The FASEB Journal • Research Communication

Aquaporin-1 retards renal cyst development in polycystic kidney disease by inhibition of Wnt signaling Weiling Wang,*,1 Fei Li,*,1 Yi Sun,* Lei Lei,* Hong Zhou,* Tianluo Lei,* Yin Xia,† A. S. Verkman,‡ and Baoxue Yang*,2 *Department of Pharmacology, School of Basic Medical Sciences, Peking University, and State Key Laboratory of Natural and Biomimetic Drugs, Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing, China; †School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China; and ‡Departments of Medicine and Physiology, University of California, San Francisco, San Francisco, California USA 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 b-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 b-catenin and cyclinD1 expression, suggesting down-regulation of Wnt signaling, and coimmunoprecipitation showed AQP1 interaction with bcatenin, glycogen synthase kinase 3b, 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 b-catenin phosphorylation and increased b-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.—Wang, W., Li, F., Sun, Y., Lei, L., Zhou, H., Lei, T., Xia, Y., Verkman, A. S., Yang, B. Aquaporin-1 retards renal cyst development in polycystic kidney disease by inhibition of Wnt signaling. FASEB J. 29, 1551–1563 (2015). www.fasebj.org ABSTRACT

Key Words: water channel • MDCK • destruction complex ADPKD

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AQUAPORIN-1 (AQP1) IS A WATER CHANNEL PROTEIN expressed highly in the kidney, as well as in erythrocytes, choroid plexus, ciliary body, alveolar microvessels, gall bladder, placenta, and various other epithelia and endothelia (1–3). In the kidney, AQP1 is expressed at the apical and Abbreviations: ADPKD, autosomal dominant polycystic kidney disease; APC, adenomatous polyposis coli; AQPs, aquaporins; 8-Br-cAMP, 8-bromoadenosine-39,59-cyclic adenosine monophosphate; BSA, bovine serum albumin; CK1, casein kinase 1; CST, cell signaling technology; DKK3, dickkopf 3; FSK, forskolin; GSK3b, glycogen synthase kinase 3b; (continued on next page)

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basolateral membranes of epithelial cells in proximal tubule and thin descending limb of Henle’s loop (4, 5), and in endothelial cells of descending vasa recta (6). AQP1 deficiency in mice reduces transepithelial osmotic water permeability in proximal tubules, descending limb of Henle and descending vasa recta, resulting in defective urinary concentrating function (1). AQP1 has been shown to facilitate cell migration (7), cell proliferation (8), and angiogenesis (9). One recent study found an interaction between AQP1 and b-catenin, a transcription cofactor with T-cell factor/lymphoid enhancer factor (TCF/LEF) in the Wnt signal pathway. However, the mechanism and physiologic significance of interaction between AQP1 and the canonical Wnt signal pathway remains unknown. AQP1 is expressed in the epithelia lining 71% renal cysts in human autosomal dominant polycystic kidney disease (ADPKD), 44% of which were derived from the proximal tubules as shown by colocalization with gp330, a marker protein of proximal tubule (10). Two-thirds of the cysts express either AQP1 or renal collecting duct water channel AQP2 in which AQP1 expression decreased with increasing cyst size, whereas expression of AQP2 was unchanged (11). These results suggest the involvement of AQP1 in renal cystogenesis in ADPKD. ADPKD, one of the most common human monogenic diseases, is caused by mutations in one of 2 genes, Pkd1 and Pkd2, which encode the proteins polycystin-1 (PC1) and polycystin-2 (PC2), respectively (12). ADPKD affects between 1 in 400 to 1000 individuals and is characterized by massive enlargement of fluid-filled cysts of renal tubular origin that compromise normal renal parenchyma and leads to renal failure (13, 14). There is no currently approved drug therapy for ADPKD. Survival of the patients with end-stage ADPKD requires lifelong hemodialysis or kidney transplantation (15). There is interest in development of new therapies for ADPKD. However, the mechanism of renal cysts development in ADPKD is 1

These authors contributed equally to this work. Correspondence: Department of Pharmacology, School of Basic Medical Sciences, Peking University, 38 Xueyuan Lu, Haidian District, Beijing 100191, China. E-mail: baoxue@ bjmu.edu.cn doi: 10.1096/fj.14-260828 2

