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Decreased expression of monocarboxylate transporter 1 and 4 in the branching airway epithelium of nitrofen-induced congenital diaphragmatic hernia
Journal of Pediatric Surgery xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Rosenkrantz Podium Session
Decreased Expression of Monocarboxylate Transporter 1 and 4 in the Branching Airway Epithelium of Nitrofen-Induced Congenital Diaphragmatic Hernia Toshiaki Takahashi a, Florian Friedmacher a, Julia Zimmer a, Prem Puri a,b,⁎ a b
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland Conway Institute of Biomolecular and Biomedical Research, School of Medicine & Medical Science, University College Dublin, Dublin, Ireland
a r t i c l e
i n f o
Article history: Received 10 February 2016 Accepted 26 February 2016 Available online xxxx Key words: Monocarboxylate transporters Lung branching morphogenesis Pulmonary hypoplasia Congenital diaphragmatic hernia Nitrofen
a b s t r a c t Background/Purpose: Monocarboxylate transporters (MCTs) are crucial for the maintenance of intracellular pH homeostasis in developing fetal lungs. MCT1/4 is strongly expressed by epithelial airway cells throughout lung branching morphogenesis. Functional inhibition of MCT1/4 in fetal rat lung explants has been shown to result in airway defects similar to pulmonary hypoplasia (PH) in congenital diaphragmatic hernia (CDH). We hypothesized that pulmonary expression of MCT1/4 is decreased during lung branching morphogenesis in the nitrofen model of CDH-associated PH. Methods: Timed-pregnant rats received nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested on D15, D18, and D21, and divided into control and nitrofen-exposed group. Pulmonary gene expression levels of MCT1/4 were analyzed by qRT-PCR. Immunofluorescence staining for MCT1/4 was combined with E-cadherin in order to evaluate protein expression in branching airway tissue. Results: Relative mRNA levels of MCT1/4 were significantly reduced in lungs of nitrofen-exposed fetuses on D15, D18, and D21 compared to controls. Confocal laser scanning microscopy confirmed markedly decreased immunofluorescence of MCT1/4 in distal bronchial and primitive alveolar epithelium of nitrofen-exposed fetuses on D15, D18, and D21 compared to controls. Conclusion: Decreased expression of MCT1/4 in distal airway epithelium may disrupt lung branching morphogenesis and thus contribute to the development of PH in the nitrofen-induced CDH model. © 2016 Elsevier Inc. All rights reserved.
Congenital diaphragmatic hernia (CDH) is a relatively common malformation, affecting 1 in 2500 live births [1]. The main cause of high mortality and morbidity in newborns with CDH is severe pulmonary hypoplasia (PH) and persistent pulmonary hypertension [2]. Hypoplastic lungs are characterized by immaturity and smaller size with a decreased number of terminal airways, thickened alveolar walls, increased interstitial tissue, diminished alveolar airspaces and reduced gas-exchange surface area [3,4]. In general, fetal lung development takes place in a relatively hypoxic environment, thus generating intracellular acids, which in turn leads to a decrease in intracellular pH [5,6]. The low oxygen environment of the fetus has been shown to be essential for normal lung branching morphogenesis [7]. Furthermore, it has been reported that disruption of the cellular response to oxygen alterations results in a significantly reduced distal airway branching [8]. Although the pathological mechanisms of PH have been extensively studied, the molecular basis
⁎ Corresponding author at: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland. Tel.: +353 1 409 6420; fax: +353 1 455 0201. E-mail address: [email protected] (P. Puri).
of disrupted lung branching morphogenesis in CDH remains largely unclear. Monocarboxylate transporters (MCTs) have been demonstrated to be crucial for the maintenance of intracellular pH homeostasis in developing fetal lungs by transporting intracellular acids across the plasma membrane [9–11]. MCT1 and MCT4 are strongly expressed in distal bronchial and primitive alveolar epithelial cells during lung branching morphogenesis, indicating their important role in the formation of fetal airways [12]. In addition, functional inhibition of MCT1 and MCT4 in fetal rat lung explants has been shown to result in severe defects in lung branching with a reduced number of distal airway buds similar to the phenotype of PH in human CDH [12]. Most of our current knowledge about the structural and molecular changes in CDH derives from experimental animal models [13]. Administration of the herbicide nitrofen (2,4-dichloro-phenyl-p-nitrophenyl ether) to pregnant rats on gestational day 9 (D9) has been demonstrated to result in PH and diaphragmatic defects in the offspring, both remarkably similar to human CDH [14,15]. We designed this study to investigate the hypothesis that pulmonary expression of MCT1 and MCT4 is decreased during lung branching morphogenesis in the nitrofen model of CDH-associated PH.
