Better microvascular function on long-term treatment with lisinopril than with nifedipine in renal transplant recipients

Anders Åsberg, Karsten Midtvedt, Trond Vassbotn and Anders Hartmann

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. The prevalence of hypertension in renal transplant recipients is high but the pathophysiology is poorly defined. Impaired endothelial function may be a factor of major importance. The present study addresses the effects of long-term treatment with either lisinopril or slow-release nifedipine on microvascular function and plasma endothelin in renal transplant recipients on cyclosporin A (CsA).

Methods. Seventy-five hypertensive renal transplant recipients were double-blind randomized to receive slow-release nifedipine (NIF, n=40) or lisinopril (LIS, n=35). Ten normotensive, age-matched recipients served as controls. All patients received CsA-based immunosuppressive therapy including prednisolone and azathioprine. Microvascular function was assessed in the forearm skin vasculature, using laser Doppler flowmetry in combination with post-occlusive reactive hyperaemia and endothelial-dependent function during local acetylcholine (ACh) stimulation.

Results. The analysis of microvascular function (AUCrh) showed that nifedipine-treated patients had significantly lower responses compared with lisinopril-treated patients (20±17 and 43±20 AUxmin respectively, P=0.0016). Endothelial function was borderline significantly lower in the NIF group compared with the LIS group (640±345 and 817±404 AUxmin respectively, P=0.056). The responses in the LIS group were comparable with those in non-hypertensive controls (AUCrh was 37±16 and AUCACh was 994±566 AUxmin). Plasma endothelin-1 concentrations were significantly higher in the NIF group compared with the LIS group (0.44±0.19 vs 0.34±0.10 fmol/ml respectively, P=0.048), and were 0.29±0.09 fmol/ml in the control patients. AUCACh was associated with plasma endothelin-1 (P=0.0053), while AUCrh was not (P=0.080).

Conclusions. The study indicates that long-term treatment with lisinopril, when compared with nifedipine, yields a more beneficial effect on microvascular function in hypertensive renal transplant recipients on CsA. The beneficial microvascular effect may be mediated in part by an endothelin-1-associated effect on the endothelium.

Keywords: cyclosporin; endothelin; hypertension; kidney; microvascular function; transplantation



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The incidence of post-transplant hypertension ranges from 60 to 90% in renal transplant recipients. The pathophysiological mechanisms involved in post-transplant hypertension are poorly understood, but impaired endothelial function may be involved. Endothelial dysfunction has been reported in renal transplant recipients [1] as well as in patients with essential hypertension [24]. Endothelin-1 (ET-1), a potent vasoconstrictive peptide, is produced by endothelial cells. Cyclosporin A (CsA) has been shown to induce increased plasma concentrations of ET-1, but also transiently upregulate vasodilative responses [5,6]. In rats, short-term treatment with CsA induces isolated endothelial dysfunction [7], while long-term treatment also attenuate endothelial-independent vascular function [8].

Calcium-channel blockers (CCB) and angiotensin-converting enzyme (ACE) inhibitors are commonly used drugs for treatment of post-transplant hypertension. CCBs and ACE inhibitors may possess ‘endothelium protective’ properties in non-transplant patients [9,10], and both drugs interact with vasomodulation through ET-1 and nitric oxide (NO) systems in vitro and in vivo.

Skin blood-perfusion monitoring (laser Doppler flowmetry) in combination with post-occlusive reactive hyperperfusion test and iontophoretic acetylcholine (ACh) administration can be used for non-invasive determination of microvascular and endothelial-specific function of the forearm skin vasculature [11].

The aim of the present study was to investigate which treatment, ACE inhibitor or CCB, can best preserve microvascular function in hypertensive renal transplant recipients on CsA. In addition, the relationship between microvascular function and plasma endothelin was addressed.



   Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients and study design
Seventy-five hypertensive renal transplant recipients were consecutively recruited between August 1997 and June 1998 from an ongoing prospective double-blinded, single-centre, block-randomized, parallel-group study, investigating the long-term effect of slow-release nifedipine and lisinopril treatment on renal function [12]. Forty patients were randomized to treatment with slow-release nifedipine (NIF), 30–60 mg s.i.d., and 35 patients to treatment with lisinopril (LIS), 10–20 mg s.i.d. within 3 days following transplant surgery. There were no significant differences in demographic data between the groups at the time of randomization. Systolic blood pressure (SBP) was 168±17 mmHg (NIF) and 169±17 mmHg (LIS) and diastolic blood pressure (DBP) was 103±5 and 104±5 mmHg respectively. Twenty-seven (68%) patients in the NIF group and 23 (66%) patients in the LIS group had received dialysis treatment for a median time of 6 (range 1–23) and 7 (range 1–22) months respectively previous to transplantation. The number of patients treated for hypertension before transplantation was 33 (82%) in the NIF group and 29 (83%) in the LIS group. In the NIF group, 17 (42%) patients used ß1-blockers and five (12%) {alpha}-blockers as additional anti-hypertensive therapy. The corresponding numbers of patients in the LIS group were respectively 13 (37%) and six (17%). Four (10%) of the patients in the NIF group and five (14%) in the LIS group used HMG-CoA reductase inhibitors. Immunosuppressive therapy consisted of CsA, prednisolone, and azathioprine in all patients. Patients with unstable renal function or ongoing acute rejection were not eligible for inclusion in this substudy.

