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Epithelial to Mesenchymal Transition and Peritoneal


Membrane Failure in Peritoneal Dialysis Patients:
Pathologic Significance and Potential Therapeutic
Interventions
Luiz S. Aroeira,* Abelardo Aguilera,* Jose A. Sanchez-Tomero, M. Auxiliadora Bajo,
Gloria del Peso, Jose A. Jimenez-Heffernan, Rafael Selgas, and Manuel Lopez-Cabrera*
*Unidad de Biologa Molecular and Servicio de Nefrologa, Hospital Universitario de la Princesa, and Servicio de
Nefrologa, Hospital Universitario La Paz, Madrid, and Departamento de Patologa, Hospital Universitario de
Guadalajara, Guadalajara, Spain

ABSTRACT
Peritoneal dialysis (PD) is a form of renal replacement and is based on the use of
the peritoneum as a semipermeable membrane across which ultrafiltration and
diffusion take place. Nevertheless, continuous exposure to bioincompatible PD
solutions and episodes of peritonitis or hemoperitoneum cause acute and chronic
inflammation and injury to the peritoneal membrane, which progressively undergoes fibrosis and angiogenesis and, ultimately, ultrafiltration failure. The pathophysiologic mechanisms that are involved in peritoneal functional impairment have
remained elusive. Resident fibroblasts and infiltrating inflammatory cells have been
considered the main entities that are responsible for structural and functional
alterations of the peritoneum. Recent findings, however, demonstrated that new
fibroblastic cells may arise from local conversion of mesothelial cells (MC) by
epithelial-to-mesenchymal transition (EMT) during the inflammatory and repair
responses that are induced by PD and pointed to MC as protagonists of peritoneal
membrane deterioration. Submesothelial myofibroblasts, which participate in inflammatory responses, extracellular matrix accumulation, and angiogenesis, can
originate from activated resident fibroblasts and from MC through EMT. This
heterogeneous origin of myofibroblasts reveals new pathogenic mechanisms and
offers novel therapeutic possibilities. This article provides a comprehensive review
of recent advances on understanding the mechanisms that are implicated in
peritoneal structural alterations, which have allowed the identification of the EMT
of MC as a potential therapeutic target of membrane failure.
J Am Soc Nephrol 18: 2004 2013, 2007. doi: 10.1681/ASN.2006111292

In the past decades, peritoneal dialysis


(PD) has become an established alternative to hemodialysis for the treatment of
ESRD, and the number of patients who
are in PD programs has increased progressively worldwide, especially in some
Asian countries. One of the most important challenges in PD is the long-term
preservation of the peritoneal membrane
integrity.1,2 Damage to the peritoneum is
2004

ISSN : 1046-6673/1807-2004

a serious event in PD, because it may


jeopardize the organ on which the whole
treatment is based.13 The morphology
of the peritoneum is simple; A single
layer of mesothelial cells (MC) covers a
submesothelial region that is composed
of connective tissue with few fibroblasts,
mast cells, macrophages, and vessels.4
The nonphysiologic nature of dialysis
fluids and the uremic status are consid-

ered the main etiologic factors that lead


to the functional decline of the peritoneal
membrane.1 They induce a sustained situation of peritoneal chronic inflammation that can be exacerbated periodically
by recurrent or acute episodes of peritonitis or hemoperitoneum. Closely linked
to the inflammatory response is the reparative process. Its activation is responsible for many of the structural abnormalities of the peritoneal membrane,
including loss of MC monolayer, submesothelial fibrosis, angiogenesis, and hyalinizing vasculopathy.5 8 Such alterations are considered the major cause of
ultrafiltration failure and loss of the dialytic capacity of the peritoneum.
There are two different pathologic
forms of PD-related fibrosis. The most
common is simple peritoneal sclerosis,
which appears in almost all patients. The
degree of fibrosis is usually mild and
shows a relation with time on dialysis.4
On the other end of the spectrum is encapsulating peritoneal sclerosis, which is
an uncommon form of sclerosis that
Published online ahead of print. Publication date
available at www.jasn.org.
Correspondence: Dr. Manuel Lopez-Cabrera, Unidad de Biologa Molecular, Hospital Universitario
de la Princesa, C/ Diego de Leon 62. 28006-Madrid,
Spain. Phone: 34-91-5202334; Fax: 34-915202374; E-mail: mlopez.hlpr@salud.madrid.org
Copyright 2007 by the American Society of
Nephrology

