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Insulin receptor is downregulated in the nitrofen-induced hypoplastic lung
Journal of Pediatric Surgery (2010) 45, 948–952
www.elsevier.com/locate/jpedsurg
Insulin receptor is downregulated in the nitrofen-induced hypoplastic lung Elke Ruttenstock, Takashi Doi, Jens Dingemann, Prem Puri ⁎ Children's Research Center, Our Lady's Children's Hospital, Dublin 12, Ireland Received 27 January 2010; accepted 3 February 2010
Key words: Insulin receptor; Nitrofen; Hypoplastic lung; Congenital diaphragmatic hernia
Abstract Purpose: The pathogenesis of pulmonary hypoplasia in congenital diaphragmatic hernia (CDH) is still poorly understood. During fetal lung development, the insulin receptor (IR) plays an important role by mediating the cellular uptake of glucose, which is a major substrate for the biosynthesis of surfactant phospholipids. In fetal rat lung, IR gene expression has been revealed on type II pneumocytes. Recent studies have demonstrated that downregulation of pulmonary IR in late gestation causes pulmonary hypoplasia by inhibition of surfactant synthesis. We hypothesized that pulmonary gene expression of IR is downregulated during the late stages of lung development in the nitrofen-induced CDH model. Methods: Timed pregnant Sprague-Dawley rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Cesarean deliveries were performed on D15, D18, and D21. Fetal lungs were divided into 3 groups: control, nitrofen without CDH (CDH[−]), and nitrofen with CDH (CDH[+]) (n = 8 at each time-point, respectively). Relative messenger RNA (mRNA) levels of IR were determined by using real time reverse transcription polymerase chain reaction. Immunohistochemistry was performed to evaluate protein expression of IR. Results: Relative expression levels of IR mRNA on D21 were significantly decreased in CDH(−) and CDH(+) group (3.99 ± 1.50 and 5.14 ± 0.99, respectively) compared to control (7.45 ± 3.95; P b .05). Immunohistochemistry showed decreased IR expression in the proximal alveolar epithelium on D21 in hypoplastic lungs compared to control lungs. Conclusion: Downregulation of IR gene and protein expression in hypoplastic lung during late stages of lung development may interfere with normal surfactant synthesis, causing pulmonary hypoplasia in the nitrofen-induced CDH model. © 2010 Elsevier Inc. All rights reserved.
Despite recent advances in prenatal diagnosis, resuscitation, and intensive care, congenital diaphragmatic hernia (CDH) still remains the most common life-threatening cause
Presented at the 41st Annual Meeting of the Canadian Association of Paediatric Surgeons, Halifax, Nova Scotia, Canada, October 1-3, 2009. ⁎ Corresponding author. Tel.: +353 1 4096420; fax: +353 1 4550201. E-mail address: [email protected] (P. Puri). 0022-3468/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2010.02.018
of severe respiratory failure in the term infant [1]. The high mortality and morbidity in CDH has been attributed mainly to the associated pulmonary hypoplasia and pulmonary hypertension [2]. The pathogenesis of pulmonary hypoplasia in CDH is not fully understood. Nitrofen-induced CDH has been extensively used as an experimental model because the occurrence of the diaphragmatic defect and associated pulmonary hypoplasia is strikingly similar to the human situation. Maternal exposure to nitrofen on day 9 of gestation in rodents results in 100% lung hypoplasia and a
Downregulation of insulin receptor in hypoplastic lung high rate (40%-80%) of CDH in the offspring [3,4]. Although the nitrofen model of CDH has been widely used, the exact pathogenesis of pulmonary hypoplasia in CDH is still poorly understood. Recently, there is increasing evidence that the insulin receptor (IR) within the insulinlike growth factor (IGF) pathway plays an important role during fetal lung development. Insulin-like growth factors are peptide growth factors involved in lung development, and they are expressed in the fetal lung of humans, rodents, and other species [5,6]. The actions of IGF-I and IGF-II are mediated by 3 receptors, the IR, the type 1 IGF receptor (IGF1R), and the type 2 IGF receptor (IGF2R) [7]. The human IR is a heterotetrameric protein that consists of 2 extracellular α subunits and 2 transmembrane β subunits linked by disulfide bonds [8]. Two major signal transduction pathways are associated with activation of the insulin receptor, the ras/mitogen-activated protein kinase pathway and the phosphatidylinositol 3-kinase pathway [9]. In fetal lung tissue, IR mediates the cellular uptake of glucose, a major substrate for the synthesis of surfactant phospholipids [10], and thus, may be an important regulator of surfactant biosynthesis. A developmentally regulated increase in fetal IR, as well as a rise in its binding affinity for its ligands, has been also demonstrated in fetal lungs [11,12]. During gestation, the IR is expressed mainly in the type II cells of the fetal lung [12]. It has recently been reported that in infants of diabetic mothers, the incidence of respiratory distress syndrome is higher because downregulation of lung IRs late in fetal life may limit the availability of glucose as a substrate for surfactant synthesis in the perinatal period. This may partially explain the increased incidence of respiratory distress syndrome in infants of poorly controlled diabetic pregnancies [13,14]. We designed this study to investigate the hypothesis that the pulmonary gene expression of IR is downregulated during the late stages of lung development in the nitrofeninduced CDH model.
