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Abstract: FR-OR48

In Vivo and In Vitro Effects of Hypoxia on NKCC2: Role of MAGE-D2

Session Information

Category: Fluid‚ Electrolyte‚ and Acid-Base Disorders

  • 1001 Fluid‚ Electrolyte‚ and Acid-Base Disorders: Basic


  • Laghmani, Kamel, Inserm, CNRS-ERL8228 , Sorbonne Universite, Paris, France
  • Bakhos Al Douaihy, Dalal, Centre de Recherche des Cordeliers, Paris, Île-de-France, France
  • Charlemagne, Thibaut, Inserm, CNRS-ERL8228 , Sorbonne Universite, Paris, France
  • Rudloff, Stefan, Universitat Bern, Bern, Bern, Switzerland
  • Seaayfan, Elie, Philipps-Universitat Marburg Fachbereich Medizin, Marburg, Hessen, Germany
  • Demaretz, Sylvie, Inserm, CNRS-ERL8228 , Sorbonne Universite, Paris, France
  • Huynh-do, Uyen, Universitat Bern, Bern, Bern, Switzerland
  • Kömhoff, Martin, Philipps-Universitat Marburg Fachbereich Medizin, Marburg, Hessen, Germany

We previously reported that MAGE-D2 mutations cause a severe but a transient form of antenatal Bartter syndrome (tBS) associated with impaired expression of the sodium–chloride cotransporters NKCC2 and NCC. However, the transient nature of the disease remained unclear. In this regard, hypoxia was of particular interest because we considered it as the major difference between the antenatal symptomatic stage of MAGE-D2 related tBS and the post-natal asymptomatic stage. Consequently, the aim of the present study was to investigate the effect of chronic hypoxia induced in vivo and in vitro, on NKCC2 biogenesis and explore the potential role of MAGE-D2 in this process.


To model chronic fetal hypoxia, timed-mated pregnant mice were exposed to 10% oxygen from E14.5 to E18.5 as previously described (Rudloff et al, Nat Commun 2021; 12:549). NKCC2 subcellular distribution was assessed using immunohistochemistry. In vitro hypoxia was induced, in HEK cells transiently or stably transfected with NKCC2, physically (1% O2) or chemically using cobalt chloride for 16-24 hours. NKCC2 stability was assessed by cycloheximide chase assay.


Chronic fetal hypoxia induced ER stress conditions as illustrated by the increase in the expression of GRP78/BiP. Importantly, this was associated with a reduction of NKCC2 surface expression in hypoxic fetal kidneys as judged by enhanced ER retention and colocalization with the ER marker BiP. Physically or chemically induced cellular hypoxia in HEK cells, marked by the stabilization of HIF-1 alpha, was correlated also with an elevation of this ER stress marker. Under these hypoxic conditions, total NKCC2 protein expression was significantly decreased. Moreover, cycloheximide chase assay revealed that in cells submitted to cellular hypoxia, NKCC2 stability and maturation are decreased. Interestingly, the detrimental effect of hypoxia on NKCC2 maturation was more profound in MAGE-D2 depleted cells.


Our data indicate that cellular hypoxia induces ER stress and impairs NKCC2 maturation and cell surface expression. Most importantly, our findings strongly suggest that MAGE-D2 protects NKCC2 against ER associated degradation induced by ER stress under cellular hypoxia, which could explain, at least in part, the transient nature of BS in carriers of MAGE-D2 mutations.


  • Government Support – Non-U.S.