Arginine, citrulline, and nitric oxide metabolism in end-stage renal disease patients

The kidneys are thought to be a major site of net de novo arginine synthesis, but the quantitative status of arginine metabolism and its substrate precursor relationship to nitric oxide (NO) synthesis in end stage renal disease (ESRD) patients have not been characterized. We have investigated kinetic aspects of whole body arginine metabolism in six patients with ESRD. They received two pre- and two post-hemodialysis intravenous tracer infusion studies with L-[guanidino-¹⁵N₂]arginine and L-[¹³C]leucine during the first study, and L-[5-¹³C]arginine and L-[5-¹³C-ureido,5,5,²H₂]citrulline during the second study. Arginine homeostasis in ESRD patients was found to be associated with a lower rate of arginine oxidation, and despite the decrease in renal function, the rate of de novo arginine synthesis appeared to be preserved. Plasma citrulline concentrations and flux were also elevated in these subjects compared with healthy adults. The rate of whole body NO synthesis was increased in the ESRD patients, but apparently not different pre- and post-hemodialysis therapy. The anatomic site(s) responsible for the maintenance of net de novo arginine synthesis and for the elevated NO synthesis and its pathophysiological importance in ESRD remain to be established.

Arginine, traditionally considered to be a nutritionally dispensable (nonessential) amino acid, serves multiple functions in addition to its role in protein synthesis (1). It is the precursor of nitric oxide (NO) (2), creatine, agmatine, and other polyamines (3, 4), and is an intermediate in the urea cycle. The kidneys are thought to be the main site of net de novo arginine synthesis, which would contribute to the maintenance of whole-body arginine homeostasis (5–10).

Despite the potential pathophysiologic significance of altered arginine metabolism in end-stage renal disease (ESRD) (11–13), in which reduced arginine synthesis is hypothesized, there are no in vivo studies of the quantitative aspects of arginine and arginine-NO kinetics for this group of patients. However, such information is necessary for the development of safe and effective therapeutic strategies aimed at modulating arginine metabolism and its several functions.

Therefore, in this study we have examined plasma kinetics of arginine and assessed the activity of the L-arginine–NO pathway in adults with ESRD. The possible effects of hemodialysis on arginine and NO kinetics were also explored. Because maintenance hemodialysis has been thought to be responsible for protein (nitrogen) catabolic stress (14, 15), we also investigated leucine kinetics as an indicator of the dynamic status of whole-body protein metabolism (16). Our findings reveal that at the whole-body level, net de novo arginine synthesis is maintained in ESRD patients, possibly due to an adaptive increase in citrulline availability and turnover. In addition, NO production in ESRD patients was significantly increased, when compared with healthy adults, but was apparently not influenced acutely by hemodialysis.

Plasma isotopic abundances (net mole fraction) pre- and post-hemodialysis for the infused L-[1-¹³C]leucine and L-[guanidino-¹⁵N₂]arginine are shown in Figure 4a. Values for L-[¹³C]arginine and L-[¹³C-ureido,5,5,²H₂]citrulline for the pre- and post-hemodialysis studies are shown in Figure 4b. The pre- and post-hemodialysis plasma enrichment values of [¹⁵N]citrulline, arising from L[guanidino-¹⁵N₂]arginine and reflecting formation of NO, are shown in Figure 5a. Enrichment of L-[¹³C-guanidino,5,5, ²H₂]arginine formed from the L-[¹³C-ureido,5,5, ²H₂]citrulline tracer, reflecting net de novo arginine synthesis, is presented in Figure 5b. In all cases, a steady-state isotopic enrichment plateau was achieved. The average isotopic enrichments of the L-[¹³C]arginine tracer, pre- and post-hemodialysis, were 0.082 and 0.079, respectively. They were higher (P < 0.05) than for the ¹⁵N₂arginine tracer (0.070 and 0.054, respectively), possibly due to appearance of [mono5-²H]arginine (M+1), from the labeled citrulline after its cycling through the arginine and ornithine pools. Although our analytical procedure does not differentiate between the two M+1 arginine species (¹³C- arginine or ²H-arginine), this would not measurably affect our kinetic values and conclusions.

