http://www.kidney-international.org original article & 2009 International Society of Nephrology see commentary on page 811 Glycyrrhetinic acid food supplementation lowers serum potassium concentration in chronic hemodialysis patients Stefan Farese1, Anja Kruse1, Andreas Pasch1, Bernhard Dick1, Brigitte M. Frey1, Dominik E. Uehlinger1 and Felix J. Frey1

1Department of Nephrology and Hypertension, Inselspital, University of Berne, Berne, Switzerland

Hyperkalemia is a common life-threatening problem in Hyperkalemia is a common and sometimes life-threatening hemodialysis patients. Because glycyrrhetinic acid (GA) problem in patients with end-stage renal disease and is a inhibits the enzyme 11b-hydroxy-steroid dehydrogenase II frequent reason for emergency dialysis in patients undergoing and thereby increases availability to the colonic chronic renal replacement therapy.1 Severe hyperkalemia mineralocorticoid receptor, it has the potential to lower (serum potassium 46.0 mmol/l) was reported to occur in serum potassium concentrations. To test this, 10 patients up to 12%2–4 of hemodialysis patients. In these patients, in a 6 month prospective, double-blind, placebo-controlled three mechanisms account for hyperkalemia: an increase in crossover study were given cookies or bread rolls potassium intake, a decreased potassium excretion and a shift supplemented with glycyrrhetinic acid or placebo. Twenty- of intracellular potassium to the extracellular fluid. Metabolic four-hour blood pressure measurements were performed at acidosis, the main mechanism for intra–extracellular potas- baseline and week 6 and 12 of each treatment period. The sium shift, is not a relevant cause of hyperkalemia at steady ratio of plasma cortisol/ was significantly increased state in adequately dialysed patients. The efficacy of a dietary in all patients on GA as compared to baseline or placebo, restriction of potassium intake as a measure to avoid indicating appropriate enzyme inhibition. Nine of the 10 hyperkalemia is often limited for nutritional and compliance patients had a persistent decrease in predialysis serum reasons.1 In patients without renal function, the gastro- potassium concentration. On GA, mean predialysis serum intestinal excretion of potassium has a pivotal role.3,5 Thus, potassium was significantly lower than at baseline or on pharmacological interventions enhancing the gastrointestinal placebo. On placebo, serum potassium was significantly excretion of potassium are of potential relevance. The elevated above the upper limit of normal in 76% compared gastrointestinal mode of disposal of potassium is enhanced to 30% of measurements during GA treatment. Furthermore, by cation exchange resins, together with a cathartic, usually on this treatment the frequency of severe hyperkalemia sorbitol.6 The efficacy of the unpleasant resin therapy has significantly decreased from 9% to 0.6%. No differences were been questioned7 and gastrointestinal side effects, including found in parameters reflecting sodium retention. Although colonic necrosis, have been observed.8,9 Thus, alternative these studies show that prolonged GA supplementation agents to enhance gastrointestinal potassium removal are persistently lowers serum potassium in dialysis patients, a warranted. long-term toxicity study will be mandatory before we Potassium secretion is an established mechanism of rectal recommend the routine use of this treatment. and colonic mucosa.10,11 This loss is regulated, at least in 12 Kidney International (2009) 76, 877–884; doi:10.1038/ki.2009.269; part, by the mineralocorticoid receptor (MR). The activa- published online 29 July 2009 tion of the MR by fludrocortisone enhances the rectal KEYWORDS: dialysis; food supplement; glycyrrhetinic acid; hyperkalemia; electrical potential difference, an effect that is mimicked by serum potassium inhibiting the enzyme 11b-hydroxysteroid dehydrogenase (11b-HSD2) in segments of normal rectal colon obtained from humans.13 The 11b-HSD2 enzyme converts endogenous cortisol into cortisone in mineralocorticoid target tissues including epithelial cells of the colon.14,15 This mechanism protects the 16–18 Correspondence: Felix J. Frey, Department of Nephrology and Hypertension, MR from promiscuous activation by cortisol (Figure 1). University of Berne Medical School, Inselspital, Freiburgstrasse 15, 3010, Therefore, we hypothesize that inhibition of 11b-HSD2 Berne, Switzerland. E-mail: [email protected] might enhance intestinal potassium loss. In a 2-week prelimi- Received 23 March 2009; revised 3 June 2009; accepted 9 June 2009; nary proof of principle study, we showed that inhibition of published online 29 July 2009 11b-HSD2 by glycyrrhetinic acid (GA), the active ingredient

