 0.54). After the administration of L-dopa at t = 90 min, the natriuretic
response to volume expansion was larger and faster in Unx than in Sham
rats. This effect was particularly evident during the next two
subsequent collection periods (t = 90-120 and
t = 120-150 min), when differences in the urinary
excretion of sodium between Unx and Sham rats attained a statistical
significance (P < 0.05). The enhanced natriuretic response in Unx rats coincided with the peak of urinary excretion of
dopamine in the period t = 90-120 min, which was
greater (P < 0.05) than that observed in Sham rats
(Fig. 5B). The accumulated urinary excretion of sodium,
dopamine, DOPAC, 3-MT, and HVA after L-dopa administration
was markedly (P < 0.05) larger in Unx than in Sham
rats (Table 6). Glomerular filtration
rate, evaluated as the clearance of [3H]inulin, in Unx
rats (1.99 ± 0.27 ml · min
1 · g
kidney wt
1) was greater (P < 0.05) than
in Sham rats (1.32 ± 0.22 ml · min
1 · g kidney wt
1).
The administration of L-dopa (0.1 mg/kg) failed to affect
glomerular filtration rate in both groups of rats (Unx, 1.85 ± 0.26; Sham, 1.31 ± 0.29 ml · min
1 · g kidney wt
1).

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Fig. 5.
Effect of L-dopa (0.1 mg/kg) administration
on the urinary excretion of sodium (A) and urinary levels of
dopamine (B) in Unx (filled symbols) and Sham (open symbols)
rats before (0-60 min), during (60-90 min), and after
(90-240 min) isotonic volume expansion. Arrow, administration of
L-dopa (0.1 mg/kg, intraduodenal; squares) or vehicle
(0.9% saline, 0.4 ml/kg; circles). Symbols represent means of 5 experiments/group, and error bars represent SE. *Significantly
different from corresponding values for Sham rats (P < 0.05; Student's t-test).
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Table 6.
Accumulated urinary excretion of sodium, L-dopa, dopamine,
DOPAC, 3-MT, and HVA in isotonic volume expanded
sham-operated and uninephrectomized rats after
L-dopa (0.1 mg/kg) administration
|
|
Na+-K+-ATPase Activity
Basal Na+-K+-ATPase activity in renal
proximal tubules of Unx rats was similar to that observed in Sham rats
(138.2 ± 8.6 vs. 133.1 ± 4.5 nmol
Pi · mg protein
1 · min
1). As shown in Fig.
6, dopamine caused a
concentration-dependent reduction in
Na+-K+-ATPase activity in proximal tubules of
Unx and Sham rats, being endowed with the same inhibitory potency in
Unx and Sham rats. In contrast to the activity observed at the kidney
level, uninephrectomy resulted in a significant reduction in
Na+-K+-ATPase activity in jejunal epithelial
cells (Fig. 7). In jejunal epithelial
cells from Sham rats, dopamine (1 µM) failed to inhibit Na+-K+-ATPase activity (175.3 ± 9.9 vs.
171.1 ± 6.3 nmol Pi · mg protein
1 · min
1), whereas in Unx rats it
produced a significant reduction (Fig. 8). Figure 8 shows the effect of dopamine
(1 µM) in the absence and in the presence of SKF-83566 (1 µM),
S-sulpiride (1 µM), or SKF-83566 (1 µM) plus
S-sulpiride (1 µM) on
Na+-K+-ATPase activity in renal proximal
tubules and jejunal epithelial cells from Unx rats. As shown in Fig. 8,
the effect of dopamine in both preparations was antagonized by the
selective D1-receptor antagonist (SKF-83566) but not by the
selective D2-receptor antagonist (S-sulpiride).

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Fig. 6.
Concentration-dependent effect of dopamine on
Na+-K+-ATPase activity in the renal proximal
tubules of Unx and Sham rats. Symbols represent means of 5 experiments/group, and error bars represent SE. Basal
Na+-K+-ATPase activity in Unx and Sham rats was
138.2 ± 8.6 and 133.1 ± 4.5 nmol
Pi · mg
protein 1 · min 1, respectively.
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Fig. 7.
Na+-K+-ATPase activity in
isolated jejunal epithelial cells from Unx and Sham rats. Bars
represent means of 5 determinations/group, and error bars represent SE.
*Significantly different from corresponding values for Sham rats
(P < 0.05; Student's t-test).
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Fig. 8.
Effect of dopamine (DA; 1 µM), dopamine (1 µM)+SKF-83566 (SKF; 1 µM), and dopamine (1 µM)+S-sulpiride (Sulp, 1 µM), and dopamine (1 µM)+SKF-83566+S-sulpiride on
Na+-K+-ATPase activity in isolated proximal
tubules (A) and jejunal epithelial cells (B)
from Unx rats (control, Ct). Bars represent means of 4-5
experiments/group; error bars represent SE. Basal renal
Na+-K+-ATPase activity in Sham rats and basal
intestinal Na+-K+-ATPase activity in Unx rats
were 131.1 ± 5.8 and 104.6 ± 12.8 nmol
Pi · mg
protein 1 · min 1, respectively.