J Am Soc Nephrol 18: 2004 2013, 2007

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evolves rapidly with intense fibrosis, inflammation, and fibrin deposits. It is a


life-threatening condition that in many
cases evolves to visceral encapsulation
and progresses even if the patient is removed from PD.13 It is well established,
however, that fibrosis is not the unique
structural alteration of the peritoneal
membrane induced by PD. Besides this
alteration, the peritoneum shows an increase of capillary number (angiogenesis) and vasculopathy.5 There is increasing evidence that fibrosis, angiogenesis,
and probably augmented vessel permeability are key determinants of ultrafiltration dysfunction.1,9,10 The relationship between peritoneal fibrosis and
angiogenesis has not been clearly defined. In animal models of PD, it has
been shown that fibrosis and angiogenesis may be two separate responses to peritoneal injury.8 However, in PD patients,
it is likely that fibrosis and angiogenesis
are intimately and closely related in the
response of the peritoneum to continuous injury.8
Recent works have begun to identify
the mechanisms that are involved in the
pathogenesis of peritoneal membrane
failure during long-term PD. Resident
stromal fibroblasts and inflammatory
cells have been classically considered
the main cells responsible for structural
and functional peritoneal alterations,
whereas MC have been considered mere
victims of peritoneal injury. However,
more recently, it has been shown that
MC also play an active role in peritoneal
membrane alteration. It has been demonstrated that, soon after PD is initiated,
peritoneal MC show a progressive loss of
epithelial phenotype and acquire myofibroblast-like characteristics by an epithelial-mesenchymal transition (EMT).11 MC
that have undergone an EMT acquire
higher migratory and invasive capacities,
which allow these cells to invade the submesothelial stroma, where they contribute
to peritoneal fibrosis and angiogenesis and
ultimately lead to peritoneal membrane
failure.1113 The myofibroblastic conversion of MC has been confirmed in an in
vivo animal model based in the injection of
an adenovirus vector that transferred active TGF-1 in rat peritoneum.14 In these
J Am Soc Nephrol 18: 2004 2013, 2007

studies, EMT appears as the central point


in the early pathogenesis of peritoneal
damage associated with PD. Given that
there is no definitive treatment for the progressive loss of the dialytic capacity of the
peritoneum, the identification of the EMT
of MC as a key process in the onset and
progression of peritoneal fibrosis and angiogenesis opens new insights for therapeutic intervention.

PATHOGENESIS OF PERITONEAL
MEMBRANE FAILURE

PD for ESRD treatment has been used for


almost 30 yr.3 The two first decades of the
modern era of PD were dedicated to
catheter design, technical aspects, and
peritonitis prevention. The third decade
was devoted to improving the biocompatibility of the solutions with the expectancy of diminishing their adverse effects on peritoneal morphology and
function.1 PD is a successful alternative
to hemodialysis, and several studies have
confirmed equivalent adequacy, mortality, and fluid balance status with both
modalities, at least for the first 4 to 5 yr.15
However, the growth of PD continues to
be limited by the membrane incapacity
to perform adequate diffusive and/or
convective transports in the long term.1,2
Peritonitis and ultrafiltration failure,
with a clinical result of extracellular volume overload and an increased cardiovascular risk, are the major factors that
contribute to technique dropouts.1,3,9
Our overview of the morpho-functional relationship in peritoneal membrane failure has changed as a result of
some recent experimental data. Some
studies of peritoneal biopsies have suggested that hyalinizing vasculopathy and
angiogenesis are the most characteristic
features in PD-related peritoneal pathology, at least in patients with severe membrane failure.5,6 In contrast, a more recent report by Sherif et al.10 showed that
in stable, uncomplicated PD patients,
vascular density does not increase,
whereas intact vessels decrease with time
of treatment and severe vasculopathy
predominate mostly in long-term PD.
Other studies have demonstrated that

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before any other lesions are evident, human peritoneal biopsies show an EMT of
the MC and the consequent environmental change, mainly represented by
the accumulation of collagen I and fibronectin.12 In fact, the submesothelial
thickening is the constant change found
in peritoneal biopsies after a time on
PD.57,12 In animal models that seem to
reproduce the sequence of phenomena
that occur in PD patients, Margetts et
al.16 demonstrated that the transduction
of TGF- into rat peritoneum causes
peritoneal sclerosis and angiogenesis,
and both processes are followed by the
EMT of MC.14
It is worthy of remark that our understanding of peritoneal functional outcome in response to PD requires a deep
knowledge of the starting point because
of initial functional diversity of the human peritoneum. It can be hypothesized
that the diversity in solute and water
transports at PD initiation may represent
different tissue responses to similar components of dialysates. Furthermore, different starting points followed by diverse
reactions to PD at midterm can generate
a wide spectrum of peritoneal morphofunctional scenarios.17 After molecular
and cellular biology studies of the intimate processes that take place into the
peritoneum, a refinement in therapeutic
strategies is expected. Therefore, it can be
proposed that the fourth decade of PD
should be conducted to explore molecular aspects of the peritoneal response.

