The role of NOS2 and NOS3 in renal protein and arginine metabolism during early endotoxemia in mice

【关键词】  arginine

    Departments of Surgery and Anatomy and Embryology, Maastricht University, and Nutrition and Toxicology Research Institute Maastricht, Maastricht, The Netherlands

    ABSTRACT

    Previously, we observed an enhanced renal protein synthesis and increased de novo arginine production in the early response to endotoxemia in wild-type Swiss mice (Hallemeesch MM, Soeters PB, and Deutz NE. Am J Physiol Renal Physiol 282: F316F323, 2002). To establish whether these changes are regulated by nitric oxide (NO) synthesized by NO synthase isoforms NOS2 and NOS3, we studied C57BL6/J wild-type (WT), NOS2-deficient (NOS2/), and NOS3-deficient (NOS3/) mice under baseline (unstimulated) and LPS-treated conditions. The metabolism of renal protein, amino acid, and arginine was studied at the whole body level and across the kidney by infusing the stable isotopes L-[phenyl-2H5]phenylalanine, L-[phenyl-2H2]tyrosine, L-guanidino-[15N2]arginine, and L-[ureido-13C,2H2]citrulline. Renal blood flow was measured using radioactive PAH extraction. Under baseline conditions, renal blood flow was significantly reduced in NOS2/ mice (0.29 ± 0.01 vs. 0.48 ± 0.07 ml?10 g body wt1?min1 in WT) (P < 0.05), and de novo arginine production was lower in NOS2/ mice. After LPS challenge, renal protein turnover and arginine production increased in all three groups (P < 0.05), even though renal de novo arginine synthesis did not increase. The expected increase in renal citrulline production and disposal after LPS was not observed in NOS2/ mice (P = 0.06). Collectively, these data show that NOS2 is constitutively expressed in the kidney and remarkably functional as it affects renal blood flow and de novo arginine production under baseline conditions and is important for the increase in renal citrulline turnover during endotoxemia. NOS3, in contrast, appears less important for renal metabolism. The increase in renal protein turnover during endotoxemia does not depend on NOS2 or NOS3 activity.

    kidney; nitric oxide; nitric oxide synthase; sepsis

    ARGININE IS AN IMPORTANT amino acid in the biosynthetic pathways of proteins, nitric oxide (NO), agmatine, creatine, urea, and polyamines and as such has an important role in cellular regeneration, wound healing, immune function, and protein turnover (8, 37). Arginine is not an essential amino acid in the strict sense, since it can be synthesized endogenously. The kidney is the main endogenous source for de novo arginine synthesis from circulating citrulline, that originates from intestinal glutamine metabolism (5, 7, 29, 35).

    Under baseline conditions, low amounts of NO are produced by the constitutively expressed nitric oxide synthase isoforms NOS1 (neuronal NOS) and NOS3 (endothelial NOS) (1, 18). In addition, NOS2 (inducible NOS) was shown to be expressed in the kidney under normal conditions (21, 24), but its functional relevance is unknown.

    Sepsis is commonly associated with acute renal failure (17). This renal failure may be the result of vasoconstriction in the kidney, resulting in a reduced glomerular filtration rate, as observed after experimental LPS (4, 19, 26, 27). Sepsis and experimental endotoxemia upregulate NOS2 (21, 23, 25). At the same time, NO production by NOS3 can be inhibited (11), as is shown locally for the kidney by diminished NOS3 mRNA expression (27). Selective NOS2 inhibition was shown to prevent the reduction of the glomerular filtration rate and to improve renal function (20, 27), findings that suggest detrimental effects of increased NOS2 activity during endotoxemia. Others, however, suggested that NO bioavailability was important for the preservation of renal function in endotoxemia, because NO can act as a scavenger of reactive oxygen species or as a microvasodilator (15, 34).

    The increased need for NO and, therefore, for the NOS substrate arginine, in sepsis makes arginine a potentially essential amino acid in this condition (16). Previously, we reported that de novo arginine production from citrulline in the kidney is increased in "early" endotoxemic mice (12), with a resultant net renal arginine release (14). In addition, renal protein synthesis was increased in this endotoxemic mice model (12). Selective NOS2 inhibition in a "late" endotoxemic rat model stimulated both renal arginine production and net renal protein synthesis (10). The increased renal protein synthesis may therefore increase the arginine need in the kidney even further.