Microvascular function was investigated in a stable phase, at a median time after transplantation and randomization of 21 months (range 10–35 months). Patients were asked to fast overnight (food, drugs, caffeine, tobacco, and alcohol) prior to investigation. Anti-hypertensive drugs were withheld for more than 24 h. Ten age-matched, untreated, normotensive renal transplant recipients served as control for anti-hypertensive drug effects on microvascular function per se. Demographic data at the time of microvascular investigations are summarized in Table 1Go.


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Table 1. Demographic data at the microvascular function investigation day

 
All patients completed the study. Specific informed consent for the microvascular study was obtained according to 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 microvascular investigations were performed in a quiet room at a constant temperature (22–24°C, not changing more than ±1°C during the day of investigations). Patients were examined in a supine position with both arms comfortably supported at the side of the body. The volar side of the forearm was gently cleaned with isopropanol and de-ionized water. Patients with an AV fistula were examined on the contralateral arm. Changes in skin blood perfusion were measured with a double-channelled laser Doppler perfusion and temperature monitor (Dual DRT4 Research, version 4.24, Moor Instruments Ltd, UK), calibrated against the manufacturers flux standard. The laser Doppler probes (DP1T/7 large-area probe, Moor Instruments) were mounted in the inner hole of ion chambers (ION1, Moor Instruments). Skin blood perfusion was measured as flux (a quantity proportional to the product of average speed of the blood cells and their concentration) obtained from the laser Doppler signal in arbitrary units (AU). A detailed description of the method has previously been published [11,13].

Post-occlusive reactive hyperaemia test combined with skin blood perfusion monitoring with laser Doppler flowmetry allows non-invasive assessment of microvascular function. Following an acclimatization period of 20 min, and a 5-min baseline measurement, the forearm blood flow was occluded for 3 min with a suprasystolic cuff pressure of 280 mmHg. The pressure was instantly released and skin blood perfusion monitoring was continued for at least 5 min after cuff deflation. The mean difference between two investigations on the same day in eight healthy volunteers for the post-occlusive reactive hyperaemic test was 48±23% (AUCrh) and 30±16% (Peakrh).

Non-invasive administration of ACh across the epidermal skin barrier by means of iontophoretic charges (anodal direct current) was combined with skin blood-perfusion monitoring 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 0.9% NaCl solution at the cathode. Monitoring of skin blood perfusion started with 5-min baseline measurement followed by consecutive 6-min periods after each charge given in the iontophoresis protocol; 0.50 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). The mean difference between two investigations on the same day in eight healthy volunteers for ACh stimulation test was 32±16% (AUCACh) and 18±15% (PeakACh).

Calculations
Changes in skin blood perfusion (flux) were analysed off-line, using DRTSOFT software package version 3.8 (Moor Instruments). Baseline values were calculated as mean flux over 3 min, just prior to the suprasystolic occlusion of the forearm circulation and the start of the iontophoresis protocol respectively. Effect parameters are given as absolute increases from baseline values [13]. The primary effect parameter for the post-occlusive reactive hyperaemia test was the area under the flux vs time curve (AUC) for the whole reactive phase (AUCrh), peak flux (Peakrh) was also determined. The primary effect parameter for the endothelial function test with ACh was defined as the AUC of the 6-min monitoring period following the 1.5 mC charge (AUCACh), peak flux after the 1.5 mC charge (PeakACh) was also determined.

Protocol for analysis and exclusion of data
Predetermined exclusion criteria for inadequate laser Doppler measurements were: Peakrh and PeakACh <20 AU, AUCrh <10 AUxmin, and AUCACh <40 AUxmin. Microvascular function data from two patients, one NIF and one LIS, were excluded from data analysis because environmental disturbances precluded accurate function assessment. One LIS patient was excluded from microvascular data analysis because of incomplete storage of data on the computer. Four ACh-assessed endothelial-dependent function investigations (3 NIF and 1 LIS) were excluded because of disturbed contact in the iontophoretic circuit and one (LIS) because of flux signals below the predetermined limits. One post-occlusive reactive hyperaemia investigation (LIS) was excluded because of incomplete storage of data on the computer and two AUCrh (NIF) were below the predetermined lower limit. One plasma ET-1 concentration was not assessable (NIF) after inappropriate sample handling.

