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Acute renal failure (ARF) is a clinical syndrome with high
morbidity and mortality. The incidence of ischemic ARF has
been rising rapidly, especially with the rapid development of
various difficult surgical operations and kidney
transplantation[1,2]. Unfortunately, up to now, there was no really
effective medicine available to treat this disease.
Several recent studies have reported that, aside from
tubular injury directly caused by renal abnormal hemodynamic
states, the recruitment and activation of numerous
inflammatory cells after ischemia, and immune inflammatory
responses mediated by the expression and secretion of
inflammatory cytokines are the main causes of ischemic acute
renal injury[3-5]. Among the various cytokines produced
during renal ischemia, interleukin-18 (IL-18),
interleukin-1b
(IL-1b) and interferon-gamma (IFN-g), a group of
proinflam-matory cytokines with closely related function, play an
important role in the inflammatory reaction and
renal tubuloin-terstitial
impairment[6-9]. Other studies have reported that
applying antibodies or soluble receptors to block the roles
of these cytokines can alleviate ischemic
ARF[10,11]. It has been demonstrated that both
IL-1b and IL-18 are members of the IL-1 family, which can be activated by
interleukin-1b-converting enzyme (ICE) via cleaved precursor peptide, and
furthermore, that IL-18 is the strongest inducing factor of
IFN-g[12,13]. We therefore postulated that it would be more
effective to protect the kidney from acute ischemic
injury by regulating the whole cytokine network, including
IL-1b, IL-18, and IFN-g, with selective ICE inhibitors, instead of only
blocking any one of the cytokines using antibodies or soluble
receptors. More significantly, several kinds of ICE
inhibitors with simple structure and low antigenicity have been
synthesized by chemical methods. The inhibitors are
superior to cytokine antibodies and soluble receptors, which are
difficult to produce, expensive, hypersensitive and require
injection.
The present study aimed at exploring the role of
selective ICE inhibitors in preventing the kidney from ischemic
ARF by the combined inhibition of IL-1b, IL-18, and
IFN-g in mouse models of ischemic ARF.
Materials and methods
Materials All reagents were obtained from Sigma (St
Louis, MO, USA), unless otherwise indicated.
Ischemic ARF model induction Male Kunming mice (SPF
grade, Experimental Animal Center of Guangdong Medical
College, Zhanjiang, China) weighing 22.1±1.3 g (20 g-25 g)
were used. After being anesthetized with an injection of
0.3% sodium pentobarbital (5 mL/kg-10 mL/kg, ip), an
abdominal mid-line incision was made and the renal pedicles
were clamped bilaterally for 45 min with non-traumatic
microaneurysm clamps. Restoration of blood flow was
confirmed when the kidneys returned to their original color after
the clamps were removed. Afterward, the abdomen was
closed and the mice were allowed to recover. The mice were
observed for 2 h after the operation was finished. A model
was considered to be developed successfully if the animal¡¯s
behavior returned to normal. Those that failed to return to
normal behavior after operation or those in which any 1 of
the kidneys failed to return to normal color after the clamps
were removed were regarded as unsuccessful models and
were removed from the experiment. The sham-operated
group consisted of the same surgical procedure except that
clamps were not applied.
Experimental groups Animals were kept in a clean
environment at 24 °C-29 °C with free access to standard food
and water after model induction. The study was composed
of 3 parts as follows. Experiment I: Thirty-nine mice were
distributed randomly into the sham-operated group, the
model control group or the therapy group. After
post-operative elimination, these groups contained 13, 13, and 11 mice,
respectively. The therapy group was administered
AC-YVAD-CMK (6.25 mg/kg, ip; Calbiochem, Darmstadt,
Germany), which was dissolved in 2% Me2SO saline, at 2 h,
8 h, and 16 h after surgery. The other 2 groups received the
vehicle; an equal volume of 2% Me2SO saline at the same
times as the therapy group. All of the mice were killed at 24 h
after surgery. Experiment II: Forty-five mice were enrolled
in this part of the experiment and were also distributed
randomly into 3 groups as in Experiment I. Each group
contained 13 mice after model construction and elimination. The
mice underwent similar procedures as those in
Experiment I, except that either AC-YVAD-CMK or the vehicle were
administered to the mice at 2 h, 8 h, 14 h, 20 h, 28 h, and 36 h,
and the mice were killed at 48 h after surgery.
Experiment III: Thirty-nine mice were distributed randomly into the 3
groups as in Experiment I and Experiment II, and each group
contained 12 mice. AC-YVAD-CMK or the vehicle was
administered in the same way as in Experiment II, but the
animals were not killed. Their clinical features were observed
and recorded twice per day for 14 d after surgery.