*Significantly different from corresponding control values
(P < 0.05; Student's t-test).
#Significantly different from values for dopamine alone
(P < 0.05; Student's t-test).
|
|
Jejunal Sodium Absorption
Jejunal sodium absorption was studied in Sham and Unx rats
subjected to jejunal perfusion in the presence of a nonabsorbable marker. As shown in Fig. 9, jejunal net
sodium absorption in Unx was significantly (P < 0.05)
lower than in Sham rats. The results presented in Fig. 9 are the
algebraic sum of net sodium absorption during the two collection
periods (20 min each, immediately after 50 min of equilibration). The
consistency of the feces in Unx rats, however, was similar to that in
Sham rats.

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Fig. 9.
Net sodium absorption (µmol · 40 min 1 · g dry tissue 1) in the
perfused jejunum from Unx and Sham rats. Results are the algebraic sum
of net sodium absorption during the 2 collection periods (t
= 0-20 and t = 20-40 min). Bars represent
means of 4 experiments/group, and error bars represent SE.
*Significantly different from corresponding values for Sham rats
(P < 0.05; Student's t-test).
|
|
 |
DISCUSSION |
In the present study, uninephrectomy was associated with the known
consequences of partial renal ablation, namely, compensatory renal
growth and changes in solute transport (18). Early after uninephrectomy, sodium transport per unit kidney was found to be
enhanced, this being due to the time lag between an increased transport
requirement and tubule growth. Several studies indicated that the early
enhancement of sodium transport, evidenced by an increase in
Na+-K+-ATPase activity, disappears with
progressive renal growth. Major increases in
Na+-K+-ATPase activity occur early after
uninephrectomy and are particularly evident in the medulla (13,
22, 35). Two weeks after uninephrectomy, Na+-K+-ATPase activity in renal proximal
tubules was found to be similar to that in corresponding controls
(27), which is in agreement with the results reported
here. More recently, the cell rubidium concentration after a 30-s
rubidium infusion, an index of Na+-K+-ATPase
activity, as well as sodium concentrations were unaltered in cells of
all nephron segments in Unx rats (32). The finding that
inhibition of renal Na+-K+-ATPase activity by
dopamine in Unx rats was similar to that observed in Sham rats agrees
with the view that enhanced natriuresis in Unx may have to do with
enhanced renal dopaminergic activity and not to differences in
Na+-K+-ATPase activity. This suggestion fits
well with the finding that treatment of Unx rats with the
D1-receptor antagonist Sch-23390 was accompanied by a
marked decrease (31% reduction) in the urinary excretion of sodium.
Recently, in a chronic renal failure experimental model in the rat, a
5/6 nephrectomy was shown to be accompanied by significant reductions
in the total kidney levels of the proximal tubule sodium transporter
isoform 3 Na+/H+ exchanger, type 2 Na-Pi cotransporter, and
Na+-K+-ATPase (24). However, the
densities per nephron of these transporters were not significantly
altered (24), which is consistent with the view that
densities of sodium transporters did not increase proportionately to
the extensive renal hypertrophy. Although reductions in total kidney
sodium transporters might justify the enhanced increase in urinary
sodium, the fact is that Isaac et al. (20, 21) showed that
the remnant kidney (2/3 reduction in renal mass) exhibits increased
urinary excretion of dopamine and exaggerated dopamine-sensitive
phosphaturic response to parathyroid hormone. Thus it is likely that
the enhanced activation of the renal dopaminergic system may also
contribute to some of the adaptations of renal function after
uninephrectomy, namely, enhanced diuresis and natriuresis, or even more
drastic reductions in renal mass.
