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Inhibition of the P2X7 Reduces Cystogenesis in PKD

Ming-Yang Chang,* Jenn-Kan Lu,† Ya-Chung Tian,* Yung-Chang Chen,* Cheng-Chieh Hung,* ʈ Yi-Hui Huang,* Yau-Hung Chen,‡ Mai-Szu Wu,§ Chih-Wei Yang,* and Yi-Chuan Cheng

*Kidney Research Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan, Taiwan; †Laboratory of Molecular Biology, Institute of Aquaculture, National Taiwan Ocean University, Keelung, Taiwan; ‡Department of Chemistry, Tamkang University, Tamsui, Taiwan; §Department of Nephrology, Chang Gung Memorial Hospital, Keelung, ʈ Taiwan; Department of Biochemistry and Molecular Biology, Chang Gung University, Taoyuan, Taiwan

ABSTRACT The P2X7 receptor participates in purinergic signaling, which may promote the progression of ADPKD. We examined the effects of a P2X7 receptor antagonist and a P2X7 receptor on cyst develop- ment in a zebrafish model of polycystic kidney disease in which we knocked down pkd2 by morpholinos. We used live wt-1b pronephric-specific GFP-expressing zebrafish embryos to directly observe changes in the pronephros. Exposure of pkd2-morphant zebrafish to a P2X7 receptor antagonist (oxidized ATP [OxATP]) significantly reduced the frequency of the cystic phenotype compared with either exposure to a P2X7 receptor agonist (BzATP) or with no treatment (P Ͻ 0.01). Histology confirmed improvement of glomerular cysts in OxATP-treated pkd2 morphants. OxATP also reduced p-ERK activity and cell proliferation in pronephric kidneys in pkd2 morphants. Inhibition of P2X7 with an additional specific antagonist (A-438079), and through morpholino-mediated knockdown of p2rx7, confirmed these effects. In conclusion, blockade of the P2X7 receptor reduces cyst formation via ERK-dependent pathways in a zebrafish model of polycystic kidney disease, suggesting that P2X7 antagonists may have therapeutic potential in ADPKD.

J Am Soc Nephrol 22: 1696–1706, 2011. doi: 10.1681/ASN.2010070728

Autosomal dominant polycystic kidney disease pathway regulated by 5Ј triphosphate (ADPKD), caused by mutations in either PKD1 or (ATP). Besides its classical role as an intracellular PKD2, is the most common genetic kidney disease, energy source, ATP has been recognized as an ex- characterized by progressive enlargement of cysts tracellular signaling molecule.3,4 It has been pro- due to excessive fluid secretion and proliferation of posed that ATP release from cyst epithelial cells in renal tubular epithelial cells.1 It is one of the major response to stimuli such as mechanical deforma- causes of end stage renal disease and there is cur- tion may contribute to cyst formation and enlarge- rently no proven treatment. ment by activating purinergic receptors.5 A previ- Multiple signaling pathways are involved in cyst formation and progression, and studies of these sig- naling pathways have led to potential treatments for Received July 14, 2010. Accepted March 31, 2011. ADPKD, such as vasopressin V2 receptor antago- Published online ahead of print. Publication date available at nists, mTOR inhibitors, and somatostatin ana- www.jasn.org. logues.1 A combination therapy targeting different Correspondence: Dr. Yi-Chuan Cheng, Department of Biochem- istry and Molecular Biology, Chang Gung University College of pathways in cystogenesis may be required to maxi- Medicine, 259 Wen-Hwa 1st Road, Kueishan, Taoyuan 333, Taiwan. mize the treatment effects and limit their side ef- Phone:ϩ886-3-2118800, Ext.3396; Fax:ϩ886-3-2118042; E-mail: fects.2 A potential treatment target that has not been [email protected] completely explored is the Copyright © 2011 by the American Society of Nephrology

