β-Catenin Overexpression in the Metanephric Mesenchyme Leads to Renal Dysplasia Genesis via Cell-Autonomous and Non–Cell-Autonomous Mechanisms
2014; Elsevier BV; Volume: 184; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2014.01.018
ISSN1525-2191
AutoresSanjay Sarin, Felix Boivin, Aihua Li, Janice Lim, Bruno Svajger, Norman D. Rosenblum, Darren Bridgewater,
Tópico(s)Renal cell carcinoma treatment
ResumoRenal dysplasia, a developmental disorder characterized by defective ureteric branching morphogenesis and nephrogenesis, ranks as one of the major causes of renal failure among the pediatric population. Herein, we demonstrate that the levels of activated β-catenin are elevated in the nuclei of ureteric, stromal, and mesenchymal cells within dysplastic human kidney tissue. By using a conditional mouse model of mesenchymal β-catenin overexpression, we identify two novel signaling pathways mediated by β-catenin in the development of renal dysplasia. First, the overexpression of β-catenin within the metanephric mesenchyme leads to ectopic and disorganized branching morphogenesis caused by β-catenin directly binding Tcf/lef consensus binding sites in the Gdnf promoter and up-regulating Gdnf transcription. Second, β-catenin overexpression in the metanephric mesenchyme leads to elevated levels of transcriptionally active β-catenin in the ureteric epithelium. Interestingly, this increase of β-catenin–mediated transcription results from a novel Ret/β-catenin signaling pathway. Consistent with these findings, analysis of human dysplastic renal tissue demonstrates that undifferentiated mesenchymal cells expressing high levels of β-catenin also express increased GDNF. Furthermore, dysplastic ureteric tubules that were surrounded by high levels of GDNF also exhibited increased levels of activated β-catenin. Together, these data support a model in which the elevation of β-catenin in the metanephric mesenchyme results in cell-autonomous and non–cell-autonomous events that lead to the genesis of renal dysplasia. Renal dysplasia, a developmental disorder characterized by defective ureteric branching morphogenesis and nephrogenesis, ranks as one of the major causes of renal failure among the pediatric population. Herein, we demonstrate that the levels of activated β-catenin are elevated in the nuclei of ureteric, stromal, and mesenchymal cells within dysplastic human kidney tissue. By using a conditional mouse model of mesenchymal β-catenin overexpression, we identify two novel signaling pathways mediated by β-catenin in the development of renal dysplasia. First, the overexpression of β-catenin within the metanephric mesenchyme leads to ectopic and disorganized branching morphogenesis caused by β-catenin directly binding Tcf/lef consensus binding sites in the Gdnf promoter and up-regulating Gdnf transcription. Second, β-catenin overexpression in the metanephric mesenchyme leads to elevated levels of transcriptionally active β-catenin in the ureteric epithelium. Interestingly, this increase of β-catenin–mediated transcription results from a novel Ret/β-catenin signaling pathway. Consistent with these findings, analysis of human dysplastic renal tissue demonstrates that undifferentiated mesenchymal cells expressing high levels of β-catenin also express increased GDNF. Furthermore, dysplastic ureteric tubules that were surrounded by high levels of GDNF also exhibited increased levels of activated β-catenin. Together, these data support a model in which the elevation of β-catenin in the metanephric mesenchyme results in cell-autonomous and non–cell-autonomous events that lead to the genesis of renal dysplasia. Human renal dysplasia is a severe form of congenital kidney malformation that affects up to 1 in 1000 of the general population1Winyard P. Chitty L.S. Dysplastic kidneys.Semin Fetal Neonatal Med. 2008; 13: 142-151Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar and is one of the leading causes of childhood end-stage kidney disease.2Neu A.M. Ho P.L. McDonald R.A. 