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Renal-Retinal Sdccag8 Regulates DNA Damage Response Signaling

† ‡ | Rannar Airik,* Gisela G. Slaats, Zhi Guo, Anna-Carina Weiss,§ Naheed Khan, †† ‡‡ Amiya Ghosh,¶ Toby W. Hurd,** Simon Bekker-Jensen, Jacob M. Schrøder, ‡ ‡‡ Steve J. Elledge, Jens S. Andersen, Andreas Kispert,§ Maddalena Castelli,§§ † || Alessandra Boletta,§§ Rachel H. Giles, and Friedhelm Hildebrandt*

*Division of Nephrology, Boston Children’s Hospital, Boston, Massachusetts; †Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht, The Netherlands; ‡Department of Genetics, Harvard Medical School, Division of Genetics, Brigham and Women’s Hospital, Boston, Massachusetts; §Institute of Molecular Biology, Hannover Medical School, Hannover, Germany; |Kellogg Eye Center, Department of Ophthalmology and Visual Sciences, University of Michigan Medical School, Ann Arbor, Michigan; ¶Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan; **Medical Research Council Human Genetics Unit, Medical Research Council Institute of Genetics and Molecular Medicine, Western General Hospital, Edinburgh, United Kingdom; ††Novo Nordisk Foundation Center for Research, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; ‡‡Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; §§Division of Genetics and Cell Biology, Dulbecco Telethon Institute, San Raffaele Scientific Institute, Milan, Italy; and ||Howard Hughes Medical Institute, Chevy Chase, Maryland

ABSTRACT Nephronophthisis-related (NPHP-RCs) are developmental and degenerative kidney diseases that are frequently associated with extrarenal pathologies such as retinal degeneration, obesity, and intellectual disability. We recently identified mutations in a gene encoding the centrosomal protein SDCCAG8 as causing NPHP type 10 in humans. To study the role of Sdccag8 in disease pathogenesis, we generated a Sdccag8 gene-trap mouse line. Homozygous Sdccag8gt/gt mice lacked the wild-type Sdccag8 transcript and protein, and recapitulated the human phenotypes of NPHP and retinal degeneration. These mice exhibited early onset retinal degeneration that was associated with rhodopsin mislocalization in the photoreceptors and reduced cone cell numbers, and led to pro- gressive loss of vision. By contrast, renal histologic changes occurred later, and no global ciliary defects were observed in the kidneys. Instead, renal pathology was associated with elevated levels of DNA damage response signaling activity. Cell culture studies confirmed the aberrant activation of DNA damage response in Sdccag8gt/gt-derived cells, characterized by elevated levels of gH2AX and phosphorylated ATM and cell cycle profile abnormalities. Our analysis of Sdccag8gt/gt mice indicates that the pleiotropic phenotypes in these mice may arise through multiple tissue-specificdisease mechanisms.

J Am Soc Nephrol 25: 2573–2583, 2014. doi: 10.1681/ASN.2013050565

Nephronophthisis-related ciliopathies (NPHP-RCs) Received May 31, 2013. Accepted February 25, 2014.

(Online Mendelian Inheritance in Man [OMIM] Published online ahead of print. Publication date available at 256100) are heterogenetic autosomal recessive dis- www.jasn.org. orders that feature nephronophthisis, a degeneration Correspondence: Dr. Friedhelm Hildebrandt, Division of Ne- 1 . disorder of the kidney. To date, mutations in 20 phrology, Boston Children’s Hospital, 300 Longwood Avenue, NPHP-RC have been identified2 that manifest HU319, Boston MA 02115. Email: Friedhelm.Hildebrandt@ nephronophthisis as part of their pathogenesis in the childrens.harvard.edu context of ciliopathy syndromes such as Senior–Loken Copyright © 2014 by the American Society of Nephrology

J Am Soc Nephrol 25: 2573–2583, 2014 ISSN : 1046-6673/2511-2573 2573 BASIC RESEARCH www.jasn.org syndrome (OMIM 266900), Bardet–Biedl syndrome (BBS; mouse embryonic fibroblasts (Supplemental Figure 1C). Im- OMIM 209900), Joubert syndrome (OMIM 213300), and orofa- munoblotting (Supplemental Figure 1D) confirmed the ab- ciodigital syndrome (OFD; OMIM 311200). sence of Sdccag8 protein from lung and kidney lysates of We recently showed that mutations in serologically defined Sdccag8gt/gt mice. Two isoforms of the Sdccag8 protein (78 colon cancer antigen 8 (SDCCAG8) cause nephronophthisis kD and 83 kD) were detected in Sdccag8wt/gt kidneys (Supple- type 10, characterized by retinal and renal degeneration, mild mental Figure 1D).3 Sdccag8gt/gt mice were present at Mende- intellectual disability, obesity, hypogonadism, and recurrent re- lian ratios at weaning age, indicating that the Sdccag8 gene-trap spiratory infections in humans.3,4 Because several of the clinical allele does not cause embryonic or early postnatal lethality. features are shared with BBS, with the exception of the absence of polydactyly, individuals with SDCCAG8 mutations are also con- Sdccag8 Is Expressed in Kidney and Lung Epithelia sidered as part of the BBS spectrum.3,4 SDCCAG8 encodes a Mutations in SDCCAG8 were previously reported to affect two coiled-coil domain protein with no additional conserved do- parenchymal organs in humans, the kidneys and the lungs, caus- mains.5 The protein localizes to the throughout the ing nephronophthisis and, infrequently, bronchiectasis.3,4 To cell cycle,3,5 to the of cilia, and also to the spermato- understand the underlying pathogenetic mechanisms, we first cytes in the rat testis.3,6 Immunohistochemical analysis of retina examined the expression pattern of Sdccag8 in these organs by has shown SDCCAG8 colocalization with retinitis pigmentosa taking advantage of the lacZ cassette in the Sdccag8 gene-trap allele. protein 1 (RP1), retinitis pigmentosa GTPase regulator (RPGR), b-Galactosidase activity staining of wild-type and Sdccag8wt/gt and retinitis pigmentosa GTPase regulator interacting protein 1 whole urogenital systems at E16.5 showed strong Sdccag8 (RPGRIP1) in the connecting of the photoreceptors.3,7 expression in the corticomedullary region of the Sdccag8wt/gt Biochemical studies have demonstrated SDCCAG8 homodime- kidneys (Figure 1A) and no staining in the wild-type control rization and direct interaction with two ciliopathy : (1) (Supplemental Figure 2A). Examination of the X-gal–stained OFD1 and (2) family with sequence similarity 161, member A kidney sections at higher resolution showed staining in the (FAM161A).3,5,8 Despite the data on SDCCAG8 protein locali- renal tubule epithelia in a pattern compatible with the distal zation and its interaction partners, the precise molecular func- convoluted tubule (DCT) and cortical collecting ducts (CCDs) tion at centrosomes and cilia remains unknown. (Figure 1B). Sdccag8 expression in the collecting ducts was also Werecentlydemonstrated that mutations inthe gene encoding observed in postnatal P14 and P100 kidneys by in situ hybrid- the centrosomal protein CEP164 cause NPHP-RC whose path- ization (Figure 1, C and D), whereas the sense probe showed no ogenesis involves defects in the DNA damage response (DDR) staining (Supplemental Figure 2, B and C). In the lung, X-gal signaling pathway.9 The same study also implicated SDCCAG8 in staining in Sdccag8wt/gt mice at E16.5 showed Sdccag8 expres- this pathway through its colocalization with CEP164 and Tat- sion in the epithelium of the developing bronchi and bron- interactive protein 60 in the .9 Indeed, there is a wealth chioles (Figure 1E). Examination of lung sections at higher of evidence in the literature that implicates centrosomal protein resolution confirmed this observation and further showed 2 function in the regulation of genome stability, including the that the blue lacZ+ cells were interspersed with lacZ cells in NPHP-RC proteins AHI1,10 NIMA-related kinase 8 (NEK8/ the bronchioles (Figure 1F). No b-galactosidase staining was NPHP9),11 and the SDCCAG8-interacting protein OFD1.12,13 detected in the epithelial cells of alveoli—the terminal ends of To study the role of Sdccag8 in the pathogenesis of NPHP- the airways, which do not have cilia (Figure 1F, delineated with RC, we generated a transgenic Sdccag8gt/gt mouse model. We dashed line). To determine whether the lacZ+ cells in the lung demonstrate that Sdccag8gt/gt mice recapitulate aspects of the epithelium represent the multiciliated cells, we performed im- human disease phenotype. Furthermore, we show that munofluorescence analysis on E16.5 CETN2-GFP mouse lung Sdccag8 is involved in cell cycle S-phase progression and its sections using the cilia marker anti-polyglutamylated tubulin loss leads to replication stress–related DDR activation. antibody and anti-SDCCAG8 antibody. CETN2-GFP fusion protein localizes to centrioles,14 which are presented in hun- dreds of copies in the multiciliated cells of the respiratory RESULTS epithelium.15 Because centriologenesis precedes multiciliogen- esis, almost no cilia were detected in E16.5 distal bronchioles Generation of Sdccag8gt/gt Mice (Supplemental Figure 2, D–F), as previously described.16 To investigate the function of the Sdccag8 gene, the embryonic However, we found that the CETN2-GFP–positive structures stem cell line OST40418 containing the gene-trap cassette (Figure 1G) in the progenitors of the multiciliated cells fully VICTR24 in the intronic region downstream of Sdccag8 exon overlapped with SDCCAG8 antibody staining in E16.5 bron- 1 (Supplemental Figure 1A) was microinjected and founders chioles (Figure 1, H and I). Together, this analysis demonstrates were bred. Allele-specific primers were used to genotype the that Sdccag8 is expressed in the embryonic and postnatal kid- mice (Supplemental Figure 1, A and B). Mice carrying the ney in a pattern that partially overlaps with the localization of gene-trap allele are referred to as Sdccag8gt. The absence of ciliated cells in these tissues. In lung Sdccag8 is expressed in the Sdccag8 mRNA was verified by quantitative RT-PCR analysis prospective multiciliated cells, whereas Sdccag8-negative cells using RNA isolated from embryonic day 13.5 (E13.5) Sdccag8gt/gt most likely represent the nonciliated intercalating goblet cells.