Haemodynamic and laboratory tests
Blood samples for determination of plasma ET-1 and BigET-1, whole blood CsA, and serum creatinine were obtained before microvascular function investigations.

Blood samples for determination of plasma ET-1 and BigET-1 were drawn into prechilled EDTA tubes on ice, centrifuged (4°C) at 2500 g for 10 min. Plasma was frozen at -70°C within 1 h from sampling and stored for 2–8 weeks before analysis. ET-1 was analysed as described elsewhere [11], and BigET-1 analysis was performed with a direct-ELISA assay (Biomedica, Vienna, Austria). The intra-assay CV was 14±6% and cross reactivity was, ET:s <1%, human BigET-1 (1-38) 100%, human BigET-1 (22-38) <1%.

Whole-blood (EDTA tubes) concentrations of non-metabolized CsA were analysed with a fluorescence polarization immunoassay (FPIA) using a relatively specific monoclonal antibody (TDx, Abbott).

Sitting blood pressures were measured manually with a sphygmomanometer, after a minimum of 5 min rest, before the microvascular function tests. A mean of at least three measurements is given.

Renal function was assessed from serum creatinine, analysed with a semi-automatic COBAS MIRA (Roche, Basel, Switzerland).

Drugs and chemicals
All laboratory chemicals were of analytical grade; acetylcholine chloride from Sigma Chemical Company, St Louis, MO, USA. De-ionized water was prepared in the laboratory with an Elgastat UHQ II (Elga Ltd, Wycombe, Bucks, UK). Nifedipine GITS© 30-mg tablets, active substance and placebo, were kindly supplied by Bayer AG (Leverkusen, Germany), and lisinopril 10 mg tablets, active substance and placebo, were kindly supplied by Zeneca Pharmaceuticals (UK).

Statistics
The results are presented as mean±SD. Differences between the treatment groups were analysed using Mann–Whitney U-test (0.05 significance level), and associations between parameters using linear regression with SPSS© for Windows 6.1.4. P values <0.05 are considered statistically significant.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients
As shown in Table 1Go, demographic data at the time of microvascular function investigation was not significantly different between groups, with the exception of s-creatinine, which was significantly lower in the NIF group compared with the LIS group (Table 1Go, P=0.0016).

Microvascular function
The NIF group showed significantly lower responses to the post-occlusive reactive hyperaemia test (AUCrh) compared with the LIS group (Table 2Go, Figure 1Go; P=0.0016). The average AUCrh response in the LIS group was similar to that in controls. Endothelial function (AUCACh) also showed a strong tendency to be lower in the NIF group compared with LIS (P=0.056), which showed responses similar to controls (Table 2Go). Peak responses in both tests showed the same tendencies as AUC data (Table 2Go).


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Table 2. Mean (±SD) microvascular function data

 


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Fig. 1. Individual responses to the post-occlusive reactive hyperaemia test (microvascular function, AUCrh) in hypertensive renal transplant recipients treated with lisinopril (LIS, n=32) or slow-release nifedipine (NIF, n=37). Age-matched, untreated normotensive recipients serve as controls (n=9). Horizontal lines indicate mean values. AUCrh is determined as the area under the curve for the whole reactive phase, corrected for baseline flux. P<0.00001.

 
Baseline fluxes were not significantly different between the treatment groups in the post-occlusive reactive hyperaemia test (P=0.13). However, in the ACh stimulation test, baseline perfusion was significantly higher in the NIF group (Table 2Go; P=0.048).

Mean local skin temperature ranged from 28.3 to 31.2°C between investigated patients, though the temperature changed less than 1.0°C during each microvascular function investigation. There were no differences between groups (data not shown).

Associations
There were no significant associations between AUCrh or AUCACh and the following parameters: recipient age, time since transplantation/randomization, blood pressure, s-creatinine, haemoglobin, trough CsA, plasma BigET-1, and baseline flux. However, endothelial function (AUCACh) showed a significant association with plasma ET-1 concentrations (n=76, r=0.32, ß=-0.425; P=0.0053). Microvascular function (AUCrh) was not significantly associated with plasma ET-1 (n=77, r=0.20, ß=-0.370; P=0.080).

Endothelin
There was a significant difference in plasma ET-1 concentrations between treatment groups (P=0.048). The NIF group showed highest levels (0.44±0.19 fmol/ml), followed by the LIS group (0.34±0.10 fmol/ml), and controls (0.29±0.09 fmol/ml).

Plasma BigET-1 concentrations were not significantly different between treatment groups (P=0.80) (NIF, 1.39±1.47 fmol/ml; LIS, 1.62±1.99 fmol/m, and controls 2.21±2.36 fmol/ml).



