A Randomized Trial of Distal Diuretics Versus Dietary Sodium Restriction for Hypertension in Chronic Kidney Disease

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A Randomized Trial of Distal Diuretics versus Dietary Sodium Restriction for Hypertension in Chronic Kidney Disease

Dominique M. Bovée,1 Wesley J. Visser,2 Igor Middel,3 Anneke De Mik–van Egmond,2 Rick Greupink,4 Rosalinde Masereeuw ,3 Frans G. M. Russel,4 A. H. Jan Danser,5 Robert Zietse,1 and Ewout J. Hoorn 1

Due to the number of contributing authors, the afﬁliations are listed at the end of this article.

ABSTRACT Background Distal diuretics are considered less effective than loop diuretics in CKD. However, data to support this perception are limited. Methods To investigate whether distal diuretics are noninferior to dietary sodium restriction in reducing BP in patients with CKD stage G3 or G4 and hypertension, we conducted a 6-week, randomized, open- label crossover trial comparing amiloride/hydrochlorothiazide (5 mg/50 mg daily) with dietary sodium restriction (60 mmol per day). Antihypertension medication was discontinued for a 2-week period before randomization. We analyzed effects on BP, kidney function, and fluid balance and related this to renal clearance of diuretics. Results A total of 26 patients (with a mean eGFR of 39 ml/min per 1.73 m2) completed both treatments. Dietary sodium restriction reduced sodium excretion from 160 to 64 mmol per day. Diuretics produced a greater reduction in 24-hour systolic BP (SBP; from 138 to 124 mm Hg) compared with sodium restriction (from 134 to 129 mm Hg), as well as a significantly greater effect on extracellular water, eGFR, plasma renin, and aldosterone. Both interventions resulted in a similar decrease in body weight and NT-proBNP. Neither approaches decreased albuminuria significantly, whereas diuretics did significantly reduce urinary angiotensinogen and b2-microglobulin excretion. Although lower eGFR and higher plasma indoxyl sulfate correlated with lower diuretic clearance, the diuretic effects on body weight and BP at lower eGFR were maintained. During diuretic treatment, higher PGE2 excretion correlated with lower free water clearance, and four patients developed mild hyponatremia. Conclusions Distal diuretics are noninferior to dietary sodium restriction in reducing BP and extracellular volume in CKD. Diuretic sensitivity in CKD is maintained despite lower diuretic clearance. Clinical Trial registry name and registration number DD-study: Diet or Diuretics for Salt-sensitivity in Chronic Kidney Disease (DD), NCT02875886

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Salt-sensitive hypertension and overhydration are hallmarks of CKD and are associated with adverse Received September 12, 2019. Accepted December 11, 2019. 1–4 outcomes. Dietary sodium restriction effectively Published online ahead of print. Publication date available at lowers BP, extracellular volume, and albuminuria www.jasn.org. in CKD.5–8 However, given the high sodium con- Correspondence: Prof. Ewout J. Hoorn, Department of Internal tent of most food products, long-term adherence to Medicine, Division of Nephrology and Transplantation, Erasmus dietary sodium restriction remains a challenge.9 Medical Center, PO Box 2040, Room Ns403, 3000 CA Rotter- Therefore, a pertinent question is whether other dam, Netherlands. Email: [email protected] approaches to reduce sodium in CKD, such as Copyright © 2020 by the American Society of Nephrology

650 ISSN : 1046-6673/3103-650 JASN 31: 650–662, 2020 www.jasn.org CLINICAL RESEARCH diuretics, are similarly effective. To inhibit sodium reabsorp- Significance Statement tion, diuretics first need to be secreted by the proximal tubule, a process that may be impaired in CKD.10 CKD is characterized by increased extracellular volume and salt- Although “high-ceiling” loop diuretics are commonly used sensitive hypertension, but it is unknown whether dietary or phar- in CKD stages G3–5, “low-ceiling” distal diuretics are consid- macologic approaches are preferable to reduce sodium in CKD, and if distal diuretics are still effective at lower eGFRs. In a ran- 11–13 ered less effective. However, it is uncertain whether these domized crossover trial in patients with CKD stage G3 or G4 and assumptions are justified. Experimental data indicate that the hypertension, the authors compared dietary sodium restriction with pharmacologic targets of thiazide diuretics and amiloride— a combination of distal diuretics (hydrochlorothiazide and ami- the sodium chloride cotransporter (NCC) and the epithelial loride). Both interventions effectively lowered 24-hour BP and ex- sodium channel (ENaC)—are upregulated in CKD.14–16 tracellular volume, with diuretics exerting a stronger effect. Al- though the tubular secretion of diuretics was impaired at a lower n5 – 17–23 Several small case series ( 5 12) and one larger study eGFR, the reductions in body weight and BP effect were main- (n560)24 analyzed the effects of thiazide or thiazide-like tained. These findings indicate that even at lower eGFRs, use of diuretics on BP in patients with CKD stages G3 to G5D. distal diuretics is as effective as dietary sodium restriction in treating The majority of these studies found that the antihyperten- hypertension and volume overload in CKD. sive effect of thiazide diuretics is preserved in CKD, except for three studies that included patients with CKD stage METHODS G5.11,22,23 Two small studies analyzed amiloride in CKD and also observed a preservation of its natriuretic and anti- Participants 25,26 kaliuretic effects. These observations provide a rationale We conducted a single-center, randomized, open-label, for a more systematic investigation of distal diuretics in crossover study (Figure 1). The study was approved by CKD. Several investigators have previously called for the Medical Ethics Committee of the Erasmus Medi- 27–31 such a study. In designing this study, we considered it cal Center (MEC-2015-576) and registered at www. rational to combine diuretics to prevent diuretic resistance clinicaltrials.gov (NCT02875886). The Consolidated Stan- secondary to upregulation of the uninhibited trans- dards of Reporting Trials flow diagram and checklist are porter.32–34 Although the efficacy of combining loop and available in Supplemental Figure 1 and Supplemental thiazide diuretics has been shown previously in CKD,33 the Table 1, respectively. Patients were recruited from the ne- effect of combining inhibitors of NCC and ENaC in CKD has phrology outpatient clinic of the Erasmus Medical Center not been analyzed. Advantages of a combination of distal . 35 between June 2016 and May 2017. Patients aged 18 years diuretics could be to maintain potassium balance and to – 36 oldwithCKDstageG3orG4(eGFR15 59 ml/min per prevent proteinuria-induced activation of ENaC. There- 2 fore, we set out to address the hypothesis that distal diuretics 1.73 m ) with hypertension were eligible for inclusion. Hy- fi 1 are noninferior to dietary sodium restriction in reducing pertension was de ned as ( ) current use of antihypertensive 2 BP in patients with CKD. To do so, we recruited patients drug; or ( ) no use of antihypertensive drugs, but a mean . with CKD stage G3 or G4 and hypertension, discontinued SBP 140 mm Hg after six consecutive measurements with their antihypertensive drugs, and subsequently performed a an oscillometric BP monitor. Exclusion criteria were previ- randomized crossover trial to compare the two sodium- ous intolerance or allergy to thiazide diuretics or amiloride, reducing strategies. In addition to the effects of both inter- pregnancy, the presence of certain diseases (nephrotic syn- ventions on clinical parameters, we also analyzed markers of drome, salt-wasting nephropathy, liver cirrhosis with ascites, fluid balance, the circulating and intrarenal renin- heart failure class III or IV), electrolyte disorders (serum angiotensin system, and renal clearance of diuretics. We sodium ,136 mmol/L, serum potassium ,3.5 or .5.5 demonstrate that distal diuretics are at least as effective as mmol/L), high likelihood of kidney replacement therapy in dietary sodium restriction for the treatment of hypertension ,4 months, and previous kidney transplantation or use of in CKD. immunosuppressive drugs.