Kidney International (2009) 76, 877–884 877 original article S Farese et al.: Glycyrrhetinic acid lowers predialysis potassium

Prereceptor control Binding Transactivation 7.0 O translocation Cortisol 6.5 C - CH2 OH CH3 HO OH 6.0 ** CH3 GR 5.5 ** O 5.0 GA 11β-HSD2 4.5 O 4.0 C - CH OH GRE Cortisone CH 2 O 3 OH Gene MRE 3.5 CH3 Serum potassium concentration (mmol/l)

O Baseline GA Placebo

Nucleus Figure 2 | Predialysis serum potassium concentrations at baseline and during glycyrrhetinic acid (GA) and placebo periods. The values displayed for the GA and placebo periods MR represent the mean of the two measurements at weeks 6 and Figure 1 | receptors (GR) and mineralocorticoid 12 on each treatment modality. Six patients (.) were on GA for receptors (MR) are ligand-inducible transcription factors. the first 12 weeks and then switched to placebo for 12 weeks, Following binding of steroid hormones these receptors whereas four patients (J) started with placebo and were translocate from the cytoplasm into the nucleus and display switched to GA. Potassium concentrations during GA their transactivation potential. The intracellular concentrations administration were lower than at baseline (**Po0.01) or during of steroid molecules available for binding to the cognate intake of placebo (**Po0.01). receptor depends on the free extracellular concentrations and an intracellular prereceptor control mechanism constituted by the 11 b-hydroxyglucocorticoid dehydrogenase (11 b-HSD) enzyme. The 11b-HSD2 enzyme acts predominantly as a dehydrogenase Table 1 | Predialysis serum potassium concentrations during and converts 11b-hydroxyglucocorticoid cortisol with a high GA and placebo periods affinity for both GR and MR into 11-ketosteroid cortisone with virtually no affinity for MR or GR. By this mechanism the 11b-HSD2 GA Placebo enzyme protects MR from promiscuous activation by the period period P-valuea glucocorticoid hormone cortisol. Inhibition of the 11b-HSD2 enzyme by glycyrrhetinic acid (GA) increases cortisol Measurements (n) 357 358 concentrations in MR-expressing cells and therefore causes a Within normal range (3.5–4.7mmol/l) 250 (70%) 84 (24%) o0.001 glucocorticoid-mediated mineralocorticoid effect (from refs. 18 and 49). Hyperkalemia (46 mmol/l) 2 (0.6%) 31 (9%) o0.001

