DRA involvement in linaclotide-stimulated bicarbonate secretion during loss of CFTR function
Jessica B. Sarthi, … , Calvin J. Kuo, Zachary M. Sellers
Jessica B. Sarthi, … , Calvin J. Kuo, Zachary M. Sellers
Published June 13, 2024
Citation Information: JCI Insight. 2024;9(14):e172364. https://doi.org/10.1172/jci.insight.172364.
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Research Article Gastroenterology

DRA involvement in linaclotide-stimulated bicarbonate secretion during loss of CFTR function

Abstract

Duodenal bicarbonate secretion is critical to epithelial protection, as well as nutrient digestion and absorption, and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy, and de novo analysis of human duodenal single-cell RNA sequencing (scRNA-Seq) data sets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of cystic fibrosis transmembrane conductance regulator (CFTR) expression (Cftr-knockout mice) or function (CFTRinh-172). Na+/H+ exchanger 3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. ScRNA-Seq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.

Authors

Jessica B. Sarthi, Annie M. Trumbull, Shayda M. Abazari, Vincent van Unen, Joshua E. Chan, Yanfen Jiang, Jesse Gammons, Marc O. Anderson, Onur Cil, Calvin J. Kuo, Zachary M. Sellers

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Figure 6

Linaclotide increases membrane expression of DRA in apical-out human duodenal enteroids.

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Linaclotide increases membrane expression of DRA in apical-out human duo...
Representative images of DRA (A and E), villin (B and F), and DRA and villin and Hoechst (C and G) in apical-out healthy duodenal enteroids during control conditions (water or DMSO), CFTRinh-172 (2 × 10–5 M), or linaclotide (10–7 M) with or without CFTRinh-172 pretreatment (2 × 10–5 M) (all 40 minutes). (D, H, and I) Quantification of DRA at the apical brush border using villin to define this cell region. Apical brush border DRA MFI following linaclotide (10–7 M, 40 minutes, n = 16), CFTRinh-172 alone (2 × 10–5 M, 40 minutes, n = 31), or CFTRinh-172+linaclotide (n = 21). Treatments were normalized to vehicle controls (water for linaclotide, DMSO for CFTRinh-172, n = 12–31). Enteroids from 3 individuals were used for each condition. Significance determined by unpaired, 2-tailed Student’s t test. *P < 0.05, ***P < 0.001. Representative images of DRA (J), villin (K), and DRA and villin and Hoechst (L) in apical-out CF (F508del homozygous) duodenal enteroids during control (water, 40 minutes) or linaclotide stimulation (10–7 M, 40 minutes). (M) Quantification of DRA present at the apical brush border using villin, similar to D. (N) Comparison of baseline DRA MFI between non-CF and CF enteroids, with data normalized to non-CF enteroids. n = 8–11 enteroids from 1 patient across 2 different passages. Significance determined by unpaired, 2-tailed Student’s t test. *P < 0.05, **P < 0.01. (O) To compare DRA membrane expression across non-CF and CF enteroids in different situations, absolute DRA membrane MFI was plotted. Data are from experiments performed in D, H, I, and M. In images, arrows assist with identifying regions of interest (scale bar, 20 µm). Columns with whiskers are mean ± SEM with each dot representing a different enteroid.