Atorvastatin improves endothelial function in renal-transplant recipients

Anders Åsberg, Anders Hartmann, Ellen Fjeldså and Hallvard Holdaas

Laboratory for Renal Physiology, Section of Nephrology, Medical Department, The National Hospital, Oslo, Norway



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Hyperlipidaemia and endothelial dysfunction are common features in cyclosporin A (CsA)-treated renal transplant recipients. Endothelial dysfunction may contribute to the risk of premature atherosclerosis and cardiovascular death in these patients. A beneficial effect of statin therapy beyond cholesterol lowering may be an improvement of endothelial function. The present study was designed to assess the effect of atorvastatin on serum lipids and endothelial function in CsA treated renal transplant recipients.

Methods. This pilot study was an open trial of 4 weeks atorvastatin (10 mg per day) treatment in renal transplant recipients (n=22). All patients received a CsA- and prednisolone-based immunosuppressive regimen. Endothelial function was assessed in the forearm skin microvasculature by acetylcholine stimulation and laser Doppler flowmetry, before and after atorvastatin treatment. Serum lipids, plasma endothelin-1 (ET-1), nitric oxide (NO), and von Willebrand factor (vWF) were also measured.

Results. Both total and LDL cholesterol were significantly reduced by 26.8 ± 8.4 and 41.5 ± 11.0% respectively, after 4 weeks of treatment. Endothelial function was significantly improved during atorvastatin treatment, area under the flux versus time curve (AUC)ACh was 538 ± 362 AUxmin before and 682 ± 276 AUxmin after treatment (P=0.042). Plasma NO levels also showed a borderline significant increase from 49 ± 30 to 57 ± 37 µmol/l during the treatment period (P=0.051), though plasma ET-1 (0.37±0.08 vs 0.37±0.12 fmol/ml) and vW (196±57 vs 197±37%) were unchanged.

Conclusion. Atorvastatin lowered serum cholesterol significantly and improved endothelial function in renal transplant recipients after 4 weeks of treatment. Plasma NO levels were increased during atorvastatin treatment, indicating a possible endothelial protective effect through an ‘endothelial–NO pathway’.

Keywords: atorvastatin; CsA; endothelial function; kidney; nitric oxide; transplantation



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The introduction of cyclosporin A (CsA) to standard immunosuppressive protocols in the 1980s has greatly improved graft survival in renal transplantation [1], but patients continue to die prematurely from cardiovascular disease [2]. Solid-organ transplant recipients receiving CsA display endothelial dysfunction [35] and elevated atherogenic lipids [6]. Both factors are likely to play an important role in development of premature cardiovascular disease.

Impaired endothelial function in CsA-treated solid-organ recipients might be a consequence of reduced bioavailability of nitric oxide (NO) [37] or an increase in endothelin-1 (ET-1) release [8,9]. There is some evidence suggesting that statins (HMG-CoA reductase inhibitors) may improve endothelial function in heart-transplanted patients treated with CsA and pravastatin [10]. However, a recent well-controlled study failed to demonstrate any beneficial effect of simvastatin on endothelial function in patients with heart disease [11]. Thus, the evidence for a beneficial class effect of statins on endothelial function is not clear.

Some studies indicate that statins may improve endothelial function that is partly independent of the cholesterol-lowering effect [1214]. Atorvastatin, having 10–15 times higher distribution volume compared with other statins, may therefore have a more substantial potential to interact with mechanisms in extrahepatic tissues, and may therefore show better endothelial protective properties. Previous studies in non-transplanted patients have demonstrated improved endothelial function after treatment with atorvastatin [15,16].

Endothelial function can be investigated by studying microvascular reactivity following specific stimulation. In the present study, changes in forearm skin blood flow were assessed by laser Doppler flowmetry following acetylcholine stimulation and during post-occlusive reactive hyperaemia [17,18]. The major advantage of this method is that it is completely non-invasive and measures real-time blood-flow. Plasma von Willebrand factor (vWF) may also be used as an indication of structural endothelial damage.

Trials with hard end-points are the gold standard for evaluation of beneficial effects of statin intervention, but examination of endothelial function may possibly provide an appropriate surrogate end-point. The present pilot trial was therefore designed to assess the effects of atorvastatin on endothelial function in renal transplant recipients. The relationship between microvascular function and plasma ET-1, NO, and vWF levels were also addressed.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients and study design
This pilot study was an open trial of atorvastatin 10 mg daily over a period of 4 weeks. Renal transplant recipients over 18 years of age having received a transplant more than 1 month ago, with total cholesterol (TC) concentrations between 4.0 and 12.0 mmol/l, were eligible for inclusion. Exclusion criteria were treatment with known interacting drugs (e.g. erythromycin, azole antifungals), known familial hypercholesterolaemia, active liver disease, or hepatic dysfunction.

