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Cystic Renal Neoplasia Following Conditional Inactivation of Apc in Mouse Renal Tubular Epithelium*

Open AccessPublished:November 18, 2004DOI:https://doi.org/10.1074/jbc.M410697200
      Alterations in Wnt/β-catenin signaling have been linked to abnormal kidney development and tumorigenesis. To gain more insights into the effects of these alterations, we created mice carrying a conditional deletion of the Apc tumor suppressor gene specifically in the renal epithelium. As expected, the loss of Apc leads to increased levels of β-catenin protein in renal epithelium. Most of these mice die shortly after birth, and multiple kidney cysts were found upon histological examination. Only rarely did these animals survive to adulthood. Analysis of these adults revealed severely cystic kidneys associated with the presence of renal adenomas. Our results confirm an important role for proper regulation of Wnt/β-catenin signaling in renal development and provide evidence that dysregulation of the pathway can initiate tumorigenesis in the kidney.
      Kidney disease is a major worldwide health problem. In the United States alone, ∼4.5% of adults over 20 years of age (7.4 million persons) have physiological evidence of chronic kidney disease as determined by a moderately or severely reduced glomerular filtration rate (
      • Initiative K/DOQI Kidney Disease Outcomes Quality
      ). Polycystic kidney diseases are one of the major types of kidney disease and are found in 5–10% of patients who require dialysis (
      • Zerres K.
      ). Another renal disease associated with high morbidity and mortality is renal cell carcinoma (RCC).
      The abbreviations used are: RCC, renal cell carcinoma; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; FITC, fluorescein isothiocyanate; H&E, hematoxylin/eosin; LCM, laser capture microdissection; Ab, antibody; GSK, glycogen synthase kinase; DBA, Dolichos biflorus agglutinin.
      1The abbreviations used are: RCC, renal cell carcinoma; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate; FITC, fluorescein isothiocyanate; H&E, hematoxylin/eosin; LCM, laser capture microdissection; Ab, antibody; GSK, glycogen synthase kinase; DBA, Dolichos biflorus agglutinin.
      Approximately 32,000 new cases of RCC are diagnosed each year in the United States (
      • Jemal A.
      • Thomas A.
      • Murray T.
      • Thun M.
      ), and almost 12,000 deaths each year are caused by this cancer. Whereas great progress has been made recently in identifying genetic changes underlying kidney diseases, more needs to be done to effectively address these important clinical problems. This includes work to characterize the signaling pathways related to these diseases and to create model systems on which the factors that initiate these diseases can be assessed. One attractive candidate for such a signaling pathway is the Wnt/β-catenin pathway.
      Wnts are a family of secreted proteins that bind to a receptor complex including a frizzled molecule and either Lrp5 or Lrp6. This binding initiates an intracellular signaling cascade that eventually results in the stabilization of the cytoplasmic β-catenin protein (
      • Nelson W.J.
      • Nusse R.
      ). β-Catenin is a multifunctional protein, which, in addition to its roles in Wnt signaling, also mediates cell-cell adhesion by linking cadherins to the actin cytoskeleton (
      • Nelson W.J.
      • Nusse R.
      ). Normally, the cytoplasmic (non-cadherin-associated) levels of β-catenin are low because of the constitutive activity of the serine/threonine protein kinase glycogen synthase kinase 3 (GSK3). In the absence of an upstream Wnt signal, GSK3 phosphorylates β-catenin on serine and threonine residues in its N terminus, targeting it for ubiquitin-dependent proteolysis. When the appropriate Wnts are present, GSK3 kinase activity is inhibited, leading to stabilization of β-catenin in the cytoplasm and the nucleus. Nuclear β-catenin associates with members of the Lef/TCF family of DNA-binding proteins, leading to up-regulation of specific target genes (
      • Nelson W.J.
      • Nusse R.
      ).
      Alterations in β-catenin regulation are very common in human tumors (
      • Moon R.T.
      • Kohn A.D.
      • DeFerrari G.V.
      • Kaykas A.
      ). One way that this occurs is via loss of the APC tumor suppressor gene. APC is necessary for the proper regulation of β-catenin; in its absence, β-catenin is not degraded via GSK3-dependent phosphorylation (
      • Moon R.T.
      • Kohn A.D.
      • DeFerrari G.V.
      • Kaykas A.
      ). APC mutations underlie familial adenomatous polyposis syndrome, in which affected patients develop hundreds of colon polyps, some of which progress to full-blown carcinomas (
      • Kinzler K.W.
      • Vogelstein B.
      ). These patients carry germline-inactivating mutations in the APC gene, and loss of the wild-type copy leads to polyp initiation. In addition, ∼85% of all sporadically occurring colon tumors have inactivation of both APC alleles (
      • Kinzler K.W.
      • Vogelstein B.
      ). In all cases, the loss of APC is associated with stabilization of the β-catenin protein in the cytoplasm. Mutations in β-catenin itself, which eliminate the sites phosphorylated by GSK3, are also found in colon tumors as well as many other tumor types (
      • Moon R.T.
      • Kohn A.D.
      • DeFerrari G.V.
      • Kaykas A.
      ).
      Several studies have linked alterations in β-catenin regulation to normal kidney development and to kidney disease (
      • Vainio S.J.
      • Uusitalo M.S.
      ), and proper regulation of Wnt signaling is necessary for normal renal development (
      • Vainio S.J.
      • Uusitalo M.S.
      ). For example, there are studies that link β-catenin to proper regulation of the PKD1 promoter (
      • Rodova M.
      • Islam M.R.
      • Maser R.L.
      • Calvet J.P.
      ). PKD1 is mutated in ∼85% of patients with autosomal dominant polycystic kidney disease (ADPKD) (
      • Wilson P.D.
      ). Also, mutations in the β-catenin gene have been identified in renal cell carcinomas and Wilms' tumors (
      • Koesters R.
      • Ridder R.
      • Kopp-Schneider A.
      • Betts D.
      • Adams V.
      • Niggli F.
