Indomethacin, Amiloride, Or Eplerenone for Treating Hypokalemia in Gitelman Syndrome

CLINICAL RESEARCH www.jasn.org

Indomethacin, Amiloride, or Eplerenone for Treating Hypokalemia in Gitelman Syndrome

†‡ | †† Anne Blanchard,* Rosa Vargas-Poussou,§ Marion Vallet,¶ Aurore Caumont-Prim,** ‡‡ || † Julien Allard, Estelle Desport,§§ Laurence Dubourg, Matthieu Monge, ‡ ‡‡ Damien Bergerot, Stéphanie Baron,*¶¶ Marie Essig, Frank Bridoux,§§ Ivan Tack,¶ and †‡ Michel Azizi*

*Université Paris Descartes, Faculté de Médecine, Sorbonne Paris Cité, Paris, France; †Assistance Publique Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Centre d’Investigation Clinique, Paris, France; ‡Institut National de la Santé et de la Recherche Médicale, Centre d’Investigation Clinique 1418, Paris, France; §Département de génétique and ¶¶Service d’explorations fonctionnelles, Hôpital Européen Georges Pompidou, Paris, France; |Institut National de la Santé et de la Recherche Médicale, UMR970, Paris-Cardiovascular Research Center, Paris, France; ¶Service des explorations fonctionnelles physiologiques, Hôpital de Rangueil, Toulouse, France; **Assistance Publique–Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Unité d’Épidémiologie et de Recherche Clinique, Paris, France; ††Institut National de la Santé et de la Recherche Médicale, Centre d’Investigation Épidémiologique 4, Paris, France; ‡‡Centre Hospitalier Universitaire Dupuytren, Service de néphrologie, Centre d’Investigation Clinique Centre d’Investigation Clinique Institut National de la Santé et de la Recherche Médicale 0801, Limoges, France; §§Centre Hospitalier Universitaire de Poitiers, Service de néphrologie, Centre d’Investigation Clinique Centre d’Investigation Clinique Institut National de la Santé et de la Recherche Médicale 1402, Université de Poitiers, Poitiers, France; and ||Hôpital Edouard Herriot, Lyon, Paris, Hospices civils de Lyon, Lyon, France

ABSTRACT Patients with Gitelman syndrome (GS), an inherited salt-losing tubulopathy, are usually treated with potassium-sparing diuretics or nonsteroidal anti-inflammatory drugs and oral potassium and magnesium supplementations. However, evidence supporting these treatment options is limited to case series studies. We designed an open-label, randomized, crossover study with blind end point evaluation to compare the efficacy and safety of 6-week treatments with one time daily 75 mg slow-release indomethacin, 150 mg eplerenone, or 20 mg amiloride added to constant potassium and magnesium supplementation in 30 patients with GS (individual participation: 48 weeks). Baseline plasma potassium concentration was 2.860.4 mmol/L and increased by 0.38 mmol/L (95% confidence interval [95% CI], 0.23 to 0.53; P,0.001) with indomethacin, 0.15 mmol/L (95% CI, 0.02 to 0.29; P=0.03) with eplerenone, and 0.19 mmol/L (95% CI, 0.05 to 0.33; P,0.01) with amiloride. Fifteen patients became normokalemic: six with indomethacin, three with eplerenone, and six with amiloride. Indomethacin significantly reduced eGFR and plasma renin concentration. Eplerenone and amiloride each increased plasma aldosterone by 3-fold and renin concentration slightly but did not significantly change eGFR. BP did not significantly change. Eight patients discontinued treatment early because of gastrointestinal intolerance to indomethacin (six patients) and hypotension with eplerenone (two patients). In conclusion, each drug increases plasma potassium concentration in patients with GS. Indomethacin was the most effective but can cause gastrointestinal intolerance and decreased eGFR. Amiloride and eplerenone have similar but lower efficacies and increase sodium depletion. The benefit/risk ratio of each drug should be carefully evaluated for each patient.

J Am Soc Nephrol 26: 468–475, 2015. doi: 10.1681/ASN.2014030293

Published online ahead of print. Publication date available at Leblanc, 75015 Paris, France. Email: anne.blanchard@egp. www.jasn.org. aphp.fr Correspondence: Dr. Anne Blanchard, Centre d’Investigations Cliniques, Hôpital Européen Georges Pompidou, 20-40 rue Copyright © 2015 by the American Society of Nephrology

468 ISSN : 1046-6673/2602-468 J Am Soc Nephrol 26: 468–475, 2015 www.jasn.org CLINICAL RESEARCH

Gitelman syndrome (GS; Mendelian Inheritance in Man tolerability of slow-release indomethacin, amiloride, and 263800) is a rare inherited autosomal recessive salt-losing eplerenone in patients with type I GS treated with stable tubulopathycharacterized byhypokalemic metabolic alkalosis, potassium and magnesium supplementation. hypomagnesemia, hypocalciuria, and secondary hyperaldosteronism.1 GS has a prevalence of heterozygotes of approximately 1% and an estimated prevalence of homozygotes of RESULTS approximately 1 in 40,000 in Caucasians.2 It is mainly related to loss-of-function mutations in the SLC12A3 gene encoding Baseline characteristics of 30 patients (17 women) randomized for the apically expressed thiazide-sensitive NaCl cotrans- in the study are shown in Table 1; all had typical GS with low porter (NCC) of the distal convoluted tubule (DCT).2 BP, hypokalemia, and hypomagnesemia with inappropriate The natural history of GS is highly variable in terms of age at urine potassium and magnesium excretion, low urine calcium clinical diagnosis, biologic phenotype, and severity of clinical excretion, high plasma renin, and normal plasma aldosterone manifestations. However, severe hypokalemic rhabdomyolysis concentrations. Supplemental Figure 1 shows the reasons for or ventricular arrhythmias may occur when acute additional exclusion or study drug discontinuation, the number of pa- extrarenal potassium and magnesium losses occur (vomiting tients randomized to each treatment sequence, and the num- or diarrhea).3–7 ber completing the study. Lifelong oral potassium and magnesium supplementation is the cornerstone of treatment aiming at relieving symptoms and Tolerability preventing severe hypokalemia-related complications, including Indomethacin caused GI intolerance (heartburn, dyspepsia, or cardiac arrhythmias.8 However, oral supplementation may be in- gastric irritation) in six patients, resulting in early discontin- sufficient to fully correct the electrolyte disorders or may have uation. Eplerenone (150 mg once a day [o.d.].) was discon- poor gastrointestinal (GI) tolerability, which can limit dos- tinued in two other patients for palpitations and dizziness ing.9,10 In the case of persistent symptomatic hypokalemia or (Supplemental Figure 1). The 20-mg o.d. dose of amiloride intolerance to supplementation, potassium-sparing diuretics, was downtitrated to 10 mg o.d. in two patients and 15 mg o.d. such as amiloride, or mineralocorticoid receptor (MR) antago- in another two patients for palpitations and dizziness. Thus, nists (spironolactone or eplerenone) usually constitute the first 22 of 30 patients actually received all three study drugs. Their therapeutic option.11–14 Another option is low-dose indometh- baseline characteristics were similar to those of the random- acin, a nonselective inhibitor of cyclooxygenase 1 (COX-1) and ized population of 30 patients (Table 1). COX-2.11,15 However, evidence is limited for all these treatment options, and no randomized controlled trial has so far compared Effects of Indomethacin, Eplerenone, and Amiloride on their efficacy, tolerability, or safety in patients with GS. Electrolytic and Hormonal Parameters and GFR We designed an open-label, randomized, crossover study to All parameters returned to their respective baseline and pre- compare the efficacy on plasma potassium concentration and treatment levels after the 6-week washout periods (Supplemental