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Springer Berlin Heidelberg, Berlin, Germany2009: 107-120Google Scholar Defects in ureteric epithelial branch patterning and subsequent collecting duct differentiation and nephron induction are major contributing factors that lead to the gross and histopathological changes observed in renal dysplasia; however, the molecular mechanisms are not well understood.4Woolf A.S. Price K.L. Scambler P.J. Winyard P.J. Evolving concepts in human renal dysplasia.J Am Soc Nephrol. 2004; 15: 998-1007Crossref PubMed Scopus (142) Google Scholar, 7Rosenblum ND, Waters AM: Overview of congenital anomalies of kidney and urinary tract (CAKUT). TK Mattoo, LS Baskin, MS Kim (Eds). In UpToDate [Internet]. 2014. Available at: http://www.uptodate.com/contents/overview-of-congenital-anomalies-of-the-kidney-and-urinary-tract-cakut, last updated January 21, 2014.Google Scholar Ureteric branching begins with the emergence of the ureteric bud, an outgrowth off the caudal Wolffian duct, in response to signals originating in the metanephric mesenchyme. The ureteric bud migrates into the adjacent metanephric mesenchyme and undergoes a process of growth, elongation, and branching to form the collecting duct system of the kidney. In a reciprocal manner, the tips of the ureteric epithelium induce a subset of mesenchyme to undergo nephrogenesis, which will form the functional units of the kidney, termed nephrons.8Saxen L. Sariola H. Early organogenesis of the kidney.Pediatr Nephrol. 1987; 1: 385-392Crossref PubMed Scopus (299) Google Scholar Central to growth of the ureteric epithelium is the GDNF/RET signaling pathway. The secreted growth factor GDNF is expressed in the metanephric mesenchyme, whereas its cell surface coreceptors RET and Gdnf family receptor α1 are co-expressed in the Wolffian duct, ureteric bud, and tips of its daughter branches.9Costantini F. Shakya R. GDNF/Ret signaling and the development of the kidney.Bioessays. 2006; 28: 117-127Crossref PubMed Scopus (242) Google Scholar, 10Bridgewater D. Rosenblum N.D. Stimulatory and inhibitory signaling molecules that regulate renal branching morphogenesis.Pediatr Nephrol. 2009; 24: 1611-1619Crossref PubMed Scopus (26) Google Scholar Binding of GDNF to its coreceptors results in the stimulation of the RET receptor and activation of numerous downstream intracellular signaling pathways, including the RAS/extracellular signal–regulated kinase mitogen-activated protein kinase, phosphatidylinositol-3-kinase, and phospholipase C-γ pathways. Together, these signaling pathways mediated by RET activation lead to changes in gene expression that promote proliferation, cell rearrangements, changes in cell shape, and cell movements leading to the outgrowth of the ureteric bud and branching morphogenesis.11Takahashi M. The GDNF/RET signaling pathway and human diseases.Cytokine Growth Factor Rev. 2001; 12: 361-373Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 12Fisher C.E. Michael L. Barnett M.W. Davies J.A. Erk MAP kinase regulates branching morphogenesis in the developing mouse kidney.Development. 2001; 128: 4329-4338Crossref PubMed Google Scholar, 13Tang M.J. Cai Y. Tsai S.J. Wang Y.K. Dressler G.R. Ureteric bud outgrowth in response to RET activation is mediated by phosphatidylinositol 3-kinase.Dev Biol. 2002; 243: 128-136Crossref PubMed Scopus (118) Google Scholar Genetic deletion of Gdnf, Ret, or Gfrα1 results in renal aplasia or hypodysplasia due to a failure in ureteric budding. Despite the critical contribution of this signaling system to kidney development, the factors regulating expression of these genes are not completely understood.14Pichel J.G. Shen L. Sheng H.Z. Granholm A.C. Drago J. Grinberg A. Lee E.J. Huang S.P. Saarma M. Hoffer B.J. Sariola H. Westphal H. Defects in enteric innervation and kidney development in mice lacking GDNF.Nature. 