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LDL –Related 6 Modulates Ret Proto- Signaling in Renal Development and Cystic Dysplasia

† † † ‡ Yongping Wang,* Arjun Stokes,* Zhijian Duan,* Jordan Hui, Ying Xu, YiPing Chen,§ † Hong-Wu Chen,* Kit Lam,* and Chengji J. Zhou*

*Department of Biochemistry and Molecular Medicine and Comprehensive Center, University of California Davis, School of Medicine, Sacramento, California; †Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children-Northern California and University of California Davis School of Medicine, Sacramento, California; ‡Cambridge-Suda Genome Resource Center, Soochow University, Suzhou, China; §Department of Cell and Molecular Biology, Tulane University, New Orleans, Louisiana

ABSTRACT Hypoplastic and/or cystic kidneys have been found in both LDL receptor–related protein 6 (Lrp6)- and b-–mutant mouse , and these are key molecules for Wnt signaling. However, the underlying mechanisms of Lrp6/b-catenin signaling in renal development and cystic formation remain poorly understood. In this study, we found evidence that diminished and increased occur before cystic dysplasia in the renal primordia of Lrp6-deficient mouse embryos. The expression of Ret proto- oncogene (Ret), a critical receptor for the glial cell line–derived neurotrophic factor (GDNF), which is required for early nephrogenesis, was dramatically diminished in the mutant renal primordia. The activities of other representative nephrogenic , including Lim1, Pax2, Pax8, GDNF,andWnt11, were subsequently diminished in the mutant renal primordia. Molecular biology experiments demonstrated that Ret is a novel transcriptional target of Wnt/b-catenin signaling. Wnt agonist lithium promoted Ret expression in vitro and in vivo. Furthermore, Lrp6-knockdown or lithium treatment in vitro led to downregulation or upregulation, re- spectively, of the phosphorylated mitogen-activated protein kinases 1 and 3, which act downstream of GDNF/ Ret signaling. Mice with single and double of Lrp6 and Ret were perinatal lethal and demonstrated dosage–dependent effects on the severity of renal hypoplasia during embryogenesis. Taken together, these results suggest that Lrp6-mediated Wnt/b-catenin signaling modulates or interacts with a signaling network consisting of Ret cascades and related nephrogenic factors for renal development, and the disruption of these genes or signaling activities may cause a spectrum of hypoplastic and cystic kidney disorders.

J Am Soc Nephrol 27: 417–427, 2016. doi: 10.1681/ASN.2014100998

The mammalian kidney develops through the pro- Either overactivation or inactivation of Wnt signal- nephros, mesonephros, and metanephros stages by ing may cause polycystic kidneys in mod- reiterated branching, patterning, and segmentation els.8–11 The gene-trapped mouse mutants of Lrp6, a on the base of reciprocal inductions and interactions between the epithelial and mesenchymal tissues originated from the intermediate .1,2 Received January 14, 2015. Accepted April 13, 2015. Wnt signaling plays crucial roles in organogenesis Published online ahead of print. Publication date available at and disease.3 Among 19 ligands, Wnt4, Wnt9b, and www.jasn.org. Wnt11 are uniquely expressed in renal tissues and Correspondence: Dr. Chengji Zhou, 2425 Stockton Blvd., Room- are required for kidney development.4–6 The ca- 602B, SHCNC, Sacramento, CA 95817. Email: cjzhou@ucdavis. nonical Wnt/b-catenin signaling is involved in edu induction and epithelial differentiation.7 Copyright © 2016 by the American Society of Nephrology

