Introduction

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Although the increase in GFR and renal blood flow after an AA load are well-known phenomena, the underlying mechanisms are unsatisfactorily understood (see for review Woods, 1993; Lang et al., 1995). Because 1) i.v. infusion of dopamine induces similar changes of renal hemodynamics as a solution of amino acids (ter Wee et al., 1986) and 2) specific inhibition of D₂ receptors abolished the AA-induced increase in GFR (Mendez et al., 1991; Mühlbauer et al., 1994), renal dopamine might be involved in this GFR response. Dopamine receptors have been classified into the D₁-like and the D₂-like family (Seeman and van Tol, 1994) and could be demonstrated in the central nervous system (Seeman, 1980) as well as various peripheral tissues (Clark, 1981; Jose et al., 1992). Earlier, D₁ receptors were suggested to mediate the renal hemodynamic effects of dopamine (Bhat et al., 1986), but D₂ receptors might be involved as well (Seri and Aperia, 1988). To test the hypothesis that pharmacological activation of D₂-like receptors mimicked the AA-induced hyperfiltration, the renal effects of QP, an agonist of the D₂ receptor family (Brodde, 1989), were compared with those of an AA infusion in anesthetized rats. In an attempt to differentiate the involvement of central and peripheral D₂ receptors, the experiments were performed also during administration of SUL, a centrally and peripherally acting D₂ receptor antagonist, or DOM that, due to its inability to cross the blood-brain barrier (Laduron and Leysen, 1979), affects only peripheral D₂ receptors.

	Methods

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Animals and microsurgical preparation. Experiments were performed in male Sprague-Dawley rats (Charles River, Sulzfeld, Germany), 220 to 290 g in weight, with free access to standard rat food (Altromin 1320, Altromin, Lage, Germany) and tap water. On the day of the clearance studies, rats were anesthetized with thiopental sodium (80 mg · kg¹ i.p.; TRAPANAL, Byk Gulden, Konstanz, Germany) and placed on a heated table (RT, Effenberger, Munich, Germany), which was thermo-controlled to maintain the rectal temperature at 37.2°C. After tracheostomy, two PE catheters were inserted into the right jugular vein for infusion. Another PE-catheter in the left carotid artery was used for drawing of blood samples and continuous monitoring of blood pressure by means of a recorder (WK 280 WKK, Kaltbrunn, Switzerland) connected to an electronic transducer (TBM4, WPI, Heidelberg, Germany). A PE-catheter, inserted deeply in the bladder, served for urine collection.

Design of clearance experiments. The time course of the experiments is shown in figure 1. After surgical preparation the animals were allowed to reach steady-state conditions, defined by stable systemic hemodynamics and constant urinary flow rate. Via the first i.v. catheter ³H-inulin (1.5 µCi · ml¹; NEN, Dreieich, Germany) dissolved in isotonic saline (0.85% NaCl) was infused at a rate of 0.6 ml · hr¹ throughout the entire experiment for assessment of GFR. In experiments in which animals were pretreated with D₂ receptor antagonists, this infusion also contained SUL (15 µg · 100 g¹ · min¹; Sigma Chemicals, Deisenhofen, Germany) or DOM (0.8 µg · 100 g¹ · min¹; Biotrend, Cologne, Germany). Via the second catheter, isotonic NaCl was infused at a rate of 2.4 ml · hr¹. After reaching steady-state, urine was collected in 20-min periods. Blood samples were drawn at the midpoint of each clearance period. After two baseline periods, NaCl infusion was continued in the CON group (n = 6), whereas all other groups (n = 6-7) received either a standard AA solution (10%; Delta-Pharma, Pfullingen, Germany) or QP (Biotrend, Cologne, Germany) at a dose of 0.3 µg · 100 g¹ · min¹. Both solutions (AA and QP) were prepared with isotonic saline. Ten minutes after onset of the AA or QP infusion two CP were performed. The composition of the AA solution (in g · liter¹) was: L-isoleucine 3.8, L-leucine 6.6, L-lysine 9.3, L-methionine 2.8, L-phenylalanine 4.1, L-threonine 4.6, L-tryptophan 1.2, L-valine 4.1, L-arginine 9.2, L-histidine 4.4, aminoacetic acid 7.7, L-alanine 14.3, L-proline 9.2, L-cysteine 0.7, L-glutamic acid 9.9, L-ornithine-L-aspartate 4.6, L-serine 5.9, L-tyrosine 0.5. 