1. Materials and methods 1.1. Animals and drugs Fetal pulmonary hypoplasia, with coexisting diaphragmatic hernia, was created by gavaging time-dated pregnant Sprague-Dawley rats with nitrofen. Adult rats were mated, and the females were checked daily for cervical plugging. The presence of spermatozoids in the vaginal smear was considered as proof of pregnancy; the day of observation was determined as gestational day 0; term was considered as day 22. Pregnant female rats were than randomly divided into 2 groups. At day 9 of gestation (D9), animals in the experimental group received 100 mg of 2,4-dichloro-4′ diphenylether (nitrofen) (WAKO Chemicals, Osaka, Japan)
949 dissolved in 1 mL of olive oil via gastric tube under short anesthesia. In control animals, the same dose of olive oil was given without nitrofen. Fetuses were harvested by cesarean delivery on D15, D18, and D21. Left lungs of the fetus were dissected after thoracotomy under microscopic inspection. Fetus exposed to nitrofen were divided into 2 groups; nitrofen without CDH group (CDH[−]) and nitrofen with CDH group (CDH[+]) (n = 8 at each time-point, respectively). The control group (n = 8 at each time-point) consisted of animals that only received olive oil. The Department of Health and Children approved the protocol of these animal experiments (ref. B100/4142) under the Cruelty to Animals Act, 1876, as amended by European Communities Regulations 2002 and 2005, and all animals were treated according to the current guidelines of animal care.
1.2. Isolation of messenger RNA, complementary DNA synthesis, and real time reverse transcription polymerase chain reaction The peripheral region of the left lung of each fetus was suspended in TRIzol Reagent (Invitrogen) immediately after dissection, quick frozen in liquid nitrogen, and stored at −20°C. After thawing frozen samples, they were homogenized using a pellet pestle and the total messenger RNA (mRNA) of the lung tissue was isolated from the TRIzol suspension using the acid guanidinium-thiocyanate-phenolchloroform extraction method [15] according to the recommended protocol. Total mRNA quantification was performed spectrophotometrically (ND-1000 UV-Vis Spectrophotometer; NanoDrop). Synthesis of complementary DNA was performed using a Transcript High Fidelity cDNA Synthesis Kit (Roche Diagnostics, Germany) according to the manufacturer's protocol. Reverse transcription was carried out at 85°C for 30 minutes (min) (denaturation), at 44°C for 60 min (annealing), and at 92°C for 10 min (reverse transcription inactivation) according to the manufacturer's protocol. Real-time polymerase chain reaction was performed using LightCycler 480SYBR Green I Master (Roche Diagnostics, Germany) according to the manufacturer's protocol. The specific primer set used in this study is listed (Table 1). After an initialization step at 95°C for 5 min, 45 cycles of amplification were carried out (denaturation at 95°C for 10 seconds [sec], annealing at 60°C for 15 sec, and extension at 72°C for 10 sec in each cycle). Relative levels of gene expression were measured by LightCycler 480 (Roche Diagnostics) according to the manufacturer's instruction. The mRNA expression levels of IR were normalized to β-actin mRNA expression levels in each sample.
1.3. Immunohistochemistry The paraffin-embedded left lungs were sectioned at a thickness of 7 μm, and the sections were deparaffined with xylene and then rehydrated through ethanol and distilled
