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A potential strategy for reducing cysts in autosomal dominant polycystic kidney disease with a CFTR corrector

  • Murali K. Yanda
    Affiliations
    Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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  • Qiangni Liu
    Affiliations
    Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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  • Liudmila Cebotaru
    Correspondence
    To whom correspondence should be addressed: The Johns Hopkins University School of Medicine, Hunterian 415, 725 N. Wolfe St., Baltimore, MD 21205.
    Affiliations
    Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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  • Author Footnotes
    3 All data generated or analyzed during this study are included in this published article and are available from the corresponding author upon reasonable request.
    2 The abbreviations used are: ADPKDautosomal dominant polycystic kidney diseaseCFTRcystic fibrosis transmembrane conductance regulatorCFcystic fibrosisNBDnucleotide-binding domainMSDmembrane-spanning domainHspheat shock proteinIPintraperitonealPNDpostnatal dayIBMX3-isobutyl-1-methylxanthineERendoplasmic reticulumCHOPC/EBP homologous proteinPCpolycystin; mitogen-activated proteinSERCAsarcolemma-endoplasmic reticulum Ca2+PNPkd1-nullPHPkd1-heterozygousAMacetoxymethylBrdUbromodeoxyuridineACadenylyl cyclase.
Open AccessPublished:June 06, 2018DOI:https://doi.org/10.1074/jbc.RA118.001846
      Autosomal dominant polycystic kidney disease (ADPKD) is associated with progressive enlargement of cysts, leading to a decline in function and renal failure that cannot be prevented by current treatments. Mutations in pkd1 and pkd2, encoding the polycystin 1 and 2 proteins, induce growth-related pathways, including heat shock proteins, as occurs in some cancers, raising the prospect that pharmacological interventions that target these pathways might alleviate or prevent ADPKD. Here, we demonstrate a role for VX-809, a corrector of cystic fibrosis transmembrane conductance regulator (CFTR), conventionally used to manage cystic fibrosis in reducing renal cyst growth. VX-809 reduced cyst growth in Pkd1-knockout mice and in proximal, tubule-derived, cultured Pkd1 knockout cells. VX-809 reduced both basal and forskolin-activated cAMP levels and also decreased the expression of the adenylyl cyclase AC3 but not of AC6. VX-809 also decreased resting levels of intracellular Ca2+ but did not affect ATP-stimulated Ca2+ release. Notably, VX-809 dramatically decreased thapsigargin-induced release of Ca2+ from the endoplasmic reticulum (ER). VX-809 also reduced the levels of heat shock proteins Hsp27, Hsp70, and Hsp90 in mice cystic kidneys, consistent with the restoration of cellular proteostasis. Moreover, VX-809 strongly decreased an ER stress marker, the GADD153 protein, and cell proliferation but had only a small effect on apoptosis. Given that administration of VX-809 is safe, this drug potentially offers a new way to treat patients with ADPKD.

      Introduction

      Autosomal dominant polycystic kidney disease (ADPKD)
      The abbreviations used are: ADPKD
      autosomal dominant polycystic kidney disease
      CFTR
      cystic fibrosis transmembrane conductance regulator
      CF
      cystic fibrosis
      NBD
      nucleotide-binding domain
      MSD
      membrane-spanning domain
      Hsp
      heat shock protein
      IP
      intraperitoneal
      PND
      postnatal day
      IBMX
      3-isobutyl-1-methylxanthine
      ER
      endoplasmic reticulum
      CHOP
      C/EBP homologous protein
      PC
      polycystin; mitogen-activated protein
      SERCA
      sarcolemma-endoplasmic reticulum Ca2+
      PN
      Pkd1-null
      PH
      Pkd1-heterozygous
      AM
      acetoxymethyl
      BrdU
      bromodeoxyuridine
      AC
      adenylyl cyclase.
      (
      • Torres V.E.
      • Harris P.C.
      Polycystic kidney disease: Genes, proteins, animal models, disease mechanisms and therapeutic opportunities.
      ) is characterized by multiple renal cysts, hypertension, and a decline in renal function that culminates in renal failure in 50% of all ADPKD patients (
      • Calvet J.P.
      • Grantham J.J.
      The genetics and physiology of polycystic kidney disease.
      ). Two proteins, PC1 and PC2, are associated with ADPKD. Mutations in pkd1 produce 85% of the cases of ADPKD (
      • Calvet J.P.
      • Grantham J.J.
      The genetics and physiology of polycystic kidney disease.
      ,
      • Al-Bhalal L.
      • Akhtar M.
      Molecular basis of autosomal dominant polycystic kidney disease.
      ). Patients with pkd2 mutations have a similar disease that is considered milder because of its later onset of symptoms (
      • Bastos A.P.
      • Onuchic L.F.
      Molecular and cellular pathogenesis of autosomal dominant polycystic kidney disease.
      ).
      Cysts can develop in each nephron segment through a combination of aberrant epithelial cell proliferation and abnormal fluid secretion (
      • Ong A.C.
      • Harris P.C.
      A polycystin-centric view of cyst formation and disease: The polycystins revisited.
      ). Cystic fluid is produced by a cAMP-dependent mechanism similar to that found in secretory epithelia (
      • Ikeda M.
      • Fong P.
      • Cheng J.
      • Boletta A.
      • Qian F.
      • Zhang X.M.
      • Cai H.
      • Germino G.G.
      • Guggino W.B.
      A regulatory role of polycystin-1 on cystic fibrosis transmembrane conductance regulator plasma membrane expression.
      ). Elevated cAMP levels have been detected in animal models of ADPKD, and this cAMP elevation provides the stimulation for fluid secretion in the cysts. An important step in cAMP-dependent fluid secretion is the activation of the cystic fibrosis transmembrane conductance regulator (CFTR), which secretes chloride into the cyst lumen followed by sodium and water (
      • Hanaoka K.
      • Devuyst O.
      • Schwiebert E.M.
      • Wilson P.D.
      • Guggino W.B.
      A role for CFTR in human autosomal dominant polycystic kidney disease.
      ).
      CFTR is a cAMP-dependent chloride channel (
      • Fuller C.M.
      • Benos D.J.
      CFTR!.
      ) best known for its role in cystic fibrosis (CF) (
      • Rosenstein B.J.
      • Zeitlin P.L.
      Cystic fibrosis.
      ). The most common mutation is ΔF508-CFTR, found in the nucleotide-binding domain 1 (NBD1) region of the CFTR molecule; this mutation affects about 90% of all CF patients (
      • Riordan J.R.
      • Rommens J.M.
      • Kerem B.
      • Alon N.
      • Rozmahel R.
      • Grzelczak Z.
      • Zielenski J.
      • Lok S.
      • Plavsic N.
      • Chou J.L.
      Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA.
      ). Correctors have been identified that act on ΔF508-CFTR either directly or indirectly to attenuate the deleterious effects of the disease (
      • Pedemonte N.
      • Lukacs G.L.
      • Du K.
      • Caci E.
      • Zegarra-Moran O.
      • Galietta L.J.
      • Verkman A.S.
      Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by high-throughput screening.
      ). One of these correctors that has is currently approved for use in patients, namely VX-809, restores the trafficking of ΔF508-CFTR to the plasma membrane (
      • Van Goor F.
      • Hadida S.
      • Grootenhuis P.D.
      • Burton B.
      • Stack J.H.
      • Straley K.S.
      • Decker C.J.
      • Miller M.
      • McCartney J.
      • Olson E.R.
      • Wine J.J.
      • Frizzell R.A.
      • Ashlock M.
      • Negulescu P.A.
      Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809.
      ). Precisely how VX-809 rescues CFTR function is still unclear. Some researchers maintain that VX-809 acts directly on CFTR, particularly within the membrane-spanning domain 1 of transmembrane domain 1 (
      • Ren H.Y.
      • Grove D.E.
      • De La Rosa O.
      • Houck S.A.
      • Sopha P.
      • Van Goor F.
      • Hoffman B.J.
      • Cyr D.M.
      VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1.
      ). We have shown that corrector C18, a close relative of VX-809 (
      • Van Goor F.
      • Straley K.S.
      • Cao D.
      • González J.
      • Hadida S.
      • Hazlewood A.
      • Joubran J.
      • Knapp T.
      • Makings L.R.
      • Miller M.
      • Neuberger T.
      • Olson E.
      • Panchenko V.
      • Rader J.
      • Singh A.
      • et al.
      Rescue of DeltaF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules.
      ), in combination with corrector C4 (
      • Pedemonte N.
      • Lukacs G.L.
      • Du K.
      • Caci E.
      • Zegarra-Moran O.
      • Galietta L.J.
      • Verkman A.S.
      Small-molecule correctors of defective DeltaF508-CFTR cellular processing identified by high-throughput screening.
      ), alters a proteostatic network of proteins (
      • Balch W.E.
      • Morimoto R.I.
      • Dillin A.
      • Kelly J.W.
      Adapting proteostasis for disease intervention.
      ) to rescue CFTR (
      • Lopes-Pacheco M.
      • Boinot C.
      • Sabirzhanova I.
      • Morales M.M.
      • Guggino W.B.
      • Cebotaru L.
      Combination of correctors rescue ΔF508-CFTR by reducing its association with Hsp40 and Hsp27.
      ).
      Heat shock proteins are abnormally regulated in ADPKD cysts (
      • Seeger-Nukpezah T.
      • Proia D.A.
      • Egleston B.L.
      • Nikonova A.S.
      • Kent T.
      • Cai K.Q.
      • Hensley H.H.
      • Ying W.
      • Chimmanamada D.
      • Serebriiskii I.G.
      Inhibiting the HSP90 chaperone slows cyst growth in a mouse model of autosomal dominant polycystic kidney disease.
      ), indicating the occurrence of a heat shock response (
      • Roth D.M.
      • Hutt D.M.
      • Tong J.
      • Bouchecareilh M.
      • Wang N.
      • Seeley T.
      • Dekkers J.F.
      • Beekman J.M.
      • Garza D.
      • Drew L.
      • Masliah E.
      • Morimoto R.I.
      • Balch W.E.
      Modulation of the maladaptive stress response to manage diseases of protein folding.
      ) in proliferating cyst cells. Indeed, the down-regulation of Hsp90 reduces cyst growth (
      • Seeger-Nukpezah T.
      • Proia D.A.
      • Egleston B.L.
      • Nikonova A.S.
      • Kent T.
      • Cai K.Q.
      • Hensley H.H.
      • Ying W.
      • Chimmanamada D.
      • Serebriiskii I.G.
      Inhibiting the HSP90 chaperone slows cyst growth in a mouse model of autosomal dominant polycystic kidney disease.
      ). Along these same lines, we provide here a novel strategy based on the use of VX-809 to reduce cyst growth in ADPKD. We found that treatment of cysts with VX-809 reduces their growth by restoring a network of proteostatic proteins that, when misregulated, contribute to cyst growth in ADPKD.