Figure 4

(a) Plasma isotopic enrichment of L-[guanidino¹⁵N₂]arginine (⁵N₂ Arg) and L-[1-¹³C]leucine (¹³C Leu) pre-hemodialysis (Pre-HD) and post-hemodialysis(post-HD). (b) Plasma isotopic enrichment, pre- and post-hemodialysis, of L-[5-¹³C]arginine (¹³C Arg) and L-[¹³C ureido, 5,5,²H₂]citrulline (M+3 Cit). Time shown is minutes of tracer infusion. Error bars are SEM.

Figure 5

(a) Plasma isotopic enrichment of [¹⁵N-ureido]citrulline (¹⁵N Cit; VI in Figure 3) derived from L-[guanidino-¹⁵N₂]arginine (IV in Figure 3), an index of NO synthesis, pre- and post-hemodialysis. (b) Plasma isotopic enrichment of L-[¹³C-guanidino, 5,5, ¹³C-guanidino,5,5,²H₂]arginine (M+3 Arg; VIII in Figure 3) derived from L-[¹³C-ureido5,5,²H₂]citrulline (VII in Figure 3), an index of arginine synthesis, pre- and post-hemodialysis. Time shown is minutes of tracer infusion. Error bars are SEM.

Table 2 summarizes the plasma fluxes of arginine, citrulline, and leucine, and the rates of arginine and NO synthesis during the post-absorptive and fed states for the pre- and post-hemodialysis groups. Values for the healthy controls (17, 18) are also given in this table. As shown here, for the pre-hemodialysis phase, the amino acid fluxes did not differ between the post-absorptive and fed conditions. Similar findings apply for the post-hemodialysis phase, except that the [¹³C]arginine flux was lower (P < 0.05) in the fed period than in the post-absorptive period. The general lack of change in fluxes between the post-absorptive and fed states in ESRD patients contrasts with the significant increases (P < 0.01) in arginine and leucine fluxes, and decrease (P < 0.01) in the citrulline fluxes with feeding in healthy controls. It is possible that these different responses in controls and ESRD patients for the post-absorptive and fed states are partly due to the fact that a somewhat shorter period of time elapsed between the 2 metabolic states in ESRD patients than in control subjects, or to differences in the mode of feeding (the controls were given small frequent meals) (17, 18), or both. Finally, the citrulline fluxes were 4–5 times higher in ESRD patients than in the healthy controls (P < 0.001). In comparison with our earlier estimates in healthy controls (17, 20), the rates of whole-body net de novo arginine synthesis appear to have been maintained in the ESRD group, despite significant renal dysfunction. The isotopically derived estimate of the rate of NO synthesis, determined from the rate of conversion of plasma [¹⁵N₂]arginine to [¹⁵N]citrulline, is also summarized in Table 2. The NO synthesis rate was 5.2 ± 3.1 μmol/kg per hour (P < 0.03 vs. healthy controls) for the pre-hemodialysis phase, and 8.7 ± 4.9 μmol/kg per hour (P < 0.017 vs. healthy controls) for the post-hemodialysis phase; NO synthesis amounted to about 45–65% of the net rate of arginine de novo synthesis in patients with ESRD. The comparative value for healthy controls is approximately 12% (18).

Table 2

Post-absorptive (PA) and fed-state plasma fluxes of L-[guanidino-¹⁵N₂]arginine, L-[¹³C-ureido,5,5,²H₂]citrulline, and L-[1-¹³C]leucine and rates of arginine and NO synthesis pre- and post-hemodialysis in 6 ESRD patients and 6 healthy adults^A

Table 3 summarizes results for ¹³C enrichment of expired air and the appearance rate of ¹³CO₂ in expired air in ESRD patients. Rates of CO₂ production (VCO₂) (mmol/kg per hour) remained constant and were similar to those observed in healthy controls (16).