950
E. Ruttenstock et al.
Table 1 Primers for quantitative real time reverse transcriptionpolymerase chain reaction
Table 2 Group
D15
D18
D21
Gene
Control CDH (−) CDH (+)
10.86 (±4.64) 11.41 (±5.50)
5.29 (±0.88) 5.00 (±1.73) 5.52 (±2.50)
7.45 (±3.95) 3.99 (±1.50) ⁎ 5.14 (±0.99) ⁎
β-Actin Forward Reverse IR Forward Reverse
Sequence (5′-3′)
Product size (base pair)
TTG GAT GCC TGT GGT CTG TC 108 TAG AGC CAC CAA TCC ACA CA 108 CTG GCG CTG TGT AAA CTT CA GGG ATG CAC TTG TTG TTG TG
108 108
water. Tissue sections were immersed in Dako Target Retrieval Solution (DakoCytomation, Calif) that was heated for 10 min at 121°C followed by incubation in Peroxidase Block (En Vision + System-HRP [DAB], DakoCytomation, Calif) for 30 min to block endogenous peroxidase activity. Sections were incubated overnight at 4°C with a 1:100 dilution of rabbit polyclonal antibodies against IR (LOT sc7953, Santa Cruz Biotechnology). Sections were then treated in Labelled Polymer-HRP Anti-Rabbit (En Vision + SystemHRP [DAB]; DakoCytomation) secondary antibody and then further processed using DAB + Substrate Buffer and DAB + Chromogen (En Vision + System-HRP [DAB]; DakoCytomation) according to the manufacturer's instruction. Finally, sections were counterstained with aqueous hematoxylin, mounted with aqueous-based Glycergel Mounting Medium (DakoCytomation) and coverslipped.
The relative mRNA expression levels of IR in lung
⁎ P b .05 vs control.
2.2. Protein expression of IR in fetal rat lungs To determine whether the quantitatively decreased amounts of IR transcripts were reflected in the qualitative amount of the protein itself in the nitrofen-induced hypoplastic lung, immunohistochemical studies were performed (Fig. 1). In D15 control and CDH lungs, there was diffuse expression of IR protein that appeared to be localized to both the proximal alveolar epithelial and the mesenchymal compartments of the lung with no differences in immunoreactivity (Fig. 1A and B). In D18 lungs, there was also diffuse expression of IR protein in both epithelial and mesenchymal compartments, with no difference in immunoreactivity between control and CDH (Fig. 1C and D). In D21 lungs, the overall immunoreactivity of IR was diminished in CDH lungs (Fig. 1F), whereas strong IR protein expression was observed predominantly in the proximal alveolar epithelium in control lungs (Fig. 1E).
1.4. Statistical analysis
3. Discussion
All numbered data are presented as means ± SD. Differences between 2 groups at each gestational day were tested by using an unpaired Student's or Welch's t test when the data had normal distribution or Mann-Whitney U test when the data deviated from a normal distribution. Statistical significance was accepted at P values of less than .05.
The lungs in pulmonary hypoplasia associated with CDH fail to develop normally and manifest both morphologic and biochemical immaturity. Morphologically, the hypoplastic lung is characterized by fewer alveoli, thickened alveolar walls, increased interstitial tissue, markedly diminished alveolar airspaces, and aberrantly muscularized pulmonary vessels. Biochemical evidence of lung immaturity comes mainly from 2 experimental models of CDH; surgically produced CDH in lambs and nitrofen-induced CDH in rodents [16-18]. It has been reported that in the bronchoalveolar lavage fluid of lambs with surgical-produced CDH, the total amount of surfactant phospholipids is significant lower compared with control animals [16]. Decreased concentrations of surfactant phospholipids has also been observed in the lungs of rat pups with nitrofen-induced CDH [18]. In humans with CDH, biochemical immaturity has been reported in the lungs of nonsurvivors with CDH, both in the amniotic fluid and in broncho-alveolar lavage samples [19,20]. The IR mediates the cellular uptake of glucose, which is a major substrate for the synthesis of surfactant phospholipids. It has been reported that IR downregulation results in the reduced uptake of glucose into type II cells in the immediate postnatal period, thereby, causing surfactant
2. Results 2.1. Relative mRNA expression levels of IR in fetal rat lungs Relative expression levels of IR mRNA on D21 were significantly decreased in nitrofen-induced hypoplastic lungs of CDH(−) and CDH(+), compared to control lungs (P b .05) (Table 2). However, there were no significant differences between CDH(−), CDH(+), and controls in the pulmonary IR gene expression levels at D15 and D18. Furthermore, there were no significant differences between CDH(−) and CDH(+) groups in the gene expression levels of IR at each time-point.
