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Nephrology, Department of Medicine, University Hospital Dresden,
Germany.
Institute of Medical Informatics and Biometry, University Hospital
Dresden, Germany.
Correspondence to Dr. Jens Passauer, University Hospital Carl Gustav Carus, Technical University Dresden, Division of Nephrology, Fetscherstraße 74, 01307 Dresden, Germany. Phone: 493514583510; Fax: 493514416237; E-mail: passauer{at}rcs.urz.tu-dresden.de
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Endothelial dysfunction has been shown recently to be a major initiating factor in atherosclerosis (reviewed in reference (8). Endothelial dysfunction is associated primarily with decreased nitric oxide (NO). However, endothelial dysfunction of uremia has received little attention. There are only a few studies of NO in uremia, and they have reached contradictory conclusions. NO is difficult to measure, and some of the discrepancies may be technique related.
To clarify the role of NO in vivo, we used the forearm perfusion technique to study endothelial function in hemodialyzed patients. This invasive method permits dissociation of endothelial mechanisms from those of the smooth muscle cell layer. This enabled us to use a more complex set of functional tests than has been used by other investigators of uremia. We generated dose—response patterns for acetylcholine (ACH), a direct stimulus of endothelial NO, and for N-monomethyl-L-arginine (LMA), a blocker of NO synthases (NOS). We also infused L-arginine (L-ARG), the substrate of NOS. Because these tests are functional tests, depending on the ability of arterial resistance vessels to respond to NO, we took specific care to establish a dose—response curve for NO itself. To do so, we infused glyceroltrinitrate (GTN). To evaluate further the principal constrictory properties of uremic resistance vessels, we also tested the vascular response to norepinephrine (NE).
In this study, we focused on normotensive patients on HD. We matched the control subjects for age, gender, height, weight, BP, and serum cholesterol.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Forearm Blood Flow Studies
Studies were performed in a quiet, temperature-controlled room (23 to
25°C) between 9 a.m. and 4 p.m. with the subjects resting supine. HD
patients were studied during their interdialytic day. All infusions were
administered in the brachial artery. In control subjects, we used the brachial
artery of the nondominant arm, whereas in HD patients, the arm opposite the
one carrying the arteriovenous fistula was cannulated. We inserted a 20-gauge
silicon catheter (Vygon; Ecouen, France) under local anesthesia (1%
lignocaine) using the Seldinger technique. A pressure transducer (Baxter;
Unterschleißheim. Germany) was connected to the arterial catheter via a
three-way stopcock for continuous monitoring of arterial BP. Forearm blood
flow (FBF) was measured by strain gauge plethysmography (Periquant 815;
Gutmann Medizinelektronik, Eurasburg, Germany) as published
(9), with modifications. The
modifications were required because HD patients have only one arm for flow
measurements (the other arm usually has the dialysis fistula and is therefore
unavailable). Accordingly, flow measurements were obtained in the cannulated
arm only in all participants. The upper arm cuff for venous congestion was
kept inflated at 40 mmHg for 10 s during each 15-s cycle of FBF measurement.
The circumferential increase of the forearm in response to the cuff-induced
venous congestion was measured by a calibrated strain gauge that was placed
around the widest part of the forearm. Arterial inflow during venous outflow
obstruction is proportional to the increase in circumference
(10). Arterial inflow is
expressed as milliliters per 100 ml of forearm volume. To provide for a rapid
venous outflow after cessation of venous congestion, the forearm was placed 10
cm above the level of the right atrium. During all recording periods, the hand
was excluded from the circulation by a wrist cuff inflated to a suprasystolic
pressure of 200 mmHg.
Determinations of baseline FBF were started 20 min after insertion of the arterial catheter. As shown in Figure 1, at least two baseline determinations of FBF were obtained before the beginning of the experimental protocol. Each determination took 2.5 min. We made 10 individual measurements in succession during each 2.5-min period of FBF determination. Determinations of FBF were repeated during all subsequent periods of drug infusion. Each drug dose was given for 4 min at a constant rate of 1 ml/min. We made 16 measurements of FBF during each dosing period. Between the infusions of different agents, a control period of 20 min was allowed, and baseline FBF was determined thereafter by six individual measurements before the infusion of any new agent (Figure 1).
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Study Protocol and Agents
After the end of the baseline period, participants received an infusion of
L-ARG at one dose (57 µmol/min = 10 mg/min). In the second part of the
protocol, ACH was given in increasing doses of 55, 110, and 220 nmol/min. In
the third part of the protocol, subjects received LMA in three doses (1, 2,
and 4 µmol/min). The study protocol was finished by observations of the
effects of three doses (2.2, 4.4, and 8.8 nmol/min) of GTN. In a different
session, six HD patients and six matched control subjects received three doses
of NE (60, 120, and 240 pmol/min).
