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Published as doi: 10.1096/fj.08-111872.
 (The FASEB Journal. 2008;22:3672-3684.)
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Aquaporin-11 knockout mice and polycystic kidney disease animals share a common mechanism of cyst formation

Shinji Okada*, Takumi Misaka*, Yasuko Tanaka{dagger}, Ichiro Matsumoto*, Kenichi Ishibashi{dagger}, Sei Sasaki{ddagger} and Keiko Abe*,1

* Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan;

{dagger} Department of Medical Physiology, Meiji Pharmaceutical University, Tokyo, Japan; and

{ddagger} Department of Nephrology, Tokyo Medical and Dental University, Tokyo, Japan

1Correspondence: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. E-mail: aka7308{at}mail.ecc.u-tokyo.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aquaporin-11 (AQP11), a new member of the aquaporin family, is localized in the endoplasmic reticulum (ER). Aqp11–/– mice neonatally suffer from polycystic kidneys derived from the proximal tubule. Its onset is proceeded by the vacuolization of ER. However, the mechanism for the formation of vacuoles and cysts remains to be clarified. Here, we show that Aqp11–/– mice and polycystic kidney disease (PKD) animals share a common pathogenic mechanism of cyst formation. We performed microarray analyses and histochemical staining to characterize the effects of the disruption of Aqp11 on kidneys of 1-wk-old mice. Microarray analyses revealed that the significantly changed functional categories in Aqp11–/– mice were similar to those in PKD animals. Histochemical studies showed expression changes of 3 genes, Myc, Egfr, Egf, which are assumed to be involved in the proliferation of cystic cells in PKD. We actually confirmed the activation of cell proliferation in the proximal tubule cells with vacuolized ER. Furthermore, three genes associated with the remodeling of the extracellular matrix, Mmp12, Timp1, Tgfb1, were up-regulated in the fibroblasts. We also demonstrated the activation of apoptosis via the ER-stress pathway in the proximal tubule cells with vacuolized ER. These results provide new insights into the physiological roles of AQP11.—Okada, S., Misaka, T., Tanaka, Y., Matsumoto, I., Ishibashi, K., Sasaki, S., Abe, K. Aquaporin-11 knockout mice and polycystic kidney disease animals share a common mechanism of cyst formation.


Key Words: subcellular aquaporin • DNA microarray • histochemical analysis • ER stress


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
AQUAPORINS (AQPs) ARE A FAMILY of channel-forming membrane proteins that facilitate the transport of water and some low-molecular-weight solutes (1) . They are widely expressed in bacteria, plants, and animals. In mammals, 13 members, AQP0-AQP12, have been identified so far, and they are generally classified into 3 subfamilies (1 2 3 4 5) . The "aquaporin" subfamily is composed of six members whose permeation is highly water selective, whereas the "aquaglyceroporin" subfamily is composed of four members that are permeable to both water and low-molecular-weight solutes such as glycerol and urea. The members of these two families have been shown to function at the plasma membrane and to be involved in osmoregulation as well as in transcellular solute transport. Recently, two new aquaporins, AQP11 and AQP12, belonging to the third subfamily have been identified (3 4 5) . They have low sequence similarities to the other mammalian AQPs and less conserved asparagine-proline-alanine (NPA) motifs, which are highly conserved in aquaporins and constitute the channel pore domain (2 , 6) . It was also reported that AQP11 is localized in the endoplasmic reticulum (ER; ref. 4 ) and that AQP12 is localized in the intracellular organelles (5) . Based on these characteristics, AQP11 and AQP12 are classified as "subcellular aquaporins" or "superaquaporins" (2 , 4) . Similar subcellular aquaporins have been found in multicellular organisms, such as vertebrates, insects, nematodes, and plants, but not in unicellular organisms (2 , 6) . Subcellular aquaporins, therefore, may contribute to the roles required by multicellular organisms, such as organogenesis and cell-to-cell signaling (2) . Currently, their physiological roles are poorly understood.

To achieve an insight into the roles of subcellular aquaporins, we generated Aqp11 knockout (Aqp11–/–) mice (4) . We reported that Aqp11–/– mice were fatal due to uremia resulting from a polycystic kidney (4) . Cysts developed from the proximal tubule where AQP11 is normally expressed at 4 wk of age. Interestingly, vacuoles were formed before cyst development. The vacuoles were derived from the ER, where AQP11 is normally expressed. A recent membrane reconstitution analysis revealed that AQP11 actually possesses water-channel activity (7) . However, the formation of vacuoles and the development of cysts in Aqp11–/– mice may not completely be explained by the loss of such channel activity. It is thus expected that the elucidation of the mechanisms for the formation of vacuoles and the development of cysts in Aqp11–/– mice will provide some insight into the physiological roles of AQP11.

