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Phosphoinositide 3-Kinase-C2a Regulates Polycystin-2 Ciliary Entry and Protects against Kidney Cyst Formation

† Irene Franco,* Jean Piero Margaria,* Maria Chiara De Santis,* Andrea Ranghino, ‡ Daniel Monteyne, Marco Chiaravalli,§ Monika Pema,§ Carlo Cosimo Campa,* ‡| Edoardo Ratto,* Federico Gulluni,* David Perez-Morga, Stefan Somlo,¶ Giorgio R. Merlo,* Alessandra Boletta,§ and Emilio Hirsch*

*Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of Torino, Turin, Italy; †Renal Transplantation Center “A. Vercellone”, Division of Nephrology, Dialysis and Transplantation, Department of Medical Sciences, Città della Salute e della Scienza, Hospital and Research Center for Experimental Medicine (CeRMS) and Center for Molecular Biotechnology, University of Torino, Turin, Italy; ‡Laboratoire de Parasitologie Moléculaire, Institut de Biologie et de Médecine Moléculaires (IBMM), Université Libre de Bruxelles, Gosselies, Charleroi, Belgium; §Division of Genetics and Cell Biology, Dibit San Raffaele Scientific Institute, Milan, Italy; |Center for Microscopy and Molecular Imaging (CMMI), Université Libre de Bruxelles, Gosselies, Belgium; and ¶Section of Nephrology, Yale University School of Medicine, New Haven, Connecticut.

ABSTRACT Signaling from the primary cilium regulates kidney tubule development and cyst formation. However, the mechanism controlling targeting of ciliary components necessary for cilium morphogenesis and signaling is largely unknown. Here, we studied the function of class II phosphoinositide 3-kinase-C2a (PI3K-C2a)inrenal tubule-derived inner medullary collecting duct 3 cells and show that PI3K-C2a resides at the recycling endo- some compartment in proximity to the primary cilium base. In this subcellular location, PI3K-C2a controlled the activation of Rab8, a key mediator of cargo protein targeting to the primary cilium. Consistently, partial re- duction of PI3K-C2a was sufficient to impair elongation of the cilium and the ciliary transport of polycystin-2, as well as to alter proliferation signals linked to polycystin activity. In agreement, heterozygous deletion of PI3K- C2a in mice induced cilium elongation defects in kidney tubules and predisposed animals to cyst development, either in genetic models of polycystin-1/2 reduction or in response to ischemia/reperfusion-induced renal damage. These results indicate that PI3K-C2a is required for the transport of ciliary components such as polycystin-2, and partial loss of this enzyme is sufficient to exacerbate the pathogenesis of cystic kidney disease.

J Am Soc Nephrol 27: 1135–1144, 2016. doi: 10.1681/ASN.2014100967

Polycystic kidney disease (PKD) represents a group Therefore, ADPKD is classified among an emerging of disorders characterized by the formation of cysts group of complex diseases called ciliopathies.5 Study in the kidney. Different forms of PKD are classified according to their causative major genetic lesion, which results in different phenotypic manifesta- Received October 4, 2014. Accepted July 14, 2015. tions. An autosomal dominant form (ADPKD) I.F. and J.P.M. contributed equally to this work. PKD1 results from mutations in either the or Published online ahead of print. Publication date available at PKD2 genes, encoding for two transmembrane www.jasn.org. 1,2 proteins collectively termed polycystins. Notably, Correspondence: Prof. Emilio Hirsch, Molecular Biotechnology ADPKD phenotypes can be observed when the poly- Center, Department of Molecular Biotechnology and Health Sciences, cystins do not reach, at sufficient levels, a specific University of Torino, Turin, Italy. Email: [email protected] cellular organelle called the primary cilium.3,4 Copyright © 2016 by the American Society of Nephrology

J Am Soc Nephrol 27: 1135–1144, 2016 ISSN : 1046-6673/2704-1135 1135 BASIC RESEARCH www.jasn.org of these syndromes helped to define the association between base.3,15–17 In this region, known as basal body, a selective polycystins, cilium-dependent signaling, and cystogenesis, al- barrier regulates entry of specific proteins inside the ciliary though the exact mechanism is still unclear.6–9 compartment.18,19 A key player in this selective entry of ciliary Primarycilia are long, thin, microtubule-based structures that components is the small guanosine triphosphatase (GTPase) protrude from most cell types. In cells characterized by an apico- Rab8.19 Consistently, silencing of Rab8 impairs transport of basal polarity, such as epithelial and endothelial cells, cilia appear proteins required for ciliogenesis,20 whereas a dominant-neg- on the apical side and project into the lumen of tubules/vessels. ative form of Rab8a prevents ciliary delivery of polycystin-2.21 This architecture allows cilia to function as mechanosensors Rab8 activation is thus a crucial step for correct cilium bio- responding to fluid flow.10,11 Increasing evidence suggests that, genesis and sensory function. in renal epithelial cells and endothelial cells, polycystins localize Guanosine triphosphate (GTP) loading of Rab8 occurs at to cilia and cooperate to transduce flow-dependent stimuli.12 the ciliary base and is mediated by the Rab GTPexchange factor Polycystin-1 acts as a mechanosensor13 activating polycystin-2, Rabin8.22,23 Localization and function of this GTP exchange which opens its channel pore and allows calcium entry.12 factor critically depends on the activation of another Rab, Polycystin-evoked calcium in turn is proposed to regulate cell Rab11.20,24 We recently showed that the phosphatidylinositol-3- polarity and to activate intracellular pathways that counteract kinase (PI3K) of class II, PI3K-C2a, acts not only in the endo- proliferation signals, such as the mammalian target of rapamycin cytic compartment25 but also at the pericentriolar recycling (mTOR) and mitogen-activated protein kinase (MAPK) path- ways, eventually avoiding cystogenesis.14 Transport of polycystins to the primary cilium is tightly regulated and several mediators of different trafficking steps have been identified to direct vesicles to the primary cilium