J Am Soc Nephrol 27: 417–427, 2016 ISSN : 1046-6673/2702-417 417 BASIC RESEARCH www.jasn.org key coreceptor in the Wnt/b-catenin signaling pathway, also morphologic changes of the mutant mesonephros compared exhibit hypoplastic and cystic kidneys as embryos,12 yet the with the littermate controls at embryonic day (E) 9.5 (Figure 1, detailed phenotypic and mechanistic information remains A and E). At E11.5, the mutants exhibit fewer mesonephric unavailable. tubules compared with the controls (Figure 1, B and F). At During early development, the receptor Ret and E18.5, all examined mutant kidneys (n.40) are hypoplastic its molecule glial cell line–derived neurotrophic factor and accompanied by multiple cysts (Figure 1, G and H). (GDNF) play critical roles in the induction and branching of the The phospho-histone H3 (pHH3) immunostaining for ureteric bud for kidney .2,13,14 In particular, Ret acts mitotic cells and the terminal deoxynucleotidyl transferase– through distinct downstream cascades, including PLCg and PI3K/ mediated digoxigenin-deoxyuridine nick-end labeling (TU- MAPK, which have been linked to different types of renal anom- NEL) for apoptotic cells were examined in Lrp6-KO mutants alies, including renal agenesis and cystic dysplasia.15–17 This may and compared with their littermate controls. The percentages provide a basis for understanding the related genetic mechanisms. of the pHH3(+) cells in the mutant renal primordia dramat- However, the upstream regulators of the Ret signaling remain un- ically decreased by 5-fold at E10.5 and 2.7-fold at E17.5 (Fig- clear. Several factors, such as Lim1 and Pax2, which ure 2, A–D). Conversely, the percentages of the TUNEL(+) are interactive with Ret signaling, also play critical roles in renal cells in the mutant renal primordia increased by 2-fold at development and related disorders.1,2 This study using Lrp6 knock- E10.5 and 1.6-fold at E17.5, with statistical significance (Fig- out mice combined with molecular and cellular approaches dem- ure 2). The diminished proliferation and elevated apoptosis onstrates that Lrp6-mediated Wnt/b-catenin signaling is required suggest a possible cellular mechanism underlying renal cystic for metanephric branching through modulation of or interaction dysplasia in Lrp6-KO embryos. with a signaling network consisting of Ret cascades and related nephrogenic factors. The double knockout analyses demonstrate Repressed Expression of Ret Followed by other the genetic interaction between Lrp6 and Ret in urogenital devel- Nephrogenic Factors in the Lrp6-KO Embryos opment. These results provide novel insights into the signaling and To address the underlying molecular mechanisms of the renal genetic mechanisms of renal development and cystic dysplasia. defects in the Lrp6-KO embryos, we performed wholemount in situ hybridization with riboprobes for representative neph- rogenic molecules that have been shown to play important RESULTS roles in early kidney development.1,2 Ret is a GDNF receptor acting as an inductive signaling molecule for renal develop- Hypoplastic and Cystic Kidneys with Diminished ment. In situ signals for Ret mRNAs are drastically lost in the Proliferation and Increased Apoptosis in the Renal mutant renal primordia at E10.5 and E12.5 (Figure 3, A, F, K, Primordia of the Lrp6-KO Embryos and P). Among crucial nephrogenic transcription factors, Histologic analyses of the sagittal sections of Lrp6ßgeo/ßgeo Lim1, Pax2,andPax8 mRNAs are moderately reduced at (knockout [KO]) mouse embryos demonstrate no obvious E10.5 (Figure 3, B–D, G–I), and are significantly diminished

Figure 1. Hypoplastic and cystic kidneys in Lrp6-KO mouse embryos. (A–C, E–G) Hematoxylin and eosin (HE) staining shows the meso- nephros at E10.5 and E11.5, and the metanephros at E18.5 of WT and mutant embryos. (D, H) Whole renal structures of the normal and mutant embryos at E18.5. Ad, adrenal gland; Glu, glomerulus; Ki, kidney; MD/MT, mesonephric duct/tubules; asterisk, cysts.

418 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 417–427, 2016 www.jasn.org BASIC RESEARCH

