Renal Hypodysplasia Associates with a Wnt4 Variant That Causes Aberrant Canonical Wnt Signaling

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Renal Hypodysplasia Associates with a Wnt4 Variant that Causes Aberrant Canonical Wnt Signaling

† † ‡ † † Asaf Vivante,* Michal Mark-Danieli, Miriam Davidovits, Orit Harari-Steinberg, Dorit Omer, † ‡ ‡| Yehudit Gnatek, Roxana Cleper, Daniel Landau,§ Yael Kovalski, Irit Weissman,¶ †† ‡‡ || Israel Eisenstein,** Michalle Soudack, Haike Reznik Wolf, Naomi Issler,§§ Danny Lotan, †|| Yair Anikster,¶¶ and Benjamin Dekel

*Department of Pediatrics, Talpiot Medical Leadership Program, Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; †Pediatric Stem Cell Research Institute, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; ‡Institute of Pediatric Nephrology, Schneider Children’s Medical Center of Israel, Petah Tiqwa, Israel; §Department of Pediatrics A and Pediatric Nephrology, Saban Children’s Hospital, Soroka Medical Center, Ben Gurion University, Beer Sheva, Israel; |Children’s Medical Center, Ramla, Israel; ¶Nephrology Unit, Western Galilee Hospital, Nahariya, Israel; **Pediatric Nephrology Unit, Meyer Children’s Hospital, Rambam Health Care Campus, Faculty of Medicine, Technion, Israel Institute of Technology, Haifa, Israel; ††Pediatric Imaging, Edmond and Lily Safra Children’s Hospital, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; ‡‡Genetic Institute, Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; §§Centre for Nephrology, Institute of Child Health, University College London, London, United Kingdom; ||Division of Pediatric Nephrology, Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; and ¶¶Metabolic Disease Unit, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Tel Hashomer, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

ABSTRACT Abnormal differentiation of the renal stem/progenitor pool into kidney tissue can The pathologic basis of RHD is the lead to renal hypodysplasia (RHD), but the underlying causes of RHD are not well disturbance of normal nephrogenesis, understood. In this multicenter study, we identified 20 Israeli pedigrees with isola- possibly due to mutations in genes that ted familial, nonsyndromic RHD and screened for mutations in candidate genes in- direct the process.1,8 Most of the genes volved in kidney development, including PAX2, HNF1B, EYA1, SIX1, SIX2, SALL1, known to be involved are transcriptional GDNF, WNT4,andWT1. In addition to previously reported RHD-causing genes, we factors and genes that encode for pro- found that two affected brothers were heterozygous for a missense variant in the teins involved in the mesenchymal to ep- WNT4 gene. Functional analysis of this variant revealed both antagonistic and ag- ithelial transition.2,8 To date, most forms onistic canonical WNT stimuli, dependent on cell type. In HEK293 cells, WNT4 inhibi- of RHD have been found negative for tedWNT3Ainducedcanonicalactivation,andtheWNT4 variant significantly abnormalities in recognized renal devel- enhanced this inhibition of the canonical WNT pathway. In contrast, in primary cul- opmental genes,3,9,10 and it is highly tures of human fetal kidney cells, which maintain WNT activation and more closely likely that other, still unreported, genes represent WNT signaling in renal progenitors during nephrogenesis, this mutation will be identified, especially genes for caused significant loss of function, resulting in diminished canonical WNT/b-catenin which renal maldevelopment has been signaling. In conclusion, heterozygous WNT4 variants are likely to play a causative role in renal hypodysplasia. Received January 28, 2012. Accepted December 6, J Am Soc Nephrol 24: 550–558, 2013. doi: 10.1681/ASN.2012010097 2012.

Published online ahead of print. Publication date available at www.jasn.org.

