Increased Renal Sodium Absorption by Inhibition of Prostaglandin Synthesis During Fasting in Healthy Man. A Possible Role of the Epithelial Sodium Channels

Thomas G Lauridsen; Henrik Vase; Jørn Starklint; Carolina C Graffe; Jesper N Bech; Søren Nielsen; Erling B Pedersen

Disclosures

BMC Nephrology. 2010;11 

In This Article

Methods

Participants

Inclusion Criteria: Both males and females; age 18–65 years; body mass index <30. Exclusion criteria: Clinical signs or history of disease in the heart, lungs, kidneys or endocrine organs; abnormal laboratory tests (blood hemoglobin, white cell count, platelet counts, plasma concentrations of sodium, potassium, creatinine, albumine, bilirubine, alanine-aminotransferase, and cholesterol; blood glucose and albumin and glucose in urine); malignancies; arterial hypertension (i.e. casual blood pressure >140/90 mmHg); alcohol abuse (more than 21 drinks per week for males and more than 14 for females); medical treatment; pregnancy; breast-feeding; lack of oral contraceptive treatment to women in the fertile age; intercurrent diseases; problems with blood sampling or urine collection; allergy to ibuprofen; medicine abuse; donation of blood less than 1 month before the study; and unwillingness to participate. Withdrawal criteria: Development of one or more of the exclusion criteria.

Ethics

The Scientific Committee of Ringkøbing, Ribe and Southern Jutland Counties (j.no.2623–04) approved the study. All participants received written information and gave their consent by signature. The study was carried out in compliance with the Helsinki Declaration.

Design

The study was randomized, placebo-controlled, double blind, and over-crossed. There was a time interval of 2 weeks between the two examinations. Each examination took three days.

Recruitment

Participants were recruited by advertisements in public and private institutions.

Diet and Fluid Intake

The normal energy requirement was calculated with the formula: weight (kg) * 100 (kJ) * activity factor (AF). AF ranged from 1.3 to 2.4 with a possible extra of 0.3 for physical activity in the spare time, e.g. 30 min. of sport 5–6 times a week. AF of 1.3 indicates no physical activity. AF of 1.4–1.5 indicates sedentary work without physical activity in the spare time. AF of 1.6–1.7 indicates work with walking during work hour and/or physical activity in the spare time, AF of 1.8–1.9 corresponds to shop assistant, (standing/walk all day), AF 2.0–2.4 indicates hard physical activity with or without physical activity in the spare time. The food had a specified energy amount with carbonhydrates (55% of the total energy), protein (15% of the total energy), and fat (30% of the total energy). The diet contained three main meals and three small meals. The participants were not allowed to add any spices or sodium to the meals or to divide the meals into bigger or smaller portions. The participants drank tap water 35 mL/kg each 24-hour and nothing else. They maintained normal physical activity during the study.

Procedure

Subjects were studied on three consecutive days. Table 1 shows the experimental design.

Day 1 (24 hours): The participants ate the specified diet, drank tap water 35 mL/kg body weight, and maintained normal physical activity.

Day 2 (24 hours): The participants fasted, drank tap water 35 mL/kg body weight maintained normal physical activity, and collected urine during 24 hours. They received a tablet of 600 mg ibuprofen or placebo at 07.00 a.m., 03.00 p.m., 11.00 p.m., and the next morning at 07.00 a.m.

Day 3: The participants arrived at 7:30 a.m. in the laboratory. An intravenous catheter was placed in fossa cubiti on each side; one for collection blood samples, the other for infusion of the 51 Cr-EDTA and hypertonic saline. Urine was collected from 7:30–9:30 to measure the effect variables after a period of 24 hours of fasting. Afterwards, urine was collected in the following seven periods: 09:30–10:00 a.m. (P-1), 10:00–10:30 a.m. (P-2), 10:30–11:00 a.m. (P-3), 11:00–11:30 a.m. (P-4), 11:30–12:00 a.m. (P-5), 12:00–12:30 p.m. (P-6) and 12:30–01:00 p.m. (P-7). Urine was analyzed for u-AQP-2, u-ENaC, u-Osm, u-Na, u-K, u-creat, u-cAMP, u-PGE2, u-51Cr-EDTA. The subjects voided in the standing or sitting position. Otherwise, they were in the supine position during the examination. Blood samples were taken every 30 min. starting at 09.30 a.m. for analysis of p-AVP, p-Osm, p-K, p-Na, p-Crea, p-albumin, p-51Cr-EDTA. In addition, blood samples for measurements of p-Ang II, p-ANP, p-BNP, p-PRC, and p-ALDO were drawn at 08.00 a.m., 11.00 a.m., 12.00 p.m., and 01.00 p.m. A total amount of 350 mL blood was drawn during the each study day. The blood drawn at blood sampling was immediately substituted with isotonic saline. From 11.00 a.m. to 11.30 a.m. (P-4), 3% saline was infused, 7 ml/kg body weight. Blood pressure and pulse rate were measured every 30 min. during the examination.

