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Research Article
Correlation between Urothelial Differentiation and
Sensory Proteins P2X3, P2X5, TRPV1, and TRPV4 in Normal
Urothelium and Papillary Carcinoma of Human Bladder
Igor Sterle,1Daša ZupanIiI,2and Rok Romih2
1Department of Urology, University Medical Centre Ljubljana, Z aloˇ
ska cesta 2, 1000 Ljubljana, Slovenia
2Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000 Ljubljana, Slovenia
Correspondence should be addressed to Rok Romih; rok.romih@mf.uni-lj.si
Received January ; Revised April ; Accepted April ; Published April
Academic Editor: Michael Winder
Copyright © Igor Sterle et al. is is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Terminal dierentiation of urothelium is a prerequisite for blood-urine barrier formation and enables normal sensory function
of the urinary bladder. In this study, urothelial dierentiation of normal human urothelium and of low and high grade papillary
urothelial carcinomas was correlated with the expression and localization of purinergic receptors (PX, and PX) and transient
receptor potential vanilloid channels (TRPV, and TRPV). Western blotting and immunouorescence of uroplakins together
with scanning electron microscopy of urothelial apical surface demonstrated terminal dierentiation of normal urothelium, partial
dierentiation of lowgrade c arcinoma, and poor dierentiation of high grade carcinoma. PX was expressed in normal urothelium
as well as in low grade carcinoma and in both cases immunolabeling was stronger in the supercial cells. PX expression decreased
in high grade carcinoma. PX expression was detected in normal urothelium and in high grade carcinoma, while in low grade
carcinoma its expression was diminished. e expression of TRPV decreased in low grade and even more in high grade carcinoma
when compared with normal urothelium, while TRPV expression was unchanged in all samples. Our results suggest that sensory
proteins PX and TRPV are in correlation with urothelial dierentiation, while PX and TRPV have unique expression patterns.
1. Introduction
e urothelium, which lines the urinary bladder, performs
two major functions. e rst one is a well-characterized
high resistance permeability barrier, and the second, not so
well understood, is a sensory function. Permeability barrier
is formed and maintained during urothelial dierentiation,
which reaches the terminal stage in supercial umbrella
cells. Umbrella cells synthesize four major transmembrane
proteins, uroplakins (UPIa, UPIb, UPII, and UPIIIa), which
form unique membrane specialization, that is, urothelial
plaques []. It was shown that uroplakins directly contribute
to the urothelial barrier function []. Aer synthesis and
modications of uroplakins in the endoplasmic reticulum
and the Golgi apparatus, respectively, urothelial plaques are
gradually assembled in post-Golgi compartments. ey are
transported to the apical plasma membrane of umbrella cells
by fusiform vesicles [–]. Urothelial plaques are encircled
by so-called hinge regions, which form microridges at the
urothelial apical surface [].
Urothelium, together with lamina propria, acts also as a
sensory web, which is able to receive, amplify, and transmit
information about its environment []. Numerous receptors
and ion channels, including purinergic PX receptors and
transient receptor potential vanilloid (TRPV) channels, have
been identied in urothelial cells. ey respond to bladder
lling, changes of urine composition, or autocrine and
paracrine mediators []. PX receptors and TRPV channels
are relatively nonselective cation channels [,]. Stretching
triggers chemically mediated activation of purinergic PX
receptors and exocytosis of fusiform vesicles []. Moreover,
stretching stimulates aerent nerve processes and may signal
the degree of bladder lling to the central nervous system
[,]. TRPV channels may also be involved in response
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BioMed Research International
Volume 2014, Article ID 805236, 9 pages
http://dx.doi.org/10.1155/2014/805236
BioMed Research International
to mechanical and chemical stimuli [,]. It has been
proposed that TRPV and TRPV are involved in bladder
lling sensation and regulation of the voiding reex [–].
