Aquaporin-11 (AQP11) is a membrane-channel protein. Although AQP11 is reported to be permeable to the water molecule,1–3 the permeability of AQP11 to other solutes remains unclear. AQP11(−/−) mice die in the neonatal period because of renal failure and retarded growth.4,5 Moreover, AQP11(−/−) mice develop renal cysts, suggesting that AQP11 can play a role in cystogenesis.4,5 However, the mechanisms of cystogenesis in AQP11(−/−) mice have yet to be clarified. One of the reasons for the difficulties in investigating AQP11 has been the lack of a good antibody for detecting endogenous AQP11 in mouse tissues.
Autosomal dominant polycystic kidney disease (PKD) is the most common inherited renal disorder, occurring in 1:400 to 1:1000 live births. It is characterized by gradual renal cyst development and expansion, ultimately resulting in massive kidney enlargement and ESRD. Among autosomal dominant PKD patients, 85%–90% of cases result from mutations in the PKD1 gene, whereas another 10%–15% of cases are accounted for by mutations in the PKD2 gene. PKD1 encodes polycystin-1 (PC-1), a 462-kD, 4303–amino acid integral membrane protein with 11 transmembrane domains, a long extracellular N terminus with multiple binding domains, and a short cytoplasmic C terminus that interacts with multiple proteins, including the protein product of PKD2, polycystin-2 (PC-2).6 PC-2 is a significantly smaller 110-kD protein with six transmembrane domains. PC-1 and PC-2 are located in the plasma membrane and cilia of renal epithelia.6–8
To enable the analyses of AQP11 in mice at the protein level in vivo, we generated AQP11 BAC transgenic mice (TgAQP11) that express AQP11 tagged with 3×hemagglutinin (HA) sequence at its N terminus and showed that AQP11 localizes to the endoplasmic reticulum (ER) of proximal tubule cells in vivo. Moreover, to investigate the mechanisms of cystogenesis in AQP11(−/−) mouse kidneys, we focused on PC-1 and PC-2. Impaired glycosylation processing and membrane trafficking of PC-1 in AQP11(−/−) mouse kidneys were found, which could represent a key mechanism of cyst formation in AQP11(−/−) mice.
Generation of 3×HA-Tagged AQP11 BAC Transgenic Mice
Transgenic mice that expressed N-terminal 3×HA-tagged AQP11 were generated. A BAC clone containing the whole exons of mouse AQP11 with its 60-kb promoter region was used. A 3×HA tag flanked by the N terminus of AQP11 was inserted in BAC (Figure 1A). As shown in Figure 1B, Southern blots were used to select founder transgenic mice, which were successfully bred to establish the low- and high-copy transgenic mouse lines. Genomic PCR was used for detection of transgene (Figure 1C). A Western blot of kidney homogenate probed with anti-HA antibody detected that the 3×HA-tagged AQP11 was expressed in the kidney, confirming that a 3×HA-tagged AQP11 transgenic mouse was generated (Figure 1D). Additionally, no significant differences in body weight were observed between wild-type mice and either low- or high-copy TgAQP11 mice (Supplemental Figure 1).
3×HA-Tagged AQP11 Transgene Rescued Renal Cyst Formation and Retarded Growth in AQP11(−/−) Mice
To clarify that the 3×HA tag does not affect the function and subcellular localization of AQP11 in vivo, AQP11(−/−)TgAQP11 mice, which express only 3×HA-tagged AQP11 protein, were generated. The 3×HA-tagged AQP11 transgene completely rescued renal cystogenesis in 3-week-old AQP11(−/−) mice (Figure 2, A and B, Supplemental Figure 2). Moreover, retarded growth in 3-week-old AQP11(−/−) mice was also rescued by the 3×HA-tagged AQP11 transgene (Figure 2C). These results showed that 3×HA-tagged AQP11 protein can function physiologically to replace native AQP11 protein in vivo.