      • von Knebel Briner J.
      • Doeberitz M.
      ,
      • Zhu X.
      • Kanai Y.
      • Saito A.
      • Kondo Y.
      • Hirohashi S.
      ,
      • Kim Y.S.
      • Kang Y.K.
      • Kim J.B.
      • Han S.A.
      • Kim K.I.
      • Paik S.R.
      ). Finally, expression of a form of β-catenin in which the N-terminal 131 amino acids are deleted induces early development of polycystic kidney disease in a mouse model (
      • Saadi-Kheddouci S.
      • Berrebi D.
      • Romagnolo B.
      • Cluzeaud F.
      • Peuchmaur M.
      • Kahn A.
      • Vandewalle A.
      • Perret C.
      ).
      Recently, promoter methylation of the APC gene associated with its down-regulation was found in human RCC (
      • Battagli C.
      • Uzzo R.G.
      • Ibanez de Dulaimi E.
      • Caceres I.
      • Krassenstein R.
      • Al-Saleem T.
      • Greenberg R.E.
      • Cairns P.
      ). While the major function of APC is thought to be its regulation of β-catenin, APC also interacts with a multitude of other cellular proteins including axin, plakoglobin, asef, kinesin superfamily-associated protein 3 (KAP3), EB1, microtubules, and the human homolog of Drosophila Discs large (hDLG) (
      • Lee E.
      • Salic A.
      • Kruger R.
      • Heinrich R.
      • Kirschner M.W.
      ,
      • Jimbo T.
      • Kawasaki Y.
      • Koyama R.
      • Sato R.
      • Takada S.
      • Haraguchi K.
      • Akiyama T.
      ,
      • Fearnhead N.S.
      • Britton M.P.
      • Bodmer W.F.
      ). Thus, APC can potentially regulate many cellular functions including intercellular adhesion (
      • Fearnhead N.S.
      • Britton M.P.
      • Bodmer W.F.
      ,
      • Faux M.C.
      • Ross J.L.
      • Meeker C.
      • Johns T.
      • Ji H.
      • Simpson R.J.
      • Layton M.J.
      • Burgess A.W.
      ), cytoskeletal organization (
      • Mogensen M.M.
      • Tucker J.B.
      • Mackie J.B.
      • Prescott A.R.
      • Nathke I.S.
      ), regulation of plakoglobin levels (
      • Shibata T.
      • Gotoh M.
      • Ochiai A.
      • Hirohashi S.
      ), regulation of the cell cycle and apoptosis (
      • Hasegawa S.
      • Sato T.
      • Akazawa H.
      • Okada H.
      • Maeno A.
      • Ito M.
      • Sugitani Y.
      • Shibata H.
      • Ji MiyazakiJ.
      • Katsuki M.
      • Yamauchi Y.
      • Yamamura Ki K.
      • Katamine S.
      • Noda T.
      ,
      • Chen T.
      • Yang I.
      • Irby R.
      • Shain K.H.
      • Wang H.G.
      • Quackenbush J.
      • Coppola D.
      • Cheng J.Q.
      • Yeatman T.J.
      ), orientation of asymmetric stem cell division (
      • Yamashita Y.M.
      • Jones D.L.
      • Fuller M.T.
      ), and control of cell polarization (
      • Etienne-Manneville S.
      • Hall A.
      ).
      Given its important role in regulating β-catenin and other cellular processes as well as its down-regulation in some RCCs, we decided to directly test whether loss of Apc leads to the development of kidney disease in a mouse model. To do this, we conducted the present study by using the cre-lox system (
      • Sauer B.
      ) to specifically inactivate the Apc gene in mouse kidney.
      Ksp-cadherin is a tissue-specific member of the cadherin family that is expressed exclusively in the tubular epithelial cells of the kidney and the developing genitourinary (GU) tract (
      • Shao X.
      • Johnson J.E.
      • Richardson J.A.
      • Hiesberger T.
      • Igarashi P.
      ). Recently, Ksp-Cre transgenic mice expressing Cre recombinase under the control of the Ksp-cadherin promoter have been established (
      • Shao X.
      • Somlo S.
      • Igarashi P.
      ). Mice containing a floxed allele of Apc (Apc580S) have been previously reported (
      • Shibata H.
      • Toyama K.
      • Shioya H.
      • Ito M.
      • Hirota M.
      • Hasegawa S.
      • Matsumoto H.
      • Takano H.
      • Akiyama T.
      • Toyoshima K.
      • Kanamaru R.
      • Kanegae Y.
      • Saito I.
      • Nakamura Y.
      • Shiba K.
      • Noda T.
      ). We generated mice homozygous for the Apc-flox allele that also contained the Ksp-Cre transgene (Ksp-Cre;Apc580S/580S). We report here that these mice develop early-onset polycystic kidney disease that results in neonatal death in the large majority of these mice. The rare surviving animals develop severe cystic disease, with some of the cysts having an appearance consistent with a diagnosis of cystic nephroblastoma or renal adenoma.

      EXPERIMENTAL PROCEDURES

      Transgenic Mice and Genotyping—Ksp-Cre transgenic mice containing 1.3 kb of the Ksp-cadherin promoter linked to the coding region of Cre recombinase were established previously (
      • Shao X.
      • Somlo S.
      • Igarashi P.
      ). Apc580S mice, carrying a pair of loxP sites that flank Apc exon 14 and a PGK-Neo cassette, (
      • Shibata H.
      • Toyama K.
      • Shioya H.
      • Ito M.
      • Hirota M.
      • Hasegawa S.
      • Matsumoto H.
      • Takano H.
      • Akiyama T.
      • Toyoshima K.
      • Kanamaru R.
      • Kanegae Y.
      • Saito I.
      • Nakamura Y.
      • Shiba K.
      • Noda T.