ESRD in children most commonly results diagnosed sporadically or with familial Correspondence: Dr. Benjamin Dekel, Pediatric from congenital anomalies of the kidney aggregation.1,3–6 For familial cases, the sug- Stem Cell Research Institute, Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Tel 1 and urinary tract. The most common gested mode of inheritance in most pedi- Hashomer, 52621, Israel. Email: binyamin.dekel@ congenital anomalies of the kidney and grees is autosomal dominant with variable sheba.health.gov.il or [email protected] urinary tract clinic-pathologic type is re- expression and reduced penetrance, esti- Copyright © 2013 by the American Society of nal hypodysplasia (RHD).2 RHD can be mated to range between 50% and 90%.7 Nephrology

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fi demonstrated in genetically modi ed A B models.11 C T GG A A G T C A T G G A C T C G G Weinvestigatedtheprevalenceof WT mutations in 9 kidney developmental genes among a selected group of 20 families with isolated nonsyndromic familial RHD. We showed that mutations are present in a significant part of this C T GG A A G T C A T G G A C T C G G >C group, and we outlined the possible role M64T of mutated WNT4 as a cause for dis- turbed early kidney development leading to the RHD phenotype. Viewing RHD as a disease of the renal stem/progenitor cell pool, we hypothesized that C D human developing kidney cell–based systems would be extremely useful for disease modeling. Our study group comprised 51 RHD- affected individuals and 91 unaffected family members from 20 unrelated families. The families’ clinical characteristics are shown in Supplemental Figure 1 and Supplemental Table 1. We identified 13 Figure 1. Mutation analysis of the p.M64T, WNT4 variant shows high degree of conservation. (A) Sequence analysis reveals the WNT4 variant caused by heterozygous transition mutation-carrying participants from (c.t191c) (lower panel, blue arrow) resulting in amino acid substitution M64T. The wild-type four unrelated families harboring muta- (WT) sequence is given for comparison (upper panel). (B) WNT4 variant is found in patients tions in three different genes: one family 1, 3, 4, and 5. Whereas patients 3 and 4 have a clear isolated renal phenotype of severe left with a PAX2 mutation, two families with renal hypodysplasia, patient 1 has an isolated left renal cyst (sized 12.837 mm) in addition HNF1B mutations (for full genetic and to normal BP and normal GFR. Patient 5, age 3 years, has normal pelvic and renal ultra- clinical characterization see Supplemen- sound results, normal BP, normal GFR, and low morning serum cortisol levels. Incomplete tal Appendix), and one family (family 4) penetrance or variable expression can be considered in this family. Squares indicate male demonstrating a novel heterozygous family members and circles female family members; black filled squares indicate that the missense variant in the human WNT4 patients are affected with RHD. Gray filled squares and circles indicate subtle clinical signs. gene. The latter family includes two af- (C) cDNA sequences of human WNT4 are compared with WNT4 orthologs in other species. fected brothers with severe left RHD (D) Conservation scale of WNT4 protein shows the amino acid methionine in position 64 (blue arrow) to be highly conserved (8 of 9), with “b” indicating a buried residue and “e” who were found to harbor a novel mis- indicating an exposed residue. Note the high degree of conservation. sense WNT4 variant, c.t191c– p.M64T. This is the first description of an associ- ation between a WNT4 heterozygous variant and isolated human RHD. Geno- [http://www.hgmd.cf.ac.uk/ac/index. is known to cause renal malformation as type-phenotype correlation in this fam- php]). Automatic computer prediction part of the general Mayer–Rokitansky- ily revealed an autosomal dominant for possible effects of amino acid substi- like syndrome that was first described pattern of inheritance with incomplete tution on the structure and function of the in an 18-year-old female presenting penetrance, similar to other renal hypo- WNT4 protein using PolyPhen software with primary amenorrhea and a single dysplasia-causing genes (Figure 1). predictions (http://genetics.bwh.harvard. kidney.13 This is strikingly similar to The WNT4 p.M64T variant affects a edu/pph)12 showed that this variant may the WNT4 knockout mouse model that highly conserved methionin residue be possibly damaging (position-specific showed defective mesenchymal-epithe- found in all living organisms for which independent count [PSIC] score, 1.79). lial transition (MET), lack of nephron the WNT4 