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complicated, and involves several intracellular signaling pathways. It is found that the Pkd1 gene mutation causes activation of the canonical Wnt/b-catenin/TCF/Lef1 signaling pathway in model organisms and cultured cells (16), which indicates that the renal cyst development of ADPKD is involved in Wnt signaling pathway and related mechanisms. We suggest that the interaction of AQP1 and b-catenin might influence renal cyst development through the Wnt signaling pathway. Here, we studied the effects of AQP1 on the renal cyst development and Wnt signaling pathway using MDCK cyst model, embryonic cyst model and PKD model. It was found that AQP1 inhibited cyst development in matrix-grown MDCK cells, embryonic kidney cultures and PKD mice. In these experimental models, AQP1 down-regulated Wnt signaling by interaction with b-catenin, GSK3b, LRP6 and Axin1. Biochemical analysis revealed decreased b-catenin phosphorylation and enhanced Wnt signaling in AQP1 null PKD mice. 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. MATERIALS AND METHODS Mice AQP1 knockout mice in a C57BL/6 genetic background were generated by targeted gene disruption, as reported previously (15). Pkd1flox/flox mice (from the Yale PKD Center) and Ksp-Cre transgenic mice (from the University of Texas Southwestern O’Brien Center) in a C57BL/6 genetic background were generated as described previously (17). Ksp-Cre mice express Cre recombinase under the control of the Ksp-cadherin promoter. PKD mice (with Pkd1flox/flox; Ksp-Cre genotype) were generated by cross-breeding Pkd1flox/flox mice with Ksp-Cre mice. The generation of Pkd1flox/flox;Ksp-Cre;AQP12/2 mice was performed in 2 steps. First, Pkd1flox/+ mice were crossed with Ksp-Cre mice. Offspring with the Pkd1flox/+;Ksp-Cre genotype was then crossed with AQP12/2 mice, generating Pkd1flox/+;Ksp-Cre;AQP1+/2 mice, which were then interbred to generate Pkd1flox/flox;Ksp-Cre; AQP12/2 mice. We used the Pkd1flox/flox;Ksp-Cre;AQP1+/2 mice as AQP1-expressing PKD mice to provide an adequate supply of litter-matched AQP1-expressing and AQP-null PKD mice. Neonatal mice (age 1 day) were genotyped by genomic PCR. Body weight was measured at ages 1, 3, 5, and 7 days. Kidneys were removed, weighed, and fixed for histologic examination. Protocols were approved by the Peking University Health Center Committee on Animal Research. Cell culture Type I MDCK cells (ATCC, Manassas, VA, USA) and AQP1MDCK cells were cultured in DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum, 1% penicillin/ streptomycin and incubated at 37°C in 5% CO2/95% air. The cells were seeded at 2 3 104 cells per 60-mm plate (Corning, (continued from previous page) HGF, hepatocyte growth factor; Lef1, line-width enhancement factor 1; LTL, lotus tetragonolobus lectin; MDCK, MadinDarby canine kidney; PC-1/2, polycystin-1/2; PKD, polycystic kidney disease; TCF, T-cell factor; Wnt, wingless-related mouse mammary tumor virus

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Corning, NY). In some experiments, cells were incubated overnight with 30 mM lithium chloride. Cell aggregation assay The cell aggregation assay was performed as described previously (18, 19). In brief, MDCK cells were trypsinized, centrifuged, and resuspended as single-cell suspensions at 2.5 3 105 cells/ml. Twenty-microliter drops of the cell suspension were pipetted onto the inside surface of 35-mm culture dish lids, and dishes were filled with 3 ml media to prevent evaporation. At specified time points, the lid was inverted, and drops were triturated 10 times through a 20 ml pipette and each drop was spread onto a glass slide. The entire coverslip was scanned and photographed using an inverted microscope (Leica Microsystems, Buffalo Grove, IL) with 310 objective, and the numbers and sizes of clusters were counted. Cell proliferation analysis The CCK-8 kit (Dojindo, Tokyo, Japan) was used to evaluate cell proliferation of MDCK and AQP1-MDCK cells (15). Cells were plated in 96-well plates at a density of 1 3 104 cells/well. At each time point, 100 ml CCK-8 solution at a dilution of 1/10 with 10% FBS DMEM was added to each well, and the cells were incubated for 2 hours at 37°C. Absorbance at 450 nm was measured with a microplate reader (MQX200; BioTek Instruments, Winooski, VT). Fluid secretion assay MDCK and AQP1-MDCK cells were plated at a density of 2.5 3 105 cells/ml on filter inserts (3493; Corning) and cultured for ~4 to 5 days. MDCK media were changed every 2 days, and transepithelial resistance (TER) was measured using a Millicell ERS-2 epithelial voltohmmeter (Millipore, Billerica, MA, USA). TER measurements are expressed as ohms/centimeter squared (V/cm2). Only polarized epithelia monolayers with resistance .500 V/cm2 were used for subsequent experiments. Cells were serum starved (0.5% serum; Gibco/Invitrogen) in basal DMEM/F12 medium for 24 hours, and the basolateral side of the cell monolayers was treated with 10 mM forskolin. Control monolayers were incubated in basal medium. Fresh medium (150 ml) was placed on the apical surface of the cells, and mineral oil was layered over the top of the medium to prevent evaporation. Cultures were incubated at 37°C for an additional 3 days, and the apical medium was collected and the volume of fluid was determined using calibrated microcapillary tubes (Drummond, Broomall, PA) (20). The volume of fluid transported across the epithelium, expressed in microliters per hour per centimeter squared, was determined from the change in volume during the experimental period. MDCK cyst model MDCK cells were cultured in an atmosphere of 5% CO2/95% air at 37°C in a 1:1 mixture of DMEM and Ham’s F-12 nutrient medium supplemented with 10% fetal bovine serum (Hyclone), 100 U/ml penicillin, and 100 mg/ml streptomycin. For cyst generation, 400 MDCK cells were suspended in 0.4 ml of ice-cold MEM containing 2.9 mg/ml collagen (PureCol; Inamed Biomaterials, Fremont, CA), 10 mM HEPES, 100 U/ml penicillin, and 100 mg/ml streptomycin (pH 7.4). The cell suspension was plated onto 24-well plates. After incubation for 90 minutes at 37°C, 1.5 ml of the MDCK cell medium containing 10 mM forskolin was then

