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Am J Physiol Renal Physiol. 2014 Sep 15; 307(6): F660–F665.Published online 2014 Jul 23. doi: 10.1152/ajprenal.00289.2014

PMCID: PMC4166726

Extrarenal citrulline disposal in mice with impaired renal functionJuan C. Marini, Inka C. Didelija, and Marta L. Fiorotto

Pediatric Critical Care Medicine, Department of Pediatrics, Baylor College of Medicine, Houston, Texas; andUSDA/ARS Children's Nutrition Research Center, Baylor College of Medicine, Houston, TexasCorresponding author.

Address for reprint requests and other correspondence: J. C. Marini, 1100 Bates St., Mail Stop BCM320, Houston, TX 77030 (e­mail:marini@bcm.edu).

Received 2014 May 23; Accepted 2014 Jul 9.

Copyright © 2014 the American Physiological Society

Abstract

The endogenous synthesis of arginine, a semiessential amino acid, relies on the production of citrulline bythe gut and its conversion into arginine by the kidney in what has been called the “intestinal­renal axis” forarginine synthesis. Although the kidney is the main site for citrulline disposal, it only accounts for ∼60–70%of the citrulline produced. Because the only known fate for citrulline is arginine synthesis and the enzymesthat catalyze this reaction are widespread among body tissues, we hypothesized that citrulline can be utilizeddirectly by tissues to meet, at least partially, their arginine needs. To test this hypothesis, we used stable andradioactive tracers in conscious, partially nephrectomized (½ and ⅚) and anesthetized acutely kidney­ligatedmouse models. Nephrectomy increased plasma citrulline concentration but did not affect citrulline synthesisrates, thus reducing its clearance. Nephrectomy (⅚) reduced the amount of citrulline accounted for as plasmaarginine from 88 to 42%. Acute kidney ligation increased the half­life and mean retention time of citrulline.Whereas the rate of citrulline conversion into plasma arginine was reduced, it was not eliminated. In addition,we observed direct utilization of citrulline for arginine synthesis and further incorporation into tissue proteinin kidney­ligated mice. These observations indicate that a fraction of the citrulline produced is utilizeddirectly by multiple tissues to meet their arginine needs and that extrarenal sites contribute to plasma arginine.Furthermore, when the interorgan synthesis of arginine is impaired, these extrarenal sites are able to increasetheir rate of citrulline utilization.

Keywords: arginine, citrulline, kidney, stable isotope

THE ENDOGENOUS SYNTHESIS OF arginine is an interorgan process in which its precursor, citrulline, issynthesized in the gut and, after entering the portal circulation, reaches different cell types, where it isconverted into arginine. The role of the kidney as the main organ involved in the synthesis of circulatingarginine has been well established (11, 14), and this process has been named the “intestinal­renal axis” forarginine synthesis (3). Using isotope tracers and arteriovenous differences, however, it has emerged that asignificant fraction of the citrulline (∼30–40%) produced by the gut cannot be accounted for as plasmaarginine (3, 17, 38). Because the only known fate for citrulline is its conversion into arginine and there iswidespread distribution of the enzymes that catalyze the conversion of citrulline into arginine, i.e., argininesuccinate synthase (ASS) and lyase (ASL) (10, 16, 37), it seems likely that plasma citrulline is utilized bydifferent cell types directly to generate local arginine. This ability for citrulline utilization to meet the localneed for arginine has been reported in endothelial cells (18), macrophages (36), and the pancreas (15) in thecontext of nitric oxide (NO) production and citrulline recycling. Furthermore, during sepsis there is areduction in the “de novo” synthesis of arginine, which has been interpreted as a reduction in overall kidneyfunction (19, 23). However, an alternative interpretation could be that some peripheral tissues increase theiruptake of citrulline, thereby reducing the amount of citrulline available for plasma arginine synthesis by thekidneys. Regardless, more citrulline becomes available for direct tissue utilization during endotoxemia, and

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General.

SURGERY.

INFUSIONS.

SAMPLE COLLECTION.

Experiment 1 (chronic renal nephrectomy model).

SURGERY.

INFUSIONS.

SAMPLE COLLECTION.

Experiment 2 (acute kidney ligation).

INFUSIONS.

Experiment 3 (acute kidney ligation).

this may also contribute to tissue arginine availability and synthesis of NO.