sequence is known (Figure 1), Finally, screening of 280 ethnically formation, and development of renal hy- suggesting possible functional impor- matched control participants (560 alleles) podysplasia,14,15 in addition to defective tance. The variant was not found in a for presence of the WNT4 variant was sexual differentiation exclusive to the fe- search of the single nucleotide polymor- negative, suggesting that this is not a com- male model. Supporting our findings, phism (SNPs) or mutation databases mon polymorphism. the male mouse model showed only iso- (dbSNP, 1000 genomes [http://brows- Further evidence supporting the cau- lated RHD, similar to both affected er.1000genomes.org/index.html]; Hu- sality of this rare WNT4 variant include males described herein.15 The interde- man Gene Mutation Database HGMD that heterozygous mutation in this gene pendence among other WNT proteins

J Am Soc Nephrol 24: 550–558, 2013 Genetics of Renal Hypodysplasia 551 BRIEF COMMUNICATION www.jasn.org involved in nephrogenesis and different significant portion of cells with renal shown that WNT/b-catenin signaling is a WNT pathways may explain why some epithelial stem/progenitor features. Ex- necessary and sufficient trigger of the early of the phenotypic characteristics of this panded cells exhibit the morphology and stages of nephron differentiation.26,27 We syndrome have incomplete penetrance.16 clonogenic growth properties of renal linked the human WNT4 variant to defec- Importantly, our in vitro studies, which epithelial stem/progenitors, express renal tive WNT signaling by two independent are described as follows, support the del- progenitormarkergenesandproteins,and model systems. In the first model, utilizing eterious effect of this variant. are devoid of hematopoietic and endothe- HEK293, WNT4 by itself does not trigger The best understood mechanism of lial markers. Furthermore, HFK-PC cells WNT/b-catenin signaling but rather WNT signaling is the canonical pathway exhibit basal canonical WNT activity and inhibits a Wnt3A-induced signal, whereas that activates the nuclear functions of therefore are a reliable and relevant disease the WNT4 variant induces significantly b-catenin, leading to changes in gene ex- modeling platform (Figure 3). exaggerated inhibition. Although these pression that influence proliferation and Next, we assessed canonical pathway data clearly show a differential response, survival.17 Consequently, using human activation using the TOPFlash reporter they might not mimic renal development embryonic kidney cell line (HEK293) assay; activation was only evident with because the WNT3A ligand is not a phys- as a first platform, we assessed the con- wild-type WNT4. Neither WNT3A nor iologic participant in the process.28 Ac- sequence of the WNT4 variant by cell the WNT4 variant was found to activate cordingly, in the second disease model proliferation assay and TOPFlash re- this pathway (Figure 4A). Finally, we utilizing early developing human kidney porter assay18 to study the canonical tested the consequence of the WNT4 cells, design principles appear to more WNT signaling pathway. We found that variant using conditioned media experi- closely simulate renal development and the WNT4 p.M64T variant leads to sig- ments. We chose this type of experiment WNT signaling due to the following find- nificantly increased cell proliferation because WNT4 is a secreted glycol- ings. First, WNT3A fails to activate the ca- compared with the wild-type WNT4 protein that acts as a short-range ligand nonical WNTsignaling. Second, wild-type (Figure 2C). Interestingly, analysis of on the cell surface. HFK-PC cells were WNT4 is overexpressed. Third, a basal Wnt/b-catenin target genes by real- stimulated with conditioned medium activation of canonical WNT pathway is time PCR after introduction of wild- from HEK293 cells transfected with present. Finally, wild-type WNT4 induces type WNT4 or WNT4 M64T failed to wild-type WNT4, mutant, or empty vec- canonical activation on protein and target show upregulation (Figure 2A). More- tor (control) (Figure 4B). In contrast to gene level, whereas the p.M64T WNT4 over, activation of the canonical WNT wild-type WNT4, both mutant WNT4 variant fails to do so. Clearly not all of pathway via the WNT3A ligand, a and control conditioned media did not the components required to study WNT known activator of the canonical path- increase activated b-catenin levels nor it signaling in human kidney development way in HEK293, showed that the wild- target gene levels in the treated HFK-PC are present in HEK293.29 Given the fact type WNT4 ligand is an inhibitor of the cells (Figure 