Table 1. Baseline characteristics of 30 randomized patients and the subpopulation of 22 patients who received all study drugs Randomized Patients Patients Who Received All Study Drugs P Value n 30 22 Age, yr 39.9613.5 40.6612.4 0.79 Body weight, kg 66.8613.2 68.6613.2 0.67 Body mass index, kg/m2 24.364.1 24.464.1 0.86 Systolic BP, mmHg 11369113690.67 Heart rate, beats/min 67610 66610 0.92 Plasma potassium, mmol/L 2.860.4 2.860.5 .0.99 Plasma magnesium, mmol/L 0.5360.08 0.5260.07 0.84 Plasma sodium, mmol/L 140.061.2 139.961.2 0.91 Plasma creatinine, mmol/L 57.7614.6 63.6617.0 0.80 eGFR, ml/min per 1.73 m2 124633 123632 0.96 Plasma renin concentration, mU/L 89 (60 to 155) 98 (49 to 162) 0.99 Plasma aldosterone, pg/ml 44 (28 to 87) 51 (28 to 87) 0.88 Urine sodium excretion, mmol/24 h 186 [148–214] 177 [148–211] 0.68 Urine potassium excretion, mmol/24 h 108 [91–142] 108 [91–134] 0.86 Urine magnesium excretion, mmol/24 h 2.0 [0.9–3.3] 2.8 [1.0–3.8] 0.59 Urine calcium excretion, mmol/24 h 1.10 [0.49–1.85] 1.21 [0.75–1.85] 0.51 Data are mean6SD, geometric mean (95% CI), or median [interquartile range]. P values were by unpaired t test. Normal values for plasma renin concentration are 20.8 mU/L (95% CI, 17.7 to 24.4) in men and 16.2 mU/L (95% CI, 13.6 to 19.2) in women. Normal values for plasma aldosterone concentration are 45 pg/ml (95% CI, 39 to 52) in men and 34 pg/ml (95% CI, 28 to 40) in women. mU/L, milli-international units per liter.

J Am Soc Nephrol 26: 468–475, 2015 Crossover Trial in Gitelman Syndrome 469 CLINICAL RESEARCH www.jasn.org

Table 1). Therefore, only treatment effects are reported for the P=0.03 versus baseline), and 3 of 28 (11%) patients normalized on-treatment population who actually received all three study their plasma potassium concentration (Figure 1). Eplerenone drugs (n=22 of 30). Treatment effects analyzed by an ANOVA did not change plasma magnesium concentration and slightly for a crossover design were significant for the following param- decreased plasma sodium concentration (n=22) (Table 2). It eters: plasma potassium, creatinine, aldosterone and renin con- markedly increased plasma aldosterone concentration com- centrations, and urine magnesium excretion. Two-by-two pared with baseline (n=22; P,0.001) (Table 2), but the increase comparisons are shown below. in plasma renin concentration was not significant (n=22) (Table 2). Indomethacin significantly increased plasma potassium by eGFR and urine potassium, magnesium, and sodium excretion 0.34 mmol/L (95% confidence interval [95% CI], 0.20 to 0.48 did not change significantly (Table 2). mmol/L) compared with the control period (n=22, P,0.001) Amiloride increased plasma potassium by 0.18 mmol/L (Table2)butdidnotsignificantly change plasma magnesium (95% CI, 0.04 to 0.32 mmol/L) compared with the control or sodium concentration (Table 2). When using the whole period (n=22; P=0.09) (Table 2). When using the whole data- dataset for the indomethacin period (n=24), the net potas- set for the amiloride period (n=30), the net potassium increase sium increase from baseline was 0.38 mmol/L (95% CI, 0.23 was 0.19 mmol/L (95% CI, 0.05 to 0.33; P,0.01 versus base- to 0.53; P,0.001 versus baseline), and 6 of 24 (25%) patients line), and 6 of 30 (20%) patients normalized their plasma normalized their plasma potassium concentration ($3.5 potassium concentration (Figure 1). Amiloride increased mmol/L) (Figure 1). As expected, indomethacin markedly re- plasma magnesium concentration and decreased plasma so- duced plasma renin concentration (n=22; P,0.001) (Table 2) dium concentration slightly (n=22) (Table 2). As expected, it but did not change plasma aldosterone concentration (n=22; markedly increased plasma aldosterone concentrations com- P.0.99) (Table 2). Compared with the control period, eGFR pared with the control period (n=22; P,0.001) (Table 2), but significantly decreased by 10.0 ml/min per 1.73 m2 (95% CI, the increase in plasma renin concentration was not significant 4.2 to 15.9 ml/min per 1.73 m2; n=22; P,0.01) (Table 2) after (Table 2). Compared with the control period, eGFR slightly the 6-week treatment with indomethacin. Urine potassium, decreased by 6.4 ml/min per 1.73 m2 (95% CI, 20.54 to 12.20 magnesium, and sodium excretion did not significantly ml/min per 1.73 m2; P=0.20) (Table 2) after the 6-week treat- change (Table 2). ment with amiloride. Urine potassium and sodium excretion Eplerenone increased plasma potassium by 0.13 mmol/L did not change significantly, but magnesium excretion signif- (95% CI, 20.01 to 0.27 mmol/L) compared with the control icantly decreased (n=22) (Table 2). period (n=22; P=0.41) (Table 2). When using the whole data- The increase in plasma potassium concentration was set for the eplerenone period (n=28), the net potassium in- significantly greater after the 6-week treatment with indo- crease from baseline was 0.15 mmol/L (95% CI, 0.02 to 0.29; methacin than eplerenone (increase of 0.21 mmol/L; 95% CI,

Table 2. Effects of 6-week treatments with 75 mg o.d. slow-release indomethacin, 150 mg o.d. eplerenone, or 20 mg o.d. amiloride in 22 patients with genetically proven GS type I who received all three treatments Control Period Indomethacin Eplerenone Amiloride ANOVA Parameters (Week 6) (Week 6) (Week 6) (Week 6) (P Value) Body weight, kg 69.3613.1 70.3613.4a 68.5612.6b 68.7613.1b ,0.001 Systolic BP, mmHg 11569 116611 115611 114610 0.24 Heart rate, beats/min 71.8610.1 73.567.6 75.868.0 75.669.7 0.07 Plasma potassium, mmol/L 2.860.4 3.260.4c 3.060.5d 3.060.5 ,0.01 Plasma magnesium, mmol/L 0.5460.09 0.5660.07 0.5660.13 0.5860.08 0.59 Plasma sodium, mmol/L 140.061.6 139.161.4 138.661.5 138.561.6 0.17 Plasma creatinine, mmol/L 61.7615.1 66.6617.6e 63.1615.6 64.8616.4 0.03 eGFR, ml/min per 1.73 m2 126628 115625e 122624 119628 0.05 Plasma renin concentration, mU/L 90 (61 to 133) 47 (32 to 69)c 113 (76 to 167)b 112 (76 to 166)b ,0.001 Plasma aldosterone, pg/ml 45 (33 to 62) 42 (30 to 59) 131 (94 to 183)b,c 155 (107 to 124)b,c ,0.001 Urine sodium excretion, mmol/24 h 187 [137–231] 187 [127–207] 186 [143–273] 193 [136–255] 0.50 Urine potassium excretion, mmol/24 h 107 [93–141] 123 [89–134] 104 [90–129] 104 [94–137] 0.95 Urine magnesium excretion, mmol/24 h 2.1 [1.0–3.3] 3.1 [1.4–3.7] 3.0 [1.5–4.6] 1.7 [0.8–3.3]a,d 0.02 Data are mean6SD, geometric mean (95% CI), or median [interquartile range]. ANOVA is P value for the treatment effect in ANOVA for a crossover study. Normal values for plasma renin concentration are 20.8 mU/L (95% CI, 17.7 to 24.4) in men and 16.2 mU/L (95% CI, 13.6 to 19.2) in women. Normal values for plasma aldosterone concentration are 45 pg/ml (95% CI, 39 to 52) in men and 34 pg/ml (95% CI, 28 to 40) in women. mU/L, milli-international units per liter. aP,0.05 versus control period. bP,0.001 versus indomethacin. cP,0.001 versus control period. dP,0.05 versus indomethacin. eP,0.01 versus control period.

