Department of Surgery, Maastricht University, 6200 MD Maastricht, The Netherlands
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ABSTRACT |
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The kidney has an important function in arginine metabolism, because the kidney is the main endogenous source for de novo arginine production from circulating citrulline. In conditions such as sepsis, nitric oxide (NO) production is increased and is dependent on extracellular arginine availability. To elucidate the adaptive role of renal de novo arginine synthesis in a condition of increased NO production, we studied renal arginine metabolism in a mouse model of endotoxemia. Because arginine flux is largely dependent on protein flux, we also measured protein metabolism in mice. Female mice were injected intraperitoneally with lipopolysaccharide; control mice received 0.9% NaCl. Six hours later, renal blood flow was measured with the use of para-aminohippuric acid. Arginine and protein metabolism were studied using organ-balance, stable-isotope techniques. Systemic NO production was increased in the endotoxin-treated mice. In addition, renal protein synthesis and de novo arginine production from citrulline were increased. However, no effect on renal NO production was observed. In conclusion, increased renal de novo arginine production may serve to sustain systemic NO production. To our knowledge, it was shown for the first time that renal protein synthesis is enhanced in the early response to endotoxemia.
citrulline; kidney; lipopolysaccharide; nitric oxide
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INTRODUCTION |
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THE AMINO ACID ARGININE HAS an important role in cellular regeneration, immune function, and protein breakdown and synthesis (7, 39). Arginine also is the precursor of urea, agmatine, and nitric oxide (NO). In past years, many studies have been undertaken that aimed at increasing or decreasing NO production, either by supplementation of arginine or NO donors (6, 25, 26) or by the administration of nitric oxide synthase (NOS) inhibitors or arginase (5, 15, 33). In general, the effects of these treatments were conflicting and confusing. More detailed information on local arginine metabolism and NO production may be helpful for an understanding of the effects of arginine supplementation or NOS inhibition.
The kidney has an important function in arginine metabolism, because the kidney is the main endogenous source for de novo arginine production (37). Arginine production involves collaboration between the gut and the kidney. Citrulline is produced from glutamine and arginine in the gut and then released into the circulation. The liver does not consume citrulline, which leaves it available for the kidney. In the kidney, citrulline is used for the production of arginine, which, in turn, is released into the circulation (37). The importance of this pathway is illustrated by the fact that arginine becomes an essential amino acid when intestinal citrulline synthesis is inhibited (34).
During sepsis and experimental endotoxemia, the expression of inducible NOS (iNOS) is increased in a variety of organs and cells (27). iNOS is dependent on the extracellular supply of arginine (2, 21), and reduced arginine availability may impair systemic NO production (2, 33). During conditions of increased arginine utilization, the kidney may increase endogenous arginine production to serve systemic NO production;however, it is well known that <5% of arginine disposal is used for NO production.
To unravel the renal metabolic response during enhanced systemic NO production, we measured renal arginine turnover in a mouse model of endotoxemia, which mimics the septic condition (11). We chose experimental endotoxemia as a model for the study of arginine metabolism because increases in plasma nitrite and nitrate data suggest that systemic NO production is increased in this condition (29). We also measured renal protein turnover because arginine turnover is mainly dependent on protein turnover (41). Finally, we measured renal de novo arginine production and related this to arginine and protein turnover.
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METHODS |
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Animals
Female Swiss mice (22-26 g) were obtained from IFFA Credo Broekman (Someren, The Netherlands). The mice were fed standard lab chow and were subject to standard 12:12-h light-dark cycle periods (7:30 AM to 7:30 PM). Room temperature was maintained at 25°C. Experiments were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals (35) and approved by the Ethical Committee of Animal Research of Maastricht University.Experimental Protocol
LPS (Escherichia coli O55:B5, 10 mg/kg body wt ip, Sigma, St. Louis, MO) was injected into the mice (n = 10) (29). Control animals (n = 10) received a corresponding volume of 150 mM NaCl (saline). Drinking water was provided, but food was withheld after the injection of endotoxin or saline to avoid influences of differences of food intake.Five hours after LPS treatment, anesthesia was induced in the mice by an intraperitoneal injection followed by a subcutaneous infusion of ketamine and medetomidine (19). Mice were sufficiently anesthetized after 10 min so that surgical procedures could begin. Catheterization of the jugular vein, abdominal aorta, carotid artery, renal vein, and caval vein was performed as described recently and took ~20 min (19). During the surgical procedures, the mice were kept at 37°C, using a temperature controller (Technical Service, Maastricht University) and heat pads (19).