4, C and D). that WNT4 does not stimulate canonical canonical pathway, in accordance with WNT4 belongs to the WNT family, a WNT signaling in HEK293, we hypoth- previous report19 (Figure 2B). Strikingly, large group of secreted glycoproteins esized that the observed inhibition of in HEK293, the mutant WNT4 (p. encoded by 19 distinct genes involved the WNT3A-induced canonical WNT M64T) led to significantly enhanced in- in the WNT signaling pathway.17 In activation by WNT4 may be a result of hibition of the canonical WNT pathway mammals, WNT4 function has mainly competitive inhibition at the receptor compared with wild-type control, been studied during nephrogenesis be- level. In line with our findings and hy- revealing a functional cell phenotype causemicelackingWNT4 die shortly pothesis, the ability of one WNT ligand (Figure 2D). after birth as a result of kidney failure to function in two distinct pathways To further investigate the functional secondary to severe bilateral RHD.20 based on receptor context was demon- effect of the WNT4 variant on the ca- The mouse model has also shown that strated with WNT5A.30 nonical WNT pathway, we performed suboptimal WNT4 signaling results in We therefore favor the mechanism functional analysis experiments using a RHD.14 During normal kidney develop- delineated in the more physiologic HFK- more physiologic disease model. We ment WNT4 plays an essential role in PC model that is congruent with data aimed for the original cells where the ge- induction of mesenchymal progenitor generated in mouse models whereby loss- netic insult first initiates its effect to pro- cells to epithelialization (MET) and gen- of-function mutation overall reduces duce RHD, hence, human developing eration of pretubular aggregates, leading WNT/b-catenin signaling, leading to kidney cells. Consequently we used pri- to renal vesicles and eventually tubule lack of proper nephron differentiation mary cultures of human fetal kidneys formation.21–23 Recapitulating normal and proliferation of undifferentiated cell (HFK-PC) obtained after curettage of development, WNT4 was also demon- types and resulting in a isolated pheno- elective abortions performed during strated to be upregulated in the proximal type of maldeveloped dysplastic kidney. the 18th week of gestation. tubules during recovery from AKI24 and Our results clearly show that studying Cell characterization of low passage has a suggested role in the pathogenesis of mutations in cell lines may pose prob- HFK-PC demonstrated that it comprises a renal fibrosis.25 Elegant studies have lems in interpretation of mechanism, as

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Figure 2. Functional analysis of the p.M64T,WNT4 variant in HEK293 shows enhanced inhibition of the canonical WNT pathway. (A) WNT4, wild- type and mutant, does not increase WNT canonical pathway target genes. Quantitative RT-PCR on HEK293 transfected cells with WNT4 wild-type (WT) and mutant (Mut) plasmids shows the mRNA expression levels of canonical WNT pathway target genes (MYC, CCND1, and AXIN). (B) WNT3A, butneitherwild-typeWNT4normutantWNT4,activatesthecanonicalWNTpathway.HEK293cellsarecotransfectedona6-wellplate,withTOPFlash reporter plasmid (4 mg) and either wild-type WNT4, mutant WNT4, or WNT3A expression plasmid (4 mg). pCMV-Renilla plasmid (0.4 mg) is used as the internal control. (C) Wild-type WNT4 and mutant variants increase cell proliferation compared with control. Mutant WNT4 leads to significantly increased proliferation compared with wild-type WNT4. *P,0.05. Results are presented as the mean absorbance at 492 nm using the MTS proliferation assay 6 SEM of at least three replicates. (D) WNT4, wild-type and mutant, inhibit the canonical WNT pathway. WNT4 mutant compared with the wild-type leads to significantly enhanced inhibition of the canonical WNT pathway. Cells are cotransfected with TOPFlash reporter plasmid (500 ng), WNT3A expression plasmid (200 ng) to activate the canonical WNT pathway, and an increasing concentration of WNT4 wild-type or WNT4 mutant expression plasmid (200, 400, 800, and 1600 ng). pCMV-Renilla plasmid is used as an internal control (50 ng). Cell lysates are measured for luciferase activity 48 hours after transfection. Activities are expressed as fold activation of the relative luciferase activity. WNT3A and WNT4 does not activate the mutant TOPFlash reporter, FOPFlash, confirming assay specificity. Asterisks indicate a significant difference of the mutant WNT4 compared with dose-equivalent WNT4 wild-type transfection. *P,0.05; **P,0.01 (t test). Data are presented as the mean 6 SEM from three separate experiments.

