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*
Renal Transplant Unit, Hospital Clínico,
Barcelona, Spain.
Hormonal Laboratory, Hospital Clínico,
Barcelona, Spain.
Nuclear Medicine Department, Hospital
Clínico, Barcelona, Spain.
§
BioStatistic Unit of the Public Health Department, University of
Barcelona, Barcelona, Spain.
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Institut d'Investigació Biomediques August
Pi i Sunyer (IDIBAPS), University of Barcelona, Barcelona, Spain.
Correspondence to Dr. Josep M. Campistol, Renal Transplant Unit, Hospital Clinic, 170, Villarroel, Barcelona 08036, Spain. Phone: 34-93-227-54-23; Fax: 34-93-227-54-98; E-mail: jmcampis{at}medicina.ub.es
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The aim of the present study was to compare the effects of an AngIIA (losartan) and a calcium-channel blocker (amlodipine) on BP control, renal hemodynamics, TGF-ß1, and endothelin-1 (ET-1) in renal transplant patients who had normal graft function and who were treated with CsA.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-blockers (doxazosin). All patients gave written
informed consent, and the protocol was approved by the Ethical Committee of
our hospital.
Study Design
The study was a randomized, open, controlled 2 x 2 cross-over trial
(11) in which the two
treatments were amlodipine and losartan
(Figure 1). It consisted of a
4-wk run-in period without medication, a wash-out period, a 6-wk active
treatment with losartan or amlodipine (period 1), a 4-wk wash-out period, and
a 6-wk treatment with the alternative treatment (period 2). At the end of the
run-in period, patients were randomized to one of the two sequences:
amlodipine/losartan (sequence 1) or losartan/amlodipine (sequence 2). The
studies were performed at baseline and at the end of either treatment period.
Plasma levels of TGF-ß1 and ET-1 were determined at the same
times and before starting period 2.
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Losartan was used at a dose of 25 mg/d for the first week and 50 mg/d for the rest of the period. Amlodipine was administered at a dose of 5 mg/d during the whole study period.
BP Measurement
BP was measured every 2 wk at the out-patient department of the Renal
Transplant Unit. BP determinations were performed according to World Health
Organization recommendations. An automatic oscillometer (HEM-711; OMRON
Healthcare, Inc., Vernon Hills, IL) was used to perform three consecutive
measurements at 2-min intervals with the patient in supine position. The mean
of the three determinations was used for calculations. Mean arterial pressure
(MAP) was calculated as [SBP + 2(DBP)]/3.
All antihypertensive drugs or treatments known to affect BP or renal hemodynamics were discontinued before the run-in period. One and eight patients required doxazosin (2 mg/12 h) to control BP in the active and the wash-out periods, respectively.
Biochemical Parameters, Hormones, and TGF-ß1
Serum creatinine, serum electrolytes (sodium and potassium), uric acid,
total protein (TP), hematocrit (Hct), and white blood cell count were measured
by routine techniques (Hitachi, Japan). Plasma renin activity (PRA) was
estimated by RIA (Clinical Assay; Baxter, Cambridge, MA) of AngI generated
after 3 h of incubation at pH 7.4 and 37°C under conditions to inhibit
further conversion of AngI. Plasma concentration of AngII was determined by
RIA (Nichols Institute, Wichjen, The Netherlands). Plasma concentration of
ET-1 was measured by RIA (Nichols Institute) after extraction on Sep-Pak C18
cartridges (Water Associates, Milford, MA). Plasma samples (1 ml) were
acidified with 4% acetic acid (4.5 ml) and applied to cartridges that were
preactivated with methanol, distilled water, and 4% acetic acid. The
cartridges were then washed with distilled water and 25% ethanol, and
immunoreactive ET was eluted twice with 1 ml of 4% acetic acid in 86% ethanol.
