Pediatric Surgery International

, Volume 24, Issue 9, pp 993–1000 | Cite as

NKCC-1 and ENaC are down-regulated in nitrofen-induced hypoplastic lungs with congenital diaphragmatic hernia

  • Andreas Ringman
  • Marina Zelenina
  • Ann-Christine Eklöf
  • Anita Aperia
  • Björn Frenckner
Original Article


Congenital diaphragmatic hernia (CDH) is accompanied by pulmonary hypoplasia and pulmonary hypertension. Fetal lung growth is dependent on the secretion of lung liquid, which normally is absorbed at partus. The ion channel NKCC-1 is involved in this secretory process, but has recently also been reported to be implicated in absorption. CDH patients show a disturbed transition from secretion to absorption. α- and β-ENaC are essential for lung liquid absorption. Common for all transcellular ion transport is the need for Na/K-ATPase as a primary driving force. The aim of the study was first to map the normal pulmonary expression of the above proteins during late gestation and secondly to see if the expression was affected in a CDH rat model. Pregnant Sprague–Dawley rat dams were given nitrofen on gestational day 9.5 to induce CDH. The fetuses were removed on gestational days E18 and E21. In addition, newborn rats were harvested postpartum on day P2. The fetuses were put into one of two groups: hypoplastic lungs without CDH (N−CDH) and hypoplastic lungs with CDH (N+CDH). The pulmonary expression of NKCC-1, α-/β-ENaC and Na/K-ATPase was then analyzed using Western blot. We found that the protein levels of NKCC-1 on gestational days E18 and E21 were significantly lower among fetuses with N+CDH as well as N−CDH compared to controls. The expression of β-ENaC was also significantly down-regulated in both the groups on E18 and E21. The protein levels of α-ENaC and Na/K-ATPase were not found to be significantly decreased, but both showed a tendency towards down-regulation. The marked down-regulation of NKCC-1 in fetal hypoplastic lungs with CDH indicates a possibly decreased lung liquid production. This may be one of the mechanisms behind the disturbed pulmonary development in CDH. We also show that β-ENaC is down-regulated. Down-regulation of β-ENaC may result in abnormal lung liquid absorption, which could be one of the mechanisms behind the respiratory distress seen in CDH patients postpartum.


Congenital diaphragmatic hernia Lung liquid Na–K–Cl cotransporter ENaC 



Congenital diaphragmatic hernia


Extracorporeal membrane oxygenation


Epithelial sodium channel


Fibroblast growth factor


Sodium-potassium-2-chloride co-transporter 1


Respiratory distress syndrome


Tracheal occlusion



This work was supported by grants from the HRH Crown Princess Lovisa and Theilmans Foundation, the Swedish Freemasons Childhood Foundation, the Swedish Heart-Lung Foundation and Karolinska Institutet. We thank Prof. S. Nielsen, Faculty of Health Sciences, University of Aarhus, Denmark for kindly providing the β-ENaC antibody and Y. Li for valuable technical assistance. The experiments comply with the current laws of Sweden.