Immunofluorescence of AQP11 in the Kidney
AQP11 localization in the TgAQP11 mouse kidney was then examined. The immunofluorescence of TgAQP11 mouse kidneys with anti-HA antibody revealed that AQP11 was present at the cortex, whereas no labeling was detected at the cortex of kidneys from wild-type littermates (Figure 3A). In the medulla, AQP11 labeling was absent in both wild-type and TgAQP11 mice (Figure 3B). As shown in Figure 3C, double immunofluorescence with anti-HA and anti-AQP1 antibodies revealed that AQP11 was localized in the cytoplasm of proximal tubule cells. However, AQP11 staining was not detected in other segments of the kidney (Figure 3C), including the primary cilia. As expected from the observed proximal tubule localization of AQP11, the cysts in AQP11(−/−) mice originated mainly from the proximal tubules (Figure 3D).
AQP11 Localizes to ER In Vivo
To the best of our knowledge, the intracellular localization of AQP11 in vivo has yet to be reported. Double immunofluorescence with anti-HA antibody and organelle markers in the kidney revealed that 3×HA-tagged AQP11 was mainly colocalized with Lys-Asp-Glu-Leu (KDEL), an ER marker, and not with GM130, a Golgi marker, and Lamp2, a lysosome marker (Figure 4A). To confirm this ER localization of AQP11 in vivo, the ER fraction of the kidney was isolated from the TgAQP11 mouse, and immunoblotting was performed. As shown in Figure 4B, robust expression of AQP11 was found in the ER fraction. These data clearly show ER localization of overexpressed transgenic AQP11 in vivo.
Loss of AQP11 Leads to Impaired Glycosylation Processing of PC-1
ER is the organelle that is involved in protein translation, translocation, protein folding, and N-glycosylation processing of many integral membrane proteins.9 Because it is possible that AQP11 localizes to ER, we hypothesized that mechanisms of cystogenesis in the kidney of the AQP11(−/−) mouse were related to impaired quality control and aberrant trafficking of PC-1 and PC-2. PC-1 is a multidomain glycoprotein that is cleaved at a G protein–coupled receptor proteolytic site and divided into an N-terminal product (NTP) and a C-terminal product.10 It has also been reported that mouse monoclonal antibody (7e12) detects endogenous PC-1 as two distinct glycoforms: endoglycosidase H-resistant (EndoH-resistant) and sensitive NTP forms.11,12 As reported,11,12 Western blot analyses of PC-1 with mouse monoclonal antibody (7e12) showed two NTP bands in mouse kidneys (Figure 5A). An increased protein expression level of PC-1 and a decreased protein expression level of PC-2 were also found in AQP11(−/−) mouse kidneys compared with wild-type mice (Figure 5). In addition, Western blot analyses of PC-1 detected an altered electrophoretic mobility of PC-1 in AQP11(−/−) mouse kidneys compared with wild-type mice, suggesting aberrant post-translational modification of PC-1, such as glycosylation (Figure 5A). In addition, the 3×HA-tagged AQP11 transgene rescued the altered electrophoretic mobility of PC-1 and the altered protein expression levels of PC-1 and PC-2 (Supplemental Figure 3). Therefore, to determine which type of protein modification is responsible for this altered electrophoretic mobility of PC-1, a deglycosylation assay was performed. Treatment of PC-1 with peptide-N-glycosidase F (PNGaseF), an enzyme that removes all N-linked sugars, reduced the size of the two products of abnormally modified PC-1 in AQP11(−/−) mice to the same molecular mass as the deglycosylated PC-1 product of wild-type mice (Figure 6A), indicating that PC-1 was abnormally N-glycosylated in AQP11(−/−) mouse kidneys. This abnormal N-glycosylation of PC-1 in AQP11(−/−) mouse kidneys was observed in the cortex but not the medulla (Supplemental Figure 4). In addition, we examined the effects of EndoH digestion on PC-1. EndoH resistance indicates the presence of mature complex glycans, which are typically required for protein trafficking to the cell surface. In wild-type mice, the upper PC-1 band was EndoH-resistant as previously reported (Figure 6A).11 In contrast, in AQP11(−/−) mice, treatment with EndoH reduced the size of the abnormally glycosylated upper and lower bands, indicating that PC-1 from the AQP11(−/−) mouse kidney cortex is EndoH-sensitive (Figure 6A). These results suggested that the membrane trafficking of the abnormally glycosylated PC-1 in the AQP11(−/−) kidney was likely impaired. In contrast, the PNGaseF and EndoH assays on PC-2 and AQP1 showed no differences in molecular mass between wild-type and AQP11(−/−) mice before and after the treatment (Figure 6, B and C).