In this study, to clarify the early events in cyst formation after the vacuolization of ER in Aqp11–/– mice, we performed a transcriptome analysis of the kidney in 1-wk-old Aqp11–/– animals in whom vacuoles are evident but cysts are absent in the kidney (4) . We further performed a clustering analysis, extraction of the regulated genes, and categorization of the extracted genes to describe the characteristics of the gene expression profiles of the kidneys in Aqp11–/– mice. The cellular events were confirmed by means of histochemical staining. Our results suggest that Aqp11–/– mice and polycystic kidney disease (PKD) share a common pathogenic mechanism for cyst formation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Aqp11+/– mice, originally generated in a 129/SvEvBrd background (4) , were back-crossed 10–11 times to a C57/BL6 background. Heterozygous mice were interbred to obtain knockout (Aqp11–/–) and wild-type littermates (Aqp11+/+). The genotypes of offsprings were analyzed by polymerase chain reaction (PCR) using tail DNA as a template. All animal experiments were approved by the Animal Care Committee of the University of Tokyo.

Total RNA extraction
One-week-old mice were sacrificed by cervical dislocation. Kidneys were rapidly excised on ice, washed in ice-cold phosphate-buffered saline (PBS), immersed in RNAlater (Applied Biosystems, Foster City, CA, USA), and stored at –20°C. Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and cleaned up using an RNeasy minikit with DNase treatment (Qiagen, Valencia, CA, USA). The RNA quality and quantity were examined by agarose-gel electrophoresis and a spectrophotometer (GE Healthcare Bioscience, Tokyo, Japan).

Microarray experiments
Five pairs of Aqp11+/– mice were interbred, and we obtained 7 Aqp11+/+ and 9 Aqp11–/– offspring. Three micrograms of total RNA obtained from each kidney were individually reverse-transcribed, amplified, and labeled using GeneChip One-Cycle target labeling and Control Reagent package (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s protocol. Labeled cRNA was hybridized to GeneChip Mouse Genome 430 2.0 Array (Affymetrix; a total of 16 samples on 16 different arrays). The arrays were washed, stained using Fluidics Station (Affymetrix), and then scanned with the GeneChip Scanner (Affymetrix). Data collection was performed using GeneChip Operating Software (Affymetrix). The quality of collected data was checked by scatter plot analysis. The data were normalized using GeneSpring GX software (Agilent Technologies, Santa Clara, CA, USA) by the gcRMA normalization method (8) . Data were deposited in the Gene Expression Omnibus repository under the accession number GSE10634.

Microarray data analysis
Hierarchical clustering with Pearson’s squared distance metric was carried out using the logarithms of gcRMA normalized signal values for all probe sets using GeneSpring GX software. The statistical analysis was performed with GeneSpring GX software. Briefly, we first selected the probe sets that had "present" or "marginal" calls in more than half the samples under any condition (4 in Aqp11+/+ or 5 in Aqp11–/–). Accordingly, 28,717 probe sets from a total of 45,101 were selected, which were defined as probe sets for kidney-expressing genes. To identify probe sets with significant expression changes, we next performed Welch’s t test with Benjamini and Hochberg false discovery rate multiple testing correction (9) using the logarithms of gcRMA normalized signal values, where the threshold of the false discovery rate (FDR) was set at 5% (q<0.05; maximum P value in the selected probe sets was 0.00693). The annotations of each probe set were obtained from the Affymetrix’s NetAffx database (version 2007/3/8; ref. 10 ). For gene classifications and further analyses, we eliminated the probe sets with low-grade annotations (grade C, E, and R) to analyze data sets with reliable annotations. The reliable probe sets were categorized by Gene Ontology (GO) terms using DAVID 2007 online software (http://david.abcc.ncifcrf.gov/; ref. 11 ). The threshold of Fisher’s exact test was set at P < 0.05.

In situ hybridization
In situ hybridization analyses were performed as described previously (12) with the following modifications: kidneys of Aqp11+/+ and Aqp11–/– animals were fixed with 4% paraformaldehyde (PFA) in PBS for 18 h at 4°C, cryoprotected with 30% sucrose in PBS, embedded in Tissue-Tek O.C.T. compound (Sakura Finetechnical, Tokyo, Japan) in liquid nitrogen, and sectioned in 7 µm thick slices. The sections were fixed with PFA, carboethoxylated, treated with 20 µg/ml proteinase K for 10 min, and postfixed with 4% PFA in PBS for 5 min. They were then hybridized with riboprobes and hybridization buffer followed by counterstaining with Nuclear Fast Red solution (Sigma-Aldrich, St. Louis, MO, USA). The antisense riboprobe for each clone was synthesized from the fragments subcloned into pBluescript II SK-(–) vector (Stratagene, LA Jolla, CA, USA) and hydrolyzed to ~500 bases length before hybridization. Negative control experiments were performed with sense riboprobes (data not shown).

Immunohistochemistry
PFA-prefixed cryosections were prepared as described above. The sections were treated with Target Retrieval Solution (Dako, Carpinteria, CA, USA) for 20 min at 95°C and then cooled for 20 min at room temperature. After the inactivation of endogenous peroxidase with methanol containing 0.3% hydrogen peroxide for 30 min at –20°C, the sections were incubated with avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA, USA), blocked with normal serum in PBS containing 0.1% Triton X-100, and incubated overnight with rabbit anti-cleaved caspase-3 antibody (1:400, Cell Signaling Technologies, Beverly, MA, USA) or goat anti-Ki-67 antibody (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C. Visualization was performed with the VectaStain Elite ABC kit (Vector Laboratories) and DAB Metal Enhanced Substrate kit (Pierce Biotechnology, Rockford, IL, USA), followed by counterstaining using hematoxylin. Negative control experiments were performed by staining without the primary antibody, and no signal could be observed in the kidneys of Aqp11+/+ or Aqp11–/– mice (data not shown).