PERITONEAL INFLAMMATORY
RESPONSE TO INJURY AND
INFECTION

Exposure to bioincompatible fluids and


episodes of bacterial and fungal infection
or hemoperitoneum induce situations of
acute and chronic inflammation that
cause damage to the peritoneal tissue.
The successful repair of injured tissue requires a tightly controlled response to
limit the structural alteration. The peritoneal immune response to injury or infection involves, among other cells, MC
and resident macrophages that work in a
coordinated manner to recruit other inEMT and Peritoneal Membrane Failure

2005

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flammatory cells, including mononuclear phagocytes, lymphocytes, and neutrophils.18 MC and infiltrating immune
cells can produce a wide number of cytokines, growth factors, and chemokines to
establish a complex network that feeds
back, resulting in acute or chronic inflammation, which leads to membrane
deterioration.18 21 Many of these inflammatory mediators, such as TNF-, IL-1,
IL-8, TGF-, and fibroblast growth factor-2 (FGF-2), may have a role in peritoneal fibrosis by stimulating resident fibroblasts proliferation and extracellular
matrix (ECM) component deposition22
and by inducing EMT of MC,11 which
further increases the number of peritoneal fibroblasts.12 In addition, IL-8,
FGF-2, and especially vascular endothelial growth factor (VEGF) may induce an
increase of peritoneal capillary number
and probably vessel permeability,13,19
causing an increase of small-solute transport, which characterize ultrafiltration
failure.
Even in the absence of infection or hemoperitoneum, it is thought that PD patients have a low-grade chronic inflammation that promotes a progressive
structural alteration of the peritoneal
membrane. This could be due to the mechanical injury during PD fluid exchanges and also to nonphysiologic composition of the fluids. Prolonged
exposure of the peritoneum to glucose,
the most common osmotic agent in PD
fluids, or to Amadori adducts, formed by
the condensation between glucose and
reactive amino groups of proteins, induces the synthesis of inflammatory, fibrogenic, and angiogenic factors by macrophages23 and MC.24,25 Glucose may
also promote peritoneal inflammation
through a leptin-dependent mechanism.26 In this context, adipocytes that
are exposed to glucose produce leptin,
which in turn promotes TGF- production by MC.27 This evidence suggest that
peritoneal inflammation is a key triggering process for peritoneal membrane
failure; therefore, the knowledge of the
mechanisms that are involved in its regulation may serve in the design of therapeutic interventions.
2006

MAJOR MEDIATORS OF
PERITONEAL MEMBRANE
DETERIORATION

Chronic and acute inflammations are believed to be key determinants in the onset
and progression of peritoneal membrane
structural and functional alteration.
Among the cytokines and growth factors
produced during peritoneal inflammation, TGF-, a strong profibrotic cytokine,28 is considered the master molecule
in the genesis of peritoneal fibrosis, because its overexpression has been correlated to worse PD outcomes.19 The relevance of TGF- in peritoneal fibrosis has
been demonstrated in an in vivo rat
model, in which TGF- gene was transduced into the peritoneal cavity with an
adenovirus vector, reproducing the
structural and functional alterations that
are observed in PD patients.16 Along with
TGF-, other proinflammatory and profibrotic cytokines as well as angiotensin
II (AngII) have been shown to be upregulated during peritonitis episodes and
may contribute in the setting of peritoneal fibrosis.8,29 In regard to peritoneal
angiogenesis, it has been shown that local
production of the proangiogenic and vasoactive factor VEGF during PD plays a
central role in increased solute transport
across the peritoneum and ultrafiltration
failure.13,30,31
The most important factor of the PD
solutions that are responsible for peritoneal deterioration seems to be glucose
degradation products, which, through
the formation of advanced glycation end
products (AGE), stimulate the production of (ECM) components as well as the
synthesis of cytokines and growth factors, including TGF- and VEGF.8 Furthermore, AGE may also induce EMT of
MC.32 Several studies have demonstrated
the appearance of AGE in the peritoneal
effluents of PD patients, which correlated with the time on PD treatment. Biopsy studies have confirmed the accumulation of AGE in the peritoneal tissues
of PD patients. The intensity of AGE accumulation is associated with fibrosis
and ultrafiltration dysfunction.8
The uremic status also contributes to
the accumulation of AGE and may affect

Journal of the American Society of Nephrology

the anatomy of the peritoneal membrane


and its transport characteristics. In this
context, the peritoneum of partially nephrectomized rats showed increased permeability associated with upregulation
of nitric oxide synthase isoforms, VEGF,
and FGF-2 and accumulation of AGE.33
Despite these findings in animal models,
the effect of uremia itself on the peritoneum in humans is controversial. Two
human peritoneal biopsy studies have
shown a modest compact zone thickening and vasculopathy in predialysis renal
patients.5,7 In contrast, in other studies,
no significant fibrosis or vasculopathy
was observed in uremic non-PD patients.12

SUBPOPULATIONS OF
PERITONEAL FIBROBLASTS

Fibroblasts represent a dynamic population of cells that show functional and


phenotypic diversity. Among the various
fibroblastic phenotypes, the myofibroblast is of the greatest importance. The
term myofibroblast defines a cell with
intermediate features between a fibroblast and a smooth muscle cell and is
characterized by the expression of
-smooth muscle actin (-SMA). Myofibroblasts have been reported as important protagonists of almost all situations
of repair and fibrosis in various pathologies. Their capacity to synthesize ECM,
growth factors, cytokines, and participation in the inflammatory response, as
well as their contractile properties, convert them in the most important fibroblastic phenotype.34
In normal peritoneum or in the peritoneum of uremic non-PD patients,
myofibroblasts are not present, but the
resident fibroblasts show an intense expression of CD34, an antigen characteristic of bone marrow stem cells (Figure
1). The expression of CD34 gradually
disappears in PD patients during the onset of peritoneal fibrosis.12 The significance of the loss of CD34 expression is
unknown but seems to correlate with the
appearance of the myofibroblastic phenotype.12 Tissue CD34 fibroblasts are
closely related to circulating CD34 fiJ Am Soc Nephrol 18: 2004 2013, 2007

