1 Department of Pediatrics, We investigated the effects of hypotonic
saline-induced modifications of extracellular volume and sodium
handling on the renal and metabolic response to amino
acids (AA). Renal hemodynamics (Inutest, p-aminohippurate
clearance), plasma AA, and glucagon levels, as well as urea and sodium
excretion, were studied in seven adult volunteers infused for 2 h, on
six separate occasions, according to the following protocols:
1) high-AA solution (300 mg · min
renal hemodynamics; urea excretion; glucagon; sodium
handling
THE INFUSION OF AMINO ACIDS (AA) increases glomerular
filtration rate (GFR) and renal plasma flow (RPF) (5, 14, 18, 21). Some
lines of evidence suggest that an intermediate step in the splanchnic
region, characterized by stimulation of gluconeogenesis, ureagenesis,
and glucagon secretion, is necessary for the renal response to AA to
take place (6, 8, 9, 11, 18). However, there is a growing body of
information indicating that intrarenal mechanisms related to
extracellular volume control, renal sodium handling, and
tubuloglomerular feedback (TGF) are important in the genesis of
AA-induced hyperfiltration: a low-sodium diet preceding AA infusion
prevents the glomerular response (19); AA-induced hyperfiltration is
associated with increased proximal sodium reabsorption in vivo (7, 16)
and in vitro (2, 21); and a protein meal fails to induce a rise in GFR
in dogs with impaired proximal tubular function due to experimental
Fanconi's syndrome or to administration of some diuretics (22, 23).
In this study, in a first set of experiments, we investigated the renal
and metabolic response to a low and a high dose of AA (compared with
the effects of a saline infusion alone). Then, in a second set, we
studied the acute modifications of this response when a low- and a
high-sodium solution were infused with the AA. The results showed that
hypotonic saline infusion inhibited AA-induced hyperfiltration.
Seven healthy male volunteers aged 22-25 yr were selected for the
study. Clinical and laboratory investigations revealed no indication of
any disease. Dietary records were obtained and urinary sodium and
nitrogen excretion were determined for 3-4 days before each test
to calculate average dietary intakes. No differences were observed
among the estimated sodium and protein intakes of each period. The
subjects underwent six experimental protocols in random order with
intervals of at least 1 mo between each of them. All tests were
performed in the morning after an overnight fast. Informed consent was
obtained from each subject, and the study was approved by the
institutional review board.
Study Design
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
1 · 1.73 m
2);
2) low-AA solution (150 mg · min
1 · 1.73 m
2);
3) low AA + 2,000 ml/1.73 m2 of 0.23% saline solution;
4) high AA + 0.23% saline;
5) high AA + 0.45% saline; and
6) 0.45% saline alone. The
glomerular filtration rate (GFR) rise induced by the high-AA solution
was similar to that induced by the low-AA solution (
GFR = +24 ± 6 and +20.2 ± 7 ml · min
1 · 1.73 m
2, respectively), whereas
the plasma AA and glucagon levels and urea excretion rate increases
were related to AA dose. The addition of 0.23% saline to the low-AA
solution and of 0.45% saline to the high-AA solution blunted the renal
hemodynamic response (
GFR = +6.6 ± 10.1 and +11.4 ± 8.3 ml · min
1 · 1.73 m
2, respectively) without
modifying the pattern of plasma AA and glucagon levels and urea
excretion observed with the AA infusion alone. Urinary sodium excretion
increased from baseline with each protocol and rose even further when
saline was added to AA. A negative correlation
(r =
0.38,
P < 0.05) was found between the changes from basal values in GFR and those in sodium excretion rate
with high-AA infusion at different levels of sodium concentration. These data suggest that AA-induced hyperfiltration might be blunted by
hypotonic saline infusion, possibly through an acute modification of
renal sodium handling and extracellular volume.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Table 1.
Characteristics of the study protocols and clinical data of the
subjects at the time of administration of each protocol
Body weight and systolic and diastolic blood pressures were measured before and at the end of each test.