Although in vitro studies with multiple cell types have identified the ability of citrulline to replace argininefor growth and proliferation (31, 33, 34), little information on the direct utilization of citrulline in vivo isavailable due to the difficulties associated with these determinations (14). The objective of this study,therefore, was to determine the magnitude of extrarenal citrulline utilization when kidney function isimpaired. To accomplish this objective, three experiments were performed: in experiment 1, we determinedthe effect of chronically nephrectomized mice on citrulline production and disposal; in experiment 2, weestablished the disposal of citrulline in acutely kidney­ligated mice; and in experiment 3, we identified theconversion of citrulline into arginine and its incorporation into multiple tissues of kidney­ligated mice.

MATERIALS AND METHODS

Animals and Treatments

Young adult male ICR (Institute of Cancer Research, Harlan Laboratories, Houston, TX) mice (6wk old) were used for all the experiments. Mice were housed under standard conditions (27). All animalprocedures were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee.

Conscious mice (initial n = 10) were studied three times:before (control, weight ± SD 30.7 ± 3.1 g) and 1 wk after ½ and ⅚ nephrectomy (1/2N and 5/6N,respectively). The weight of the mice was not affected as a result of the surgery or infusions (30.6 ± 2.9 and31.0 ± 2.8 g, for the 1/2N and 5/6N, respectively).

Mice underwent two surgical procedures, 1 wk apart. During the first procedure, the right kidneywas ligated and removed to produce the 1/2N model. One week later, the left kidney was exposed and two­thirds were removed by polectomy, resulting in the 5/6N model (24).

Mice were studied after a postsurgical 7­day recovery period. On the day of the infusion, feedwas removed at 7:00, and the lateral tail vein was catheterized for infusion of tracers (25). A primed­continuous infusion of tracers was started at 10:00 and lasted for 4 h. To determine the entry rate of citrullineand arginine and the conversion of citrulline into arginine, [ureido­ N]citrulline (prime: 7 μmol/kg;continuous infusion: 7 μmol·kg ·h ) and [ N ]arginine (prime: 45 μmol/kg; continuous infusion: 45μmol·kg ·h ) in 0.9% NaCl were infused.

At the end of the infusion, blood was collected from the submandibular bundle usinga lancet. In addition, spot urine was collected to determine citrulline loss by this route.

Mice (n = 5; weight ± SD: 35.5 ± 1.9 g) were studied under anesthesiaafter ligation of the renal vasculature.

Mice were anesthetized with 2% isoflurane in oxygen. An arterial catheter (carotid) and a venouscatheter (lateral tail vein) were implanted for blood sampling and infusion, respectively. After performance ofa midline laparotomy, a braided silk suture was placed around the renal vasculature of both kidneys and waseither ligated (LIG) or left untied in the control group (SHAM). This procedure was completed in <25 min.

A bolus dose of [ureido­ N]citrulline (1.2 μmol in 300 μl) was delivered with an infusionpump at a rate of 50 ml/h, followed by a continuous infusion of [ N ]arginine at a rate of 15 μmol·kg ·h.

Approximately 15 μl of blood were collected from the arterial catheter into apreweighed 1.5­ml Eppendorf tube before and 1.5, 2.5, 4, 6, 8, 11, 15, 20, 30, 40, 50, 60, and 70 min afterthe start of the infusion. After weighing of the tube containing the blood sample, 80 μl of an internal standard([U­ C ]arginine and [1,2,3,4,5 C ]citrulline) in saline (0.9 g NaCl/l) was added, and the weight wasrecorded. Samples were then centrifuged, and the supernatant was kept at −80°C until analysis.

A preparation similar to that in experiment 2 was used (n = 5; weight ±SD: 33.6 ± 1.8 g).

A bolus dose of [ C]citrulline (3 μCi in 300 μl saline) was delivered using the tail catheter as

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SAMPLE COLLECTION.

described above.

Sixty minutes after tracer administration, the mice were euthanized by decapitationand the tissues were collected to determine the incorporation of the C label into protein. Tissues (liver,kidney, pancreas, spleen, small and large intestine, lung, thymus, testis, heart, gastrocnemius muscle, skin,and epididymal and brown fat) were snap frozen in liquid nitrogen and weighed. Samples were stored at−80°C until analysis.