http://dx.doi.org/10.1016/j.jpedsurg.2016.02.050 0022-3468/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Takahashi T, et al, Decreased Expression of Monocarboxylate Transporter 1 and 4 in the Branching Airway Epithelium of Nitrofen-Induced Congenital Diap..., J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.02.050
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T. Takahashi et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx
1. Material and methods 1.1. Animals, drugs and experimental design Pathogen-free adult Sprague–Dawley rats® (Harlan Laboratories, Shardlow, UK) were mated overnight, and females were checked daily for spermatozoids in their vaginal smear. The day of observation was considered as proof of pregnancy and was defined as embryonic day 0.5 (D0.5). Timed-pregnant animals were randomly divided into two experimental groups: “Nitrofen” and “Control”. On gestational day 9 (D9), dams were briefly anesthetized with 2% volatile isoflurane (Piramal Healthcare Ltd., Morpeth, UK) and either 100 mg of nitrofen (WAKO Chemicals GmbH, Neuss, Germany), dissolved in 1 ml of olive oil, or vehicle alone was administered via oral-gastric lavage. On the selected time-points D15, D18 and D21, animals were anesthetized and their fetuses were delivered via caesarean section. After laparotomy, fetal diaphragms were inspected under a Leica S8AP0 stereomicroscope (Leica Microsystems AG, Heerbrugg, Switzerland) for CDH (Fig. 1). Whole lungs of nitrofen-exposed fetuses with a diaphragmatic defect (n = 12 per time-point) and controls (n = 12 per time-point) were dissected under sterile conditions via thoracotomy and stored either in a TRIzol® reagent (Invitrogen, Carlsbad, USA) for total RNA isolation or fixed in 10% paraformaldehyde (PFA) (Santa Cruz Biotechnology Inc., Heidelberg, Germany) for immunofluorescence-double-staining. All animal procedures were carried out according to the current guidelines for management and welfare of laboratory animals and the experimental protocol was approved by the local research ethics committee (REC668b) and the Department of Health and Children (Ref. B100/4378) under the Cruelty to Animals Act, 1876 (as amended by European Communities Regulations 2002 and 2005). 1.2. Total RNA isolation from fetal rat lungs Total RNA was isolated from fetal lungs with the acid guanidinium thiocyanate-phenol-chloroform extraction method using a TRIzol® reagent according to the manufacturer's protocol. Spectrophotometrical quantification of total RNA was performed with a NanoDrop ND-1000 UV–Vis® Spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, USA). 1.3. Complementary DNA synthesis and quantitative real-time polymerase chain reaction Reverse transcription of total RNA was carried out at 85 °C for 3 min (denaturation), at 44 °C for 60 min (annealing), and at 92 °C for 10 min (reverse transcriptase inactivation) using a Transcript High Fidelity cDNA Synthesis Kit® (Roche Diagnostics, Grenzach-Whylen, Germany) according to the manufacturer's instruction. The resulting
cDNA was used for quantitative real-time polymerase chain reaction (qRT-PCR) using a LightCycler® 480 SYBR Green I Master Mix (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol. Gene-specific primer pairs are listed in Table 1. After an initialization phase at 95 °C for 5 min, 55 amplification cycles were carried out. Each cycle included an initial denaturation step at 95 °C for 10 s, an annealing step at 60 °C for 15 s and an elongation step at 72 °C for 10 s. The final elongate temperature was 65 °C for 1 min. Relative mRNA expression levels of MCT1 and MCT4 were measured with a Light Cycler® 480 instrument (Roche Diagnostics, West Sussex, UK) and gene levels were normalized to the housekeeping gene β-actin. All experiments were run duplicated for each sample and primer pair.
1.4. Immunofluorescence double staining and confocal laser scanning microscopy Following overnight fixation in 10% PFA, fetal lungs were paraffinembedded, transversely sectioned at a thickness of 5 μm, and mounted on polylysine-coated slides (VWR International, Leuven, Belgium). Tissue sections were deparaffinized with xylene and rehydrated through ethanol and distilled water. To improve cell permeabilization, sections were incubated with phosphate-buffered saline (PBS) containing 1.0% Triton X-100 (Sigma Aldrich Ltd., Arklow, Ireland) for 20 min at room temperature. Sections were then washed in PBS + 0.05% Tween (Sigma Aldrich, Saint Louis, USA) and subsequently blocked with 3% bovine serum albumin (Sigma Aldrich, Saint Louis, USA) for 30 min to avoid non-specific absorption of immunoglobulin. The blocking solution was rinsed off and sections were incubated with primary antibodies either against MCT1 (goat polyclonal, sc-14,917, 1:100) or MCT4 (rabbit polyclonal, sc-50,329, 1:100) and E-cadherin (mouse polyclonal, sc8426, 1:100) (Santa Cruz Biotechnology Inc., Heidelberg, Germany) overnight at 4 °C. On the next day, sections washed in PBS + 0.05% Tween and incubated with corresponding secondary antibodies (donkey anti-goat Alexa 555-A21432, 1:250, donkey anti-rabbit Alexa 647A150067, 1:250 and donkey anti-mouse Alexa 488-A150109, 1:250) (Abcam plc, Cambridge, UK) for 1 h at room temperature. After another washing step in PBS + 0.05% Tween, sections were counterstained with a DAPI antibody (10,236,276,001, 1:1000) (Roche Diagnostics GmbH, Mannheim, Germany) for 10 min, washed again, and mounted with glass coverslips using Sigma Mounting Medium (Sigma-Aldrich, St. Louis, MO, USA). All sections were scanned with a ZEISS LSM 700 confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) and independently evaluated by two investigators.