We have shown previously that uroplakin expression
decreases during bladder carcinogenesis [–], which is
reected in partial urothelial dierentiation and barrier dis-
ruption [,]. Compromised permeability barrier results
in lower urinary tract symptoms (LUTS), which are divided
into three categories: storage, voiding, and postmicturition
symptoms. Storage symptoms include increased micturition
frequency, nocturia, urinary urgency, and urinary inconti-
nence []. Common voiding symptoms contain slow or
weak stream, hesitancy, and terminal dribble. Postmicturi-
tion symptoms include the sensation of incomplete emptying
and postmicturition dribble [,]. Although the aetiology
of LUTS is multifactorial, bladder carcinomas represent one
ofthepossiblecauses[]. Since PX receptors are implicated
in the bladder sensation mediated by aerent nerves, it is
likely that sensory web plays an important role in some
bladder diseases accompanied by LUTS []. Currently, very
little is known about the roles of PX receptors and TRPV
channels in human bladder tumours. It is assumed that PX
receptors, activated by ATP, have a signicant antineoplastic
action and might be involved in urothelial dierentiation in
high grade supercial bladder cancer []. Regarding TRPV,
its downregulation was reported in supercial and muscle
invasive urothelial cancers [,].
Here we report on the expression and localization of
PX, PX, TRPV, and TRPV in normal human urothe-
lium and in low and high grade papillary urothelial carcino-
mas. e results are compared with uroplakin expression and
urothelial apical surface ultrastructure. Our results suggest
correlation between sensory function of the urothelium and
urothelial dierentiation.
2. Material and Methods
2.1. Patients and Sampling. e study was conducted in
accordance with the Helsinki Declaration and approved by
the Slovenian National Medical Ethics Committee number
//. Eighteen patients with papillary urothelial carci-
noma who underwent transurethral resection of the bladder
were included in the study. Informed consent was obtained
from all patients. Two samples were acquired by cold-cup
biopsies from each patient: (i) the urothelial tumour and (ii)
the normal urothelium cm posterior from the interureteric
ridge. Biopsies captured urothelium and lamina propria. For
pathological staging and grading, EAU Guidelines on Non-
muscle-invasive Bladder Cancer []wereused.Urothelial
tumours were diagnosed as low grade papillary urothelial
carcinoma with no lamina propria invasion: pTa ( patients;
to years old; mean age . years), high grade papillary
urothelial carcinoma with lamina propria invasion: pT or
with muscularis propria invasion: pT ( patients; to
years old; mean age . years). Regarding normal samples,
only those showing no signs of hyperplasia or dysplasia were
further processed ( samples). Each sample was processed
for Western blotting, immunouorescence, and scanning
electron microscopy.
2.2. Western Blotting. Samples were homogenized in ice-
cold buer (. M Tris-HCl, .% SDS, and mM phenyl-
methylsulfonyl uoride). e lysates were centrifuged and
the protein concentration in the supernatant was determined
by using a BCA protein assay kit (Pierce, Rockford, IL).
From each patient, the protein sample ( 𝜇g/lane) from the
normal urothelium was loaded next to the protein sample
( 𝜇g/lane) from the urothelial tumour. Proteins were size
fractionated on .%, %, or % SDS-polyacrylamide gels
and then transferred to Hybond ECL nitrocellulose mem-
branes (Amersham Biosciences, Buckinghamshire, UK) by
electroblotting. Aer blocking overnight at ∘Cin%skim
milk in phosphate buer saline with .% Tween (PBS-
Tween), membranes were incubated for hours at room
temperature with rabbit polyclonal anti-uroplakin ( : .;
kindly provided by Tung-Tien Sun, New York University
Medical School, USA), guinea pig polyclonal anti-PX
( : ; cat. number NB-, Novus Biologicals, Little-
ton, USA), goat polyclonal anti-PX ( : ; cat. number sc-
, Santa Cruz Biotechnology, Dallas, USA), rabbit poly-
clonalanti-TRPV(:;cat.numberACC-,Alomone
Labs, Jerusalem, Israel), or rabbit polyclonal anti-TRPV
( :; cat. number ab, Abcam, Cambridge, UK).