1996; 382: 73-76Crossref PubMed Scopus (1003) Google Scholar, 15Schuchardt A. D'Agati V. Larsson-Blomberg L. Costantini F. Pachnis V. Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret.Nature. 1994; 367: 380-383Crossref PubMed Scopus (1416) Google Scholar Kidney formation is also regulated by members of the Wnt family of secreted glycoproteins.16Carroll T.J. Park J.S. Hayashi S. Majumdar A. McMahon A.P. 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Wnt/beta-catenin signaling: components, mechanisms, and diseases.Dev Cell. 2009; 17: 9-26Abstract Full Text Full Text PDF PubMed Scopus (4151) Google Scholar β-Catenin is a multifunctional protein involved in cell-cell adhesion by associating with E-cadherin and α-catenin and linking the actin cytoskeleton with adherens junctions, whereas in the nucleus, it acts as a transcriptional activator with Tcf/Lef family members. During kidney development, both nephrogenic induction and ureteric morphogenesis require β-catenin signaling. Loss of β-catenin from nephron progenitors inhibits nephron formation due to reduced expression of key genes required in nephrogenesis, including Fgf8, Pax8, Wnt4, and Lhx1.19Park J.S. Valerius M.T. McMahon A.P. Wnt/beta-catenin signaling regulates nephron induction during mouse kidney development.Development. 2007; 134: 2533-2539Crossref PubMed Scopus (258) Google Scholar In the ureteric epithelium, homozygous β-catenin deficiency results in premature differentiation of the collecting system, and a deficiency in the expression of Emx2, Lim1, Pax2, Ret, and Wnt11, genes required for branching morphogenesis.22Bridgewater D. Cox B. Cain J. Lau A. Athaide V. Gill P.S. Kuure S. Sainio K. Rosenblum N.D. Canonical WNT/beta-catenin signaling is required for ureteric branching.Dev Biol. 2008; 317: 83-94Crossref PubMed Scopus (114) Google Scholar, 23Marose T.D. Merkel C.E. McMahon A.P. Carroll T.J. Beta-catenin is necessary to keep cells of ureteric bud/Wolffian duct epithelium in a precursor state.Dev Biol. 2008; 314: 112-126Crossref PubMed Scopus (120) Google Scholar, 24Grote D. Boualia S.K. Souabni A. Merkel C. Chi X. Costantini F. Carroll T. Bouchard M. Gata3 acts downstream of beta-catenin signaling to prevent ectopic metanephric kidney induction.PLoS Genet. 2008; 4: e1000316Crossref PubMed Scopus (110) Google Scholar We have previously shown that β-catenin is overexpressed in the ureteric epithelium in human dysplastic renal tissue and in distinct genetic mouse models of renal dysplasia.3Hu M.C. Piscione T.D. Rosenblum N.D. Elevated SMAD1/beta-catenin molecular complexes and renal medullary cystic dysplasia in ALK3 transgenic mice.Development. 2003; 130: 2753-2766Crossref PubMed Scopus (63) Google Scholar, 19Park J.S. Valerius M.T. McMahon A.P. Wnt/beta-catenin signaling regulates nephron induction during mouse kidney development.Development. 2007; 134: 2533-2539Crossref PubMed Scopus (258) Google Scholar, 25Bridgewater D. Di Giovanni V. Cain J.E. Cox B. Jakobson M. Sainio K. Rosenblum N.D. beta-Catenin causes renal dysplasia via upregulation of Tgfbeta2 and Dkk1.J Am Soc Nephrol. 2011; 22: 718-731Crossref PubMed Scopus (32) Google Scholar Generation of a mouse model in which β-catenin levels were elevated exclusively in the ureteric epithelium during kidney development resulted in a block in ureteric differentiation and up-regulation of Tgfβ2 and Dkk1, resulting in the genesis of renal dysplasia.25Bridgewater D. Di Giovanni V. Cain J.E. Cox B. Jakobson M. Sainio K. Rosenblum N.D. beta-Catenin causes renal dysplasia via upregulation of Tgfbeta2 and Dkk1.J Am Soc Nephrol. 