Hypokalemia (o3.5 mmol/l) 1 (0.3%) 1 (0.3%) NS of licorice, decreased serum potassium concentrations in aFor GA period compared with placebo period. GA, glycyrrhetinic acid. patients on dialysis,19 an observation suggesting that this xenobiotic has the potential to be a potassium-lowering agent in dialysis patients. To determine the efficacy and safety of GA, we designed a trial with a 3-month treatment period and difference in serum potassium concentrations between placebo a 3-month placebo period. and GA (0.15±0.6 mmol/l and 0.95±0.45 mmol/l, respec- tively, Po0.001) as compared with baseline values. RESULTS Of all the serum potassium measurements obtained Effect of GA on potassium concentrations in blood, urine, and routinely before every dialysis session, 70% of the measure- dialysate and on the serum ratios of cortisol/cortisone and ments were in the defined normal range of 3.5–4.7mmol/l /renin while patients were on GA, whereas only 24% were within Predialysis serum potassium concentrations were significantly these limits in the placebo period (Po0.001, Table 1). lower during periods with GA ingestion. In Figure 2 the values Furthermore, GA treatment reduced the frequency of severe obtained after 6 and 12 weeks on GA or placebo were hyperkalemia (46 mmol/l) when compared with placebo combined and compared with baseline measurements. Mean (0.6 vs 9%, Po0.001, Table 1). GA treatment induced a rapid observed serum potassium values were 5.5±0.6 mmol/l during (within days) and sustained decline in predialysis serum baseline, 4.4±0.6 mmol/l (week 6) and 4.6±0.6 mmol/l potassium concentrations (Figure 3). For the whole study (week 12), whereas on GA and 5.4±0.7 mmol/l (week 6) period, mean dialysate potassium concentrations were higher and 5.3±0.7 mmol/l (week 12) during the placebo period. on GA than on placebo (3.1±0.4 mmol/l vs 2.8±0.4 mmol/l, Predialysis potassium concentrations during the GA period Po0.05, Figure 3). were significantly lower when compared with the concentra- From 4 out of the 10 patients who dropped out during the tions at baseline or during the placebo periods (Po0.01 for study at least one blood sample during the GA period was all). A combined linear model relating absolute individual obtained. When the predialysis serum potassium concentra- differences to drug exposure and time revealed a significant tions of these samples were compared with those obtained at

878 Kidney International (2009) 76, 877–884 S Farese et al.: Glycyrrhetinic acid lowers predialysis potassium original article

baseline or during the placebo period, a uniform decline was changes in blood pressure or weight gain occurred during observed in all patients while on GA (5.7±0.4 mmol/l vs the observation time (data shown below). 4.5±0.3 mmol/l, Po0.01). Furthermore, no significant During the GA period, the ratio of plasma cortisol/ cortisone increased in all patients and was significantly 6 – GA higher than at baseline or during the placebo phase of the + GA 5 trial (Table 2). When these values were plotted against Dialysate K+ concentration the predialysis potassium concentrations given in Figure 2, 4 a significant inverse correlation (Po0.002) was found (mmol/l) 3 (Figure 4). The increase in the cortisol/cortisone ratio

Serum and dialysate resulted from a uniform decrease in plasma cortisone in 2 potassium concentrations Week1–12 with GA Week 1–12 with placebo the GA period (Table 2). Plasma aldosterone concentrations and the aldosterone/renin ratios decreased significantly Figure 3 | Mean cumulative predialysis serum (J, K) and during GA phases when compared with baseline and placebo dialysate (D) potassium concentrations. Each dot represents measurements (Table 2). the mean of all 10 patients at a given treatment session. A total of 357 measurements were performed during the GA and 358 during In an attempt to determine whether GA enhances urinary the placebo period. The J and D with the bars represent potassium excretion, 24 h urine samples were obtained from the mean (±s.d.) baseline serum and dialysate potassium the day before dialysis at baseline and after 6 and 12 weeks on concentrations of the 10 patients. A sustained decrease in blood each treatment modality. As expected, these values showed a potassium concentrations was observed 3–5 days after starting GA treatment (K), and the concentrations in the dialysate increased high variability among patients with some residual renal slightly (D) to avoid . When GA was discontinued, function (baseline 46±39 mmol/24 h; GA 40±27 mmol/ potassium concentrations returned to baseline levels within 1 24 h; placebo 43±30 mmol/24 h). The variability of urinary week. Blood potassium concentrations on GA were markedly lower in the presence of higher dialysate potassium sodium excretion was even more pronounced (baseline concentrations (D) when compared with placebo. GA, 97±113 mmol/24 h; GA 158±184 mmol/24 h; placebo glycyrrhetinic acid. 157±133 mmol/24 h).

