1 The Renal Unit and 2 Department of Cardiology, Western Infirmary, Glasgow, UK
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Abstract |
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Methods. Sixteen uraemic patients (pre-dialysis and continuous ambulatory peritoneal dialysis) and 18 controls were studied. Forearm plethysmography was used to measure forearm blood flow and the changes induced by carbachol (endothelium-dependent vasodilator) and sodium nitroprusside (SNP; endothelium-independent vasodilator). The order of drugs infused was randomized between subjects. Dose response curves were constructed for each agent and area under the curve (AUC) calculated (arbitrary units).
Results. Overall, vasodilatation to SNP and carbachol was similar between uraemic patients and controls. However, it became apparent that there was a marked order effect for the drugs infused, such that infusion of SNP as the first agent blunted the subsequent response to carbachol. When only those patients and controls who received carbachol followed by SNP were studied (10 in each group), the response to carbachol in uraemic patients was attenuated compared to controls: AUC (median(range)) for uraemic patients 529.0 (150.9834.7) compared to AUC for controls 703.9 (583.51576.6); P=0.028. Vasodilatation to SNP was, however, similar between groups: AUC for uraemic patients 1475.0 (857.84717.1) compared to AUC for controls 1328.1 (216.63311.4); P=0.545.
Conclusions. This study has demonstrated a marked drug order effect not previously described for forearm plethysmography. When the order effect was taken into account, this study demonstrated reduced vasodilatation to carbachol in uraemic patients with a preserved response to SNP. This pattern indicates impaired endothelium-dependent vasodilatation in uraemic patients, a defect that may predispose this group to accelerated atherosclerosis.
Keywords: cardiovascular disease; forearm plethysmography; hypertension; nitric oxide; uraemia; vascular endothelium
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Introduction |
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The vascular endothelium plays a pivotal role in controlling blood vessel tone and preventing the development of atherosclerosis, principally through the production and release of vasoactive compounds such as nitric oxide (NO). Nitric oxide has several anti-atherogenic roles including vasodilatation [4], inhibition of vascular smooth muscle cell proliferation, inhibition of platelet adhesion and aggregation and inhibition of monocyte adhesion and migration. The production of NO by the vascular endothelium is continuous, and contributes to the resting tone of the vessel. NO is produced by the constitutive enzyme NO synthase (eNOS), the substrate being L-arginine. Production is stimulated by circulating or locally released substances such as acetylcholine and bradykinin, and in conditions of hypoxia and increased shear stress. The activity of the enzyme is inhibited by L-arginine analogues such as NG-monomethyl L-arginine and asymmetric dimethylarginine (ADMA), endogenous substances known to accumulate in CRF [5].
In addition to NO, the vascular endothelium produces other vasodilating substances including prostacyclin and endothelium-derived hyperpolarizing factor, which also contribute to resting vascular tone. Stimulation of the vascular endothelium with an agonist such as acetylcholine (or carbachol as in this study) will release endothelium-derived hyperpolarizing factor as well as NO.
This present study was designed to examine endothelium-dependent vasodilatation in adult uraemic patients compared to controls using bilateral forearm strain-gauge plethysmography.
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Subjects and methods |
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The patient and control groups were comparable for age, sex, and body mass index, and had similar smoking habits and serum cholesterols. Background characteristics for patients and controls are detailed in Table 1.
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All subjects were studied in the morning after an overnight fast, and were asked to omit anti-hypertensive medication for at least 48 h before the study. Subjects were asked to avoid caffeine and alcohol, and to refrain from smoking, for at least 12 h prior to the study.
Plethysmography
Forearm strain-gauge plethysmography works on the principle that during short-term venous occlusion of the forearm, the rate of distension of the forearm is proportional to the rate of arterial inflow into the forearm. Forearm blood flow (FBF) can be measured at rest and after infusion of drugs.
All forearm studies were performed in a sealed, sound-proofed, vascular research laboratory maintained at 2426°C. Subjects lay supine with arms supported above heart level on foam blocks. Paediatric cuffs (Hokanson TMC-7, PMS Instruments, Maidenhead, UK) were placed around the wrists, and inflated to at least 40 mmHg above systolic blood pressure for 3 min during each recording, to exclude the hand circulation from the system. Collecting cuffs (Hokanson SC10) were placed around the upper arms and inflated (40 mmHg) and deflated in 15-s cycles. Rapid cuff inflation was achieved using a Hokanson AG101 air source linked to two Hokanson E20 rapid cuff inflators. Blood pressure was measured at the end of each blood flow recording, using a semiautomatic oscillometric sphygmomanometer (Critikon Dinamap Plus, FL, USA) placed around the dominant arm, over the collecting cuff.