Analysis

Arginine and citrulline plasma concentrations and enrichments were determined by liquid chromatography­tandem mass spectrometry as their dansyl derivatives as described elsewhere (26). Tissue was homogenized,and protein was precipitated with perchloric acid (PCA) to determine the incorporation of C into protein.After the protein pellet was thoroughly washed to remove any traces of soluble intra­ and extracellularcontaminants, including free tracer, the pellet was solubilized with Soluene­350 (PerkinElmer, Waltham,MA) at 60°C for 4 h. Then, the scintillation cocktail Ultima Gold (PerkinElmer) was added, and Cradioactivity was measured using a Liquid Scintillation Analyzer (Packard TriCarb 3180 TR). Tissue ofanimals that underwent similar surgical preparation but no [ C]citrulline infusion was used for backgroundcorrection.

Calculations

For the continuous infusion of tracers (experiment 1), steady­state conditions and isotopic plateau enrichmentwere assumed based on our previous work (24). The rate of appearance (Ra; Eq. 1) of circulating citrullineand arginine was calculated from the isotopic dilution of the corresponding infused tracers as

(1)

where Ra is the Ra of the unlabeled metabolite M (μmol kg h ), iIV is the intravenous (iv) infusionrate of the tracer (μmol kg h ), E is the enrichment of the infused iv tracer, and E is the plasmaenrichment of metabolite M at isotopic plateau enrichment (mpe).

The rate of conversion (Rc; Eq. 2) of these amino acids was calculated based on the transfer of the label fromthe precursor to its product as previously described (25).

(2)

where Rc is the rate of conversion of a precursor into its product (μmol kg h ), Ra is theRa of the product, determined from the steady­state enrichments of the iv infused tracer, and E and Eare the respective plasma enrichments of the precursor and product due to the infusion of the labeledprecursor.

The conversion of infused [ N ]arginine into [ N ]citrulline is a proxy for NO synthesis, and theconversion of infused [ureido­ N]citrulline into [imino­ N]arginine was used to estimate the de novoarginine synthesis. Under steady­state conditions, the Ra of the amino acids is identical to their rate ofdisposal; thus clearances (ml·kg ·min ) were calculated by dividing the Ra by the respective plasmaconcentration.

For the bolus dose of [ N]citrulline (experiment 2), steady­state conditions cannot be assumed, especially inthe case of the kidney­ligated animals due to the rapid expansion of the citrulline pool. For this reason, theconcentration of the [ N]citrulline tracer at the different time points was calculated based on the enrichment

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R = iI ∙ ( − 1)am VMEiIV

EIVM

M· −1· −1

M· −1· −1

iIV IVM

R = R ∙ ( )cprec→prod aprod/ (100 − )Eprod Eprod

/ (100 − )Eprec Eprec

prec→prod· −1· −1

prod

prec prod

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of citrulline and plasma citrulline concentration. The following biexponential model was then applied to theresulting data

(3)

where y is the concentration of tracer in blood (μmol [ureido­ N]citrulline/l) at time t, and A, b, C, and dare the parameters of the equation. The first term of the equation corresponds to distribution of citrullinewithin the body, and the second one to the disposal of citrulline. Goodness of fit of the model employed wasdetermined by graphical residual analysis.

The area under the curve from zero to infinity (AUC ), the area under the curve from the 0­ to 70­min(AUC period of sampling and observation), and the area under the moment curve (AUMC) werecalculated as in the following

(4)

(4')

and

(5)

where AUC is expressed as [ureido­ N]citrulline·l ·min ); AUMC is expressed [ureido­ N]citrulline·l·min ); A, b, C, and d are the parameters calculated previously; and E is the [ureido­ N]citrulline

concentration at 70 min.