The participants were weighed before (day 3 at 07:30 a.m.) and after the trial (01:30 p.m.).

Effect Variables

Main effect variables were u-AQP2 and u-ENaC. The other effect variables were u-PGE2, u-CAMP, p-AVP, CH2O urine volume, FENa, p-Osm, u-Osm, p-PRC, p-Aldo, p-ANP, p-BNP and p-Ang II.

Number of Participants

An increase in u-AQP2 of 0.2 ng/min was considered the relevant difference between ibuprofen and placebo. The standard deviation was estimated to be 0.15 ng/min. With a level of significance of 5% and a power of 90%, 14–15 healthy subjects needed to be included in the trial.

Test Substance

The Hospital Pharmacy conducted the randomization and prepared the medication. The tablets were kept in a sealed container with tablets for one day. Each tablet contained either ibuprofen 600 mg or placebo.

Measurements

Glomerular filtration rate was measured using the constant infusion clearance technique with 51Cr-EDTA as a reference substance.

U-AQP-2 was measured by radioimmunoassay as previously described, and antibodies were raised in rabbits to a synthetic peptide corresponding to the 15 COOH-terminal amino acids in human AQP2 to which was added an NH2-terminal cystein for conjugation and affinity purification.[10] Minimal detection level was 32 pg/tube. The coefficients of variation were 11.7% (inter-assay) and 5.9% (intra-assay).

U-c-AMP was measured using a kit obtained from R & D Systems, Minneapolis, MN, USA. Minimal detection level was 12.5 pmol/tube. The coefficients of variation were 6.9% (inter-assay) and 5.3% (intra-assay).

ENaC β was measured by a newly developed RIA. Urine samples were kept frozen at -20°C until assayed. ENaCβ was synthesized and purchased by Lofstrand Labs Limited - Gaithersburg, Maryland, USA, see Appendix. U-PGE 2 was measured by a kit from Assay Designs, Inc., Ann Arbor, MI, USA. The coefficients of variations were 10.9% (inter-assay) and 6.3% (intra-assay).

Blood samples were centrifuged for 15 minutes at 3000 rpm at 4°C. Plasma was separated from blood cells and kept frozen at -20°C until assayed. AVP, ANP, BNP, and Ang II were extracted from plasma with C18 Sep-Pak (Water associates, Milford, MA, USA), and subsequently determined by radioimmunoassays.[11,12] The antibody against AVP was a gift from Professor Jacques Dürr, Miami, FL., USA. Minimal detection level was 0.5 pmol/L. The coefficients of variation were 13% (inter-assay) and 9% (intra-assay). Rabbit anti-ANP antibody was obtained from Department of Clinical Chemistry, Bispebjerg Hospital, Denmark. Minimal detection level was 0.5 pmol/L, coefficients of variation were 12% (inter-assay) and 10% (intra-assay). Rabbit anti-BNP antibody without cross-reactivity with urodilatin and α-ANP was used. Minimal detection level was 0.5 pmol/L plasma. The coefficients of variation were 11% (inter-assay) and 6% (intra-assay). The antibody against Ang II was obtained from Department of Clinical Physiology, Glostrup Hospital, Denmark. Minimal detection level was 2 pmol/L. The coefficients of variation were 12% (inter-assay) and 8% (intra-assay).

Aldosterone in plasma was determined by radioimmunoassay using a kit from Diagnostic Systems Laboratories Inc., Webster, Texas, USA. Minimal detection level was 22 pmol/L. The coefficients of variations were 8.2% (inter-assay) and 3.9% (intra-assay).

PRC is determined by radioimmunoassay using a kit from CIS Bio International, Gif-Sur-Yvette Cedex, France. Minimal detection level was 1 pg/ml. The coefficients of variations were 14.5% (inter-assay) and 4.5% (intra-assay).

Plasma and urinary osmolality were measured by freezing-point depression (Advanced Model 3900 multisampling osmometer).

Blood pressure was measured with UA-743 digital blood pressure meter (A&D Company, Tokyo, Japan)

Plasma and urinary concentrations of sodium and potassium were measured by routine methods at the Department of Clinical Biochemistry, Holstebro Hospital, Denmark.

All clearances were standardized to a body surface area of 1.73 m2

Statistics

Statistical level of significance was P < 0.05 in all analyses. We used A General Linear Model with Repeated Measures for comparison between ibuprofen treatment and placebo during fasting when several measurements were done during the examination. A paired t-test was used for comparison between two groups. Bonferroni correction was used when appropriate. Values are given as mean ± SD

Comments

3090D553-9492-4563-8681-AD288FA52ACE
Comments on Medscape are moderated and should be professional in tone and on topic. You must declare any conflicts of interest related to your comments and responses. Please see our Commenting Guide for further information. We reserve the right to remove posts at our sole discretion.

processing....