Modulation of the Secretory Pathway Rescues Zebrafish Polycystic

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Modulation of the Secretory Pathway Rescues Zebraﬁsh Polycystic Kidney Disease Pathology

† Stéphanie Le Corre,* David Eyre, and Iain A. Drummond*

*Nephrology Division, Massachusetts General Hospital, Charlestown, Massachusetts; and †Department of Orthopedics and Sports Medicine, University of Washington, Seattle, Washington

ABSTRACT Mutations in polycystin 1 and polycystin 2 are responsible for autosomal dominant polycystic kidney disease, the most common heritable human disease. Polycystins function as calcium ion channels, but their impact on cell physiology is not fully known. Recent findings suggest that polycystins could function in the maintenance of extracellular matrix integrity. In zebrafish, polycystin 2 knockdown induces kidney cysts, hydrocephalus, left/right asymmetry defects, and strong dorsal axis curvature. Here, we show that increased notochord sheath collagen deposition in polycystin 2–deficient embryos is directly linked to axis defects. Increased collagen II protein accumulation did not associate with increased col2a1 mRNA or a decrease in matrix metalloproteinase activity but, instead, it associated with increased expression of the endoplasmic reticulum/Golgi transport coat protein complex II Sec proteins. sec24D knockdown prevented dorsal axis curvature and kidney cystogenesis in polycystin 2 morphants. Nontoxic doses of brefeldin A also prevented the dorsal axis curvature formation in polycystin 2 morphants and curly up polycystin 2 mutants. Brefeldin A treatment after the onset of polycystin deficiency phenotypes reversed the curved axis phenotype but not kidney cyst progression. Our results suggest that polycystin 2 deficiency causes increased collagen II synthesis with upregulation of secretory pathway coat protein complex II components. Restoration of normal rates of secretory protein synthesis and secretion may be a new target in the treatment of autosomal dominant polycystic kidney disease.

J Am Soc Nephrol 25: 1749–1759, 2014. doi: 10.1681/ASN.2013101060

Autosomal dominant polycystic kidney disease and the cellular functions of polycystins has been (ADPKD) is the most common heritable human elusive. disease, with an incidence of 1 in 800 births, and it is Polycystin 1 and polycystin 2 are multipass caused by mutations in polycystic kidney disease 1 transmembrane proteins that function together (PKD1)andPKD2 encoding polycystin 1 and poly- as a receptor–ion channel complex,1 leading to cystin 2.1 ADPKD is characterized by the presence the widely held view that primary cellular defects of epithelial cysts in both kidneys as well as cysts in in polycystin-deficient cells are caused by a disrup- the liver and pancreas. In addition to cystic pathol- tion of normal Ca2+ signaling. The polycystin 1/2 ogy,ADPKDisalsolinkedtoabdominalhernia, complex in apical cilia participates in mechanosen- and it is one of the best defined heritable disorders sory Ca2+ signaling in response to fluid flow,6 sug- associated with intracranial aneurysm2 (conditions gesting that loss of either polycystins or cilia associated with both altered extracellular matrix (ECM) composition and integrity). Other manifes- tations of ADPKD include gastrointestinal cysts Received October 9, 2013. Accepted December 11, 2013. 3 and cardiac valve defects. Aortic aneurysm and Published online ahead of print. Publication date available at vascular fragility are also observed in mouse models www.jasn.org. 4,5 of ADPKD. These features of ADPKD suggest Correspondence: Dr.IainA.Drummond,Nephrology Division, that altered tissue structure or ECM integrity Massachusetts General Hospital, 149 13th Street, Room 149- could play an important role in this disease; 8000, Charlestown, MA 02129. Email: [email protected] however, a mechanism linking ECM abnormalities Copyright © 2014 by the American Society of Nephrology