      ) were generated from ES cells obtained from Tetsuo Noda. Genotyping was performed by PCR analysis of tail DNA. The primers used for Cre recombinase genotyping were as follows: 5′-CGATGCAACGAGTGATGAGGTTC-3′ (CreF) and 5′-GCACGTTCACCGGCATCAAC-3′ (CreR). Apc580S genotyping was carried out using the following primers: 5′-GTTCTGTATCATGGAAAGATAGGTGG-3′(APC-flox-P3) and 5′-CACTCAAAACGCTTTTGAGGGTTGAT-3′ (Apc-flox-P4).
      Histology, Immunohistochemistry, and Immunofluorescent Staining—The mice were mainly euthanized by continuous inhalation of CO2. In some cases, 2.5% avertin (2,2,2-tribromoethanol), 0.5–0.6 ml, was injected intraperitoneally for immobilization of the mice, followed by the intraventricular perfusion of phosphate-buffered saline (PBS) containing 4% paraformaldehyde for tissue fixation. The kidneys were isolated and fixed in 10% neutral buffered formalin overnight, embedded in paraffin, and sectioned at 3- to 4-μm thickness. A part of the sections were stained in hematoxylin/eosin (H&E). For immunohistochemical staining, the sections were deparaffinized and epitope retrieval was performed by heating the sections at 95 °C in 10 mm citrate buffer pH 6.0 for 25 min. After treating in 0.3% hydrogen peroxide for 30 min in room temperature, the sections were blocked by normal sera according to the instructions for the VECTASTAIN Elite ABC kit (POD; Vector Laboratories), incubated with primary antibodies at 4 °C overnight, visualized by VECTASTAIN Elite ABC kit, and counterstained in hematoxylin. The primary antibodies used were rabbit anti-mouse APC polyclonal Ab (1:100, SC-896, Santa Cruz Biotechnology), anti-β-catenin mouse monoclonal Ab (1:100, cat. no. 610154, BD Biosciences Pharmingen), and rat anti-mouse Ki-67 monoclonal Ab (1:25, TEC-3, DAKO, Glostrup, Denmark).
      For the immunofluorescent and lectin staining, the paraformaldehyde-fixed, paraffin-embedded sections were dewaxed, rehydrated in graded ethanol, washed in PBS, and then pretreated with 10 mm sodium citrate (pH 6.0) in a microwave oven for 20 min or PBS containing 20 μg/ml proteinase K at room temperature for 15 min. Sections were blocked in PBS containing 10% goat serum, 0.1% bovine serum albumin, and/or 1:5 mouse IgG, then incubated with the following antibodies: rabbit anti-Tamm Horsfall protein at 1:100 dilution (gift from J. Hoyer, University of Pennsylvania), rabbit anti-Na-K-Cl cotransporter (NKCC2) at 1:400 dilution (gift from M. Knepper, National Institutes of Health), rabbit anti-Na-Cl cotransporter (NCC) at 1:100 dilution (gift from M. Knepper) (
      • Kim G.H.
      • Masilamani S.
      • Turner R.
      • Mitchell C.
      • Wade J.B.
      • Knepper M.A.
      ), mouse anti-E-cadherin at 1:200 dilution (BD Biosciences), rabbit anti-aquaporin-2 at 1:400 dilution (gift from M. Knepper), rabbit anti-aquaporin-3 at 1:400 dilution (Chemicon), rabbit anti-polycystin-2 (YCC2) at 1:150 dilution, rabbit anti-polycystin-1 at 1:200 dilution (Zymed Laboratories), rabbit anti-HNF-1β at 1:200 dilution (gift from M. Pontoglio, Pasteur Institute) (
      • Gresh L.
      • Fischer E.
      • Reimann A.
      • Tanguy M.
      • Garbay S.
      • Shao X.
      • Hiesberg T.
      • Fiette L.
      • Igarashi P.
      • Yaniv M.
      • Pontoglio M.
      ), and/or mouse anti-acetylated tubulin at 1:1000 dilution (Sigma-Aldrich). In some experiments (Fig. 10, F and H), the antibody was preincubated in PBS containing 25 μg/ml blocking peptide (Zymed Laboratories Inc.) for 30 min at room temperature. After incubation with the primary antibody for 2 h at room temperature or overnight at 4 °C, sections were washed in PBS and incubated with 1:400 dilution of anti-rabbit IgG or anti-mouse IgG conjugated to cyanine 3 (Jackson ImmunoResearch Laboratories), Alexa Fluor 488 (Molecular Probes), or Alexa Fluor 594 (Molecular Probes). Some sections were co-stained with TRITC-coupled Dolichos biflorus agglutinin or FITC-coupled Lotus tetragonolobus agglutinin (Vector Laboratories), as described previously (
      • Sauer B.
      ). Slides were mounted in Vectashield (Vector) containing 1:1000 DAPI and photographed under epifluorescence illumination using a Zeiss Axioplan 2 microscope. Images were analyzed using OpenLab software (Improvision). Sections from mutant and wild-type animals were treated identically and photographed under identical exposure conditions.
      Sections for double immunofluorescent staining were deparaffinized, rehydrated, and then boiled in 10 mm sodium citrate buffer (pH 6.0) for 10 min to increase antigen exposure. Nonspecific antibody-binding sites in the tissue sections were blocked by incubation in 5% donkey serum (30 min at room temperature). The sections were then incubated overnight at 4 °C with both the rabbit anti-Apc (1:50) and mouse anti-β-catenin (1:50) antibodies described above. The sections were then washed and incubated in rhodamine-conjugated secondary donkey anti-rabbit (1:150) and FITC-conjugated secondary donkey anti-mouse antibody (1:150) (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min at room temperature. Confocal fluorescent microscopy was performed to evaluate the slides.
      Laser Capture Microdissection (LCM) and PCR Amplification—To confirm that Apc exon 14 had been deleted in the neoplastic renal epithelium, LCM was performed on both neoplastic tissue and histologically normal glomeruli. Sections (8 μm) were cut from the paraffin blocks, stained with H&E, dehydrated through a graded ethanol series to xylene, and then allowed to air dry in a desiccator. LCM was then performed using a PixCell IIe LCM system (Arcturus Engineering, Inc., Mountain View, CA) following manufacturer's protocols. Captured cells attached to the polymer film surface on the CapShur LCM caps (Arcturus Engineering, Inc.) were put directly into 0.5-ml microcentrifuge tubes and incubated with 150 μl of digestion buffer from PicoPure DNA extraction kit (Arcturus Engineering, Inc.) at 65 °C for 24 h. After boiling for 10 min to inactivate the proteinase K in the buffer, the DNA was ready for PCR amplification.