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ous study has shown that primary cultures of ADPKD tor is unique in both its tissue localization and functional epithelial cells release two- to fivefold more ATP under basal response, their activation requiring 10- to 100-fold higher and hypotonicity challenge compared with non-ADPKD concentrations of ATP than the P2X1 to 6 receptors.9 A cells.6 In ADPKD cysts, ATP is present in significant quantities comparative expression study of zebrafish P2X receptors (up to 10 ␮M) sufficient to stimulate purinergic receptors.6 has shown that the other P2X receptor family members are Thus, ATP release and signaling could contribute detrimen- specifically localized to the nervous system, but the expres- tally to the gradual expansion of cyst fluid volume that is a sion of P2X7 is more ubiquitous in other organs,19 suggest- hallmark of polycystic kidney disease.6,7 However, in vivo data ing a possibility of its localization in the zebrafish prone- are still lacking to support this hypothesis. phros. First, we determined the mRNA expression of P2X7 Two groups of purinergic receptors regulated by ATP (ATP-gat- receptors in pkd2 morphants with a specific focus on pro- ing P2X receptors and ATP-sensing P2Y G--coupled recep- nephros. In addition to diffuse brain and spinal cord stain- tors) have been identified and their autocrine and paracrine functions ing, as illustrated in two previous studies,18,19 we found ev- have been reviewed.4,7 In kidneys, these receptors may regulate water idence of P2X7 receptor expression in the pronephric and ion transport in different nephron segments.7 The P2X7 receptor region (Figure 1). Notably, the p2rx7 mRNA expression is is unique in its function in cytolytic pore formation and modulating earlier (at 24hpf) and stronger in pkd2 morphants com- release, proliferation, and .8,9 The P2X7 receptor is pared with controls (Figure 1 A-F), suggesting P2X7 recep- only activated at high ATP concentrations, allowing Ca2ϩ and Naϩ tors may be involved in the formation of cystic phenotype of influx and Kϩ efflux, and thus is regarded as mediating a danger path- pkd2 morphants. A dorsal view of the pkd2 morphant em- way in the face of cell injury.10 In addition, P2X7 receptor expression bryo confirmed the expression of P2X7 receptors in the two has been shown in dilating collecting ducts and cysts in human auto- pronephric ducts (Figure 1 G). The pattern of p2rx7 mRNA somal recessive polycystic kidney disease (ARPKD).8 P2X7-express- expression was very similar to the pkd2 mRNA expression ing collecting duct cysts dominate kidney histology of cpk/cpk mice (Figure 1 H), suggesting a possible functional link between from two weeks after birth until they die from uremia.11 However, the pkd2 and P2X7 receptors. possibility that P2X7 receptors contribute to cyst formation of AD- PKD has not been examined. OxATP Inhibits Pronephric Cyst Formation in pkd2 The zebrafish (Danio rerio) has become a popular model of Morphants various diseases not only for the study of functions but also Pkd2 morphants showed a curly-tail phenotype and a cystic for screening small molecules because of the ease of waterborne phenotype including glomerular cyst, pronephric duct dila- treatments.12 Knockdown of polycystic kidney disease 2 (pkd2) tion, and peritubular edema,14,15 which could be used as pa- with morpholinos (MO) in zebrafish embryos led to the forma- rameters for assessment of treatment effects on drug screen- tion of kidney cysts,13–17 which is the first orthologous zebrafish ing.20 First, we examined the effects of P2X7 receptor model for polycystic kidney disease. Moreover, the zebrafish inhibition with OxATP and stimulation with BzATP. No sig- P2X receptor subunit , which are orthologs of mam- nificant phenotype was observed in wild-type embryos treated malian P2X receptors, have been characterized.18 This in with OxATP (100␮M) or BzATP (100␮M). We observed the vivo model provides a unique opportunity to examine the early changes in the pronephros (48 hpf) using wt-1b proneph- hypothesis that blocking purinergic receptors may affect ric-specific GFP zebrafish embryos with morpholino-medi- cyst development in ADPKD. ated knockdown of pkd221. Pkd2 morphant embryos devel- In this work, we evaluated whether P2X7 receptors contrib- oped cysts in the region of the glomerulus and tubules, which uted to cyst formation and progression in zebrafish knock- were directly observed under a fluorescence microscope (ar- down of pkd2. We have examined the effects of oxidized ATP rows in Figure 2 A, B). The frequency of cystic pronephros was (OxATP), a P2X7 receptor antagonist, and BzATP, a P2X7 36/48 (75%) in pkd2 morphant embryos. Treatment of pkd2 receptor agonist, on the development of cystic phenotype in morphant embryos with BzATP to stimulate the P2X7 recep- the PKD2 zebrafish model. In addition, we have assessed tor resulted in cysts in 41/52 (78.8%) fish, while inhibition of whether knockdown of P2X7 receptors in pkd2 morphants the receptor with OxATP decreased the frequency significantly may prevent the progression of cyst. From these observations, (P Ͻ 0.01) to 18/52 (34.6%; Figure 2C). Histologic examina- we describe the first in vivo evidence that P2X7 receptor inhi- tion of H and E stained sections revealed profound glomerular bition can modify cyst formation in an ADPKD model. cystic dilation and pronephric tubular dilation in pkd2 mor- phants at 5 dpf, whereas the cystic dilation was markedly im- proved in embryos incubated with OxATP (Figure 2D). The RESULTS frequency of abdominal edema in pkd2 morphants at 5 dpf was significantly reduced in the OxATP-treated embryos (50.9 Ϯ Expression of P2X7 Receptors in the Zebrafish 7.1%, n ϭ 87, from three independent experiments) as com- Pronephros pared with the BzATP-treated embryos (92.7%Ϯ2.0, n ϭ 82) We chose to study the role of purinergic receptor signaling in a or the untreated controls (75.9%Ϯ3.0, n ϭ 81; P Ͻ 0.01; Sup- well-characterized zebrafish pkd2 model.13,15 The P2X7 recep- plemental Figure S1). These data support a role of OxATP in