The eluted ET was then concentrated to dryness (Speed Vac Concentrator; Savant
Instruments Inc., Farmingdale, NY) and reconstituted for RIA. The recovery
rate for the extraction procedure was 85%, as determined by the addition of
labeled ET-1 (3500 cpm) to plasma. Cross-reactivity of the antiserum for ET-1,
-2, -3, and big ET was 100%, 52%, 96%, and 7%, respectively. Intra-assay and
interassay coefficients of variation were 6.9% and 12.1%, respectively. To
measure TGF-ß1 plasma levels, peripheral venous blood was
obtained from the patients with ethylenediaminetetraacetate as an
anticoagulant and was centrifuged at 1000 x g for 30 min. The
plasma was isolated and stored at -70°C until the assay was performed. To
activate latent TGF-ß1 into immunoreactive
TGF-ß1 detectable by the immunoassay test, we acidified the
samples (to 0.1-ml sample, add 0.1 ml of 2.5 N acetic acid/10 M urea) and then
neutralized (adding 0.1 ml of 2.7 N NaOH/1 M HEPES) to yield a pH of 7.2 to
7.6. Three sample dilutions (1:12, 1:24, and 1:48) were performed with the
calibrator Diluent RD6M. TGF-ß1 was determined using a solid
phase TGF-ß1—specific sandwich ELISA (Quantykine;
R&D Systems, Minneapolis, MN). TGF-ß1 standard curve was
constructed using 2000, 1000, 500, 250, 62.5, and 31.25 pg/ml recombinant
human TGF-ß1 protein. The minimum detectable level of
TGF-ß1 with the test was 7 pg/ml. Thrombomodulin was
determined in some random samples to excluded TGF-ß1 platelet
contamination.
Renal Hemodynamics and Calculations
Samples of 4 ml of heparinized blood were taken at 5, 10, 15, 20, 25, 30,
40, 50, 60, 90, 120, 150, 180, and 240 min after the injection of a bolus of
70 µmCi of 131I-orthoiodophippurate and 50 µmCi de
125I-iodothalamate to determine the effective renal plasma flow (ERPF) and
GFR, respectively. The compartmental model of Sapirstein et al.
(12) and Blufoux et
al. (13) was used to
study the kinetics of 125I-iodothalamate and
131I-orthoiodohippurate.
Gomez's equations were used to calculate afferent and efferent arteriolar resistance (AAR and EAR, respectively) (14,15). Data such as MAP, GFR, ERPF, renal blood flow (RBF), Hct, and TP were used to estimate quantitatively the intrarenal hemodynamics.
RBF was calculated as RBF(ml/min) = ERPF/(1 — Hct). The filtration
fraction (FF) was calculated as FF = (GFR/ERPF) x 100. Glomerular
hydrostatic pressure (PG) was calculated from Gomez's equations:
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AAR and EAR were calculated from the following equations:
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Statistical Analyses
The study had a standard AB/BA crossover design (in this study, AL/LA: A,
amlodipine; L, losartan) (16),
with baseline measurements taken at the beginning of the first period for all
variables and, in the case of TGF-ß1, also before the second
period. A logarithmic transformation was applied for TGF-ß1
values because TGF-ß1 baseline data showed a non-normal
distribution (Kolmogogorov Dmax = 0.299; P < 0.05). The
treatment effect, period effect, and treatmentperiod interaction for logarithm
of TGF-ß1 were tested by the two-sample t test
(17). The analysis of
treatment effect, allowing for period effects, was performed by means of a
spreadsheet, following the Hills-Armitage approach. The same analysis was used
for each variable in the study.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Effects on BP
The control of BP was excellent with both treatments; all patients reached
the target BP (<130/85 mmHg). Only one patient required administration of
doxazosin during the active treatment period (losartan). Statistically
significant differences from baseline were observed in both treatment groups;
from baseline MAP, 107.9 ± 6 to 96 ± 10 mmHg with losartan
(P < 0.0005) and 92 ± 7 mmHg with amlodipine (P
< 0.0001). No significant differences with regard to the control of BP were
observed between the two treatments (P = 0.124).
Effects on Renal Function and Biochemical Parameters
During the study, no significant changes of biochemical parameters (serum
sodium, potassium, uric acid, albumin, TP) were observed with either
treatment. Serum creatinine tended to increase slightly during losartan
treatment (1.29 to 1.39 mg/dl), but this change was not statistically
significant. Creatinine clearance tended to decrease from baseline on losartan
and tended to increase on amlodipine, but this was not statistically
significant. The difference between the two treatment periods was significant
(P = 0.023), however. No significant changes in proteinuria were
observed between baseline (108 ± 103 mg/dl) and the end of both
treatment periods (88 ± 75 with losartan and 132 ± 90 with
amlodipine), although there was a significant difference between the two
treatment periods (P < 0.01). No changes of the hemoglobin
concentration were observed. CsA doses and blood levels did not change during
the study periods (baseline, 97 ± 16 ng/ml; with losartan, 101.5
± 32; with amlodipine, 103 ± 28; P = NS).
