Sorting nexin 27 (SNX27), a PDZ (Postsynaptic density-95/Discs large/Zonula occludens 1) domain-containing protein, cooperates having a retromer complex, which regulates intracellular trafficking and the abundance of membrane proteins. 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 BH3I-1 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. gene transcription [2,6,10,11]. The AQP2c is subjected to post-translational modification, e.g., phosphorylation and ubiquitination [6,12,13,14]. In particular, the last four-amino acid sequence in the AQP2c (residues 268C271) corresponds to a class I PDZ (Postsynaptic density-95/Discs large/Zonula occludens 1) domain-binding motif [X-(S/T)-X-, where X is any amino acid and is any hydrophobic residue] [15,16,17,18]. A previous study revealed that signal-induced proliferation-associated gene-1 (SPA-1) is a PDZ domain-containing protein that mediates AQP2 trafficking to the apical plasma membrane [15]. Depletion of SPA-1 reduced apical AQP2 expression, indicating that SPA-1 is likely to be directly bound to AQP2 and regulates AQP2 trafficking [15]. BH3I-1 Moreover, signal-induced proliferation-associated 1 like 1 (Sipa1I1), another PDZ domain-containing protein, mediates AQP2 endocytosis in the absence of vasopressin [19]. The retromer complex is a crucial component of the endosomal protein sorting machinery [20,21,22]. The complex is composed of the cargo-selective trimer Vps26-Vps29-Vps35 (hVps26, hVps29, and hVps35 in human being) as well as the membrane-associated heterodimer BH3I-1 of two sorting nexin (SNX) proteins Vps5-Vps17 (SNX1 and SNX2 in human being) [20]. In mammals, the retromer BH3I-1 complicated can be recruited to endosomes, where it facilitates cargo retrieval from endosomes towards the trans Golgi network. Furthermore, the retromer complicated plays a part in the cargo sorting in the first endosomes before cargo delivery to many intracellular compartments, like the recycling of membrane protein towards the plasma membrane. We previously proven that vacuolar proteins sorting-associated proteins 35 (Vps35) interacts using the AQP2c, as well as the depletion of Vps35 was connected with reduced AQP2 trafficking and improved lysosomal degradation of AQP2 [23]. Regularly, a recent research also proven that AQP2 gathered in the recycling endosomes without apical AQP2 trafficking in response to Vps35 knockdown [24]. The sorting nexins participate in a family group of protein characterized by the current presence of a PX (Phox homology) site. They are indicated through the entire endosomal system, taking part in many trafficking pathways [25]. Among the sorting nexins, sorting nexin 27 (SNX27) may be the just member creating a PDZ site and it SERP2 is among three sorting nexins including an atypical FERM (C-terminal 4.1/ezrin/radixin/moesin)-like domain [26]. Earlier studies show that SNX27 cooperates using the retromer complicated by interacting straight using the retromer subunit Vps26 from the Vps26:Vps29:Vps35 trimer and is important in the rules of endosomal recycling and proteins great quantity [27,28,29]. SNX27 was recognized to connect to transmembrane protein BH3I-1 including Asn-Pro-Xaa-Tyr (NPxY) sequences and in addition using the transmembrane protein having the course I PDZ domain-binding motifs [X-(S/T)-X-] through its PDZ site [30]. After interacting with target transmembrane proteins having the PDZ domain-binding motif, SNX27 cooperates with the retromer complex, preventing the entry of transmembrane proteins into the lysosomal pathway, and activating the retromer-tubule-based recycling to the plasma membrane [31]. Since AQP2c has a class I PDZ domain-binding motif, we hypothesized that SNX27 interacts with AQP2c through its PDZ domain, and regulates intracellular trafficking as well as the protein abundance of AQP2. The aim of the present study was, therefore, to examine the role of SNX27 in the vasopressin-mediated regulation of AQP2 in the kidney collecting duct cells, which provides new insights into the AQP2 regulatory mechanism. 2. Materials and Methods 2.1. cDNA Construction of Rat SNX27 The SNX27 gene was amplified by PCR using primers from the cDNA (complementary DNA) of rat kidney inner medulla (Table 1). The amplified PCR products.