The Influence of Rosiglitazone on the Diuretic Effect of Furosemide and Amiloride
Condition: Insulin Resistance
MeSH terms: Insulin Resistance
Study type: Interventional

Summary: 
      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.
    

Detailed description: 
      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.
    

Intervention name(s): Rosiglitazone versus placebo; response (sodium excretion) to amiloride infusion; response (sodium excretion) to furosemide infusion
Intervention type(s): Drug; Drug; Drug
Intervention MeSH terms: Rosiglitazone; Amiloride; Furosemide
Allocation: Randomized
Intervention model: Crossover Assignment
Masking: Triple (Participant, Care Provider, Investigator)
Primary purpose: Treatment
Keywords: Rosiglitazone; Furosemide; Amiloride; Epithelial sodium channel; Sodium excretion