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Diverse diuretics regimens differentially enhance theantialbuminuric effect of reninangiotensin blockers

in patients with chronic kidney diseaseEnrique Morales1, Jara Caro1, Eduardo Gutierrez1, Angel Sevillano1, Pilar Aun1, Cristina Fernandez2 andManuel Praga1,3

1Department of Nephrology, University Hospital 12 de Octubre, Madrid, Spain; 2Research and Clinical Epidemiology Unit, Department of

Preventive Medicine, Hospital Clinic, San Carlos, Madrid, Spain and 3Department of Medicine, Complutense University, Madrid, Spain

The addition of spironolactone or hydrochlorothiazide

enhances the antialbuminuric effect of reninangiotensin

blockers. However, comparative studies on the effect of

different diuretics are lacking. We conducted a prospectiverandomized crossover study to compare the effects of

spironolactone (25 mg/day), hydrochlorothiazide (50 mg/day)

without/with amiloride (5 mg/day) on top of enalapril

treatment in 21 patients with CKD stages 13 and a urinary

albumin-to-creatinine ratio (UACR) over 300 mg/g. Treatment

periods lasted 4 weeks. The UACR showed a significant

reduction with the diuretics: spironolactone, 34% or

hydrochlorothiazide without/with amiloride 42% or 56%,

respectively. Reduction of the UACR was significantly greater

with hydrochlorothiazide without/with amiloride when

compared with spironolactone. The percentage of patients

who achieved UACR reductions greater than 30% and 50%

was greater with hydrochlorothiazide without/with amiloride

(81% and 57%, and 81% and 66%, respectively) when

compared with spironolactone alone (57% and 28%,

respectively). Glomerular filtration rate (GFR), blood pressure,

and body weight decreased with the three diuretic regimens.

A significant correlation was found between the UACR

reduction and GFR and blood pressure changes. Thus, diverse

diuretic regimens differentially enhance albuminuria

reduction, an effect likely associated with the degree of GFR

reduction.

Kidney International (2015) 88, 14341441; doi:10.1038/ki.2015.249;

published online 26 August 2015

KEYWORDS: albuminuria; amiloride; diuretics; hydrochlorothiazide; RAASblockade; spironolactone

The most important therapeutic strategies for slowing theprogression of chronic kidney disease (CKD) and reducingthe disproportionate cardiovascular risk of CKD patients are

controlling blood pressure (BP) and reducing albuminuria.1

7Reninangiotensinaldosterone system (RAAS) blockers(angiotensin-converting enzyme inhibitors (ACEIs) andangiotensin II receptor blockers (ARBs)) are the backboneof these therapies due to their efficacy in controlling BP andtheir known antialbuminuric effect. The favorable influenceof these drugs on the progression of chronic diabetic andnondiabetic nephropathy has been demonstrated in severalprospective controlled studies.39 This favorable influence hasa close relationship with the reduction of albuminuria; themore intense the reduction in albuminuria the greaterthe reduction in the risk of progression of CKD.4,7 However,

the antialbuminuric effect of RAAS blockers is mild ornegligible in a substantial number of CKD patients. The so-called residual albuminuria (i.e., the level of albuminuriathat persists after reaching the maximum tolerated dosage ofRAAS blockers and proper BP control) is considered one ofthe most significant factors in the progression of kidneydamage.1013 Therefore, the search for new alternatives thatenhance the antialbuminuric effect of ACEIs and ARBs is ofparamount importance.

In recent years, several studies have demonstrated theantialbuminuric potential of aldosterone receptor antagonists(spironolactone (SR) and eplerenone).1421 Likewise, observa-tional studies have suggested that this reduction in albumi-

nuria, as occurs with ACEIs and ARBs, is associated with asignificant reduction in the risk of progression of CKD.18,2023

Nevertheless, prospective controlled studies have not beenperformed with the duration necessary to demonstrate therenoprotective effect of aldosterone antagonist diuretics.Moreover, the combination of these diuretics with ACEIs orARBs increases the risk of hyperkalemia, especially in patientswith reduced glomerular filtration.18,20,21,23

Compared with the extensive experimental and clinicalresearch performed on aldosterone antagonist diuretics, the

possible antialbuminuric effect of other diuretics has been

scarcely studied. However, several clinical studies have shown

c l i n i c a l t r i a l http://www.kidney-international.org

2015 International Society of Nephrology

Correspondence: Manuel Praga, Servicio de Nefrologa, Hospital 12 de

Octubre, Avenida Crdoba s/n., Madrid 28041, Spain.