      Results

      VX-809 reduces cyst growth and improves renal function in Pkd1fl/fl;Pax8rtTA;TetO-cre mice

      To show that VX-809 is effective in reducing cyst growth in vivo, we injected the drug into the intraperitoneal (IP) space in Pkd1fl/fl;Pax8rtTA;TetO-cre model mice. When treated with doxycycline, these mice express Cre, causing knockout of PC1 (
      • Traykova-Brauch M.
      • Schönig K.
      • Greiner O.
      • Miloud T.
      • Jauch A.
      • Bode M.
      • Felsher D.W.
      • Glick A.B.
      • Kwiatkowski D.J.
      • Bujard H.
      • Horst J.
      • von Knebel Doeberitz M.
      • Niggli F.K.
      • Kriz W.
      • Gröne Koesters R.
      An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice.
      ). As has shown previously, an injection of doxycycline results in the development of multiple large cysts in these mice at ∼3 weeks of age (
      • Traykova-Brauch M.
      • Schönig K.
      • Greiner O.
      • Miloud T.
      • Jauch A.
      • Bode M.
      • Felsher D.W.
      • Glick A.B.
      • Kwiatkowski D.J.
      • Bujard H.
      • Horst J.
      • von Knebel Doeberitz M.
      • Niggli F.K.
      • Kriz W.
      • Gröne Koesters R.
      An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice.
      ,
      • Piontek K.
      • Menezes L.F.
      • Garcia-Gonzalez M.A.
      • Huso D.L.
      • Germino G.G.
      A critical developmental switch defines the kinetics of kidney cyst formation after loss of Pkd1.
      ) (Fig. 1A). In sharp contrast, when this strain of mice was injected daily with VX-809 (30 mg/kg) from postnatal day (PND) 10 to PND20, they showed significantly less cyst growth (∼60.4%) (Fig. 1, A and B). Kidney weight (Fig. 1C) and kidney-to-body weight ratios (Fig. 1D) were also lower than those of the control mice. There was no difference in overall body weight (Fig. 1E) between the treated and untreated groups. The administration of VX-809 improved renal function, as evidenced by lower blood urea nitrogen (Fig. 1F, BUN) and creatinine (Fig. 1G) values in the VX-809–treated mice than in the DMSO-treated mice. The dosage of VX-809 injected was lower than the human pediatric dose, which is 500 mg of lumacaftor/day. The adult dose is 800 mg/day.
      Figure thumbnail gr1
      Figure 1VX-809 slows cyst growth and improves renal function in pkd1−/− mice. A, representative images of PND21 kidney sections from DMSO- and VX-809–treated mice. B–D, significant reductions occurred in cyst index (B), kidney weight (C), and kidney-to-body weight ratio (D). E, no differences were noted in body weight. Also reduced was blood urea nitrogen (BUN) (F) and creatinine (G) levels. The total kidney area and total cystic area were measured with ImageJ (provided by National Institutes of Health). Cystic index = 100 × (total cystic area/total kidney area) and is expressed as a percentage. Columns represent mean ± S.E. for DMSO (vehicle)-treated (n = 4–5) and VX-809–treated mice. *, p < 0.05; **, p < 0.01 (for all graphs). Statistical analysis was performed using an unpaired two-tailed Student's t test.

      VX-809 reduces cyst growth in vitro

      We conducted our experiments using a model ADPKD cell line (PH, pkd1+/− heterozygote control; PN, pkd−/− knockout or Pkd1-null) clonally isolated from single parental clones obtained from a pkdfl/− mouse that was manufactured in an ImmortoMouse containing the H-2Kb-tsA58 gene. The null cells (PN) stably express the Cre recombinase. All of the cells are of proximal tubule origin (
      • Joly D.
      • Ishibe S.
      • Nickel C.
      • Yu Z.
      • Somlo S.
      • Cantley L.G.
      The polycystin 1-C-terminal fragment stimulates ERK-dependent spreading of renal epithelial cells.
      ,
      • Wei F.
      • Karihaloo A.
      • Yu Z.
      • Marlier A.
      • Seth P.
      • Shibazaki S.
      • Wang T.
      • Sukhatme V.P.
      • Somlo S.
      • Cantley L.G.
      Neutrophil gelatinase-associated lipocalin suppresses cyst growth by Pkd1 null cells in vitro and in vivo.
      ).
      One of the key questions we addressed was how VX-809 reduces cyst growth. For this purpose, we grew cysts in the presence of forskolin (Fig. 2). After treatment with forskolin, the cysts obtained were larger than those in the control cells, indicating that cyst growth is indeed cAMP-dependent as shown previously (
      • Hanaoka K.
      • Guggino W.B.
      cAMP regulates cell proliferation and cyst formation in autosomal polycystic kidney disease cells.
      ). Fig. 2 shows the effect on cyst growth of C18 or VX-809 when administered every other day, as well as the same treatment administered from days 9–16 in mice with already established cysts. Importantly, this drug treatment regime was effective in dramatically reducing cyst size, even in the presence of the hyperstimulatory environment created by forskolin. Also, VX-809 and a related compound, C18 (
      • Lopes-Pacheco M.
      • Boinot C.
      • Sabirzhanova I.
      • Morales M.M.
      • Guggino W.B.
      • Cebotaru L.
      Combination of correctors rescue ΔF508-CFTR by reducing its association with Hsp40 and Hsp27.
      ), were both able to reduce cyst growth equally well in the presence and absence of forskolin (Fig. 2). This important finding shows that VX-809 can overcome the powerful stimulatory effect of forskolin-induced increases in cAMP levels and still reduce cyst growth. Taken together with the animal data
      All data generated or analyzed during this study are included in this published article and are available from the corresponding author upon reasonable request.
      provided in Fig. 1, these in vitro results clearly indicate that CFTR correctors are effective in reducing cyst growth.
      Figure thumbnail gr2
      Figure 2VX-809 inhibits cyst growth in the presence of forskolin. A and B, VX-809 inhibits cyst growth in the presence of forskolin. Cells were treated with DMSO (control), VX-809, or C18 (10 μm) plus or minus forskolin on days 0, 2, 4, 6, 8, 10, 12, and 14. C and D, VX-809 reduces the size of established cysts in the presence of forskolin. Cells were treated with DMSO (control), C18, or VX-809 (10 μm) from 9–16 (
      • Hanaoka K.
      • Devuyst O.
      • Schwiebert E.M.
      • Wilson P.D.
      • Guggino W.B.
      A role for CFTR in human autosomal dominant polycystic kidney disease.
      ) days. All pictures were taken on day 16. Columns represent means ± S.E. (n = 6). The average cyst size from the control group was considered 100%, and the sizes of the rest of the cysts were compared with this average. ****, p < 0.0001. Cyst size was estimated from the cross-sectional area. Scale bar = 200 μm.