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Figure 1. Subpopulations of peritoneal fibroblasts in peritoneal membranes of normal


and peritoneal dialysis (PD) patients. (A) The morphology of normal peritoneum is simple,
with a single layer of mesothelial cells (MC) that covers a submesothelial region that is
composed of connective tissue with few fibroblasts and some vessels. In normal peritoneum, myofibroblasts are not present, whereas resident fibroblasts show an intense
expression of CD34, an antigen that is characteristic of bone marrow stem cells. Peritoneal CD34 fibroblasts do not express fibrocyte markers, suggesting that they are simply
residual embryonic mesenchymal cells that remained in the peritoneal tissue after organogenesis. (B) In PD patients, there are many structural abnormalities of the peritoneal
membrane, including loss of MC monolayer, increased number of fibroblasts, submesothelial fibrosis, and augmented vessel number. In contrast to normal peritoneum,
myofibroblasts can be easily detected in the peritoneal membrane of many patients who
undergo PD. The myofibroblasts may originate from activated resident fibroblasts, from
circulating cells (fibrocytes), and from MC through an epithelial-to-mesenchymal transition (EMT). The myofibroblastic conversion of MC can be observed by immunohistochemical analysis, which reveals the presence of fibroblast-like cells embedded in the
compact zone expressing mesothelial markers such as cytokeratins, intercellular adhesion
molecule-1, and calretinin. Peritoneal myofibroblasts express vascular endothelial growth
factor (VEGF), extracellular matrix (ECM), and cyclooxygenase-2 (COX-2), indicating that
these cells are implicated in peritoneal structural alterations that are induced by PD. The
proportions of peritoneal myofibroblasts that originated from resident fibroblasts, circulating fibrocytes, and MC remain to be established.

brocytes and reflect a bone marrow origin. Fibrocytes were described as a small
subpopulation of circulating leukocytes
that express collagen I, CD45RO, CD13,
J Am Soc Nephrol 18: 2004 2013, 2007

CD11b, CD34, CD86, and MHC class II,


which transform into myofibroblasts
when exposed to TGF- in vitro.35 However, peritoneal CD34 fibroblasts do

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not express these fibrocyte markers, suggesting that they are simply residual embryonic mesenchymal cells that remained in the peritoneal tissue after
organogenesis (Figure 1). In contrast to
normal peritoneum, myofibroblasts can
be easily detected in the peritoneal membrane of many patients who undergo PD
treatment,6 even at very early stages of
PD and preceding the appearance of fibrosis (Figure 1).12
There is evidence that myofibroblasts
may originate from different sources.
First, they may differentiate from resident fibroblasts.34 Second, they may derive from circulating cells (fibrocytes)
that are recruited to injured tissues.35 Finally, they can originate from nearby epithelial cells through an EMT. The contribution of EMT as an important source
of myofibroblasts was initially described
in animal models of renal fibrosis.36 In
the peritoneum, the proportions of myofibroblasts that originated from resident
fibroblasts, circulating fibrocytes, and
MC remain to be established. Similarly,
the dynamics in which EMT takes place
has to be defined. Most probably, it is not
a uniform process, and several factors
may result in variations of its intensity.
Among others, the episodes of peritonitis
must have an influence on EMT.12

KEY CELLULAR AND MOLECULAR


EVENTS DURING EMT

Turning an epithelial cell into a mesenchymal cell is a complex and step-wise


process that requires alterations in cellular architecture and behavior and a profound molecular reprogramming with
new biochemical instruction.36 38 As illustrated in Figure 2, the EMT starts with
the dissociation of intercellular junctions, as a result of downregulation of adhesion molecules such as E-cadherin,
claudins, occludins, zona occludens-1,
and desmoplakin, and with the loss of
microvilli and apical-basal polarity.
Then, the cells adopt a front-back polarity, as a result of cytoskeleton reorganization, and acquire -SMA expression and
increased migratory capacity. In the latest stages of EMT, the cells acquire the
EMT and Peritoneal Membrane Failure

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EMT
Tight-junction dissociation
Loss of microvilli

Adherant-junction and
desmosome dissociation
Loss of apical-basal polarity

EMT effectors

SMA expression
Cytoskeleton reorganization
Front-back polarity
Migration

MMPs up-regulation
Basement membrane
degradation
Invasion

EPITHELIAL MARKERS

MESENCHYMAL MARKERS

E-Cadherin
Claudins
Occludins
ZO-1
Desmoplakin
Cytokeratins

N-Cadherin
Fibronectin
Collagen I/III
Snail
SMA
Vimentin

Figure 2. Key events during EMT. The diagram shows four key steps that are essential for the completion of the entire EMT course and
the most commonly used epithelial and mesenchymal markers.

capacity to degrade the basement membrane and to invade the fibrotic stroma
by upregulating the expression of matrix
metalloproteinases (MMP). Other commonly used molecular markers for EMT
include the downregulation of cytokeratins; upregulation of vimentin, N-cadherin, and transcription factor snail; and

increased production of ECM components.


