Deletion of ADP Ribosylation Factor-Like Gtpase 13B Leads to Kidney Cysts

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Deletion of ADP Ribosylation Factor-Like GTPase 13B Leads to Kidney Cysts

† † † Yuanyuan Li,* Xin Tian, Ming Ma, Stephanie Jerman,* Shanshan Kong,* Stefan Somlo,* and Zhaoxia Sun*

Departments of *Genetics and †Internal Medicine, Yale University School of Medicine, New Haven, Connecticut

ABSTRACT The gene for ADP ribosylation factor–like GTPase 13B (Arl13b) encodes a small GTPase essential for cilia biogenesis in multiple model organisms. Inactivation of arl13b in zebrafish leads to a number of phenotypes indicative of defective cilia, including cystic kidneys. In mouse, null mutation in Arl13b results in severe patterning defects in the neural tube and defective Hedgehog signaling. Human mutations of ARL13B lead to Joubert syndrome, a ciliopathy. However, patients with mutated ARL13B do not develop kidney cysts. To investigate whether Arl13b has a role in ciliogenesis in mammalian kidney and whether loss of function of Arl13b leads to cystic kidneys in mammals, we generated a mouse model with kidney– specific conditional knockout of Arl13b. Deletion of Arl13b in the distal nephron at the perinatal stage led to a cilia biogenesis defect and rapid kidney cyst formation. Additionally, we detected misregulation of multiple pathways in the cystic kidneys of this model. Moreover, valproic acid, a histone deacetylase inhibitor that we previously showed slows cyst progression in a mouse cystic kidney model with neonatal inactivation of Pkd1, inhibited the early rise of Wnt7a expression, ameliorated fibrosis, slowed cyst progression, and improved kidney function in the Arl13b mutant mouse. Finally, in rescue experiments in zebrafish, all ARL13B allele combinations identified in patients with Joubert syndrome provided residual Arl13b function, supporting the idea that the lack of cystic kidney phenotype in human patients with ARL13B mutations is explained by the hypomorphic nature of the mutations.

J Am Soc Nephrol 27: 3628–3638, 2016. doi: 10.1681/ASN.2015091004

The cilium is a cell surface organelle that is uniquely by Polycystin inactivation, suggesting that Polycystins positioned to function as a cellular antenna that de- inhibit a novel cilia–dependent, cyst–promoting tects environmental signals and couples these signals pathway.10 Although the precise relationship between to cellular responses.1–6 Ciliary defects lead to a wide Polycystins and cilia is complex and not well under- range of human diseases collectively referred to as stood, all of these studies suggest that the cilium is a ciliopathies, including polycystic kidney diseases critical organelle in the pathogenesis of PKD. (PKDs). Autosomal dominant polycystic kidney dis- The gene for ADP ribosylation factor–like GTPase fi ease (ADPKD) is the most common form of PKD 13B (Arl13b), encoding a small GTPase, was rst fi – and caused by mutations in PKD1 or PKD2 encoding identi ed as a cystic kidney gene in a large scale Polycystin 1 and Polycystin 2, respectively.7,8 The role of cilia in PKD pathogenesis is highlighted by the Received September 10, 2015. Accepted March 8, 2016. facts that disruption of cilia biogenesis in mouse Published online ahead of print. Publication date available at models almost inevitably leads to kidney cyst forma- www.jasn.org. tion5,9 and that Polycystins are targeted to the cilium; fi fi Correspondence: Prof. Zhaoxia Sun, Department of Genetics, this speci ctraf cking highly correlates with the in Yale University School of Medicine, SHM I-329A, PO Box 208005, vivo function of the Polycystins. Interestingly, a 333 Cedar Street, New Haven, CT 06520. Email: zhaoxia.sun@ recent study revealed unexpectedly that complete re- yale.edu moval of cilia ameliorated cyst progression triggered Copyright © 2016 by the American Society of Nephrology

