this was a double - blind , randomized , crossover trial in accordance with the declaration of helsinki and good clinical practice .
the primary objective was to assess albuminuria during different treatments compared with that with placebo ; secondary objectives were to assess effect on 24-h blood pressure , glomerular filtration rate ( gfr ) , biomarkers , and raas components .
after informed consent , patients attended a screening visit comprising laboratory tests and evaluations of inclusion / exclusion criteria .
slow - release furosemide in a fixed dose ( mean dose 109 mg/24 h , range 60360 mg/24 h ) was prescribed to prevent blood pressure elevation and fluid retention .
patients used an electronic blood pressure device ( ua-779 ; a&d instruments , abingdon , u.k . ) to measure home blood pressure throughout the study .
change in uaer ( percentage ) versus placebo during treatment with 300 mg aliskiren daily , 300 mg irbesartan daily , or the combination ( p < 0.001 vs. placebo for all treatments ) . from a list of prescreened candidates , 41 patients were screened for study participation ( supplementary fig .
1 , available in an online appendix at http://care.diabetesjournals.org/cgi/content/full/dc09-0168/dc1 ) . nine of these were screen failures mainly due to albuminuria levels below the randomization requirement .
of the 10 randomly assigned patients who left the study before completion , 2 died , 1 was lost to follow - up , 4 had an adverse event that led to exclusion ( diarrhea , severe hypertension , recurrent urinary tract infection , and dizziness ) , and 3 withdrew consent .
twenty - six patients had the primary end point , albuminuria assessed after randomization , and were included in the final analysis ; the remaining six patients dropped out shortly after random assignment and were not included in the final analysis .
after washout , patients attended a randomization visit before 2 months of treatment with placebo , 300 mg aliskiren once daily , 300 mg irbesartan daily , or the combination of the two , in random order .
patients with type 2 diabetes ( world health organization criteria ) aged 3080 years were eligible for randomization with baseline urinary albumin excretion rate ( uaer ) > 100 mg/24 h , hypertension ( baseline office blood pressure > 135/85 mmhg ) , and baseline gfr > 40 ml / min per 1.73 m. exclusion criteria included major cardiovascular disease ( within 6 months ) , heart failure ( new york heart association class ii 
rather , we used active washout : during the first 14 days of all treatments , every patient received 150 mg aliskiren daily to avoid risk of hypotension or a drastic increase in blood pressure in the switch from placebo to combination treatment or vice versa . during the last 3 days of each treatment
, patients collected three consecutive 24-h urine samples for assessment of geometric mean uaer . at the last day of each treatment period , patients attended our clinic for assessment of gfr and mounting of standard takeda 24-h blood pressure devices ( tm2421 , version 7 ; a&d medical , tokyo , japan ) .
measurements were performed every 15 min from 7 to 23 h ( daytime ) and every 30 min from 23 to 7 h ( nighttime ) .
urinary albumin and creatinine concentrations were determined on a turbidimetric hitachi 912 system ( roche diagnostics , mannheim , germany ) .
samples for prorenin , plasma renin activity ( pra ) , high - sensitivity pra ( hs - pra ) , immunoreactive plasma renin concentration ( ir - prc ) , angiotensinogen , ang i , ace activity , ang ii , and aldosterone levels were determined after 30 min of supine rest , and the plasma was frozen after centrifugation ( 80c ) .
raas components and biomarkers were measured at baseline and at the end of treatment periods .
biomarkers of inflammation , endothelial dysfunction , and cardiovascular risk were measured : high - sensitivity c - reactive protein ( hs - crp ) ( enzyme immunosorbent assay [ eia ] ; dako , glostrup , denmark ) ; serum soluble vascular adhesion molecule-1 and serum soluble intercellular adhesion molecule-1 ( eia ; diaclone , besanon , france ) ; plasma plasminogen activator inhibitor-1 ( hyphen biomed kit ; andresy , france ) ; serum nh2-terminal - probrain natriuretic peptide ( eia kit ; biomedica , wien , austria ) ; fibrinogen ( immunoturbidimetry ) ; and plasma asymmetrical dimethyl arginine ( high - performance liquid chromatography ) .
total renin concentration , prorenin concentration , plasma angiotensinogen , pra , and biomarkers of inflammation and endothelial dysfunction were measured using methodology described previously ( 4 ) .
ir - prc was measured with an immunoradiometric kit ( renin iii ; cisbio , gif - sur - yvette , france ) .
hs - pra was measured by in - house radioimmunoassay of ang i formed during incubation of 25 l plasma and 50 pmol sheep angiotensinogen for 3 h at 37c in a total reaction volume of 100 l .