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Figure 3. Human fetal kidney cells harbor renal epithelial stem/progenitor characteristics and exhibit basal canonical WNT activity. (A–D) HFK- PC cells express renal stem/progenitor markers. (A) Cellular appearance of cultured fetal kidney cells (upper panel) and clone formation by human fetal kidney cells, demonstrating their high clonogenic capacity. Culturing of 0.3 cells per well results in 5%–10% of single clone formation (lower panel). (B) Gene expression analysis of renal stem/progenitor genes. Quantitative RT-PCR (qat-PCR) analysis of Vimentin, PAX2, and SIX2, three representative genes expressed early during nephrogenesis. Normalization is performed against control GAPDH expression and real-time quantitative is calculated relative to the well differentiated HAK-PC cells. (C) Immunofluoresence staining for the expression of two representative early nephrogenesis transcriptional factors: WT1 and SIX2 (320 and 3100). All nuclei are stained with DAPI (blue). The green florescence signal in the upper and lower panels corresponds to anti-SIX2 protein and anti-WT1 protein staining respectively. Both clearly show nuclear staining (representative stained nuclei are indicated with white arrows). (D) Flow cytometric analysis for CD34, CD45, and CD56/NCAM1 expression and corresponding isotype controls in HFK-PC cells. Results show negligible levels of CD34 (a well known marker of hematopoietic stem cells and vascular endothelial cells) and CD45 (leukocyte common antigen). Moreover, the HFK-PC contains 44% of CD56/ NCAM1-positive cells. NCAM1 has been previously shown to be a stem/progenitor cell marker in the human fetal kidney.31 (E–G) HFK-PC cells harbor basal canonical WNT activity. (E) qRT-PCR analysis of WNT4 and frizzled 7 (FZD7), a representative receptor of the WNT signaling li- gands. (F) Because WNT/b-catenin and SIX2 pathways have opposing actions (commitment and self-renewal of renal stem/progenitors, respectively), we sought to manipulate this balance so as to provide additional support that our HFK-PC cells contain a significant portion of cells with early renal stem/progenitor characteristics. Consequently, qRT-PCR analysis of SIX2, in HFK-PC after the addition of three different WNT pathway antagonists—dickkopf-related protein 1 (DKK1), secreted frizzled-related protein 1 (sFRP1), and Wnt inhibitory factor (WIF)—shows significant SIX2 increment compared with the control. Normalization is performed against control GAPDH expression and real-time quantitative is calculated relative to the well differentiated HAK-PC cells. Data are presented as the mean 6 SEM from three separate experiments. (G) HFK-PC immunofluorescence staining for anti-active b-catenin (green) discloses its cytoplasmic and nuclear presence. All nuclei are stained with DAPI (blue). *P,0.05; **P,0.01 (t test). DAPI, 4’,6-diamidino-2-phenylindole; NCAM-1, neural cell adhesion molecule 1. opposed to human fetal kidney–derived CONCISE METHODS units in Israel enrolled in a prospective trial. primary cell models that can be further The study comprised Israeli pedigrees iden- generalized to study mechanisms of Patients tified using index pediatric patients with other mutations affecting renal develop- A nationwide multicenter collaboration study isolated, nonsyndromic, familial RHD. The mental diseases. was conducted, with six pediatric nephrology diagnosis of isolated RHD was made by

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Figure 4. Functional analysis of the p.M64T, WNT4 variant in HFK-PC shows significant loss of function and diminished canonical WNT/ b-cateninsignaling. (A) Wild-typeWNT4,but neither WNT3Anor mutant WNT4, activates the canonicalWNT pathwayinHFK-PC.HFK-PCcells are cotransfected on a 6-well plate, with TOPFlash reporter plasmid (4 mg) and wild-type WNT4, mutant WNT4, or WNT3A expression plasmid (4 mg). pCMV-Renilla plasmid (0.4 mg) is used as internal control. *P,0.05 (t test). Data are presented as the mean 6 SEM from three separate experiments. (B) Illustration of the conditioned media experiments. HEK293 cells are transfected separately with wild-type WNT4, mutant WNT4, or empty vector (control). Conditioned media containing WNT proteins were applied on HFK-PC 24 hours after transfection. Cells are harvested for total RNA and total protein 6–24 hours later. (C) Results of a representative Western blot analysis of the HFK-PC cells after they are treated with wild-type WNT4,mutantWNT4, and control conditioned media, with the use of anti-active b-catenin antibody. Cbl (95 kD) is used as a loading control. Active b-catenin protein levels are adjusted to Cbl and are quantified compared with the control (empty vector). Three separate experiments yield a similar result in which wild-type WNT4 induces a significant increase in active b-catenin as opposed to the p.M64T Wnt4 variant, which results in a nonsignificant change. (D) Quantification by real-time RT-PCR of the expression of mRNA axin2 and ccnd1 in HFK-PC after treatment with wild-type WNT4, mutant WNT4, and control conditioned media.