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added to each well, and plates were maintained in a humidified atmosphere of 5% CO2/95% air at 37°C. The medium containing 10 mM forskolin was incubated for 14 days and changed every 12 h. Micrographs of the same cysts in collagen gels were taken on days 4, 6, 8, 10, 12, and 14 after seeding. For analysis of cyst growth, diameters of cysts that were $50 mm were measured using the NIH ImageJ software (National Institutes of Health, Bethesda, MD, USA). MDCK branch model To prepare conditioned medium, 3T3 cells were seeded into 15-cm dishes (Corning) at 104 cells/cm2 and grown to confluence, at which time 30 ml of fresh complete medium was added. After further 3-day incubation, the conditioned medium was collected, centrifuged to remove floating cells and cell debris, and stored at 220°C. MDCK cells were suspended at 1000 cells/ml in 0.4 ml ice-cold MEM containing 2.9 mg/ml collagen (PureCol), 10 mM HEPES, 100 U/ml penicillin, and 100 mg/ml streptomycin (pH 7.4). The cell suspension was plated onto 24-well plates. After incubation for 90 minutes at 37°C, 1.5 ml 3T3 conditioned medium was added to each well, and plates were maintained in 5% CO2/95% air humidified atmosphere at 37°C. Branches were photographed every 2 days. On day 12, the number of branches was quantified from the number of branch ends as a correlative measure of the number of branching events (21). Tubules of length less than 200 mm were excluded from the analysis. Embryonic kidney cyst model Mouse embryonic kidneys at embryonic day 13.5 (E13.5) were dissected and placed on transparent Falcon 0.4 mM diameter porous cell culture inserts as described previously (22). The lower chambers were filled with a 1:1 mixture of DMEM/Ham’s F-12 nutrient medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, 5 mg/ml insulin, 5 mg/ml transferrin, 2.8 nM selenium, 25 ng/ml prostaglandin E, 32 pg/ml T3, 250 U/ml penicillin, 250 mg/ml streptomycin, and 100 mM 8-Br-cAMP. Medium was replaced every 12 hours. Kidneys were photographed using a Nikon inverted microscope (Nikon TE 2000-S) equipped with 310 objective lens, 520 nm band-pass filter, and high-resolution PixeLINK color CCD camera. Cyst area was calculated by dividing the total cyst area by total kidney area. Immunofluorescence Kidneys were collected and fixed in 4% paraformaldehyde for 16 hours at 4°C and then equilibrated in 30% sucrose overnight before embedding in Optimal Cutting Temperature (OCT; Sakura Finetek,Tokyo, Japan). Kidney sections were cut at 6 mm thickness on a cryostat. The sections were blocked with 5% (w/v) bovine serum albumin containing 0.3% Triton X-100 overnight at 4°C. The sections then were incubated with primary monoclonal antibodies to AQP1 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), b-catenin (1:500; Epitomics, Burlingame, CA, USA) or E-cadherin (1:200; Bioworld, Nanjing, China) at 4°C followed by secondary antibody (tetramethylrhodamine isothiocyanate-goat-anti-rabbit, 1:200) or lotus tetragonolobus lectin (LTL) (1:400; Vector Laboratories, Burlingame, CA, USA) for 1 hour. Hoechst dye 33342 (1:1000; Leagene, Beijing, China) was used to label nuclei. Images were captured by Leica fluorescence microscope. The dilated tubules, with diameters of more than 50 mm, were considered cysts. The numbers of cysts in 5 sections per mouse from 5 mice were averaged. Fluorescence intensity was quantitated using ImageJ software (U.S. National Institutes of Health) (23).