the assay was double calibrated against ang i and the international reference preparation of renin , 68/356 , from nibsc ( hertfordshire , u.k . ) .
plasma ang i and ang ii were measured using in - house radioimmunoassays and ethanol extraction of plasma samples .
ace activity was determined using a commercial radioenzymatic assay ( ace direct ; bhlmann - laboratories , schnenbuch , switzerland ) .
randomization was blinded to all investigators , and the study drugs were packed and labeled before delivery to the site .
it was estimated that 20 patients completing the study could provide 80% power to demonstrate a significant difference between two treatments in antiproteinuric effect ( uaer ) if the true difference was 15% .
this was based on the assumption that intrasubject coefficient of variation for the uaer was 13% .
the log - transformed values of uaer were analyzed by a proc mixed model with sequence , treatment , and period as fixed factors and subject ( nested in sequence ) as a random factor . for 24-h blood pressure data , daytime average , nighttime average , and 24-h average values for systolic blood pressure and diastolic blood pressure
were analyzed using a proc mixed model with sequence , treatment , and period as fixed factors and subject ( nested in sequence ) as a random factor .
extra analyses were performed to test the assumption of no carryover effect by fitting a carryover effect term into the model . gfr results and all other laboratory assessment data were analyzed similarly to blood pressure data . a two - sided p value < 0.05 was considered significant
the correlation between changes in albuminuria and changes in hs - pra or ang ii were assessed by a nonparametric spearman correlation coefficient .
correlations between changes in albuminuria and changes in ir - prc were assessed by linear regression analysis within each active treatment ( aliskiren , irbesartan , and aliskiren / irbesartan combination ) and for all active treatments combined .
fractional clearance was calculated using urine samples collected during gfr measurements ( urinary albumin excretion / serum albumin concentration  gfr ) , and log - transformed levels were compared using a paired t test .
statistical analyses were performed using sas ( version 8.2 or higher ; sas institute , cary , nc ) and spss ( version 14.0 ; spss , chicago , il ) .
it was estimated that 20 patients completing the study could provide 80% power to demonstrate a significant difference between two treatments in antiproteinuric effect ( uaer ) if the true difference was 15% .
this was based on the assumption that intrasubject coefficient of variation for the uaer was 13% .
the log - transformed values of uaer were analyzed by a proc mixed model with sequence , treatment , and period as fixed factors and subject ( nested in sequence ) as a random factor . for 24-h blood pressure data , daytime average , nighttime average , and 24-h average values for systolic blood pressure and diastolic blood pressure were analyzed using a proc mixed model with sequence , treatment , and period as fixed factors and subject ( nested in sequence ) as a random factor .
extra analyses were performed to test the assumption of no carryover effect by fitting a carryover effect term into the model . gfr results and all other laboratory assessment data were analyzed similarly to blood pressure data .
a two - sided p value < 0.05 was considered significant . the correlation between changes in albuminuria and changes in hs - pra or ang ii
correlations between changes in albuminuria and changes in ir - prc were assessed by linear regression analysis within each active treatment ( aliskiren , irbesartan , and aliskiren / irbesartan combination ) and for all active treatments combined .
fractional clearance was calculated using urine samples collected during gfr measurements ( urinary albumin excretion / serum albumin concentration  gfr ) , and log - transformed levels were compared using a paired t test .
statistical analyses were performed using sas ( version 8.2 or higher ; sas institute , cary , nc ) and spss ( version 14.0 ; spss , chicago , il ) .
baseline demographic data are shown in table 1 . primary and secondary objectives were met . during placebo treatment
geometric mean uaer was 258 ( range 842,361 ) mg / day , mean  sd 24-h blood pressure was 140/73  15/8 mmhg , daytime blood pressure was 144/76  17/9 , and nighttime blood pressure was 130/68  17/9 .
gfr was 89  27 ml / min per 1.73 m and mean serum creatinine was 88
demographics of the 32 randomly assigned patients with type 2 diabetes , hypertension , and albuminuria and the 26 included in the final analysis data are means sd , n , or mean ( range ) .
aliskiren treatment led to a significant reduction in albuminuria by 48% ( 95% ci 2762 ) compared with placebo ( p < 0.001 ) but not significantly different from irbesartan , lowering uaer by 58% ( 4270 ) ( p < 0.001 vs. placebo ) .
combination treatment reduced albuminuria by 71% ( 5979 ) ( p < 0.001 ) compared with placebo , significantly more than with either monotherapy ( p < 0.001 and p = 0.028 ) .
