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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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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]beta -phenylethylamine (beta -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 gamma -glutamyl transferase (gamma -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]beta -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]beta -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.


    RESULTS
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MATERIALS AND METHODS
RESULTS
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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

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; gamma -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

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]beta -PEA. Deamination of [14C]beta -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

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).

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).

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).

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma -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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