Twenty-two patients on CsA- and prednisolone-based immunosuppressive therapy (after basiliximab induction) were included in the study. Fourteen (64%) patients used calcium-channel blockers, five (23%) ß-blockers, two (9%) ACE inhibitors, and one (5%) an {alpha}-blocker. There were no changes in the doses of these drugs during the study. Demographic data at the time of inclusion are shown in Table 1Go.


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Table 1. Average (range) demographic data at inclusion

 
Endothelial function was investigated before and after 4 weeks of atorvastatin therapy. Samples for fasting serum lipids, serum creatinine, plasma ET-1, plasma NO (total nitrate and nitrite concentration), plasma vWF, and whole blood CsA were taken before each microvascular function investigation. Patients received standard hospital diet during the study and were asked to fast overnight (food, drugs, caffeine, tobacco, and alcohol), and antihypertensive drugs were withheld for at least 24 h.

All patients gave their informed consent in accordance with the Declaration of Helsinki. The study was approved by the Regional Ethics Committee of Health Region II and by the Norwegian Medicines Control Authority, Oslo, Norway.

Microvascular function
The investigations were performed in a quiet room at a constant temperature (22–25°C, not changing more than ±0.4°C during specific investigations) as earlier described [19]. Patients with an arteriovenous fistula were examined on the contralateral arm.

Non-invasive administration of acetylcholine (ACh) across the epidermal skin barrier by means of iontophoresis was combined with skin blood-perfusion monitoring (laser Doppler flowmetry) to assess endothelial function. The ion chambers were filled with test or control solutions; 1% ACh dissolved in de-ionized water at the anode and 5 mol/l NaCl solution at the cathode to eliminate ‘non-specific’ iontophoresis-induced vasodilatation [17]. Patients were given an acclimatization period of 20 min. Subsequent monitoring of skin blood perfusion started with baseline measurements for 5 min, followed by four consecutive 6-min periods after each charge given. The iontophoresis protocol consisted of the following charges: 0.50 milliColoumb (mC) (50 µA for 10 s), 0.75 mC (75 µA for 10 s), 1.0 mC (100 µA for 10 s) and 1.5 mC (150 µA for 10 s).

A post-occlusive reactive hyperaemia test was performed following a new acclimatization period of 5 min and 5 min baseline measurement. The forearm blood flow was occluded for 3 min with a suprasystolic cuff pressure of 280 mmHg, followed by instant release of the occlusion. Laser Doppler measurements continued for at least 5 minutes after cuff deflation. Skin blood perfusion was measured as flux (velocityxconcentration, obtained from the laser Doppler signal, Dual DRT4 Research version 4.24, Moor Instruments Ltd. UK) in arbitrary units (AU).

Calculations and data analysis
Changes in skin blood perfusion (flux) measured by laser Doppler flowmetry were analysed off-line, using DRTSOFT software package version 3.8 (Moor Instruments Ltd., UK). Baseline values were calculated as mean flux over 3 min, just prior to the start of the iontophoresis protocol and suprasystolic occlusion of the forearm circulation, respectively. Effect parameters are given as absolute increases from baseline values [17]. The primary effect parameter for the endothelial function test with ACh was predefined as the 6-min area under the flux versus time curve (AUC) following the 1.5-mC charge (AUCACh) [17]. Peak flux (Peakrh) after the 1.5-mC charge (PeakACh) was also determined. The primary effect parameter for the post-occlusive reactive hyperaemia test was the AUC for the whole reactive phase (AUCrh), and (Peakrh) was determined in addition [17].

Protocol for analysis and exclusion of data
Predetermined exclusion criteria for inadequate laser Doppler measurements were; PeakACh and Peakrh <20 AU, AUCACh <40 AUxmin, and AUCrh <10 AUxmin.