Aer washing in PBS-Tween, membranes were incubated
for hour, depending on primary antibody either with
horseradish peroxidase-conjugated goat anti-rabbit ( : ;
Santa Cruz Biotechnology), goat anti-guinea pig ( : ;
Santa Cruz Biotechnology), or donkey anti-goat ( : ;
Jackson ImmunoResearch Laboratories, West Baltimore Pike,
USA). Membranes were nally probed with enhanced chemi-
luminescence reagent (ECL; Amersham Biosciences, Buck-
inghamshire, UK) and exposed to X-ray lms. To conrm
equal protein loading, the blots were stripped with Restore
Western Blot Stripping Buer (Pierce, Rockford, IL) and
reprobed with anti-actin antibody (diluted : ; Sigma,
Tauirchen, Germany).
2.3. Immunouorescence. Samples were xed with %
formaldehyde in PBS for . hours at ∘C. ey were washed
and impregnated with % sucrose, embedded in OCT
mounting medium (Tissue Tek, Sakura Finetek Europe B.V.,
e Netherlands), and frozen in liquid nitrogen. Frozen
sections were cut in cryostat at −∘C, collected on glass
slides, and air dried. Sections were incubated in % BSA
in PBS for hour. Immunolabeling was performed using
the same antibodies as for Western blotting: antibodies
against uroplakins ( : .), PX ( : ), PX ( : ),
TRPV ( : ), and TRPV ( : ). Aer washing with
PBS, sections were incubated for minutes either with
rabbit anti-goat AlexaFluor for uroplakins (Molecular
Probes), donkey anti-guinea pig TRITC or FITC for
PX (Jackson ImmunoResearch Europe Ltd.), rabbit
anti-goat AlexaFluor for PX (Invitrogen), or goat anti-
rabbit AlexaFluor for TRPV and TRPV (Molecular
Probes). All secondary antibodies were diluted : in
.% bovine serum albumin (BSA) in PBS. A series of
negative controls were performed, omitting the primary
antibody or incubating sections with nonrelevant antibodies.
Sections were washed, stained with DAPI, and immersed in
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Vectashield embedding medium. Slides were examined with
a uorescence microscope Eclipse TE (Nikon).
2.4. Scanning Electron Microscopy. Samples were xed in
.% paraformaldehyde and % glutaraldehyde for hours.
e samples were postxed in osmium tetroxide, dehydrated
in ethanol, and critical-point dried. Aer sputter-coating with
gold, they were examined at kV with a Jeol JSM A
scanning electron microscope (Jeol Ltd., Tokyo, Japan).
3. Results
3.1. Normal Urothelium. Uroplakins, dierentiation depen-
dent and urothelium-specic transmembrane proteins, were
detected by polyclonal anti-uroplakin antibody, which reacts
strongly with UPIIIa (kDa). e expression of UPIIIa
in all samples of normal human urothelium was positive
(Figure ). To localize uroplakins within urothelium we used
immunouorescence. Normal urothelium showed strong
uroplakin labelling of the supercial cells (Figure (a)).
Scanning electron microscopy revealed large, polygonal cells
covering urothelial surface (Figure (b)). ey had scalloped
appearance with microridges, demonstrating the presence
of urothelial plaques. All features indicated above provided
evidence that umbrella cells formed the supercial cell layer
of normal urothelium, and they were therefore considered
to be terminally dierentiated. Conrming this, we further
analysed the expression and distribution of four nonselective
ion channels.
Western blotting conrmed the expression of PX
(approximately kDa) in the normal urothelium (Figure ).
Antibodies against PX labelled all urothelial cell layers in
all samples of normal urothelium, with more intense labelling
inthesuperciallayer,whereumbrellacellsarelocated
(Figure (c)). Regarding PX, the expression of monomer
protein ( kDa) was conrmed, while dimmer protein
( kDa) was not detected by Western blotting (Figure ). An
additional band of approximately kDa was also obser ved
with anti-PX antibody. In immunouorescence, anti-PX
antibody labelled all urothelial cell layers in all samples of
normal urothelium, with weaker labelling intensity in the
basal than in the supercial layer (Figure (d)).
Data on TRPV molecular weight range from to
kDa. Anti-TRPV antibody, which was used in this study,
revealed the most intense band for TRPV at approximately
kDa and weak band at approximately kDa (Figure ).
