Perit Dial Int
29(2):
227-230
2009
© 2009 International Society for Peritoneal Dialysis
Chronic Exposure of Mouse Peritoneum to Peritoneal Dialysis Fluid: Structural and Functional Alterations of the Peritoneal Membrane
Guadalupe T. González-Mateo1,
Jesús Loureiro2,
José A. Jiménez-Hefferman3,
M.-Auxiliadora Bajo1,
Rafael Selgas1,
Manuel López-Cabrera2,4,a and
Luiz S. Aroeira1,a,*
Unidad de Investigación y Servicio de
Nefrología1 Hospital Universitario de la Paz
Unidad de Biología Molecular2 Hospital
Universitario de la Princesa Hospital Universitario Puerta de
Hierro3 Centro de Biología Molecular Severo
Ochoa4 Consejo Superior de Investigaciones
Científicas Red Española de Investigación Renal
(REDinREN) del Instituto de Salud Carlos III, RETICS 06/0016 Madrid,
Spain
*
e-mail:
lstark.hlpr{at}salud.madrid.org
An alternative to hemodialysis, peritoneal dialysis (PD) is a form of renal
replacement therapy based on the use of the peritoneum as a semipermeable
membrane across which ultrafiltration and diffusion take place
(1). Permanent exposure to
bioincompatible PD fluids (PDFs) and episodes of infections cause inflammation
and injury to the peritoneal membrane (PM), which progressively undergoes f
ibrosis and angiogenesis, and ultimately its dialytic capacity fails
(1). The pathophysiologic
mechanisms involved in PM deterioration are still poorly understood. However,
it is known that myofibroblasts play important roles in inflammatory response,
extracellular matrix accumulation, and angiogenesis
(2,3).
Animal models have added important insight into understanding the pathogenesis
of membrane failure. The rat model is the most commonly employed to study
fibrosis (4), fluid
biocompatibility
(5,6),
and inflammation (7), but it
has not improved our knowledge of mechanisms involved in peritoneal damage.
The utilization of genetically manipulated mice creates the possibility of
better dissection of the mechanisms involved in PD-induced peritoneal damage
and ultrafiltration failure
(8). The aim of the present
work is the development of a mouse model of chronic PM exposure to PDF
instilled through a catheter. The results demonstrate that daily exposure of
mouse peritoneum to PDF induces morphological and functional alterations of
the PM similar to those observed in PD patients, demonstrating that this model
is suitable to study the mechanisms involved in PDF-induced peritoneal
damage.
 |
MATERIAL AND METHODS
|
|---|
Experimental Animals, Surgery, and Exposure to PDF: This study was
performed in nonuremic female C57BL/6 mice (n = 35, age 12 – 14
weeks; Harlan Interfauna Iberica, Barcelona, Spain). The dropout percentage in
our two experimental groups was 30%; dropout was due mainly to catheter
displacement, trapping, and hemoperitoneum. The mice were housed under
standard conditions and with free access to food and water. The experimental
protocol was approved by the Animal Ethics Review Committee of the Unidad de
Cirurgía Experimental of Hospital La Paz, Madrid. During the
experiment, the health of the mice was checked daily by a veterinary doctor.
The mice were weighed every week and those presenting more than 10% weight
loss, any sign of port infection, or abnormal activity were excluded from the
experiment. Following sacrifice, peritoneum was carefully examined for any
sign of bleeding or infection.
Catheter and Installation: A customized vascular access port (ROP;
Access Technologies, Skokie, IL, USA) was implanted into the mice. The
catheters had 10 holes located within 1 cm of the tip to help the fluid exit
and to prevent obstructions [Figure
1(a)]. The animals were anaesthetized with intraperitoneal 100
mg/kg ketamine and 10 mg/kg xylazine. Afterwards, we performed an incision in
the skin in the right flank of the animal. The skin was separated from the
muscle layer below. Through another incision in this second layer, we
introduced the end of the catheter into the peritoneal cavity
[Figure 1(b)]. The port was
displaced at the subcutaneous space of the mouse's back
[Figure 1(c)].