Laboratory analysis
Blood samples for determination of ET-1 were drawn into pre-chilled EDTA tubes on ice and analysed as described elsewhere [19]. Total plasma NO activity was determined as plasma nitrite and nitrate concentrations as previously described [19]. Plasma vWF was analysed by an immunoturbidimetric assay (IL vWF : Ag, Instrumentation Laboratory Company, Lexington, MA, USA). Whole-blood (EDTA tubes) concentrations of non-metabolized CsA was analysed with a fluorescence polarization immunoassay (FPIA) using a relatively specific monoclonal antibody (TDx, Abbott).

Supine blood pressure was measured manually with a sphygmomanometer, after a minimum of 5 min rest, following the microvascular function investigations. A mean of at least three measurements is given.

Drugs and chemicals
All laboratory chemicals were of analytical grade; acetylcholine chloride from Sigma Chemical Co., St Louis, MO, USA. De-ionized water was prepared in the laboratory with an Elgastat UHQ II, Elga Ltd, Wycombe Bucks, UK. Atorvastatin 10 mg tablets were supplied by Pfizer AS (Oslo, Norway).

Statistics
Absolute values and individual changes are presented as means±SD. Relative changes in effect parameters (log-transformed for microvascular function parameters to obtain normal distribution) were analysed with two-tailed Student's t-test, using SPSS for Windows, release 9.0.0. P values <0.05 were considered statistically significant.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
One patient did not conclude the study because of gastric discomfort. Four patients had their daily CsA dose reduced by an average of 1.00 ± 0.94 mg/kg/day during the study. One patient increased the daily dose of CsA by 0.72 mg/kg/day and the daily prednisolone dose by 5 mg/day.

Two AUCACh, one PeakACh, and three AUCrh recordings were excluded because of flux signals below the predetermined limits. Storage of measurements on the computer was incomplete for two patients.

Lipids
Four weeks of atorvastatin treatment induced significant reductions in both total cholesterol by 26.8 ± 8.4% (from 6.50 ± 1.11 to 4.71 ± 0.70 mmol/l, n=21, P<0.0001) and in LDL cholesterol by 41.5 ± 11.0% (from 4.12 ± 1.01 to 2.37 ± 0.56 mmol/l, n=18, P<0.0001) (Figure 1). TG were lowered by 7.5 ± 42.1% (from 1.79 ± 0.89 to 1.63 ± 1.08 mmol/l, n=21 P=0.42), while HDL cholesterol remained unchanged during the study (1.62 ± 0.53 before and 1.61 ± 0.61 mmol/l after atorvastatin, n=20, P=0.73).



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Fig. 1. Mean (+SD) total cholesterol (n=21) and LDL-cholesterol (n=18) before and after 4 weeks of treatment with 10 mg atorvastatin in renal transplant recipients. P<0.0001 vs baseline.

 

Microvascular function
ACh-assessed endothelial function (AUCACh and PeakACh) showed a significant improvement following atorvastatin treatment (Figure 2Go and Table 2Go), while the post-occlusive reactive hyperaemia test (AUCrh and Peakrh) did not show any significant changes (Table 2Go).



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Fig. 2. Individual endothelial function (AUCACh) before and after 4 weeks of 10 mg atorvastatin treatment in renal transplant recipients (n=17, P=0.042). Horizontal lines indicate mean values.

 

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Table 2. Mean (±SD) microvascular function data before and after 4 weeks of atorvastatin treatment

 
No significant associations were found between microvascular function (AUCACh and AUCrh) and the following parameters; TC, LDL cholesterol, HDL cholesterol, TG, plasma ET-1, plasma NO, plasma vWF, or blood pressure (P>0.37).

Baseline fluxes were similar between the two investigations of ACh stimulation and post-occlusive reactive hyperaemia (Table 2Go). Mean local skin temperature ranged from 28.2 to 32.9°C between the investigated patients, but the average change in local skin temperature during a single microvascular function test was only 0.4 ± 0.4°C, with no significant difference between investigations (P=0.72).

Vasoactive mediators and blood pressure
Plasma ET-1 concentrations did not change during atorvastatin treatment. Average plasma ET-1 concentration was 0.37 ± 0.08 before and 0.37 ± 0.12 fmol/ml after treatment (n=21, P=0.72).

Plasma NO concentrations, estimated from total plasma nitrite and nitrate concentrations, showed a borderline significant increase from 49 ± 30 µmol/l to 57 ± 37 µmol/l during 4 weeks of atorvastatin treatment (n=21, P=0.051).

Plasma vWF was not altered by atorvastatin treatment, but the values were high both before (196 ± 57%) and after (197 ± 37%) treatment (n=21, P=0.32).