Immunolabeling of TRPV was evident in all urothelial cell
layers in all samples of normal urothelium (Figure (e)).
Anti-TRPV antibody used in this study detected band at
kDa, as predicted. TRPV immunolabeling was weak in
basal and intermediate cell layers and moderate in umbrella
cells in all samples of normal urothelium (Figure (f)).
3.2. Low Grade Papillary Urothelial Carcinoma. In low grade
papillary urothelial carcinoma, decreased or abolished uro-
plakin expression was detected (Figure )whencompared
to normal urothelium. In all samples of low grade pap-
illary urothelial carcinoma, immunolabeling of uroplakins
Normal
Low grade
High grade
(kDa)
55
55
55
55
55
35
35
70
70
70
130
95
Actin
TRPV4
TRPV1
P2X5
P2X3
UPIIIa
pT1
pT1
pT1
pT2
pT2
pT2
F : e expression pattern of uroplakins, PX, PX, TRPV,
and TRPV in normal human urothelium and in low and high grade
papillary urothelial carcinomas as determined by Western blotting.
In the protein samples of normal urothelium, UPIIIa, PX, PX,
TRPV, and TRPV are expressed. In low grade carcinoma, there
is no expression of uroplakins. PX and TRPV are expressed as
in normal urothelium, while PX is greatly diminished. TRPV
expression is decreased in comparison to normal urothelium. In
the protein samples of high grade carcinoma with lamina propria
invasion (pT) and those with muscularis propria invasion (pT),
the expression patterns were similar and therefore three examples
of each are presented here. e expression of uroplakins is negative
and expressions of PX and TRPV are substantially decreased
compared to normal urothelium. e expressions of PX and
TRPV are the same as in normal urothelium. Western blots were
done in duplicate. Molecular weights are shown in kilodaltons
(kDa).
was heterogeneous and we were able to discriminate (i)
regions with uroplakin positive labelling of all supercial
cells (Figure (a)), (ii) regions with uroplakin positive and
uroplakin negative supercial cells (Figure (b)), and (iii)
regions with only uroplakin negative supercial cells. Urothe-
lial apical surface displayed altered appearance in comparison
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UP
L
U
LP
(a)
SEM
(b)
L
U
LP
P2X3
(c)
LU
LP
P2X5
(d)
L
U
LP
TRPV1
(e)
L
U
LP
TRPV4
(f)
F : Normal human urothelium (U). (a) Strong uroplakin (UP) immunolabeling (red) is restricted to umbrella cells. (b) Scanning
electron microscopy (SEM) shows large polygonal umbrella cells with microridges on the urothelial apical surface. (c) Antibodies against
PX and (d) PX label (red) all layers of the normal urothelium. e reaction is stronger in umbrella cells than in basal cells. (e) Anti-
TRPV antibody labels (green) all urothelial cell layers. (f) Anti-TRPV labelling (green) is weak in basal and intermediate cell layers and
moderate in umbrella cells. In (a), and (c)–(f) nuclei are labelled blue with DAPI. L = lumen, LP = lamina propria. Scale bars = 𝜇m.
to normal urothelium with disrupted supercial cell layer
and gaps between adjacent supercial cells (Figure (c)).
Supercial cells were not covered with microridges and they
weresmallerincomparisontoumbrellacells.Takentogether,
these results indicated partial dierentiation of supercial
cells in low grade carcinoma.
In the protein samples of low grade carcinoma strong
expression of PX was detected (Figure ). In all sam-
ples of low grade papillary urothelial carcinoma, PX
immunolabeling was similar as in normal urothelium; that
is, the labelling of the supercial cell layer was the strongest
(Figure (d)). Western blotting revealed that PX expression
wasmuchweakerinthelowgradecarcinomathaninnormal
urothelium (Figure ). By PX immunolabeling, two types
of regions were observed in all samples: (i) regions with
immunolabeling of supercial cells and individual inter-
mediate cells (Figure (e)) and (ii) regions with negative
immunolabeling in all cell layers, except weak labelling in
individual supercial cells (Figure (f)).
