Reduced NHE3-mediated Na+ absorption increases survival and decreases the incidence of intestinal obstructions in cystic fibrosis mice

Abstract

In cystic fibrosis, impaired secretion resulting from loss of activity of the cystic fibrosis transmembrane conductance regulator (CFTR) causes dehydration of intestinal contents and life-threatening obstructions. Conversely, impaired absorption resulting from loss of the NHE3 Na+/H+ exchanger causes increased fluidity of the intestinal contents and diarrhea. To test the hypothesis that reduced NHE3-mediated absorption could increase survival and prevent some of the intestinal pathologies of cystic fibrosis, Cftr/Nhe3 double heterozygous mice were mated and their offspring analyzed. Cftr-null mice lacking one or both copies of the NHE3 gene exhibited increased fluidity of their intestinal contents, which prevented the formation of obstructions and increased survival. Goblet cell hyperplasia was eliminated, but not the accumulation of Paneth cell granules or increased cell proliferation in the crypts. Microarray analysis of small intestine RNA from Cftr-null, NHE3-null, and double-null mice all revealed downregulation of genes involved in xenobiotic metabolism, including a cohort of genes involved in glutathione metabolism. Expression of energy metabolism genes was altered, but there were no changes in genes involved in inflammation. Total intracellular glutathione was increased in the jejunum of all of the mutants and the ratio of reduced to oxidized glutathione was reduced in Cftr-null mutants, indicating that CFTR deficiency affects intestinal glutathione metabolism. The data establish a major role for NHE3 in regulating the fluidity of the intestinal contents and show that reduced NHE3-mediated absorption reverses some of the intestinal pathologies of cystic fibrosis, thus suggesting that it may serve as a potential therapeutic target.

Fig. 1.

Survival of offspring from Cftr^+/−Nhe3^+/− breeders. Double heterozygous mice were bred and survival of mice for 5 of the resulting 9 genotypes (see Table 1) was analyzed. The numbers of mice and genotypes analyzed were: 44 Cftr^+/+Nhe3^+/+, 39 Cftr^+/+Nhe3^−/−, 44 Cftr^−/−Nhe3^−/−, 81 Cftr^−/−Nhe3^+/−, and 36 Cftr^−/−Nhe3^+/+. Cftr^−/−Nhe3^+/+ mice exhibited 100% mortality by 6 wk of age, but survival improved dramatically in Cftr^−/−Nhe3^+/− and Cftr^−/−Nhe3^−/− mice. All curves, except those for Cftr^−/−Nhe3^+/− and Cftr^+/+Nhe3^−/− mice, were significantly different (P < 0.009) from each other based on a Kaplan-Meier log rank test.

Fig. 2.

Effects of Cftr- and Nhe3-null mutations on body weight. Average body weights of mice of the 5 genotypes shown were analyzed at weaning (A) and adulthood (B). The weight of Cftr^−/−Nhe3^+/+ and Cftr^+/+Nhe3^−/− mice was significantly lower than that of wild-type (WT) mice at weaning, and this was moderated in Cftr^−/−Nhe3^+/− and Cftr^−/−Nhe3^−/− mice. At 8 wk of age, all mice, except Cftr^+/+Nhe3^−/− mice, had relatively normal body weights. The Cftr^−/−Nhe3^+/+ mice in B were maintained on the osmotic laxative Colyte, which prevents desiccation of intestinal contents. In each graph, n = 6–16 mice per genotype. Groups with the same letters (a or b) did not differ significantly; those with different letters were significantly different (P < 0.05) based on a 1-way ANOVA with a post hoc Tukey's t-test.

Fig. 3.

Gross anatomy of the intestinal tract of Nhe3 and Cftr mutants. Note the swollen intestine, cecum, and colon in mice lacking NHE3 (B and E) and the abnormal “corkscrew” ceca (see Supplementary Fig. S1) in Cftr^−/−Nhe3^+/+ (C) and Cftr^−/−Nhe3^+/− mice (D). The midpoint of 2 impactions (dark arrows) and the points at which the blockages occurred (white arrows) are shown in the Cftr^−/−Nhe3^+/+ intestine (C); one blockage occurred in the proximal ileum and the other near the ileal-cecal junction. The Nhe3^−/− phenotype, characterized by high fluidity of intestinal, cecal, and colonic contents and expansion of the cecum, is dominant in Cftr^−/−Nhe3^−/− mice. In each image, the stomach (St) is oriented in the upper left and the cecum (Cec) in the upper right. All mice were 7–8 wk of age except in C, which was 3 wk old.

Fig. 4.

Fluidity of intestinal and colonic contents is increased in adult mice lacking NHE3. Luminal contents of the small intestine and colon were collected, and both wet weight and dry weight of the contents were determined. Fluidity was calculated as the weight ratio of water content to dry content (water wt/dry wt) in small intestine (A) and colon (B). *P < 0.05 compared with WT based a 1-way ANOVA with a post hoc Tukey's t-test; n = 6–9 mice per genotype.

Fig. 5.