Impaired Membrane Trafficking of PC-1 in AQP11(−/−) Mouse Kidneys
Because PC-1 was found to be abnormally glycosylated in AQP11(−/−) mice, we hypothesized that membrane trafficking of PC-1 was impaired. To confirm this hypothesis, immunohistochemistry could not be used because of the lack of a good antibody. Therefore, ER and membrane compartments were prepared by density gradient centrifugation from wild-type and AQP11(−/−) kidney homogenates. Although PC-1 mainly localized at the ER, as previously reported,13–15 protein expression of PC-1 in the plasma membrane fraction was clearly decreased in AQP11(−/−) mouse kidneys compared with wild-type mouse kidneys, suggesting that PC-1 trafficking to the plasma membrane was impaired (Figure 7, A and B). In addition, we used an in vivo protein biotinylation assay and showed that cell surface expression of PC-1 in vivo was clearly decreased in AQP11(−/−) kidneys compared with wild type (Figure 7C; Supplemental Figure 5). In contrast, membrane trafficking of PC-2 was still observed in AQP11(−/−) mice, although PC-2 levels in the membrane fraction were decreased (Supplemental Figure 6).
The Pkd1(+/−) Background Results in Increased Severity of PKD in the AQP11(−/−) Mouse
To confirm that loss of PC-1 function is a key mechanism involved in cystogenesis in AQP11(−/−) mice, the dosage of PC-1 was reduced using Pkd1(+/−) background in AQP11(−/−) mice. As expected, histologic examination of AQP11(−/−) mouse kidneys clearly revealed that the severity of cystic disease was markedly increased on the Pkd1(+/−) background in AQP11(−/−) littermates at postnatal day 12, indicating that loss of function of PC-1 is involved in the mechanism of cystogenesis in AQP11(−/−) mice (Figure 8A). We confirmed that the segmental origin of the cysts in Pkd1(+/−)AQP11(−/−) mice is mainly in the proximal tubules, consistent with AQP11(−/−) mice (Figure 8B). In addition, the kidney-to-body weight ratio (Figure 8C) and BUN (Figure 8D) showed significant increases in Pkd1(+/−)AQP11(−/−) mice compared with their counterparts.
Primary Cilia of Proximal Tubules Are Elongated in AQP11(−/−) Mice
Abnormal ciliary length has been reported in many renal cystic diseases.11,16–21 Therefore, we examined the ciliary length of proximal tubules in AQP11(−/−) mice. Elongated primary cilia of proximal tubules were observed in AQP11(−/−) mice (Figure 9). In addition, 3×HA-tagged AQP11 transgene expression normalized the length of primary cilia in the AQP11(−/−) kidney, indicating that AQP11 may play a role in controlling ciliary length (Figure 9). However, the Pkd1(+/−) background did not alter the ciliary length of proximal tubules in AQP11(−/−) mice.
In this study, we generated TgAQP11 mice to show that AQP11 localizes to the ER in vivo. Furthermore, we also confirmed that there were aberrant glycosylation processing and defective membrane trafficking of PC-1 in AQP11(−/−) mice, resulting in cyst formation in the kidney (Figure 10).