Table 2 | Laboratory values at baseline, during glycyrrhetinic acid (GA) and placebo treatment Baseline mean (±s.d.) GA mean (±s.d.) Placebo mean (±s.d.) Cortisol (ng/ml) 101±25 116±38 102±56 Cortisone (ng/ml) 12.7±3 8.0±1.7** 11.2±3.2 Ratio cortisol/cortisone 8.1±2.1 14.3±3.3*** 9.7±3.3$$

Renin (ng/l) 18±18 23±14 27±22 Aldosterone (pmol/l) 1030±1539 356±301** 689±709$ Ratio aldosterone/renin 88.7±67 18±13** 37±36*

Sodium (mmol/l) 136±2.9 136±3.8 137±3.9 Bicarbonate (mmol/l) 23±2.6 23±1.4 23±2.6 Urea reduction rate (%) 74.7±4.3 75.8±5.5 77.5±4.3 Predialysis urea (mmol) 22.1±5.9 22.2±7.5 24.2±7.0 Alanine aminotransferase (U/l) 13±722±11* 14±8$ g-Glutamyl transpeptidase (U/l) 40±23 64±48* 46±51$

Parathyroid hormone (pg/ml) 234±177 219±122 217±174 1.25-OH-Vitamin D (pmol/l) 41±15 36±17 41±20

Fasting glucose (mmol/l) 5.9±1.4 5.5±1.2 6.1±1.8 Insulin (mmol/l) 16±23 12±16 18±21 Homeostatic Model Assessment Index 4.6±8 3.2±5 5.2±8 HbA1c (%) 4.8±0.6 6.1±0.9** 6.0±0.8*

Cholesterin (mmol/l) 4.2±0.9 3.8±0.7* 4.3±0.9 HDL (mmol/l) 1.6±0.5 1.6±0.5 1.6±0.5 Triglycerides (mmol/l) 1.6±1.2 1.6±1 1.7±1.1

Hemoglobin (g/l) 115±5.7 116±8.8 115±11.3 White cells (G/l) 6±1 6.9±3 6.4±1.4 Platelets (G/l) 221±84 239±89 220±70 Values presented in the GA and placebo columns represent the mean of the two measurements at week 6 and 12 on each modality. *Po0.05 as compared with baseline measurements. **Po0.01 as compared with baseline measurements. ***Po0.001 as compared with baseline measurements. $Po0.05 as compared with GA period. $$Po0.01 as compared with GA period.

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7 In addition, GA appears to diminish the concentrations of aldosterone, a steroid considered to be relevant for

6 myocardial fibrosis. The mechanism underlying the uniform effect on serum potassium concentrations with a clinically significant reduc- 5 tion of hyperkalemic episodes deserves some pathophysio- logical considerations. Although a shift of potassium from

4 the extracellular to the intracellular compartment might be a potential mechanism accounting for lowering serum