Sample collection and
pretreatment Blood samples were obtained via orbital cavity after removing eyeballs. The blood
samples were then centrifuged to separate the serum. The
kidneys were collected, each of which was divided into 4
equal sections. One section was fixed in 10% neutral forma
lin solution while the others were frozen immediately in
liquid nitrogen and preserved at -72 °C until use.
Blood biochemical parameters
assay The levels of blood urea nitrogen (BUN) and serum creatinine (Scr) were
measured using an automatic biochemical analyzer (Beckman
Instruments, Fullerton, CA, USA).
Renal histological examination The 4%
paraformaldehyde-fixed and paraffin-embedded kidney samples were
sectioned at 3 µm and stained with hematoxylin-eosin (HE) and
periodic acid-Schiff (PAS) using standard methods.
Histological examinations were carried out in a single blinded
fashion. Histological changes due to acute renal
tubulo-interstitial injury were quantitated using the method of Paller
et al[14]. Five fields (×200) were reviewed for each slide. The
scores included: diffuse tubular epithelial cell flattening and
tubular lumen dilatation (1 point), lesion of brush border (1
point), loss of brush border (2 points), cell membrane bleb
formation (1 point), cytoplasmic vacuolization (1 point),
interstitial edema (1 point), necrotic cells in tubular lumen but
no cast formation (1 point), and casts or cell pieces
formation (2 points). Higher scores represented more severe
damage.
Renal ICE activity assay Renal ICE activity was
measured using a ICE fluorescent assay kit (Calbiochem),
according to the manufacture¡¯s protocols. Briefly, 1 mL of
tissue lytic buffer (containing 1×phosphate-buffered saline,
1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium
dodecylsulfate, 10 µg/mL phenylmethylsulfonylfluoride and
10 µL/mL Aprotinin) was added to approximately 100 mg
renal tissue. The renal tissues were then homogenized
artificially and incubated on ice for 1 h. The tissue lysate was
then centrifuged at 4 °C at
12 000×g for 5 min. The
supernatant was collected in a separate microfuge tube and stored
immediately at -72 °C until use. The protein concentration of
each tissue lysate was measured by spectrophotometry
after being dyed with Coomassie brilliant blue G250.
Supernatants were adjusted to a final protein concentration of 150µg/µL-200 µg/µL. The enzymatic activity was measured as
follows: 50 µL of 2×ICE assay buffer, 5 µL of substrate
(1 mmol/L YVAD-AFC) and 50 µL protein solution were added
to a 96-well polyvinyl plate. The reaction system, kept in
darkness, was incubated at 37 °C for 90 min. The
absorbance of each well was then read by a DA620 fluorescence
microplate reader (Bio-Tek, Winooski, Vermont, USA) at an
excitation wavelength of 380 nm and an emission wavelength
of 460 nm. Tissue lytic buffer was replaced with tissue
lysate as a negative control. A ICE standard curve was
determined for the experiment at the same time. ICE activity was
expressed as units per mg protein.
Assay for expression of mature renal IL-18
protein The level of mature IL-18 protein expressed in renal tissue was
measured using a mouse IL-18 enzyme-linked immunosorbent
assay kit (MBL, Nagoga, Japan), which was only specific for
mature IL-18. Renal tissue total protein was extracted as
described above. The assay procedures strictly followed
the manufacturer¡¯s instructions. Every sample was measured
by double parallel wells, and was remeasured when the
intra-error was over 10%.
Assay for renal IFN-g mRNA
expression Approximately 100 mg of frozen kidney sample was homogenized in a muller
by adding liquid nitrogen to crisp the tissue. Total RNA was
extracted from kidney tissue using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized
with the SuperScriptionTM First-Strand Synthesis System
(Invitrogen) according to the manufacturer¡¯s protocol. The
primers used for the IFN-g polymerase chain reaction (PCR)
were as follows: 5¡¯-AGG AAC TGG CAA AAG GAT GGTG-3¡¯
(sense), and 5¡¯-GTG CTG GCA GAA TTA TTC TTA TTG-3¡¯
(anti-sense), and the product was 353 bp. The primers used
for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR were as follows: 5¡¯-AAC GAC CCC TTC ATT
GAC-3¡¯ (sense), and 5¡¯-TCC ACG ACA TAC TCA GCAC-3¡¯
(anti-sense), and the product was 191 bp. Primers were
synthesized and purified by Shanghai Biological Engineering
(Shanghai, China). The PCR-amplified system included
1.2 µL of 25 mmol/L
MgCl2, 0.4 µL of 10 mmol/L dNTP mix,
0.8 µL of 5 µmol/L sense and anti-sense primers, 2 U of
Taq DNA polymerase, 1 µL of cDNA and sterile purified water to
20 µL. The IFN-g PCR was carried out in a thermal cycler
(Eppendorf, New York city, New York, USA) at 95 °C for 60 s,
58 °C for 60 s, and 72 °C for 60 s. The GAPDH PCR was
carried out at 94 °C for 60 s, 58 °C for 60 s, and 72 °C for 60 s.