      VX-809 reduces proliferation

      A hallmark of the cysts in ADPKD is the increase in proliferation in response to cAMP (
      • Yamaguchi T.
      • Nagao S.
      • Wallace D.P.
      • Belibi F.A.
      • Cowley B.D.
      • Pelling J.C.
      • Grantham J.J.
      Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys.
      ). Therefore, we asked whether VX-809 would affect proliferation. We found that administration of VX-809 at 30 mg/kg (see Fig. 1) to mice (Fig. 3, A–C) or to cells (Fig. 3D) at 10 μm did significantly inhibit cell proliferation when compared with that of DMSO-treated mice or cells.
      Figure thumbnail gr3
      Figure 3Cell proliferation is reduced. A and B, proliferation in kidneys of Pkd1fl/fl;Pax8rtTA;TetO-cre mice. Representative images of Ki67 (a cellular marker for proliferation) staining of PN21 kidney sections from DMSO- and VX-809–treated mice. Arrows indicate Ki67-positive cells. Pictures were acquired with a Zeiss microscope equipped ×20 objective. C, summary data for Ki67-positive cells. Columns represent averages ± S.E. of DMSO (vehicle)-treated (n = 3) and VX-809–treated (n = 3) mice. Statistical analysis was performed using a two-tailed Student's t test. D, proliferation in PN cells. PN cells were treated with VX-809 or DMSO. The bromodeoxyuridine (BrdU) concentration in the cells was measured by using a BrdU cell proliferation assay kit (MilliporeSigma 2750) according to the manufacturer's protocol. Columns represent averages ± S.E. for the OD of BrdU at 450/550 nm. Data were analyzed using Student's t test with n = 5–7. Methods were described previously (
      • Cebotaru L.
      • Liu Q.
      • Yanda M.K.
      • Boinot C.
      • Outeda P.
      • Huso D.L.
      • Watnick T.
      • Guggino W.B.
      • Cebotaru V.
      Inhibition of histone deacetylase 6 activity reduces cyst growth in polycystic kidney disease.
      ). *, p > 0.05;**, p < 0.01.

      VX-809 down-regulates cAMP levels

      We have shown previously in both animal and cell culture models of ADPKD that cells lacking functional PC1 have elevated cAMP levels when compared with those that contain functional PC1 (
      • Cebotaru L.
      • Liu Q.
      • Yanda M.K.
      • Boinot C.
      • Outeda P.
      • Huso D.L.
      • Watnick T.
      • Guggino W.B.
      • Cebotaru V.
      Inhibition of histone deacetylase 6 activity reduces cyst growth in polycystic kidney disease.
      ). To assess the effect of CFTR correctors, we measured cAMP activity in PC1-conditional knockout mouse kidneys and in PN cells that were either left untreated or treated with VX-809. We found that administration of VX-809 (Fig. 4, A and B) significantly decreased the cAMP levels. Fig. 4B shows that PH (pkd1 heterozygote) cells have lower resting cAMP compared with PN cells, as we also have shown in previous findings (
      • Yanda M.K.
      • Liu Q.
      • Cebotaru V.
      • Guggino W.B.
      • Cebotaru L.
      Histone deacetylase 6 Inhibition reduces cysts by decreasing via cAMP and Ca2+ in knockout mouse models of polycystic kidney disease.
      ). Next, we treated the cells with forskolin, which increased cAMP levels drastically, as expected (Fig. 4B). It should be noted that VX-809 reduced the forskolin-induced increase in cAMP, indicating a direct action of VX-809 on adenylyl cyclase activity. To determine whether VX-809 also inhibits phosphodiesterase activity, we treated the cells with forskolin or with forskolin in combination with 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, to maximally stimulate adenylyl cyclase activity. Treating PN cells with forskolin or IBMX (Fig. 4B) increased the cAMP activity by ∼10-fold. However, VX-809 did not affect the forskolin- or IBMX-induced increases in adenylyl cyclase activity (Fig. 4B, compare data bars 5 and 9), indicating that VX-809 does not affect phosphodiesterase activity. These data suggest that under basal conditions, one way that VX-809 reduces cyst growth (as shown in Fig. 1) is most likely by reducing the resting cAMP levels. However, the results presented above, indicating that VX-809 can inhibit cyst growth even in the presence of forskolin (which elevates cAMP activity almost 500-fold), strongly suggest that other mechanisms are also involved.
      Figure thumbnail gr4
      Figure 4cAMP levels are reduced. A, cAMP levels in mouse tissue were reduced by VX-809. The kidney samples (n = 6) analyzed were taken from those depicted in . B, confluent PN and PH cells were treated with VX-809 (10 μm) or DMSO for 16 h and then treated with forskolin (100 μm) for 30 min before the cells were harvested for the assay. Columns represent means ± S.E. Statistical analysis was performed using a two-tailed Student's t test. Each set of data is from three individual wells. Note that resting cAMP is greater in PN versus PH cells as we showed previously (
      • Yanda M.K.
      • Liu Q.
      • Cebotaru V.
      • Guggino W.B.
      • Cebotaru L.
      Histone deacetylase 6 Inhibition reduces cysts by decreasing via cAMP and Ca2+ in knockout mouse models of polycystic kidney disease.
      ). Also note that forskolin caused a large increase in cAMP. A smaller increase occurred with IBMX. However, the increase induced by forskolin alone was similar to that caused by IBMX plus forskolin. VX-809 treatment reduced the levels of cAMP when compared with untreated PN cells in either the presence or absence of forskolin. C–E, adenylyl cyclase expression. C, Western blotting showing the expression of AC6 and AC3 in treated and control PN cells. D and E, columns represent means ± S.E. of the AC3 and AC6 expression. The data were analyzed by nonparametric t test. The experiment was repeated four times. For all graphs, *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

      VX-809 reduces AC3 but not AC6

      Ca2+-dependent adenylyl cyclase (AC) activity has been shown to play a role in cyst growth in ADPKD (
      • Yamaguchi T.
      • Pelling J.C.
      • Ramaswamy N.T.
      • Eppler J.W.
      • Wallace D.P.
      • Nagao S.
      • Rome L.A.
      • Sullivan L.P.
      • Grantham J.J.
      cAMP stimulates the in vitro proliferation of renal cyst epithelial cells by activating the extracellular signal-regulated kinase pathway.
      ). There are two classes of adenylyl cyclases that are regulated by intracellular Ca2+: one class is activated and the other inhibited. Thus, we chose to focus on one member from each of these two classes: AC3 and AC6. AC6 activity is inhibited by Ca2+, whereas that of AC3 is enhanced by increases in intracellular Ca2+ (
      • Rieg T.
      • Kohan D.E.
      Regulation of nephron water and electrolyte transport by adenylyl cyclases.
      ). Both AC3 and AC6 are expressed in the proximal tubules of rat kidneys (
      • Shen T.
      • Suzuki Y.
      • Poyard M.
      • Miyamoto N.
      • Defer N.
      • Hanoune J.
      Expression of adenylyl cyclase mRNAs in the adult, in developing, and in the Brattleboro rat kidney.
      ), and AC6 is already known to play a role in ADPKD (
      • Rieg T.
      • Kohan D.E.
      Regulation of nephron water and electrolyte transport by adenylyl cyclases.
      ). We have now shown (Fig. 4, C–E) that AC3 and AC6 are both expressed in PN cells. Surprisingly, the AC3 levels were decreased following treatment of the PN cells with VX-809. This result is consistent with our observation that VX-809 reduces both basal and forskolin-stimulated cAMP levels.