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p-ERK showed increased expression in pronephric epithelial cells of pkd2 mor- phants that was suppressed by OxATP treatment (Figure 3 C–E), suggesting that pronephros contributed to the increased level of p-ERK in pkd2 morphants. Con- sistent with these results, stimulation of the P2X7 receptor with BzATP increased the level of p-ERK in the morphants (Fig- ure 3 C–E). We also observed prominent p- ERK staining on the apical surface (brush border) of the pronephric ducts, suggesting that activation of p-ERK might have a role in mediating flow-induced mechanosensation and ion transport.23

OxATP Reduces Cell Proliferation of Pronephric Ducts in pkd2 Morphants Increased cell proliferation has been re- ported in mammalian PKD models.24,25 We observed a twofold increase in anti- phospho-H3 staining cells in the prone- phros of pkd2 morphants (5.1 Ϯ 0.4) at 5 dpf compared with wild-type embryos (2.5 Ϯ 0.4; P Ͻ 0.05; Figure 4). Increased proliferation was also observed in BzATP- treated embryos (5.2 Ϯ 0.5; P Ͻ 0.05 ver- sus wild-type controls). However, cell pro- liferation of pronephric ducts was reduced in OxATP-treated pkd2 morphants (3.9 Ϯ 0.8; P Ͼ 0.05 versus wild-type controls; Figure 4 E). We also examined apoptosis using the TUNEL assay but there were too few apoptotic cells in the pronephros for meaningful comparison (data not Figure 1. Increased expression of p2rx7 mRNA in pkd2 morphants. Whole-mount in situ shown). hybridization for p2rx7 mRNA in 24 hpf (A, B) and 48 hpf (C, D) zebrafish embryos. Note expression of p2rx7 in pronephric ducts (arrow) in wild-type siblings (C) and pkd2 mor- IL-1 Beta mRNA Expression in phants (B, D). Diffuse staining in brain and spinal cord was also noted. Quantitative pkd2 Morphants real-time PCR showing p2rx7 mRNA upregulation in pkd2 morphants at 24 hpf (E) and 48 hpf (F) (n ϭ 3 and 4, respectively). *P Ͻ 0.05. (G) Dorsal view of pkd2 morphant embryos Inflammatory have been pro- showing localization of p2rx7 mRNA in pronephric ducts (arrowheads). (H) In situ hybrid- posed to promote cyst progression in ization showing a similar expression pattern of pkd2 to p2rx7 mRNA. ADPKD.26,27 Consistent with this, we ob- served a twofold increase of IL-1 beta suppressing the early formation of pronephric cyst in pkd2 mRNA expression in 5 dpf pkd2 morphants (Supplemental Figure morphant embryos. S2). Strikingly, stimulation of purinergic receptors with BzATP led to a significant increase of IL-1 beta expression. However, the OxATP Inhibits pERK Activation in pkd2 Morphants higher IL-1 beta expression in pkd2 morphants was not sup- The ERK pathway has been implicated in mediating proliferation pressed by OxATP. These results suggest that OxATP does not and fluid secretion in various PKD models.22 We therefore looked suppress cyst progression through an anti-inflammatory effect. at the activation of the ERK pathway in the pkd2 morphants and the effect of OxATP treatment. We observed a 2.5-fold increase of P2X7 Knockdown Reduces Cystic Phenotype of pkd2 p-ERK expression in pkd2 morphants by denstiometric analysis of Morphants Western blot, which was suppressed by 53% with OxATP treat- To further confirm if inhibition of P2X7 receptors is responsi- ment (Figure 3 A and B). Consistently, immunostaining for ble for the beneficial effect of OxATP in pkd2 morphants, we

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trafficking and function.9 No significant cystic phenotype was observed in wild- type siblings or the embryos injected with p2rx7 morpholinos. Consistent with the ef- fects of OxATP, p2rx7 knockdown (with any one MO) significantly reduced the frequency of cystic phenotype in pkd2 morphants (Figure 5 A). The frequency of cystic pheno- type in pkd2 morphants was significantly re- duced from 69/83 (83.1%) to 24/43 (55.8%) with p2rx7 ATG-MO (P Ͻ 0.01); 33/68 (48.5%) with p2rx7 exon 2 splice-MO (P Ͻ 0.01); and 31/71 (43.7%) with p2rx7 exon 14 splice-MO (P Ͻ 0.01; Figure 5 B, C). Similar to OxATP treatment, P2X7 knockdown re- duced cell proliferation and p-ERK expres- sion in pronephric ducts in pkd2 morphants (Figure 6).