chromatin immunoprecipitation assays on both L cells and the freshly isolated kid- ney cells from the E14.5 wild-type (WT) mouse . Our results demonstrate that the R4 binding site (22640 bp) in the upstream Ret promoter was specifically amplified by PCR after chromatin immuno- precipitation with anti–b-catenin antibodies (Figure 5B), suggesting that b-catenin/Tcf transcriptional complex was recruited to the R4 binding site of the Ret promoter. We made a series of deletion constructs with four binding sites in the luciferase reporter pGL2 vectors (including the mu- tated R4 binding site, shown in Figure 5A). Our results demonstrate that the construct containing the WT binding site R4 in the Ret gene was activated by co-transfection with the persistent active b-catenin and Lef1 cDNA constructs (Figure 5C). Other constructs, including one containing the mutant sequences of the R4 binding site, were not activated by active b-catenin/ Figure 2. Diminished proliferation and elevated apoptosis in renal primordia or em- Lef1. We also found that the luciferase ac- bryonic kidneys of Lrp6-KO mice. Micrographs (A, B) or histograms of the percentages tivities of the WT R4 construct is dose- (C, D) of the mitotic cells immunolabeled for pHH3, or apoptotic cells detected by dependently responsive to stimulation by TUNEL in the mesonephric tissues at E10.5 or in the kidney at E17.5. Asterisks in B Wnt3a (Figure 5D) or Wnt agonist lithium indicate cysts with varied sizes in the mutant kidney. Asterisks in C, D: *P,0.05; (Figure 5E). These results suggest that Ret , **P 0.01. is a transcriptional target of Wnt/b-catenin signaling. at E12.5 in the mutant renal primordia (Figure 3, L–N, Q–S). Ret Expression and MAPK1/3 Can Be Among Wnt ligands expressed in renal primordia, Wnt9b ex- Modulated by Wnt Agonists and/or Lrp6-Knockdown pression is relatively conserved in the mutant mesonephros at We performed real-time PCR assays to detect whether Wnt- E10.5 (Figure 3, E and J). Wnt11 expression is absent in the signaling activation can modulate Ret expression in vivo and in mutant metanephros at E12.5 (Figure 3, O and T). By contrast, vitro. Lithium is a well known small-molecule Wnt-signaling the expression of Gfra1, a GDNF coreceptor, was relatively con- agonist, acting through inhibition of Gsk3b in the b-catenin served in the mutant renal primordia at E10.5 and E12.5 (Figure degradation complex.20 Compared with the control treat- 4, A and B). GDNF expression is also detectable, but diminished ments with sodium chloride, intraperitoneal injection of lith- in the mutant metanephros at E12.5 (Figure 4B). In addition, ium chloride upregulated 4-fold of Ret mRNAs in the E12.5 Bmp4 and Bmp7 mRNAs are unchanged, and Fgfr2 mRNAs are embryonic kidneys (Figure 6A). The upregulation of Ret moderately diminished in the mutant mesonephros at E10.5 mRNA levels stimulated by lithium demonstrated a dose- (Figure 4A). Wnt4 expression is still detectable in the mutant dependent fashion in the murine inner medullary collecting metanephros at E12.5 (Figure 4B). Because the Ret/GDNF and duct–3 (mIMCD3) cells (Figure 6B). In order to get further Wnt11 pathway is critical for ureteric budding and branching,18 details about Ret mRNA upregulation, we designed different our in situ results suggest a possible mechanism of this pathway primers to amplify all of the Ret splicing isoforms. The Ret acting downstream of Lrp6/b-catenin signaling. intracellular isoform encodes a tyrosine kinase domain, while three variants of Ret9 (short), Ret43 (middle), and Ret51 Ret is a Transcriptional Target of Wnt/b-Catenin (long) are encoded in the C-terminal tail. After lithium stim- Signaling ulation, the expression levels of most Ret isoforms (except We searched the promoter sequence of the mouse Ret gene19 Ret51) increased in a dose-dependent manner, while the Ret and identified several conserved Tcf/Lef1-binding sites, or the intracellular isoform shows the highest expression level com- Wnt-responsive elements, in the upstream of the Ret promoter pared with other variants (Figure 6C). Because of the consider- (Figure 5A). To examine whether the b-catenin/Tcf complex ably low level of Ret expression in mIMCD3 cells, we performed can bind to these sites in vitro and in vivo,weperformed Western blots on Neuro-2a, another mouse cell line, which has a

J Am Soc Nephrol 27: 417–427, 2016 Lrp6 and Ret in Renal Disorders 419 BASIC RESEARCH www.jasn.org