1.5. Statistical analysis All numerical data are presented as means ± standard error of the mean. Differences between two groups were tested using an unpaired Student's t test when the data had normal distribution or a Mann–Whitney U test when the data deviated from normal distribution. Statistical significance was accepted at P values of less than 0.05.
2. Results 2.1. Relative mRNA expression levels of MCT1 and MCT4 in fetal rat lungs
Fig. 1. Diaphragmatic defect in the nitrofen-induced rat on gestational day 21.
Following qRT-PCR, the relative mRNA expression levels of MCT1 and MCT4 were significantly reduced in CDH lungs of nitrofenexposed fetuses on D15 (0.23 ± 0.10 vs. 0.67 ± 0.37; P b 0.05 and 0.17 ± 0.04 vs. 0.25 ± 0.08; P b 0.05), D18 (0.44 ± 0.23 vs. 0.69 ± 0.23; P b 0.05 and 0.10 ± 0.07 vs. 0.18 ± 0.04; P b 0.05) and D21 (0.24 ± 0.13 vs. 0.61 ± 0.37; P b 0.05 and 0.10 ± 0.05 vs. 0.19 ± 0.06; P b 0.05) compared to controls.
Please cite this article as: Takahashi T, et al, Decreased Expression of Monocarboxylate Transporter 1 and 4 in the Branching Airway Epithelium of Nitrofen-Induced Congenital Diap..., J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.02.050
T. Takahashi et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx Table 1 Primer sequences for quantitative real-time polymerase chain reaction. Gene MCT1 Forward Reverse MCT4 Forward Reverse β-actin Forward Reverse
Sequence (5ۥ-3)ۥ
Product size (bp)
ATG TTG TCC TGT CCT CCT GG TAG AGG CCT GCG ATG ATG AG
114
CCA GCA TGC TCC TAA GAC CT GCG GTT TGG TGT TTC TGT CT
122
TTG CTG ACA GGA TGC AGA AG TAG AGC CAC CAA TCC ACA CA
108
2.2. Immunofluorescence evaluation of MCT1, MCT4 and E-cadherin in fetal rat lungs Immunofluorescence staining for MCT1 and MCT4 was combined with the epithelial marker E-cadherin in order to evaluate MCT1 and MCT4 protein expression and localization in the branching airway tissue of the fetal rat lungs. Confocal laser scanning microscopy revealed a co-expression of MCT1 and MCT4 with E-cadherin, and confirmed the qRT-PCR results by showing a markedly diminished MCT1 (Fig. 2) and MCT4 (Fig. 3) expression in the distal bronchial and primitive alveolar epithelium of nitrofen-exposed CDH lungs on D15, D18 and D21 compared to controls. 3. Discussion Hypoxia is a normal component of fetal lung development and has been demonstrated to be one of the most important extracellular factors that stimulate the complex process of distal airway branching [10]. The low oxygen level during intrauterine fetal growth is essential for a normal lung epithelial branching morphogenesis in the developing fetus [16,17]. Disruption of the cellular response to oxygen alterations has been shown to result in a significant reduction of branching airways [8]. In a hypoxic environment, increased glycolytic metabolism generates intracellular acids, thus leading to a decrease in intracellular pH [5,6]. These acids have an important role in cell metabolism and their rapid transport across the plasma membrane is crucial for pH homeostasis [18]. This transport is carried out by a family of transporters, designated by MCT isoform 1 and 4 [19]. Several studies have demonstrated the expression of MCTs during fetal development of humans and rodents [9]. The importance of MCTs has been described in numerous organs, including the lung, during fetal rat maturation [20]. Granja et al. [12] have recently reported on the expression pattern of MCT1 and MCT4 during lung branching morphogenesis and performed studies using a fetal rat lung explant model in order to understand the influence of MCT inhibition on airway
3
formation. They demonstrated that in normal fetal lungs, MCT1 and MCT4 isoforms are strongly expressed in distal bronchial and primitive alveolar epithelial cells throughout lung branching morphogenesis, indicating their important role in fetal airway development [12]. Furthermore, functional inhibition of MCT1 and MCT4 in fetal rat lung explants has been shown to result in severe defects in lung branching with reduced number of distal airway buds similar to the phenotype of PH in human CDH [12]. In the present study, we found that the relative mRNA levels of MCT1 and MCT4 were significantly reduced in CDH lungs of nitrofen-exposed fetuses on D15, D18 and D21 compared to controls. In addition, immunofluorescence staining demonstrated a markedly diminished MCT1 and MCT4 expression in the distal bronchial and primitive alveolar epithelium within branching airways of nitrofen-exposed CDH lungs on D15, D18 and D21. Hence, these results confirmed that the quantitative decrease in pulmonary MCT1 and MCT4 mRNA transcripts were also translated to the protein level. A decreased expression of MCT1 and MCT4 in the distal airway epithelium may therefore disrupt the fetal lung