MP005INCREASED LYSOSOMAL DEGRADATION OF AQP2 IN MPKCCDC14 CELLS WITH KNOCKDOWN OF VACUOLAR PROTEIN SORTING-ASSOCIATED PROTEIN 35

Introduction and Aims: Carboxyl-terminus of AQP2 (AQP2c) undergoes post-translational modifications, including phosphorylation and ubiquitination, for the regulation of aquaporin-2 (AQP2) translocation and protein expression. We aimed to identify novel proteins interacting with AQP2c. Methods: Recombinant AQP2c protein was made in E. coli BL21 (DE3) by exploiting the pET32 TrxA fusion system. Lysates of rat kidney inner medullary collecting duct tubule suspensions were interacted with rat AQP2c bound to Ni2+-resin. Results: LC-MS/MS proteomic analysis demonstrated 18 proteins, including vacuolar protein sorting-associated protein 35 (Vps35). Co-immunoprecipitation assay demonstrated that Vps35 interacted with AQP2c. Immunohistochemistry of rat kidney revealed that AQP2 and Vps35 were co-localized at the intracellular vesicles in the collecting duct cells. The changes of intracellular translocation and protein expression of Vps35, however, were not seen in response to water restriction in rats. The role of Vps35 in the dDAVP-induced AQP2 regulation was examined in mpkCCDc14 cells. Cell surface biotinylation assay demonstrated that dDAVP-induced apical translocation of AQP2 was significantly decreased under the siRNA-mediated Vps35 knockdown. Importantly, dDAVP-induced AQP2 up-regulation was seen to a lesser extent in the cells with Vps35 knockdown, when compared with control. Moreover, AQP2 expression was decreased to a greater extent during the withdrawal period after dDAVP stimulation under the Vps35 knockdown, which was significantly inhibited by chloroquine (a blocker of the lysosomal pathway) treatment, but not by MG132 (a proteasome inhibitor) treatment.