  1. 1.
    Langham MR Jr, Kays DW, Ledbetter DJ, Frentzen B, Sanford LL, Richards DS (1996) Congenital diaphragmatic hernia. Epidemiology and outcome. Clin Perinatol 23:671–688PubMedGoogle Scholar
  2. 2.
    Yang W, Carmichael SL, Harris JA, Shaw GM (2006) Epidemiologic characteristics of congenital diaphragmatic hernia among 2.5 million California births, 1989–1997. Birth Defects Res A Clin Mol Teratol 76:170–174. doi: 10.1002/bdra.20230 PubMedCrossRefGoogle Scholar
  3. 3.
    Bohn D, Tamura M, Perrin D, Barker G, Rabinovitch M (1987) Ventilatory predictors of pulmonary hypoplasia in congenital diaphragmatic hernia, confirmed by morphologic assessment. J Pediatr 111:423–431. doi: 10.1016/S0022-3476(87)80474-2 PubMedCrossRefGoogle Scholar
  4. 4.
    Folkesson HG, Chapin CJ, Beard LL, Ertsey R, Matthay MA, Kitterman JA (2006) Congenital diaphragmatic hernia prevents absorption of distal air space fluid in late-gestation rat fetuses. Am J Physiol Lung Cell Mol Physiol 290:L478–L484. doi: 10.1152/ajplung.00124.2005 PubMedCrossRefGoogle Scholar
  5. 5.
    Adamson TM, Boyd RD, Platt HS, Strang LB (1969) Composition of alveolar liquid in the foetal lamb. J Physiol 204:159–168PubMedGoogle Scholar
  6. 6.
    Olver RE, Strang LB (1974) Ion fluxes across the pulmonary epithelium and the secretion of lung liquid in the foetal lamb. J Physiol 241:327–357PubMedGoogle Scholar
  7. 7.
    Gillie DJ, Pace AJ, Coakley RJ, Koller BH, Barker PM (2001) Liquid and ion transport by fetal airway and lung epithelia of mice deficient in sodium-potassium-2-chloride transporter. Am J Respir Cell Mol Biol 25:14–20PubMedGoogle Scholar
  8. 8.
    Haas M, McBrayer DG, Yankaskas JR (1993) Dual mechanisms for Na-K-Cl cotransport regulation in airway epithelial cells. Am J Physiol 264:C189–C200PubMedGoogle Scholar
  9. 9.
    Blott M, Greenough A, Nicolaides KH (1990) Fetal breathing movements in pregnancies complicated by premature membrane rupture in the second trimester. Early Hum Dev 21:41–48. doi: 10.1016/0378-3782(90)90109-V PubMedCrossRefGoogle Scholar
  10. 10.
    Hummler E, Barker P, Gatzy J, Beermann F, Verdumo C, Schmidt A et al (1996) Early death due to defective neonatal lung liquid clearance in alpha-ENaC-deficient mice. Nat Genet 12:325–328. doi: 10.1038/ng0396-325 PubMedCrossRefGoogle Scholar
  11. 11.
    Harrison MR, Adzick NS, Flake AW, VanderWall KJ, Bealer JF, Howell LJ et al (1996) Correction of congenital diaphragmatic hernia in utero VIII: Response of the hypoplastic lung to tracheal occlusion. J Pediatr Surg 31:1339–1348. doi: 10.1016/S0022-3468(96)90824-6 PubMedCrossRefGoogle Scholar
  12. 12.
    Kitano Y, Kanai M, Davies P, von Allmen D, Yang EY, Radu A et al (2001) BAPS prize-1999: lung growth induced by prenatal tracheal occlusion and its modifying factors: a study in the rat model of congenital diaphragmatic hernia. J Pediatr Surg 36:251–259. doi: 10.1053/jpsu.2001.20683 PubMedCrossRefGoogle Scholar
  13. 13.
    Goodman BE, Kim KJ, Crandall ED (1987) Evidence for active sodium transport across alveolar epithelium of isolated rat lung. J Appl Physiol 62:2460–2466PubMedGoogle Scholar
  14. 14.
    Ye X, Norlin A, Folkesson HG (2004) Stimulation of distal airspace fluid clearance in guinea pigs involves bumetanide-sensitive ion transport. Am J Obstet Gynecol 191:340–345. doi: 10.1016/j.ajog.2003.09.074 PubMedCrossRefGoogle Scholar