branching morphogenesis and contribute to the development of PH in the nitrofen-induced CDH model. These findings thus provide new insights into the pathomechanisms underlying PH in CDH. Given the frequency with which CDH occurs, an understanding of the genetic, cellular and morphogenetic mechanisms regulating lung development, both normally and during herniation, is critical. Many fundamental questions about lung development remain unanswered. The nitrofen model currently appears to be the best available experimental model to study the pathogenesis of CDH. The timing of the developmental insult is remarkably similar to the human situation as well as the PH. The research using nitrofen model should help determine the potential mechanisms that lead to the development of diaphragmatic defect and associated PH in CDH. As with other congenital anomalies, an improved understanding of the pathogenesis of PH may help to design new treatment modalities targeted at specific developmental insults. In the present study, we demonstrated that decreased expression of MCT1 and MCT4 in the distal airway epithelium may disrupt fetal lung branching morphogenesis and contribute to the development of PH in the nitrofen-induced CDH model. It has been reported that HIF-1 upregulates MCT4 and c-Myc up-regulates MCT1 [21,22]. It has also been described that MCT1 and 4 require type I transmembrane glycoprotein CD147/BASIGIN (BSG) for proper folding, stability and trafficking to the cell surface [19].We now propose to evaluate the pulmonary expression of HIF-1 and C-Myc in this model to confirm that MCT pathway involved in lung branching morphogenesis is disrupted in the nitrofen model. Further studies are needed focusing on the investigation of the MCT pathway and associated intracellular pH homeostasis as a potential therapeutic target for the treatment of PH in CDH.
Fig. 2. Immunofluorescence evaluation of pulmonary tissue for MCT1 (red staining) and E-cadherin (green staining) with DAPI (blue staining). Confocal laser scanning microscopy demonstrated a co-expression of MCT1 and E-cadherin mainly in primitive alveolar epithelial cells and further showed a markedly diminished MCT1 immunofluorescence in nitrofen-exposed CDH lungs on D15, D18 and D21 compared to controls.
Please cite this article as: Takahashi T, et al, Decreased Expression of Monocarboxylate Transporter 1 and 4 in the Branching Airway Epithelium of Nitrofen-Induced Congenital Diap..., J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.02.050
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T. Takahashi et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx
Fig. 3. Immunofluorescence evaluation of pulmonary tissue for MCT4 (red staining) and E-cadherin (green staining) with DAPI (blue staining). Confocal laser scanning microscopy showed co-localization of MCT4 and E-cadherin primarily in distal bronchial epithelial cells and revealed strikingly diminished MCT4 immunofluorescence in nitrofen-exposed CDH lungs on D15, D18 and D21 compared to controls.
References [1] McGivern MR, Best KE, Rankin J, et al. Epidemiology of congenital diaphragmatic hernia in Europe: a register-based study. Arch Dis Child Fetal Neonatal Ed 2015; 100:F137–44. [2] Keijzer R, Puri P. Congenital diaphragmatic hernia. Semin Pediatr Surg 2010;19: 180–5. [3] Kotecha S, Barbato A, Bush A, et al. Congenital diaphragmatic hernia. Eur Respir J 2012;39:820–9. [4] Sabharwal AJ, Davis CF, Howatson AG. Post-mortem findings in fetal and neonatal congenital diaphragmatic hernia. Eur J Pediatr Surg 2000;10:96–9. [5] Lee YM, Jeong CH, Koo SY, et al. Determination of hypoxic region by hypoxia marker in developing mouse embryos in vivo: a possible signal for vessel development. Dev Dyn 2001;220:175–86. [6] Groenman FA, Rutter M, Wang J, et al. Effect of chemical stabilizers of hypoxiainducible factors on early lung development. Am J Physiol Lung Cell Mol Physiol 2007;293:L557–67. [7] Gebb SA, Fox K, Vaughn J, et al. Fetal oxygen tension promotes tenascin-Cdependent lung branching morphogenesis. Dev Dyn 2005;234:1–10. [8] van Tuyl M, Liu J, Wang J, et al. Role of oxygen and vascular development in epithelial branching morphogenesis of the developing mouse lung. Am J Physiol Lung Cell Mol Physiol 2005;288:L167–78. [9] Herubel F, El Mouatassim S, Guerin P, et al. Genetic expression of monocarboxylate transporters during human and murine oocyte maturation and early embryonic development. Zygote 2002;10:175–81. [10] Truog WE, Xu D, Ekekezie II, et al. Chronic hypoxia and rat lung development: analysis by morphometry and directed microarray. Pediatr Res 2008;64:56–62. [11] Halestrap AP, Price NT. The proton-linked monocarboxylate transporter (MCT) family: structure, function and regulation. Biochem J 1999;343:281–99.