A primed constant infusion of L-[guanidino-15N2]arginine, L-[ureido-13C-2H2]citrulline, L-[ring-2H5]phenylalanine, and L-[ring-2H2]tyrosine (Mass Trace, Woburn, MA) was given in the jugular vein of each mouse (prime [15N2]arginine: 850 nmol; [13C,2H2]citrulline: 215 nmol; [2H5]phenylalanine: 340 nmol; [2H2]tyrosine: 215 nmol; infusion [15N2]arginine: 1,700 nmol/h; [13C,2H2]citrulline: 430 nmol/h; [2H5]phenylalanine: 680 nmol/h; and [2H2]tyrosine 430 nmol/h). Steady state of tracers and metabolites was reached within 20 min (not shown).
For flow measurements, the indicator dilution method with
[glycyl-1-14C]p-aminohippuric acid
([14C]PAH, NEN Life Science Products, Boston, MA) was
used. Briefly, a primed (20 µl/10 g body wt of 3 µCi/ml
[14C]PAH) continuous (1 ml · 10 g body
wt1 · h
1 3 µCi/ml
[14C]PAH) infusion was given in the abdominal aorta. When
steady state had been reached, blood (0.2 ml/catheter) was sampled from the carotid artery and the renal vein and collected in heparinized cups
(Sarstedt, Nümbrecht, Germany) on ice. Blood collection took
place 6 h after LPS treatment. The right kidney was freeze-clamped in liquid nitrogen and stored at
80°C. In the present model, renal
metabolism and hindquarter metabolism are measured in one animal
(19). Data on hindquarter metabolism will be reported elsewhere.
In whole blood, hematocrit and [14C]PAH were determined
as described (19). Blood was centrifuged to obtain plasma,
because we have recently shown that plasma sampling is required in
organ-balance metabolic studies using amino acid tracers that do not
equilibrate well in blood cells (18). For determination of
amino acid concentrations and tracer-to-tracee ratios (TTRs), 80 µl
of plasma were added to 7 mg of dry sulfosalicylic acid, vortexed,
frozen in liquid nitrogen, and stored at 80°C. For determination of
plasma urea and ammonia concentrations, 20 µl of plasma were added to
80 µl of 1% trichloroacetic acid. Plasma urea and ammonia were
determined on a Cobas Mira S (Roche Diagnostica, Hoffman La Roche,
Basel, Switzerland) by standard enzymatic methods, using commercially available kits as described previously (9). For
determination of tissue amino acid concentrations, tissue was
homogenized and deproteinized as described before (8).
Approximately 80 mg of tissue were added to 400 µl of 5%
sulfosalicylic acid (SSA), with 300 mg of glass beads, and beaten for
30 s (8). Plasma and tissue amino acid concentrations
and enrichments were measured with a fully automated liquid
chromatography-mass spectrometry system, using precolumn
derivatization with o-phthalaldehyde (32). Plasma NO
For Western blot determination of iNOS protein in renal tissue, ~100 mg of tissue were homogenized in homogenization buffer [1:10 wt/vol, 20 mM Tris · HCl, 1 mM EDTA, 0.25 M sucrose, pH 7.4, supplemented with protease inhibitor cocktail (Complete Mini EDTA-free, Boehringer Mannheim, Mannheim Germany)]. Five micrograms of protein (Bio-Rad Protein Assay, Bio-Rad Laboratories, Munich, Germany) was separated on 5-15% pseudogradient SDS polyacrylamide gels and blotted onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). Mouse macrophage lysate (Transduction Laboratories, Lexington, KY) was used as a positive control. Membranes were blocked in 5% nonfat milk in PBS and subsequently incubated with primary antibody (anti-iNOS antibody, 1:10,000, Transduction Laboratories). Goat anti-mouse IgG conjugated to horseradish peroxidase (1:2,000) was used as secondary antibody.
Calculations
Whole body arginine and protein metabolism.
Plasma arginine, citrulline, phenylalanine, and tyrosine fluxes
were calculated from the arterial isotope enrichment values of,
respectively, L-[15N2]arginine,
L-[13C, 2H2]citrulline,
[2H5]phenylalanine and
[2H2]tyrosine, using the steady-state isotope
dilution equation
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Renal plasma flow and net substrate fluxes.