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Time course of the clearance experiments in anesthetized rats. Animals were allowed to recover from surgery for 60 to 90 min. 3H-inulin infusion (in 0.85% NaCl) for assessment of GFR contained no additional drug for vehicle animals; other groups were pretreated with S()-sulpiride (SUL) or domperidone (DOM), respectively. NaCl was infused throughout the entire experiment in time control animals. In all other groups, after two baseline clearance periods (CP 1 and CP 2) either quinpirole (QP) or amino acid (AA) solution was administered with 10 min break until performance of experimental clearance periods (CP 3 and CP 4).

Analyses. Urine volume was measured gravimetrically. Blood samples were centrifugated, and the hematocrit was assessed. Both plasma and urine samples were stored at 80°C until analysis. Sodium was determined by flame photometry (ELEX 6361, Eppendorf, Hamburg, Germany), ³H-inulin radioactivity by a liquid scintillation counter (2550 TR®, Canberra Packard, Frankfurt, Germany). Dopamine was measured by HPLC with electrochemical detection (Sykam, Gilching, Germany) as described previously (Mühlbauer et al., 1997b). In brief, dihydroxybenzylamine was added to the urine sample as internal standard. After pH was adjusted to 8.6, neutral alumina oxide was added. After this absorption step, the samples were washed twice with bidistilled water and finally eluted with phosphoric acid. The eluate was applied onto the reversed phase HPLC system. The mobile phase consisted of a citrate buffer, octane sulfonic acid (sodium salt), methanol and acetonitrile in bidistilled water. Internal standard-corrected recovery of dopamine added to the urine averaged 96 to 104%.

Calculations and statistics. GFR as renal clearance of inulin and fractional sodium excretion were calculated according to the standard formulas. Means of the two base-line and experimental periods were calculated individually. Statistical significance of the differences between baseline (NaCl) and experimental periods (AA or QP infusion) within groups was assessed by the paired two-sided t test. Statistical analysis of the differences among groups was performed by the analysis of variance. P < .05 was considered to be statistically significant. All values are expressed as means of groups ± S.E.M.

	Results

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Time controls and baseline values of all groups. In the CON group no significant changes in GFR, UV, U_NaV as well as FE_Na were observed during the entire experiments. U_DAV, MAP and HR remained unchanged as well (tables 1 and 2). When comparing the baseline values observed in CON animals with those of the other groups, no significant variations among the different pretreatment protocols could be detected.

Renal response to AA or QP infusion in vehicle-treated rats. AA infusion in the AA-VHC group increased GFR significantly by 20 ± 4% (fig. 2). UV, U_NaV and FE_Na were elevated during infusion of AA by 1.5- to 2.5-fold compared to baseline; also mean U_DAV was significantly increased by 2.0-fold (table 1). QP infusion in the QP-VHC group caused a significant increase of GFR by 20 ± 2% (fig. 3). QP increased UV, U_NaV and FE_Na by factors 1.6 to 2.0, whereas U_DAV was slightly reduced compared to baseline (table 1). Neither AA nor QP administration significantly affected MAP, Hct or Na_Plasma. There was a slight but significant decrease in HR due to QP whereas no change was observed during amino AA infusion (table 2).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effect of amino acid (AA) infusion on glomerular filtration rate expressed as mean of individual differences (GFR). In the time control group (CON) isotonic saline (NaCl) was infused throughout the entire experiment. Animals in the vehicle group (AA-VHC) received an AA solution without pretreatment, whereas S()-sulpiride (SUL) or domperidone (DOM) was continuously administered in the other groups. See table 1 for absolute values. Statistical comparison with baseline periods was performed using the paired two-sided t test (* P < .05).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effect of quinpirole (QP) infusion on glomerular filtration rate expressed as mean of individual differences (GFR). In the time control group (CON) isotonic saline (NaCl) was infused throughout the entire experiment. Animals in the vehicle group (QP-VHC) received QP without pretreatment, whereas S()-sulpiride (SUL) or domperidone (DOM) was continuously administered in the other groups. See table 1 for absolute values. Statistical comparison with baseline periods was performed using the paired two-sided t test (* P < .05).

