Am J Physiol Renal Physiol 300: F1375–F1384, 2011.
First published March 23, 2011; doi:10.1152/ajprenal.00482.2010.
Thiazolidinediones inhibit MDCK cyst growth through disrupting oriented
cell division and apicobasal polarity
Zhiguo Mao,1,2 Andrew J. Streets,1 and Albert C. M. Ong1
1
Kidney Genetics Group, Academic Nephrology Unit, The Henry Wellcome Laboratories for Medical Research, University
of Sheffield Medical School, Sheffield, United Kingdom; and 2Kidney Institute of PLA, Division of Nephrology, Changzheng
Hospital, Second Military Medical University, Shanghai, China
Submitted 17 August 2010; accepted in final form 15 March 2011
PPAR␥; polarity; Cdc42; lumen; PKD
AUTOSOMAL DOMINANT POLYCYSTIC kidney disease (ADPKD) is
the most common inherited renal disease with a reported
incidence of 1 in 1,000 live births. It is the fourth most
common cause for end-stage renal failure. ADPKD is caused
by mutations in two genes, PKD1 (85%) and PKD2 (15%),
and is characterized by the formation and expansion of
fluid-filled cysts which eventually destroyed kidney structure and function (23).
The pathogenesis of cyst formation in ADPKD has been
intensively investigated, and several potential mechanisms
suggested by clinical and experimental studies. These include
increased cell proliferation and apoptosis, enhanced fluid secretion, abnormal cell-matrix interactions, alterations in cell
polarity, and abnormalities in ciliary structure or function (5).
At present, there are no effective treatments for ADPKD in
humans although a number of compounds have been found to
Address for reprint requests and other correspondence: A. C. M. Ong,
Kidney Genetics Group, Academic Unit of Nephrology, The Henry Wellcome
Laboratories for Medical Research, Univ. of Sheffield Medical School, Beech
Hill Rd., Sheffield S10 2RX, UK (e-mail: [email protected]).
http://www.ajprenal.org
work in rodent PKD models (5). Among these are peroxisome
proliferator-activated receptor-␥ (PPAR␥) agonists or thiazolidinediones (TZDs). TZDs, including rosiglitazone and pioglitazone, are synthetic ligands for the PPAR␥ receptor and are in
widespread clinical use for the treatment of type 2 diabetes due
to their insulin-sensitizing properties. It has also been recognized that TZDs also have multiple anti-inflammatory, antifibrotic, and vascular effects independent of blood glucose
lowering (17). Of relevance to PKD, maternal administration
of pioglitazone improved the postnatal survival of Pkd1 null
mouse embryos, and this was associated with a reduction in
renal cystic disease by uncertain mechanisms (21). More recent
studies have shown a survival benefit in conditional (principal
cell) Pkd1 null mice associated with a reduction in systemic
blood pressure (but without an effect on cyst formation) and
Han:SPRD rats, with a moderate effect on kidney and liver cyst
volumes in female Pck rats (2, 6, 25). Nonetheless, the mechanism of these reported effects remains unclear, with one study
reporting no effect on cell proliferation or apoptosis, another
reporting reductions in the expression of profibrotic [transforming growth factor (TGF)-␤1] and inflammatory (MCP-1)
cytokines and Wnt (␤-catenin) signaling, and finally decreased
CFTR expression by cyst epithelia (and thus Cl⫺ secretion) (2,
6, 25).
Recently, long-term cardiac safety concerns have been
raised for TZDs which are clinically significant given their
widespread use. In view of the potential beneficial effects of
TZDs in ADPKD, a useful approach would be to clarify their
underlying therapeutic mechanism on cyst expansion so as to
minimize off-target effects. In this study, we sought to investigate the potential mechanisms of PPAR␥ activation on cystogenesis using the well-established three-dimensional (3D) Madin-Darby canine kidney (MDCK) model.
MATERIALS AND METHODS
Materials and antibodies. All chemicals were purchased from
Sigma (Poole, UK) unless otherwise stated. Alexa 594-phalloidin was
purchased from Invitrogen (Carlsbad, CA). The gp135 mAb was a gift
of G. Ojakian (SUNY, New York). Antibodies against E-cadherin and
calnexin were purchased from BD Transduction Labs (Oxford, UK),
cdc42 from Santa Cruz Biotechnology (Santa Cruz, CA), ␥-tubulin
and acetylated ␣-tubulin from Sigma-Aldrich, Ki-67 from Dianova
(Hamburg, Germany), and cleaved caspase-3 (Cell Signalling Technology, NEB, Hitchin, Hertfordshire, UK). Goat anti-mouse FITC and
rabbit anti-mouse IgG (H⫹L) FITC were from Southern Biotech,
(Birmingham, AL), and donkey anti-goat IgG (H⫹L) Alexa Fluor 568
was from Invitrogen. The GST-CRIB-PAK1 plasmid (34) was obtained from A. Rajasekaran (gift of Dr. Y Zheng). Parental MDCK II
(T23) and stable transfectants expressing tetracycline-inducible myctagged dominant-negative Cdc42 (Cdc42N17) or constitutively active
1931-857X/11 Copyright © 2011 the American Physiological Society
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Mao Z, Streets AJ, Ong AC. Thiazolidinediones inhibit MDCK
cyst growth through disrupting oriented cell division and apicobasal
polarity. Am J Physiol Renal Physiol 300: F1375–F1384, 2011. First
published March 23, 2011; doi:10.1152/ajprenal.00482.2010.—Thiazolidinediones have been reported to retard cystic disease in rodent
models by uncertain mechanisms. We hypothesized that their major
effect in retarding cystogenesis was through inhibiting cell proliferation or stimulating apoptosis. In the Madin-Darby canine kidney cell
(MDCK) model, rosiglitazone inhibited cyst growth in a time- and
dose-dependent manner and this was accompanied by a reduction in
basal proliferation and an increase in apoptosis. Unexpectedly, we
also observed a striking abnormality in lumen formation resulting in
a characteristic multiple lumen or loss of lumen phenotype in treated
cells at doses which did not inhibit cell proliferation. These changes
were preceded by mislocalization of gp135 and Cdc42, misorientation
of the mitotic spindle, and retardation in centrosome reorientation
with later changes in primary cilia length and mislocalization of
E-cadherin. Cdc42 activation was unaffected by rosiglitazone in
monolayer culture but was profoundly inhibited in three-dimensional
culture. MDCK cells stably expressing mutant Cdc42 showed a
similar mislocalization of gp135 expression and multilumen phenotype in the absence of rosiglitazone. We conclude that rosiglitazone
influences MDCK cyst growth by multiple mechanisms involving
dosage-dependent effects on proliferation, spindle orientation, centrosome migration, and lumen formation. Correct spatial Cdc42 activation is critical for lumen formation, but the effect of rosiglitazone is
likely to involve both Cdc42 and non-Cdc42 pathways.