Terminal deoxynucleotidyl transferase digoxigenin-dUTP nick end labeling (TUNEL) assay
PFA-prefixed cryosections were prepared as described above. The sections were treated with target retrieval solution for 20 min at 95°C and then cooled for 20 min at room temperature and incubated with 1% hydrogen peroxide for 10 min. TUNEL was then carried out using the DeadEnd Colometric TUNEL System (Promega, Madison, WI, USA) and digoxigenin-16-dUTP (Roche Diagnostics, Tokyo, Japan). Visualization was performed with peroxidase-conjugated antidigoxigenin antibody (Roche) and DAB Metal Enhanced Substrate kit, followed by counterstaining with hematoxylin. As a positive control, the sections were pretreated with DNase I. Negative control experiments were performed by staining without the TdT enzyme, and no nuclear staining could be observed in the kidneys of Aqp11+/+ or Aqp11–/– mice (data not shown).

Semiquantitative reverse transcription-PCR (RT-PCR)
Total RNA obtained from 4 Aqp11+/+ and Aqp11–/– mice each at age 1 wk were used. One microgram of total RNA was reverse-transcribed using SuperScript III First Strand SuperMix (Invitrogen) and an oligo-dT primer, according to the manufacturer’s protocol. One microliter of 0.1-diluted cDNA was amplified in 25 µl of reaction mixture containing 0.5 µM of each primer, 800 µM dNTPs, 1x Phusion HF buffer, and 20 U/ml Phusion DNA polymerase (New England Biolabs, Beverly, MA, USA). The nucleotide sequence used for each primer was as follows: for Casp12: sense primer, 5'-CCACTGCTGATACAGATGAGG-3' and antisense primer, 5'-GGACCTCAAATGAGTGTTGAAC-3'; for Pkhd1: sense primer, 5'-AGATTGGCTGTCTTGATCTCTG-3' and antisense primer, 5'-AGCCTGGGATTCAGATATGTC-3'; and for Gapdh: sense primer, 5'-TCACTCAAGATTGTCAGCAATG-3' and antisense primer, 5'-CATCATACTTGGCAGGTTTCTC-3'. The amplified fragment size of Casp12, Pkhd1, and Gapdh was 240, 258, and 343 bp, respectively. Gapdh was used as an internal reference in each PCR reaction. After a denaturing step (30 s at 98°C), amplifications (10 s at 98°C, 30 s at 65°C, and 10 s at 72°C) were performed. In preliminary experiments, we performed the amplification over a variety of PCR cycles (20–35 cycles) to determine the linear range of amplification for each primer set. Thus, 31 cycles for Casp12, 28 cycles for Pkhd1, and 22 cycles for Gapdh were adopted for the amplification. The PCR products obtained were electrophoresed on 2% agarose gels and visualized by ethidium-bromide staining. Band intensities of the PCR products were quantified using ImageJ software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Gene expression profiles of the kidneys in Aqp11+/+ and Aqp11–/– mice
To achieve an insight into the mechanisms underlying the formation of cysts in Aqp11–/– mice, we performed a transcriptome analysis on the kidneys of 1-wk-old Aqp11–/– and Aqp11+/+ littermates. The vacuolization of the ER was evident, but cysts were absent in the Aqp11–/– kidneys at this age (4) . We first performed a hierarchical clustering analysis using all probe sets (45,101). As shown in Fig. 1 , correlation coefficients between each pair of 16 samples were ≥0.987, suggesting the good reliability of our microarray data. Sixteen samples were classified into two clusters based on their genotypes but not on their parents or sexes (Fig. 1) . Thus, the lack of Aqp11 made a dramatic effect on the gene expression profile in the kidney at 1 wk after birth.


Figure 1
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  		Figure 1. Hierarchical clustering analysis for the microarray data obtained from the kidneys of 1-wk-old Aqp11+/+ and Aqp11–/– mice. Clustering analysis was carried out using normalized data for all 45,101 probe sets obtained from 16 mice. Each sample name represents a serial number of a pair of parents and that of their offspring along with sex and genotype. Numbers at nodes represent Pearson’s correlation coefficients. F, female; M, male.

We then performed a statistical analysis to extract the genes with significant changes in their expression levels. First, we selected 28,717 probe sets representing 16,065 genes expressed in the kidney, as described in Materials and Methods. Using these probe sets, we performed Welch’s t test for FDR multiple testing correction, where the threshold of FDR was set at 5% (q<0.05). As a result, a total of 3106 genes (3991 probe sets), which corresponded to ~19% of the genes expressed in the kidney, demonstrated significant changes in their expression levels at the age of 1 wk. A total of 1584 genes (2060 probe sets) were observed to be up-regulated,and 1534 genes (1,991 probe sets) were down-regulated in Aqp11–/– mice. The number of up-regulated genes was almost the same as that of the down-regulated genes.