The calculation of plasma flow across the kidney has been described
recently (19). Organ substrate fluxes are calculated by
multiplying the renal venous arterial concentration difference with the mean plasma flow and are expressed in nanomoles per 10 gram
body weight per minute. A positive flux indicates net release, and a
negative flux reflects net uptake.
Organ-balance tracer methodology. Amino acid stable isotope tracers are used to obtain additional information regarding net fluxes. With the use of these tracers, organ disposal and production rates can be calculated. However, the mathematical formulas that are used to calculate disposal and production rates can only be applied if a few criteria are met. The most important criterion is that sampling should occur from a pool in which the tracer mixes freely (38). We have recently shown that arginine tracers do not mix with blood cell cytoplasm and that erroneous results are obtained when organ disposal and production rates are calculated using whole blood in this condition (18). Although it is known that red blood cells do play a role in renal metabolism (18, 31), the use of stable isotopes requires plasma sampling (18).
Renal protein metabolism. The phenylalanine tracer was used to measure whole body (see above) and renal protein turnover. Previously, we have shown that phenylalanine, valine, and leucine yield similar results with regard to renal protein synthesis as the percentage of whole body protein synthesis (18). If phenylalanine and tyrosine tracers are used to estimate protein synthesis and breakdown, additional information is received on renal phenylalanine-to-tyrosine hydroxylation, an important aspect of renal metabolism (1).
The rates of phenylalanine and tyrosine metabolism in the kidney were evaluated using, in principle, the models described previously for quantifying leucine metabolism in the splanchnic region (13). Calculation of renal hydroxylation of phenylalanine to form tyrosine (Phe
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Renal arginine metabolism.
Renal arginine turnover was calculated, in principle, as described in
detail by Yu et al. (40), with the adaptation that renal
[15N2]arginine tracer net balance was
corrected for [2H2]arginine production in the
kidney. The rationale for this correction is that the
[13C,2H2]citrulline tracer
leads to the formation of
[13C,2H2]arginine (see below).
[13C,2H2]arginine, in turn, may
be catabolized by arginase, leading to
[2H2]ornithine, which will give rise to
[2H2]citrulline and
[2H2]arginine. Circulation of M+2 tracers
does not disturb the measurements, except in the kidney, where
[2H2]citrulline is used for the production of
[2H2]arginine. Correction of
[15N2]arginine tracer net balance for
[2H2]arginine production is performed in the
following way
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Statistical Analysis
Results are presented as means ± SE. Significance between groups was tested by the Mann-Whitney U-test. Net substrate fluxes across the kidney were tested against zero using the Wilcoxon test to test whether a significant amount of substrate was produced or consumed by the kidney. Significance was considered present at P < 0.05. ![]() |
RESULTS |
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The arterial concentrations of nitrate, urea, ammonia, and many
amino acids, including glutamine, citrulline, ornithine, tyrosine and
phenylalanine, were increased after LPS treatment (Table
1). The arterial concentration of
arginine, however, was not changed in LPS-treated mice.
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LPS treatment resulted in an increase in whole body protein turnover,
as shown by the increase in plasma phenylalanine and tyrosine flux
(Table 2). Moreover,
phenylalanine-to-tyrosine labeling, indicating net protein breakdown,
was increased. Plasma arginine and citrulline flux were not
significantly changed by LPS treatment. In addition, whole body de novo
arginine synthesis (Cit Arg) was not significantly changed. Whole
body NO production (Arg
Cit) was increased after endotoxin
challenge.
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Under baseline conditions, no iNOS protein could be detected in renal
tissue. In addition, stimulation with LPS did not induce iNOS protein
in the kidney (Fig. 1). Most renal
intracellular amino acid concentrations, including arginine, and
ornithine, were not significantly changed by LPS treatment (Table
3). However, renal citrulline was
increased in LPS-treated animals (Table 3). Renal plasma flow was not
significantly increased after LPS treatment (Table
4). Moreover, LPS treatment caused an
increase in net renal citrulline uptake and an increase in net arginine
release. Besides an increase in citrulline uptake, an increase in
uptake of glutamine, phenylalanine, and tyrosine was also present in the kidney after LPS treatment.