2011; 22: 718-731Crossref PubMed Scopus (32) Google Scholar However, it is not clear if a functional role for β-catenin in the genesis of renal dysplasia exists in other cells of the kidney. Herein, we demonstrate that the transcriptionally active form of β-catenin localizes to the nucleus of ureteric epithelium, stroma, and mesenchyme, in dysplastic human renal tissue. To understand the pathological significance of β-catenin overexpression in renal dysplasia, we generated mutant mice in which β-catenin is overexpressed in the kidney mesenchyme and demonstrated histopathological changes remarkably consistent with human renal dysplasia. The histopathological changes observed in our mutant mice resulted from the activation of two novel genetic pathways, both mediated by β-catenin. In the metanephric mesenchyme, the overexpression of β-catenin directly regulates Gdnf expression and also activates a novel Ret/β-catenin–mediated signaling pathway within the ureteric epithelium. Together, these novel pathways resulted in the genesis of renal dysplasia through ectopic and disorganized ureteric branching. Rarβ2-Cre (Cre expression limited to the metanephric mesenchyme) (Supplemental Figure S1) male mice were crossed with female mice containing LoxP sites flanking exon 3 of the β-catenin allele (β-catΔ3/Δ3)26Harada N. Miyoshi H. Murai N. Oshima H. Tamai Y. Oshima M. Taketo M.M. Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of beta-catenin.Cancer Res. 2002; 62: 1971-1977PubMed Google Scholar to generate β-catenin gain-of-function mutant progeny, termed β-catGOF-MM. The Cre transgene was detected in mouse tail DNA using primers 5′-GCGGCATGGTGCAAGTTGAAT-3′ and 5′-CGTTCACCGGCATCAACGTTT-3′. Tcf–β-galactosidase reporter mice27Cheon S.S. Cheah A.Y. Turley S. Nadesan P. Poon R. Clevers H. Alman B.A. beta-Catenin stabilization dysregulates mesenchymal cell proliferation, motility, and invasiveness and causes aggressive fibromatosis and hyperplastic cutaneous wounds.Proc Natl Acad Sci U S A. 2002; 99: 6973-6978Crossref PubMed Scopus (272) Google Scholar were crossed with β-catΔ3/Δ3 to generate female Tcf/LacZ;β-catΔ3/+mice. Female Tcf/LacZ;β-catΔ3/+ mice were then crossed with male Rarβ2-Cre mice to generate TcF/LacZ;β-catGOF-MM mice and used to visualize β-catenin transcriptional activity in the ureteric epithelium.22Bridgewater D. Cox B. Cain J. Lau A. Athaide V. Gill P.S. Kuure S. Sainio K. Rosenblum N.D. Canonical WNT/beta-catenin signaling is required for ureteric branching.Dev Biol. 2008; 317: 83-94Crossref PubMed Scopus (114) Google Scholar, 25Bridgewater D. Di Giovanni V. Cain J.E. Cox B. Jakobson M. Sainio K. Rosenblum N.D. beta-Catenin causes renal dysplasia via upregulation of Tgfbeta2 and Dkk1.J Am Soc Nephrol. 2011; 22: 718-731Crossref PubMed Scopus (32) Google Scholar Rarβ2-Cre male mice were crossed with female mice containing LoxP sites flanking exons 2 through 628Brault V. Moore R. Kutsch S. Ishibashi M. Rowitch D.H. McMahon A.P. Sommer L. Boussadia O. Kemler R. Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development.Development. 2001; 128: 1253-1264Crossref PubMed Google Scholar to generate Rarβ2-Cre; β-cat+/− males. These males were then mated to homozygous β-catenin conditional knockout females to generate homozygous β-catenin loss-of-function mutants in the mesenchyme, termed β-catLOF-MM. The Cre transgene was detected in mouse tail DNA using primers 5′-GCGGCATGGTGCAAGTTGAAT-3′ and 5′-CGTTCACCGGCATCAACGTTT-3′. To detect the β-catenin floxed allele, primers 5′-AAGGTAGAGTGATGAAAGTTGTT-3′ and 5′-CACCATGTCCTCTGTCTATTC-3′ were used.28Brault V. Moore R. Kutsch S. Ishibashi M. Rowitch D.H. McMahon A.P. Sommer L. Boussadia O. Kemler R. Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development.Development. 