Fig 2: Vasopressin freed a-actinin 4 from F-actin for nuclear translocation in the mpkCCD cells. (A) Confocal immunofluorescence micrographs of a-actinin 4 (Actn4, green) and F-actin (red). Polarized cells grown on Transwell® were exposed (24 h) to vehicle, 1 nM dDAVP, or 1 nM dDAVP plus 50 nM jasplakinolide (JAS) that binds and induces F-actin polymerization (Bubb et al., 2000). The nuclei were stained with DAPI (blue). Shown at the bottoms are signal profiles along the dotted lines. Bars indicate 10 µm. (B,C) Summaries of Actn4 colocalization with F-actin and DAPI, respectively. Numbers are means ± SEM, summarized from three independent experiments and about 330 cells per experiment. Asterisks indicate statistical significance, t-test p < 0.05. (D,E) Representative immunoblotting and summary of Actn4 in the cytosol vs. nuclei of the cells exposed to vehicle (V) vs. dDAVP (D). Numbers are means ± SEM, summarized from three independent experiments. Gapdh and H2A were markers for cytosol and nuclei, respectively. Protein intensities were corrected against those of Gapdh or H2A to avoid variations in the sample preparation. M, marker.

Fig 3: A model illustrates dual functions of a-actinin 4 in vasopressin short-term and long-term actions in the kidney collecting duct cells. ABD, actin-binding domain; CaM, calmodulin-like domain; Dex, dexamethasone.

Fig 4: Isolation and validation of urine EVs. Urine (40 ml) was centrifuged to deplete cell debris, followed by depletion of apoptotic bodies by 1.0 µm and 0.22 µm filters, and finally to isolate EVs from solution by another 0.03 µm filter (A). Urine EVs (UEV) showed a prominent CD9 expression in contrast to plasma EVs (PEV) expressing a higher CD81, and no exosomal markers (CD9, CD63, CD81 or HSP90) was detected in the drop through solution (Soln) in a normal adult. The PEV isolated from the young adult (Y1) by ExoQuick contained some albumin contamination, but UEV, isolated from the young adult (Y1) by a series of membrane filtrations as shown in (A), contained neglectable albumin contamination (B). Further studies showed that similar levels of CD9/CD81/HSP90/syntenin expression were found in the EVs from young adults (n = 4; Y1, Y2, Y3 and Y4) and the EVs from old adults (n = 4; O1, O2, O3 and O4), but some extent of higher CD63/actinin-4 expression in the EVs from the elderly (C). The blots presented here are derived from the images of original western blots which are shown in Supplementary Fig. 1B and C. The flow cytometric analyses of CD63, CD9 and CD47 expression using anti-CD63-beads enriched UEVs showed that CD9 co-expressed with CD63, but not CD47, on UEVs in three replicable experiments (D). We also performed fluorescent nanoparticle tracking analyses (NTA), gating the size between 30 and 300 nm, to visualize the UEVs derived from old (O1) and young (Y1) adults in three replicable experiments (E).

Fig 5: Vasopressin-induced glucocorticoid receptor nuclear translocation was independent of a-actinin 4. (A,B) Confocal immunofluorescence micrographs of glucocorticoid receptor in control (shCtrl) and a-actinin 4 knockdown (shActn4-1 and shActn4-2) mpkCCD cells grown on Transwell® under vehicle vs. dDAVP (1 nM, 24 h) conditions. Bars indicate 10 µm. The primary antibody to glucocorticoid receptor was omitted in (B). (C) Summary of the confocal imaging results. Numbers are means ± SEM from three independent experiments, about 330 cells in each experiment. Asterisks indicate statistical significance, t-test p < 0.05.