Aquaporin-1 retards renal cyst development in polycystic kidney disease by inhibition of Wnt signaling

Abstract

Water channel aquaporin-1 (AQP1) is expressed at epithelial cell plasma membranes in renal proximal tubules and thin descending limb of Henle. Recently, AQP1 was reported to interact with β-catenin. Here we investigated the relationship between AQP1 and Wnt signaling in in vitro and in vivo models of autosomal dominant polycystic kidney disease (PKD). AQP1 overexpression decreased β-catenin and cyclinD1 expression, suggesting down-regulation of Wnt signaling, and coimmunoprecipitation showed AQP1 interaction with β-catenin, glycogen synthase kinase 3β, LRP6, and Axin1. AQP1 inhibited cyst development and promoted branching in matrix-grown MDCK cells. In embryonic kidney cultures, AQP1 deletion increased cyst development by up to ∼ 40%. Kidney size and cyst number were significantly greater in AQP1-null PKD mice than in AQP1-expressing PKD mice, with the difference mainly attributed to a greater number of proximal tubule cysts. Biochemical analysis revealed decreased β-catenin phosphorylation and increased β-catenin expression in AQP1-null PKD mice, suggesting enhanced Wnt signaling. These results implicate AQP1 as a novel determinant in renal cyst development that may involve inhibition of Wnt signaling by an AQP1-macromolecular signaling complex.

Keywords: ADPKD; MDCK; destruction complex; water channel.

Figure 1.

AQP1 expression decreases E-cadherin and inhibits cell adhesion. A) Aggregation assay, with aggregation calculated by dividing the difference between the particle number before and after the assay by the number of cells at the start of the assay. Left: Representative light micrographs of MDCK and AQP1-MDCK cells after the aggregation assay. Right: Histogram of aggregation index. B) Western blots (left) of lysates of MDCK, AQP1-MDCK and AQP3-MDCK cells. Bar graph (right) shows the density ratio of E-cadherin to β-actin. Mean ± sem, n = 3, *P < 0.05 compared with MDCK cells. C) E-cadherin immunostaining. Bar graph (right) shows immunofluorescence ratios of E-cadherin to β-actin. Means ± sem, n = 3, **P < 0.01 compared with MDCK cells.

Figure 2.

AQP1 inhibits Wnt signaling. A) β-catenin immunostaining in MDCK and AQP1-MDCK cells. Bar graph (right) shows immunofluorescence ratios of β-catenin. Means ± sem, n = 3, **P < 0.01 compared with MDCK cells. B) Western blots (left) of lysates of MDCK, AQP1-MDCK and AQP3-MDCK cells analyzed with indicated antibodies. Bar graph (right) shows the ratios of p-β-catenin/β-catenin, p-GSK3β/GSK3β and cyclinD1/β-actin. Means ± sem, n = 3, *P < 0.05, **P < 0.01 compared with MDCK cells.

Figure 3.

AQP1 interacts with destruction complex proteins. A) Western blots (left) of MDCK and AQP1-MDCK cells treated with 30 mM LiCl overnight analyzed with indicated antibodies. Bar graph (right) shows the ratios of p-β-catenin/β-catenin and p-GSK3β/GSK3β. Means ± sem, n = 3, **P < 0.01 compared with MDCK cells. B) Coimmunoprecipitation with anti-AQP1 showing AQP1 interaction with β-catenin, GSK3β, LRP6 and Axin1 in AQP1-MDCK cells. C) Coimmunoprecipitation with anti-β-catenin and anti-Axin1 show β-catenin and Axin1 interaction with AQP1 in AQP1-MDCK cells. M, MDCK cells; A, AQP1-MDCK cells; NC, negative control. D) Western blot analyses of cytosolic and membrane fractions of AQP1-MDCK cells. C, cytosolic fraction. M, membrane fraction.

Figure 4.

AQP1 inhibits cyst formation in matrix-grown MDCK cells. A) Light micrographs of cysts formed by control and AQP1-transfected MDCK cells on the day 14 after culture with forskolin. Scale bars, 500 μm. B) Light micrographs of cysts on the days 4–14 formed by non-transfected MDCK, AQP1-MDCK and AQP3-MDCK cells. Each series of photographs shows the same cyst on successive days in culture. Scale bars, 200 μm. C) Hoechst dye 33342 staining of cysts on the day 8 formed by non-transfected MDCK, AQP1-MDCK and AQP3-MDCK cells. Scale bars, 100 μm. D) Cyst formation percentage of MDCK and AQP1-MDCK cells on day 14. E) Cyst diameters of MDCK and AQP1-MDCK cells on day 14. Means ± sem; > 30 cysts analyzed, *P < 0.05 compared with MDCK cells.

Figure 5.