Figure 2. PI3K-C2a is required for Rab8-mediated cilium elonga- tion. (A) Representative images of cilia by immunofluorescence of acetylated-a-tubulin (red) in 48 hours-starved control and Sh1- Figure 1. PI3K-C2a promotes localization of active Rab8a at the IMCD3 cells. Bar=3 mm. (B) Measurement of primary cilium length primary cilium. Immunofluorescence analysis of control and Sh1- assessed by immunofluorescence in control, Sh1 and Sh2-IMCD3 IMCD3 cells transfected with either GFP-Rab8 or the constitutively cells (n=150/group). (C) Rescue of ciliary length defects in Sh1- active mutant GFP-Rab8Q67L(green). Cells were starved for 24 hours IMCD3 cells by expression of the constitutively active mutant of and forming cilia were stained with anti-acetylated tubulin (red) and Rab8 (GFP-Rab8Q67L). Transfection of either empty-EGFP or wild- analyzed for the presence of Rab8. In control cells both the wild- type GFP-Rab8 is used as control (n=40 cilia/group). (D) Repre- type Rab8 and Rab8Q67L were localized in cilia. On the contrary, in sentative images showing that Sh1-IMCD3 cells transfected with Sh1-IMCD3 cells, only the constitutively active Rab8Q67L could be GFP-Rab8Q67L (green) display longer cilia (red) than non-transfected found at cilia. Bar=3 mm. PI3K-C2a, phosphoinositide 3-kinase- cells (right panel), while wild-type GFP-Rab8 does not affect cilium C2a; IMCD3, inner medullary collecting duct 3. elongation (left panel). EGFP, enhanced green fluorescent protein.

1136 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 1135–1144, 2016 www.jasn.org BASIC RESEARCH endosomal compartment (PRE) at the base of the primary than Sh1 (Supplemental Figure 2A), severity of cilium short- cilium where it promotes Rab11 activation upstream of ening in Sh2-IMCD3 cells correlated with the extent of Rab8.26,27 Consistently, genetic inactivation of Pik3c2a, the mu- Pik3c2a silencing (Figure 2, A and B). In addition, a specific rine gene encoding PI3K-C2a, leads to embryonic lethality rescue of ciliary length was detected in Sh1-IMCD3 transfec- with typical signs of ciliary dysfunction such as laterality de- ted with constitutively active Rab8Q67L but not wild-type Rab8 fects and impaired Sonic hedgehog pathway activation.26 The (Figure 2, C and D). In agreement with Rab8 localizing to the severity of the phenotype has precluded the analysis of ciliary ciliary shaft during early ciliogenesis (Figure 1), but rarely to dysfunction in adult organs but mouse mutants weakly completely elongated cilia,24 neither wild-type Rab8 nor Rab8 expressing a truncated form of PI3K-C2a display kidney ab- Q67L were observed on cilia of 48 hours-starved control and normalities including tubular dilations.28 Although this con- Sh1-IMCD3 cells. These results demonstrate that PI3K-C2a dition typically precedes kidney cyst formation, the effects of controls primary cilium elongation by promoting the Rab11/ PI3K-C2a loss on cystogenesis are currently unknown. Rab8 cascade activation. To determine whether changes in PI3K-C2a expression Rab8 is required to deliver ciliary components, including could impact on kidney tubule primary cilia and renal cysto- polycystin-2.21 Thus, polycystin-2 positive cilia were scored in genesis, we analyzed the effects of PI3K-C2a downregulation the same cells where ciliary length was measured. In agree- in vitro in inner medullary collecting duct 3 (IMCD3) cells and ment with a dose-dependent effect of PI3K-C2a on protein in vivo in heterozygous null mice. We found that decreased PI3K-C2a expression impairs Rab8-dependent transport of ciliary components, including polycystin-2. Consistently, PI3K-C2a heterozygous mice showed morphologic defects of kidney cilia and enhanced renal cyst formation when chal- lenged with kidney ischemic insults or crossed with either Pkd2 or Pkd1 heterozygous mutants, thus indicating that PI3K-C2a reduction can predispose to kidney cystic disease.

RESULTS

PI3K-C2a Colocalizes with Rab8 and Promotes its Ciliary Localization In ciliated IMCD3 murine kidney-derived cells, PI3K-C2a was enriched in vesicular structures surrounding the base of the primary cilium (Supplemental Figure 1A). This localization overlapped with that of two markers of the PRE, Rab11 and Rab8 (Supplemental Figure 1, B and C). Rab11 and Rab8 are activated in cascade20,24 and PI3K-C2a promotes triggering of Rab11.26 Consistently, shRNA-mediated PI3K-C2a silencing in IMCD3 cells caused a significant de- crease in Rab11 activity, assessed by a FIP3 pull-down assay.26 The use of two sh-RNAs able to target the Pik3c2a transcript with different efficacy (Supplemental Figure 2A) showed a correlation between the severity of PI3K-C2a loss and the impairment of Rab11 activation (Supplemental Figure 2B). Ac- tive Rab11 is required for Rab8 triggering and subsequent cil- Figure 3. PI3K-C2a is required for Rab8-mediated ciliary locali- iogenesis.23 Consistently, loss of PI3K-C2a prevented Rab8 zation of polycystin-2. (A) Representative immunofluorescence of ciliary targeting, but constitutive activation of Rab8 (through polycystin-2 (PC2, red) localization at cilia (acetylated-a-tubulin, the expression of Rab8Q67L)wassufficient to overcome this de- green) of 48 hours starved control and Sh1-IMCD3 cells. Bar=3 m fi fect (Figure 1). m. (B) Quanti cation of the percentage of PC2 positive cilia in control, Sh1 and Sh2-IMCD3 cells (n=40). (C) Rescue experiment a of PC2 ciliary localization in Sh1-IMCD3 cells. Cells transfected PI3K-C2 is Required for Rab8-Dependent Targeting with either empty-EGFP, wild-type GFP-Rab8, or GFP-Rab8Q67L of Ciliary Components, Including Polycystin-2 were starved for 48 hours, then PC2-positive cilia were visualized 23 Inactivation of Rab8 is expected to cause ciliary shortening. by immunofluorescence as shown in Supplementary Figure 3. Consistently, cilium length was significantly reduced in Sh1- The percentage of PC2 positive cilia was calculated as: the IMCD3 cells after 48 hours of starvation (Figure 2, A and B, number of PC2 positive cilia/ten GFP-positive ciliated cells. At Supplemental Figure 3A). Given that Sh2 was more effective least 60 cilia per group were scored.