A P2X7 Specific Antagonist A-438079 Significantly Reduces the Frequency of Pronephric Cyst Formation in pkd2 Morphants We have tested a novel P2X7 specific an- tagonist,28 A-438079, to confirm the effect of pharmacologic inhibition of P2X7 on pkd2 morphants by OxATP. Consistently, A-438079 (0.1␮M) showed similar effects to OxATP in reducing the frequency of cystic phenotype in pkd2 morphant em- bryos. The frequency of cystic phenotype was reduced from 25/27(92.6%) to 15/42 (35.7%; P Ͻ 0.01), confirming the benefi- cial effect of P2X7 blockade on pkd2 mor- phants (Figure 6 A). Similarly, p-ERK expression and cell proliferation of pronephric ducts were suppressed in Figure 2. Disruption of P2X7 receptor signaling attenuates cystic dilation in pkd2 A-438079-treated pkd2 morphants (Fig- morphants. The wt-1b pronephric-specific GFP zebrafish embryos were microinjected ure 6 B-D). Expression of P2X7 protein in with a pkd2 ATG morpholino. Cystic phenotype was observed directly in lived pkd2 morphants was analyzed by Western embryos at 2 dpf. (A) Pronephric cysts (arrows) in a pkd2 morphant embryo (dorsal blotting using a custom rabbit polyclonal view, anterior to the top); pd, pronephric duct. (B) Restoration of normal glomerular antibody, and upregulation of P2X7 recep- structure (white circle) in a pkd2 morphant treated with OxATP. (C) The frequency of tors in pkd2 morphants was confirmed cystic phenotype in pkd2 morphants (n ϭ 48) and those treated with 100␮M BzATP (n ϭ 52) or 100␮M OxATP (n ϭ 52). Data were pooled from three independent (Figure 6 E). The expression of P2X7 re- experiments. *P Ͻ 0.01. (D) Transverse sections of a pkd2 morphant at 5 dpf show ceptors was not significantly changed by profound glomerular and tubular dilations indicated by the mark * (upper panel). OxATP and BzATP (Figure 6 E), but it was Minimal glomerular cyst and no tubular dilation in a pkd2 morphant incubated slightly upregulated with A438079, sug- continuously with 100␮M OxATP (lower panel). gesting a possible compensatory effect. designed three different morpholinos to specifically target P2X7 is Expressed in the Primary Cilia in Human p2rx7: on the ATG site that blocks translation and on the splic- Kidney Cells ing sites at the intron 1/exon 2 and the intron 13/exon 14 Primary cilium signaling defects is one of the pathogenic mecha- boundaries. Exon 2 of p2rx7 contains the five positive charged nisms of cyst formation in PKD.1 Therefore, we asked whether amino acid residues important for binding, and P2X7 receptors are located in the primary cilia of renal tubular exon 14 of p2rx7 contains domains responsible for receptor epithelial cells. We performed a double immunocytochemistry

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inhibition of P2X7 receptors with OxATP and A-438079. Similarly, knockdown of P2X7 receptors suppressed the cystic phe- notype of pkd2 morphants. Our results suggest important roles for P2X7 recep- tors in modifying cyst formation in AD- PKD. One mechanism by which the P2X7 receptor blockade reduces cyst forma- tion may be through inhibiting P2X7 re- ceptor-mediated ERK activation. Acti- vation of ERK by P2X7 receptors has been demonstrated in various epithelial cells, such as rat pancreas.29 P2X7 recep- tors are also present in the rat lacrimal gland, where stimulation with BzATP increases ERK 1/2 activation.30 P2X7 re- ceptors are also involved in fluid flow- induced ERK activation in osteoblasts.31 Furthermore, ERK activation has an im- portant role in determining the rate of cyst enlargement in ADPKD through its actions to stimulate cellular prolifera- tion and transepithelial solute and fluid secretion.32–35 Consistent with such a role, we found activation of ERK path- way and increased tubular cell prolifera- tion in the pronephric kidneys of pkd2 morphants, which was suppressed by blockade of P2X7 receptors with Ox- Figure 3. OxATP inhibits phospho-ERK1/2 activation in pkd2 morphants at 5dpf. (A) ATP. Although originally thought as a Western blot analysis with antibody for phospho-ERK1/2 (p-ERK) and total ERK1/2 (t-ERK) death receptor, a trophic role of P2X7 shows increased p-ERK expression in pkd2 morphants and reduced expression with 5-d receptor has recently been shown.10 Our of OxATP (100␮M) incubation. (B) Denstiometric analysis shows that OxATP significantly results are consistent with a role of P2X7 reduced p-ERK/t-ERK expression (n ϭ 4). (C) Cross-sections of immunostaining for p-ERK in ERK activation and cell proliferation, on anterior pronephric duct show increased expression of p-ERK in pkd2 morphants, which may contribute to cyst develop- which is suppressed by OxATP. (D) DAPI nuclear staining for the anterior pronephric ment and progression in ADPKD. ducts. The location of anterior pronephric ducts are circled with a white line. (E) Merged Reduced fluid flow or obstruction of images of p-ERK and DAPI staining show p-ERK expression (arrows) in both nucleus and pronephric duct (without apparent cili- cytoplasm. ary abnormality) has been shown in pkd2 morphants.14,15 With P2X7 recep- for P2X7 and acetyl-␣-tubulin in the human kidney epithelial tor blockade, we observed a significant reduction of proneph- cells (HK-2), and the result showed the expression of P2X7 recep- ric cystic dilation and peritubular edema. This suggests that tors was indeed localize in the primary cilia (Figure 7), suggesting P2X7 receptor blockade can reduce the ATP stimulation of salt that P2X7 receptors may participate in the mechanosensory sig- and water fluxes into the lumen of the pronephric ducts. naling in regulating cyst formation. Whether this process was driven by and/or the cystic fibrosis transmembrane-conductance-regulator (CFTR), as proposed previously, is worthy of further studies.7,36 Interest- DISCUSSION ingly, we have found evidence of P2X7 expression at the pri- mary cilia in cultured tubular epithelial cells, suggesting that To explore whether inhibiting P2X7 receptors reduces cyst forma- P2X7 receptors may play a role in the defective cilium signaling tion in ADPKD, we hypothesized that blocking P2X7 receptors in PKD. How P2X7 interact with PKD2 in the cilium signaling would be effective in reducing renal cyst formation in a zebrafish is still unclear. Since PKD2 has been shown to interact with model of PKD2. We showed that the cystic dilation of pronephros many other membrane ,1 we speculate that a compen- in pkd2 knockdown embryos was prevented by pharmacologic satory upregulation of P2X7 to overcome the deficiency of

