Scandinavian Journal of Gastroenterology. 2015; 50: 454–461

ORIGINAL ARTICLE

Urinary aquaporin-2 excretion before and after transjugular intrahepatic portosystemic shunt insertion for refractory ascites

MARIANNE GEILSWIJK1, KAREN LOUISE THOMSEN1, ERLING BJERREGAARD PEDERSEN2, HENDRIK VILSTRUP1 & HENNING GRØNBÆK1 1

Department of Hepatology and Gastroenterology, Aarhus University Hospital, 44 Nørrebrogade, 8000 Aarhus C, Denmark, and 2Department of Medical Research, Regional Hospital Jutland West and Aarhus University, University Clinic in Nephrology and Hypertension, 12 Lægårdsvej, 7500 Holstebro, Denmark

Abstract Objective. The role of renal aquaporin-2 (AQP2) water channel turnover in patients with liver cirrhosis, portal hypertension and water retention remains unclear. Transjugular intrahepatic portosystemic shunt (TIPS) insertion reduces portal hypertension, improves water excretion and lowers plasma vasopressin. The aim of this study was to establish whether TIPS insertion decreases urinary AQP2 excretion (uAQP2) in parallel with improved water excretion. Material and methods. Fourteen cirrhosis patients with refractory ascites were studied before TIPS insertion and 4 and 12 weeks after insertion. A 24-h urine collection was followed by an oral water load (20 ml/kg body weight) with a 4-h blood and urine sampling. Results. TIPS reduced the portal pressure gradient from a median 18(4) (25–75% InterQuartile-range) to 7(2) mmHg, p < 0.05 and the need for diuretics (p < 0.05). TIPS increased plasma sodium from 136(6) mmol/l to 139(4), (p < 0.05) and diuresis from 1650(1043) ml/24 h to 2230(560) (p < 0.05), although the 24-h urinary sodium excretion did not change. There was no change in the baseline uAQP2 before 274(249) ng/(mmol creatinine/24 h) and 12 weeks after TIPS 242(201). There were no systematic changes in uAQP2, plasma vasopressin or other vasoactive substances during the water loads, before or after TIPS. Conclusion. The effective amelioration of portal hypertension improved the patient’s water excretion and plasma sodium, but there was no change in renal AQP2 trafficking or vasopressin. These findings do not support a primary role for renal AQP2 water channels in portal hypertensive water retention.

Key Words: aquaporin2, ascites, liver cirrhosis, portal hypertension, portosystemic shunt, transjugular intrahepatic

Introduction Ascites in cirrhosis is a marker of poor prognosis and is associated with increased morbidity and mortality [1,2]. In decompensated cirrhosis, increased portal pressure with ongoing stimulation of the vasoconstrictor systems causes renal hypoperfusion and water retention with ascites formation [3–6]. Renal aquaporin-2 (AQP2) water channels have been proposed to play an important role, but the correlations between AQP2 and portal hypertensive water retention have not been fully established [7–13]. Renal trafficking of AQP2 is partly mediated by vasopressin,

with cAMP as a second messenger. Urinary cAMP has been shown to correlate with renal vasopressin susceptibility [14]. Other factors also influence renal AQP2 trafficking, including prostaglandin E2 and so-called vasopressin uncoupling [15]. A part of the renal AQP2 is shed and excreted in the urine, and animal studies have suggested that the excreted fraction of the total renal AQP2 is constant [8,16]. It has been reported that renal AQP2 is increased in cirrhosis with water retention and that urinary AQP2 excretion increases with advanced disease stage in cirrhosis [11]. Transjugular intrahepatic portosystemic shunt (TIPS) insertion is used to treat complications of

Correspondence: Henning Grønbæk, Professor, MD PhD, Department of Hepatology and Gastroenterology, Aarhus University Hospital, 44 Nørrebrogade, 8000 Aarhus C, Denmark. Tel: +4526833436. Fax: +4578462820. E-mail: [email protected]

(Received 14 August 2014; accepted 25 August 2014) ISSN 0036-5521 print/ISSN 1502-7708 online  2015 Informa Healthcare DOI: 10.3109/00365521.2014.962610

