Intestinal current measurement detects age-dependent differences in CFTR function in rectal epithelium

Abstract

Objective: Intestinal current measurement (ICM) provides a sensitive bioassay for assessment of cystic fibrosis transmembrane conductance regulator (CFTR) function in rectal biopsies ex vivo and is used as a diagnostic tool for cystic fibrosis (CF). Furthermore, ICM was shown to be sensitive to detect pharmacological rescue of CFTR function by CFTR modulators in people with CF carrying responsive CFTR mutations. Results from clinical trials of CFTR modulators across age groups indicate that CFTR function in the sweat duct may be age-dependent with children reaching higher levels than adults. However, little is known about age dependency of CFTR function in the intestinal epithelium.

Methods: We investigated CFTR-mediated chloride secretion in rectal biopsies from 258 people without CF and 72 people with pancreatic-insufficient CF from 1 month to 68 years of age. Change in transepithelial short-circuit current in response to cyclic adenosine monophosphate (cAMP)-mediated (100 μM IBMX, 1 µM forskolin, basolateral) and cholinergic (100 μM carbachol, basolateral) stimulation was assessed as a readout for CFTR function using perfused micro-Ussing chambers. Furthermore, quantitative real-time PCR of CFTR and morphometric analysis of epithelial cells lining the crypts and surface of the rectal mucosa were performed to assess regulation at the levels of gene expression and epithelial cell densities.

Results: We found that CFTR-mediated chloride secretion across rectal tissues, as determined from cAMP-mediated as well as cholinergic chloride-secretory responses was highest during infancy and early childhood and declined with age in people without CF (both P < 0.001). Although, there was no difference in cAMP-mediated currents in people with CF, potassium-secretory responses induced by cholinergic stimulation were also reduced with increasing age. Transcript analyses showed that CFTR mRNA expression was slightly increased with increasing age in people without CF (P < 0.05). Morphometric analyses demonstrated that CFTR expressing colonocytes at the crypt base were decreased with age (P < 0.05). A secondary analysis of the ICM data of our previous studies on the effects of lumacaftor/ivacaftor on CFTR function in F508del -homozygous people with CF aged 12 years and older and 2-11 year old children showed correlations of the change in cAMP-mediated and cholinergic chloride secretory response with the age of people with CF (P < 0.01 and P < 0.05, respectively).

Conclusion: These results demonstrate that CFTR function in the rectal epithelium is reduced with increasing age and indicate that this change is likely due to a decline in the number of secretory colonocytes at the crypt base. These findings suggest that differences in CFTR expressing cells may explain increased functional responses to CFTR modulator therapies in children compared to adult people with CF.

Keywords: CFTR; CFTR modulator therapy; age-dependency; intestinal current measurement; rectal epithelium; secretory diarrhea.

FIGURE 1

The mechanisms of ion transport in colonic enterocytes. Forskolin increases cytosolic cAMP which enhances CFTR (cystic fibrosis transmembrane conductance regulator)-conductance and increases basolateral K⁺ conductance. Carbachol (CCH) increases cytosolic Ca²⁺ concentration that activates K⁺ channels in the luminal and basolateral membrane, increasing the driving force for chloride secretion via CFTR. In people with CF, CFTR channels cannot be activated and Ca²⁺ mobilizing agonists enhance K⁺ secretion.

FIGURE 2

Chloride secretion in human rectal tissue is age-dependent. (A, B) Original recordings of the effects of cAMP-dependent (100 μmol/L IBMX and 1 μmol/L forskolin, basolateral) and cholinergic (100 μmol/L carbachol, basolateral) activation on V_te and R_te in rectal tissue from (A) A 3 year old and (B) A 27 year old person without CF. (C, D) Summary of the effects of (C) cAMP-induced (IBMX/forskolin) and (D) Carbachol-induced short-circuit current (I_sc) in rectal tissues from people without CF. (E, F) Summary of the effects of (E) cAMP-induced and (F) carbachol-induced short-circuit currents in rectal tissues from people without CF in different age groups. Experiments were performed in the presence of indomethacin and amiloride. n = 258; *, P < 0.05 vs. 0–4 years, †, P < 0.05 vs. 5–10 years, #, P < 0.05 vs. 11–21 years.

FIGURE 3

Ion transport in cystic fibrosis rectal tissue in different age groups. (A, B) Original recordings of the effects of cAMP-dependent (100 μmol/L IBMX and 1 μmol/L forskolin, basolateral) and cholinergic (100 μmol/L carbachol, basolateral) activation on V_te and R_te in rectal tissue from (A) A 4 year old and (B) a 33 year old person with CF. (C, D) Summary of the effects of (C) cAMP-induced (IBMX/forskolin) and (D) Carbachol-induced short-circuit current (I_sc) in rectal tissues from people with CF. (E, F) Summary of the effects of (E) cAMP-induced and (F) carbachol-induced short-circuit currents in rectal tissues from people with CF in different age groups. Experiments were performed in the presence of indomethacin and amiloride. n = 72; *, P < 0.05 vs. 0–4 years, †, P < 0.05 vs. 5–10 years.

FIGURE 4

Morphometric analyses of the intestinal crypts in children compared to adults. (A) Morphology of crypts in rectal biopsies of people without CF with the age of 3 years and 44 years. Sections were stained with hematoxylin and eosin (H&E). Scale bars = 50 µm. (B–D) Total cell, goblet cell and non-goblet cell counts from (B) The total crypt (C) the upper crypt half and (D) The lower crypt half of people without CF aged 0–4 years and ≥22 years. n = 9; *, P = 0.05.

FIGURE 5

Response to CFTR modulator therapy is age-dependent. (A, B) Change in cAMP-induced (A) and carbachol-induced (B) short circuit current in rectal tissue of people with CF on lumacaftor-ivacaftor therapy compared to baseline (n = 61). Data was reanalyzed from Graeber et al. (2018); Berges et al. (2023).