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examined the extent of renal fibrosis in Sdccag8gt/gt kidneys using Masson tri- chrome staining (Figure 3, A–D). In a majority of P100 Sdccag8gt/gt kidneys, no collagen deposits were detected (Figure 3, B and E), which is in agreement with the limited amount of interstitial infiltrate seen by histologic staining (Figure 2G). By con- trast, the P250 Sdccag8gt/gt kidneys dis- played extensive fibrosis (Figure 3D), which was significantly increased between P100 and P250 kidneys (Figure 3E). To identify the tubular origin of the cysts, we stained P100 kidney sections using neph- ronsegment-specificantibodies.Stainingwith aproximaltubulemarkerLotus tetragonolobus agglutinin lectin (LTA; Figure 3, F–I), a DCT marker, and thiazide sensitive Na-Cl Figure 1. Sdccag8 is expressed in kidney and lung epithelia. b-Galactosidase activity cotransporter (TSC) (Figure 3, J and K), staining in the urogenital system at E16.5 demonstrates Sdccag8 expression in the cor- wt/gt a distal tubule/collecting duct marker ticomedullary region (arrows) and in the CCDs (arrowheads) in Sdccag8 kidneys Dolichos biflorus agglutinin (DBA) (Figure 3, (A and B). (C and D) Sdccag8 expression is maintained in the collecting ducts of the F and G), revealed that the cysts in Sdccag8gt/gt kidneys in two-week-old (C) and adult (D) mice. (E) b-Galactosidase activity staining on kidneys originated from two sites—the E16.5 whole lung demonstrates Sdccag8 expression in the bronchi and bronchioles. (F) Analysis of a cross-section through the X-gal–stained lung at higher resolution confirms CCD (Figure 3G) and the DCT (Figure 3K). Sdccag8 expression localization to the epithelial layer of the bronchioles and its absence Costaining with an antibody against a-smooth from alveoli (delineated with a dashed line) and surrounding stromal mesenchyme. No- muscle actin that labels the fibrogenic gt/gt tice the presence of lacZ-negative cells (white arrows) in the bronchioles. (G–I) CETN2- myofibroblasts in Sdccag8 kidneys GFP expression in the bronchiole epithelium (G) overlaps (I) with SDCCAG8 antibody demonstrates that both CCD and DCT staining (H) in the E16.5 mouse lung section, indicating that Sdccag8-positive cells cor- cysts were surrounded by interstitial fibrosis respond to the progenitors of the multiciliated cells in the lung. Bar, 1 mm in A and B; (Figure 3, I and K). We examined whether – 400 mm in C; 2 mm in D and E; 200 mminF;25mminG I. Sdccag8gt/gt mice have defective cilia in the cystic CCDs, by staining kidney sections with antibodies against the cilia marker Sdccag8gt/gt Mice Develop Late-Onset acetylated tubulin and CCD marker aquaporin 2. No global Nephronophthisis ciliary abnormalities in the Sdccag8gt/gt CCDs were observed Given the Sdccag8 expression pattern in the kidney, we next (Supplemental Figure 3, A and B). Deregulation of cytoskeletal examined whether loss of Sdccag8 causes nephronophthisis. rearrangements has been implicated in the pathomechanisms The first histologic signs of cyst formation and interstitial in- of polycystic kidney disease.17 We tested this hypothesis on filtration, features that are characteristic of nephronophthisis, Sdccag8gt/gt mouse embryonic fibroblasts using a wound heal- were detected at P100 in Sdccag8gt/gt kidneys (Figure 2, A–C). ing assay, but found no changes in their ability to regulate The initial cyst formation occurred primarily in the cortical cytoskeletal rearrangements when grown in two-dimensional region of the kidneys (Figure 2, B and C), with either no culture (Supplemental Figure 3, C and D). amount (Figure 2G) or a small amount (Figure 2H) of inter- Taken together, our data show that Sdccag8gt/gt mice present stitial infiltrate surrounding the dilated tubules. At P250, the late-onset nephronophthisis characterized by cyst formation renal histology appeared progressively deteriorated with en- in the DCT, CCD, and glomeruli. Cyst formation was not larged cortical cysts, cyst formation in the corticomedullary associated with global ciliary defects in the kidney. There region, dedifferentiation of the corticomedullary junction, was a marked fibrosis around the renal cysts. and replacement of the renal parenchyma by interstitial infil- trate (Figure 2, D and E). In addition to tubular cysts, glomer- Loss of Sdccag8 Causes Photoreceptor Degeneration ular cysts were also observed in Sdccag8gt/gt kidneys at P250 in Sdccag8gt/gt Mice (Figure 2, I–K). There was no difference in kidney weight to Mutations in SDCCAG8 cause retinal degeneration in hu- body weight ratio in Sdccag8gt/gt mice at P100; however, the mans.3 To study the disease progression in Sdccag8gt/gt mice, mutant kidneys were significantly enlarged by P250 (Figure we performed retinal histologic analysis in mice aged P30, 2L). Similarly, the mean value of the cystic index was signifi- P100, and P250 days (Figure 4, A–C). Although the retina cantly increased between P100 and P250 (Figure 2M). We next appeared to have formed normally, we observed a slight