Besides these classic markers, there
are some other markers as well as a number of biochemical changes that define
the EMT (summarized in Table 1). EMT
can be easily engaged by combinations of
a wide spectrum of extracellular stimuli,

including cytokines, growth factors,


AGE, MMP, and ECM components such
as collagen I. It is worthwhile to point out
that most of these EMT regulators have
been identified by using in vitro cell culture system in which epithelial cells were
treated with purified factors, either alone
or in combination, at high concentra-

Table 1. Molecular and functional patterns of EMTa


Inducers/promoters of EMT
TGF-
FGF-2
EGF
AngII
PDGF
IL-1
AGE
MMP-2 and -3
collagen I
Proteins activated
ILK
Wnt
MAPK
PI3-K
Src
Ras/Rho GTPases
ROCK
Proteins inhibited
GSK-3

Proteins upregulated
N-Cadherin
Snail
vimentin
TGF-
fibronectin
collagen I/III
-SMA
FGF-1 and -2
MMP-2 and -9
FSP-1
PAI-1
Proteins downregulated
E-cadherin
cytokeratins
claudins
occludins
desmoplakin
ZO-1
mucin-1
tPA

Proteins in the nucleus


-catenin
LEF1/TCF
Snail
Smad-2/3
NF-B
FGF-2/FGFR-1
Negative regulators
Smad co-repressors
Smad-5
Smad-7
Cellular changes
front-back polarity
increased migration
increased invasion
increased scattering
degreased fibrinolysis
growth arrest
survival

AGE, advanced glycation end products; AngII, angiotensin II; EMT, epithelial-to-mesenchymal transition; FGF, fibroblast growth factor; FGFR-1, fibroblast
growth factor receptor-1; FSP-1, fibroblast-specific protein-1; GSK-3, glycogen synthase kinase-3; ILK, integrin-linked kinase; LEF1/TCF, lymphoid enhancer
factor/T-cell factor; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; PAI-1, plasminogen activator inhibitor-1; PI3-K,
phosphatidylinositol-3-kinase; ROCK, Rho-activated kinase; -SMA, -smooth muscle actin; tPA, tissue plasminogen activator; ZO-1, zona occludens-1.

2008

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tions. These fractionated studies, although necessary to define the potential


role of each individual factor in regulating EMT, did not mimic the real situation in vivo, in which the EMT regulators
are present at low concentrations. Therefore, an EMT in vivo may result from an
integration of diverse signals triggered by
multiple factors, being difficult to assign
priorities or hierarchy.
Receptor-mediated signaling in response to these factors triggers the activation of a complex network of intracellular effector molecules, such as Ras/Rho
GTPases, Rho-activated kinase, tyrosinekinase Src, integrin-linked kinase (ILK),
Wnt-1, Smad 2 and 3, the mitogen-activated protein kinases p38 and extracellularsignal regulated kinases, and phosphatidylinositol-3-kinase (Table 1).
These effectors orchestrate the dissociation of intercellular adhesion complexes,
the changes in cytoskeletal organization,
and the acquisition of migratory and invasive capacities that occur during
EMT.36 38
A central target of some of these signaling pathways is glycogen-synthase kinase-3 (GSK-3), which has been
shown to phosphorylate -catenin and
the transcriptional repressor Snail, leading to their ubiquitinization and degradation via the proteasome. The phosphorylation of GSK-3 by extracellular
signal regulated kinases, ILK, Wnt-1, or
phosphatidylinositol-3-kinase leads to

its functional inhibition. As a result,


-catenin is stabilized and localizes to
the nucleus, where it feeds into the Wnt
signaling pathway by interacting with
lymphoid enhancer factor-1/T cell factor. In addition, the inhibition of
GSK-3 drives the stabilization and nuclear translocation of Snail, a potent
transcriptional repressor of E-cadherin
and other intercellular adhesion molecules, and inducer of cell growth arrest
and survival.39 The regulation of signaling pathways also results in the activation
and nuclear translocation of other transcription factors, such as Smads 2 and 3,
NF-B, and ligand-bound FGF receptor
1, which in conjunction with -catenin,
lymphoid enhancer factor-1/T cell factor, and Snail, repress the expression of
epithelial markers and engage the EMT
transcriptome.36 38 It is important to
point out that EMT is a reversible process, at least in the early stages. Therefore, molecules that negatively regulate
EMT and that promote mesenchymalto-epithelial transition must exist (Table
1). In this context, two endogenous factors, namely hepatocyte growth factor
(HGF) and bone morphogenetic protein-7 (BMP-7), have been demonstrated to block and reverse EMT by
inducing the expression of the transcriptional co-repressors, in the case of HGF,
or by activating Smad-5, in the case of
BMP-7, which interfere with TGF-activated Smad-2/3. Furthermore, Smad-7