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using scanning electron microscopy imag- ing showed a 30% reduction of ciliary length in proximal and a 20% shortening in distal tubules (Figure 4B). Altogether, these data show that heterozygous deletion of Pik3c2a is sufficient to induce defects in cilium morphology, thus indicating that PI3K-C2a is haploinsufficient in kidney tubules. Although low antibody sensitivity pre- vented the analysis of polycystin-2 localiza- tion in kidney cilia, in vivo study of ciliary polycystin-2 succeeded on ventral node cilia of mouse embryos (Supplemental Figure 5). 2/2 With this staining, Pik3c2a embryos showed a severe defect of polycystin-2 cil- iary localization compared with wild-type controls. This demonstrates that the lack of PI3K-C2a affects ciliary targeting of polycystin-2 not only in IMCD3 cells (Figure 3), but also in vivo (Supplemental Figure 5). 2 Figure 4. Pik3c2a+/ mice show shorter cilia in proximal and distal renal tubules. (A) Polycystin-2 deficiency is associated with +/2 Representative micrographs of cilia in wild-type and Pik3c2a kidney tubules, de- kidney cystic phenotype and partial reduc- tected by immunostaining with acetylated-a-tubulin (red) and DNA (DAPI, blue). tion of polycystin-2 levels is a weak stimulus Bar=2 mm. Graph shows the quantification of ciliary length in tubules of wild-type and +/2 to cyst formation. However, spontaneous de- Pik3c2a mice. n=100 cilia per /genotype. (B) Imaging by scanning electron mi- velopment of a few cysts can occur in Pkd2 croscopy of proximal and distal tubule primary cilia in kidneys of 6-week-old wild-type 29 +/2 heterozygous mice. To test if PI3K-C2a and Pik3c2a mice. Quantification of 20 (proximal tubule, PT) and 75 (distal tubule, m and polycystin-2 functionally interact in DT) cilia per genotype are provided on the right. Bar=1 m. 2 kidney cyst development, Pik3c2a+/ mice were crossed with heterozygous mutants 2 entry into cilia, 40% and 50% reduction in the number (Pkd2+/ mice) for a polycystin-2 knock-out allele.29 Histology of polycystin-2 positive cilia was observed in Sh1 and of the kidney at 3 months of age did not reveal a spontaneous 2 Sh2-IMCD3 cell lines, respectively (Figure 3, A and B). Inter- propensity of Pik3c2a+/ mice to develop cysts, while two out 2 estingly, polycystin-2 positivity and length were inversely cor- of eight Pkd2+/ mice showed overt monolateral kidney cysts. 2 related, with polycystin-2 being detected on the rare longest However, Pik3c2a and Pkd2 double heterozygotes (Pik3c2a+/ ; 2 cilia (Supplemental Figure 3A). To test whether the observed Pkd2+/ ) showed a significantly enhanced rate of cystogenesis, localization defect was due to impaired Rab8 activation, rescue with detection of cysts in six (three monolateral and three bi- experiments were attempted using the constitutively active lateral) out of ten double heterozygous mice. In further form of the protein (Supplemental Figure 3B). Transfection agreement, a detailed blind scoring of the number of tubular of Rab8Q67L was able to restore the number of polycystin-2 dilation, microcysts and cysts showed that those lesions were 2 2 positive cilia in Sh1-IMCD3 cells, while a wild-type Rab8 was significantly more frequent in Pik3c2a+/ ; Pkd2+/ double mu- not (Figure 3C). These results indicate that PI3K-C2a pro- tants than in single heterozygotes (Figure 5A). In agreement with motes Rab8-dependent localization of polycystin-2 into cilia. polycystin-1 and -2 being strictly associated, heterozygous loss of 2 Pik3c2a enhanced cyst development not only in Pkd2+/ but also +/2 Heterozygous Loss of Pik3c2a Induces Ciliary Defects in Pkd1 mice30 carrying a heterozygous deletion of the gene and Enhances Cyst Formation in Polycystin encoding for polycystin-1 (Supplemental Figure 6, A and B). This Heterozygous Mutants genetic evidence demonstrates that PI3K-C2a functionally co- Ciliary defects due to reduced PI3K-C2a levels were next in- operates with the polycystin complex to prevent cyst formation. vestigated in vivo in the kidney of Pik3c2a heterozygous mice. +/2 Pik3c2a mutants normally reached adulthood and Ischemia/Reperfusion-Induced Cysts in the Kidneys of 2 showed a 50% decrease in the amount of PI3K-C2a in the Pik3c2a+/ Adults majority of tissues, including brain, testis, pancreas, muscle, Heterozygous loss of polycystins not only predisposes to cyst fat, and kidney (Supplemental Figure 4). Inspection of cilia in formation31 but also exacerbates cystogenesis in response to 2 renal tubules of 2-month-old Pik3c2a+/ mice showed an tubular-cell damage such as renal ischemia/reperfusion overall reduction of ciliary length (Figure 4A). Specific analysis (I/R).32,33 To test whether heterozygous mutation of Pik3c2a

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not wild-type controls developed overt cysts (Supplemental Figure 9A). To provide a quantitative assessment of kid- ney remodeling, tubular dilations, micro- cysts and cysts were scored (Figure 5B). Tubular dilations and microcysts were sig- 2 nificantly more abundant in Pik3c2a+/ than in wild-type controls in both outbred and inbred backgrounds (Figure 5C, Sup- plemental Figure 9B). Furthermore, overt cysts could only be scored in heterozygous mutants (Figure 5C, Supplemental Figure 9B), thus indicating that the heterozygous loss of PI3K-C2a is sufficient to predis- pose to cyst development in response to ischemic injury.