Effect of S()-sulpiride on the renal response to AA or QP infusion. As shown in figure 2 and table 1, pretreatment with SUL completely abolished the AA-induced hyperfiltration (AA-SUL group), in contrast, UV, U_NaV and FE_Na were still elevated 1.5- to 2.5-fold in response to AA administration. The increase of mean U_DAV during infusion of AA in AA-SUL animals was similar to that observed in the AA-VHC animals (table 1). SUL also blocked the QP-induce GFR-rise (QP-SUL group; fig. 3), with baseline levels of GFR slightly higher compared to CON animals. The QP-induced increase in UV, U_NaV and FE_Na (factors 1.5 to 3.0) in SUL-treated rats was similar compared to the VHC group, although the difference was not statistically significant; U_DAV did not change during infusion of QP and was similar to those observed in the CON group (table 1). In SUL-treated animals neither MAP, HR, Hct nor Na_Plasma showed significant changes due to administration of AA or QP (table 2).

Effect of domperidone on the renal response to AA or QP infusion. After DOM pretreatment the AA-induced increase in GFR was attenuated to 12 ± 4% (AA-DOM; fig. 2). In contrast, UV and FE_Na increased 2-fold, U_NaV by a factor of 3.5 during AA infusion; U_DAV was also significantly enhanced (table 1). Compared to VHC, infusion of DOM also attenuated the QP-induced increase in GFR which was 10 ± 2% (QP-DOM; fig. 3). In contrast, DOM did not influence the effects of QP on UV, U_NaV and FE_Na which were 2- to 3-fold increased, whereas U_DAV remained unchanged. As observed in the VHC group, QP also reduced HR slightly but significantly in DOM-treated animals. Neither MAP, Hct, nor Na_Plasma were affected by QP or AA administration during pretreatment with DOM (table 2).

	Discussion

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Our study showed that the increase in GFR during infusion of AA was mimicked in a quantitatively similar manner by QP which has been decribed as an agonist for receptors of the D₂-like family (Brodde, 1989). This result is in accordance with the increase in single nephron GFR in anesthetized rats during systemic QP administration reported by Seri and Aperia (1988). In contrast to these studies, Siragy et al. (1992) found a significant decrease in GFR due to QP in conscious dogs. Possible reasons for this discrepancy might be that the dose of QP used by Siragy et al. (1992) was, although given directly into the renal artery, lower by orders of magnitude compared to the other studies. Whether species differences and, more important, the possible influence of anesthesia might additionally contribute to the contrasting observations has to be clarified in further studies.

To substantiate the involvement of D₂-like receptors in the AA-induced GFR increase, rats were pretreated with the D₂ receptor antagonist SUL. In fact, both AA- and QP-induced hyperfiltration were completely abolished by continuous administration of SUL. These observations are in accordance with the studies by Mendez et al. (1991) and by our group (Mühlbauer et al., 1994, 1997b), in which the changes in renal hemodynamics during AA infusion were inhibited by D₂ receptor blockade. Concerning the action of the D₂-like agonist, Seri and Aperia (1988) found that the QP-induced single nephron-hyperfiltration was abolished in the presence of SUL indicating a specific D₂ receptor action. Taken together, the data strongly support the involvement of D₂ receptors in the AA-induced hyperfiltration. Because QP might also possess affinity to the D₃ subtype of the D₂-like receptor family (Seeman et al., 1991), a possible contribution of this receptor to the hyperfiltration needs further investigation. Due to the lack of selective agonists, the role of D₃ receptors in the regulation of renal function has not been determined so far.

