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. Author manuscript; available in PMC: 2017 Jul 1.
Published in final edited form as: Kidney Int. 2016 Mar 25;90(1):90–99. doi: 10.1016/j.kint.2016.01.026

INHIBITION OF HISTONE DEACETYLASE 6 ACTIVITY REDUCES CYST GROWTH IN POLYCYSTIC KIDNEY DISEASE

Liudmila Cebotaru 1, Qiangni Liu 2, Murali Yanda 3, Clement Boinot 3, Patricia Outeda 2, David L Huso 4, Terry Watnick 2, William B Guggino 3, Valeriu Cebotaru 2,§
PMCID: PMC4912414  NIHMSID: NIHMS791931  PMID: 27165822

Abstract

Abnormal proliferation of cyst-lining epithelium and increased intra-cystic fluid secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) are thought to contribute to cyst growth in autosomal dominant polycystic kidney disease (ADPKD). Histone deacetylase 6 (HDAC6) expression and activity are increased in certain cancers, and neurodegenerative diseases, and in Pkd1-mutant renal epithelial cells. Inhibition of HDAC6 activity with specific inhibitors slows cancer growth. Here we studied the effect of tubacin, a specific HDAC6 inhibitor, on cyst growth in polycystic kidney disease. Treatment with tubacin prevented cyst formation in MDCK cells, an in vitro model of cystogenesis. Cyclic AMP stimulates cell proliferation and activates intra-cystic CFTR-mediated chloride secretion in ADPKD. Treatment with tubacin down-regulated cyclic AMP levels, inhibited cell proliferation, and inhibited cyclic AMP-activated CFTR chloride currents in MDCK cells. We also found that tubacin reduced cyst growth by inhibiting proliferation of cyst-lining epithelial cells, down-regulated cyclic AMP levels, and improved renal function in a Pkd1-conditional mouse model of ADPKD. Thus, HDAC6 could play a role in cyst formation and could serve as a potential therapeutic target in ADPKD.

Keywords: Autosomal dominant polycystic kidney disease, renal cyst growth, histone deacetylase 6 inhibitor, cyclic AMP

INTRODUCTION

Autosomal dominant polycystic kidney disease (ADPKD), a hereditary disorder affecting 1:1000 to 1:500 people, is characterized by the formation of fluid-filled cysts that arise from renal tubules.1 These cysts progressively enlarge and displace the renal parenchyma, leading to end-stage renal disease in nearly half of all ADPKD patients by the fifth decade of life. ADPKD is a heterogeneous disease that results from mutations in either the PKD1 or PKD2 gene.2, 3 Mutations in PKD1 or PKD2 lead to dysregulation of multiple signaling pathways that may be responsible for cyst growth.4 Although it has been shown that targeting of specific pathways can slow cyst growth in PKD animal models,5, 6 currently there is no cure for ADPKD.

Abnormal proliferation of cyst-lining epithelium and increased intra-cystic fluid secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) are thought to contribute to cyst growth in ADPKD.7, 8 CFTR, a cyclic AMP (cAMP) activated chloride channel, is expressed in the apical epithelia in ADPKD cystic tissue and is functional as indicated by cAMP-mediated activation of chloride currents.8 Increased levels of cAMP have been reported in animal models for PKD.9-11 Besides activating CFTR, cAMP also stimulates cell proliferation in ADPKD.12

HDACs are a family of small molecules that remove acetyl groups from histones and non-histone proteins, leading to modulation of gene expression.13, 14 HDACs play an essential role in many important cellular processes.15 HDAC6 is predominantly localized to the cytoplasm and has unique substrate specificity for non-histone proteins.13 HDAC6 regulates a number of important biological processes, including transcription, cell migration and proliferation, cell signaling, the immune response, and protein degradation. Interestingly, mice lacking HDAC6 are viable, fertile, and have no gross morphological abnormalities.16 There are an increasing number of reports indicating that HDAC6 expression and activity are dysregulated in a wide range of disease states.17-20 Hence, HDAC6 has been designated as a potential therapeutic target, and a number of specific HDAC6 inhibitors (HDAC6i) have been developed. Preclinical data suggest that small molecule-specific HDAC6 inhibitors may play a role in treatment of certain cancers, neurodegenerative diseases, and autoimmune disorders.17 Hideshima et al. have shown that tubacin, a specific HDAC6i, inhibits multiple myeloma cell growth without cytotoxicity to peripheral blood mononuclear cells,21 suggesting that HDAC6i could be used in non-oncologic disorders.

There is evidence to suggest that HDAC6 may play a role in cyst formation in ADPKD. For example, HDAC6 expression and activity are increased in Pkd1-mutant renal epithelial cells.22 HDAC6 is an α-tubulin deacetylase. HDAC6-mediated tubulin deacetylation leads to increased cell motility resulting from microtubule instability.23 Inhibition of HDAC6 activity with specific inhibitors increases microtubule acetylation and decreases microtubule growth.23 Knockdown of HDAC6 inhibits anchorage-independent proliferation in cancer cell lines, whereas overexpression of HDAC6 promotes anchorage-independent proliferation.24

Polycystin-1 (PC1) and polycystin-2 (PC2) are gene products of PKD1 and PKD2, respectively.2, 3, 25 Either malfunction or dysregulation of the expression of PC1 or PC2 leads to cyst formation in ADPKD.26-28 We have shown previously that PC1 negatively regulates PC2 expression via the aggresome/autophagsome pathway.29 HDAC6 transports misfolded proteins towards the aggresome for degradation,30 and inhibition of HDAC6 activity with tubacin prevents PC2 degradation when PC1 is overexpressed.29 These observations and published data showing increased HDAC6 expression in Pkd1-mutant renal epithelial cells22 have led us to explore HDAC6’s role in cyst formation in ADPKD. We hypothesized that targeting HDAC6 activity with a specific HDAC6 inhibitor (HDAC6i) could alter cyst growth in ADPKD. Thus, in this report, we investigated the role of tubacin, a specific HDAC6i, on cyst growth in cell culture and in an animal model of PKD.