2 Pik3c2a+/ Kidneys Show Unrestrained Proliferative Signals after I/R To understand the trigger for cyst forma- tion, cystogenic pathways known to be upregulated in the cyst lining epithelia of Pkd1 and Pkd2 mutants were initially 2 analyzed in protein extracts from Pik3c2a+/ 2 Figure 5. Heterozygous loss of Pik3c2a enhances cysts formation in Pkd2+/ mice and and control kidneys of BALB/c mice at after I/R. (A) Quantification, using the TD/cyst index, of tubular dilations (TD, left), 48 hours after I/R. Phosphorylation of Erk 2 microcysts (mCysts, middle) and cysts (right). n=7 wild-type, n=7 Pik3c2a+/ , n=8 (p42–44 MAPK) and S6rp were increased 2 2 2 2 Pkd2+/ and n=10 Pik3c2a+/ ;Pkd2+/ mice. Mice were analyzed at 3 months of age. twofold in Pik3c2a+/ kidneys (Figure 6, (B) Representative micrographs of tubular dilations (left), a microcyst (middle), or a cyst A and B), thus demonstrating that the 2 (right panel), recovered in Pik3c2a+/ outbred mice. Tc, tubular cyst. Bar=100 mm. (C) MAPK and mTOR pathways are more ac- Quantification, using the TD/cyst index, of tubular dilations (TD, left), microcysts tive in these organs. Polycystins also in- (mCysts, middle), and cysts (right). Ctrl, untreated kidney; I/R, ischemia reperfusion. +/2 hibit proliferation by upregulating the n=7 wild-type and n=8 Pik3c2a outbred mice. cyclin-dependent kinase inhibitor p21.34 Consistent with impaired polycystin sig- 2 enhanced the propensity to develop cysts consequent to I/R, naling in Pik3c2a+/ kidneys, protein levels of p21 were signif- the monolateral renal artery was temporarily occluded for 35 icantly decreased in mutant organs after I/R (Figure 6, A and B). minutes. To confirm result specificity, the effect of Pik3c2a Furthermore, the amount of proliferating tubular cells in sec- 2 haploinsufficiency after I/R treatment was evaluated in both tions from Pik3c2a+/ kidneys, although equal to wild-type outbred C57BL6/129Sv and inbred mice, obtained by back- numbers at 48 hours, was two times higher than controls at crossing to BALB/c mice for eight generations. 6 weeks after I/R (Figure 6, C and D). In line with these find- 2 First, the extent of tissue damage was evaluated by quan- ings, the majority of cysts from Pik3c2a+/ kidneys stained pos- tifying tubular necrosis in tissue sections of wild-type and itive for pErk and pS6rp (Figure 6E). Altogether, these results 2 Pik3c2a+/ kidneys 48 hours after ischemia (Supplemental show that proliferative signals normally limited by polycystins 2 Figure 7A). The number of necrotic tubules (NT) and casts are uncontrolled in Pik3c2a+/ kidneys, thus generating a (CS) was similar in wild-type and mutant kidneys in both condition known to favor cyst development. backgrounds (Supplemental Figure 7, B and C), thus indicat- ing that a reduction of PI3K-C2a doesnotaffecttheinitial Reduction of PI3K-C2a Levels Impairs Polycystin- damage due to the ischemic treatment. Mediated Control of Proliferative Signals in Renal Next, I/R treated kidneys were analyzed after 6 weeks of Tubular Cells recovery. Ischemic damage in both genotypes caused a re- Analysis of proliferative pathways in Sh1 and Sh2-IMCD3 ductioninkidneyweight(SupplementalFigure8A)andan showed an increased phosphorylation of Erk and S6K, 2 increase in fibrosis (Supplemental Figure 8, B and C). mirroring the condition of Pik3c2a+/ kidneys (Supplemen- However, while both wild-type and heterozygous mice tal Figure 10). Given the effect of Rab8 activation on PI3K- showed a general enlargement of tubules, mutant mice but C2a-dependent polycystin-2 trafficking to the primary

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cilium, Pik3c2a-silenced IMCD3 cell lines stably expressing either green fluorescent protein (GFP)-Rab8Q67L or the empty GFP control vector were established. Anal- ysis of the mTOR and MAPK pathways showed that loss of PI3K-C2a in control cells induced the expected upregulation in Erk and S6K phosphorylation (Figure 7A). Conversely, expression of Rab8Q67L in Pik3c2a knockdown cells significantly re- duced these phosphorylation events, rescu- ing Erk and S6K phosphorylation to the levels of PI3K-C2a-competent cells (Figure 7A). To determine whether Rab8Q67L rescues overactivation of the mTOR and MAPK pathways by either restoring polycystin transport to the cilium or promoting cil- ium elongation, wild-type and Pik3c2a knock-down IMCD3 cells were stimulated with the polycystin-2 agonist triptolide (Figure 7, B and C). This small molecule enhances polycystin-2 activity35 but has neg- ligible effects on primary cilium length36 (4.460.3 and 4.560.2 mmintriptolide- treated and untreated IMCD3 cells, re- spectively). The response to triptolide was evaluated in IMCD3 cells grown either with or without serum, to promote deci- liation or ciliogenesis, respectively. In the concomitant presence of serum and ab- sence of cilia, mTOR, MAPK, and cAMP- responsive element binding protein (CREB) were equally phosphorylated in either wild-type or Sh1-IMCD3 cells (Figure 7B). On the contrary, under se- rum deprivation, only Sh1-IMCD3 cells showed mTOR, MAPK, and CREB hyper- 2 Figure 6. Uncontrolled proliferative signaling in the tubules of Pik3c2a+/ kidneys phosphorylation. Treatment with triptolide after I/R. (A and B) Western blot analysis of pathways regulating proliferation in wild- did not change the background phos- 2 type and Pik3c2a+/ samples from either control (ctrl) or ischemic (I/R) kidneys at 48 phorylation in deciliated cells but was hours after treatment. Activation of the MAPK pathway is investigated through analysis able to rescue the hyperphosphorylation of pErk, while phosphorylation of S6rp shows the activation of the mTOR pathway. p21 in ciliated Sh1-IMCD3 only (Figure 7C). is a cyclin-dependent kinase inhibitor upregulated after ischemic injury of the kidney. These results show that the sole potenti- A representative western blot (A) as well as protein quantification (B) of n=8 wild-type ation of ciliary polycystin-2 signaling is +/2 and n=8 Pik3c2a mice are shown. (C and D) Analysis of proliferation was achieved able to restore normal mTOR, MAPK, and by immunohistochemical staining of PCNA in sections of either control (ctrl) or is- 2 CREB pathway activation in Pik3c2a- chemic (I/R) kidney from wild-type and Pik3c2a+/ mice. Representative sections of +/2 silenced cells. Altogether, these observa- a wild-type and a Pik3c2a kidney at 6 weeks after I/R is shown (C). Graphs provided a in (D) represent the percentage of tubular cells showing a PCNA positive nucleus over tions further indicate that PI3K-C2 is the number of tubular cells per section at either 48 hours or 6 weeks after I/R. For all crucially required for the transport to groups n=6 mice. (E) Representative micrographs showing that the majority of cystic the cilium of polycystin-2 where it relays 2 lesions is positive for pErk and pS6rp in sections from a Pik3c2a+/ kidney at 6 weeks signals protecting from cystogenesis after I/R. PCNA, proliferative cell nuclear antigen. (Figure 8).