Table 3

Enrichment of ¹³CO₂ in expired air, carbon dioxide production (VCO₂), ¹³CO₂ output (V¹³CO₂), and oxidation and intakes of arginine and leucine after [¹³C]arginine and [¹³C]leucine infusions in ESRD patients and in healthy controls during the post-absorptive and fed states^A

Arginine oxidation rates averaged 6.5 ± 1.6 μmol/kg per hour and 6.9 ± 1.1 μmol/kg per hour, respectively, for the entire pre- and post-hemodialysis phases; these are lower (P < 0.01) than those in healthy controls (17). There were some differences in the estimated dietary arginine and leucine intakes between the ESRD patients and the healthy controls (17, 18), but these would not explain the lower rate of oxidation in patients during the post-absorptive state, or the lower leucine oxidation after hemodialysis in the fed state. Thus, it appears that rates of leucine and arginine oxidation are reduced in ESRD, due to endogenous rather than dietary factors.

Data for plasma amino acid concentrations are summarized in Table 4. There were no marked differences in arginine concentrations between the ESRD patients and healthy controls, but there were dramatic differences for citrulline, reflecting the differences in endogenous citrulline metabolism between the 2 populations. The fed-state plasma leucine concentrations were lower in ESRD patients than in controls, which is consistent with the lower fed-state rate of leucine oxidation seen in ESRD patients than in healthy subjects.

Table 4

Plasma amino acid concentrations (μM; mean ± SD) in ESRD patients for the post-absorptive and fed states during the periods pre- and post-hemodialysis and in healthy controls

We have investigated in vivo aspects of whole-body arginine and citrulline metabolism and rates of NO production in patients with ESRD. Our findings are evaluated in part with reference to results obtained previously in our laboratories, using comparable analytical and tracer techniques in healthy adults. We should point out, however, that there were some specific differences in experimental design between this and our earlier studies, including amounts of amino acids ingested and meal pattern, partly because it was too difficult to achieve strict dietary control with the ESRD patients who were awaiting a kidney transplant. Nevertheless, we can draw a number of new and important conclusions about the comparative aspects of arginine metabolism in ESRD patients and healthy adults.

With respect to whole-body leucine kinetics, used here as an index of the general status of body protein turnover, we did not detect profound differences between the ESRD patients and healthy controls, or evidence of a major catabolic state. The somewhat lower rates of leucine oxidation in ESRD patients during the post-absorptive phase may reflect an adaptive response to a possibly chronically lower protein intake than for our healthy subjects, as seen in our earlier long-term feeding studies of renal patients given a low-protein diet (28). Lim et al. (29) recently reported lowered post-absorptive values of leucine oxidation in non-acidotic, chronic renal failure patients compared with controls, but protein intakes in those patients were about 75% of those in normal subjects.

Arginine homeostasis is maintained through a cooperative metabolic interaction between the intestine, liver, and kidneys. It is thought that the intestine is the net site of citrulline synthesis from which the amino acid enters the circulation (6–10) to be taken up by the kidneys. Here the conversion of citrulline to arginine (de novo arginine synthesis) takes place, almost exclusively in the proximal convoluted tubule, through the action of the cytosolic enzymes argininosuccinate synthetase (EC 6.3.4.5) and argininosuccinate lyase (EC 4.3.2.1) (6–8). The kidneys, therefore, have been considered to be the main organ site for net de novo arginine synthesis in the maintenance of the whole-body arginine economy. This view is supported by the findings of Tizianello et al. (30), who measured arteriovenous renal gradients of amino acids in healthy humans and in patients with chronic renal failure. They showed that in the latter individuals, a net citrulline uptake was balanced by an equal net arginine output, which overall was 40% less than in healthy subjects.

Our study has revealed maintenance in the net rate of de novo arginine synthesis in patients with ESRD, notwithstanding that renal function was severely compromised, as demonstrated by a creatinine clearance of less than 10% of a normal value. The mechanism (or mechanisms) responsible for this maintenance of arginine synthesis remains to be established. Dhanakoti and coworkers (31) demonstrated in rats that renal arginine production is independent of dietary arginine, but that availability of citrulline was the limiting factor for renal arginine synthesis. Also, studies in a rat model of chronic renal failure, induced by 5/6 nephrectomy (32), showed that arginine synthesis was maintained. Three possible causes given for this were hypertrophy of the proximal tubule in remnant nephrons, which might double the arginine synthesis capacity per nephron; hyperfiltration, which increases the amount of citrulline filtered per nephron; and increased plasma citrulline concentrations. In our ESRD patients, the plasma rate of appearance of citrulline flux was significantly elevated, together with a markedly higher than normal plasma citrulline concentration. The latter has been reported by other researchers (30) in patients with ESRD, together with an observed inverse correlation between the plasma citrulline concentrations and renal function (30). According to the findings of Bouby and coworkers (32), the higher citrulline turnover may be a peripheral adaptation, enabling a reduced mass of functional tissue in the kidney to maintain a constant rate of arginine synthesis.