Table 2. Molecular markers of mesothelial cells and fibroblastsa


Molecular Marker
E-cadherin
N-cadherin
Snail (mRNA)
ICAM-I
Cytokeratins
Vimentin
Calretinin
CA125
VEGF
COX-2
Fibronectin
Collagen I
-SMA

Peritoneal Dialysis Effluent


Omentum

Epithelioid

Fibroblast-Like

Fibroblast

ICAM-1, intercellular adhesion molecule-1; CA125, cancer antigen 125; VEGF, vascular endothelial
growth factor; COX-2, cyclooxygenase-2.
In activated fibroblasts.

J Am Soc Nephrol 18: 2004 2013, 2007

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is another molecule that negatively regulates EMT by blocking Smad-2 phosphorylation and activation.36 38

EVIDENCE FOR EMT OF MC IN PD


PATIENTS

The presence of EMT in the peritoneum


of PD patients was first demonstrated in
a landmark paper that published in
2003.11 It was described that confluent
MC cultures from PD effluents may
show epithelioid and nonepithelioid (fibroblast-like) morphologies. The frequency of nonepithelioid MC was associated with time on PD and with episodes
of peritonitis or hemoperitoneum. MC
from effluents showed high expression of
intercellular adhesion molecule-1 independent of their morphology. In contrast, this mesothelial marker was negligible on cultured peritoneal fibroblasts,
supporting that effluent nonepithelioid
cells had a mesothelial origin and were
not fibroblast contaminations. The analysis of the epithelial markers cytokeratins
and E-cadherin was also important to
determine more precisely the nature of
effluent-derived cells. High expression of
cytokeratins and E-cadherin was observed only in omentum-derived MC,
whereas effluent-derived cells showed a
progressive reduction in the expression
of these molecules, although even fibroblast-like MC maintained a small population of positive cells. Fibroblasts were
completely negative for these two markers. The phenotype changes in effluent
MC were indicative of an EMT. Additional evidence that the PD-induced
changes of the MC were due to an EMT
came from the analysis of the expression
of the transcription factor Snail, a potent
inducer of EMT. Omentum-derived MC
did not express Snail mRNA, whereas a
progressive expression of this mRNA was
observed in effluent MC preparations
along the transdifferentiation process.11
Besides these molecules, there are some
other markers that may help to distinguish effluent MC from contaminating
fibroblasts as well as to establish the various stages of transdifferentiation of MC
(summarized in Table 2).
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Glucose (AGEs/GDPs)
Low pH
Mechanical injury
Hemoperitoneum
Inflammation:
(TGF-, IL-1, FGF2, TNF-)
Angiotensin II

VEGF

Angiogenesis

EMT
tPA (Less fibrinolysis)
5
2

3
4

Peritoneal
membrane
failure

ECM (Fibrosis)

MMPs
Invasion

Figure 3. Therapeutic strategies for peritoneal membrane failure based on EMT of MC.
EMT of MC in vivo results from integrated signals that are induced by multiple stimuli.
These include elevated glucose and glucose degradation products (GDP) and concentration of PD fluids, which through the formation of advanced glycation-end products
(AGE) stimulate the transdifferentiation of MC. The formation of AGE may also be due to
the uremic status of the PD patients. The low pH of the dialysates and the mechanical
injury during PD fluid exchanges may cause tissue irritation and contribute to chronic
inflammation of the peritoneum, which promote EMT of MC. Episodes of bacterial or
fungal infections or hemoperitoneum cause acute inflammation and upregulation of
cytokines and growth factors such as TGF-, IL-1, fibroblast growth factor-2 (FGF-2),
TNF-, and angiotensin II (AngII), among others, which are strong inducers of EMT. The
therapeutic strategies may be designed either to prevent or to reverse the EMT itself or
to treat its effects such as cellular invasion, fibrosis, or angiogenesis. The diagram
illustrates six steps related to the EMT process of the MC that can be clinically managed,
alone or in combination, to prevent peritoneal membrane failure. See text for details.