      Primers were designed to amplify the region flanked by loxP sites in the 580S allele. A third primer specific for the PGK-neo cassette in the Apc580S allele was also used. Sequences were as follows: 5′-CCTGTTCTGCAGTATGTTATCATTC-3′ (P1); 5′-AAGACACCAAGTCCAAAGCACAC-3′ (P2); and 5′-CTATCAGGACATAGCGTTGG-3′ (P3). PCR was performed using Platinum PCR SuperMix High Fidelity (Invitrogen) and 300 nm each primer. Three DNA samples were analyzed: 1) laser-captured renal neoplastic epithelium from a Ksp-Cre(+)/Apc580S/580S mouse; 2) tail DNA from the same mouse, and 3) tail DNA from a mouse homozygous for the wild-type Apc allele. The PCR conditions were the following: 94 °C/2:00 (94 °C/15 s; 55 °C/30 s; 68 °C/2:30) (35×). PCR products were analyzed on a 1% agarose ethidium bromide gel.
      Blood Chemistry—The blood samples from twelve 1- to 2-day-old neonates in two litters were collected. Among them, there were four Apc580S/580S neonates, five Ksp-Cre/Apc580S/+ neonates, and three Ksp-Cre/Apc580S/580S neonates. The blood samples from four adult litters at the ages of 5–12 weeks old with each of the three different genotypes were also collected. The plasma of the mice was separated by centrifuging the heparinized blood at 3,500 × g for 15 min. For quantitative determination of BUN, the VetScan Chemistry Analyzer (Abaxis, Inc., Union City, CA) was used. The plasma of neonates was diluted 1:5 with saline prior to the measurement. The plasma of adult mice was diluted 1:3.
      Statistics—Mann-Whitney nonparametric test was applied for the comparisons of the BUN levels. A one-tailed p value <0.05 was considered statistically significant.

      RESULTS

      Ksp-cre/Apc580S/580S mice develop severe cystic kidney disease associated with kidney neoplasia. The expression of Cre recombinase in Apc580S mice creates an allele that encodes a protein that prematurely truncates the Apc protein at codon 580, creating a functionally null allele (
      • Shibata H.
      • Toyama K.
      • Shioya H.
      • Ito M.
      • Hirota M.
      • Hasegawa S.
      • Matsumoto H.
      • Takano H.
      • Akiyama T.
      • Toyoshima K.
      • Kanamaru R.
      • Kanegae Y.
      • Saito I.
      • Nakamura Y.
      • Shiba K.
      • Noda T.
      ). Mice expressing cre under the control of the kidney-specific cadherin (Ksp-Cre) promoter were described previously (
      • Shao X.
      • Somlo S.
      • Igarashi P.
      ). These mice were crossed to mice carrying the floxed allele of Apc (Apc580S/580S)to generate Ksp-cre/Apc580S/+ mice, which were subsequently crossed to Apc580S/580S mice. These matings would be predicted to generate litters in which one-fourth of the mice have the Ksp-Cre;Apc580S/580S genotype. Upon routine monitoring of these litters, we found that several neonates became moribund, necessitating sacrifice. PCR analysis identified these animals as the Ksp-Cre;Apc580S/580S genotype. Analysis of blood urea nitrogen (BUN) levels in these mice showed significant elevation in the neonatal period (Fig. 1). Histological analysis of kidneys from these neonates revealed the presence of multiple cysts (Fig. 1), consistent with the idea that kidney dysfunction leads to the neonatal death.
      Figure thumbnail gr1
      Fig. 1Ksp-Cre/Apc580S/580S mice display evidence of kidney dysfunction and develop renal cysts as neonates. A, H&E-stained kidney tissues of 2-day-old control littermate (Apc580S/580S) mouse. B, H&E stained 2-day-old neonate kidney tissue of a Ksp-Cre/Apc580S/580S mouse, showing the polycystic renal abnormality involving both nephrogenic zone (N) and renal cortex (C). C, blood urea nitrogen (BUN) levels are elevated in Ksp-Cre/Apc580S/580S mice relative to control littermates. BUN levels in Ksp-Cre/Apc580S/580S neonates are significantly higher than that of Apc580S/580S neonate (p = 0.0285), and that of Ksp-Cre/Apc580S/+ neonates (p = 0.018). There was no significant difference between Apc580S/580S and Ksp-Cre/Apc580S/+ neonates (p = 0.2065). The BUN level in the rare Ksp-Cre/Apc580S/580S mice that survived to adulthood was significantly higher than that of Apc580S/580S adults (p = 0.007), and Ksp-Cre/Apc580S/+ adults (p = 0.016). There was no significant difference between Apc580S/580S and Ksp-Cre/Apc580S/+ adults (p = 0.221). Columns, means; bars, ± S.D.
      Most Ksp-Cre/Apc580S/580S mice die within 2–3 days of birth. We genotyped 540 weanling mice (3 weeks of age) resulting from crosses between Ksp-Cre/Apc580S/+ and Apc580S/580S mice. If there were no neonatal lethality associated with loss of Apc in the kidney, one would predict that one-fourth of the resulting offspring present at weaning would be Ksp-Cre/Apc580S/580S. Consistent with the observed neonatal lethality, only 6 of the 540 mice surviving until weaning were of the Ksp-Cre/Apc580S/580S genotype. These animals were noticeably smaller than their littermates (3–4 grams less in weight). Plasma BUN levels were elevated in these animals and they became moribund at 8–12 weeks of age (Fig. 1). Necropsy of these animals revealed the presence of severely enlarged kidneys containing multiple cysts (Fig. 2A). Histological examination of the kidneys showed they contained simple cysts lined by a single layer of epithelial cells; glomerular cysts; multilayered cysts lined by a hyperplastic epithelium; and adenomas (Fig. 2, B and C). Examination of other organs revealed no other abnormalities, and no evidence of metastatic disease was found in the adult Ksp-Cre/Apc580S/580S mice.