It is well known that PKD gene products localize to the primary cilia.22 However, through the analysis of TgAQP11 mice, AQP11 was found to be present in ER in vivo but not cilia. To date, only two nonciliary proteins other than AQP11 have been reported to be responsible for cystogenesis (glucosidase IIβ (GIIβ) and SEC63p) which are autosomal dominant polycystic liver disease–causing gene products. In ER, glycans are trimmed by glucosidases, and it has been reported that certain proteins fold improperly and fail to reach the Golgi in the absence of GII.9 Loss of GIIβ and SEC63p led to cystogenesis in mouse liver and kidney.22 Interestingly, both of them localize to ER as well as AQP11. GIIβ plays a role in glycosylation processing of PC-1 at the ER.9,22 However, both kidney homogenates from GIIβ knockout mice and immortalized epithelial cells from kidney tubules of SEC63p knockout mice showed decreased protein expression of PC-1, which was opposite to the increased expression of PC-1 in AQP11(−/−) mice.22 Although GIIβ protein was checked in AQP11(−/−) mouse kidneys, no difference was found in the GIIβ protein expression level in AQP11(−/−) mouse kidneys (data not shown). Taken together, AQP11 may play a role in glycosylation processing of PC-1 at the ER in a different manner from GIIβ and SEC63p. However, the reduction of EndoH-resistant PC-1 and impaired trafficking of PC-1 were observed in both AQP11(−/−) and GIIβ knockout mice.22 It is possible that aberrantly glycosylated PC-1 in the AQP11(−/−) mouse is retained in the ER, similar to the GIIβ knockout mouse. Although PC-1 protein levels in the kidneys of AQP11(−/−) mice were increased, loss of function of PC-1, due to, impaired membrane localization, is considered to be the main cause of renal cystogenesis in AQP11(−/−) mice, which could represent a common mechanism of cystogenesis for ER proteins. In addition, PC-1 mRNA levels were not increased.23 These data indicated that aberrantly glycosylated PC-1 might fail to enter the degradation pathway in AQP11(−/−) kidney because of the inability of PC-1 to exit the ER in the AQP11(−/−) kidney. Additional investigation will be required to clarify this issue.
In this study, it was shown that PCs are involved in the mechanism of cystogenesis in AQP11(−/−) mice; however, the true function of AQP11 at the ER remains unclear. It has been reported that proximal tubule cells exhibited ER vacuolization in AQP11(−/−) mouse kidneys.4 In addition, increased ER stress response and oxidative stress in AQP11(−/−) mice were recently reported.23–25 Therefore, AQP11 may play an important role in the homeostasis of the ER, and the absence of AQP11 could lead to ER dysfunction. However, to the best of our knowledge, there are no reports showing that renal cystogenesis is caused by nonspecific disruption of ER function. In this study, we clarified a defective glycosylation in PC-1 in the proximal tubules of AQP11(−/−) mice but not other proteins, such as AQP1 and PC-2. Therefore, the absence of AQP11 in the ER might specifically lead to aberrant glycosylation of PC-1.
In this study, in contrast to PC-1, we observed decreased protein expression levels of PC-2 in the AQP11(−/−) kidney. It was previously reported that PC-2 mRNA levels are not altered significantly in AQP11(−/−) kidneys compared with wild type.23 PC-2 could be misfolded, less stable, and susceptible to degradation because of the loss of AQP11 protein in the ER, and the mechanism of degradation for PC-1 and PC-2 could differ. In addition, although it is well known that inactivation of PKD2 leads to renal cystogenesis,26–29 Wu et al.26 reported that Pkd2(+/−) mice showed only limited renal cytogenesis in the age range of 9–17 weeks. Considering that the membrane trafficking of PC-2 is still observed in AQP11(−/−) mouse kidneys, the decreased PC-2 protein level in the kidneys of AQP11(−/−) mice at 2 weeks old might not be the main cause of renal cystogenesis in the AQP11(−/−) kidney. Additional investigation will be required.
Interestingly, although AQP11 does not localize to primary cilia, elongated primary cilia of proximal tubules in AQP11(−/−) mouse kidneys were observed (Figure 9). To the best of our knowledge, this case is the first in which ER protein was found to regulate the ciliary length of kidney tubules. Hopp et al.11 recently reported that the ciliary length increase could be a compensatory response to reduced functional PC-1, suggesting that elongation of cilia is caused by loss of PC-1 function in AQP11(−/−) mice. However, the Pkd1(+/−) background did not change the ciliary length of proximal tubules in AQP11(−/−) mice (Figure 9). These data indicate that elongation of cilia in AQP11(−/−) mice might not be solely dependent on PC-1. In addition, these data also indicate that elongated primary cilia of proximal tubules in the kidneys of AQP11(−/−) mice might not play a major role in the mechanism of renal cystogenesis, although it is very interesting that an ER protein was found to regulate the ciliary length of kidney tubules. Additional investigation will be required.