RESULTS

The HDAC6i tubacin prevents cyst formation in vitro

To determine whether HDAC6 does indeed play a role in cyst formation, we grew canine renal epithelial cells (MDCK.2 cells) in three-dimensional (3D) matrigel-collagen I gels that were treated either with tubacin (HDAC6i) or DMSO (vehicle). MDCK.2 cells spontaneously form cysts in 3D gels. As a first step, we confirmed that the MDCK.2 cells spontaneously made cysts in 3D culture (Fig. 1A [parts a and f within]). We then treated the cells with tubacin on days 0, 2, 4, 6, 8, 10, 12 and 14, and found that this treatment did indeed prevent cyst formation (Fig. 1A [parts b and g within] and 1B). In order to define this effect further, we treated the cells only once with tubacin, on day 0. Importantly, this single treatment alone slowed the cyst growth (Fig. 1A [parts c and h within] and 1B). Finally, we let the cysts grow for 10 days and treated them with tubacin on days 10, 12, and 14, examining them on day 16 (Fig. 1A [parts d and i within] and 1B). Treatment with HDAC6i not only arrested cyst growth but also shrank the cysts (Fig. 1A [parts d and i within] and 1B vs. [parts e and j within] and 1B). The effect of tubacin is dose-dependent (Supplemental Figure 1). Furthermore, a second specific HDAC6 inhibitor, tubastatin-A (TSA), has a similar effect in inhibiting cyst formation in MDCK.2 cells in dose a dose dependent manner (Supplemental Figure 2). Taken together these data show convincingly that specific inhibitors of HDAC6 activity reduce cyst growth in vitro.

Figure 1. (A) In vitro cystogenesis.

Figure 1

Figure 1

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MDCK.2 cells grown in Matrigel/collagen I gel. On days 0, 2, 4, 6, 8, 10 and 12 tubacin (10 μM) was added to (b) and (g), and DMSO (vehicle) to (a) and (f). Tubacin (10 μM) was added once on day 0 to (c) and (h). Cells in (d) and (i) were treated with tubacin (10 μM) on days 10, 12 and 14. Cells in (e) and (j) were treated with DMSO on days 0, 2, 4, 6 and 8. Photos above were taken on day 16, except for (e) and (j) (taken on day 10). Scale bar: 150 μm (top panel) and 50 μm (bottom panel). (B) Summary data for in vitro cystogenesis experiment. Columns represent averages ± standard errors (n=3). Average cyst from DMSO group was considered 100% and the rest of the cysts were compared to this cyst. ***P<0.001. Data were analyzed by one-way ANOVA followed by Turkey’s multiple-comparison test. Abbreviations: D - DMSO, T - tubacin, d - day, d0 - cells treated once on day 0.

HDAC6 inhibitors increased tubulin acetylation in vitro

Tubulin deacetylation is one of the main downstream targets of HDAC6. In order to verify this in our experiments, we evaluated the acetylation of tubulin before and after HDAC6 inhibition. The data show that inhibition of HDAC6 activity with tubacin correlates with increased tubulin acetylation in postnatal Pkd1 heterozygous PH2 (PH2) and Pkd1 homozygous (PN18) renal epithelial cells (Fig. 2A,B,C,D). Similar results were obtained in MDCK.2 cells, where tubacin treatment dramatically increases tubulin acetylation in a dose dependent manner (Supplemental Figure 3). Finally, inhibition of HDAC6 activity with tubastatin-A, second specific HDAC6i, also increases tubulin acetylation in a dose dependent manner in MDCK.2 cells (Supplemental Figure 4). Clearly, in our experimental system tubacin is having its predicted effect to increase the acetylation of tubulin.

Figure 2. Acetylated tubulin expression in (A) PH2 and (C) PN18 cells.

Figure 2

Figure 2

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Western blot showing expression of acetylated tubulin in postnatal Pkd1 heterozygous PH2 (PH2) and Pkd1 homozygous (PN18) cells. PH2 and PN18 cells were treated with DMSO or tubacin (10μM) for 17 h. Summary data for acetylated tubulin expression in (B) PH2 and (D) PN18 cells. Columns represent averages ± standard errors of acetylated tubulin expression. Acetylated tubulin bands were normalized to β-actin. ***P<0.001. Statistical analysis was performed using a two-tailed Student’s t test. Experiments were repeated 3 times and all samples were run on the same gel.