the relative difference between aliskiren and combination treatment was 31% . to adjust for treatment - induced changes in gfr and the potential influence on albuminuria reduction , we calculated fractional clearance , which was reduced by 46% versus placebo during aliskiren treatment ( p = 0.021 ) , by 56% versus placebo during irbesartan treatment ( p = 0.002 ) , and by 67% versus placebo during combination treatment ( p = 0.001 ) .
systolic / diastolic 24-h blood pressure was reduced 3/4 mmhg by aliskiren ( ns / p = 0.009 ) , 12/5 mmhg by irbesartan ( p < 0.001/p = 0.002 ) , and 10/6 mmhg by the combination ( p = 0.001/p < 0.001 ) versus placebo .
a correlation was found between change in albuminuria and change in 24-h diastolic blood pressure during all treatments ( p = 0.039 ) .
seated office systolic / diastolic blood pressure was reduced 7/4 mmhg by aliskiren , 6/4 mmhg by irbesartan , and 12/8 mmhg by the combination , all statistically significant compared with placebo , except for diastolic blood pressure during irbesartan treatment .
gfr was significantly reduced 4.6 ( 95% ci 8.80.3 ) ml / min per 1.73 m by aliskiren ( p = 0.037 ) , 8.0 ( 12.33.6 ) ml / min per 1.73 m by irbesartan ( p < 0.001 ) , and 11.7 ( 15.97.4 ) ml / min per 1.73 m by the combination ( p < 0.001 ) compared with placebo .
aliskiren significantly reduced hs - pra , ang i , and ang ii by 87 , 75 , and 52% , respectively , compared with placebo ; irbesartan had the opposite effect ( table 2 ) .
when combined , the activating effect of irbesartan was counteracted by aliskiren , reducing hs - pra , ang i , and ang ii by 88 , 78 , and 56% , respectively , compared with irbesartan monotherapy . whereas combination treatment caused a 1,068% increase in ir - prc versus a 279% increase during aliskiren monotherapy and a 178% increase during irbesartan monotherapy , hs - pra was reduced 47% compared with placebo after combination therapy .
pra measured by a conventional method was affected similarly to hs - pra , although the changes were smaller ( table 2 ) .
the renin - specific activity ( renin bioactivity / total renin mass ) was 3 and 4% after aliskiren and combination therapy compared with placebo , respectively , thereby confirming that 9697% of renin was aliskiren bound . during irbesartan monotherapy ,
changes in raas components and cardiovascular biomarkers versus placebo all treatments were administered once daily .
adma , asymmetrical dimethyl arginine ; icam-1 , intracellular adhesion molecule-1 ; nt - probnp , nh2-terminal - probrain natriuretic peptide ; pai-1 , plasminogen activator inhibitor-1 ; vcam-1 , vascular adhesion molecule-1 ; vwf , von willebrand factor .
a significant correlation between reduction in albuminuria and increase in ir - prc was observed for all active treatments combined ( r = 0.597 , p = 0.0001 ) .
there was a significant correlation between changes in albuminuria and increase in ang ii during irbesartan treatment ( correlation coefficient 0.486 , p = 0.022 ) ; no significant correlations were observed in the other treatment groups .
hs - crp was reduced 35% from the placebo level with aliskiren ( p = 0.047 ) and 35% with irbesartan ( p = 0.043 ) .
other statistically significant changes from placebo levels were a 6% reduction in soluble intracellular adhesion molecule-1 ( p = 0.017 ) observed with the combination treatment and a 7% reduction in fibrinogen during aliskiren treatment ( p = 0.037 ) .
no treatment led to significant changes from placebo levels in any of the other cardiovascular biomarkers ( table 2 ) .
the most frequent adverse events were urinary tract infection ( four patients , one male ) , pneumonia ( three patients ) , and cough ( three patients ) , occurring during different treatments .
anemia and hypomagnesemia were detected in two patients during the combination treatment . compared with each monotherapy
, combination treatment showed an increase in plasma potassium by 0.2 mmol / l ( p = 0.036 ) .
one patient dropped out during the placebo period after several systolic blood pressure readings > 180 mmhg .
two patients died before the first measurement of albuminuria after the randomization and were not included in the final end point analysis .
the first death was that of a 42-year - old obese man ( bmi 42 kg / m ) with a history of ischemic heart disease , myocardial infarction , and hypertension 4 years before study entry .
the patient experienced sudden cardiac arrest , seemingly after a myocardial infarction during aliskiren treatment .
the second death was that of a hypertensive , obese , 73-year - old man with diabetes duration of 16 years .
the deaths were instantly reported to relevant authorities and were not suspected as being related to any of the drugs studied .