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amplitudes from P100 to P250, which were in tight correlation with the degree of histologic defects at the respective ages (Supplemental Figure 4, A and B). SDCCAG8 localization to the photorecep- tor connecting cilium is regulated by 2 2 RPGRIP1.7 Because Rpgrip1 / mice show a defect in photoreceptor protein traffick- ing,18,19 we hypothesized that loss of SDCCAG8 may also lead to impaired pro- tein trafficking in the photoreceptors. To test this hypothesis, we stained Sdccag8gt/gt ret- inas with an antibody against rhodopsin that localizes to the photoreceptor outer segment and against CEP164 that labels the photoreceptor basal body. Consistent with a defect in protein trafficking, rhodopsin accumulated to the plasma membrane of the photoreceptor inner segment and cell bodies in Sdccag8gt/gt retina at P30 (Figure 4, D and E) andP100(SupplementalFigure4,DandE). Peanut agglutinin lectin staining of photo- receptor cone sheath at P30 revealed a re- duced number of cones in Sdccag8gt/gt mice (Figure4,FandG,SupplementalFigure 4C). Thus, our data indicate that Sdccag8 is not essential for retina development and photoreceptor formation, but is required for the maintenance of both rod and cone gt/gt – Figure 2. Sdccag8 mice develop nephronophthisis. (A C) H&E staining of sagittal photoreceptor cells. Loss of Sdccag8 leads to sections of P100 kidneys shows the formation of cortical cysts in Sdccag8gt/gt kidneys progressive retinal degeneration and blindness. (B and C). (D and E) Analysis of H&E staining on sagittal sections of P250 kidneys shows loss of corticomedullary differentiation and progressive cyst formation in Sdccag8gt/gt kidneys (E). (F–H) H&E-stained Sdccag8wt/wt (F) and Sdccag8gt/gt (G and Loss of Sdccag8 Leads to Impaired H) kidney sections demonstrate the variation in tubular cyst size and amount of in- S-Phase Progression terstitial infiltrate in Sdccag8gt/gt kidneys at P100 (G and H). (I–K) By P250, kidney cysts We and others have shown that the patho- and interstitial infiltrate have replaced most of the renal parenchyma in Sdccag8gt/gt genesis of renal ciliopathies and renal fibrosis kidneys (J and K). Glomerular cysts are marked with asterisks. (L) The kidney weight to may involve mutations in genes that modu- body weight ratio is not changed in Sdccag8gt/gt mice at P100 (mean wt/wt latecellularresponsetoDNAdamage,suchas 1.66760.09516, gt/gt 1.73260.09247); however, the ratio is significantly increased in ZNF423, CEP164, FAN1, and NEK8.9,11,20 gt/gt 6 6 P250 Sdccag8 mice (mean wt/wt 1.554 0.07007, gt/gt 2.151 0.08878; We recently demonstrated that SDCCAG8 , gt/gt *P 0.05). (M) The kidney cyst index is low in P100 Sdccag8 mice (mean colocalizes with CEP164 and Tat-interactive 4.27062.024), but is greatly increased and with noticeable variation in P250 mice protein 60, an activator of the DDR regulator (mean 21.9168.662; *P,0.05). H&E, hematoxylin and eosin; wt, Sdccag8 wild-type 9,21 allele; gt, Sdccag8 gene-trap allele; KW/BW, kidney weight to body weight ratio. Bar, ATM. However, it remained unaddressed 2mminA–E; 100 mminF–K. whether SDCCAG8 functions in the context of DNA damage response signaling. To test this hypothesis, we first examined whether loss of Sdccag8 causes cell cycle abnormalities reduction of the photoreceptor layer at P30 (Figure 4A). No as a result of checkpoint activation by performing FACS analysis other retinal abnormalities were detected at this stage. The on conditionally immortalized adult kidney cells from Immorto; retinal phenotype became more severe at P100 (Figure 4B) Sdccag8wt/wt and Immorto;Sdccag8gt/gt mice. To perform the ex- and complete loss of the photoreceptor cell layer occurred periments, cells were grown at 37°C without g-IFN to exclude by P250 (Figure 4C). We performed electroretinography any interference from the large T antigen (Supplemental Figure measurements to study the physiologic consequences of the 5A). Cell cycle analysis showed that Sdccag8gt/gt cells accumulated observed histologic changes in the retina. Sdccag8gt/gt mice in the S phase. The number of bromodeoxyuridine (BrdU)-positive displayed a progressive decline in electroretinography wave S-phase cells was increased from 18% in wild-type cells to

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synchronized the cells with a double thymi- dine block and treated them with 4.5 U/ml bleomycin or 30 J UV light. After release from the block, Sdccag8gt/gt cells progressed in the S phase more slowly and remained in the S phase longer than wild-type cells (Figure 5C). Treatment with 30 J UV light further extended the S-phase duration, and bleomycin had a slightly milder effect (Fig- ure 5C). Interestingly, G2/M checkpoint acti- vation was not impaired in Sdccag8gt/gt cells because treatment with bleomycin or UV light arrested both wild-type and Sdccag8gt/gt cells in the G2/M checkpoint (Figure 5D). There was also no difference in the levels of apoptosis between wild-type and mutant cell lines after treatment with bleomycin or UV light, al- though treatment with 30 J UV increased the apoptotic index compared with nontreated cells (Supplemental Figure 5, B and C). We next examined whether replication stress in Sdccag8gt/gt cells leads to the acti- vation of DDR signaling. There was a significant increase in the levels of phos- phorylated H2AX (gH2AX) and ATM (S1987) proteins in Sdccag8gt/gt cells, indi- cating activation of DDR signaling pathway in these cells (Figure 5, E and F). Treatment with bleomycin drastically increased the phosphorylation of either protein, both in wild-type and Sdccag8gt/gt cells. In addition, Sdccag8gt/gt cells demonstrated increased proliferating cell nuclear antigen protein gt/gt fi – Figure 3. Sdccag8 mice develop brosis. (A D) Masson trichrome staining reveals levels, consistent with their prolonged gt/gt fi no collagen deposits in Sdccag8 kidneys at P100 (B); however, extensive brosis is stay in the S phase (Figure 5E). visible in P250 Sdccag8gt/gt kidneys (D). (E) The fibrosis index is significantly increased We next asked whether the in vitro find- in Sdccag8gt/gt mice between P100 (mean 10.50610.50) and P250 (mean 6 , ings of abnormal DDR signaling activation 47.00 12.41; *P 0.05). (F and G) LTA-FITC and DBA-rhodamine staining on kidney gt/gt sections demonstrates the formation of cysts in the CCDs (DBA) and not in the in Sdccag8 cells can also be detected under gt/gt proximal tubules (LTA) of Sdccag8gt/gt kidneys at P100. (H and I) Immunofluorescence in vivo conditions. We stained Sdccag8 staining with a-SMA-rhodamine and LTA-FITC demonstrate the presence of myofi- and control kidneys with gH2AX antibody broblasts in the proximity of a nascent cyst (I, white arrow). (J and K) Immunofluo- at P100 (Figure 5, G–J) and P250 (Figure 5, rescence staining with aSMA-FITC and DCT marker TSC show that besides CCDs, K–N) and indeed found increased nuclear cysts also originate from DCTs (marked with white asterisks) in P100 Sdccag8gt/gt gH2AX staining in the tubular and intersti- kidneys (K). aSMA-FITC staining shows the myofibroblasts surrounding the affected tial compartments of Sdccag8gt/gt kidneys. tubules. wt, Sdccag8 wild-type allele; gt, Sdccag8 gene-trap allele; a-SMA, a-smooth The gH2AX-positive nuclei were observed – – muscle actin. Bar, 100 mminA D; 100 mminF K. in the dilated tubules at P100 (Figure 5, I andJ),whereasnondilatedtubuleswere also positive for gH2AX staining at P250 (Figure 5, M and N), likely indicating a 24.5% in Sdccag8gt/gt cells (Figure 5, A and B), and this figure was more general tubular stress in more fibrotic kidneys.20 Together, further increased from 23.4% in wild-type cells to 33.6% in these data suggest that Sdccag8 has an important role in DNA Sdccag8gt/gt cells after treatment with 30 J ultraviolet (UV) light, damage signaling and its loss leads to replication stress and indicating that loss of Sdccag8 affects primarily S-phase progres- slower S-phase progression during the normal cell cycle. Fur- sion and makes the cells more sensitive to UV light–induced thermore, Sdccag8gt/gt kidneys display elevated levels of gH2AX, DNA damage. To study the cell cycle defect in more detail, we implicating impaired DDR in their pathogenesis.