2 weeks 2 weeks 2 weeks 2 weeks

Amiloride/HCTZ Amiloride/HCTZ (5/50 mg/day) (5/50 mg/day) V V V V Run-in Washout (off BP drugs) V Na+ restriction Na+ restriction (60 mmol/day) (60 mmol/day) V V V V

1 Figure 1. Overview of the study design. HCTZ, hydrochlorothiazide; Na , sodium; V, visit.

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Study Design quantifies angiotensin I generation in the presence of excess The study started with a 2-week run-in period during which angiotensinogen and recombinant renin, respectively.40,41 all antihypertensive medication was discontinued, except for Plasma and urine aldosterone were measured by RIA (Deme- b-blockers (for cardiac reasons). Patients were provided with a ditec, Kiel, Germany). PGE2 and its metabolite were measured home BP monitor (Omron HBP-1300; Omron Healthcare, using an ELISA (Cayman Chemicals). Plasma and urine Hoofddorp, The Netherlands) and instructed to measure BP hydrochlorothiazide and amiloride concentrations were mea- twice daily. If the SBP was $160 mm Hg during three consec- sured using liquid chromatography–mass spectrometry utive measurements, treatment with amlodipine was started (Waters), as previously described with minor modifications.42 (5 mg once daily with possible uptitration to 10 mg once Renal clearance of hydrochlorothiazide and amiloride was cal- daily). Subsequently, patients were randomly assigned to start culated based on their concentration in 24-hour urine and with sodium restriction (60 mmol/d) or amiloride/hydrochlo- plasma samples which were collected immediately after rothiazide (combination preparation of 5/50 mg once daily). urine collection. Plasma indoxyl sulfate was measured using Allocation to treatment order was done by randomization liquid chromatography–mass spectrometry (Agilent Tech- using sequentially numbered, opaque, sealed envelopes. Treat- nologies, Santa Clara, California), as described.43 During ment periods lasted for 2 weeks and were separated by a the treatments, the following objective side effects were 2-week washout period (Figure 1). All patients received die- monitored: orthostatic hypotension (20 or 10 mm Hg de- tary counseling from a renal dietitian at the start of treatment crease in SBP or diastolic BP within 3 minutes of stand- with sodium restriction. In addition, saltfree bread was pro- ing after 5 minutes of supine rest44), gout, hyponatremia vided for the complete duration of the dietary intervention. (plasma sodium ,136 mmol/L), hypo- and hyperkalemia After 1 week of treatment, the dietitian called patients to (plasma potassium ,3.5 or .5.5 mmol/L), and hyperurice- increase adherence to the diet and provide additional coun- mia (plasma uric acid .7.1 mg/dl). seling, if necessary. Compliance to sodium restriction was monitored with 24-hour urinary sodium excretion and Statistics adherence was defined as .10% reduction. Two patients re- The primary outcome was the change in mean 24-hour SBP peated the 2-week period of dietary sodium restriction. Ad- from baseline. Secondary end points included change in ex- herence to diuretics was evaluated using drug accountability tracellular volume, body weight, albuminuria, and adverse (counting pills) and the measurement of urinary diuretic effects. All end points were analyzed per protocol and inten- concentrations. tion to treat. A power calculation based on previous studies indicated that a minimum of 22 patients was required to Measurements establish noninferiority of diuretics compared with sodium BP, body weight, and body composition were measured and restriction (a50.05; b590%; expected effect of sodium re- blood and urine were collected before and after each interven- striction, 28.7568.5 mm Hg5; expected effect of diuretics, tion. The 90217A Ultralite (Spacelabs Healthcare) with 21068.5 mHg18; correlation coefficient between effects of masked screen was used to perform 24-hour ambulatory BP both treatments, 0.8; variance of difference in treatment effect, measurements. BP was measured at 15-minute intervals dur- 5.38; noninferiority margin, 22 mm Hg). The omnibus K2 ing daytime (16 out of 24 hours) and at 30-minute intervals test was used to screen for normality. Results are presented as during nighttime (8 out of 24 hours). The starting time mean6SD for normally distributed data, and median with of daytime and nighttime measurements was set based on interquartile range for non-normally distributed data. Non- the patient’s sleeping habits. A 24-hour ambulatory BP mea- normally distributed data were log transformed for statistical surement was considered successful when $70% of expected analysis. The Grubbs test was used to detect outliers. One out- measurements were valid (45 valid awake, 11 valid asleep).37 lier in the urinary PGE2 data was not included in the analysis Extracellular water was measured using a bio-impedance because the result suggested the presence of semen in urine, a spectroscopy monitor (Body Composition Monitor; Fresenius known cause of very high urinary PGE2 levels.45 Primary and Medical Care, Bad Homburg, Germany). All urinary measure- secondary outcomes were analyzed by two-way repeated mea- ments were performed in 24-hour urine samples. Compliance sures ANOVAthat also included treatment order as a between- of 24-hour urine collection was determined by creatinine ex- subject factor. A pretest was performed and indicated that the cretion/weight ratio (Supplemental Figure 2).38 Plasma and assumption of negligible carryover effects was met.46 A paired urine electrolytes, albumin, creatinine, and b2-microglobulin t test was performed to analyze if the effects of diuretics or were measured at the Department of Clinical Chemistry at the dietary sodium restriction affected the baseline parameters Erasmus Medical Center. eGFR was calculated using the CKD after washout. The possibility of a period effect was analyzed Epidemiology Collaboration equation.39 eGFR was also recor- and found to be absent.47 Adverse events were analyzed by ded up to 1 year after completion of the study. Plasma renin the McNemar test. Correlations were analyzed on normally was measured using a radioimmunometric assay (Cisbio, distributed or log-transformed data using the Pearson corre- Codolet, France). Urinary renin and angiotensinogen were lation coefficient. If a correlation was present between nor- measured using an in-house enzyme-kinetic assay that mally distributed data and log-transformed data, nonlinear