Downregulation of insulin receptor in hypoplastic lung
951
Fig. 1 Insulin receptor immunohistochemistry. Diffused IR protein expression at D15 control (A) and CDH (B) in epithelial and mesenchymal departments, showing no difference in immunoreactivity. At D18, diffused expression in control (C) and CDH (D) in epithelial and mesenchymal departments. In D21, strong IR expression is seen in proximal alveolar epithelium in control (E), whereas overall immunoreactivity of IR is diminished in CDH lungs (F).
phospholipid deficiency [13]. In infants of diabetic mothers, who develop under high-glucose/high-insulin conditions, the occurrence of biochemical lung immaturity late in gestation is reported to be 5-fold higher than in the general neonatal population [11,13]. Lung hypoplasia with arrest of alveolar development is an almost universal finding in CDH and remains one of the major determinants of CDH-associated morbidity and mortality. Studies in the nitrofen model of CDH have shown that pulmonary hypoplasia develops early in gestation, before normal closure of the diaphragm takes place. Using the nitrofen murine model, Guilbert et al [21] showed that nitrofen-treated fetuses had an early abnormality in the extent of lung branching morphogenesis, which was initiated before completion of the diaphragm. A “dual-hit hypothesis” of pulmonary hypoplasia proposed that the first hit was primary pulmonary underdevelopment and that the second hit was mechanical compression because of the upward displacement of abdominal contents into the chest [22]. The combination of 2 hits accounts for the fact that pulmonary hypoplasia is bilateral but more severe in the lung ipsilateral to the diaphragmatic defect [23]. Previous studies have shown that lung development in CDH appears to be stalled not only early but also late in gestation [21,23,24]. It has also been reported that in the fetal lung, augmented synthesis of pulmonary surfactant is initiated toward the end of gestation, and inadequate surfactant synthesis by the lungs of premature infants can result in delayed pulmonary maturity [25]. In the present study, we examined the mRNA and protein expression of the IR in hypoplastic lungs in CDH at different stages of development. We observed significant downregulation of IR mRNA gene expression in the hypoplastic lungs on D21, at a stage when IR is required for proper phospholipid synthesis and lung maturation. Immunohisto-
chemical studies also demonstrated that IR protein expression was also decreased on D21 in the proximal alveolar epithelium in the nitrofen-induced hypoplastic lungs compared to controls. We suggest that the downregulation of the IR gene in the hypoplastic lung during the late stages of fetal lung development may interfere with normal surfactant phospholipid synthesis, causing delayed lung maturity and therefore pulmonary hypoplasia in the nitrofen-induced CDH model. Further studies of the IR signaling in the nitrofen-induced hypoplastic lung should provide new insights into the pathogenesis of altered lung morphogenesis in CDH.
References [1] Stage G, Fenton A, Jaffray B. Nihilism in the 1990s: the true mortality of congenital diaphragmatic hernia. Pediatrics 2003;112:532-5. [2] Santos S, Moura R, Gonzage S. Embryonic essential myosin light chain regulates fetal lung development in rats. Am J Respir Cell Mol Biol 2007;37:330-8. [3] Noble B, Babiuk R, Clugston R. Mechanisms of the congenital diaphragmatic hernia-inducing teratogen nitrofen. Am J Physiol Lung Cell Mol Physiol 2007;293:1079-87. [4] Montedonico S, Nakazawa N, Puri P. Congenital diaphragmatic hernia and retinoids: searching for an etiology. Pediatr Surg Int 2008;24: 755-61. [5] Schuller A, van Neck J, Beukenholdt R, et al. IGF, type I IGF receptor and IGF-binding protein mRNA expression in the developing mouse lung. J Mol Endocrinol 1995;14:349-55. [6] Maitre B, Clement A, Williams M, et al. Expression of insulin-like growth factor receptors 1 and 2 in the developing lung and their relation to epithelial cell differentiation. Am J Respir Cell Mol Biol 1995;12:56-64. [7] Jones J, Clemmons D. Insulin-like growth factors and their binding proteins: biological actions. Endocr Rev 1995;16:3-34. [8] Tavare J, Siddle K. Mutational analysis of insulin-receptor function: consensus and controversy. Biochem Biophys Acta 1993;1178:21-39.