470 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 468–475, 2015 www.jasn.org CLINICAL RESEARCH

DISCUSSION

The results of this crossover trial showed that the moderate-to- severe hypokalemia of patients with genetically proven GS was corrected in the short term but only partially by 75 mg/d slow- releaseindomethacin, 20mg/damiloride, or150mg/deplerenone given for 6 weeks combined with a constant oral potassium and magnesium supplementation. At the selected doses, indomethacin was more effective in increasing mean plasma potassium concentration than amiloride or eplerenone, which were of similar efficacy. The effects of the treatments on plasma magnesium concentration were either negligible (amiloride) or ab- sent(indomethacinandeplerenone).Asexpected,indomethacin and the two diuretics had opposite effects on plasma renin and Figure 1. Effect of 6-week treatments with 75 mg o.d. slow-release indomethacin, 150 mg o.d. eplerenone, or 20 mg o.d. amiloride on aldosterone concentrations as well as the slight treatment- fi plasma potassium concentration in patients with genetically proven induced BP changes. eGFR signi cantly decreased with in- GStypeI.Alldatasetsofvalidperiodsoftreatmentsforindomethacin domethacin and less so with amiloride but did not change with (n=24), eplerenone (n=28), and amiloride (n=30) are used to connect eplerenone. Clinical tolerability of the drugs was inversely re- plasma potassium values under treatment with corresponding pre- lated to their efficacy, with indomethacin being the least tol- treatment values. *p, 0.05; **p,0.01; ***p,0.001. erated because of GI intolerance leading to early discontinuation in 6 of 30 (20%) patients, despite coprescription of a proton pump inhibitor. The crossover nature of the study design with rotation of the 0.07 to 0.35 mmol/L; P=0.03; n=22) but not amiloride three drugsshowedthat 40% of patients whowereintermediate (P=0.15; n=22) (Figure 1, Table 2). responders, nonresponders, or patients intolerant to indo- There was no significant difference between amiloride and methacin became full responders by switching to amiloride or eplerenone (P.0.99) (Figure 1, Table 2). eplerenone. Thus, 73% of patients who tolerated the three There was no correlation between the changes in plasma treatments were responders to at least one of three treatments. potassium concentration in the three possible pairs of Our primary end point was treatment-induced increase in treatment responses (Supplemental Figure 2). In patients plasma potassium concentration in patients with GS. Hypo- who received all three treatments (n=22), 16 (73%) patients kalemia is associated with the intensity of musculoskeletal were responders (increase of at least 0.3 mmol/L in plasma symptoms and the occurrence of more severe complications, potassium concentration) to at least one of three treatments; and it has previously been shown to respond to each of three 6 of 22 patients (27%) had no response to any of the three study drugs in preliminary reports.11,15,16 Hypokalemia in pa- drugs. tients with GS is caused by secondary consequences of the Among 15 intermediate responders, nonresponders, or inherited inactivation of NCC in the proximal DCT, including patients intolerant to indomethacin, 6 patients became full increased sodium delivery to late DCTand cortical connecting responders by switching to amiloride (n=3) or eplerenone duct and secondary renin-dependent hyperaldosteronism (n=3). Among 22 intermediate responders or nonresponders triggered by sodium depletion. This biologic and hormonal to amiloride, 13 patients became full responders by switching phenotype mimics the electrolytic consequences of chronic to one of the other drugs (indomethacin alone, n=3; eplerenone administration of thiazide diuretics, which specifically block alone, n=2; both treatments, n=8). Among 19 intermediate res- NCC in the kidney.12–14 The rationale behind administering ponders, nonresponders, or patients intolerant to eplerenone, the epithelial sodium channel inhibitor amiloride and the MR 10 patients became full responders to one of the other drugs antagonist eplerenone is, thus, to neutralize the compensatory (indomethacin alone, n=3; amiloride alone, n=3; both treat- mechanisms in the distal nephron that are responsible for ments, n=4). hypokalemia in patients with GS. The dose of 20 mg amiloride has previously been shown to Effects of Treatments on BP, Heart Rate, Body Weight, reverse the hydrochlorothiazide-induced hypokalemia and and Quality of Life increase plasma magnesium concentration more effectively The slight changes in systolic/diastolic BP (decrease with the than 100 mg spironolactone in healthy volunteers.17 Because two potassium-sparing diuretics and increase with indometh- eplerenone is approximately 75% less potent than spironolac- acin) were as expected and nonsignificant (Table 2). The tone,18 the selected dose was 150 mg on the basis of the healthy changes in body weight were as expected (Table 2). volunteer study mentioned above.17 Eplerenone was uptitrat- None of the treatments significantly improved or worsened ed weekly from 50 to 100 mg o.d. and then, to the maximum quality of life (Supplemental Table 3). dose of 150 mg o.d. administered for 4 weeks. A 6-week

J Am Soc Nephrol 26: 468–475, 2015 Crossover Trial in Gitelman Syndrome 471 CLINICAL RESEARCH www.jasn.org administration of either 20 mg o.d. amiloride or 150 mg o.d. In contrast to amiloride and eplerenone, indomethacin eplerenone was equally effective in increasing plasma potassium decreased plasma renin concentration. Indeed, indomethacin concentration. However, amiloride and eplerenone normalized is known to inhibit renin secretion by inhibition of PGE2 plasma potassium concentration in only 20% and 11% of the synthesis in the juxtaglomerular apparatus.20 Indomethacin patients, respectively, when given combined with potassium and blunted the expected physiologic increase in plasma aldoste- magnesium supplementation. Amiloride slightly increased rone concentration in response to the increase in plasma po- plasma magnesium concentration and significantly reduced tassium concentration. Moreover, indomethacin has been urine magnesium excretion, indicating the stimulation of tubu- shown experimentally to inhibit the diuretic effect of thiazides lar magnesium reabsorption. This observation is in line with a and stimulate sodium reabsorption in the loop of Henle.22 previous report in healthy volunteers.17 It is not known Accordingly, systolic BP increased slightly by approximately whether a higher dose of amiloride or eplerenone could be 2 mmHg, suggesting that the potassium-sparing effect of in- more effective in correcting hypokalemia in patients with GS, domethacin was associated with a slight correction of hypo- but this option would be limited by tolerability. Indeed, the use volemia. In summary, the effect of indomethacin in patients of amiloride or eplerenone is, in part, paradoxical, because both with GS may be caused by (1) the increase in loop NaCl re- drugs may worsen sodium depletion and hypovolemia in these absorption, leading, in turn, to a decrease in NaCl delivery to patients with a salt-losing nephropathy.8 In fact, both drugs DCT,23 and (2) the inhibition of renin release by the juxtaglo- induced a marked increase in plasma aldosterone concentrations merular apparatus as observed in rats treated with hydrochlo- secondary to (1) diuretic-induced sodium depletion associated rothiazide.24 with a mild decrease in systolic BP (approximately 2 mmHg) and Indomethacin normalized plasma potassium concentration eGFR (amiloride only), although the rise in plasma renin was in only 25% of the patients who tolerated the drug. A titration nonsignificant, and (2) partial correction of hypokalemia. The of indomethacin dosage by monitoring the effect on plasma progressive titration of amiloride and eplerenone to their highest renin concentration or urinary excretion rates of prostaglan- respective doses may explain the satisfactory clinical and biologic dins may have improved efficacy and/or tolerability of in- tolerability of the two drugs. Amiloride was downtitrated in only domethacin. However, whether a higher dose of indomethacin two patients, and eplerenone was discontinued in only two would have been more effective in correcting hypokalemia in patients for worsened hypovolemia. Whether additional MR ac- these patients is not known,15 but the use of slow-release in- tivation by high aldosterone concentrations triggered by amilor- domethacin (even at the low dose of 75 mg o.d.) for only 6 ide may contribute to long-term cardiovascular fibrosis and weeks had limited tolerability. First, eGFR decreased by 10.0 remodeling and renal fibrosis in patients with a salt-losing tu- ml/min per 1.73 m2, despite the mild increase in systolic BP bulopathy is unknown. Indeed, aldosterone has a number of after 6 weeks of treatment. The indomethacin-induced de- nonclassic, MR-mediated actions, such as tissue remodeling, crease in eGFR was slightly greater than that observed with modulation of vascular tone, and stimulation of inflammation amiloride, and both decreases were reversible when the drugs and fibrosis, including the kidney, in the setting of high salt in- were stopped. This result is probably related to the specific take.19 Such effects would be neutralized by the use of the MR effects of COX-2 inhibition on renal hemodynamics, which antagonist eplerenone. are specifically enhanced in sodium- and volume-depleted We also investigated whether the nonselective COX-1 and patients with GS.25,26 The risk of a more marked decrease in COX-2 inhibitor, indomethacin, would increase plasma po- GFR may be increased with longer-term indomethacin use in tassium concentration without worsening sodium and volume the setting of (1) additional extrarenal sodium loss or (2) depletion in patients with GS. By inhibiting prostaglandin E2 drug-induced interstitial nephritis.27–30 Second, six patients (PGE2) synthesis in the kidney,20 indomethacin corrects hy- suffered from GI intolerance related to the indomethacin- pokalemia in patients with Bartter syndrome, another salt- induced inhibition of COX-1 at the level of the GI mucosa, losing tubulopathy caused by mutations in genes coding for leading to early discontinuation, despite coprescription of a proteins responsible for salt reabsorption in the loop of proton pump inhibitor. The risk of severe GI adverse events is Henle.21 Thedosageregimen(75mg)wasonthebasisof increased with long-term use of nonsteroidal anti-inflammatory the low indomethacin doses (approximately 1 mg/kg) cur- drugs, including indomethacin.31,32 Third, there may also be a rently used in Bartter syndrome21 and preliminary reports theoretical increased risk of cardiovascular events with pro- in GS.15 We showed that a 6-week administration of 75 mg longed exposure to the drug.32 However, this risk is related to o.d. slow-release indomethacin significantly increased plasma the degree of COX-2 inhibition33 and thus, remains probably potassium concentration from baseline but had no effect on low at a dose of 75 mg o.d. slow-release indomethacin. In ad- plasma magnesium concentration in patients with GS. This dition, chronic use of a proton pump inhibitor could worsen result suggests a role of renal PGE2 in the pathogenesis of GS magnesium depletion by decreasing net intestinal absorption as shown for loop disorders.21 The net potassium increase of magnesium.34 after 6 weeks of treatment with indomethacin was greater, The principle limitations of this study were (1) the short although nonsignificantly, than that achieved with amiloride duration of exposure to treatments, which may not have been or eplerenone. sufficient to fully assess tolerability and sustained efficacy for a