Protocol 1: High AA. After the
baseline period, a 2-h intravenous infusion of AA (10% Freamine III,
Baxter) was administered at the rate of 3 ml · min1 · 1.73 m
2, corresponding to 300 mg · min
1 · 1.73 m
2 (2,550 µmol · min
1 · 1.73 m
2) of AA.
Protocol 2: Low AA. This protocol was
like protocol 1 except that the
concentration of the AA solution was 5%, so that AA were infused at
the rate of 150 mg · min1 · 1.73 m
2 (1,275 µmol · min
1 · 1.73 m
2).
Protocol 3: Low AA, 0.23% (low) saline
concentration. This protocol was like
protocol 2 except that 2,000 ml/1.73 m2 (17 ml · min1 · 1.73 m
2) of a 40 mmol/l
(0.23%) solution of sodium chloride was infused for 2 h together with
the 5% AA (150 mg · min
1 · 1.73 m
2).
Protocol 4: High AA, 0.23% (low) saline
concentration. This protocol was like
protocol 1 except that 2,000 ml/1.73 m2 (17 ml · min1 · 1.73 m
2) of 40 mmol/l (0.23%)
sodium chloride was infused for 2 h together with the 10% AA (300 mg · min
1 · 1.73 m
2).
Protocol 5: High AA, 0.45% (high) saline
concentration. This protocol was like
protocol 1 except that a solution of
2,000 ml/1.73 m2 (17 ml · min1 · 1.73 m
2) of 80 mmol/l (0.45%)
sodium chloride was infused for 2 h together with the 10% AA (300 mg · min
1 · 1.73 m
2).
Protocol 6: 0.45% saline alone. An 80 mmol/l (0.45%) solution of sodium chloride alone was infused for 2 h
at the rate of 20 ml · min1 · 1.73 m
2.
Analysis, Calculations and Statistics
We calculated Inutest and PAH clearances on the basis of their urinary concentrations at the collection times and the means of the plasma concentrations measured in each sample drawn during each collection period. Hematocrit (Hct), plasma urea, electrolytes, glucagon, and AA were measured in the last sample drawn during each period; urinary urea, osmolality, and electrolytes were measured during each urine collection period. All determinations were performed in duplicate. The blood samples for the determination of Inutest, PAH, sodium, and osmolality were collected in lithium-heparin, those for AA determinations in sodium-heparin, and those for glucagon determinations in EDTA + Trasylol.Plasma and urinary Inutest and PAH were measured as previously described (7), AA was measured by ion-exchange chromatography, glucagon was measured by radioimmunoassay (RIA-Mat; Byk Gulden), and urinary urea was measured by standard automated laboratory procedures using Hitachi 737 equipment. Plasma and urinary electrolytes were analyzed by flame photometry. Plasma and urinary osmolality were measured using a Fiske osmometer (freezing-point technique).
Filtration fraction was calculated as GFR/RPF, and fractional Na excretion (FENa) was calculated as UNa/PNa × Pinulin/Uinulin, where UNa/PNa is the ratio of urinary to plasma sodium concentration, and Pinulin/Uinulin is the ratio of plasma to urinary inulin concentration.
Data are reported as group means ± SD. Differences within each
protocol were determined comparing data at baseline and 1 and 2 h of
treatment; differences between protocols were analyzed as changes from
baseline of variables at 1 and 2 h (: value at 1 h minus baseline
and at 2 h minus baseline). The statistical significance of differences
was evaluated by analysis of variance for randomized blocks for
repeated measures, followed by Duncan's multiple range test.
Correlations between variables were performed using linear multiple
regression analysis.
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RESULTS |
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The two protocols (protocols 1 and 2) in which AA solutions were infused without sodium did not cause variations in body weight, blood pressure, or Hct, whereas there was a significant increase in weight and a fall in Hct at the end of protocols 5 and 6 (high sodium content). A significant reduction in Hct was found at the end of protocols 3 and 4 (low sodium content). Blood pressure, plasma sodium, and plasma osmolality never changed (Table 1).