Functional classification of genes with significant changes in expression level
To find common molecular functions among the significantly changed genes, we classified them into the categories of the GO Biological Process. Table 1 and Supplemental Table S1 show the significantly enriched GO terms identified by Fisher’s exact test (P<0.05). We first used the GO terms in GO levels 1–3 for a brief classification (Table 1) and then used the GO terms in all the levels for a detailed classification (Supplemental Table S1). The up-regulated genes were classified into many GO terms, whereas the down-regulated genes were classified into far fewer GO terms (Table 1 and Supplemental Table S1). The up-regulated genes were classified into 56 GO terms in GO levels 1–3 (Table 1) and 209 GO terms in all levels (Supplemental Table S1A), whereas the down-regulated genes were classified into 10 GO terms in GO levels 1–3 (Table 1) and 129 GO terms in all levels (Supplemental Table S1B). Furthermore, the up-regulated and down-regulated genes were classified into considerably different categories from each other. In detail, the up-regulated genes were classified into the categories related to various biological processes, such as immune response/response to stress, chemotaxis, cell proliferation, translation/protein biosynthesis, actin cytoskeleton, cell adhesion, cell death/apoptosis, cell differentiation/development, cellular localization (protein localization/protein transport), phosphate transport/anion transport, and ER-associated protein catabolism (Table 1 and Supplemental Table S1A). In contrast, the down-regulated genes were classified mainly into the categories related to cellular homeostasis such as ion homeostasis (cation transport/anion transport), localization, cellular metabolism, and sensory perception of mechanical stimulus (Table 1 and Supplemental Table S1B). There were only six GO terms (amino acid metabolism, amino acid derivative metabolism, anion transport, biosynthesis, establishment of localization, and localization) common to the up-regulated and down-regulated genes (Supplemental Table S1).


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  		Table 1. GO Biological Process terms considered significantly enriched for the genes regulated in the kidney in Aqp11–/– mice

Then, the significantly changed genes were also classified into the categories of GO Cellular Component to obtain information about the subcellular localization. The categories into which the up-regulated and down-regulated genes were classified also differed from each other, although with certain similarities (Table 2 ). The up-regulated genes were classified into the categories of extracellular region/extracellular matrix (ECM)/ collagen, membrane/plasma membrane, ER, cytoskeleton/actin cytoskeleton/intermediate filament, nucleolus, and lysosome, whereas the down-regulated genes were classified into the categories of mitochondrion, peroxisome, membrane/plasma membrane/organelle membrane, and extracellular region/collagen type IV (Table 2) . Both the up-regulated and down-regulated genes were classified into the categories of extracellular region and membrane/plasma membrane (Table 2) . This result revealed that the lack of AQP11, which is localized in the ER membrane, influences the expressions of gene products localized not only in the ER but also in other organelles. Many products of the down-regulated genes were localized in the organelles associated with cellular metabolism.


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  		Table 2. GO Cellular Component terms considered significantly enriched for the genes regulated in the kidney in Aqp11–/– mice

As for the formation of cysts, the animals suffering from PKD and nephronophthisis show characteristics similar to those of Aqp11–/– mice (13) . In PKD animals, abnormal cell proliferation, remodeling of ECM, activation of apoptosis, nondifferentiation of renal cells, and up-regulation of immune response, have been reported for the pathogenic mechanisms (14 , 15) . Thus, the GO classification clearly showed that the biological process categories changed in the noncystic kidneys of Aqp11–/– mice were similar to those changed in the polycystic kidneys of PKD animals.

Cell proliferation
Based on the GO classification, we hypothesized that the mechanisms for cyst formation in Aqp11–/– mice and PKD animals were common to both. In addition, although no cysts are present in the kidneys of 1-wk-old Aqp11-null mice, cystogenesis would have already been initiated. To evaluate the hypotheses, we first focused on the proliferation of tubular cells, because abnormal cell proliferation is one of the key mechanisms underlying cyst formation in PKD (14 15 16) . The genes related to cell proliferation were up-regulated in the kidneys of Aqp11–/– mice (Table 1 and Supplemental Table S1A). We then evaluated whether the proliferation of the proximal tubule cells with vacuolized ER was increased in Aqp11–/– mice by immunostaining for Ki-67 antigen, a nuclear protein present only in cycling cells. We observed a limited number of Ki-67-positive cells in the proximal tubule in Aqp11+/+ mice (Fig. 2A , arrowheads). On the other hand, we observed intense Ki-67 immunoreactivity in many proximal tubule cells in Aqp11–/– mice (Fig. 2B , arrowheads). This result indicates an increased proliferation of the proximal tubule cells in Aqp11–/– mice.