2001; 128: 1253-1264Crossref PubMed Google Scholar Rarβ2-Cre mice were crossed with Gt(ROSA)26Sor(ROSA) mice to analyze the spatial Cre recombinase activity.29Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain.Nat Genet. 1999; 21: 70-71Crossref PubMed Scopus (4153) Google Scholar In all instances, 12 pm on the day of vaginal plug detection was considered to be E0.5. Animal studies were done in accordance with animal care and institutional guidelines. One-year post-natal and 38-week dysplastic human renal tissues were obtained from the McMaster University (Hamilton, ON, Canada) pathology department in accordance with research ethics (Research Ethics Board approval 13-160-T). Whole kidney tissue was fixed in 4% paraformaldehyde for at least 24 hours at 4°C. Kidneys were paraffin embedded, divided into sections (4 μm thick), mounted onto Superfrost Plus slides (Thermo Fisher Scientific, Waltham, MA), and incubated overnight at 37°C. Sections were deparaffinized using xylene washes and rehydrated using graded ethanol washes (100%, 95%, 75%, 50%, H20) and stained with H&E (Sigma, St. Louis, MO). Antigen retrieval was performed for 5 minutes in 10 mmol/L sodium citrate solution (pH 6.0) in a pressure cooker, followed by blocking with 7.5% normal goat serum/4.5% bovine serum albumin. Sections were incubated with primary antibodies to Six2 (Proteintech Group, Chicago, IL; 1:250 dilution), Pax2 (Covance, Montreal, QC, Canada; 1:200 dilution), cytokeratin (Sigma; 1:200 dilution), Wt-1 (SantaCruz, Dallas, TX; 1:200 dilution), or nephrin (Sigma; 1:200 dilution) overnight at 4°C. Tissue sections were washed in PBS (pH 7.4), incubated with secondary antibodies Alexa Fluor 488 or 568 (Invitrogen, Carlsbad, CA; 1:1000 dilution) for 1 hour at room temperature, stained with DAPI (Sigma; 1:1000 dilution) for 5 minutes, and coverslipped using Fluoromount (Sigma). Immunohistochemistry (IHC) was performed using the Vectastain elite avidin-biotin complex kit (Vector Labs, Burlingame, CA). After antigen retrieval, endogenous peroxidase activity was blocked using 3% H2O2 for 10 minutes, followed by blocking endogenous biotin-binding activity using a biotin/avidin blocking kit (Vector Labs), as per company protocol. Incubation with primary antibodies, including activated β-catenin (Millipore, Billerica, MA; 1:100 dilution), Pax2 (Covance; 1:200 dilution), and Gdnf (R&D Systems, Minneapolis, MN; 1:100 dilution), was performed overnight at 4°C. Sections were washed and incubated in biotinylated secondary antibodies (Vector Labs) for 30 minutes at room temperature. Color reaction was visualized using diaminobenzidine (Vector Labs), and slides were coverslipped using Permount (Thermo Fischer Scientific) and imaged on a Nikon 90i-eclipse upright microscope. Kidneys were flattened on 0.4-μm transwell filters (BD, Franklin Lakes, NJ) for 4 hours at 37°C in wells containing Dulbecco's minimal essential medium (DMEM) and stored in 100% methanol at −20°. Kidneys were washed 3× with PBS and blocked in 10% normal goat serum, followed by incubation with primary antibodies to cytokeratin (Sigma; 1:200 dilution) or Pax2 (Covance; 1:200 dilution) at 37°C for 1.5 hours. Kidneys were washed in PBS (pH 7.4), incubated with secondary antibodies Alexa Fluor 488 or 568 (Invitrogen; 1:200 dilution) for 1.5 hours at 37°C, and imaged on a Nikon 90i-eclise inverted microscope. Kidneys were fixed in 0.2% glutaraldehyde, 100 mmol/L EGTA (pH 7.3), 1 mol/L MgCl2, 0.1 mol/L sodium phosphate buffer (pH 7.3), and 1.5% formaldehyde for 5 minutes. Kidneys were washed in 1 mol/L MgCl2, 0.1 mol/L sodium phosphate buffer (pH 7.3), and 0.25% Nonidet P-40, and whole kidney tissue was incubated with X-Gal stain [25 mg/mL X-Gal (Bethesda Research Labs, Burlington, ON, Canada), 0.25 mmol/L potassium ferrocyanide, and 0.25 mmol/L potassium ferricyanide]. Kidneys were washed and fixed in 4% paraformaldehyde and visualized on