Cystic fibrosis growth retardation is not correlated with loss of Cftr in the intestinal epithelium

Abstract

Maldigestion due to exocrine pancreatic insufficiency leads to intestinal malabsorption and consequent malnutrition, a mechanism proposed to cause growth retardation associated with cystic fibrosis (CF). However, although enzyme replacement therapy combined with increased caloric intake improves weight gain, the effect on stature is not significant, suggesting that growth retardation has a more complex etiology. Mouse models of CF support this, since these animals do not experience exocrine pancreatic insufficiency yet are growth impaired. Cftr absence from the intestinal epithelium has been suggested as a primary source of growth retardation in CF mice, a concept we directly tested by generating mouse models with Cftr selectively inactivated or restored in intestinal epithelium. The relationship between growth and functional characteristics of the intestines, including transepithelial electrophysiology, incidence of intestinal obstruction, and histopathology, were assessed. Absence of Cftr exclusively from intestinal epithelium resulted in loss of cAMP-stimulated short-circuit current, goblet cell hyperplasia, and occurrence of intestinal obstructions but only slight and transient impaired growth. In contrast, specifically restoring Cftr to the intestinal epithelium resulted in restoration of ion transport and completely protected against obstruction and histopathological anomalies, but growth was indistinguishable from CF mice. These results indicate that absence of Cftr in the intestinal epithelium is an important contributor to the intestinal obstruction phenotype in CF but does not correlate with the observed growth reduction in CF.

Fig. 1.

Survival and growth of cystic fibrosis (CF) mice with and without FABP-CFTR. Survival (A) and growth of mice (B) were assessed in CF mice (Cftr^tm1Unc, n = 24), CF mice in which human cystic fibrosis transmembrane regulator (CFTR) was expressed by the fatty acid binding protein (FABP) promoter (Cftr^tm1Unc+FABP-CFTR, n = 28), and control littermates (C57Bl/6J, n = 96) up to 40 days of age. Weight was significantly decreased in Cftr^tm1Unc and Cftr^tm1Unc+FABP-CFTR compared with control animals at every age measured (P < 0.0001) with equal numbers of each sex used (data represent means ± SD). Incremental loss of Cftr^tm1Unc mice led to decreasing numbers of weight measurements for this group (e.g., by day 40 only 4 mice were available for weights). The Cftr^tm1Unc+FABP-CFTR and C57Bl/6J groups contained consistent numbers of mice in each group throughout the growth curve.

Fig. 2.

Schematic of 2 conditional Cftr alleles. A: conditional Cftr allele consisting of exon 10 surrounded by 2 loxP sites in the same orientation (Cftr^fl10). The presence of Cre recombinase induces recombination between the 2 loxP sites, resulting in deletion of exon 10 (Cftr^Δ10) and thus nonfunctional Cftr. B: conditional Cftr allele consisting of an inverted exon 10, and thus nonfunctional Cftr, surrounded by 2 loxP sites in opposite orientation (Cftr^invfl10). The presence of Cre recombinase induces recombination between the 2 loxP sites, resulting in the inversion of exon 10 and thus producing functional Cftr (Cftr^fl10′). The inversion of exon 10 is a reversible reaction in the presence of Cre recombinase that results in approximately equal presence of exon 10 sense and antisense alleles (functional and nonfunctional Cftr, respectively).

Fig. 3.

Intestinal epithelium-specific recombination of conditional Cftr alleles. A: DNA amplification of the region surrounding exon 10 from various tissues of a mouse homozygous for Cftr^fl10 with the villin-Cre transgene. Cre recombinase expression specifically in intestinal epithelium results in deletion of exon 10 (Cftr^Δ10, 148 bp) in the intestinal epithelium (ie) and the whole intestine (i). Exon 10 is still present in lung (lu), heart (h), liver (li), and brain (b), as well as the whole intestine, which includes cell types other than epithelium (Cftr^fl10- 408 bp). Cre expression was also absent in kidney and bone (data not shown). B: DNA amplification of the region surrounding exon 10 from various tissues of a mouse homozygous for Cftr^invfl10 with the villin-Cre transgene. Cre recombinase expression specifically in intestinal epithelium results in inversion of exon 10 (Cftr^fl10′, 408 bp) in the intestinal epithelium and the whole intestine. Exon 10 remains inverted (Cftr^invfl10, 563 bp) in the lung, heart, liver, and brain. The inverted form of exon 10 is also observed in the intestinal tissue due to the reversible nature of this reaction.