The terminal half­life (λ) after reaching pseudoequilibrium (min) and mean residence time (MRT; min) werecalculated as

(6)

and

(7)

Because of the different kinetics between the LIG and SHAM groups for the arginine produced from thecitrulline bolus dose, no single equation could be fitted. Thus the trapezoidal rule was utilized to obtain theAUC between 0 and 70 min for the N and N arginine data. The actual amount of arginine producedfrom the citrulline bolus dose can be approximated as follows

(8)

= A ∙ Exp (−b∙  time) + C ∙ Exp (−d∙  time)yt

t15

0­∞

0–70min

AU = +C0−∞A

b/ C

d/

AU = + −C0−70 minA

b/ C

d/ E

d/

AUMC = +Ab2/ C

d2/

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λ = Ln(2)d

/

MRT = AUMCAUC/

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(8)

where AUC and AUC are the AUC for the arginine produced from citrulline and the arginineinfused, respectively, and infused N Arginine is the total amount of arginine infused during the experiment.Note that this equation does not represent a precursor­product relationship as Eq. 2. It represents the ratiobetween the AUC for two isotopologs of arginine, and because one was infused (and thus the total amountinfused is known), the total conversion of the bolus [ N]citrulline to [ N]arginine can then be estimated.

Statistical Analysis

Results from the chronic renal nephrectomy model were analyzed as a mixed model with mouse as therandom variable of the model using the “proc MIXED” procedure of SAS (v. 9.2, SAS, Cary, NC). In thisway, comparisons were made within animals. For the acute kidney ligation models, the “proc NLIN”procedure was utilized to calculate the parameters of the biexponential equation. Homogeneity of thevariances was tested using Levene's test; for those variables with heterogeneous variance, a logtransformation of the variable was performed before statistical analysis. For these variables, the mean andconfidence intervals are presented. Otherwise, means ± SE are reported.

RESULTS

Arginine and Citrulline Kinetics in Chronically Nephrectomized Mice

Partial nephrectomy reduced the clearance rate of arginine and citrulline and increased the plasmaconcentration of these amino acids without affecting their rates of appearance (Table 1). The rate ofconversion of arginine to citrulline, a proxy for NO production, was not different among the three treatments(Table 1). The conversion of citrulline to arginine (de novo arginine production), which was 88% of thecitrulline produced in control mice, was reduced to 64 and 42% in nephrectomized animals (1/2N and 5/6N,respectively). Only traces of citrulline were found in urine regardless of the extent of kidney ablation.

Citrulline Disposal During Acute Kidney Ligation

Kidney ligation increased (P < 0.001) plasma citrulline concentration (Fig. 1) and reduced the rate ofdisposal of the citrulline tracer injected (Fig. 2). This resulted in a greater AUC of the [ N]citrulline tracerand a longer half­life and MRT of the citrulline pool in the LIG mice (Table 2). The ligation of the kidneysresulted in a 65 and 84% increase in these two parameters, respectively. The appearance of [imino­N]arginine (resulting from [ureido­ N]citrulline) followed a different pattern in the two treatment groups (

Fig. 3). In the SHAM group [ N]arginine appeared almost immediately after the citrulline tracer wasadministered, and its plasma concentration declined over time. In the LIG group, [ N]arginine appeared in aslow, but steady fashion. Whereas the AUC for the [ N ]arginine tracer continuously infused was notdifferent between the two groups (P = 0.597, data not shown), the AUC for [ N]arginine in the LIG groupwas ∼24% of the area calculated for the SHAM group (Table 2). This translated into a reduction in the Nlabel recovered in arginine in the LIG mice. The percentage of the infused N label recovered as[ N]arginine was 14.2 and 61.7% for the LIG and SHAM group, respectively. This represented a fivefoldincrease (from 13.6 to 72.7 μmol·kg ·h ) in the amount of citrulline disposed by extra renal tissues.

Citrulline Utilization in Kidney­Ligated Mice

The C label originally in citrulline was incorporated to a different extent in the different tissues analyzed inthe LIG and SHAM group (Fig. 4). In the SHAM group, the pancreas and kidney had the highest level ofC incorporation. Kidney ligation reduced (P < 0.05) the incorporation of radioactivity in some tissues

(spleen, stomach, small and large intestine, thymus, heart, gastrocnemius muscle, brain, and brown adiposetissue), with no incorporation in the kidney as expected. There was no difference between the SHAM andLIG groups for liver (P = 0.211), pancreas (P = 0. 485), lungs (P = 0.201), testis (P = 0.933), skin (P =0.372), and epididymal fat pad (P = 0.478).

