Perit Dial Int
29(Supplement_2):
202-205
2009
© 2009 International Society for Peritoneal Dialysis
Part 7: Protection of Peritoneal Membrane |
CAN N-ACETYLCYSTEINE PRESERVE PERITONEAL FUNCTION AND MORPHOLOGY IN ENCAPSULATING PERITONEAL SCLEROSIS?
Devrim Bozkurt1,
Ender Hur1,
Burcu Ulkuden2,
Murat Sezak3,
Hasim Nar2,
Ozlem Purclutepe2,
Sait Sen3 and
Soner Duman1
Divisions of Nephrology,1 Internal
Medicine,2 and Pathology,3 Ege
University, Izmir, Turkey
Correspondence to: S. Duman, Ege Üniversitesi Tip Fakültesi,
Nefroloji Bilim Dali, Bornova, Izmir 35100 Turkey.
dumans{at}mail.ege.edu.tr,
sonerduman{at}hotmail.com
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ABSTRACT
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Long-term use of the peritoneum as a dialysis membrane results in
progressive irreversible dysfunction, described as peritoneal fibrosis.
Oxidative stress during peritoneal dialysis has been established in many
studies. Generation of reactive oxygen species (ROS) by conventional
peritoneal dialysis solutions, regardless of whether produced by high glucose,
angiotensin II, or glucose degradation products may be responsible for
progressive membrane dysfunction.
The well-known antioxidant molecule N-acetylcysteine (NAC) is
capable of direct scavenging of ROS. The aim of the present study was to
investigate the effect of NAC therapy on both progression and regression of
encapsulating peritoneal sclerosis (EPS).
We divided 49 nonuremic Wistar albino rats into four groups: Control
group—2 mL isotonic saline intraperitoneally (IP) daily for 3 weeks; CG
group—2 mL/200 g 0.1% chlorhexidine gluconate (CG) and 15% ethanol
dissolved in saline injected IP daily for a total of 3 weeks; Resting
group—CG (weeks 1 – 3), plus peritoneal resting (weeks 4 –
6); NAC-R group—CG (weeks 1 – 3), plus 2 g/L NAC (weeks 4 –
6).
At the end of the experiment, all rats underwent a 1-hour peritoneal
equilibration test with 25 mL 3.86% PD solution. Dialysate-to-plasma ratio
(D/P) urea, dialysate white blood cell count (per cubic milliliter),
ultrafiltration (UF) volume, and morphology changes of parietal peritoneum
were examined.
The CG group progressed to encapsulating peritoneal sclerosis,
characterized by loss of UF, increased peritoneal thickness, inflammation, and
ultimately, development of fibrosis. Resting produced advantages only in
dialysate cell count; with regard to vascularity and dialysate cell count, NAC
was more effective than was peritoneal rest. Interestingly, we observed no
beneficial effects of NAC on fibrosis. That finding may be a result of our
experimental severe peritoneal injury model. However, decreased inflammation
and vascularity with NAC therapy were promising results in regard to membrane
protection.
KEY WORDS: NAC; encapsulating peritoneal sclerosis; membrane protection.
Inadequate solute clearance and ultrafiltration failure (UF) in long-term
peritoneal dialysis (PD) patients as a result of peritoneal fibrosis is a
major problem. Encapsulating peritoneal sclerosis (EPS) is the most
dangerous—very rare, but highly fatal—form of peritoneal fibrosis.
The main factors contributing to the development of EPS are bioincompatible
dialysis solutions with high glucose content, recurrent peritonitis attacks,
and long PD duration. Oxidative stress under high glucose media during PD has
been established in many studies
(1). Also interactions of
angiotensin II, protein kinase C, transforming growth factor β1
(TGFβ1), and reactive oxygen species (ROS) with peritoneal mesothelial
cells have been reported
(2).
The well-known mucolytic drug N-acetyl-L-cysteine (NAC)
is widely used in respiratory diseases. It is also capable of scavenging ROS
and replenishing antioxidant molecule levels in the body. In several
experimental models of fibrosis, NAC has been shown to have antifibrotic
properties
(3,4).
No therapy has been established for EPS other than preventive modalities. The
aim of the present study was to evaluate the effects of NAC therapy in an
experimental EPS model in rats.
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MATERIALS AND METHODS
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We carried out the study in 39 female nonuremic Wistar albino rats,
weighing 160 – 180 g. The rats were housed in polycarbonate cages at
24°C room temperature with a 12-hour light/dark cycle and were fed a
standard laboratory diet. The Animal Ethics Committee of Ege University
Hospital approved the study design.
We divided the 39 rats into four groups:
- Control group: 2 mL isotonic saline injected intraperitoneally (IP) daily,
for 3 weeks
- CG group: 2 mL/200 g 0.1% chlorhexidine gluconate (CG) and 15% ethanol
dissolved in saline injected IP daily for a total of 3 weeks
- Resting group: CG (weeks 1 – 3), plus peritoneal rest (weeks 4
– 6)
- NAC-R group: CG (weeks 1 – 3), plus 2 g/L NAC [200 mg pill (Temmler
Pharma, Marburg, Germany)] (weeks 4 – 6)
At the end of the study period, all rats underwent a 1-hour peritoneal
equilibration test with 25 mL 3.86% PD solution (Dianeal:
Eczacibasi–Baxter Healthcare, Istanbul, Turkey). At 1 hour of the test,
ketamine HCl anesthesia (60 mL/kg body weight) was applied, and blood samples
were collected immediately through direct cardiac puncture. Dialysate samples
were obtained through a midline incision, using a shortened dialysis catheter
to prevent leakage of any dialysate solution.