AQP1 REDUCES RENAL CYST DEVELOPMENT

Immunofluorescence staining of cysts was done as described previously (24). In brief, collagen gels were rinsed twice in PBS plus Ca2+ and Mg2+ (PBS+) and briefly digested first with 50 U/ml collagenase IA (Sigma-Aldrich, St. Louis, MO, USA) in PBS+ at 37°C for 30 minutes in the presence of protease inhibitors. Cysts then were fixed with 4% paraformaldehyde in PBS for 30 minutes at room temperature and quenched in 50 mM NH4Cl in PBS for 30 minutes. Cysts were blocked in 5% normal goat serum in PBS/Triton X-100 for another 30 minutes at room temperature and then incubated with 10 mg/ml Hoechst dye 33342. Finally, the gels were washed in PBS/Triton X-100 4 times, followed by PBS once and dH2O once. Cysts were photographed on Leica fluorescence microscope. Western blot analysis Tissues or cells were homogenized in radioimmunoprecipitation assay lysis buffer (89901; Thermo Fisher Scientific, Waltham, MA, USA) containing protease inhibitor cocktail (11873580001; Roche, Indianapolis, IN, USA). The extract was homogenized using a Dounce homogenizer and spun at 12,000 3 g for 20 minutes at 4°C. Total protein was measured by BCA (Pierce Biotechnology, Rockford, IL, USA) and resolved by SDS– polyacrylamide gel electrophoresis. Proteins were blotted to polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, NJ, USA). Blots were incubated with polyclonal antibody against AQP1 (Santa Cruz Biotechnology), b-catenin (Epitomics), p-b-catenin (Epitomics), E-cadherin (Bioworld), GSK3b (CST), p-GSK3b (CST), cyclin D1 (Bioworld), LRP6 (CST), and Axin1 (CST). Goat anti-rabbit IgG (Abcam, Cambridge, MA, USA) and goat anti-mouse IgG (Santa Cruz Biotechnology) were added, and the blots were developed with ECL Plus Kit (Amersham Biosciences). Relative protein expression was quantified by optical density. To determine protein localization in cytosolic and membrane fractions, cells were grown in 60 mm dishes and were collected with 100 ml cold hypotonic buffer (10 mM Tris-HCl, pH 7.5, 0.2 mM MgCl2, with proteinase inhibitor cocktail) using a cell scraper. The samples were Dounce homogenized and spun at 15,000 g for 45 minutes at 4°C, and the supernatants were collected as the cytosolic fraction. The pellets were resuspended in 100 ml lysis buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1% Triton X-100, proteinase inhibitor cocktail) on ice for 10 min and spun at 10,000 g for 10 minutes at 4°C, and the supernatants were collected as the membrane fraction. Both fractions were incubated with Laemmli sample buffer containing 5% b-mercaptoethanol at 100°C for 5 minutes before loading onto the gel for Western blot. Immunoprecipitation MDCK and AQP1-MDCK cells were homogenized in radioimmunoprecipitation assay lysis buffer (89901; Thermo Fisher Scientific) containing protease inhibitor cocktail (11873580001; Roche). The extract was Dounce homogenized and spun at 12,000 g for 20 minutes at 4°C. Precleared lysates were incubated at 4°C overnight with anti-AQP1 agarose, anti-b-catenin agarose, or anti-Axin1-agarose (Santa Cruz Biotechnology). Beads were collected by centrifugation, and the pellets were washed in lysis buffer 3 times for 10 minutes with rotation at 4°C. Immunocomplexes were washed with cold lysis buffer 6 times, resuspended in SDS sample buffer, and subjected to SDSpolyacrylamide gel electrophoresis and Western blot analysis. Statistical analyses All results are expressed as mean 6 SEM. For multiple comparisons, statistical analysis was performed by using Student’s t test or

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one-way ANOVA followed by the Tukey’s multiple comparison tests. P values ,0.05 were considered statistically significant.

transcription complex-regulated protein, was reduced in AQP1-MDCK cells. Together, these results suggest that AQP1 inhibits Wnt and its downstream signaling.

RESULTS AQP1 inhibits cell adhesion in MDCK cells To confirm the effect of AQP1 on cell-cell adhesion, we performed a cell aggregation assay and found that AQP1 overexpression reduced cell adhesion (Fig. 1A). Because cadherins are responsible for cell-cell adhesion (24), we assayed the expression of E-cadherin in MDCK and AQP1MDCK cells. As expected, the expression of E-cadherin was reduced in AQP1-MDCK cells (Fig. 1B). As a control, expression of E-cadherin was not affected by AQP3. AQP1 overexpression did not affect E-cadherin localization (Fig. 1C). AQP1-MDCK cells were larger than MDCK cells. AQP1 inhibits Wnt signaling in MDCK cells Because the cytoplasmic domain of E-cadherin interacts with b-catenin and regulates stability of b-catenin (25), we investigated whether the expression and localization of b-catenin was changed by AQP1 overexpression. Immunofluorescence confirmed reduced expression of b-catenin in AQP1-MDCK cells compared with control MDCK cells and a larger cell size (Fig. 2A). In addition, b-catenin appeared to have a punctate pattern on the cell membrane, suggesting b-catenin aggregation (Fig. 2A). Western blot showed reduced b-catenin expression in AQP1-MDCK cells (Fig. 2B). Interestingly, b-catenin phosphorylation in AQP1-MDCK cells was much higher than in control cells. The ratio of p-b-catenin to b-catenin was nearly 15. In addition, expression of cyclinD1, a b-catenin/TCF

AQP1 interacts with “destruction complex” Lithium chloride inhibits GSK3b-mediated phosphorylation of a downstream transcription factor b-catenin and prevents its subsequent degradation by the proteasome complex (26, 27). We used lithium chloride to mimic the effects of canonical Wnt signaling. MDCK and AQP1MDCK cells were incubated with 30 mM lithium chloride overnight to activate the Wnt pathway. Expression of b-catenin was lower, and p-b-catenin was higher in AQP1MDCK cells than in MDCK cells (Fig. 3A), suggesting that AQP1 may have a similar effect on b-catenin and p-b-catenin after Wnt activation. To investigate the possibility of a physical interaction between AQP1 and b-catenin, lysates prepared from MDCK and AQP1-MDCK cells were subjected to immunoprecipitation. b-Catenin and GSK3b, which compose a destruction complex with casein kinase 1 (CK1) and adenomatous polyposis coli (APC), were coprecipitated with AQP1 (Fig. 3B). LRP6, a membrane protein that could recruit Axin1, was also coprecipitated with AQP1 (28). AQP1 was coprecipitated with b-catenin and Axin1 as well (Fig. 3C). Cell fractionation analyses showed that b-catenin, GSK3b, and Axin1 existed in both cytosolic fraction and membrane fraction. LRP6 and AQP1 were just detected in the membrane fraction (Fig. 3D), which is the same as the experimental results reported in previous studies (29, 30). We considered that the destruction complex was located in cell membrane. These data suggest the interaction of AQP1 with the destruction complex to

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 AQP3MDCK cells. Bar graph (right) shows the density ratio of E-cadherin to b-actin. Mean 6 SEM, n = 3, *P , 0.05 compared with MDCK cells. C) E-cadherin immunostaining. Bar graph (right) shows immunofluorescence ratios of E-cadherin to b-actin. Means 6 SEM, n = 3, **P , 0.01 compared with MDCK cells.