[12] Granja S, Morais-Santos F, Miranda-Goncalves V, et al. The monocarboxylate transporter inhibitor alpha-cyano-4-hydroxycinnamic acid disrupts rat lung branching. Cell Physiol Biochem 2013;32:1845–56. [13] van Loenhout RB, Tibboel D, Post M, et al. Congenital diaphragmatic hernia: comparison of animal models and relevance to the human situation. Neonatology 2009;96: 137–49. [14] Montedonico S, Nakazawa N, Puri P. Congenital diaphragmatic hernia and retinoids: searching for an etiology. Pediatr Surg Int 2008;24:755–61. [15] Noble BR, Babiuk RP, Clugston RD, et al. Mechanisms of action of the congenital diaphragmatic hernia-inducing teratogen nitrofen. Am J Physiol Lung Cell Mol Physiol 2007;293:L1079–87. [16] Shimoda LA, Semenza GL. HIF and the lung: role of hypoxia-inducible factors in pulmonary development and disease. Am J Respir Crit Care Med 2011;183:152–6. [17] Gebb SA, Jones PL. Hypoxia and lung branching morphogenesis. Adv Exp Med Biol 2003;543:117–25. [18] Harding EA, Gibb CA, Johnson MH, et al. Developmental changes in the management of acid loads during preimplantation mouse development. Biol Reprod 2002;67: 1419–29. [19] Halestrap AP. The monocarboxylate transporter family–structure and functional characterization. IUBMB Life 2012;64:1–9. [20] Harding EA, Day ML, Gibb CA, et al. The activity of the H + −monocarboxylate cotransporter during pre-implantation development in the mouse. Pflugers Arch 1999;438:397–404. [21] Doyen J, Trastour C, Ettore F, et al. Expression of the hypoxia-inducible monocarboxylate transporter MCT4 is increased in triple negative breast cancer and correlates independently with clinical outcome. Biochem Biophys Res Commun 2014;451: 54–61. [22] Pinheiro C, Longatto-Filho A, Azevedo-Silva J, et al. Role of monocarboxylate transporters in human cancers: state of the art. J Bioenerg Biomembr 2012;44:127–39.
Please cite this article as: Takahashi T, et al, Decreased Expression of Monocarboxylate Transporter 1 and 4 in the Branching Airway Epithelium of Nitrofen-Induced Congenital Diap..., J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.02.050
Contents lists available at ScienceDirect
Journal of Pediatric Surgery journal homepage: www.elsevier.com/locate/jpedsurg
Rosenkrantz Podium Session
Decreased Expression of Monocarboxylate Transporter 1 and 4 in the Branching Airway Epithelium of Nitrofen-Induced Congenital Diaphragmatic Hernia Toshiaki Takahashi a, Florian Friedmacher a, Julia Zimmer a, Prem Puri a,b,⁎ a b
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin, Ireland Conway Institute of Biomolecular and Biomedical Research, School of Medicine & Medical Science, University College Dublin, Dublin, Ireland
a r t i c l e
i n f o
Article history: Received 10 February 2016 Accepted 26 February 2016 Available online xxxx Key words: Monocarboxylate transporters Lung branching morphogenesis Pulmonary hypoplasia Congenital diaphragmatic hernia Nitrofen
a b s t r a c t Background/Purpose: Monocarboxylate transporters (MCTs) are crucial for the maintenance of intracellular pH homeostasis in developing fetal lungs. MCT1/4 is strongly expressed by epithelial airway cells throughout lung branching morphogenesis. Functional inhibition of MCT1/4 in fetal rat lung explants has been shown to result in airway defects similar to pulmonary hypoplasia (PH) in congenital diaphragmatic hernia (CDH). We hypothesized that pulmonary expression of MCT1/4 is decreased during lung branching morphogenesis in the nitrofen model of CDH-associated PH. Methods: Timed-pregnant rats received nitrofen or vehicle on gestational day 9 (D9). Fetuses were harvested on D15, D18, and D21, and divided into control and nitrofen-exposed group. Pulmonary gene expression levels of MCT1/4 were analyzed by qRT-PCR. Immunofluorescence staining for MCT1/4 was combined with E-cadherin in order to evaluate protein expression in branching airway tissue. Results: Relative mRNA levels of MCT1/4 were significantly reduced in lungs of nitrofen-exposed fetuses on D15, D18, and D21 compared to controls. Confocal laser scanning microscopy confirmed markedly decreased immunofluorescence of MCT1/4 in distal bronchial and primitive alveolar epithelium of nitrofen-exposed fetuses on D15, D18, and D21 compared to controls. Conclusion: Decreased expression of MCT1/4 in distal airway epithelium may disrupt lung branching morphogenesis and thus contribute to the development of PH in the nitrofen-induced CDH model. © 2016 Elsevier Inc. All rights reserved.
