(743.17) Serum and Glucocorticoid-Inducible Kinase 1 (SGK1): An Important Contributor to Diarrhea and Malabsorption in Microvillus Inclusion Disease (MVID)

Poster Board Number: E344

Md Kaimul Ahsan (Yale University), Diego Carlos dos Reis (Yale University), Andrea Barbieri (Yale University), Kaelyn Sumigray (Yale University), Timothy Nottoli (Yale University), Pedro Salas (University of Miami), Nadia Ameen (Yale University, Yale University)

Microvillus Inclusion Disease (MVID) is a lethal congenital diarrheal disease resulting from loss of function mutations in the actin motor myosin VB (MYO5B) that regulates apical traffic. MVID remains without treatment to reverse the severe diarrhea that leads to death. MVID diarrhea results from both increased fluid secretion and malabsorption of ions and carbohydrates in the small intestine. Serum and Glucocorticoid-inducible kinase 1 (SGK1), is a potent regulator of ion transporters including cystic fibrosis transmembrane conductance regulator (CFTR) in the intestine. Since SGK1 upregulates CFTR function in the intestine, we hypothesized that loss of SGK1 could potentially reduce MVID diarrhea by decreasing CFTR fluid secretion.

Using CRISPR-Cas 9 approaches, we first generated MYO5B-floxed mice and to generate conditional MYO5B-KO (R26creER;MYO5Bf/f) mice, we cross bred with R26creER mice. Tamoxifen-treated R26creER;MYO5Bf/f mice resulted in characteristic features of human MVID including severe diarrhea, Microvillus Inclusions (MIs), defective apical traffic, depolarization of proteins, and ion transporters including CFTR, NHE3 and DRA. MYO5B-KO mice also showed increased phosphorylation of SGK1 and PDK1 in the intestine. SGK1-floxed (SGK1f/f) mice were crossed with R26creER;MYO5Bf/f mice to generate conditional MYO5B/SGK1 DcKO (R26creER;MYO5Bf/f;SGK1f/f) upon Tamoxifen induction. Surprisingly, tamoxifen treatment (3 day) of MYO5B/SGK1 DcKO mice resulted in more severe diarrhea compared to MYO5B (R26creER;MYO5Bf/f) mice. Immunoblots of intestinal lysates revealed decreased CFTR, increased alpha- and beta-ENaC, increased phosphorylation of PDK1, Nedd4-2 and pPKCιThr563 in MYO5B/SGK1 DcKO vs. MYO5B ScKO mice. Finally, fecal glucose loss was higher with reduced SGLT1 and GLUT2 by immunoblot in MYO5B/SGK1 DcKO vs MYO5B ScKO mice. We conclude that activation of SGK1 pathway and CFTR contribute to worsening of diarrhea and carbohydrate malabsorption in MVID.

North American Society For Pediatric Gastroenterology, Hepatology amp;amp; Nutrition (NASPGHAN)