      VX-809 down-regulates resting intracellular Ca2+ levels and release of Ca2+ from the ER

      Misregulation of Ca2+-dependent signal transduction is associated with cyst formation (
      • Yamaguchi T.
      • Pelling J.C.
      • Ramaswamy N.T.
      • Eppler J.W.
      • Wallace D.P.
      • Nagao S.
      • Rome L.A.
      • Sullivan L.P.
      • Grantham J.J.
      cAMP stimulates the in vitro proliferation of renal cyst epithelial cells by activating the extracellular signal-regulated kinase pathway.
      ,
      • Mangolini A.
      • de Stephanis L.
      • Aguiari G.
      Role of calcium in polycystic kidney disease: From signaling to pathology.
      ). To determine whether VX-809 alters Ca2+ movement, we treated the cells with ATP, which stimulates purinergic receptors (
      • Menzies R.I.
      • Tam F.W.
      • Unwin R.J.
      • Bailey M.A.
      Purinergic signaling in kidney disease.
      ) and found (Fig. 5, A–C) that VX-809 caused a small reduction in resting Ca2+ but had no effect on intracellular Ca2+ movement in response to ATP. We suggested previously that PC2 is a positive regulator of ER Ca2+ release (
      • Li Y.
      • Santoso N.G.
      • Yu S.
      • Woodward O.M.
      • Qian F.
      • Guggino W.B.
      Polycystin-1 interacts with inositol 1,4,5-trisphosphate receptor to modulate intracellular Ca2+ signaling with implications for polycystic kidney disease.
      ,
      • Santoso N.G.
      • Cebotaru L.
      • Guggino W.B.
      Polycystin-1, 2, and STIM1 interact with IP(3)R to modulate ER Ca release through the PI3K/Akt pathway.
      ). To address the effect of VX-809 on this Ca2+ release, we treated the cells with thapsigargin, a specific inhibitor of the ER Ca2+-ATPase which, when applied, allows Ca2+ to leak out of the ER through independent Ca2+-permeable pathways (
      • Thastrup O.
      • Cullen P.J.
      • Drøbak B.K.
      • Hanley M.R.
      • Dawson A.P.
      Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase.
      ). Again, we saw a small effect of VX-809 on resting Ca2+. Importantly, VX-809 dramatically reduced the thapsigargin-induced release of Ca2+ from the ER (Fig. 5, D–F).
      Figure thumbnail gr5
      Figure 5Intracellular Ca2+ and ER Ca2+ are affected. A, representative traces of intracellular Ca2+ release in response to ATP (100 μm) in PN cells and cells treated with VX-809 (10 μm). B and C, graphs summarizing resting calcium levels (B) and the average amplitude of Ca2+ release (C) in response to ATP. D, representative traces of ER Ca2+ release in response to thapsigargin (4 μm) in PN cells treated with VX-809. E and F, resting calcium levels (E) and the average amplitude of Ca2+ release (F) in response to thapsigargin. (F340/F380) levels obtained by ratiometric Fura-2 AM analysis of PN cells treated with VX-809 (10 μm) for 16 h. Amplitude was measured as the standard deviation of the signal base to peak Δf/f. Significance between the two groups was analyzed using Student's t test (n = 4–5). *, p < 0.05; ***, p < 0.001.

      VX-809 has no effect on PC2 but increases CFTR expression

      PC2 is a 968–amino acid protein with an approximate molecular mass of 100 kDa in its monomeric form. To determine whether VX-809 affects PC2 protein expression, we conducted Western blotting experiments on PN cells to assess the resting levels of endogenous PC2. Our results indicated (Fig. 6, A and B) that VX-809 has no effect on PC2 expression.
      Figure thumbnail gr6
      Figure 6PC2 is unchanged and CFTR expression is increased. A, Western blotting showing expression of PC2 in treated or control cells. B, columns represent the means ± S.E. of the PC2 expression. The data were analyzed by nonparametric t test. The experiment was repeated six (control) or seven times. Note that PC2 levels are unchanged after VX-809 treatment. C, Western blotting showing expression of CFTR in VX-809 treated or control PN cells. D, columns represent averages ± S.E. of the CFTR expression. Data were analyzed by nonparametric t test. Experiment was repeated 4–5 times. **, p < 0.01. Note that VX-809 treatment enhances the CFTR expression in PN cells compared with untreated control cells.
      Fig. 6, C and D, shows that VX-809 did significantly increase the levels of CFTR in PN cells. It is known that the processing of WT CFTR is increased following CFTR corrector treatment by increasing its processing (
      • Lopes-Pacheco M.
      • Boinot C.
      • Sabirzhanova I.
      • Morales M.M.
      • Guggino W.B.
      • Cebotaru L.
      Combination of correctors rescue ΔF508-CFTR by reducing its association with Hsp40 and Hsp27.
      ).

      VX-809 reduces the steady-state levels of heat shock proteins

      To facilitate their growth in the kidney, cysts develop an altered network of proteins that most likely serves to protect them from stress (
      • Torres V.E.
      • Rossetti S.
      • Harris P.C.
      Update on autosomal dominant polycystic kidney disease.
      ). Thus, one may ask whether restoring this network to its normal state would reduce cyst growth. We have shown previously that CFTR-based correctors rescue ΔF508-CFTR by altering the proteostatic network of proteins involved in its degradation (
      • Lopes-Pacheco M.
      • Boinot C.
      • Sabirzhanova I.
      • Morales M.M.
      • Guggino W.B.
      • Cebotaru L.
      Combination of correctors rescue ΔF508-CFTR by reducing its association with Hsp40 and Hsp27.
      ). Therefore, we asked whether VX-809 would alter the levels of Hsp in pkd−/− mice and in PN cells. Fig. 7 shows the results in mice. The first thing to note is that Hsp27, -70, and -90 are all elevated in cystic kidneys compared with normal controls. Treatment with VX-809 of kidneys in which PC1 levels had been knocked out resulted in significantly less expression of Hsp27, Hsp70, and Hsp90. There was no change in Hsp40. Likewise, PN cells treated with VX-809 (Fig. 8, A–D) showed reduced expression of Hsp27, Hsp70, and Hsp90 following treatment. Both sets of data are consistent with an alteration in the heat shock response to cyst growth. A comparison of the PH cells, which are pkd+/−, with the PN cells, which are pkd−/−, shows that Hsp27 and -90 are elevated and Hsp70 reduced in the PN versus the PH cells (Fig. 8, F–H). The higher levels of HSP70 in PH versus PN cells (Fig. 8G) do not recapitulate what we saw in the mice (Fig. 7D), which had higher levels in the kidneys, where PC1 is absent. However, Hsp70 was reduced further when PN cells were treated with VX-809, as shown in Fig. 8C. Thus, although there are some differences between cell and animal models regarding the resting levels of Hsp70, both models show a reduction in Hsp70 when treated with VX-809.
      Figure thumbnail gr7
      Figure 7Chaperone expression is altered in mice. A and B, representative Western blot images of HSP27, -70, –90, and -40 in lysates of kidney tissue from no cyst induced (ND), cyst induced with doxycycline (D), or cyst-induced pkd−/− mice treated with VX-809 (D+VX809) 30 mg/kg body weight. C–F, columns represent averages ± S.E. of HSP27, -70, -90, and -40 expression. Data were analyzed by nonparametric t test. n = 4 for each treated and control group. Vertical black lines between the Western blot lanes are representative of experiments from the same gel. *, p < 0.05; **, p < 0.01. Although lines appear between some of the lanes, the data were extracted from the same gel.
      Figure thumbnail gr8
      Figure 8Chaperone expression is altered in PH and PN cells. A, Western blotting showing expression of HSP27, HSP70, and HSP90 in VX-809 treated or control PN cells. B–D, columns represent averages ± S.E. of the HSP27, -70, and -90 expression and show that VX-809 reduces the expression of HSP27, -70, and -90. E, Western blotting of HSP27, -70, and -90 in PH versus PN cells. F–H, columns represent averages ± S.E. of the HSP27, -70, and -90 expression and show that the expression of HSP27 and -90 are higher in PN versus PH cells, whereas Hsp70 is higher in PH versus PN cells. Data were analyzed by nonparametric t test. The experiment was repeated 4–5 times. Cells were grown in 10-cm culture dishes under permissive conditions (33 °C) with γ-interferon in the culture medium. Cells were then transferred to nonpermissive conditions at 37 °C in γ-interferon–free culture medium and evaluated at full confluence. At day 4, cells were treated with VX-809 (10 μm) for 16 h and harvested on the fifth day for assay. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