Figure 2
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  		Figure 2. Expression of cell proliferation-related genes and immunostaining for Ki-67 in the renal tubules of Aqp11+/+ and Aqp11–/– mice. A, B) Sections of kidneys of 1-wk-old Aqp11+/+ (A) and Aqp11–/– (B) mice were stained with anti-Ki-67 antibody (brown). Sections were counterstained with hematoxylin (blue). An enlarged image of the cortical region of each section is shown. Arrowheads indicate a part of Ki-67-immunoreactive cells. C–H) Sections of kidneys of 1-wk-old Aqp11+/+ (C, E, G) and Aqp11–/– (D, F, H) mice were hybridized with Myc (C, D), Egfr (E, F), and Egf (G, H) probes (dark blue). Sections were counterstained with nuclear fast red (red). An enlarged image of the cortical region of each section is shown. Note that Myc was expressed in some proximal tubule cells with no clear vacuoles in Aqp11–/–mice (D, arrowheads). Scale bar = 100 µm.

We then investigated the expression of c-Myc (Myc), epidermal growth factor (Egf) and epidermal growth factor receptor (Egfr) genes, all of which are assumed to be involved in the proliferation of cystic cells in PKD animals (14 , 17 18 19 20) . In the microarray data, the expression of Myc and Egfr was observed to be up-regulated 1.92-fold (from 133.1±14.0 to 255.3±40.1) and 1.50-fold (from 158.0±35.9 to 237.7±24.9) in Aqp11–/– mice, respectively, whereas Egf was down-regulated 0.19-fold (from 603.9±159.9 to 114.3±36.0) in Aqp11–/– mice. The expression profiles of these genes were similar to those in the animals with PKD (14 , 17 18 19 20) .

We then performed in situ hybridization analyses using probes for these three genes. The signals for Myc were not observed in the kidneys of Aqp11+/+ mice, whereas Myc signals were clearly localized in the proximal tubule cells with vacuolized ER in Aqp11–/– mice (Fig. 2C, D ). Furthermore, weak Myc signals were also localized in some proximal tubule cells with no clear vacuoles in Aqp11–/– mice (Fig. 2D , arrowheads). Egfr was expressed weakly in the collecting duct in Aqp11+/+ mice, whereas it was expressed strongly in the proximal tubule cells with vacuolized ER and weakly in the collecting duct in Aqp11–/– mice (Fig. 2E, F ). In contrast to these genes, strong signals for Egf were localized in the distal tubule in Aqp11+/+ mice, whereas its signals were not observed in Aqp11–/– mice (Fig. 2G, H ). These results strongly suggest that Myc, Egfr, and Egf are involved in cyst formation in the kidneys of Aqp11–/– mice as in the case of PKD animals.

ECM
The remodeling of the ECM is assumed to be responsible for cyst formation in PKD diseases (14 , 15 , 21) . By the GO classification, both up-regulated and down-regulated genes were enriched in the "extracellular region" category (Table 2) . The microarray data showed that a large portion of ECM components, including collagens, fibronectin, elastin, and laminins, were significantly changed in Aqp11-null mice (Supplemental Table S2). In detail, 18/29 genes of collagens, 1/1 gene of fibronectin, 1/1 gene of elastin, and 1/11 gene of laminin demonstrated alterations in the microarray data. In addition, 3/11 of the genes of matrix metalloproteinases (Mmp) that degrade ECM were up-regulated, and all 3 genes of the tissue inhibitor of metalloproteinases (Timp), an inhibitor of Mmp, were changed in their expression levels (Supplemental Table S2). Furthermore, 2/3 of the genes of Tgf-β, a cytokine regulating ECM remodeling (22 , 23) , were up-regulated (Supplemental Table S2).

We then performed in situ hybridization to examine the expressions of the Mmp, Timp, and Tgf-β genes that demonstrated substantial changes in the microarray data. Riboprobes for Mmp12 (23.87-fold in the microarray data), Timp1 (8.66-fold in microarray), and Tgfb1 (1.72-fold in microarray) were used. The signals of none of the three genes could be observed in the Aqp11+/+ kidneys, whereas their signals were observed in the Aqp11–/– kidneys (Fig. 3 ). In this case, their signals were not localized to the renal tubules but to the fibroblasts around the proximal tubules with vacuolized ER in Aqp11–/–mice (Fig. 3B, D, F ). Only a few renal fibroblasts in Aqp11–/–mice expressed the ECM-related genes, and the population of the Mmp12-expressing cells was smaller than that of the cells expressing the other genes (Fig. 3B, D, F ). Similarly, the expression changes in ECM-related genes in fibroblasts around cysts have been reported in PKD animals (24 , 25) . It is thus suggested that the changes in ECM are an important event in cyst formation in the kidneys of Aqp11–/– mice, similar to that in PKD animals.


Figure 3
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  		Figure 3. Genes related to the remodeling of the ECM are up-regulated in renal fibroblasts in Aqp11–/– mice. Sections of kidneys of 1-wk-old Aqp11+/+ (A, C, E) and Aqp11–/– (B, D, F) mice were hybridized with Mmp12 (A, B), Timp1 (C, D), and Tgfb1 (E, F) probes (dark blue). Sections were counterstained with nuclear fast red (red). An enlarged image of cortical region of each section is shown. Scale bar = 100 µm.

