EHD4 Regulates Prostaglandin E2 Synthesis in Renal Collecting Duct Principal Cells: Potential Implications for AQP2

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Eps15 homology domain‐containing protein 4 (EHD4) regulates endocytic recycling. We previously reported that global EHD4 knockout (EHD4‐KO) mice have a higher urine flow and lower urine osmolality than wild‐type (WT) mice. Further, the diuretic phenotype in EHD4‐KO mice is associated with a reduced accumulation of aquaporin 2 (AQP2) in the apical membrane of principal cells of EHD4‐KO mice. However, the mechanism by which EHD4 regulates AQP2 localization is not known. EHD4 and AQP2 share common protein interacting partners, therefore we hypothesized that EHD4 interacts with AQP2, thereby regulating its trafficking. To test this hypothesis, we transfected mouse cortical collecting duct principal (mpkCCD) cells with a GFP‐tagged EHD4 plasmid and visualized the localization of EHD4 and AQP2. EHD4 was found to co‐localize with AQP2 in mpkCCD cells. To assess the interaction between EHD4 and AQP2, co‐immunoprecipitation of AQP2 was performed and the presence of EHD4 in the immunecomplex was measured by Western blotting. EHD4 was not detected in the immune‐complex containing AQP2, suggesting that these two proteins may not interact directly. Pointing to a potential indirect mechanism of regulation, evidence from the literature suggests that the paralog EHD1 binds to cytosolic phospholipase A2α, a key enzyme for eicosanoid synthesis. Prostaglandin E2 (PGE2) is a known negative regulator of AQP2 trafficking. Therefore, we hypothesize that EHD4 deletion increases PGE2 synthesis in principal cells, thereby contributing to the reduced membrane accumulation of AQP2. To test our hypothesis we examined urinary PGE2 excretion by female mice and found it to be significantly higher in EHD4‐KO mice (3595 ± 581 pg/day, n = 6) compared to WT mice (1042 ± 131 pg/day, n = 4; P = 0.008). To exclude a role for flow‐mediated stimulation of PGE2 synthesis, we examined the role of EHD4 in PGE2 synthesis in the mpkCCD cell line. PGE2 concentrations in the surrounding media of EHD4‐siRNA treated mpkCCD cells was significantly higher than of scrambled siRNA‐treated cells (84 ± 7 versus 53 ± 5 pg/ml respectively; n = 6; P = 0.0002). Together, these data suggest that EHD4 regulates collecting duct PGE2 production, providing a possible mechanism by which EHD4 regulates AQP2 localization and urine excretion in vivo.Support or Funding InformationAmerican Heart Association Grant‐In‐Aid 17GRNT33661008This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.