J Am Soc Nephrol 24: 550–558, 2013 Genetics of Renal Hypodysplasia 555 BRIEF COMMUNICATION www.jasn.org pediatric nephrologistsand radiologists based for gene deletions by the multiplex ligation-de- Billerica, MA) antibodies overnight in 4C. on sonographic imaging studies. RHD was pendent probe amplification (MLPA) method Cells were washed and then incubated with defined as familial when at least a second case using the SALSA MLPA kit P153EYA1 (MCR- Alexa-488 conjugated anti-mouse or anti- of RHD was reported and verified in the Holland, Amsterdam, The Netherlands) in rabbit IgG secondary antibody for 60 minutes. proband’s immediate family. Excluded from the conditions suggested by the manufac- Mounting containing 4’,6-diamidino-2- the study were patients with RHD associated turer. Amplified samples were denatured and phenylindole (ProLonged Gold; Invitrogen, with one of the following: posterior urethral separated by capillary electrophoresis on an Grand Island, NY) was applied. The cells valve, primary bladder abnormalities, previ- ABI 3130 sequencer (Applied Biosystems, were analyzed by Olympus BX51 fluorescence ously identified or complex syndromes, and Carlsbad, CA). microscope using Olympus DP72 camera and extrarenal major malformations. cellSens standard software. The study was approved by the national Expression Studies and local Helsinki committees, and informed Plasmids Cell Proliferation Assay assent and/or consent for genetic screening The full-length WNT4 cDNAwas cloned into The viability of transfected HEK293 cells was obtained from the patients and/or pa- pCMV6-AC-GFP (OG-RC-209205; OriGene carrying wt/mutant WNT4 was colorimetri- rents, as appropriate. Technologies Inc, Rockville, MD). The cally determined using a cell proliferation as- WNT4 variant c.t191c (p.M64T) was intro- say kit (Cell-Titter 96 AQueous One Solution Genetic Analyses duced to pCMV6-MYC-GFP -WNT4 by a Cell Proliferation Assay; Promega, Madison, Mutation analysis was carried out on DNA ex- QuikChange II XL Site-Directed Mutagenesis WI) in a 96-well tissue culture plate, with 20 tracted according to standard methods from kit (200521; Agilent Technologies, Santa ml Cell-Titter 96 AQueous One Solution peripheral blood obtained from 20 probands. Clara, CA) using 59-GCGGAACCTG- Reagent containing 3-(4,5-dimethylthiazol- If mutations were detected, all additional fam- GAAGTCACGGACTCGGTGCGCCGCG-39 2-yl)-5-(3-carboxymeth-oxyphnyl)-2-(4- ily members were sequenced to look for seg- and 59-CGCGGCGCACCGAGTCCGT- sufophnyl)-2H-terasolium (MTS) and regation. Amplified products were initially GACTTCCAGGTTCCGC-39 primers. The phenazine ethosulfate added to each well. screened using denaturing high-performance plasmids were verified by direct sequencing. Cultured cells were incubated at 37°C in a liquid chromatography (DHPLC). All 57 exons humidified, 5% CO atmosphere for 4 hours. of nine candidate genes—PAX2, HNF1B, 2 The absorbance of soluble formazan pro- EYA1, SIX1, SIX2, SALL1, WNT4, GDNF,and Cell Culture and Transfections Renal HEK293 cells were grown in DMEM duced by cellular reduction of the MTS was WT1—were individually amplified using supplemented with 10% FCS, penicillin (100 measured at 492 nm using an ELISA plate exon-flanking primers. DNA sequencing was U/ml), and streptomycin (100 mg/ml) at 37°C reader. conducted on the DHPLC-positive PCR prod- and 5% CO . HEK293 cells were transfected ucts. After positive DHPLC products were 2 using calcium phosphate with equal amounts sequenced, synonymous SNPs or variants Clonogenic Potential Evaluation of wild-type and mutant WNT4 DNA or See the Supplemental Methods. previously reported in public SNP databases with a combination of each with WNT3A. were excluded from further analysis. Expression of the encoded proteins was ana- Luciferase Reporter Assay DHPLC lyzed 2 days after transfection. To assay the canonical WNT4 pathway, we Scanning for DNA mutations and variants used the TOPFlash-TCF reporter plasmid, as 18 fl using DHPLC involves subjecting PCR prod- RT-PCR previously described. Brie y, HEK293 cells 3 5 ucts to chromatography using an ion-pair Total RNA was