Abstract: SA-PO0252
Necroptosis Is Modulated by AQP2 in Kidney Epithelial Cells by Regulation of MLKL Membrane Accumulation
Session Information
- AKI: Mechanisms - Cell Signaling
October 24, 2026 | Location: Exhibit Hall A, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Tchakal-Mesbahi, Asma, Mass General Brigham Inc, Boston, Massachusetts, United States
- Huang, Huihui, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States
- Bouley, Richard, Mass General Brigham Inc, Boston, Massachusetts, United States
- Brown, Dennis, Mass General Brigham Inc, Boston, Massachusetts, United States
Group or Team Name
- Program in Membrane Biology
Background
In addition to affecting systemic water balance, we found that AQP2 deficiency leads to acute tubular injury and renal dysfunction with neo-mortality, even in heterozygous AQP2-deficient mice, suggesting a role in cell survival. Necroptosis is a form of lytic cell death mediated by phosphorylation of the mixed lineage kinase domain-like protein (MLKL). Once activated, MLKL oligomerizes and accumulates at the plasma membrane in permeabilization domains (hotspots) that disrupt membrane integrity and trigger release of inflammatory DAMPs. In addition, MLKL is regulated by vesicular trafficking, which determines cell fate.
Methods
To uncover a role of AQP2 in necroptosis, we exposed cells to a “TCZ cocktail” and applied co-immunoprecipitation (IP) and immunostaining of p-MLKL. Cell viability was assessed with propidium iodide or an MTT assay of metabolic activity, and MLKL accumulation was monitored in cells transfected with an inducible active MLKL construct (MLKL-2Fv-Venus) to express a fusion protein containing the N-terminus of MLKL with 2 binding domains of FKBP-12 (Fv). The dimerizer B/B (AP20187: 25 nM) was used to activate the fusion protein and cause membrane accumulation of MLKL-Venus.
Results
AQP2-expressing epithelial cells were protected from TCZ-induced necroptosis compared to non-expressing cells. Phosphorylated MLKL showed robust plasma membrane accumulation in non-expressing cells, whereas AQP2-expressing cells had reduced membrane MLKL. Co-IP showed association of AQP2 and MLKL, suggesting a common pathway of trafficking and/or a functional association. We found that AQP2 phosphorylation status modulates necroptosis: constitutively membrane-localized AQP2 (S256D mutation) enhanced MLKL membrane accumulation, while the S256A mutant that recycles constitutively conferred even greater resistance to necroptosis than WT AQP2, possibly due to rapid co-internalization of AQP2 and MLKL. A 2–5 minute B/B treatment of LLCPK1-AQP2 cells expressing MLKL-Venus caused membrane accumulation of MLKL-Venus, and membrane hotspots. A so-called “death bubble” appeared around 20 minutes, and many cells began to lyse.
Conclusion
AQP2 is a regulator of necroptosis in renal epithelial cells, a novel mechanism of injury that may reveal a therapeutic target in kidney disease.
Funding
- NIDDK Support