952 [9] Saltiel A. Diverse signaling pathways in the cellular actions of insulin. Am J Physiol 1996;270:375-85. [10] Felts J. Biochemistry of the lung. Health Phys 1964;10:973-9. [11] Ulane R, Graeber J, Hansen J, et al. Insulin receptors in developing fetal lung. Life Sci 1982;31:3017-22. [12] Snyder JG, Miakotina O. Insulin and lung development. In: Mendelson CR, editor. Endocrinology of the lung development and surfactant synthesis. Totowa (NJ): Human Press Inc; 2000. p. 181-200. [13] Gewolb I, O'Brien J, Palese T, et al. High glucose and insulin decrease fetal lung insulin receptor mRNA and tyrosine kinase activity in vitro. Biochem Biophys Res Comm 1994;202:694-700. [14] Zmora E, Gewolb IH, Shapiro DL. Effects of insulin and glucose on pulmonary insulin receptors in late gestation fetal rats. Exp Lung Res 1992;18:247-58. [15] Chomczynski P, Sacchi N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Protoc 2006;1:581-5. [16] Glick PL, Stannard VA, Leach CL, et al. Pathophysiology of congenital diaphragmatic hernia II: the fetal lamb CDH model is surfactant deficient. J Pediatr Surg 1992;27:382-7. [17] Wilcox DT, Glick PL, Karamanoukian HL, et al. Contributions by individual lungs to the surfactant status in congenital diaphragmatic hernia. Pediatr Res 1997;41:686-91.
E. Ruttenstock et al. [18] Suen H, Catlin E, Ryan D, et al. Biochemical immaturity of the lung in congenital diaphragmatic hernia. J Pediatr Surg 1993;28:471-5. [19] Sullivan K, Hawgood S, Flake A, et al. Amniotic fluid phospholipids analysis in the fetus with congenital diaphragmatic hernia. J Pediatr Surg 1994;29:1020-3. [20] Ijsselstijn H, Zimmermann L, Bunt J, et al. Prospective evaluation of surfactant composition in bronchioalveolar lavage fluid in infants with congenital diaphragmatic hernia and of age matched controls. Crit Care Med 1998;26:573-80. [21] Guilbert T, Gebb S, Shannon J. Lung hypoplasia in the nitrofen model of congenital diaphragmatic hernia occurs early in development. Am J Physiol Lung Cell Mol Physiol 2000;279:L1159-L1171. [22] Keijzer R, Liu J, Deimling J. Dual-hit hypothesis explains pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia. Am J Pathol 2000;156:1299-306. [23] Alfonso LF, Vilanova J, Aldazabal P, et al. Lung growth and maturation in the rat model of experimentally induced congenital diaphragmatic hernia. Eur J Pediatr Surg 1993;3:6-11. [24] Iritani I. Experimental study on embryogenesis of congenital diaphragmatic hernia. Anat Embryol (Berl) 1984;169:133-9. [25] Mendelson CR, Boggaram V. Regulation of pulmonary surfactant protein synthesis in fetal lung: a major role of glucocorticoids and cyclic AMP. Trends Endocrinol Metab 1989;1:20-5.
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Insulin receptor is downregulated in the nitrofen-induced hypoplastic lung Elke Ruttenstock, Takashi Doi, Jens Dingemann, Prem Puri ⁎ Children's Research Center, Our Lady's Children's Hospital, Dublin 12, Ireland Received 27 January 2010; accepted 3 February 2010
Key words: Insulin receptor; Nitrofen; Hypoplastic lung; Congenital diaphragmatic hernia
Abstract Purpose: The pathogenesis of pulmonary hypoplasia in congenital diaphragmatic hernia (CDH) is still poorly understood. During fetal lung development, the insulin receptor (IR) plays an important role by mediating the cellular uptake of glucose, which is a major substrate for the biosynthesis of surfactant phospholipids. In fetal rat lung, IR gene expression has been revealed on type II pneumocytes. Recent studies have demonstrated that downregulation of pulmonary IR in late gestation causes pulmonary hypoplasia by inhibition of surfactant synthesis. We hypothesized that pulmonary gene expression of IR is downregulated during the late stages of lung development in the nitrofen-induced CDH model. Methods: Timed pregnant Sprague-Dawley rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Cesarean deliveries were performed on D15, D18, and D21. Fetal lungs were divided into 3 groups: control, nitrofen without CDH (CDH[−]), and nitrofen with CDH (CDH[+]) (n = 8 at each time-point, respectively). Relative messenger RNA (mRNA) levels of IR were determined by using real time reverse transcription polymerase chain reaction. Immunohistochemistry was performed to evaluate protein expression of IR. Results: Relative expression levels of IR mRNA on D21 were significantly decreased in CDH(−) and CDH(+) group (3.99 ± 1.50 and 5.14 ± 0.99, respectively) compared to control (7.45 ± 3.95; P b .05). Immunohistochemistry showed decreased IR expression in the proximal alveolar epithelium on D21 in hypoplastic lungs compared to control lungs. Conclusion: Downregulation of IR gene and protein expression in hypoplastic lung during late stages of lung development may interfere with normal surfactant synthesis, causing pulmonary hypoplasia in the nitrofen-induced CDH model. © 2010 Elsevier Inc. All rights reserved.