The results presented here clearly show that Unx rats have in fact an
increased renal dopaminergic activity and respond to HS intake with a
further increase in dopamine synthesis, which is accompanied by a
marked increase in urinary sodium excretion, this being sensitive to
D1 receptor blockade. This increased renal dopaminergic
activity and enhanced natriuresis, in the presence of unaltered
sensitivity of Na+-K+-ATPase to inhibition by
dopamine, suggest that renal dopamine may play an important role in
keeping Unx rats within sodium balance. High AADC activity, and high
dopamine and amine metabolite levels in urine, evidenced the increased
renal dopaminergic activity in Unx rats. Compensatory renal growth is
most likely not a determining factor in this increase, because the
activities of other enzymes, such as MAO-A, MAO-B, COMT,
Na+-K+-ATPase, ALKP, and
-GT, localized in
the same cellular structures, were found not to differ between Unx and
Sham rats. It is interesting to note that AADC activity in jejunal
epithelial cells failed to change after uninephrectomy, suggesting that
the increase in enzyme activity observed in renal proximal tubules is
the result of local adaptations. On the other hand, the presence of
increased dopaminergic activity in Unx rats was not accompanied by an
increase in the tissue levels of either dopamine or of its precursor,
L-dopa. This lack of correlation between renal AADC
activity and tissue levels of dopamine contrasts with the positive
correlation between renal AADC activity and the high urinary levels of
dopamine, and particularly those of DOPAC, in Unx. The most likely
explanation for this apparent discrepancy might have to do with the
nature of this nonneuronal dopaminergic system. Both the amine storage structures normally present in monoaminergic neuronal systems and the
classic mechanisms for the regulation of amine formation and release do
not appear to be present or in operation (40); the basic
mechanisms for the regulation of this system appear to depend on the
availability of L-dopa, its fast decarboxylation into
dopamine, and in precise and accurate cell- outward amine transfer
mechanisms (38, 39, 43). Newly formed dopamine, on the
other hand, is extensively deaminated to DOPAC, the increased levels of
which indicate the presence of enhanced formation of the parent amine
(40). The finding that the urinary excretion of DOPAC
followed quite closely the urinary excretion of the parent amine
suggests that deamination of dopamine was not compromised and that HS
intake did not interfere with this process. This further supports our
previous suggestion that urinary DOPAC is a good marker of renal
production of dopamine and, simultaneously, a good index of cell
integrity and viability (8, 28, 29). The finding that
treatment of Unx rats with the D1-receptor antagonist Sch-23390 was accompanied by a marked decrease (31% reduction) in the
urinary excretion of sodium is in agreement with the suggestion that
renal dopamine exerts a tonic effect in reducing renal sodium absorption. The effect of Sch-23390 in Sham rats was a 19% reduction in the urinary excretion of sodium, a difference that was not statistically significant. In this respect it is interesting to note that Unx dogs, but not Sham dogs, responded to Sch-23390 with
antinatriuresis (37); this corresponded to the first
observation on the tonic role of endogenous renal dopamine as a local
natriuretic hormone.
The finding that uninephrectomy resulted in a significantly lower
jejunal net sodium absorption and reduced jejunal
Na+-K+-ATPase activity, with recovered
sensitivity to inhibition by dopamine, suggests that a decreased
jejunal absorption of sodium may take also place in response to partial
renal ablation, as an example of renal-intestinal concerted action. At
the intestinal level, previous studies have shown that the inhibitory
effects of dopamine on jejunal sodium absorption and
Na+-K+-ATPase activity in rat jejunal
epithelial cells are limited to animals under 20 days of age, adult
animals being insensitive to the inhibitory effects of dopamine
(12, 44, 46). Intestinal function has a great impact
during early postnatal life, not only on the uptake of nutrients but
also on the maintenance of electrolytes and water metabolism (19,
50). In fact, although nephrogenesis is completed at birth,
renal tubular function continues to develop postnatally, and the kidney
has a limited capacity to regulate fluids and electrolyte homeostasis
(34). The lack of effect of dopamine on jejunal
Na+-K+-ATPase activity in adult animals
coincided with the period in which renal function has reached
maturation (12, 46). The finding that uninephrectomy was
accompanied by a significant decrease in jejunal net sodium absorption
and jejunal Na+-K+-ATPase activity further
suggests that adaptations to reduction in renal mass, to keep Unx rats
within sodium balance, may also involve a reduction in intestinal
sodium absorption. In this respect, it is interesting to verify that
Na+-K+-ATPase in jejunal epithelial cells
recovered the sensitivity to inhibition by dopamine, suggesting that
the dopaminergic influence may play a role in this process of
renal-intestinal cross talk. Furthermore, the similarity of effects of
dopamine on Na+-K+-ATPase in proximal renal
tubules and jejunal epithelial cells, namely, the intensity of the
inhibitory effect and the subtype of receptor involved
(D1), strongly suggests that the dopaminergic influence on
sodium transport across epithelia assumes particular importance when
mechanisms involved in maintenance of sodium balance are disturbed. In
this context, it is interesting to stress the suggestion that the small
intestine contains a natriuretic factor that may be also involved in
sensing sodium (16, 26). However, because the consistency
of the feces in Unx rats was similar to that in Sham rats, it is quite
possible that the decrease in water and sodium absorption in the
jejunum of Unx rats may have been compensated for at a more distal
level. Studies are in progress to evaluate this aspect.
In conclusion, it is suggested that uninephrectomy results in increased
renal dopaminergic activity that responds to HS intake with a further
increase in dopamine synthesis. This increased renal dopaminergic
activity and enhanced natriuresis indicate that renal dopamine may play
an important role in keeping Unx rats within sodium balance. The
significant reduction in jejunal Na+-K+-ATPase
activity, with recovered sensitivity to inhibition by dopamine, also
suggests that decreased jejunal absorption of sodium may take place in
response to partial renal ablation, as an example of renal-intestinal
cross talk.
 |
ACKNOWLEDGEMENTS |
This study was supported by Grants PECS/S/SAU/29/95 and
PRAXIS/2/2.1/SAU/1386/95 from the Fundação para a
Ciência e a Tecnologia.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: P. Soares-da-Silva, Institute of Pharmacology and Therapeutics, Faculty of
Medicine, 4200 Porto, Portugal (E-mail:
patricio.soares{at}mail.telepac.pt).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 25 February 2000; accepted in final form 19 July 2000.
 |
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