The mesenchymal conversion of MC


could also be observed in vivo in the peritoneum as a response to PD. Immunohistochemical analysis of peritoneal biopsies from PD patients revealed the
presence of fibroblast-like cells embedded in the compact zone expressing mesothelial markers. In addition, these peritoneal biopsies showed expression of
-SMA in the fibrotic stroma, especially
in the upper submesothelial level, and
in many cases, these myofibroblasts
showed coexpression of cytokeratins
(Figure 1).12 These results indicated that
new myofibroblastic cells may arise from
local conversion of MC by EMT during the
repair responses that take place in PD. Recently, the myofibroblastic conversion of
MC was confirmed in vivo by injection of
an adenovirus vector that transferred active TGF-1 in rat peritoneum.14
2010

PATHOLOGIC SIGNIFICANCE OF
EMT IN PERITONEAL FIBROSIS
AND ANGIOGENESIS

It has been shown that during the progression of EMT, MC acquire higher ex
vivo and in vitro ability to synthesize
components of the matrix, such as fibronectin and collagen I.13 In addition,
MC that undergo an EMT express high
levels of cyclooxygenase-2 (unpublished
data, Aroeira et al.), which has been implicated in tissue remodeling and fibrosis
processes.40 Furthermore, it can be observed that the grade of peritoneal fibrosis and small-solute transport rate of PD
patients correlate with the expression
of mesothelial markers within the fibrotic stroma (unpublished data, del
Peso et al.). These results support the notion that transdifferentiated MC play an
essential role in the initiation of fibrosis

Journal of the American Society of Nephrology

and subsequent peritoneal functional


decline.
Besides fibrosis, an increased number
of capillaries is related to peritoneal
membrane failure.1,8 It was proposed
that local production of VEGF during
PD plays a central role in the processes
that lead to peritoneal angiogenesis and
functional decline.30,31 However, the
main source of VEGF in the PD patients
as well as the mechanisms that are implicated in VEGF upregulation during PD
remained elusive. A number of studies
have shown that MC from omentum
have the capacity to produce VEGF in
vitro in response to a variety of stimuli.13,41 A recent study demonstrated that
transdifferentiated MC are an important
source of VEGF in PD patients and that
the underlying mechanism of VEGF upregulation in MC is the mesenchymal
conversion of these cells.13 It was shown
that MC from effluents with fibroblastlike phenotype produce much more
VEGF ex vivo than MC with epithelial
phenotype and that patients who drain
fibroblast-like cells have higher blood
VEGF levels than patients with MC with
epithelial phenotype in their effluents.
Moreover, a correlation between ex vivo
and in vivo VEGF levels and the rate of
peritoneal transport in patients who
were on PD could be demonstrated.13
These results suggest a direct and active
role of MC not only in fibrosis but also in
peritoneal angiogenesis. In addition,
these studies indicated that the EMT of
MC reflects peritoneal functional decline
better than previous tests, such as measurement of effluent cancer antigen 125
(CA125).13 Although CA125 has been
classically used as an index of MC mass
and peritoneal health in PD patients, the
value of effluent CA125 has been questioned recently because it correlates with
peritoneal transport rate only in shortterm but not in long-term PD.42

THERAPEUTIC INTERVENTION ON
EMT

The insights of the molecular mechanisms that regulate chronic inflammation that is induced by peritoneal injury
J Am Soc Nephrol 18: 2004 2013, 2007

www.jasn.org

will allow the identification of potential


therapeutic targets. However, from the
clinical nephrologists point of view, perhaps the most important aspect is the
identification of the EMT of MC as a key
event in peritoneal membrane failure,
because this process can be manipulated
with a wide range of agents and pharmaceutical products.37 The therapeutic
strategies may be designed either to prevent or to reverse the EMT itself or to
treat its effects such as cellular invasion,
ECM accumulation, or angiogenesis factor synthesis.
It can be proposed that at least six
steps that are related to the EMT process
of the MC can be clinically managed,
alone or in combination, to prevent peritoneal membrane failure (Figure 3). Because EMT is reversible, treatments may
be tailored either to prevent (step 1) or to
reverse (step 2) this process. These therapeutic approaches have been proved to
be effective in animal models of renal fibrosis. The endogenous factors HGF and
BMP-7 have been demonstrated to block
EMT both in vitro and in vivo and to prevent renal interstitial fibrosis.37 In addition, these two proteins reversed the phenotypic conversion of tubular epithelial
cells that was induced by TGF- by restoring E-cadherin expression and
downregulating the expression of mesenchymal markers such as -SMA, vimentin, and fibronectin.37 The reversion
from mesenchymal to epithelial phenotype by BMP-7 has also been demonstrated for transdifferentiated MC.43
Mechanistically, HGF interferes with
TGF-mediated EMT by inducing the
expression of the transcriptional co-repressors such as SnoN and TG interacting factor that interact with activated
Smad-2/4 complex and block the expression of Smad-dependent genes, including ILK.37 The underlying mechanism of
BMP-7 blockade of EMT is by activation
of Smad-5 protein that counteracts with
TGF-activated Smad-2/3.36
Besides targeting EMT by supplementation of endogenous factors, this process can be disrupted by using small molecule inhibitors. For instance, the
inhibition of Rho-activated kinase, a
downstream effector kinase of RhoA, reJ Am Soc Nephrol 18: 2004 2013, 2007