Goblet cell hyperplasia of the ileal villi of cystic fibrosis (CF) mice is corrected by loss of one or both copies of the Nhe3 gene. Representative Alcian blue and periodic acid-Schiff (AB/PAS)-stained sections of ileum from Cftr^+/+Nhe3^+/+ (A), Cftr^+/+Nhe3^−/− (B), Cftr^−/−Nhe3^+/+ (C), Cftr^−/−Nhe3^+/− (D), and Cftr^−/−Nhe3^−/− (E) mice. Arrows indicate goblet cells, which appear dark blue (containing acidic mucins) or magenta (containing neutral or a mix of neutral and acidic mucins). Normal goblet cell numbers were observed in Cftr^+/+Nhe3^+/+ (A) and Cftr^+/+Nhe3^−/− (B) mice. In contrast, an increase in both the numbers and apparent size of the goblet cells is evident in Cftr^−/−Nhe3^+/+ mice (C). Goblet cell hyperplasia is corrected in Cftr^−/−Nhe3^+/− (D) and Cftr^−/−Nhe3^−/− (E) mice. F: Cftr^−/−Nhe3^+/+ crypts also exhibit severe goblet cell hyperplasia. Scale bars represent 50 μm. G: cell counts of villus goblet cells (normalized to 100 μm) indicate a ∼3-fold increase in goblet cell numbers in Cftr^−/−Nhe3^+/+ mice relative to other genotypes. *P < 0.05 compared with WT based on a 1-way ANOVA with a post hoc Tukey's t-test; n = 3–6 mice per genotype.

Fig. 6.

Crypt and villus heights do not differ significantly among WT and mutant mice. Measurements of the heights of crypt-villus axes (CVA; A), villi (B), and crypts (C) in the ileum of 25-day-old mice revealed no significant differences among genotypes. Only those axes in which both the villus and its adjacent crypt were full length were measured; a minimum of 10 crypt-villus axes were measured for each mouse. *P < 0.05 compared with WT based on a 1-way ANOVA with a post hoc Tukey's t-test; n = 4–6 mice per genotype.

Fig. 7.

Paneth cell granules accumulate in the crypts of mice lacking CFTR, even in the absence of NHE3. Hematoxylin and eosin-stained sections of crypts from ileum of Cftr^+/+Nhe3^+/+ (A), Cftr^+/+Nhe3^−/− (B), Cftr^−/−Nhe3^+/+ (C), Cftr^−/−Nhe3^+/− (D), and Cftr^−/−Nhe3^−/− mice (E). F: higher magnification of a crypt from a Cftr^−/−Nhe3^+/+ mouse ileum. Arrowheads point to crypt lumens; undissolved granules are highly eosinophilic (dark red). Original images were taken at ×40 magnification. G: the percent of crypts containing undissolved granules was greatly increased in all Cftr^−/− mice, regardless of Nhe3 genotype. Crypts were analyzed in at least 10 ×400 fields of view; n = 3–6 mice per genotype. *P < 0.05 compared with WT based on a 1-way ANOVA with a post hoc Tukey's t-test.

Fig. 8.

Increased cell proliferation in crypts of Cftr-null and Nhe3-null mice. Cell proliferation in duodenal crypts was assessed by quantitation of cells staining positive for proliferating cell nuclear antigen (PCNA), a nuclear marker of proliferation. Bars represent the number of PCNA-positive nuclei as a fraction of the total number of nuclei. Nuclei were counted in at least 10 whole crypts per mouse; n = 4–6 mice per genotype. *P < 0.05 compared with WT and †P < 0.05 compared with Cftr^−/−Nhe3^+/+ based on a 1-way ANOVA with a post hoc Tukey's t-test.

Fig. 9.

Northern blots verify the downregulation of several xenobiotic metabolism genes identified by microarray analysis. Because similar patterns of gene expression changes were identified in all 3 sets of microarrays, expression changes for the genes indicated on the left were verified by using WT and Cftr^−/−Nhe3^−/− (KO) small intestine total RNA (10 μg/lane, 3 mice of each genotype). The blots were hybridized with PCR-amplified cDNA probes, and expression was normalized to the L32 ribosomal subunit mRNA. All four genes were significantly altered (GSTα4, −2.35 ± 0.07; GSTκ1, −2.90 ± 0.05; Cyp3a11, −3.17 ± 0.07; Cyp4v3, −4.24 ± 0.59; P < 0.05 compared with WT based on a Student's t-test).

Fig. 10.

Intestinal glutathione levels. Total and oxidized (GSSG) glutathione levels were measured in samples of intestine from mice of each of the indicated genotypes (n = 3–5 mice per genotype). A: tissue levels of total glutathione (solid bars) and GSSG (hatched bars) were increased in all mutant mice compared with WT controls; *P < 0.05 compared with WT based on a Student's t-test. B: when all 3 CF genotypes were pooled, the ratio of GSH to GSSG in mice lacking a functional Cftr gene (and WT, heterozygous, or null mutant with respect to Nhe3) was significantly different (P = 0.009 based on a Student's t-test) compared with pooled values from mice that were WT with respect to Cftr (and either WT or null mutant with respect to Nhe3).