Fig 2: Expression of AQP1 is associated with AQP9 protein level in oligohydramnios patients. Immunohistochemical staining of AQP1, AQP3, AQP8 and AQP9 was performed in placenta in normal AF volume group and oligohydramnios group (a–h). Immunohistochemical staining of AQP1, AQP3, AQP8 and AQP9 was conducted in amnion epithelial cells and chorion in normal AF volume group and oligohydramnios group (i–p). Bar 50 μm

Fig 3: Transepithelial water transport in the mouse mesentery and role of AQP1. (A) Schematic representation of isolation of mouse mesentery patches for measurements of the osmotically induced transcellular water flux with TEA+-sensitive microelectrodes; (B) Characterization of mouse mesentery by immunofluorescence analysis: in confocal sections AQP1 and mesothelin co-localized at the plasma membrane. bar = 10 µm; (C) Osmotically induced transepithelial water transport in mouse mesentery in response to Physioneal 40 2.27%, Physioneal 40 3.86% and Extraneal. Slopes (solid lines) and 95% confidence interval (shaded areas, CI) for the regression of [TEA+] calculated during the first 60 s of the response. Statistical analysis of the differences between slopes is reported within each panel. The value of the slopes is reported on the bottom of each panel; (Physioneal 2.27%: n = 3; Physioneal 3.86%: n = 5; Extraneal: n = 3); (D) Osmotically induced transepithelial water transport in mouse mesentery in response to Physioneal 40 3.86%, in the presence or in the absence of HgCl2. The value of the slopes is reported on the bottom of each panel (Physioneal 3.86% without HgCl2: n = 3; Physioneal 3.86% with HgCl2: n = 3).

Fig 4: Cellular localization and protein expression of peritoneum AQP1 was altered in NaClO-injured pigs. (A) Immunofluorescent staining showed that AQP1 appeared on both mesothelial cells (A1) and endothelial cells (A2) that surrounded vessels. AQP1 was double stained with vWF to confirm the identity of an endothelial cell. In the vehicle control group, AQP1 mainly appeared on the apical and adluminal region of cells (white arrowheads); however, after 0.1%*2 NaClO injury, signals of AQP1 on both types of cells became weakened (A3) and more scattered into the cytosol (A4). (B) Quantitative analyses on whole peritoneal tissue homogenates showed that total protein expression of AQP1 on parietal peritoneum was significantly decreased (80% less) in 0.1%*2 NaClO-injured pigs. Eukaryotic elongation factor 2 (EEF2) was used as protein loading control, AQP1 signal was first corrected with loading control EEF2 before fold change comparisons to control animals. L lumen of vessels. Representative images from 3–4 individual pigs of each group were presented. For tissue sampling, 2 samples from different regions of the peritoneum were taken from every individual for further analysis. Whole membrane images of western blotting could be found in Figure S4. Asterisks indicate significant differences between groups (** p < 0.01).

Fig 5: Increased expression of Piezo1 protein and mRNA in UUO mice.(A and B) Representative immunoblots and corresponding densitometry analysis of Piezo1 protein abundance in the kidney of sham and UUO mice at 30 minutes, the first hour, and the third hour after surgery. Data are shown as mean ± SEM (n = 6 in each group). (C and D) Representative immunoblots and corresponding densitometry analysis of Piezo1 protein abundance in the kidney of sham, 3UUO, and 7UUO mice. Data are shown as mean ± SEM (n = 4 in each group). (E) mRNA levels of Piezo1, TRPC1, TRPC6, and TRPV4 in the kidney of sham, 3UUO, and 7UUO mice. Data are shown as mean ± SEM (n = 4 in each group). (F) Colocalization of Piezo1 (red) and AQP1 (green) or AQP2 (green) in the proximal tubules or inner medullary collecting ducts of sham and 7UUO mice, detected by immunofluorescence. Scale bars: 500 μm, 208 μm (insets). *P < 0.05 when compared with sham mice by 1-way ANOVA with Student-Newman-Keuls test. UUO, unilateral ureteral obstruction; 3UUO, UUO for 3 days; 7UUO, UUO for 7 days; TRPC, transient receptor potential canonical; TRPV, transient receptor potential vanilloid.