IFN-g was amplified for 35 cycles and GAPDH for 28 cycles.
Equal volumes of each PCR product were loaded into gels
and electrophoresis was carried out. The ethidium
bromide-stained gels were analyzed semiquantitatively using a gel
imaging system (UVP, Cambridge, UK). The quantity of
IFN-g expression in every renal tissue was the integral absorbency
of the IFN-g amplification band divided into GAPDH
amplification band.
Statistical analysis SPSS version 11.0 software was used
to obtain data statistics. Measurement data were expressed
as mean±SD. One-way ANOVA was used for statistical
analysis among multiple groups. Kaplan-Meier was used for
statistical analysis of survival rates among multiple groups.
Results
Effect of AC-YVAD-CMK on renal function in ischemic
ARF mice The levels of Scr and BUN in model controls
were increased significantly compared with sham-operated
groups both in experiment I and experiment II. The levels of
Scr decreased significantly but that of BUN did not decrease
significantly in the AC-YVAD-CMK therapy group compared
with the vehicle-treated group in experiment I; the
levels of both Scr and BUN decreased significantly in the
therapy group in experiment II (Table 1).
Effect of AC-YVAD-CMK on renal tubulointerstitial
injury in ischemic ARF mice Kidneys from sham-operated
groups were normal in form and rubicund color while those
from model control groups were swollen with pale cortex and
congested medulla. These anatomical changes were also
observed but were significantly less in kidneys from
AC-YVAD-CMK therapy groups. Renal tissues from
sham-operated groups had a basically normal microcosmic
structure except for focal vacuolation of tubular epithelial cells
(TECs). Renal tissues from model control groups showed
severe damage in microcosmic structure, including
extensive TECs vacuolation, scattered TECs brush border flatting,
shrinkage and loss as well as focal TECs nucleus nakedness
and necrosis. Dilated lumens and cast formation were also
found in parts of the tubules. Interstitial inflammatory cell
infiltration was also found, especially with neutrophil cells.
Renal tissue damage was relieved significantly in
AC-YVAD-CMK therapy groups. The main histological changes
included extensive TECs vacuolation, focal TECs nucleus
nakedness and very rare TECs necrosis. The basement
membranes were basically integrated; casts were seldom.
Inflammatory infiltration in interstitial areas was slight. The mean
histological score for the kidney tissue of model control
groups was significantly higher than that of the
sham-operated groups, both in experiment I and experiment II.
The mean score for the AC-YVAD-CMK therapy groups was much
lower than that for the vehicle-treated groups, again in both
experiment I and experiment II (Figure 1, Table 2).
Effect of AC-YVAD-CMK on survival rate in ischemic
ARF mice In Experiment III, Sham-operated groups had no
apparent abnormal appearance, activity or ingestion during
the whole observation period, and all of them had survived
at the end of observation. Model controls were cachexia in
different extents, with reduced activity and ingestion.
Anasarca and dyspnea occurred gradually. The survival rate
was 83.3% on d 7, 58.3% on d 10, and only 8.3% on d 14.
However, the clinical features were relatively improved in
the AC-YVAD-CMK therapy group during the period of
observation compared with model controls. The survival
rate was 100.0% on d 7, 91.6% on d 10, and 25.0% on d 14.
All together, the 2-week accumulated survival rate in the
AC-YVAD-CMK therapy group was higher than that of the
vehicle-treated group by survival analysis for time
(P<0.01, Figure 2).
Effect of AC-YVAD-CMK on renal ICE activity in
ischemia ARF mice ICE activity in renal tissue from model
controls was 347.0±97.5 U/mg protein in experiment I and
536.1±43.0 U/mg protein in experiment II, both much higher
than that for the sham-operated groups, which was 239.5±
56.5 U/mg protein in experiment I and
237.2±27.4 U/mg protein in experiment II. The
ICE activity for the therapy group was
314.0±56.0 U/mg protein in experiment I and 412.2±
12.5 U/mg protein in experiment II. ICE
activity was decreased significantly compared with the model control group in
experiment II, but not in experiment I (Figure 3).
Effect of AC-YVAD-CMK on expression of renal mature
IL-18 protein in ischemia ARF mice Interleukin-18
expression in renal tissue from model controls was 28.9±11.6
pg/mg protein in experiment I and
15.2±9.4 pg/mg protein in experiment II, both
much higher than for the sham-operated groups, which was
13.1±3.5 pg/mg protein in experiment I and
7.3±3.5 pg/mg protein in experiment II. Mature IL-18
protein expression was 16.7±4.8 pg/mg
protein in experiment I and 6.9±3.5 pg/mg
protein in experiment II in AC-YVAD-CMK therapy groups; both were decreased significantly
compared with model control groups (Figure 4).
