Fig 2: ZIKV-induced IL-1β decreased the expression of aquaporins in renal cells. (A,B) Immunohistochemistry analysis of kidney sections showed comparable low immunoreactivity in the AQP1 and AQP2 labeling in both cortex and inner medulla (IM) of kidneys in newborn mice (A) and adult mice (B) at 7 days post-infection. The black arrows depicted AQP1and AQP2 expression. Scale bars, 40 μm. (C–H) The expression of AQP1 and AQP2 in the kidneys of newborn mice (C–E) and adult mice (F–H) were assessed by western blot at 7 days post-infection. The statistical graphs were shown as the mean ± SEM; N = 6 mice/group, *p < 0.05 vs. mock group; #p < 0.05 vs. ZIKV group.

Fig 3: Erlotinib treatment increases urinary concentration and enhances AQP2 abundance in the apical membrane.(A) Quantitative plots show decreased urine volume after administration of Erlotinib (100 mg/kg) in Pdcd10-deficient mice but not the control mice (Pdcd10fl/fl, n = 4–6; Pdcd10KspKO, n = 5). (B) Quantitative plots show increased urine osmolality after administration of Erlotinib, but not vehicle, in Pdcd10-deficient mice (n = 4–6). (C) Representative Western blots show the increase of Aqp2 and pS256-Aqp2 proteins in the whole kidney lysates of Pdcd10-deficient mice after administration of Erlotinib for 3 days. (D) Immunofluorescence staining of pS256-Aqp2 (green) and Ezrin (red) on sections of kidney medulla of control and Pdcd10-deficient mice show the increased p-Aqp2 expression is correlation with the decrease of Ezrin expression after administration of Erlotinib for 3 days. ***P < 0.001; *P < 0.05. Original magnification, ×630 (D).

Fig 4: Inhibitory effects of FOXO1 overexpression on viability, migration and fibrosis in TGF-β1-induced SV-HUC-1 cells are partly abolished by AQP2 knockdown. (A) FOXO1 protein expression was measured using western blotting in SV-HUC-1 cells following TGF-β1 stimulation. **P<0.01 vs. control. (B) shRNA-NC and shRNA-AQP-1/2 were used to transfect SV-HUC-1 cells and then AQP2 protein expression was detected by western blotting. **P<0.01 and ***P<0.001 vs. control. (C) SV-HUC-1 cells were treated with TGF-β1 and transfected with pcDNA-FOXO1 alone or co-transfected with pcDNA-FOXO1 and shRNA-NC/shRNA-AQP2. Subsequently, cell viability was measured using Cell Counting Kit-8 assay. (D and E) Cell migration was measured using wound healing assay. (F and G) E-cadherin, N-cadherin, cytokeratin, FN and α-SMA protein expression levels were detected and analyzed using western blotting. *P<0.05 and ***P<0.001 vs. control; #P<0.05, ##P<0.01 and ###P<0.001 vs. TGF-β1+Oe-NC; $P<0.05, $$P<0.01 and $$$P<0.001 vs. TGF-β1+Oe-FOXO1+shRNA-NC. FOXO1, forkhead box protein O1; AQP2, aquaporin 2; shRNA, short hairpin RNA; NC, negative control; FN, fibronectin; α-SMA, α-smooth muscle actin.

Fig 5: Pdcd10-deficient mice maintain proper physiological responses to dDAVP stimulation and water deprivation (WD).(A and B) Quantitative plots of urine volume (A) and osmolality (B) before and after administration of dDAVP (i.p. 1 ng/g) to control and Pdcd10-deficient mice (n = 5 for each group). (C) Relative expression level of Aqp2, Aqp3, and Aqp4 genes in the kidney of control and Pdcd10-deficient mice 6 hours after administration with saline or dDAVP (1 ng/g) (Pdcd10fl/fl + saline, n = 6; Pdcd10fl/fl + dDAVP, n = 3–4; Pdcd10KspKO + saline, n = 4; Pdcd10KspKO + dDAVP, n = 3). (D) Quantitative plots of urine osmolality in control and Pdcd10-deficient mice 24 hours after water deprivation (Pdcd10fl/fl, n = 5–6; Pdcd10KspKO, n = 8). (E) The fold change of osmolality in control and Pdcd10-deficient mice 24 hours before and after water deprivation (Pdcd10fl/fl, n = 6; Pdcd10fl/fl + WD, n = 5; Pdcd10KspKO, n = 8; Pdcd10KspKO + WD, n = 8). (F) Relative expression level of Avpr2 gene in the kidney of control and Pdcd10-deficient mice before and 24 hours after water deprivation (n = 3 for each group) (G) Relative expression level of Aqp2, Aqp3, and Aqp4 genes in the kidney of control and Pdcd10-deficient mice before and 24 hours after water deprivation (n = 3–4 for each group). (H) Representative Western blots of Aqp1, Aqp2, and pS256-Aqp2 proteins in the whole kidney lysates of control and Pdcd10KspKO mice after administration of dDAVP or saline for 6 hours (n = 3). (I–P) Immunofluorescence staining of Aqp2 (green) and pS256-Aqp2 (red) on sections of kidney medulla of control and Pdcd10-deficient mice after administration of saline or dDAVP for 6 hours. Data are presented as mean ± SEM using unpaired t test (D) or 1-way ANOVA (A–C,E–G). **P < 0.01; *P < 0.05. Original magnification, ×630 (I–P).