Congenital diaphragmatic hernia (CDH) is a relatively common malformation, affecting 1 in 2500 live births [1]. The main cause of high mortality and morbidity in newborns with CDH is severe pulmonary hypoplasia (PH) and persistent pulmonary hypertension [2]. Hypoplastic lungs are characterized by immaturity and smaller size with a decreased number of terminal airways, thickened alveolar walls, increased interstitial tissue, diminished alveolar airspaces and reduced gas-exchange surface area [3,4]. In general, fetal lung development takes place in a relatively hypoxic environment, thus generating intracellular acids, which in turn leads to a decrease in intracellular pH [5,6]. The low oxygen environment of the fetus has been shown to be essential for normal lung branching morphogenesis [7]. Furthermore, it has been reported that disruption of the cellular response to oxygen alterations results in a significantly reduced distal airway branching [8]. Although the pathological mechanisms of PH have been extensively studied, the molecular basis
⁎ Corresponding author at: National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland. Tel.: +353 1 409 6420; fax: +353 1 455 0201. E-mail address: [email protected] (P. Puri).
of disrupted lung branching morphogenesis in CDH remains largely unclear. Monocarboxylate transporters (MCTs) have been demonstrated to be crucial for the maintenance of intracellular pH homeostasis in developing fetal lungs by transporting intracellular acids across the plasma membrane [9–11]. MCT1 and MCT4 are strongly expressed in distal bronchial and primitive alveolar epithelial cells during lung branching morphogenesis, indicating their important role in the formation of fetal airways [12]. In addition, functional inhibition of MCT1 and MCT4 in fetal rat lung explants has been shown to result in severe defects in lung branching with a reduced number of distal airway buds similar to the phenotype of PH in human CDH [12]. Most of our current knowledge about the structural and molecular changes in CDH derives from experimental animal models [13]. Administration of the herbicide nitrofen (2,4-dichloro-phenyl-p-nitrophenyl ether) to pregnant rats on gestational day 9 (D9) has been demonstrated to result in PH and diaphragmatic defects in the offspring, both remarkably similar to human CDH [14,15]. We designed this study to investigate the hypothesis that pulmonary expression of MCT1 and MCT4 is decreased during lung branching morphogenesis in the nitrofen model of CDH-associated PH.
http://dx.doi.org/10.1016/j.jpedsurg.2016.02.050 0022-3468/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Takahashi T, et al, Decreased Expression of Monocarboxylate Transporter 1 and 4 in the Branching Airway Epithelium of Nitrofen-Induced Congenital Diap..., J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.02.050
2
T. Takahashi et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx
1. Material and methods 1.1. Animals, drugs and experimental design Pathogen-free adult Sprague–Dawley rats® (Harlan Laboratories, Shardlow, UK) were mated overnight, and females were checked daily for spermatozoids in their vaginal smear. The day of observation was considered as proof of pregnancy and was defined as embryonic day 0.5 (D0.5). Timed-pregnant animals were randomly divided into two experimental groups: “Nitrofen” and “Control”. On gestational day 9 (D9), dams were briefly anesthetized with 2% volatile isoflurane (Piramal Healthcare Ltd., Morpeth, UK) and either 100 mg of nitrofen (WAKO Chemicals GmbH, Neuss, Germany), dissolved in 1 ml of olive oil, or vehicle alone was administered via oral-gastric lavage. On the selected time-points D15, D18 and D21, animals were anesthetized and their fetuses were delivered via caesarean section. After laparotomy, fetal diaphragms were inspected under a Leica S8AP0 stereomicroscope (Leica Microsystems AG, Heerbrugg, Switzerland) for CDH (Fig. 1). Whole lungs of nitrofen-exposed fetuses with a diaphragmatic defect (n = 12 per time-point) and controls (n = 12 per time-point) were dissected under sterile conditions via thoracotomy and stored either in a TRIzol® reagent (Invitrogen, Carlsbad, USA) for total RNA isolation or fixed in 10% paraformaldehyde (PFA) (Santa Cruz Biotechnology Inc., Heidelberg, Germany) for immunofluorescence-double-staining. All animal procedures were carried out according to the current guidelines for management and welfare of laboratory animals and the experimental protocol was approved by the local research ethics committee (REC668b) and the Department of Health and Children (Ref. B100/4378) under the Cruelty to Animals Act, 1876 (as amended by European Communities Regulations 2002 and 2005). 1.2. Total RNA isolation from fetal rat lungs Total RNA was isolated from fetal lungs with the acid guanidinium thiocyanate-phenol-chloroform extraction method using a TRIzol® reagent according to the manufacturer's protocol. Spectrophotometrical quantification of total RNA was performed with a NanoDrop ND-1000 UV–Vis® Spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, USA). 1.3. Complementary DNA synthesis and quantitative real-time polymerase chain reaction Reverse transcription of total RNA was carried out at 85 °C for 3 min (denaturation), at 44 °C for 60 min (annealing), and at 92 °C for 10 min (reverse transcriptase inactivation) using a Transcript High Fidelity cDNA Synthesis Kit® (Roche Diagnostics, Grenzach-Whylen, Germany) according to the manufacturer's instruction. The resulting
cDNA was used for quantitative real-time polymerase chain reaction (qRT-PCR) using a LightCycler® 480 SYBR Green I Master Mix (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's protocol. Gene-specific primer pairs are listed in Table 1. After an initialization phase at 95 °C for 5 min, 55 amplification cycles were carried out. Each cycle included an initial denaturation step at 95 °C for 10 s, an annealing step at 60 °C for 15 s and an elongation step at 72 °C for 10 s. The final elongate temperature was 65 °C for 1 min. Relative mRNA expression levels of MCT1 and MCT4 were measured with a Light Cycler® 480 instrument (Roche Diagnostics, West Sussex, UK) and gene levels were normalized to the housekeeping gene β-actin. All experiments were run duplicated for each sample and primer pair.