472 Journal of the American Society of Nephrology J Am Soc Nephrol 26: 468–475, 2015 www.jasn.org CLINICAL RESEARCH chronic disease such as GS; (2) the selected doses of the three Design of the Study drugs, which may have not been maximal to fully assess effi- This study was a seven-period, three-treatment, open-label, random- cacy; (3) the lack of consideration of other treatment options, ized, crossover study with blind end point evaluation. The initial such as renin-angiotensin system blockers (however, increas- baseline assessments were performed after a 2- to 6-week washout ing the doses of each study drug or using a renin-angiotensin period to stop any COX inhibitor (2 weeks) or potassium-sparing system blocker would have been limited by tolerability in these diuretic (6 weeks) effects. Patients were then randomly assigned to sodium- and volume-depleted patients); and (4)thelackof sequentially receive slow-release indomethacin (75 mg for 6 weeks) consideration of sodium chloride supplementation as a more combined with a proton pump inhibitor (omeprazole at 20 mg/d) or physiologic way to decrease renin and aldosterone secretion. eplerenone (uptitrated weekly from 50 to 100 mg o.d. and then, to the However, extending the duration of each active period or in- maximum dose of 150 mg o.d. administered for 4 weeks) or amiloride creasing the number of periods would have increased the risk (uptitrated weekly from 10 to 15 mg o.d. and then, to the maximum of loss to follow-up of patients, who were already included in dose of 20 mg o.d. administered for 4 weeks) in addition to oral this crossover study for 38–42 weeks. Finally, another option potassium and magnesium supplementation. would have been to carry out a long-term parallel group study, but this option would have required more patients to include Randomization and follow-up, a difficult task in patients with a rare disease, The randomization sequence was generated by a computer without such as GS. stratification using randomized blocks of small size and permutation In conclusion, this study is the first and largest randomized of treatments within each block. Investigators, patients, and research controlled study performed in patients with GS providing staff were blinded to the randomization list. evidence of moderate efficacy of three treatments currently used on the basis of limited case series. Although the three drugs Study Evaluations have different mechanisms of action, they all increase plasma Biochemical, hormonal, hemodynamic, and safety assessments were potassium concentration on the short term, which however, is performed after each 6-week washout and active treatment periods. normalized in a minority (20%–25%) of patients with GS. Blood was sampled at approximately 9:00 a.m. in fasting conditions Because we did not specifically address the long-term after the patient rested for 1 hour in a semirecumbent position. Seated benefits and risks with the three treatments used in our study, BP and heart rate were measured at home with a validated electronic these treatments still remain the last resort in patients with GS device (OMRON M6; Omron Co., Kyoto, Japan) as described who remain with severe or symptomatic hydroelectrolytic previously.35 In addition, we assessed quality of life using the Short abnormalities, despite potassium, magnesium, and possibly, Form Health Survey self-administered questionnaire adapted for the salt supplementation at optimally titrated and tolerated doses. French population36 at randomization and the end of each active The decision should also take into consideration the clinical treatment period. setting (e.g., the need for nonsteroidal anti-inflammatory drugs for chondrocalcinosis), the specific contraindications Laboratory Methods of each drug, and the patient’s informed choice. BP and Biochemical and hormone measurements were performed blind to plasma potassium, magnesium, and creatinine concentrations the randomization sequence in a centralized laboratory. Plasma and need to be monitored to accurately assess efficacy and tolera- urinary electrolytes and creatinine were measured as described bility. Patients should be informed of the possibility of renal previously.37 eGFR was calculated by the Modification of Diet in Re- failure or hyperkalemia occurring with long-term treatment nal Disease formula.38 Plasma renin and aldosterone concentrations during episodes of dehydration or with concomitant use of were measured as described previously.39 potentially nephrotoxic and/or hyperkalemic drugs. Addi- tional studies are needed to address the long-term efficacy Statistical Analyses and tolerability of these treatment options. The primary objective of the study was to determine whether slow-release indomethacin would significantly increase the plasma potassium concentration compared with the control period, which CONCISE METHODS was defined as the first 6-week period after the 2- to 6-week washout period. Using awithin-patient SD of 0.3 mmol/L, we calculated that 30 Detailed methods are given in Supplemental Material. patients were needed to detect a 0.3-mmol/L difference in plasma potassium concentration between the indomethacin and control Participants periods with 80% power and 5% a-error for a two-tailed test. Eligible patients were men and women (18–60 years old) diagnosed Because eight patients had to stop one of the study drugs for with genetically proven GS at five tertiary French hospitals. All pa- intolerance, leading to a missing period for each of these patients (see tients gave written informed consent before participating in the Results), data were analyzed by an ANOVA for a crossover design on study. The protocol (ClinicalTrials.gov Identifier: NCT01146197) the on-treatment population who actually received all three study was approved by the Comité de Protection des Personnes, Paris-Île drugs (n=22 of 30).40 The model included treatment, sequence, and de France III, France and adhered to the Declaration of Helsinki. period as fixed effects and subject nested within sequence as the