J Am Soc Nephrol 25: 1749–1759, 2014 ISSN : 1046-6673/2508-1749 1749 BASIC RESEARCH www.jasn.org structure would lead to acute loss of flow sensation and cyst collagen type II protein knockdown rescued the pkd2 axis formation. However, the relatively slow and sporadic develop- curvature phenotype, suggesting that collagen type II overpro- ment of kidney cysts in polycystin 1 and cilia protein condi- duction may be causally linked to axis curvature or alterna- tional knockout models has suggested that other factors may tively, that collagen type II may simply be required to stabilize contribute to cyst formation. Polycystin 1 and polycystin 2 are an altered axial structure induced by other factors in pkd2- also localized to the endoplasmic reticulum (ER) membrane, deficient embryos. To distinguish between these possibilities, where they function to regulate ER calcium stores, calcium we used lower doses of col2a1a morpholino and attempted to release, and intracellular calcium homeostasis.7–9 The ER and restore collagen type II protein to near wild-type levels in pkd2 Golgi apparatus are also the main vesicular organelles associ- morphants. Partial col2a1a knockdown almost completely ated with secretory protein synthesis and vesicle trafficking; rescued dorsal axis curvature (Figure 1, A–C). Collagen type altered ER calcium handling in ADPKD and the involvement II immunostaining revealed that partial col2a1a knockdown of multiple calcium binding proteins in vesicular trafficking successfully restored the amount of collagen type II to wild- suggest that ADPKD could affect secretion. Indeed, early stud- type levels in the notochord sheath of pkd2 morphants as ies of ADPKD patient cells revealed defects in ECM proteo- shown in longitudinal views of the notochord (Figure 1, D– glycan synthesis in ADPKD cells,10–13 whereas more recently, L). Phenotype quantification showed that nearly all (96.5%) of Golgi function and basolateral exocytosis have been shown to the pkd2 morphants exhibited moderate or severe dorsal axis be altered in PKD1 mutant epithelial cells,14 with altered in- curvature at 48 hours postfertilization (hpf), whereas embryos tracellular vesicle trafficking linked to mislocalization of sec6, coinjected with col2a1a and pkd2 morpholinos showed only sec8, and E-cadherin proteins. Although the data are sugges- mild or no curvature (Figure 1M). Knockdown of col2a1a in tive of a link between altered secretory protein production and the ventrally curved kidney cyst mutant double bubble19 had no disease pathology, it remains to be shown that modulating the effect on mutant curvature (Supplemental Figure 2). Taken secretory pathway can impact tissue morphogenesis defects together, these results suggest that matrix overproduction is associated with ADPKD. directly linked to the axis curvature phenotype and that this We and others have modeled ADPKD in the zebrafish by mechanism is specifictopkd2 deficiency. knockdown or mutation of orthologous pkd genes.15–17 Poly- cystin loss of function results in left–right asymmetry, kidney Altered Collagen Type II Accumulation Occurs Post- cysts, hydrocephalus, and a strong dorsal axis curvature. Dor- Transcriptionally sal axis curvature is a fully penetrant phenotype associated To test whether collagen overaccumulation in the notochord with overaccumulation of collagen type II protein in the no- sheath of pkd2 morphants was caused by increased collagen tochord sheath.18 Here, we show that accumulation of colla- mRNA expression or other mechanisms, we assessed col2a1a gen type II in the notochord sheath is a consequence of an and col9a2 expression by quantitative RT-PCR. Surprisingly, increased protein production and secretion linked to upregu- expression of col2a1a (Figure 1N) and col9a2 mRNA (Figure lation of the cytoplasmic coat protein II (COPII) components 1O) was similar in pkd2 morphants compared with wild-type sec23B, sec24C,andsec24D. Inhibition of the secretory path- embryos at 24 and 48 hpf, suggesting a post-transcriptional waybyknockdownofsec24D or chemical inhibition with mechanism for overaccumulation of collagen II protein. Con- brefeldin A prevents the dorsal axis curvature and cyst for- sistent with this idea, low, relatively nontoxic doses of the mation observed in polycystin 2–deficient embryos. Our data protein synthesis inhibitor cycloheximide (20 mg/ml) were suggest a causative role for increased ECM synthesis and sufficient to prevent the pkd2 axis curvature phenotype (Sup- secretion in the initiation of pkd2 deficiency phenotypes plemental Figure 3). These results suggested that overaccu- and raise the possibility that vesicle trafficking may be a tar- mulation of notochord collagen II protein was most likely get to ameliorate disease pathogenesis in human ADPKD linked to enhanced collagen translation, secretion, or reduced patients. degradation.

Collagen Type II Accumulation Is Linked to Increased RESULTS Expression of COPII Components Collagen mRNA translation, fibril assembly, and secretion are Altered Collagen Type II Accumulation Mediates Axis subject to multiple processing steps that could affect overall Defects in pkd2-Deficient Embryos collagen abundance or integrity of the ECM. A critical step of Polycystin 1a/b and polycystin 2 deficiency in zebrafish results collagen processing is its vesicular trafficking from the ER, in kidney cysts, hydrocephalus, and strong dorsal axis curva- where it is synthesized,tothe Golgiapparatusforsecretion. The ture (Figure 1, A and B, Supplemental Figure 1).15,16,18 Dorsal COPII complex that consists of multiple Sec proteins mediates axis curvature is the most penetrant and consistent phenotype, ER to Golgi vesicle traffic. Interestingly, quantitative RT-PCR affecting almost 100% of zebrafish pkd2 morphants/mutants, expression analysis of mRNA encoding the COPII components and it is associated with overexpression of notochord sheath sec23A, sec23B, sec24C,andsec24D indicated that the level of collagen type II.18 We have previously shown that complete sec23B, sec24C,andsec24D mRNAs (Figure 2, B–D) was

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significantly increased in pkd2-deficient embryos compared with wild type at 48 hpf, whereas sec23A mRNA expression was not affected (Figure 2A). These data suggestthataccumulationofcollagen type II could be linked to an increased rate of secretion. Other factors, such as expression of the collagen crosslinking lysyl oxidase genes lox, loxl1,andloxl5b20 (Sup- plemental Figure 4, A–C) or the cross- linked status of collagen type II protein itself (Supplemental Figure 4, D–F), were not affected. Also, it is unlikely that decreased collagen degradation could account for increased collagen type II accumulation; matrix metalloprotease (MMP) mRNA expression did not decrease in pkd2 morphants (Supplemental Figure 5), and multiple different MMP (GM6001, doxycycline, and ascorbic acid) and proteasome (MG132 and MLN4924) inhibitors did not induce axis curvature in wild-type embryos (data not shown). Increased accumulation of collagen type II protein could, however, be de- tected in Western blots of pkd2 morphants, confirming our immunostaining results (Supplemental Figure 4, D–F).