  15. 15.
    Takayasu H, Nakazawa N, Montedonico S, Puri P (2007) Reduced expression of aquaporin 5 water channel in nitrofen-induced hypoplastic lung with congenital diaphragmatic hernia rat model. J Pediatr Surg 42:415–419. doi: 10.1016/j.jpedsurg.2006.10.029 PubMedCrossRefGoogle Scholar
  16. 16.
    Kluth D, Kangah R, Reich P, Tenbrinck R, Tibboel D, Lambrecht W (1990) Nitrofen-induced diaphragmatic hernias in rats: an animal model. J Pediatr Surg 25:850–854. doi: 10.1016/0022-3468(90)90190-K PubMedCrossRefGoogle Scholar
  17. 17.
    Finley N, Norlin A, Baines DL, Folkesson HG (1998) Alveolar epithelial fluid clearance is mediated by endogenous catecholamines at birth in guinea pigs. J Clin Invest 101:972–981. doi: 10.1172/JCI1478 PubMedCrossRefGoogle Scholar
  18. 18.
    Norlin A, Folkesson HG (2001) Alveolar fluid clearance in late-gestational guinea pigs after labor induction: mechanisms and regulation. Am J Physiol Lung Cell Mol Physiol 280:L606–L616PubMedGoogle Scholar
  19. 19.
    Jiang X, Ingbar DH, O’Grady SM (1998) Adrenergic stimulation of Na+ transport across alveolar epithelial cells involves activation of apical Cl- channels. Am J Physiol 275:C1610–C1620PubMedGoogle Scholar
  20. 20.
    Tchepichev S, Ueda J, Canessa C, Rossier BC, O’Brodovich H (1995) Lung epithelial Na channel subunits are differentially regulated during development and by steroids. Am J Physiol 269:C805–C812PubMedGoogle Scholar
  21. 21.
    Watanabe S, Matsushita K, Stokes JB, McCray PB Jr (1998) Developmental regulation of epithelial sodium channel subunit mRNA expression in rat colon and lung. Am J Physiol 275:G1227–G1235PubMedGoogle Scholar
  22. 22.
    O’Brodovich H, Canessa C, Ueda J, Rafii B, Rossier BC, Edelson J (1993) Expression of the epithelial Na+ channel in the developing rat lung. Am J Physiol 265:C491–C496PubMedGoogle Scholar
  23. 23.
    Talbot CL, Bosworth DG, Briley EL, Fenstermacher DA, Boucher RC, Gabriel SE et al (1999) Quantitation and localization of ENaC subunit expression in fetal, newborn, and adult mouse lung. Am J Respir Cell Mol Biol 20:398–406PubMedGoogle Scholar
  24. 24.
    Farman N, Talbot CR, Boucher R, Fay M, Canessa C, Rossier B et al (1997) Noncoordinated expression of alpha-, beta-, and gamma-subunit mRNAs of epithelial Na+ channel along rat respiratory tract. Am J Physiol 272:C131–C141PubMedGoogle Scholar
  25. 25.
    Matsushita K, McCray PB Jr, Sigmund RD, Welsh MJ, Stokes JB (1996) Localization of epithelial sodium channel subunit mRNAs in adult rat lung by in situ hybridization. Am J Physiol 271:L332–L339PubMedGoogle Scholar
  26. 26.
    Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD et al (1994) Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367:463–467. doi: 10.1038/367463a0 PubMedCrossRefGoogle Scholar
  27. 27.
    McDonald FJ, Yang B, Hrstka RF, Drummond HA, Tarr DE, McCray PB Jr et al (1999) Disruption of the beta subunit of the epithelial Na+ channel in mice: hyperkalemia and neonatal death associated with a pseudohypoaldosteronism phenotype. Proc Natl Acad Sci USA 96:1727–1731. doi: 10.1073/pnas.96.4.1727 PubMedCrossRefGoogle Scholar
  28. 28.