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Figure 4. Sdccag8 is required for photoreceptor maintenance. (A–C) Histologic analysis of H&E-stained wild-type and Sdccag8gt/gt retinas at P30 (A), P100 (B), and P250 (C) show progressive degeneration of photoreceptor inner segments and outer segments, as well as severe reduction of the outer nuclear layer in Sdccag8gt/gt mice. (D and E) Immunolocalization of CEP164 and rhodopsin in the P30 retina shows rhodopsin accumulation (white arrows) at the photoreceptor inner segment plasma membrane and cell bodies in Sdccag8gt/gt retina (E). (F and G) PNA-FITC lectin staining of cone sheaths at P30 demonstrates a reduction in cone cell number in Sdccag8gt/gt retina (G). H&E, hematoxylin and eosin; RPE, retinal pigment epithelium; ROS, retinal outer segment; IS, inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; ISL, inner plexiform layer; GCL, ganglion cell layer; wt, Sdccag8 wild-type allele; gt, Sdccag8 gene-trap allele; PNA, peanut agglutinin lectin. Bar, 100 mminA–C; 10 mm in D and E; 50 mm in F and G.

DISCUSSION in individuals with SDCCAG8 mutations.3 Our ability to de- tect lacZ expression only in the ciliated cells most likely reflects We generated Sdccag8gt/gt mice to study the role of Sdccag8 in higher Sdccag8 promoter activity in ciliated cells. It does not, nephronophthisis type 10. Sdccag8gt/gt mice faithfully recapit- however, exclude that Sdccag8 is not expressed in nonciliated ulate the human SDCCAG8 loss of function phenotypes of cells, whose levels may be below the detection limit of our retinal degeneration and nephronophthisis. Through mor- b-galactosidase staining activity.3 phologic analysis of Sdccag8gt/gt mice and functional studies Our histologic analysis of the kidneys showed that Sdccag8gt/gt using cell culture assays, we show that SDCCAG8 is important mice displayed a classic nephronophthisis phenotype at P100, for photoreceptor maintenance and cell cycle regulation, im- with the cystic dilations originating from the DCTs and CCDs. pairment of which leads to blindness and aberrant DDR sig- Compared with other mouse models of nephronophthisis,22–25 naling activation through replication stress. the onset of the renal phenotype in Sdccag8gt/gt mice occurred relatively late. The process of cyst formation appeared to coin- Sdccag8 and Degenerative Phenotypes cide with interstitial fibrosis that surrounded the glomeruli and Sdccag8 expression was previously localized to the retina and nephrons. In progressively later stages, tubular dilations were testis.3,6–8 Our studies using the Sdccag8 lacZ gene-trap allele also observed in the Bowman’s capsule of glomeruli. Curiously, and SDCCAG8 antibody staining show Sdccag8 expression in no global cilia defects were observed in the cystic renal tubule the multiciliated cells of the respiratory epithelium and in the cells. Similar results were obtained in in vitro studies using DCTs and collecting ducts of the kidney. These expression a spheroid assay, in which loss of Sdccag8 did not affect cilia domains are consistent with the recorded tissue pathologies numbers.3,26 However, it remains to be studied whether the cilia

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Figure 5. Sdccag8 inactivation causes S-phase delay and replication stress. (A and B) The cell cycle profile of Immorto;Sdccag8wt/wt and Immorto;Sdccag8gt/gt nonsynchronized cells demonstrates significant accumulation of Sdccag8gt/gt cells in the S phase (P,0.05; n=3) compared with the wild-type control (P,0.05, n=3). The number of cells in the S phase is further increased after treatment with 30 J UV light. (C) Sdccag8gt/gt cells synchronized with double thymidine block show a delay in S-phase progression, which is further in- creased after treatment with UV light. (D) Sdccag8gt/gt cells are competent in G2/M check point activation, both in response to UV light and bleomycin treatment. (E) Sdccag8gt/gt cells have increased PCNA levels, reflecting their extended duration in the S phase. PCNA levels do not change in response to bleomycin treatment, which is in agreement with the cell cycle analysis data. Increased gH2AX levels in cultured Sdccag8gt/gt cells are a sign of replication stress. H2AX and b-actin are used as loading controls. (F) Phosphorylated ATM levels are low in cultured wild-type cells, which become elevated upon bleomycin treatment. By contrast, Sdccag8gt/gt cells display high phosphorylated ATM levels even without treatment. ATM is used as a loading control. (G–J) Immunofluorescence staining

J Am Soc Nephrol 25: 2573–2583, 2014 Loss of Sdccag8 Causes NPHP-RC 2579 BASIC RESEARCH www.jasn.org in Sdccag8gt/gt collecting ducts have deficiencies in some aspects model in which Sdccag8 has an important role in S-phase pro- of cilia function, such as mechanosensation as shown for poly- gression during the normal cell cycle, in which it could facilitate cystic kidney disease or in loss of transition zone integrity.27 DNA replication from the stalled replication forks; however, Retinal degeneration in Sdccag8gt/gt mice progresses slower than the molecular analysis required to answer this question is exten- in the mouse model of its protein interactor RPGRIP1,19 but sim- sive and will be the focus of future work. It remains to be tested ilarly to that of its other interaction partners RP1 and RPGR.28,29 whether treatment of Sdccag8gt/gt mice with cyclin-dependent This suggests that RPGRIP1 functions upstream of Sdccag8 in kinase inhibitors, such as R-roscovitine or S-CR8, reverses or photoreceptor cilia, which is in agreement with a recent report attenuates the renal cyst progression in Sdccag8gt/gt mice as has 2 2 2 2 of RPGRIP1-dependent ciliary localization of SDCCAG8.7 Al- been shown for jck/Nphp9 / and Pkd1 / mice.38,39 though photoreceptor formation in Sdccag8gt/gt mice appeared intact, the absence of Sdccag8 led to accumulation of rhodopsin in the photoreceptor inner segments and cell bodies at P30. This CONCISE METHODS study did not address whether rhodopsin mislocalization was caused by structural defects in the connecting cilium or whether Mouse Breeding and Maintenance SDCCAG8 is directly involved in rhodopsin trafficking. Rho- The experimental protocol was reviewed and approved by the Animal dopsin mislocalization to photoreceptor inner segments is Care Committee of the University of Michigan. Embryonic stem cell known to be toxic for the photoreceptors due to membrane line OST40418 containing gene-trap vector VICTR24 in intron 1 of crowding resulting in retinal degeneration.30,31 Our data are Sdccag8 was obtained from Texas Institute for Genomic Medicine and consistent with this mode of retinal pathogenesis and cultured as described with the use of ESGRO (EMD Millipore).40 indicate a role for Sdccag8 in intraciliary trafficking. Immuno- Chimeric mice were prepared by blastocyst microinjection and histochemical and functional analysis showed that both rod and bred with C57BL/6J mice to obtain germline transmission. Genotyp- cone photoreceptors were affected in Sdccag8gt/gt mice, demon- ing primers and PCR conditions are available upon request. strating that Sdccag8 is essential for the survival and mainte- CETN2-GFP mice were purchased from The Jackson Laboratory nance of both rods and cones, similar to RPGR function.29 (stock number 008234). ImmortoMouse mice were purchased from Charles River Laboratories (strain code 238). Sdccag8 wild-type or het- Sdccag8 and DDR Signaling erozygous littermates were used as controls for mutant mice. For timed Examination of the molecular defects underlying the pathogenesis matings, noon on the day a plug was found was designated as E0.5. in Sdccga8gt/gt mice using cell culture revealed abnormalities in cell cycle progression as a result of activation of DDR signaling, char- Histologic Analyses acterized by hyperactivation of ATM and elevation of gH2AX Tissues were fixed in 4% (w/v) paraformaldehyde in PBS at 4°C. All levels. Increased DDR signaling activity was also observed in tissues were then dehydrated through an ethanol series and em- Sdccag8gt/gt kidneys, implicating DDR signaling in the pathogen- bedded in paraffin. Sections were taken at 5 mm. Hematoxylin and esis of NPHP-RCs. Impaired DDR signaling is associated with eosin staining followed standard protocols. b-Galactosidase activity degenerative diseases such as Seckel syndrome, which is caused staining was carried out on whole tissues as previously described.41 by mutations in genes encoding for other centrosomal and DDR b-Galactosidase–stained tissues were cleared in glycerol for imaging proteins such as ATR,32 pericentrin,33 CEP152,34 or CEP63.35 or embedded in paraffin and sectioned at 10 mm. Pathologically, Seckel syndrome shares with NPHP-RC the phe- notype of premature degeneration and fibrosis of parenchymal Cystic Index and Fibrosis Index Calculation organs, including the kidney.36 Because kidneys are constantly Representative images of hematoxylin and eosin–stained or Masson exposed to genotoxins, they are especially prone to lesions caused trichrome–stained kidneys were acquired. A grid was placed over the by impaired DDR signaling. Increased susceptibility to genotoxins images, and the cystic index or fibrosis index was calculated as the 2 2 was previously reported for the Ahi1 / mouse model of NPHP- percentage of grid intersection points that bisect cystic and noncystic RC,10 and genome instability because of the loss of survivin was or fibrotic and nonfibrotic areas. 2 2 2 2 reported in Pkd1 / , Pkd2 / ,andTg737Orpk/Orpk isolated pri- mary cells.37 Mutations in a related ciliopathy NEK8/NPHP9 Electroretinography were recently shown to lead to replication stress–related To assess rod- and cone-mediated function, electroretinograms were DDR signaling activation.11 Our data are consistent with a performed using the Espion e2 recording system (Diagnosys) as