a Leica EZ4D microscope (Leica, Wetzlar, Germany). In situ hybridization was performed using digoxigenin-labeled chromosomal RNA probes on whole kidney tissue, as previously described. Tissue was hybridized with sense (control) or antisense RNA transcripts for the following genes: Gdnf, Ret, and Wnt11 (gifts from N.D.R.). Kidneys were imaged using a Leica EZ4D microscope. Mean staining intensity and domain were measured using NIS elements software (version AR 3.22 64-bit; Nikon, Melville, NY). E14.5 kidney tissue was dissected and cross-linked in 4% formaldehyde. Cross-linked tissue was homogenized in lysis buffer (1% SDS, 10 mmol/L EDTA, and 50 mmol/L Tris, pH 8.0) and sonicated to generate DNA fragments of 200 to 1000 bp. Soluble chromatin was precleared by protein G agarose/salmon sperm DNA beads (Millipore). Immunoprecipitation was performed using 2 μg of mouse anti–β-catenin antibody (BD), or isotype-specific IgG (negative control) was added to every 100 μL of soluble chromatin to immunoprecipitate β-catenin–binding DNA. Immune complexes were separated with G agarose/salmon sperm DNA beads, followed by a series of washing buffer with protein inhibitors. Cross-linked protein-DNA compound was disassociated by incubation with DNase-free proteinase K, and RNA residual was removed by RNase A. DNA was purified with a Zymo chromatin immunoprecipitation (ChIP) DNA clean and concentrated kit (Zymo Research, Irvine, CA). Three fragments containing the Tcf-binding sites identified in silico using MULAN were examined by PCR. We designed primers to a site within the Gdnf coding region with no consensus β-catenin–binding site as the non-specific internal control site. All primers were designed with Primer 3 software version 0.4.0 (http://bioinfo.ut.ee/primer3-0.4.0, last accessed March 14, 2014). Real-time PCR amplification was performed using the 7900HT fast RT-PCR system (Applied Biosystems, Burlington, ON, Canada). Real-time PCR reaction mix contained 0.5 ng of each pull-down DNA sample, SYBR Green PCR Master Mix (Applied Biosystems), and 300 nmol/L of each primer to a total volume of 25 μL. Primers for the 4.9-, 2.25-, and 2.1-kb β-catenin consensus binding sites were designed using the Primer 3 software version 0.4.0 (http://primer3.sourceforge.net/webif.php, last accessed March 14, 2014) and verified using the University of California, Santa Cruz, genome bioinformatics website (http://genome.ucsc.edu, last accessed January 10, 2011). Primer design was restrictive, the annealing temperature was restricted to 59°C to 60°C, and the length of the PCR product was set between 100 and 200 bp. Specificity of the amplification was performed by agarose gel electrophoresis. Relative levels of pull-down DNA were performed in triplicate using the 2(−ΔΔCT) method.30Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (123392) Google Scholar Individual expression values were normalized by comparison to input DNA. Primer sequences were as follows: 4.9-kb site on 5′ untranslated region (UTR) (forward) 5′-TCTCAGCTGCTGTGCCTATG-3′ and (reverse) 5′-GGCAGGTCAGGAGTAAGCAA-3′; 2.25-kb site on 5′ UTR (forward) 5′-GCAAACCAGCTCTTTCAACA-3′ and (reverse) 5′-AATTGCTGGACTGAACATGGA-3′; 2.3-kb site on 5′ UTR (forward) 5′-GGGTAATGTGTGTGGCAATG-3′ and (reverse) 5′-CTTCCCTGCAAGGTGTTGTT-3′; and GDNF coding region (forward) 5′-ACATGCCTGGCCTACTTTGT-3′ and (reverse) 5′- GACTTGGGTTTGGGCTATGA-3′. A 560-bp MluI/HindIII fragment of the Gdnf promoter, including the Tcf-binding site TTCAAAG, was amplified from human 293 genomic DNA with primers (forward) 5′-ACGCGTTGAGGGGACATTCCAGGCT-3′ and (reverse) 5′-AAGCTTATCTCCCTTGCCAATGTCAGGA-3′. The amplified fragment was cloned into pGLuc-Basic vector from New England BioLabs (Ipswich, MA). TATA box oligonucleotides were annealed and ligated into the BamHI site to generate the pGluc-4.9kb-TATA. All