labeling (green) in the notochord of a (D and G) wild-type embryo, (E and H) pkd2 morphant, and (F and I) col2a1a/pkd2 double morphants (pkd2+col2a1a MO) at 30 hpf. (J–L) Merged images of collagen type II and wga; (D–F) co- l2a1a partial knockdown in pkd2 morphants reduces the collagen type II level close to wild- type level. Stacks were acquired through the whole notochord, and images from the middle of the notochord were selected. (M) Quantifi- cation of the percentage of embryos showing no curvature, mild curvature, moderate curvature, and severe curvature in the different experimental groups from a single represen- tative experiment (of four replicates). Wild- type embryos (n=89); pkd2 morphants (n=57); pkd2+col2a1a morphants (n=101). (N and O) Quantitative RT-PCR measurements of co- l2a1a and col9a2 mRNA abundance showing no difference in wild-type embryos (white bars) and pkd2 morphants (black bars) at 24 and 48 hpf. Expression is shown relative to the EIF1a mRNA level (n=4 per group). Differ- ences in col2a1a mRNA levels were not sta- Figure 1. Collagen type II overexpression plays a direct role in dorsal axis curvature in tistically significant. A small increase in col9a2 pkd2-deficient embryos. (A) Wild-type embryo and (B) pkd2 morphant (pkd2 MO) at mRNA was significant at 24 hpf (**P=0.003) 48 hpf; (C) col2a1a knockdown in pkd2 morphants prevents the dorsal axis curvature. but not 48 hpf. WT, wild-type. Scale bars, (D–L) Confocal images showing collagen type II immunostaining (red) and wga 10 mm.

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Figure 2. Overexpression of sec genes contributes to dorsal axis curvature and cystogenesis in pkd2-deficient embryos. (A–D) Quantitative RT-PCR measurements of sec genes showing an increased expression of (B) sec23B,(C)sec24C, and (D) sec24D in pkd2 morphants (blacks bars) compared with wild-type embryos (white bars) at 48 hpf (n=4 per group; *P,0.05; **P,0.01; ***P,0.001). (E) Wild-type embryo, (F) pkd2 morphant, and (G) sec24D knockdown in pkd2 morphant at 48 hpf. (H) Quantification of the number of embryos with severe dorsal axis curvature in three independent experiments revealed that sec24D knockdown can prevent the dorsal axis curvature in pkd2 morphants. Wild-type embryos and sec24D morphants never displayed any curvature. Wild type (n=512); sec24D morphants (n=175); pkd2 morphants (n=179); pkd2+sec24D morphants (n=129). #P=0.04. (I–K) Dorsal view of zebrafish pronephros in a (I) control Tg(wt1b:GFP) embryo, (J) Tg(wt1b:GFP) pkd2 morphant, and (K) Tg(wt1b:GFP) sec24D MO in pkd2 morphants at 48 hpf. (L) The quantification of three independent experiments shows that the injection of sec24D morpholino in pkd2 morphants decreases cyst formation compared with pkd2 morphants. Wild type (n=41); sec24D morphants (n=31); pkd2 morphants (n=43); pkd2+sec24D morphants (n=41). MO, morpholino oligo–injected; WT, wild-type. sec24D Knockdown Prevents the Dorsal Axis 48 hpf (Figure 2, E–G). Quantification revealed that 86% of Curvature and Cyst Formation in pkd2-Deficient pkd2 morphants had severe curvature compared with 38% of Embryos the double morphants, indicating that the secretory pathway To determine if increased Sec gene expression might be linked playsacrucialroleinthisphenotype(Figure2H).Totestif to the increased collagen production observed in pkd2-deficient increased sec gene expression was also linked to pronephric embryos, we knocked down sec24D in pkd2 morphants and cyst formation, we monitored kidney cyst formation in the monitored dorsal axis curvature as a readout of altered ECM. Tg(wt1b:GFP) line after knocking down sec24D in pkd2 Significantly, the severity of curvature in the pkd2/sec24D dou- morphants. Cyst formation was decreased in double mor- ble morphants was reduced compared with pkd2 morphants at phants compared with pkd2 knockdown alone (Figure 2,

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I–K). Cyst quantiﬁcation revealed that 49% of pkd2 morphants exhibited cysts compared with 17% of the double morphants (Figure 2L). Cyst rescue was not associated with any increase in apoptosis in double morphants (Supplemental Figure 6, A–D).