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fective in reducing cyst growth,37 but P2X7 specific antagonists were not examined in that study. In a model of autosomal recessive kid- ney disease (ARPKD) using suspension culture of dissociated cpk/cpk kidneys cells, BzATP was found to reduce the number but not the size of cysts through an as-yet-undefined mechanism not re- lated to proliferation and apoptosis,8 which is contrary to our results. These dif- ferent results may be due to differences in the polycystic kidney models used (ADPKD versus ARPKD), the mecha- nisms of cyst development (zebrafish pro- nephric cyst versus 3-dimensional cyst culture), the efficacy of antag- onists, or the tissue expression of P2 recep- tor subtypes.5,11,37 Zebrafish has been used as a model for screening drug candidates for differ- ent diseases.38 A recent study has vali- dated that chemical modifiers identified by zebrafish screening assays could be applicable to a mouse PKD model.39,40 Our results suggest that P2X7 receptor antagonists may hold promise as a po- tential therapy for ADPKD. P2X7 antag- onists have provided promising results in clinical trials for the treatment of rheumatoid arthritis, and these data raise hopes that new analgesic and anti- inflammatory treatments that target Figure 4. OxATP reduces cell proliferation of pronephric kidneys in pkd2 morphants. P2X receptors will be available.41 In ani- ␣ Embryos at 5 dpf were stained for anti-PH3 (red) and anti- 6F (green). Images were mal models of spinal cord injury42 merged for counting proliferating cells (orange) in the pronephric kidneys. (A) A wild-type and experimental glomerulonephri- sibling at 5 dpf showing a proliferative cell in the pronephric kidney (arrow). (B) A pkd2 43 morphant with increased proliferating cells in the pronephric kidney (arrows). (C) A tis, P2X7 receptor antagonists have BzATP-treated pkd2 morphant showing multiple proliferating cells in the pronephric been shown to be effective. Interest- kidney (arrows). (D) Reduced proliferating cells in an OxATP-treated pkd2 morphant ingly, as the P2X7 receptor may en- embryo (arrow). (E) Counts for anti-PH3 and anti-␣6F positive cells in the pronephric hance nociceptive transmission asso- kidneys. n ϭ 16 to 26 for each group. * indicates significant different means compared ciated with microglial activation and with the sibling controls with P Ͻ 0.05 by Kruskal-Wallis test and Dunn’s multiple secretion of IL-1 beta in the dorsal comparison test. horn, a CNS-penetrant P2X7 receptor antagonists is beneficial for the treat- PKD2 may trigger an injury pathway leading to more severe ment of persistent pain,44 which might improve pain man- cyst formation. The link between PKD2 deficiency and P2X7 agement in ADPKD patients if proven in further studies. overexpression warrants further study. Although our study has shown promising effects of P2X7 re- Our finding that P2X7 receptor blockade may suppress cyst ceptor blockade in the PKD2 zebrafish model, further studies on formation is consistent with a previous in vitro study using other animal models are required to confirm the results. Animal MDCK-derived cyst cultures.37 Removal of ATP with apyrase and studies on different models of PKD, such as PKD1 or ARPKD, are the use of nonspecific P2 receptor inhibitors reduced the growth also needed to clarify the effects. Effects of other purinergic recep- of cysts by blocking cAMP-dependent activation of the ERK path- tor antagonists on other P2X or P2Y receptors may need further way,37 indicating a beneficial effect of blocking purinergic signal- studies. However, the current study provides the first in vivo ing pathways on cyst formation. In the same study, a nonselective evidence for a beneficial effect of P2X7 receptor blockade in P2X receptor inhibitor, G, seemed inef- ADPKD.

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Figure 5. Coinjection of morpholinos against pkd2 and p2rx7 in zebrafish embryos inhibits the cystic phenotype. (A) Note the cystic dilation (arrow) of the pronephros in pkd2 morphants were rescued by p2rx7 knockdown. The glomerular structure is circled in white. (B) Reduced frequencies of cystic phenotype in pkd2 morphants with microinjection of morpholinos against p2rx7 at the ATG site or the splicing sites of exon 2 and exon 14. *P Ͻ 0.01. n ϭ 43 to 83 in each groups. (C) RT- PCR from lysates of 48-hpf wild-type and pkd2 morphant embryos coinjected with the splicing-morpholinos targeted against p2rx7 exon 2 and exon 14, showing the splicing is blocked in the p2rx7 transcripts. P2rx7 was amplified using primers directed to exon 1 to 13 (818 bp) and exon 9 to 14 (538 bp).