    The aim of this study was to investigate the role of NOS2 and NOS3 in arginine and protein metabolism during early endotoxemia in mice. In this study, we specifically questioned whether NOS2 and NOS3 are important for renal arginine and protein metabolism under normal/baseline conditions and whether the increased de novo arginine production and increased renal protein synthesis during endotoxemia that we observed previously are mediated by NO from NOS2 or NOS3.

    MATERIALS AND METHODS

    Animals

    Female C57BL6/J (wild-type; WT), C57BL6/J NOS2/ mice, and C57BL6/J NOS3/ mice [1626 g (mean 20 g/group), 23 mo old] were originally obtained from Jackson Laboratories and bred at the Department of Anatomy and Embryology (AMC, Amsterdam, The Netherlands). The mice were fed standard lab chow (Hope Farms, Woerden, The Netherlands) and were subjected to standard 12:12-h light-dark cycle periods (7:30 AM-7:30 PM). Room temperature was maintained at 25°C. Water was provided ad libitum throughout the experiment. Experiments were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals (33) and approved by the Ethical Committee of Animal Research of Maastricht University.

    Experimental Protocol

    Six different groups were discriminated in the protocol: WT (n = 8), NOS2/ (n = 8), NOS3/ (n = 9), WT+LPS (n = 8), NOS2/+LPS (n = 9), and NOS3/+LPS (n = 9). All experiments started between 8:00 AM and 10:00 PM. LPS (Escherichia coli O55:B5, 100 μg/10 g in 200 μl saline, Sigma, St. Louis, MO) was administered by intraperitoneal injection to mice (28). Control animals received a corresponding volume of saline. After the animals were injected with LPS or saline, food was withheld but drinking water was provided ad libitum.

    Five hours after LPS treatment, anesthesia and fluid maintenance were performed as described before (13). During the surgical procedures, the mice were kept at 37°C using a temperature controller (Technical Service, Maastricht University) and heat pads. Catheterization of the jugular vein, carotid artery, right renal vein, and inferior caval vein was performed as described (13).

    A primed-constant infusion of stable isotopes (Mass Trace, Woburn, MA) was given in the jugular vein: L-[guanidino-15N2]arginine ([15N2]Arg; 850 nmol/mouse prime, 1,700 nmol/h continuous); L-[ureido-13C; 2H2]citrulline ([13C;2H2]Cit; 215 nmol/mouse prime, 430 nmol/h continuous); L-[ring-2H5]phenylalanine ([2H5]Phe; 340 nmol/mouse prime, 680 nmol/h continuous); and L-[ring-2H2]tyrosine ([2H2]Tyr; 215 nmol/mouse prime, 430 nmol/h continuous). Plasma flow across the kidney was measured using an indicator extraction technique with [glycyl-1-14C]PAH ([14C]PAH; New England Nuclear Life Science Products, Boston, MA) (13).

    Blood was collected from the renal vein (venous blood), carotid artery (arterial blood), and caval vein (venous blood for hindquarter metabolism; published elsewhere) as described (13). Amino acid concentrations and tracer/tracee ratios (TTR) were determined in plasma as described using a fully automated LC-MS system after precolumn derivatization with o-phthaldialdehyde (30, 31). Plasma urea was determined as described (6).

    Calculations

    Renal substrate fluxes (net balances) were calculated by multiplying the renal venous arterial concentration difference with the mean renal plasma flow of the group and are expressed in nanomoles per 10 grams body weight per minute (6, 13). A positive flux indicates net release, and a negative flux reflects net uptake.

    Whole body rate of appearance (WbRa) of arginine, citrulline, phenylalanine, and tyrosine in plasma were calculated from the arterial isotope TTR (TTRA) values of [15N2]Arg, [13C,2H2]Cit, [2H5]Phe, and [2H2]Tyr, respectively, (12) as

    NO production was calculated as plasma arginine-to-citrulline flux, and de novo arginine production was calculated as plasma citrulline-to-arginine flux (12)

    Whole body protein breakdown, net protein breakdown, protein synthesis, and phenylalanine hydroxylation were calculated as described (12)

    (PheTyr) represents the hydroxylation of phenylalanine to tyrosine, which equals net protein breakdown, and is calculated as

    Renal tracer net balance (nb) and disposal and production rates were calculated by multiplying TTR with substrate fluxes (3)

    where [V] and [A] are the venous and arterial plasma concentrations of substrates, and TTRA and TTRv are the TTR of the measured amino acid in the arterial and venous plasma, respectively.

    The venous TTR (TTRv) was used as a surrogate precursor pool enrichment, because the venous TTR, compared with the arterial TTR, more closely resembles the precursor pool TTR (36).