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Table 1. Baseline characteristics of study participants (n526) RESULTS Characteristic Value Age, yr 61614 Patient Characteristics and Study Men 17 (65) Compliance Diabetes mellitus 5 (19) A total of 1563 patients were assessed for Number of antihypertensive medications 1.861.1 eligibility: 1274 did not meet inclusion Renin-angiotensin system blockers 23 (89) criteria and 262 declined to participate b-Blockers 8 (31) (Supplemental Figure 1). A total of 27 Calcium channel blockers 8 (31) patients entered the study protocol, one Diuretic 7 (27) of which discontinued during the run-in 2 6 Body mass index, kg/m 28.0 4.8 phase (because of study burden). There- 2 6 eGFR (ml/min per 1.73 m )3913 fore, 26 patients finished both treat- Office SBP, mm Hg 140617 ments; their baseline characteristics are Office diastolic BP, mm Hg 88615 Albuminuria, mg/24 h 145 (10, 1050) shown in Table 1. In seven out of 26 pa- . Urine sodium, mmol/24 h 135 (100, 207) tients, SBP increased to 160 mm Hg Data are presented as n (%), mean6SD, or median (interquartile range). during the run-in phase and amlodipine was given until the end of the study protocol (average dose 5.862.0 mg). During regression using a linear-logarithmic model was used to fitthe the treatment phase with diuretics, there was 100% drug ac- original data. Data were analyzed using SPSS Statistics version countability and all patients had detectable plasma and urine 24.0 (IBM) and Graphpad Prism version 7 (GraphPad Soft- diuretic concentrations. The response in urine-electrolyte ex- ware, San Diego, CA). P,0.05 was considered statistically cretion confirmed study compliance to dietary sodium restric- significant. tion in all patients (Figure 2). Urine sodium decreased from

A * B * 400 * NS 15 * *

300 10 (mmol/day) (mmol/day) + 200 2+ 5 100 Urinary Na Urinary Ca 0 0 Before After Before After Before After Before After Na+ restriction Diuretics Na+ restriction Diuretics C

200 NS NS

150

(mmol/day) 100 +

50 Urinary K 0 Before After Before After Na+ restriction Diuretics

Figure 2. Only dietary sodium (Na1) restriction reduced urinary Na1 excretion, while diuretics reduced urinary calcium (Ca21)ex- 1 cretion more than Na1 restriction. Two-way repeated measures ANOVA was used for analysis. (C) Urine potassium (K ) was normally 1 1 distributed, whereas (A) urine Na and (B) calcium (Ca2 )werenot.*P,0.05 for difference before versus after treatment, and for difference between treatments.

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160 to 64 mmol/d during dietary sodium restriction (mean with the 24-hour urine collection (n54) did not change these difference, 295.3 mmol; 95% CI, 67.6 to 123.1; P,0.001), ﬁndings. whereas it remained similar during treatment with amiloride/hydrochlorothiazide (154–153 mmol/24 h; mean differ- Effect on BP and Kidney Function ence, 20.8 mmol/24 h; 95% CI, 26.9 to 28.6; P.0.99). Urine Both treatments reduced mean 24-hour SBP from 134 to calcium decreased signiﬁcantly with both interventions, but 129 mm Hg for sodium restriction (mean difference, more so with amiloride/hydrochlorothiazide. Finally, urine 25 mm Hg; 95% CI, 21to29; P,0.05) and from 138 to potassium did not change during both interventions, indicat- 124 mm Hg for amiloride/hydrochlorothiazide (mean differ- ing stable dietary potassium intake during the study period. A ence, 214 mm Hg; 95% CI, 210 to 218; P,0.001; Figure 3). subanalysis excluding patients who appeared noncompliant The treatment effect of amiloride/hydrochlorothiazide on

A * B * 200 **110 NS *

180 100 90 160 80 140 70 120 60 mean 24h SBP (mmHg) mean 24h DBP (mmHg) 100 50 Before After Before After Before After Before After Na+ restriction Diuretics Na+ restriction Diuretics

C NS NS D * NS NS

) 80 * * 5 2

4 60 3

40 2

1 eGFR (ml/min/1.73m 20 Albuminuria (g/24h) 0 Before After FU Before After FU Before After Before After Na+ restriction Diuretics Na+ restriction Diuretics

E 6.0 NS NS

5.5

5.0 (mmol/L) + 4.5

4.0 Plasma K 3.5 Before After Before After Na+ restriction Diuretics

Figure 3. Sodium (Na1) restriction and diuretics lowered blood pressure and kidney function, but had no effect on albuminuria and plasma potassium (K1). Two-way repeated measures ANOVA was used for analysis. (A) SBP and (B) diastolic BP (DBP), (C) eGFR, and (E) 1 plasma K were normally distributed, whereas (D) albuminuria was not. *P,0.05 for difference before versus after treatment, and for difference between treatments. FU, follow-up.