Effects on Renal Hemodynamics
GFR (iothalamate clearance) was unchanged during losartan treatment (71 to
69 ml/min; P = NS). A statistically significant increase was observed
during amlodipine treatment, from 71 to 79 ml/min (P = 0.02). The
difference between the two treatments reached statistical significance
(P = 0.01). No statistically significant changes were observed in
ERPF values during the study periods.
AAR decreased significantly with losartan and amlodipine (10.584 ± 4331 to 7917 ± 4065 and 7073 ± 3479 dyn/s per cm-5, respectively; P = 0.0002 and 0.00003). There was no difference between the two treatment periods (P = 0.14). A completely different behavior was observed with respect to EAR. EAR at baseline was 2976 ± 951. The EAR significantly decreased with losartan (P = 0.05) and tended to increase with amlodipine (3137 ± 705; P = NS). There was a significant difference between the two treatment periods (P = 0.002).
Estimated PG was 51.4 mmHg at baseline, 52 mmHg with losartan (P = NS), and 54.5 mmHg with amlodipine (P = 0.0014). The difference between the two treatment groups was significant (P = 0.02).
Significant differences in FF were observed with both treatments. FF at baseline was 23.7% and decreased to 21.4% with losartan (P = 0.07) and to 25.5% with amlodipine treatment (P = NS). The difference between the two treatment periods was significant (P = 0.008).
Effects on PRA, AngII, and ET-1
There was a significant increase in PRA during losartan treatment (0.42
± 0.3 to 2.2 ± 2.6 ng/ml per h; P = 0.008) but not
during amlodipine treatment (0.75 ng/ml per h; P = 0.109). The
difference between the two treatment periods was significant (P =
0.009). There was also a significant increase in the AngII concentration with
both treatments from a baseline value of 23.7 ± 7.3 to 54.6 ± 42
with losartan (P = 0.008) and 32.4 ± 7 with amlodipine
(P = 0.01). The difference between the two treatment periods was
significant (P = 0.04). No significant changes in the ET-1 values
from baseline were observed in study period, but the difference between these
two periods was significant (P = 0.03; ET-1 at baseline, 7.3 ±
2.9 pg/ml; 6.6 ± 2 pg/ml with losartan; 8.2 ± 2.5 pg/ml with
amlodipine).
Effects on TGF-ß1
The presence of a treatment effect on TGF-ß1 plasma levels
was documented (P = 0.011; 95% confidence interval, 1.09 to 1.82).
Losartan decreased TGF-ß1 plasma levels between 1.09 to 1.8
times more than amlodipine. The possible presence of a period effect in the
sequence of the treatments could be excluded by the results of a period effect
test (P = 0.129); the order in the treatments (A/L or L/A) did not
change the results. The existence of a treatment-period interaction could also
be excluded by the results of a carryover test (P = 0.6, t = -0.54),
i.e., the results in the first study did not condition the second
study. TGF-ß1 plasma levels decreased significantly during
losartan therapy (P = 0.018), reaching normal levels after 6 wk of
therapy (5.1 ± 4 to 2.63 ± 1.07 ng/ml). During amlodipine
therapy, the TGF-ß1 plasma levels tended to decrease (5.3
± 4 to 4.4 ± 4.1 ng/ml), but this was not statistically
significant (P = 0.09). Basal levels at the beginning of both
treatment periods were similar, without any significant differences (5.3 and
5.1 ng/ml). The decrease in TGF-ß1 levels during losartan
therapy was significantly greater than that observed with amlodipine
(P = 0.045).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hypertension is a very important cardiovascular risk factor in renal transplant patients and has been related clearly to the development of chronic allograft nephropathy. Opelz et al. (3) demonstrated the negative impact of hypertension on graft survival. An inverse relationship was found between the severity of arterial hypertension and graft survival. Following the World Health Organization recommendations (18), control of BP in this specific high-risk population must to be strict. SBP and DBP should be lowered to values less than 130/80 mmHg, respectively (19). Although the control of BP is essential in renal transplant patients, many issues remain unresolved. Calcium channel blockers are the most frequently used antihypertensive drugs in renal transplantation, essentially because they are highly effective in the control of BP, are easy to use, and have an acceptable side effect profile (20). The risk of renal artery stenosis and initial reports on transplant patients with a slight increase of serum creatinine on ACEi have limited the use of ACEi or AngII receptor blockers (21,22). In the present study, the control of BP was excellent with both calcium channel blocker and AngII receptor blocker, without any significant difference between the two drugs. Only one patient needed doxazosin during the study period to reach the target BP. Both treatments were tolerated without any relevant side effects. Biochemical parameters or blood levels of CsA did not change during either treatment. With both treatments, BP control was excellent and well tolerated.
