Animals
Male Wistar rats (Experimental Medicine Center, Lublin, Poland) weighing 150–200 g, were housed under standard laboratory conditions (12 h light-dark cycle, standard humidity and temperature) with food and water available without limits. Every experimental procedure was performed between 7 a.m. and 1 p.m. All procedures presented in this study were in accordance with the international, national and institutional guidelines for the care and use of animals.
Chemical substances
L-kynurenine (sulfate salt), fenofibrate, gemfibrozil, dimethyl sulfoxide (DMSO), components of Krebs-Ringer buffer: sodium chloride, potassium chloride, magnesium sulfate heptahydrate, calcium chloride anhydrous, sodium phosphate dibasic dodecahydrate, sodium phosphate monobasic dihydrate, glucose, distilled water; substances for KATs analysis: Trizma base, acetic acid, pyridoxal 5′-phosphate hydrate, 2-mercaptoethanol, pyruvate and glutamine were purchased from Sigma-Aldrich. Substances used for high-performance liquid chromatography (HPLC) were from J.T. Baker Chemicals and from Sigma-Aldrich.
The analysis of KYNA production in rat kidney in vitro
Animals kidneys were collected immediately after decapitation and placed on ice. Every kidney was weighed and homogenized in preformed oxygenated Krebs-Ringer buffer at pH 7.4 (1:4; w/v). After that, 100 µL of kidney homogenate was put into test tubes, pre-filled with oxygenated Krebs-Ringer buffer (800 µL in every tube). Then, the homogenate was incubated for 2 h at 37°C in the presence of 10 µM Lkynurenine (50 µL) and one from the tested compound: fenofibrate or gemfibrozil (50 µL). Six concentrations of each drug were tested: 1 µM, 10 µM, 50 µM, 100 µM, 500 µM and 1 mM. Due to limited solubility each drug was dissolved in DMSO. For each set of experiments at least six independent tissue samples were analyzed. Homogenate incubation was stopped on ice by adding 1 N HCl (100 µL per each sample). All samples were centrifuged (15 133 × g, 15 min), then obtained supernatants were examined by the HPLC (Thermo Fisher Scientific HPLC system, ESA catecholamine HR-80, 3 µm, C18 reverse-phase column) and the KYNA level was assessed fluorometrically.
KAT I and KAT II activity analysis in rat kidney in vitro
KATs activity in rat kidney in vitro were quantified as described previously (Gramsbergen et al. 1992) with minor modifications. Shortly, to evaluate KAT I and KAT II activity, rat kidneys were immediately homogenized in dialysate buffer (1:9; w/v) containing 5 mM Tris-acetate buffer (pH 8.0) with 50 µM pyridoxal 5’phosphate and 10 mM 2-mercaptoethanol. Afterwards, kidney homogenate was centrifuged (15 133 × g, 15 min) and the obtained supernatant was dialyzed against 4 L of the dialysate buffer for 12 hours at 8°C in cellulose membrane dialysis tubing. Later, the obtained enzyme sample was incubated for 2 h at 37°C with L-kynurenine (2 µM), glutamine (2 mM) and selected fibrate (at 1 µM, 10 µM, 50 µM, 100 µM, 500 µM and 1 mM concentration) at pH 9.5 or 7.0, optimal for KAT I or KAT II activity, respectively. KAT I inhibitor, glutamine (2 mM) was given to samples to assess KAT II activity. The reaction was ended by putting all samples into ice cold bath. At the end samples were centrifuged and tested by the means of HPLC as samples from KYNA synthesis analysis. All assays were carried out in triplicates.
Statistical analysis
Presented data were expressed as mean ± standard deviation (SD), until stated otherwise. Differences between the means of the treatments were examined using one-way analysis of variance (one-way ANOVA) followed by Tukey’s multiple comparison test. Data were statistically analyzed with the use of GraphPad Prism 6. p < 0.05 was set as statistically significant.