Figure 3. Wholemount in situ hybridization results demonstrate diminished expression of Ret followed by Lim1, Pax2, Pax8, Wnt9b,and Wnt11 in the renal primordia of Lrp6-KO embryos. (A–J) Lateral views of whole renal primordia of the WT controls and mutants at E10.5. (A9–J9) Sagittal vibrotome sections of respective renal primordia at E10.5. (K–T) Front or back views of whole metanephric primordia of E12.5 control and mutant embryos. Arrowheads, mesonephric tubules/ducts (MT/MD); arrows, ureteric buds (UB); asterisks, spinal cords. Relative mRNA signal intensities: +/2, almost none or very weak; +, weak; ++, moderate; +++, strong; ++++, very strong. higher level of Ret expression. We found that both Ret and phos- Akt and PLCg (Figure 7 and data not shown). phorylated Ret (Y1062) increased dose-dependently in response These results suggest that Lrp6-mediated Wnt/b-catenin signal- to lithium treatment in Neuro-2a cells (Figure 6, D–F). ing preferably acts through Ret and MAPK1/3 cascade for sig- To determine the downstream cascades of Ret signaling in naling transduction in renal and neuronal cells in vitro. response to Wnt/b-catenin signaling modulation, we carried out further Western blots of PLCg,PI3K-AKT,andMAPK1/3 Genetic Interactions Between Lrp6 and Ret in (Erk2/1) kinases on both mIMCD3 and Neuro-2a cells after Renal Disorders Lrp6-knockdown (KD), with or without stimulation by lith- To examine whether Lrp6 and Ret are genetically interactive, ium, which acts downstream of Lrp6 to activate b-catenin we examined the renal phenotypes in the compound mutants signaling. Our results demonstrate that Lrp6-KD significantly of Lrp6 and Ret knockout mice (Figure 8). Single heterozygous 2 2 2 2 diminished, while Wnt/b-catenin signaling activation by lith- Lrp6+/ or Ret+/ and double heterozygous Lrp6+/ ;Ret+/ ium significantly restored, the phosphorylation levels of mice are viable and fertile without obvious defects in urogen- 2 2 MAPK1/3 in both cell lines compared with their controls after ital organs. All of the examined single homozygous Lrp6 / normalization to the levels of b- loading controls (Figure 7). embryos exhibit hypoplastic urogenital organs, including However, neither Lrp6-KD nor lithium treatment had effects on two kidneys, a bladder, and two gonads. The compound

420 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 417–427, 2016 www.jasn.org BASIC RESEARCH

Figure 4. Wholemount in situ hybridization for the expression of the less affected nephrogenic factors in Lrp6-KO mutants. (A) Gfra1, Bmp4, Bmp7,andFgfr2 mRNA signals are shown in vibrotome sections of E10.5 mesonephric structures. (B) Diminished, but still expressed mRNA signals of Gfra1, GDNF, Bmp7,andWnt4 in the whole renal organs or vibrotome sections (for GDNF inserts and Wnt4 panels) at E12.5.

2 2 2 mutants of Lrp6 / ;Ret+/ mice exhibit more severe urogen- lip/palate, cardiac outflow tract defects, defects, 2 2 ital defects than in the single Lrp6 / embryos, including 19% and urogenital disorders as the consequences of the inactiva- (4 of 21) compound mutants with only one small cystic kidney tion of Wnt/b-catenin signaling during organogenesis.12,21–25 and a bladder, 14.3% (3 of 21) without kidneys but with a b-catenin has been shown to regulate nephron induction, to bladder, and 47.6% (10 of 21) with neither kidneys nor a blad- maintain the precursor state of the Wolffian ducts and the der. The latest phenotype is also observed in 100% (5 of 5) ureteric bud, and is also required for ureteric branching mor- 2 2 2 2 double homozygous Lrp6 / ;Ret / embryos (Figure 8). phogenesis.7,11,26 However, b-catenin has dual roles in Wnt 2 2 Thus, removal of one or two copies of Ret genes in Lrp6 / signaling and cell adhesion.27 The consistent renal phenotypes mice synergistically increased the severities of urogenital de- of Lrp6 and b-catenin mutants suggest that the defective tran- fects. By contrast, the adrenal primordia remain in all of these scriptional function of b-catenin is a main cause of renal dis- single or compound mutant embryos. orders. Several studies further support the critical role of Wnt/ b-catenin signaling in polycystic kidney including cystic kid- ney .28 Genetic activation of the oncogenic form of DISCUSSION b-catenin in the renal epithelial cells causes polycystic lesion in neonatal mice.8 Conditional inactivation of APC, a negative Lrp6-Mediated Wnt/b-Catenin Signaling in Renal regulator in Wnt/b-catenin signaling, leads to cystic renal Development and Cystic Dysplasia neoplasia.10 These observations suggest that overactivation This study shows that genetic ablation of Lrp6 resulted in of Wnt/b-catenin signaling is a condition of cystic kidney cili- multicystic dysplastic kidneys. Lrp6 is a key coreceptor that opathy. Conversely, conditional removal of b-catenin from the mediates Wnt/b-catenin signaling. Lrp6-deficient mice mouse Wolffian duct epithelium by Hoxb7-Cre also causes exhibit a spectrum of structural disorders, including limb de- cystic kidneys.11 Downregulation of Wnt/b-catenin activities fects, developmental brain disorders, ocular coloboma, cleft and cystic kidney ciliopathy are observed in the KO mice of