E-mail:[email protected]

Received 5 April 2015; revised 20 June 2015; accepted 25 June 2015;

published online 26 August 2015

1434 Kidney International (2015) 88 , 14341441
http://dx.doi.org/10.1038/ki.2015.249http://www.kidney-international.org/mailto:[email protected]:[email protected]://www.kidney-international.org/http://dx.doi.org/10.1038/ki.2015.249


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that hydrochlorothiazide (HCT), at dosages of 2550 mg/day,induces powerful albuminuria reductions in patients with orwithout diabetes who have residual albuminuria despitemaximum dosages of ACEIs or ARBs.2426 This antialbumi-nuric effect was similar to that achieved with a low-sodiumdiet. The combination of the two measures (HCT plus a low-sodium diet) achieved a very significant reduction inalbuminuria, greater than that achieved by each measure inisolation.24,26 Other studies have shown that furosemide canalso boost the antialbuminuric effect of ACEIs and ARBs.27,28

There is no clinical information available on amilorideconcerning its possible antialbuminuric effect, but experi-mental models have suggested a possible nephroprotectiverole of this diuretic.29,30

To the best of our knowledge, there are no clinical studiesthat have compared the antialbuminuric efficacy of varioustypes of diuretics. This information would be extremelyimportant for the design of renoprotective clinical strategies,given that the use of various types of diuretics is standard

practice for controlling BP and volume overload in CKDpatients. Aim of our study was to compare the antialbumi-nuric effect of SR, HCT, and amiloride. However, amiloride isusually marketed in combination with HCT in mostcountries, including ours. We therefore designed a prospec-tive, randomized crossover study to compare the antialbu-minuric effect of SR, HCT, and HCT+amiloride (A) forpatients with CKD and a urinary albumin-to-creatinine ratio(UACR) 4300 mg/g.

RESULTS

Of the 29 initially selected patients, 21 patients provided their

informed consent and started the study. Three patients wereexcluded for presenting UACR o300 mg/g, three patientswere excluded because of lack of motivation to continue withthe study, and two patients were excluded for other causes(Figure 1).

Table 1 reflects baseline clinical and biochemical char-acteristics at the end of the run-in period for the 21randomized patients. Almost half of the patients had diabetesand the other half had various glomerular conditions. In all,3 patients had stage 1 CKD, 10 patients stage 2 CKD, and 8patients stage 3 CKD. During the study, there was very goodtreatment adherence to the various types of diuretics in allpatients (490% of SR, HCT, and HCT+A pills during the

three treatment periods). Two patients had to reduce theenalapril dosage (20 mg/day) because of excessive BP controlin the HCT+A group.

Main objective

As can be seen in Table 2, UACR showed a significantreduction with the three types of diuretics: SR, 34% (95%confidence interval (CI)= 21 to 47; P= 0.001); HCT, 42% (95% CI = 28 to 56; P= 0.001); and HCT+A, 56% (95% CI = 44 to 67; P= 0.001). UACR reductionwas significantly greater with HCT and HCT+A whencompared with SR.

Secondary objectives

The percentage of patients who achieved UACR reductions430 and 450% was also greater with HCT and HCT+A whencompared with SR, although these differences did not reachstatistical significance (Table 2). There was a 430% reductionin UACR in 12 patients (57%) treated with SR and in 17patients (81%) treated with HCT or HCT+A. The percentage ofpatients with 450% UACR reduction was greater in theHCT+A group (14 patients (66%)) compared with the HCTgroup (12 patients (57%)) and SR group (6 patients (28%)).24-h proteinuria and 24-h albuminuria also showed significantreductions with the three types of diuretics, without significantbetween-group differences (Table 2).