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treatment with collagenase (100 U/ml, Sigma) for 10 min at 37°C.
After quenching with 0.15 M glycine in PBS, they were permeabilized
with 0.1% Triton X-100 for 60 min and blocked with 5% BSA for 60
min. The gels were then incubated with primary antibodies diluted in
5% BSA at 4°C for 24 h. After extensive washing for 4 h with several
changes of PBS, the samples were incubated with fluorescenceconjugated secondary antibodies for another 24 h. Finally, the gels
were mounted with Vectashield with 0.5% DAPI after extensive
washing.
For studies on monolayer cultured cells, cells grown on collagen
I-coated glass coverslips were rinsed twice with PBS and fixed with
4% PFA or ice-cold 100% methanol (for ␥-tubulin). The rest of the
procedure followed the standard protocol.
Images were acquired on an Olympus inverted IX71 microscope
using SimplePCI imaging software (Hamamatsu) and analyzed using
ImageJ software (NIH). Z-stacks were taken with a z-axis section
thickness of 0.1 ␮m, and the images were processed by Autoquant X2
software (Media Cybernetics, Bethesda, MD). 3D reconstruction was
performed using Imaris 6.3.1 image-analysis software (Bitplane, Zurich, Switzerland).
Cell proliferation assays. The proliferation of MDCK cells in
monolayer culture was assessed using a BrdU incorporation assay
according to the manufacturer’s instructions (30). The proliferation of
MDCK cells in gels was evaluated by Ki-67 staining and the total cell
counts. Ki-67 staining (antibody dilution 1:100) for MDCK cells
grown in collagen gels was modified for 3D culture from that
described for monolayer cultures (33). For total cell counts, MDCK
cells in triplicate collagen gels were released with an equal volume of
collagenase (2 mg/ml, Sigma) at 37°C for 20 min. Cells released were
pelleted by centrifugation, and a single-cell suspension was made in
PBS. The total cell number was assessed using a hemocytometer, and
the DMSO-treated group was set as a control (100%).
Cell apoptosis assays. MDCK cells were seeded on collagen
I-coated glass coverslips and treated with rosiglitazone (20 ␮M) or
DMSO for 24 h. After washing with PBS twice, cells were fixed with
4% PFA and stained with antibody to cleaved caspase-3 (1:100) and
DAPI. Cleaved caspase-3-positive cells were counted in 50 random
fields (⫻600) without overlap in each group, and the results were
adjusted by the total cell numbers in each field. Similarly, MDCK
cells in collagen gels treated with rosiglitazone (20 ␮M) or DMSO
before (day 3) or after (day 6) lumen formation were fixed with 4%
PFA and stained with an antibody to cleaved caspase-3 (1:100). To
quantify the degree of apoptosis, 50 random fields (⫻400) without
overlap were selected in each group, and the percentage of cysts with
cleaved caspase-3-positive cells were calculated.
To assess of the location of caspase-3-positive cells in each group,
costaining with gp135 was performed and DIC images were simultaneously captured. Caspase-3-positive cells were considered to be
“luminal” if they was clearly located in the central cyst lumen in both
gp135 and DIC images. The number of cysts with luminal apoptotic
cells were quantified and expressed as a percentage of the total
number of cysts with apoptotic cells counted.
Measurement of mitotic spindle angles. To increase the number of
cells undergoing mitosis, a thymidine block was introduced as previously described with modification for 3D culture (10). Briefly, MDCK
cells in gels were cultured in complete medium for 72 h to form
microcysts. They were then incubated with medium containing 3 mM
thymidine (Sigma) for 48 h to arrest cells at the early S phase and
simultaneously treated with rosiglitazone (20 ␮M) or DMSO. To
release cells from the S phase, the gels were extensively washed with
PBS (6 ⫻ 10-min washes) to remove thymidine. They were then
further incubated with medium containing rosiglitazone or DMSO for
another 24 h to permit entry into the late G2 phase.
Gels were fixed with 4% PFA and stained with ␣-tubulin antibody
(1:800) and phalloidin to visualize the mitotic spindle and actin
cytoskeleton. To measure the spindle angles, z-stack images of cells
undergoing metaphase and early anaphase cells were captured in the
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Cdc42 (Cdc42V12) were a gift of W. J. Nelson (Stanford, CA;
obtained through J. H. Lipschutz) (27).
