1 Departments of Medicine, 3 Basic Pharmaceutical Sciences, and 2 Physiology, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia 26506
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To test the hypothesis that nitric oxide (NO) deficiency occurs in end-stage renal disease (ESRD), NO oxidation products (NO2 + NO3 = NOx) and cGMP were measured in blood, urine, and dialysate effluent of peritoneal dialysis (PD) patients and compared with blood and urine of healthy subjects. All subjects were on a controlled low-nitrate diet (~330 µmol/day). NOx and cGMP outputs were significantly reduced in PD patients (334 ± 50 µmol/24 h and 55 ± 13 nmol/24 h, respectively) vs. controls (823 ± 101 µmol/24 h and 149 ± 46 nmol/24 h). Plasma arginine was borderline low, plasma citrulline was elevated and plasma levels of the endogenous NO synthase inhibitor asymmetric dimethylarginine were approximately five time higher in PD patients (2.2 ± 0.3 µM) vs. controls (0.4 ± 0.1 µM). Although blood pressure (BP) was not different between groups at the time of study, 10 of 11 PD patients were on medication for hypertension. These studies demonstrate that total NO production is low in ESRD, and with appropriate caution, we conclude that this NO deficiency may contribute to the increased BP that occurs in ESRD.
hypertension; arginine; citrulline; guanosine 3',5'-cyclic monophosphate; endogenous nitric oxide synthase inhibitors
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CHRONIC, experimentally induced nitric oxide (NO) deficiency in animals produces hypertension, and there is evidence that regional vascular endothelial NO production may be defective in some individuals with primary and secondary hypertension (3). Hypertension is prevalent in patients with end-stage renal disease (ESRD), irrespective of the etiology of their renal failure. Many ESRD patients are refractory to multiple antihypertensive agents even in conjunction with fluid restriction and maintenance dialysis fluid removal (15, 16). NO deficiency has been reported in children and adults with chronic progressive renal disease (5, 8) and has been implicated in ESRD, based on indirect evidence (14, 19). There have, however, been no clinical studies that directly assess the NO status in ESRD. Accordingly, we conducted the present experiments to specifically test the hypothesis that NO deficiency occurs in peritoneal dialysis (PD)-dependent patients. Several indexes of the L-arginine:NO system were measured, including NO2 + NO3 (NOx), the stable inert oxidation products of NO, the second messenger cGMP, as well as plasma levels of arginine, citrulline, and the endogenous NO synthase (NOS) inhibitors asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and N-monomethyl-L-arginine (L-NMA). Of importance, these experiments were conducted under conditions of controlled dietary NOx intake, the only situation in which NOx levels in body fluids can give insight into NO production (4).
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Informed consent to participate in the study for measurement of 24-h
NOx production was obtained from
11 PD patients and 11 normotensive, healthy subjects. Demographic
information on both groups is given in Table
1. Criteria for controls were as follows: normotensive, healthy individuals with no known illnesses
and taking no cardiovascular medications. Of the 11 PD patients, 6 had
been diagnosed with diabetic nephropathy, 2 with hypertensive nephrosclerosis, and 1 each with glomerulonephritis, renovascular disease and obstructive uropathy. All but one of the PD patients were
taking antihypertensive medication.
|
For 48 h, all participants consumed nutritionally complete (~2,000 calories/24 h), low NOx content (330 µmol, i.e., ~15-30% of normal dietary NO3 intake; Ref. 4) food which was also low in sodium (60 meq/day), and potassium and phosphorus restrictions appropriate for dialysis-dependent patients were also maintained. All participants received complete preweighed packages of food and reported any food not consumed. Control patients collected all urine (with boric acid as preservative), and PD patients collected all peritoneal dialysate effluent plus any urine made, during the second 24-h period of low dietary NOx intake. Blood pressure (BP) was measured while subjects were quietly seated, and a blood sample was obtained at the end of the 48-h diet. Plasma for cGMP analysis was stored with the phosphodiesterase inhibitor IBMX to prevent breakdown of cGMP in vitro.