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Figure 7. Reduction of PI3K-C2a levels impairs polycystin-mediated control of proliferative signals in IMCD3 cells. (A) Western blot analysis of the activation of the MAPK (pErk) and mTOR (p70S6K) pathways in control and Pik3c2a-silenced (Sh1-IMCD3) cells ex- pressing either GFP-Rab8Q67L or a control plasmid (GFP-empty). Constitutive Rab8 activity is able to rescue excessive proliferative pathways in Sh1-IMCD3. Graph provides the mean quantification value of n=5 independent experiments. (B and C) Western blot analysis of CREB, MAPK, and mTOR pathways in Sh1-IMCD3 cells in serum (B) and 48 hours-starved (C) conditions. Treatment with the polycystin-2 agonist triptolide at a concentration of 25 nM for either 6 (CREB) or 48 hours (MAPK, mTOR) was able to rescue abnormal activation of these pathways.

DISCUSSION IMCD3 cells resulted in impaired targeting of polycystin-2 and reduced cilium length. Importantly, both defects could Signals from the primary cilium are essential to ensure the be rescued by the expression of a constitutively active form correct development of kidney tubules and to prevent cyst of Rab8. Altogether these results provide the genetic proof that formation.1,14 Our results indicate that PI3K-C2a modulates PI3K-C2a is functionally required to promote Rab8-dependent trafficking of primary cilium-bound cargo proteins such as cargo trafficking to the primary cilium. polycystin-2, thus allowing polycystin-mediated control of A link between Rab8, polycystin-2 ciliary targeting, and proliferative signals in kidney tubular cells. This function is kidney cyst development is suggested by studies on the Exocyst strictly dose-dependent and a 50% reduction in PI3K-C2a complex3,22,38 and on other genes epistatic to Rab8.39,40 De- expression is sufficient to cause primary cilium abnormalities spite the reduced expression of PI3K-C2a leading to decreased in kidney tubules as well as to predispose to renal cyst forma- Rab8 activation and a significant decline in ciliary polycystin- tion. 2, heterozygosity for a Pik3c2a-null allele did not directly in- In mouse embryonic fibroblasts, PI3K-C2a activity con- duce kidney cyst development in mice. This suggests that the trols the Rab11/Rab8 cascade at the PRE and regulates a levels of polycystin-2 (or of the polycystin1/polycystin-2 com- 2 vesicular trafficking process, targeting cargo proteins to the plex) in Pik3c2a+/ cilia remain above a threshold sufficient to primary cilium.20,23,24,26 Consistently, silencing of PI3K- protect from spontaneous cyst formation. Consistent with this C2a in collecting-duct derived IMCD3 cells impaired Rab11 hypothesis, a further decrease in ciliary polycystin-2 levels 2 2 activation in a dose-dependent manner and prevented the cil- obtained by crossing Pik3c2a+/ with Pkd2+/ mice was suf- iary localization of Rab8. This Rab GTPase represents a key ficient to enhance the frequency of cyst formation. The anti- hub regulating selective entry of proteins inside the ciliary cystogenic function of polycystin-2 requires the formation of a compartment21,32,37 and delivery of material required for cil- dimer with polycystin-1 on the ciliary membrane,41 and a ium elongation.23 In agreement, silencing of PI3K-C2a in concomitant reduction of polycystin-1 and polycystin-2 levels

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polycystin-2 ciliary translocation via a constitutively active Rab8 and the hyperstimulation with triptolide of the low levels of ciliary polycystin-2 present in PI3K-C2a knock-down cells. Therefore, these observations demonstrate that a failure in polycystin-2 function, rather than a general primary cil- ium shortening, is the major cause of the alterations in pro- liferative signaling pathways detected after down-modulation of Pik3c2a. Furthermore, this evidence clearly links the polycystin-2 trafficking defect to unrestricted tubular prolif- eration in Pik3c2a heterozygous kidneys. Altogether, these results highlight a PI3K-C2a/Rab8-mediated targeting route to the primary cilium that critically contributes to protect the kidney from cyst formation and indicate Pik3c2a as a disease modifier gene in PKD.

