KIF7 Expression Is Decreased in the Diaphragmatic and Pulmonary Mesenchyme of Nitrofen-Induced Congenital Diaphragmatic Hernia

Friday, October 10, 2014
Marina Ballroom Salon E-F (San Diego Marriott Marquis )
Toshiaki Takahashi, MD, Florian Friedmacher, MD, MSc, Alejandro Hofmann, MD, Hiromizu Takahashi and Prem Puri, Our Lady's Children's Hospital, National Children's Research Centre, Dublin, Ireland

Purpose: The origin of congenital diaphragmatic hernia (CDH) is assumed to lie in a malformation of the amuscular primordial diaphragm, allowing intrathoracic herniation of abdominal viscera and thus disturbing normal lung development. It has further been demonstrated that regular diaphragmatic and pulmonary morphogenesis requires the structural integrity of mesenchymal tissue, and developmental mutations that inhibit proper mesenchymal formation have been shown to cause CDH with associated hypoplastic lungs. Kinesin family member 7 (Kif7), an essential component of the sonic hedgehog signaling cascade, has recently been identified to play a crucial role in diaphragmatic and pulmonary development by controlling the proliferation of mesenchymal cells. In addition, the loss of Kif7 has been reported to result in diaphragmatic defects and hypoplastic lungs. Although the pathological mechanisms of diaphragmatic defects and associated pulmonary hypoplasia have been extensively studied, the molecular basis of the malformed amuscular mesenchymal components in CDH remains unclear. Therefore, we designed this study to investigate the hypothesis that diaphragmatic and pulmonary Kif7 expression is decreased in the nitrofen-induced CDH model.

Methods: After obtaining ethical approval (REC668b), timed-pregnant Sprague-Dawley rats were exposed to either nitrofen or vehicle on gestational day 9 (D9), and fetuses were harvested on selected time-points D13, D15 and D18. The dissected fetal diaphragms (n=72) and lungs (n=48) were divided into two groups: control and nitrofen-exposed samples (n=12 per specimen and time-point, respectively). Diaphragmatic and pulmonary gene expression levels of Kif7 were analyzed by quantitative real-time polymerase chain reaction. Immunohistochemical staining was performed to evaluate Kif7 protein expression in fetal diaphragms and lungs.

Results: Relative mRNA expression of Kif7 was significantly reduced in pleuroperitoneal folds/premordial lung buds of nitrofen-exposed fetuses on D13 (0.010.01 vs. 0.060.01; P<0.05), developing diaphragms and lungs of nitrofen-exposed fetuses on D15 (0.020.02 vs. 0.060.02; P<0.05 and 0.010.01 vs. 0.040.02; P<0.05), and fully muscularized diaphragms and differentiated lungs of nitrofen-exposed fetuses on D18 (0.010.01 vs. 0.040.02; P<0.05 and 0.010.01 vs. 0.020.01; P<0.05) compared to controls. Diaphragmatic and pulmonary immunoreactivity/-fluorescence of Kif7 was markedly decreased in amuscular mesenchymal components of nitrofen-exposed fetuses on D13, D15 and D18 compared to controls (Figure).

Conclusions: Decreased mesenchymal Kif7 expression during diaphragmatic development and pulmonary maturation may interfere with normal mesenchymal cell proliferation, leading to malformed amuscular mesenchymal components and contributing to the diaphragmatic defects and hypoplastic lungs in the nitrofen-induced CDH model.