Tertiary objectives

Estimated glomerular filtration rate (eGFR) was reduced withthe three types of diuretics, as shown in Table 3. Thisreduction was statistically significant with HCT (8.5%(95% CI= 3.8 to 13.3; P= 0.002)) and with HCT+A(

12% (95% CI=

5.9 to

18.1; P= 0.001)), whereasit did not reach statistical significance with SR (6% (95%CI= 0.9 to 11.9)). There were no statistically significantbetween-group differences. As shown inTable 3, BP (systolicBP, diastolic BP, and mean arterial pressure) decreased withthe three types of diuretic treatment. This decrease achievedstatistical significance with SR and with HCT+A. Body weightalso decreased with the three types of diuretics (Table 3),reaching statistical significance with SR and HCT+A. Therewere no significant between-group differences regarding BPand body weight changes.

Other parameters

There were no significant changes in plasma sodium levelswith any of the diuretics, whereas serum potassium levelsexperienced a significant increase with SR and HCT+A(Table 4). Urinary excretions of sodium and potassiumshowed no significant changes (Table 4). Uric acid levelsincreased significantly with all study diuretics, with nobetween-group differences (Table 4). As expected, renin andaldosterone levels showed an increase with all types ofdiuretics, which was significant in all cases except for theincrease in aldosterone levels in patients treated with HCT(Table 4). No significant between-group differences

were found.

Correlations and multivariate analysis

As shown in Figure 2, we found a significant correlationbetween changes in UACR and eGFR when analyzing alltreatment periods (r= 0.50, P= 0.002) and in each of thethree types of diuretics separately. Changes in UACR alsoshowed a significant correlation with BP changes whenanalyzing all treatment periods (Figure 3), although it did notreach statistical significance when the various diuretics wereanalyzed separately. No significant correlation between UACRand weight changes was found.

E Moraleset al.: Antialbuminuric effect of diuretics c l i n i c a l t r i a l

Kidney International (2015) 88 , 14341441 1435


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Adverse effects

Tolerance to the three types of diuretics was good, withouthypotension episodes. As shown inTable 4, serum potassiumshowed a significant increase with SR and HCT+A, althoughno episodes of severe hyperkalemia (serum potassium45.5 mEq/l) were observed. The number of patients showing

a serum potassium of 5 mEq/l or higher was 7 (33%) with SR,2 (9%) with HCT, and 9 (42%) with HCT+A. None of thepatients discontinued the study during the follow-up. Allrandomized patients completed the three treatment periods.

DISCUSSION

Our study is the first head-to-head comparison of the

antialbuminuric effect of three different types of diuretics inpatients who had UACR4 300 mg/g despite maximal dosesof ACEI and an acceptable control of BP. Our data show asignificant reduction in albuminuria with the three types ofdiuretics used: SR, HCT, and HCT+A. Regarding SR, ourstudy confirms its antialbuminuric properties, which havebeen previously demonstrated in both observational studiesand prospective controlled trials.1421 We found a 34%(95% CI= 21 to 47) reduction in UACR baseline valuesafter 4 weeks of treatment with 25 mg/day of SR. This dosehas been the most commonly used in previous studies thatexamined the antialbuminuric effect of SR.1421

But the most important and novel findings of our study

are that HCT, at doses of 50 mg/day, achieved a reductionin UACR (42%; 95% CI= 28 to 56), which wassignificantly higher than that obtained with SR, and that theUACR reduction was even more marked when patients weretreated with the combination HCT+A (56%; 95% CI= 44to 67). Similarly, the percentage of patients who presentedUACR reductions greater than 30 and 50% of baseline valueswas higher with HCT or HCT+A as compared with SR(Table 2), although these differences were not statisticallysignificant. It should be stressed that the percentageof patients who reduced their UACR in more than 50% ofbaseline values was 57% with HCT and 66% with HCT+A.

41 Patients eligible and subsequentlyinvited for participation

12 Declined participation

29 Enrolled in the run-in period

8 Excluded

3 Lack of motivation to adhere to study procedures

3 Albumin:creatinine excretion (mg/g)


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Previous studies had demonstrated an antialbuminuriceffect of HCT in chronic nephropathies.2426 Vogt et al.24

showed in a prospective, randomized, crossover study

conducted in non-diabetic patients with proteinuria thatlosartan, at a dose of 100 mg/day, reduced proteinuria by30%. The addition of a low-salt diet increased theanti-proteinuric effect of losartan up to 55%, and that ofHCT (25 mg/day) up to 56%. When a low-salt diet and HCTwere simultaneously added to losartan, proteinuria reductionsreached 70% of baseline values. Another crossover, prospec-tive, and randomized study showed that the administration of50 mg/day of HCT for 6 weeks had the same effect on residualalbuminuria of diabetic patients treated with maximal ACEIdose than a low-sodium diet (42% reduction for bothmeasures), whereas the simultaneous application of both

