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NDT Advance Access originally published online on December 5, 2005
Nephrology Dialysis Transplantation 2006 21(3):683-689; doi:10.1093/ndt/gfi310
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© The Author [2005]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Original Articles: Clinical Nephrology

Effect of low-dose dual blockade of renin–angiotensin system on urinary TGF-ß in type 2 diabetic patients with advanced kidney disease

Joon Ho Song1,4, Seok Ho Cha2,4, Hun Jae Lee3, Seoung Woo Lee1,4, Geun Ho Park1, Seung Won Lee1 and Moon-Jae Kim1,4

1 Division of Nephrology and Hypertension, Department of Internal Medicine, 2 Department of Pharmacology and Toxicology, 3 Department of Preventative and Social Medicine and 4 Kidney Disease Research Group, Inha University College of Medicine, Incheon, Republic of Korea

Correspondence and offprint requests to: Moon-Jae Kim, Division of Nephrology and Hypertension, Department of Internal Medicine, Inha University College of Medicine, Incheon, Republic of Korea. Email: nhkimj{at}inha.ac.kr



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. We evaluated the renoprotective effects of dual blockade of renin–angiotensin system (RAS) by using a low-dose combination of ACE inhibiter and angiotensin II receptor blocker in type 2 diabetic patients with advanced kidney disease. The amount of proteinuria and the urinary levels of bioassayable TGF-ß1 were used as surrogate markers of renal injury and sclerosis.

Methods. We performed a prospective double-blinded randomized crossover trial consisting of three 16-week treatment periods with ramipril alone (10 mg/day), candesartan alone (16 mg/day), and ramipril (5 mg/day) plus candesartan (8 mg/day) combination therapy. Twenty-one type 2 diabetic patients with overt nephropathy with a 24 h urinary protein excretion rate (UPER) of >1.0 g/24 h and creatinine clearance (Ccr) of 30 to 59 ml/min/1.73 m2 completed the entire study.

Results. Subjects consisted of 10 female and 11 male patients with a mean age of 49±8 years and duration of diabetes ranging from 4 to 13 years. At baseline, 24-h blood pressures (BPs) were 133±6/81±7 mmHg, Ccr 40.6±4.1 ml/min/1.73 m2, 24-h UPER 4.1±1.9 g/24 h, and urinary TGF-ß1 level 28.4±16.1 pg/mg creatinine (cr). Although there was no comparable change in BP and plasma/urinary biochemical parameters, 24-h UPER was significantly reduced by the combination therapy (2.9±1.4 g/24 h) compared with that of ramipril (3.5±1.8 g/24 h) and of candesartan (3.3±2.0 g/24 h) single therapy (P<0.05). Urinary TGF-ß1 level was reduced in all three therapies compared with that of the control (28.4±16.1 pg/mg cr) (P<0.05). However, the combination therapy showed the most significant change (combination 19.6±10.6 pg/mg cr; ramipril 24.7±13.3 pg/mg cr; candesartan; 23.4±11.7 pg/mg cr). No significant or irreversible adverse effect was observed in the 21 patients who completed the entire study.

Conclusions. The dual blockade of RAS with low-dose ramipril plus candesartan was found to be safe and offered additive benefits with respect to reducing proteinuria and urinary TGF-ß1 excretion in diabetic patients with advanced kidney disease. These benefits were evident as compared with single ramipril and candesartan therapies at doses two-fold greater. Further study on the dose-titration is mandatory in terms of safety and especially for maximizing renoprotection in this patient population.

Keywords: ACE inhibitors; angiotensin II antagonists; diabetic nephropathy; renin–angiotensin aldosterone system; TGF-ß



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 References
 
The pharmacological blockade of the renin–angiotensin system (RAS) using either angiotensin-converting enzyme (ACE) inhibitors or angiotensin (AT) II receptor blockers (ARBs) has renoprotective effects and retards the progression of diabetic nephropathy [1,2]. The combination of ACE inhibitors and ARBs may maximize the RAS blockade since they block RAS at the two different levels, i.e., at AT II synthesis and at AT II receptor binding [3,4]. A series of recent clinical reports have suggested that this method may further enhance the anti-proteinuric effects compared with the use of either ACE inhibitors or ARBs alone [3,4].