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24-hour SBP was significantly greater compared with sodium patients that first received sodium restriction (n512) con- restriction (P,0.001). Intention-to-treat analysis similarly firmed that diuretics had a stronger antihypertensive showed that both treatments reduced mean 24-hour SBP effect than sodium restriction (138 to 132 mm Hg for sodium from 134 to 130 mm Hg for sodium restriction (mean differ- restriction, P50.06; 140 to 125 mm Hg for amiloride/ ence, 24 mm Hg; 95% CI, 0 to 29; P,0.05) and 138 to hydrochlorothiazide, P,0.001; P,0.05 for interaction). 124 mm Hg for amiloride/hydrochlorothiazide (mean difference, 214 mm Hg; 95% CI, 210 to 218; P,0.001). The Effect on Fluid Balance and Volume Markers 24-hour diastolic BP also decreased by both treatments, but Both interventions decreased body weight (21.661.1 kg for this change was only significant for the diuretic treatment. SBP sodium restriction, P,0.001; 21.961.5 kg for amiloride/ was reduced by diuretics in all patients and by dietary sodium hydrochlorothiazide, P,0.001) and N-terminal–pro B-type restriction in 19 patients. The effects of both interventions on natriuretic peptide similarly (Figure 5). day and night BP are shown in Supplemental Table 2. Albu- Diuretics had a significantly greater effect on extracellular minuria and plasma renin measured at the start of the first water, plasma renin, and plasma aldosterone compared with treatment period did not correlate with SBP responses to both dietary sodium restriction. Fluid balance and volume markers treatments (data not shown). eGFR and creatinine clearance did not correlate with the 24-hour SBP response to both treat- decreased with both treatments and this effect was signifi- ments (data not shown). cantly greater with diuretics compared with dietary sodium restriction (Figure 3, Supplemental Figure 2). Follow-up Clearance of Distal Diuretics in CKD eGFRs obtained after the study showed that eGFR returned Lower eGFRs were associated with lower diuretic clearance, in to baseline. No significant change in albuminuria was detected a nonlinear manner, indicating reduced tubular secretion of for both treatments. Plasma potassium remained constant diuretics at lower eGFR (Figure 6). To explore this further, with both interventions. No statistically significant carryover plasma indoxyl sulfate concentrations were measured, based effects of both treatments were present. However, a persisting on previous data showing that this uremic toxin competes effect of the diuretics on BP and plasma potassium after wash- with the tubular secretion of diuretics in the proximal tu- out was observed (Figure 4). A sensitivity analysis including bule.10 Indeed, higher plasma indoxyl sulfate concentrations

A B * NS 160 * 200 *

150 180 140 160 130 140 120

24h SBP (mmHg) 120 24h SBP (mmHg) 110 100 100 Before After After Before Na+ After Na+ After diuretics diuretics washout restriction restriction washout

C * D NS NS NS 6.0 6.0 5.5 5.5 5.0 (mmol/L) (mmol/L)

+ 4.5 + 5.0 4.0 4.5 3.5 Plasma K Plasma K 3.0 4.0 Before After After Before Na+ After Na+ After diuretics diuretics washout restriction restriction washout

Figure 4. Diuretics but not sodium (Na1) restriction caused persistent effects on BP and plasma potassium (K1) after washout. The 1 data show that the effect of diuretics (A C) and but not Na restriction (B and D) persists after discontinuation of their use. A paired t test was used for analysis. *P,0.05 for difference before versus after treatment, or before treatment versus after washout.

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A NS B * 140 **30 **

120 25

100 20 80

Body weight (kg) 15

60 Extracellular water (L) 10 Before After Before After Before After Before After Na+ restriction Diuretics Na+ restriction Diuretics

C NS D * ** **160 150 140 120 100 100 80 60 50 40

Plasma renin (ng/L) 20 NT-pro BNP (pmol/L) 0 0 Before After Before After Before After Before After Na+ restriction Diuretics Na+ restriction Diuretics

E * ** 4000

3000

2000

1000

Plasma aldosterone (ng/L) 0 Before After Before After Na+ restriction Diuretics

Figure 5. Both sodium (Na1) restriction and diuretics reduced indices of fluid balance and increased plasma renin and aldosterone. (A–E) All data were normally distributed. Two-way repeated measures ANOVA was used for analysis. *P,0.05 for difference before versus after treatment, and for difference between treatments. NT-pro-BNP, N-terminal–pro B-type natriuretic peptide. were associated with significantly lower clearance of both di- baseline did not predict the BP response to diuretics (data uretics (Figure 6). To analyze whether these pharmacokinetic not shown). effects also had pharmacodynamic consequences, we analyzed the diuretic response on body weight and BP across Thiazide-Induced Hyponatremia the different levels of eGFR. Of note, lower eGFR was as- Diuretic treatment was generally well tolerated with a compa- sociated with a greater reduction in body weight and a sim- rable incidence of adverse effects (Table 2). The only exception ilar reduction in BP. Before diuretic treatment, lower eGFR was mild hyponatremia, which developed in four patients af- correlated with higher N-terminal–pro B-type natriuretic ter diuretic treatment (plasma sodium 13561 mmol/L). Be- peptide (P,0.01, r520.5), suggesting more fluid over- cause thiazide-induced hyponatremia was recently linked to load. In contrast to patients with hypertension and normal PGE2,48 we measured the excretion of PGE2 and its metabolite kidney function,35 plasma renin and albuminuria at (Figure 7). Diuretics but not dietary sodium restriction

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A B 600 P < 0.001 300 P < 0.01 R = 0.65 R = 0.51

400 200 (mL/min) (mL/min) 200 100 HCTZ Amiloride Cl Cl 0 0 0 10203040506070 0 10203040506070 eGFR (mL/min/1.73m2) eGFR (mL/min/1.73m2)

C D 600 P < 0.01 300 P = 0.04 R = -0.54 R = -0.40

400 200 (mL/min) (mL/min) 200 100 HCTZ Amiloride Cl Cl 0 0 0102030 0102030 Plasma indoxyl sulfate (μmol/24h) Plasma indoxyl sulfate (μmol/24h)

