Abstract P204: Epithelial Sodium Channel Stimulation by Glucose

Parijat S Joy; Peter M. Snyder

doi:10.1161/hyp.68.suppl_1.p204

There is a link between diabetes mellitus and hypertension, but the underlying mechanisms are poorly understood. The epithelial Na + channel ENaC plays an important role in blood pressure control; ENaC mutations cause Liddle’s syndrome, an inherited form of hypertension. Previous work suggests that ENaC abundance is increased in diabetes mellitus, but the underlying mechanisms are unclear. Here we tested the effect of glucose on ENaC regulation. In Ussing chamber experiments using mouse kidney collecting duct cells (mCCD) and primary cultures of human lung epithelia, elevated glucose increased ENaC-mediated short-circuit current by 2-3 times in a dose-dependent manner from 100mg/dl to 400mg/dl of glucose. This was caused by an increase in ENaC abundance at the cell surface. We hypothesized that hyperglycemia might enhance ENaC cell surface abundance by altering activity of Nedd4-2, an E3 ubiquitin-protein ligase that binds to PY motifs within ENaC. Consistent with this hypothesis, we found that mutation of the PY motifs abolished ENaC stimulation by elevated glucose. Moreover, using a biotinylation assay, we found that elevated glucose (300 mg/dl) slowed ENaC endocytosis and reduced its degradation in the endocytic pathway. These changes in trafficking are explained by our finding that glucose reduced ENaC binding to Nedd4-2, and hence, reduced ENaC ubiquitination. O-GlcNAcylation plays a role in insulin signaling and glucose toxicity due to increased O-GlcNAcylation of target proteins. To test a role for O-GlcNAcylation in ENaC stimulation by glucose, we used 6-Diazo-5-oxo-l-norleucine (DON) to inhibit O-GlcNAcylation. DON abolished ENaC stimulation by elevated glucose. Using anti-O-GlcNAc antibody, we found that Nedd4-2 is a substrate for O-GlcNAcylation, and this modification was increased by elevated glucose. DON also reversed the reduction in binding of Nedd4-2 to ENaC at high glucose levels. Together, our data suggest a model in which hyperglycemia stimulates ENaC through O-GlcNAcylation of Nedd4-2, increasing ENaC abundance at cell surface thus increasing epithelial sodium absorption.

Abstract P204: Epithelial Sodium Channel Stimulation by Glucose

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