Conclusion the Institutional Animal Care and Use Committee of Chang Gung This study demonstrates that inhibition of P2X7 receptors University. can reduce cystic phenotype in pkd2 knockdown zebrafish. Our studies provide support for the hypothesis that modi- Morpholino Injection fying purinergic-signaling pathways may affect cyst forma- One- to two-cell stage embryos were microinjected with 0.125 mM tion in PKD. Our results further imply that purinergic re- antisense morpholino oligos (MO) at the amount of approximately ceptor blockade could be a potential therapeutic modality 4.45 ng per embryo (Gene Tools, Philomath, OR). For knockdown of for ADPKD. pkd2, we used pkd2 ATG-MO (5Ј-AGGACGAACGCGACTG- GAGCTCATC-3Ј) as described previously.13 The pkd2 morphants showed consistent cystic dilation of pronephros and severe edema by CONCISE METHODS 5 d postfertilization suitable for drug screening. For knockdown of P2X7 receptors (p2rx7), an ATG-MO (5Ј-AGAGGTTCAGCAA- Fish Maintenance AACGCAAGGCAT-3Ј) designed by Gene Tools, and two MOs against Zebrafish and embryos were maintained by standard methods.45 splicing sites at the intron/exon boundaries of p2rx7 exon2 (5Ј-CAT- Embryos were staged according to hours post fertilization (hpf). GATACTGGAAAGTAAAGACATT-3Ј) and p2rx7 exon14 (5Ј-TCATTCT- The transgenic line wt1b:: GFP with pronephros specific GFP ex- GAGAACAGACATGAGGTA-3Ј) were used at a concentration of 0.25 pression21 was kindly provided by Dr. Christoph Englert (Fritz mM. The knockdown efficacy of the p2rx7 splice-MOs was checked by Lipmann Institute, Jena, Germany). The study was approved by reverse transcriptase PCR. A morpholino standard control oligo (5Ј-

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Figure 6. A P2X7 specific antagonist A-438079 reduces the frequency of pronephric cyst formation in pkd2 morphants. (A) Overlays of transmission (gray) and fluorescence (green) images (dorsal view, anterior to the top) showing effects of A438079 (0.1␮M) on pronephric cysts (arrows) in pkd2 morphant embryos at 2 dpf; comparative frequencies of cystic phenotype in pkd2 morphants at 2 dpf with (n ϭ 27) and without A-438079 treatment (n ϭ 42). Results were pooled from two independent experiments. *P Ͻ 0.01. (B) Merged images of p-ERK and DAPI staining showing decreased p-ERK expression in pronephric ducts (white circle) with A-438079 treatment and p2rx7 MO knockdown. (C) Counts for anti-PH3 and anti-␣6F double positive cells (orange; arrows) in the pronephric ducts at 5 dpf. n ϭ 26 to 29 for each group. *P Ͻ 0.05 compared with control. (D) Western blot showing increased p-ERK activities in pkd2 morphants and the effects of A438079 and p2rx7 MO knockdown. (E) Western blot showing increased expression of P2X7 protein in pkd2 morphants and the effects of treatments. Data shown are representative of two experiments with similar results.

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(JB-4; Polyscience), and cut at 4 ␮m. Slides were stained with Hematoxylin and Eosin.