Apoptosis and ER-stress response
Activation of apoptosis is also assumed to be one of the key mechanisms underlying cyst formation in PKD (14 , 15) . In the microarray data, the genes related to apoptosis were up-regulated in the kidneys of Aqp11–/– mice (Table 1 and Supplemental Table S1A, GO terms including "death," "cell death," or "apoptosis"). We then examined whether apoptosis was facilitated in the kidneys of Aqp11–/–mice by means of the TUNEL assay and caspase-3 staining. Few TUNEL-positive cells were observed in Aqp11+/+ mice, whereas many TUNEL-positive cells were observed in Aqp11–/– mice (Fig. 4A, B ). The rate of apoptotic cells was apparently higher in Aqp11–/– mice than in Aqp11+/+ mice. In addition, a large number of the proximal tubule cells with vacuolized ER were TUNEL positive in Aqp11–/–mice (Fig. 4B ). Immunohistochemical analysis of cleaved caspase-3 further confirmed a marked increase of apoptotic cells and apoptosis of the proximal tubule cells with vacuolized ER in Aqp11–/– mice (Fig. 4C, D ).


Figure 4
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  		Figure 4. Detection of apoptosis in the kidneys of Aqp11+/+ and Aqp11–/– mice. A, B) TUNEL staining of sections of kidneys of 1-wk-old Aqp11+/+ (A) and Aqp11–/– (B) mice (brown). Sections were counterstained with hematoxylin (blue). An enlarged image of the cortical region of each section is shown. Arrowheads indicate a part of TUNEL-positive cells. C, D) Sections of kidneys of 1-wk-old Aqp11+/+ (C) and Aqp11–/– (D) mice were stained using anti-cleaved caspase-3 antibody (brown). Sections were counterstained with hematoxylin (blue). An enlarged image of the cortical region of each section is shown. Arrowheads indicate a part of cleaved caspase-3-immunoreactive cells. Scale bar = 100 µm.

Next, we investigated the apoptotic pathway in the proximal tubule cells with vacuolized ER. By the GO Cellular Component classification, the expression of some of the gene products localized in ER was significantly increased in the kidneys of Aqp11–/– mice (Table 2) . By the GO Biological Process classification, the genes related to "response to unfolding protein" and "ER-associated protein catabolism" were up-regulated in Aqp11–/– mice (Supplemental Table S1A). Therefore, it is likely that the vacuolization of ER induces ER stress in the proximal tubule and that a sustained ER-stress response induces the activation of a specific apoptotic pathway.

We then evaluated the changes in expression of the genes related to the ER-stress response. In the microarray data, 45/88 genes (69/198 probe sets) of ER-stress-related genes were significantly changed; 39 genes were up-regulated, whereas 6 genes were down-regulated (Supplemental Table S3). The dominant number of the up-regulated genes agreed with our speculation. Next, we examined the changes in expression of the genes related to ER-stress and ER-associated degradation (ERAD) by means of histochemical experiments. Hspa5 (Bip, Grp78, 1.56-fold in microarray) and Hsp90b1 (Grp94, 1.35-fold in microarray) are the major genes related to ER-stress response, and Sec61a (1.26-fold in microarray) and Derl3 (7.82-fold in microarray) are those related to retro-translocation and ubiquitination of unfolding proteins in the ERAD pathway (26) . Using antisense riboprobes for these four genes, we performed in situ hybridization analyses of kidney sections. No signals for these genes could be observed in Aqp11+/+ mice, whereas intense signals were observed in the proximal tubule cells with vacuolized ER in Aqp11–/– mice (Fig. 5A-H ). Further, no signals could be observed in the proximal tubule cells without any ER vacuolizations in Aqp11-null mice (Fig. 5B, D, F, H ). These results suggest that the ER-stress response including the activation of the ERAD pathway actually occurs in the proximal tubule cells with vacuolized ER in Aqp11–/– mice.


Figure 5
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  		Figure 5. Expressions of ER-stress-responsive genes in the kidneys of Aqp11+/+ and Aqp11–/– mice. A–L) Sections of kidneys in 1-wk-old Aqp11+/+ (A, C, E, G, I, K) and Aqp11–/– (B, D, F, H, J, L) mice were hybridized with Hspa5 (A, B), Hsp90b1 (C, D), Sec61a1 (E, F), Derl3 (G, H), Ddit3 (I, J), and Casp12 (K, L) probes (dark blue). Sections were counterstained with nuclear fast red (red). An enlarged image of the cortical region of each section is shown. Scale bar = 100 µm. M) Semiquantitative RT-PCR analysis of the mRNA expression of Casp12 in kidneys of 1-wk-old Aqp11+/+ Aqp11–/– mice (n=4). Gapdh was used as an internal control for RT-PCR. A pair of representative gel images from 3 independently performed experiments are shown.

To validate the ER-stress-induced apoptosis in Aqp11–/– mice, we examined the expression changes of Ddit3 and Casp12. Ddit3 (Chop) is a transcription factor involved in the process of ER-stress-induced apoptosis (27 , 28) , and Casp12 (caspase-12) is a caspase associated with ER-stress-induced apoptosis (29 , 30) . In the microarray data, the expressions of Ddit3 and Casp12 were up-regulated 3.13- and 2.00-fold, respectively (Supplemental Table S3). We then performed in situ hybridization analyses using probes for Ddit3 and Casp12. No signal for Ddit3 could be observed in Aqp11+/+ mice, while intense signals were observed in the proximal tubule cells with vacuolized ER in Aqp11–/–mice (Fig. 5I, J ). On the other hand, no clear signal of the Casp12 probe could be observed in the kidneys of either Aqp11+/+ or Aqp11–/–animals (Fig. 5K, L ). We therefore examined the change in expression of Casp12 by semiquantitative RT-PCR. The semiquantitative RT-PCR confirmed that the expression of Casp12 was increased in the kidneys of Aqp11–/– mice as in the case of the microarray analysis (Fig. 5M ).