Tubacin inhibits proliferation in vitro

As mentioned above, HDAC6 regulates cell proliferation in cancer.24 Therefore, we asked whether HDAC6 would affect the proliferation of renal epithelial cells. We found that administration of HDAC6i, tubacin, did indeed significantly inhibit the proliferation of PH2 and PN18 cells when compared to DMSO-treated cells (Fig. 3). Inhibition of proliferation in PH2 and PN18 cells was dose dependent (Fig. 3). Next we showed that tubacin significantly inhibited the proliferation of MDCK.2 cells (Supplemental Figure 5). Furthermore, we showed that tubastatin-A, second HDAC6i, significantly inhibited the proliferation of PH and PN cells when compared to DMSO-treated cells (Supplemental Figure 6).

Figure 3. BrdU cell proliferation assay in (A) PH2 and (B) PN18 cells.

Figure 3

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PH2 and PN18 cells were treated with DMSO or decreasing doses of tubacin (1, 5 or 10μM) for 17 h. Columns represent averages ± standard errors of the percentage of BrdU incorporation into PH2 or PN18 cells. #P>0.05,**P<0.01,***P<0.001. Data were analyzed by one-way ANOVA followed by Turkey’s multiple-comparison test. Abreviations: D – DMSO, T - tubacin. This experiment was repeated 3 times.

Tubacin downregulates cAMP levels in MDCK.2 cells

It has been shown that cAMP contributes to cyst growth in ADPKD cells by stimulating cell proliferation and CFTR-driven fluid secretion.7, 8 We therefore asked whether tubacin would affect the cAMP levels in MDCK.2 cells. To our surprise, the administration of tubacin significantly decreased the cAMP levels in these cells (Fig. 4).

Figure 4. Levels of cAMP in MDCK.2 cells.

Figure 4

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MDCK.2 cells were plated in a 96-well plate for 24 h. Confluent cells were treated with tubacin (10 μM) or DMSO for 17 h before cAMP assay. cAMP is expressed in pmole/μg of protein (n=3). Columns represent averages ± standard errors. *P<0.05. Statistical analysis was performed using a two-tailed Student’s t test.

Tubacin inhibits CFTR-dependent short-circuit currents in MDCK.2 cells

Next, we determined whether HDAC6 was able to regulate the chloride channel activity of the CFTR. For this purpose, we grew MDCK.2 cells for 7 days on Snapwell filters and measured CFTR-dependent short-circuit currents (ISC), treating the cells with tubacin or DMSO for 17 h before ISC measurement. Given that CFTR-mediated chloride secretion is positively regulated by cAMP,31, 32 we added the adenylate cyclase activator forskolin (10 μM) and potentiated the CFTR-mediated chloride secretion with the tyrosine kinase inhibitor genistein (30 μM). Thiazolidonone, a specific CFTR inhibitor (CFTRinh172),33, 34 was added at 10 μM to inhibit the ISC, indicating that the measured current was indeed generated by the CFTR. Pretreatment with tubacin significantly reduced the CFTR-dependent ISC in MDCK.2 cells when compared to DMSO-treated cells (Fig. 5A,B). Tubacin had no effect on transepithelial resistance (Fig. 5C) indicating that it is not cytotoxic.

Figure 5. Short-circuit current measurements in MDCK.2 cells.

Figure 5

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(A) Original short-circuit current recording after incubation with DMSO or tubacin (10 μM) for 17 h. Data are expressed as the CFTRinh172-sensitive short-circuit current (ΔISC), calculated by subtracting the ISC after CFTRinh172 treatment from the peak forskolin-genistein-stimulated ISC and normalized using the DMSO condition. (B) Corresponding normalized Δ ISC responses. Columns represent the means of the normalized ΔISC response ± standard error in tubacin-treated (n=11) and DMSO-treated (n=5) cells. (C) Transepithelial resistance. Columns represent averages ± standard errors in tubacin-treated (n=11) and DMSO-treated (n=5) cells. ***P<0.001, NS, no significant difference (for all graphs). Statistical analysis was performed using a two-tailed Student’s t test.

Tubacin slows renal cyst growth in Pkd1fl/fl;Pax8rtTA;TetO-cre mice

Based on the evidence presented above that the HDAC6i tubacin prevents cyst formation and inhibits cell proliferation in MDCK.2 cells, an in vitro model of cystogenesis, we asked whether inhibiting HDAC6 activity would also delay cyst growth in vivo. For this purpose we used the Pkd1fl/fl;Pax8rtTA;TetO-cre mouse model, which allowed us to inactivate Pkd1 in renal tubules with doxycycline at specific time points. Pkd1fl/fl;Pax8rtTA;TetO-cre mice were injected intraperitoneally (IP) with doxycycline on post-natal days (PN) 11, 12, and 13. This treatment produces a very aggressive murine model of PKD that features the development of giant polycystic kidneys at 3 weeks of age. Pkd1fl/fl;Pax8rtTA;TetO-cre mice were injected daily with tubacin (5 mg/kg) or DMSO from PN10 to PN20, and kidneys were harvested on PN21. We found that the administration of tubacin significantly reduced the kidney growth in Pkd1fl/fl;Pax8rtTA;TetO-cre mice, as assessed by the kidney-to-body weight ratio (Fig. 6A). The average kidney-to-body weight ratio was lowered by 52% in the tubacin-treated group when compared to controls. In addition, the administration of tubacin significantly decreased the renal cyst burden in the Pkd1fl/fl;Pax8rtTA;TetO-cre mice when compared to the DMSO-treated mice, as indicated by the cystic index (Fig. 6B,C). In this mouse model, the majority of the surviving offspring were males, and therefore we could not separate the animals into male and female groups for power analysis. The cause of this disparity in the male/female ratio is unknown.

