Abstract: SA-PO0474
Pre-Golgi Regulation of NKCC2 by Gαs and Cyclic AMP (cAMP) Signaling Pathway During Hypoxia and Endoplasmic Reticulum (ER) Stress Conditions
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Basic Research
November 08, 2025 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid, Electrolytes, and Acid-Base Disorders
- 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic
Authors
- Bakhos Al Douaihy, Dalal, Centre de Recherche des Cordeliers, Paris, France
- Demaretz, Sylvie, Centre de Recherche des Cordeliers, Paris, France
- Rudloff, Stefan, Bern University Hospital, Bern, BE, Switzerland
- Huynh-Do, Uyen, Bern University Hospital, Bern, BE, Switzerland
- Laghmani, Kamel, Centre de Recherche des Cordeliers, Paris, France
Background
MAGE-D2 mutations cause the most severe, but transient, form of antenatal Bartter syndrome (BS type 5), associated with impaired expression of Na-Cl cotransporters NKCC2 and NCC. Cellular hypoxia is of particular interest in elucidating the molecular basis of the transient nature of the disease, as it constitutes one of the main differences between the prenatal symptomatic period of BS type 5 and the postnatal asymptomatic phase. In support of this hypothesis, it was found that under hypoxia, MAGE-D2 is required for the appropriate expression of Gsα and thus for the activation of the cAMP signaling pathway, a major regulator of NKCC2 and NCC. All previous studies have focused on the involvement of Gsα and cAMP signaling in the post-Golgi regulation of these Na-Cl cotransporters. The present study aimed to determine whether this signaling pathway is also involved in the regulation of NKCC2 at the pre-Golgi level, particularly under hypoxia and ER stress conditions.
Methods
Cellular hypoxia was induced chemically by incubating HEK cells with cobalt chloride. NKCC2 stability and maturation were assessed by cycloheximide chase assay. NKCC2 protein expression was monitored by immunoblotting in HEK cells transiently transfected with NKCC2.
Results
Similar to MAGE-D2, Gsα knockdown (KD) enhanced NKCC2 ER-associated degradation and strikingly decreased total NKCC2 abundance, an effect abolished by the proteasome inhibitor MG132. Conversely, incubating cells with the adenylate cyclase activator forskolin produced the opposite effect and reversed Gsα KD effect on NKCC2. Subjecting cells to hypoxia significantly decreased NKCC2 stability and maturation. Most importantly, similar to MAGE-D2, the effect of hypoxia on NKCC2 was more severe following Gsα KD. Interestingly, forskolin reversed, at least partially, the effect of hypoxia on NKCC2 in MAGE-D2-depleted cells, which is consistent with an involvement of the cAMP signaling in NKCC2 protection by MAGE-D2 during hypoxia.
Conclusion
Our data indicate that Gsα and cAMP signaling promote NKCC2 protein expression at the pre-Golgi level, particularly under ER stress. Most importantly, our findings reveal that Gsα is involved in the protection of NKCC2 by MAGE-D2 against ERAD induced by cellular hypoxia, which could also contribute to the transient nature of BS type 5.
Funding
- Government Support – Non-U.S.