Immunohistochemistry Embryos were fixed overnight in 4% parafor- maldehyde at 4 °C. Immunostaining for phospho-ERK (extracellular signal regulated kinases) was performed according to pub- lished protocols.47 An antibody to phospho- ERK 1/2 (1: 100) was purchased from Cell Signaling. Immunostaining was performed in whole-mount embryos and then embed- ded in OTC medium and cryosectioned. Sec- tions were mounted in Vectashield (Vector Laboratories) with DAPI. Double staining was performed in whole- mount embryos using an anti- phospho-H3 antibody (1:100, Upstate) to label proliferat- ing cells and an anti-NA/K ATPase ␣-1 sub- unit (␣6F) antibody (1:200, Developmental Figure 7. P2X7 receptors are expressed in the primary cilium of cultured human proximal Studies Hybridoma Bank) to label proneph- tubular cells (HK-2). Cilia were stained with anti-acetylated-␣-tubulin antibodies (green). ric epithelial cells. Secondary antibodies used P2X7 were stained with anti-P2X7 antibodies (red). Nuclei were stained with DAPI (blue). were Alexa Fluor 594 goat anti-rabbit IgG P2X7 (red) and acetylated-␣-tubulin (green) are colocalized, as shown in the merged and Alexa Fluor 488 goat anti-mouse IgG (1: image (yellow). 500, Molecular Probes). Images for anti- phospho-H3 and anti-␣6F staining were CCTCTTACCTCAGTTACAATTTATA-3Ј), pkd2 5-mismatch MO taken and merged for counting proliferating cells in the pronephric (5Ј-AGCACCAACCCGACTGCACCTCATC-3Ј), p2rx7 exon2 5-mis- kidneys. match MO (5Ј-TCATTCTGAGAACAGACATGAGGTA-3Ј) and p2rx7 exon14 5-mismatch MO (5Ј-CATGATACTGGAAAGTAAAGACATT- Whole-mount In Situ Hybridization Ј 3 ) were applied as negative controls. Embryos were fixed overnight in 4% paraformaldehyde at 4 °C. Whole-mount in situ hybridization was performed according to pub- Drug Treatment lished protocols.48 The digoxigenin-labeled antisense probe for P2X7 A purinergic P2X7 receptor agonist BzATP (3Ј-O-(4-benzoylben- receptors was prepared from a pCRII-TOPO-p2rx7 construct (a kind zoyl)-ATP) and a P2X7 receptor blocker, oxidized ATP, were added gift from Dr Lior Appelbaum, Stanford University) using the most 3Ј directly into E3 medium (5mM NaCl, 0.17mM KCl, 0.4mM CaCl2, 600 bp fragment of p2rx7.19 and 0.16mM MgSO4) in 6-cm Petri dishes. The final concentration of each molecule was 100 ␮M. Pkd2 morphants embryos were incubated from 4 hpf (after microinjection) till 5 dpf at 28.5 °C in the dark.12 Western Blot Analysis Proteins extracted from whole embryos were analyzed by Western OxATP and BzATP were purchased from Sigma. We have chosen blotting following the standard method.49 Antibodies used include OxATP initially because all of the effects of P2X7 receptors, including anti-phospho-ERK1/2 (1:1000, Cell Signaling), anti-total ERK1/2 (1: the ATP-mediated cytotoxicity, can be abrogated by this antagonist.46 1000, Santa Cruz),47 anti-tubulin (clone DM1a, Sigma), and a cus- Another highly selective P2X7 receptor antagonist (A-438079, Tocris tom-made rabbit polyclonal antibody raised against a N-terminal Bioscience, Bristol, UK) was used at a concentration of 0.1 ␮Mto peptide (EKGFSDQHSHGVQTGAC) of zebrafish P2X7 receptor confirm the results.28 (Kelowna, Taiwan). Direct Observation of Pronephric Cyst in Live Embryos The wt-1b:: GFP transgenic fish line21 was microinjected with pkd2 Immunocytochemistry MO for direct observation of the cystic phenotype. At 48 hpf, embryos HK-2 cells (immortalized human proximal tubular cell line, Ameri- were anesthetized with tricaine (0.2 mg/ml) and oriented in 5% can Type Culture Collection, Manassas, VA) were grown in 8-well methyl cellulose (Sigma). The presence of pronephric cysts was deter- culture chamber slide for 8 d. Slides were washed in PBS, fixed in 4% mined using fluorescence microscope. paraformaldehyde, blocked with 1% BSA, and incubated with pri- mary antibody, P2X7 (1:100, APR-008; Alomone) and acetylated-␣- Histology tubulin (1:100, clone 6-11-B1; Sigma) overnight at 4 °C. After washes Zebrafish embryos were fixed in 4% paraformaldehyde in Phosphate in PBS, the cells were incubated with the secondary antibody Alexa Buffered Saline (PBS) overnight at 4 °C, embedded in glycolmethacrylate Fluor 594 donkey anti-rabbit IgG and Alexa Fluor 488 goat anti-