CitArg = ∙ infuse   ArginineAUC15NArgAUC15N4Arg

/ d15N4

15NArg 15N4Arg15

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DISCUSSION

The synthesis of citrulline from ornithine takes place in only two cell types, the enterocyte and thehepatocyte. In the liver, citrulline functions as part of the urea cycle in the detoxification of ammonia, andbecause of the channeling of urea cycle intermediates (7), little or no citrulline escapes the liver. The citrullineproduced in the small intestine, however, enters the portal vein, escapes liver extraction, and appears in theperipheral circulation, serving as a precursor for arginine synthesis. The contribution of other pathways suchas NO synthesis, degradation of citrullinated proteins, and demethylation of methylarginines are likely tocontribute a small fraction of the total flux of citrulline. Because most of the circulating citrulline is of enteralorigin, plasma citrulline concentration has been proposed as a marker for gut mass and function (9, 32).Conversely, because citrulline utilization takes place mainly in the kidney, plasma citrulline can also beconsidered a marker of renal function (21). Whereas the determination of citrulline synthesis rate and itsprecursors is relatively straightforward (25), the determination of citrulline disposal and utilization is morecomplex. For this reason, we utilized two different and complementary approaches. In chronicallynephrectomized mice, the rate of appearance of citrulline was determined, which under the (pseudo) steady­state conditions of the experiment is equivalent to its rate of disposal. In kidney­ligated mice, a bolus dose oflabeled citrulline was administered and the rate of disposal of the tracer (and tracee) was determined directly.

The role of the kidney has been established by arteriovenous differences in rats (11) and dogs (38).However, a significant fraction of citrulline disposal (∼40%) cannot be accounted for by renal metabolism(38). Similarly, the de novo arginine production determined with stable isotopes fails to account for 25–40%of the rate of appearance of citrulline (4, 29). Because the only known metabolic fate of citrulline is itsconversion into arginine (through argininosuccinate), it is likely that this unaccounted fraction is utilized forarginine synthesis in “hidden” compartments that do not equilibrate with plasma. Initially, to study thenonrenal disposal of citrulline, we crossed ASL mice (13) with an improved Cre recombinase (iCre)under the control of the kidney androgen­regulated protein (KAP) promoter (22). However, we were unableto alter the de novo synthesis of arginine with this model, even after providing exogenous testosterone tomale mice. Subsequent immunohistochemical analysis showed that ASL was still present in the proximaltubules of the kidney. For this reason, we resorted to other well­established models of kidney functionimpairment to disrupt the interorgan metabolism of citrulline and arginine. These models, however, havesome limitations: chronic partial nephrectomy is a well­established model for kidney disease, and this mayhave an independent impact on arginine metabolism. Acute kidney ligation is usually done under generalanesthesia, which also can have an effect on the metabolism of citrulline and arginine. Regardless, the mainlimitation of this approach is that the disappearance of citrulline is what is being measured and not itsutilization for arginine synthesis. For this reason, we used a complementary approach to determine the tissueutilization of citrulline for arginine synthesis when renal function is impaired. To determine this local process,we measured the incorporation of the ureido carbon of citrulline into the acid­precipitable fraction of differenttissues. This approach assumes that 1) there is no tRNA for citrulline and thus no (direct) incorporation ofcitrulline into protein; 2) the new arginine that is formed from citrulline can then be incorporated into protein,and, in kidney­ligated mice, this process reflects a local phenomenon; 3) protein is the main component ofthe acid­precipitable fraction; and 4) the main fate of the ureido carbon of citrulline is urea, which is lost fromthe system, i.e., the ureido carbon does not recycle (this is also true for other minor pathways, e.g., agmatineand creatine synthesis).

Chronic Nephrectomy Reduces Clearance of Citrulline and De Novo Synthesis of Arginine

Given the central role of the kidney in the disposal of citrulline (11, 14), the increase in plasma citrullineconcentration following partial nephrectomy was expected (1, 5). Although the increase in plasma arginineconcentration seems counterintuitive, this phenomenon has been previously reported in mice (1).Theobserved increase in plasma citrulline concentration in chronically nephrectomized animals was due toimpaired citrulline disposal, because the rate of appearance of citrulline was unaffected (Table 1). However,the rate of disposal of citrulline has to match the rate of appearance to maintain a (pseudo) steady state. Thedisposal of citrulline takes place by conversion into argininosuccinate and then arginine or alternatively byexcretion into the urine. We have only detected traces of citrulline in the urine, which is consistent withprevious observations in partially nephrectomized mice (1) and with the great ability of the kidney toreabsorb citrulline after an overload with this amino acid (30). Thus, in the absence of urinary citrullineexcretion, it seems likely that citrulline was utilized for arginine synthesis by nonrenal tissue. Chronic partial