Agents were obtained from the following: L-ARG and LMA from Clinalfa (Läufelfingen, Switzerland), ACH as Acetylcholinum ophthalmicum dispersa from Ciba Vision (Germering, Germany), GTN as Perlinganit from Schwarz-Pharma (Monheim, Germany), and NE as Arterenol from Hoechst Marion Roussel (Frankfurt, Germany). All drug solutions were prepared fresh before each study using isotonic saline to obtain the required concentrations.
Statistical Analyses
For each determination of FBF, the mean of the last 10 individual FBF
measurements was calculated (in several control periods, the mean of six
individual measurements was calculated)
(Figure 1). Observations are
reported in percentage of the FBF baseline value (FBF observed/baseline FBF
x 100) ± SEM. For the baseline FBF, we used the one determined
just before the period of observation. As shown in the studies using GTN, the
response of FBF to defined doses of NO was different between HD patients and
control subjects; we therefore report the results of observations involving
L-ARG, ACH, and LMA corrected for this difference. For these corrections, we
used the equation 100 + {[FBF response to test agent (in percentage of
baseline) - 100]/[FBF response to GTN 3 (in percentage of baseline) - 100]}
x 100.
Comparisons between HD and control groups were done by two-way ANOVA for repeated measurements. The data for baseline FBF as well as those for observations under L-ARG were evaluated by t test. P < 0.05 was considered statistically significant. Statistical calculations were performed by means of the computer software Statistical Package for the Social Sciences, version 8.0.1.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Baseline FBF values in the second half of the protocol before LMA and GTN in HD patients and control subjects differed slightly from initial baseline FBF (before L-ARG). However, this variation was similar in HD patients and control subjects (baseline before LMA: 142 ± 11% [SEM] of initial baseline in control subjects versus 131 ± 11% in HD patients, P = 0.1; baseline before GTN: 91 ± 7% in control subjects versus 77 ± 4% in HD patients, P = 0.44).
In this section, the presentation of our observations is given in a sequence other than that of the actual experiments to facilitate interpretation of results. Table 2 lists all observed raw data. Table 3 basically reports the same data as Table 2 in percentage of baseline.
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GTN infusion (Figure 2) resulted in a significant and dosedependent increase in FBF. Control subjects: 183 ± 20% (GTN 1); 246 ± 26% (GTN 2); and 338 ± 29% (GTN 3); HD patients: 161 ± 7% (GTN 1); 206 ± 12% (GTN 2); and 262 ± 24% (GTN 3). The HD group had a significantly smaller response to GTN than did control subjects (P = 0.032).
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Because the effects of L-ARG, ACH, and LMA depended on the ability of arterial resistance vessels to respond to NO and because HD patients showed a significantly smaller response to defined doses of NO than control subjects did, we decided to report all results of those observations after correction Figures (3,4,5) for a standard NO-mediated dilation (GTN 3).
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L-ARG infusion (Figure 3) resulted in an increase in FBF to 108 ± 4% in control subjects and 103 ± 4% in HD patients. There was no difference between both groups (P = 0.36).
ACH infusion (Figure 4) was followed by a dose-dependent rise in FBF. Control subjects: 246 ± 32% (ACH 1), 340 ± 40% (ACH 2), and 465 ± 52% (ACH 3); HD patients: 251 ± 55% (ACH 1), 244 ± 36% (ACH 2), and 318 ± 50% (ACH 3). Comparison of both groups revealed significantly less ACH-induced vasodilation in the HD group than in the control group (P = 0.002).
NE induced a significant and dose-dependent decrease of FBF in both groups. Control subjects: 83 ± 5% (NE 1), 67 ± 4% (NE 2), and 56 ± 5% (NE 3); HD patients: 85 ± 3% (NE 1), 71 ± 5% (NE 2), and 52 ± 5% (NE 3). These responses statistically were not different (P = 0.15), thus indicating similar endothelium-independent vasoconstriction in both groups.
LMA reduced FBF in a dose-dependent manner (Table 2). We reasoned that the impairment of response to defined doses of NO (GTN experiment) most likely would prevail in the LMA observations as well. We therefore corrected the FBF response to LMA accordingly (i.e., reporting the FBF data as fraction of the standard NO-induced vasodilation [GTN 3]). The results (90 ± 2% [LMA 1], 83 ± 2% [LMA 2], and 74 ± 4% [LMA 3] in control subjects versus 84 ± 3% [LMA 1], 73 ± 3% [LMA 2], and 64 ± 4% [LMA 3] in HD patients) (Figure 5) demonstrated a significant difference of the calculated data. This probably shows an increased constrictor response to LMA in the HD group (P = 0.037).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four different aspects of the NO system were studied: (1) We infused L-ARG to assess the role(s) of potential L-ARG deficiency and of possible endogenous NOS inhibitors and to obtain an indication of the effect of L-ARG on NO formation. (2) We infused ACH to stimulate endothelial NOS (eNOS, NOS III). (3) LMA was given to block the activity of all isoforms of NOS. This was used to explore the contribution of endogenous NO to basal FBF. (4) We infused GTN to determine the underlying capacity of resistance vessels to respond to defined doses of (exogenous) NO. (5) In an additional experiment, we tested vascular responsiveness to the endothelium-independent vasoconstrictor NE for comparison with the results obtained under LMA infusion.