In summary, impaired glycosylation processing and aberrant membrane trafficking of PC-1 could be key mechanisms of cystogenesis in AQP11(−/−) mice. The pathogenesis underlying PKD phenotypes remains unclear. It has been reported that aberrant PC-1 post-translational modifications, such as glycosylation, phosphorylation, and cleavage, are related to cyst formation.10,22,30–33 Additional investigation of post-translational modifications of PC-1 will be required to clarify the pathogenesis of cystogenesis in PKD.
AQP11 BAC transgenic mice were generated by PhoenixBio (Utsunomiya, Tochigi, Japan). BMQ33M09, a 160-kb BAC clone containing the whole exons of mouse AQP11 with its promoter region was prepared. The galactokinase selection system was used for BAC recombineering,34 and a 3×HA tag flanked by the N terminus of AQP11 of BAC was inserted. To select transgenic mice by Southern blotting and select AQP11(−/−)TgAQP11 mice by PCR and EcoRI digestion, the modified BAC also contained a new EcoRI site in intron 1. The transgene was injected into fertilized eggs of C57BL/6J mice, and transgenic mice were obtained. Founder transgenic mice were identified by PCR and Southern blot analysis, and offspring were genotyped by PCR. PCR primers were designed on both sides of the 3×HA tag. The forward primer was 5-AGGTCACATCTGCACAGCGC-3, the reverse primer was 5-ACGGGCCTGTGTAGCTGTTG-3, and the resulting amplification product was 380 bp. AQP11(+/−) mice4 and Pkd1(+/−) mice35,36 were used as previously reported. AQP11(−/−)TgAQP11 mice were genotyped by PCR and EcoRI digestion. PCR primers were designed on both sides of the new EcoRI site. The forward primer was 5-TACTGCTGTGGCATGAGCAG-3 and the reverse primer was 5-GTTCCAAGGTATCCAGGGC-3. All animal studies were performed using the procedures approved by the Institutional Animal Care and Use Committee at the Tokyo Medical and Dental University.
Kidneys were dissected as previously reported.37 Kidney homogenates of 2-week-old mice without the nuclear fraction (600 g) were prepared to measure the levels of PC-1, PC-2, and AQP1. The crude membrane fractions (17,000-g pellet) of 3-week-old mouse kidneys were prepared to measure the levels of AQP11. Semiquantitative immunoblotting was performed as described previously.38 The relative intensities of immunoblot bands were determined by densitometry with YabGelImage (free software). The primary antibodies used were rat anti-HA (Roche Applied Science, Penzberg, Germany), goat anticalnexin (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-glyceraldehyde 3-phosphate dehydrogenase (Santa Cruz Biotechnology), mouse anti–PC-1 (7e12; Santa Cruz Biotechnology), rabbit anti–PC-2 (H-280; Santa Cruz Biotechnology), mouse anti-KDEL (Enzo Life Sciences, Farmingdale, NY), rabbit anti–Na-K-ATPase (Santa Cruz Biotechnology), rabbit anti-AQP1 (Sigma-Aldrich, St. Louis, MO), mouse anti-GM130 (BD Transduction Laboratories, San Jose, CA), guinea pig anti-NCC,37 and rabbit anti–UT-A1 antibodies.39 Alkaline phosphatase-conjugated anti-IgG antibodies (Promega, Madison, WI) and WesternBlue (Promega) were used to detect the signals. Deglycosylation assays by PNGaseF and EndoH (New England Biolab, Beverly, MA) were performed according to the manufacturer’s protocol.