extracted from transected/ were seeded at 1 10 cells per well in a 24- reversed-phase cartridge (PCR primers are treated/primary cell cultures using the High well plate 24 hours before transfection. Cells available upon request). PCR products are Pure RNA Isolation Kit (Roche Diagnostics, were transfected with the indicated vector, denaturated and allowed to re-anneal. Under Mannheim, Germany). Total cDNA was syn- along with pTOPFlash/pFOPFlash and conditions of partial denaturation with a linear thesized using the Reverse-iT 1st Strand Syn- pCMV-Renilla plasmids. At 48 hours after fi acetonitrilegradient,heteroduplexesfromPCR thesis Kit (ABgene, Surrey, UK) and ampli ed transfection, the cells were harvested and samples with internal sequence variation dis- using Taq polymerase, Q solution (QIAGEN, subjected to Dual-Luciferase Reporter Assay fi play reduced column retention time relative to Valencia, CA), and intron-crossing speci c System (E1910; Promega) according to man- ’ their homoduplex counterparts. The elution primers. ufacturer s instructions. profile for a heterozygous sample is typically quite distinct from that of either homozygous Immunofluorescence Establishment of Primary Cultures from fi sequence, making identification of heterozy- HFK-PC cells were xed with 2% PFA 3% Human Fetal Kidney and Human Adult gous mutations relatively straightforward. sucrose in PBS for 10 minutes and washed Kidney with PBS. Cells were blocked with 5% human Normal human 18-week gestation kidneys Multiplex Ligation-Dependent Probe serum and 1% BSA in PBS-Tween (0.05%) for (HFK) were obtained after curettage of elec- Amplification 30 minutes followed by incubation with SIX2 tive abortions. Normal human adult kidney Deletion screening of HNF1B was carried out (Norus), WT1 (Santa Cruz Biotechnology, (HAK) samples were retrieved from borders as follows: All index patients were screened Santa Cruz, CA), active b-catenin (Millipore, of renal cell carcinoma tumors from partial

556 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 550–558, 2013 www.jasn.org BRIEF COMMUNICATION nephrectomy patients. The tissues were han- 5% skim milk, incubated with the relevant an- 2. Sanna-Cherchi S, Caridi G, Weng PL, Scolari dled within 1 hour after the procedure.31,32 tibody, and then followed by incubation with a F, Perfumo F, Gharavi AG, Ghiggeri GM: Genetic approaches to human renal agene- All studies were approved by the local ethics secondary, peroxidase-conjugated antibody sis/hypoplasia and dysplasia. Pediatr Neph- – – – – committee and informed consent was pro- (1:10,000, #115 035 146 and #115 035 144; rol 22: 1675–1684, 2007 vided by the patients involved in this research Jackson ImmunoResearch, West Grove, PA). 3. Weber S, Moriniere V, Knüppel T, Charbit M, according to the Declaration of Helsinki. Peroxidase activity was detected by exposure Dusek J, Ghiggeri GM, Jankauskiené A, Mir Both HFK and HAK tissues were washed of the membrane to chemiluminescence solu- S, Montini G, Peco-Antic A, Wühl E, Zurowska AM, Mehls O, Antignac C, Schaefer F, with cold Hanks’ balanced salt solution (In- tion containing 150 mM Tris-HCl (pH 8.9), Salomon R: Prevalence of mutations in renal vitrogen, Carlsbad, CA) and minced into 0.22 mg/ml Luminol (Sigma, St. Louis, MO), developmental genes in children with renal approximately 1-mm cubes using sterile sur- 0.033 mg/ml paracoumaric acid (Sigma), and hypodysplasia: Results of the ESCAPE study. – gical scalpels. The dissected tissue was then 0.015% H2O2. Bands were visualized and JAmSocNephrol17: 2864 2870, 2006 incubated for 2 hours at 37°C with Iscoves’s quantified using densitometer software. 4. Eccles MR, Schimmenti LA: Renal-coloboma syndrome: A multi-system developmental modified Dulbecco’s medium (IMDM; Invi- disorder caused by PAX2 mutations. Clin trogen) supplemented with 0.1% collagenase FACS Analyses Genet 56: 1–9, 1999 IV (Invitrogen). The digested tissue was then See the Supplemental Methods. 