      VX-809 enhances the disappearance of Hsp70 and Hsp90

      To gain more insight into the mechanism whereby VX-809 alters Hsp protein levels, we inhibited translation by using cycloheximide (
      • Obrig T.G.
      • Culp W.J.
      • McKeehan W.L.
      • Hardesty B.
      The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes.
      ) and monitored the disappearance of the three Hsp. Fig. 9, A–D, clearly shows that the steady-state levels of each of the Hsp was unchanged over the 8 h immediately following cycloheximide treatment, suggesting that they are long-lived, stable proteins. In contrast, after treatment with VX-809, the Hsp70 levels dropped over the 8-h period to approximately half the level observed at time 0. Hsp90 was reduced by ∼25%, and Hsp27 was unchanged. These data suggest that VX-809 reduces the half-life of two key Hsp, most likely by increasing their rates of degradation.
      Figure thumbnail gr9
      Figure 9Hsp27, HSp70, and Hsp90 expression in PN cells treated with VX809 and/or cycloheximide indicates altered degradation. A, Western blot showing expression of Hsp27, Hsp70, and Hsp90, in cycloheximide-treated (25 μm) or control PN cells for indicated time points before being harvested for the assay. B–D, columns represent averages ± standard errors of Hsp27, Hsp70, and Hsp90 expression in cells treated with cycloheximide. E, Western blot showing expression of Hsp27, Hsp70, and Hsp90 in PN cells treated with VX809 (16 h)+cycloheximide or control PN cells. F–H, columns represent averages ± standard errors of Hsp27, Hsp70, and Hsp90 expression in cells treated only with cycloheximide or in control PN cells. Data were analyzed by non-parametric t test. The experiments were repeated 3–5 times. *, p < 0.05, **, p < 0.01.

      VX-809 has a small effect on apoptosis

      Apoptosis is associated with the absence of PC1 in ADPKD (
      • Boletta A.
      • Qian F.
      • Onuchic L.F.
      • Bhunia A.K.
      • Phakdeekitcharoen B.
      • Hanaoka K.
      • Guggino W.
      • Monaco L.
      • Germino G.G.
      Polycystin-1, the gene product of PKD1, induces resistance to apoptosis and spontaneous tubulogenesis in MDCK cells.
      ). To assess whether apoptosis is altered directly by VX-809, we monitored the caspase-3 activity in PN cells. Fig. 10 shows that there was a small but significant reduction in caspase-3 activity in the PN cells after treatment with VX-809 indicative of a small effect on apoptosis.
      Figure thumbnail gr10
      Figure 10VX-809 has a small effect on apoptosis. Apoptosis was measured using the EnzChek caspase-3 assay kit (Invitrogen, E13184). Cells were treated either with VX-809 or with DMSO at the indicated concentrations for 16 h or left untreated (control). Both treated and control cells were then harvested, lysed, and assayed as described in the manufacturer's protocol. Reactions were carried out at room temperature, and fluorescence was measured in a fluorescence microplate reader (SpectraMax M3) with excitation at 496 nm and emission detection at 520 nm. Background fluorescence, determined for a no-enzyme control, was subtracted from each value. Ac-DEVD-CHO was used as an inhibitor control (Inhib-cntrl) to confirm the correlation between signal detection and caspase-3–like protease activity. Camptothecin (Cam) was purchased from Selleckchem (catalog No. S1288). It is known to induce apoptosis (
      • Pommier Y.
      Topoisomerase I inhibitors: Camptothecins and beyond.
      ) and was added as a positive control. Data were analyzed using one-way analysis of variance multiple comparisons. n = 4–5. *, p < 0.05; ****, p < 0.0001.

      VX-809 dramatically reduces the ER stress-related protein GADD153

      Given that VX-809 alters an ensemble of heat shock proteins, we asked whether it also alters proteins associated with ER stress. To address this question, we measured the levels of three ER stress-associated proteins, 78-kDa glucose-regulated protein (GRP78), ER oxidoreductin 1 (Ero1), and DNA damage-inducible protein 3 (GADD153), also known as C/EBP homologous protein (CHOP) (
      • Akerfelt M.
      • Morimoto R.I.
      • Sistonen L.
      Heat shock factors: Integrators of cell stress, development and lifespan.
      ,
      • Krebs J.
      • Agellon L.B.
      • Michalak M.
      Ca2+ homeostasis and endoplasmic reticulum (ER) stress: An integrated view of calcium signaling.
      ). The results depicted in Fig. 11 indicate no changes in GRP78 or Ero1 in response to VX-809. In sharp contrast, there was a dramatic increase in GADD153 when cysts were induced in the mice compared with their normal littermates and an equally dramatic reduction in GADD153 reduction when the cyst containing mice were treated with VX-809. Immunostaining for GADD153 in the mice kidneys also shows a strong reduction when mice are treated with VX-809 (Fig. 12).
      Figure thumbnail gr11
      Figure 11Expression of ER stress markers GRP78 and ErO1 are unchanged, but GADD153 level is altered in Pkd1−/−mice. A and D, representative Western blot images of GRP78, ErO1, and GADD153 in lysates of kidney tissue from no cyst induced (ND), cyst induced with doxycycline (D), or cyst induced in pkd−/− mice treated with VX-809 (D+VX809), 30 mg/kg body weight. B, C, and E, columns represent averages ± S.E. of GRP78, ErO1, and GADD153 expression. Data were analyzed by nonparametric t test. n = 4 for each treated and control group. Black lines between lanes indicate that experiments are from the same gel but loaded in different lanes. **, p < 0.01; ***, p < 0.001. A and B are representative images extracted from the same gel.
      Figure thumbnail gr12
      Figure 12GADD153 expression is reduced in kidneys of Pkd1fl/fl;Pax8rtTA;TetO-cre mice. Immunofluorescence images representing GADD153 expression (green) in DMSO (A)- or VX-809 (B)-treated PN21 Pkd1−/− mouse. C, columns represent the % area of GADD153 immunopositive (green) cells in DMSO- or VX-809–treated PN21 Pkd1−/− mouse kidney sections. Data are expressed as mean ± S.E. of DMSO (vehicle)-treated (n = 4) and VX-809–treated (n = 4) mice. Statistical analysis was performed using a 2-tailed Student t test. Kidneys were fixed in 4% paraformaldehyde as described (
      • Cebotaru L.
      • Liu Q.
      • Yanda M.K.
      • Boinot C.
      • Outeda P.
      • Huso D.L.
      • Watnick T.
      • Guggino W.B.
      • Cebotaru V.
      Inhibition of histone deacetylase 6 activity reduces cyst growth in polycystic kidney disease.
      ). Used here were GADD153 antibody (Sc-7351), goat anti-rabbit (A21429, Alexa Fluor 555, 1:1,000, Life Technologies), and 4′,6-diamidino-2-phenylindole (H-1200, Vector Laboratories, Burlingame, CA). Pictures were acquired with a Zeiss microscope equipped with a ×20 objective. Cells positive for GADD153 and the total number of cells were measured with ImageJ. The results are expressed as percentages. *, p < 0.05.

      Discussion

      CFTR and cyst growth

      Here we report the surprising finding that VX-809, the drug designed to rescue ΔF508-CFTR in CF, also inhibits cyst growth in ADPKD both in vivo and in vitro. The CFTR protein is found in the apical membrane in the cysts of CF, so treatment with VX-809 might be expected to increase cyst size in ADPKD. Thus, the opposite result is particularly remarkable; in similar in vivo mouse studies in which tubacin was injected IP using a protocol identical to that described in Fig. 1, the tubacin treatment reduced the cyst index by ∼15% compared with a 65% reduction following VX-809 treatment. How can this dual function be explained? The effective reduction in cyst index in the mice and in vitro in the PN cells occurred through a series of factors that work together to reduce cyst growth. The inhibition of cyst growth both in vivo in the mouse and in vitro in the PN cells suggests that the CFTR correctors have a direct effect on the cysts rather than having an overall global effect on mouse physiology.