UAQP2 in TIPS treated patients portal hypertension in patients with liver cirrhosis, including ascites refractory to pharmacological treatment [17,18]. TIPS treatment effectively reduces the portal pressure, improving the decreased sodium and water excretion and lowering the increased plasma levels of such vasoactive substances as vasopressin, renin, angiotensin-II, aldosterone (e.g., the renin– angiotensin–aldosterone system) and catecholamines [17,19]. The treatment may prolong a patient’s life expectancy [20]. Thus, TIPS treatment offers a clinical intervention model to study whether portal hypertensive water retention is related to renal AQP2 trafficking. To strengthen the signals of any possible changes in the AQP2 system, we subjected each subject to a water load before and after TIPS. A priori, we hypothesized that TIPS insertion, together with the improved water clearance and normalized P-sodium, would decrease the renal urinary AQP2 excretion and decrease the first and second aquaporin messengers. Methods Ethics statement The study was designed as a descriptive follow-up study and was conducted with approval from The Regional Ethics Committee (no. 20070127, Central Denmark Region, the 28th of September 2007). The study conformed with the Second Declaration of Helsinki. Participants were recruited at the Department of Hepatology & Gastroenterology, Aarhus University Hospital, Denmark, and all gave written consent before examination. Eligibility criteria were ages between 20 and 70 years, verified cirrhosis (liver biopsy or classical ultrasonographic, clinical and biochemical findings) with portal hypertension and treatment-refractory ascites with need for frequent paracenteses. We included 14 participants, 6 of whom had diuretic-resistant ascites and 8 who had diuretic-intractable ascites. Exclusion criteria were cardiopulmonary or endocrine disease, arterial hypertension, portal vein thrombosis, neoplasm, renal insufficiency with plasma creatinine above 150 mmol/l or ongoing treatment with cyclo-oxygenase inhibitors. All participants were abstinent from alcohol for at least 3–6 months prior to the TIPS procedure. Each patient’s clinical status was assessed according to the Model for End-stage Liver Disease (MELD) and the Child-Pugh score. All participants were examined before the TIPS procedure and again 4 and 12 weeks after TIPS. Each examination consisted of a baseline clinical and biochemical evaluation and an acute water load. The participants suspended

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diuretic treatment for 24 h before examination and submitted to a 24-h urine collection. Urine volume was measured, and the samples were frozen and stored for further analysis. On the day of the examination, the participants fasted from midnight. Encephalopathy and ascites scores, body weight, blood pressure and pulse were assessed. At time -15 min, routine and special blood samples (specified below), as well as urine samples, were drawn. At time 0 to 20 min, the participants were given an oral water load of 20 ml per kg body weight. Urine samples were collected at times 30, 60, 120, 180 and 240 min. The urine volume was registered, and the samples were stored at -20 C for later analysis. At times 60 and 240 min, special blood samples were drawn; blood pressure and pulse were also registered. In addition to the oral water load, the participants were not allowed to eat or drink, and during the examination, they were resting in bed. Movement was allowed, and strict horizontal positioning was not obtained for the entire study period. One participant died during follow-up, and one did not complete all examinations. Thus, of the 14 patients remaining, all underwent the examination before TIPS, and 13 were examined at 4 weeks and/or 12 weeks after TIPS. Routine blood samples Routine blood samples were analyzed for routine hematological, hepatological and renal parameters on the day of the examination using standard laboratory methods at the Department of Clinical Biochemistry, Aarhus University Hospital. Special blood samples Special blood samples were analyzed using specific methods and assays. To determine plasma angiotensin-II, tubes were prepared with EDTA and O-phenathrolin; for plasma renin and aldosterone, tubes were prepared with K+-EDTA; for plasma arginine vasopressin (AVP), tubes were prepared with sodium and creatinine with lithium heparin; and for serum osmolality, glasses without preparation were used. Blood samples were (except for s-osmolality) kept on ice after withdrawal and centrifuged within 15 min at 4 C at 3000 g for 10 min, after which the plasma was stored in plastic tubes at -20 C (angiotensin-II, Na+, creatinine) or at -80 C (renin, aldosterone, AVP) for later analysis. To determine serum osmolality, blood samples was centrifuged at 20 C at 3000 g for 10 min, and the serum was pipetted and stored in plastic tubes at -20 C.

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The below analysis was performed at University Clinic in Nephrology and Hypertension, Holstebro Hospital. Angiotensin-II and AVP were extracted using C18 Sep-Pak (Water associates, Milford, MA, USA) and were determined by radioimmunoassay [21]. Angiotensin-II antibody was obtained from the Department of Clinical Physiology, Glostrup Hospital, Denmark. The lower limit of detection was 2 pmol/l, and the coefficients of variation were 12% (inter-assay) and 8% (intra-assay), with a normal range of 4.6–18.4 pg/ml. The AVP antibody was a gift from Professor Jacques Dürr, Miami, FL, USA. The lower limit of detection was 0.5 pmol/l. The coefficients of variation were 13% (inter-assay) and 9% (intra-assay), and the normal range was < 2.5 pg/ ml. Plasma aldosterone was determined by radioimmunoassay (Diagnostic Systems Laboratories Inc., Webster, Texas, USA). The lower limit of detection was 22 pmol/l, and the coefficients of variation were 8.2% (inter-assay) and 3.9% (intra-assay), with a normal range (supine) of 81–447 pmol/l. Plasma renin was determined by radioimmunoassay (CIS Bio International, Gif-Sur-Yvette Cedex, France) with a lower limit of detection 1 pg/ml and coefficients of variation at 14.5% (inter-assay) and 4.5% (intraassay). The normal range (supine) was 1.2–20.2 pg/ ml in subjects older than 40 years. Serum osmolality was measured using freezing point depression (Advanced Model 3900 multisampling osmometer). Plasma sodium and creatinine were determined using standard methods. Urine samples Urine samples were analyzed in the University Clinic of Nephrology and Hypertension, Holstebro Hospital, for urine AQP2, urine Na+, urine creatinine, urine osmolality and urine cAMP. Urine AQP2 was determined by radioimmunoassay as previously described using rabbit anti-AQP2-antibody from Professor Søren Nielsen, Water and Salt Research Centre, Institute of Biomedicine, Aarhus University [22]. The lower limit of detection was 32 pg/tube, and the coefficients of variation were 11.7% (inter-assay) and 5.9% (intra-assay). Urine osmolality was measured using freezing point depression (Advanced Model 3900 multisampling osmometer). Urine cAMP was determined by radioimmunoassay (Biomedical Technologies Inc., Stoughton, MA. USA). Urine sodium and creatinine were measured using standard methods. SPSS 11.0 was applied for statistical analysis using nonparametric testing with Friedman’s two-way analysis of variance by ranks and Wilcoxon signed ranks test. We used Spearman’s rho for the correlation