The following analyses were conducted:
NOx concentrations of plasma,
urine, and peritoneal dialysate were measured using the Griess assay
after conversion of NO3 to
NO2 with the
NO3 reductase enzyme, as described
previously by us (18). In preliminary studies, we obtained complete
recovery of NO3 added to PD
fluid. Plasma and urine cGMP was measured by the Cayman
Chemicals ELISA kit. Plasma arginine and citrulline were measured by
reverse-phase HPLC with precolumn derivatization and fluorescent
detection using a modification of the AccQ Tag system for amino acid
analysis (Waters, Milford, MA). With -aminobutyric acid as an
internal standard, plasma samples were ultrafiltered with a 10,000 mol wt cutoff and then derivatized at 55°C with
6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (7). Dual pumps (Waters model 510) delivered the binary
mobile phase (140 mM sodium acetate, with 17 mM
tetraethylammonium titrated to pH 5.05; and 60%
acetonitrile) at 1 ml/min using the AccQ Tag gradient table to a
C18 column (Nova-Pak, 4 µm, 3.9 × 150 mm, Waters), maintained at 60°C to allow resolution of
citrulline and threonine. Ten-microliter samples were
injected by autosampler (Waters model 717 plus) with the eluting
products measured with a fluorescence detector (Waters model 474) at
excitation and emission wavelengths of 250 and 395 nm (gain 100), respectively.
For measurement of the endogenous NOS inhibitors ADMA, SDMA, and L-NMA, the reverse-phase HPLC with AccQ Tag method of Anderstam et al. (1) was used with minor modifications. Standards of L-NMA, ADMA, and SDMA were run in the concentration range from 0.625 to 10.0 µM. Recoveries of spiked synthetic ADMA, SDMA, and L-NMA were ~100%. Concentrations of arginine, citrulline, and methylarginines were calculated using Millennium chromatography manager (version 2.10) software for integrations and calculations based on the established standard curves for each run.
The methylarginine method was established by us after the NOx production studies were completed, and therefore measurements were made on separate groups, all n = 8. All controls were a subset of the NOx production series; PD group contained two of the original patients and six others, three with diabetic nephropathy, two with hypertensive nephrosclerosis, and one each with glomerulonephritis, membranous nephropathy, and scleroderma. Demographics are given in Table 1 in parentheses.
Plasma and urine sodium and potassium was measured by flame photometer, creatinine concentrations were by colorimetric assay of the Janovski complex (Sigma kit no. 555-A).
All data are reported as means ± SE, and statistics were by paired and unpaired t-test and one way ANOVA. Statistical significance was defined where P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Table 1 summarizes baseline information on the subjects in both groups in the NOx production and the ADMA studies. Generally, the groups were well matched, although the controls were younger than PD patients in the ADMA series and the gender mix was variable.
The 24-h output of NOx in normal
controls was significantly higher than in PD patients
(P < 0.001) (Table
2). Dietary
NOx intake was similar in both
groups, but the difference between output and intake, which provides a
qualitative estimate of total NO production, was high in
controls and lower, not different from zero, in PD patients. This does
not mean that there was zero NO production in PD patients but rather
provides a nonquantitative index of low NO production (8). The 24-h
output of cGMP (the major second messenger of NO) was greater in
controls than PD patients (Table 2, P < 0.05). The BP values for PD patients were not significantly
different vs. controls but 10/11 PD patients were on antihypertensive
medication at the time of study. Weight loss due to fluid removal
averaged 1.20 ± 0.32 kg per 24-h treatment period in PD patients.
PD patients had mean Kt/V values (i.e., urea clearance × time of
dialysis/volume of distribution) of 2.0 ± 0.2 per week, and the
24-h creatinine clearance in the control group was 129 ± 8 ml/min.
|
Plasma creatinine values were high in ESRD patients vs. controls as
expected (Table 3). Plasma sodium was
similar and plasma potassium was lower in PD patients vs. controls.
Despite reduced production of NOx
and cGMP (indicated by low 24-h
NOx and cGMP outputs; Table 2),
plasma concentrations of NOx and
cGMP were higher in PD vs. controls (P < 0.001), reflecting inadequate plasma clearance (Table 3). Plasma
arginine values were reduced in PD patients
(P < 0.05; Table 3),
whereas plasma levels of citrulline (P < 0.001) and the endogenous NOS inhibitor ADMA were elevated approximately five times the control value in PD patients (Table 3).