      Figure thumbnail gr2
      Fig. 2The rare Ksp-Cre/Apc580S/580S mice that survive to adulthood have severe cystic kidney disease. A, gross appearance of a Ksp-Cre/Apc580S/580S mouse showing massive bilateral cystic renal abnormalities. B, renal adenoma found in the 8-week-old Ksp-Cre/Apc580S/580S mice, star indicates central necrosis. C, image showing the spectrum of renal abnormalities observed: simple cysts (S-Cy) lined by a single layer of epithelial cells, glomerular cysts (G-Cy), and multilayered cysts (M-Cy) lined by a hyperplastic epithelium. All six adult Ksp-Cre/Apc580S/580S mice examined displayed severe cystic kidney disease, while at least two animals displayed clear evidence of renal adenoma.
      Confirmation of Apc Loss in the Kidneys of Ksp-Cre/Apc580S/580S Mice—To verify that cre-mediated recombination results in loss of Apc in the kidney, we performed immunohistochemistry (IHC) and PCR-based assays. IHC for Apc protein revealed expression in tubules of control animals (Fig. 3, B and C), and loss of Apc expression in the hyperplastic epithelial lining of cysts (Fig. 3D). Consistent with the incomplete penetrance of cre expression in kidney tubules, mosaic expression of cre is observed in some kidney tubules (Fig. 3D). To verify on the genomic level that Apc was lost in kidney cells, we isolated cells in the hyperplastic cyst walls by laser capture microdissection and performed PCR analysis (Fig. 3F). Deletion of exon 14 in the Apc gene (and thus inactivation of the protein) was confirmed in the hyperplastic epithelial cells (Fig. 3G).
      Figure thumbnail gr3
      Fig. 3Demonstration of loss of Apc in Ksp-cre/Apc580S/580S mice at both the DNA and protein level. A, kidney tissue stained with no primary antibody. B, Apc immunohistochemistry in a control littermate. C, anti-Apc immunostaining in the kidney of Ksp-Cre/Apc580S/+ mouse. D, Ksp-Cre/Apc580S/580S kidney, showing the absence of APC protein in the cyst wall epithelium (arrow) forming the neoplasm. Note the presence of mosaic expression of Apc in some kidney tubules (denoted by a star). E, proliferative epithelia from a Ksp-Cre/Apc580S/580S adult mouse were isolated by using LCM. F, schematic diagram of the PCR strategy used to verify loss of Apc. Three designed primers (P1, P2, and P3) were used for the PCR amplification of exon 14 in the Apc gene in the proliferative renal epithelium isolated as indicated in E. G, PCR amplification confirmed the deletion of exon 14 of the Apc gene in the hyperplastic epithelia of the involved mouse kidney.
      Cytoplasmic Levels of β-Catenin Are Elevated in Kidney Cells of Ksp-Cre/Apc580S/580S Mice—We predicted that loss of Apc would lead to elevated levels of cytoplasmic β-catenin. We performed IHC for β-catenin and found that its level was dramatically elevated in Ksp-Cre/Apc580S/580S kidney tissue (Fig. 4). This included elevated levels in the hyperplastic lining of cysts. Again, we observed a mosaic pattern of overexpression in some kidney tubules, consistent with the mosaicism observed for loss of Apc expression (Figs. 3D and 4, E–G). We also anticipated that cell proliferation would be increased in these cystic kidneys. To test this, we stained kidney tissue for the proliferation marker Ki67 and found a dramatic increase in proliferating cells within cysts of Ksp-Cre/Apc580S/580S mice, consistent with deregulated growth (Fig. 4J).
      Figure thumbnail gr4
      Fig. 4Cytoplasmic levels of β-catenin and Ki-67 immunoreactivity are both increased in the kidneys of 8-week-old Ksp-Cre/ Apc580S/580S mice. A–D, β-catenin immunohistochemistry. A, kidney tissue of control (Apc580S/580S) mouse. B, kidney tissue of control Ksp-Cre/Apc580S/+ mouse. C, kidney tissue of Ksp-Cre/Apc580S/580S mouse in lower magnification, showing the nuclear accumulation of β-catenin in the cyst wall epithelium (arrow), which is unusually proliferative (star). D, higher magnification view of the area within the frame in C, showing the nuclear localization of the accumulated β-catenin. Note the presence of tubules with mosaic expression of β-catenin (*). E, immunofluorescent detection of Apc protein in a kidney tubule from a Ksp-Cre/Apc580S/580S mouse. F, immunofluorescent staining of β-catenin protein in the same kidney tubule shown in E. G, merging of images shown in E and F. Note the inverse correlation between Apc (red) and B-catenin (green) staining. H–J are Ki-67 immunohistochemistry with arrows indicating the cells stained with Ki-67. H, kidney tissue of a control Apc580S/580S mouse showing rarely proliferative cells in the renal tubules (arrow). I, control Ksp-Cre/Apc580S/+ littermate, showing similar histological Ki-67 staining (arrow). J, Ki-67 staining of Ksp-Cre/Apc580S/580S littermate. Note the dramatic increase in proliferative cells in the hyperplastic epithelia lining the renal cyst walls (arrows).
      Tubular Origin of the Simple Cysts—To assess the tubular origins of the simple cysts, sections from neonate (2-day-old) and adult (8-week-old) Ksp-Cre/Apc580S/580S kidneys were stained by antibodies or lectins that label specific nephron segments. The markers included Lotus tetragonolobus agglutinin (LTA), which labels proximal tubules (
      • Shao X.
      • Johnson J.E.
      • Richardson J.A.
      • Hiesberger T.
      • Igarashi P.