      CFTR correctors

      High-throughput screening has identified CFTR correctors that can rescue the folding and trafficking of CFTR (
      • Van Goor F.
      • Hadida S.
      • Grootenhuis P.D.
      • Burton B.
      • Stack J.H.
      • Straley K.S.
      • Decker C.J.
      • Miller M.
      • McCartney J.
      • Olson E.R.
      • Wine J.J.
      • Frizzell R.A.
      • Ashlock M.
      • Negulescu P.A.
      Correction of the F508del-CFTR protein processing defect in vitro by the investigational drug VX-809.
      ). Correctors also increase the steady-state levels of WT CFTR protein and increase CFTR-generated chloride currents when administered to airway cells (
      • Lopes-Pacheco M.
      • Boinot C.
      • Sabirzhanova I.
      • Morales M.M.
      • Guggino W.B.
      • Cebotaru L.
      Combination of correctors rescue ΔF508-CFTR by reducing its association with Hsp40 and Hsp27.
      ). Relevant to the present study is the mechanism by which the correctors rescue the trafficking mutants of CFTR. Some investigators have suggested that VX-809 may bind directly to CFTR (
      • Wang Y.
      • Loo T.W.
      • Bartlett M.C.
      • Clarke D.M.
      Correctors promote maturation of cystic fibrosis transmembrane conductance regulator (CFTR)-processing mutants by binding to the protein.
      ) and have its major effect on membrane-spanning domain (MSD) 1, thereby enhancing the interactions between NBD1 and MSD1 and improving the interaction between NBD1 and MSD2 (
      • Ren H.Y.
      • Grove D.E.
      • De La Rosa O.
      • Houck S.A.
      • Sopha P.
      • Van Goor F.
      • Hoffman B.J.
      • Cyr D.M.
      VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1.
      ). Others have suggested that misfolded CFTR causes an unfolded protein response in the cell that unleashes a storm of heat shock proteins (
      • Roth D.M.
      • Balch W.E.
      Modeling general proteostasis: Proteome balance in health and disease.
      ) and that modifying this effect in the direction of normal levels promotes CFTR folding (
      • Peters K.W.
      • Okiyoneda T.
      • Balch W.E.
      • Braakman I.
      • Brodsky J.L.
      • Guggino W.B.
      • Penland C.M.
      • Pollard H.B.
      • Sorscher E.J.
      • Skach W.R.
      • Thomas P.J.
      • Lukacs G.L.
      • Frizzell R.A.
      CFTR Folding Consortium: Methods available for studies of CFTR folding and correction.
      ). Along these lines, we have shown previously that a combination of correctors, namely C18 + C4, rescues ΔF508-CFTR by decreasing the binding of CFTR to Hsp27 and Hsp40 (
      • Lopes-Pacheco M.
      • Boinot C.
      • Sabirzhanova I.
      • Morales M.M.
      • Guggino W.B.
      • Cebotaru L.
      Combination of correctors rescue ΔF508-CFTR by reducing its association with Hsp40 and Hsp27.
      ). We took this result further by showing that silencing Hsp27 or Hsp40 has the same effect as applying correctors, suggesting that the correctors alter the interaction between CFTR and the cell's quality control machinery rather than directly affecting CFTR. Given this background information, it does not seem counterintuitive for VX-809 to reduce cyst growth by restoring cellular proteostasis (
      • Balch W.E.
      • Morimoto R.I.
      • Dillin A.
      • Kelly J.W.
      Adapting proteostasis for disease intervention.
      ).

      VX-809 alters Hsp levels

      It is well known that many signal transduction and transcription factor pathways are altered in cystic kidneys as compared with normal kidneys. Recent global gene profiling has indicated a down-regulation of epithelium-associated genes and an up-regulation of genes involved in development, mitogen-activated proliferation, cell cycle progression, epithelial–mesenchymal progression, hypoxia, aging, and the inflammatory response (
      • Song X.
      • Di Giovanni V.
      • He N.
      • Wang K.
      • Ingram A.
      • Rosenblum N.D.
      • Pei Y.
      Systems biology of autosomal dominant polycystic kidney disease (ADPKD): Computational identification of gene expression pathways and integrated regulatory networks.
      ). We propose that some of these altered gene profiles are a direct effect of the loss of polycystin (PC) protein function, and others result from an adaptation of the cyst cells to growth in an abnormal and diseased renal environment. For example, the up-regulation of Hsp in cancer cells contributes to the folding of overexpressed proteins, and these Hsp are targets for anticancer drugs (
      • El-Hariry I.
      • Proia D.
      • Vukovic V.
      Treating cancer with HSP90 inhibitory compounds.
      ).
      Along these lines, Hsp90 is overactive in ADPKD cells, and reducing its function decreases cyst growth (
      • Seeger-Nukpezah T.
      • Proia D.A.
      • Egleston B.L.
      • Nikonova A.S.
      • Kent T.
      • Cai K.Q.
      • Hensley H.H.
      • Ying W.
      • Chimmanamada D.
      • Serebriiskii I.G.
      Inhibiting the HSP90 chaperone slows cyst growth in a mouse model of autosomal dominant polycystic kidney disease.
      ,
      • Proia D.
      • Golemis E.
      • Seeger-Nukpezah T.
      Treating polycystic kidney disease with hsp90 inhibitory compounds.
      ). Seeger-Nukpezah et al. (
      • Seeger-Nukpezah T.
      • Proia D.A.
      • Egleston B.L.
      • Nikonova A.S.
      • Kent T.
      • Cai K.Q.
      • Hensley H.H.
      • Ying W.
      • Chimmanamada D.
      • Serebriiskii I.G.
      Inhibiting the HSP90 chaperone slows cyst growth in a mouse model of autosomal dominant polycystic kidney disease.
      ) have shown that the application of the small-molecule inhibitor STA-2842 over a 10-week period inhibits the initial formation of cysts and also slows the progression of pre-existing cysts, not unlike the situation we observed for VX-809. Based on their studies, the VX-809–induced decrease in Hsp90 levels that we noted here would certainly have a negative influence on cyst growth. We have now shown that VX-809 reduces Hsp90 levels in the kidneys of PC1 conditional knockout mice and in PN cells. Both of these observations suggest that VX-809 has a major effect on the protein levels of Hsp90.
      In contrast to Hsp90, little is known about the role of Hsp70 or Hsp27 in ADPKD cyst growth. Stress-inducible Hsp70 (HspA1A) has an ATP-dependent mechanism and plays an important role in protein folding. Hsp70 is also up-regulated in cancer cells and is associated with a negative outcome (
      • Murphy M.E.
      The HSP70 family and cancer.
      ,
      • Gyrd-Hansen M.
      • Nylandsted J.
      • Jäättelä M.
      Heat shock protein 70 promotes cancer cell viability by safeguarding lysosomal integrity.
      ). It is known that silencing of the gene encoding HSP70-1 (HSPA1A) is cytotoxic to cancerous but not to normal cells. Hsp70 has been identified as a promising cancer drug target, stimulating intense interest in identifying and characterizing HSP70 inhibitors for cancer therapy (
      • Murphy M.E.
      The HSP70 family and cancer.
      ). Our data here show that VX-809 dramatically reduces Hsp70 in the kidneys of the conditional PC1 knockout mice and in PN cells, most likely by increasing its turnover.
      Hsp27 (HspB1), a member of the small heat shock protein family, is thought to guard against protein aggregation during stress (
      • Kim Y.E.
      • Hipp M.S.
      • Bracher A.
      • Hayer-Hartl M.
      • Hartl F.U.
      Molecular chaperone functions in protein folding and proteostasis.
      ). In addition to its role as a chaperone and checkpoint for targeting CFTR for degradation, it also plays a role in p38 MAP kinase-dependent inflammation (
      • Bobo L.D.
      • El Feghaly R.E.
      • Chen Y.S.
      • Dubberke E.R.
      • Han Z.
      • Baker A.H.
      • Li J.
      • Burnham C.A.
      • Haslam D.B.
      MAPK-activated protein kinase 2 contributes to Clostridium difficile-associated inflammation.
      ). Hsp27 is phosphorylated by p38 MAP kinase, which promotes dimerization of the protein and inhibits its multimerization (
      • Mymrikov E.V.
      • Seit-Nebi A.S.
      • Gusev N.B.
      Large potentials of small heat shock proteins.
      ). In many aggressive cancers, Hsp27 promotes drug resistance, metastasis, and poor patient outcomes. The strong protective effect of Hsp27 is mainly a function of its blockade of apoptosis (
      • Wang X.
      • Chen M.
      • Zhou J.
      • Zhang X.
      HSP27, 70 and 90, anti-apoptotic proteins, in clinical cancer therapy.
      ). Our data show that VX-809 indeed induces a profound reduction in Hsp27 in both the kidneys of the conditional PC1 knockout mice and in PN cells. Clearly, the down-regulation by VX-809 of key heat shock proteins implicated in the growth of cancer cells (Hsp27, Hsp70, and Hsp90) (
      • Wang X.
      • Chen M.
      • Zhou J.
      • Zhang X.
      HSP27, 70 and 90, anti-apoptotic proteins, in clinical cancer therapy.
      ) is a key factor in reducing cyst growth in ADPKD.