The studies on ‘non-diabetic’ renal diseases have consistently demonstrated that the combined use of ACE inhibitor and ARB is superior to the dose escalation of either drug alone [4–7]. Meanwhile, such comparisons have not been made in ‘diabetic’ nephropathy [4,8–10]. In most cases, the benefits of the combination therapy have been compared with single therapies of the same dosage. In several studies, one drug was added on top of the other, and the results were compared before and after the addition. Moreover, in most studies performed in patients with diabetic nephropathy, the benefit from the blood pressure-lowering effect was not completely excluded from that of the anti-proteinuric effect.

Transforming growth factor-ß (TGF-ß) is a fibrogenic cytokine that contributes to renal tissue fibrosis [11]. TGF-ß induces the synthesis of extracellular matrix, retards its degradation, and stimulates the synthesis of integrin matrix receptors. Of its isoforms, TGF-ß1 is known as the major mediator in the hypertrophic and sclerotic changes of diabetic kidney disease. The access of urinary bioassayable TGF-ß1 levels showed increase in the net local production of TGF-ß1 protein in diabetic nephropathy [12,13]. It is of clinical interest that inhibition of RAS may protect the kidney by lowering local TGF-ß1 production.

Although the dose titration of RAS-inhibiting agents is a common clinical practice to establish a maximal antiproteinuric effect [1,2], one important concern is that patients with renal dysfunction may not tolerate high-doses of these drugs. Therefore, studies on the dual blockade of RAS in patients with advanced diabetic renal disease are scarce. The aim of the present study was to examine the efficacy and safety of the dual blockade of RAS using low-dose ramipril plus candesartan combination therapy in type 2 diabetic patients with advanced kidney disease. We compared ramipril plus candesartan combination therapy with either ramipril or candesartan single therapy of twofold higher doses. Proteinuria and the bioassayable TGF-ß1 urinary levels were used as surrogate markers of renal injury and sclerosis [1,2,12].



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 References
 
Subjects
The study was conducted at Inha University Hospital, Incheon, Korea with the approval of the hospital ethics committee. The subjects were type 2 diabetes patients, as defined by WHO criteria, with overt nephropathy who had already been administered either 5 mg or more of ramipril or 8 mg or more of candesartan without complications. We selected subjects who met the following inclusion criteria: (i) 24-h urinary protein excretion rate (24-h UPER) >1.0 g/24 h, (ii) creatinine clearance (Ccr) 30 to 59 ml/min/1.73 m2, (iii) blood pressure (BP) maintained at <140/90 mmHg with or without additional antihypertensives for at least 3 months prior to the study. The exclusion criteria were: a history of noticeable side effects or hypersensitivity to ACE inhibitors or ARBs, an age <18 years, pregnant women, serum potassium >5.5 mmol/l, the absence of retinopathy, or the presence of nondiabetic renal disease, renal artery stenosis, type IV renal tubular acidosis, morbid cardiac, vascular diseases or malignancy, or uncontrolled diabetes.

Design
The study was of a prospective double-blinded randomized crossover design. All subjects underwent an 8-week run-in period to ensure (i) the safety of ramipril or candesartan and (ii) the efficacy of the agents in maintaining BP within the goal of <140/80 mmHg. RAS inhibiting drug administered before the enrollment was maintained at low fixed-doses, i.e. either of 5 mg of ramipril or 8 mg of candesartan. As a result, of 25 initially enrolled subjects, 13 subjects received 5 mg ramipril and 12 subjects received 8 mg candesartan during the run-in period (Figure 1). Other antihypertensive drugs including calcium channel blocker, {alpha}- or ß-blockers and/or diuretics were added if necessary to achieve the BP goal during the study period. The measurements at the end of the run-in period were used as the baseline values.


Figure 1
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  		Fig. 1. A schematic diagram of the crossover trial. aDue to a drug-related side effect; bdue to a non-drug-unrelated side effect; Ram=ramipril; Can = Candesartan.

 
After the run-in period, all subjects were randomly allocated to one of six treatment order sequence permutations to prevent period effects. Each treatment consisted of 16-week treatment periods of: 10 mg/day ramipril single therapy, 16 mg/day candesartan single therapy, and 5 mg/day ramipril plus 8 mg/day candesartan combination therapy (Figure 1). Ramipril was started at a dose of 5 mg and increased by 1.25 mg/day biweekly to a target level of 10 mg/day. Candesartan was started at 8 mg/day and increased by 2 mg/day biweekly to a target level of 16 mg/day. The combination therapy was started at 2.5 mg/day of ramipril and 4 mg/day of candesartan and increased alternately by 1.25 mg/day and 2 mg/day biweekly to target doses of 5 mg/day and 8 mg/day, respectively. The target dose was established within 8 weeks in most patients.