1704 Journal of the American Society of Nephrology J Am Soc Nephrol 22: 1696–1706, 2011 www.jasn.org BASIC RESEARCH mouse IgG (1:300, Molecular Probes) for 30 min at room tempera- ATP release contributes to absence of flow-sensitive, purinergic ture. DAPI was used to counterstain nuclei. Cai2ϩ signaling in human ADPKD cyst epithelial cells. Am J Physiol Renal Physiol 296: F1464–1476, 2009 6. Wilson PD, Hovater JS, Casey CC, Fortenberry JA, Schwiebert EM: Real-time PCR ϭ ATP release mechanisms in primary cultures of epithelia derived from Embryos from each treatment group (n 15 to 30) were mixed and the cysts of polycystic kidneys. J Am Soc Nephrol 10: 218–229, 1999 grinded in liquid nitrogen. RNA-Bee isolation kit (TEL-TEST, Inc., 7. Hovater MB, Olteanu D, Welty EA, Schwiebert EM: Purinergic signal- Friendswood, TX) was used for total RNA extraction. First strand ing in the lumen of a normal nephron and in remodeled PKD encap- cDNA synthesis kit for RT-PCR (AMV; Roche Diagnostics, Germany) sulated cysts. Purinergic Signal 4: 109–124, 2008 was used for cDNA synthesis. Real-time PCR was performed with the 8. Hillman KA, Woolf AS, Johnson TM, Wade A, Unwin RJ, Winyard PJ: The P2X7 ATP receptor modulates renal cyst development in vitro. ABI PRISM 7700 sequence detection system (Applied Biosystems) in Biochem Biophys Res Commun 322: 434–439, 2004 the presence of SYBR Green. PCR primers for zebrafish IL-1␤ were 9. Lopez-Castejon G, Young MT, Meseguer J, Surprenant A, Mulero V: 5Ј-CAT TTG CAG GCC GTC ACA-3Ј(forward) and 5Ј-GGA CAT Characterization of ATP-gated P2X7 receptors in fish provides new GCT GAA GCG CAC TT-3Ј(reverse).50 PCR primers for p2rx7 were insights into the mechanism of release of the leaderless cytokine 5Ј-CTG CCG AGA CCT TAC AGG AG-3Ј (forward) and 5Ј-AAA interleukin-1 beta. Mol Immunol 44: 1286–1299, 2007 Ј 10. Monif M, Reid CA, Powell KL, Smart ML, Williams DA: The P2X7 GCG TTC TCC AGG ACA GA-3 (reverse). Zebrafish ß-actin was receptor drives microglial activation and proliferation: A trophic role used as internal control. Reaction was performed in triplicate for each for P2X7R pore. J Neurosci 29: 3781–3791, 2009 sample. 11. Hillman KA, Johnson TM, Winyard PJ, Burnstock G, Unwin RJ, Woolf AS: P2X(7) receptors are expressed during mouse nephrogenesis and in Statistical Analyses collecting duct cysts of the cpk/cpk mouse. Exp Nephrol 10: 34–42, 2002 Values are expressed as means Ϯ SEM. Comparison between groups 12. Murphey RD, Zon LI: Small molecule screening in the zebrafish. Meth- ods 39: 255–261, 2006 was performed by ANOVA followed by Dunnett multiple comparison 13. Sun Z, Amsterdam A, Pazour GJ, Cole DG, Miller MS, Hopkins N: A test. Values that were not normally distributed were analyzed by genetic screen in zebrafish identifies cilia genes as a principal cause of Kruskal-Wallis test followed by Dunn multiple comparison test. The cystic kidney. Development 131: 4085–4093, 2004 comparison of binary variables was performed by Fisher’s exact t test 14. Sullivan-Brown J, Schottenfeld J, Okabe N, Hostetter CL, Serluca FC, or Chi-square test. P Ͻ 0.05 was considered statistically significant. Thiberge SY, Burdine RD: Zebrafish mutations affecting cilia motility share similar cystic phenotypes and suggest a mechanism of cyst formation that differs from pkd2 morphants. Dev Biol 314: 261–275, 2008 15. Obara T, Mangos S, Liu Y, Zhao J, Wiessner S, Kramer-Zucker AG, ACKNOWLEDGMENTS Olale F, Schier AF, Drummond IA: Polycystin-2 immunolocalization and function in zebrafish. J Am Soc Nephrol 17: 2706–2718, 2006 16. Schottenfeld J, Sullivan-Brown J, Burdine RD: Zebrafish curly up en- We are grateful to Christoph Englert for the gift of wt-1b GFP codes a pkd2 ortholog that restricts left-side-specific expression of zebrafish line; Lior Appelbaum for the p2rx7 plasmid; Thomas T. southpaw. Development 134: 1605–1615, 2007 Chen for helpful discussions; Chia-Hsien Fan for help with in situ 17. Bisgrove BW, Snarr BS, Emrazian A, Yost HJ: Polaris and Polycystin-2 hybridization; Yi-Ching Ko, Ya-Tzu Lin, Hsiang-Chi Huang and in dorsal forerunner cells and Kupffer’s vesicle are required for spec- Chiung-Tseng Huang for their excellent technical assistance; and Paul ification of the zebrafish left-right axis. Dev Biol 287: 274–288, 2005 18. Kucenas S, Li Z, Cox JA, Egan TM, Voigt MM: Molecular characteriza- J. Scotting for critical reading of the manuscript. This work was sup- tion of the zebrafish P2X receptor subunit gene family. Neuroscience ported by grants from the National Science Council and Chang Gang 121: 935–945, 2003 Medical Research Projects, Taiwan. 19. Appelbaum L, Skariah G, Mourrain P, Mignot E: Comparative expres- sion of p2x receptors and ecto-nucleoside triphosphate diphospho- hydrolase 3 in hypocretin and sensory neurons in zebrafish. Brain Res 1174: 66–75, 2007 DISCLOSURES 20. Cao Y, Semanchik N, Lee SH, Somlo S, Barbano PE, Coifman R, Sun Z: None. Chemical modifier screen identifies HDAC inhibitors as suppressors of PKD models. Proc Natl Acad Sci USA, 2009 21. Perner B, Englert C, Bollig F: The Wilms tumor genes wt1a and wt1b REFERENCES control different steps during formation of the zebrafish pronephros. Dev Biol 309: 87–96, 2007 22. Omori S, Hida M, Fujita H, Takahashi H, Tanimura S, Kohno M, Awazu 1. Torres VE, Harris PC: Autosomal dominant polycystic kidney disease: M: Extracellular signal-regulated kinase inhibition slows disease pro- The last 3 years. 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