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Figure 2. AQP1 inhibits Wnt signaling. A) b-catenin immunostaining in MDCK and AQP1MDCK cells. Bar graph (right) shows immunofluorescence ratios of b-catenin. Means 6 SEM, n = 3, **P , 0.01 compared with MDCK cells. B) Western blots (left) of lysates of MDCK, AQP1MDCK and AQP3-MDCK cells analyzed with indicated antibodies. Bar graph (right) shows the ratios of p-b-catenin/b-catenin, p-GSK3b/ GSK3b and cyclinD1/b-actin. Means 6 SEM, n = 3, *P , 0.05, **P , 0.01 compared with MDCK cells.

increase the stability of b-catenin and maybe also of its destruction complex. AQP1 inhibits cyst formation and enlargement in an MDCK cyst model The Wnt/b-catenin pathway is associated with cell proliferation and differentiation and is involved in ADPKD

development (31). An established MDCK cyst model (15, 32, 33) was used to study whether AQP1 overexpression inhibits cyst development. In the presence of 10 mM forskolin, cysts were observed on day 4 and progressively expanded over the next 10 days. To investigate whether AQP1 water transport was involved in cytogenesis, we also studied MDCK cells transfected with AQP3, a different water channel that is expressed in collecting duct. As shown in the light microscope photo and immunofluorescence of

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-b-catenin/b-catenin and p-GSK3b/GSK3b. Means 6 SEM, n = 3, **P , 0.01 compared with MDCK cells. B) Coimmunoprecipitation with anti-AQP1 showing AQP1 interaction with b-catenin, GSK3b, LRP6 and Axin1 in AQP1-MDCK cells. C) Coimmunoprecipitation with anti-b-catenin and anti-Axin1 show b-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.

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Hoechst dye 33342, the MDCK and AQP3-MDCK cell cysts consisted of a monolayer of polarized cells enclosing a central lumen (Fig. 4A–C). It is remarkable that AQP1expressing MDCK cells formed cyst-like cell clusters that had no discernible lumens (Fig. 4A–C). This phenotype was similar to that reported with E-cadherin knockdown (24). The percentage of AQP1-MDCK cells that formed cysts was 36 6 6%, significantly lower than in control MDCK cells (53 6 4%) (Fig. 4D). The diameter of cysts formed by AQP1-MDCK cells (225 6 25 mm) was significantly lower than in MDCK cells (520 6 125 mm) (Fig. 4E). AQP1 promotes MDCK cell branching Under appropriate growth factor or hormonal influence, renal epithelial cells cultured in collagen gels form branching tubular elements, reminiscent of metanephric tubulogenesis (34). We used conditioned media from 3T3 fibroblasts as the source of hepatocyte growth factor (HGF) to induce branching and evaluated the role of AQP1 on promoting tubulogenesis of cyst epithelial cells. Initially, some of the control MDCK cells formed basal extensions that grew into processes or cords. Later, small nascent lumens formed within the processes or cords, which merged and eventually connected with the central lumen (Fig. 5A). AQP1-MDCK cells formed a greater number of branches compared with control MDCK cells (Fig. 5A, B). The number of branches formed by AQP1-MDCK cells was 3.2-fold greater than of MDCK cells (Fig. 5C). AQP1 inhibits cell proliferation but has no effect on fluid secretion The effects of AQP1 on MDCK cell proliferation measured by CCK-8 assay are presented in Fig. 6A. Compared with

MDCK cells, AQP1-MDCK cells had a significantly lower cell proliferation rate at 12 h and later times (P , 0.01). To test whether transepithelial fluid transport was influenced by AQP1, and MDCK and AQP1-MDCK cells were cultured in transwells. When TER was greater than 500V/cm2 (TERMDCK =22936414V/cm2,TERAQP1-MDCK = 515 6 10 V/cm2), the basolateral side of the cell monolayers was treated with 10 mM forskolin for 3 days. Apical fluid on cell layer was collected under forskolin stimulation. The values of fluid secretion were 42 6 4 ml in MDCK cells and 36 6 2 ml in AQP1-MDCK cells, respectively, showing that AQP1 does not increase the fluid secretion in MDCK cells (Fig. 6B). AQP1 deficiency promotes cyst development in embryonic kidneys To study the effect of AQP1 gene deletion on renal cytogenesis in embryonic kidneys, which is not confounded by the polyuria in AQP1-null mice, we first confirmed AQP1 expression in embryonic kidneys by immunoblot (Fig. 7A). There were 28 and 34 kDa AQP1 proteins found in embryonic kidneys from E12.5 to E17.5. The 34 kDa form of AQP1 is smaller than a glycosylated AQP1 band in adult mice and should be a glycosylation form of AQP1 at embryonic stage. Embryonic kidneys from AQP1-expressing and AQP1null mice at E13.5 were cultured for 6 days in the presence of 100 mM 8-Br-cAMP. Under basal culture conditions, the E13.5 renal explants from both wild-type and AQP1 nullmice increased in size over 6 days in culture (Fig.7B) (35, 36). Treatment with 8-Br-cAMP produced numerous dilated tubules, which enlarged over several days in culture, resulting in greatly expanded cyst-like structures throughout the kidneys (Fig. 7B). Kidneys from AQP1-null mice