    Khan AM, Lally KP (2005) The role of extracorporeal membrane oxygenation in the management of infants with congenital diaphragmatic hernia. Semin Perinatol 29:118–122. doi: 10.1053/j.semperi.2005.04.005 PubMedCrossRefGoogle Scholar
  29. 29.
    West KW, Bengston K, Rescorla FJ, Engle WA, Grosfeld JL (1992) Delayed surgical repair and ECMO improves survival in congenital diaphragmatic hernia. Ann Surg 216:454–460. doi: 10.1097/00000658-199210000-00009 discussion 460–452PubMedCrossRefGoogle Scholar
  30. 30.
    TCDHS Group (1999) Does extracorporeal membrane oxygenation improve survival in neonates with congenital diaphragmatic hernia? J Pediatr Surg 34:720–725. doi: 10.1016/S0022-3468(99)90363-9 CrossRefGoogle Scholar
  31. 31.
    Kizilcan F, Tanyel FC, Cakar N, Buyukpamukcu N, Hicsonmez A (1995) The effect of low amniotic pressure without oligohydramnios on fetal lung development in a rabbit model. Am J Obstet Gynecol 173:36–41. doi: 10.1016/0002-9378(95)90166-3 PubMedCrossRefGoogle Scholar
  32. 32.
    Hooper SB, Harding R (1995) Fetal lung liquid: a major determinant of the growth and functional development of the fetal lung. Clin Exp Pharmacol Physiol 22:235–247. doi: 10.1111/j.1440-1681.1995.tb01988.x PubMedCrossRefGoogle Scholar
  33. 33.
    Moessinger AC, Harding R, Adamson TM, Singh M, Kiu GT (1990) Role of lung fluid volume in growth and maturation of the fetal sheep lung. J Clin Invest 86:1270–1277. doi: 10.1172/JCI114834 PubMedCrossRefGoogle Scholar
  34. 34.
    Adzick NS, Harrison MR, Glick PL, Villa RL, Finkbeiner W (1984) Experimental pulmonary hypoplasia and oligohydramnios: relative contributions of lung fluid and fetal breathing movements. J Pediatr Surg 19:658–665. doi: 10.1016/S0022-3468(84)80349-8 PubMedCrossRefGoogle Scholar
  35. 35.
    Davey MG, Hooper SB, Cock ML, Harding R (2001) Stimulation of lung growth in fetuses with lung hypoplasia leads to altered postnatal lung structure in sheep. Pediatr Pulmonol 32:267–276. doi: 10.1002/ppul.2008.abs PubMedCrossRefGoogle Scholar
  36. 36.
    Davey MG, Hedrick HL, Bouchard S, Mendoza JM, Schwarz U, Adzick NS et al (2003) Temporary tracheal occlusion in fetal sheep with lung hypoplasia does not improve postnatal lung function. J Appl Physiol 94:1054–1062PubMedGoogle Scholar
  37. 37.
    Kitano Y, Davies P, von Allmen D, Adzick NS, Flake AW (1999) Fetal tracheal occlusion in the rat model of nitrofen-induced congenital diaphragmatic hernia. J Appl Physiol 87:769–775PubMedGoogle Scholar
  38. 38.
    Alcorn D, Adamson TM, Lambert TF, Maloney JE, Ritchie BC, Robinson PM (1977) Morphological effects of chronic tracheal ligation and drainage in the fetal lamb lung. J Anat 123:649–660PubMedGoogle Scholar
  39. 39.
    Grubb BR, Pace AJ, Lee E, Koller BH, Boucher RC (2001) Alterations in airway ion transport in NKCC1-deficient mice. Am J Physiol Cell Physiol 281:C615–C623PubMedGoogle Scholar
  40. 40.
    Starrett RW, de Lorimier AA (1975) Congenital diaphragmatic hernia in lambs: hemodynamic and ventilatory changes with breathing. J Pediatr Surg 10:575–582. doi: 10.1016/0022-3468(75)90359-0 PubMedCrossRefGoogle Scholar
  41. 41.
    Nobuhara KK, Wilson JM (1996) The effect of mechanical forces on in utero lung growth in congenital diaphragmatic hernia. Clin Perinatol 23:741–752PubMedGoogle Scholar