E F 0 2 P = 0.019 P = 0.2 1 R = 0.47 -10 0 -1 -20 -2

-30 -3

Δ Body weight (kg) -4 Δ 24h SBP (mmHg) -40 -5 0 10203040506070 0 10203040506070 eGFR (mL/min/1.73m2) eGFR (mL/min/1.73m2)

Figure 6. Lower eGFR reduced diuretic clearance and increased plasma indoxyl sulfate, while maintaining diuretic effects on BP and body weight. (A–F) Clearances were not normally distributed. Pearson correlation coefficient was calculated. D, change in; ClAmiloride, clearance of amiloride; ClHCTZ, clearance of hydrochlorothiazide. increased urine PGE2 excretion. Higher urine PGE2 excretion the change in the fractional excretions of renin. This analysis was associated with lower free water clearance. showed that diuretics selectively reduced the fractional excretion of renin. Effects on Urinary Renin, Angiotensinogen, and b2-microglobulin CKD may activate the intrarenal renin-angiotensin system DISCUSSION with urinary angiotensinogen and renin as potential markers for the activity of this system.49,50 Therefore, we analyzed This is the first study to investigate the effects of the distal whether our interventions changed these parameters. To ac- diuretics hydrochlorothiazide and amiloride in patients with count for changes in the tubular reabsorption of filtered pro- CKD. The effects of distal diuretics on BP and extracellular teins, we also measured b2-microglobulin. Of interest, dietary volume were analyzed in the absence of renin-angiotensin in- sodium restriction selectively increased urinary renin, hibition and compared with dietary sodium restriction as the whereas diuretics selectively decreased urinary angiotensino- active comparator. We showed that distal diuretics are non- gen and b2-microglobulin (Figure 8). To account for the con- inferior to dietary sodium restriction in reducing BP and ex- current changes in plasma renin and eGFR, we also analyzed tracellular volume. In fact, diuretics appear to exert a stronger

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Table 2. Adverse effects acute experiment, Reubi et al.11 showed fi Dietary Sodium Restriction Amiloride/Hydrochlorothiazide that only at very low ltration rates (GFR Side Effect (n526) (n526) ,15 ml/min) was the saluretic effect of fi Orthostatic 4(15) 6(23) chlorothiazide impaired. It is dif cult to hypotension directly compare the BP response in our Gout 0 (0) 1 (4) study to previous studies because we dis- Hyponatremia 0 (0) 4 (15) continued most other antihypertensive Hypokalemia 0 (0) 0 (0) drugs. However, both interventions Hyperkalemia 0 (0) 1 (4) showed a clinically relevant BP response. Hyperuricemia 17 (65) 22 (85) A novel finding is that the antihyper- Data are presented as n (%). No significant differences by the McNemar test. Hyponatremia was tensive effect of distal diuretics is main- defined as plasma sodium ,136 mmol/L, hypo- and hyperkalemia as plasma potassium ,3.5 or .5.5 mmol/L, and hyperuricemia as plasma uric acid .7.1 mg/dl. tained at lower eGFR. Diuretics are secreted by organic anion transporters (OATs) in the proximal tubule.34 Renal antihypertensive effect than dietary sodium restriction, al- clearance of diuretics is reduced in CKD, an observation that though the noninferiority design of our study precludes a de- was also confirmed by our study. Several mechanisms can finitive conclusion. In addition, a longer treatment period contribute to reduced diuretic clearance in CKD, including than 2 weeks may be necessary to obtain the full response to lower nephron number and competition for peritubular up- dietary sodium restriction on BP and total peripheral take through OATs.10 One of the metabolites that can compete resistance.8,51 with diuretics for OAT is the uremic toxin and organic anion The diuretic effects were preserved at lower eGFR despite a indoxyl sulfate.54 We measured plasma indoxyl levels and in- lower clearance of diuretics. Overall, both dietary sodium re- deed found a negative correlation with diuretic clearance. The striction and distal diuretics were well tolerated, except for observation that the BP response to diuretics was independent mild diuretic-induced hyponatremia in four patients. of eGFR may be explained by several mechanisms. First, at Thiazide diuretics are often considered ineffective in CKD, lower eGFR, the reduction in renal diuretic clearance may especially with eGFR ,30 ml/min.52 Several small case series have been leveraged by increased diuretic sensitivity. Second, and pilot studies have challenged this assumption by showing single-nephron diuretic concentrations may have been higher that thiazide diuretics can still lower BP when added to other in patients with lower eGFR because of a lower nephron num- antihypertensive drugs.17–19,21,53 A larger study by Cirillo ber. Third, nonrenal mechanisms such as vasodilation may et al.24 also showed that chlorthalidone effectively reduced have contributed to the antihypertensive effects, although BP, but restricted inclusion to an eGFR between 30 and we did not measure vascular tone. The possibility that thiazide 60 ml/min per 1.73 m2.Ourstudyconfirms that thiazide di- diuretics can cause vasodilation is supported by the demon- uretics in combination with amiloride are still effective in stration of thiazide-induced vasodilation in patients with CKD across a wide eGFR range. Bennett et al.22 did show Gitelman syndrome, who lack functional NCC.55 In one that some residual kidney function is required because thia- study, the vasodilatory effect of thiazide diuretics was observed zide diuretics had no effect in patients on hemodialysis. In an only at high plasma concentrations.56 This could imply that

AB 15000 NS * 1 P = 0.02 R = -0.46 0 10000 -1 (mL/min)

5000 H2O -2 Cl

-3

Urinary PGE2 + M (ng/24h) 0 Before After Before After 0 5000 10000 15000 Na+ restriction Diuretics Urinary PGE2 + M (ng/24h)

Figure 7. Diuretics but not sodium (Na1) restriction increased urinary PGE2 and this reduced free water clearance (ClH2O). Effects of 1 dietary sodium (Na ) restriction and diuretics on the excretion of (A) PGE2 and its metabolite (PGE21M), and (B) the correlation between urinary PGE21M excretion with free water clearance (ClH2O) in patients treated with diuretics. ClH2O was normally distributed, whereas PGE21M was not. Two-way repeated measures ANOVA and Pearson correlation coefﬁcient were used for analysis. One patient had ten- to 100-fold higher PGE2 values and this outlier was excluded from the analysis; we suspect that his urine was con- taminated with semen, which contains high PGE2 levels.45 *P,0.05, difference before versus after treatment.

