Sorting Nexin 27 Regulates the Lysosomal Degradation of Aquaporin-2 Protein in the Kidney Collecting Duct.

Hyo-Jung Choi,Hyo-Ju Jang,Stine Julie Tingskov,Hyun Jun Jung,Rikke Nørregaard,Euijung Park,Tae-Hwan Kwon

doi:10.3390/cells9051208

Abstract

Sorting nexin 27 (SNX27), a PDZ (Postsynaptic density-95/Discs large/Zonula occludens 1) domain-containing protein, cooperates with a retromer complex, which regulates intracellular trafficking and the abundance of membrane proteins. Since the carboxyl terminus of aquaporin-2 (AQP2c) has a class I PDZ-interacting motif (X-T/S-X-Φ), the role of SNX27 in the regulation of AQP2 was studied. Co-immunoprecipitation assay of the rat kidney demonstrated an interaction of SNX27 with AQP2. Glutathione S-transferase (GST) pull-down assays revealed an interaction of the PDZ domain of SNX27 with AQP2c. Immunocytochemistry of HeLa cells co-transfected with FLAG-SNX27 and hemagglutinin (HA)-AQP2 also revealed co-localization throughout the cytoplasm. When the PDZ domain was deleted, punctate HA-AQP2 labeling was localized in the perinuclear region. The labeling was intensively overlaid by Lysotracker staining but not by GM130 labeling, a cis-Golgi marker. In rat kidneys and primary cultured inner medullary collecting duct cells, the subcellular redistribution of SNX27 was similar to AQP2 under 1-deamino-8-D-arginine vasopressin (dDAVP) stimulation/withdrawal. Cell surface biotinylation assay showed that dDAVP-induced AQP2 translocation to the apical plasma membrane was unaffected after SNX27 knockdown in mpkCCD cells. In contrast, the dDAVP-induced AQP2 protein abundance was significantly attenuated without changes in AQP2 mRNA expression. Moreover, the AQP2 protein abundance was markedly declined during the dDAVP withdrawal period after stimulation under SNX27 knockdown, which was inhibited by lysosome inhibitors. Autophagy was induced after SNX27 knockdown in mpkCCD cells. Lithium-induced nephrogenic diabetes insipidus in rats revealed a significant downregulation of SNX27 in the kidney inner medulla. Taken together, the PDZ domain-containing SNX27 interacts with AQP2 and depletion of SNX27 contributes to the autophagy-lysosomal degradation of AQP2.

Highlights

Aquaporin-2 (AQP2) is a water channel protein expressed in the kidney connecting tubule and collecting ducts and plays an essential role in vasopressin-induced water reabsorption andCells 2020, 9, 1208; doi:10.3390/cells9051208 www.mdpi.com/journal/cellsCells 2020, 9, 1208 urinary concentration [1,2,3,4]
To examine the endogenous interaction between AQP2 and Sorting nexin 27 (SNX27), we performed immunoprecipitation assay from a rat kidney Inner Medullary Collecting Duct (IMCD) tubule suspension
Since lithium is known to induce autophagy in a number of cell types [43,44], we directly examined whether lithium-induced nephrogenic diabetes insipidus (NDI) is associated with downregulation of SNX27 in the kidney

Summary

Introduction

Aquaporin-2 (AQP2) is a water channel protein expressed in the kidney connecting tubule and collecting ducts and plays an essential role in vasopressin-induced water reabsorption andCells 2020, 9, 1208; doi:10.3390/cells9051208 www.mdpi.com/journal/cellsCells 2020, 9, 1208 urinary concentration [1,2,3,4]. Aquaporin-2 (AQP2) is a water channel protein expressed in the kidney connecting tubule and collecting ducts and plays an essential role in vasopressin-induced water reabsorption and. AQP2 is regulated by vasopressin on a short-term or a long-term basis for collecting duct water reabsorption. Short-term regulation is dependent on the translocation of AQP2 from subapical vesicles to the apical plasma membrane, associated with phosphorylation of the serine residues (serine 256, serine 264, and serine 269) in the carboxyl terminus of AQP2 (AQP2c) [1,7,8,9]. Long-term regulation is based on the prolonged half-life of AQP2 protein and the induction of Aqp gene transcription [2,6,10,11]. The last four-amino acid sequence in the AQP2c (residues 268–271)

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