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nephrectomy reduced the efficiency with which citrulline was removed from the system, as indicated by theincrease in plasma citrulline concentration and the decrease in its clearance rate. The amount of citrullinerecovered as plasma arginine (de novo arginine synthesis) was also reduced in this nephrectomy model, andthe amount disposed of by extrarenal tissues increased fivefold. The conversion of arginine into citrulline, aproxy for NO production, was not affected by partial nephrectomy in this study. Other authors have reporteda decrease in the production of NO in chronic kidney disease (2, 6). The reason for the discrepancy betweenour data and previous reports may be due to the higher plasma arginine concentration observed in ouranimals, or that more time after the renal injury is needed for the development of a full renal dysfunctionmodel to become established.

Acute Kidney Ligation Increases the Half­Life of Citrulline

The abrupt ligation of the kidneys also resulted in a reduction in the disposal of citrulline, which translatedinto a greater plasma concentration, AUC, longer half­life, and residence time of this amino acid (Fig. 1 and Table 2). The appearance of [ N]arginine in blood in the LIG mice (Fig. 3) indicates that nonrenal tissuesproduce and export arginine synthesized from plasma citrulline. Assuming that this process is also present inthe SHAM group, we can estimate that this nonrenal source accounts for ∼24% of de novo argininesynthesis. The amount of citrulline infused, recovered as plasma arginine in the SHAM group, was ∼60%,which falls within the range previously reported (28). For the LIG group, however, only 14% of the citrullineinfused was recovered as plasma arginine (Table 2). This nonrenal source of plasma arginine, to the best ofour knowledge, has not been described previously.

Plasma Citrulline Is Incorporated in Tissues Despite the Presence of a Metabolically Active Kidney

The ureido­ C originally in the citrulline administered was readily incorporated into the acid­precipitablefraction of multiple tissues in both the LIG and SHAM groups. In animals without a functional kidney, thisutilization represents the uptake of plasma citrulline, its conversion into arginine, and, finally, itsincorporation into protein. The difference in incorporation among the different tissues is likely to represent(for the most part) their rate of protein synthesis, rather than their ability to convert citrulline into arginine.Nonetheless, these measurements indicate that the conversion of citrulline into arginine is widely presentamong body tissues. Interestingly, a reduced incorporation was found in the liver of SHAM and LIG mice,despite its high protein synthesis rate; this response is consistent with the poor uptake of citrulline by thisorgan reported by early researchers (8, 20). Not surprisingly, LIG mice had a reduced C incorporation intoprotein in some tissues, since these animals had to rely on the local conversion of citrulline to arginine,whereas SHAM mice in addition were also able to utilize plasma [ C]arginine. The pancreas, however,despite a very high fractional protein synthesis rate (>400%/day, Marini JC, unpublished observations),seems to be able to produce enough arginine from citrulline to sustain its high protein synthesis rate.

In conclusion, when taken together, the data indicate that a fraction of the citrulline produced is utilizeddirectly by multiple tissues to meet their arginine needs and that extrarenal sources contribute to plasmaarginine. Furthermore, when the interorgan synthesis of arginine is impaired due to nephrectomy or kidneyligation, these extrarenal sites are able to increase their rate of citrulline utilization. This direct citrullineutilization may be the reason why citrulline supplementation has been more effective than argininesupplementation in improving the microcirculation during endotoxemia (35) and restoring NO synthesis inpatients with mitochondrial disorders (12).

GRANTS

This work was supported by funds from the US Department of Agriculture, Agricultural Research Service,under Cooperative Agreement Number 58­6250­6­001, and the National Institutes of Health (Grant K01RR024173 to J. C. Marini).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Author contributions: J.C.M. provided conception and design of research; J.C.M., I.C.D., and M.L.F.

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performed experiments; J.C.M. analyzed data; J.C.M. and M.L.F. interpreted results of experiments; J.C.M.prepared figures; J.C.M. drafted manuscript; J.C.M. and M.L.F. edited and revised manuscript; J.C.M.,I.C.D., and M.L.F. approved final version of manuscript.