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A B 80 * NS 5 NS *

4 60 3 40 2

20 FE renin (%) 1 Urinary renin (ng/24h) 0 0 Before After Before After Before After Before After Na+ restriction Diuretics Na+ restriction Diuretics

C D

800 NS * 50 NS *

40 600 30 400 20 200 10 Urinary AGT ( μ g/24h) 0 0 Before After Before After Before After Before After Urinary β 2-microglobulin (mg/24h) Na+ restriction Diuretics Na+ restriction Diuretics

Figure 8. Diuretics but not sodium (Na1) restriction reduced (fractional) renin, angiotensinogen (AGT), and ß2-microglobulin excretion. (A–D) All data were normally distributed. Two-way repeated measures analysis was used for analysis. FE, fractional excretion. *P,0.05, difference before versus after treatment. these vasodilatory mechanisms are more prominent in CKD, renoprotective, this raises the possibility that thiazides may because it raises the plasma concentrations of diuretics. also help preserve eGFR in CKD. Several experimental Whether amiloride can also cause vasodilation is less clear, and clinical studies suggest a possible renoprotective effect although ENaC is expressed in endothelial cells and involved of thiazide diuretics in CKD, especially in combination with in vascular tone.57 Endothelial ENaC can increase vascular renin-angiotensin inhibition.63–69 Clinical trials, however, are stiffness and reduce nitric oxide.58 Therefore, it would be in- lacking. teresting to analyze whether the diuretics increased the nitric In contrast to previous studies, we did not observe a sig- oxide indices. nificant decrease in albuminuria by distal diuretics or sodium Both interventions decreased eGFR, and this has also been a restriction.5,59 However, previous studies usually combined consistent finding in previous studies.5,59 Although this may dietary sodium restriction or diuretics with an inhibitor of be interpreted as progression of CKD, the effect on eGFR was the renin-angiotensin system.8,70–72 reversible and is therefore most likely a hemodynamic effect. Diuretics did decrease the excretion of angiotensinogen This is supported by the observed neurohumoral activation and b2-microglobulin. Because both proteins are reabsorbed and restoration of eGFR after follow-up. Bank et al.17 also in the proximal tubule, this suggests that diuretics increase observed an initial decrease in eGFR with thiazide diuretics proximal tubular reabsorption. This would also be in agree- in CKD, but showed that eGFR subsequently remained con- ment with the hypocalciuric effect of diuretics.73 Of note, uri- stant or rose toward pretreatment levels. Cakalaroski et al.60 nary renin increased after dietary sodium restriction. Some showed that long-term treatment with a thiazide diuretic investigators consider urinary angiotensinogen and renin (3 months) did not change eGFR in patients with CKD. markers for the intrarenal renin-angiotensin system and pos- Longer-term studies powered for hard end points would be tulate local production by the kidney. However, we previously required to analyze if thiazide-induced reduction in BP and showed that an increase in urinary renin reflects increased extracellular volume offset the decrease in eGFR. The initial glomerular filtration or reduced reabsorption rather than con- diuretic-induced eGFR decrease is reminiscent of the effects version of prorenin to renin in the tubular fluid.74 The data of angiotensin-converting enzyme and sodium-glucose trans- in this study also support this conclusion, because dietary port protein 2 inhibitors.61,62 Because these drugs are sodium restriction did not change the fractional excretion of