Average systolic blood pressure was not altered and averaged 144 ± 18 mmHg before and 144 ± 21 mmHg after atorvastatin treatment (n=21, P=0.99). The respective diastolic blood pressures were 90 ± 11 and 88 ± 11 mmHg (n=21, P=0.32).



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
This study indicates that treatment with atorvastatin in CsA-treated renal-transplant recipients is effective in both reducing atherogenic lipids and improving endothelial function. Peripheral plasma NO concentrations also showed a borderline increase during the treatment period, suggesting a possible mechanism for the improvement in endothelial function.

Statins lower cholesterol levels significantly but with some differences in efficacy. The endothelial protective effect of statins has been attributed to the general cholesterol-lowering effect [20,21], although some studies also indicate improvements in endothelial function beyond the lipid lowering effect [1214]. In the general population, statin therapy has been shown to improve endothelial dysfunction in some studies [12,22], but not all [11]. Due to chemical and pharmacokinetic differences, it is uncertain whether all statins will show the same degree of such a hypothetical effect dissociated from the cholesterol-lowering effect. However, both pravastatin and atorvastatin have now been shown effective in solid-organ transplant recipients, using the same method for endothelial-function investigation [10].

Only endothelial function assessed by ACh stimulation, but not the post-occlusive reactive hyperaemia test, showed a significant improvement during atorvastatin treatment. The reason for this is not clear since both are considered to investigate endothelial function. It might indicate that statins have a specific effect on the ‘ACh–NO pathway’ in the endothelium without influence on other factors. Peripheral plasma levels of NO were accordingly increased during atorvastatin treatment. The patients were on standard hospital diet during the study, and were also fasting overnight before investigation. Therefore a true increase in NO production seems likely. Several experimental studies indicate that a central mechanism for the endothelial dysfunction associated with dyslipidaemia and CsA may be decreased NO availability due to overproduction of superoxide anion, which in turn degrades NO. Statins may exert part of their endothelial ‘protective’ effect by scavenging superoxide anion, increasing eNOS activity or reducing ET-1 secretion. No change in plasma ET-1 concentrations were however found in the present study. Plasma vWF was well above normal levels, both before and after atorvastatin treatment (NS), thus atorvastatin does not seem to influence this surrogate marker of endothelial cell function during this short period of treatment.

By design the present study was open and not randomized and we did not include untreated controls, leaving a possibility for bias. Considering the relatively short time period and due to the fact that most patients did not change their medication during the study, a time-dependent bias seems unlikely. A sub-analysis of only those patients who did not change CsA or prednisolone dose during the study also gave the same overall results, although they were not significant because of the reduced number of patients (data not shown). We have previously also found that the vasodilatation caused by ACh (AUCACh) did not change significantly from one week to another (823 ± 453 to 721 ± 443 AUxmin, P=0.41).

The applicability of the present results to other vascular beds may be questioned and needs further investigation. However, a previous study has shown a close relationship between coronary and brachial-artery responses to stimulation of the endothelium [23]. Although these findings may not be universal, it indicates that different vascular beds may share common regulation mechanisms.

In conclusion, treatment of renal transplant recipients with 10 mg atorvastatin significantly lowers plasma cholesterol levels and improves endothelial function in as little as 4 weeks. Plasma NO levels were increased during the investigation (just reaching significance), suggesting that atorvastatin may improve endothelial function through an ‘endothelial–NO pathway’. The dual effect of safe lipid lowering and improvements in endothelial function makes atorvastatin a potentially important adjunct for use after solid-organ transplantation. A large double-blind placebo-controlled trial will be required to further assess the findings of this pilot study.



   Acknowledgments
 
The authors wish to thank all personnel at the Laboratory for Renal Physiology for skilled technical assistance. Dr Ee Chye Ng and co-workers at the Laboratory for Hematology are acknowledged for performing the vWF analysis. In addition we would like to thank Ingar Holme and co-workers at the Institute for Medical Statistics, Ullevål Hospital for performing the statistical analyses. Pfizer Norway AS is acknowledged for sponsoring and monitoring the study. Dr Åsberg was partly financed by a grant from Medinnova SF.



   Notes
 
Correspondence and offprint requests to: Anders Åsberg, Laboratory for Renal Physiology, Section of Nephrology, C1 1027, Medical Department, The National Hospital, N-0027 Oslo, Norway. Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 19. 2.01
Revision received 3. 5.01.