measures (low-sodium diet+50 mg/day of HCT) reducedalbuminuria by 61%.26 Our study confirms the strongantialbuminuric effect of HCT on top of maximal ACEI

doses and without changes in salt intake, and is the first todemonstrate that the HCT-induced reduction in albuminuriais greater than that caused by SR at doses of 25 mg/day andthat the addition of amiloride to HCT increases furtheralbuminuria reduction.

The exact mechanisms by which the anti-aldosteronediuretics (SR, eplerenone) induce a significant reduction ofproteinuria/albuminuria have not been clarified.1421 Experi-mental studies have shown that aldosterone infusions causedirect damage to podocytes, which ultimately leads to theappearance of proteinuria and glomerulosclerosis.31,32 SR oreplerenone administration in these same experimental models

Table 2 |Effects of diuretics on albuminuria and proteinuria

Spironol actone Hydrochlorothiazide H ydrochl orothiazide+amiloride

Baseline 4 Weeks Baseline 4 Weeks Baseline 4 weeks

UACR (mg/g) 810 (6011020) 742* (2411244) 1011 (8031218) 566*+ (205927) 1135 (9261344) 398*+ (212584)% UACR reduction 34 (2147) 42 (2856) 56 (4467)

Patients with 430% UACR reduction (%) 12 (57) 17 (81) 17 (81)

Patients with 450% UACR reduction (%) 6 (28) 12 (57) 14 (66)

24-H Albuminuria (mg) 1600 (10472152) 1125.2* (5001750) 1417 (8681965) 935* (2661603) 1882 (13252440) 577* (300855)

24-H Proteinuria (g) 1.7 (1.32.2) 1.5* (0.82.3) 1.7 (1.32.1) 1.3* (0.62) 2.4 (1.92.8) 0.9* (0.61.2)

Abbreviation: UACR, urinary albumin-to-creatinine ratio.

*Po0.05 for intragroup comparison; +Po0.05 for between-group comparison.

Table 3 |Effects of diuretics on renal function, blood pressure, and weight

Spironolactone Hydrochlorothiazide Hydrochlorothiazide+amiloride

Baseline 4 Weeks Baseline 4 Weeks Baseline 4 Weeks

Serum creatinine (mg/dl) 1.17 (0.4) 1.25 (0.4) 1.15 (0.3) 1.26 (0.4)* 1.21 (0.4) 1.35 (0.4)*eGFR ml/min per

1.73 m266 (26) 62 (26) 66 (25) 60 (24)* 64 (24) 55 (20)*

% eGFR reduction 6 (0.911.9) 8.5 (3.813.3) 12 (5.918.1)

SBP (mm Hg) 130 (18) 125 (20)* 129 (18) 124 (19) 128 (20) 121 (15)*DBP (mm Hg) 76 (13) 72 (10)* 75 (12) 71 (7) 74 (12) 70 (8)*

MAP (mm Hg) 87 (5) 85 (5)* 86 (3) 85 (4) 86 (4) 84 (4)*

% MAP reduction 2.1 (0.63.6) 1 (0.62.6) 2.7 (0.84.6)

Weight (kg) 89.1 (15.6) 88.1 (15.6)* 89.1 (16.2) 88.5 (15.2) 88.6 (15.3) 87.3 (15.1)*

% Weight reduction 1.2 (0.22.1) 0.4 (0.51.4) 1.3 (0.52.1)

Abbreviations: DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate; MAP, mean arterial pressure; SBP, systolic blood pressure.

*Po0.05 for intragroup comparisons.

