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Abstract: FR-PO615

MAGE-D2 and HSP40 Protect NKCC2 Against Hypoxia-Induced Endoplasmic Reticulum-Associated Degradation

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Nait meddour, Kahina, CRC, INSERM-U1138, CNRS ERL8228, Sorbonne Université, Paris, France
  • Shaukat, Irfan, CRC, INSERM-U1138, CNRS ERL8228, Sorbonne Université, Paris, France
  • Demaretz, Sylvie, CRC, INSERM-U1138, CNRS ERL8228, Sorbonne Université, Paris, France
  • Bronji, Zaineb, CRC, INSERM-U1138, CNRS ERL8228, Sorbonne Université, Paris, France
  • Seaayfan, Elie, Philipps University Marburg, Marburg, HE, Germany
  • Kömhoff, Martin, Philipps University Marburg, Marburg, HE, Germany
  • Laghmani, Kamel, CRC, INSERM-U1138, CNRS ERL8228, Sorbonne Université, Paris, France
Background

We recently showed that MAGED2 mutations cause polyhydramnios with prematurity and a severe but transient form of antenatal Bartter’s syndrome associated with inappropriate expression of the sodium–chloride transporters NKCC2 and NCC (Laghmani et al, N Engl J Med. 2016 May 12;374(19):1853-63). However, the transient nature of the disease remains unclear. We speculated that through its interaction with HSP40, MAGE-D2 protects the co-transporters from endoplasmic reticulum (ER) associated degradation activated by ER stress due to tissue hypoxia during early pregnancy, an effect that becomes less relevant later. In support of this notion, we found that HSP40 interacts with NKCC2 at the ER. Consequently, the aim of the present study was to investigate the effect of hypoxia on NKCC2 biogenesis and explore the potential role of MAGE-D2 and HSP40 in this process.

Methods

Cellular hypoxia was induced chemically by incubating HEK cells for 16-24 hours with cobalt chloride (CoCl2). NKCC2 protein expression was monitored by immunoblotting in HEK cells transfected with the co-transporter. NKCC2 stability was assessed by cycloheximide chase assay.

Results

Similar to MAGE-D2, HSP40 co-expression increases also NKCC2 biogenesis. Subjecting HEK cells, transfected either stably or transiently with NKCC2, to a hypoxic microenvironment chemically induced by CoCl2 (200-600 μM) significantly decreased total NKCC2 protein expression in a dose-dependent fashion. Cycloheximide chase assay revealed that in cells subjected to chemical hypoxia, NKCC2 stability and maturation are decreased. Interestingly, the effect of hypoxia on NKCC2 maturation was more severe following HSP40 or MAGE-D2 knockdown. Even more impressive, simultaneous knockdown of MAGE-D2 and HSP40 strikingly aggravated the effect of cellular hypoxia on NKCC2 maturation (> 99%).

Conclusion

Our data indicate that cellular hypoxia chemically induced by CoCl2 alters NKCC2 stability and maturation. Most importantly, our findings strongly suggest that MAGE-D2 cooperates with HSP40 to protect NKCC2 against ER associated degradation induced by cellular hypoxia, which could explain, at least in part, the transient nature of antenatal Bartter's syndrome in carriers of MAGED2 mutations.

Funding

  • Government Support - Non-U.S.