Few published studies have attempted to delineate the role of the NO system in uremic patients in vivo. Most of them used noninvasive techniques. Several authors measured vasodilation in arterial conduit vessels in response to postischemic flow and to sublingual GTN (12,13,14) by high-resolution ultrasound. Flow-dependent vasodilation in conduit vessels is known to be dependent on endothelium-derived NO (15). In another study, the vasomotion of dorsal hand veins in response to local infusion of ACH, GTN, and L-ARG was tested (16). For several reasons, it is difficult to compare our results with the studies mentioned previously. First, NO effects were studied in different types of vessels. Second, the age of the participants as well as the duration and state of uremia in the individual study groups differed. Third, none of the techniques mentioned is able to provide information on the contribution of NO to the basal vascular tone. Compared with these methods, the technique that we used may offer a technically more direct and a pharmacologically better defined approach to arterial vascular NO function. In a recently published abstract (17), the authors also documented use of our technique in patients with renal insufficiency; however, the patients were not undergoing dialysis. In comparison with these studies, we offer the following comments concerning our results.
Response to GTN
We considered the dilatory properties of NO in the muscular vessel wall
per se. As shown in Figure
2, the response to GTN was reduced consistently and significantly
in the HD patients. Recently, Adams et al.
(18) performed a large,
ultrasound-based study of endothelium-dependent and -independent vasodilation
in 800 volunteers. They demonstrated that the vasodilator response to
exogenous NO is impaired in subjects who are at risk for atherosclerosis
(cigarette smoking, diabetes mellitus, hypercholesterolemia) independently
from endothelium-dependent vasodilation. Joannides et al.
(14) and Hand et al.
(17) in their studies found
the response to GTN to be decreased in uremic subjects. Conversely, Kari
et al. (12), van
Guldener et al. (13),
and Hand et al. (16)
found in their patients a response to GTN that was not significantly different
from that in control subjects. We are unable to explain these different
findings. They may be related in part to methodologic aspects and different
study populations as mentioned previously. It is conceivable that the
preuremic state and/or the young age of subjects in the study of Kari et
al. is associated with lesser influences on the vasodilatory effects to
exogenous NO than those to be found in fully established uremia of adults.
There are several conceivable explanations for the diminished response to exogenous NO in HD patients. Oxidative stress—believed to be present in HD patients (19,20)—may have contributed to inactivation of NO (21,22). This possibility might be tested using antioxidants. Furthermore, there may be a defect in the vascular smooth muscle cell signaling pathway downstream from NO. For instance, an impairment of the activity of guanylate cyclase could result in a reduced vasodilation in response to exogenous and endogenous NO. Finally, fibrosis of the arterial wall may cause increased stiffness and reduced distensibility of the vessels in HD patients. Such observations have been reported by Mourad et al. (7). However, this observation was made in conduit vessels, and it is unclear to what extent their findings might apply to resistance vessels (that have been studied by us) as well. Recent, albeit preliminary, studies in our laboratory have tested the effects of adenosine (75, 150, and 300 µg/min) instead of GTN as a vasodilator. Under these conditions, resistance vessels of HD patients dilated comparably with control subjects. These preliminary findings therefore may indicate that a defect in the cellular signaling cascade of NO or increased breakdown of NO may be involved in the deficient response to GTN in HD patients. Our finding of a decreased vascular response to exogenous NO may have implications for nitrate therapy in uremic patients.