Table 4 |Effects of diuretics on other biochemical parameters

Spironolactone Hydrochlorothiazide Hydrochlorothiazide+amiloride

Baseline 4 Weeks Baseline 4 Weeks Baseline 4 Weeks

Sodium (mEq/l) 141 (2.8) 140 (1.8) 140 (3.4) 140 (2.3) 141 (1.9) 140 (2.7)

Potassium (mEq/l) 4.7 (0.4) 5 (0.6)* 4.6 (0.4) 4.5 (0.4) 4.6 (0.5) 5 (0.6)*

Urinary sodium (mEq/24 h) 184 (152261) 227 (183271) 216 (175256) 240 (146333) 194 (160227) 208 (164253)

Urinary potassium (mEq/24 h) 73 (5987) 79 (6791) 77 (6788) 80 (56105) 80 (6694) 76 (6791)

Uric acid (mg/dl) 6.3 (1.4) 6.8 (1.8)* 6.5 (1.5) 7.3 (1.6)* 6.4 (1.5) 7.6 (1.7)*

Renin (pg/ml) 47 (2866) 82* (37126) 46 (2765) 106* (50163) 34 (1354) 168* (95241)Aldosterone (pg/ml) 150 (110198) 203* (162244) 166 (127205) 182 (142222) 119 (80158) 298* (198398)

*Po0.05 for intragroup comparisons.




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largely prevents aldosterone detrimental effects.31,33 Relativeto amiloride, experimental studies have shown reductions inproteinuria and a regression of glomerular lesions inhypertensive rats29 and improvements in podocyte lesionsand glomerulosclerosis in the 5/6 nephrectomy rat modeltreated with this drug, probably mediated by an inhibition ofurokinase receptor.30 However, to our knowledge, no clinicalstudies have previously evaluated the effect of amiloride onpatients with proteinuria.

Regarding HCT, no specific mechanisms that may explainits antialbuminuric effect have been elucidated. It has beensuggested25,26 that HCT-induced antialbuminuric effect maybe caused by volume depletion, thereby decreasing theintraglomerular hypertension that typically accompaniesCKD. Although no concrete data to support such hypothesishave been reported, the similar antialbuminuric effect of HCTand salt-free diet, as well as the synergistic effect of the twomeasures24,26 support this possibility. If this hypothesis provesto be true, the same mechanism (reduced glomerularintracapillary pressure secondary to diuretic-induced volumedepletion) could be shared by any type of diuretic, including

SR, eplerenone or amiloride, independently of drug-specificantialbuminuric mechanisms. In favor of a possible anti-albuminuric overall effect of diuretics, some studies haveshown that furosemide also potentiates the antialbuminuriceffect of RAAS blockade.27,28

Data from our study are also in agreement with a possibleinfluence of volume depletion in the antialbuminuric effect ofdiuretics. In line with previous studies,2426,34 we found adecrease in BP, eGFR, and body weight with the three types ofdiuretics, a decrease that reached statistical significance withseveral of them (Table 3). As shown inFigures 2and3, bothBP and eGFR decrease showed a statistically significantcorrelation with the reduction in albuminuria. The differenteffects of SR, HCT, and HCT+A on albuminuria coulddepend, therefore, on their potency to reduce plasma volume.In addition, many studies have shown that both BP loweringand weight loss induce significant albuminuria reduction.35,36

However, it is unlikely that BP and eGFR lowering alone canexplain the antialbuminuric effect of diuretics. Mean BP

reduction was 2.1%, 1%, and 2.7% with SR, HCT, andHCT+A, respectively, and eGFR reduction was 6%, 8.5%, and12% in the same periods, whereas UACR reduction achieved34%, 42%, and 56% of the baseline values with SR, HCT, andHCT+A, respectively (Tables 2and3). BP, eGFR, and bodyweight decrease, as well as the significant increase in uric acid,plasma renin, and plasma aldosterone (Table 4), are likelyconsequences of the volume depletion induced by diuretics.Studies are needed to accurately analyze the relationshipbetween hemodynamic changes induced by diuretics andtheir effect on albuminuria and glomerular filtrationrate (GFR).

Our study shows that both SR and HCT or HCT+Acombination are powerful drugs to enhance the antialbumi-nuric effect of RAAS blockade. Given the far-reachinginfluence of the amount of albuminuria on the progressionof renal diseases,4,7,1013 reductions in albuminuria by anytherapeutic measure are usually equated to a renoprotectiveinfluence. However, we believe that the potential renopro-tective effect of diuretics should be evaluated by means ofprospective studies of sufficient duration to establish renaloutcomes. Although diuretic tolerance was good in ourpatients and reduced GFR reversible in all the cases, theefficacy and safety of their long-term administration needs tobe demonstrated. Serum potassium showed a significant

increase with SR and HCT+A, so using these medications forlonger than 4 weeks may exacerbate the risk of serioushyperkalemia. On the other hand, previous studies haveshown that an initial reduction of GFR in patients treatedwith ARB or SR23,37 predicts a subsequent long-termrenoprotective effect, probably reflecting a salutary reversalof glomerular hyperfiltration.