J Am Soc Nephrol 26: 468–475, 2015 Crossover Trial in Gitelman Syndrome 473 CLINICAL RESEARCH www.jasn.org random effect. When the F test was significant (P,0.05) and there 9. McMahon FG, Ryan JR, Akdamar K, Ertan A: Upper gastrointestinal was no period, sequence, or carryover effect, paired comparisons lesions after potassium chloride supplements: A controlled clinical trial. – were made between treatments by the Holm method.41 Lancet 2: 1059 1061, 1982 10. Peters M, Jeck N, Reinalter S, Leonhardt A, Tönshoff B, Klaus G G, We also performed an analysis restricted to main criteria, i.e., Konrad M, Seyberth HW: Clinical presentation of genetically defined plasma potassium concentration, using paired t tests comparing patients with hypokalemic salt-losing tubulopathies. Am J Med 112: 6-week on-treatment versus pretreatment values on all datasets of 183–190, 2002 valid periods of treatments. 11. Monnens L, Bindels R, Grünfeld JP: Gitelman syndrome comes of age. Nephrol Dial Transplant 13: 1617–1619, 1998 12. Ito Y, Yoshida M, Nakayama M, Tsutaya S, Ogawa K, Maeda H, Miyata M, Oiso Y: Eplerenone improved hypokalemia in a patient with Gitelman’s ACKNOWLEDGMENTS syndrome. Intern Med 51: 83–86, 2012 13. Morton A: Eplerenone in the treatment of Gitelman’ssyndrome.Intern Med J 38: 377, 2008 We thank Prof. P. Houillier at the Georges Pompidou Hospital for 14. Morton A, Panitz B, Bush A: Eplerenone for gitelman syndrome in helping with patient recruitment. We thank the patients for partici- pregnancy. Nephrology (Carlton) 16: 349, 2011 pating in the study. We thank the nursing staff, Jeanne Meunier, and 15. Liaw LC, Banerjee K, Coulthard MG: Dose related growth response to Valérie Paquet of the Clinical Investigation Center of Georges Pompidou indometacin in Gitelman syndrome. Arch Dis Child 81: 508–510, 1999 Hospital, the nursing staff and Debohra Postil of the Clinical Inves- 16. Colussi G, Rombolà G, De Ferrari ME, Macaluso M, Minetti L: Correc- tion of hypokalemia with antialdosterone therapy in Gitelman’s syn- tigation Center of Limoges, and the nursing staff, Pierre Jean Saulnier, drome. Am J Nephrol 14: 127–135, 1994 and Elodie Rogeon of the Clinical Investigation Center of Poitiers. We 17. Murdoch DL, Forrest G, Davies DL, McInnes GT: A comparison of the thank Chantal Andrieux from the Clinical Research Unit for helping potassium and magnesium-sparing properties of amiloride and spi- us with regulatory processes. ronolactone in diuretic-treated normal subjects. Br J Clin Pharmacol 35: – This work was supported by French Ministry of Health and As- 373 378, 1993 18. Weinberger MH, Roniker B, Krause SL, Weiss RJ: Eplerenone, a se- sistance Publique des Hôpitaux de Paris Grant PHRCAOM08193. A.B. lective aldosterone blocker, in mild-to-moderate hypertension. Am J “ ’ ” was the recipient of a Contratd Interface Grant from Institut Na- Hypertens 15: 709–716, 2002 tional de la Santé et de la Recherche Médicale. 19. Brown NJ: Contribution of aldosterone to cardiovascular and renal in- The funding sources had no involvement in study design; the flammation and fibrosis. Nat Rev Nephrol 9: 459–469, 2013 collection, analysis, and interpretation of data; the writing of the 20. Matzdorf C, Kurtz A, Höcherl K: COX-2 activity determines the level of report; or the decision to submit the paper for publication. renin expression but is dispensable for acute upregulation of renin expression in rat kidneys. Am J Physiol Renal Physiol 292: F1782– F1790, 2007 21. Seyberth HW, Schlingmann KP: Bartter- and Gitelman-like syndromes: DISCLOSURES Salt-losing tubulopathies with loop or DCT defects. Pediatr Nephrol 26: 1789–1802, 2011 None. 22. Kirchner KA: Indomethacin antagonizes furosemide’s intratubular effects during loop segment microperfusion. J Pharmacol Exp Ther 243: 881–886, 1987 REFERENCES 23. Kirchner KA, Brandon S, Mueller RA, Smith MJ, Bower JD: Mechanism of attenuated hydrochlorothiazide response during indomethacin ad- – 1. Knoers NV, Levtchenko EN: Gitelman syndrome. Orphanet J Rare Dis ministration. Kidney Int 31: 1097 1103, 1987 3: 22, 2008 24. Kammerl MC, Nüsing RM, Richthammer W, Krämer BK, Kurtz A: In- 2. Simon DB, Nelson-Williams C, Bia MJ, Ellison D, Karet FE, Molina AM, hibition of COX-2 counteracts the effects of diuretics in rats. Kidney Int – Vaara I, Iwata F, Cushner HM, Koolen M, Gainza FJ, Gitleman HJ, Lifton 60: 1684 1691, 2001 RP: Gitelman’s variant of Bartter’s syndrome, inherited hypokalaemic 25. Breyer MD, Harris RC: Cyclooxygenase 2 and the kidney. Curr Opin – alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl co- Nephrol Hypertens 10: 89 98, 2001 transporter. Nat Genet 12: 24–30, 1996 26. Stichtenoth DO, Frölich JC: COX-2 and the kidneys. Curr Pharm Des 6: – 3. Malafronte C, Borsa N, Tedeschi S, Syrèn ML, Stucchi S, Bianchetti MG, 1737 1753, 2000 Achilli F, Bettinelli A: Cardiac arrhythmias due to severe hypokalemia 27. Palmer BF: Renal complications associated with use of nonsteroidal in a patient with classic Bartter disease. Pediatr Nephrol 19: 1413–1415, anti-inflammatory agents. JInvestigMed43: 516–533, 1995 2004 28. Passmore AP, Copeland S, Johnston GD: The effects of ibuprofen and 4. Ahmed S, Qayyum M, Farooq F: Quadriparesis in an adult—Gitelman indomethacin on renal function in the presence and absence of fruse- syndrome. JPakMedAssoc61: 182–184, 2011 mide in healthy volunteers on a restricted sodium diet. Br J Clin Phar- 5. Scognamiglio R, Negut C, Calò LA: Aborted sudden cardiac death in macol 29: 311–319, 1990 two patients with Bartter’s/Gitelman’s syndromes. Clin Nephrol 67: 29. Sandhu GK, Heyneman CA: Nephrotoxic potential of selective cyclo- 193–197, 2007 oxygenase-2 inhibitors. Ann Pharmacother 38: 700–704, 2004 6. Slyper AH: Growth, growth hormone testing and response to growth 30. Schlondorff D: Renal complications of nonsteroidal anti-inflammatory hormone treatment in Gitelman syndrome. J Pediatr Endocrinol Metab drugs. Kidney Int 44: 643–653, 1993 20: 257–259, 2007 31. Ofman JJ, MacLean CH, Straus WL, Morton SC, Berger ML, Roth EA, 7. Pachulski RT, Lopez F, Sharaf R: Gitelman’s not-so-benign syndrome. Shekelle P: A metaanalysis of severe upper gastrointestinal complications NEnglJMed353: 850–851, 2005 of nonsteroidal antiinflammatory drugs. J Rheumatol 29: 804–812, 2002 8. Park PG Jr, Karet Frankl FE: Gitelman syndrome. BMJ 344: e3590–851, 32. Bhala N, Emberson J, Merhi A, Abramson S, Arber N, Baron JA, 2012 Bombardier C, Cannon C, Farkouh ME, FitzGerald GA, Goss P, Halls H,