In an attempt to compare the role of central and peripheral D₂ receptors in the modulation of glomerular filtration, we administered DOM, a peripherally acting D₂ receptor antagonist. DOM attenuated both the AA- and the QP-induced hyperfiltration; however, the increase in GFR was not completely abolished as it was in SUL-treated animals. It might be objected that the dose of SUL was markedly higher compared to DOM. However, the dose of SUL used in our study was orientated on data of recent experiments showing a complete inhibitory effect of SUL on the renal hemodynamic response to AA infusion; in that study the SUL effect was dose-dependent suggesting a specific action (Mühlbauer et al., 1997b). In preliminary experiments, the 5-fold higher dose of DOM as used in our study produced no additional inhibitory effect on AA-induced hyperfiltration but affected the systemic hemodynamics (data not shown). Although similar K_B values of SUL and DOM in the rabbit ear artery and rectococcygeus muscle, respectively, have been reported (Brodde, 1989) higher doses of SUL compared to DOM were used in vivo (Brooks and Weinstock, 1991) or in vitro (Starke et al., 1983; Rump et al., 1991) by other investigators. Taken together, dose differences are unlikely to be the reason for the varying modulation of the AA- or QP-induced hyperfiltration by SUL and DOM. Concerning the higher lipid solubility of SUL compared to DOM, it may be argued that the disparity between both D₂ receptor antagonists might be due to greater penetration of SUL into peripheral neural or epithelial compartments. However, continuous administration of both antagonists was initiated approximately 2 hr in advance of the functional experiments which should be a sufficient time span for achieving constant tissue levels of the drugs. Therefore, such an explanation appears unlikely. Taken together, the data suggest that the increase in GFR is mediated by central as well as peripheral D₂-like receptors. Recently we could demonstrate that the increase in GFR due to systemic AA load was completely blocked by chronical renal denervation (Mühlbauer et al., 1997b). Thus, activation of a dopaminergic mechanism by AA infusion might modulate GFR via the neuronal route. Because renal nerves contain both afferent and efferent nerve fibers the exact site of action of the proposed neuronal D₂ mechanism needs further investigation.

Urinary dopamine excretion has been described to rise after protein intake (Williams et al., 1986; Kaufman et al., 1989) and AA load (Mühlbauer et al., 1997a). Renal dopamine is mainly formed in the cells of the proximal tubules from filtered L-DOPA by L-amino acid decarboxylase (Hayashi et al., 1990). Only the catecholamine precursors, L-tyrosine and L-phenylalanine, are responsible for the AA-induced increase in urinary dopamine excretion (Mühlbauer et al., 1997a). In our experiments, urinary dopamine was elevated during infusion of AA which contained the catecholamine precursors but was unaffected by the D₂ receptor antagonists. However, because the latter affected the AA-induced hyperfiltration the response of urinary dopamine excretion and of GFR to AA appears to be dissociated. This observation is in correspondence with a previous study (Mühlbauer et al., 1997b), in which systemic AA elevated GFR and renal dopamine excretion simultaneously; however, if L-tyrosine was omitted from the AA solution, urinary dopamine remained at baseline despite the increase in filtration rate. The enhancement of GFR by QP without affecting urinary dopamine as observed in our study further argues against the idea that dopamine released into the tubular lumen might influence renal hemodynamics. However, urinary excretion of dopamine does not reflect its potential release at other intrarenal sites than the tubular lumen. Thus, an additional paracrine action of dopamine cannot be excluded. As for the gross release of dopamine into the proximal tubule, a role in the regulation of renal hemodynamics appears unlikely.

We conclude that, in the anesthetized rat, dopamine D₂ receptors are involved in the AA-induced glomerular hyperfiltration. Both central and peripheral dopamine receptors appear to contribute to this renal response whereas dopamine excreted into the urine does not seem to play a functional role.