5. Bingham C, Bulman MP, Ellard S, Allen LI, forced through a 100-mmcellstrainerto Lipkin GW, Hoff WG, Woolf AS, Rizzoni G, achieve a single cell suspension and after re- Statistical Analyses Novelli G, Nicholls AJ, Hattersley AT: Muta- tions in the hepatocyte nuclear factor-1beta moval of the digesting medium, it was then Functional in vitro experiments were inter- gene are associated with familial hypoplastic resuspended in growth medium (IMDM con- preted using an unpaired, two-sided t test glomerulocystic kidney disease. Am J Hum taining 10% FBS [Invitrogen], 100 ng/ml comparing effect and appropriate controls. Genet 68: 219–224, 2001 epidermalgrowthfactor,100ng/mlbasic Data are reported as mean and SEM. 6. Roodhooft AM, Birnholz JC, Holmes LB: Fa- fibroblast growth factor, and 10 ng/ml stem milial nature of congenital absence and severe dysgenesis of both kidneys. NEnglJ cell factor [R&D Systems Inc, Minneapolis, Med 310: 1341–1345, 1984 fl ACKNOWLEDGMENTS MN]) and plated in asks. HFK-PC cells 7. McPherson E, Carey J, Kramer A, Hall JG, (passages 0–1) were incubated and upon Pauli RM, Schimke RN, Tasin MH: Dominantly 90% confluence, cells were subjected to We thank all of the families for their generous inherited renal adysplasia. Am J Med Genet – both luciferase reporter assay (TOPFlash) participation in our study. We also thank 26: 863 872, 1987 8. Kerecuk L, Schreuder MF, Woolf AS: Renal tract and conditioned media experiments. For con- Professor Elon Pras for providing 280 control DNA samples, as well as Dr. Rina Rosin-Ar- malformations: Perspectives for nephrologists. ditioned media, HEK293 cells were transfec- NatClinPractNephrol4: 312–325, 2008 ted with 4 mg empty vector, wild-type Wnt4, besfeld and Dr. Michal Caspi for their generous 9. Saisawat P, Tasic V, Vega-Warner V, Kehinde or mutant WNT4, grown to near confluence. support with the TOPFlash reporter assay. In EO, Günther B, Airik R, Innis JW, Hoskins BE, fi The media were then removed and used to re- addition, we thank Dr. Robert Kleta and Dr. Hoefele J, Otto EA, Hildebrandt F: Identi - cation of two novel CAKUT-causing genes by place the media from untransfected HFK-PC Detlef Bockenhauer from University College massively parallel exon resequencing of fl London (London, UK) for their support and cells grown to near con uence in separate candidate genes in patients with unilateral flasks. These cells were grown for 6–24 hours thoughtful comments. We also thank Sigal renal agenesis. Kidney Int 81: 196–200, 2012 in the conditioned media and then harvested Koren and Dr. Dani Bercovich from MIGAL 10. Thomas R, Sanna-Cherchi S, Warady BA, for protein and RNA extraction. Galilee Technology Center (Kiryat Shmona, Furth SL, Kaskel FJ, Gharavi AG: HNF1B and Israel) for technical assistance. This work was PAX2 mutations are a common cause of renal carried out in partial fulfillment of the PhD hypodysplasia in the CKiD cohort. Pediatr Protein Extraction and Western Blot Nephrol 26: 897–903, 2011 Analyses degree requirements for A.V. 11. Woolf AS: Renal hypoplasia and dysplasia: Cultured cells were washed twice in cold PBS This work was supported in part by the Starting to put the puzzle together. JAmSoc – and resuspended in lysis buffer (1% Triton- Israeli Society of Clinical Pediatrics, Bretler Nephrol 17: 2647 2649, 2006 12. Ramensky V, Bork P, Sunyaev S: Human non- X100, 20 mM Tris-HCl, 120 mM NaCl) and a Foundation, Sackler School of Medicine, Tel Aviv University, Israel Scientific Foundation synonymous SNPs: Server and survey. Nu- tablet of protease inhibitors (Complete Mini cleic Acids Res 30: 3894–3900, 2002 Protease Inhibitor Cocktail; Roche), incubated (ISF) 11/910 (B.D.), and the Israeli Ministry 13. Biason-Lauber A, Konrad D, Navratil F, for 1 hour on ice and centrifuged at 16,1003g of Health. Schoenle EJ: A WNT4 mutation associated for 20 minutes. The cell supernatant was col- with Müllerian-duct regression and viriliza- tionina46,XXwoman.NEnglJMed351: lected, concentration was determined using DISCLOSURES 792–798, 2004 the Bradford method (Pierce), and the super- 14. Iglesias DM, Hueber PA, Chu L, Campbell R, None. natant was then resuspended in loading buffer Patenaude AM, Dziarmaga AJ, Quinlan J, and denatured in 95°C for 5 minutes. 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