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Hawk E, Hawkey C, Hennekens C, Hochberg M, Holland LE, Kearney PM, activity and renal calcium handling. Clin J Am Soc Nephrol 2: 320–325, Laine L, Lanas A, Lance P, Laupacis A, Oates J, Patrono C, Schnitzer TJ, 2007 Solomon S, Tugwell P, Wilson K, Wittes J, Baigent C; Coxib and traditional 38. Levey AS, Eckardt KU, Tsukamoto Y, Levin A, Coresh J, Rossert J, De NSAID Trialists’ (CNT) Collaboration: Vascular and upper gastrointestinal Zeeuw D, Hostetter TH, Lameire N, Eknoyan G: Definition and classi- effects of non-steroidal anti-inflammatory drugs: Meta-analyses of individual fication of chronic kidney disease: A position statement from Kidney participant data from randomised trials. Lancet 382: 769–779, 2013 Disease: Improving Global Outcomes (KDIGO). Kidney Int 67: 2089– 33. García Rodríguez LA, Tacconelli S, Patrignani P: Role of dose potency in 2100, 2005 the prediction of risk of myocardial infarction associated with non- 39. Nguyen G, Blanchard A, Curis E, Bergerot D, Chambon Y, Hirose T, steroidal anti-inflammatory drugs in the general population. JAmColl Caumont-Prim A, Tabard SB, Baron S, Frank M, Totsune K, Azizi M: Cardiol 52: 1628–1636, 2008 Plasma soluble (pro)renin receptor is independent of plasma renin, 34. Famularo G, Gasbarrone L, Minisola G: Hypomagnesemia and proton- prorenin, and aldosterone concentrations but is affected by ethnicity. pump inhibitors. Expert Opin Drug Saf 12: 709–716, 2013 Hypertension 63: 297–302, 2014 35. Bobrie G, Frank M, Azizi M, Peyrard S, Boutouyrie P, Chatellier G, 40. Senn S: Cross-Over Trials in Clinical Research, Chichester, United Laurent S, Menard J, Plouin PF: Sequential nephron blockade versus Kingdom, Wiley, 2000 sequential renin-angiotensin system blockade in resistant hyperten- 41. Aickin M, Gensler H: Adjusting for multiple testing when reporting re- sion: A prospective, randomized, open blinded endpoint study. JHy- search results: The Bonferroni vs Holm methods. Am J Public Health 86: pertens 30: 1656–1664, 2012 726–728, 1996 36. Leplège A, Ecosse E, Verdier A, Perneger TV: The French SF-36 Health Survey: Translation, cultural adaptation and preliminary psychometric evaluation. J Clin Epidemiol 51: 1013–1023, 1998 37. Blanchard A, Azizi M, Peyrard S, Stern N, Alhenc-Gelas F, Houillier P, This article contains supplemental material online at http://jasn.asnjournals. Jeunemaitre X: Partial human genetic deficiency in tissue kallikrein org/lookup/suppl/doi:10.1681/ASN.2014030293/-/DCSupplemental.

J Am Soc Nephrol 26: 468–475, 2015 Crossover Trial in Gitelman Syndrome 475 Supplemental Table 1: Recovery after each treatment period. The parameters were measured just before the treatment, at the end of the preceding washout treatment (WO‐pre‐tt) and after a 6‐week washout post‐treatment period (WO post‐tt). Full recovery was obtained for the three treatments.

Indomethacin Eplerenone Amiloride

WO pre‐tt WO post‐tt P‐valuea WO pre‐tt WO post‐tt P‐valuea WO pre‐tt WO post‐tt P‐valuea PLASMA Sodium mmol/L 139 ± 1 139 ± 1 0.2822 140 ± 2 139 ± 1.5 0.2000 140 ± 1.5 139 ± 2 0.4183 Potassium mmol/L 2.8 ± 0.4 2.8 ± 0.4 0.9057 2.8 ± 0.4 2.8 ± 0.3 0.8764 2.8 ± 0.4 2.8 ± 0.4 0.7957 Magnesium mmol/L 0.55 ± 0.08 0.55 ± 0.08 0.7851 0.55 ± 0.08 0.55 ± 0.09 0.9824 0.55 ± 0.08 0.55 ± 0.07 0.8944 Renin mU/L 82 [62;109] 82 [62;109] 0.4716 78 [59;105] 80 [60 ;106] 0.9472 86 [64;117] 82 [62;109] 0.8655 Aldosterone pg/mL 54 [40;73] 54 [40;73] 0.7917 54 [39;73] 57 [40;81] 1.0000 61 [44;85] 60 [43;85] 0.8953

URINE Sodium mmol/24h 160 [123;208] 165 [136;200] 0.8526 185 [161;213] 171 [131;224] 0.8926 183 [155;214] 170 [145;199] 0.4836 Potassium mmol/24h 106 [91;124] 103 [88;121] 0.6761 114 [99;132] 110 [95;127] 0.9620 111 [97;128] 104 [91;119] 0.6455 Magnesium mmol/24h 1.5 [1.0;2.1] 1.3 [0.9;1.9] 0.6875 1.3 [0.9;2.0] 1.9 [1.3;2.8] 0.1587 1.8 [1.3;2.6] 1.4 [0.9;2.2] 0.5219 Creatinine mmol/24h 10.3 [9.0;11.8] 10.2 [8.7;12.0] 0.8164 10.9 [9.8;12.2] 11.0 [9.8;12.4] 0.9683 11.0 [9.8;12.4] 10.6 [9.5;12.0] 0.6098 a : pre‐treatment vs. post‐treatment washout periods.

Supplemental Table 2. Genetic data obtained in the 33 patients included in the study. Patient Sex Status Allele 1 Allele 1 References Nucleotide Protein§ Exon/ Intron Nucleotide Protein§ Exon/ Intron 1 F Ho1 c.625C>T p.Arg209Trp 5 c.625C>T p.Arg209Trp 5 1 2 F Ho1 c.625C>T p.Arg209Trp 5 c.625C>T p.Arg209Trp 5 1 3 F Ho2 c.3077C>T p.Thr1026Ile 26 c.3077C>T p.Thr1026Ile 26 2 4 M CH c.947G>T p.Gly316Val 7 c.2191G>A p.Gly731Arg 18 3/4 5 F CH c.947G>T p.Gly316Val 7 c.2191G>A p.Gly731Arg 18 3/4 6 F CH c.1195C>T p.Arg399Cys 10 c.2221G>A p.Gly741Arg 18 2/1 7$ F CH c.1195C>T p.Arg399Cys 10 c.2221G>A p.Gly741Arg 18 2/1 8 M CH c.965C>T p.Ala322Val 8 c.1946C>T p.Thr649Met 16 5/6 9 F CH c.1805_1806del3c.2 p.Tyr602CysfsX313 14 c.2747+1G>A p.? splice defect 23 7/8/5 660+1G>A p.? splice defect 22 10 M CH c.2576T>C p.Leu859Pro 22 c.2581C>T p.Arg861Cys 22 1/9 11 M CH c.1046C>T p.Pro349Leu 8 c.1-?_964+?del3 p.? 1 to 7 1/5 12 M CH c.2221G>A p.Gly741Arg 18 c.2581C>T p.Arg861Cys 22 1/9 13 F CH c.533C>T p.Ser178Leu 4 c.2221G>A p.Gly741Arg 18 10/1 14 M2 Ho c.2883+1G>T p.? splice defect 24 c.2883+1G>T p.? splice defect 24 1 15 F CH c.1143G>A p.Trp381* 9 c.2981G>A p.Cys994Tyr 26 5/3 16 F CH c.1336-1G>C p.? splice defect 10 c.2581C>T p.Arg861Cys 22 5/9 17 F CH c.2581C>T p.Arg861Cys 22 c.1-?_964+?del3 p.? 1 to 7 9/5 18 M CH c.1046C>T p.Pro349Leu 8 c.1519C>T p.Arg507Cys 12 1/5 19$ M CH c.2581C>T p.Arg861Cys 22 c.2929C>T p.Arg977* 25 9/1 20 F CH c.852+1G>A p.? splice defect 6 c.1963C>T p.Arg655Cys 16 New mutation/9 21 F CH c.852+1G>A p.? splice defect 6 c.1963C>T p.Arg655Cys 16 New mutation /9 22 M CH c.911C>T p.Thr304Met 7 c.2576T>C p.Leu859Pro 22 5/1 23$ M CH c.1180+1G>T p.? splice defect 9 c.1763C>T p.Ala588Val 14 11/1 24 M CH c.2576T>C p.Leu859Pro 22 c.2993T>G p.Leu998Arg 26 1/ New mutation 25 F CH c.938C>T p.Ala313Val 7 c.2581C>T p.Arg861Cys 22 10/9 26 M Ho2 c.2576T>C p.Leu859Pro 22 c.2576T>C p.Leu859Pro 22 1 27 F CH c.56_57dup p.Phe20Alafs*8 1 c.1-?_964+?del3 p.? 1 to 7 New mutation /5 28 M CH c.2581C>T p.Arg861Cys 22 c.3006G>C p.Trp1002Cys 26 9/5 29 M Ho2 c.2687G>A p.Arg896Gln 23 c.2687G>A p.Arg896Gln 23 12 30 F Ho2 c.2576T>C p.Leu859Pro 22 c.2576T>C p.Leu859Pro 22 1 31 M CH c.897C>A p.Asn299Lys 7 c.1568C>T p.Ala523Val 13 New mutations 32 F CH c.1196_1202dup p.Ser402* 10 c.1881C>G p.Tyr627* 15 4/5 33 F Ho2 c.938C>T p.Ala313Val 7 c.938C>T p.Ala313Val 7 10 M: Male; F: female. $Patients included, not randomized. § Numbering is according to the cDNA sequence (GenBank : NM_000339.2). The A of the ATG of the initiator methionine codon is denoted as nucleotide. Mutations are described using the HGVS recommendations CH: compound heterozygous, Ho : homozygous. 1Both parents are heterozygous 2 No deletions detected by MLPA 3Heterozygous deletion detected par MLPA, exon1 to exon 7 deletion without breakpoint characterization. New mutation: not previously published mutation. The following mutations were previously expressed in vitro: p.Arg209Trp, p.Gly741Arg, p.Pro349Leu and p.Thr1026Ile (2) ; p.Gly316Val and p.Ala588Val (13) ; p.Cys994Tyr (14) . The others were predicted to be pathogen in silico.

