Thiazolidinedione derivates (TZD's) are Peroxisome-Proliferator-Activated-Receptor-γ agonists (PPARγ-agonists) and enhance insulin sensitivity. One of the side effects, however, is the fact that subjects treated with these drugs seem to be more prone to fluid retention. The precise mechanism of rosiglitazone-related fluid retention is unknown, but it is clear that either primary or secondary renal sodium retention is part of the mechanism. Furthermore in observational studies, TZD-related oedema seems to be resistant to loop diuretic therapy. The recent finding that rosiglitazone induces upregulation of the epithelial sodium channel (ENaC) in the kidney could be the explanation for TZD-related fluid retention and the observed resistance to loop diuretics. In the present human in-vivo study the following hypothesis will be tested:

Rosiglitazone treatment stimulates the activity of ENaC in the distal nephron, which enhances the natriuretic effect of amiloride and decreases the natriuretic effect of furosemide in parallel.

This is a randomized, placebo-controlled, double-blind, single-centre, cross-over study with 4 weeks of wash out comparing placebo with rosiglitazone 4 mg bid for 9 weeks treatment periods. Randomization of the treatment sequence will be computer-generated, with a sequentially driven allocation. Randomization and blinding will be performed at the department of Clinical Pharmacy. After 8 (furosemide) and 9 (amiloride) weeks in each period the end-point experiments will be performed. During all visits (week 0, 4, 8, 9) of each period, adverse events and pill compliance will be recorded. In addition, physical examination, foot volume and bio-impedance measurements will be performed and safety chemical, and hematological profiles will be determined. Only at start and at 8 weeks in each period, glucose, insulin and HbA1c are measured. All visits and interventions will be performed at the Clinical Research Center Nijmegen (CRCN).

Furosemide end-point experiment Each participant will attend the hospital at 8 a.m. after an overnight fast and abstinence of alcohol and caffeine for 20 hours, delivering a 24-hour urine collection and the present morning voiding. The previous three days each participant will adhere to an individualized diet containing 150 mmol of sodium and 80 mmol of potassium prescribed by a dietician. First, blood will be collected to measure fasting glucose and insulin concentrations. Then the subject will be given an individualized breakfast including 1 cup of water. Afterwards a brachial vein will be cannulated and connected to a Braunpump (10 ml/hr NaCl 0.9%), followed by blood drawing for safety and vascular hormone measurements (aldosterone, Atrial Natriuretic Peptide (ANP), Brain Natriuretic Peptide (BNP), Vascular Endothelial Growth Factor (VEGF) and renin).

A bolus of furosemide (40 mg) will be injected through a small cannule in a vein of the contra-lateral arm, just after bladder emptying. Venous blood samples will be drawn at 0, 15, 30, 45, 60, 90,120, 150, 180, 240, 300, 360, 420 and 480 minutes after bolus injection to measure plasma furosemide levels. The participants will be asked to urinate regularly, at least hourly. The exact time of voiding and the urine volume will be recorded. Two urine samples will be taken. In one sample, sodium and creatinine concentrations will be measured while the other sample will be light-protected and immediately frozen for measurement of furosemide levels later on. To prevent dehydration each participant will be asked to drink tap water equal to the volume of diuresis in the previous hour. During the test the participant will be sitting on a bed. At noon the participant will be offered an individualized lunch. After 8 hours each participant will leave the hospital with the instruction to adhere to the diet without fluid restrictions and to collect the urine for up to 24 hours after start of the experiment.

Amiloride end-point experiment Until amiloride infusion the procedures will be similar. At time point 0, venous infusion of a loading dose of amiloride will be started (150 μg/kg in 60 minutes) followed by maintenance infusion (0.20 μg/kg/min) for 4 hours. Amiloride will be obtained as a sterile powder in the form of amiloride HCl/2H2O . Directly before use, the powder will be dissolved in NaCl 0,9% up to a concentration of 1 mg/ml and the solution was filtered through a 0.22 μm Millipore filter. Venous blood for measurement of the amiloride concentration will be sampled at 60, 180, 300 and 420 minutes. All the other procedures will be similar to the furosemide experiment.

Pharmacokinetic considerations on the amiloride-dose The peak plasma levels 3-4 hours after intake of 10 or 20 mg amiloride are 20 μg/L (32) and 38-40 μg/L respectively(33). These concentrations are well below the half maximal inhibitory concentration (IC50) of amiloride for Na+/H+ and Na+/Ca2+-transporters and the α1-receptor, but well above the IC50 for ENaC(34). Using the pharmacokinetic characteristics of amiloride(35) we calculated the required amiloride infusion in order to reach a steady-state concentration between 30-45 μg/L.

Exosome extraction:

Urinary exosomes will be isolated by ultracentrifugation and ENaC abundance will be measured by immunoblotting as previously described (19;36) and normalized to urine creatinine levels. 4 µg of protein lysed in Laemmli buffer will be loaded on 8% SDS-PAGE. PAGE, blotting and blocking of the PVDF membranes will be done as previously described. Membrane will be incubated with 1:4000-diluted affinity-purified rabbit α-ENaC antibody (Rossier BC, Lausanne, Switzerland), followed by 1:5,000-diluted goat anti-rabbit IgG's as secondary antibody coupled to horseradish peroxidase. Blotting signals will be visualized using enhanced chemiluminescence. The samples will be normalized for the expression level of α-ENaC in placebo treatment and indicated as percentage.

Inclusion Criteria:

• Healthy but with 2 features of the metabolic syndrome (AHA/NHLBI) (16)
• Willing and able to provide a signed and dated written informed consent.
• Male or female subject aged between 30 and 70 years

Exclusion Criteria:

• Fasting glucose > 7,0 mmol/L or the use of hypoglycaemic agents. If fasting plasma glucose is between 6.1 and 7,0 mmol/L,an oral 75 g glucose test will be performed to exclude diabetes mellitus.
• Exposure to a PPAR-g agonist during the last 4 months or a documented significant hypersensitivity to a PPAR-g agonist.
• Participant in another study.
• Angina or heart failure (NYHA I-IV).
• Clinically significant liver disease (3 times the upper normal limit of ALAT, ASAT, AF, γGT or LDH)
• Clinically significant anaemia (male Hb < 6,9 mmol/L, female < 6,25 mmol/L)
• Creatinin clearance < 40 mL/min
• Pregnancy, lactation
• Alcohol or drug abuse. Liquorice