3628 ISSN : 1046-6673/2712-3628 JAmSocNephrol27: 3628–3638, 2016 www.jasn.org BASIC RESEARCH genetic screen in zebrafish.4 The identified mutant scorpion P14 (Figure 1C). The mutant mice die at around P60 (not shows body curvature and cystic kidney, typical of ciliary defects shown). Histologic sections of the kidney failed to detect cysts in zebrafish. Moreover, Arl13b protein is highly enriched within at P1 (not shown), minimal cysts at P7, significant cysts in the cilium and essential for cilia biogenesis in all tissues analyzed both the medulla and cortex region at P14, and severely cystic in zebrafish.4,11 Arl13b in mouse was also found to be essential kidneys at P17 (Figure 1D). Quantification of cystic index for cilia biogenesis, and it plays an important role in the pat- verified that cyst formation is already significant in mutant terning of the neural tube through regulation of Hedgehog mice at P14 (Figure 1E). To analyze kidney function in mutant signaling.12 mice, we perform BUN assay. Results show that the BUN level To study the function of Arl13b in the mammalian is significantly increased in mutant mice at P14 (Figure 1E), fl fl kidney, we generated Arl13b ox/ ox;Ksp-Cremice to achieve indicating a decline in kidney function. Combined, these re- kidney–specific conditional knockout. Deletion of Arl13b at sults suggest a rapid progression of PKD in the mutant mouse. the perinatal stage leads to rapid kidney cyst formation. We further show that the histone deacetylase (HDAC) inhibitor Arl13b Is Essential for Cilia Biogenesis in Collecting valproic acid (VPA) suppresses kidney disease progression in Duct Epithelial Cells fl fl Arl13b ox/ ox;Ksp-Cremice and that Wnt7a is an early target To confirm that cell type–specific knockout of Arl13b was of VPA treatment. achieved, immunostaining for Arl13b was performed and ver- In humans, mutations of ARL13B have been linked to ified that Arl13b expression is absent in the collecting duct Joubert syndrome (JS), an autosomal recessive disorder labeled by Dolichos Biflorus Agglutinin (DBA) in knockout characterized by a distinct cerebellar malformation.13,14 mice at P14 (Figure 2A, left panel). We then investigated cilia However, patients with JS do not develop cystic kidneys. biogenesis in the conditional knockout model. At P14, DBA– Using complementation experiments in zebrafish, we positive ductal cells were ciliated in control mice as shown by complete a dataset that shows that all ALR13B pathogenic the cilia marker antiacetylated tubulin (Figure 2A, right variants associated with human JS provide residual activity panel). In contrast, in mutant mice, most DBA-positive cells of Arl13b. In totality, our results support a conserved func- were devoid of cilia (Figure 2A, right panel). To further con- tion of Arl13b in cilia biogenesis and development in verte- firm this result, we performed immunostaining using anti– brate kidneys. g-tubulin as a basal body marker. At P7, DBA-positive cells were frequently ciliated as shown by antiacetylated tubulin in wild–type control mice (Figure 2B). In knockout mice, some RESULTS DBA-positive regions were already dilated (Figure 2B). Inter- estingly, in dilated regions, cilia were largely absent, whereas Inactivation of Arl13b at the Perinatal Stage in the cilia were still present in undilated DBA–positive regions (Figure Distal Nephron Leads to Rapid Cyst Formation and 2B), suggesting that cilia biogenesis defect in the mutant kidney Renal Failure in Mice correlates with kidney cyst formation. To generate a conditional allele of Arl13b,weacquired Arl13btm1a embryonic stem cell clones generated by the Euro- Cyst Formation Is Confined to the Distal Nephron in flox/flox pean Conditional Mouse Mutagenesis Program (EUCOMM). Arl13b ; Ksp-Cre Mice In this allele, exon 3 of Arl13b is flanked by two LoxP sites, To define the cellular origin of kidney cysts in this model, we and an FRT-LacZ-neo-FRT selection cassette is located in in- labeled P14 kidney sections with the collecting duct marker tron 2 (Figure 1A). Germline transmission of Arl13btm1a from DBA, the proximal tubule marker Lotus Tetragonolobus injected chimeras was confirmed by long-range PCR (not Lectin (LTL), and the thick ascending limb marker anti-Tamm– shown). Arl13btm1a/+ mice were crossed with a transgenic Horsfall protein (THP). At P14, cysts mainly form in the fl strain carrying an actin-driven FLPe15 to generate the Arl13b ox collecting duct (Figure 2C, columns 1 and 2). Consistent fl fl allele. Arl13b ox/ ox mice are viable with no discernible pheno- with the lack of Ksp-Cre expression in the proximal tubule, types (not shown). this segment labeled by LTL does not appear to be cystic (Figure To generate kidney-specific knockout of Arl13b, we crossed 2C, column 1). Interestingly, the THP–positive thick ascending fl Arl13b ox/+ mice with a Ksp-Cre strain.16 Ksp-Cre mediates limb, which expresses Cre, does not appear to be cystic at this recombination in distal segments of the nephron after stage (Figure 2C, column 2). However, at P28, cysts are de- fl fl E11.5.17 Arl13b ox/ ox;Ksp-Cremice were born in normal tected in the THP–positive thick ascending limb and the distal Mendelian ratios but show progressively enlarged kidneys convoluted tubule labeled with anti-Parvalbumin (Figure 2C, (Figure 1B). At postnatal day 1 (P1) and P7, the size of the columns 3 and 4). Combined, the overall distribution pattern kidney in mutants is similar to that in the control (data not of cysts is consistent with the expression pattern of Ksp-Cre.17 shown). From P14, the size is obviously enlarged, and by P17, The difference in cyst progression in different segments may the size is significantly increased (Figure 1B). Consistent with represent differential Cre expression in these regions. Alterna- the enlargement of the kidney, the kidney-to-body weight tively, the collecting duct may be more susceptible to cyst (KBW) ratio is significantly increased in mutant mice by growth.18

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of DBA–positive cyst–lining cells displayed nuclear BrdU incorporation compared with around 2.8% positive nuclei in DBA- positive cells in control kidneys (Figure 3, A and B), suggesting a signiﬁcant increase of cell proliferation.