      ); Tamm-Horsfall protein (THP), which labels thick ascending limbs of loops of Henle and distal tubules (
      • Shao X.
      • Somlo S.
      • Igarashi P.
      ); Na-K-Cl cotransporter type 2 (NKCC2), which labels thick ascending limbs of loops of Henle (
      • Takahashi N.
      • Chernavvsky D.R.
      • Gomez R.A.
      • Igarashi P.
      • Gitelman H.J.
      • Smithies O.
      ); Na-Cl cotransporter (NCC), which labels distal convoluted tubules (
      • Wyse B.
      • Ali N.
      • Ellison D.H.
      ); and Dolichos biflorus agglutinin (DBA), which labels collecting ducts (
      • Zolotnitskaya A.
      • Satlin L.M.
      ). This analysis showed that cysts were derived from Bowman's capsule, proximal tubules, thick ascending limbs, distal convoluted tubules, and collecting ducts (Fig. 5). These findings are consistent with the known expression of Cre in Ksp-Cre mice in all tubular epithelial cells extending from Bowman's capsule to collecting ducts (
      • Shao X.
      • Somlo S.
      • Igarashi P.
      ). Importantly, NKCC2 and NCC, which are normally located in the apical membrane of renal tubular cells (Fig. 5D), were also located in the apical membrane of cyst epithelial cells (Fig. 5, C and E). These results suggested that a major defect in epithelial polarity was not present in kidneys containing Apc-deficient cells.
      Figure thumbnail gr5
      Fig. 5Origins of renal cysts (cy) in APC knock-out mice. Antibody or lectin staining (A–F) of kidneys from 2-day-old and 8-week-old knock-out mice. A, staining with FITC-coupled Lotus tetragonolobus agglutinin (LTA, green), which labels proximal tubules. B, staining with antibody to Tamm-Horsfall protein (green), which labels thick ascending limbs of loops of Henle and distal convoluted tubules. C, staining with antibody to the Na-K-Cl cotransporter NKCC2 (green), which labels thick ascending limbs of loops of Henle. D, expression of NKCC2 in wild-type loops of Henle (tal). E, staining with antibody to the Na-Cl cotransporter NCC (green), which labels distal convoluted tubules. F, staining with TRITC-coupled DBA (red), which labels collecting ducts. Nuclei were counterstained with DAPI (blue).
      To assess the origins of the cysts, adjacent sections from neonate and adult Ksp-Cre/Apc580S/580S kidneys were co-stained with LTA, THP, NKCC2, and DBA. The percentages of simple cysts that were labeled with each marker were then measured. Only those cysts that could be unequivocally identified in adjacent sections were scored. At 2 days, 43% of cysts were labeled with LTA, indicating proximal tubule origin; 19% expressed THP and/or NKCC2, indicating thick ascending limb of loop of Henle origin; and 34% were labeled with DBA, indicating collecting duct origin. 5% of the cysts were not labeled with any of the markers tested. At 8 weeks of age, 7% of cysts were labeled with LTA, 19% expressed THP and/or NKCC2, 9% were labeled with DBA, and 65% were not labeled with any of the markers. The larger number of cysts that did not express any markers at 8 weeks of age compared with 2 days of age suggests that the cyst epithelial cells were progressively de-differentiated. Simple cysts, multilayered cysts, and adenomas all expressed E-cadherin in the plasma membrane at sites of cell-cell contact, indicating that they were epithelial in origin (Fig. 6). Antibody staining showed that simple cysts derived from collecting ducts (evidenced by labeling with DBA) expressed aquaporin-2 (Fig. 6C) and aquaporin-3 (Fig. 6D). Moreover, similar to what is found in normal collecting ducts, aquaporin-2 was localized exclusively in the apical membrane, whereas aquaporin-3 was localized exclusively in the basolateral membrane, further indicating the absence of a defect in epithelial cell polarity. In contrast, the large multilayered cysts and adenomas observed in the older kidneys were labeled with DBA, indicating that they originated from collecting ducts. However, the cells comprising these structures did not express aquaporin-2 or aquaporin-3, indicating that they were relatively undifferentiated.
      Figure thumbnail gr6
      Fig. 6Abnormalities of tubular differentiation. Simple cyst (A–D), multilayered cyst (E–H), and adenoma (I–L) in kidneys from 8-week-old knock-out mice. A, E, I, staining with H&E. B, F, and J, staining with antibody to E-cadherin (red). Arrowheads indicate localization in the basolateral membrane. C, G, and K, co-staining with antibodies to aquaporin-2 (green) and TRITC-coupled DBA (red). Arrowheads indicate localization of aquaporin-2 in the apical membrane of cystic collecting ducts and absence of expression in collecting duct-derived multilayered cysts and adenomas. D, H, and L, co-staining with antibodies to aquaporin-3 (green) and TRITC-coupled DBA (red). Arrowheads indicate localization of aquaporin-3 in the basolateral membrane of cystic collecting ducts and absence of expression in collecting duct-derived multilayered cysts and adenomas. Nuclei were counterstained with DAPI (blue). Cy, cyst; ad, adenoma.
      Expression of Polycystin-2, Polycystin-1, Hepatocyte Nuclear Factor-1β, and the Presence of Cilia—Given the potential relationship between Wnt signaling and polycystins (
      • Wilson P.D.
      ,
      • Kim E.
      • Arnould T.
      • Sellin L.K.
      • Benzing T.
      • Fan M.J.
      • Gruning W.
      • Sokol S.Y.
      • Drummond I.
      • Walz G.
      ), we evaluated the expression of these proteins in Ksp-Cre/Apc580S/580S mice. Polycystin-2 was highly expressed in many of the cysts (Fig. 7, A and B), including both simple cysts and multilayered cysts. Higher magnification images showed that polycystin-2 was localized in the basal cytoplasm, similar to what has been observed in wild-type renal tubules (Fig. 7, C and D). Polycystin-1 was expressed in renal tubules but was absent from glomeruli and blood vessels in control mice, consistent with previous reports (
      • Ward C.J.