These results strongly suggest that the sustained ER stress caused by the vacuolization of ER facilitates apoptosis via a caspase-12-mediated pathway in the proximal tubule of Aqp11–/–mice.

Genes responsible for PKD and nephronophthisis
We evaluated the expression of the genes responsible for PKD and nephronophthisis, which are diseases with characteristics similar to those observed in the kidneys of Aqp11–/–mice (31) . Nine responsible genes have been identified. Pkd1 and Pkd2 are the responsible genes for autosomal dominant polycystic kidney disease (ADPKD; ref. 32 33 34 ); Pkhd1, for autosomal recessive polycystic kidney disease (ARPKD; ref. 35 36 37 ); and Nphp1–Nphp6, for nephronophthisis (38 39 40 41 42 43 44) . As shown in Table 3 , no gene had altered significantly in the microarray data. Then, we performed a semiquantitative RT-PCR and in situ hybridization for Pkhd1 because the FDR q value for Pkhd1 (q=0.058) was close to the threshold. However, both failed to detect the expression change of Pkhd1 (Supplemental Fig. S1). The PCR product for Pkhd1 in Aqp11–/– animals was not less than that in Aqp11+/+ ones (Supplemental Fig. S1A). By in situ hybridization, no apparent differences were observed between the Aqp11+/+ and Aqp11–/– mice with respect to gene expression (Supplemental Fig. S1B, C).


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  		Table 3. Expression of the genes responsible for polycystic kidney diseases and nephronophthisis in Aqp11–/– mice

Aquaporins
We evaluated whether the loss of Aqp11 influences the expression of the other aquaporins. On the microarray, there were 24 reliable probe sets for all 12 subtypes of mouse aquaporins, and 16 probe sets for 7 aquaporins were selected as probe sets for kidney-expressing genes (Supplemental Table S4). Reasonably, the expression of Aqp11 was greatly decreased in Aqp11-null mice and Aqp11 was shown to be one of the most down-regulated genes (Supplemental Table S4). Except for Aqp11, three aquaporins, Aqp1, Aqp2, and Aqp3, were significantly changed in Aqp11–/–mice (Supplemental Table S4). Aqp1 was down-regulated, and Aqp2 and Aqp3 were up-regulated. It is well known that Aqp1 is expressed in the proximal tubules and descending thin limbs, while Aqp2 and Aqp3 are expressed in the collecting ducts (45) . The lack of Aqp11, which is expressed in proximal tubules only, influenced the expression of aquaporins in collecting ducts as well as in proximal tubules.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrate for the first time the gene expression profile of the kidneys of Aqp11–/–mice. In our previous study (4) , we showed that 1) AQP11 is localized to the ER, 2) cysts are restricted to the proximal tubule in Aqp11–/– mice, and 3) cyst development occurs after ER vacuolization in Aqp11–/– mice. Therefore, we considered that the pathogenic mechanisms responsible for cyst formation in Aqp11–/–mice differ from those involved in PKD in other animals because 1) all the gene products responsible for PKD and nephronophthisis are localized to the primary cilia of renal tubules (35 , 39 , 42 , 43 , 46 47 48) ; 2) cysts originate mainly from the collecting duct in PKD (31) ; and 3) cyst development after ER vacuolization has not been reported in any case of PKD or nephronophthisis cases (31) . However, the results of this study strongly imply that cyst formation in Aqp11–/– mice and in PKD animals is mediated by a common mechanism, at least in its early stages.

Gene expression profile of the kidneys in Aqp11–/–mice
Approximately 19% of the genes expressed in the kidney differed significantly between Aqp11–/– and Aqp11+/+ animals (FDR<5%). Such a large number of extracted genes appears reasonable since Schieren’s group (49) has reported a major change in the gene expression profile of PKD animals. They performed a transcriptome analysis of the kidneys in ADPKD patients and selected ~16% of the genes identified by microarray technology by statistical analysis (P<0.0005; ref. 49 ).

Microarray analyses and histochemical staining in this study revealed abnormal cell proliferation, remodeling of the ECM, activation of apoptosis, and up-regulation of the immune response, as the common events in cyst formation in both Aqp11–/– mice and PKD animals. On the other hand, our study also revealed some discrepancies in the mechanism of cyst formation between Aqp11–/– mice and the PKD animals and also among the PKD animals themselves. ER stress-induced apoptosis was facilitated in Aqp11–/– mice (Figs. 4 and 5) , whereas there have been no reports of an ER-stress-induced apoptotic pathway in PKD animals. Comparing our results with those of the transcriptomal analysis of ADPKD patients reported by Schieren et al. (49) , only 23 of the 85 genes that they selected demonstrated a regulation pattern similar to those of the genes analyzed in our study. In the case of the transcriptomal analysis of cultured cells derived from ADPKD patients by Lee et al. (50) , as few as 7 of the 52 genes selected showed a regulation pattern similar to that of the genes analyzed by us; moreover, 12 genes demonstrated opposite regulation. In addition, Mmp12 that was observed to be greatly up-regulated (23.87-fold) in the kidneys of Aqp11–/– mice in our study (Fig. 3) was strongly down-regulated (0.23-fold) in their study (50) . Such variations in individual gene expression may be due to difference in the time points of investigation, the methods used, or phenotypical differences such as age of onset and life span.