Arl13b Inactivation Leads to Kidney Fibrosis Cystic kidneys are frequently fibrotic. To investigate whether Arl13b inactivation eventually leads to fibrosis in the kidney, we performed trichrome staining on kidney sections to analyze potential collagen deposition. At P21, we failed to detect any significant increase of trichrome signal in the mutant kidney (data not shown). In contrast, at P28, increase of trichrome staining was obvious in the mutant kidney (Figure 3C). We further stained kidney sections with anti-smooth muscle actin, a marker of myofibroblasts. Results showed dramatic increase in the mutant kidney at P28 (Figure 3D). Combined, these results show that cyst formation caused by Arl13b inactivation precedes overt kidney fibrosis in the later stage of the disease progression.

Multiple Signaling Pathways Are Misregulated in the Cystic Kidney of flox/flox Arl13b ; Ksp-Cre Mice Multiple pathways have been implicated in cystic kidney progression. In the Ift140 model, targets and components of the Wnt pathway, HH pathway, and Hippo pathway are upregulated.19 To investigate Figure 1. Distal nephron–specific inactivation of Arl13b in Arl13bflox/flox;Ksp-Cre potential mechanisms for cyst progres- fl fl mice leads to rapid progression of PKD. (A) Diagram of targeting vector. Exons are sion in Arl13b ox/ ox;Ksp-Cremice, we showed by black boxes. FRT indicates FLPE recombinase sites, and loxP indicates Cre performed time course for the expres- flox/flox recombinase sites. (B) Gross morphology of kidneys from P14 and P17 Arl13b ; sion level of candidate genes through Ksp-Cre mice compared with control siblings. (C) Increasing KBW ratio in mutants quantitative PCR (qPCR) using P7–P28 during the neonatal stage (n=3 for each stage and each genotype). (D) Hematoxylin/ whole-kidney lysates. At P28, the expres- eosin-stained sections from control and mutant kidneys on P7, P14, and P17. Scale bar, 2 mm. (E) Significant cyst formation quantified by cystic index and increased BUN sion of two Wnt ligands, Wnt7a and levels in knockout mice at P14 (n=3 for each genotype). Control indicates the wild Wnt10a, is dramatically increased (Figure type. KO, knockout. *P,0.05; **P,0.01. 4). The Wnt pathway targets Lef1 and Axin2 are also significantly upregulated. The expression of c-Myc, a potential target Arl13b Inactivation Leads to Cell Overproliferation of the Wnt pathway that has been implicated in PKD,20–23 Because cell overproliferation is one of the mechanisms of cyst also increases dramatically. In addition, the Hedgehog path- progression, we investigated cell proliferation in Arl13b mutant way ligands Shh, Ihh,andDhh are upregulated as well as HH kidney compared with controls using a bromodeoxyuridine pathway targets Gli1, Gli2,andGli3.Similarly,theexpression (BrdU) incorporation assay. At P14, kidney samples were col- of Hippo pathway targets Birc3, Ctgf,andInhbA also rises. lected 3 hours after intraperitoneal (ip) injection of BrdU. Finally, we analyzed the expression level of complement C3,an Immunostaining on kidney sections revealed that around 14% innate immune response gene that is upregulated in both

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Wnt7a and Wnt10a, shows relatively stable or even slightly increased expression in control kidneys between P7 and P14 followed by a decrease at later time points (Figure 4). Their expression in the mutant kidney increases more dramatically at P14 and persists at P28. Complement C3 expression represents yet another pattern: although it increases from P7 to P14 and relatively stabilizes afterward in controls, it rises dramatically over the entire period in mutant kidneys (Figure 4). Combined, these results suggest that multiple pathways are misregulated in the mutant kidney in a complex pattern. Com- pared with age–matched wild–type kidneys,inmutantkidneys,theexpression of c-Myc, Wnt7a, Wnt10a, Gli3,andInhbA is signiﬁcantly higher by P14, the expression of Axin2, RhoU, Ihh, Dhh, Gli2, Ctgf, and Birc3 is elevated by P21, and the rest become signiﬁcantly increased at P28 (Figure 4).