The maximal circumference of the forearm was measured and a mercury-in-silastic strain gauge (Hokanson forearm set) chosen 2 cm shorter than the circumference. Strain gauges were calibrated electrically while in position using a built-in calibration method.
Under local anaesthesia (1 ml 1% lignocaine), a sterile 27-gauge dental needle (Terumo, Japan) was inserted into the brachial artery of the non-dominant forearm. The needle was connected to an IVAC infusion pump using a modified 16-gauge epidural giving set (Portex, Hythe, UK), and drugs or 0.9% saline were infused throughout the study at 1 ml/min. Baseline measurements of FBF were obtained at 10-min intervals for 30 min to allow acclimatization to inflation and deflation of the cuffs. The final baseline reading was used as the baseline FBF.
Infusion protocol
After an acclimatization period of 30 min with infusion of 0.9% saline, incremental infusions of carbachol (0.1, 0.3, 1.0, 3.0 µg/min) and sodium nitroprusside (SNP) (0.3, 1.0, 3.0, 10.0 µg/min) were commenced. There was a 30-min washout period with 0.9% saline between drug infusions and the order of drugs infused was randomized between subjects in blocks of 10. The protocol is illustrated in Figure 1. All drug solutions were prepared in 0.9% saline in the sterile pharmacy productions unit in the hospital. Each drug was infused for 7 min with FBF measurements taken from min 36, and blood pressure recorded in the seventh minute. During each drug infusion, several FBF measurements were made; the mean of the final five readings was taken as the FBF achieved for each dose of drug.
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Statistical comparison
Results are expressed as median (range) unless otherwise specified. For each dose response curve, area under the curve (AUC) was calculated using the trapezoid rule [8]. This simply involves measuring the area under each section of a dose-response curve, and calculating an overall area under the curve. The benefit of this method is that a single summary measure is given for each dose-response curve, allowing easy statistical comparison between groups by MannWhitney test. Statistical significance was defined at the 5% level.
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Results |
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Effect of infusion of carbachol and SNP in all subjects
Overall there was no difference in response of the two groups to infusion of carbachol or SNP. The area under the curve for infusion of carbachol in uraemic patients was 487.9 (150.81180.3) compared to 598.4 (191.51576.6) for controls, P=0.463. The AUC for infusion of SNP in uraemic patients was 1867.4 (216.63521.4) compared to 1734.6 (857.84717.1) for controls, P=0.986. The higher AUC values for SNP compared to carbachol simply reflect the higher doses of drug used.
When performing the studies it became clear that there was a drug order effect which was introducing error. Despite a 30-min wash-out period, infusion of SNP as the first drug appeared to have an unusually prolonged effect, such that the second baseline was rarely equivalent to the first. The infusion of SNP as the first drug therefore blunted the subsequent response to carbachol. This is illustrated in Figure 2, where patients and controls combined as a group are split into those who received carbachol as the first or second drug. It can be clearly seen that the response to carbachol is markedly attenuated if it is infused after SNP.
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Effect of infusion of carbachol and SNP in subgroup of subjects in whom SNP was infused last
When those patients (n=10) and controls (n=10) who received carbachol first followed by SNP are examined as a subgroup, results are clearly different. The background characteristics of this subgroup are detailed in Table 2; patients and controls are again matched for age, sex, smoking habits, and serum cholesterol. The response to infusion of carbachol is depicted in Figure 3
and to SNP in Figure 4
. For infusion of carbachol, the AUC was significantly reduced in uraemic patients: 529.0 (150.9834.7) for uraemic patients compared to 703.9 (583.51576.6) for controls; P=0.028. For infusion of SNP, the AUC did not differ significantly between the two groups: 1328.1 (216.63311.4) for uraemic patients compared to 1475.0 (857.84717.1) for controls; P=0.545).
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Blood pressure
As the doses of drugs were chosen to act only on the forearm, blood pressure did not change significantly throughout the studies (Tables 3 and 4
) except for a small decrease in diastolic blood pressure after infusion of SNP in controls.
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Discussion |
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Results from all subjects demonstrated similar vasodilatation to both carbachol (endothelium-dependent vasodilator) and SNP (endothelium-independent vasodilator) in both uraemic patients and controls without renal disease. However, the infusion of SNP first appeared to blunt the subsequent response to carbachol, perhaps owing to a more prolonged effect of SNP. This feature has not previously been described in plethysmography, and is important for planning future studies. We therefore excluded those patients and controls who had received SNP first from subsequent analysis. Ten patients and controls were compared, in whom carbachol was infused first followed by SNP, and these groups were again comparable in age, sex, cholesterol, and smoking habits (Table 2). In this substudy the pattern of normal vasodilatation to SNP but impaired vasodilatation to carbachol indicates a reduced ability of the vascular endothelium in uraemic patients to produce and release vasodilator substances upon stimulation.