Figure 6. Treatment with the HDAC6i tubacin slows cyst growth in Pkd1fl/fl;Pax8rtTA;TetO-cre mice.

Figure 6

Figure 6

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(A) Kidney-to-body weight ratio (%). Columns represent averages ± standard errors of DMSO (vehicle)-treated (n=8) and tubacin-treated (n=8) mice. (B) Kidney histology. Representative images of PN21 kidney sections from DMSO- and tubacin-treated Pkd1fl/fl;Pax8rtTA;TetO-cre mice. (C) Cystic index. The total kidney area and total cystic area were measured with ImageJ (provided by NIH). Cystic index = 100 X (total cystic area/total kidney area) and is expressed as a percentage. Columns represent averages ± standard errors of DMSO (vehicle)-treated (n=8) and tubacin-treated (n=8) mice. *P<0.05; ***P<0.001 (for all graphs). Statistical analysis was performed using a paired two-tailed Student’s t test.

Tubacin improves renal function in Pkd1fl/fl;Pax8rtTA;TetO-cre mice

Administration of the HDAC6i improved the renal function in Pkd1fl/fl;Pax8rtTA;TetO-cre mice, as indicated by lower serum urea nitrogen (SUN) and serum creatinine values in the tubacin-treated group than in the DMSO-treated group (Fig. 7A,B).

Figure 7. Treatment with tubacin improves renal function in Pkd1fl/fl;Pax8rtTA;TetO-cre mice.

Figure 7

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(A) SUN measurement. Columns represent SUN averages ± standard errors of DMSO (vehicle)-treated (n=8) and tubacin-treated (n=8) mice (PN21). (B) SCr measurement. Columns represent SCr averages ± standard errors of DMSO (vehicle)-treated (n=7) and tubacin-treated (n=8) mice (PN21). *P<0.05; **P<0.01. Abreviations: SUN – serum urea nitrogen, SCr – serum creatinine. Statistical analysis was performed using a two-tailed Student’s t test.

Tubacin inhibits proliferation in the cyst-lining epithelia of the kidneys of Pkd1fl/fl;Pax8rtTA;TetO-cre mice

Next, we asked whether tubacin would inhibit the epithelial proliferation in vivo as well as in vitro. Indeed, we found that tubacin significantly decreased the proliferation of the cyst-lining epithelia in the kidneys of Pkd1fl/fl;Pax8rtTA;TetO-cre mice, as indicated by Ki67 staining, when compared to DMSO-treated mice (Fig. 8A,B).

Figure 8. Treatment with tubacin inhibits proliferation in the cyst-lining epithelia in kidneys of Pkd1fl/fl;Pax8rtTA;TetO-cre mice.

Figure 8

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(A) Ki67 staining. Representative images of Ki67 staining of PN21 kidney sections from DMSO- (a,c) and tubacin-treated (b,d) mice. Arrows indicate Ki67-positive cells. (B) Summary data for Ki67-positive cells. Columns represent averages ± standard errors of DMSO (vehicle)-treated (n=6) and tubacin-treated (n=6) mice. **P<0.01. Statistical analysis was performed using a two-tailed Student’s t test.

Tubacin downregulates cAMP levels in the kidneys of Pkd1fl/fl;Pax8rtTA;TetO-cre mice

In some animal models for PKD, the levels of cAMP are increased.9-11 Having shown that tubacin had an anti-proliferative effect on the cyst-lining epithelia and decreased the CFTR-dependent ISC in MDCK.2 cells, we asked whether administration of tubacin would affect the cAMP levels in the kidneys of Pkd1fl/fl;Pax8rtTA;TetO-cre mice. We confirmed that (as in our in vitro experiments) administration of tubacin significantly decreased the cAMP levels in Pkd1fl/fl;Pax8rtTA;TetO-cre mouse kidneys when compared to DMSO-treated mouse kidneys (Fig. 9).

Figure 9. Treatment with tubacin downregulates cAMP levels in the kidneys of Pkd1fl/fl;Pax8rtTA;TetO-cre mice.

Figure 9

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Columns represent cAMP averages ± standard errors of DMSO (vehicle)-treated (n=8) and tubacin-treated (n=8) mice (PN21). **P<0.01. Statistical analysis was performed using a two-tailed Student’s t test.

DISCUSSION

In this study, we have shown that tubacin prevented the formation of cysts in MDCK cells, an in vitro model of cystogenesis, and slowed renal cyst growth in Pkd1fl/fl;Pax8rtTA;TetO-cre mice, a mouse model of ADPKD showing aggressive renal cyst formation. Pkd1fl/fl;Pax8rtTA;TetO-cre mice develop giant polycystic kidneys at 3 weeks of age, and there is only a very short therapeutic window for slowing down cyst growth. Administration of tubacin reduced the growth of renal cysts in the Pkd1fl/fl;Pax8rtTA;TetO-cre mice, as assessed by kidney-to-body weight ratio and kidney cystic index. Consistent with the reduction in cystic index was an improvement in renal function in Pkd1fl/fl;Pax8rtTA;TetO-cre mice treated with tubacin, as indicated by SUN and serum creatinine levels. These are significant findings in an aggressive ADPKD mouse model with such a short therapeutic window. Prior studies have shown that inhibiting class I HDACs with valproic acid, or class I & II with trichostatin A, reduces cyst progression.35, 36 However, studies utilized non-specific inhibitors. Likewise, inhibition of non-specific deacetylase Sirtuin 1 delays cyst progression.37 Here we clearly show that inhibition of HDAC6 whose function is clearly defined by the specific inhibitor, tubacin is effective in reducing cyst growth suggesting that the mechanism of action is via the acetylation of tubulin.