VPA, an HDAC Inhibitor, Can Partially Suppress PKD Progression in flox/flox Arl13b ;Ksp-CreMice Figure 2. Arl13b is essential for cilia biogenesis, and its depletion leads to cyst for- In a previous study, we found that the mation in mouse kidney. (A) P14 kidney sections shown in the collecting duct are histone deacetylase inhibitor (HDACI) labeled by DBA (red) and Arl13b signal (green; left panel), and cilia signal labeled by VPA slowed cyst progression and improved antiacetylated tubulin (A-tub; green; right panel) is lacking in the mutant kidney kidney function in a mouse ADPKD model (knockout [KO]). Nuclei are labeled with DAPI in blue. Although insets in control show on the basis of neonatal inactivation of cilia in the collecting duct, insets in KO show cilia outside of the collecting duct. Arrows 26 point to cilia. Cy, cyst. (B) P7 kidney sections show basal body labeled with anti– Pkd1. We, therefore, investigated the ex- g-tubulin (g-tub; green) and cilia labeled with antiacetylated tubulin (A-tub; red) in pression level of Hdac1 in the mutant control and mutant (KO) mice. Arrow points to a cilium in DBA-positive (blue) seg- kidney. Our results show that, at P28, the ment. Arrowheads point to basal bodies without cilium in a dilated DBA–positive expression level of Hdac1 is significantly region (Di). Inset shows a basal body without cilia. (C) Cyst formation is confined to the increased, although there is no significant distal nephron in Arl13bflox/flox;Ksp-Cremice. DBA marks the collecting duct. The change at P7 (Figure 5A). We further in- proximal tubule is labeled with LTL, the medullary thick ascending limb is labeled by vestigated whether VPA can ameliorate dis- fl fl THP, and the distal convoluted tubule is labeled with anti-Parvalbumin (Pvalb). Nuclei ease progression in Arl13b ox/ ox; Ksp-Cre are labeled with DAPI in blue. Control indicates the wild type. Scale bars, 20 mmin mice. Results show that mutant mice treated A and B; 100 mminC. with VPA from P10 to P25 show significantly reduced kidney size (Figure 5, B autosomal recessive PKD and ADPKD models,24,25 and found and C) and KBW ratio (Figure 5D) compared with vehicle- that it is significantly increased as well (Figure 4). treated samples. Moreover, BUN level is also significantly Consistent with previous findings,19 our results show that reduced, indicating improved kidney function (Figure 5D). the expression of most of these signaling pathway components Furthermore, the expression of the Wnt, Hedgehog, and steadily decreases during normal kidney development at the Hippo pathway components analyzed above is all reduced neonatal stage, including c-Myc, Lef1, Axin2, RhoU, and many by this treatment in mutant kidneys (Supplemental Figure Hedgehog pathway components (Figure 4). Within this group 1). In contrast, the effect of the same VPA treatment of genes, c-Myc is unique in that its expression increases by P14 has minimal effect on kidney size, KBW ratio, or gene ex- in the mutant kidney (Figure 4). In contrast, some of the pression in wild-type controls (Figure 5, E and F, Supple- Hedgehog pathway components, including Gli1, Gli2,and mental Figure 1). Combined, these results show that VPA fl fl Shh, show a decrease between P7 and P21 and rise at P28 in can slow down disease progression in Arl13b ox/ ox;Ksp-Cre mutant kidneys (Figure 4). A second group of genes, including mice.

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function of Y86C and formally test the function of the truncation mutation W82X, we performed zebrafish rescue experiments. As shown in our previous study, injection of mRNA encoding wild-type Arl13b fused with eGFP fully rescues the zebrafish scorpionhi459 mutant (Figure 6A). In contrast, arl13b-W82X mRNA could not rescue either body curvature or kidney cyst phenotypes (Figure 6A). Expression of Y86C reduced but did not fully eliminate the percentage of phenotypic embryos (Figure 6A). To verify that the reduction of phenotypic embryos is caused by partial recue of the mutants, we genotyped embryos with nor- Figure 3. Increased cell proliferation and fibrosis in the kidney of Arl13bflox/flox;Ksp- mal body axis and no kidney cyst. Results Cre mice. (A) Kidney sections stained with anti-BrdU (green) and DBA (red) at P14. (B) showed that some of the embryos with Quantitation of BrdU-positive nuclei in DBA-positive cells (n=3 animals of each ge- wild-type appearance are rescued homo- notype). (C) Trichrome stain indicates collagen deposition (green) in mutant kidneys at zygous mutants (Figure 6B), supporting the P28. (D) Interstitial cells show increased smooth muscle actin (SMA; green) expression hypothesis that Y86C is, indeed, a hypomor- in mutant kidney compared with controls at P28. The collecting duct is labeled with DBA in red. Control indicates the wild type. cy, Cyst; KO, knockout. Scale bars, 50 mm phic mutation that retains residual activity. in A; 20 mm in C and D. **P,0.01. Thus, all ARL13B genotypes associated with JS could provide partial activity of Arl13b. Western blot using anti-eGFP showed VPA Treatment Ameliorates Fibrosis and Suppresses that wild-type Arl13b and the three missense mutants are flox/flox Early Transcriptional Changes in Arl13b ; Ksp-Cre expressed at similar levels when equal amounts of mRNAwere Mice injected, whereas the level of W82X is much higher than the Because HDACIs have been shown to suppress fibrosis, includ- wild type (Figure 6C). ing renal fibrosis,27–37 we investigated the effect of VPA treat- We further investigated the ability of Y86C and W82X to fl fl ment on fibrosis in Arl13b ox/ ox; Ksp-Cre mice. VPA treatment rescue cilia biogenesis defects in scorpionhi459 mutant using a from P10 to P25 significantly reduced collagen deposition in previously established assay.11 In the wild–type zebrafish kid- mutant mice as shown by trichrome staining (Figure 5G). ney duct, cilia bundle together and appear as a line between Smooth muscle actin was also significantly decreased in the in- somite and yolk in whole-mount embryos stained with terstitium of VPA–treated mutant kidneys (Figure 5H). antiacetylated tubulin. In contrast, in mutant embryos, stun- To tease out the potential mechanism for HDACI–mediated ted cilia appear as spots (Figure 6D, column 1). Injection of disease inhibition, we sought to identify early transcriptional wild-type and Y86C arl13b mRNA restored cilia bundle, changes in the mutant kidney. qPCR using P10 kidney for genes whereas W82X mRNA failed to do so (Figure 6D). Combined, that are misregulated at P14 revealed that a single gene, Wnt7a, the ability of different versions of arl13b to rescue the mor- was significantly upregulated at the whole–kidney lysate level phologic defects of scorpionhi459 mutant correlates with their in mutants (Figure 5I). The expression of Wnt10a was also in- ability to restore ciliogenesis in this mutant. creased but did not reach a statistically significant level (Figure Previous cell culture study suggests that R79Q and R200C 5I). Interestingly, VPA treatment from P7 to P10 effectively could still traffic to cilia. We further investigated the subcel- abolished the increase of Wnt7a and Wnt10a expression specif- lular localization pattern of all of the mutant proteins in vivo in ically in the mutant kidney (Figure 5J). developing zebrafish embryos by injecting mRNA into wild- type embryos. Immunostaining with anti-eGFP on injected The Nature of ARL13B Mutations Associated with JS in embryos showed that all three missense mutant proteins Humans could still traffic effectively to cilia, similar to wild-type Three mutant genotypes, R79Q/R79Q, W82X/R200C, and Arl13b (Figure 6E). In contrast, W82X is diffusely localized Y86C/Y86C, have been linked to JS.13,14 Although R79Q, throughout the cell (Figure 6E). R200C, and Y86C are single-amino substitutions, W82X is a truncation. It was reported that, although R79Q and R200C can partially rescue both the body curvature and cystic kidney DISCUSSION phenotypes of the zebrafish scorpionhi459 mutant, Y86C was not able to rescue the body curvature phenotype, although it reduced Although originally isolated as a cystic kidney gene in zebrafish, the percentage of kidney cyst formation.13,14 To clar ify the the role of Arl13b in mammalian kidney development and