CONCISE METHODS Figure 8. Graphical representation of the role of PI3K-C2a in the prevention of kidney cyst formation. PI3K-C2a localizes in the pericentriolar recycling compartment surrounding the base of Animal Models Pik3c2a2/2 et al 26 the primary cilium in cells of the kidney epithelium. In this vesicular mice were generated as described in Franco . Chi- compartment, PI3K-C2a promotes the Rab11/Rab8 activation cas- meric mice were either intercrossed to maintain a mixed C57/BL6- cade, which allows Rab8 targeting to the primary cilium. Ciliary SV129 background or backcrossed for eight generations in the Rab8 regulates cilium elongation and entry of ciliary proteins, in- BALB/C background; wild-type littermates from heterozygous 2 cluding polycystin-2. High levels of polycystin-2 on the ciliary crosses were always used as controls. Pkd2+/ were kindly provided 2 membrane are necessary for counteracting intracellular pathways, by Dr. S. Somlo (Yale University),29 while Pkd1+/ were provided by such as the mTOR and MAPK pathways, which constitutively induce Dr. A. Boletta (San Raffaele).30 Double heterozygous animals were 2 2 proliferation of renal cells and favor the development of kidney obtained by intercrossing either Pkd1+/ or Pkd2+/ (both in the cysts. PRE, pericentriolar recycling compartment. +/2 C57/BL6 background) and Pik3c2a mice backcrossed for 12 gen- erations into the C57/BL6 background. increases cysts formation in mouse kidneys.31 Consistently, Silencing of Pik3c2a and Cell Treatments reduction of PI3K-C2a levels exacerbated spontaneous cyst Silencing was achieved through lentiviral infection as described in formation also in a Pkd1 heterozygous background. Given that Franco et al.26 Triptolide (Invivogen, San Diego, California) was polycystin-1 and -2 mutually influence their ciliary transloca- added to either serum treated or 48-hour serum-starved cells at a tion from the Golgi apparatus,41,42 enhanced cystogenesis in concentration of 25 nMol. 2 2 Pik3c2a+/ ;Pkd2+/ mice is in agreement with an effect of PI3K-C2a on the ciliary trafficking of both proteins of the Plasmids and Antibodies polycystin dimer. GFP-Rab8a was generated using PCR amplification and cloning into Although the onset of spontaneous cystic lesions in the pEGFP-C3 expression vector. GFP-Rab8aQ67L was kindly provided kidney of either polycystin-1 or -2 heterozygous mutants is a by Dr Peranen (Helsinki University, Helsinki, Finland). Primary rare event,29,32,43 these mice are prone to develop cysts in re- antibodies: anti-acetylated-tubulin monoclonal (6–11B-1; Sigma- sponse to an insult such as renal ischemia.32,33 This indicates Aldrich, St. Louis, Missouri), anti-acetylated-tubulin polyclonal that subtle changes in polycystin dosage are sufficient to D20G3 (Cell Signaling Technology, Danvers, Massachusetts), anti- weaken the anti-proliferative effect exerted by this pathway PI3K-C2a monoclonal (anti p170 #611046; BD Transduction Labo- in kidney tubules and to increase susceptibility to cyst forma- ratories, Franklin Lakes, New Jersey), anti-polycystin2 D3 monoclonal tion after damage. In line with this view, ischemic damage of (Santa Cruz Biotechnology, Dallas, Texas). Pik3c2a heterozygous kidneys caused exaggerated activation of proliferative signals that are considered to be under polycystin Renal Damage control, such as the mTOR and MAPK cascades. This is in A microvascular clamp was applied, in 6-week-old male mice, at the agreement with a reduction in the levels of p21/cdk, a negative renal pedicle of the left kidney for 35 minutes to induce a transient regulator of cell cycle induced by polycystin signaling.34 ischemia; the right kidney was left untouched and used as a control. Downregulation of PI3K-C2a in IMCD3 cells mirrored the For quantification of tubule dilation, cortical sections were stained dysregulation of MAPK and mTOR pathways observed in vivo. with hematoxylin-eosin, three 203 fields/kidney were photographed However, these alterations could be avoided by restoring poly- and the TD/cystic index was calculated as described32: a grid com- cystin-2 function using two different strategies: the rescue of posed of 13.6 mm-spaced dots was overlapped on the images, the

1142 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 1135–1144, 2016 www.jasn.org BASIC RESEARCH tubules were counted/scored according to the number of dots present rapid cyst formation after cilia disruption. JAmSocNephrol24: 456– in their lumen. Tubular dilation (Td), two dots; microcysts (MC), 3–9 464, 2013 dots; cyst, more than nine dots. 10. Nauli SM, Alenghat FJ, Luo Y, Williams E, Vassilev P, Li X, Elia AE, Lu W, Brown EM, Quinn SJ, Ingber DE, Zhou J: Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells. Nat Genet 33: Statistical Analyses 129–137, 2003 Values were reported as the mean6SEM. Statistical significance was cal- 11. Torres VE, Harris PC: Autosomal dominant polycystic kidney disease: culated with one-way ANOVA and Bonferroni post hoc tests. One, two the last 3 years. Kidney Int 76: 149–168, 2009 fi fi P 12. Nauli SM, Kawanabe Y, Kaminski JJ, Pearce WJ, Ingber DE, Zhou J: and three asterisks (*) in all the gures indicate signi cance with a value fl , , , Endothelial cilia are uid shear sensors that regulate calcium signaling 0.05, 0.01 and 0.001, respectively. All the analyses were performed and nitric oxide production through polycystin-1. Circulation 117: with the software PRISM5 (GraphPad Software Inc., La Jolla, CA). 1161–1171, 2008 13. Forman JR, Qamar S, Paci E, Sandford RN, Clarke J: The remarkable mechanical strength of polycystin-1 supports a direct role in mecha- notransduction. JMolBiol349: 861–871, 2005 ACKNOWLEDGMENTS 14. Abdul-Majeed S, Nauli SM: Calcium-mediated mechanisms of cystic expansion. Biochim Biophys Acta 1812: 1281–1290, 2011 15. Bae YK, Qin H, Knobel KM, Hu J, Rosenbaum JL, Barr MM: General and The authors would like to thank Dr. J. Peranen (Helsinki University) cell-type specific mechanisms target TRPP2/PKD-2 to cilia. Develop- Q67L for providing the GFP-Rab8a plasmid and Mossino M. and ment 133: 3859–3870, 2006 Fioravanti A. for technical help with histology. 16. Kaplan OI, Molla-Herman A, Cevik S, Ghossoub R, Kida K, Kimura Y, This work was supported by grants from Progetto Ateneo Compagnia Jenkins P, Martens JR, Setou M, Benmerah A, Blacque OE: The AP-1 San Paolo, Italy, (EH), AIRC, Italy (EH), Regione Piemonte, Italy (EH), clathrin adaptor facilitates cilium formation and functions with RAB-8 in C. elegans ciliary membrane transport. JCellSci123: 3966–3977, 2010 Leducq Foundation, France (EH) and Telethon Foundation, Italy 17. Follit JA, Tuft RA, Fogarty KE, Pazour GJ: The intraflagellar transport (AB and GRM). protein IFT20 is associated with the Golgi complex and is required for The CMMI is supported by the European Regional Development cilia assembly. Mol Biol Cell 17: 3781–3792, 2006 Fund and the Walloon Region. 18. 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1144 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 1135–1144, 2016 Supplementary Information

Materials and Methods:

Cell culture and treatments. IMCD3 cells were maintained in DMEM-F12 supplemented with 10% calf serum. Starvation and cilium assembly were achieved by 24/48 hrs of serum deprivation. IMCD3 at 80% confluency were transfected on coverslips in 24-well plates with 0.5 µg of plasmid DNA using the Effectene Transfection Reagent (Qiagen, Hilden, Germany). GFP- and GFP- Rab8aQ67L IMCD3 stable cell lines were obtained by Geneticin selection. Briefly, IMCD3 cells were plated in 12-well plates and transfected with either EGFP-C3 or GFP- Rab8aQ67L plasmid. After 36 hours, cells were plated in a 9 cm plate and medium was replaced with medium containing Geneticin G418 Sulphate (Life Technologies). Resistant cells were then plated at single cell dilution to allow clonal selection of GFP-positive clones.