SDMA levels were also higher in ESRD vs. controls, but
L-NMA was undetectable in most
plasma samples, irrespective of group, and is therefore not reported.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study was designed to test the hypothesis that impaired NO production occurs in ESRD, using NOx removal via urine and/or peritoneal dialysate as our index. We observed a marked reduction in the total rate of removal of the stable NO oxidation products, NOx, in patients on PD, indicating that total NO production is low in ESRD. These measurements were made during controlled low dietary NOx intake, a uniquely important aspect of this study. As discussed in detail by us elsewhere (4), dietary NOx intake varies between subjects and within one individual from day to day. Since exogenous (dietary) NOx and NOx formed from endogenously produced NO, cannot be differentiated in measurements of total NOx, NOx intake must be controlled when using NOx levels to measure NO production.
The present studies indicate that total NO production is low in ESRD patients. The difference between NOx intake and output was close to zero in ESRD, although presumably these patients do make some NO. Even subjects with normal renal function on a high NOx intake excrete less NOx in their urine than is ingested (4), reflecting other routes by which NOx is lost from the body. Furthermore, we assume that the proportion of NOx cleared from the body by PD, as a surrogate of functioning kidneys, is equivalent to that removed in the urine in controls, and this may not be correct. These considerations highlight the fact that NOx output, from the kidney or surrogate, even when measured under ideal conditions, gives only a "qualitative" index of total NO production (4). Of note, it is unlikely that PD directly affected NO production, since inducible NOS is not stimulated by the native peritoneum, at least in the absence of peritonitis (17). One point that we cannot address in this study is whether total NOx production necessarily reflects production of the "hemodynamically active" component of total body NO. The general assumption in the literature is that these variables are interchangeable, although there is no direct evidence to support this (4). There is widespread and heterogeneous tissue distribution of the various NOS isoforms, which will vary in disease (13); thus the relationship between total NO and "hemodynamically active" NO is likely to be variable and unpredictable.
Despite our finding from the NOx output data that total NO production is reduced in ESRD, plasma NOx levels, even after strict dietary nitrate restriction and an overnight fast, were higher in ESRD patients compared with controls. Since plasma NOx is influenced by dietary NOx intake as well as the time since eating and importantly, by renal clearance, plasma NOx levels may reflect little about overall NO production (4). This is an important point, because investigators have begun to report that high plasma NOx values automatically reflect increased systemic NO production, flawed conclusion.
Soluble guanylyl cyclase in an important receptor for NO (10), and cGMP levels often change in the same direction as changes in NO activity in the vasculature. In the PD patients, the decline in NOx production was paralleled by a fall in cGMP, a reassuring finding. However, we caution against assuming a "cause-and-effect" relationship, since NO also signals by non-cGMP mechanisms, cGMP acts as second messenger for other agonists, and cGMP is removed from the body by a combination of excretion and metabolism.
What causes the deficiency in total NO production in ESRD? Arginine used for NO synthesis throughout the body is derived both from the diet and from that made endogenously (11, 14). Lack of functional renal mass will compromise the main endogenous source of arginine generation and could lead to arginine deficiency and impaired NO synthesis. Indeed, experimentally induced arginine deficiency in rats is associated with decreased NO production and hypertension (20). Citrulline is the substrate for intrarenal arginine generation (11), and this accumulates in "anephric" patients (14). Citrulline levels were high in PD patients in the present study, which is suggestive of reduced renal arginine synthesis as well as reduced renal clearance.
Plasma arginine levels have been variously reported as normal or low in ESRD patients (14, 19) and in the present study were subnormal in PD patients. Given that clearance from the plasma is reduced as a result of the renal failure, low plasma arginine suggests a substantial reduction in available arginine in ESRD. However, the low plasma arginine levels in PD patients are still above the Km of the various NOS enzymes (6) and should not lead to substrate-dependent falls in NO production, unless plasma arginine levels do not reflect intracellular arginine availability. In this regard, we recently reported that plasma from ESRD patients, and uremic levels of urea in artificial serum, inhibits arginine transport into cultured endothelial cells (21, 22). Therefore, there may be cellular arginine depletion in ESRD, and in support of this, orotic acid (a marker for relative arginine deficiency) is increased in renal failure (14).