Each 16-week treatment period was separated by an 8-week washout period to rule out the residual effect of the previous treatment, so-called carryover effect. This was done based on previous findings that the effects of ACE inhibitors and ARBs on proteinuria and glomerular permselectivity are fully reversible within 4 weeks [14]. During washout period, ramipril and candesartan were stopped and, if necessary, other antihypertensive agents were titrated to maintain optimal BP. Patients visited an outpatient clinic every fortnight during the washout period and during the first 8 weeks of each treatment period, and visited every 4 weeks for the final 8 weeks of each treatment period.

Measurement of clinical parameters
Clinical parameters were measured at the end of run-in period and at the end of each active treatment period to determine baseline values. BP was monitored using a sphygmomanometer (phase I/V) in the sitting position after at least 10 min rest, and 24-h ambulatory BPs were measured at the last week of each period using an oscillometric BP monitor (90207, SpaceLabs Inc, Redmond, WA, USA). Mean values of 24-h systolic and diastolic BP were used for analysis. Fasting blood samples and first voiding fresh urine were taken for TGF-ß1 determinations during the morning of the final outpatient visit of each treatment period. Serum biochemistries were measured using routine methods. The 24-h urine samples were collected from the morning of the final visit for urinary protein, sodium, urea and creatinine (cr) excretion. Urinary protein was measured using a benzethoium chloride based turbidimetric method, and the other tests were performed using routine methods. Ccr was calculated based on urine volume and creatinine concentration in serum and in 24-h urine samples.

Measurement of urinary TGF-ß1 levels
First void mid-stream urine samples were collected aseptically. To remove particulate matter, each urine sample was centrifuged at 1500 rpm for 10 min. A 2.0 ml aliquot of clear supernatant was then immediately stored at –80°C and thawed for assay on the same day. The urinary total TGF-ß1 levels were determined by enzyme-linked immunosorbent assays using commercially available kits (Human TGF-ß1 immunoassay; Quantikine; R & D system, Minneapolis, MN) according to the manufacturers’ instructions. Briefly, urine samples were incubated with 1.0 N HCl for 10 min to activate latent TGF-ß1 to the immunoreactive form and then neutralized with 1.2 N NaOH/0.5 M HEPES buffer (pH 7.2–7.6). A standard curve was prepared in duplicate by serially diluting RD5I calibrator to concentrations of 2000, 100, 500, 250, 125, 62.5 and 31.2 pg/ml. Absorbance was measured at 450 nm using a plate reader. Concentrations read off the standard curve were multiplied by the dilution factor 1.4.

The minimum detectable level was 7 pg/ml. TGF-ß1 values are presented as picograms (of TGF-ß1) per mg of creatinine (pg/mg cr), to correct for variations in urine amount. Urine samples from normal healthy volunteers in our institute were used as controls; 0.5±0.1 pg/mg creatinine (n = 9, range: 0.2–1.2). The intra- and interassay coefficients of variation were 7.5 and 12.3%, respectively.

Statistics
Values are expressed as mean±SD unless stated otherwise. By choosing a type I error of 5%, a statistical power of 80% (type II error of 20%), and by assuming a 20% drop out rate, the minimal sample size required to detect clinically significant changes was determined to be 21 (standardized effect size = 1). The paired Student t-test was used to test the significances of changes in continuous variables due to treatment from baseline and between continuous variables (n = 21; by per protocol analysis). Pearson correlation analysis was used to analyse relations between 24-h urinary protein excretion rate, urinary TGF-ß1 level, and other clinical parameters. Statistical analysis was performed using SPSS for Windows release 10.0 (SPSS Inc., Chicago, IL). Two-tailed P values of <0.05 were considered statistically significant.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 References
 
Twenty-five patients were initially enrolled, and 21 patients completed the study (Figure 1). Two patients withdrew due to drug-related adverse effects; one for dizziness and a severe headache during the ramipril period, and the other for a rapid irreversible increase in creatinine (from 1.9 to 4.3 mg/dl) during the candesartan period. The other two patients withdrew for reasons not related to the treatment: one experienced traumatic cerebral haemorrhage and the other had foot gangrene. All patients were ethnic Koreans, 10 women and 11 men, mean age 49±8 years (range 35–66), and mean duration of diabetes treatment 8±3 years (range 4–13). BP was optimally maintained at the time of enrollment in all subjects (134±7/80±6 mmHg) (Table 1). Of the 21 subjects who completed the study, 11 patients had ongoing treatment of ramipril and 10 were under candesartan treatment before the enrollment. Accordingly, baseline data obtained at the end of run-in period corresponded to the states of receiving 5 mg ramipril/day for 11 patients and 8 mg candesartan/day for 10 patients. The baseline characteristics of these two sets of patients were similar for all clinical parameters (data not shown). Sixteen patients (76.2%) required additional antihypertensives at some stage to achieve the BP goal.