Immunofluorescence. Immunofluorescence of PFA 4% fixed IMCD3 cells followed standard procedures. Raw images were digitally processed only to normalize the background and enhance the contrast. Cells were stained with DAPI and examined with either Leica TCS-II SP5 confocal microscope, or a Zeiss Observer-Z1 microscope, equipped with Apotome. Z-stacks were acquired and processed with the Maximum Projection tool. 3D morphometric measurement of ciliary length was performed with Filament tool of Imaris (BitPlane, Zurich, Switzerland). In situ localization of Polycystin-2 and acetylated-Tubulin in nodal cilia was done on embryos at the presomite stage essentially as described in 1. Primary antibodies: anti-Acetylated-Tubulin monoclonal (6- 11B-1, Sigma, St. Louis MO, USA), anti-Acetylated-Tubulin polyclonal D20G3 (Cell Signaling Technology, Boston, MA, USA), anti-PI3K-C2α monoclonal (anti p170 #611046 BD Transduction Laboratories), anti-Rab11a polyclonal (Abcam, Inc Cambridge MA), anti- Polycystin2 D3 monoclonal (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibodies were: goat anti-rabbit and goat anti-mouse, labelled with Alexa-488 or -568 (Molecular Probes/Invitrogen).

Scanning Electron Microscopy. Samples were fixed ON at 4°C in 2.5% glutaraldehyde, 0.1M cacodylate buffer (pH 7.2) and post-fixed in 2% OsO 4 in the same buffer. After serial dehydration in increasing ethanol concentrations, samples were dried at critical point, coated with platinum by standard procedures, and examined in a ESEM Tecnai Quanta 200 FEG (FEI).

Histological Analyses and Immunohistochemistry. Kidneys were isolated and fixed overnight with paraformaldehyde 4%. Samples were dehydrated, paraffin embedded and sectioned into 3 µm thick slices. Standard protocols where followed for the quantification of tubular dilations 2. Briefly, cortical sections were stained with Hematoxylin-Eosin, three 20x fields/kidney were photographed and the TD/cystic index was calculated: a grid composed of 13,6 μm-spaced dots was overlapped on the images, the tubules were counted/scored according to the number of dots present in their lumen (Tubular dilation, 2 dots; microcysts, 3-9 dots; Cyst, >9 dots). To visualize extracellular matrix deposition, sections were stained with Picrosirius red (Fluka, Buchs, Switzerland). The percentage of fibrosis (red staining) over the total tissue was calculated by Metamorph software (Universal Imaging Corporation, Downingtown, PA, USA). 5 fields were quantified for each kidney. For immunohistochemical analyses rabbit monoclonal antibodies against pErk1/2 (Phospho-p44/42 MAPK Thr202/Tyr204, Cell Signaling #4376) and pS6rp (Phospho S6Rp Ser240/244, Cell Signaling #2217) were used on paraffin sections. Rabbit anti-rat IgG (Dako Cytomation, Milano, Italy) was used as the biotinylated secondary antibody. PCNA staining was performed using mouse monoclonal antibodies against PCNA (SantaCruz Biotechnology,) and the Vector M.O.M. Immunodetection Kit (Vector Laboratories, Burlingame, CA, USA). Immunoreactivity was detected with the streptABCComplex/HRP system (DakoCytomation; Milano, Italy) and developed with DAB (methanol 3,3 diamino- benzide, Roche Diagnostic Corp., Milano, Italy). Sections were analyzed on an Olympus BX41 microscope (objective Olympus Plan 4X, 10X or 40X) equipped with an Olympus DP50 camera for images acquisition (Olympus, Milan, Italy). For quantifications, 5 fields were analysed for each kidney.

Western blotting. Protein extraction, SDS-PAGE and western blotting followed standard procedures. Antibodies: anti-PI3K-C2α monoclonal (anti p170 #611046 BD Transduction Laboratories), anti-Rab11a monoclonal (BD Bioscience #610657), anti-phosphoS6K polyclonal (Phospho-p70 S6 Kinase (Thr389) Cell Signaling #9205), anti-S6K polyclonal (p70 S6 kinase α (C-18) sc-230), anti-phosphoS6 polyclonal (Phospho-S6 Ribosomal Protein (Ser235/236) Cell Signaling #2211), anti-phosphoERK polyclonal (Phospho-p44/42 MAPK (Erk1/2)(Thr202/Tyr204) Cell Signaling #9101), anti-ERK polyclonal (p44/42 MAPK (Erk1/2) Cell Signaling #9102), anti-p21 monoclonal (P21 (F-5) sc-6246 Santa Cruz), anti- phosphoCREB monoclonal (Phospho-CREB (Ser133) Cell Signaling #9198). Anti-GFP and Anti-Vinculin are homemade produced.

References: 1. Franco, I, Gulluni, F, Campa, CC, Costa, C, Margaria, JP, Ciraolo, E, Martini, M, Monteyne, D, De Luca, E, Germena, G, Posor, Y, Maffucci, T, Marengo, S, Haucke, V, Falasca, M, Perez-Morga, D, Boletta, A, Merlo, GR, Hirsch, E: PI3K Class II alpha Controls Spatially Restricted Endosomal PtdIns3P and Rab11 Activation to Promote Primary Cilium Function. Dev Cell, 28: 647-658, 2014. 2. Bastos, AP, Piontek, K, Silva, AM, Martini, D, Menezes, LF, Fonseca, JM, Fonseca, II, Germino, GG, Onuchic, LF: Pkd1 haploinsufficiency increases renal damage and induces microcyst formation following ischemia/reperfusion. J Am Soc Nephrol, 20: 2389-2402, 2009.