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Figure 4. Multiple pathways are misregulated in cystic kidneys of Arl13bflox/flox;Ksp-Cremice. qPCR analysis of genes involved in Wnt (cMyc, Wnt7a, Wnt10a, Lef1, Axin2,andRhoU), Hedgehog (Shh, Ihh, Dhh, Ptch1, Ptch2, Gli1, Gli2,andGli3), Hippo (InhbA, Ctgf,and Birc3), and innate immunity (C3) pathways in control (Con) and knockout (KO) kidneys at P7, P14, P21, and P28. Gene expression is normalized with that of Gapdh. Unit 1 is defined as the expression level in wild-type samples at P7 (n=4 kidneys from two mice). Error bars show SDs. *P,0.05; **P,0.01. homeostasis was unclear until very recently. The lack of expression of 2%–10% of expressed genes, with roughly kidney symptoms in human patients with defective equal numbers of genes either up- or downregulated.40–45 Arl13b raises the possibility that there may be tissue and Past studies provide some tantalizing clues for potential tar- species variations in its function. To investigate the func- gets of HDACI treatment. It was shown that, in response to tion of Arl13b in mammalian kidney development directly, flow, Hdac5 is exported out of the nucleus, leading to acti- we generated a conditional knockout mouse of Arl13b.De- vation of Mef2c-mediated transcription.39 Moreover, letion of Arl13b in the distal nephron at the perinatal stage among the genes that we analyzed, c-Myc shows abnormally leads to rapid progression of cystic kidney disease, consis- increased expression in Arl13b mutant kidney at P14. A re- tent with a very recent report that conditional inactivation cent study reported that JQ1, a small molecule inhibitor of of Arl13b via Nestin-Cre leads to kidney cyst,38 showing its the epigenetic regulator Brd4, reduces c-Myc expression and essential role in developing and maintaining the elaborate slows disease progression in an orthologous mouse ADPKD three–dimensional epithelial structure of the mammalian model.46 kidney. HDACIs have been used to suppress fibrosis, including We previously showed that VPA ameliorated cyst progres- renal fibrosis.27–37 Our results show that VPA treatment sup- fl fl sion and improved kidney function in a neonatal mouse presses interstitial fibrosis in Arl13b ox/ ox;Ksp-Cremice. VPA fl fl ADPKD model based on conditional inactivation of Pkd1.26 treatment also inhibited the rise of Wnt7a in Arl13b ox/ ox; Additionally, trichostatin A, another HDACI, was shown to Ksp-Cre mice at a very early stage during cystogenesis. Inter- suppress cyst progression in a mouse Pkd2 knockout model.39 estingly, Wnt7a is upregulated in renal epithelial cells in a 2 2 In this study, we provide evidence that VPA is also effective Pkd1 / model.47 Wnt signaling has been reported to pro- fl fl against PKD progression in the Arl13b ox/ ox; Ksp-Cre model. mote renal fibrosis,48 and increasing evidence supports the Combined, these results show that HDACIs can slow disease notion that inflammation and fibrosis contribute to PKD pro- progression in not only ADPKD models based on inactivation gression.49–51 Combined, these results suggest that Wnt7a is of Polycystins but also, PKD models based on ciliogenesis de- an early target of HDACI treatment. fects. A remaining question is why patients with JS and ARL13B Through chromatin remodeling, HDACs regulate tran- mutations do not develop kidney cyst. Species difference could scription directly or indirectly. From the budding yeast be a factor. Alternatively, the identified mutations could be to mammalian cells, HDACIs were reported to change the hypomorphic alleles, and the residual activity could support