Despite recent advances in prenatal diagnosis, resuscitation, and intensive care, congenital diaphragmatic hernia (CDH) still remains the most common life-threatening cause
Presented at the 41st Annual Meeting of the Canadian Association of Paediatric Surgeons, Halifax, Nova Scotia, Canada, October 1-3, 2009. ⁎ Corresponding author. Tel.: +353 1 4096420; fax: +353 1 4550201. E-mail address: [email protected] (P. Puri). 0022-3468/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2010.02.018
of severe respiratory failure in the term infant [1]. The high mortality and morbidity in CDH has been attributed mainly to the associated pulmonary hypoplasia and pulmonary hypertension [2]. The pathogenesis of pulmonary hypoplasia in CDH is not fully understood. Nitrofen-induced CDH has been extensively used as an experimental model because the occurrence of the diaphragmatic defect and associated pulmonary hypoplasia is strikingly similar to the human situation. Maternal exposure to nitrofen on day 9 of gestation in rodents results in 100% lung hypoplasia and a
Downregulation of insulin receptor in hypoplastic lung high rate (40%-80%) of CDH in the offspring [3,4]. Although the nitrofen model of CDH has been widely used, the exact pathogenesis of pulmonary hypoplasia in CDH is still poorly understood. Recently, there is increasing evidence that the insulin receptor (IR) within the insulinlike growth factor (IGF) pathway plays an important role during fetal lung development. Insulin-like growth factors are peptide growth factors involved in lung development, and they are expressed in the fetal lung of humans, rodents, and other species [5,6]. The actions of IGF-I and IGF-II are mediated by 3 receptors, the IR, the type 1 IGF receptor (IGF1R), and the type 2 IGF receptor (IGF2R) [7]. The human IR is a heterotetrameric protein that consists of 2 extracellular α subunits and 2 transmembrane β subunits linked by disulfide bonds [8]. Two major signal transduction pathways are associated with activation of the insulin receptor, the ras/mitogen-activated protein kinase pathway and the phosphatidylinositol 3-kinase pathway [9]. In fetal lung tissue, IR mediates the cellular uptake of glucose, a major substrate for the synthesis of surfactant phospholipids [10], and thus, may be an important regulator of surfactant biosynthesis. A developmentally regulated increase in fetal IR, as well as a rise in its binding affinity for its ligands, has been also demonstrated in fetal lungs [11,12]. During gestation, the IR is expressed mainly in the type II cells of the fetal lung [12]. It has recently been reported that in infants of diabetic mothers, the incidence of respiratory distress syndrome is higher because downregulation of lung IRs late in fetal life may limit the availability of glucose as a substrate for surfactant synthesis in the perinatal period. This may partially explain the increased incidence of respiratory distress syndrome in infants of poorly controlled diabetic pregnancies [13,14]. We designed this study to investigate the hypothesis that the pulmonary gene expression of IR is downregulated during the late stages of lung development in the nitrofeninduced CDH model.
1. Materials and methods 1.1. Animals and drugs Fetal pulmonary hypoplasia, with coexisting diaphragmatic hernia, was created by gavaging time-dated pregnant Sprague-Dawley rats with nitrofen. Adult rats were mated, and the females were checked daily for cervical plugging. The presence of spermatozoids in the vaginal smear was considered as proof of pregnancy; the day of observation was determined as gestational day 0; term was considered as day 22. Pregnant female rats were than randomly divided into 2 groups. At day 9 of gestation (D9), animals in the experimental group received 100 mg of 2,4-dichloro-4′ diphenylether (nitrofen) (WAKO Chemicals, Osaka, Japan)
949 dissolved in 1 mL of olive oil via gastric tube under short anesthesia. In control animals, the same dose of olive oil was given without nitrofen. Fetuses were harvested by cesarean delivery on D15, D18, and D21. Left lungs of the fetus were dissected after thoracotomy under microscopic inspection. Fetus exposed to nitrofen were divided into 2 groups; nitrofen without CDH group (CDH[−]) and nitrofen with CDH group (CDH[+]) (n = 8 at each time-point, respectively). The control group (n = 8 at each time-point) consisted of animals that only received olive oil. The Department of Health and Children approved the protocol of these animal experiments (ref. B100/4142) under the Cruelty to Animals Act, 1876, as amended by European Communities Regulations 2002 and 2005, and all animals were treated according to the current guidelines of animal care.