View larger version (13K):
[in this window]
[in a new window]
|
Figure 1 — Catheter installation and peritoneal dialysis fluid instillation in
mice. The catheter contains 10 holes located within 1 cm of the tip to help
the fluid exit and to prevent obstructions (A). The catheter installation
surgical procedure consists of a small incision in the right flank, where the
catheter is sutured (B). At the end of the procedure, the catheter port is
located in the back of the mouse in the subcutaneous space (C).
|
|
During the first week post surgery, 0.2 mL saline was instilled in order to
prevent catheter trapping. Thereafter, during the experimental procedure, 1.5
mL standard PDF (lactate with 4.25% glucose; Fresenius Medical Care, Bad
Homburg, Germany) or physiologic saline solution was instilled daily for 5
weeks. This volume was chosen because it is almost equivalent to a single
exchange in PD patients, relative to body weight, and does not affect mouse
respiration. A third group, age matched and not manipulated, was used as
control. On the last day of the study, in order to perform the functional
analysis of the PM, we instilled PDF (1.5 mL) into the saline- and
lactate-instilled groups. After 90 minutes, the mice were anesthetized by
isoflurane inhalation and sacrificed by cervical dislocation. Total peritoneal
fluid was collected and weighed on a precision balance to calculate drained
volume. Peritoneal ultrafiltration capacity was determined by the percentage
of peritoneal liquid recovered after 90 minutes.
Histological Analyses: Tissue collected from parietal and visceral
peritoneum was fixed in neutral-buffered formalin, embedded in paraffin, and
cut into 5-µm sections. The sections were stained with hematoxylin &
eosin and analyzed microscopically (Leica CTR6000, with a Leica Microsystems
LAS-AF6000; Witzlar, Germany) to determine the thickness of the mesothelial
layer. To determine the presence of blood vessels we stained the sections with
anti-CD31 mAb (Becton & Dickinson, Franklin Lakes, NJ, USA) by
immunohistochemistry. The thickness of the PM and the number of blood vessels
were achieved as the median of seven measurements in different fields of each
animal sample.
Statistical Analysis: Statistical analysis of data was performed
using GraphPad Prism 4 software (GraphPad Software, La Jolla, CA, USA).
Comparison among groups was made with Mann–Whitney test. A p
value < 0.05 was considered statistically significant.
|
RESULTS AND DISCUSSION
|
|---|
Compared to non-manipulated mice, chronic instillation of PDF and saline
induced thickening and inflammation of the submesothelial compact zone
[Figure 2(a)]. The thickening
of PM was due mainly to matrix collagen deposition and increased numbers of
cells [Figure 2(c)].
Measurement of the submesothelial fibrotic stroma revealed that the
PDF-instilled group had significantly increased peritoneal thickness compared
with the saline-instilled group (p < 0.01)
[Figure 2(c)]. This
demonstrates that instillation of peritoneal liquid induces morphological
alterations in PM, as happens in patients submitted to PD. In mice, we
observed a strong thickening of the mesothelial membrane in a relatively short
period, probably because of the high metabolism of these animals. No
significant increase in thickness was observed in PM surrounding the
intestines (data not shown). In addition to morphological changes,
instillation of PDF also induced functional alterations in peritoneum. Chronic
instillation of PDF induced alteration of membrane permeability since the
quantity of PDF drained on the last day of the experiment was lower than that
in mice chronically instilled with saline
[Figure 2(d)].
Immunohistochemistry with anti-CD31 antibodies
[Figure 2(b)] revealed that
chronically instilled-PDF mice had a higher number of blood vessels at the
submesothelial space than the saline-instilled mice
[Figure 2(e)]. This strongly
suggests that alterations in peritoneal permeability are dependent on
angiogenesis at the submesothelial space. Again, this observation is in
agreement with observations in patients submitted to PD, where the increase in
the numbers of blood vessels plays an important role in PM transport rate and
ultrafiltration failure
(9).
View larger version (289K):
[in this window]
[in a new window]
|
Figure 2 — Exposure to peritoneal dialysis fluid (PDF) induces morphological
and functional alterations in the peritoneal membrane. After 5 weeks of fluid
instillation, peritoneal biopsies were collected and stained with hematoxylin
& eosin. Representative microphotographs of the control (no liquid
instillation), saline-instilled, and PDF-instilled groups are shown (A). The
staining of CD31-positive blood vessels of both experimental groups are also
shown (B). Arrows indicate CD31-positive blood vessels. Structural
measurements of the thickness of the submesothelial space reveal that PDF
instillation induces thickening of the submesothelial space (C) (n =
14 for PDF-instilled and n = 7 for saline-instilled mice). Peritoneal
function was determined as the percentage of peritoneal effluent recovered 90
minutes after instillation (D) (n = 8 for PDF-instilled and
n = 5 for saline-instilled mice). The numbers of vessels per field of
submesothelium were determined in biopsy stained with anti-CD31 (E)
(n = 14 for PDF-instilled and n = 7 for saline-instilled
mice).