The rate of citrulline appearance (and disappearance) was about 65–80 μmol/kg per hour in the ESRD patients. This is about 4 or 5 times higher than in healthy controls (20). It also exceeds the net rate of de novo arginine synthesis, which approximates 13 μmol/kg per hour. Because the only route of citrulline catabolism is arginine synthesis, this must indicate a significant compartmentation of citrulline metabolism in vivo. Clearly, further detailed studies of citrulline metabolism in ESRD would be worthwhile, possibly using compartmental modeling (33).

We observed that the rate of arginine oxidation during the post-absorptive state in patients with ESRD was about 30% of that for healthy controls. In healthy subjects we have shown an adaptive reduction in arginine oxidation when they were fed an arginine-free diet (17, 22), which is associated with a lowered plasma arginine concentration (17, 22; unpublished data). In contrast, the lowered rate of arginine oxidation in the ESRD patients was associated with a normal plasma arginine concentration. This implies a diet-independent change in arginine metabolism in patients with severe renal dysfunction.

It can be seen from Table 3 that the rate of intake of arginine exceeded the estimated rate of arginine oxidation over the combined post-absorptive and fed states. On this basis, the patients were in marked positive arginine balance during the tracer study. Because a positive arginine balance would not be sustained over a prolonged period, the possibility exists that we have underestimated the rate of arginine oxidation. However, because this limitation would probably also apply to oxidation values for healthy controls, it seems that there are differences between the patient and control groups. Similarly, leucine oxidation for the combined post-absorptive and fed states indicated a positive leucine balance that tended to be greater during the post-hemodialysis phase. In comparison, the healthy subjects were close to leucine balance, again suggesting a retention of body protein in ESRD patients during the period of the tracer studies.

Finally, we have observed increased rates of NO synthesis in ESRD patients, although acute hemodialysis treatment did not appear to change the rate of NO production. Yokokawa and coworkers (34) demonstrated increased production of NO, determined by plasma nitrite and nitrate concentrations, in patients with ESRD and hemodialysis-induced hypotensive episodes. However, NO production evaluated in this way did not appear to increase in their ESRD patients who were undergoing hemodialysis but were without hypotensive episodes. In contrast, our ESRD patients receiving hemodialysis treatment remained hemodynamically stable, but presented a significant increase in NO production. Wever et al. (35), using L-[guanidino-¹⁵N₂]arginine in a tracer paradigm comparable to the one used here for determination of NO production, reported a reduced rate of NO production in patients with chronic renal failure who were not on hemodialysis. Thus, the consequences for NO production of ESRD appear to be complex, and this will require additional study.

In summary, our findings indicate that arginine homeostasis under conditions of ESRD is supported by maintenance in the rate of net de novo arginine synthesis, apparently due to an adaptive increase in the rate of citrulline synthesis and maintenance of high plasma levels and turnover. The anatomic site or sites responsible for net arginine synthesis under these conditions remain to be established. Arginine may also be conserved in part by a lower post-absorptive rate of oxidation. In addition, the rate of NO synthesis in patients with ESRD was significantly elevated, and accounted for a much higher fraction of arginine disposal than in healthy subjects. In comparison with healthy subjects, the altered dynamic relationships in ESRD among arginine and citrulline metabolism on the one hand, and NO synthesis on the other, indicate the need to further investigate the pathophysiological implications of arginine and citrulline metabolism in patients with severe renal disease.

This study was supported by funding from the Anesthesia Research Foundation, by the Children’s Hospital, the Lynne Reid Fellowship at Harvard Medical School, and by National Institutes of Health grants RR-88, DK-15856, DK-40561, and P30-DK40561.

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