Induction of UCP2 expression in the lung and in the spleen upon analgesia and sedation.

<p><b>A</b>, Western blot analysis of UCP2 (upper panel) and COX I (lower panel) expression in the lung of C57B6/J mice. Mice (5 per group) were injected with ketamine (20 mg/kg) in combination with the α2 adrenergic receptor agonist medetomidine (0.2 mg/kg). Fifteen minutes after induction of anaesthesia, the mice were awoken by injection of 0.4 mg/kg of the α2 adrenergic receptor antagonist atipamezole and euthanatized 2, 3 and 4 hours after arousal from anaesthesia. The lungs were collected and mitochondrial preparation and Western blot analysis were performed. <b>B</b>, Graphic representation of the UCP2/COX I ratio from the experiment in panel A. <b>C</b>, UCP2/COX I ratio in the lung of mice 3 hours after injection of 0.2 mg/kg, medetomidine (Met.), 30 min after intravenous injection of 20 mg/kg of ketamine (Ket. <i>i.v.</i>), 3 hours after intramuscular injection of 20 mg/kg ketamine (Ket. <i>i.m.</i>), or 3 hours after 5 min inhalation of 3.6% isoflurane (Isof.). COX I subunit was used to normalize the amount of mitochondrial proteins. <b>D</b>, UCP2 expression in the spleen of mice 3 hours after injection of 0.2 mg/kg medetomidine (Met.), 200 mg/kg of ketamine (Ket. 200), 20 mg/kg of ketamine (Ket. 20) or 5 min inhalation of 3.6% isoflurane (Isof.). Porin was used to normalize the amount of mitochondrial proteins.</p

UCP2 induction correlates with sedation-induced hypothermia.

<p>Mice were intramuscularly injected with medetomidine (0.2 mg/kg). A, Rectal body temperature was recorded at indicated time after injection. B, Mice (5 on each time point) were euthanized and lung mitochondria were analyzed for their UCP2/porin protein content by Western blot using the anti UCP2-605 and the anti porin antibodies.</p

Immune cells contribute to UCP2 expression in lung during analgesia or sedation.

<p><b>A</b>, Irradiated C57B6/J mice were transplanted with the bone marrow from <i>Ucp2^+/+</i> (C57B6t(<i>Ucp2^+/+</i>)) or <i>Ucp2^−/−</i> (C57B6t(<i>Ucp2^−/−</i>)) mice as described in (6). Two months after transplantation, the mice were intramuscularly injected with medetomidine (0.2 mg/kg) and ketamine (20 mg/kg). Fifteen minutes after anaesthesia, the mice were awoken by injection of atipamezole and euthanized 3 hours later. UCP2/COX I protein ratio was established by Western blot using the anti UCP2-605 and the anti COX I antibodies. <b>B</b>, Three hours after induction of anaesthesia or sedation, the awoken mice were intraperitoneally injected with pentobarbital (50 mg/kg) and bronchoalveolar lavage was immediately performed. Monocytes were identified and counted under a microscope.</p

Locomotion and body temperature of <i>Ucp2^−/−</i> mice during medetomidine induced sedation.

<p>One week before the start of the experiment, transponders were implanted in mice (five per group). A, Activity and B, body temperature of <i>Ucp2^+/+</i> and <i>Ucp2^−/−</i> 24 hours before medetomidine injection. Data were plotted every 2 hours. Mice were subsequently injected with medetomidine (180 µg/kg) and C, the physical activity and D, the body temperature were recorded every 5 min for 7 hours. Time 0 in panels B and D corresponds to the time of medetomidine injection. Note, using this measurement system, the detection limit of temperature was 30°C. * P<0.05 between genotypes for a given time.</p

<i>Ucp2</i>^−/− mice recover faster than wild type mice upon arousal from isoflurane-induced anaesthesia.

<p>Graphic representation of arousal and locomotion times of <i>Ucp2</i>^+/+ and <i>Ucp2</i>^−/− mice after isoflurane anaesthesia. Mice inhaled isoflurane 3.6% for five minutes in an anesthetic chamber. Anaesthesia was interrupted by taking the mice out of the chamber. The mice were laid down on their back and from this time-point, arousal (rotation of the mice onto their feet) and locomotion times were recorded.</p

Expression pattern of MITF-A during kidney development.