An increased proliferation index of cyst-lining epithelium is thought to contribute to cyst growth in ADPKD.38 HDAC6 deacetylates α-tubulin and regulates microtubule-associated processes, and overexpression of HDAC6 leads to tubulin hypoacetylation as well as increased cell motility and proliferation.23, 24 Conversely, inhibition of HDAC6 activity causes hyperacetylation of α-tubulin and microtubules, prevents cell motility, and inhibits cell proliferation.23, 24 We have now shown that administration of tubacin can inhibit proliferation in MDCK.2, PH2 and PN18 cells and in cyst-lining epithelial cells in the kidneys of Pkd1fl/fl;Pax8rtTA;TetO-cre mice.

An increase in intra-cystic CFTR-regulated fluid secretion occurs in ADPKD. Hanaoka et al.8 have shown that CFTR is expressed in the apical epithelia in ADPKD cystic tissue. In addition, these authors have demonstrated that the CFTR expressed in ADPKD cystic tissue is functional, as indicated by cAMP-mediated activation of chloride currents. MDCK.2 cells are known to secrete chloride when stimulated with adenylyl cyclase agonists.31 We have shown here that tubacin inhibits CFTR-dependent short-circuit currents, and therefore chloride secretion, in MDCK.2 cells. Thus, tubacin apparently slows renal cyst growth not only by inhibiting cell proliferation in cystic epithelium but also by downregulating chloride secretion. In some animal models for PKD, increased levels of cAMP are present.9-11 It is thought that dysregulation of Ca2+ homeostasis as a result of mutations in the PKD1 or PKD2 gene lead to the increased cAMP levels. Cyclic AMP contributes to cyst growth and fluid accumulation in ADPKD by stimulating cell proliferation and activating CFTR-driven chloride secretion.8 We have shown here that administration of tubacin decreases cAMP levels in MDCK.2 cells and in Pkd1fl/fl;Pax8rtTA;TetO-cre mouse kidneys.

To the best of our knowledge, we are the first to show that inhibition of HDAC6 activity downregulates cAMP levels. It is not clear how tubacin downregulates cAMP, and the possible mechanisms will need to be studied in the future. Based on our current data, we propose a model in which PKD1 mutations result in an upregulation of HDAC6, which leads to cyst growth in ADPKD (Fig. 10A). HDAC6i slows renal cysts growth by lowering cAMP levels, inhibiting cell proliferation and CFTR-mediated chloride currents (Fig. 10B).

Figure 10. Schematic diagram depicting how an HDACi slows cyst growth in PKD.

Figure 10

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A) PKD1 mutations result in upregulation of HDAC6, leading to cyst growth in ADPKD. (B) An increased proliferation index of cyst-lining epithelium leads to cyst growth in ADPKD. Elevated levels of cAMP contribute to cyst growth and fluid accumulation in ADPKD via stimulation of cell proliferation and activation of CFTR-mediated chloride secretion. HDAC6i slows renal cyst growth by inhibiting cell proliferation, cAMP levels, and CFTR-mediated chloride currents.

There is growing evidence to suggest that HDAC6 may play a role in cyst formation in ADPKD: First, HDAC6 expression and activity are increased in Pkd1-mutant renal epithelial cells. A lack of primary cilia in renal epithelial cells leads to cyst formation in mice,39 and it is of interest that HDAC6 regulates cilia disassembly during the cell cycle,40 further indicating that HDAC6 may play a role in cyst formation. ADPKD is associated with abnormal activation of the Wnt/β-catenin-dependent pathway and nuclear translocation of β-catenin.41-43 Epidermal growth factor (EGF) also induces nuclear translocation of β-catenin.44 It has been shown that HDAC6 regulates EGF induced nuclear translocation of β-catenin.45 EGF receptor activity is increased, and the receptor is mis-localized to the apical membrane in Pkd1 knockout (ko) mice, whereas inhibition of HCAC6 activity in Pkd1 ko mice restores EGF localization to the basolateral cell membrane.22 In addition to protein mis-localization, planar cell polarity and apico-basal cell polarity are also defective in ADPKD. It is also interesting that HDAC6 has been shown to play a role in the establishment of functional neuronal polarity.46 It is not yet clear whether HDAC6 also plays a role in planar cell polarity and apico-basal cell polarity in ADPKD, and this is a subject for further study in the future.

In conclusion, we report for the first time that the specific HDAC6 inhibitor, tubacin, reduces cyst growth in MDCK.2 cells and in a mouse model for PKD via a joint reduction in cAMP and proliferation, two hallmarks of ADPKD. Currently, multiple HDAC6 inhibitors are being developed, and some are being tested in clinical trials for lymphoproliferative disorders. Our results provide compelling evidence that HDAC6 inhibitors may open a new avenue in ADPKD drug discovery.

METHODS

Cell culture

MDCK.2 cells were purchased from ATCC (CRL-2936) and cultured as previously described29. Pkd1 heterozygous (PH2) and homozygous (PN18) proximal tubule conditionally immortalized cells, provided by Stefan Somlo through the George M. O’Brien Kidney Center at Yale University (New Haven, Connecticut, USA), were cultured as previously described.47 PH2/PN18 cells were grown for 5-7 days under non-permissive conditions (37°C, without γ-interferon) before experimentation.

