Abnormal Function of the Vasopressin-cyclic-AMP-aquaporin2 Axis During Urine Concentrating and Diluting in Patients with Reduced Renal Function. A Case Control Study

Erling B Pedersen; Ingrid M Thomsen; Thomas G Lauridsen


BMC Nephrology. 2010;11(26) 

In This Article



Healthy Control Subjects Inclusion criteria: Both men and women; age 18- 65 years; body mass index < 30. Exclusion criteria: Clinical signs or history of diseases 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; 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, and problems with blood sampling or urine collection.

Patients Inclusion criteria: Both men and women; age 18- 75 years; a diagnosis of hypertension or an estimated GFR (e-GFR) between 15–90 ml/min. Exclusion criteria: heart failure; heart arrhythmias; pulmonary insufficiency; liver disease with plasma alanine aminotransferase >100 U/L; previous cerebrovascular insult; diabetes mellitus; other endocrine diseases than diabetes mellitus not satisfactorily controlled; malignancies; alcohol abuse (more than 21 drinks per week for males and more than 14 for females); abuse of medicine; pregnancy; other diseases or conditions prohibiting participation in the trial; unwillingness to participate. Withdrawal criteria: Development of one or more of the exclusion criteria, and problems with blood sampling or urine collection.


The investigation was as a case-control study.


The local Medical Ethics Committee approved the study (j. no. 2580–04). All participants received written information and gave their consent by signature. The study was carried out in compliance with the Helsinki Declaration.


Healthy participants were recruited by advertisements in public and private institutions in Holstebro. Patients with primary hypertension or chronic renal failure were recruited from the Out-patients' Clinic, Department of Medicine, Section of Nephrology, Holstebro Hospital.

Experimental Procedure

An ambulatory 24 hours blood pressure was made before the participants began the study procedure.

The study started 36 hours before the participants arrived in the laboratory. Part 1(Control period): Urine was collected during 24 hours from 07.30 p.m.-07.30 p.m. The subjects maintained their normal daily activities, ate their usual diet, and had usual fluid intake. They followed their normal medical prescriptions. Part 2: (Urine concentrating test): From 07.30 p.m., the night before arriving in the laboratory, until 07. 30 a.m. the next day, a 12 hours urine collection was done, followed by one hour urine collection from 07.30 to 08.30, while the participants fasted and thirsted. Part 3 (Urine dilution test): Urine was collected in the following 6 periods: 08.30–09. 30 a. m.; 09.30–10. 30 a. m.; 10.30–11.30 a. m.; 11.30 a.m. -00.30 p. m.; 00.30–01.30 p.m.; 01.30–02.30 p.m. An oral water load (tap water, 20 ml per body kg weight) was given from 9.30- 9.45 a.m.

The participants arrived at 7.30 a.m. in the laboratory. An intravenous catheter was inserted in a vein in fossa cubiti in one arm for collecting blood samples every hour. The participants were in the supine position except during voiding, which took place in the sitting or standing position. A light meal consisting of one slice of toast with jam was served at 08.30 a.m. All morning medication was postponed to the end of the study.

Blood samples were drawn for measurements of p-AVP, p-Osm, p-creatinine every hour, starting at 08.30 a.m. In addition, blood samples were drawn for determination of b-hemoglobin, p-sodium, p-potassium, p-albumin, p-glucose, p-ALAT, and for women a pregnancy test, at 08.30 a.m.

Urine samples were analyzed for u-AQP2, u-Osm, u-creatinine, and u-c-AMP. Blood pressure and pulse rate were measured once every hour during the examination.

Effect Variables

The main effect variable was u-AQP2, and other effect variables were u-Osm, p-AVP, and u-c-AMP, UV and CH2O.

Number of Subjects

Using a significance level of 5% and a power of 90%, it could be calculated that the number of subjects in each group should 10–15, when the minimal relevant difference in u- AQP2 was 0.3 ng/min and SD was 0.2 ng/min.


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.[13] 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).

Blood samples for determination of p-AVP 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 was extracted from plasma with C18 Sep-Pak (Water associates, Milford, MA, USA), and subsequently determined by radioimmunoassay.[14] 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).

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 creatinine was measured by routine methods at the Department of Clinical Biochemistry, Holstebro Hospital, Denmark.

The estimated glomerular filtration rate (e-GFR) was calculated according to the following formulas:[15]

Free water clearance was calculated according to the formula CH2O =UV*u-Osm/p-Osm, where CH2O is free water clearance in ml/min, UV is urine volume in ml/min, u-Osm is urine osmolarity mosmol/l, and p-Osm is plasma osmolarity in mosmol/l.


Statistical level of significance was P < 0.05 in all analyses. Kruskal-Wallis's test and Friedman's tests were used to analyze differences between several unpaired and paired groups, respectively. Mann-Whitney's test and Wilcoxon's signed rank test were used to analyze differences between two unpaired and paired groups, respectively. For the urine diluting test, we used A General Linear Model with Repeated Measures for comparison between and within groups. Spearman's rho test or Pearson's test were used for analysis of correlations between to variables. Multiple regression analysis was used to measure the influence of several effect variables on the amount of water excreted during urine diluting test. Values are given as medians with interquartile ranges in brackets, or as median with 25 and 75 quartiles. SPSS version 11.5 was used for the statistical analyses.


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