JASN 31: 650–662, 2020 Diuretic vs. Na+ Restriction in CKD 659 CLINICAL RESEARCH www.jasn.org renin, although this was reduced by thiazide diuretics. Thus, we FUNDING propose that the changes in urinary angiotensinogen and renin represent changes in renal tubular handling rather than changes This study was funded by a Nierstichting (Dutch Kidney Foundation) re- in the activity of the intrarenal renin-angiotensin system. search grant (KSP-14OK19 to Prof. Hoorn). Both interventions were generally well tolerated, but diuretics did cause mild hyponatremia in four patients. Hyponatremia is a well characterized side effect of thiazide diuretics.75 Ware SUPPLEMENTAL MATERIAL et al.48 recently linked thiazide-induced hyponatremia to increased production of PGE2. We also found a negative correla- This article contains the following supplemental material online at tion between urinary PGE2 and free water clearance. A final http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2019090905/-/ observation is that patients who first received the diuretics had DCSupplemental. significantly lower BPs and plasma potassium after the 2-week Supplemental Table 1. CONSORT checklist of information for washout than the patients who first received dietary sodium re- reporting randomized trials. striction. This suggests that the effects of distal diuretics tempo- Supplemental Table 2. Effects of sodium restriction and diuretics rarily persist after their discontinuation. This “legacy” effect may on day and night BPs. be related to the distal tubule remodeling that was described in Supplemental Figure 1. CONSORT diagram. mice lacking NCC phosphorylation.76 Supplemental Figure 2. Creatinine clearances. Our study has a number of limitations. First, we used a combination treatment and therefore it is not clear if both diuretics REFERENCES equally contributed to the observed effects. Second, we excluded patients with CKD stage G5 and, therefore, we were not able to study the possibility that distal diuretics become ineffective at a 1. Faucon AL, Flamant M, Metzger M, Boffa JJ, Haymann JP, Houillier P, et al.; NephroTest Study Group: Extracellular fluid volume is associated certain level of eGFR. A sensitivity analysis did show that patients with incident end-stage kidney disease and mortality in patients with , 2 with an eGFR 30 ml/min per 1.73 m had a similar BP re- chronic kidney disease. Kidney Int 96: 1020–1029, 2019 sponse that occurred independently of eGFR. Finally, we did not 2. Hung SC, Kuo KL, Peng CH, Wu CH, Lien YC, Wang YC, et al.: Volume specifically select patients based on salt sensitivity or an ex- overload correlates with cardiovascular risk factors in patients with – panded extracellular volume. However, sodium retention is a chronic kidney disease. Kidney Int 85: 703 709, 2014 1,3,4 3. 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McMahon EJ, Campbell KL, Bauer JD, Mudge DW: Altered dietary salt intake for people with chronic kidney disease. Cochrane Database Syst Rev 2: CD010070, 2015 ACKNOWLEDGMENTS 6. Saran R, Padilla RL, Gillespie BW, Heung M, Hummel SL, Derebail VK, et al.: A randomized crossover trial of dietary sodium restriction in stage 3-4 CKD. Clin J Am Soc Nephrol 12: 399–407, 2017 We thank all patients who participated in this trial, our colleagues 7. Campbell KL, Johnson DW, Bauer JD, Hawley CM, Isbel NM, Stowasser who helped with the inclusion of patients, and the research nurse who M, et al.: A randomized trial of sodium-restriction on kidney function, fl helped with data collection (Ms. Brigitte Nome). uid volume and adipokines in CKD patients. BMC Nephrol 15: 57, 2014 Research idea and study design: Prof. Hoorn. Generation random 8. McMahon EJ, Bauer JD, Hawley CM, Isbel NM, Stowasser M, Johnson allocation sequence, enrolling patients, and assigning patients to DW, et al.: A randomized trial of dietary sodium restriction in CKD. JAm interventions: Dr. Bovée. Data collection: Dr. Bovée, Mr. Visser, Mrs. Soc Nephrol 24: 2096–2103, 2013 De Mik–van Egmond, Dr. Greupink, Dr. Middel. Data analysis/in- 9. Henney JE, O’Hara JA 3rd., Taylor CL: Sodium-intake reduction and the – terpretation: Dr. Bovée, Prof. Hoorn. Statistical analysis: Dr. Bovée, food industry. NEnglJMed381: 201 203, 2019 10. Nigam SK, Wu W, Bush KT, Hoenig MP, Blantz RC, Bhatnagar V: Prof. Hoorn. Supervision: Prof. Hoorn. Each author contributed Handling of drugs, metabolites, and uremic toxins by kidney proximal important intellectual content during the writing and revision of the tubule drug transporters. Clin J Am Soc Nephrol 10: 2039–2049, 2015 manuscript. 11. Reubi FC, Cottier PT: Effects of reduced glomerular filtration rate on responsiveness to chlorothiazide and mercurial diuretics. Circulation 23: 200–210, 1961 DISCLOSURES 12. Izzo JL Jr., Tobe SW: Is there a preferred diuretic class for patients with renal impairment and hypertension? JAmSocHypertens10: 282–284, 2016 13. Schreiner GE: Chlorothiazide in renal disease. Ann N Y Acad Sci 71: None. 420–429, 1958

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Lenders M, Hofschröer V, Schmitz B, Kasprzak B, Rohlmann A, Missler teinuria in Patients with Chronic Kidney Disease and Hypertension M, et al.: Differential response to endothelial epithelial sodium channel (ILOHA) Study Investigators: Impact of combined losartan/hydrochlo- inhibition ex vivo correlates with arterial stiffness in humans. rothiazide on proteinuria in patients with chronic kidney disease and JHypertens33: 2455–2462, 2015 hypertension. Hypertens Res 37: 993–998, 2014 59. Morales E, Caro J, Gutierrez E, Sevillano A, Auñón P, Fernandez C, 69. Hoshino T, Ookawara S, Miyazawa H, Ito K, Ueda Y, Kaku Y, et al.: et al.: Diverse diuretics regimens differentially enhance the anti- Renoprotective effects of thiazides combined with loop diuretics in albuminuric effect of renin-angiotensin blockers in patients with chronic patients with type 2 diabetic kidney disease. Clin Exp Nephrol 19: kidney disease. Kidney Int 88: 1434–1441, 2015 247–253, 2015 60. 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AFFILIATIONS

1Divisions of Nephrology and Transplantation, 2Dietetics, and 5Vascular Medicine and Pharmacology, Department of Internal Medicine, Erasmus Medical Center, University Medical Center Rotterdam, Rotterdam, The Netherlands; 3Division of Pharmacology, Department of Pharmaceutical Sciences, University Utrecht, Utrecht, The Netherlands; and 4Department of Pharmacology and Toxicology, Radboud University Medical Center, Nijmegen, The Netherlands

662 JASN JASN 31: 650–662, 2020 1

Supplemental Material

A Randomized Trial of Distal Diuretics versus Dietary Sodium Restriction

for Hypertension in Chronic Kidney Disease

Dominique M. Bovée, Wesley J. Visser, Igor Middel, Anneke De Mik – van Egmond,

Rick Greupink, Rosalinde Masereeuw, Frans G.M. Russel,

A.H. Jan Danser, Robert Zietse, Ewout J. Hoorn

Table of Contents:

• Supplemental Methods

• Supplemental Table 1: Consolidated Standards of Reporting Trials (CONSORT) 2010

checklist of information.

• Supplemental Table 2: Effect of sodium restriction and diuretics on day and night

systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure

(MAP).

• Supplemental Figure 1: Consolidated Standards of Reporting Trials (CONSORT)

diagram.

• Supplemental Figure 2: Effect of sodium (Na+) restriction and diuretics on creatinine

clearance.

Supplemental Methods

Urinary aldosterone measurements were performed using a commercial assay (Demeditec,

Germany). The detection limit and mean coefficient of variation of the within-run and between-run variability of this assay are 1.44 pg/mL, 3.3 % and 8.4 %, respectively.

Moreover, the antibody used in the immunoassay is highly specific for aldosterone. Extremely low cross-reactivities were obtained against other naturally occurring steroids (cortisone, corticosterone, DHEAs, etc.). Urinary renin and angiotensinogen measurements were performed using an in-house developed enzyme-kinetic assay. The detection limit and mean coefficient of variation of the within-run and between-run variability of this assay are 2 pg/mL, 2.9% and 12.6% for renin measurements and 0.12 ng/mL, 4% and 10% for angiotensinogen measurements. Please note that in every enzyme-kinetic assay, we measure a set of standard samples allowing us to determine the within- and between-run variability.