Fig. 4.

Cftr mRNA expression in isolated intestinal epithelium from conditional Cftr mice with and without villin-Cre Cftr expression in intestinal epithelial isolates from the duodenum (D), jejunum (J), ileum (I), cecum (C), and colon (Co) from Cftr^fl10, Cftr^fl10 + villin-Cre, Cftr^invfl10, and Cftr^invfl10 +villin-Cre. Expression is reported as a percentage of Cftr expression in control mice (Cftr^fl10) in the corresponding intestinal section. Each measurement is an average of 3 mice completed in replicates (data represent means ± SE).

Fig. 5.

Exon 10 manipulation in the intestinal epithelium does not alter nasal potential difference (NPD) of mice with Cftr function [Cftr^fl10 (A) and Cftr^fl10 + villin-Cre (B)] and without Cftr function [Cftr^invfl10 (C) and Cftr^invfl10 + villin-Cre (D)] in the nasal epithelium. Arrow indicates addition of forskolin (10 μM) and chloride-free HEPES-buffered Ringer solution. A bi-ionic junction potential of −12.5 mV was observed (step increase in voltage coincident with solution change) independent of Cftr function and was excluded from the calculation of change in NPD.

Fig. 6.

Exon 10 manipulation in the intestinal epithelium leads to either inactivation or activation of Cftr function as assessed by change in intestinal short-circuit current (I_sc). I_sc response to the addition of forskolin (10 μM) and IBMX (100 μM) (indicated by arrows) in ileal sections from Cftr^fl10 (A), Cftr^fl10 + villin-Cre (B), Cftr^invfl10 (C), and Cftr^invfl10 + villin-Cre (D). Vertical deflections (at 1-min intervals) result from voltage-clamp to nonzero values to measure transepithelial resistance.

Fig. 7.

Histology of ileal sections in conditional Cftr mice with and without Cftr in the intestinal epithelium. Ileal sections from mice with functional Cftr in the intestinal epithelium, Cftr^fl10 (A) and Cftr^invfl10 + villin-Cre (D), displayed a normal number of goblet cells (purple circles) in the crypts and villi. Intestinal sections from mice without functional Cftr in the intestinal epithelium, Cftr^fl10 + villin-Cre (B) and Cftr^invfl10 (C), displayed goblet cell hyperplasia along the villi and within the crypts. Ileal photomicrographs are ×200 magnification.

Fig. 8.

Survival and growth of conditional Cftr mice with and without Cftr in the intestinal epithelium. Percentage of mice surviving up to 40 days of age for Cftr^fl10 + villin-Cre (n = 31) and Cftr^fl10 (n = 60) (A) and Cftr^invfl10 + villin-Cre (n = 28) and Cftr^invfl10 (n = 31) (B). Survival of Cftr^fl10 + villin-Cre and Cftr^invfl10 was significantly reduced compared with littermates (P < 0.0001). Average weight up to 40 days of age from Cftr^fl10 + villin-Cre and Cftr^fl10 (C) and Cftr^invfl10 + villin-Cre, Cftr^invfl10, and control littermates (D). Cftr^fl10 + villin-Cre mice displayed significantly reduced weight from 20–30 days of age compared with control littermates (P < 0.05). Cftr^invfl10 + villin-Cre and Cftr^invfl10 mice displayed significantly reduced weight at every age compared with control littermates (P < 0.001) but were not significantly different from each other. Weights include mice that died before 40 days of age. Thus the incremental loss of Cftr^invfl10 mice led to decreasing numbers of weight measurements for this group (e.g., by day 40 only 10 mice were available for weights). Average body length from 6-wk-old mice (n ≥ 8 for each group) from Cftr^fl10 + villin-Cre and Cftr^fl10 (E) and Cftr^invfl10 + villin-Cre, Cftr^invfl10, and control littermates (F). Cftr^invfl10 + villin-Cre and Cftr^invfl10 mice displayed significantly reduced body length compared with control littermates (*P < 0.005) but were not significantly different from each other (all data represent means ± SD).