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Figures and Tables

Table 1.

Citrulline and arginine kinetics in control and nephrectomized mice

Control 1/2N 5/6N P

Plasma concentration, μmol/l

    Arginine 37.7 ± 4.0 32.5 ± 4.8 119.4 ± 32.6 <0.0002

    Citrulline 20.4 ± 1.8 37.4 ± 3.6 111.0 ± 34.9 <0.0003

Rate of appearance, μmol·kg ·h

    Arginine 434.1 ± 25.6 406.8 ± 36.0 504.2 ± 37.0 <0.2112

    Citrulline 121.5 ± 5.0 116.5 ± 11.5 126.5 ± 11.6 <0.7976

Rate of conversion, μmol·kg ·h

    Arginine to citrulline 0.88 ± 0.12 0.74 ± 0.06 1.09 ± 0.06 <0.0943

    Citrulline to arginine 107.9 ± 11.9 74.9 ± 14.6 53.8 ± 9.3 <0.0430

Clearance, ml·kg ·min

    Arginine 197.7 228.7 83.2 <0.0105

    Citrulline 103.1 ± 6.5 55.7 ± 6.4 30 ± 10.6 <0.0001

Values are means ± SE; control and ½ nephrectomy (½N), n = 10; ⅚ nephrectomy (⅚N), n = 7. For arginineclearance, the variance was not homogenous across treatments, and thus this variable was log transformed.95% confidence intervals were 166–236, 146–357, and 45­154 ml·kg ·h for control, 1/2N, and 5/6Ntreatment, respectively.

Values with different superscripts differ (P < 0.05).

Fig. 1.

b b a

b b a

−1 −1

−1 −1

a ab b

−1 −1

a a b

a b b

−1 −1

a,b

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Plasma citrulline concentration in mice after kidney ligation (LIG) or sham surgery (SHAM). Values are means ± SE; n = 5.Arrow and *P < 0.05, for treatment differences.

Fig. 2.

Plasma [ureido­ N]citrulline concentration after a bolus dose of this tracer in mice that underwent kidney ligation (LIG)or SHAM surgery. Biexponential curves were fitted to the data (LIG = 49.6 ± 2.4·e + 26.7 ± 2.2·e

, R = 0.98; LIG = 59.8 ± 1.1·e + 16.3 ± 1.3·e , R = 0.94).

Table 2.

[ureido­ N]citrulline and [ N ]arginine kinetics in sham (SHAM) and kidney­ligated (LIG) mice

SHAM LIG P

[ureido­ N]citrulline

    AUC , μmol/min 1,012 ± 98 1,726 ± 155 <0.011

    AUC , μmol/min 998 ± 99 1,588 ± 77 <0.003

    AUMC, μmol/min 27,340 ± 5,076 86,845 ± 15,310 <0.021

    λ, min 23.5 ± 3.0 38.7 ± 3.3 <0.014

    MRT, min 26.7 ± 3.5 49.2 ± 4.3 <0.007

[ N]arginine

15

t(−0.27 ± 0.03·t) (−0.02 ±

0.00·t) 2t

(−0.22 ± 0.01·t) (−0.03 ± 0.00·t) 2

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15

0­∞

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    AUC , μmol/min 351 ± 8.5 85 ± 3.6 <0.001

    Recovered, μmol 0.74 ± 0.06 0.17 ± 0.01 <0.001

    As % infused [ N]citrulline 61.7 ± 5.22 14.2 ± 0.48 <0.001

[ N ]arginine

    AUC , μmol/min 297 ± 18 315 ± 27.2 <0.597

Values are means ± SE; n = 5.

AUC, area under the curve; AUMC, area under the moment curve; λ, half­life; MRT, mean retention time.

Fig. 3.

Plasma [ N]arginine concentration after a bolus dose of [ureido­ N]citrulline in LIG or SHAM mice.

Fig. 4.

Tissue protein C radioactivity after a bolus dose of [ureido­ C]citrulline in LIG or SHAM mice. Values are means ± SE;n = 5. *P < 0.05, for treatment differences.

Articles from American Journal of Physiology ­ Renal Physiology are provided here courtesy of AmericanPhysiological Society

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