References to supplemental Table 2: 1. Simon, DB, Nelson-Williams, C, Bia, MJ, Ellison, D, Karet, FE, Molina, AM, Vaara, I, Iwata, F, Cushner, HM, Koolen, M, Gainza, FJ, Gitleman, HJ & Lifton, RP: Gitelman's variant of Bartter's syndrome, inherited hypokalaemic alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransporter. Nat Genet, 12:24-30, 1996. 2. Kunchaparty, S, Palcso, M, Berkman, J, Velazquez, H, Desir, GV, Bernstein, P, Reilly, RF & Ellison, DH: Defective processing and expression of thiazide-sensitive Na-Cl cotransporter as a cause of Gitelman's syndrome. Am J Physiol, 277:F643-9, 1999. 3. Syren, ML, Tedeschi, S, Cesareo, L, Bellantuono, R, Colussi, G, Procaccio, M, Ali, A, Domenici, R, Malberti, F, Sprocati, M, Sacco, M, Miglietti, N, Edefonti, A, Sereni, F, Casari, G, Coviello, DA & Bettinelli, A: Identification of fifteen novel mutations in the SLC12A3 gene encoding the Na-Cl Co-transporter in Italian patients with Gitelman syndrome. Hum Mutat, 20:78, 2002. 4. Mastroianni, N, De Fusco, M, Zollo, M, Arrigo, G, Zuffardi, O, Bettinelli, A, Ballabio, A & Casari, G: Molecular cloning, expression pattern, and chromosomal localization of the human Na-Cl thiazide-sensitive cotransporter (SLC12A3). Genomics, 35:486-93, 1996. 5.Vargas-PoussouR, Dahan K, KahilaD, Venisse A, Riveira-MunozE, Debaix H, Grisart B, BridouxF, UnwinR, Moulin B, HaymannJP, VantyghemMC, Rigothier C, Dussol B, Godin M, NivetH, Dubourg L, TackI, Gimenez-Roqueplo AP, Houillier P, BlanchardA, DevuystO and Jeunemaitre X. Spectrum of mutations in Gitelman syndrome. J Am Soc Nephrol 22:693- 703, 2011. 6. Ji, W, Foo, JN, O'Roak, BJ, Zhao, H, Larson, MG, Simon, DB, Newton-Cheh, C, State, MW, Levy, D & Lifton, RP: Rare independent mutations in renal salt handling genes contribute to blood pressure variation. Nat Genet, 40:592-9, 2008. 7. Maki, N, Komatsuda, A, Wakui, H, Ohtani, H, Kigawa, A, Aiba, N, Hamai, K, Motegi, M, Yamaguchi, A, Imai, H & Sawada, K: Four novel mutations in the thiazide-sensitive Na- Cl co-transporter gene in Japanese patients with Gitelman's syndrome. Nephrol Dial Transplant, 19:1761-6, 2004 8. Fava, C, Montagnana, M, Rosberg, L, Burri, P, Jonsson, A, Wanby, P, Wahrenberg, H, Hulthen, UL, Aurell, M, Guidi, GC & Melander, O: Novel mutations in the SLC12A3 gene causing Gitelman's syndrome in Swedes. DNA Seq, 18:395-9, 2007. 9. Lemmink, HH, Knoers, NV, Karolyi, L, van Dijk, H, Niaudet, P, Antignac, C, Guay-Woodford, LM, Goodyer, PR, Carel, JC, Hermes, A, Seyberth, HW, Monnens, LA & van den Heuvel, LP: Novel mutations in the thiazide-sensitive NaCl cotransporter gene in patients with Gitelman syndrome with predominant localization to the C-terminal domain. Kidney Int, 54:720-30, 1998. 10. Cruz, DN, Shaer, AJ, Bia, MJ, Lifton, RP & Simon, DB: Gitelman's syndrome revisited: an evaluation of symptoms and health-related quality of life. Kidney Int, 59:710-7, 2001. 11. Coto, E, Rodriguez, J, Jeck, N, Alvarez, V, Stone, R, Loris, C, Rodriguez, LM, Fischbach, M, Seyberth, HW & Santos, F: A new mutation (intron 9 +1 G>T) in the SLC12A3 gene is linked to Gitelman syndrome in Gypsies. Kidney Int, 65:25-9, 2004. 12. Kurschat, C, Heering, P & Grabensee, B: [Gitelman's syndrome: an important differential diagnosis of hypokalemia]. Dtsch Med Wochenschr, 128:1225-8, 2003. 13. Riveira-Munoz E(1), Chang Q, Godefroid N, Hoenderop JG, Bindels RJ, Dahan K, Devuyst O; Belgian Network for Study of Gitelman Syndrome. Transcriptional and functional analyses of SLC12A3 mutations: new clues for the pathogenesis of Gitelman syndrome. J Am Soc Nephrol. 2007 Apr;18(4):1271-83. 14. De Jong JC(1), Van Der Vliet WA, Van Den Heuvel LP, Willems PH, Knoers NV, Bindels RJ. Functional expression of mutations in the human NaCl cotransporter: evidence for impaired routing mechanisms in Gitelman's syndrome. J Am Soc Nephrol. 2002 Jun;13(6):1442-8.

Supplemental Table 3: Results of the Short Form (Health Survey (SF36) quality of life self‐questionnaire at randomization and at the end of each active treatment period.