    Renal protein metabolism was estimated from the [2H5]Phe and [2H2]Tyr tracers (3)

    and

    (PheTyr) represents the hydroxylation of phenylalanine to tyrosine in the kidney and is calculated as

    where PF is renal plasma flow. The term (1 FE) represents the fraction of total amino acids that bypasses metabolism in the kidney and appears in the output

    and

    NO production and de novo arginine production in the kidney were calculated using [15N2]Arg and [13C,2H2]Cit tracers (3). The rates of conversion of [15N2]Arg to [15N]Cit (NO production) and of [13C,2H2]Cit to [13C,2H2]Arg (de novo arginine production) were calculated essentially as written for the conversion of Phe to Tyr. Since renal NO production was at the limit of our detection level, these data are not shown.

    Statistical Analysis

    Results are presented as means ± SE. Two-way ANOVA was used to compare differences between treatment groups, using "group" with three levels (WT, NOS2/, and NOS3/) and "LPS" with two levels (saline, LPS) as the factors. When significant differences were observed between groups, further analysis with contrast methods was used. Significance was defined as P < 0.05.

    RESULTS

    Role of NOS2 in Baseline Metabolism

    Under baseline conditions, whole body NO production was 3.1 ± 0.4 nmol?10 g body wt1?min1 in WT mice. The absence of NOS2 did not change systemic NO production (see Table 2), although arterial nitrate concentration was reduced to 50% (29 ± 3 μM in WT and 13 ± 2 μM in NOS2/) (P < 0.01; Table 1). Whole body protein and arginine metabolism (Table 2) were not different between NOS2/ and WT mice. Arterial amino acid concentrations only differed for serine, which was decreased to 85% of control levels in NOS2/ mice (Table 1).

    View this table:

    View this table:

    Renal plasma flow in NOS2/ mice was significantly reduced (0.29 ± 0.01 vs. 0.48 ± 0.07 ml?10 g body wt1?min1 in WT mice) (P < 0.05; Fig. 1). No differences in renal fluxes (Table 3) and renal protein metabolism (Fig. 2) were observed between NOS2/ and WT mice, although net renal release of arginine tended to be lower (P = 0.06; Table 3) and renal de novo arginine production was significantly lower in NOS2/ mice (Fig. 3).

    View this table:

    Role of NOS3 in Baseline Metabolism

    Absence of NOS3 under baseline conditions significantly lowered whole body de novo arginine production (P < 0.05; Table 2).

    Renal plasma flow (Fig. 1) and metabolism (Figs. 24) were not changed in NOS3/ mice compared with WT mice.

    LPS Effect in WT Mice

    In WT mice, LPS reduced whole body protein synthesis to 90% of baseline values and de novo arginine production to 60% (Table 2). Whole body protein breakdown tended toward a decrease (P = 0.07; Table 2). Whole body rate of appearance of tyrosine decreased significantly after LPS (42.6 ± 2.2 baseline vs. 37.2 ± 4.9 nmol?10 g body wt1?min1 after LPS; P < 0.05). No changes in whole body arginine and citrulline production were observed (Table 2), whereas whole body NO production tended toward an increase (P = 0.07; Table 2). LPS increased arterial plasma levels of nitrate, urea, and most amino acids, although plasma arginine and glycine levels did not change (Table 1).

    LPS did not affect renal plasma flow but did result in enhanced arginine and citrulline production and disposal rates (Figs. 3 and 4). Uptake of phenylalanine by the kidney was also increased (Table 3). This could be related to the increased phenylalanine hydroxylation in the kidney after LPS (Fig. 2). In addition, renal protein breakdown, protein synthesis, and net protein synthesis increased after LPS (Fig. 2).

    LPS Effect in NOS2/ Mice

    In NOS2/ mice, whole body changes in protein metabolism after LPS were similar to WT mice (Table 2). However, whole body citrulline production was decreased in NOS2/ mice after LPS, whereas citrulline production did not change in WT mice (Table 2). In accordance, arterial plasma citrulline levels did not increase after LPS in NOS2/ mice as in endotoxemic WT mice (Table 1). In contrast to the fivefold increase in plasma nitrate levels in WT mice, plasma nitrate levels did not change in endotoxemic NOS2/ mice (Table 1). Plasma glycine was reduced after LPS in NOS2/ mice, but not in WT mice (Table 1).