AQP1 promotes MDCK tubulogenesis. A) Light micrographs showing branches following MDCK culture with 3T3 fibroblast-conditioned media. Light micrographs were taken every other day from days 4 to 12. Each series of photographs shows the same area on successive days in culture. Scale bar, 200 μm. B) Percentage of branches formed in MDCK and AQP1-MDCK cells on day 12. C) Numbers of branches on day 12. Means ± sem; > 30 tubules analyzed, **P < 0.01 compared with MDCK cells.

Figure 6.

AQP1 inhibits cell proliferation, but has no effect on fluid secretion. A) Curves show the cell proliferation as indicated, measured by CCK-8 assay. Means ± sem, n = 3, *P < 0.05, **P < 0.01 compared with MDCK cells. B) The fluid secretion rate of MDCK and AQP1-MDCK cells with forskolin stimulation. Means ± sem, n = 3.

Figure 7.

AQP1 deficiency promotes cyst development in embryonic kidney cyst model. A) Western blot of the embryonic kidneys from wild-type mice on embryonic day 12.5, 13.5, 15.5, 17.5, and at age 8 wk. B) Light micrographs of embryonic kidney cultures in transwells maintained for 6 days. Embryonic day 13.5 kidneys were exposed to 0 (control) or 100 μM 8-Br-cAMP. Each series of photographs shows the same kidney on successive days in culture. C) Percentage cyst area in AQP1^+/− and AQP1^−/− embryonic kidneys on day 6 in culture. Means ± sem; n = 6–10, *P < 0.05 compared with AQP1^+/− kidneys. D) AQP1 and LTL immunofluorescence control and 8-Br-cAMP-treated embryonic kidneys on day 6 in culture.

Figure 8.

AQP1 deficiency promotes proximal tubule-derived cyst development in PKD mice. A) Representative images of kidneys from mice with wild-type phenotype (P+A+, Pkd1^flox/+;Ksp-Cre; AQP1^+/− genotype), AQP1 expressing PKD (P-A+, Pkd1^flox/flox;Ksp-Cre;AQP1^+/− genotype) and AQP1 null PKD (P-A-, Pkd1^flox/flox;Ksp-Cre;AQP1^−/− genotype) mice at age 1, 3, 5, and 7 days. Bottom: Histogram of kidney index (ratio of kidney to body weight). Means ± sem; n = 6–10, *P < 0.05, **P < 0.01 compared with AQP1-expressing PKD mice. B) Hematoxylin and eosin (H&E) stained sections. Bottom: Histogram of cystic index. Means ± sem; n = 6–10, *P < 0.05 compared with AQP1 expressing PKD mice.

Figure 9.

AQP1 reduced proximal tubule cyst development. A) LTC immunofluorescence in AQP1-expressing PKD (P-A+) kidneys at age 3, 5, and 7 days. B) AQP2 and LTL immunofluorescence of AQP1-expressing PKD (P-A+) and AQP1 null PKD (P-A-) kidneys at age 7 days. The sections were stained with AQP2 antibody to mark collecting duct (red), LTL antibody to mark proximal tubule marker (green), and Hoechst dye 33342 to mark nuclei (blue). C) AQP1 and LTL immunofluorescence of AQP1-expressing PKD and AQP1 null PKD kidneys at age 7 days. The sections were stained with AQP1 antibody (red), LTL antibody (green) and Hoechst dye 33342 (blue). C, cysts. D) Percentage of cysts derived from collecting duct. E) Percentage of cysts derived from proximal tubule. F) Diameters of cysts (≥50 μm) derived from collecting duct. G) Diameters of cysts (≥50 μm) derived from proximal tubule. Mean ± sem; n = 5, *P < 0.05 compared with AQP1-expressing PKD mice.

Figure 10.

AQP1 deficiency upregulates Wnt signaling. A) Western blots (left) of lysates of wild-type and AQP1 null kidneys. Bar graph (right) shows the ratios of AQP1 and β-catenin to β-actin. Mean ± sem, n = 3, *P < 0.05, **P < 0.01 compared with wild-type mice. B) Western blot of lysates of wild-type phenotype (P+A+), AQP1-expressing PKD (P-A+) and AQP1-null PKD (P-A-) kidneys. Left graph shows representative blots. Bar graph (right) shows the ratios of p-β-catenin/β-catenin, p-GSK3β/GSK3β and AQP1/β-actin. Means ± sem, n = 3, *P < 0.05 compared with wild-type phenotype mice, ^#P < 0.05 compared with AQP1 expressing PKD mice.

Figure 11.

Schematic of proposed β-catenin (β-cat) regulation by AQP1. A) AQP1 may interact with the “destruction complex” and increase the stability of the “destruction complex”. Then βb-catenin's phosphorylation is increased. p-β-catenin is then recognized by bβ-TrCP and ubiquitinated (Ub). Subsequently, ubiquitinated β-catenin is rapidly degraded by the proteasome. B) If there is no AQP1 in the cells, the stability of the “destruction complex” is decreased and the ubiquitination of bβ-catenin would be blocked, which leads to βb-catenin accumulation and translocation into the nucleus and binding to TCF. The bβ-cat/TCF complex promotes transcription of Wnt target genes.