of the inserts were verified by sequencing. Human embryonic kidney (HEK) 293 cells were obtained from ATCC (Manassas, VA) and cultured in DMEM-Hyclone (Thermo Scientific, Waltham, MA) with 4 mmol/L l-glutamine, supplemented with 10% fetal bovine serum (PAA, Etobicoke, ON, Canada) at 37°C in a 5% CO2 water-jacketed incubator. HEK293 cells were seeded into a 24-well plate at 1.0 × 105 per well in 0.5 mL of growth medium without antibiotics. Twenty-four hours later, HEK293 cells were transfected with pGluc-Basic-TATA or pGluc-4.9kb-TATA per Lipofectamine 2000 manufacturer's instructions (Invitrogen). Twenty-four hours after transfection, conditioned media were collected. Cells were then serum starved overnight (16 hours) and subsequently activated by 40 mmol/L lithium chloride (LiCl; Sigma). Media were collected at 0, 6, 8, 10, 12, and 24 hours. Cells were lysed in lysis buffer (150 mmol/L NaCl, 20 mmol/L Tris, and 1% Nonidet P-40, pH 8.0), and lysates were harvested for protein assay. Gaussia luciferase assays were performed with BioLux Gaussia Luciferase Flex Assay Kit from New England BioLabs, as per manufacturer's instructions. Culture medium (20 μL) was added into a 96-well opaque plate, and 50 μL of Gluc substrate working solution was injected and relative light units were measured by an LB luminometer (Thermo Fisher, Waltham). Gaussia luciferase activity was calculated as total relative light units in the medium and standardized by the total protein of the cells in corresponding wells. E14.5 kidneys were resected and grown onto transwell filters at the air-media interface. Kidneys were incubated in DMEM supplemented with 1% fetal bovine serum. Culture media were switched to DMEM supplemented with or without 100 ng/mL of GDNF for 3 hours. Protein was isolated from kidney explants by homogenizing in TENT++ buffer [0.1 mol/L Tris-Cl (pH 8.0), 0.01 mol/L EDTA (pH 8.0), 1 mol/L NaCl, 0.2% Triton X-100, protease inhibitor, and phosphatase inhibitor]. Protein concentration was determined by Bradford protein assay, according to the manufacturer's instructions (Bio-Rad, Hercules, CA). For immunoprecipitations, lysates were incubated with a 1:40 dilution of Ret C-19 or C-20 antibody (Santa Cruz Biotech, Dallas, TX) and immunoprecipitation matrix (Santa Cruz Biotech) at 4°C overnight on a rotator. A sample with no protein lysate was used as a negative control. Immunoprecipitates were pelleted and resuspended in SDS-PAGE sample buffer, and Western blot analysis was performed by incubating for 1 hour at 37°C with primary β-catenin antibody (BD Transduction Laboratories, Mississauga, ON, Canada) and horseradish peroxidase–conjugated secondary antibodies. The reaction was visualized using an enhanced chemiluminescence detection system (Pierce; Thermo Scientific, Rockford, IL). Analysis included determination of mean signal intensity and positive staining area of Gdnf expression in β-catGOF-MM mutants and wild type (WT) using NIS elements software (version AR 3.22 64-bit; Nikon). The data were analyzed using a two-tailed Student's t-test using GraphPad Prism software version 5.0c (Graphpad, La Jolla, CA). P < 0.05 was considered to indicate statistical significance. Values are given as means ± SEM. Studies have demonstrated that β-catenin is overexpressed in the ureteric epithelium in dysplastic kidneys and regulates a genetic program that results in an inhibition of branching morphogenesis, resulting in renal dysplasia.3Hu M.C. Piscione T.D. Rosenblum N.D. Elevated SMAD1/beta-catenin molecular complexes and renal medullary cystic dysplasia in ALK3 transgenic mice.Development. 2003; 130: 2753-2766Crossref PubMed Scopus (63) Google Scholar However, it is not known if β-catenin is overexpressed in other cell types in dysplastic human kid
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