Brefeldin A Prevents Dorsal Axis Curvature in pkd2 Mutants and Morphants Brefeldin A (BFA) blocks ER to Golgi vesicular trafficby stabilizing the Arf-GDP:ArfGEF intermediate and preventing Arf cycling.21,22 High doses of BFA (10 mg/ml) completely block ER/Golgi trafficinzebrafish embryos, inducing the ER stress response and apoptosis.23 We reasoned that low, nontoxic doses of BFA might reduce the rate of vesicle trafficking without inducing cell stress. We, therefore, treated wild-type zebrafish embryos and pkd2 morphants with low to intermediate doses of BFA (from 0.5 to 2 mg/ml) and monitored collagen accumulation. Collagen type II is primarily localized in the notochord sheath ECM in wild-type embryos and pkd2 Figure 3. BFA blocks collagen type II secretion in pkd2 morphants. morphants at 30 hpf. (Figure 3, A, B, E, F, I, J, M, and N). Confocal images showing (A–D) collagen type II immunostain- However, after BFA treatment, collagen accumulated in a ve- ing(red)and(E–H) wga labeling (green) in the notochord of a (A sicular pattern, indicating that low-dose BFA inhibits secre- and E) wild-type embryo, (B and F) pkd2 morphant, (C and G) tion (Figure 3, C, D, G, H, K, L, O, and P) and can reduce BFA-treated wild-type embryo, and (D and H) BFA-treated pkd2 collagen type II accumulation in the ECM of pkd2 morphants morphant at 30 hpf. (I–L) Merged images of collagen type II and to wild-type levels (Figure 3, D, H, L, and P). Notably, collagen wga. (A and B) Collagen type II staining was localized in the II–containing vesicles were always much larger in pkd2 mor- notochord sheath in wild-type and pkd2 morphants, with a phants, consistent with an ECM overproduction phenotype higher expression in pkd2 morphants, whereas (C and D) it accumulates in a vesicular pattern in BFA-treated embryos, (Figure 3D). Wild-type embryos treated with BFA exhibited showing that BFA inhibits collagen type II sheath accumulation a shortened body axis, especially at the highest dose tested – m close to wild-type levels in pkd2 morphants. Arrowheads in A D (2 g/ml) (Supplemental Figure 7), consistent with previous designate the notochord sheath ECM. (M–P) Line scan analysis studies reporting a correlation between disrupted collagen se- of A–D (at the yellow lines in A–D) shows (N) increased colla- cretion and reduced body length.24,25 Strikingly, early (24 hpf) gen II immunoreactivity in pkd2 morphants and (O and P) BFA treatment of pkd2 morphant embryos resulted in a dose- reduction in notochord sheath collagen II (arrowheads) in BFA- dependent decrease in dorsal axis curvature at 48 hpf (Figure treated embryos. Confocal stacks were acquired of the whole 4, A, B, E, and F), with the highest BFA doses tested nearly notochord, and images from the middle of the notochord were completely preventing development of axis curvature (Figure selected. Scale bars, 10 mm. MO, morpholino oligo–injected; WT, 4D). Another ER/Golgi transport small molecule inhibitor, wild-type. CI-976, also induced a significant dose-dependent rescue of the pkd2 axis curvature phenotype (Supplemental Figure 8). addition of BFA at 30 hpf reversed axis curvature pathology To confirm the effects of BFA in a genetic model of pkd2 de- (Figure 4, I–K), such that after 3 hours of BFA treatment, ficiency, we treated embryos from an incross of the zebrafish only 17% of pkd2 morphants exhibited curvature compared pkd2 mutant curly up (cup)17 with BFA. Untreated cup em- with 61% before treatment (Figure 4L). The effect persisted bryos from clutches of incrossed heterozygotes exhibited the at 48 hpf, when only 27% of pkd2 morphants treated with typical Mendelian ratio (25%) of moderate to severe dorsal BFA displayed curvature (Figure 4L). The results show that axis curvature (Figure 4, C and H, upper panel). BFA treat- BFA treatment cannot only prevent development of axis cur- ment eliminated severe axis curvature and induced a signifi- vature but also, restore a straight axis in previously curved cant shift to the mild curvature class (Figure 4, G and H, lower embryos. panel). These results confirm that modulation of the secretory pathway can rescue pkd2 mutant phenotypes and further sug- BFA Reduces Kidney Cyst Formation in pkd2 gest causal links between increased collagen synthesis, in- Morphants creased secretion, and pkd pathology. Totest whether BFA treatment could also prevent pkd2-associated To test whether BFA treatment could restore normal cystogenesis, we quantified cysts in Tg(wt1b:GFP); pkd2 mor- morphology after axis curvature had started to form, we phants treated with BFA (2 mg/ml). Similar to the effect of initiated BFA treatment at 30 hpf, when more than 60% of pkd2 sec24D knockdown, kidney cyst formation was reduced in morphants already exhibit dorsal axis curvature. Strikingly, BFA-treated pkd2 morphants compared with nontreated