analysis. The data are presented as medians and interquartile ranges (25–75%). p < 0.05 was considered to be significant for two-sided tests. Results In Table I, the demographic and clinical characteristics of the study population are presented. Twelve participants were male and two female with a median age of 54 years. In all cases, cirrhosis was caused by excessive alcohol intake. At inclusion, the median Child-Pugh and MELD scores were 7.0 and 8.5, respectively. TIPS insertion significantly reduced the portal pressure gradient from 18 to 7 mmHg (p < 0.05). The study revealed no significant effect of TIPS on the Child-Pugh or MELD scores. Throughout the study, the hemodynamic parameters stayed within normal ranges. Systolic blood pressure increased significantly after TIPS treatment, whereas diastolic blood pressure was unchanged. Heart rate increased significantly after TIPS and remained at this level during follow-up. The TIPS procedure was followed by significant reductions in diuretic dosage, and there was no further need for ascites drainage. Propranolol treatment was stopped after TIPS insertion. The baseline biochemical characteristics of the participants, before and after TIPS, are presented in Table II. Twelve weeks after TIPS insertion, we observed a significant decrease in plasma coagulation factors II, VII, X (p < 0.05), although the INR was unchanged. Plasma bilirubin increased during followup (p < 0.05). Plasma sodium increased significantly during follow-up from 136 mmol/l to 139 (Figure 1A), and plasma creatinine decreased from 80 mmol/l to 64 (p < 0.05). The estimated eGFR increased significantly from baseline to 4 weeks (p = 0.021) and showed a trend for increase after 12 weeks (p = 0.075). In addition, diuresis increased from 1650 ml/24 h before TIPS to 2130 after TIPS (p < 0.05) (Figure 1B). There were no significant changes in urinary sodium, creatinine, osmolality or cAMP during the study period. Furthermore, there was no significant change in 24 h UAQP2 or 24 h UAQP2 per creatinine (Figure 1C). There were no significant changes in the vasoactive substances from the baseline assessment and during the study period. Correlations We observed a positive correlation between the MELD score and 24 h urine volume (rho = 0.49, p = 0.09) and a significantly negative correlation between the MELD score and 24 h UAQP per creatinine (rho = -0.56, p = 0.048). Other correlations

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UAQP2 in TIPS treated patients

Table I. Demographic and clinical characteristics at baseline and at 4 and 12 weeks after the TIPS procedure (data are medians and interquartile range (25–75%)). Baseline Gender (male/female) Etiology (alcohol/other) Age Weight (kg) Child-Pugh (A/B/C) Child-Pugh score MELD score CRT index BP (systolic) (mmHg) BP (diastolic) (mmHg) HR (beats/minute) Portal pressure (mmHg) Spironolactone I (mg/day) Furosemide (mg/day) Propranolol (mg/day)

12/2 14/0 54 74.7 3/9/2 7.0 8.5 2.4 117 78 66 18 200 80 80

Four weeks 10/2

(1.3) (9.5) (1.7) (15) (13) (16) (4) (100) (120) (0–160)

74.0 3/9/0 8.0 9.0 2.2 125 69 83 – 100 40 0

Difference – – –

11/2 –

(9) (22.0)

Twelve weeks

(26.5) (1.7) (8.5) (1.4) (21) (19) (12) (50) (40) (0)

– – 78.0 (22.3) 6/6/1 7.0 (2.5) 8.0 (6.5) 2.2 (1.1) 127 (38) 68 (12) 80 (9) 7 (2) 100 (100) 20 (80) 0 (0)

NS – p< NS NS p< NS p< p< p< p< p