Cell culture. MDCK II cells were cultured in DMEM (GIBCO)
with a supplement of 1% antibiotic/antimycotic (AA, GIBCO), 1%
L-glutamine, and 10% fetal bovine serum. Cells were maintained at
37°C in a humidified atmosphere of 5% CO2, and the medium was
changed every 2 days.
Cytotoxicity assays. LDH release and crystal violet assays were
used to establish the therapeutic/toxic dose range for rosiglitazone.
For the LDH release assay, a Cytotoxicity Detection KitPLUS LDH
(Roche) was used according to the manufacturer’s instructions.
The crystal violet assay was performed as previously described
(13). Briefly, MDCK cells were incubated for 24 h on 96-well plates
and then incubated for 24 h with rosiglitazone (ranging from 0.05 to
1,000 ␮M). DMSO treatment were set as a control. The medium was
removed, and adherent cells were fixed with 100 ␮l/well ice-cold
methanol/acetone (1:1 vol) and stained for 60 min with 0.2% crystal
violet in 2% methanol. Plates were washed with distilled water and air
dried thoroughly. Stain was extracted with 100 ␮l/well 10% acetic
acid at room temperature with shaking for 30 min, and absorbance
was measured at 570 nm.
Cystogenesis assays. Cystogenesis assays were performed as previously described with minor modifications (28). Briefly, MDCK II
cells were detached with trypsin/EDTA (Invitrogen) and pipetted into
ice-cold type I collagen-based medium (70% vol/vol, 3.84 mg/ml
collagen I, 20% 11.76 mg/ml NaHCO3 solution and 10% 10⫻ MEM)
to make a single-cell suspension at a final plating density of 1 ⫻ 104
cells/ml. The collagen-based medium with cells was aliquoted into
96-well plates at 100 ␮l/well, and the plates were incubated at 37°C
for 20 min to promote gelation. Two hundred microliters of medium
was introduced into each well after gelation. Cells were maintained at
37°C in a humidified atmosphere of 5% CO2, and the medium was
changed every 2 days.
To induce Cdc42 expression in MDCK lines expressing Cdc42N17
or Cdc42V12, tetracycline was first removed, and the cells were
treated with rosiglitazone (20 ␮M) or DMSO 24 h after plating into
collagen and cultured for 7 days.
To monitor the growth rate of individual cysts, each cell was
identified with a unique reference number relative to a grid placed
under the plate after seeding (32). Phase-contrast images of cells were
taken by an inverted microscopy (Olympus IX71) from day 2 and
followed every 2 days. At least 20 cysts/group were followed, and the
cyst section with the widest transverse diameter was chosen for image
analysis. The overall transverse area of each cellular structure at
different time points was measured using ImageJ [National Institutes
of Health (NIH)]. The growth rate of each cellular structure was
calculated as (change of cyst area between two time points/cyst area
at former time point) ⫻ 100%.
For the end point assay, gels were treated in triplicate with
rosiglitazone concentrations ranging from 0.5 to 100 ␮M (based on
the results of cytotoxicity assay). DMSO-treated cells served as
controls. After 13 days, the gels were fixed with 4% paraformaldehyde (PFA; pH 7.4) and stained with Alexa 594-phalloidin (1:50) and
DAPI. The cyst image with the widest transverse area was used for
morphological analysis.
Ten fields (⫻200 magnification) without overlay were selected
randomly for each gel, and images of all cellular structures were taken
for analysis. All MDCK cysts were classified into three groups
according to morphological definitions: single-lumen cyst, multiplelumen cyst, and cell aggregate. A single-lumen cyst was defined as a
cyst with a single central lumen lined by a monolayer, a multiplelumen cyst as a cyst with ⱖ2 visible lumens, and a cell aggregate as
a cellular mass without discernable lumen formation by phase contrast
microscopy at ⫻200 magnification.
Immunofluorescence. Cells cultured in collagen gels were fixed and
stained as previously described (24). Briefly, the gels were rinsed
twice with PBS and fixed with 4% PFA (pH 7.4) for 60 min after
ROSIGLITAZONE-ORIENTED CELL DIVISION AND LUMEN FORMATION
fixed and stained for acetylated-␣-tubulin and DAPI. Cilia lengths and
numbers were measured and counted with Image J, and the number of
cilia was normalized to the number of nuclei for both groups.
Cdc42 activation assay. MDCK cells on monolayer culture were
rinsed with ice-cold PBS after a 24-h incubation with rosiglitazone or
DMSO, and total cell lysates were prepared on ice and clarified by
ultracentrifugation. Fifty microliters of cell supernatant was set aside
for determination of total cdc42 and protein concentrations, and the
rest was used to determine GTP-bound Cdc42 using a pull-down
assay (see below). For cyst assays, cells were plated in collagen gels
at a density of 4 ⫻ 104 cells/ml, treated with rosiglitazone (20 ␮M) or
DMSO 24 h after plating, and cultured for a further 6 days. Cystcontaining collagen gels were incubated in calcium-free HBSS containing 4,000 U/ml collagenase, 0.005% DNase, and protease inhibitor cocktail for 45 min at 37°C with gentle rotation to digest the
collagen matrix. Intact cysts were then pelleted from the collagenasetreated mixture by centrifugation at 3,000 g for 3 min, washed twice
with PBS, and then lysed in IP buffer.