Normal values (*) Baseline Indomethacin Eplerenone Amiloride

Physical component summary Physical functioning 84 ± 21 75 ± 22 70 ± 24 68 ± 21 72 ± 23 Role limitations (physical) 81 ± 32 48 ± 43 58 ± 39 50 ± 44 53 ± 36 Bodily pain 73 ± 24 53 ± 24 62 ± 25 52 ± 25 54+/‐ 24 General health perception 69 ± 19 45 ± 19 45 ± 20 44 ± 18 44 ± 20

Mental component summary Vitality 60 ± 18 37 ± 19 42 ± 22 34 ± 18 41 ± 19 Social functioning 82 ± 21 54 ± 20 65 ± 24 55 ± 28 55 ± 24 Role limitations (emotional) 82 ± 32 64 ± 44 63 ± 42 56 ± 42 59 ± 41 General mental health 68 ± 18 57 ± 15 57 ± 18 54 ± 19 56 ± 17

Physical functioning: Measurement of limitations in physical activities such as walking, climbing stairs, bending forward, lifting and significant and moderate physical effort. Role limitations because of physical health problem: Measurement of discomfort, due to the physical condition, in daily activities; measurement of limitations in certain activities or difficult to achieve them. Bodily pain: Measurement of the intensity of the pain and inconvenience. Vitality: Self‐assessment of vitality, energy, fatigue. Social functioning: Measurement of limitations in social activities due to physical problems and mental health. Role limitations because of emotional problems: evaluation of discomfort due to psychological problems in daily activities; time spent on less important work, sloppy work. Mental health: Self‐rated mental health: anxiety, depression, well‐being (happiness?). (*) Normal values are those available for established in a French population of 209 healthy subjects (Leplege, A, Ecosse, E, Verdier, A, Perneger, TV: The French SF‐36 Health Survey: translation, cultural adaptation and preliminary psychometric evaluation. Journal of clinical epidemiology, 51: 1013‐1023, 1998.) Supplemental Figure 1: Flow chart of the study

5 Supplemental Figure 2: Correlations between treatment‐induced changes in plasma potassium concentration (ΔK mmol/L). The correlation between responses to indomethacin and amiloride was significant (panel A, r2=0.1713, 0.0444). The correlation between responses to indomethacin and eplerenone (B panel) and between responses to eplerenone and amiloride (C panel) were not significant. ABC

1.2 1.2 1.2

0.9 0.9 0.9

0.6 0.6 0.6

0.3 0.3 0.3 K, mmol/l K, mmol/l K, mmol/l Δ Δ Δ Eplerenone Indomethacin Indomethacin 0.0 0.0 0.0

-0.3 -0.3 -0.3

-0.6 -0.6 -0.6 -0.6 -0.3 0.0 0.3 0.6 0.9 1.2 -0.6 -0.3 0.0 0.3 0.6 0.9 1.2 -0.6 -0.3 0.0 0.3 0.6 0.9 1.2 ΔK, mmol/l ΔK, mmol/l ΔK, mmol/l Amiloride Eplerenone Amiloride

DETAILED METHODS

Participants

Eligible patients were men and women aged 18 to 60 years diagnosed with genetically proven GS at five tertiary French hospitals. Patients had either homozygous (n=8) or compound heterozygous for point mutations or large rearrangements in the SLC12A3gene

(n=27) (see supplemental Table 2). Exclusion criteria were: known intolerance to the study drugs, significant cardiac arrhythmia, no contraception in women of child bearing potential, pregnancy, or an estimated glomerular filtration rate (eGFR) < 60 ml/min/1.73 m2. All patients gave written informed consent before participating in the study. The protocol

(ClinicalTrials.gov Identifier: NCT01146197) was approved by the “Comité de Protection des

Personnes”, Paris‐Île de France III, France.

Design of the study

This was a seven‐period, three‐treatment, open‐label, randomized, crossover study with blind end‐point evaluation. The initial baseline assessments were performed after a 2‐ to 6‐week washout period to stop any COX inhibitor (2 weeks) or potassium‐sparing diuretic

(6 weeks) effects. Patients were then randomly assigned to sequentially receive slow release indomethacin, eplerenone or amiloride for 6 weeks each in addition to oral potassium and

7 magnesium supplementation. Each active period was intercalated with a 6‐weeks washout period during which the potassium and magnesium supplementation was maintained.

Slow release indomethacin was given at a dose of 75 mg o.d. for 6 weeks in combination with a proton pump inhibitor (omeprazole 20 mg/day) for gastric protection.

This dosage regimen was based on the low indomethacin doses (≈ 1 mg/kg) currently used in

Bartter syndrome 16 and preliminary reports in GS. 15

Amiloride was uptitrated weekly from 10 mg o.d. to 15 mg o.d. and then to the maximum dose of 20 mg o.d. administered for 4 weeks. This dose of 20 mg amiloride has previously been shown to reverse the hydrochlorothiazide‐induced hypokalemia and to increase plasma magnesium concentration more effectively than 100 mg spironolactone in healthy volunteers. 17

Eplerenone was uptitrated weekly from 50 mg o.d. to 100 mg o.d. and then to the maximum dose of 150 mg o.d. administered for 4 weeks. Since eplerenone is ≈75% less potent than spironolactone 18, the selected dose was 150 mg based on the healthy volunteer study mentioned above. 17. The amiloride and eplerenone doses could be down‐titrated if symptomatic hypotension occurred.

The oral potassium and magnesium supplementation doses were adjusted for each patient during the first 6‐week washout period to maintain plasma K concentration at either

≥ 2.8 mmol/L or 0.2 to 0.4 mmol/L above their usual plasma K concentration. The mean dose

8 of slow release potassium chloride was 4.0±1.7 g, and that of magnesium element was 226 ±

77 mg /day. Thereafter, the dose was kept constant throughout the study.

Randomisation

The randomisation sequence was generated by computer without stratification, using randomised blocks of small size and permutation of treatments within each block.

Investigators, patients, and research staff were blinded to the randomisation list.

Study evaluations

Biochemical, hormonal, hemodynamic, and safety assessments were performed after each 6‐week washout and active treatment periods. Blood was sampled at ≈ 09:00 a.m. in fasting conditions after the patient rested for one hour in a semi‐recumbent position.

Seated blood pressure (BP) and heart rate (HR) was measured at home with a validated electronic device (OMRON M6®, Omron Co., Kyoto, Japan) as described previously. 19 In addition, we assessed quality of life using the Short Form Health Survey (SF36) self‐ administered questionnaire adapted for the French population 36 at randomization and the end of each active treatment period.

Laboratory methods

Biochemical and hormone measurements were performed blind to the randomization sequence in a centralized laboratory. Plasma and urinary electrolytes and creatinine were measured as described previously.20 Estimated GFR was calculated by the

9 Modification of Diet in Renal Disease(MDRD) formula.21 Plasma renin and aldosterone concentrations were measured as described previously.22

Statistical analysis

The primary objective of the study was to determine whether slow release indomethacin would significantly increase the plasma potassium concentration compared to the control period, defined as the first 6‐week period following the 2‐ to 6‐week washout period. Using a within‐patient standard deviation (SD) of 0.3 mmol/L, we calculated that 30 patients were needed to detect a 0.3 mmol/L difference in plasma potassium concentration between the indomethacin and control period with 80% power and 5% alpha error for a two‐tailed test.The secondary objectives were to compare the potassium‐ and magnesium‐ sparing effects as well as the hemodynamic and hormonal effects and tolerability of the three study drugs.

Patients with a clinically significant treatment‐induced increase of at least 0.3 mmol/L in plasma potassium concentration were considered as full responders, those with

0.10 to 0.29 mmol/L increases were intermediate responders and those with less than 0.10 mmol/L increases were non responders.

Since eight patients had to stop one of the study drugs for intolerance leading to a missing period for each of these patients (see results), data were analyzed by an ANOVA for a crossover designon the on‐treatment population who actually received all three study

10 drugs (n=22/30).23 The model included treatment, sequence, and period as fixed effects and subject nested within sequence as the random effect. When the F test was significant

(P<0.05), and when there was no period, sequence or carryover effect, paired comparisons were made between treatments by Holm's method.24 The assumptions of ANOVA

(homogeneity of variance and normality) were checked for each variable and natural logarithmic transformation was applied where appropriate. We also performed an analysis restricted to plasma potassium concentration using paired t‐tests comparing 6‐week on‐ treatment vs. pre‐treament values on all data sets of valid periods of treatments.

Correlations were estimated using Pearson coefficients. SAS software version 9.3 (SAS

Institute Inc., Cary, NC, US) was used for statistical analyses. Data are expressed as geometric means with 95 % confidence intervals (CI) or medians (interquartile range, IQR) for non‐normal parameters and as means ± one SD for normally distributed data. Two‐sided

P values of <0.05 were considered to be significant.