194 N-Glycosylation Is Essential for Ileal ASBT Function and Protection Against Proteases

Abstract

those patients. Total and surface expression of NHE3 were reduced in rat ileum treated with rapamycin, along with suppressed S6 protein phophorylation and increased ratio of LCII/I, confirming inhibition of mTOR and subsequent activation of autophagy by rapamycin. We further demonstrated that genetic inactivation of autophagy in mouse intestinal epithelium resulted in a marked accumulation of NHE3 and attenuated the rapamycin-induced down-regulation of NHE3, demonstrating a critical role for autophagy in maintaining NHE3 homeostasis. However, in addition to causing autophagic down-regulation of NHE3, we also demonstrate that rapamycin has a second mode of action: at high doses which mimics the sharp rise in serum rapamycin in non-infectious diarrhea, it causes acute inhibition of NHE3 surface expression in mouse ileum and NHE3 transporter activity in fibroblast PS120 cells, lacking endogenous NHE3, when stably transfected with human NHE3. Together, our data implicate two pathophysiological mechanisms of NHE3 down-regulation in the profound non-infectious diarrhea induced by rapamycin: 1) increased autophagic turnover of NHE3 by chronic exposure to rapamycin, and 2) reduced surface expression of NHE3 by acute exposure to a high concentration (spike) of rapamycin. Our findings not only lead to new insights into NHE3 regulation, but also provide new strategies to manage diarrhea in organ transplantations.