Impaired endothelium-dependent vasodilatation in uraemia

Scott T. W. Morris1,, John J. V. McMurray2, R. Stuart C. Rodger1 and Alan G. Jardine1

1 The Renal Unit and 2 Department of Cardiology, Western Infirmary, Glasgow, UK



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Patients with chronic renal failure (CRF) have a substantially increased risk of cardiovascular death, the proposed mechanisms being arrhythmias (left ventricular hypertrophy) and accelerated atherosclerosis. The vascular endothelium protects against the development of atherosclerosis principally by releasing vasoactive substances such as nitric oxide (NO) and endothelium-derived hyperpolarizing factor. In CRF there is accumulation of endogenous inhibitors of NO synthesis. In this present study we assessed endothelium-dependent vasodilatation in patients with advanced uraemia.

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 (150.9–834.7) compared to AUC for controls 703.9 (583.5–1576.6); P=0.028. Vasodilatation to SNP was, however, similar between groups: AUC for uraemic patients 1475.0 (857.8–4717.1) compared to AUC for controls 1328.1 (216.6–3311.4); 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



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The paradox of successful developments in dialysis and renal transplantation is that patients with chronic renal failure (CRF) now die prematurely of cardiovascular disease rather than of uraemia. Overall, patients with CRF have a 20-fold increased risk of cardiovascular death compared with age- and sex-matched controls; substantially worse than the 3-fold excess risk associated with diabetes [1]. The mechanisms underlying this greatly increased risk are poorly understood, but are likely to reflect changes in both the heart and the vasculature. Conventional cardiovascular risk factors such as hypertension, smoking, and dyslipidaemia are prevalent in the renal failure population, but do not appear to carry the same prognostic weight as they do in the general population. Studies in the 1970s suggested that uraemia is associated with accelerated atherosclerosis [2,3] and led to the concept of an as yet unidentified atherogenic ‘uraemic factor(s)’.

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.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Subjects
Patients with advanced renal failure (median creatinine 786 µmol/l) were recruited from the pre-dialysis (n=10) and continuous ambulatory peritoneal dialysis (CAPD, n=6) clinics at the Western Infirmary, Glasgow. Controls without renal disease were obtained from hospital staff and by advertisement. All subjects gave written informed consent, and the protocol was approved by the local ethics committee.

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 1Go.


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Table 1. Background characteristics for uraemic patients and controls

 
Patients were excluded if they were currently anti-coagulated, had a history of diabetes mellitus or vasculitis, or were taking anti-anginal medications or statins. Patients were also excluded if they had severe hypertension such that anti-hypertensive drugs could not be withheld prior to the study, or if they had previously been treated by haemodialysis and had functioning AV fistulae. For those patients on CAPD the median (range) time on dialysis was 11.5 (7–32) months. Nine of the 16 patients were taking anti-hypertensive drugs: seven were taking a calcium-channel blocker, six were taking a beta-blocker, and one patient was on doxazosin. No patients or controls were taking aspirin.

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 24–26°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 1Go. 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 3–6, 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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Fig. 1. Infusion protocol for plethysmography. Each dose of drug infused for 7 min. Saline or drugs (dissolved in saline) infused at 1 ml/min for duration of study.

 
FBF is expressed as millilitres blood flow/min/ 100 ml forearm [6]. Bilateral FBF measurements were made, and results expressed as a ratio of infused:non-infused arm, since this method is more reproducible than unilateral plethysmography [7], and corrects for background variation in FBF. The response to a particular drug infusion rate was calculated as the percentage change in FBF ratio from baseline.

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 Mann–Whitney test. Statistical significance was defined at the 5% level.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The two groups had a similar age and sex distribution, and there was a similar number of cigarette smokers in each group (Table 1Go). Total serum cholesterol was similar in patients and controls, although the fasting serum triglyceride level was significantly greater in patients. Both systolic and diastolic blood pressures were higher in uraemic patients, and the uraemic patients had lower haemoglobin concentrations.

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 (150.8–1180.3) compared to 598.4 (191.5–1576.6) for controls, P=0.463. The AUC for infusion of SNP in uraemic patients was 1867.4 (216.6–3521.4) compared to 1734.6 (857.8–4717.1) 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 2Go, 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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Fig. 2. Dose-response curves for infusion of carbachol in all subjects, divided into those who received carbachol as first drug infused and those who received carbachol second, after infusion of SNP. Results expressed as means±SEM with comparison of AUC by Mann–Whitney test.

 

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 2Go; patients and controls are again matched for age, sex, smoking habits, and serum cholesterol. The response to infusion of carbachol is depicted in Figure 3Go and to SNP in Figure 4Go. For infusion of carbachol, the AUC was significantly reduced in uraemic patients: 529.0 (150.9–834.7) for uraemic patients compared to 703.9 (583.5–1576.6) for controls; P=0.028. For infusion of SNP, the AUC did not differ significantly between the two groups: 1328.1 (216.6–3311.4) for uraemic patients compared to 1475.0 (857.8–4717.1) for controls; P=0.545).


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Table 2. Background characteristics for subgroup of uraemic patients and controls in whom SNP was infused last

 


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Fig. 3. Dose-response curves for infusion of carbachol in only those patients (n=10) and controls (n=10) who received carbachol followed by SNP. Results expressed as means±SEM with comparison of AUC by Mann–Whitney test.

 


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Fig. 4. Dose-response curves for infusion of SNP in only those patients (n=10) and controls (n=10) who received carbachol followed by SNP. Results expressed as means±SEM with comparison of AUC by Mann–Whitney test.

 

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 3Go and 4Go) except for a small decrease in diastolic blood pressure after infusion of SNP in controls.


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Table 3. Blood pressure values before and after infusion of carbachol

 

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Table 4. Blood pressure values before and after infusion of SNP

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The vascular endothelium provides a strategic barrier between the circulating blood and the tissues. Endothelial dysfunction is thought to be a key initial event in the development of atherosclerosis, a principal mechanism being through a loss or reduction in effect of the anti-atherogenic molecule NO. This study was designed to examine endothelium-dependent vasodilatation in adult patients with advanced uraemia.

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 2Go). 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 2–3 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.



   Acknowledgments
 
Dr Morris is funded through a British Heart Foundation Junior Fellowship.



   Notes
 
Correspondence and offprint requests to: Dr Scott Morris, Renal Unit, Western Infirmary, Dumbarton Road, Glasgow G11 6NT, UK. Back



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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 30. 7.99
Revision received 23. 3.00.