1.2. Isolation of messenger RNA, complementary DNA synthesis, and real time reverse transcription polymerase chain reaction The peripheral region of the left lung of each fetus was suspended in TRIzol Reagent (Invitrogen) immediately after dissection, quick frozen in liquid nitrogen, and stored at −20°C. After thawing frozen samples, they were homogenized using a pellet pestle and the total messenger RNA (mRNA) of the lung tissue was isolated from the TRIzol suspension using the acid guanidinium-thiocyanate-phenolchloroform extraction method [15] according to the recommended protocol. Total mRNA quantification was performed spectrophotometrically (ND-1000 UV-Vis Spectrophotometer; NanoDrop). Synthesis of complementary DNA was performed using a Transcript High Fidelity cDNA Synthesis Kit (Roche Diagnostics, Germany) according to the manufacturer's protocol. Reverse transcription was carried out at 85°C for 30 minutes (min) (denaturation), at 44°C for 60 min (annealing), and at 92°C for 10 min (reverse transcription inactivation) according to the manufacturer's protocol. Real-time polymerase chain reaction was performed using LightCycler 480SYBR Green I Master (Roche Diagnostics, Germany) according to the manufacturer's protocol. The specific primer set used in this study is listed (Table 1). After an initialization step at 95°C for 5 min, 45 cycles of amplification were carried out (denaturation at 95°C for 10 seconds [sec], annealing at 60°C for 15 sec, and extension at 72°C for 10 sec in each cycle). Relative levels of gene expression were measured by LightCycler 480 (Roche Diagnostics) according to the manufacturer's instruction. The mRNA expression levels of IR were normalized to β-actin mRNA expression levels in each sample.
1.3. Immunohistochemistry The paraffin-embedded left lungs were sectioned at a thickness of 7 μm, and the sections were deparaffined with xylene and then rehydrated through ethanol and distilled
950
E. Ruttenstock et al.
Table 1 Primers for quantitative real time reverse transcriptionpolymerase chain reaction
Table 2 Group
D15
D18
D21
Gene
Control CDH (−) CDH (+)
10.86 (±4.64) 11.41 (±5.50)
5.29 (±0.88) 5.00 (±1.73) 5.52 (±2.50)
7.45 (±3.95) 3.99 (±1.50) ⁎ 5.14 (±0.99) ⁎
β-Actin Forward Reverse IR Forward Reverse
Sequence (5′-3′)
Product size (base pair)
TTG GAT GCC TGT GGT CTG TC 108 TAG AGC CAC CAA TCC ACA CA 108 CTG GCG CTG TGT AAA CTT CA GGG ATG CAC TTG TTG TTG TG
108 108
water. Tissue sections were immersed in Dako Target Retrieval Solution (DakoCytomation, Calif) that was heated for 10 min at 121°C followed by incubation in Peroxidase Block (En Vision + System-HRP [DAB], DakoCytomation, Calif) for 30 min to block endogenous peroxidase activity. Sections were incubated overnight at 4°C with a 1:100 dilution of rabbit polyclonal antibodies against IR (LOT sc7953, Santa Cruz Biotechnology). Sections were then treated in Labelled Polymer-HRP Anti-Rabbit (En Vision + SystemHRP [DAB]; DakoCytomation) secondary antibody and then further processed using DAB + Substrate Buffer and DAB + Chromogen (En Vision + System-HRP [DAB]; DakoCytomation) according to the manufacturer's instruction. Finally, sections were counterstained with aqueous hematoxylin, mounted with aqueous-based Glycergel Mounting Medium (DakoCytomation) and coverslipped.
The relative mRNA expression levels of IR in lung
⁎ P b .05 vs control.
2.2. Protein expression of IR in fetal rat lungs To determine whether the quantitatively decreased amounts of IR transcripts were reflected in the qualitative amount of the protein itself in the nitrofen-induced hypoplastic lung, immunohistochemical studies were performed (Fig. 1). In D15 control and CDH lungs, there was diffuse expression of IR protein that appeared to be localized to both the proximal alveolar epithelial and the mesenchymal compartments of the lung with no differences in immunoreactivity (Fig. 1A and B). In D18 lungs, there was also diffuse expression of IR protein in both epithelial and mesenchymal compartments, with no difference in immunoreactivity between control and CDH (Fig. 1C and D). In D21 lungs, the overall immunoreactivity of IR was diminished in CDH lungs (Fig. 1F), whereas strong IR protein expression was observed predominantly in the proximal alveolar epithelium in control lungs (Fig. 1E).
1.4. Statistical analysis
3. Discussion
All numbered data are presented as means ± SD. Differences between 2 groups at each gestational day were tested by using an unpaired Student's or Welch's t test when the data had normal distribution or Mann-Whitney U test when the data deviated from a normal distribution. Statistical significance was accepted at P values of less than .05.