Blood and dialysate urea were determined using the enzymatic kinetic method
(Randox Laboratories, San Francisco, CA, U.S.A.). The D/P urea was then
calculated. Net ultrafiltration (UF) was calculated as the difference between
the instilled and the drained dialysate volumes. Dialysate cell count was
taken as a white blood cell count (WBC) per cubic milliliter of dialysate.
The peritoneal membrane samples were fixed in 4% formalin and embedded in
paraffin. Paraffin blocks were divided into 5-µm sections and were then
stained with hematoxylin–eosin and Masson trichrome. All samples were
examined by the same pathologist, who was unaware of the nature of the groups
from which the samples originated. Peritoneal thickness, neovascularization,
and inflammation were evaluated. Thickness was measured with an ocular
micrometer, and neovascularization and inflammation were defined by counting
capillaries and mononuclear cells per high-power field at 400x
magnification.
Results are reported as mean ± standard error of the mean. The
statistical analyses were performed using analysis of variance, unpaired
t-test, and Mann–Whitney U-test. A p value of
less than 0.05 was considered significant.
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RESULTS
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Table 1 summarizes the
results. The CG group showed severely disturbed peritoneal functional and
structural properties, characterized by UF failure, increased peritoneal
thickness, and neovascularization as compared with control animals
(–2.46 ± 0.94 mL vs. 7.81 ± 0.37 mL, 117 ± 10 µm
vs. 8 ± 0.3 µm, and 5.6 ± 0.8 vessels vs. 0 vessels
respectively, p < 0.05). Peritoneal rest had a beneficial effect
on UF failure and dialysate cell count as compared to results in the CG group
(2.86 ± 0.54 mL vs. –2.46 ± 0.94 mL and 593
cells/mm3 vs 1006 cells/mm3 respectively, p
< 0.05). In the NAC-R (regression) group, NAC therapy significantly
improved UF failure and neovascularization as compared with peritoneal rest
(4.45 ± 1.46 mL vs. 2.86 ± 0.54 mL and 7.14 ± 1.94
vessels vs. 12 ± 3 vessels respectively, p < 0.05).
In histology micrographs (Control, CG, Resting, and NAC-R), CG disrupted
the mesothelial cell layer, and the expanded submesothelial compact zone
showed increased vascularity, cellularity, and fibrosis. Therapy with NAC had
beneficial effects on peritoneal structure, which showed decreased
vascularity, thickness, and cellular activity as compared with the Resting
group (Figure 1).
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Figure 1 — A thin submesothelial compact zone with intact mesothelial cell
layer is shown in the Control micrograph. An extremely increased
submesothelial compact zone with increased vascularity and fibrosis is shown
in the chlorhexidine gluconate (CG) micrograph. Increased peritoneal thickness
with neovascularization, inflammation, and fibrosis is seen in the Resting
micrograph. A marked decrease in vascularity and inflammation in the
peritoneum is seen in the N-acetylcysteine with resting (NAC-R)
micrograph.
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DISCUSSION
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Administration of CG for 3 weeks ultimately resulted in experimental EPS as
described previously by Ishii et al.
(5). In PD, ROS activity is
known to play a pivotal role in the complex fibrosing process, meaning that
NAC therapy might have therapeutic value. In the regression group in our
study, NAC therapy had significant benefit with respect to UF failure,
possibly as a result of decreased neovascularization. That finding accords
with the report of Noh et al.
(2) in which NAC significantly
inhibited vascular endothelial growth factor (VEGF). Those authors also showed
that NAC therapy significantly decreased the activity of angiotensin II and
TGFβ1 in human peritoneal mesothelial cells after exposure to high
glucose concentrations. The main interest with regard to the molecular
mechanism of peritoneal membrane damage is focused on growth factors, and in
particular, TGFβ1 and VEGF, which are the inducer cytokines most
responsible for epithelial-to-mesenchymal transition (EMT)
(6). Decreases in dialysate
cell count and in peritoneal vascularity and an increase in UF are promising
findings, suggesting that NAC may have a therapeutic value in EPS.
Peritoneal rest is an alternative approach in long-term PD patients with UF
failure (7); however, several
reports also showed that the fibrosing process is dynamic, and that once it
starts, it tends to continue
(8). In our study, peritoneal
vascularity and thickness clearly continued to increase during the resting
period, as shown in Table 1 and
the histology pictures of a resting peritoneum.
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CONCLUSIONS
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Therapy with NAC may have some advantages over peritoneal rest in
peritoneal membrane protection.
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ACKNOWLEDGMENTS
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The authors thank Figen Cetin and Sultan Ozkan for their kind technical
assistance.
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ABSTRACT
MATERIALS AND METHODS
