Intestinal CFTR expression alleviates meconium ileus in cystic fibrosis pigs
David A. Stoltz, … , David K. Meyerholz, Michael J. Welsh
David A. Stoltz, … , David K. Meyerholz, Michael J. Welsh
Published May 8, 2013
Citation Information: J Clin Invest. 2013;123(6):2685-2693. https://doi.org/10.1172/JCI68867.
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Intestinal CFTR expression alleviates meconium ileus in cystic fibrosis pigs

Abstract

Cystic fibrosis (CF) pigs develop disease with features remarkably similar to those in people with CF, including exocrine pancreatic destruction, focal biliary cirrhosis, micro-gallbladder, vas deferens loss, airway disease, and meconium ileus. Whereas meconium ileus occurs in 15% of babies with CF, the penetrance is 100% in newborn CF pigs. We hypothesized that transgenic expression of porcine CF transmembrane conductance regulator (pCFTR) cDNA under control of the intestinal fatty acid–binding protein (iFABP) promoter would alleviate the meconium ileus. We produced 5 CFTR–/–;TgFABP>pCFTR lines. In 3 lines, intestinal expression of CFTR at least partially restored CFTR-mediated anion transport and improved the intestinal phenotype. In contrast, these pigs still had pancreatic destruction, liver disease, and reduced weight gain, and within weeks of birth, they developed sinus and lung disease, the severity of which varied over time. These data indicate that expressing CFTR in intestine without pancreatic or hepatic correction is sufficient to rescue meconium ileus. Comparing CFTR expression in different lines revealed that approximately 20% of wild-type CFTR mRNA largely prevented meconium ileus. This model may be of value for understanding CF pathophysiology and testing new preventions and therapies.

Authors

David A. Stoltz, Tatiana Rokhlina, Sarah E. Ernst, Alejandro A. Pezzulo, Lynda S. Ostedgaard, Philip H. Karp, Melissa S. Samuel, Leah R. Reznikov, Michael V. Rector, Nicholas D. Gansemer, Drake C. Bouzek, Mahmoud H. Abou Alaiwa, Mark J. Hoegger, Paula S. Ludwig, Peter J. Taft, Tanner J. Wallen, Christine Wohlford-Lenane, James D. McMenimen, Jeng-Haur Chen, Katrina L. Bogan, Ryan J. Adam, Emma E. Hornick, George A. Nelson IV, Eric A. Hoffman, Eugene H. Chang, Joseph Zabner, Paul B. McCray Jr., Randall S. Prather, David K. Meyerholz, Michael J. Welsh

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Figure 2

CFTR is expressed and functional in CFTR–/–;TgFABP>pCFTR piglets.

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CFTR is expressed and functional in CFTR–/–;TgFABP>pCFTR piglets. (...
(A) Range of meconium ileus severity in the different CFTR–/–;TgFABP>pCFTR transgenic lines. Normal, absence of meconium ileus (CFTR+/+ intestine). 3+, severe meconium ileus phenotype (CFTR–/– intestine). Refer to Methods for further details of scoring system. (B) CFTR mRNA expression in ileal segments from CFTR+/+, CFTR–/–, and CFTR–/–;TgFABP>pCFTR newborn piglets. CFTR mRNA levels were determined with quantitative RT-PCR (relative to β-actin), and values are expressed as a percentage relative to CFTR+/+ mRNA levels. Symbols represent individual animals except for CFTR+/+, which is the mean. (C) CFTR localizes to ileal crypts (arrows). Confocal microscopy images show staining for β-catenin (a component of adherens junctions, red), CFTR (green), and nuclei (DAPI, blue). Scale bars: 10 μm. (D) Changes in short-circuit current (Isc) following addition of forskolin/IBMX (ΔIscF&I) to freshly excised ileal segments from newborn CFTR+/+, CFTR–/–, and CFTR–/–;TgFABP>pCFTR piglets. Prior to forskolin/IBMX treatment, tissues were exposed to amiloride. Symbols represent individual animals. Line represents mean ± SEM. (E) Representative Isc tracings from freshly excised ileal segments mounted in Ussing chambers for electrophysiological studies. Ileal samples were obtained from newborn piglets. The following agents were added sequentially: apical 100 μM amiloride (amil), apical 10 μM forskolin/100 μM IBMX (F/I), apical 100 μM GlyH, basolateral 100 μM carbachol (CCH), apical 100 μM DIDS, apical 5 mM dextrose (Dex), and apical 200 μM phlorizin (Phz).