As a large proportion of CKD patients are treated withdiuretics for the treatment of hypertension, volume overload,or heart failure, our data showing that thiazide diuretics andamiloride portend an important antialbuminuric effect, evenhigher than that of SR, are of considerable practical interest

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HCT HCT+A SR

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Figure 2 |Relationship between changes in glomerular filtrationrate (GFR) and changes in urinary albumin-to-creatinine ratio

(UACR).


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for the design of therapeutic strategies for these patients. Forexample, hyperkalemia is a serious potential risk in patientstreated with ACEI, ARB, or antialdosterone diuretics,particularly when kidney function is impaired.1421,38 Accord-ing to our data, HCT could be an attractive therapeuticalternative, owing to its antialbuminuric and kaliuretic effects,in patients with residual albuminuria in whom RAASblockade optimization is hampered by hyperkalemia.Combining HCT and SR is another interesting alternative in

patients prone to develop hyperkalemia.Our study has important limitations. We analyzed short-

term effects of diuretics on intermediate end points, thus noconclusions can be drawn about the sustainability of theantialbuminuric effect or the possible long-term renoprotec-tive effect of these drugs. As in our country there are onlydrugs with amiloride in combination with HCT, we could notevaluate the separate antialbuminuric effect of amiloride. Thesmall sample size prevented from adjustment of additionalfactors that affect the outcome. However, the prospective,randomized, and crossover design of the study, whichincluded washout periods to more properly analyze the

separate effects of each diuretic, reinforces the robustness ofthe data.

In conclusion, the addition of SR, HCT, or HCT+A topatients with UACR4 300 mg/day on top of maximum doseof ACEI, induces a significant antialbuminuric effect,which was even higher with HCT or HCT+A as comparedwith SR, likely associated with a more profound reduction ofGFR in HCT or HCT+A groups. Further prospective studiesare needed to evaluate the long-term renoprotectiveinfluence of these albuminuria-lowering effects of diuretictreatment.

METHODS

This was an open, single-center, randomized crossover study that

compared the antiproteinuric effect of three types of diuretics, SR,

HCT, HCT+A in patients with CKD and a UACR 4300 mg/g

(EudraCT No: 2011-001929-24).

Patients

Candidates were identified at the Nephrology Division of theHospital 12 de Octubre. The study protocol was approved by the

Ethics Committee of Hospital 12 Octubre, and all study patients

read and signed the informed consent document before starting the

study. The included patients met the following criteria: male or

female older than 18 years, chronic diabetic or nondiabetic

nephropathies, UACR 4300 mg/g, stable renal function during

the last 3 months, GFR 430 ml/min per 1.73 m2 and treatment

with ACEIs or ARBs in stable dosages during the last 3 months.

Patients were excluded from the study if they had poorly controlled

BP (systolic BP4160 mm Hg or diastolic BP4100 mm Hg),

a history of cardiovascular events (stroke, ischemic heart disease)

in the past 6 months, or were on treatment with nonsteroidal anti-

inflammatory drugs, corticosteroids, or other immunosuppres-

sants. The other exclusion criteria were a history of renovasculardisease, obstructive uropathy, autoimmune disease, cancer,

pregnancy or currently breastfeeding, and allergies or intolerance

to HCT, SR, or amiloride.

The patients who met the inclusion/exclusion criteria in the

screening underwent a 3-month run-in period (Figure 1) during

which the ACEIs or ARBs they were taking were replaced by

enalapril, the dosage of which was progressively increased until the

maximum dosage of 40 mg/day was reached. This dosage remained

fixed at that point and throughout all trial periods.

Randomization

All participants who by the end of the run-in period continued to

meet the inclusion/exclusion criteria were randomly assigned to oneof the three types of diuretic treatment, in a 1:1:1 ratio. The

randomized assignment list was generated by a computer at the

Clinical Research Unit of Hospital 12 de Octubre. All participants

underwent the three types of diuretic treatment; the sequence of

these treatments was established randomly, with cleansing periods

between each of the three distinct treatments (Figure 1).