TRPV expression determined by Western blotting was
weaker in the low grade carcinoma than in normal urothe-
lium (Figure ). Immunouorescence of TRPV revealed
stronger immunolabeling in the basal and in the intermediate
cell layers than in the supercial cell layer (Figure (g)). In
the low grade carcinoma, TRPV expression was stronger or
equaltotheexpressionofTRPVinthenormalurothelium
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UP
L
(a)
UP
L
U
LP
(b)
SEM
∗
(c)
P2X3
(d)
L
P2X5
(e)
L
P2X5
(f)
L
TRPV1
(g)
L
TRPV4
(h)
L
U
TRPV4
(i)
F : Low grade papillary urothelial carcinoma (pTa). (a) Uroplakin (UP) immunolabeling (red) is detected either continuously
throughout the urothelial (U) supercial cell layer or (b) as regions where some supercial cells are uroplakin positive (red) and some
uroplakin negative. (c) Scanning electron microscopy (SEM) reveals altered appearance of the urothelial apical surface in comparison to
normal urothelium. Some neighbouring supercial cells are separated from one another (arrows) and underlying intermediate cell can be
seen (asterisk). (d) PX immunolabeling (red) is positive in all urothelial cell layers with the strongest immunolabeling in the supercial
cells (arrows). (e) Some regions are intensely immunolabeled with anti-PX antibody (red) in the urothelial supercial cell layer and in
individual intermediate cells (arrows), (f) while other regions are PX negative. (g) TRPV immunolabeling (green) is seen in basal and
intermediate cells, but not in supercial cells. (h) In some regions, supercial cells (arrows) are TRPV positive (green), while (i) in other
regions all urothelial cells are TRPV negative. TRPV positive immunolabeling is seen in the compartments of the lamina propria (arrow).
In images (a)-(b) and (d)–(I), nuclei are labelled blue with DAPI. L = lumen, LP = lamina propria. Scale bars = 𝜇m.
(Figure ). Regarding TRPV immunolabeling, two types of
regions were discriminated in all samples: (i) regions with
positive immunolabeling of supercial and intermediate cell
layers (Figure (h)) and (ii) regions with negative reaction
in all urothelial cell layers (Figure (i)). Positive TRPV
immunolabeling was seen in the lamina propria.
3.3. High Grade Papillary Urothelial Carcinoma. We did not
observe any dierence between pT and pT high grade
papillary urothelial carcinoma with respect to the protein
expression and localization studied here. Western blotting
showed that there was no uroplakin expression in these
samples (Figure ) and uroplakin immunolabeling was also
negative in all samples of pT and pT (Figure (a)). Scanning
electron microscopy revealed supercial cells of dierent
sizes, but prevailing ones were smaller than in normal
urothelium (Figure (b)). ey were covered with microvilli
(Figure (b)),whicharefoundonlyonpoorlydierentiated
supercial urothelial cells [].
e expression of PX was greatly decreased in all
samples of the high grade carcinoma when compared to
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UP
L
pT1
(a)
SEM
(b)
P2X3pT2
(c)
P2X5pT2
(d)
TRPV1pT1
(e)
U
LP
TRPV4pT1
(f)
F : High grade papillary urothelial carcinomas (pT or pT). (a) Uroplakin (UP) labelling (red) is negative. (b) Scanning electron
microscopy (SEM) shows that supercial urothelial cells are small and polymorphic. ey have microvilli on their apical surface. (c) Antibody
againstPXweaklylabels(green)urothelialcells.(d)PXlabelling(red) is present in all urothelial cells. Nuclei of some cells are also labelled.
(e) TRPV labelling is negative. (f) Weak labelling of TRPV in urothelial cells (U) is seen, but strong TRPV labelling (arrows) is seen in the
compartments of the lamina propria (LP). In images (a) and (c)–(f), nuclei are labelled blue with DAPI. L = lumen. Scale bars = 𝜇m.
normal urothelium or to low grade carcinoma (Figure ). In
all samples of high grade carcinoma, antibodies against PX
weakly labelled all cell layers (Figure (c)). Western blotting
conrmed the expression of monomer form of PX, which
was similar in pT and pT protein samples (Figure ). As
observedbyWesternblotting,theexpressionofPXwas
higher in high grade than in low grade carcinoma. PX
was labelled in all cell layers of all pT and pT samples
(Figure (d)). By Western blotting, the expression of TRPV
was the lowest in high grade carcinoma in comparison to
normal urothelium and to low grade carcinoma (Figure ).
