Mechanisms of Regulation of Intestinal Cl Absorption in IBD Associated Diarrhea

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Diarrhea is the hallmark symptom of inflammatory bowel diseases (IBD). Recurring diarrheal episodes not only significantly affect treatment options, but also the quality of life of the US veteran patients with IBD. IBD associated diarrhea results from a decrease in luminal NaCl and water absorption as well as derangements in barrier integrity. Major route of NaCl absorption in the human intestine involves concerted operation of Na+/H+ and Cl-/HCO3- exchangers. SLC26A3 or DRA (Down-Regulated in Adenoma) is the key transporter involved in intestinal Cl- absorption. DRA knockout mice have been shown to exhibit diarrheal phenotype resembling human CLD (congenital chloride diarrhea) and recent genome-wide association studies (GWAS) have linked the dysregulated DRA expression to IBD pathogenesis. Further, we have recently demonstrated that DRA deficiency in mice enhances intestinal permeability and compromises barrier integrity. Thus, DRA has emerged as an important novel target for intervention in diarrheal disorders and IBD. Therefore, it is vital to characterize molecular mechanisms involved in DRA regulation in health and disease and to identify agents that can activate DRA and/or counteract its downregulation. In this regard, glucocorticoids (GCs) are important anti-inflammatory agents and are first line therapeutics for the induction of remission in IBD. Also, GCs have been shown to stabilize epithelial barrier function and exert antidiarrheal effects by restoring electrolyte and water absorption in chronically inflamed epithelium. However, to date, the direct effects of GCs on DRA expression and chloride absorption have not been investigated. Our current preliminary data provide strong evidence that synthetic GC such as Dexamethasone (DEX) via glucocorticoid receptor (GR) can increase DRA expression via transcriptional activation. DEX treatment also increased DRA expression in mouse intestine. Therefore, we hypothesized that DEX exerts antidiarrheal effects by upregulating DRA expression and chloride absorption. Also, in light of our preliminary data demonstrating a key role of DRA in intestinal barrier integrity, we further hypothesized that DEX mediated upregulation of DRA will not only ameliorate diarrhea but will also restore epithelial barrier integrity. The current application is, therefore, designed to investigate the mechanisms of regulation of DRA gene expression by DEX, role of GR receptor and the associated co-activators under normal and inflammatory conditions utilizing both in-vitro cell culture, ex-vivo human apical-out enteroids, and in-vivo mouse models including DRA-KO mice and mice with intestine specific deletion of GR. The Specific Aims are: 1. Elucidate the mechanisms of DEX-induced transcriptional regulation of DRA expression and function and its role in counteracting inhibitory effects of TNF- on DRA gene expression; 2. Elucidate the mechanisms underlying DRA up-regulation by DEX in-vivo utilizing wild type and intestine specific GR knockout mice; and 3. Examine the role of DEX in DRA upregulation as a novel therapeutic approach in reversal of diarrhea and restoration of barrier integrity in mice with experimental colitis. Our proposed studies will yield mechanistic information vital for a better understanding of the mechanisms of regulation of DRA function and expression by GCs such as DEX and are of critical importance in advancing our knowledge to develop newer and better therapeutic tools to improve the health of veterans.