To detect active or GTP-bound Cdc42, a GST pull-down assay
using a GST-PAK fusion protein (which binds to GTP-Cdc42 but not
GDP-Cdc42) was performed as previously described (9). In brief, 2
␮g of GST (control) or GST-PAK protein was added to 500 ␮g of
DMSO or rosiglitazone-treated cyst cell lysate, and the samples were
incubated with rotation for 1 h at 4°C. The GST protein was pulled
down by incubation with glutathione 4B Sepharose beads for 2 h at
4°C. Beads were washed three times with PBS and boiled for 5 min
in 40 ␮l SDS-PAGE loading buffer before loading on the gel. GTP␥S
(100 ␮M) or GDP (1 mM) loading of cell lysates in vitro were used
as positive and negative controls. In vivo stimulation will activate
⬃10% of the available Cdc42, whereas in vitro GTP␥S protein
loading will activate nearly 100% of available Cdc42. Equal concentrations of lysates were added to two tubes, and EDTA was added to
a final concentration of 10 mM. Samples were then incubated at 30°C
Fig. 1. Growth rate of Madin-Darby canine kidney cell (MDCK) II cysts was reduced by rosiglitazone (Rosi). Individual MDCK cysts were monitored every 2 days after plating by phase-contrast
microscopy, and the cyst area was measured sequentially. A: the average area of MDCK II cysts
was significantly reduced by rosiglitazone (20
␮M). Values are means ⫾ SE (n ⫽ 20 –27).
B: example of an MDCK II cyst treated with
DMSO (n ⫽ 20) or rosiglitazone 20 ␮M (n ⫽ 27)
monitored serially from day 0 to day 12. Scale
bar ⫽ 100 ␮m. *P ⬍ 0.05, **P ⬍ 0.01 vs.
DMSO.
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middle region of the cysts and merged to ensure that both poles of a
spindle were in the same image. The centroid was defined as the point
of intersection between lines representing the longest and shortest
diameters of the cyst. The center of the spindle axis was defined as the
midpoint of a line connecting the two poles of the spindle. The angle
between the spindle axis and the line connecting the centroid of the
cyst and the center of the spindle axis was analyzed, as shown in Fig.
5C (12). Cells were defined as being in metaphase when chromosomes
were aligned at the cell equator. Early anaphase was defined as the
point at which sister chromatids had just started to separate, but cell
elongation and membrane invagination had not yet occurred.
Reorientation of centrosomes. To assess the reorientation of centrosomes, cells treated with rosiglitazone or control were seeded on
collagen I-coated coverslips in six-well plates and incubated with
complete medium for 4, 24, or 72 h. The seeding cell density was
chosen to ensure that cells would reach complete confluence but not
be overconfluent at 4 (100 ⫻ 104 /well)-, 24 (50 ⫻ 104/well)-, or 72-h
(30 ⫻ 104/well) experiments. Cells were then fixed with ice-cold
100% methanol and stained for ␥-tubulin (antibody dilution 1:800)
and DAPI.
Z-stack images of centrosomes stained with ␥-tubulin were obtained with a z-axis thickness of 0.1 ␮m. The distance between the
centrosomes and basal membrane was measured to assess the reorientation rate of centrosomes to the apical side of the nucleus (8).
Measurement of cilia length and number. Measurement of cilia
length was modified from a previous report (31). In brief, MDCK
cysts were fixed on days 12, 14, and 17 and stained with ␣-acetylated
tubulin (antibody dilution 1:800) and phalloidin to visualize the
primary cilia and cystoskeleton. Thirty cysts were randomly selected
from each group, and images of cysts were taken at different focal
planes to visualize clearly the entire cilia length.
To measure cilia number and length in monolayer culture, cells
were seeded on collagen-coated coverslips, cultured for 7 days, then
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for 15 min with agitation, and a GST pull-down was carried out with
the GST-PAK as described above.
Samples were blotted with anti-Cdc42 or anti-GST antibodies.
Total Cdc42 was shown to be equal in both DMSO- and rosiglitazonetreated cells following loading of equal concentrations of cell lysate
(20 ␮g/lane).
Statistical analysis. All data are expressed as means ⫾ SE. Student’s unpaired t-test was applied with GraphPad Prism software.
RESULTS
Fig. 2. Rosiglitazone inhibited proliferation, induced apoptosis, and decreased the percentage of single central lumen cysts in MDCK II cells. A: total cell number
of MDCK II cysts from triplicate wells after 13 days following treatment with rosiglitazone (0.5–100 ␮M) or DMSO. *P ⬍ 0.05 vs. DMSO (100%).
B: rosiglitazone (20 ␮M for 8 days) significantly suppressed cell proliferation compared with DMSO-treated cysts, as quantified as the number of Ki-67-positive
cells per cyst. C: rosiglitazone significantly inhibited cell proliferation in monolayer cultured MDCK cells. A small but significant dose-dependent effect was
seen compared with monolayer cells by BrDU incorporation assays at doses ⱖ5 ␮M. Data shown represent the mean of three experiments performed in triplicate.
*P ⬍ 0.05 vs. DMSO control. D: there was a significant increase in the percentage of cysts with apoptotic cells (cleaved caspase-3 positive) following
rosiglitazone (20 ␮M) treatment in MDCK cysts before (day 3) or after (day 6) lumen formation. E: rosiglitazone (20 ␮M) did not significantly alter the apoptotic
rate (cleaved caspase-3 positive) of monolayer MDCK cells. Data are shown from 3 experiments performed in triplicate. F: MDCK II cells formed cysts with
a well-developed central lumen following plating into type I collagen gels. Incubation with rosiglitazone (0.5–100 ␮M for 13 days) induced MDCK cysts with
multiple ectopic lumen and cellular aggregates without a discernable lumen in a dose-dependent manner. *P ⬍ 0.01 vs. DMSO-treated cells for single-lumen
cysts.