1.4. Immunofluorescence double staining and confocal laser scanning microscopy Following overnight fixation in 10% PFA, fetal lungs were paraffinembedded, transversely sectioned at a thickness of 5 μm, and mounted on polylysine-coated slides (VWR International, Leuven, Belgium). Tissue sections were deparaffinized with xylene and rehydrated through ethanol and distilled water. To improve cell permeabilization, sections were incubated with phosphate-buffered saline (PBS) containing 1.0% Triton X-100 (Sigma Aldrich Ltd., Arklow, Ireland) for 20 min at room temperature. Sections were then washed in PBS + 0.05% Tween (Sigma Aldrich, Saint Louis, USA) and subsequently blocked with 3% bovine serum albumin (Sigma Aldrich, Saint Louis, USA) for 30 min to avoid non-specific absorption of immunoglobulin. The blocking solution was rinsed off and sections were incubated with primary antibodies either against MCT1 (goat polyclonal, sc-14,917, 1:100) or MCT4 (rabbit polyclonal, sc-50,329, 1:100) and E-cadherin (mouse polyclonal, sc8426, 1:100) (Santa Cruz Biotechnology Inc., Heidelberg, Germany) overnight at 4 °C. On the next day, sections washed in PBS + 0.05% Tween and incubated with corresponding secondary antibodies (donkey anti-goat Alexa 555-A21432, 1:250, donkey anti-rabbit Alexa 647A150067, 1:250 and donkey anti-mouse Alexa 488-A150109, 1:250) (Abcam plc, Cambridge, UK) for 1 h at room temperature. After another washing step in PBS + 0.05% Tween, sections were counterstained with a DAPI antibody (10,236,276,001, 1:1000) (Roche Diagnostics GmbH, Mannheim, Germany) for 10 min, washed again, and mounted with glass coverslips using Sigma Mounting Medium (Sigma-Aldrich, St. Louis, MO, USA). All sections were scanned with a ZEISS LSM 700 confocal microscope (Carl Zeiss MicroImaging GmbH, Jena, Germany) and independently evaluated by two investigators.
1.5. Statistical analysis All numerical data are presented as means ± standard error of the mean. Differences between two groups were tested using an unpaired Student's t test when the data had normal distribution or a Mann–Whitney U test when the data deviated from normal distribution. Statistical significance was accepted at P values of less than 0.05.
2. Results 2.1. Relative mRNA expression levels of MCT1 and MCT4 in fetal rat lungs
Fig. 1. Diaphragmatic defect in the nitrofen-induced rat on gestational day 21.
Following qRT-PCR, the relative mRNA expression levels of MCT1 and MCT4 were significantly reduced in CDH lungs of nitrofenexposed fetuses on D15 (0.23 ± 0.10 vs. 0.67 ± 0.37; P b 0.05 and 0.17 ± 0.04 vs. 0.25 ± 0.08; P b 0.05), D18 (0.44 ± 0.23 vs. 0.69 ± 0.23; P b 0.05 and 0.10 ± 0.07 vs. 0.18 ± 0.04; P b 0.05) and D21 (0.24 ± 0.13 vs. 0.61 ± 0.37; P b 0.05 and 0.10 ± 0.05 vs. 0.19 ± 0.06; P b 0.05) compared to controls.
Please cite this article as: Takahashi T, et al, Decreased Expression of Monocarboxylate Transporter 1 and 4 in the Branching Airway Epithelium of Nitrofen-Induced Congenital Diap..., J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.02.050
T. Takahashi et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx Table 1 Primer sequences for quantitative real-time polymerase chain reaction. Gene MCT1 Forward Reverse MCT4 Forward Reverse β-actin Forward Reverse
Sequence (5ۥ-3)ۥ
Product size (bp)
ATG TTG TCC TGT CCT CCT GG TAG AGG CCT GCG ATG ATG AG
114
CCA GCA TGC TCC TAA GAC CT GCG GTT TGG TGT TTC TGT CT
122
TTG CTG ACA GGA TGC AGA AG TAG AGC CAC CAA TCC ACA CA
108
2.2. Immunofluorescence evaluation of MCT1, MCT4 and E-cadherin in fetal rat lungs Immunofluorescence staining for MCT1 and MCT4 was combined with the epithelial marker E-cadherin in order to evaluate MCT1 and MCT4 protein expression and localization in the branching airway tissue of the fetal rat lungs. Confocal laser scanning microscopy revealed a co-expression of MCT1 and MCT4 with E-cadherin, and confirmed the qRT-PCR results by showing a markedly diminished MCT1 (Fig. 2) and MCT4 (Fig. 3) expression in the distal bronchial and primitive alveolar epithelium of nitrofen-exposed CDH lungs on D15, D18 and D21 compared to controls. 3. Discussion Hypoxia is a normal component of fetal lung development and has been demonstrated to be one of the most important extracellular factors that stimulate the complex process of distal airway branching [10]. The low oxygen level during intrauterine fetal growth is essential for a normal lung epithelial branching morphogenesis in the developing fetus [16,17]. Disruption of the cellular response to oxygen alterations has been shown to result in a significant reduction of branching airways [8]. In a hypoxic environment, increased glycolytic metabolism generates intracellular acids, thus leading to a decrease in intracellular pH [5,6]. These acids have an important role in cell metabolism and their rapid transport across the plasma membrane is crucial for pH homeostasis [18]. This transport is carried out by a family of transporters, designated by MCT isoform 1 and 4 [19]. Several studies have demonstrated the expression of MCTs during fetal development of humans and rodents [9]. The importance of MCTs has been described in numerous organs, including the lung, during fetal rat maturation [20]. Granja et al. [12] have recently reported on the expression pattern of MCT1 and MCT4 during lung branching morphogenesis and performed studies using a fetal rat lung explant model in order to understand the influence of MCT inhibition on airway