Renal and Metabolic Effects of High-AA Infusion and of 0.45% Saline Solution: Protocols 1 and 6
As shown in Table 2, high-AA infusion (protocol 1) induced a significant increase in GFR and RPF; with saline (protocol 6), neither GFR nor RPF changed. High-AA infusion caused a significant increase in plasma AA, plasma glucagon levels, and the urinary urea excretion rate at 1 h, followed by a further significant rise at 2 h (Table 3). Diuresis, sodium excretion rate and FENa were significantly modified only at 2 h (Table 4). On the contrary, saline solution did not change the above metabolic indexes (Table 3) but induced a brisk increase in diuresis, sodium excretion, and FENa at 1 h and a further significant rise at 2 h of the infusion (Table 4).
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|
|
AA Dose-Related and Saline Infusion-Related Effects on Renal and Metabolic Response to AA: Protocols 2-5
Overall analysis and results of the data obtained with protocols 2-5 are shown in Tables 2-4 and in Figs. 1 and 2. The low-AA infusion (protocol 2) induced a GFR and RPF increase only at 2 h (Table 2), but the greatest GFR increase (between 1 and 2 h) was not significantly different from that obtained with the high-AA infusion (+20.2 ± 7 and +24 ± 6 ml · min
|
|
The addition of a 40 mmol/l (0.23%) sodium solution to the low AA dose
(protocol 3) blunted the renal
hemodynamic response (the greatest GFR increase, 6.6 ± 10.1 ml · min1 · 1.73 m
2) (Table 2; Fig. 1);
the pattern of the metabolic data did not differ with
protocol 2 (Table 3). When the 0.23%
sodium solution was infused together with the high AA dose
(protocol 4), the glomerular response was not affected (the greatest GFR increase, 20.8 ± 18 ml · min
1 · 1.73 m
2) (Table 2; Fig. 1);
the rise in plasma AA and plasma glucagon was significantly greater
than in protocols 2 and
3 (Table 3). With
protocol 5, the addition of a higher
sodium dose (80 mmol/l, 0.45%) to a high AA dose blunted the GFR (the
greatest GFR change, +11.4 ± 8.3 ml · min
1 · 1.73 m
2) and RPF rise (Table
2; Fig. 1) without modifying the pattern of the metabolic indexes
observed with protocol 4 (Table 3).
Diuresis increased with all of the protocols except protocol 2, and the increase was already observed at 1 h with protocols 4 and 5. Urinary sodium excretion (UNaV) and FENa showed an increase from baseline at 2 h with protocols 2 and 3, whereas with protocols 4 and 5 the increase was already significant at 1 h. With all of the protocols, the increase in UNaV at 2 h was significantly greater than that observed at 1 h. Protocol 5 produced the greatest increase in UNaV of all the protocols, at both 1 and 2 h (Table 4).
A negative correlation (r = 0.38, P < 0.05) between the
change from basal values in GFR (
GFR) and in sodium excretion rate (
UNaV), was found with high-AA
infusion administered with different amounts of sodium
(protocols 1,
4, and
5) (Fig.
3).
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DISCUSSION |
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The most important finding of this study was that hypotonic saline infusion reduced the renal hemodynamic effects of AA. Furthermore, the renal response to AA was not affected with a higher AA dose and was blunted when hypotonic saline of higher concentration was added to the high-AA infusion.