J Am Soc Nephrol 27: 417–427, 2016 Lrp6 and Ret in Renal Disorders 421 BASIC RESEARCH www.jasn.org

b-catenin– and mTOR (or mechanistic tar- get of rapamycin)–dependent pathways,34 which is partly consistent with our results. GDNF is a Ret ligand and activates Ret kinase domains in the via phos- phorylation.35 We also detected the dimin- ished expression of GDNF in the renal primordia of Lrp6 mutant embryos. A re- cent study revealed that b-catenin directly regulates GDNF expression in renal mesen- chyme, while b-catenin may also recipro- cally interact with Ret in renal epithelia,36 which supports a possible role of Ret in mediating Wnt/b-catenin signaling during Figure 5. Transcriptional activation of Ret promoter by Wnt/b-catenin signaling. (A) nephrogenesis. The residual expression of Schematic structure of the mouse Ret promoter with four putative Tcf/lef1-binding Ret in the renal primordia of Lrp6-null em- sites or Wnt-responsive elements (R1–R4). (B) Chromatin immunoprecipitation dem- bryos indicates that Ret may also be regulated onstrates the specific amplification of the R4 site immunoprecipitated with b-catenin by other factors, either Wnt-independent antibodies, not the control IgG, from either E14.5 WT embryonic kidneys or L cells. (C) factors or the Wnt coreceptor Lrp5; the latter Luciferase assay results show specificresponseofWTPrw-luc, but not the mutant Prm- plays a functionally redundant role with luc constructs, co-transfected with the persistent active b-catenin and Lef1 constructs. Lrp6 during .37,38 (D) Dose-dependent activation of Prw-luc construct treated with Wnt3a. (E) Dose- Our double KO analyses demonstrate the dependent activation of Prw-luc construct treated with Wnt agonist lithium chloride. genetic interaction of Lrp6 and Ret on neph- , , *P 0.05; **P 0.01. rogenesis and related urogenital develop- ment. The enhanced urogenital defects in 2 2 2 Ahi1 that encodes a cilial protein Jouberin and may be genet- the Lrp6 / ;Ret+/ double mutants also indicate that Lrp6 ically interactive with Lrp6 in polycystic kidney disease (PKD) and Ret may synergistically or in parallel regulate renal and related cystogenesis.29 In support of this, a recent study development. revealed that LRP5 mutations and canonical Wnt signaling are associated with hepatic cystogenesis.30 However, Wnt/ An Lrp6-Regulated Signaling Network in b-catenin signaling is not involved in renal cystic formation Early Nephrogenesis of the mutant mice of Invs that encodes a cilial protein inversin Our results show that several transcription factors are also or nephrocystin-2.31 b-catenin/TCF signaling activity is also severely affected in the mutant renal primordia of Lrp6-null not ectopically activated in PKD1 and PKD2 mutants, two embryos. Pax2, Pax8,andLim1 play indispensable roles in different models of polycystic kidney, or renal cystic ciliop- early mesodermal regionalization, and genetic ablation of athy.32 Together, these contradictory observations suggest that these factors causes renal agenesis.39–41 Intriguingly, both not all cystic kidney have abnormal Wnt/b-catenin Pax2 and Pax8 can activate the Ret promoter.42 It is unclear signaling, but disruptions of Wnt/b-catenin signaling may trig- whether the diminished Pax2 and Pax8 expression in the Lrp6- ger cystic dysplasia in kidneys and related organs. deficient renal primordia is a direct consequence of the inac- tivated Wnt/b-catenin signaling or indirectly affected by the Ret as a Downstream Effecter of Lrp6-Mediated loss of Ret signaling. Mice deficient in Lim1 also display renal Wnt/b-Catenin Signaling in Renal Development agenesis associated with diminished expression of Ret in the Our results demonstrate that among a panel of critical ureteric bud.43 These results suggest a potential feedback in- nephrogenic factors, Ret expression is mostly diminished in teraction among Ret and Pax2, Pax8,orLim1 downstream of the mutant renal primordia at E10.5 onwards. The ureteric Lrp6/b-catenin signaling (Figure 9). We also show that Wnt11 buds start to outgrow and invade the metanephric mesen- was downregulated in the Lrp6-deficient metanephros at chyme at E10.5 and branch robustly by E12.5, and loss of E12.5. Wnt11 may regulate GDNF in metanephric develop- Ret signaling can prevent these critical processes and cause ment.18 It remains unknown whether Wnt11 acts through renal agenesis or severe renal defects.33 Our results showed a Lrp6 to regulate GDNF. We further show that Wnt4 and dose-dependent upregulation of Ret and its isoforms under Wnt9b expression were less affected in the renal primordia the Wnt agonist lithium treatment in vivo or in renal-derived of Lrp6-KO mice. Wnt9b acts upstream of Wnt4 to induce mIMCD3 cells. ChIP and luciferase assays also support that the metanephric to undergo a mesenchymal to Ret is a downstream target and effector of the Wnt/b-catenin epithelial transition (MET) for the formation of the epithelial signaling in nephrogenesis. A recent study reports that lithium renal vesicles.5 Interestingly, Ret expression does not change, upregulates Ret expression in mIMCD3 cells through both but GDNF, Wnt11,andWnt4 are downregulated in the