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  		Table 1. Baseline Characteristicsa

 
No treatment significantly changed 24-h BPs or other parameters, i.e. haematocrit, haemoglobin A1C, electrolytes, creatinine, albumin, or urinary sodium excretion vs baseline or vs the other treatments (Table 2). On the other hand, 24-h UPER and urinary TGF-ß1 levels were reduced significantly by all treatments compared with that of the baseline. 24-h UPER was reduced from 4.1±1.9 g/24 h at baseline to 2.9±1.4 g/24 h by combination therapy (5 mg ramipril plus 8 mg candesartan), to 3.5±1.8 g/24 h by 10 mg ramipril single therapy, and to 3.3±2.0 g/24 h by 16 mg candesartan single therapy. Within the treatment group, 24-h UPER was significantly lower in patients with the combination therapy compared with that of either of single therapies (P<0.05). Urinary TGF-ß1 levels were reduced from 28.4±16.1 pg/mg cr at baseline to 19.6±10.6 pg/mg cr by combination therapy, to 24.7±13.3 pg/mg cr by ramipril single therapy, and to 23.4±11.7 pg/mg cr by candesartan single therapy; and this was significantly lower for the combination therapy than for either of the two single therapies (P<0.05).


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  		Table 2. Comparison of clinical parameters among baseline and each treatmenta

 
Mean % reductions in arterial pressure by ramipril single therapy, candesartan single therapy, and combination therapy were 0.2±6.1%, 0.8±4.9% and 1.5±5.0% and mean % reductions in Ccr were 0.2±7.2%, 3.3±12.0% and 4.0±10.3%, respectively, but these differences were without significance (all P = NS) (Figure 2). The mean % 24-h UPER reduction by combination therapy was 28.2±11.3%, and this was significantly larger than the 14.6±17.8% of ramipril (mean difference vs combination therapy 13.6%, 95% CI, 5.7–21.6, P<0.05) and remarkably larger than the 19.8±21.5% of candesartan single therapy (8.4%, 1.2–18.0, P = 0.08). The mean % reduction in urinary TGF-ß1 level achieved by combination therapy was 29.3±16.6%, and again this was significantly larger than the 12.5 + 18.9% achieved by ramipril (mean difference vs combination therapy 16.8%, 95% CI, 9.8–23.9, P<0.05) or the 10.1±34.7% by candesartan single therapy (–19.3%, 1.4–37.0, P<0.05).


Figure 2
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  		Fig. 2. Mean % reductions in mean arterial pressure, creatinine clearance, 24-hr proteinuria and urinary TGF-ß1 level induced by the treatments. The combination therapy reduced 24-h UPER and urinary TGF-ß1 levels more so than ramipril or candesartan single therapy at doses 2-fold greater without significantly changing mean arterial pressure or creatinine clearance. Of 21 subjects, 11 were administered 5 mg ramipril and 10 were administered 8 mg candesartan at baseline. *P<0.05 vs ramipril single therapy. #P<0.05 vs candesartan single therapy.

 
The 24-h UPER and urinary TGF-ß1 levels were significantly correlated with the duration of diabetes (n = 21, r2 = 0.887; P<0.001 for 24-h UPER; r2 = 0.659 P = 0.001 for urinary TGF-ß1) and systolic BP (n = 21, r2 = 0.617, P = 0.003 for 24-h UPER, r2 = 0.534; P = 0.013 for urinary TGF-ß1) at baseline, but these significances disappeared after treatment. Other plasma and urinary parameters showed no significant correlation with 24-h UPER or urinary TGF-ß1 at baseline or at the end of each treatment. 24-h UPER and urinary TGF-ß1 levels were significantly correlated with each other at baseline (n = 21, r2 = 0.757; P<0.001) and maintained weakened but significant correlations after each treatment (r2 = 0.624, 0.630 and 0.736 for combination, ramipril, and candesartan, respectively; P<0.05 for all). Percentage changes in 24-h UPER or urinary TGF-ß1 levels by each treatment were not significantly correlated to those in mean systolic or diastolic BP, plasma creatinine or Ccr.