    In endoxemic NOS2/ mice, renal plasma flow increased slightly (P = 0.08 vs. NOS2/ controls; Fig. 1). Renal phenylalanine uptake, protein breakdown, and synthesis increased, similar to WT mice (Table 3; Fig. 2). LPS tended to increase renal citrulline disposal (P = 0.06) but did not increase renal citrulline production in NOS2/ mice, as in WT mice (Fig. 4). After LPS, renal serine release was lower in NOS2/ mice than in WT mice (Table 3).

    LPS Effect in NOS3/ Mice

    In NOS3/ mice, whole body changes in protein metabolism after LPS were similar to WT mice (Table 2). Like in NOS2/ mice, but in contrast to WT mice, whole body citrulline production decreased after LPS in NOS3/ mice (Table 2). In contrast to a decrease in whole body de novo arginine production in WT and NOS2/ mice, de novo arginine production remained unchanged in NOS3/ mice (Table 2). Although plasma nitrate increased in NOS3/ mice, the increase was less than in WT mice (Table 1). Similar to NOS2/ mice, but in contrast to WT mice, arterial plasma citrulline levels remained unchanged after LPS. In contrast to WT and NOS2/ mice, plasma tyrosine, glutamine, and the sum of amino acids did not increase after LPS in NOS3/ mice (Table 1). Plasma glycine was reduced after LPS in NOS3/ mice similar to NOS2/ mice, but different from WT mice (Table 1).

    Renal plasma flow did not change after LPS in NOS3/ mice (Fig. 1). Both net renal citrulline uptake and arginine release after LPS in NOS3/ mice were lower than in WT mice (Table 3). Renal serine release was lower in NOS3/ after LPS compared with WT mice (Table 3). Changes in renal protein, arginine, and citrulline metabolism after LPS in NOS3/ were similar to WT mice (Figs. 24).

    DISCUSSION

    The present in vivo study shows that NOS2 is remarkably important for renal blood flow under baseline conditions, whereas renal blood flow was not affected by the absence of NOS3. The absence of NOS2 also reduced renal de novo arginine production under baseline conditions and prohibited the increase in renal citrulline turnover during endotoxemia. The absence of NOS3 only affected the degree of net renal citrulline uptake and arginine release. These findings therefore show that NOS2, rather than NOS3, is constitutive and functional in renal metabolism. However, because the studies were conducted in knockout mice, which, by definition, have compensatory mechanisms, it should be kept in mind that the importance of NOS2 and relative unimportance of NOS3 may be confounded.

    Baseline Renal Metabolism

    NOS2. The absence of NOS2 reduced renal blood flow under basal conditions. This effect is probably related to the presence of NOS2 in the outer medulla of the kidney, as shown in untreated rats (24). The authors of the aforementioned study (24) suggested that in the setting of the medulla, a sustained production of NO could be necessary to ensure adequate blood flow, but they could not provide evidence. Studies by Brezis et al. (2) showed that NO regulates renal blood flow, preferentially within the medullary vasculature and is involved in the regulation of renal medullary oxygenation, but the authors did not discriminate between specific NOS isoforms. De novo arginine production in the kidney under basal conditions was lower in NOS2/ mice, although renal citrulline disposal data were not accordingly affected. This again suggests functional involvement of constitutive NOS2 in the kidney. Finally, NOS2/ mice had 15% lower plasma serine levels, but because the decrease in renal serine flux was not significant, this effect probably originated outside the kidney.

    NOS3. The absence of NOS3 did not affect renal blood flow under basal conditions in our study but decreased net renal citrulline uptake and net renal arginine release slightly. The reduced net citrulline uptake could be the result of a reduced citrulline delivery to the kidneys due to impaired citrulline production in the intestinal region (5, 35) or to increased intrarenal citrulline production or reduced utilization. For the latter, we could not find evidence from our data. As whole body citrulline production and arterial concentration were not different in NOS3/ mice, data on intestinal citrulline production are needed as a possible explanation for the reduced net renal citrulline uptake.

    Although the reduction in net renal arginine release seems in line with the reduced net renal citrulline uptake, arginine production from protein breakdown or from citrulline (de novo synthesis) were not accordingly reduced. It is also possible that the arginine formed within the kidney is not completely released into the blood but is used in other metabolic pathways within the kidney, like protein synthesis, NO production, or the production of guanidioacetate in the synthesis pathway of creatine (see Ref. 38 for a review). Our arginine disposal data, however, cannot confirm this either. The role of NOS3 in renal citrulline and arginine metabolism, therefore, warrants further investigation.