There is also evidence that circulating levels of endogenous NOS inhibitors increase in renal failure (19). Of these NOS inhibitors, ADMA acts as a potent nonselective NOS inhibitor, whereas SDMA is ineffective (19). When plasma ADMA levels rise and arginine levels fall, NOS will be inhibited, if the rise in ADMA is of sufficient magnitude. According to Vallance and colleagues (19), plasma ADMA levels increase in uremia by about eight times, which brings the circulating ADMA concentration (~9 µmol/l) into a range where functional NOS inhibition is expected (19). Subsequently, other workers have also observed increases in plasma ADMA levels in ESRD, but report lower absolute values and smaller increases, such that plasma ADMA levels rise to 1.25 µmol/l or less, a value which would have little impact on NOS activity (1, 9, 12). In the present study, we observed marked rises in plasma ADMA levels in PD patients to values that, although lower than originally reported (19), are sufficient to exert some NOS inhibitory effect. Therefore, although this area remains controversial, the present study supports the possibility that a rise in the ADMA:arginine ratio contributes to NO deficiency in patients with ESRD. In further support of our findings, NO deficiency has been reported in children and adults with chronic renal failure (5, 8). Furthermore, chronic oral supplementation of L-arginine in rats with chronic renal failure and hypertension improved BP control, in addition to improvement of renal function (2).
In conclusion, although simple chemical indexes remain an imperfect measure of NO-dependent BP control, this carefully controlled clinical study provides evidence that total NO generation is substantially lower in ESRD patients treated for hypertension vs. normotensive controls. Production rate of the major second messenger, cGMP, also declines in uremic patients as does plasma substrate (arginine) levels, whereas the endogenous NOS inhibitor ADMA, increases. Low NO generation likely contributes to the hypertensive state in ESRD.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Gerald Hobbs, Phd., Associate Professor of Biostatistics, West Virginia University (WVU) Department of Community Medicine, for statistical guidance and support. In addition, we are grateful to Jennifer Domico, Kevin Engels, Glenn Kuenzig, and Lennie Samsell for excellent technical assistance, and we thank all patients and control subjects.
![]() |
FOOTNOTES |
---|
This work was supported by Biomedical Research Support Grant 9410 (424) from WVU School of Medicine, Office of the Dean (to R. J. Schmidt), a National Kidney Foundation of Western Pennsylvania Grant-in-Aid (to R. J. Schmidt), and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-45517 (to C. Baylis).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. Baylis, Dept. of Physiology, PO Box 9229, West Virginia Univ., Morgantown, WV 26505-9229 (E-mail: cbaylis{at}wvu.edu).
Received 7 January 1999; accepted in final form 12 March 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Anderstam, B.,
K. Katzarski,
and
J. Bergstrom.
Serum levels of NG,NG-dimethyl-L-arginine, a potential endogenous nitric oxide inhibitor in dialysis patients.
J. Am. Soc. Nephrol.
8:
1437-1442,
1997[Abstract].
2.
Ashab, I.,
G. Peer,
M. Blum,
Y. Wollman,
T. Chernihovsky,
A. Hassner,
D. Schwartz,
S. Cabili,
D. Silverberg,
and
A. Iaina.
Oral administration of L-arginine and captopril in rats prevents chronic renal failure by nitric oxide production.
Kidney Int.
47:
1515-1521,
1995[Medline].
3.
Baylis, C.,
and
P. Vallance.
Nitric oxide and BP: effects of NO deficiency.
Curr. Opin. Nephrol. Hypertens.
5:
80-88,
1996[Medline].
4.
Baylis, C.,
and
P. Vallance.
Measurement of nitrite and nitrate (NOx) levels in plasma and urine: what does this measure tell us about the activity of the endogenous nitric oxide?
Curr. Opin. Nephrol. Hypertens.
7:
1-4,
1998[Medline].
5.
Blum, M.,
T. Yachnin,
Y. Wollman,
T. Chernihovsky,
G. Peer,
I. Grosskopf,
E. Kaplan,
D. Silverberg,
S. Cabili,
and
A. Iaina.
Low nitric oxide production in patients with chronic renal failure.
Nephron
79:
265-268,
1998[Medline].
6.
Bredt, D. S.,
and
H. H. H. W. Schmidt.
The citrulline assay.
In: Methods in Nitric Oxide Research, edited by M. Feelisch,
and J. S. Stamler. Chichester: Wiley, 1996, p. 249-255.
7.