  42. 42.
    Inanlou MR, Baguma-Nibasheka M, Kablar B (2005) The role of fetal breathing-like movements in lung organogenesis. Histol Histopathol 20:1261–1266PubMedGoogle Scholar
  43. 43.
    Fewell JE, Lee CC, Kitterman JA (1981) Effects of phrenic nerve section on the respiratory system of fetal lambs. J Appl Physiol 51:293–297PubMedGoogle Scholar
  44. 44.
    Wigglesworth JS, Desai R (1979) Effect on lung growth of cervical cord section in the rabbit fetus. Early Hum Dev 3:51–65. doi: 10.1016/0378-3782(79)90020-3 PubMedCrossRefGoogle Scholar
  45. 45.
    Jesudason EC, Smith NP, Connell MG, Spiller DG, White MR, Fernig DG et al (2005) Developing rat lung has a sided pacemaker region for morphogenesis-related airway peristalsis. Am J Respir Cell Mol Biol 32:118–127. doi: 10.1165/rcmb.2004-0304OC PubMedCrossRefGoogle Scholar
  46. 46.
    Smith PG, Janiga KE, Bruce MC (1994) Strain increases airway smooth muscle cell proliferation. Am J Respir Cell Mol Biol 10:85–90PubMedGoogle Scholar
  47. 47.
    Maksym GN, Deng L, Fairbank NJ, Lall CA, Connolly SC, Smith PG et al (2005) Beneficial and harmful effects of oscillatory mechanical strain on airway smooth muscle. Can J Physiol Pharmacol 83:913–922. doi: 10.1139/y05-091 PubMedCrossRefGoogle Scholar
  48. 48.
    Khan PA, Cloutier M, Piedboeuf B (2007) Tracheal occlusion: a review of obstructing fetal lungs to make them grow and mature. Am J Med Genet C Semin Med Genet 145:125–138. doi: 10.1002/ajmg.c.30127 Google Scholar
  49. 49.
    Iwamoto LM, Fujiwara N, Nakamura KT, Wada RK (2004) Na-K-2Cl cotransporter inhibition impairs human lung cellular proliferation. Am J Physiol Lung Cell Mol Physiol 287:L510–L514. doi: 10.1152/ajplung.00021.2004 PubMedCrossRefGoogle Scholar
  50. 50.
    Panet R, Eliash M, Pick M, Atlan H (2002) Na(+)/K(+)/Cl(-) cotransporter activates mitogen-activated protein kinase in fibroblasts and lymphocytes. J Cell Physiol 190:227–237. doi: 10.1002/jcp. 10055 PubMedCrossRefGoogle Scholar
  51. 51.
    Jiang G, Klein JD, O’Neill WC (2001) Growth factors stimulate the Na-K-2Cl cotransporter NKCC1 through a novel Cl(-)-dependent mechanism. Am J Physiol Cell Physiol 281:C1948–C1953PubMedGoogle Scholar
  52. 52.
    Acosta JM, Thebaud B, Castillo C, Mailleux A, Tefft D, Wuenschell C et al (2001) Novel mechanisms in murine nitrofen-induced pulmonary hypoplasia: FGF-10 rescue in culture. Am J Physiol Lung Cell Mol Physiol 281:L250–L257PubMedGoogle Scholar
  53. 53.
    Teramoto H, Yoneda A, Puri P (2003) Gene expression of fibroblast growth factors 10 and 7 is downregulated in the lung of nitrofen-induced diaphragmatic hernia in rats. J Pediatr Surg 38:1021–1024. doi: 10.1016/S0022-3468(03)00183-0 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Andreas Ringman
    • 1
    • 2
  • Marina Zelenina
    • 1
  • Ann-Christine Eklöf
    • 1
  • Anita Aperia
    • 1
  • Björn Frenckner
    • 2
  1. 1.Department of Woman and Child Health, Astrid Lindgren Children’s Hospital Research Laboratory Q2:09, Nordic Centre of Excellence for Research in Water Imbalance Related Disorders (WIRED)Karolinska InstitutetStockholmSweden
  2. 2.Division of Pediatric Surgery, Department of Woman and Child HealthKarolinska InstitutetStockholmSweden

Personalised recommendations