Physiological functions of AQP11
Our study provides new insights into the physiological roles of AQP11 in the kidney. Expression changes in a large number of genes in the kidney of Aqp11–/– mice suggest an important role of AQP11 in the organ function of the kidney. The gene expression profile of the Aqp11–/– mice (Table 1 and Supplemental Table S1) suggests abnormal cellular homeostasis in the proximal tubule cells due to the lack of AQP11. Moreover, the activation of the ER stress response was also confirmed in the proximal tubule cells of Aqp11–/– mice (Fig. 5) . ER stress response is induced by accumulation of unfolding protein (26) . Therefore, it may be likely that AQP11 is involved in maintaining an environment suitable for translation or protein foldings in the ER lumen to prevent the accumulation of unfolding protein by localizing to the ER. Because the proximal tubule reabsorbs large amounts of water, its intracellular environment should change dynamically. Accordingly, in the proximal tubule cells, a water channel in the ER membrane may be required to maintain the environment of the ER lumen.

Initiation of cystogenesis in the kidneys of Aqp11-null mice
Our results strongly suggest that in Aqp11–/– mice, cystogenesis is already initiated in the kidneys at the age of 1 wk, although cysts are absent at this age. ER vacuolization and cystogenesis may progress simultaneously because the up-regulation of Myc was observed to occur in the proximal tubule cells with no clear vacuoles (Fig. 2) .

The event that triggers cystogenesis in cells lacking AQP11 is currently not clear. However, this event may be similar to that in PKD since, in this study, the mechanisms for cyst formation in Aqp11–/–mice were shown to be similar to those in PKD. Although our study showed that the expressions of genes responsible for PKD and nephronophthisis were not altered in the kidney of Aqp11–/–mice (Table 3 and Supplemental Fig. S1), several possible mechanisms can be proposed for the triggering of cystogenesis. Our study suggests the involvement of AQP11 in maintaining an environment suitable for protein folding. Therefore, one possible cause of cystogenesis may be the protein misfolding of gene products responsible for PKD or of other ciliary proteins. Excessive misfolding may cause a reduction in the number of normal gene products, and such a reduction could trigger cystogenesis. Interestingly, two genes responsible for polycystic liver disease, Prkcsh and Sec63, encode the components of the molecular machinery involved in the translocation, folding, and quality control of newly synthesized glycoproteins in the ER (51) . Our study also showed that the genes related to cellular homeostasis, including ion homeostasis, were down-regulated in the kidneys of the Aqp11–/–mice (Table 1 and Supplemental Table S1). It has been reported that polycystin-2, a product of Pkd2, which is one of the genes responsible for ADPKD, functions as a channel in the ER membrane and that the impairment of Ca2+ homeostasis caused by mutations in polycystin-2 may be responsible for cystogenesis (52) . Therefore, another possible cause of cystogenesis is the impairment of Ca2+ homeostasis in renal tubular cells. Deletion of AQP11 may cause the impairment of Ca2+ homeostasis in the proximal tubule cells; this impairment may trigger cystogenesis.

On the other hand, the activation of the ER stress response in the kidney of Aqp11–/– mice suggests another plausible initiation event that is not similar to that involved in PKD. It is well known that the expression of various genes are induced by transcriptional factors such as ATF4, ATF6, XBP1, and CHOP (DDIT3) (53) . In fact, we demonstrated that the expression levels of these transcriptional factors were altered in Aqp11–/– mice (Fig. 5 and Supplemental Table S2). It is thus possible that the products of certain genes induced by ER stress trigger cystogenesis.

In summary, we investigated the gene expression profile of the kidney in 1-wk-old Aqp11–/– mice and found that the mechanisms underlying cyst formation in these mice and in PKD animals were similar. The present study is apparently the first to suggest the physiological roles of AQP11 in special reference to the pathogenic mechanisms of PKD. However, to completely elucidate the mechanisms underlying cyst formation in Aqp11–/– mice, further analyses are required, including detailed observations over a longer period of time. The further study of Aqp11–/– mice will help clarify the physiological roles of AQP11 and the pathogenic mechanisms of PKD.


   ACKNOWLEDGMENTS
 
The work was partly supported by grants-in-aid 16108004 (to K.A.) and 17780099 (to S.O.) from the Ministry of Education, Culture, Sports, Science and Technology, and 0745 (to S.O.) from the Salt Science Research Foundation.

Received for publication April 10, 2008. Accepted for publication June 5, 2008.


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 DISCUSSION
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