    Renal Metabolism During Endotoxemia

    Renal blood flow, measured with the PAH indicator-extraction technique, did not change during endotoxemia in our study. However, it is very possible that arteriovenous shunting increases during endotoxemia, resulting in a fall in tissue perfusion and renal function (19). Since the PAH extraction method is based on the measurement of arteriovenous differences in the indicator [14C]PAH (13), local changes in microcirculatory perfusion may go undetected. Moreover, fluid administration during the surgical procedure could have added to the maintenance of an adequate flow.

    Whole body NO synthesis. Endotoxemia tended to increase whole body NO production in our female mice, although plasma nitrate levels increased 5-fold in WT mice, whereas no increase in plasma nitrate was seen in NOS2/ mice and there was a 3.5-fold increase in NOS3/ mice. We noticed such a discrepancy between the change in plasma nitrate and systemic NO production in another study (12). The discrepancy between both measurements may be related to an effect of endotoxemia on renal function and clearance. Moreover, the absence of an increase in plasma nitrate in NOS2/ mice may also be related to an effect on intestinal bacterial nitrate production in these mice. In addition, strain and sex differences seem to exist, since we observed, using the same experimental protocol, a 2.5-fold increase in systemic NO synthesis after LPS in Swiss mice (12) and a similar increase in male C57BL6/J mice (32).

    Protein turnover. In contrast to the slight decrease in whole body protein synthesis, renal protein synthesis and breakdown were increased during early endotoxemia, with an increase in net protein synthesis as a result. The absence of NOS2 or NOS3 did not affect this response to LPS. The increased renal protein synthesis in WT mice is in agreement with our previous observation in Swiss mice and suggests that this change in renal metabolism is specific for the early (6 h) response (12) compared with late (24 h) response to endotoxemia (9). The discrepancy between whole body and renal metabolism might indicate that other organs, e.g., the gut, importantly downregulate their protein metabolism during early endotoxemia. In male C57BL6/J mice, we indeed observed a reduction in gut protein breakdown and synthesis (32).

    Phenylalanine hydroxylation. Phenylalanine-to-tyrosine hydroxylation at the whole body level did not change during endotoxemia. In the kidney, phenylalanine hydroxylation increased during endotoxemia, and the contribution of the kidney to whole body hydroxylation therefore increased from 1726% under control conditions to 3759% during endotoxemia. As a consequence, phenylalanine hydroxylation in other parts of the body, mainly in the liver (22), probably contributes less during endotoxemia. These effects were NOS2 and NOS3 independent.

    De novo arginine production. De novo arginine production contributes to a great extent (73%) to total arginine production in the kidney of C57/BL6/J female mice under baseline conditions compared with only 38% in Swiss mice (12). During endotoxemia, renal de novo arginine production remained unchanged, in contrast to the increase that we observed previously in Swiss mice (12). Since total arginine production in the kidney is increased after endotoxin challenge, this increase can only be attributed to an increase in renal protein breakdown, as we indeed observed.

    Citrulline turnover. Regarding the more sustained reduction of citrulline production on whole body level during endotoxemia in NOS2/ mice, and the absence of an increase in arterial plasma citrulline and renal citrulline production in NOS2/ mice, NOS2 must be largely responsible for citrulline production from arginine (NO synthesis route) in the kidney. Similar effects on whole body citrulline production were present in NOS3/ mice, whereas the absence of NOS3 did not affect renal citrulline production during endotoxemia. Since both arterial plasma citrulline and glutamine levels did not rise in NOS3/ mice, this may suggest that NOS3 is more involved in intestinal citrulline synthesis during endotoxemia, but this requires further study.

    In conclusion, NOS2 expression in the kidney is constitutive and functional as it affects renal blood flow and de novo arginine production. De novo renal arginine production did not change during endotoxemia. NOS2 and NOS3 did not affect baseline protein metabolism, whereas the increase in renal protein turnover did not depend on either NOS2 or NOS3 activity. NOS2 is important for the increase in renal citrulline production during LPS, whereas NOS3 is not involved in changes in renal metabolism during endotoxemia.

    GRANTS

    This study was supported by Grants 902-23-098 and 902-23-239 from the Dutch Association of Scientific Research.

    ACKNOWLEDGMENTS

    The authors thank Jean Scheyen and Dr. Hans van Eijk for expert LC-MS measurements. The assistance of Gabrie ten Have in the performance of animal experiments is gratefully acknowledged.

    FOOTNOTES

    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.

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