(203) Re-Evaluating the Expression Profile of SLC26A3 (DRA) Chloride/Bicarbonate Exchanger in the Small Intestine using Human Models: Implications for Cystic Fibrosis

Background: Small intestinal bicarbonate secretion aids in epithelial protection, digestion, and absorption. The cystic fibrosis transmembrane conductance regulator (CFTR), together with putative anion transporter-1 (PAT-1) and down-regulated in adenoma (DRA) chloride/bicarbonate exchangers, regulate intestinal bicarbonate transport in the small intestine. Prior mouse studies have suggested that PAT-1 is the primary chloride/bicarbonate exchanger in the small intestine, with DRA predominant in the colon. There is limited data on the expression profiles of PAT-1 and DRA in human small intestine. This is especially relevant in CF, where we have previously identified that chloride/bicarbonate exchange can compensate for loss of CFTR-mediated bicarbonate transport in the duodenum. The aim of the current study is to examine the expression profile of PAT-1 and DRA in the human small intestine and determine if these are altered in CF.

Methods: We analyzed existing single cell RNA sequencing (sc-RNAseq) metadata annotations on human small intestine and colon (J Exp Med 2020 Feb;217:e20191130; Nature 2021, 597:250-5; Cell Rep 2021, 9;34:108819) using Seurat R toolkit. Data available for each segment was as follows: duodenum (5944 cells, 5 subjects), ileum (6,167 cells, 2 subjects), and colon (4,472 cells, 2 subjects). CFTR, PAT-1, and DRA mRNA and protein expression was also examined in human enteroids and biopsies using qPCR and confocal immunofluorescence. CFTR_inh-172 was used for CFTR inhibition.

Results: Duodenal and ileal datasets showed high expression of DRA mRNA compared to PAT-1 mRNA (~3-4x in duodenum, ~2x in ileum). The expression profile across all segments was DRA >CFTR >PAT1. DRA expression in the colon was comparable to the duodenum, but higher than the ileum. PAT-1 was higher in the duodenum and ileum compared to colon. Both DRA and PAT-1 were predominantly expressed in enterocytes. DRA was also expressed in BEST4+ cells. DRA protein was robust in villus enterocytes in human duodenal biopsies (n=3). In patient-derived duodenal enteroids, DRA was expressed in both crypt-like (undifferentiated) and villus-like (differentiated) enteroids (n=3 each), with 20-fold greater mRNA expression in the Day5 differentiated enteroids. DRA mRNA expression was ~10-fold higher than PAT-1 in Day5 differentiated enteroids. Upon CFTR inhibition (CFTR_inh-172, 20mM, 40 mins) in apical-out enteroids, there was a significant increase in DRA membrane localization.

Conclusions: In humans, DRA is expressed throughout the small intestine, in the duodenum at levels comparable to the colon. Thus, in humans, DRA may play a more prominent role in small intestinal bicarbonate secretion than previously appreciated based on mouse data. Our observations that DRA is increased upon loss of CFTR function is consistent with a recent report of increased DRA in CF ileum (Bijvelds et al. J Cyst Fibros 2021 Dec online). Ongoing work is examining DRA and PAT-1 expression and function in non-CF and CF intestine in humans to identify CFTR-independent means to restore defective intestinal bicarbonate secretion in CF.

Acknowledgements: This work was supported by NIDDK and CFF.

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