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Abstract: TH-PO495

MAGE-D2 Promotes Expression and Stability of Pendrin Protein

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Seaayfan, Elie, University Children's Hospital Marburg, Marburg, Germany
  • Renigunta, Aparna, University Children's Hospital Marburg, Marburg, Germany
  • Weber, Stefanie, University Children's Hospital Marburg, Marburg, Germany
  • Laghmani, Kamel, INSERM-CNRS, Paris, France
  • Kömhoff, Martin, University Children's Hospital Marburg, Marburg, Germany
Background

We recently showed that mutations in MAGE-D2 cause polyhydramnios with preterm delivery of the affected baby and a severe but transient form of antenatal Bartter’s syndrome (tBS). Although reduced total and apical expression of the sodium–chloride transporters NKCC2 and NCC was shown in vivo and in vitro, which explains massive salt losing (Laghmani et al, N Engl J Med. 2016), additional transporters may also be impaired. One potential candidate is Pendrin, because its loss increases salt losing and because it is expressed in the distal tubule as MAGE-D2. Consequently, the aim of the present study was to investigate the potential role of MAGE-D2 in Pendrin biogenesis.

Methods

Pendrin protein expression was monitored in transiently transfected HEK293 cells by immunoblotting, which allows the detection of both the immature (core-glycosylated) and mature Pendrin protein. Stability of Pendrin protein was assessed by cycloheximide chase assay. We studied the effects of wild type MAGE-D2 and mutant R446CMAGE-D2 on Pendrin expression. In addition, the role of endogenously expressed MAGE-D2 role was investigated by small interfering RNA. To assess the specificity of our findings, we also analyzed the effects of MAGE-D2 overexpression on NHE3 biogenesis.

Results

MAGE-D2 co-expression robustly increased expression of immature and total cellular Pendrin protein in a dose dependent manner. Cycloheximide chase assays (CHX) showed that in cells over expressing MAGE-D2, stability of immature Pendrin is increased. In contrast to MAGE-D2 wild type, R446CMAGE-D2, a missense mutation identified in a tBS patient, co-expression significantly decreases Pendrin stability. Analogous to R446CMAGE-D2, knockdown of endogenous MAGE-D2 by small interfering RNA also decreases Pendrin stability. In contrast to Pendrin, our preliminary data did not reveal an effect of MAGED2 on the expression of total NHE3.

Conclusion

Our findings show that MAGED2 specifically promotes the expression of Pendrin. Importantly, our data indicate that MAGED2 affects multiple salt transporters in the distal tubule, thus explaining the severe phenotype.