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Rosiglitazone inhibits the overall growth of MDCK cysts.
Preliminary assays showed that rosiglitazone had no effect on
cell viability (crystal violet) or toxicity (LDH release) at doses
of up to 100 ␮M in monolayer culture (not shown). Sequential
analysis of cyst area from days 2–12 showed that 20 ␮M
rosiglitazone significantly inhibited MDCK cyst growth from
day 6 compared with DMSO-treated controls (Fig. 1, A and B).
Rosiglitazone inhibits proliferation and induces apoptosis of
MDCK cysts. Cell counts in MDCK cysts after 13-day incubation showed a significant dose-dependent decrease above 10
␮M (Fig. 2A). Since this reduction could be the consequence of
a decrease in proliferation and/or increase in apoptosis, we first
investigated cell proliferation in MDCK cysts using the proliferation marker Ki-67. Rosiglitazone treatment (20 ␮M, 8
days) decreased the number of Ki-67 positive cells per cyst by
70% (from 47.9 ⫾ 2.6 to 14.5 ⫾ 1.0 positive cells/cyst, P ⬍
0.0001, Fig. 2B). The inhibitory effect of rosiglitazone (ⱖ5
␮M) on basal proliferation was confirmed in MDCK monolayer cultures using a BrdU incorporation assay (Fig. 2C).
To study changes in apoptosis, we stained cysts using an
antibody to cleaved caspase-3 before (day 3) or after (day 6)
the onset of lumen formation (day 4). As shown in Fig. 2D,
rosiglitazone increased the percentage of cysts with apoptotic
cells by over twofold at both time points. Another striking
difference was a major shift in the location of apoptotic cells
away from luminal (DMSO) to multiple nonluminal areas
following rosiglitazone treatment (Supplementary Fig. S1; all
supplemental material for this article is accessible online at the
journal web site). Rosiglitazone did not increase the basal
ROSIGLITAZONE-ORIENTED CELL DIVISION AND LUMEN FORMATION
central lumen in DMSO controls (Fig. 3B). At intermediate
time points (day 3, day 6, day 8), we observed gp135 expression at areas of membrane protrusions rather than in the central
lumen or preluminal membranes (Supplementary Fig. S2).
This alteration in gp135 localization was accompanied by
the mislocalization of the basolateral protein E-cadherin in
some cysts. By day 8, ⬃30% of rosiglitazone-treated cysts
displayed apical E-cadherin staining in addition to basolateral
staining (Supplementary Fig. S3).
Rosiglitazone randomizes mitotic spindle orientation. Misalignment of the mitotic spindle between dividing cells has
been reported to result in multiple lumen cysts through the
targeting of apical patches to aberrant sites (12). We studied
the effects of rosiglitazone on the mitotic spindle orientation of
MDCK cells in collagen gels, by measuring the spindle angles
in dividing cells as shown in Fig. 4B. In control cysts, the
majority of the mitotic spindles are oriented within the tissue
plane (perpendicular to the centroid of the cyst) with a mean
angle of 73.8 ⫾ 3.5° (n ⫽ 30) (Fig. 4, A and C). Following
rosiglitazone treatment, mitotic spindle angles became randomized, with a mean angle of 44.0 ⫾ 5.1° (n ⫽ 27) (Fig. 4,
A and C).
Rosiglitazone retards the reorientation of centrosomes. Initial experiments indicated that rosiglitazone did not alter the
gross morphology of the microtubular network in MDCK cells
(data not shown). However, we hypothesized that rosiglitazone
might alter specific microtubular functions such as centrosome
migration and polarity. Normally, the centrosomes are located
randomly following seeding onto collagen I-coated coverslips.
As apical polarity is established through cell-matrix cues,
cellular organelles including centrosomes and Golgi will reorient to the apical side of the nucleus.
Fig. 3. Rosiglitazone induced ectopic expression of apical marker gp135 in MDCK II cells in 3-dimensional (3D) culture. A: z-scans (i, ii, and iii) and 3D
reconstructed images (iv) of day 2 MDCK II cells treated with DMSO or rosiglitazone showing that gp135 is normally localized between dividing cells but shows
a randomized location after rosiglitazone treatment. B: central lumen apical gp135 (green) expression in DMSO-treated cysts on day 12 compared with gp135
expression on multiple ectopic lumens after rosiglitazone. F-actin (red) staining highlights lumen structures. Scale bar ⫽ 20 ␮m.
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apoptotic rate in MDCK monolayers (Fig. 2E). This suggests
that the increase in apoptosis observed in 3D culture might be
secondary to changes in cell polarity (see below).
Rosiglitazone disrupts central lumen formation in MDCK
cysts. Besides the effects on proliferation and apoptosis,
rosiglitazone-treated cells displayed a clear defect in central
lumen formation. Rosiglitazone reduced the percentage of
single central lumen cysts (91.8% in the DMSO group vs.
0 –53.3% in the rosiglitazone groups, P ⬍ 0.01). There was a
corresponding increase in the percentage of multiple lumen
cysts (1% in the DMSO group vs. 12.8 –24.4% in the rosiglitazone groups, P ⬍ 0.01) and cell aggregates without obvious
lumen (7.23% in DMSO vs. 24.3– 87.24% in rosiglitazone
groups, P ⬍ 0.01, Fig. 2F). This effect was observed at doses
below 10 ␮M (as low as 0.5 ␮M) and therefore distinguishable
from the effects on cell proliferation and apoptosis.
In addition to multiple lumens, we observed short irregular
tubular protrusions in a minority (⬃20%) of rosiglitazonetreated cysts. These were not present in control cells and varied
in length from 5 to 40 ␮m (Supplementary Fig. S2, for
example).
