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Abstract: FR-PO606

MAGE-D2 Is Required for a Normal Cell Surface Expression of NHE3 Protein

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic


  • Seaayfan, Elie, Philipps University Marburg, Marburg, HE, Germany
  • Renigunta, Aparna, Philipps University Marburg, Marburg, HE, Germany
  • Ruttkowski, Lars lennart, Philipps University Marburg, Marburg, HE, Germany
  • Herrmann, Tom Leonard, Philipps University Marburg, Marburg, HE, Germany
  • Schallopp, Nadine, Philipps University Marburg, Marburg, HE, Germany
  • Weber, Stefanie, University Children's Hospital Marburg, Marburg, Germany
  • Laghmani, Kamel, INSERM-CNRS, Paris, France
  • Kömhoff, Martin, Philipps University Marburg, Marburg, Germany

We recently showed that mutations in MAGE-D2 cause polyhydramnios leading to preterm birth and a severe but transient form of antenatal Bartter’s syndrome (transient aBS). Reduced expression of the sodium–chloride transporters NKCC2 and NCC was shown in vivo and in vitro and explains massive salt losing (Laghmani et al, N Engl J Med. 2016). The absence of metabolic alkalosis however, which is pathognomonic feature of aBS, suggests that additional transporters may be affected. One possible explanation is that MAGE-D2 also regulates NHE3, the predominant isoform responsible for apical membrane Na(+)/H(+) exchange in the proximal tubule and thick ascending limb. Consequently, the aim of the present study was to investigate the potential role of MAGE-D2 in NHE3 biogenesis.


Protein-protein interaction was assessed by co-immunoprecipitation (COIP) assay and proximity ligation assay (PLA). NHE3 protein expression was monitored in transiently transfected HEK293 cells by immunoblotting. Stability of NHE3 protein was assessed by cycloheximide chase assay. We studied the effects of wild type MAGED2 and mutant R446CMAGE-D2 on total and cell surface expression of NHE3. In addition, the role of endogenously expressed MAGE-D2 role was investigated by small interfering RNA.


CO-IP assays in renal cells showed that MAGE-D2 WT interact physically with NHE3. Accordingly, PLA revealed that MAGE-D2 is close to NHE3 in renal cells. Interestingly, R446CMAGE-D2, a missense mutation identified in a transient aBS patient, co-expression significantly decreased NHE3 cell surface expression (48.8% from the control, P value = 0.0172). Analogous to R446CMAGE-D2, knockdown of endogenous MAGE-D2 by small interfering RNA also decreases NHE3 cell surface abundance. Interestingly, in contrast to NKCC2 and NCC, MAGE-D2 co-expression did not affect total NHE3 protein expression. Moreover, cycloheximide chase assays (CHX) showed that in cells over expressing MAGE-D2, stability of NHE3 is not affected.


Our findings show that MAGED2 specifically promotes the cell surface expression of NHE3. Importantly, our data indicate that MAGED2 differentially affects multiple salt transporters in the nephron, thus explaining the severe phenotype as well as the absence of metabolic alkalosis.