Furthermore, we always include buffer samples to correct for background noise. As an example, when measuring renin in the presence of excess angiotensinogen, the same amount of excess angiotensinogen is incubated with buffer, and the generated amount of angiotensin I is subtracted from the amount of angiotensin I generated in the actual sample. This needs to be taken into account, particularly when measuring samples with low renin levels like urine.1

In previous studies we have added fixed amounts of (pro)renin and angiotensinogen to urine samples, and found recovery to be >95%.2, 3 This is not surprising, since the levels of degrading enzymes in urine are negligible. Finally, regarding cross-reactivity, it is important to note that the assays rely on the detection of angiotensin I making use of an angiotensin I- directed antibody. Cross-reactivity of this antibody with other angiotensin metabolites is

<0.1%.4 In this regard, the correction for background noise as explained above seems to be of greater importance, since the assay is performed in the presence of angiotensinase inhibitors, not allowing the generation of metabolites other than angiotensin I. 3

Supplemental Table 1: Consolidated Standards of Reporting Trials (CONSORT) 2010

checklist of information.

Reported Item on page Section/Topic No Checklist item No Title and abstract 1a Identification as a randomized trial in the title 1 1b Structured summary of trial design, methods, results, and conclusions (for specific guidance see CONSORT for abstracts) 3 Introduction Background and 2a Scientific background and explanation of rationale 4-5 objectives 2b Specific objectives or hypotheses 5 Methods Trial design 3a Description of trial design (such as parallel, factorial) including allocation ratio 6-7 3b Important changes to methods after trial commencement (such as eligibility criteria), with reasons NA Participants 4a Eligibility criteria for participants 6 4b Settings and locations where the data were collected 6 Interventions 5 The interventions for each group with sufficient details to allow replication, including how and when they were actually administered 6-7 Outcomes 6a Completely defined pre-specified primary and secondary outcome measures, including how and when they were assessed 7-8 6b Any changes to trial outcomes after the trial commenced, with reasons NA Sample size 7a How sample size was determined 8 7b When applicable, explanation of any interim analyses and stopping guidelines NA Randomization: Sequence 8a Method used to generate the random allocation sequence 7 generation 8b Type of randomization; details of any restriction (such as blocking and block size) 7 Allocation 9 Mechanism used to implement the random allocation sequence (such as concealment sequentially numbered containers), describing any steps taken to conceal mechanism the sequence until interventions were assigned 7 Implementation 10 Who generated the random allocation sequence, who enrolled participants, and who assigned participants to interventions 18 Blinding 11a If done, who was blinded after assignment to interventions (for example, participants, care providers, those assessing outcomes) and how NA 11b If relevant, description of the similarity of interventions 4 Statistical methods 12a Statistical methods used to compare groups for primary and secondary outcomes 8-9 12b Methods for additional analyses, such as subgroup analyses and adjusted analyses 8-9 Results 4

Participant flow (a 13a For each group, the numbers of participants who were randomly assigned, diagram is strongly received intended treatment, and were analyzed for the primary outcome 10 recommended) 13b For each group, losses and exclusions after randomization, together with reasons 10 Recruitment 14a Dates defining the periods of recruitment and follow-up 6 14b Why the trial ended or was stopped NA Baseline data 15 A table showing baseline demographic and clinical characteristics for each group 19 Numbers analyzed 16 For each group, number of participants (denominator) included in each analysis and whether the analysis was by original assigned groups 8 & 10 Outcomes and 17a For each primary and secondary outcome, results for each group, and the estimation estimated effect size and its precision (such as 95% confidence interval) 10-12 17b For binary outcomes, presentation of both absolute and relative effect sizes is recommended - Ancillary analyses 18 Results of any other analyses performed, including subgroup analyses and adjusted analyses, distinguishing pre-specified from exploratory 10-12 Harms 19 All important harms or unintended effects in each group (for specific guidance see CONSORT for harms) 20 Discussion Limitations 20 Trial limitations, addressing sources of potential bias, imprecision, and, if relevant, multiplicity of analyses 16 Generalizability 21 Generalizability (external validity, applicability) of the trial findings 13-17 Interpretation 22 Interpretation consistent with results, balancing benefits and harms, and considering other relevant evidence 13-17

Other information Registration 23 Registration number and name of trial registry 6 Protocol 24 Where the full trial protocol can be accessed, if available NA Funding 25 Sources of funding and other support (such as supply of drugs), role of 18 funders

Supplemental Table 2: Effect of sodium restriction and diuretics on day and night systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP).

Na+ P for Distal P for P for

restriction treatment diuretics treatment interaction

effect effect

Day SBP (mmHg) –6 ± 2 P < 0.01 –14 ± 2 P < 0.01 P < 0.01

Day DBP (mmHg) –2 ± 1 P = 0.2 –5 ± 1 P < 0.01 P = 0.09

Day MAP (mmHg) –4 ± 1 P < 0.05 –8 ± 1 P < 0.01 P < 0.05

Night SBP (mmHg) –3 ± 2 P = 0.4 –14 ± 2 P < 0.01 P < 0.01

Night DBP (mmHg) 0 ± 2 P = 1 –5 ± 2 P < 0.01 P < 0.05

Night MAP (mmHg) –1 ± 2 P = 0.8 –8 ± 2 P < 0.01 P < 0.01

SBP dipping (%) +2.1 ± 3.4 P = 1 +1.4 ± 3.4 P = 1 P = 1

Treatment effect and treatment interaction were analyzed by two-way repeated measures

ANOVA that included treatment order as between-subject factor.

DBP, diastolic blood pressure; MAP, mean arterial pressure; Na+, sodium; SBP, systolic blood pressure.

Supplemental Figure 1: Consolidated Standards of Reporting Trials (CONSORT) diagram.

Legend: Treatment order 1: first treatment period diuretics, second treatment period dietary sodium restriction. Treatment order 2: first treatment period sodium restriction, second treatment period diuretics. 7

Supplemental Figure 2: Effect of sodium (Na+) restriction and diuretics on creatinine clearance.

Legend: Data are normally distributed and two-way repeated measures ANOVA was used for analysis. * P < 0.05 for difference before versus after treatment.

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