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normal kidney development, whereas some other tissues (e.g., the cerebellum) could be more sensitive to the level of Arl13b activity. Our results in zebrafish suggest that all of the identified JS genotypes provide some level of Arl13b activity, supporting the hypomorphic allele hypothesis, which is also consistent with the observation that a complete or severe loss of function of Arl13b in either zebrafish or mouse is incompatible for sur- vival beyond early embryonic stages.4,12 Arl13b is required for cilia biogenesis in multiple tissues and multiple organisms. Here, we show that the mammalian renal epithelial cell is no exception. Previous work suggests that Arl13b is highly enriched on cilia and that its ability to traffic to cilia is essential for the in vivo function of this protein.4,11 Combined, these results suggest that defective cilia biogenesis is a major mechanism for kidney cyst formation in Arl13b mutants. Arl13b is an ARF family small GTPase with the GTPase domain at the N terminus followed by a C-terminal region containing a coiled coil domain. Both regions are essential for its ciliary localization and in vivo function.11 Not surprisingly, the W82X truncation, which removes more than one half of the GTPase domain and the entire C–terminal region, abolishes its function. Previous structural study on the Arl13b homolog in Chlamydomonas reinhardtii suggests that R79 and R200 res- idues may be involved in stabilizing the conformation of activated Arl13b.52 Inter- estingly, although located in the small Figure 5. The HDACi VPA suppresses disease progression in Arl13bflox/flox;Ksp- GTPase domain and conserved from Cre mice. (A) qPCR on Hdac1 expression in wild-type (WT; Con) and mutant (KO) Caenorhabditis elegans to human, Y86 is fi kidney. Unit 1 is de ned as the expression in WT sample at P7 (n=3 mice). (B) not conserved in C. reinhardtii.Theef- Kidney morphology of vehicle and VPA-injected mutant (KO) mice at P25. (C) fective localization of Y86C to cilia sug- Representative hematoxylin/eosin–stained sections of kidneys from vehicle and gests that its reduced function is not caused VPA-treated mutant mice at P25. (D) KBW ratio, cystic index, and BUN in VPA and fi vehicle-treated mutant (KO) mice at P25 (n=3 mice). (E) Kidney morphology in by traf cking defects. Moreover, all three vehicle and VPA–injected WT mice at P25. (F) KBW ratio in vehicle and VPA– missense mutants seem to be as stable as injected WT mice at P25. (G) Trichrome staining of P25 kidney section of vehicle the wild-type protein. Agents stabilizing (Con) and VPA–treated Arl13bflox/flox; Ksp-Cre mice. (H) P25 kidney section of the conformation of the GTP-bound vehicle (Con) and VPA–treated Arl13bflox/flox; Ksp-Cre mice stained with anti- form of Arl13b could potentially increase smooth muscle actin (green) and DBA (red). (I) Wnt7a expression is upregulated in or restore the function of these missense mutant (KO) kidney at P10 as shown by qPCR analysis. Gene expression is nor- mutants. malized with that of Gapdh.Unit1isdefined as the expression level in WT samples (n=3 kidneys from three mice). (J) qPCR analysis of Wnt7a and Wnt10a expression in mutant (KO) and WT kidney treated with vehicle or VPA. Unit 1 is CONCISE METHODS defined as the expression level in vehicle–treated WT samples (n=3 kidneys from three mice). Error bars show SDs. cy, Cyst. Scale bars, 20 mminGandH.*P,0.05; **P,0.01. Animal Care Ethics All zebrafish and mouse experiments were performed in Yale University School of Medicine

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fl Arl13b ox allele by mating with the FLPe transgenic mice. Ksp-Cre was used to delete exon 3, creating Arl13b knockout specifically in the kidney. Genotyping of Arl13b alleles was performed using primers 59-CTCTTTGCAGGATGCCCTTC-39 and 59-AAGGGTTTTTCTCTGTAGCAC-39. Although the wild-type allele is identified by a fl 437-bp band, the Arl13b ox allele is identified by a 594-bp band.

Zebraﬁsh Husbandry Zebraﬁsh were maintained according to standard protocols.53 Embryos were obtained through natural spawning.