with anti-gH2AX antibody on P100 Sdccag8wt/wt and Sdccag8gt/gt kidneys shows no gH2AX staining in control kidneys (G and H), whereas the dilated tubules (red dashed lines) in Sdccag8gt/gt kidneys are positive for gH2AX (I and J). (K–N) P250 Sdccag8wt/gt kidneys are negative for gH2AX staining (K and L), while Sdccag8gt/gt kidneys show positive staining in dilated and nondilated nephron epithelium (red dashed lines) and fibrotic mesenchyme (M and N). wt, Sdccag8 wild-type allele; gt, Sdccag8 gene-trap allele; PCNA, proliferating cell nuclear antigen. Bar, 25 mminG–J; 20 mminK–N.

2580 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2573–2583, 2014 www.jasn.org BASIC RESEARCH previously described.42 After overnight dark adaptation, mice were Data were normalized to Gapdh. Primer sequences are available anesthetized with an intraperitoneal injection of ketamine (93 mg/kg) upon request. and xylazine (8 mg/kg). Body temperature was maintained at 37°C with a heating pad. After pupil dilation with topical phenylephrine Western Blotting (2.5%) and tropicamide (1.0%), corneal electroretinograms were Lung and kidney tissues were lysed in radioimmunoprecipitation recorded from both eyes using gold wire loops and a drop of 2% assay lysis buffer (Pierce) and homogenized with a douncer. Cleared methylcellulose for corneal hydration. A gold wire loop placed tissue lysates were produced by centrifugation of the resulting samples in the mouth was used as reference, and the ground electrode was at 16,0003g for 30 minutes at 4°C. Gel electrophoresis of tissue ly- placed on the tail. The dark-adapted electroretinogram was recorded sates and immunoprecipitation eluents were performed using the 2 from 25.8 to +1.09 log cdzszm 2/flash in steps of 0.5 log units. After NuPAGE system (Invitrogen). Samples were resolved on 4%–12% 2 10 minutes of light adaptation to a white 32 cdzm 2 rod-suppressing Bis-Tris gels in 3-(N-morpholino)propanesulfonic acid buffer and background, light-adapted electroretinograms were recorded from transferred to a nitrocellulose membrane that was then probed for 2 20.91 to +2.0 log cdzs zm 2. Ten to 25 responses were recorded at the protein of interest using antibodies diluted in Tris-buffered saline 3–60 seconds depending upon the stimulus intensity intervals. containing 5% milk and 0.1% Tween-20 (Sigma-Aldrich).

Antibodies Apoptosis FACS Forimmunostaining,tissuesweredeparaffinizedandhydratedthrougha To quantify apoptosis, wild-type or Sdccag8gt/gt cells were plated and graded ethanol series. Antigen retrieval was carried out in heated antigen incubated with or without thymidine. After two overnight thymidine retrievalbuffer(pH8,SIG-31910-50;Covance).Primaryantibodiesused blocks, cells were released and damage was induced by 1-hour 4.5 were as follows: rabbit polyclonal anti-CEP164 (gift from Erich Nigg, U/ml bleomycin or 30 J UV exposure. After 24 hours, cells were University of Basel, Switzerland), rabbit polyclonal anti-SDCCAG8 harvested and washed once with 1% BSA-PBS. Cells were collected (13471-1-AP; Proteintech), rabbit polyclonal anti-SDCCAG8 in FACS tubes in 200 ml 1% BSA-PBS containing Vybrant DyeCycle (ab101969; Abcam, Inc.), mouse monoclonal anti–g-tubulin (GTU- Violet Stain (V35003, 1:1000; Invitrogen) to stain living and apo- 88; Sigma-Aldrich), anti-acetylated tubulin (6-11B-1; Sigma-Aldrich), ptotic cells (7 minutes at 37°C) and 7-AAD viability stain (00-6993, rabbit anti-Aquaporin 2 (ab15081; Abcam, Inc.), rabbit polyclonal anti- 1:60; eBioscience) to stain dead cells (10 minutes on ice). Cells were TSC (AB3553; EMD Millipore), peanut agglutinin lectin-FITC (Vector measured (20,000 events) with a BD FACSCanto II flow cytometer Laboratories), lectin Lotus tetragonolobus agglutinin (Vector Laborato- and analyzed using BD FACSDiva Software. ries), anti–aSMA-FITC (1A4; Sigma-Aldrich), anti-gH2AX (9718; Cell Signaling), rabbit anti-ATM (S1981, 5883; Cell Signaling Technology), BrdU FACS mouse monoclonal anti-rhodopsin (R5403; Sigma-Aldrich), rat anti– To quantify cell cycle phase distribution, wild-type or Sdccag8gt/gt cells proliferating cell nuclear antigen (ABIN334654; Antibodies Online), were plated and incubated with or without thymidine. After double mouse anti–b-actin (AC-15, A5441; Sigma-Aldrich), rabbit anti- overnight thymidine block, cells were released and damage was in- histone H2A.X (070627; EMD Millipore), and mouse monoclonal duced by 30 J UV exposure. After 6- or 10-hour release of thymidine anti-polyglutamylated tubulin (T9822; Sigma-Aldrich). Secondary block, cells were incubated with 10 mM BrdU for 30 minutes and antibodies were goat anti-mouse Alexa Fluor 488 (Molecular fixed in 70% EtOH. Samples were stained for FACS analysis with Probes) and goat anti-rabbit Alexa Fluor 594 (Molecular Probes). BrdU mouse mAb (clone MoBU-1), Alexa Fluor 647 conjugate Samples were mounted in ProlongGold (Molecular Probes) and (1:200; Invitrogen) in 0.1% BSA-PBS-T for 1 hour on ice, and images were captured on a Leica TSC 5SP X confocal microscope 49,6-diamidino-2-phenylindole (1:2000) in PBS, and were measured (Leica Microsystems). (10,000 events) with a BD FACSCanto II flow cytometer and ana- lyzed using BD FACSDiva Software. Generation of Mouse Embryonic Fibroblasts and Immortalized Adult Kidney Cells Statistical Analyses Mouse embryonic fibroblasts were established from wild-type and The t test was used to compare data between two groups. Significance Sdccag8gt/gt E13.5 embryos and cultured in DMEM with 10% FBS and was determined at P,0.05. Where appropriate, data are presented as penicillin/streptomycin. Isolation of immortalized adult kidney cells the mean6SEM. from P50 Immorto;Sdccag8wt/wt and Immorto;Sdccag8gt/gt mouse kid- neys was performed as previously described.43 ACKNOWLEDGMENTS RNA Extraction and Quantitative RT-PCR RNA was isolated from wild-type, Sdccag8wt/gt,andSdccag8gt/gt We thank Erich Nigg for providing the CEP164 antibody. We also mouse embryonic fibroblast cells and adult kidney cells using the thank Thomas L. Saunders, Elizabeth Hughes, Keith Childs, Galina RNeasy Mini Kit (Qiagen), and RT was performed using Super- Gavrilina, and Debra Vanheyningen for preparation of the embryonic script III (Invitrogen). Quantitative real-time PCR was carried stem cell mouse chimeras from gene-trapped embryonic stem cell out using SYBR Green (Qiagen) and run on a MyiQ Single-Color clone OST40418 and the Transgenic Animal Model Core of the Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). University of Michigan’s Biomedical Core Facilities.