Procedures

Treatment periods with SR (25 mg/day), HCT (50 mg/day), and

HCT (50 mg/day)+A (5 mg/day) lasted 4 weeks each and were

separated by washout periods of the same length (Figure 2) to avoid a

carry-over effect. The HCT dose was selected because amiloride is

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Figure 3 | Relationship between changes in mean arterial pressure

(MAP) and changes in urinary albumin-to-creatinine ratio (UACR).




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marketed in combination with the same HCT dosage (50 mg).

Enalapril dosage was kept fixed (40 mg/day) throughout the trial,

and the patients remaining standard medication was also main-

tained without changes. The study drug was administered in the

morning. At the start and end of each of the three different

treatments (SR, HCT, HCT+A), patients body weight, body mass

index, BP, and heart rate were measured. The blood tests performed

at the start and end of each treatment period were as follows: serumcreatinine, glucose, glycated hemoglobin (for patients with diabetes),

sodium, potassium, calcium, chloride, total cholesterol, high-density

lipoprotein-cholesterol, low-density lipoprotein-cholesterol (calcu-

lated according to the Friedewald formula), triglycerides, uric acid,

liver enzymes, plasma renin, and aldosterone. Patients were

instructed to collect 24-h urine during the day before visit.

Albuminuria, proteinuria, sodium, potassium, urea, and creatinine

were measured in this urine sample. A first morning urine sample

was collected to determine UACR.

Body mass index was calculated as weight/height squared. BP was

measured after 5 min of rest with the participant seated, with

automatic equipment (OMRON, Lake Forest, IL), and the mean of

three readings was recorded. Mean arterial pressure was calculated as

the sum of one-third of the systolic BP and two-third of the diastolic

BP. Plasma renin activity and aldosterone concentrations were

measured by radioimmunoassay. The eGFR was measured using the

MDRD-4 formula.

Objectives

The main study objective was the percentage change in the median of

UACR between the baseline and final value of each treatment period.

The secondary objectives were the proportion of patients who

achieved a 430 or 450% reduction in the UACR and the

percentage change in the median of the 24-h albuminuria and

proteinuria in the three different treatment periods. The tertiary

objectives were the percentage change between the baseline valuesand the end values of each treatment period for the following

parameters: eGFR, systolic BP, diastolic BP, mean arterial pressure,

and weight. The tolerance and adverse effects for each treatment

period were recorded.

Statistical analysis

Normally distributed continuous variables are presented as mean

standard deviation or median and interquartil range (P25P75).

Variables were analyzed for a normal distribution with the

KolmogorovSmirnov test. Categorical variables are expressed as

frequencies and percentages. CIs and all tests of statistical

significance for treatment comparisons were evaluated at a

two-tailed significance level of 0.05.

All statistical comparisons of variables between treatment groups

for the primary and secondary objectives with continuous variables

were conducted using a linear mixed-effect model for repeated

measures. In this model, baseline levels of percentage change in the

median of proteinuria between the baseline and final value of each

treatment period were considered as covariance, participants as

random effects, and treatment group, sequence, and treatment-

group-by-sequence (treatment by period interaction to test for carry-

over effects) as fixed effects. Method used to fit such mixed models is

that of expectationmaximization algorithm where the variance

components (intragroup and between group comparison) are treated

as unobserved nuisance parameters in the joint likelihood.

All analyses were conducted in all randomized participants who

were treated with at least one dose of the study drug. Statistical

analyses were performed using SPSS/PC 17 (SPSS Inc. Chicago, IL).

DISCLOSURE

The authors declare no conflict of interest.

ACKNOWLEDGMENTSThis study was supported by grants from Ministerio de Sanidad yPoltica Social (Ministry of Health and Social Policy; 1392-H-199),REDinREN (RD012/0021), FIS (Fondo de Investigaciones Sanitarias)10/02668 and 13/02502 and Asociacin para la Investigacin yTratamiento de la Enfermedad Renal (AITER, Association for theResearch and Treatment of Kidney Disease). We thank Rosa MaraVega Viaa for her cooperation throughout the preparation andmanagement of the clinical trial.

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