subsequently , home blood pressure measurement frequency was increased from twice weekly to twice daily , and patients were instructed to contact the investigator by direct phone ( available around the clock ) , if any measurement was > 160/100 mmhg .
extra measurements of sodium , potassium , and creatinine were introduced 3 weeks into each treatment period .
in this exploratory study , we demonstrated that treatment with 300 mg aliskiren once daily was as efficient in reducing albuminuria as standard therapy with 300 mg irbesartan once daily . when we combined the two treatments at the same doses , the reduction in albuminuria
was enhanced . the added antiproteinuric effect with combination treatment compared with aliskiren alone was 31% .
given that the reductions in 24-h systolic blood pressure with aliskiren were unexpectedly small compared with those with placebo relative to 24-h diastolic blood pressure changes and were substantially lower than the office systolic blood pressure measurements in this study , we conducted a thorough review of potential flaws in data collection , storage , device calibration , reporting , and calculation .
there was no evidence to indicate that the ambulatory data collection , storage , or reporting was flawed , and the unexpected results could be a chance occurrence .
our study suggested that the combination of aliskiren and irbesartan had an additional raas blocking effect compared with monotherapy because a synergistic increase in ir - prc was observed with the combination , which was related to the antiproteinuric effect , whereas hs - pra was reduced 50% compared with the reduction with placebo .
as opposed to the aliskiren in the evaluation of proteinuria in diabetes ( avoid ) study ( 5 ) , which showed an additional 20% albuminuria reduction after 24 weeks of treatment with aliskiren compared with placebo added to the maximal recommended dose of losartan and optimal antihypertensive therapy , this is the first study with a head - to - head comparison between aliskiren and irbesartan treatment .
no other antihypertensive drugs except for furosemide were allowed in our study , thereby offering a clearer picture of the effect of the two compounds used , compared with the avoid study , in which aliskiren was combined with losartan and a mixture of other antihypertensive drugs .
this study is different with regard to patient population , with a lower mean baseline uaer compared with that in the avoid study .
in addition , we assessed the effect of renin inhibition on gfr , a measurement that is more precise than the estimated gfr used in the avoid study .
raas blockade is believed to reduce proteinuria through several different mechanisms : the mean transcapillary hydraulic pressure difference , the glomerular surface area , and the size and charge selectivity of the glomerular filter . in diabetic nephropathy several of these variables
are abnormal , and raas blockade has been demonstrated to normalize directly measured or estimated glomerular hydraulic pressure ( 79 ) , to reduce the shunt - like defects in the membrane , at least in part ( 10 ) , and to restore the charge - selectivity properties of the glomerular membrane ( 11 ) .
aliskiren is thought to reduce albuminuria by the same mechanism as during treatment with ace inhibitors or arbs .
recently , fisher et al . ( 12 ) have shown that aliskiren treatment increases renal plasma flow to a larger extent than the ace inhibitor captopril .
the increase in renal plasma flow may be a response to angiotensin at1 receptor  dependent reduction of the vascular tone in the efferent arteriole . reduced vascular tone in the efferent glomerular arteriole
could be responsible for the decrease in intraglomerular pressure , leading to the reduction in albuminuria and gfr as demonstrated in our study .
although the combination reduced gfr up to 12 ml / min ( 95% ci 15.97.4 ) , we interpreted this as an reversible hemodynamic change and not as an indication of nephrotoxicity ( 13 ) .
in fact , it has been shown that an early hemodynamic reduction in gfr can translate into long - term renoprotection ( 13 ) .
when the albuminuria reduction was adjusted during combination treatment for changes in gfr ( fractional clearance ) , it was 11% higher than during irbesartan treatment , a nonsignificant change that was possibly due to a small sample number .
signs of more effective raas blockade were evident from the synergistic effect of combination treatment on ir - prc .
this conclusion is based on the fact that renin release into plasma is proportional to the interruption of the permanent negative feedback loop of ang ii on renin secretion ( 14 ) . combining aliskiren with
irbesartan provided a 12-fold increase in ir - prc , but still with a 50% reduction in hs - pra compared with changes during the placebo period .
this renin rise could reflect a high degree of intrarenal raas blockade during combination treatment as compared with that for the monotherapies , as has been suggested in nondiabetic patients ( 15 ) .
the reductions in albuminuria in our study were correlated with the rise in ir - prc , supporting the concept of increased intrarenal raas blockade underlying the additional effects observed during combination treatment .
compared with other studies of dual raas blockade , the rise in ir - prc is higher in dual raas blockade using aliskiren than in dual raas blockade with an ace inhibitor and an arb ( 5,16,17 ) . such marked increases in renin during aliskiren treatment have been noted before ( 18 ) .