Generation of Zebraﬁsh arl13b Mutant Constructs Zebraﬁsh arl13b-R79Q-GFP, arl13b-Y86C-GFP, arl13b-R200C-GFP,andarl13b-W82X-GFP were generated through site-directed mutagenesis using previously described pCS2+arl13b11 as a template.

Zebrafish Embryo Microinjection and Rescue Assay Capped mRNA was synthesized in vitro using the mMESSAGE mMACHINE Kit (Ambion) following the manufacturer’s instructions. One cell– stage zebrafish embryos from arl13b+/hi459 incross were injected with 250 pg capped mRNA. Ventral body curvature and pronephric cyst were scored at 3 days postfertilization. Genotyping PCR was fi Figure 6. Function and localization of four mutant Arl13b identi ed in patients with JS performed using three primers: one viral specific assayed in zebrafish. (A) Y86C-GFP could rescue the body curvature (left panel) and (59-CTGTTCCATCTGTTCCTGAC-39) and two kidney cyst (enlarged in the right panel; white arrows) of scorpionhi459 mutants. The for genomic regions flanking the proviral inser- truncated form W82X could not rescue either phenotype. (B) PCR genotyping results. 9 9 Although the wild-type (WT) band identifies the WT allele, the mutant (Mu) band tion (5 -GCACTTAACGCCTCCTCTTTGTATG-3 9 9 identifiesthemutantallele.Eachlaneisforasingle embryo with WT morphology. (C) and 5 -CCGTGCGCACAGTCAGGG-3 ). Mutant Western blot showing the protein levels of eGFP-tagged WT and mutant Arl13b at product is around 400 bp, whereas wild-type band 12-hour postfertilization. Anti–b-tubulin (tub) is used as a loading control. (D) Side is around 350 bp. views of the trunk region of day 2 scorpionhi459 mutant embryos subjected to mRNA injection. Arrows point to cilia bundles in the kidney duct. (E) Side views of the Immunostaining pronephric duct in embryos injected with mRNA encoding GFP-tagged WT and Mouse kidneys were fixed by heart perfusion mutant Arl13b. Cilia are labeled with antiacetylated tubulin in red. WT inj, Y86C inj, with 4% PFA, fixed in 4% PFA overnight at 4°C, and W82X inj indicate injected with WT, Y86C, and W82X Arl13b-GFP mRNA, re- embedded in OCT, and cryosectioned at 5 mm spectively. A-tub, antiacetylated tubulin; uninj, uninjected scorpionhi459 mutant. thickness. Scale bar, 10 mm. The following primary antibodies and lectins are used: Rhodamine DBA (RL-1032; in accordance with Yale University Institutional Animal Care and Use 1:100;VectorLaboratories,Burlingame,CA),FluoresceinLTL(FL- Committee guidelines. 1321; 1:300; Vector Laboratories), THP (AF5175; 1:500; R&D Sys- tems, Minneapolis, MN), anti-Parvalbumin mAb (MAB1572; Mouse Breeding 1:200; EMD Millipore), Rabbit anti-Arl13b (17711–1-AP; 1:100; Arl13btm1a embryonic stem cell clones were purchased from the Proteintech), mouse monoclonal antiacetylated tubulin (clone 6– EUCOMM (http://www.mousephenotype.org) and then, injected 11B-1; 1:5000; Sigma-Aldrich, St. Louis, MO), mouse a–smooth into C57/BL6J blastocysts to generate chimeric mice. Chimeric ani- muscle actin (ab7817; 1:100; Abcam, Inc., Cambridge, MA), and mals were mated to C57/BL6J mice. Arl13btm1a was converted to anti–g-tubulin (T3559; 1:500; Sigma-Aldrich).