This pattern of endothelial dysfunction has previously been described in hypercholesterolaemia [9], smoking [10], and diabetes [11] and is thought to predispose to the development of atherosclerosis. In our study, diabetics were excluded and patients had similar smoking habits and serum cholesterol levels to controls. However, blood pressure was significantly higher in the uraemic patients, as would be expected, although the median blood pressure in this group was only 140/81 mmHg. It is widely believed that hypertension is associated with endothelial dysfunction, although it is not known whether the endothelial dysfunction is a primary defect (unlikely) or whether it occurs as a response to the elevated blood pressure. While a number of studies have demonstrated endothelial dysfunction in essential hypertension [12,13], one plethysmography study by Cockcroft et al. [14] failed to demonstrate this, and the issue remains controversial. We were unable in this present study to match patients and controls for blood pressure, but further larger studies are warranted to compare endothelial function in uraemic and essential hypertensive patients.
Our patient and control groups also differed with respect to triglyceride and haemoglobin levels. Hypertriglyceridaemia, while being an independent cardiovascular risk factor, is not thought to affect endothelial function adversely [15]. In contrast, as NO binds avidly to the haem moiety of haemoglobin, changes in haemoglobin concentration could theoretically alter the response to endothelium-dependent vasodilators. However, in our study the uraemic patients had lower haemoglobin concentrations and would, if anything, be expected to have an exaggerated response to carbachol, as NO would theoretically have a longer half-life. This was not seen, and the effects of anaemia in this situation are therefore not clear.
The pattern of endothelial dysfunction demonstrated by this technique in uraemic patients may reflect a direct inhibitory effect of circulating factors such as ADMA on eNOS, although there is debate about whether ADMA levels reach physiological significance. It is also possible that the impaired endothelium-dependent vasodilatation is secondary to the effects of higher blood pressures in the uraemic patients as opposed to control subjects, or to the damaging effects of increased oxidative stress.
Another potential atherogenic uraemic factor may be homocysteine, an amino acid that accumulates in renal failure. In this study, homocysteine levels were considerably higher in the uraemic group compared to controls. Hyperhomocysteinaemia is associated with increased cardiovascular mortality in the general [16] and CRF [17] populations, and hyperhomocysteinaemia impairs endothelium-dependent vasodilatation [18]. Hyperhomocysteinaemia is 100 times more common in dialysis patients than the general population, and may therefore be one link explaining endothelial dysfunction and accelerated atherosclerosis in uraemia.
Our results are in keeping with those reported from non-invasive studies in uraemic children [19] and in adult haemodialysis patients [20]. We believe that it is likely that endothelial dysfunction develops at the earliest stages of renal disease when glomerular filtration rate begins to fall and blood pressure rises. This raises the issue of what can be done to treat or reverse endothelial dysfunction, and at what stage therapy should be commenced. Several drugs are known to improve endothelial function, including angiotensin-converting enzyme (ACE) inhibitors [21], statins [9], L-arginine [20], and vitamin C [22]. Surprisingly, a study in CAPD patients with folic acid supplementation (which lowers homocysteine significantly) failed to improve endothelial function [23]. Lifestyle modification, for example, cessation of smoking, may also reverse endothelial dysfunction [10].
Forearm plethysmography is a powerful technique with which to study the effects of vasoactive drugs in vivo. It has drawbacks, however, in that intra-subject variability has been estimated to be around 19% [7]. We did not measure intra-subject variation in this study, as it would have necessitated two arterial cannulations. Our results are also limited by small numbers, although plethysmography studies are often small, as recruitment of subjects can be difficult because of the arterial puncture and the requirement for subjects to lie still for 23 h.
In summary, this study has demonstrated a marked drug order effect with the technique of forearm plethysmography such that randomization of the drug order when using SNP is inadvisable, and SNP should be infused at the end of a protocol. When this order effect was taken into account, we demonstrated impaired endothelium-dependent vasodilatation in uraemic patients compared to controls, the exact mechanisms involved being unclear. It is likely that endothelial dysfunction develops at an early stage in renal disease, and that this predisposes to the development of atheroma. Several treatments including ACE inhibitors, statins, anti-oxidants, folate, and L-arginine may modulate endothelial function, and should be assessed further in this high-risk population.
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Acknowledgments |
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Notes |
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References |
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