J Am Soc Nephrol 25: 2573–2583, 2014 Loss of Sdccag8 Causes NPHP-RC 2581 BASIC RESEARCH www.jasn.org

Core support was provided by the Diabetes Research and Training BA, Cervenka I, Ganji RS, Bryja V, Arts HH, van Reeuwijk J, Oud MM, Center and the O’Brien Renal Core Center, which are funded by Letteboer SJ, Roepman R, Husson H, Ibraghimov-Beskrovnaya O, grants from the National Institutes of Health (NIH) (DK20572 and Yasunaga T, Walz G, Eley L, Sayer JA, Schermer B, Liebau MC, Benzing T, Le Corre S, Drummond I, Janssen S, Allen SJ, Natarajan S, O’Toole P30-DK08194, respectively). This research was also supported by JF, Attanasio M, Saunier S, Antignac C, Koenekoop RK, Ren H, Lopez I, grants from the NIH (1K99-DK099434-01 to R.A., and DK068306 Nayir A, Stoetzel C, Dollfus H, Massoudi R, Gleeson JG, Andreoli SP, and RC4-DK090917 to F.H.) and the European Commission Seventh Doherty DG, Lindstrad A, Golzio C, Katsanis N, Pape L, Abboud EB, Al- Framework Programme (HEALTH-F4-2008-201648/PROSPECTS Rajhi AA, Lewis RA, Omran H, Lee EY, Wang S, Sekiguchi JM, Saunders to J.S.A.). G.G.S. and R.H.G. were supported by grants from the R, Johnson CA, Garner E, Vanselow K, Andersen JS, Shlomai J, Nurnberg G, Nurnberg P, Levy S, Smogorzewska A, Otto EA, Hildebrandt F: Exome European Union FP7/2009 Consortium “SYSCILIA” (241955) and capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies the Dutch Kidney Foundation Kouncil Consortium (CP11). A.-C.W. to DNA damage response signaling. Cell 150: 533–548, 2012 and A.K. were supported by a grant from the German Research 10. Lancaster MA, Louie CM, Silhavy JL, Sintasath L, Decambre M, Nigam Foundation (DFG Ki728/9-1). F.H. is an investigator of the Howard SK, Willert K, Gleeson JG: Impaired Wnt-beta-catenin signaling dis- Hughes Medical Institute. rupts adult renal homeostasis and leads to cystic kidney ciliopathy. Nat Med 15: 1046–1054, 2009 11. Choi HJ, Lin JR, Vannier JB, Slaats GG, Kile AC, Paulsen RD, Manning DISCLOSURES DK, Beier DR, Giles RH, Boulton SJ, Cimprich KA: NEK8 links the ATR- regulated replication stress response and S phase CDK activity to renal None. ciliopathies. Mol Cell 51: 423–439, 2013 12. Ferrante MI, Zullo A, Barra A, Bimonte S, Messaddeq N, Studer M, Dollé P, Franco B: Oral-facial-digital type I protein is required for primary cilia REFERENCES formation and left-right axis specification. Nat Genet 38: 112–117, 2006 13. Giorgio G, Alfieri M, Prattichizzo C, Zullo A, Cairo S, Franco B: Func- 1. Hildebrandt F, Benzing T, Katsanis N: Ciliopathies. NEnglJMed364: tional characterization of the OFD1 protein reveals a nuclear localiza- 1533–1543, 2011 tion and physical interaction with subunits of a chromatin remodeling 2. Hurd TW, Hildebrandt F: Mechanisms of nephronophthisis and related complex. Mol Biol Cell 18: 4397–4404, 2007 ciliopathies. Nephron, Exp Nephrol 118: e9–e14, 2011 14. Higginbotham H, Bielas S, Tanaka T, Gleeson JG: Transgenic mouse 3. Otto EA, Hurd TW, Airik R, Chaki M, Zhou W, Stoetzel C, Patil SB, Levy S, line with green-fluorescent protein-labeled Centrin 2 allows visualiza- Ghosh AK, Murga-Zamalloa CA, van Reeuwijk J, Letteboer SJ, Sang L, tion of the centrosome in living cells. Transgenic Res 13: 155–164, 2004 Giles RH, Liu Q, Coene KL, Estrada-Cuzcano A, Collin RW, McLaughlin 15. Vladar EK, Stearns T: Molecular characterization of assembly HM, Held S, Kasanuki JM, Ramaswami G, Conte J, Lopez I, Washburn J, in ciliated epithelial cells. JCellBiol178: 31–42, 2007 Macdonald J, Hu J, Yamashita Y, Maher ER, Guay-Woodford LM, 16. Jain R, Pan J, Driscoll JA, Wisner JW, Huang T, Gunsten SP, You Y, Neumann HP, Obermüller N, Koenekoop RK, Bergmann C, Bei X, Lewis Brody SL: Temporal relationship between primary and motile cilio- RA, Katsanis N, Lopes V, Williams DS, Lyons RH, Dang CV, Brito DA, genesis in airway epithelial cells. Am J Respir Cell Mol Biol 43: 731–739, Dias MB, Zhang X, Cavalcoli JD, Nürnberg G, Nürnberg P, Pierce EA, 2010 Jackson PK, Antignac C, Saunier S, Roepman R, Dollfus H, Khanna H, 17. Boca M, D’Amato L, Distefano G, Polishchuk RS, Germino GG, Boletta Hildebrandt F: Candidate exome capture identifies mutation of A: Polycystin-1 induces cell migration by regulating phosphatidylino- SDCCAG8 as the cause of a retinal-renal ciliopathy. Nat Genet 42: 840– b 850, 2010 sitol 3-kinase-dependent cytoskeletal rearrangements and GSK3 - – 4. Schaefer E, Zaloszyc A, Lauer J, Durand M, Stutzmann F, Perdomo- dependent cell cell mechanical adhesion. MolBiolCell18: 4050 4061, Trujillo Y, Redin C, Bennouna Greene V, Toutain A, Perrin L, Gérard M, 2007 Caillard S, Bei X, Lewis RA, Christmann D, Letsch J, Kribs M, Mutter C, 18. Zhao Y, Hong D-H, Pawlyk B, Yue G, Adamian M, Grynberg M, Godzik Muller J, Stoetzel C, Fischbach M, Marion V, Katsanis N, Dollfus H: A, Li T: The retinitis pigmentosa GTPase regulator (RPGR)-interacting Mutations in SDCCAG8/NPHP10 cause Bardet-Biedl syndrome and are protein: Subserving RPGR function and participating in disk morpho- – associated with penetrant renal disease and absent polydactyly. Mol genesis. Proc Natl Acad Sci U S A 100: 3965 3970, 2003 Syndromol 1: 273–281, 2011 19. Won J, Gifford E, Smith RS, Yi H, Ferreira PA, Hicks WL, Li T, Naggert JK, 5. Kenedy AA, Cohen KJ, Loveys DA, Kato GJ, Dang CV: Identification Nishina PM: RPGRIP1 is essential for normal rod photoreceptor outer – and characterization of the novel centrosome-associated protein segment elaboration and morphogenesis. Hum Mol Genet 18: 4329 CCCAP. Gene 303: 35–46, 2003 4339, 2009 6. Kamio T, Asano A, Hosaka YZ, Khalid AM, Yokota S-i, Ohta M, Ohyama 20. Zhou W, Otto EA, Cluckey A, Airik R, Hurd TW, Chaki M, Diaz K, Lach FP, K, Yamano Y: Expression of the centrosomal colon cancer autoantigen Bennett GR, Gee HY, Ghosh AK, Natarajan S, Thongthip S, Veturi U, gene during spermatogenesis in the maturing rat testis. Biosci Bio- Allen SJ, Janssen S, Ramaswami G, Dixon J, Burkhalter F, Spoendlin M, technol Biochem 74: 1466–1469, 2010 Moch H, Mihatsch MJ, Verine J, Reade R, Soliman H, Godin M, Kiss D, 7. Patil H, Tserentsoodol N, Saha A, Hao Y, Webb M, Ferreira PA: Selective Monga G, Mazzucco G, Amann K, Artunc F, Newland RC, Wiech T, loss of RPGRIP1-dependent ciliary targeting of NPHP4, RPGR and Zschiedrich S, Huber TB, Friedl A, Slaats GG, Joles JA, Goldschmeding SDCCAG8 underlies the degeneration of photoreceptor neurons. Cell R, Washburn J, Giles RH, Levy S, Smogorzewska A, Hildebrandt F: Death Dis 3: e355, 2012 FAN1 mutations cause karyomegalic interstitial nephritis, linking 8. Di Gioia SA, Letteboer SJF, Kostic C, Bandah-Rozenfeld D, Hetterschijt chronic kidney failure to defective DNA damage repair. Nat Genet 44: L, Sharon D, Arsenijevic Y, Roepman R, Rivolta C: FAM161A, associated 910–915, 2012 with retinitis pigmentosa, is a component of the cilia-basal body com- 21. Ciccia A, Elledge SJ: The DNA damage response: Making it safe to play plex and interacts with proteins involved in ciliopathies. Hum Mol with knives. Mol Cell 40: 179–204, 2010 Genet 21: 5174–5184, 2012 22. Attanasio M, Uhlenhaut NH, Sousa VH, O’Toole JF, Otto E, Anlag K, 9. Chaki M, Airik R, Ghosh AK, Giles RH, Chen R, Slaats GG, Wang H, Hurd Klugmann C, Treier AC, Helou J, Sayer JA, Seelow D, Nürnberg G, TW, Zhou W, Cluckey A, Gee HY, Ramaswami G, Hong CJ, Hamilton Becker C, Chudley AE, Nürnberg P, Hildebrandt F, Treier M: Loss of