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For Arl13b-GFP localization in zebrafish, 24-hour postfertilization (Bio-Rad, Hercules, CA). Previously published PCR primers19 embryos were fixed in Dent’s fixative (80% methanol and 20% were used for Gapdh, c-Myc, RhoU, Wnt7a, Wnt10a, Lef1, Axin2, DMSO). Goat anti–GFP antibody (600–132–215; Rockland Immu- C3, Birc3, Ctgf, InhbA, Gli1, Gli2,andGli3, NCBI primer blast was nochemicals Inc., Gilbertsville, PA) was used at 1:500. Embryos used to design primers for the rest of the genes. They include were flat mounted using Fluoro-Gel (17985–1; Electron Microscopy Hdac1: 59-CTCACCGAATCCGCATGACT-39 and 59-GCTTCA- Sciences). CAGCACTTGCGAC-39; Shh: 59-AGACCGGCTGATGACTCA- For cilia biogenesis rescue experiment, zebrafish embryos from GAG-39 and 59-GCTCGACCCTCATAGTGTAGAGAC-39; Ptch1: crosses between heterozygous carriers of scorpionhi459 were injected 59-TGGCCGCATTGATCCCTATC and 59-ACACAGGGGCTTGT- with mRNA encoding different versions of Arl13b and fixed in Dent’s GAAACA-39; Ptch2:59-TGGTAATCCTCGTGGCCTCT-39 and fixative at day 2 postfertilization. Mouse antiacetylated tubulin was 59-AACCAGCAAGCATGAGCAGA-39; Ihh:59-CGGCTTCGACTG- used at 1:2000, and HRP goat anti–mouse IgG antibody (Pierce, GGTGTATT-39 and 59-CATCACTGAAGGTGGGGGTC-39;and Rockford, IL) was used at 1:1000. DAB reaction was used for detec- Dhh:59-CGTACCCAACTACAACCCCG-39 and 59-GTGGAGT- tion of HRP signal. Brown color was visualized and photographed in GAATCCTGTGCGT-39. Gene expression was normalized to that of bright field under a dissecting scope. Gapdh. All immunofluorescence pictures were taken with a Zeiss Axioplan 2 Microscope System (Carl Zeiss GmbH, Jena, Germany) VPA Treatment using AxioVision Rel. 4.8 software. VPA was purchased from Acros Organics. Daily ip injection was performed during the duration described in specific experiments. Protein Extraction and Western Blot The VPAtreatment group received 200 mg/kg VPA in 15% DMSO and Zebrafish embryo lysate was obtained as published before.54 Briefly, 85% saline, whereas the vehicle control group received the same embryos were deyolked, homogenized in SDS sample buffer, and volume of 15% DMSO and 85% saline without VPA. In total, six mice boiled at 95°C for 10 minutes, and resultant lysates were cleared (three for VPA and three for vehicle) were analyzed. by centrifugation at 12,0003g for 5 minutes. Blots were incubated with primary antibodies (anti–b-tubulin; T4026; 1:5000; Sigma- Statistical Analyses Aldrich; mouse anti-GFP; clones 7.1 and 13.1; 1:1000; Roche, The t test between paired samples (wild-type versus mutant or Basel, Switzerland) followed by HRP–conjugated secondary anti- vehicle versus VPA treated) was performed using Excel (Microsoft, bodies (Jackson ImmunoResearch Laboratories, West Grove, PA), Redmond, WA). and signals were developed by Western Lightning Plus ECL Kit (PerkinElmer, Waltham, MA). ACKNOWLEDGMENTS Proliferation Assay Proliferation was assayed through BrdU incorporation. Kidneys were harvested 3 hours after ip injection of 100 mg/kg body wt BrdU We thank members of the Brueckner laboratory, the laboratory of solution (10280879001; Roche). Kidneys were fixed with 4% PFA S.S., and the laboratory of Z.S. for helpful discussions; D. Shao and and embedded in OCT. Cryosections were cut at 5 mm, and BrdU S. Mentone for histology assistance; and the Yale Mouse Metabolic incorporation was detected using anti-BrdU antibody (347580; BD Phenotyping Center (grant U24 DK059635) for BUN analysis. Biosciences, San Jose, CA). About 1300 DBA-positive cells were counted This work was supported by the National Institutes of Health to calculate the BrdU–positive cell ratio in the collecting duct. (NIH) grant R01 DK092808, research grant RSG-10-247-01-DDC from the American Cancer Society (to Z.S.), NIH grant 1P30DK090744 Animal Core, NIH grant R01 DK100592 (to S.S.), and research grant Cystic Index Measurement Cystic index was measured as previously published.26 Briefly, cyst 191G14a (to Z.S.) from the PKD Foundation. S.J. is supported by NIH formation was quantified in sagittal sections of kidneys. Two sections grant T32 DK07356 Investigative Training in Hepatology. were analyzed for each animal. Hematoxylin/eosin-stained sections were scanned using the scan slide module in Metamorph v.7.1 acqui- sition software (Universal Imaging). Cystic and noncystic areas are DISCLOSURES measured by the integrated morphometric feature in Metamorph. None. Cystic index is defined as cystic area to total kidney area in each section. REFERENCES qPCR analysis Mouse kidneys were harvested at P7, P10, P14, P21, and P28. Total 1. Hildebrandt F, Attanasio M, Otto E: Nephronophthisis: Disease – RNA isolation and first–strand cDNA synthesis were performed mechanisms of a ciliopathy. J Am Soc Nephrol 20: 23 35, 2009 54 2. Pazour GJ, Baker SA, Deane JA, Cole DG, Dickert BL, Rosenbaum JL, as described previously. qPCR reactions were performed with Witman GB, Besharse JC: The intraflagellar transport protein, IFT88, is 3 the KAPA SYBR Fast qPCR Kit Master Mix (2 ) Universal (Kapa essential for vertebrate photoreceptor assembly and maintenance. 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3638 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 3628–3638, 2016 Supplemental materials:

Figure S1. The impact of VPA treatment on gene expression in mutant (KO) or control mice at P25. Gene expression is normalized with that of Gapdh. Unit 1 is defined as the expression level in vehicle treated samples. Error bars show standard deviations. n=3 mice. **p<0.01, *p<0.05.