Figure 4. AQP1 inhibits cyst formation in matrix-grown MDCK cells. A) Light micrographs of cysts formed by control and AQP1transfected MDCK cells on the day 14 after culture with forskolin. Scale bars, 500 mm. 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 mm. 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 mm. 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 6 SEM; . 30 cysts analyzed, *P , 0.05 compared with MDCK cells.

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diameter in AQP1-null PKD mice was 86 6 10 mm, 29% greater than in kidneys with AQP1 expression (Fig. 9G). There was no significant difference in the number or size of cysts derived from collecting ducts (Fig. 9B, D, F). The diameter of AQP1-expressing cysts (67 6 14 mm) was much smaller than those from collecting ducts (481 6 88 mm) (Fig. 9C, F, G). AQP1 deficiency increases Wnt signaling in kidney

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 mm. B) Percentage of branches formed in MDCK and AQP1-MDCK cells on day 12. C) Numbers of branches on day 12. Means 6 SEM; . 30 tubules analyzed, **P , 0.01 compared with MDCK cells.

had more and larger cysts than from wild-type mice (Fig. 7B). The cyst index of AQP1 null mice was 11 6 2%, significantly higher than in wild-type mice (6.6 6 0.8%) (Fig.7C), although overall kidney growth was similar. Immunofluorescence of AQP1 and LTL, a selective marker of proximal tubules, showed colocalization (Fig. 7D), indicating that the cysts were derived from proximal tubules, with AQP1 localization in cells lining the cysts. AQP1 deficiency promotes cyst enlargement in PKD mice AQP1-null PKD mice, with Aqp1 and Pkd1 double gene knockout (Pkd1flox/flox;Ksp-Cre;AQP12/2) in a C57BL/6 background, were generated by intercross of Pkd1flox/+;KspCre mice and AQP1+/2 mice. Kidney size in AQP1 null PKD mice was significantly greater than in AQP1-expressing PKD mice (Pkd1 flox/flox;Ksp-Cre;AQP1+/2 mice) on postnatal days 3 and 5, as seen from the ratio of kidney weight to body weight (kidney index, Fig. 8A). Image analysis of hematoxylin and eosin-stained sections showed significantly greater cyst index (percentage area occupied by cysts) in AQP1-null PKD mice than in AQP1-expressing PKD mice on postnatal days 3 and 5 (Fig. 8B). There was no significant difference in kidney size or cyst index on postnatal days 1 and 7. To investigate whether the effects of AQP1 gene deletion were selective for cyst development in proximal tubules, we stained sections with LTL. Immunofluorescence of aquaporin-2 (AQP2) was used to label cysts from collecting ducts. Figure 9A shows that 17 6 3% of cysts were derived from proximal tubules in AQP1-expressing PKD mice (Fig. 9B, E). The proportion of proximal tubule cysts was 23 6 3% in AQP1-null PKD mice, 36% higher than that in AQP1-expressing PKD mice (Fig. 9E). The average cyst AQP1 REDUCES RENAL CYST DEVELOPMENT

We compared Wnt signaling between wild-type and AQP1 null mice. AQP1 deficiency increased b-catenin protein expression (Fig. 10A). PKD mice had 16-fold higher b-catenin and 15-fold lower p-b-catenin than littermate wild-type mice, and AQP1 deficiency further increased b-catenin protein by 56-fold compared to wild-type mice. The ratio of p-b-catenin to b-catenin was significantly decreased in PKD mice and further decreased by AQP1 deletion (Fig. 10B). GSK3b, one of the major regulators of b-catenin turnover (37), is a serine threonine kinase that can be inhibited by phosphorylation at Ser9. It was found that the expression of GSK3b was reduced and p-GSK3b was increased in Pkd1flox/flox;Ksp-Cre mice (Fig. 10B). The ratio of p-GSK3b/ GSK3b was significantly increased in Pkd1flox/flox;Ksp-Cre mice. However, there was no difference in GSK3b and pGSK3b between Pkd1flox/flox;Ksp-Cre;AQP12/2 and Pkd1flox/flox; Ksp-Cre;AQP1+/2 mice (Fig. 10B). These results suggest that GSK3b activity is reduced in the PKD mice, which is consistent with previous studies (38, 39). However, AQP1 may not influence the GSK3b activity in PKD mice. DISCUSSION The motivation of present study based on that AQP1 interacts with b-catenin (40, 41) and is expressed in renal cyst epithelia (14). We found that the Wnt/b-catenin signaling pathway was down-regulated in AQP1-MDCK cells comparing to MDCK cells. AQP1-overexpressing MDCK cells formed fewer and smaller spherical cysts than control MDCK cells. Also, AQP1-null PKD mice had significantly larger kidneys and more cysts than littermate AQP1expressing PKD mice. Cultured embryonic AQP1-null

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 6 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 6 SEM, n = 3.