Serum potassium (mmol/l) potassium concentrations during a short-term intervention, 3 as in our previous study,19 for mass balance reasons this mechanism cannot explain the present diminished plasma 5101520 potassium levels lasting 3 months under steady-state Ratio cortisol/cortisone conditions. Furthermore, the main driving forces of such a Figure 4 | Relation between plasma cortisol/cortisone ratios shift, that is, increased insulin concentrations or metabolic and predialysis serum potassium concentrations. A significant acidosis, were not observed when patients were on GA. The (Po0.002) inverse correlation between cortisol/cortisone ratios and serum potassium concentrations was found. decline in serum potassium concentrations on GA is also not explained by changes in dietary potassium intake because: (i) predialytic and postdialytic weight did not change during Side effects of GA the study and dialysis prescriptions remained constant, and Mean pre- and post dialysis systolic/diastolic blood pressure (ii) predialytic parameters, including urea, phosphate, values were comparable on GA and placebo (142/66± bicarbonate, and creatinine concentrations, were not affected 14/11 mm Hg vs 142/65±13/10 mm Hg and 137/66± by intake of GA. Thus, the most likely mechanism for the 12/8 mm Hg vs 139/66±14/9 mm Hg, respectively). Simi- diminished plasma potassium concentration is enhanced larly, no differences were observed in the 24 h ambulatory extrarenal disposal. With respect to the anatomic site of systolic and diastolic blood pressure measurements (baseline: potassium loss, we can only speculate. It is possible that GA 132/80±15/11 mm Hg; GA: 130/80±14/13 mmHg; placebo: enhanced rectal and colonic loss of potassium, most likely by 128/77±10/10 mm Hg). Mean 24 h ambulatory heart rate activation of the MR by as a consequence was also unaffected by the treatment modality (70±13 vs of the inhibition of 11b-HSD2 by GA.16,17 Alternatively, GA 72±14 vs 71±14 b.p.m.). might have modulated potassium loss by sweat.20 Pre- and post dialysis weight as well as the interdialytic The effect of GA on 11b-HSD2 was reflected by an weight gain, were assessed at every treatment session. increased ratio of cortisol/cortisone in all patients. Such an No significant differences were found in these parameters inhibition of the gate-keeper enzyme of the MR allows when the GA period was compared with the placebo period cortisol to bind to the MR and deploy a mineralocorticoid (predialysis weight: 76±15 kg vs 76±14 kg; interdialytic action, which causes renal sodium retention, volume weight gain: 2.02±0.63 kg vs 2.07±0.55 kg, respectively). overload and hypertension in humans with functioning Orally administered GA was well tolerated in all patients kidneys.17,18,21,22 Therefore, in this study, special attention who concluded the trial. Reasons for dropout during the was paid to interdialytic weight gain and blood pressure study were mostly GA-unrelated and are given in the assessed by 24 h ambulatory and in-clinic pre- and post Materials and Methods section. It is noteworthy that three dialysis recordings. Both body weight and blood pressure patients (two on GA, one on placebo) terminated the study were unaffected by GA. The observed absence of hyper- prematurely because of diarrhea (up to five bowel move- tension is in line with previous studies where patients ments/day in one patient on GA). were treated with the MR agonist fludrocortisone.23 It is Although the HOMA index for insulin sensitivity tended conceivable that an escape mechanism for sodium retention to be lower during GA treatment, HbA1c concentrations is operative in the colonic epithelial cells, similar to that increased during the GA and placebo periods (Table 2). Total observed in the kidney following prolonged administration serum cholesterol concentrations were significantly reduced and/or secretion of aldosterone.24 during GA administration (Table 2). A moderate increase in Aldosterone and the serum aldosterone/renin ratio blood enzyme concentrations of ALAT and g-GT was decreased on administration of GA (Table 2), an effect observed during the GA period (Table 2). Other serum and best explained by a diminished kalemia-related release of hematology parameters were not influenced by GA (Table 2). aldosterone.25,26 A moderated release of aldosterone is of potential clinical benefit as both the Randomized Aldactone DISCUSSION Evaluation Study27 and the Post-Acute Myo- We show here that the potassium-lowering effect of GA in cardial Infarction Heart Failure Efficacy and Survival Study28 patients with chronic renal failure persists for 3 months indicate that inhibition of aldosterone action by spirono- without inducing hypertension or other relevant side effects. lactone or eplerenone is beneficial in humans with heart