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Renal Protein Metabolism
In control mice, renal phenylalanine disposal was 7 nmol · 10 g body wt
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After LPS treatment, renal phenylalanine disposal is increased due to
an increase in true protein synthesis and unchanged phenylalanine-to-tyrosine hydroxylation (17 4 = 13 nmol · 10 g body
wt
1 · min
1) (Fig. 2). The increase
in protein synthesis in endotoxin-treated mice was also detected when
tyrosine tracer was used (Fig. 3). A three and fourfold increase in
protein synthesis was measured using the phenylalanine and tyrosine
tracers, respectively. These observations are in agreement with
the increase in total amino acid uptake by the kidney. Because protein
breakdown, measured as phenylalanine and tyrosine production, is not
changed after endotoxin treatment (Figs. 2 and 3), net protein
synthesis is increased in mouse kidney after endotoxin challenge.
Renal Arginine Metabolism
In control mice, renal arginine disposal was 11 nmol · 10 g body wt
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Theoretically, arginine production in the kidney can be obtained from
protein breakdown and de novo arginine production from citrulline. In
control mice, de novo arginine production (11 nmol · 10 g body
wt1 · min
1) contributes 38% to
total arginine production (29 nmol · 10 g body
wt
1 · min
1) in the kidney (Fig. 4).
It is noteworthy that de novo arginine production is similar to
citrulline disposal (16 ± 3 nmol · 10 g body
wt
1 · min
1) in the kidney.
After endotoxin challenge, renal arginine production was increased.
Also, de novo arginine production and citrulline disposal (27 ± 4 nmol · 10 g body
wt1 · min
1) were increased in
endotoxin-treated mice. Thus the LPS-induced increase in net arginine
release from the kidney results from an increase in de novo arginine
production from citrulline, not from an increase in renal protein degradation.
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DISCUSSION |
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The results of the present study indicate that renal arginine and protein metabolism change in the early response to sepsis. For the first time, we report that endotoxin challenge induces a combined increase in renal protein synthesis and renal de novo arginine synthesis. The increases in renal protein synthesis and renal de novo arginine production were independent of renal NO production.
In the present experiment, renal arginine metabolism was measured in a short-term mouse model of endotoxemia. Although it is also interesting to obtain further information on LPS-induced changes in renal metabolism in time, in our opinion this should be performed in an animal model that allows repeated blood sampling (28). The mouse model is well established, because of its iNOS-induced increase in NO production (29) and because it mimics the septic condition (11). Usually, increased NO production is assessed from changes in plasma nitrate or the sum of nitrite and nitrate. We indeed detected an increase in plasma nitrate. In addition, we measured an increase in systemic NO production using the stable isotope technique. The increase in plasma nitrate was sixfold, whereas the increase in stable isotope-measured NO production was approximately twofold. Many studies have used urinary nitrate excretion as a parameter of systemic NO production. It has been shown, that under baseline conditions, urinary nitrate excretion is linearly related to the nitrate plasma concentration (14) and that the urinary nitrate excretion rate is highly correlated with systemic NO production measured using stable isotopes (3). However, the discrepancy between the increase in plasma nitrate and systemic NO production indicates that this is probably not true in disease states like experimental endotoxemia. In our opinion, quantification of systemic NO production in the disease state is only possible using stable isotope technology.
Septic patients are characterized by a catabolic state. In our
endotoxemic mice, net protein breakdown, measured as whole body Phe Tyr, was increased. In this model of endotoxemia, phenylalanine hydroxylation to tyrosine was increased in LPS-treated mice from 8.2 to
12.6 nmol · 10 g
1 · min
1,
indicating an increase in net protein breakdown in this condition.
Despite the increase in net whole body protein loss, renal protein synthesis was markedly increased after LPS treatment. In a previous study in which renal amino acid metabolism was studied after 16 h in rats, renal uptake of total amino acids was decreased, which may indicate that renal protein synthesis is decreased at this time (16). It appears that the increase in renal protein synthesis is specific for the time point studied. Which specific proteins, if any, are made at this time is unknown. The intestines also produce proteins in response to LPS injection (see review in Ref. 12). With respect to general protein metabolism, it seems that the kidney and intestine respond similarly to early endotoxemia. The differences between the present 6-h study and the previous 16-h study may well be caused by the difference in timing, and we therefore introduced the terms "late endotoxemia" for the previous study and "early endotoxemia" for the present study.
Although renal protein synthesis was increased by LPS treatment, renal arginine disposal was unchanged. This apparent discrepancy can be explained by the fact that arginine has several other metabolic fates besides protein synthesis. For instance, the kidney also contains arginase II (24), arginine:glycine amidinotransferase (36), and arginine decarboxylase (10). It is possible that protein synthesis is increased and (some of) the other pathways of arginine disposal are reduced in response to endotoxin treatment.