|
|
The results presented here indicate that PM of mice chronically exposed to
PDF suffers alterations similar to those observed in patients, and that this
mouse model is suitable for the study of mechanisms involved in peritoneal
deterioration. The use of an indwelling catheter for chronic instillation of
PDF makes our model similar to PD in patient treatment and reduces the risk of
infections and hemoperitoneum in chronic treatment. The surgery and catheter
did not significantly affect the structure and function of PM. We observed
that contact of the catheter with PM produced only localized inflammation and
fibrosis, which did not affect peritoneal function. In order to prevent
interference by catheter-induced damage in the analyses, we avoided taking
samples from areas near the catheter.
This model provides a valuable tool to unravel the molecular basis of PM
deterioration through the utilization of genetically manipulated mice, and
will open the possibility of developing and establishing efficient new
therapies.
|
DISCLOSURE
|
|---|
The authors declare that no financial conflict of interest exists.
|
ACKNOWLEDGMENTS
|
|---|
This work was supported by grants SAF2007-61201 (Ministerio de
Educación y Ciencia) to M. López-Cabrera, FIS PI 06/0098 and
RETICS 06/0016 to R. Selgas, and FIS PI050618 to M.A. Bajo. G.T.
González-Mateo received financial support from Gambro Europe.
We thank Javier B. de la Víbora (DVM) and Carlota L. Aramburu (DVM,
PhD) for their assistance with the care of the mice.
|
FOOTNOTES
|
|---|
a These two authors contributed equally to this work. 
|
REFERENCES
|
|---|
- Krediet RT. The peritoneal membrane in chronic peritoneal dialysis.
Kidney Int 1999;55
: 341-56.[Medline]
- Yanez-Mo M, Lara-Pezzi E, Selgas R, Ramirez-Huesca M,
Dominguez-Jimenez C, Jimenez-Heffernan JA, et al. Peritoneal dialysis
and epithelial-to-mesenchymal transition of mesothelial cells. N
Engl J Med 2003; 348:403
-13.[Abstract/Free Full Text]
- Jimenez-Heffernan JA, Aguilera A, Aroeira LS, Lara-Pezzi E, Bajo
MA, del Peso G, et al. Immunohistochemical characterization of
fibroblast subpopulations in normal peritoneal tissue and in peritoneal
dialysis-induced fibrosis. Virchows Arch2004; 444:247
-56.[Medline]
- Wieczorowska K, Khanna R, Moore HL, Nolph KD, Twardowski ZJ. Rat
model of peritoneal fibrosis: preliminary observations. Adv Perit
Dial 1995; 11:48
-51.[Medline]
- Hekking LH, Zareie M, Driesprong BA, Faict D, Welten AG, de Greeuw
I, et al. Better preservation of peritoneal morphologic features and
defense in rats after long-term exposure to a bicarbonate/lactate-buffered
solution. J Am Soc Nephrol 2001;12
: 2775-86.[Abstract/Free Full Text]
- Zareie M, Hekking LH, Welten AG, Driesprong BA, Schadee-Eestermans
IL, Faict D, et al. Contribution of lactate buffer, glucose and
glucose degradation products to peritoneal injury in vivo. Nephrol
Dial Transplant 2003; 18:2629
-37.[Abstract/Free Full Text]
- Mortier S, Lameire NH, De Vriese AS. The effects of peritoneal
dialysis solutions on peritoneal host defense. Perit Dial
Int 2004; 24:123
-38.[Abstract]
- Nishino T, Ni J, Devuyst O. Transgenic mouse models.
Perit Dial Int 2007;27
: 625-33.[Abstract/Free Full Text]
- Williams JD, Craig KJ, Topley N, Von Ruhland C, Fallon M, Newman
GR, et al. Morphologic changes in the peritoneal membrane of patients
with renal disease. J Am Soc Nephrol2002; 13:470
-9.[Abstract/Free Full Text]
TOP
MATERIAL AND METHODS
RESULTS AND DISCUSSION