Adverse effects
As described above, two patients dropped out before analysis due to drug-related adverse effects (Figure 1). Of the 21 patients who completed this study, an increase in creatinine of more than 30% occurred in one patient during ramipril treatment and in two during candesartan treatment (Table 3). All these cases were reversible and recovered to serum creatinine levels within 30% of baseline. Mild transient hypotensive symptoms occurred in one patient during candesartan treatment and in two during combination treatment. Hyperkalaemia of >5.5 mEq/l developed in one patient during ramipril treatment, and in two during combination treatment, and both were resolved by increasing the doses of furosemide or potassium-binding resin.


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  		Table 3. Incidences of adverse effects during study period (n = 21)a

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
References
 
The present study suggested that dual blockade of RAS by ramipril and candesartan combination therapy produced superior efficacy in reducing proteinuria and urinary TGF-ß1 excretion compared with that of the either single therapy at twofold higher doses. To our knowledge, this information was previously unavailable for diabetic nephropathy patients with mild to moderate renal dysfunction [4,8–10]. The present study has the additional advantage that urinary TGF-ß, a major fibrogenic cytokine responsible for renal sclerosis, was also measured as a surrogate marker in addition to proteinuria.

Since the time that the first study was conducted on human renal diseases [15], there has been a continued focus on the benefits of the dual blockade of RAS in chronic renal diseases [3,4]. The first large clinical study reported was the candesartan and lisinopril microalbuminuria (CALM) study [8]. In this study, a 12-week combination of 16 mg candesartan and 20 mg lisinopril was compared with 16 mg candesartan and 20 mg lisinopril single therapies in hypertensive and microalbuminuria type 2 diabetic patients. The results obtained showed that urinary albumin excretion was reduced more by the combination therapy. However, a significant reduction in BP was also shown concomitantly and thus it was difficult to exclude the BP-reducing effect. The results were similar in subsequent studies on ‘diabetic’ nephropathy [4,9–10]. In a study on type 1 diabetic patients with albuminuria >300 mg/day [9], for example, 20 mg benazepril plus 80 mg valsartan induced a 43% reduction in albuminuria in association with a reduction of 7/6 mmHg in BP as compared with single therapies of 20 mg benazepril or 80 mg valsartan. In a study on type 2 diabetes patients [10], a 25% reduction in albuminuria was achieved in association with a 10 mmHg reduction in 24-h systolic BP by adding 8 mg candesartan to maximal doses of various ACE inhibitors. Moreover, no comparison was made of the combination with higher dose single therapies in the previous studies on diabetic nephropathy.

The superiority of combination therapy over single therapies at higher doses was firstly reported in non-diabetic renal diseases by Russo et al. [5]. They suggested that 10 mg enalapril and 50 mg losartan combination therapy offered more renoprotection than the single therapies at double these dosages. In animal study, Komine et al. [16] demonstrated that the combination of captopril and losartan further reduced renal tissue AT II levels vs single therapies at triple doses. Subsequent studies on non-diabetic renal diseases have produced similar results [6,7,13,17]. The results of the present study extend these findings to diabetic nephropathy.

TGF-ß1 is a fibrogenic cytokine that contributes to renal tissue fibrosis [11]. One major advantage of measuring TGF-ß1 in urine is that its urinary level reflects the net local renal production of TGF-ß1 and it is not influenced by platelet degranulation [12,13]. Clinically it is of interest whether the dual blockade of RAS reduces local TGF-ß1 production more than a single blockade. This issue was first investigated by Agarwal and colleagues [13]. They demonstrated that the addition of losartan to full-dose lisinopril significantly reduced urinary TGF-ß1 in various renal diseases. Since this occurred despite no reduction in proteinuria, they suggested that urinary TGF-ß1 might respond to combination therapy more sensitively than proteinuria. We also previously demonstrated that the addition of candesartan to ramipril reduced TGF-ß1 significantly in IgA nephropathy patients (28.9%, n = 14) and markedly in diabetic nephropathy patients (14.3%, n = 18) [17]. Since the primary aim of our previous study was to compare the efficacy of combination therapy between two diseases, IgA nephropathy vs diabetic nephropathy, the patients selection criteria and drug dosing schedules used were designed for IgA nephropathy rather than diabetic nephropathy patients. In the present study, we targeted diabetic nephropathy at higher drug doses and found a definite benefit for combination therapy in terms of reducing TGF-ß1 levels. Importantly, the present study shows that the combination therapy was beneficial vs double-dose single therapies.