By immunouorescence, all samples of high grade carcinoma
were TRPV negative (Figure (e)). TRPV expression in
all samples of high grade carcinoma was similar to TRPV
expressioninnormalurotheliumaswellasinlowgradecarci-
noma (Figure ). Immunolabeling was negative or weak in all
cell layers of all high grade carcinoma samples (Figure (f)).
Positive TRPV immunolabeling was detected in the lamina
propria.
4. Discussion
Unique dierentiation of normal urothelium has been inves-
tigated since the s, while its sensory role has been discov-
ered only recently [,]. Among sensory proteins, members
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Normal Low grade High grade
UPIIIa
Apical surface Large cells
microridges
Large and small cells
no microridges
Small cells
microvilli
Dierentiation Terminal Partial Poor
P2X3
P2X5
TRPV1
TRPV4
Correlation with
dierentiation
Positive
No
Positive
No
F : Summarised results of immunouorescence, Western blotting, and sc anning electron microscopy in normal human urothelium and
in low and high grade papillary urothelial carcinomas. Uroplakin UPIIIa expression and apical surface appearance indicate dierentiation
stages of urothelial cells. PX, PX, TRPV, and TRPV expressions are illustrated and their correlations with dierentiation stage of
urothelial cells are presented. Black squares indicate high protein expression, dashed squares denote moderate protein expression, and white
squares represent no protein expression.
of purinergic PX receptors and transient receptor potential
vanilloid (TRPV) channels are under intense investigation.
e majority of studies have been conducted on animal
tissues or cell culture models, while very little is known about
their distribution and function in normal human urothelium
or in urothelial tumours. We investigated the correlation
between urothelial dierentiation and sensory function-
related proteins PX, PX, TRPV, and TRPV in normal
human urothelium and in low and high grade papillary
urothelial carcinomas. All our results are summarised in
Figure .
First we evaluated the dierentiation of normal urothe-
lium and that of low and high grade carcinomas. Two
well-established criteria were used to determine urothelial
cell dierentiation. One is the expression and localization
of uroplakins and the second criterion is the appearance
of urothelial apical surface. In normal urothelium, there
was strong expression of uroplakins, which were localized
in supercial urothelial cell layer. Apical surface was scal-
loped, with microridges covering the umbrella cells. ese
characteristics demonstrate terminal dierentiation of the
urothelium, as proposed previously []. In low grade car-
cinoma decrease in uroplakin expression was detected and
altered urothelial apical surface appearance was observed.
Both criteria provide evidence for partial dierentiation of
low grade carcinoma [,]. In high grade carcinoma there
was no expression of uroplakins and supercial cells, which
were covered with microvilli, were small and polymorphic.
All these features point to poor dierentiation of high grade
carcinoma. Decreased urothelial dierentiation is usually
associated with incomplete barrier formation and bladder
dysfunction. Since urothelial carcinoma may cause LUTS, we
hypothesised that this involves also changes of some sensory
proteins expression and localization.
Since the rst demonstration of the PX receptor in the
human urothelium [], only few studies conrmed the pres-
ence of PX protein [], while expression analyses revealed
no PX mRNA in the human urothelium []. ese could
be assigned to factors such as translational regulation, mRNA
stability, and half-life of a protein []. In the present
study, PX immunoreactivity was observed throughout all
urothelial cell layers of normal urothelium. Umbrella cells,
which were terminally dierentiated, were labelled stronger
than cells in other layers, which is in coincidence with
previous results []. Since dierentiation of the urothelium
progresses from basal cells to umbrella cells, it seems that
PX expression is related to cell dierentiation stage. In
low grade carcinoma we conrmed the expression of PX.