<p><b>A-B)</b><i>In situ</i> hybridization of <i>Mitf-A</i> of E13.5 kidneys from wild-type (WT) and homozygous (HO) MITF-A transgenic embryos using an antisense RNA probe directed against a sequence encompassing exon 1A, specific for <i>Mitf-A</i>, and exon 1B common to <i>Mitf-A</i>, <i>Mitf-H</i>, <i>Mitf-C</i>, <i>Mitf-J</i> and <i>Mitf-Mc</i> isoforms. The inset shows the staining of E13.5 kidneys using the sense RNA probe. Magnifications are X100 (left panels), X200 (middle panels) and X400 (right panels). In WT kidneys <b>(A)</b> a weak staining is observed in branches of UB (black arrow), in S-shaped body (blue arrow) and in metanephric mesenchyme (asterisk). Consistent with the use of the Ksp-cadherin promoter, the signal in MITF transgenic kidneys <b>(B)</b> was strongly increased in UB and tips (black arrow), in ureteric tip (black arrow) and to a lesser extent in S-shaped body (blue arrow). <b>C)</b> <i>In situ</i> hybridization of <i>Mitf-A</i> in transgenic HO kidneys after laminin immunohistochemistry (red). Note <i>Mitf</i> expression in ureteric bud and tip (black arrow), in and S-shaped body (blue arrow). Magnification X400. Sections are representative images of 4 kidneys per genotype. <b>D</b>) Immunostaining of MITF-A in WT and HO MITF-A transgenic metanephroi at E13.5. Note the increase of MITF-A expression in UB stalks, tips and S-bodies. Magnification X400.</p

Characterization of MITF-A transgenic mice.

<p>Characterization of MITF-A transgenic mice.</p

<i>Mitfa</i> inactivation results in reduced glomeruli number.

<p><b>A)</b> Schematic representation of the targeting strategy used to inactivate <i>Mitfa</i>. <b>B-C)</b> <i>Mitf-A</i> <b>(B)</b> and total <i>Mitf</i> mRNA <b>(C)</b> expression evaluated by quantitative RT-PCR in kidneys from 2 months-old <i>Mitfa</i>^<i>+/+</i> and <i>Mitfa</i>^<i>-/-</i> mice. <b>D)</b> Glomerular number in kidneys from 2 months-old <i>Mitfa</i>^<i>+/+</i> and <i>Mitfa</i>^<i>-/-</i> mice. Data are means ± SEM, n = 8–10 per each genotype. Mann-Whitney test; <i>Mitfa</i>^<i>-/-</i> <i>versus Mitfa</i>^<i>+/+</i>: *** P < 0.001.</p

Generation of MITF-A transgenic mice.

<p><b>A)</b> Schematic representation of the Ksp-cadherin-FLAG-MITF-A transgene. <b>B)</b> <i>Mitf-A</i> mRNA expression evaluated by quantitative RT-PCR in kidneys from wild-type (WT), heterozygous (HE) and homozygous (HO) MITF-A transgenic mice (line 42) 2 months after birth. Data are means ± SEM; n = 4–6 per each genotype. ANOVA followed by Tukey-Kramer test; transgenic <i>versus</i> wild-type mice: ** P < 0.01, *** P < 0.001. <b>C)</b> MITF-A protein expression evaluated by western blot on kidney nuclear protein extracts from WT, HE and HO MITF-A transgenic mice 2 months after birth. This is a representative image of three experiments. Nuclear protein extracts from <i>Mitfa</i>^-/- kidneys were used as a negative control; crude extracts from renal cells transfected with either FLAG-MITF-A plasmid (lane 1) or MITF-A plasmid (lane 2) were used as a positive control. Lamin A/C was used as control of nuclear protein amount. IB = immunoblot.</p

Expression of candidate MITF-A targets in E13.5 kidneys.

<p><b>A)</b><i>In situ</i> hybridization of <i>Bmp7</i>, <i>Pax2</i> and <i>Wnt9b</i> in wild-type (WT) and homozygous (HO) MITF-A transgenic kidneys at E13.5 (magnification X200, n = 5–6 per genotype). <b>B)</b> Quantitative RT-PCR analysis of <i>Bmp7</i>, <i>Pax2</i> and <i>Wnt9b</i> mRNA expression in E13.5 kidneys of WT, heterozygous (HE) and HO MITF-A transgenic embryos (n = 6–9 per genotype). <b>C)</b> <i>In situ</i> hybridization of <i>Re</i>t, <i>Wnt11</i> and <i>Spry1</i> in WT and HO MITF-A transgenic kidneys at E13.5 (magnification X200, n = 5–6 per genotype). Note the increased staining of <i>Re</i>t mRNA in transgenic kidneys at E13.5. <b>D)</b> Quantitative RT-PCR analysis of <i>Re</i>t, <i>Wnt11</i> and <i>Spry1</i> mRNA expression in E13.5 kidneys of WT, HE and HO MITF-A transgenic embryos (n = 6–9 per genotype). Data are means ± SEM. ANOVA followed by Tukey-Kramer test; transgenic <i>versus</i> wild-type mice: * P < 0.05, ** P < 0.01.</p