Supplementary Figures:

Supplemental Figure 1. PI3K-C2α localizes around the base of the primary cilium. (A) Immunofluorescence analysis of controol-IMCD3 cells stained with antti-PI3KC2α antibody (red). Cilium (green) and nuclei (blue)) staining is provided. Bar= 3μm. (B-C) Confocal images of control-IMCD3 cells transfected with either GFP-Rab11 (B) or GFP-Rab8 (C) and stained with anti-PI3KC2α antibody (red), show partial co-localizattion of PI3KC2α with these markers of the pericentriolar recycling endosome. Bar= 3μm.

Supplemental Figure 2. PI3K-C2α regulates Rab11 activation in a dose dependent manner in IMCD3 cells. (A) Western blot analysis with anti-PI3K-C2α antibody in IMDC3 cells infected with either a control sequence (empty-pGIPZ) or shRNAs downmodulating PI3K-C2α (Sh1 and Sh2). Different efficiency of silencing produced two cell lines showing 40% (Sh1) or 20% (Sh2) PI3K-C2α levels of control cells. (B) Pull down experiment showing the endogenous content of Rab11-GTP in IMCD3 cells downnmodulated for PI3K- C2α (Sh1 and Sh2). Lower levels off PI3K-C2α (Sh2) correspondded to more severe impairment of Rab11 activation. N=6 independent experiments.

Supplemental Figure 3. (A) Measurement of ciliary length and Polycystin-2 positive staining, obtained in parallel for each primary cilia (n=50 cilia for each condition), analysing wild-type (IMCD3) or Sh1-treated IMCD3 (Sh1-IMCD3) cells after 48-hour starvation. Percent frequency of cilia in a specific length range (Total) is shown superimposed to percent frequency of Polycystin-2 positive cilia in the same length range (PC2+). (B) Rescue of Polycystin-2 ciliary localization by transfection of constitutively active Rab8. Representative immunofluorescence of Polycystin-2 (PC2, red) on cilia (acetilated-α- tubulin, blue) of 48 hours-starved Sh1-IMCD3 cells, after transfection with either a control plasmid (empty-EGFP, green, upper panels) or constitutively active Rab8 (GFP-Rab8Q67L, green, lower panels). Sh1-IMCD3 cells transfected with the control plasmid show a defective accumulation of PC2 on cilia, while the transfection of GFP-Rab8Q67L rescues this defect. Bar= 3 μm.

Supplemental Figure 4. Analysis of Pik3c2a+/- adult mice. Western blot analysis of PI3K- C2α protein levels in wild-type and Pik3c2a+/- adult tissues. Equal loading was monitored by vinculin. Reduction of PI3K-C2α protein levels is observed in most heterozygous tissues including the kidney (first row).

Supplemental Figure 5. Polycystin-2 fails to localize to ventral node cilia in Pik3c2a-/- embryos. Whole mount immunofluoresccence of wild-type and Pik3c2a-/- embryos at the early somite stage. Cilia of the ventral node were stained for PC2 (red) and acetyl-tubulin (green) and the percentage of PC2 positive cilia was calculated in n=3 wild type and n=3 Pik3c2a-/- embryos.

Supplemental Figure 6. Heterozygous loss of Pik3c2a enhances cysts formation in Polycystin-1 and 2 heterozygous mutants. (A) Quantification, using thhe TD/cysts index, of tubular dilations (TD, left), microcysts (µCysts, middle) and cysts (right) observed in n=10 wild type, n=11 Pik3c2a+/-, n=8 Pkd1+/- and n=12 double heterozygotes Pik3c2a+/-;Pkd1+/- outbred mice. 3-months old mice were analysed. (B) Representative low magnification images (22X) from Hematoxylin/eosin stained whole kidneys (top) with the specified genotypes; boxed regions are enlarged in the bottom panel. Arrowheads indicate cysts. Scale barrs, 1 mm (top); 13.6 μm (bottom).

Supplemental Figure 7. I/R-induced tubular necrosis at 48 hours after treatment. (A) Hematoxylin/eosin staining of wild-type (left) and Pik3c2a+/- (right) outtbred kidneys, either untreated (Control) or subjected to Ischemia/Reperfusion (I/R) followwed by a 48 hours recovery. Bar=100 μm. (B-C) Quantification of necrotic tubules and casts in wild-type and Pik3c2a+/- kidneys in either outbred (B) or inbred BALB/c (C) backgrouund.

Supplemental Figure 8. I/R-induced kidney remodelling in Pik3c2a+/- mice. (A) Kidney weight loss in wild-type and Pik3c2a+/- mice after I/R and 6 weeks recovery. Kidney weight (KW) is normalized on mouse body weight (BW). The left kidney (untreated) of each animal is used as control. While weight reduction of I/R compared tto control kidneys is evident in mice of outbred background (leftward graph), BALB/c mice are less sensitive to kidney injury caused by I/R (rightward graph). (B-C) Analysis of kidney fibrosis 6 weeks after I/R. (B) Representative micrographs of Picrosirius red stained kidneys from wild-type (left) and Pik3c2a+/- (right) outbred micee. (C) Quantification of the percentage of fibrosis in the kidney tissue of either untreated (Control) or ischemized (I/R) kidneys from wild-type and Pik3c2a+/- mice both in the outbreed or inbred BALB/c background. n=6, all groups. Bar=100 μm

Supplemental Figure 9. I/R-induced tubular dilations in Pik3c2a+/- mice at 6 weeks after treatment. (A) Low magnification images from Hematoxylin/eosin stained sections of wild- type (left) and (right) outbred kidneys, either untreated (Control) or subjected to Ischemia/Reperfusion (I/R). A cyst is evident in the section from Pik3c2a+/- kidney. Bar=100 μm. (B) Quantification, using the TD/cyst index, of tubular dilations (TD, left), microcysts (µCysts, middle) and cysts (right) in n=8 wild type and n==7 Pik3c2a+/- Balb/c mice.

Supplemental Figure 10. PI3K-C2α regulates the activation of prolifferative pathways in IMCD3 cells. Western blot analysis of the activation of the MAPK andd mTOR pathways in control and Pik3c2a-silenced IMCD3 cells (Sh1 and Sh2-IMCD33). A representative western blot showing increased phosphorylation of ERK and p70S6K in Sh1 and Sh2- IMCD3 cells is shown on the left. Protein quantification of n=6 independent experiment is provided on the right.