Rosiglitazone induces mislocalization of apical and basolateral membrane proteins in MDCK cysts. The protein gp135/
podocalyxin is expressed at the apical surface before lumen
formation is visible and marks the site of future lumen formation. In view of the defect in apical lumen formation, we
followed the expression of this protein in cells from day 2 after
plating. As reported previously, we observed localization of
gp135 to the cell-cell borders of dividing cells in the DMSO
controls (18). With rosiglitazone, however, gp135 became
mislocalized to multiple intracellular vesicles and to the cellular edges of outer protrusions (Fig. 3A). By day 12, multiple
gp135-positive lumen structures had formed, unlike a single
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To quantify the reorientation rate of centrosomes, we measured the distance between centrosomes and cell basal surfaces
at different time points in rosiglitazone-treated and DMSO
control cells. As shown in Fig. 5, 4 h after seeding, most cells
in both group had attached firmly to the collagen I-coated
coverslips, and the average distance between the centrosomes
and basal cell surfaces was 2.43 ⫾ 0.03 ␮m in rosiglitazonetreated cells and 2.48 ⫾ 0.03 ␮m in control cells (P ⫽ 0.23).
Twenty-four hours after seeding, centrosomes in rosiglitazonetreated cells moved more slowly toward the apical surface
(2.59 ⫾ 0.03 ␮m) than in control cells (3.02 ⫾ 0.03 ␮m, P ⬍
0.001). Seventy-two hours after seeding, the difference between rosiglitazone-treated (3.02 ⫾ 0.03 ␮m) and control
(3.04 ⫾ 0.03 ␮m) cells was no longer significant (P ⫽ 0.65).
There was no difference in the distance between the apical
nuclear surface and basal surface between both groups at all
three time points (DMSO: 2.8 ⫾ 0.02 ␮m vs. rosiglitazone:
2.8 ⫾ 0.03 ␮m), excluding changes in cell height as a trivial
explanation.
Rosiglitazone alters primary cilia expression and length.
Primary cilia are enucleated from the mother centriole or
basal body. In view of the defects in spindle orientation and
centrosome reorientation, we hypothesized that rosiglitazone might result in abnormalities in cilia structure. Preliminary experiments indicated that primary cilia in MDCK
cysts were only visible from day 10 to day 11 in 3D culture
(data not shown). Cilia length was similar at day 12 in
control and treated cysts. However, treated cysts expressed
significantly shorter cilia at days 14 and 17 (Supplementary
Fig. S4). In monolayer culture, MDCK cells expressed
longer cilia compared with 3D culture and at an earlier time
AJP-Renal Physiol • VOL
point (Supplementary Fig. S4, B and D). Rosiglitazone
similarly reduced cilia length in MDCK monolayers
(DMSO: 12.45 ⫾ 0.36 ␮m vs. rosiglitazone: 7.49 ⫾ 0.17
␮m, Supplementary Fig. S4D) and increased the percentage
of cells without detectable primary cilia by 20% (DMSO:
8% vs. rosiglitzone: 28%, Supplementary Fig. S4E).
Rosiglitazone inhibits activation of the small GTPase Cdc42
and alters its subcellular localization in MDCK cysts. The
small GTPase Cdc42 has been recently shown to play a critical
role in the establishment and maintenance of apical polarity
and lumen formation in epithelial cells. In particular, knockdown of Cdc42 induced a multiple lumen phenotype in MDCK
and Caco-2 cells (12, 18). We next investigated whether
alterations in Cdc42 localization, expression, or activation
might underlie the disruption of apical lumen formation induced by rosiglitazone.
As shown in Fig. 6A, Cdc42 in control cells localized in
an almost identical pattern to gp135, i.e., between dividing
cells before (day 3), and lining the luminal surface after,
lumen formation (day 6) as previously reported (18). Similar
to its effect on gp135 (Fig. 3), rosiglitazone randomized
Cdc42 localization initially to protrusions at the edges (day
3) and to multiple lumen surfaces (day 6). Although total
Cdc42 expression was unchanged, Cdc42 activation (GTPbound) in treated cysts was profoundly inhibited by rosiglitazone (Fig. 6B). By contrast, rosiglitazone had no effect on
Cdc42 activation in MDCK monolayer cells (Supplementary
Fig. S5).
Inducible expression of Cdc42 mutants interferes with apical
expression of gp135 and central lumen formation. In a previous study, it was reported that MDCK cells inducibly express-
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Fig. 4. Rosiglitazone induced randomization of the mitotic spindle angle in MDCK II cysts. A: examples of
mitotic spindles in MDCK II cysts following DMSO
(top) or rosiglitazone (bottom) treatment. B: diagram
illustrating how mitotic spindle angles in cells undergoing metaphase or early anaphase were measured relative
to the centroid of cysts. C: Mitotic spindle angles of
MDCK II cells in DMSO and rosiglitazone groups.
Rosiglitazone treatment led to a significant randomization of the spindle angle compared with DMSO-treated
MDCK II cysts: DMSO: 73.8 ⫾ 3.5° (n ⫽ 30), rosiglitazone: 44.0 ⫾ 5.1° (n ⫽ 27). *P ⬍ 0.001 vs. DMSO.