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Figure 4. BFA treatment prevents dorsal axis curvature in pkd2-deficient embryos. (A) Wild-type embryo, (B) pkd2 morphant, and (C) cup embryo at 48 hpf. (E–G) Treatment with BFA (E) does not induce curvature in wild-type embryos but reduces dorsal axis curvature in (F) pkd2 morphants and (G) cup embryos when added at 2 mg/ml at 24 hpf. (D) Percentage of embryos with severe curvature in pkd2 morphants at 48 hpf (n=3 independent experiments). (H) Percentage of embryos with mild curvature (light gray bars), moderate curvature (dark gray bars), and severe curvature (black bars) in cup embryos (upper panel) and BFA-treated cup embryos (lower panel) at 48 hpf (n=3 independent experiments). pkd2 morphants (n=71); pkd2 morphants+BFA (n=57 at 0.5 mg/ml, n=75 at 1 mg/ml, n=73 at 1.5 mg/ml, n=60 at 2 mg/ml); cup mutants (n=298); cup mutants+BFA (n=222). The increase in pkd2 morphant embryos with mild curvature in the BFA sample was statistically significant (*P=0.05). (I) Wild-type embryo, (J) pkd2 morphant, and (K) BFA-treated pkd2 morphant at 48 hpf. (L) Percentage of embryos with moderate and severe curvature at 48 hpf in pkd2 morphants (black line) and pkd2 morphants treated with BFA at 30 hpf (red line). The quantification of two independent experiments at 33 hpf and three independent experiments at 48 hpf shows that BFA treatment can rescue preexisting curvature in pkd2 morphants. Wild type (n=270 at 30 hpf, n=73 at 33 hpf, n=71 at 48 hpf); pkd2 morphants (n=210 at 30 hpf, n=65 at 33 hpf, n=57 at 48 hpf); wild type+BFA (n=63 at 30 hpf, n=57 at 48 hpf); pkd2 morphants+BFA (n=68 at 33 hpf, n=53 at 48 hpf). *Percentage with severe curvature in BFA treated at 33 and 48 hpf was significantly different from untreated pkd2 morphants at 33 hpf; **percentage with severe curvature in BFA treated at 33 and 48 hpf was significantly different from untreated pkd2 morphants at 48 hpf; #percentage with severe curvature in BFA treated at 33 and 48 hpf was significantly different from the starting time point in both cases. MO, morpholino oligo–injected; WT, wild-type. pkd2 morphants (15.7% versus 43%) (Figure 5, A–D). The critical cyst initiating events or alternatively, that BFA may alter dose of BFA used in these experiments was not sufficient to the progression of cyst development. We, therefore, tested induce general apoptosis26,27 (Supplemental Figure 5), indicat- the efficacyofBFAincystinhibitionwhenitwasaddedata ing that the reduction in cyst formation was not caused by later stage in development. BFA treatment at 30 hpf did not generally toxicity. significantly prevent cystogenesis compared with non- The reduction in cyst frequency byearly treatment with BFA treated pkd2 morphants (Figure 5, E–G). In this experi- suggested that modulating the secretory pathway may block ment, we found that 37% of BFA-treated pkd2 morphants

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Figure 5. BFA treatment inhibits cyst initiation in pkd2 morphants. (A) Kidney of a control Tg(wt1b:GFP) embryo, (B) pkd2 morphant with kidney cysts, and (C) kidney of a pkd2 morphant treated with BFA at 24 hpf. (D) Percentage of embryos showing kidney cysts per group. Control Tg(wt1b:GFP) (n=32); Tg(wt1b:GFP) pkd2 morphants (n=28); BFA-treated Tg(wt1b:GFP) embryos (n=22); BFA-treated Tg(wt1b:GFP) pkd2 morphants (n=25). Data represent three independent experiments; pkd2 MO+BFA showed significantly fewer cysts than pkd2 MO alone (*P,0.05). (E) Kidney of a control Tg(wt1b:GFP), (F, arrows) kidney cyst in pkd2 morphant, and (G, arrows) kidney cyst in pkd2 morphant treated with BFA at 30 hpf. (H) Percentage of embryos with kidney cysts at 48 hpf. Control Tg(wt1b:GFP) embryos (n=43); Tg(wt1b:GFP) pkd2 morphants (n=39); BFA-treated Tg(wt1b:GFP) embryos (n=26); BFA-treated Tg(wt1b:GFP) pkd2 morphants (n=44). Data represents three independent experiments. MO, morpholino oligo–injected; WT, wild-type. were cystic compared with 43% of nontreated pkd2 mor- primary in disease initiation or a secondary consequence of phants (Figure 5H). This result suggests that BFA is most likely tissue pathology. Our previous work suggested that the most acting at the time of cyst initiation and not, for instance, block- penetrant zebrafish PKD phenotype, dorsal axis curvature, ing transport of proteins that are required to maintain cystic was caused by notochord deformity induced by abnormal ex- distension. pression of ECM collagen type II.18 Using axis curvature as a readout of pkd2 deficiency, we show here that polycystin2 knockdowninzebrafish causes overproduction of colla- DISCUSSION gen type II protein in the notochord sheath. Moreover, re- establishing wild-type levels of collagen type II protein in Despite advances in our understanding of the functions of the notochord ECM restores a normal axis morphology, sup- polycystins, mechanisms underlying ADPKD pathogenesis porting the idea that collagen overproduction plays a causal remain unclear. The polycystin complex is proposed to role in zebrafish PKD axis curvature. Collagen overproduction function in multiple cellular contexts, including the primary was also associated with increased expression of secretory cilium as a mechanosensor,6 the ER as a calcium release chan- pathway COPII components. Sec protein knockdown or nel,7,8,28 and the cell membrane as an adhesion complex.29–32 chemical inhibition of ER to Golgi transport was also suffi- Different features of ADPKD pathology, including failed flow cient to reverse PKD axis curvature and kidney cyst formation, sensing, increased fluid secretion, and cell proliferation, may highlighting the secretory pathway as a potentially novel target well involve polycystin function in different subcellular loca- for treating PKD. tions. In addition to cystic epithelial pathology in the kidney, Increased collagen type II protein production in pkd2- pancreas, and liver, ADPKD has been linked to changes in deficient embryos could be caused by several mechanisms, in- ECM composition in multiple organs.4,33–35 Increased cell ad- cluding transcriptional or translational regulation of collagen hesiveness, decreased cell migration, and abnormal expression expression. Whole-embryo quantitative RT-PCR mRNA quan- of integrins have also been observed in ADPKD cells.29,36 tification showed no difference in overall col2a1a or col9a2 However, it remains unclear whether these pathologies are mRNA expression between pkd2-deficient and wild-type