Basal NO Tone
After correction for standard NO-induced vasodilation (GTN 3), LMA induced
greater vasoconstriction in HD patients than in control subjects
(Figure 5). Studies using
intrabrachial infusions of LMA in uremic patients have not been reported in
the literature. Given that the principal constrictory properties are
comparable in both groups (response to NE), our result suggests an increased
baseline vascular NO release in uremic patients. A note of caution may be
appropriate. On the basis of Figure
2 (dose—response curve for GTN), we made the assumption that
both curves would continue steadily in the range of very low NO and even at
FBF levels below 100% of baseline. Although this assumption seems justified
from the results in Figure 2,
it has not been tested directly (infusion of NO scavengers). Studies of NO
production in uremia are controversial. Most laboratories performed
measurements of stable NO metabolites—as markers of NO—in uremic
patients. They were usually found to be elevated
(23,24,25,26),
but contrary results have been reported, too. A recent study determined
whole-body NO production by radionuclide-based measurement of arginine to
citrullin conversion and reported a reduced release of NO in uremic patients
compared with matched control subjects
(27). However, the
participants of this study were markedly older and at least some of them had
hypertension and atherosclerosis. Thus, conditions other than uremia may have
contributed to the reduced NO production in these patients. In addition, these
individuals were not undergoing renal replacement therapy. It has been
suggested that the frequent blood-membrane contact in HD patients might be
associated with NOS activation and hence increased NO production
(28). Thus, the results of
this study (27) may not be
representative of the population reported here. Furthermore, endothelial cells
in culture produced more NO after exposure to uremic than to normal serum
(29). In addition, Aiello
et al. (30) found an
increased baseline NO production in the systemic circulation of rats with
renal insufficiency compared with control subjects. This was associated with
an increased vascular expression of eNOS and inducible NOS. It is unclear
whether this finding also applies to uremia in humans. Taken together, uremia
in normotensive HD patients seems not to be a state of NO deficiency
under baseline conditions but rather is a state of NO excess. Uremia has been
described as a state of chronic endothelial injury, as shown by a typical
pattern of endothelial markers in the sera of HD patients
(31). It is conceivable that
an elevated baseline NO production in the presence of oxidative stress would
result in increased peroxynitrite formation, thus leading to maintained
endothelial damage. Future studies should be directed at the isoform(s) of NOS
in uremia and its stimuli. In addition, it might be useful to test whether
uremic toxins or factors related to the mode of the dialysis treatment are
involved.
In our study, we did not measure L-ARG analogues—such as dimethyl-L-arginine—directly. It has been proposed that L-ARG analogues accumulate in renal failure, causing competitive inhibition of NOS (32). In the present studies, however, the effects of LMA in uremic patients were not reduced, as would be expected in the presence of significant levels of L-ARG analogues. Our observations therefore did not show a major role of these compounds in normotensive patients on HD. A similar result was reached by Anderstam et al. (33). However, these observations do not exclude a potential role of L-ARG analogues in the setting of hypertensive uremia.
Intrabrachial Infusion of L-ARG
These infusions, delivered at a dose of 10 mg/min, led to a small increase
in FBF in control subjects. In HD patients, FBF tended to increase as well,
but this did not become significant. The mechanism(s) by which L-ARG induces
vasodilation has not been clarified conclusively. One suggestion is that L-ARG
stimulates NO via its effect to increase the release of insulin
(34), but other mechanisms
also may be involved
(35,36,37).
Imaizumi et al. (38)
described a threshold dose for L-ARG infusions of 20 mg/min in healthy
volunteers. We cannot explain the differences in threshold dose; however, it
is obvious that the dose that we used was a relatively small one. It has been
suggested in the literature that uremia is a state of potential L-ARG
deficiency (39). We did not
measure L-ARG in the plasma of our participants. However, the response to
L-ARG in HD patients (Figure 3)
was not greater than that of control subjects, as we would have expected in
L-ARG deficiency. In fact, the response seemed to be less in HD patients than
in control subjects. Therefore, our observations fail to support the
possibility of L-ARG deficiency in uremic patients on HD, at least under
baseline conditions.
Stimulation of eNOS by ACH
The FBF response to intrabrachial infusion of ACH was considerably
decreased in the HD group compared with control subjects
(Figure 4). These observations
are basically in accordance with previous reports in the literature
(12,13,14,
16). The techniques used and
the vascular tributaries examined by these studies were different from our own
approach. Nonetheless, the summarized data suggest reduced ACH-triggered
endothelium-dependent vasodilation in uremia. ACH stimulates the endothelial
release of NO, vasodilating and/or constricting prostaglandins, and
endothelium-derived hyperpolarizing factor(s)
(8). On the basis of our study,
we cannot ascribe the reduced responsiveness of uremic resistance vessels to
ACH to the absence or presence of one of these factors alone. Additional human
studies are necessary to delineate the potential role of vasoconstrictor
prostaglandins and of LMA-resistant vasodilation.
With respect to the NO system, it would be useful to test whether L-ARG can
improve the blunted vascular response to ACH, which has been shown in human
dorsal hand veins (16) but
could not be demonstrated in experimental renal failure
(40). Similarly, a reduced
availability of critical cofactors for NO synthesis, such as
tetrahydrobiopterin, might be involved. Furthermore, proinflammatory cytokines
(e.g., tumor necrosis factor 
), which usually are present in
HD patients
(41,42),
may cause endothelial dysfunction
(43). Alternative explanations
include changes in the expression of eNOS and/or inducible NOS
(44). Additional studies would
be useful to delineate the role of these potentially important factors in
uremia.
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