880 Kidney International (2009) 76, 877–884 S Farese et al.: Glycyrrhetinic acid lowers predialysis potassium original article

failure. Although the mechanism of this beneficial effect of other hand deploys its mineralocorticoid effects specifically aldosterone blockade in the Randomized Aldactone Evalua- in MR-expressing target tissues, such as in the cortical tion Study and Eplerenone Post-Acute Myocardial Infarction collecting duct or in the colonic epithelial cells, because the Heart Failure Efficacy and Survival Study trials is still MR-protecting enzyme 11b-HSD2 is selectively expressed in debated, whether it is due to some genomic or non-genomic cells with MR.16,17 The rigorous control of the 11b-HSD2 effects of aldosterone,29 the concept that high aldosterone gene has recently been elucidated by our group and is at least concentrations induce cardiac hypertrophy and fibrosis in part attributable to the presence of defined positive appears to be accepted knowledge.30 Thus, the present regulatory components and differential methylation of CpG observation of a decline of aldosterone concentrations after islands in the promoter.43–46 GA dosing can be interpreted as a potentially beneficial effect. The moderate increase of the serum liver enzymes However, cardiac fibrosis and hypertrophy are observed ASAT and g-GT when on GA indicates liver toxicity. when the MR is activated by aldosterone,31 and therefore Interestingly, GA has been used in humans with hepatitis C cardiac fibrosis is of concern when the MR is activated by infection47,48 and animals with different liver toxicities as a the glucocorticoid cortisol as a consequence of 11b-HSD2 hepatoprotective agent.33 In this study, the increased liver inhibition (Figure 1). Because GA appears to show enzymes returned to baseline values during the placebo antioxidative properties in various tissues including the period. Thus, a severe hepatotoxic effect of GA appears to be heart,32,33 and oxidative stress is a relevant mechanism in unlikely. Nevertheless, the effect of GA on liver enzymes in MR activation-induced fibrosis, a GA effect blunting the dialysis patients deserves further consideration in future MR-induced fibrosis might exist. Although in our trial no studies. cardiac side effects have been observed, the issue of structural In conclusion, the present investigation indicates that the heart damage by GA deserves further investigations. long-term administration of GA to dialysis patients lowers Recent accumulating evidence from studies in animals and predialysis potassium concentrations and reduces the fre- humans suggest a potential beneficial effect of 11b-HSD1 quency of dangerous hyperkalemic episodes without indu- inhibition on the metabolic syndrome.34 Because GA not cing weight gain or hypertension. Before GA can be only inhibits 11b-HSD2, but also 11b-HSD1,35 the enzyme recommended as a potassium-lowering agent, the pharma- catalyzing the transformation from inactive cortisone into cokinetics and the chronic toxicity have to be evaluated active cortisol, we investigated the effect of GA on glucose, formally in dialysis patients. HbA1c, insulin-sensitivity and blood lipids. Surprisingly, HbA1c increased slightly on both GA and placebo. The MATERIALS AND METHODS reason for this increase is unknown. The number of cookies Study design and patients B B ( 50 g/d) or bread rolls ( 30 g/d) consumed during both The study was approved by the Ethics Committee of the University study periods appears to be insufficient to explain this of Berne, Switzerland and registered at www.clinicaltrial.gov (NCT increase. In line with previous investigations using a specific 00384384). Based on an expected serum potassium concentration 11b-HSD1 inhibitor, insulin sensitivity (HOMA index) decrease of 0.6±0.6 mmol/l during treatment with GA,19 a tended to improve while the patients were taking GA.34,36 minimum number of 10 patients was estimated to provide a This is probably best explained by a diminished intracellular statistical power of 80%. ratio of biologically active 11b-hydroxyglucocorticoids to Twenty maintenance hemodialysis patients were randomly inactive 11-keto-steroids.37 allocated by a concealed randomization procedure to start either Previously, the mineralocorticoid hormone fludrocorti- with GA (n ¼ 10) or placebo (n ¼ 10) and enrolled in a prospective, placebo-controlled 6 months crossover study (Figure 5). All patients sone has been given to lower plasma potassium concentra- 4,23,38–40 gave written informed consent. No patients with nephrotic tions in patients on hemodialysis. These studies were 49,50 4 syndrome or cholestasis were included. Exclusion criteria were unblinded and comprised no placebo group. Kaisar et al. age o18 years, dialysis vintage of less than 3 months, therapy with compared prospectively patients with and without fludro- , insulin-treated diabetes mellitus and changes in cortisone for 3 months in an open-label trial and did not find antihypertensive medication in the 2 weeks before inclusion. All a significantly lower midweek predialysis serum potassium patients were asked to keep their fluid and dietary potassium intake concentration in patients on fludrocortisone. The dose of as constant as possible during the study period. Intake of licorice 21,51 fludrocortisone given in the latter study was lower than that and flavonoids were not allowed. Dropout reasons during the in the previous reports.23,38 Thus, the dose of fludrocortisone study were: Death (one patient on placebo), kidney transplantation showing a potential clinically relevant effect appears to be (one patient on placebo), diarrhea (two patients on GA, one on unknown. If relatively high doses of fludrocortisone are placebo), improvement of renal function with no further need of dialysis (one patient on GA), hospitalization for low back pain (one required in the future, side effects might be anticipated. patient on placebo), withdrawal of consent (one patient on GA, one shows a 10 times higher glucocorticoid 41 on placebo), relocation (one patient on placebo). Ten patients potency than cortisol. Thus, systemic glucocorticoid effects completed the study and were available for analysis. Patient might appear especially in patients on dialysis because 80% of characteristics are shown in Table 3. fludrocortisone is recovered in urine following an oral or Blood samples were obtained at baseline and at week 6 and 12 of intravenous dose in healthy normal volunteers.42 GA on the the GA and the placebo period. All patients were dialyzed three