3
formation. They demonstrated that in normal fetal lungs, MCT1 and MCT4 isoforms are strongly expressed in distal bronchial and primitive alveolar epithelial cells throughout lung branching morphogenesis, indicating their important role in fetal airway development [12]. Furthermore, functional inhibition of MCT1 and MCT4 in fetal rat lung explants has been shown to result in severe defects in lung branching with reduced number of distal airway buds similar to the phenotype of PH in human CDH [12]. In the present study, we found that the relative mRNA levels of MCT1 and MCT4 were significantly reduced in CDH lungs of nitrofen-exposed fetuses on D15, D18 and D21 compared to controls. In addition, immunofluorescence staining demonstrated a markedly diminished MCT1 and MCT4 expression in the distal bronchial and primitive alveolar epithelium within branching airways of nitrofen-exposed CDH lungs on D15, D18 and D21. Hence, these results confirmed that the quantitative decrease in pulmonary MCT1 and MCT4 mRNA transcripts were also translated to the protein level. A decreased expression of MCT1 and MCT4 in the distal airway epithelium may therefore disrupt the fetal lung branching morphogenesis and contribute to the development of PH in the nitrofen-induced CDH model. These findings thus provide new insights into the pathomechanisms underlying PH in CDH. Given the frequency with which CDH occurs, an understanding of the genetic, cellular and morphogenetic mechanisms regulating lung development, both normally and during herniation, is critical. Many fundamental questions about lung development remain unanswered. The nitrofen model currently appears to be the best available experimental model to study the pathogenesis of CDH. The timing of the developmental insult is remarkably similar to the human situation as well as the PH. The research using nitrofen model should help determine the potential mechanisms that lead to the development of diaphragmatic defect and associated PH in CDH. As with other congenital anomalies, an improved understanding of the pathogenesis of PH may help to design new treatment modalities targeted at specific developmental insults. In the present study, we demonstrated that decreased expression of MCT1 and MCT4 in the distal airway epithelium may disrupt fetal lung branching morphogenesis and contribute to the development of PH in the nitrofen-induced CDH model. It has been reported that HIF-1 upregulates MCT4 and c-Myc up-regulates MCT1 [21,22]. It has also been described that MCT1 and 4 require type I transmembrane glycoprotein CD147/BASIGIN (BSG) for proper folding, stability and trafficking to the cell surface [19].We now propose to evaluate the pulmonary expression of HIF-1 and C-Myc in this model to confirm that MCT pathway involved in lung branching morphogenesis is disrupted in the nitrofen model. Further studies are needed focusing on the investigation of the MCT pathway and associated intracellular pH homeostasis as a potential therapeutic target for the treatment of PH in CDH.
Fig. 2. Immunofluorescence evaluation of pulmonary tissue for MCT1 (red staining) and E-cadherin (green staining) with DAPI (blue staining). Confocal laser scanning microscopy demonstrated a co-expression of MCT1 and E-cadherin mainly in primitive alveolar epithelial cells and further showed a markedly diminished MCT1 immunofluorescence in nitrofen-exposed CDH lungs on D15, D18 and D21 compared to controls.
Please cite this article as: Takahashi T, et al, Decreased Expression of Monocarboxylate Transporter 1 and 4 in the Branching Airway Epithelium of Nitrofen-Induced Congenital Diap..., J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.02.050
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T. Takahashi et al. / Journal of Pediatric Surgery xxx (2016) xxx–xxx
Fig. 3. Immunofluorescence evaluation of pulmonary tissue for MCT4 (red staining) and E-cadherin (green staining) with DAPI (blue staining). Confocal laser scanning microscopy showed co-localization of MCT4 and E-cadherin primarily in distal bronchial epithelial cells and revealed strikingly diminished MCT4 immunofluorescence in nitrofen-exposed CDH lungs on D15, D18 and D21 compared to controls.
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Please cite this article as: Takahashi T, et al, Decreased Expression of Monocarboxylate Transporter 1 and 4 in the Branching Airway Epithelium of Nitrofen-Induced Congenital Diap..., J Pediatr Surg (2016), http://dx.doi.org/10.1016/j.jpedsurg.2016.02.050