apart from reflecting more complete ( intrarenal ) raas blockade , they may also be due to the detection of prorenin as renin ( 19 ) or a change in the renin half - life after its binding to aliskiren ( 20 ) .
pra was measured both by a conventional method and by a new high - sensitivity assay ( hs - pra ) , which is independent of endogenous substrate variation ( 21 ) . because high pra levels confer the risk of cardiovascular disease ( 22 ) , it will be interesting to evaluate long - term effects of direct renin inhibition in the ongoing aliskiren trial in type 2 diabetes using cardio - renal endpoints ( altitude ) , providing data on hard cardiovascular and renal end points ( 23 ) .
the antihypertensive effect of aliskiren was smaller than that found in previous larger studies ( 16 ) , although the office blood pressure reduction did not differ from that caused by irbesartan .
the ongoing telmisartan alone and in combination with ramipril global endpoint trial ( ontarget ) investigators ( 24 ) concluded that in a cardiovascular risk population , dual raas blockade with the arb telmisartan and the ace inhibitor ramipril is equivalent in reducing cardiovascular events compared with either as monotherapy , although with more frequent adverse events , including renal adverse events .
almost 3,000 of the participating 25,260 patients had microalbuminuria at baseline , and substudies of albuminuria effects are expected .
several short - term studies using dual raas blockade in diabetic nephropathy have shown promising antiproteinuric effects , as reviewed by rossing ( 17 ) , but the largest study so far ( 25 ) did not show additional benefits of a combination of ramipril and irbesartan , compared with ramipril monotherapy , in terms of albuminuria reduction after 20 weeks .
the size was , however , sufficient to demonstrate the likely beneficial effect of combination therapy with aliskiren and irbesartan , although we evaluated a surrogate end point .
in addition , the discrepancy between 24 h and office blood pressure readings complicates interpretation of the results .
studies evaluating mortality and morbidity are ongoing and will provide further information on dual raas blockade with aliskiren . in summary , we demonstrate an antiproteinuric effect of dual raas blockade with aliskiren and irbesartan in combination compared with either treatment alone in patients with type 2 diabetes , hypertension , and albuminuria .
the synergistic effect on ir - prc illustrates a higher degree of intrarenal raas blockade during combination treatment .
 objectivewe investigated whether the antiproteinuric effect of the direct renin inhibitor aliskiren is comparable to that of irbesartan and the effect of the combination.research design and methodsthis was a double - blind , randomized , crossover trial . 
 after a 1-month washout period , 26 patients with type 2 diabetes , hypertension , and albuminuria ( > 100 mg / day ) were randomly assigned to four 2-month treatment periods in random order with placebo , 300 mg aliskiren once daily , 300 mg irbesartan once daily , or the combination using identical doses . 
 patients received furosemide in a stable dose throughout the study . 
 the primary end point was a change in albuminuria . 
 secondary measures included change in 24-h blood pressure and glomerular filtration rate ( gfr).resultsplacebo geometric mean albuminuria was 258 mg / day ( range 842,361 ) , mean  sd 24-h blood pressure was 140/73  15/8 mmhg , and gfr was 89  27 ml / min per 1.73 m2 . 
 aliskiren treatment reduced albuminuria by 48% ( 95% ci 2762 ) compared with placebo ( p < 0.001 ) , not significantly different from the 58% ( 4279 ) reduction with irbesartan treatment ( p < 0.001 vs. placebo ) . 
 combination treatment reduced albuminuria by 71% ( 5979 ) , more than either monotherapy ( p < 0.001 and p = 0.028 ) . 
 fractional clearances of albumin were significantly reduced ( 46 , 56 , and 67% reduction vs. placebo ) . 
 twenty - four - hour blood pressure was reduced 3/4 mmhg by aliskiren ( ns / p = 0.009 ) , 12/5 mmhg by irbesartan ( p < 0.001/p = 0.002 ) , and 10/6 mmhg by the combination ( p = 0.001/p < 0.001 ) . 
 gfr was significantly reduced 4.6 ( 95% ci 0.38.8 ) ml / min per 1.73 m2 by aliskiren , 8.0 ( 3.612.3 ) ml / min per 1.73 m2 by irbesartan , and 11.7 ( 7.415.9 ) ml / min per 1.73 m2 by the combination.conclusionsthe combination of aliskiren and irbesartan is more antiproteinuric in type 2 diabetic patients with albuminuria than monotherapy . 
RESEARCH DESIGN AND METHODS
Statistical analysis
RESULTS
CONCLUSIONS
Supplementary Material