It is well-known that the use of ACE inhibitors or ARBs may cause hyperkalaemia or aggravate renal dysfunction, especially in patients with renal failure. Safety was one of the major concerns of the present study since information was lacking in the advanced stage of overt diabetic nephropathy. Therefore, we selected subjects who had already been administered either ramipril or candesartan, in order to avoid the risks of the first use, and ensure tolerability during the run-in period. This strategy may have contributed to relatively low incidence of adverse effects in the present study.

Maximal tolerable and renoprotective doses have not yet been established for most ACE inhibitors and ARBs. Although the maximal pharmaceutical dose recommended for ramipril is 20 mg/day in the general population, a 50 to 75% reduction is required in patients with a Ccr of <50 ml/min [18]. Dosing guidelines have not been established for of candesartan in cases of renal failure. Some have recommended that the dose be halved in the patients with a Ccr of <20 ml/min in view of the finding that more than 60% candesartan is excreted by kidneys [18]. Moreover, it has been argued that lower concentrations of RAS inhibitors may be appropriate in Asian patients, who usually have lower body weights than their Caucasian counterparts. In addition, a recent study on type 2 diabetic nephropathy demonstrated that no further renoprotective benefit was obtained by dosing candesartan at >16 mg [19]. Therefore, the doses of ramipril and candesartan administered in the present study do not appear submaximal in patients with a baseline Ccr of 38.3±5.6 ml/min.

After completing this study, we continued the final treatment regimen of each patient to evaluate tolerability to higher doses (data not shown). During this stage, doses were gradually elevated up to 15 mg for ramipril single therapy (n = 8), 24 mg for candesartan single therapy (n = 8), and 10 mg ramipril and 16 mg candesartan for combination therapy (n = 7) (data not shown). In total, five of 21 patients (23.8%) failed to tolerate these dose elevations; creatinine incrementation of more than 30% of baseline occurred in four patients, one on ramipril, two on candesartan, and one on the combination, and intractable dizziness also occurred in one on the combination. We stopped further evaluation for safety reasons. Until confirmatory information on dose-safety relations in renal failure patients is obtained, the combination therapy with dose reduction is probably a better choice than maximal single drug elevation in terms of both efficacy and safety.

In addition to inter-treatment group comparisons, we compared the results of each treatment vs baseline data obtained when patients were on either 5 mg ramipril or 8 mg candesartan alone. Although this comparison is likely to show a weaker effect of subsequent treatment, it has the advantage of demonstrating the superiority of combination therapy vs low-dose single therapy, and thus the synergic benefits of the combination therapy. One important limitation of the present study is that subjects were not treated with the same drug at baseline. We presumed that 5 mg of ramipril and 8 of candesartan are equivalent in terms of treatment efficacy. This strategy is open to criticism. We believe, however, it cannot change the primary results of our study concerning comparative treatment effects.

We allocated patients to one of the six permutations of the three treatment regimens and an 8-week washout period was used to avoid the residual effects of previous treatments. The duration of washout period was determined based on a previously report that effects of ACE inhibitors and ARBs on proteinuria and glomerular permselectivity are fully reversible within 4 weeks of treatment discontinuation [14]. Although we believe that the crossover design used enhanced study statistical power, we concede that the relatively small number of subjects enrolled is a major study limitation. We hope that our finding will be confirmed by a large-scale clinical trial like COOPERATE in the future [20]. Moreover, it is evident that a future study with a focus on dose-titration with respect to safety and maximal renoprotection in advanced renal disease patients is essential.

In conclusion, dual RAS blockade by low-dose combination ramipril and candesartan therapy was found to be safe and beneficial in type 2 diabetic patients with overt diabetic nephropathy and renal dysfunction. Proteinuria and urinary TGF-ß1 were additively reduced by this combination therapy vs ramipril or candesartan single therapies at doses two fold greater.



   Acknowledgments
 
This work was supported by INHA University Research Group (INHA-31581).

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Received for publication: 14. 4.05
Accepted in revised form: 11.11.05


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