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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 mM 8-Br-cAMP. Each series of photographs shows the same kidney on successive days in culture. C) Percentage cyst area in AQP1+/2 and AQP12/2 embryonic kidneys on day 6 in culture. Means 6 SEM; n = 6–10, *P , 0.05 compared with AQP1+/2 kidneys. D) AQP1 and LTL immunofluorescence control and 8-Br-cAMP-treated embryonic kidneys on day 6 in culture.

kidneys also developed more and larger cysts than kidneys from wild-type mice. The data implicate AQP1 as a novel determinant in renal cystogenesis, with AQP1 expression retarding cyst development.

Previous studies have reported AQP1 interaction with Lin-7/b-catenin in human melanoma and endothelial cells (41, 42). b-Catenin is the key regulator in the canonical Wnt signaling pathway. Cytoplasmic b-catenin is

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+, Pkd1flox/+;KspCre; AQP1+/2 genotype), AQP1 expressing PKD (P-A+, Pkd1flox/flox;Ksp-Cre;AQP1+/2 genotype) and AQP1 null PKD (P-A-, Pkd1flox/flox;Ksp-Cre; AQP12/2 genotype) mice at age 1, 3, 5, and 7 days. Bottom: Histogram of kidney index (ratio of kidney to body weight). Means 6 SEM; n = 6–10, *P , 0.05, **P , 0.01 compared with AQP1expressing PKD mice. B) Hematoxylin and eosin (H&E) stained sections. Bottom: Histogram of cystic index. Means 6 SEM; n = 6–10, *P , 0.05 compared with AQP1 expressing PKD mice.

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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 AQP1expressing 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 mm) derived from collecting duct. G) Diameters of cysts ($50 mm) derived from proximal tubule. Mean 6 SEM; n = 5, *P , 0.05 compared with AQP1-expressing PKD mice.

phosphorylated by GSK3b in the absence of Wnt, and degraded in a proteasome-dependent manner. Upon stimulation with Wnt ligand, b-catenin phosphorylation and turnover are inhibited. The accumulated b-catenin enters the nucleus and activates the expression of downstream target genes. Abnormal activation of Wnt signaling has been reported in ADPKD. The C-terminal of PC1 bound to b-catenin inhibits the ability of both b-catenin and Wnt ligands to activate TCF, a major effector of the canonical Wnt signaling pathway (38, 43). The endogenous Wnt inhibitor Dickkopf 3 (DKK3) antagonizes Wnt/b-catenin signaling, which may modulate renal cyst growth (44). Studies with conditional Wnt mutant mice, such as mice with b-catenin overexpression (45) or APC inactivation (46), have revealed that overactivation of the canonical Wnt pathway leads to renal cystic phenotypes. Here, we found significantly reduced b-catenin in AQP1-MDCK cells compared with MDCK cells. As bcatenin is phosphorylated by GSK3b and then degraded (47), we tested the phosphorylation of b-catenin. p-b-Catenin in AQP1-MDCK cells was nearly 2-fold greater than in MDCK cells. Expression of cyclinD1, a target gene of Wnt signaling, was reduced in AQP1-MDCK cells. Furthermore, deletion of AQP1 increased b-catenin expression in kidneys. Together, these results suggest that AQP1 inhibits renal cytogenesis by inhibition of Wnt signaling. AQP1 REDUCES RENAL CYST DEVELOPMENT

The present study showed that AQP1-MDCK cells formed cyst-like cell clusters that had no discernible lumens, similar to the cells with knockdown of E-cadherin (24). b-Catenin has been shown to bind E-cadherin and participates in the formation of E-cadherin-dependent adhesive junctions (31, 48, 49). It was also found that b-catenin and E-cadherin protein levels were positively correlated (50). The loss of b-catenin from the E-cadherin/ b-catenin complex results in decreased E-cadherin expression and cell-cell adhesion (51). Consistent with these previous observations, we found here that AQP1 expression was associated with decreased b-catenin and E-cadherin expression, and cell adhesion. These in vivo and in vitro experiments suggest that altered Wnt signaling may be responsible for the action of AQP1 on cyst formation and enlargement. It was reported that membrane-localized b-catenin is nondegradable (52). Here we found that b-catenin was localized to the cell membrane where it colocalized with AQP1. However, the degradation of b-catenin was increased. We conclude that AQP1 causes a fraction of b-catenin to be degraded. Previous studies suggested that stabilization of b-catenin following Wnt activation may occur in some mechanisms, such as GSK3b sequestration (53), complex disassembly (54), membrane sequestration of Axin1-GSK3b (55), inhibition of GSK3b kinase activity 1559

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 b-catenin to b-actin. Mean 6 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+), AQP1expressing PKD (P-A+) and AQP1-null PKD (P-A-) kidneys. Left graph shows representative blots. Bar graph (right) shows the ratios of p-b-catenin/b-catenin, p-GSK3b/GSK3b and AQP1/b-actin. Means 6 SEM, n = 3, *P , 0.05 compared with wild-type phenotype mice, # P , 0.05 compared with AQP1 expressing PKD mice.