      VX-809 dramatically reduces the protein expression of GADD153

      GADD153 is a transcription factor that is well known to be up-regulated in response to genotoxic challenges such as UV radiation. However, it is also induced by nutrient depletion, including glucose deprivation and amino acid starvation (
      • Oyadomari S.
      • Mori M.
      Roles of CHOP/GADD153 in endoplasmic reticulum stress.
      ). We show here that VX-809 dramatically down-regulated GADD153 in the kidneys of PC1-conditional knockout mice. In contrast, two other indicators of ER stress, Ero1 and GRP78, were unchanged in response to VX-809. Ero1 is tightly linked to protein overload in the ER (
      • Zeeshan H.M.
      • Lee G.H.
      • Kim H.R.
      • Chae H.J.
      Endoplasmic reticulum stress and associated ROS.
      ). Likewise, GRP78 is a Ca2+-dependent chaperone associated with the unfolded protein response (
      • Xu C.
      • Bailly-Maitre B.
      • Reed J.C.
      Endoplasmic reticulum stress: Cell life and death decisions.
      ). The observation that VX-809 does not affect these two stress-related proteins suggests that it not involved in regulating ER stress in our mouse model of ADPKD. Further evidence of this hypothesis is the observation that VX-809 had a small effect on apoptosis, another indicator of ER stress.
      GADD153 is often cited as a key regulator of apoptosis (
      • Oyadomari S.
      • Mori M.
      Roles of CHOP/GADD153 in endoplasmic reticulum stress.
      ). One question is why apoptosis was hardly changed in response to VX-809 despite the large decrease in GADD153? The reason is that GADD153 is associated with both cell death and cell survival. This dichotomy of function has been demonstrated nicely in myelinating cells during development in mice overexpressing GADD153. In this model, overexpression of GADD153 did not promote cell death but instead was suggested to be prosurvival through a dynamic regulatory cycle involving the unfold protein response. The higher expression levels of GADD153 in the cystic mice may be another indicator of the prosurvival pathways that allow cysts to grow in the kidney at the expense of healthy tissue (
      • Gurlo T.
      • Rivera J.F.
      • Butler A.E.
      • Cory M.
      • Hoang J.
      • Costes S.
      • Butler P.C.
      CHOP contributes to, but is not the only mediator of, IAPP induced β-cell apoptosis.
      ,
      • Southwood C.M.
      • Fykkolodziej B.
      • Maheras K.J.
      • Garshott D.M.
      • Estill M.
      • Fribley A.M.
      • Gow A.
      Overexpression of CHOP in myelinating cells does not confer a significant phenotype under normal or metabolic stress conditions.
      ). Thus, its down-regulation by VX-809 is likely the result of the reduction in cyst burden.

      VX-809 reduces Ca2+ movement out of the ER

      Misregulation of Ca2+ is associated with cyst formation in ADPKD (
      • Torres V.E.
      • Rossetti S.
      • Harris P.C.
      Update on autosomal dominant polycystic kidney disease.
      ), with some investigators reporting that disruption of Ca2+ signaling is the primary event that supports increased cyst growth (
      • Mangolini A.
      • de Stephanis L.
      • Aguiari G.
      Role of calcium in polycystic kidney disease: From signaling to pathology.
      ). Several studies have shown that a reduction in the function of either PC1 or PC2 leads to dysregulation of Ca2+ signaling (see Ref.
      • Torres V.E.
      Vasopressin receptor antagonists, heart failure, and polycystic kidney disease.
      for a review). We show here that VX-809 causes a small reduction in resting Ca2+, but it does not increase intracellular Ca2+ in response to ATP application in PN cells. ATP stimulates purinergic receptors that are themselves Ca2+ channels (
      • Menzies R.I.
      • Tam F.W.
      • Unwin R.J.
      • Bailey M.A.
      Purinergic signaling in kidney disease.
      ), and thus in our study Ca2+ movements via these receptors were not affected by VX-809. In sharp contrast, VX-809 has a profound effect on the thapsigargin-induced release of Ca2+ from the ER. Thapsigargin inhibits the SERCA pump; therefore, the magnitude of the release of Ca2+ from the ER depends on the Ca2+ gradient between the lumen of the ER and the cytoplasm and on the permeability of the ER membrane for Ca2+. Misregulation of Ca2+ with an up-regulation of store-operated Ca2+ entry (SOCE) (
      • Umemura M.
      • Baljinnyam E.
      • Feske S.
      • De Lorenzo M.S.
      • Xie L.H.
      • Feng X.
      • Oda K.
      • Makino A.
      • Fujita T.
      • Yokoyama U.
      • Iwatsubo M.
      • Chen S.
      • Goydos J.S.
      • Ishikawa Y.
      • Iwatsubo K.
      Store-operated Ca2+ entry (SOCE) regulates melanoma proliferation and cell migration.
      ,
      • Prevarskaya N.
      • Skryma R.
      • Shuba Y.
      Calcium in tumour metastasis: New roles for known actors.
      ) is also a risk factor for a poor outcome in cancer. Thus, we propose that VX-809's inhibition of ER Ca2+ release robs ADPKD cysts of the ability to respond to growth stimuli.

      Reduced ER Ca2+ movement is associated with reduced cAMP levels

      Our data show that although VX-809 causes only a small reduction in resting Ca2+, the dramatic reduction in ER Ca2+ release should affect Ca2+ signaling in the cells. Here, we focused on two adenylyl cyclases in renal cells that respond to Ca2+, AC3 and AC6 (
      • Menzies R.I.
      • Tam F.W.
      • Unwin R.J.
      • Bailey M.A.
      Purinergic signaling in kidney disease.
      ,
      • Persson A.E.
      • Carlström M.
      Renal purinergic signalling in health and disease.
      ). Western blotting confirmed that the PN cells contained both AC3 and AC6. Interestingly, VX-809 reduced the steady-state level of AC3 without producing any change in the AC6 level. Measurement of cAMP showed that VX-809 reduced cAMP levels by ∼50%. Thus, the reduction in cAMP level resulting from VX-809 application is most likely the result of its reduction of AC3 levels. To determine whether a reduction in cAMP levels is the means by which VX-809 reduces cyst size, we then treated cells with forskolin, which caused a massive 12.5-fold increase in intracellular cAMP levels that drove an ∼2.5-fold increase in cyst growth. Clearly, then, cyst growth in PN cells depended on cAMP levels. Interestingly, VX-809 caused an ∼20% decrease in forskolin-dependent cAMP production; however, cAMP levels after VX-809 treatment in the presence of forskolin were still 10 times higher than control levels. The fact that VX-809 still dramatically reduced cyst growth in the presence of forskolin strongly suggests that VX-809 inhibits the ability of cAMP to stimulate cyst growth.

      Conclusion

      We have shown here that VX-809 has the surprising ability to restore renal cells in ADPKD to a noncyst-forming phenotype, including negating the ability of cAMP to sustain and stimulate cyst growth. A key result is that VX-809 reduced Hsp27, Hsp70, and Hsp90, which are known to be up-regulated in cancer cells to protect the proteins in abnormally dividing cells (
      • Hartl F.U.
      Molecular chaperones in cellular protein folding.
      ). Although the CFTR is a major driver of cyst growth, it is apparently not a major player in the normal kidney, because patients with CF do not have kidney disease. VX-809, in removing the cyst's ability to grow in the presence of high levels of cAMP and inhibiting Ca2+ release by the ER, turns off or reduces two major signal transduction pathways that fuel cyst growth (see Fig. 13). Given that administration of VX-809 has already been shown to be safe, we conclude that it could offer a new approach for treating patients with ADPKD.
      Figure thumbnail gr13
      Figure 13Schematic representation of proposed mechanism of action of VX-809 on cyst growth. A, interestingly, gene profiling of human cysts shows an increase in HSF1 expression as compared with normal kidneys (
      • Dai C.
      • Sampson S.B.
      HSF1: Guardian of proteostasis in cancer.
      ), HSF1 activation leads to the transcriptional up-regulation of several HSP, which we speculate most likely drives the increase in heat shock factors noted here and in other studies (
      • Seeger-Nukpezah T.
      • Proia D.A.
      • Egleston B.L.
      • Nikonova A.S.
      • Kent T.
      • Cai K.Q.
      • Hensley H.H.
      • Ying W.
      • Chimmanamada D.
      • Serebriiskii I.G.
      Inhibiting the HSP90 chaperone slows cyst growth in a mouse model of autosomal dominant polycystic kidney disease.
      ). We have shown (
      • Yanda M.K.
      • Liu Q.
      • Cebotaru V.
      • Guggino W.B.
      • Cebotaru L.
      Histone deacetylase 6 Inhibition reduces cysts by decreasing via cAMP and Ca2+ in knockout mouse models of polycystic kidney disease.
      ) previously that thapsigargin-induced Ca2+ release from the ER is enhanced in PN cells versus PH cells, leading to an increase in cAMP via adenylyl cyclase 3. Thapsigargin inhibits the SERCA pump (
      • Thastrup O.
      • Dawson A.P.
      • Scharff O.
      • Foder B.
      • Cullen P.J.
      • Drøbak B.K.
      • Bjerrum P.J.
      • Christensen S.B.
      • Hanley M.R.
      Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage.
      ) causing Ca2+ to leak out of the ER via the IP3R (inositol triphosphate receptor). The combination of elevated cAMP, enhanced release of Ca2+ from the ER, and increased heat shock factor expression fuels cyst growth. B, one of the factors that up-regulates HSF1 is aberrant Ca2+ regulation (
      • Krebs J.
      • Agellon L.B.
      • Michalak M.
      Ca2+ homeostasis and endoplasmic reticulum (ER) stress: An integrated view of calcium signaling.
      ). Thus, VX-809 reduces Hsp27 expression either directly (
      • Lopes-Pacheco M.
      • Boinot C.
      • Sabirzhanova I.
      • Morales M.M.
      • Guggino W.B.
      • Cebotaru L.
      Combination of correctors rescue ΔF508-CFTR by reducing its association with Hsp40 and Hsp27.
      ) or via a reduction in thapsigargin-induced ER Ca2+ release. We showed previously that inhibiting thapsigargin-induced Ca2+ release is associated with reduced cAMP via calmodulin regulation of AC3. VX-809, by inhibiting thapsigargin-induced ER Ca2+ release, thus reducing heat shock proteins and cAMP, robs the cyst of several components that fuel cyst growth.