J Am Soc Nephrol 25: 1749–1759, 2014 PKD and the Secretory Pathway 1755 BASIC RESEARCH www.jasn.org embryos, suggesting a post-transcriptional mechanism. Re- both pkd2 morphants and cup pkd2 mutants and prevented cently, we determined that our prior findings of increased kidney cyst formation in pkd2 morphants. At the concentra- col2a1a or col9a2 mRNA expression (by in situ hybridization) tions used, BFA did not induce apoptosis or major defects in in pkd2-deficient embryos could be accounted for by increased zebrafish embryos, except a shortened body length. Most embryo sensitivity to proteinase treatment used in the in situ importantly, we showed that BFA treatment could straighten hybridization protocol and enhanced in situ probe penetration embryos that already had a mild dorsal axis curvature before (data not shown). These results are consistent with a general treatment, suggesting that reversal of collagen overproduc- ECM defect in pkd2-deficient embryos that renders them tion could allow realignment of ECM to a normal axial mor- more fragile and permeant to probes. Equivalent expression phology. However, BFA treatment was unable to rescue cyst of collagen type II mRNA coupled with increased protein formation in pkd2 morphants after cysts were formed. The accumulation in pkd-deficient embryos points strongly to dif- results suggest that ECM defects could play a role in the ferences in translation or post-translational collagen type II initiation of cystogenesis and dorsal axis curvature but processing as the cause of axis curvature in pkd morphant perhaps, could not play a role in progression of the cystic zebrafish. General inhibition of MMPs or the proteasome phenotype. was not sufficient to induce axis defects, suggesting a principle Wehave focused here on the secretory pathway as a potential role for increased collagen production in driving the zebrafish target to reverse collagen overaccumulation in the ECM of pkd phenotype. Pkd2-deficient embryos. However, it is also clear from our The simplest interpretation of our data may be that work that collagen overaccumulation must also involve an polycystin 2 activity in the ER either directly or indirectly increased rate of col2a1a mRNA translation. The observed regulates secreted protein translation or vesicle trafficking. The changes in COPII component gene expression could be a altered expression of sec23B, sec24C,andsec24D that we ob- compensatory event to support increased secretion. Although serve supports the idea that ER to Golgi secretory trafficmay the Tor pathway is known to be activated in ADPKD cells48 be generally increased in pkd2 morphants. The role of Sec24D and could, in principle, signal increased protein synthesis, we in the secretion of collagen type II has already been shown in did not observe an increase in ribosomal phospho-S6 protein the zebrafish. Bulldog and feelgood mutants, in which sec24D is levels (Supplemental Figure 9), a readout of Tor signaling. disrupted, fail to secrete collagen type II, leading to abnormal Although additional studies will be required to determine cranial morphogenesis and shorter body length, likely because the mechanism of increased collagen translation, our finding of notochord defects.24,25 Previous studies have reported that modulation of the secretory pathway prevented and even altered secretion of heparin sulfate proteoglycans12,13 and de- reversed some ADPKD phenotypes in zebrafish suggests that fective basolateral vesicle trafficking in ADPKD cells.37 Poly- the COPII complex may be a new target in the treatment of cystin 2 itself interacts with and is regulated by Syntaxin5, an ADPKD. essential regulator of vesicle trafficking.38 Consistent with a role for polycystin 2 specifically in the ER, expression an ER- retained mutant polycystin 2 protein in zebrafish pkd2 mor- CONCISE METHODS phants has been shown to rescue the dorsal axis curvature phenotype39 that we have linked to excess collagen produc- Zebrafish Lines tion. Could polycystin 2 deficiency directly modify the secre- Wild-type TüAB and Tg(wt1b:GFP) transgenic zebrafish were main- tory pathway? PKD mutant cells are known to exhibit reduced tained and raised as described previously.49 Dechorionated embryos agonist-stimulated calcium release from the ER and in some were kept at 28.5°C in E3 solution with or without 0.003% 1-phenyl- cases, show enhanced amounts of ER calcium.40–42 In local- 2-thiourea (Sigma-Aldrich) to suppress pigmentation and staged ac- ized transport between the ER and Golgi, calcium binding cording to hpf.49 proteins regulate vesicle trafficking. For instance, calcium regulates the binding between Sec31A and Apoptosis-linked gene Morpholino Antisense Oligonucleotide Injections 2, a penta-EF-hand protein that regulates secretion by stabi- Wild-type (TüAB) or WT1:GFP embryos at the one- to two-cell lizing the COPII complex at the ER.43–46 Additional studies stages were microinjected with 4.6 nl 0.05 mM col2a1 or 0.25 mM will be required to determine whether altered calcium release pkd2 or sec24D antisense morpholino oligonucleotide solution (Gene from the ER in pkd-deficient embryos could influence vesicle Tools LLC) in 200 mM KCl with 0.1% Phenol Red using a nano- transport rates or the specific vesicle cargo sorting processes ject2000 microinjector (World Precision Instruments). Morpholino required for collagen secretion.47 sequences were Regardless of whether pkd2 knockdown directly or indi- fi rectly affects secretion, our nding that sec24D knockdown pkd2 exon 12: 59-caggtgatgtttacacttggaactc-39; or BFA treatment was sufficient to prevent pkd phenotypes in zebrafish argues strongly that altered protein secretion is col2a1a exon 1: 59-tgaaaaactccaacttacggtcatc-39;and one critical component of pkd pathology. BFA treatment resulted in a dose-dependent rescue of dorsal axis curvature in sec24D 59 untranslated region: 59-cttactgttatacctctttcattcc-39.