Although an increase in renal hemodynamics following AA infusion is a well-established physiological phenomenon (5, 7, 14, 18, 24), the precise mechanism underlying this response is still under discussion. The demonstration that direct infusion of three nonessential AA (serine, alanine, and proline) in the renal artery of dogs did not affect renal hemodynamics (18), whereas the GFR increased after systemic infusion of these AA, and the close relation between AA-induced hyperfiltration and the rise of some indexes of splanchnic metabolism such as the plasma glucagon/insulin ratio and ureagenesis (9) might suggest that an intermediate splanchnic step is necessary to induce hyperfiltration in vivo. These splanchnic factors, stimulated by hyperaminoacidemia, interact with an ultimate intrarenal pathway to induce AA-dependent hyperfiltration. The finding that hyperfiltration may be induced by AA in the isolated perfused kidney of the rat demonstrates the importance that an intrarenal system may have in the genesis of this phenomenon (2, 13). In the theoretical model recently proposed by Woods (24), such a mechanism has been identified with TGF and is consistent with most (although not all; see Ref. 4) of the literature data; AA-induced variations of sodium reabsorption proximally to the juxtaglomerular apparatus lead to modulation of TGF activity and GFR. This hypothesized role of TGF is supported by a number of observations: AA-induced hyperfiltration is associated with increased proximal sodium reabsorption in vitro (21) and in vivo (7, 16), and the renal response of conscious dogs to a protein meal is inhibited when the reabsorption ability of the proximal tubule is impaired [as in experimental maleic acid-induced Fanconi syndrome (22) or after the administration of diuretics such as acetazolamide that act selectively on the proximal tubule (23)]. Furthermore, it has been shown that TGF activity is modified by dietary protein (20). It has been suggested that glucagon (12), hepatic cAMP (1), and the action of vasopressin on urea recycling (3) could modify NaCl concentration in the diluting segment and in the fluid reaching the macula densa, and thus vary TGF activity.
In the present study, we showed that the increase in GFR and RPF was associated with rises of 60% in plasma AA concentration and of 25-30% in plasma glucagon and urinary urea excretion; further increases, as observed with the higher AA dose, were not associated with any greater stimulation of renal hemodynamics but with a faster response (as early as 1 h). When hypotonic saline was present in the infusion, higher plasma AA and glucagon levels and urinary urea excretion were necessary to stimulate glomerular function. These levels in turn were ineffective when the sodium content of the solution was higher. Therefore, the acute modification of the extracellular volume and renal sodium handling induced by the hypotonic saline infusion seemed to modify the responsiveness of the intrarenal mechanism to the metabolic signal.
Other studies have demonstrated the role of extracellular volume on the mechanism leading to AA-induced hyperfiltration. Ruilope et al. (19) have shown that a volume contraction caused by sodium depletion inhibits AA-induced vasodilation, probably through an excess of vasoconstrictor factors as angiotensin II. Hadj-Aissa et al. (15) observed that protein-induced hyperfiltration is partially blunted by a high water intake, possibly by a direct or indirect effect on urine concentrating activity. Finally, Woods and Cobb (25) found that infusing isotonic saline before and during the administration of glycine prevented the GFR rise in the rat.
The reduced responsiveness of the intrarenal mechanism to the metabolic signal observed by us with the hypotonic saline infusion could be due to an increase in sodium delivery to the distal segment of the nephron secondary to the saline-induced reduction in proximal sodium reabsorption (10) and the consequent insensitivity of the TGF (17). The significant negative correlation that we found between AA-induced GFR variations and modifications of sodium excretion caused by saline infusion is in line with this hypothesis.
In conclusion, the addition of hypotonic saline to the AA infusion reduced the response of renal hemodynamics to the metabolic stimuli produced by AA infusion, possibly through an increase in urinary sodium excretion. These conclusions, derived from an experimental situation characterized by maximal diuresis (and then far from physiological levels), may not be generalized without reservation to the more normal conditions.
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ACKNOWLEDGEMENTS |
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We thank A. M. Green for revision of the English text.
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FOOTNOTES |
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This study received financial support from the Associazione per il Bambino Nefropatico.
Address for reprint requests: A. Claris-Appiani, Clinica Pediatrica De Marchi, Via Commenda 9, I-20122 Milan, Italy.
Received 31 December 1997; accepted in final form 30 September 1998.
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