422 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 417–427, 2016 www.jasn.org BASIC RESEARCH

Figure 6. Ret expression modulated by small-molecule Wnt agonists in vivo and in vitro. (A) Real-time PCR results show in vivo upregulation of Ret mRNAs in E12.5 kidney primordia after maternal intraperitoneal injection of the Wnt agonist lithium chloride. (B) Real-time PCR results demonstrate a dose-dependent upregulation of Ret mRNAs stimulated by lithium chloride in the mouse kidney- derived mIMCD3 cells. (C) RT-PCR results show the expressions of different Ret isoforms upon different dosages of lithium chloride treatments in mIMCD3 cells. (D–F) Western blot results show the changes of Ret and phosphorylated (p)-Ret (Y1062) expressions upon different dosages of lithium treatments in Neuro-2a cells. *P,0.05; **P,0.01.

Wnt9b-KO mice.5 The Ret expression is also unchanged in the activation of downstream signaling, such as PLCg,PI3Kand Wnt4-KO mice.4 These results indicate that Wnt4 and Wnt9b mitogen-activated protein kinases (MAPK).15,17,47 Inactiva- may not act though Lrp6 and thus do not regulate Ret signal- tion of Ret-mediated MAPK signaling caused renal hypoplasia ing in ureteric bud branching, or that these Wnt ligands may or agenesis, while repression of Ret-mediated PLCg signaling have functional redundancy in regulating Lrp6/Ret signaling, resulted in cystic kidneys.15 Although we detected only MAPK which warrants future investigation. signaling that is regulated by Lrp6-mediated Wnt signaling in vitro, both Ret-mediated MAPK and PLCg cascades would be Mechanisms of Renal Cystic Dysplasia in Lrp6 and repressed in vivo as the Ret expression is dramatically dimin- Ret Mutants ished in the renal primordia of Lrp6 mutants. This data Our results show that mitotic cells decreased, while apoptotic supports a consistent role of Lrp6 and Ret in renal develop- cells increased significantly in the renal primordia of Lrp6- ment and also suggests that the repression of Ret signaling deficient embryos. These observations suggest that defective cascades is a key molecular mechanism underlying renal cystic Lrp6 signaling alters kinetics and apoptosis, which dysplasia in Lrp6 mutants. may subsequently disrupt metanephric branching and mor- In conclusion, our results provide strong evidence for the phogenesis, and thus may cause renal hypoplasia. Dysregula- role of Lrp6-mediated Wnt/b-catenin pathway in modulation ted epithelial cell proliferation and apoptosis have also been of a signaling network consisting of Ret cascades and related linked with cystic kidneys in and animal models.44–46 nephrogenic factors to control cell proliferation, survival, ure- At the molecular level, diminished Ret expression may cause teric bud branching, and related nephrogenic processes (Fig- renal cystic dysplasia in Lrp6 mutants. Ret has multiple tyro- ure 9). These novel results may also provide a molecular and sine residues and mediates cascades for cell proliferation, mi- genetic basis for interpreting the mechanisms of congenital gration, differentiation and growth by phosphorylation and renal defects and anomalies.

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Davis). The Ret knockout mice generated by the Costantini laboratory13 were acquired through The Jackson Laboratory (Stock #009085) and were mated with the Lrp6ßgeo mice to generate the double knockout mutants for the study. All mice were housed in the vivar- ium of the UC Davis. All research procedures using mice were approved by the UC Davis An- imal Care and Use Committee and conformed to the National Institutes of Health guidelines.