The lungs in pulmonary hypoplasia associated with CDH fail to develop normally and manifest both morphologic and biochemical immaturity. Morphologically, the hypoplastic lung is characterized by fewer alveoli, thickened alveolar walls, increased interstitial tissue, markedly diminished alveolar airspaces, and aberrantly muscularized pulmonary vessels. Biochemical evidence of lung immaturity comes mainly from 2 experimental models of CDH; surgically produced CDH in lambs and nitrofen-induced CDH in rodents [16-18]. It has been reported that in the bronchoalveolar lavage fluid of lambs with surgical-produced CDH, the total amount of surfactant phospholipids is significant lower compared with control animals [16]. Decreased concentrations of surfactant phospholipids has also been observed in the lungs of rat pups with nitrofen-induced CDH [18]. In humans with CDH, biochemical immaturity has been reported in the lungs of nonsurvivors with CDH, both in the amniotic fluid and in broncho-alveolar lavage samples [19,20]. The IR mediates the cellular uptake of glucose, which is a major substrate for the synthesis of surfactant phospholipids. It has been reported that IR downregulation results in the reduced uptake of glucose into type II cells in the immediate postnatal period, thereby, causing surfactant
2. Results 2.1. Relative mRNA expression levels of IR in fetal rat lungs Relative expression levels of IR mRNA on D21 were significantly decreased in nitrofen-induced hypoplastic lungs of CDH(−) and CDH(+), compared to control lungs (P b .05) (Table 2). However, there were no significant differences between CDH(−), CDH(+), and controls in the pulmonary IR gene expression levels at D15 and D18. Furthermore, there were no significant differences between CDH(−) and CDH(+) groups in the gene expression levels of IR at each time-point.
Downregulation of insulin receptor in hypoplastic lung
951
Fig. 1 Insulin receptor immunohistochemistry. Diffused IR protein expression at D15 control (A) and CDH (B) in epithelial and mesenchymal departments, showing no difference in immunoreactivity. At D18, diffused expression in control (C) and CDH (D) in epithelial and mesenchymal departments. In D21, strong IR expression is seen in proximal alveolar epithelium in control (E), whereas overall immunoreactivity of IR is diminished in CDH lungs (F).
phospholipid deficiency [13]. In infants of diabetic mothers, who develop under high-glucose/high-insulin conditions, the occurrence of biochemical lung immaturity late in gestation is reported to be 5-fold higher than in the general neonatal population [11,13]. Lung hypoplasia with arrest of alveolar development is an almost universal finding in CDH and remains one of the major determinants of CDH-associated morbidity and mortality. Studies in the nitrofen model of CDH have shown that pulmonary hypoplasia develops early in gestation, before normal closure of the diaphragm takes place. Using the nitrofen murine model, Guilbert et al [21] showed that nitrofen-treated fetuses had an early abnormality in the extent of lung branching morphogenesis, which was initiated before completion of the diaphragm. A “dual-hit hypothesis” of pulmonary hypoplasia proposed that the first hit was primary pulmonary underdevelopment and that the second hit was mechanical compression because of the upward displacement of abdominal contents into the chest [22]. The combination of 2 hits accounts for the fact that pulmonary hypoplasia is bilateral but more severe in the lung ipsilateral to the diaphragmatic defect [23]. Previous studies have shown that lung development in CDH appears to be stalled not only early but also late in gestation [21,23,24]. It has also been reported that in the fetal lung, augmented synthesis of pulmonary surfactant is initiated toward the end of gestation, and inadequate surfactant synthesis by the lungs of premature infants can result in delayed pulmonary maturity [25]. In the present study, we examined the mRNA and protein expression of the IR in hypoplastic lungs in CDH at different stages of development. We observed significant downregulation of IR mRNA gene expression in the hypoplastic lungs on D21, at a stage when IR is required for proper phospholipid synthesis and lung maturation. Immunohisto-
chemical studies also demonstrated that IR protein expression was also decreased on D21 in the proximal alveolar epithelium in the nitrofen-induced hypoplastic lungs compared to controls. We suggest that the downregulation of the IR gene in the hypoplastic lung during the late stages of fetal lung development may interfere with normal surfactant phospholipid synthesis, causing delayed lung maturity and therefore pulmonary hypoplasia in the nitrofen-induced CDH model. Further studies of the IR signaling in the nitrofen-induced hypoplastic lung should provide new insights into the pathogenesis of altered lung morphogenesis in CDH.
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