      Methods

      Cell culture and reagents

      PN and PH cells, cultured as described previously (
      • Joly D.
      • Ishibe S.
      • Nickel C.
      • Yu Z.
      • Somlo S.
      • Cantley L.G.
      The polycystin 1-C-terminal fragment stimulates ERK-dependent spreading of renal epithelial cells.
      ,
      • Wei F.
      • Karihaloo A.
      • Yu Z.
      • Marlier A.
      • Seth P.
      • Shibazaki S.
      • Wang T.
      • Sukhatme V.P.
      • Somlo S.
      • Cantley L.G.
      Neutrophil gelatinase-associated lipocalin suppresses cyst growth by Pkd1 null cells in vitro and in vivo.
      ), were obtained from the Mouse Genetics and Cell Line Core of the Yale O'Brian Center. Forskolin (catalog No. 11018) was purchased from Sigma (SC23950); VX-809 (S1565) was purchased from Selleck Chemicals, Houston, TX. Ezrin (SC58758), adenylate cyclase 3 (SC588), PC2 (SC28331), Hsp27 (SC13132), Hsp70 (SC66048), anti-GADD153 antibody (SC7351), anti-ErO1 antibody (SC365526), and β-actin (SC47778) were purchased from Santa Cruz Biotechnology. Hsp90 (ADI-SPA-830F) was purchased from Enzo Life Sciences. AC6 (GTX47798) was purchased from GeneTex, Irvine, CA. Anti-GRP78 BiP antibody was purchased from Abcam (catalog No. ab21685).

      Mouse strain and treatment

      All animal use complied with the guiding principles of The Johns Hopkins University Institutional Animal Care and Use Committee, and the protocols for this work were approved by that Committee. Pkd1fl/fl;Pax8rtTA;TetO-cre mice on a C57BL/6 background (
      • Ma M.
      • Tian X.
      • Igarashi P.
      • Pazour G.J.
      • Somlo S.
      Loss of cilia suppresses cyst growth in genetic models of autosomal dominant polycystic kidney disease.
      ) were provided by the Baltimore Polycystic Kidney Disease Center. Mice of both sexes were used in this study. Mice were generally fed a diet containing low fiber (5%), protein (20%), and fat (5–10%). The pelleted feed was supplied as regular, breeder-certified, irradiated, or autoclavable. Mice were supplied food and water free choice. Water was supplied using automatic waterers. Mouse rooms were maintained at 30–70% relative humidity and a temperature of 18–26 °C with at least 10 room air changes/h. The mice were housed in standard shoebox cages without filter tops. Cages as described above were usually changed once or twice a week. Mice were provided with bedding in the shoebox cages. Bedding could be paper, wood shavings, wood chips or corncob.
      To induce cyst formation, mice were injected IP with doxycycline (Sigma, D9891) (4 μg of doxycycline/g body weight) on PND11, PND12, and PND13 to produce very rapid and aggressive cyst growth (
      • Traykova-Brauch M.
      • Schönig K.
      • Greiner O.
      • Miloud T.
      • Jauch A.
      • Bode M.
      • Felsher D.W.
      • Glick A.B.
      • Kwiatkowski D.J.
      • Bujard H.
      • Horst J.
      • von Knebel Doeberitz M.
      • Niggli F.K.
      • Kriz W.
      • Gröne Koesters R.
      An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice.
      ,
      • Piontek K.
      • Menezes L.F.
      • Garcia-Gonzalez M.A.
      • Huso D.L.
      • Germino G.G.
      A critical developmental switch defines the kinetics of kidney cyst formation after loss of Pkd1.
      ). On PND21, the mice were euthanized. Three groups of animals were used: 1) animals injected with doxycycline and with VX-809 (30 mg/kg); 2) animals injected with doxycycline and DMSO; 3) control animals not injected with doxycycline. Animal experiments were repeated 4–5 times to establish statistical significance.

      In vitro cystogenesis

      To induce differentiation, cells were kept at 37 °C for at least 6 days without γ-interferon. After 1 week, the cells were used for 3D culture or other experiments. Growth factor–reduced Matrigel (Corning, catalog No. 354230) was used, and cell dilutions were prepared so that there were 6000 cells in 25 μl of medium; 25 μl of cell preparation was mixed with 50 μl of Matrigel (see Ref.
      • Yanda M.K.
      • Liu Q.
      • Cebotaru V.
      • Guggino W.B.
      • Cebotaru L.
      Histone deacetylase 6 Inhibition reduces cysts by decreasing via cAMP and Ca2+ in knockout mouse models of polycystic kidney disease.
      ). Pictures were taken with a Zeiss Axio microscope. Cystic areas were analyzed with ImageJ (provided by National Institutes of Health).

      cAMP assay

      Confluent cells were treated with VX-809 (10 μm) or DMSO for 16 h before being harvested for assay. cAMP levels were measured with a direct cAMP enzyme immunoassay kit (Sigma, CA200) according to the manufacturer's protocol. The results are expressed as pmol/ml. Statistical analysis was performed using a two-tailed Student's t test.

      Fura-2 Ca2+ imaging assay

      On day 5 of cell culture, the cells were loaded with the cell-permeant acetoxymethyl (AM) ester of the calcium indicator Fura-2 AM at 37 °C for 90 min. Measurements were made on a Zeiss inverted microscope equipped with a Sutter Lambda 10-2 controller and filter wheel assembly. For ATP stimulation experiments, the cells were exposed to 100 μm ATP diluted in the imaging buffer. A Zeiss FluorArc mercury lamp was used to excite the cells at 340 and 380 nm, and the emission response was measured at 510 nm. Cell fluorescence was measured in response to excitation for 1000 ms at 340 nm and 200 ms at 380 nm once every 4 s. Image acquisition, image analysis, and filter wheel control were performed with IPLab software (see Ref.
      • Yanda M.K.
      • Liu Q.
      • Cebotaru V.
      • Guggino W.B.
      • Cebotaru L.
      Histone deacetylase 6 Inhibition reduces cysts by decreasing via cAMP and Ca2+ in knockout mouse models of polycystic kidney disease.
      ).

      Statistics

      All experiments were replicated until statistically significant at p < 0.05. All experiments were unblinded.

      Author contributions

      M. Y. and Q. L. data curation; M. Y., Q. L., and L. C. formal analysis; M. Y., Q. L., and L. C. investigation; M. Y., Q. L., and L. C. methodology; L. C. conceptualization; L. C. supervision; L. C. funding acquisition; L. C. validation; L. C. writing-original draft; L. C. project administration; L. C. writing-review and editing.

      Acknowledgments

      We thank William B. Guggino for reading the manuscript and Deborah McClellan for editorial assistance. We also extend special thanks to Cristina Cebotaru for work on the experiments.

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