Previous studies have suggested that systemic NO production is dependent on extracellular arginine supply (2, 21) and that reduced arginine availability may impair systemic NO production (2, 33). It therefore appears important to regulate arginine availability. The adaptive role of the kidney in increasing de novo arginine production was questioned (4). However, in agreement with the LPS-induced increase in renal argininosuccinate lyase mRNA (20), renal citrulline uptake and arginine release were increased, and the stable isotope measurements confirmed that de novo arginine production from citrulline was indeed increased. Possibly, renal arginine production was upregulated to sustain systemic NO production during endotoxemia. In the present study, renal protein synthesis was increased, because phenylalanine disposal and the net uptake of total amino acids were increased. In a previous study in rats where measurements were performed 16 h after the onset of endotoxemia, renal arginine release was increased, but renal citrulline uptake was not (16). These results indicate that the increase in arginine production by the kidney did not originate from citrulline. Possibly, in this study of late endotoxemia, the increase in arginine release was caused by an increase in renal protein breakdown, because the net uptake in total amino acids by the kidney was decreased in these animals (16). The fact that glutamine uptake was decreased in the rat study without any effect on ammonia may also indicate that there was no need for glutamine uptake because glutamine was probably released from protein.
Although systemic NO production was increased during endotoxemia, we were not able to detect renal NO production in LPS-treated mice. In agreement with this result, we and others (30) were unable to detect iNOS protein in the kidneys of endotoxin-treated mice. The amount of iNOS RNA and protein induced by activation is highly differential. The animal species and strain, the host organism of LPS, the serotype and dose of LPS, the lagtime after LPS administration, and the coadministration of proinflammatory cytokines all influence the expression level of iNOS. The results of the study may have been different had iNOS been induced.
In control mice, renal phenylalanine production contributed 6% to the
systemic phenylalanine flux, which is comparable to the value obtained
in postabsorptive rats (7%) (18). Renal tyrosine production and renal phenylalanine-to-tyrosine hydroxylation
contributed 13 and 36%, respectively, to the systemic tyrosine and Phe
Tyr fluxes, indicating the importance of the mouse kidney in
tyrosine metabolism. Similar results have recently been demonstrated in the human kidney (1). Endotoxin treatment did not change
the contribution of the kidney to the plasma fluxes of phenylalanine, tyrosine, and Phe
Tyr. Under baseline conditions, the contribution of the kidney to plasma arginine flux was 38%, again comparable to the
value obtained in postabsorptive rats (44%) (18). Renal citrulline disposal contributed 64% to the systemic flux of citrulline and was comparable to renal de novo arginine production, indicating that whole body citrulline disposal is mainly determined by renal de
novo arginine production. Renal de novo arginine production was similar
to the plasma Cit
Arg flux, suggesting that de novo arginine
production from citrulline takes place mainly in the kidney. After
endotoxin challenge, the renal contribution to plasma arginine flux
increased to 51%, again indicating the importance of the kidney in
arginine production during endotoxemia in mice.
In the present study, we measured systemic NO production and renal arginine and protein metabolism under baseline conditions and during endotoxemia in mice. These stable isotope measurements can now be used to obtain new information on the metabolic role of the kidney in a variety of circumstances by applying them to transgenic and knockout mice.
In conclusion, the kidney has an anabolic function in the early response to endotoxemia. In addition, renal de novo arginine synthesis from citrulline is increased. This increase in arginine production may serve to sustain systemic NO production.
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ACKNOWLEDGEMENTS |
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The authors thank Jean Scheyen and Dr. Hans van Eijk for expert LC-MS measurements. The assistance of Gabrie Ten Have with the animal experiments is gratefully acknowledged.
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FOOTNOTES |
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This study was supported by Grants 902-23-098 and 902-23-239 from the Dutch Association for Scientific Research (NWO).
Address for reprint requests and other correspondence: N. E. P. Duetz, Dept. of Surgery, Maastricht Univ., PO Box 616, 6200 MD Maastricht, The Netherlands (E-mail: NEP.Deutz{at}ah.unimaas.nl).
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.
First published August 21, 2001; 10.1152/ajprenal.00039.2001
Received 8 February 2001; accepted in final form 4 October 2001.
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