1756 Journal of the American Society of Nephrology J Am Soc Nephrol 25: 1749–1759, 2014 www.jasn.org BASIC RESEARCH

The control morpholino sequence was 59-cgtccattgtgtaaaagtg- Drug and Small Molecule Treatments taacca-39 (irrelevant sequence). Collagen crosslinking was inhibited by treating embryos in E3 water with the lysyl oxidase inhibitor b-aminopropionitrile (30 mM) from Quantitative RT-PCR postgastrulation (6–8 hpf) to 48 hpf. The secretory pathway from the RNAs were obtained from 24-, 48-, or 72-hpf embryos using the ER to the Golgi apparatus was inhibited using BFA or CI-976. BFA Qiagen RNeasy mini kit. RNAs were treated with DNase (DNase I, was dissolved in ethanol and used at 0.5, 1, 1.5, and 2 mg/ml in E3 egg RNase-free; Roche Diagnostics) and reverse-transcribed using oligo water containing 0.25% ethanol. CI-976 was dissolved in DMSO and dT according to the manufacturer’s protocols (Superscript III; In- used at 1–10 mM in E3 egg water containing 0.5% DMSO. Embryos vitrogen). Quantitative RT-PCR was performed using SYBR Green. were treated with BFA from 24 or 30 to 48 hpf as indicated or CI-976 Primer sequences are listed in Table 1. col2a1a, col9a2, lox, loxl1, from 24 to 30 hpf. Concentrations of MMP inhibitors used (treat- loxl5b, sec23A, sec23B, sec24C,andsec24D expressions were measured ment starting at gastrulation) were 10 mM GM6001, 10 mM, 100 mM, in control and pkd2-deficient embryos (n=4–5 samples per group; and 1 mM doxycycline, and 50, 100, 200, and 500 mM ascorbic acid in each RNA sample is obtained from 10 embryos) by quantitative E3 water. Proteasome inhibitors MG132 and MLN-4924 were used at RT-PCR. Gene expression was normalized with the housekeeping 10, 20, and 50 mM in E3 water. gene EIF1a. Quantification of Dorsal Axis Curvature Collagen Crosslinking Embryos were sorted as having a curvature of less than 90° (mild), a Collagen type IIwas extracted from 500 desiccated wild-type embryos curvature of more than 90° (moderate), or a curvature with a tail or pkd2 morphants with 4 M guanidine HCl and 0.05 M TriszHCl (pH crossing the body axis (severe) at 48 hpf (Supplemental Figure 1). 7.4) with 2 mM PMSF added as a proteases inhibitor for 48 hours at 4°C. The washed residue was digested with pepsin at 4°C and rocked Statistical Analysis for 24 hours, and the reaction was then stopped by the addition of A t test was performed to compare the different experimental groups pepstatin. Solubilized type II collagen was recovered in the 0.7 M NaCl (*P,0.05; **P,0.01; ***P,0.001). precipitate. The samples were run on a 6% SDS-PAGE gel and trans- blotted to a polyvinylidene difluoride membrane for Western blot de- Western Blotting tection of collagen type II chains with a monoclonal antibody 1C10 Anti–phospho-S6 rabbit monoclonal (Cell Signaling Technology) that recognizes a sequence-specific epitope in the a1(II) triple helical was used at 1:2000 dilution and standardized using anti-S6 mouse domain (residues 934–945).50 monoclonal (Cell Signaling Technology) used at 1:500 dilution.