Reagents

collagen I (#A10483-01), MEM 10X (#11430), HEPES (15630-106), and NaHCO3 (25080) from Gibco; growth factor-reduced Matrigel (#354230) from BD Biosciences; tubacin (#SML0065) and tubastatin-A (#SML0044) from Sigma; and forskolin (#11018), genistein (#10005167), and CFTR inhibitor 172 (#15545) from Cayman Chemical.

In vitro cystogenesis

Confluent MDCK.2 cells were split 1:10 in 10-cm dishes. After 24 h, the cells were again split, resuspended in 10 ml of medium, and pelleted. They were resuspended in 2 ml of medium, and 2×104 cells were mixed with growth factor-reduced Matrigel (1.5%) and collagen I (1.5%), MEM (1x), HEPES (20 μM), and NaHCO3 (0.24%). The Matrgel/collagen I/cell mixture was plated in 24-well plates (450 μl/well) and allowed to solidify for 30 min at 37°C before being overlaid with 500 μl of medium. Cells were treated with tubacin, tubastatin-A or DMSO dissolved in medium on days 0, 2, 4, 6, 8, 10, 12 and 14 after old medium was removed. Pictures were taken with a Zeiss Axio microscope. Cystic areas were analyzed with ImageJ (provided by the NIH).

Western blotting

Cells cultured in 6 well plates were treated with tubacin, tubastatin-A or DMSO for 7-17 h. Cells were harvested and processed as previously described.29 The cell lysates were spun at 10,000xg for 10 min at 4°C to pellet insoluble material, and the supernatants were collected. The supernatants were run on 3-8% SDS-PAGE gels (Thermo Scientific, #EA03785) before transferring to a polyvinylidene fluoride membrane (Bio-Rad). The membranes were incubated with primary antibodies against acetylated tubulin (Sigma-Aldrich, T7451) overnight and then washed with TBS-Tween 20 buffer. An HRP-conjugated secondary antibody from GE Healthcare (NA934V; 1:10,000) was incubated for 1 h, and then ECL Prime (GE Healthcare) was used for detection on a film from Denville Scientific (E3018). The membranes were then stripped using Restore Western blot buffer (Pierce, VWR) and reprobed with mouse anti-β-actin from Sigma-Aldrich (A5316, 1:50,000) or anti-ezrin from Santa Cruz Biotechnology (sc-58758, 1:5000).

Quantification of Western blot data

The density of the acetylated tubulin or β-actin bands was measured with ImajeJ (provided by NIH). The acetylated tubulin level was standardized to that of the β-actin or ezrin. Data were analyzed by the Student’s t-test or by one-way ANOVA followed by Turkey’s multiple-comparison test with Igor Pro 5.03 software (WaveMetrics, Inc).

Short-circuit current measurements

The short-circuit current was measured in a Ussing-type chamber system (Physiologic Instruments, San Diego, CA). MDCK.2 cells were grown on Snapwell filters (Corning Costar, Acton, MA; 3407) for 7 days to establish polarized monolayers. Cells were treated with tubacin (10 μM) or DMSO for 17 h before ISC measurement. ISC was measured with a VCCMC6 multichannel voltage-current clamp amplifier (Physiologic Instruments). Transepithelial resistance (TER) was measured by periodically applying a 5 mV bipolar voltage pulses, recording the change in short-circuit current (Isc), and applying Ohm’s law. The cell monolayers were bathed on both sides with a solution containing 120 mM NaCl, 2 mM CaCl2, 5 mM KCl, 2 mM MgCl2, 10 mM HEPES, and 10 mM D-glucose (adjusted to pH 7.3 with NaOH). The solution was maintained at 37°C and bubbled gently with 95%O2/5%CO2. To inhibit apical Na+ absorption through epithelial sodium channel, amiloride (100 μM) was added to the medium. After equilibration, CFTR-mediated secretion was activated with forskolin (10 μM) and genistein (30 μM). The experiment was concluded by applying 10 μM of specific CFTR inhibitor 172 (CFTRinh172). Data were acquired on an 1.71-GHz PC running Windows XP (Microsoft, Redmond, WA) and equipped with DI-720 (DATAQ Instruments, Akron, OH), with Acquire and Analyze version 2.3.159 (Physiologic Instruments) software.

BrdU cell proliferation assay

Cells were seeded into 96-well plates and cultured for 24 h. They were then treated with tubacin, tubastatin-A or DMSO for 17 h before BrdU assay. Proliferation assays were carried out with a BrdU Cell Proliferation Kit (2750, Millipore) according to the manufacturer’s protocol.

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 this committee. Pkd1fl/fl;Pax8rtTA;TetO-cre mice on a C57BL/6 background48 were provided by the Baltimore PKD Center and used to test the role of tubacin on cyst growth. Pkd1fl/fl;Pax8rtTA;TetO-cre mice were generated by crossing 3 mouse strains (Jackson Lab stocks 10671, 7176 and 6234). Mice of both sexes were used in this study. Pkd1fl/fl;Pax8rtTA;TetO-cre mice were injected IP with doxycycline resuspended in sterile water (1 μg of doxycycline/5 g of body weight) on PN11, PN12, and PN13. This treatment produces a very aggressive murine model of PKD49, 50. Pkd1fl/fl;Pax8rtTA;TetO-cre mice were injected daily with tubacin (5 mg/kg) or DMSO from PN10 to PN20. On PN21, mice were euthanized with 3-5% isoflurane/oxygen (Baxter), and thoracotomy was performed. Serum was collected to measure serum urea nitrogen (SUN) and creatinine, and kidneys were harvested for histology (right kidney) and cyclic AMP assays (left kidney). SUN and creatinine were measured by the Molecular and Comparative Pathobiology Laboratory of the Johns Hopkins University.