Moreover, the expression and localization of PX in partially
dierentiatedlowgradecarcinoma,wheresometerminally
dierentiated supercial cells were preserved, were similar as
in normal urothelium. In high grade carcinoma PX expres-
sion was decreased when compared to normal urothelium
and low grade carcinoma. Since high grade carcinoma was
poorly dierentiated, these results conrmed our abovemen-
tioned assumption that positive correlation exists between
the expression of PX and urothelial dierentiation.
e expression of PX in normal urothelium was con-
rmed, which is in coincidence with other reports []. e
majority of low grade carcinoma samples exhibited no PX
expression, while only few showed positive immunolabeling
in the supercial urothelial cells. In poorly dierentiated
highgradecarcinomastrongexpressionofPXinall
cell layers was detected. Western blotting revealed similar
level of PX expression in the protein samples of highly
dierentiated normal urothelium and of poorly dierentiated
highgradecarcinoma.ItwasshownthatATPsignicantly
reduced cell proliferation in high grade bladder cancer and
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pharmacological proling implicated PX receptor in this
antineoplastic response []. To our knowledge we showed
for the rst time that PX receptors are expressed by the high
grade papillary urothelial carcinoma.
It is known that bladder distension causes urothelial ATP
release, which can directly depolarize and initiate ring in
sensory nerves by activating PX receptors []. Sensation
and micturition are complex and not yet fully understood
processes, which are altered during progression of diverse
bladder diseases and related to various LUTS. erefore, we
suppose that decreased expression of PX and increased
expression of PX detected in high grade carcinoma might
be involved in pathogenesis and LUTS manifestations of this
kind of carcinoma.
e remarkable nding that TRPV is not only expressed
byaerentnervesbutalsointheurothelium[,]gave
rise to intensive studies of its expression and localization.
Our results showed TRPV expression in normal urothelium
with terminally dierentiated umbrella cells. TRPV was
not restricted to umbrella cells, as reported previously [].
Moreover, downregulation of TRPV in urothelial cancers of
human bladder was determined [,]andthehypothesis
that TRPV is involved in dierentiation was postulated
[]. Our results support this hypothesis, since in partially
dierentiated low grade and in poorly dierentiated high
grade carcinoma its expression was decreased and abolished,
respectively.
Several studies localized TRPV throughout all urothelial
cell layers of normal urothelium [,,]. Our results
conrmed this data and showed variability among the levels
of TRPV expression in the normal urothelium. We could
not nd any pattern in these ndings that would point to any
correlation between urothelial dierentiation and TRPV
channels. To our knowledge, there are no data about TRPV
expression in urinary bladder tumours. Although Western
blotting showed no variations in the expression of TRPV in
normalurotheliumandinlowandhighgradecarcinomas,
immunouorescence revealed great diversity among dierent
parts of the carcinomas. Some parts exhibited strong TRPV
immunolabeling, while in others immunolabeling was weak
and even parts with negative reaction were detected. It seems
that TRPV was not correlated with urothelial dierentiation
and further research is necessary to clarify its role in bladder
carcinogenesis.
5. Conclusions
Our results show that PX is in correlation with urothelial
dierentiation and might be involved in high grade papillary
carcinoma pathogenesis. We also conrm the correlation
of TRPV with urothelial dierentiation stage. Moreover,
our study supports previous proposal that TRPV recep-
torshouldbeacceptedasanegativeprognosticfactorin
patients with urothelial carcinoma. Regarding PX and
TRPV no direct correlation between their expression and
urothelial dierentiation is demonstrated. Nevertheless, new
aspects concerning their localization variability in urothelial
papillary carcinoma emerged indicating that they have a role
in bladder functioning during pathogenesis.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
Acknowledgments
e authors are grateful to Nada Pavlica, Sabina ˇ
Zeleznik,
Sanja ˇ
Cabraja, and Linda ˇ
Strus for their technical assistance.
ey thank Professor T.T. Sun of New York University School
of Medicine for donating primary antibodies used in this
study. is study was supported by a Grant from Ministry
of Higher Education, Science and Technology, Slovenia (P-
).
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