ROSIGLITAZONE-ORIENTED CELL DIVISION AND LUMEN FORMATION
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ing (Tet-Off) either dominant-negative (Cdc42N17) or constitutively active Cdc42 (Cdc42V12) (Fig. 7A) had a reversal of
apicobasal polarity in 3D culture, as reflected by phalloidin
staining (27). However, gp135 localization or the nature of
lumen formation was not reported. Using the same clones, we
found gp135 staining at the basolateral surface following
Cdc42 induction (Fig. 7B). There was also a significant reduction in the number of single central lumen cysts compared with
controls (85.5% in control cells, 1.33% in Cdc42N17 cells, and
4.667% in Cdc42V12 cells, P ⬍ 0.001). Loss of luminal gp135
expression was more profoundly seen in Cdc42N17 cells
(73.8% in Cdc42N17 cells vs. 35.3% in Cdc42V12 cells, P ⬍
0.01). These results suggest that basal Cdc42 activity is essential for apical gp135 expression and central lumen formation
(Fig. 7B).
Rosigliazone induces a multilumen phenotype in Cdc42expressing cells. Similar to MDCK II cells, MDCK T23
cells displayed a clear defect in central lumen formation
following rosiglitazone exposure (20 ␮M for 7 days) (Fig.
7C). The percentage of single lumen cysts (85.5% DMSO
vs. 7.7% rosiglitazone, P ⬍ 0.01) was reduced with a
corresponding increase in the percentage of multilumen
cysts and cell aggregates without obvious lumen.
Rosiglitazone treatment had little effect on lumen formation
in Cdc42V12 cells. However, in Cdc42N19 cells, it increased
the proportion of multiple lumen cysts (25% untreated vs. 62%
treated) compared with a higher percentage of aggregates
present in untreated cells (Fig. 7C). This was associated with
relocalization of gp135 from the basolateral surface to the
internal lumen (Fig. 7B).
AJP-Renal Physiol • VOL
DISCUSSION
The genesis of epithelial polarity is critical for the formation of tubular structures during development. Two major
events seem to be important for epithelial morphogenesis.
Initial cell-ECM interaction orientates the apicobasal cell
axis. This is followed by the formation of an apical lumen
through the genesis, polarized delivery, and exocytosis of
apically destined vacuoles or vesicles. Formation of the
central lumen can occur by either cavitation (necessitating
luminal apoptosis) or hollowing (exocytosis of preformed
apical vesicles) depending on the model used (19).
In this study, we found that rosiglitazone inhibited
MDCK cyst growth by inhibiting proliferation and, unexpectedly, by interfering with central lumen formation. Incubation with rosiglitazone resulted in misorientation of the
mitotic spindle in dividing cells and a mistargeting of
gp135, an apical marker protein at a very early stage of cyst
formation. These two effects resulted in the formation of
multiple lumens instead of a single central lumen.
The Rho GTPase Cdc42 is considered a master regulator
of cell polarity (11). In fibroblasts, Cdc42 acts upstream of
Rac and Rho to control the formation of actin-based structures and thus cell motility (22). Cdc42 has also been shown
to regulate the constitutive exit of proteins from the transGolgi network (20). Recently, several new lines of evidence
indicate that Cdc42 also plays an essential role in the
establishment of the apical surface. First, it regulates the
spatial exocytosis of apical proteins via phosphatidylinositol
4,5-bisphosphate-dependent annexin 2 targeting (18). Sec-
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Fig. 5. Rosiglitazone retarded centrosome reorientation
in MDCK II cells after reseeding. A: z-scans of centrosomes (green) in MDCK II cells treated with DMSO or
20 ␮M rosiglitazone at 4, 24, and 72 h after seeding on
collagen I-coated coverslips. B: distance between the
centrosomes and basal cell surface at 4, 24, and 72 h
after seeding. Rosiglitazone retarded the reorientation of
centrosomes to the apical side of the nucleus. *P ⬍
0.001.
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ROSIGLITAZONE-ORIENTED CELL DIVISION AND LUMEN FORMATION
ond, it controls spindle orientation during cell division in 3D
culture (12). In vivo, Cdc42 has been shown to be essential
for pancreatic tubulogenesis and indirectly specifies cell fate
in pancreatic development (14).
Our results clearly indicate that Cdc42 activation is inhibited and its normal apical localization in 3D culture
randomized by rosiglitazone treatment. This is likely to
result in loss of apical targeting of gp135 as well as changes
in oriented cell division. In previous studies, knockdown of
Cdc42 by small interfering RNA delayed fusion of gp135containing vesicles with the plasma membrane (18). Cdc42
is also known to be required for the exocytosis of secretory
vesicles in neuroendocrine cells (16) and yeast (1). These
data place Cdc42 as an upstream regulator for gp135 targeting.
Recently, a role of Cdc42 in regulating the fidelity of mitotic
spindle alignment and asymmetric abscission between dividing
epithelial cells has been shown (12). Oriented cell division in
this model was critical for the maintenance of a central lumen
since misalignment of the mitotic spindles resulted in a multilumen phenotype (as observed in our study) through the
targeting of apical patches to aberrant sites (12). E-cadherinmediated cell contacts are known to activate Cdc42 and to
regulate spindle orientation in MDCK cells in an APC-dependent but Par3/aPKC-independent manner (10, 15). It seems
likely that multiple mechanisms could be involved in correct
spindle positioning in epithelial cells.
AJP-Renal Physiol • VOL
Both of these functions of Cdc42 rely on the correct
spatiotemporal localization of Cdc42 (3, 7, 18, 26). Indeed,
we observed that Cdc42 localized to regions of membrane
protrusions following rosiglitazone treatment (Fig. 6), a
feature not observed in control cells. Although these tubular
protrusions did not elongate continuously, they may represent the response to ectopic Cdc42 localization such as that
described at the leading edge of migrating cells (35) or
polarized growth (11). Of relevance, these early structures
were transient and multiple ectopic lumen were visible later.