      • Turley H.
      • Ong A.C.
      • Comley M.
      • Biddolph S.
      • Chetty R.
      • Ratcliffe P.J.
      • Gattner K.
      • Harris P.C.
      ). The staining was blocked by preincubation of the antibody with the peptide immunogen, indicating that the staining was specific (Fig. 7F). Polycystin-1 was expressed in the multilayered cysts of Ksp-Cre/Apc580S/580S mice, and the level of expression was comparable to an adjacent non-cystic tubule. Fig. 7H shows peptide blocking photographed under identical exposure conditions.
      Figure thumbnail gr7
      Fig. 7Expression of polycystin-2, polycystin-1, HNF-1β, and primary cilia in APC knock-out mice. A–D, staining with an antibody to polycystin-2 (red) in kidneys from 8-week-old knock-out mice. Arrowheads indicate localization in the basal cytoplasm of cyst epithelial cells. E–H, staining with an antibody to polycystin-1 (red) in kidneys from 8-week-old wild-type mice (E and F) and knock-out mice (G and H) showing expression in renal tubules (tu) and multilayered cysts (cy). No expression is observed in glomeruli (gl). F and H, preincubation with the peptide immunogen blocks staining of tubules and cysts but does not block the nonspecific fluorescence of red blood cells (rc). I–L, staining with an antibody to HNF-1β (red) in kidneys from 8-week-old knock-out mice (I, J, and L) and wild-type littermates. K, arrowheads indicate expression in the nuclei of renal tubules (tu) and cysts (cy). The space between the cyst epithelium and tubules in L is an artifact. M–P, primary cilia in knock-out mice (M, N, P) and wild-type littermates (O) stained with an antibody to acetylated tubulin (red). Cilia (arrowheads) were present on cells lining simple cysts (M and N) in 2-day-old knock-out mice and renal tubules (tu) in a wild-type littermate (O). Cilia were absent in multilayered cysts of 8-week-old knock-out mice (P). Nuclei were counterstained with DAPI (blue).
      Next, we examined the expression of hepatocyte nuclear factor (HNF)-1β in the kidneys of Ksp-Cre/Apc580S/580S mice. Mutations of HNF-1β have been shown to produce glomerular cysts and multilayered hyperplastic cysts (
      • Bingham C.
      • Bulman M.P.
      • Ellard S.
      • Allen L.I.
      • Lipkin G.W.
      • Hoff W.G.
      • Woolf A.S.
      • Rizzoni G.
      • Novelli G.
      • Nicholls A.J.
      • Hattersley A.T.
      ,
      • Mache C.J.
      • Preisegger K.H.
      • Kopp S.
      • Ratschek M.
      • Ring E.
      ) similar to those seen in the Ksp-Cre/Apc580S/580S mice. Fig. 7I demonstrates HNF-1β expression in a Ksp-Cre/Apc580S/580S kidney. Expression of HNF-1β was seen in the nuclei of cells lining the simple cysts (Fig. 7J), and is also seen in the nuclei of tubules from a wild-type kidney (Fig. 7K). HNF-1β was also expressed in the nuclei of the multilayered cysts (Fig. 7L) at a comparable level to that seen in adjacent non-cystic tubules.
      Defects in cilia regulation or function are associated with cystic kidney disease (
      • Ong A.C.
      • Wheatley D.N.
      ). To gain more insight into the nature of the cysts, cilia were stained with an antibody to acetylated tubulin (Fig. 7, M–P). Cilia were present on the apical surface of tubules from control mice and in cysts from 2-day-old Ksp-Cre/Apc580S/580S kidneys. In the 8-week-old Ksp-Cre/Apc580S/580S kidneys, fewer cilia were present, but they could be clearly seen on the surface of some cyst epithelial cells. However, the large multilayered cysts completely lacked cilia. Taken together, these results suggest that the development of renal cysts and neoplasms in Ksp-Cre/Apc580S/580S mice was not caused by the loss of polycystin-1, polycystin-2, or HNF-1β.

      DISCUSSION

      In this study, we shown that kidney-specific inactivation of the Apc gene (Ksp-Cre/Apc580S/580S mice) leads to the development of early-onset cystic disease associated with perinatal lethality. Rare surviving animals develop large, multilayered cysts with high levels of proliferative cells. These hyperplastic regions also contained areas of pleiotropy and focal necrosis, consistent with a diagnosis of renal adenoma or renal carcinoma in situ. However, we did not see evidence for invasion of these hyperplastic cells into regions of more normal kidney, and no evidence of metastatic progression to other organs was seen. These results are consistent with previous reports (
      • Saadi-Kheddouci S.
      • Berrebi D.
      • Romagnolo B.
      • Cluzeaud F.
      • Peuchmaur M.
      • Kahn A.
      • Vandewalle A.
      • Perret C.
      ), where expression of a mutant, proteolysis-resistant form of β-catenin led to cystic renal disease. This work extends this observation to show that inactivation of Apc (and associated up-regulation of endogenous β-catenin) leads to early-onset renal failure in most animals. In addition, these studies reveal the presence of multilayered cysts lined with hyperplastic cells with characteristics of neoplasia. Thus, dysregulation of the canonical Wnt signaling pathway leads to developmental abnormalities in the kidney that can predispose to the subsequent development of renal cancer.
      The cysts in Ksp-Cre/Apc580S/580S kidneys were derived from multiple cell types in the kidney including Bowman's capsule, proximal tubules, thick ascending limbs, distal convoluted tubules, and collecting ducts. These abnormalities are consistent with the expression pattern of Ksp-Cre transgene (
      • Shao X.
      • Somlo S.
      • Igarashi P.
      ). A predisposition to polycystic kidney disease can be inherited in an autosomal dominant or, less commonly, in a recessive pattern (
      • Wilson P.D.