Cohen, S. A.,
K. DeAntonis,
and
D. P. Michaud.
Compositional protein analysis using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, a novel derivatization reagent.
In: Techniques in Protein Chemistry. Academic, 1993, vol. IV, p. 289-298.
8.
Kari, J. A.,
A. E. Donald,
P. T. Vallance,
K. R. Bruckdorfer,
A. Leone,
M. J. Mullen,
T. Bunce,
B. Dorado,
J. E. Deanfield,
and
L. Rees.
Physiology and biochemistry of endothelial function in children with chronic renal failure.
Kidney Int.
52:
468-472,
1997[Medline].
9.
Marescau, B. G.,
G. Nagels,
I. Possemiers,
M. E. De Broe,
I. Becaus,
J. M. Billiouw,
W. Lornoy,
and
P. P. De Deyn.
Guanidino compounds in serum and urine of nondialysed patents with chronic renal insufficiency.
Metabolism
46:
1024-1031,
1997[Medline].
10.
Moncada, S.,
R. M. J. Palmer,
and
E. A. Higgs.
Nitric oxide: physiology, pathophysiology, and pharmacology.
Pharmacol. Rev.
43:
109-141,
1991[Medline].
11.
Morris, S. M., Jr.
Regulation of enzymes of urea and arginine synthesis.
Annu. Rev. Nutr.
12:
81-101,
1992[Medline].
12.
Pettersson, A.,
L. Uggla,
and
V. Bachman.
Determination of dimethylated arginines in human plasma by high pressure liquid chromatography.
J. Chromatogr. B Biomed. Appl.
692:
257-262,
1997.
13.
Raij, L.,
and
C. Baylis.
Nitric oxide and the glomerulus.
Kidney Int.
48:
20-32,
1995[Medline].
14.
Reyes, A. A.,
I. E. Karl,
and
S. Klahr.
Role of arginine in health and in renal disease.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F331-F346,
1994
15.
Rostand, S. G.,
J. D. Brunzell,
R. O. Cannon III,
and
R. G. Victor.
Cardiovascular complications in renal failure.
J. Am. Soc. Nephrol.
2:
1053-1062,
1991[Abstract].
16.
Schmidt, R. J.,
and
F. Dumler.
Diabetic nephropathy: diagnostic techniques and follow up evaluation.
In: Contemporary Issues in Nephrology: Diagnostic Techniques in Renal Disease, edited by R. G. Narins,
and J. H. Stein. New York: Churchill-Livingstone, 1992, p. 119-144.
17.
Suh, H.,
T. Peresleni,
N. Wadhwa,
M. McNurlan,
P. Garlick,
and
M. S. Goligorsky.
Amino acid profile and nitric oxide pathway in patients on continuous ambulatory peritoneal dialysis: L-arginine depletion in acute peritonitis.
Am. J. Kidney Dis.
29:
712-719,
1997[Medline].
18.
Suto, T.,
G. Losonczy,
C. Qiu,
C. Hill,
L. Samsell,
J. Ruby,
N. Charon,
R. Venuto,
and
C. Baylis.
Acute changes in urinary excretion of nitrite+nitrate (UNOXV) do not predict renal vascular NO production.
Kidney Int.
48:
1272-1277,
1995[Medline].
19.
Vallance, P.,
A. Leone,
A. Calver,
J. Collier,
and
S. Moncada.
Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure.
Lancet
339:
572-575,
1992[Medline].
20.
Wakabayashi, Y.,
E. Yamada,
T. Yoshida,
and
H. Takahashi.
Deficiency of endogenous arginine synthesis provokes hypertension by exhausting substrate arginine for nitric oxide synthesis.
Biochem. Biophys. Res. Commun.
205:
1391-1398,
1994[Medline].
21.
Xiao, S.,
M. Howton,
and
C. Baylis.
High plasma urea inhibits arg transport in to vascular endothelial cells (Abstract).
J. Am. Soc. Nephrol.
9:
359,
1998.
22.
Xiao, S.,
R. Schmidt,
M. Howton,
and
C. Baylis.
Nitric oxide (NO) deficiency in uremia is mediated by inhibition of nitric oxide synthase (NOS) and L-arginine (L-ARG) transport in endothelial cells (Abstract).
J. Am. Soc. Nephrol.
9:
625,
1998.