Production and genotyping of COMT gene deleted mice
The COMT gene-deleted strain was produced as previously described [28]. In brief, the gene encoding COMT was disrupted by replacing part of the fragment with a cassette including the neo gene. The construct was introduced into the genome of embryonic stem (ES) cells of 129/Sv mice by means of homologous recombination. ES cells with positive homologous recombination were selected by neomycin resistance (~15 %) and were injected into C57B6 early mouse embryos in the blastocyst stage. Chimeric males (mice containing both cells from the microinjected ES cell and the host embryo) were mated with C57B6 females and in cases where the injected recombinant ES cells had contributed to the germ line of the chimeric mouse, the mating resulted in heterozygous (HT) 129/Sv/C57B6 offspring with respect to the target gene. HT animals were mated and mice of all three genotypes were obtained (F1 generation). Each mouse was then typed by southern blot analysis [28]. DNA from tail biopsies was extracted and digested with Eco RV, and fragments were separated on a gel. The fragments were blotted with two different radioactively marked probes and on the exposed films an 11.5 kb fragment was seen in wild type (WT) samples, a 3.5 kb signal in homozygous (HM) genotypes and both of these in HT genotypes.
Animal procedures
The experiments were approved by the Ethics Committee for Animal Experimentation at the University of Uppsala. The study was performed on a total of 66 mice: 11 WT, 13 HT and 8 HM male COMT gene deleted mice, weighing 31.0 ± 0.9, 30.8 ± 1.0 and 30.4 ± 1.3 g, respectively, and 10 WT, 15 HT and 9 HM female such mice, weighing 23.7 ± 0.5, 24.4 ± 0.5 and 23.0 ± 0.3 g, respectively. Up to the day of the experiment all animals had free access to tap water and standardised chow (R3, Ewos, Södertälje, Sweden) containing 0.3 % sodium, 0.8 % potassium and 21 % protein. Anaesthesia was induced by placing the mice in a polystyrene box with isoflurane flowing through it (ForeneR, Abbott Scandinavia AB, Kista, Sweden). After induction, the animals were placed in the supine position on a thermostatically controlled surgical table to maintain the body temperature at 37°C and a breathing mask was placed over the head. The inhalation gas contained ~2 % isoflurane in air with 37.5 % O2 during surgery and the amount of isoflurane was subsequently adjusted to maintain the respiratory rate and MAP. Polyethylene catheters were inserted into the right femoral vein and artery, the former for infusion of isotonic saline (0.15 M NaCl) and the latter for continuous monitoring of mean arterial blood pressure (MAP) and for blood sampling. The urinary bladder was catheterised through a suprapubic incision for urine collection. After the experimental procedures described below and excision of the kidneys, all animals were euthanised by an intravenous injection of saturated KCl.
Protocol
After completion of the surgical procedures, the mice received an intravenous bolus infusion of 0.08 ml isotonic saline to replace fluid losses during surgery. This bolus dose contained 0.5 μCi of [3H]methoxy-inulin. A maintenance infusion of 0.15 M NaCl containing 2.5 μCi·ml-1 of inulin was administered at a rate of 0.5 ml·h-1. After a 45 min stabilisation period and 60 min of control sampling (2 × 30 min, C1-C2), the mice were divided into two groups. One group was given isotonic saline (0.15 M NaCl, n = 11 WT, n = 14 HT and n = 8 HM) continuously at a rate of 1.5 ml·h-1 (ECV expansion, 5% of body weight) during four 30-min observation periods (E1-E4). The other group, the control animals, (n = 10 WT, n = 12 HT and n = 9 HM) received isotonic saline at the maintenance infusion rate (0.5 ml·h-1) throughout the study (E1-E4). Because of the small blood volume in the mouse (~1.5 ml), blood samples for GFR estimation (~15 μl) were only drawn at two time points during the experiment (C2 and E4). Before the animal was killed, the kidneys were excised, placed on carbon-dioxide ice and sliced under a microscope into sections of cortex, outer medulla and papilla; the pieces were frozen in a mixture of alcohol and carbonic ice and subsequently stored at -70°C pending assay of COMT and MAO activities.
Urine analysis
The urine volumes were measured gravimetrically. The urinary sodium concentration (UNa) was determined by flame photometry (FLM3, Radiometer, Copenhagen, Denmark). For assay of DA, DOPAC and norepinephrine (NE), urine samples were immediately transferred to polyethylene vials containing 1 ml 0.4 M perchloric acid (PCA), 0.1 ml 10 % EDTA-Na2 and 0.05 ml 5 % Na2S2O5 and immediately frozen to -70°C. The DA, DOPAC and NE contents were measured electrochemically following alumina adsorption and ion-pair, reverse-phase high-performance liquid chromatography (HPLC, [29]). An internal standard (3,4-dihydroxybenzylamine) was used and all values were corrected for its recovery. Recovery through the alumina extraction step was determined from aqueous mixtures of catecholamines.
The amount of [3H]methoxy-inulin in samples of plasma and urine was determined in a liquid scintillation counter. The glomerular filtration rate (GFR) was estimated from the clearance of [3H]methoxy-inulin (Cin) according to the equation:
Cin = (Uin · V) / Pin
where Uin and Pin are the urinary and plasma concentrations, respectively, of inulin and V is the urine flow rate.
Measurements of COMT and MAO activities
COMT and MAO activities were measured in renal sections of cortex, outer medulla and papilla (prepared as described under Protocol).
COMT activity was measured by electrochemical detection of the reaction products formed from the substrate dihydroxybenzoic acid by COMT using HPLC, as previously described in detail [30, 31]. In short, 100 μl enzyme preparation was incubated for 30 min at 37°C in the presence of 240 μM dihydroxybenzoic acid, 5 mM MgCl2 and 200 μM SAM in 100 mM sodium phosphate buffer, pH 7.4. The reaction was terminated with ice-cold PCA (4 M, 25 μl) and centrifuged for 10 min at 5530 × g at 4°C. The supernatants were subjected to HPLC analysis for vanillic acid and isovanillic acid.
Determination of MAO activity was based on the detection of hydrogen peroxide in a horseradish peroxidase-coupled reaction using 10-acetyl-3,7-dihydroxy-phenoxazine, a probe for hydrogen peroxide [32, 33]. An AmplexTM Red Monoamine Oxidase Assay Kit (A-12214, Molecular Probes) was used for measurement of the activity. One hundred microlitres of supernatant was mixed with 100 μl reaction buffer containing 50 mM phosphate buffer, pH 7.4, horseradish peroxidase 2 U·ml-1, 400 μM 10-acetyl-3,7-dihydroxy-phenoxazine and 2 mM p-tyramine as a substrate of both MAO-A and B. The increase in fluorescence intensity (excitation 530/25 nm, emission 645/40 nm) was measured with a fluorescence microplate reader, with resorufin used as standard.
Statistical analysis
All data are presented as means ± 1 standard error of the mean (SEM). Differences within and between groups were tested for significance by two-way analysis of variance (ANOVA) followed by the Tukey test (STATISTICA, StatSoft, Tulsa, OK, USA) or by an unpaired t-test. A p value of less than 0.05 was adopted as statistically significant. No gender differences in excretory data or enzymatic activities were found, and results for males and females of the same genotype and treatment group were therefore pooled. Mice with an MAP below 70 mmHg were excluded from the study.