Introduction

Renin–angiotensin–aldosterone system (RAAS) blockade is an established therapy for incipient and overt diabetic nephropathy. Randomised trials have investigated ACE inhibitors, angiotensin II receptor blockers (ARBs) [1] and a direct renin inhibitor (DRI) [2]. The trials used antihypertensive doses not necessarily optimal for renoprotection. Several dosage studies [3, 4] have demonstrated dose-dependent antiproteinuric effects. After the introduction of the renin inhibitor aliskiren and evidence of its antiproteinuric effects [2, 5] we aimed to investigate the antiproteinuric effect of increasing doses of aliskiren, by comparing urinary albumin excretion rate (UAER) during placebo and after 2 months of treatment with aliskiren 150 mg, 300 mg or 600 mg once daily. In addition we measured biochemical data of the RAAS and biomarkers of cardiovascular risk.

Methods

This was a double-blind, randomised, crossover trial. The design of this exploratory study was placebo-controlled, in order to be able to investigate the direct effects of different doses of aliskiren. Careful safety measures were taken; slow-release furosemide was prescribed to prevent fluid retention and BP elevation. Patients used an electronic blood pressure device (UA 779; A&D Instruments Ltd, Abingdon, UK) to measure home BP daily throughout the study. If home BP at any time exceeded 170/105 mmHg, the patient was excluded.

The protocol was approved by the local ethics committee and the Danish Medicine Agency. Patients with type 2 diabetes mellitus were recruited from Steno Diabetes Center, Denmark. After giving informed consent, patients underwent a 1-month run-in period, in which all antihypertensive treatment was stopped prior to randomisation. A total of 26 patients were randomised and 19 patients completed the study.

Patients attended a randomisation visit before starting treatment periods of 2 months with placebo or aliskiren 150, 300 and 600 mg once daily, in random order (Fig. 1).

Fig. 1
figure 1

Study design. Ali, aliskiren

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Patients were considered eligible for randomisation if they had baseline UAER >100 mg/24 h and <2,000 mg/24 h, baseline office BP above 135/85 mmHg and baseline GFR >40 ml min−1 1.73 m−2. Exclusion criteria included recent cardiovascular disease, heart failure (New York Heart Association class II–IV), HbA1c >11% and malignancy.

After each treatment period, patients collected three consecutive 24-h urine samples for assessment of geometric mean UAER. We performed assessment of GFR (measured as plasma clearance of 51Cr-EDTA) [6] and mounted standard Takeda 24-h BP devices (TM2421; A&D Medical, Tokyo, Japan). BP measurements were performed every 15 min from 7:00 to 23:00 and every 30 min from 23:00 to 7:00 for 24 h.

Laboratory methods

UAER was determined on a turbidimetric Hitachi 912 System (Roche Diagnostics, Mannheim, Germany) and creatinine using the enzymatic Jaffé method. Cardiovascular biomarkers, PRC, prorenin, angiotensinogen, PRA, Ang I, ACE activity and Ang II were measured using previously described methodology [7]. Prior to the study, it was anticipated that conventional PRA assays would not have the required sensitivity to detect very low levels of plasma renin activity during the 600 mg treatment. Consequently, we developed a trapping high sensitivity PRA assay with increased sensitivity. Trapping hsPRA (ThsPRA) was carried out like hsPRA with the modification that Ang I antiserum was added at the start of the enzymatic reaction at a quarter of the concentration employed in hsPRA.

Investigators were blinded to randomisation using a validated computer system. Blinded study medications were packed and labelled prior to delivery to the site.

Statistical analysis

It was estimated that 20 patients who completed the study could provide 80% power to demonstrate a significant difference between two treatments in antiproteinuric effect (measured as UAER) if the true difference was 15%. This was based on the assumption that intra-individual CV for UAER was 13%. The log-transformed values of UAER were analysed by a PROC MIXED model with sequence, treatment, and period as fixed factors and patient (nested in sequence) as a random factor. Twenty-four-hour average values for systolic blood pressure (SBP) and diastolic blood pressure (DBP) were analysed using a PROC MIXED model with sequence, treatment, and period as fixed factors and patient (nested in sequence) as a random factor. GFR results and all other laboratory assessment data were analysed in a similar way to BP data. A two-sided p value <0.05 was considered significant.

Statistical analyses were performed using SAS version 8.2 or higher (SAS Institute Inc., Cary, North Carolina, USA).

Results

Clinical and biochemical results are shown in Table 1. Treatment with aliskiren 150, 300 and 600 mg daily reduced UAER significantly by 36% (95% CI 17–51), 48% (33–60) and 52% (38–63) respectively compared with placebo (p < 0.001 for all). The 600 mg dose had a significantly larger reduction compared with 150 mg, but not compared with 300 mg.

