MAGED2 Enhances Sodium Chloride Cotransporter (NCC) Expression at the Plasma Membrane by Inhibiting Its Ubiquitination and Lysosomal Degradation under Hypoxia

Background: Transient antenatal Bartter's syndrome (tBS) is characterized by massive polyhydramnios, impaired expression of the cAMP-sensitive renal salt-transporters NKCC2 and NCC, marked perinatal mortality and preterm birth followed by spontaneous recovery from polyuria in survivors. tBS is caused by mutations in the gene encoding MAGED2, which inhibits endocytosis of Gαs under hypoxia, a prerequisite for generation of cAMP by membrane-bound adenylate cyclase (mAC). As spontaneous recovery from renal salt wasting overlaps with developmental increase of renal oxygenation, lack of MAGED2 dependent cAMP generation under hypoxia explains, at least in part, the severe but transient renal salt loss in tBS patients. The present study aims to elucidate how MAGED2 controls NCC-trafficking. Methods: Cell-surface biotinylation, endocytosis, exocytosis and activity of NCC were analyzed in HEK293 and HeLa cells under hypoxia with or without MAGED2-depletion (siMAGED2). Results were assessed using western blotting, immunocytochemistry and CoroNa Green, a sodium ion indicator. Results: In renal cells exposed to hypoxia, siMAGED2 significantly reduced the expression of total and plasma membrane-bound NCC as well as its activity. Conversely, MAGED2 overexpression promoted expression of both total and plasma membrane-bound NCC, whereas Gαs depletion led to the opposite. Interestingly, forskolin abrogated the effect of siMAGED2 on NCC expression and localization at the plasma-membrane, the latter of which was recapitulated by immunocytochemistry in HeLa cells. Of note, MAGED2 overexpression did not rescue NCC expression in Gαs-depleted cells, confirming functioning of Gαs downstream of MAGED2. Reduced expression of NCC at the cell surface resulted from decreased NCC exocytosis and enhanced endocytosis in MAGED2-depleted cells. Additionally, siMAGED2 enhanced NCC lysosomal degradation by increasing its ubiquitination. Conclusion: In conclusion, our study demonstrates the crucial role of MAGED2 under hypoxic conditions in regulating NCC lysosomal degradation and its trafficking to the plasma membrane. This sheds light on the underlying mechanisms of tBS and offers potential avenues for therapeutic intervention by targeting MAGED2-mediated pathways. Funding: Private Foundation Support, Government Support – Non-U.S.