ACKNOWLEDGMENTS Table 1. Quantitative RT-PCR primers used Gene Sequence The authors thank MaryAnn Weis and Lammy S. Kim for the collagen col2a1a Forward 59-tctccagcaggtccagtcaa-39 II analyses and Dr. Ela Knapik for advice and insight on zebraﬁsh sec col2a1a Reverse 59-gcgtggtgaaagaggtttcc-39 gene function. 9 col9a2 Forward 5 -gcaggacaaaacggaagacc-3 This work was supported by National Institutes of Health Grants 9 9 col9a2 Reverse 5 -cacccttaacccccacttca-3 AR037318 (to D.E.) and DK070263 (to I.A.D.). lox Forward 59-tgcacctcccacactcagggtt-39 lox Reverse 59-actccggcacctggtaactggga-39 loxl1 Forward 59-cggtgctccgtatgaccaagtttca-39 loxl1 Reverse 59-ctccacccgtgcctgccgaa-39 DISCLOSURES loxl5b Forward 59-tccacccggcggtaggacgat-39 None. loxl5b Reverse 59-ggcacaagatccggcagaccatt-39 mmp2 Forward 59-ggccactcacaccccaccagt-39 mmp2 Reverse 59-tcagccgtcctgaagaggaatctgt-39 REFERENCES mmp9 Forward 59-gatgggctgctggctcacgctt-39 9 9 mmp9 Reverse 5 -gcgggtttgaatggctggtccag-3 1. Sutters M, Germino GG: Autosomal dominant polycystic kidney dis- 9 9 mmp14 Forward 5 -cgggctcaggcgattcgctct-3 ease: Molecular genetics and pathophysiology. J Lab Clin Med 141: mmp14 Reverse 59-gcattgccgagagcgtagctgga-39 91–101, 2003 sec23A Forward 59-aggtggacgtggagcaatac-39 2. van Dijk MA, Chang PC, Peters DJ, Breuning MH: Intracranial aneur- sec23A Reverse 59-cgagaacgtctcggagaaac-39 ysms in polycystic kidney disease linked to chromosome 4. JAmSoc sec23B Forward 59-atgctgggactgatgaaacc-39 Nephrol 6: 1670–1673, 1995 sec23B Reverse 59-tcctgtgtttgggaaagtcc-39 3. Hossack KF, Leddy CL, Johnson AM, Schrier RW, Gabow PA: Echo- ﬁ sec24C Forward 59-cagggaagagagtggactgc-39 cardiographic ndings in autosomal dominant polycystic kidney disease. NEnglJMed319: 907–912, 1988 sec24C Reverse 59-gtcttcagctcctggcaaac-39 4. Hassane S, Claij N, Lantinga-van Leeuwen IS, Van Munsteren JC, Van sec24D Forward 59-tttgctgacaccaacgagag-39 Lent N, Hanemaaijer R, Breuning MH, Peters DJ, DeRuiter MC: Path- sec24D 9 9 Reverse 5 -tgattggggaacaggaagag-3 ogenic sequence for dissecting aneurysm formation in a hypomorphic

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J Am Soc Nephrol 25: 1749–1759, 2014 PKD and the Secretory Pathway 1759 Supplementary figure 1: Range of severity in dorsal axis curvature. We used a range of severity in dorsal axis curvature to score embryos at 48 hpf and considered it as straight when there was no curvature (A), mild when it was less than 90 degrees (B), moderate when it was more than 90 degrees (C) and severe when the tail was crossing the body axis (D). Supplementary figure 2: (A-C) col2a1a morphants, double bubble embryo (C) and col2a1a knock-down in double bubble mutant (C) at 96 hpf, showing no rescue of the ventral curvature. Supplementary figure 3.Low dose cycloheximide is sufficient to rescuepkd2 morphant axis curvature. (A) Control 30 hpf embryo. (B) pkd2 morphant at 30 hpf showing moderate axis curvature. (C) Quantification of the number ofpkd2 morphant embryos showing moderate, mild or no curvature. (D) Control cycloheximide (20 µg/ml) treated 30 hpf embryo. (E) 30 hpf pkd2 morphant embryo treated with 20 µg/ml cycloheximide starting at 24 hpf. (F) Quantification of number of cycloheximide treated pkd2 morphant embryos showing moderate, mild or no curvature. Most cycloheximide treated embryos showed no curvature (*; p=0.02) Supplementary figure 4: Collagen type II cross-linking is not affected in pkd2-deficient embryos. (A-C) Quantita- tive RT-PCR measurements of lox (A), loxl1 (B) and loxl5b (C) showing no difference in their expression between wildtype embryos (white bars) and pkd2 morphants (black bars) at 24, 48 and 72 hpf. (D-F) SDS-PAGE analysis of collagen type II serially extracted from wildtype embryos, pkd2 morphants and bAPN-treated embryos by 3% acetic acid (D), 4M guanidine- HCl (E) and pepsin digestion (F) at 48 hpf. bAPN-treated embryos mostly have soluble collagen type II while most is cross- linked in wildtype embryos and pkd2 morphants based on the relative extractabilities. Arrows indicate the size of α1 chains of collagen type II. Supplementary figure 5: Collagen accumulation is not due to a lack of mmp gene expression. Quantitative RT-PCR measurements of mmp2 (A), mmp9 (B) and mmp14 (C) showing an increased expression in pkd2 morphants (black bars) compared to wildtype embryos (white bars) at 48 hpf. n = 4 per group. Supplementary figure 6: sec24D knock-down and BFA treatment do not induce apoptosis. (A-F) Acridine orange staining in wildtype embryos (A), pkd2 morphants (B), sec24D morphant (C), sec24D + pkd2 morphant (D), BFA-treated wildtype embryo (E) and BFA-treated pkd2 morphant (F) showing no apoptosis at 48 hpf. Supplementary figure 7: BFA-treated embryos have shortened body length. BFA-treated wildtype embryos have reduced body length (B) compared to non-treated embryos (A) at 48 hpf. Supplementary figure 8: Vesicle trafficking inhibitor Cl-976 rescues axis curvature in pkd2 morphants. 24 hpf pkd2 morphant embryos were treated with the indicated concentrations of CI-976 and scored for curvature at 30 hpf. 5 µM and 10 µM Cl-976 caused a significant reduction in embryos with moderate curvature compared to control embryos (* : p=0.0268; ** : p=0.0073) Supplementary figure 9.Western blot quantification of phospho-S6 levels inpkd2 morphants. phospho-S6 was compared to total S6 protein in control and pkd2 morphant embryos at 24 hpf (left panel) and 48 hpf (right panel). Bar graphs show two independent experiments performed in triplicate for each condition.