Cystic index

Kidneys were fixed in 4% paraformaldehyde (PFA) in 1X phosphate buffer (PBS) for 24 h and then paraffin-embedded and sectioned. Sections were deparaffinized with xylene and rehydrated with a descending ethanol series into PBS, then stained with hematoxylin and eosin. Whole-kidney images were obtatined with a Nikon Eclipse e600 microscope equipped with Infinity Analyze, version 5.0.3 (Lumenera Corporation). The total kidney area and total cystic area were measured using ImageJ (provided by NIH). Cystic index = 100 X (total cystic area/total kidney area), and the results are expressed as percentages.

Ki67 staining

Kidneys were fixed in 4% PFA as described above. We used rabbit aniti-Ki67 antibody (ab15580, Acam, 1:500), goat anti-rabbit (A21429, AlexaFluor 555, Life Technology, 1:1,000) and DAPI (H-1200, Vector Laboratories). Pictures were acquired with a Zeiss Axio microscope equipped with AxioVision, version 4.8.1. Six areas from each kidney section were analyzed. Cells positive for Ki67 and the total number of cells were measured with ImageJ. Ki67-positive cells = 100 X (total number of positive cells/total number of cells), and the results are expressed as percentages.

Cyclic AMP assay

MDCK.2 cells were plated in a 6-well plate for 24 h. Confluent cells were treated with tubacin (10 μM) or DMSO for 17 h before cAMP assay. Cyclic AMP assays were carried out with a direct cAMP Enzyme Immunoassay Kit (Sigma, #CA200) based on the manufacturer’s protocol. Results are expressed as pmole/μg of protein.

Frozen kidneys were ground to powder under liquid nitrogen in a porcelain jar and homogenized in 0.1M HCl. After centrifugation at 600 X g for 10 min, the supernatants were collected and assayed with a direct cAMP Enzyme Immunoassay Kit (Sigma, #CA200). Results are expressed as nmole/μg of protein.

Statistical Analyses

Data are presented as means±SEM. Differences between two groups were assessed by two-tailed Student’s t test. Multiple groups were analyzed by one way analaysis of variance followed by Turkey multiple-comparison test. A P value <0.05 was considered statistically significant.

Supplementary Material

01

Supplemental Figure 1. In vitro cystogenesis (top panel). MDCK.2 cells grown in Matrigel/collagen I gel. DMSO (vehicle) or Tubacin (0.1, 1 or 10μM) were added on days 0, 2, 4 and 6. Photos were taken on day 7. Summary data for in vitro cystogenesis experiment (bottom panel). Columns represent averages ± standard errors (n=3). Average cyst from DMSO group was considered 100% and the rest of the cysts were compared to this cyst. *P<0.05, **P<0.01, ***P<0.001. Data were analyzed by one-way ANOVA followed by Turkey’s multiple-comparison test. Abbreviations: D - DMSO, T - tubacin.

Supplemental Figure 2. In vitro cystogenesis (top panel). MDCK.2 cells grown in Matrigel/collagen I gel. DMSO (vehicle) or Tubastatin-A (0.25, 2.5 or 25μM) were added on days 0, 2, 4 and 6. Photos were taken on day 7. Summary data for in vitro cystogenesis experiment (bottom panel). Columns represent averages ± standard errors (n=3). Average cyst from DMSO group was considered 100% and the rest of the cysts were compared to this cyst. *P<0.05, **P<0.01, ***P<0.001 Data were analyzed by one-way ANOVA followed by Turkey’s multiple-comparison test. Abbreviations: D - DMSO, TSA – tubastatin-A.

Supplemental Figure 3. (A) Acetylated tubulin expression. Western blot showing expression of acetylated tubulin in MDCK.2 cells treated with DMSO (vehicle) or decreasing doses of tubacin (10, 1, 0.1μM) for 7 h. (B) Summary data for acetylated tubulin expression. Columns represent averages ± standard errors of acetylated tubulin expression. Acetylated tubulin bands were normalized to Ezrin. **P<0.01. Data were analyzed by one-way ANOVA followed by Turkey’s multiple-comparison test. Abreviations: D – DMSO, T - tubacin. Experiment was repeated 3 times and all samples were run on the same gel.

Supplemental Figure 4. (A) Acetylated tubulin expression. Western blot showing expression of acetylated tubulin in MDCK.2 cells treated with DMSO (vehicle) or decreasing doses of tubastatin-A (25, 2.5, 0.25μM) for 7 h. (B) Summary data for acetylated tubulin expression. Columns represent averages ± standard errors of acetylated tubulin expression. Acetylated tubulin bands were normalized to Ezrin. **P<0.01, ***P<0.001. Data were analyzed by one-way ANOVA followed by Turkey’s multiple-comparison test. Abreviations: D – DMSO, TSA – tubastatin-A. Experiment was repeated 3 times and all samples were run on the same gel.