Opposite effects on renal hemodynamics were observed: the calcium channel blockers acted exclusively through vasodilatation of the afferent arteriole and the AngIIA through vasodilatation of both the afferent and efferent arterioles. Although the Gomez equations have a limited accuracy, they represent the only available method for determining glomerular hemodynamics in humans (14,15). The vasodilatation of the afferent arteriole achieved with amlodipine tended to be more intensive than that with losartan, but the effects on the efferent arteriole were diametrically opposed: losartan significantly decreased EAR, whereas amlodipine tended to increase it. These contrasting effects on EAR explain the contrasting effects on glomerular hemodynamics. Amlodipine significantly increased GFR in renal transplant patients, whereas losartan maintained GFR. Presumably the increase in GFR during amlodipine was caused by the marked vasodilatation of afferent arterioles, without any change of efferent arterioles, thus increasing FF and estimated PG. In contrast, treatment with losartan was associated with a decrease in FF and estimated PG. These different effects of losartan and amlodipine on glomerular hemodynamics could be important in the genesis of chronic allograft nephropathy, a condition characterized by high FF and PG. High FF and PG play a role in the genesis of glomerulosclerosis, and this deleterious effect may in part be mediated through the synthesis of several growth factors, such as TGF-ß1, AngII and ET-1 (23,24). The use of only one kidney for renal transplantation, the immunosuppressive therapy with nephrotoxic side effects, and the progressive increase in the age of organ donors are factors that facilitate the development of a hyperfiltration syndrome. In this scenario, antihypertensive agents that decrease the FF and the PGc could have beneficial effects on the long-term results of renal transplantation.
TGF-ß1 has also been implicated in the development of chronic allograft nephropathy, inducing interstitial fibrosis and glomerulosclerosis (6). We recently demonstrated that treatment with losartan significantly decreased the plasma levels of TGF-ß1 in renal transplant patients with chronic allograft nephropathy. The decrease of TGF-ß1 observed was approximately 50% from the initial levels (25), similar to what is found in different experimental models of renal damage (9). The same effect on TGF-ß1 plasma levels was also recently demonstrated with ACEi (captopril) in diabetic patients with proteinuria (26). In the present study, we documented that losartan has a similar effect on TGF-ß1 plasma levels in renal transplant patients with normal function. The decrease in the TGF-ß1 plasma levels was approximately 50% from the initial level. We emphasize that after 6 wk of treatment with losartan, the plasma levels of TGF-ß1 were within the normal range. This is the first demonstration that a pharmacologic treatment normalizes the plasma levels of TGF-ß1. In contrast, no significant changes were observed with amlodipine. The different effects of losartan and amlodipine on this important growth factor and profibrogenic cytokine also could be important in the prevention of chronic allograft nephropathy. Moreover, an opposite effect on ET-1 was observed between losartan and amlodipine. Losartan decreased plasma levels of ET-1; amlodipine tended to increase the concentration of this potent vasoconstrictor and remodeling factor. The different effects of losartan and amlodipine on TGF-ß1 could partially explain the different effects on ET-1, because in vivo and in vitro studies have demonstrated that AngII and TGF-ß1 are important stimuli for the synthesis of ET-1 (23,27,28).
In summary, the present study demonstrated that losartan and amlodipine were highly effective in the control of hypertension in renal transplant patients with normal renal function. Analysis of the effects on renal hemodynamics and the plasma concentration of profibrogenic cytokines (TGF-ß1 and ET-1) suggest that losartan could be more beneficial than amlodipine in interfering with progression of chronic allograft nephropathy than amlodipine. Prospective studies with the use of these drugs in the immediate posttransplant period with consecutive renal biopsies would be the only way to confirm our hypothesis that the blockade of the AngII receptor with losartan could reduce or even prevent the development of chronic allograft nephropathy in renal transplant patients who are treated with calcineurin inhibitors.
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Acknowledgments |
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