2582 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 2573–2583, 2014 www.jasn.org BASIC RESEARCH

GLIS2 causes nephronophthisis in humans and mice by increased ap- 34. Kalay E, Yigit G, Aslan Y, Brown KE, Pohl E, Bicknell LS, Kayserili H, Li Y, optosis and fibrosis. Nat Genet 39: 1018–1024, 2007 Tüysüz B, Nürnberg G, Kiess W, Koegl M, Baessmann I, Buruk K, 23. Atala A, Freeman MR, Mandell J, Beier DR: Juvenile cystic kidneys (jck): Toraman B, Kayipmaz S, Kul S, Ikbal M, Turner DJ, Taylor MS, Aerts J, A new mouse mutation which causes polycystic kidneys. Kidney Int 43: Scott C, Milstein K, Dollfus H, Wieczorek D, Brunner HG, Hurles M, 1081–1085, 1993 Jackson AP, Rauch A, Nürnberg P, Karagüzel A, Wollnik B: CEP152 is a 24. Bergmann C, Fliegauf M, Brüchle NO, Frank V, Olbrich H, Kirschner J, genome maintenance protein disrupted in Seckel syndrome. Nat Schermer B, Schmedding I, Kispert A, Kränzlin B, Nürnberg G, Becker Genet 43: 23–26, 2011 C, Grimm T, Girschick G, Lynch SA, Kelehan P, Senderek J, Neuhaus TJ, 35. Sir J-H, Barr AR, Nicholas AK, Carvalho OP, Khurshid M, Sossick A, Stallmach T, Zentgraf H, Nürnberg P, Gretz N, Lo C, Lienkamp S, Reichelt S, D’Santos C, Woods CG, Gergely F: A primary microcephaly Schäfer T, Walz G, Benzing T, Zerres K, Omran H: Loss of nephrocystin- protein complex forms a ring around parental centrioles. Nat Genet 43: 3 function can cause embryonic lethality, Meckel-Gruber-like syn- 1147–1153, 2011 drome, situs inversus, and renal-hepatic-pancreatic dysplasia. Am J 36. Ruzankina Y, Pinzon-Guzman C, Asare A, Ong T, Pontano L, Cotsarelis Hum Genet 82: 959–970, 2008 G, Zediak VP, Velez M, Bhandoola A, Brown EJ: Deletion of the de- 25. Sugiyama N, Yokoyama T: Sustained cell proliferation of renal epithelial velopmentally essential gene ATR in adult mice leads to age-related cells in mice with inv mutation. Genes Cells 11: 1213–1224, 2006 phenotypes and stem cell loss. Cell Stem Cell 1: 113–126, 2007 26. Sang L, Miller JJ, Corbit KC, Giles RH, Brauer MJ, Otto EA, Baye LM, 37. AbouAlaiwi WA, Ratnam S, Booth RL, Shah JV, Nauli SM: Endothelial Wen X, Scales SJ, Kwong M, Huntzicker EG, Sfakianos MK, Sandoval W, cells from humans and mice with polycystic kidney disease are Bazan JF, Kulkarni P, Garcia-Gonzalo FR, Seol AD, O’Toole JF, Held S, characterized by polyploidy and segregation defects Reutter HM, Lane WS, Rafiq MA, Noor A, Ansar M, Devi AR, Sheffield through survivin down-regulation. Hum Mol Genet 20: 354–367, VC, Slusarski DC, Vincent JB, Doherty DA, Hildebrandt F, Reiter JF, 2011 Jackson PK: Mapping the NPHP-JBTS-MKS protein network reveals 38. Bukanov NO, Smith LA, Klinger KW, Ledbetter SR, Ibraghimov- ciliopathy disease genes and pathways. Cell 145: 513–528, 2011 Beskrovnaya O: Long-lasting arrest of murine polycystic kidney disease 27. Cantiello HF: A tale of two tails: Ciliary mechanotransduction in with CDK inhibitor roscovitine. Nature 444: 949–952, 2006 ADPKD. Trends Mol Med 9: 234–236, 2003 39. Bukanov NO, Moreno SE, Natoli TA, Rogers KA, Smith LA, Ledbetter 28. Gao J, Cheon K, Nusinowitz S, Liu Q, Bei D, Atkins K, Azimi A, Daiger SR,OumataN,GalonsH,MeijerL,Ibraghimov-BeskrovnayaO:CDK SP, Farber DB, Heckenlively JR, Pierce EA, Sullivan LS, Zuo J: Progressive inhibitors R-roscovitine and S-CR8 effectively block renal and hepatic photoreceptor degeneration, outer segment dysplasia, and rhodopsin cystogenesis in an orthologous model of ADPKD. Cell Cycle 11: 4040– mislocalization in mice with targeted disruption of the retinitis pigmentosa- 4046, 2012 1(Rp1)gene.Proc Natl Acad Sci U S A 99: 5698–5703, 2002 40. Hansen GM, Markesich DC, Burnett MB, Zhu Q, Dionne KM, Richter LJ, 29. Huang WC, Wright AF, Roman AJ, Cideciyan AV, Manson FD, Gewaily Finnell RH, Sands AT, Zambrowicz BP, Abuin A: Large-scale gene DY, Schwartz SB, Sadigh S, Limberis MP, Bell P, Wilson JM, Swaroop A, trapping in C57BL/6N mouse embryonic stem cells. Genome Res 18: Jacobson SG: RPGR-associated retinal degeneration in human X-linked 1670–1679, 2008 RP and a murine model. Invest Ophthalmol Vis Sci 53: 5594–5608, 2012 41. Airik R, Bussen M, Singh MK, Petry M, Kispert A: Tbx18 regulates the 30. Humphries MM, Rancourt D, Farrar GJ, Kenna P, Hazel M, Bush RA, development of the ureteral mesenchyme. JClinInvest116: 663–674, Sieving PA, Sheils DM, McNally N, Creighton P, Erven A, Boros A, Gulya 2006 K, Capecchi MR, Humphries P: Retinopathy induced in mice by tar- 42. Thompson DA, Khan NW, Othman MI, Chang B, Jia L, Grahek G, Wu Z, geted disruption of the rhodopsin gene. Nat Genet 15: 216–219, 1997 Hiriyanna S, Nellissery J, Li T, Khanna H, Colosi P, Swaroop A, 31. Deretic D: A role for rhodopsin in a signal transduction cascade that Heckenlively JR: Rd9 is a naturally occurring mouse model of a common regulates membrane trafficking and photoreceptor polarity. Vision Res form of retinitis pigmentosa caused by mutations in RPGR-ORF15. PLoS 46: 4427–4433, 2006 ONE 7: e35865, 2012 32. Murga M, Bunting S, Montaña MF, Soria R, Mulero F, Cañamero M, Lee 43. Grupp C, Begher M, Cohen D, Raghunath M, Franz H-E, Müller GA: Y, McKinnon PJ, Nussenzweig A, Fernandez-Capetillo O: A mouse Isolation and characterization of the lower portion of the thin limb of model of ATR-Seckel shows embryonic replicative stress and acceler- Henle in primary culture. Am J Physiol 274: F775–F782, 1998 ated aging. Nat Genet 41: 891–898, 2009 33. Griffith E, Walker S, Martin C-A, Vagnarelli P, Stiff T, Vernay B, Al Sanna N, Saggar A, Hamel B, Earnshaw WC, Jeggo PA, Jackson AP, O’Driscoll M: Mutations in pericentrin cause Seckel syndrome with defective ATR- This article contains supplemental material online at http://jasn.asnjournals. dependent DNA damage signaling. Nat Genet 40: 232–236, 2008 org/lookup/suppl/doi:10.1681/ASN.2013050565/-/DCSupplemental.