Immunohistochemical analyses were performed on cryostat sections of TgAQP11 and control littermate kidneys as well as formalin-fixed sections of AQP11(−/−) mice and their counterparts as previously reported.40 The primary antibodies included rat anti-HA (Roche Applied Science), rabbit anti-HA (EMD Millipore, Billerica, MA), rabbit anti-AQP1 (Sigma-Aldrich), guinea pig anti-NCC,37 mouse anti-KDEL (Enzo Life Sciences), mouse anti-GM130 (BD Transduction Laboratories), rat anti-Lamp2 (Developmental Studies Hybridoma Bank, Iowa City, IA), and mouse anti–α-acetylated tubulin (Sigma-Aldrich). Alexa fluor (Molecular Probes; Invitrogen, Carlsbad, CA) was used for secondary antibodies. Fluorescent dolichos biflorus agglutinin and lotus tetragonolobus lectin were purchased from Vector Laboratories (Burlingame, CA). Immunofluorescent images were obtained using an LSM510 laser-scanning confocal microscope system (Carl Zeiss, Oberkochen, Germany).
Isolation of ER from the Kidney
For detailed analysis of the intracellular localization of AQP11, ER was isolated from TgAQP11 mouse kidneys, and immunoblotting was performed. To obtain the ER from the mouse kidneys, an ER extraction kit (Imgenex, San Diego, CA) was used following the manufacturer’s instructions as previously reported.41
Kidney lysates of 2-week-old mice were fractionated by density gradient centrifugation as previously reported.13 After kidney homogenization in ice-cold homogenization buffer (250 mM sucrose, 1 mM EDTA, and 10 mM Tris-HCl, pH 7.5) containing protease inhibitors (protease inhibitor cocktail; Roche), a postnuclear supernatant was prepared by centrifugation at 1000 rpm for 10 minutes. Postnuclear supernatant was then layered onto a continuous 0%–15% Optiprep (Axis Shield PoC AS, Oslo, Norway) gradient and centrifuged at 200,000×g for 3 hours at 4°C using a swinging bucket rotor. The individual fractions were recovered from the top with a piston gradient fractionator. Equal amounts of samples were loaded and analyzed for PC-1, PC-2, KDEL, and Na-K-ATPase expressions. Finally, the ER and plasma membrane fractions were analyzed for PC-1, PC-2, KDEL, and Na-K-ATPase expressions.
In Vivo Protein Biotinylation
Proteins from 2-week-old mice were biotinylated in vivo as previously reported.42 In brief, mice were anesthetized, the chest was opened, the left ventricle of the heart was punctured with a perfusion needle, and a small cut was made in the right atrium to allow outflow of the perfusion solutions. After blood components were washed away with prewarmed PBS supplemented with 10% (wt/vol) dextran 40 (GE Healthcare, Little Chalfont, United Kingdom), the mouse was perfused with biotinylation solution. The perfusion solution contained 1 mg/ml sulfo-NHS-LC-biotin (Pierce, Rockford, IL) in PBS (pH 7.4) and 10% (wt/vol) dextran 40. To neutralize unreacted biotinylation reagent, the mouse was then perfused with 50 mM Tris in PBS with 10% (wt/vol) dextran 40. After perfusion, kidneys were excised and freshly snap-frozen for preparation of organ homogenates. After homogenization with ice-cold homogenization buffer (1% Triton X-100, 150 mM NaCl, and 10 mM Tris-HCl, pH 7.5) containing protease inhibitor (protease inhibitor cocktail), homogenates were centrifuged at 16,100×g for 5 minutes at 4°C, and 3 mg total protein extract was added to a Streptavidin-Sepharose slurry (100 μl/sample; GE Healthcare). The biotinylated proteins were captured overnight at 4°C in a rotating mixer. The resin was washed three times and resuspended in 100 µl 2× SDS sample buffer at 60°C for 30 minutes.
Comparisons between the two groups were performed using unpaired t tests. An ANOVA with Tukey post hoc test was used to evaluate significance in comparisons among multiple groups. P values <0.05 were considered significant. Data are presented as means±SEMs.
We thank C. Iijima for help in the experiments.
This study was supported, in part, by Grants-in-Aid for Scientific Research (S, A) from the Japan Society for the Promotion of Science; a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology of Japan; a Health and Labor Sciences Research Grant from the Ministry of Health, Labor, and Welfare; Salt Science Research Foundation Grant 1228; the Takeda Science Foundation; and a Banyu Foundation Research Grant.
Published online ahead of print. Publication date available at www.jasn.org.
This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2013060614/-/DCSupplemental.
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