Kidney International (2009) 76, 877–884 881 original article S Farese et al.: Glycyrrhetinic acid lowers predialysis potassium

times weekly. Blood samples were drawn before dialysis after the time points mentioned above. Moreover, serum potassium long hemodialysis interval of 3 days and after 10 min supine rest. concentrations were measured before each dialysis session with a Standard laboratory methods were used to determine sodium, ‘point of care’ AVL 988-4 device (AVL Medical Instruments, potassium, bicarbonate, creatinine, urea, liver enzymes, lipids, Schaffhausen, Switzerland). parathyroid hormone (PTH), renin (substrate), insulin, glycosylated Pre- and post dialysis body weight and blood pressure hemoglobin (HbA1c), aldosterone, cortisol and cortisone at the were recorded at every treatment session and oscillometric 24 h ambulatory BP measurements (Profilomat II, Disetronic, Burgdorf, Switzerland) were performed at baseline and week 6, 12, 18 and 24.

Assessed for Administration of GA and placebo eligibility (n=33) Pharmaceutical quality 18-b-glycyrrhetinic acid dried powder (Vital-Chem, Zhuhai, PR China, GMP Certificate number F0012, Excluded (n =13) Certificate of analysis/batch number JV050708) was administered Not meeting inclusion orally with permission from the Swiss authorities (Swissmedic). criteria (n =10) According to the patients’ preference, either cookies or bread rolls Refused to paricipate (n=2) Other reasons (n=1) were supplemented with 500 mg GA or placebo (500 mg dextrose)

Enrollment and given twice per day. Selection of this dose was based on the results of our previously published pilot study.19 The cookies and Randomized (n =20) bread rolls were manufactured by our own hospital kitchen and To start either with GA or placebo baked at a temperature of 80 1C (GA remains stable up to 160 1C).

Prescription of dialysis and antihypertensives Allocated to GA Allocated to placebo Intraindividual dialysis prescription parameters, including duration (n=10) (n=10) of dialysis, blood flow, dialysate flow, conductivity and filter Received allocated Received allocated