J Am Soc Nephrol 25: 2573–2583, 2014 Loss of Sdccag8 Causes NPHP-RC 2583 Supplemental Information

Supplementary Figure 1. Generation of Sdccag8gt/gt mice. (A) Diagram of the insertion point of the gene-trap cassette VICTR24 between exons 1 and 2 into the wild type Sdccag8 locus. Genotyping primer location indicated as F - Sdccag8-F, LTR-R - Sdccga8-LTR-R, LTR-F - Sdccag8-LTR-F and R - Sdccga8-R. Exons indicated with numbers. (B) Genotyping PCR products from tail tips. Black arrows indicate Sdccag8 PCR bands. Primers F and R generate a 490 bp product from wild type allele, primers F and LTR_R generate 349 bp product and primers LTR_F and R generate a 290 bp product from a gene-trapped allele. Red arrows indicate internal PCR control bands (210 bp) confirming that DNA was present in each reaction mixture, except the water control. (C) qRT-PCR analysis on MEFs (n=4) demonstrates absence of any Sdccag8 transcript in the homozygous gene-trapped animals. (D) Western blot analysis on Sdccag8wt/gt and Sdccag8gt/gt kidney and lung lysates confirms the absence of both - 78 kDa and 83 kDa Sdccag8 protein isoforms from Sdccag8gt/gt tissues. β-actin was used as a loading control. wt, Sdccag8 wild type allele; gt, Sdccag8 gene-trap allele.

Supplementary Figure 2. Sdccag8 expression in kidney and lung. (A) No X-gal staining is observed in E16.5 wild type urogenital system. (B and C) No signal is visible in P14 (B) and P100 (C) kidneys hybridized with Sdccag8 sense probe. (D - F) CETN2- GFP expressing bronchiole epithelium cells (D) are negative for the cilia marker poly- glutamylated tubulin (E, white arrows) in E16.5 mouse lung section (F). Nuclei are stained with DAPI. Scale bar: (A) 400 µm; (B) 800 µm; (C) 2 mm; (D - F) 20 µm. wt, Sdccag8 wild type allele.

Supplementary Figure 3. Sdccag8gt/gt kidneys do not show global ciliary malformations, and Sdccag8gt/gt MEFs display normal cytoskeletal rearrangements in the wound healing assay. (A and B) Cortical collecting ducts (stained with AQP2 - red) bear normal looking cilia (stained with acetylated antibody - green) in Sdccag8gt/gt kidneys (B). (C) Immunofluorescence staining of wild type, Sdccag8wt/gt and Sdccag8gt/gt MEFs show similar Golgi (green) orientation towards the leading edge of the cell in the wound healing assay. DAPI labels nuclei, and α-tubulin stains microtubules. (D) Graphical representation of the percentage of cells showing Golgi orientation towards the leading edge of the cell 24 hours after wound scratch (each genotype n=3). Scale bar: (A) 7.5 µm, (B) 10 µm; (C) 40 µm. wt, Sdccag8 wild type allele; gt, Sdccag8 gene trap allele.

Supplementary Figure 4. Sdccag8gt/gt mice develop a progressive loss of vision and have reduced cone numbers. (A–B) Representative dark-adapted and light-adapted ERG waveforms are shown for increasing stimulus intensities for the Sdccag8wt/gt and Sdccag8gt/gt mice at P100 (A) and P250 (B). The Sdccag8wt/gt mice had normal dark- and light-adapted ERGs while the ERG responses of Sdccag8gt/gt were highly reduced in amplitude with varying degrees of loss of rod and cone function. (C) Number of PNA- FITC labeled cone cells within a 150µm area of retina. Values are mean numbers from 5 wild type and 4 Sdccag8gt/gt P30 mouse retinas. *P<0.05. (D, E) Immunolocalization of CEP164 and rhodopsin in the P100 retina shows rhodopsin accumulation (white arrows) at the photoreceptor inner segment plasma membrane and cell bodies in Sdccag8gt/gt retina (E). Scale bars: (D, E) 10 µm. ROS, retinal outer segment; IS, inner segment; ONL, outer nuclear layer; wt, Sdccag8 wild type allele; gt, Sdccag8 gene trap allele.

Supplementary Figure 5. Western blot analysis of SV-40 large T-antigen expression and quantitation of apoptotic index in Sdccag8gt/gt cells. (A) SV-40 large T-antigen protein is expressed in wild type and Sdccag8gt/gt isolated adult kidney cells when grown at 33°C in the presence of γ-interferon, and is absent from cells grown at 37°C without γ- interferon. β-actin was used as a loading control. Sdccag8gt/gt cells’ apoptosis indices are comparable to those of wild type cells both in synchronized (B) and unsynchronized (C) cultures after treatment with a low dose of bleomycin (4.5U/ml) or 30J UV-light. wt, Sdccag8 wild type allele; gt, Sdccag8 gene-trap allele.