sulted in suppression of -SMA expression and renal interstitial fibrosis in a


mouse model of ureteral obstruction.37
Pharmacologic inhibition of AngII, a potent EMT promoter, also attenuated
EMT and reduced renal fibrosis.37 More
recently, it was shown that paricalcitol, a
vitamin D analogue, blocked the mesenchymal conversion of tubular epithelial
cells and ameliorated renal fibrosis in animal models of obstructive nephropathy.44
Whereas these endogenous factors
and pharmacologic compounds have
been widely demonstrated to block
transdifferentiation of tubular epithelial
cells and to ameliorate renal fibrosis,
their effects on EMT of MC and in the
prevention of peritoneal membrane failure are just starting to be tested in vitro
and in animal models of PD. In this context, it has been demonstrated that AngII
induces the expression of TGF- and fibronectin in MC and that inhibitors of
angiotensin-converting enzyme and AngII type 1 receptor attenuate the production of VEGF in these cells.29,45 In addition, it was previously shown that
intraperitoneal administration of an inhibitor of angiotensin-converting enzyme attenuated structural and functional alteration of the peritoneum in a
rat PD model.46
It should be taken into consideration
that EMT of MC is a physiologic process
that is necessary for wound healing during the aggression of the peritoneal
membrane induced by PD; therefore, it is
plausible that chronic blockade of EMT
would result in inefficient tissue repair.
Therefore, alternative therapeutic approaches may be addressed to treat the
consequences of the EMT of MC, such as
cellular invasion (step 3), fibrosis (steps 4
and 5), or angiogenesis (step 6; Figure 3).
Induction of EMT of MC is accompanied by upregulated expression of MMP
such as MMP-2 and MMP-9, which
would degrade the basal membrane and
the connective tissue, allowing the submesothelial invasion by the transdifferentiated cells.14 It can be expected that
MMP inhibitors may prevent the accumulation MC-derived myofibroblasts in
the submesothelial compartment, which

BRIEF REVIEW

in turn would diminish the structural alteration of the peritoneal membrane


(step 3).
MC that have undergone an EMT
produce higher amounts of ECM components, including fibronectin and collagen I,13 and display less fibrinolytic capacity as a result of unbalanced ratio
between tissue plasminogen activator
and plasminogen activator inhibitor-1.47
Therapeutic interventions of peritoneal
fibrosis may be designed either to prevent ECM synthesis (step 4) or to increase fibrinolysis (step 5). A number of
antifibrotic drugs, including pentoxifylline, dipyridamole, troglitazone, diltiazem, and emodin, have direct inhibitory effects on ECM protein synthesis or
on TGF- expression and activity in
MC.48 52 The statin simvastatin is another candidate molecule for therapeutic
treatment of peritoneal fibrosis because
it exhibits a dual effect: (1) Inhibition of
ECM synthesis and (2) fibrinolytic activity by inducing tissue plasminogen activator synthesis and inhibiting plasminogen activator inhibitor-1 expression.53
Finally, it has been demonstrated that
during the progression of EMT, MC produce higher amounts of VEGF, which
correlated with increased peritoneal
transport.13 Therefore, therapeutic intervention may also be directed to prevent
peritoneal angiogenesis and vessel permeability, via interrupting VEGF expression or its effects on endothelial cells
(step 6). For instance, the inhibitors of
angiogenesis TNP-470 and endostatin
have been shown to suppress the progression of peritoneal deterioration in
mouse experimental models.54,55

CONCLUSION

Recent studies using ex vivo cultures of


effluent-derived MC, in conjunction
with immunohistochemical analysis of
peritoneal biopsies, have allowed the
identification of the EMT of MC as a key
process in peritoneal membrane failure.
Although it can be argued that effluent
MC are not representative of the mesothelium because they might be cells
that are committed to detach from the
EMT and Peritoneal Membrane Failure

2011

BRIEF REVIEW

www.jasn.org

tissue, there is increasing evidence that


MC that are released into PD effluent are
representative of the MC population that
remains attached to the peritoneum. In
fact, it could be demonstrated that effluent-derived MC reflected more precisely
the functional status of the peritoneal tissue than previous tests.
It can be expected that proteomics
and functional genomics analysis of the
EMT process of MC will provide fine biomarkers for accurate follow-up of the
progressive peritoneal membrane deterioration and for the identification of master molecules that govern the mesenchymal conversion of MC. These molecular
profiles of the EMT process might also
become excellent tools to test the biocompatibility of new PD fluids, both in
in vitro studies and in animal models of
PD. In addition, the increasing knowledge of the events that culminate in EMT
and its consequences will provide future
hope for long-term preservation of peritoneal membrane through the development of preventive and therapeutic approaches. However, the design of new
therapeutic strategies still requires extensive analysis, in vitro and in animal models, of the various drugs and factors with
potential effects on EMT and its deleterious consequences before starting with
clinical trials.

3.

4.

5.

6.

7.

8.

9.

10.

11.

ACKNOWLEDGMENTS
This work was supported by grant FIS
03/0599 to R.S. and by grant SAF 2004-07855
to M.L.-C. This work was also partially supported by Fresenius Medical Care and by a
research award from Fundacion Instituto de
Credito Oficial to M.L.-C.

12.

DISCLOSURES
None.

13.

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