Allocation intervention (n=10) intervention (n=10) were kept constant during the whole study period. Changes in ultrafiltration rate and, if necessary, dialysate potassium concentrations were allowed by protocol and registered for analysis. All patients followed a conventional three times weekly dialysis Discontinued intervention Discontinued intervention before 6 weeks on GA before 6 weeks on placebo schedule. Dialysis dose was measured as equilibrated Kt/V (Kt/Ve) (n=2) (n=4) and urea reduction rate (Table 2 and Table 3). Prescription and dose alterations of ‘potassium-relevant’ drugs (antihypertensives and exchange resins) are shown in Table 4. Follow-up Dosage of calcium channel blockers (given in 4 out of 10 patients) Switched from placebo to GA Switched from GA to placebo was not changed, with the exception of one patient (No. 2) who at week 12 at week 12 stopped 5 mg of during the placebo period. The doses of (n =6) (n =8) furosemide, a drug known to inhibit 11b-HSD in high doses, was Discontinued (n=2) Discontinued (n=2) kept constant.52,53

Analysis of plasma cortisol and cortisone by gas Analyzed (n=10) chromatography-mass spectrometry

Analysis Excluded from analysis (n=0) 2.5 mg of medroxyprogesterone (Steraloids, Newport, RI, USA) was added to 0.75 ml of plasma as a recovery standard and the samples Figure 5 | Patient recruitment and study flow schema. were extracted with 10 ml of methylene chloride (Merck KGaA,

Table 3 | Patient characteristics Renal Residual clearance Residual urine Dialysis vintage a b c Patient no. Sex Age diagnosis (ml/min) output (ml/24 h) Comorbidities (months) Kt/Ve 1 F 78 1 2.5 1200 6 23 1.31 2 F 75 4 0 0 4 11 2.06 3 M 71 4 4.1 1800 7 56 1.44 4 M 81 2 1.3 400 5 51 1.48 5 M 54 3 0 0 4 91 1.35 6 F 73 2 1.8 400 5 56 1.35 7 M 73 4 5.7 1500 3 6 1.24 8 F 61 3 3.3 1500 5 4 1.34 9 M 81 4 0 0 4 66 1.36 10 F 68 3 0 0 7 18 1.53 a1 = diabetes, 2 = nephroangiosclerosis, 3 = glomerulonephritis, 4 = other or undefined. bMeasured creatinine clearance (ml/min). cCharlson comorbidity index.

882 Kidney International (2009) 76, 877–884 S Farese et al.: Glycyrrhetinic acid lowers predialysis potassium original article

Table 4 | Prescription of potassium-relevant drugs during the study ACE inhibitor/ATII blocker Diuretics b-blockers Exchange resins Patient no. Baseline Changes Baseline Changes Baseline Changes Baseline Changes 1 + None + None None None 2 + None None + None + Dose kwith GA 3 None + None + None None 4 + None + None + None None 5 + None None + None None 6 None + None + None None 7 None + None + None None 8 None + None + None None 9 None None None + Dose kwith placebo 10 None None None None , amiloride, and triamteren were not prescribed for any patient either before or during the study period. k: dose decreased during the indicated treatment period. ACE, angiotensin-converting enzyme; ATII, angiotensin II.

Darmstadt, Germany) on a rotator for 20 min. After centrifugation Mann–Whitney U-test for nonparametric analyses and Spearman’s at 3000 r.p.m. for 5 min, the two layers were separated and the correlation. organic layer was transferred into a new tube. Methylene chloride was evaporated under a stream of nitrogen at 45 1C. DISCLOSURE Stigmasterol (Steraloids) was added as a standard for derivati- All the authors declared no competing interests. zation and chromatography (2.5 mg in methanol). The solvent was evaporated under nitrogen at 60 1C and the samples were ACKNOWLEDGMENTS derivatized to form methyloxime–trimethylsilyl ethers. 100 mlof This work was supported by the Swiss National Foundation for methoxy amine hydrochloride 2% in pyridine (Pierce Biotechnology Scientific Research 3100A0-102153 and 320030-122135/1 Rockford, IL, USA) was added and the extracts were heated for (FJF and BMF). 1 h at 60 1C for the formation of methyloximes. 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Kidney International (2009) 76, 877–884 883 original article S Farese et al.: Glycyrrhetinic acid lowers predialysis potassium

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