Hematoxylin and Eosin Staining, Immunofluorescence, and TUNEL Assays Hematoxylin and eosin staining and immuno- fluorescence were carried out on 10 mm frozen or paraffin-embedded tissue sections according to standard protocols. Primary antibodies against phospho-histone H3 (pHH3) (1:50; Technology) and appropriate Alexa fluorescence-conjugated secondary anti- bodies (1:500; Molecular Probes) were used. DAPI (1:500; Molecular Probes) was used for the nuclear counterstaining. TUNEL assays Figure 7. Alterations of Ret downstream factors modulated by Lrp6 knockdown and were performed using the Dead End Fluoromet- restored by Wnt agonists. Western blots were carried out for p-MAPK1/3, p-Akt, and p-PLCg in mIMCD3 (A, B) and Neuro-2a (C, D) cells after Lrp6-knockdown using ric TUNEL System (Promega) following the ’ siRNAs and treated with or without lithium chloride. Scrambled siRNAs are used as the manufacturer s instructions. The micrograph negative control for the knockdown. Only phosphorylated (p)-MAPK1/3 shows sig- images were acquired with a Nikon C1 confocal nificant changes after different treatments in both cell lines (B, D). *P,0.05. microscope. The total numbers of pHH3(+) or

Figure 8. Genetic interactions between Lrp6 and Ret in renal development. From the left to right panels, started with normal renal 2 2 2 2 2 2 2 organs in Lrp6+/ ;Ret+/ , increasingly more severe renal defects are seen in the single Lrp6 / ,thecompoundLrp6 / ;Ret+/ (with 2 2 2 2 varied severities), and the double KOs of Lrp6 / ;Ret / mutant mice around E17.5. Ad, adrenal; Bl, bladder; Go, gonads; Ki, kidney.

CONCISE METHODS TUNEL(+) cells were counted on each photographed renal primor- dial section and then were divided by the total cell numbers on the Animal Models same section area as the percentages of the positive cells. In each The Lrp6ßgeo mice were generated through a gene-trap approach by experiment at least five sections from three mutants or three litter- the Skarnes laboratory12 and conserved by the Mutant Mouse Re- mate controls at the same age were counted and averaged for statis- gional Resource Center at the University of California, Davis (UC tical analyses.

424 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 417–427, 2016 www.jasn.org BASIC RESEARCH

RNA Isolation and Real-Time PCR Total RNA was isolated from the E12.5 kidneys. Semiquantitative PCR wascarriedoutaccordingtothemanualoftheRoche480Real-TimePCR system using Faststart Universal SYBR GREEN master (ROX) (Roche Diagnostics, Zurich, Switzerland). Levels of mRNAs were normalized to glyceraldehyde-3- dehydrogenase (Gapdh)toallowcompar- isons among different experimental groups using the DCt method.23

Chromatin Immunoprecipitation and Luciferase Reporter Assay Chromatin immunoprecipitation (ChIP) assays were performed using isolated E14.5 kidneys from WT mice (in vivo) and the L cell line (in vitro) as described previously.23 Chromatin extraction and immunoprecipitation were performed according to the manufactur- Figure 9. Schematic summary of Lrp6 and Ret signaling in- er’s protocols using a ChIP assay (Upstate Biotechnology, Lake teraction in renal development and cystic dysplasia. We dem- Placid, NY). The equivalent of chromatin was immunoprecipitated onstrate that Lrp6-mediated Wnt/b-catenin signaling modulates the expression of Ret that subsequently activates MAPK1/3 with anti-b-catenin (Santa Cruz Biotechnology, Dallas, TX). Normal (Erk2/1) and PLCg signaling to promote ureteric budding, rabbit IgG was used as a negative control. Four Tcf/Lef1-binding sites branching, growth, and maturation. A recent study suggests (R1–R4; Figure 5A) in the Ret promoter were amplified by PCR with a reciprocal interaction between b-catenin and Ret during renal specific primers (Supplemental Table 1) after ChIP. Each assay was development. Lrp6 may also regulate GDNF (a ligand molecule repeated three to five times using different chromatin pools. for Ret) through Wnt11 or through b-catenin. Moreover, Lrp6- For luciferase assays, a serial of fragments of 2.4 kb, 1.3 kb, 800 bp, mediated Wnt/b-catenin signaling may regulate the expression 400bp, 180 bpflanking the R3, R2, R1, and SP1 binding site and 677 bp of nephrogenic transcription factors, such as Lim1, Pax2, and region flanking the R4 binding site (or the mutant promoter that was Pax8, which also interact with Ret. Disruption of the signaling mutated in the binding site) of mouse Ret promoter (Figure 5A) were network may cause a spectrum of renal defects, such as renal cloned to a luciferase reporter pGL2-Basic vector. These constructs agenesis, renal hypoplasia, and multicystic dysplastic kidney. were transiently transfected into HEK293T cells together with the Another Wnt coreceptor Lrp5 is involved in cytogenesis, while it remains unknown whether Lrp5 also plays a role in renal Renilla construct using the Lipotectamine 2000 reagent (Invitrogen). development and cystic dysplasia. The Wnt agonist lithium ion HEK293T cells were also transiently transfected with the expression can modulate the Ret signaling at downstream Lrp6 and up- vector pGL2-basic or constitutively active b-catenin/Lef1 construct. stream b-catenin. Solid arrows, demonstrated by the current Twenty-four hours after transfection, luciferase activities were detected study; dashed arrows, demonstrated by previous studies; X, using the Dual-Luciferase assay kit (Promega, Madison, WI) according disrupted gene activities; ?, potential but as yet unverified. to the manufacturer’s instruction.