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In the present study, hypertensive renal transplant recipients treated with lisinopril showed higher vasodilative responses in the forearm skin microvasculature as compared with nifedipine-treated recipients. It is possible that lisinopril ‘preserves’ microvascular function comparable to the responses observed in normotensive controls. The beneficial microvascular effect is possibly in part mediated by an ET-1-associated endothelial effect. However, the present study only shows a cross-sectional expression of microvascular function following long-term treatment with lisinopril or nifedipine. Therefore the results cannot be conclusive about possible longitudinal changes in microvascular function. The groups were, however, randomly selected, and demographic data were not different at baseline between the treatment groups, so no difference in baseline microvascular function should be expected.

Post-occlusive reactive hyperaemia test measures a combined function of the microvasculature (including endothelial function) while ACh stimulation more directly investigates endothelial function. The present study showed significant changes with the post-occlusive hyperaemia test, while ACh stimulation was only just significant. It is possible that the lack of significant effect in the more direct endothelial function investigation may be due to a type II error, and that it would have reached significance if more patients had been included. In concordance with these results, studies in essential hypertensive patients have shown a beneficial effect of ACE inhibitors (lisinopril and captopril) on endothelium-dependent vasodilatation in large conduit vessels [14]. Lisinopril and enalapril have also been reported to improve attenuated endothelial function in NIDDM patients [15,16], and to prevent CsA-induced impairment of ACh-mediated vasodilatation in the rat [17]. Another ACE inhibitor, quinapril, has been shown to improve endothelial function in normotensive patients [18] and in patients with chronic heart failure [19]. In these studies, enalapril did not show any significant effect on endothelial function, indicating that different ACE inhibitors may possibly exert different effects on microvascular function. The literature is more conflicting regarding the effect of nifedipine on microvascular function. Millgård et al. [14] did not find any acute effects of nifedipine on endothelial dependent vasodilatation in essential hypertensive patients, while Muiesan et al. [20] reported a beneficial endothelial effect following 2 months of nifedipine treatment.

The results of the present and earlier studies are not directly comparable because of differences in patient population and methodology. In the present study, small resistance vessels of the skin (laser Doppler flowmetry) were investigated in renal transplant recipients, while in general large conduit vessels in non-transplanted patients have been investigated in previous studies (venous occlusion plethysmography and high-resolution ultrasound measured brachial artery diameter) [14,16,19,20]. It is possible that different vascular beds are not uniformly regulated, but this remains to be shown, as also does any correlation between responses obtained with different methods.

Endothelial dysfunction is generally associated with increased risk of cardiovascular disease. It is feasible that microvascular function investigation can be used as a surrogate parameter for cardiovascular disease. However, only large studies including hard end-points can confirm the validity of this approach. Since CsA administration acutely induces increased endothelial reactivity [11], the effects seen in the present study may not be applicable for other patient groups.

The facts that plasma ET-1 concentrations were significantly associated with endothelial function, and higher concentrations were found in slow-release nifedipine-treated patients, compared with lisinopril-treated patients and controls, indicates a possible mechanism for an endothelial protective effect of lisinopril. Both CsA and angiotensin II may activate the vasoconstrictive and proliferative ET-1 system, with a negative impact on endothelial function [6]. It has been shown that ACE inhibitors have the ability to ameliorate angiotensin II induced ET-1 activation, while CCBs only counteract the vasoconstrictive effect of ET-1 but not its activation per se. The higher plasma ET-1 concentrations found in the nifedipine group may, however, also be a result of endothelial dysfunction per se, since such dysfunction may be associated with decreased nitric oxide (NO) activity, and NO inhibits ET-1 synthesis. Another possible explanation of the higher plasma ET-1 concentration and lower endothelial-dependent responses in the nifedipine group is that angiotensin II lowers NO bioavailability by free-radical-induced degradation of NO [21]. However, when interpreting plasma ET-1 and BigET-1 data, it should be taken into consideration that the ET system is an autocrine/paracrine system [22], and peripheral plasma concentrations may not reflect true local activity, but rather a ‘spill over’ from local vascular production.

In conclusion, the present study indicates that long-term anti-hypertensive treatment with lisinopril yields a more beneficial effect on microvascular function when compared with slow-release nifedipine in renal-transplant recipients. Lisinopril-treated patients show microvascular functions comparable to those in normotensive recipients. The beneficial microvascular effect may be mediated, at least in part, by an ET-1-associated endothelial effect. These findings need to be further evaluated in clinical studies, preferably with vascular determination of ET-1.



   Acknowledgments
 
The authors thank Kirsten K. Lund for skilled technical assistance.



   Notes
 
Correspondence and offprint requests to: Anders Åsberg, Laboratory for Renal Physiology, Section of Nephrology, C11027, Medical Department, The National Hospital, N-0027 Oslo, Norway. Back



   References
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 17. 8.00
Revision received 9. 2.01.