This suggests that the ectopic expression of Cdc42 itself is
insufficient to stimulate continuous cell migration but that
the intracellular ectopic localization of Cdc42 was sufficient to stimulate lumen formation. The latter may explain both the
overall increase in apoptotic rate and the ectopic “extraluminal” nature of apoptotic cells seen (Fig. 2D, Supplementary Fig. S1).
Cdc42 also regulates centrosome reorientation in migrating fibroblasts by both actin and microtubular pathways (4,
29). These could explain the defects in centrosomal migration and shorter cilia length we observed in longer cultures.
Defects in centrosome migration and ciliary structure or
function have been linked to human diseases associated with
a cystic phenotype. We suggest that correct centrosome
orientation and polarity are linked to proper cilia development and apical lumen formation. Potentially, changes in
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Fig. 6. Rosiglitazone induced ectopic localization of Cdc42 in MDCK cysts and suppressed its activation. A: MDCK II cysts
treated with DMSO or rosiglitazone (20 ␮M)
for 3 days (before lumen formation) or 6
days (after lumen formation). Cdc42 localization (red) was randomized by rosiglitazone. treatment in a similar pattern to gp135
(green). Scale bar ⫽ 20 ␮m. B: levels of
active and total Cdc42 in MDCK cysts
treated with DMSO or rosiglitazone (20 ␮M)
for 6 days as determined by GST pulldown
and Western blotting. Representative blot is
shown of 2 independent experiments. A
GTP-␥-treated sample served as a positive
control and a GDP-treated sample as a negative control. The GST blot shows equal
loading of GST and GST-Pak in each sample. Rosiglitazone treatment profoundly inhibited active Cdc42 levels but did not affect
total Cdc42 expression.
ROSIGLITAZONE-ORIENTED CELL DIVISION AND LUMEN FORMATION
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the rate of centrosomal migration could have contributed to
loss of oriented cell division in our model.
To confirm the role of Cdc42, we examined MDCK cells
expressing either dominant-negative (Cdc42N19) or constitutively active (Cdc42V12) Cdc42. Similar to rosiglitazonetreated control cells, both mutants showed mislocalization of
gp135 and loss of a single lumen phenotype, supporting a
critical role for Cdc42 in both functions. However, rosiglitazone did increase the percentage of cells with a multilumen
phenotype in Cdc42N19 cells, accompanied by relocalization
of gp135 to these multiple lumens. By contrast, there was no
significant change in the Cdc42V12 cells with rosiglitazone.
These data would be compatible with the involvement of a
partially effective non-Cdc42 salvage pathway for lumen formation (and gp135 targeting) in Cdc42N19 cells. Alternatively,
rosiglitazone could act on an effector downstream of Cdc42
activation.
In summary, we report two potential mechanisms by
which PPAR agonists can retard cyst formation in MDCK
AJP-Renal Physiol • VOL
cells, i.e., by disrupting oriented cell division and lumen
formation. These changes are likely to underlie the inhibitory effects of these compounds on cyst formation in vivo.
ACKNOWLEDGMENTS
We thank G. Ojakian, Y. Zheng, and W. J. Nelson for generous gifts of
reagents and A. Muesch for helpful discussions and comments on the manuscript.
Rosiglitazone was the kind gift of Dr. Steve Smith (GlaxoSmithKline, UK).
GRANTS
This project was funded in part by the PKD Foundation and a Walls
Bursary (Renal Association UK) to A. C. M. Ong. Z. Mao was supported
by a Fellowship from the International Society of Nephrology (ISN), a
grant from “Outstanding Young Scholars” Changzheng Hospital, Second
Military Medical University, and a grant from the National Natural Science
Foundation of China (81000281). A. J. Streets is a Research Councils UK
Academic Fellow, and A. C. M. Ong is a Wellcome Trust Research Leave
Senior Fellow.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
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Fig. 7. Inducible expression of mutant Cdc42 leads to mislocalization of gp135 and loss of a central-lumen phenotype. A: control (T23) and Cdc42 mutant
MDCK cells showing the absence of Cdc42-myc in the presence of tetracycline (⫹) but its induction following tetracycline withdrawal (⫺). Lysates were
blotted with an anti-myc (top) and anti-Na⫹-K⫹-ATPase (bottom) antibody as a loading control. B: control (T23) and Cdc42 mutant MDCK cells in 3D
culture at 7 days following DMSO or rosiglitazone (20 ␮M) treatment for 6 days. Immunofluorescence for gp135 (green) and phase-contrast microscopy
(lumen) is shown in adjacent panels for the same cysts. Both Cdc42V12 and Cdc42N17 mutants show mislocalization of gp135 to the basolateral surface
and a multilumen phenotype. Rosiglitazone had little effect in Cdc42V12 cells but increased the proportion of Cdc42N17 cells with a multilumen
phenotype. C: quantification of cyst lumen phenotype in all 3 lines following DMSO or rosiglitazone treatment for 6 days. Individual cysts were classified
as either single lumen, multiple lumen, or aggregates (no lumen) by phase-contrast microscopy and expressed as a percentage of the total numbers of cysts
counted (n ⫽ 25). Cdc42N17 cells formed a higher percentage of aggregates than Cdc42V12 and a lower percentage of single-lumen cysts. Following
rosiglitazone treatment, there was a higher percentage of Cdc42N17 cells with a predominant multiple-lumen phenotype but no significant change in
Cdc42V12 cells.
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ROSIGLITAZONE-ORIENTED CELL DIVISION AND LUMEN FORMATION
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