Supplemental Figure 5. BrdU cell proliferation assay in MDCK.2 cells. Cells were seeded into 96-well plates and cultured for 24 h. They were then treated with tubacin (10μM) or DMSO for 17 h before BrdU assay. Columns represent the percentage of BrdU incorporation into tubacin-treated (10μM) cells (n=4) as compared to DMSO (vehicle)-treated cells (n=4). Averages ± standard errors are presented. ***P<0.001. Statistical analysis was performed using a two-tailed Student’s t test.

Supplemental Figure 6. BrdU cell proliferation assay in PH2/PN18 cells. Columns represent the percentage of BrdU incorporation into tubastatin-A-treated (25μM) cells (n=3) as compared to DMSO (vehicle)-treated cells (n=3). Averages ± standard errors are presented. ***P<0.001. Statistical analysis was performed using a two-tailed Student’s t test.

Acknowledgments

We are grateful to S. Somlo for kindly providing PH2 and PN18 cell though the George M. O’Brien Kidney Center at Yale University, NIH grant P30 DK079310. This work was supported by a National Institutes of Health Grant K08 K08DK103078-01 (to V.C.), by National Institutes of Health Grant DK072084 (to W.G.). These studies utilized resources provided by the NIDDK-sponsored Baltimore Polycystic Kidney Disease Research and Clinical Core Center, P30 DK090868. We thank Deborah McClellan, Ph.D., for editorial assistance. We also thank Jie Deng and Hua Wang for experimentation.

Footnotes

Author contributions: L.C., W.B.G., and V.C. designed and supervised research; L.C., Q.L., M.Y., C.B., P.O. and V.C. performed research; D.H. provided advice for animal studies; L.C., T.W., W.B.G., and V.C. reviewed and analyzed data, and reviewed the manuscript; L.C., W.B.G., and V.C. wrote the manuscript.

Disclosures: None

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01

Supplemental Figure 1. In vitro cystogenesis (top panel). MDCK.2 cells grown in Matrigel/collagen I gel. DMSO (vehicle) or Tubacin (0.1, 1 or 10μM) were added on days 0, 2, 4 and 6. Photos were taken on day 7. Summary data for in vitro cystogenesis experiment (bottom panel). Columns represent averages ± standard errors (n=3). Average cyst from DMSO group was considered 100% and the rest of the cysts were compared to this cyst. *P<0.05, **P<0.01, ***P<0.001. Data were analyzed by one-way ANOVA followed by Turkey’s multiple-comparison test. Abbreviations: D - DMSO, T - tubacin.

Supplemental Figure 2. In vitro cystogenesis (top panel). MDCK.2 cells grown in Matrigel/collagen I gel. DMSO (vehicle) or Tubastatin-A (0.25, 2.5 or 25μM) were added on days 0, 2, 4 and 6. Photos were taken on day 7. Summary data for in vitro cystogenesis experiment (bottom panel). Columns represent averages ± standard errors (n=3). Average cyst from DMSO group was considered 100% and the rest of the cysts were compared to this cyst. *P<0.05, **P<0.01, ***P<0.001 Data were analyzed by one-way ANOVA followed by Turkey’s multiple-comparison test. Abbreviations: D - DMSO, TSA – tubastatin-A.

Supplemental Figure 3. (A) Acetylated tubulin expression. Western blot showing expression of acetylated tubulin in MDCK.2 cells treated with DMSO (vehicle) or decreasing doses of tubacin (10, 1, 0.1μM) for 7 h. (B) Summary data for acetylated tubulin expression. Columns represent averages ± standard errors of acetylated tubulin expression. Acetylated tubulin bands were normalized to Ezrin. **P<0.01. Data were analyzed by one-way ANOVA followed by Turkey’s multiple-comparison test. Abreviations: D – DMSO, T - tubacin. Experiment was repeated 3 times and all samples were run on the same gel.

Supplemental Figure 4. (A) Acetylated tubulin expression. Western blot showing expression of acetylated tubulin in MDCK.2 cells treated with DMSO (vehicle) or decreasing doses of tubastatin-A (25, 2.5, 0.25μM) for 7 h. (B) Summary data for acetylated tubulin expression. Columns represent averages ± standard errors of acetylated tubulin expression. Acetylated tubulin bands were normalized to Ezrin. **P<0.01, ***P<0.001. Data were analyzed by one-way ANOVA followed by Turkey’s multiple-comparison test. Abreviations: D – DMSO, TSA – tubastatin-A. Experiment was repeated 3 times and all samples were run on the same gel.

Supplemental Figure 5. BrdU cell proliferation assay in MDCK.2 cells. Cells were seeded into 96-well plates and cultured for 24 h. They were then treated with tubacin (10μM) or DMSO for 17 h before BrdU assay. Columns represent the percentage of BrdU incorporation into tubacin-treated (10μM) cells (n=4) as compared to DMSO (vehicle)-treated cells (n=4). Averages ± standard errors are presented. ***P<0.001. Statistical analysis was performed using a two-tailed Student’s t test.

Supplemental Figure 6. BrdU cell proliferation assay in PH2/PN18 cells. Columns represent the percentage of BrdU incorporation into tubastatin-A-treated (25μM) cells (n=3) as compared to DMSO (vehicle)-treated cells (n=3). Averages ± standard errors are presented. ***P<0.001. Statistical analysis was performed using a two-tailed Student’s t test.

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