(56, 57), Axin1 degradation (58), and dissociation of Axin1 and/or APC from GSK3b or b-catenin. These studies suggest that the dissociation of the destruction complex is a prerequisite for b-catenin stabilization. Our results support the conclusion that AQP1 acts as a scaffold for a plasma membrane associated multiprotein complex involved in cytoskeleton build-up, adhesion and motility (40). AQP1 may interact with the destruction complex, as found for another scaffold protein, Axin1, where it inhibits destruction complex dissociation. Further experiments revealed that AQP1 interacts with b-catenin and other components including GSK3b, a member of the destruction complex (59–61), and LRP6, a Wnt coreceptor located in cell membrane (59–61). We speculate that AQP1 may interact with GSK3b, LRP6 and Axin1, as well as other proteins such as serine/threonine kinases (CK1) and APC, and increase the stability of the “destruction complex,” resulting in increased b-catenin

phosphorylation. Phosphorylated b-catenin is then recognized by b-TrCP, a component of the E3 ubiquitin ligase complex, and ubiquitinated (Ub). Subsequently, ubiquitinated b-catenin is rapidly degraded by the proteasome (Fig. 11A). If there is no AQP1 in the cells, the stability of the destruction complex is decreased and the ubiquitination of b-catenin would be blocked, which leads to bcatenin accumulation and translocation into the nucleus and binding to TCF. The b-catenin/TCF complex promotes transcription of Wnt target genes (Fig. 11B). A recent study demonstrated that the integrity of the destruction complex persists after Wnt ligand binding (62), which is the reason why AQP1 still promoted b-catenin degradation even after Wnt had been activated. AQP1 deletion increased cystic dilation of proximal tubules, which may be related to decreased cell migration. ADPKD epithelia have been shown to exhibit multiple alterations, including impaired migration in response to

Figure 11. Schematic of proposed b-catenin (b-cat) regulation by AQP1. A) AQP1 may interact with the “destruction complex” and increase the stability of the “destruction complex”. Thenbb-catenin’sphosphorylation is increased. p-b-catenin is then recognized by bb-TrCP and ubiquitinated (Ub). Subsequently, ubiquitinated b-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 bb-catenin would be blocked, which leads to bb-catenin accumulation and translocation into the nucleus and binding to TCF. Thebb-cat/TCFcomplexpromotes transcription of Wnt target genes.

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growth factors when compared with normal renal epithelial cells (63–65). It has been postulated that branch morphogenesis requires cell migration prior to the formation of lumen-containing tubules (66). An earlier study on IMCD cells showed that impaired migration could disrupt normal tubulomorphogenesis and induce cystogenesis when cells were grown in three dimensional cultures (67). Consist with this, we found that AQP1 deletion promoted cyst formation and enlargement. Furthermore, AQP1 overexpression in MDCK cells promoted process formation and branching and reduced cystogenesis. As a scatter factor, HGF could induce branching. The downstream response to induction of epithelial branch morphogenesis presumably involves cell migration and changes in cell adhesion, intercellular junctions, and cell polarity (68). AQP1 has been shown to promote migration in proximal tubule epithelial cells (69, 70), with migration of AQP1 deficient cells reduced by more than 50% (71). We speculate that the greater cyst formation rate and larger cystic diameter during a brief window of postnatal development in AQP1-null PKD mice compared with AQP1-expressing PKD mice is in part due to impaired cell migration. Cre-mediated Pkd1 deletion occurs only in Kspcadherin-positive cells, the vast majority of which are in the distal nephron. As Ksp-cadherin is also expressed in proximal tubules (72) (73), Ksp-cadherin-Cre could be used to delete genes in proximal tubule. Our work confirmed that there were cysts derived from proximal tubule in Pkd1flox/flox; Ksp-Cre mice and that AQP1 deletion promoted proximal tubule cysts but not collecting duct cysts. In embryonic kidney model, immunofluorescence revealed that the cysts were mainly derived from proximal tubule and AQP1 located in the cells lining the cysts, which is consistent with previous data (74). These data suggest that AQP1 promoted cyst development in proximal tubule. In summary, this study demonstrates that AQP1 deficiency promotes cyst growth and enlargement in PKD mice, embryonic kidney cultures, and MDCK cyst models. Multiple mechanisms appear to be involved, including stabilization of the destruction complex in the Wnt signaling pathway, and promoting epithelial cell differentiation and migration. These findings reveal a previously unrecognized role of AQP1 in ADPKD and hence may provide new therapeutic targets. This work was supported by the National Natural Science Foundation of China (Grants 31200869, 81261160507, 81330074 and 81170632); the Research Fund for the Doctoral Program of Higher Education (Grant 20100001110047); the 111 Project; and International Science & Technology Cooperation Program of China (Grant 2012DFA11070). This work was also supported by the U.S. National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases Grant DK35124. The authors thank Peter Igarashi and Stefan Somlo for the Ksp-Cre and Pkd1flox/flox mouse strains.

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The FASEB Journal x www.fasebj.org

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AQP1 REDUCES RENAL CYST DEVELOPMENT

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