      ). Autosomal dominant polycystic kidney disease (ADPKD) occurs in about 1 of every 1000 individuals and is characterized by bilateral renal cyst formation (). Mutations in the PKD1 gene account for ∼85% of ADPKD. A majority of the remaining cases (10%) are associated with mutations in the PKD2 gene. Polycystin-1 (PC-1), the protein expressed from the PKD1 locus, encodes a large (462 kDa) 11-pass transmembrane protein (). There is evidence suggesting that β-catenin/TCF complexes can regulate the PKD1 promoter (
      • Rodova M.
      • Islam M.R.
      • Maser R.L.
      • Calvet J.P.
      ) and that dysregulation of β-catenin signaling is observed in polycystic kidney disease (
      • Lin F.
      • Hiesberger T.
      • Cordes K.
      • Sinclair A.M.
      • Goldstein L.S.
      • Somlo S.
      • Igarashi P.
      ,
      • Muto S.
      • Aiba A.
      • Saito Y.
      • Nakao K.
      • Nakamura K.
      • Tomita K.
      • Kitamura T.
      • Kurabayashi M.
      • Nagai R.
      • Higashihara E.
      • Harris P.C.
      • Katsuki M.
      • Horie S.
      ). In addition, polycystin-1 can physically interact with β-catenin (
      • Huan Y.
      • van Adelsberg J.
      ). We assessed whether the regulation of polycystin-1 or other proteins associated with cystic renal disease were altered in Ksp-Cre/Apc580S/580S mice. Surprisingly, the absence of Apc in our animal model did not result in the loss of polycystin-1, polycystin-2, HNF-1β, or cilia in renal tubular epithelia. This implies that the formation of the cysts in the present model was due to a novel but unknown mechanism.
      Germline mutations in the APC gene are found in the majority of familial adenomatous polypopsis (FAP) patients (
      • Kinzler K.W.
      • Vogelstein B.
      ). In animal models, inactivation of APC in colorectal epithelium results in rapid formation of adenomas (
      • Shibata H.
      • Toyama K.
      • Shioya H.
      • Ito M.
      • Hirota M.
      • Hasegawa S.
      • Matsumoto H.
      • Takano H.
      • Akiyama T.
      • Toyoshima K.
      • Kanamaru R.
      • Kanegae Y.
      • Saito I.
      • Nakamura Y.
      • Shiba K.
      • Noda T.
      ). In addition, there are a number of hyperplastic and neoplastic diseases associated with the mutation of the APC gene in FAP patients, including the congenital hypertrophy of the retinal pigment epithelium, periampullary carcinoma, Gardner's syndrome with epidermoid skin cysts and benign osteoid tumors of the mandible and long bones, benign fibromatosis, hepatoblastoma, papillary carcinoma of the thyroid, adrenocortical tumors, and Turcot syndrome with multiple colorectal polyps and cerebellar medulloblastoma (
      • Fearnhead N.S.
      • Britton M.P.
      • Bodmer W.F.
      ). Moreover, APC mutations have been found to be associated with other types of neoplastic diseases, including pancreatic acinar cell carcinomas (
      • Abraham S.C.
      • Wu T.T.
      • Hruban R.H.
      • Lee J.H.
      • Yeo C.J.
      • Conlon K.
      • Brennan M.
      • Cameron J.L.
      • Klimstra D.S.
      ), fibromatoses of the breast (
      • Abraham S.C.
      • Reynolds C.
      • Lee J.H.
      • Montgomery E.A.
      • Baisden B.L.
      • Krasinskas A.M.
      • Wu T.T.
      ), and oral squamous cell carcinomas (
      • Kok S.H.
      • Lee J.J.
      • Hsu H.C.
      • Chiang C.P.
      • Kuo Y.S.
      • Kuo M.Y.
      ). Promoter methylation and/or LOH of APC were also found in breast cancer (
      • Sarrio D.
      • Moreno-Bueno G.
      • Hardisson D.
      • Sanchez-Estevez C.
      • Guo M.
      • Herman J.G.
      • Gamallo C.
      • Esteller M.
      • Palacios J.
      ,
      • Virmani A.K.
      • Rathi A.
      • Sathyanarayana U.G.
      • Padar A.
      • Huang C.X.
      • Cunnigham H.T.
      • Farinas A.J.
      • Milchgrub S.
      • Euhus D.M.
      • Gilcrease M.
      • Herman J.
      • Minna J.D.
      • Gazdar A.F.
      ), lung cancer (
      • Virmani A.K.
      • Rathi A.
      • Sathyanarayana U.G.
      • Padar A.
      • Huang C.X.
      • Cunnigham H.T.
      • Farinas A.J.
      • Milchgrub S.
      • Euhus D.M.
      • Gilcrease M.
      • Herman J.
      • Minna J.D.
      • Gazdar A.F.
      ,
      • Usadel H.
      • Brabender J.
      • Danenberg K.D.
      • Jeronimo C.
      • Harden S.
      • Engles J.
      • Danenberg P.V.
      • Yang S.
      • Sidransky D.
      ), and kidney cancer (
      • Battagli C.
      • Uzzo R.G.
      • Ibanez de Dulaimi E.
      • Caceres I.
      • Krassenstein R.
      • Al-Saleem T.
      • Greenberg R.E.
      • Cairns P.
      ). The evidence presented here confirms that loss of Apc and associated stabilization of the β-catenin protein can lead to renal tumor development.

      Acknowledgments

      We thank Jason Martin, Bryn Eagleson, and the rest of the VARI vivarium staff for assistance in animal husbandry; Pam Swiatek and the VARI Mouse Germline Modification Core Facility for assistance; and Bree Berguis, Eric Hudson, Jim Resau, and the rest of the VARI Histology Core Facility for support. We also thank Sheri Holmen for constructive discussion on the project and Sunshine Kucholtz and Cassandra Zylstra for technical support. We thank Rod Bronson and Robert Sigler for advice on histology. We thank Mark Knepper, Steven Somlo, John Hoyer, and Marco Pontoglio for antibodies. We thank David Nadziejka for critically reading this manuscript. We also thank Tetsvo Noda for Apc580S ES cells.

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