Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells

Abstract

We previously showed that in polarized Madin-Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.Cells 2020, 9, 1057 2 of 18 mechanisms of water channel accumulation in the apical plasma membrane of collecting duct cells have been of particular interest, the regulation of basolateral AQP2 has also been investigated in several studies [22][23][24][25][26]. In the cortical connecting segment, basolateral AQP2 is usually readily detectable [23], and AQP2 has varying levels of basolateral expression in collecting duct principal cells from kidney cortex to medulla, with the most obvious basolateral expression in the inner medulla. In MDCK cells, derived from canine kidney, transfected AQP2 usually accumulates in the apical membrane upon intracellular cAMP elevation. However, AQP2 instead concentrates on the basolateral membrane after forskolin (FK) treatment under hypertonic conditions [26]. Importantly, Sec6, Sec8, and RalA, all components of the exocyst, which targets proteins to the basolateral plasma membrane [27], were associated with AQP2-containing vesicles upon mass spectrometry analysis [28]. Furthermore, we showed that AQP2 is necessary for cell migration and tubule morphogenesis through its RGD-regulated interaction with basolateral integrin-β1 [29]. These previous findings all pointed to the existence of an AQP2 basolateral targeting pathway, and indeed we demonstrated the existence of a transcytotic pathway for AQP2 in MDCK cells by blocking basolateral AQP2 during its transit through this membrane domain using low temperature [30]. After initial basolateral targeting, AQP2 is retrieved into Rab5-positive vesicles, then undertakes microtubule-dependent transcytosis to apical recycling vesicles [30]. This novel trafficking concept is supported by an apical plasma membrane proteomics study using mpkCCD cel...

Actin-related protein 2/3 complex plays a critical role in the aquaporin-2 exocytotic pathway

“…At the time, the data were reasonably interpreted as an increase in AQP2 exocytosis (75). However, in light of more recent work showing the important role of endocytosis in determining AQP2 plasma membrane residence time (9,12,18,22,23,25,60), we suggest that the effects seen in earlier studies could equally well be due to the inhibitory effect of actin depolymerization on AQP2 endocytosis.…”

“…However, we also show here that the plasma membrane endocytosis mechanism is not affected by Arp2/3 inhibition, implying that different actin-related mechanisms are in place at these different cellular locations. This would not be surprising, since there is considerable evidence in other systems that different cellular pools of actin are differentially regulated (80)(81)(82), including apical and basolateral pools in the epithelial cells (83), and including these pools in AQP2expressing cells (25). Deletion of Arp2/3 affects specific intracellular protein trafficking events but not other important functions of actin (84).…”

“…The accumulation of plasma membrane AQP2 was quantified as previously described (25,61). In brief, after AQP2 labeling, cell membranes were costained with rhodamineconjugated wheat germ agglutinin (2 μg/mL, Lectin Kit, Vector Laboratories) for 8 min.…”

“…AQP2 also interacts with ERM (Ezrin, Radixin and Moesin) family proteins (22,24), key in cross-linking actin filaments with the plasma membrane. To add another layer of complexity, we recently found that apical and basolateral actin networks regulate AQP2 trafficking differently (25). Finally, immunoisolated AQP2-bearing intracellular vesicles from rat inner medullary collecting ducts show the presence of a large variety of actin-related cytoskeletal proteins such as actin-related protein (Arp)2/3, b-actin and c-actin, myosin isoforms, tubulin, Rab GTPases, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins (26), suggesting a complex network of proteins that interact with actin during various steps of AQP2 vesicular trafficking.…”

The Barrier-Enhancing Function of Soluble Yam (Dioscorea opposita Thunb.) Polysaccharides in Rat Intestinal Epithelial Cells as Affected by the Covalent Se Conjugation

“…A slightly modified version of the previously described [ 34 ] rhodamine-phalloidin labeling was used to observe the distribution of F-actin in the treated cells. In detail, the cells inoculated at 4 × 10 6 cells/mL in 1 mL were placed in 12-well plates, treated with the samples at a dose of 40 µg/mL for 12 and 24 h, washed twice with the PBS (0.1 mol/L, pH 7.2), fixed with 4% paraformaldehyde for 15 min, and then washed twice with 0.1% Triton X-100 in PBS for 5 min.…”