RNA Knockdown and Western Blot Wholemount in situ Hybridization Lrp6 siRNA oligonucleotides were purchased from Thermo Fisher Sci- Embryos or whole renal organs were fixed in 4% paraformaldehyde entific (ON-TARGETplus SMARTpool siRNA; Thermo Fisher Scientific. (PFA) in diethylpyrocarbonate-treated PBS overnight and dehydrated The RNA oligonucleotides were transfected using Lipotectamine 2000 in graded methanol solutions and kept in 100% methanol at 280°C (Invitrogen) according to the manufacturer’sprotocolsata25nMfinal until used. Wholemount in situ hybridization was carried out as pre- concentration. Each target includes four siRNA oligonucleotides for Lrp6, viously described,48 using the DIG-labeled cRNA riboprobes (Sup- sequence 1: CGACAAAACUCCAUACGAA; sequence 2: GAGAUUA- plemental Table 1). Samples were photographed using a Carl Zeiss GAUGGACGAUCA; sequence 3: UAUCAAAGUUGGACGGACA; se- stereological microscope (Lumar V12). quence 4: GCUGUUAGCCCGACGGACA. Western blot was carried out on lysates prepared in modified radioimmunoprecipitation assay Maternal Administration of Lithium Chloride (RIPA) buffer using the following antibodies: rabbit anti-phospho-p44/42 In vivo stimulation of Wnt/b-catenin signaling pathway by lithium (MAPK1/3) (1:1000; Cell Signaling Technology), rabbit anti-phospho- was carried out as described.23 Pregnant dams were injected intra- Akt (1:1000; Cell Signaling Technology), rabbit anti-phospho-PLCg peritoneally with 200 mg/kg/day LiCl or the control NaCl solution on (1:500; Cell Signaling Technology), rabbit anti-Ret (1:200; Santa Cruz E8.5 to E11.5. The embryonic kidneys were dissected out at E12.5 for Biotechnology), rabbit anti-phospho-Ret (Y1062) (1:200; R&D Systems) subsequent experiments. and mouse anti-b-actin (1:1000; Santa Cruz Biotechnology). All antibod- ies were diluted in 5% BSA and incubated overnight at 4°C. Cell Lines The mIMCD3, L, and Neuro-2a cells were obtained from ATCC Statistical Analyses (Manassas, VA) and were grown in the Dulbecco’smodified eagle Two to five mutant embryos or littermate controls were used for each medium (DMEM, Invitrogen) with 10% fetal bovine serum. nonquantitative experiment. At least three mutant embryos or

J Am Soc Nephrol 27: 417–427, 2016 Lrp6 and Ret in Renal Disorders 425 BASIC RESEARCH www.jasn.org littermate controls were used in each quantitative analysis for 16. Richardson DS, Rodrigues DM, Hyndman BD, Crupi MJ, Nicolescu AC, statistical significance. Student’s t test was used for statistical compari- Mulligan LM: Alternative splicing results in RET isoforms with distinct fi – sons when appropriate, and differences were considered significant at traf cking properties. Mol Biol Cell 23: 3838 3850, 2012 , 17. Hoshi M, Batourina E, Mendelsohn C, Jain S: Novel mechanisms of P value 0.05. early upper and lower urinary tract patterning regulated by RetY1015 docking tyrosine in mice. Development 139: 2405–2415, 2012 18. Majumdar A, Vainio S, Kispert A, McMahon J, McMahon AP: Wnt11 and Ret/Gdnf pathways cooperate in regulating ureteric branching during ACKNOWLEDGMENTS metanephric kidney development. 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