Table 1 Clinical results and changes in RAAS components and cardiovascular biomarkers
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Twenty-four-hour SBP was reduced by 4.5 (−0.2, 9.2), 8.0 (3.2–12.6) and 9.2 (4.5–13.9) mmHg compared with placebo, significant reductions for the 300 and 600 mg group vs placebo (p = 0.001 and p < 0.001), but with no significant difference between 300 and 600 mg (p = 0.598). Twenty-four-hour DBP was reduced by 3.0 (0.5–5.4), 4.1 (1.7–6.6) and 4.4 (2.0–6.9) mmHg respectively; all doses were significantly different compared with placebo (p = 0.019, p = 0.001 and p < 0.001), and there was no significant difference between 300 and 600 mg (p = 0.814).

GFR was reduced by 3.0 (−0.6, 6.6), 5.1(1.5–8.8) and 6.5 (2.8–10.1) ml min−1 1.73 m−2, significant reductions compared with placebo for the 300 and 600 mg doses (p = 0.007 and p < 0.001), and no difference between doses (p = 0.469).

During the washout period the UAER increased towards placebo levels. After 6 weeks the UAER was reduced by 10% compared with placebo (NS) and after 8 weeks it was similar to placebo (332 mg/day vs 330 mg/day). After the 8-week washout mean 24-h BP was 136/77 mmHg, as compared with 137/81 mmHg at baseline.

We found a clear dose-dependent effect on the RAAS components as shown in Table 1.

The biomarkers of cardiovascular risk displayed only minor and insignificant changes during the different aliskiren treatments compared with placebo levels, except for aldosterone, which was significantly reduced during treatment with 150 mg and 600 mg, and total adiponectin, which showed a significant increase during 600 mg treatment.

All doses of aliskiren were well tolerated. During the study four patients had a total of five serious adverse events (SAEs). One patient was diagnosed with colon cancer, one patient was hospitalised twice for syncope, one patient developed macroscopic haematuria and one patient was hospitalised due to lower limb wound infection requiring i.v. antibiotic therapy. None of the SAEs were suspected to be related to or caused by the aliskiren treatment. No events of hyperkalaemia were observed. The most frequent side effects were dizziness (four patients, one in each treatment group) and fatigue (two patients, one in the 300 mg and one in the 600 mg group). There were no gastrointestinal adverse events during treatment with 600 mg aliskiren.

Discussion

In this study we investigate the antiproteinuric effect of increasing doses of aliskiren. Although there is no further clinical benefit from increasing the aliskiren dose above the maximal recommended antihypertensive dose, 300 mg (indicating a plateau of the dose–response curve), there are signs of a higher degree of RAAS blockade, reflected by the larger increase in plasma prorenin and plasma renin concentration, and the further decrease in plasma renin activity.

The matter of dosing is important, since most antiproteinuric treatments are applied using antihypertensive doses. Dosing of antiproteinuric RAAS blockade was recently discussed [8] and has so far been investigated in a fraction of the treatments used today [3, 9].

When comparing 300 mg and 600 mg, we found no significant differences in the clinical measures (UAER, 24-h BP), but the changes in RAAS markers imply that 600 mg aliskiren treatment achieved a higher degree of RAAS blockade compared with treatment with 300 mg aliskiren. The increase in PRC is of interest, as it is regarded as a response to the feedback from the Ang II receptors and is thus a sign of increased intrarenal inhibition of the RAAS. Also prorenin levels were significantly higher during 600 mg treatment. Long-term beneficial effects of renin inhibition may in part work by mitigating the negative effects of this (pro)renin receptor pathway. Blocking this (pro)renin receptor using a decoy peptide has so far shown diverging results [10]. Levels of biomarkers of cardiovascular risk measured in our study were mostly unchanged during aliskiren treatment. This is in agreement with previous short-term studies [5, 7]. Of note, aldosterone levels tended to decrease with all doses, which warrants further investigation, since this is a small study and the connection between RAAS blockade (i.e. aliskiren treatment) and cardiovascular biomarkers has not been well described. The antiproteinuric effect of aliskiren treatment in type 2 diabetes mellitus has been demonstrated as an add-on to standard treatment [2]. The consequences of a high degree of RAAS blockade remain to be further investigated.

This was a small exploratory study of short duration, limiting the potential interpretations. However, the crossover study design was appropriately powered to detect a clinically meaningful difference in UAER (15%) between doses which can now be excluded, and thus 600 mg should not be used in clinical practice. In conclusion, we found no additional antiproteinuric effect of 600 mg aliskiren once daily as compared with the standard dose of 300 mg daily.