Effect of AC-YVAD-CMK on renal IFN-g mRNA
expression in ischemia ARF mice Model controls had a
significantly higher expression of IFN-g mRNA in renal tissues
than sham-operated groups. The expression level in the
AC-YVAD-CMK therapy group was decreased significantly
compared with the vehicle-treated group (Table 3, Figure 5).
Discussion
The present study developed a successful ischemic ARF
model according to the dynamic changes of the kidneys color
during operation and the changes in renal function and
morphological structure after operation. However, we also found
that renal function parameters, especially the level of Scr,
and renal histological scores in experiment I were higher than
those in experiment II in the sham-operated group. This
phenomenon may be caused by muscle lesion during
peritoneotomy and incomple recovery from stress after anesthesia
and surgical operation. The difference might also result from
the variation between experiments at different times.
Based on a successful mouse model of ischemic ARF, we
first studied the renal protective effects of AC-YVAD-CMK
dynamically using a renal function assay and renal morpho
logical study at different times. The results showed that
renal function and morphological impairment were
significantly improved in the ischemic ARF mouse model after
being treated with specific ICE inhibitor. The findings
demonstrate that a specific ICE inhibitor can exert remarkable
renal-protective effects against acute ischemic lesion.
However, the ultimate objective of ischemic ARF therapy
is to increase patient survival from the disease. In the past
decade, different kinds of medicines, such as prostaglandins,
"renal-dose" dopamine and atrial natriuretic factor, have been
tried to treat patients with ischemic ARF and these animal
models. Although some of these agents have improved
renal function, urine output and even renal histological
impairment in patients and animal models, few studies have
demonstrated that these medicines can reduced the mortality of
the disease[15-18]. In the present study, we found that
applying this specific ICE inhibitor in the initial term of the disease
remarkably increased the survival rate in this animal model.
If it were applied continually for a longer period of time, the
model¡¯s survival rate might be further improved. Thus, we
speculate that this medicine might be superior in some way
to traditional medicines with regard to protection against
ischemic ARF.
Caspases are a group of protein-cleaving enzymes, and
play an important role in cell apoptosis and inflammatory
responses. ICE, also known as interleukin-1b-converting
enzyme, is a member of the inflammatory group in the caspase
family. It is a key initiative factor and an important inducer of
inflammatory chain responses secondary to an organ¡¯s
ischemic impairment[19]. Kaushal
et al[20] reported that
expression of the ICE gene and protein were up-regulated in an
ischemic ARF rat model. In the present study, ICE activity
was also increased in renal tissue of an ischemic ARF mouse
model. All of the evidence above strongly revealed that
gene transcription, protein synthesis and enzyme activation
of ICE were up-regulated during the progression of ischemic
ARF. Melnikov et al[11] reported that renal tubular
impairment and renal interstitial inflammatory infiltration were much
slighter in ICE gene knock-out mice than in wild-type mice
after renal ischemia reperfusion. The present study also
found that ICE inhibitor alleviated the impairment of renal
tissue and down-regulated the activity of renal ICE. We
conclude that excessive ICE activity prompts the
development of ischemic ARF, and selectively inhibiting
sham-operated groups ICE activity can exert renal-protective effects
on the disease. ICE activity was not suppressed to normal
levels by the dose of inhibitor used in the present study. We
presume that a better protective effect would be achieved if
a larger dose were administered.
Precursors of IL-18 and IL-1b are the main substrates of
ICE[12,13], and many reports have proved these 2 cytokines
can prompt acute ischemic organ
impairment[21-23]. Recent reports seem to emphasize the role of IL-18. Mice with
ischemic ARF have increased renal mature IL-18 expression,
and IL-18 antiserum can significantly improve the state of
the illness[11,22]. It was reported recently by Parikh
et al[7] that urinary IL-18 level was increased almost 50-fold in
patients with ARF, and the urinary IL-18 level in patients
transplanted with corpse kidney was 10-fold that in patients
transplanted with living kidney. These studies prove that IL-18
plays an important role in prompting renal impairment during
acute ischemia. Does ICE mediate the acute renal
impairment through prompting activation of IL-18 and
IL-1b? In the present study we detected the expression of mature
IL-18 in renal tissue. The results showed that expression of
activated IL-18 increased remarkably in model controls and
decreased significantly after the models were treated with
ICE inhibitor, which demonstrates that the excessive
activation of IL-18 resulting from ICE hyperactivity is one of the
main mechanisms causing renal tissue impairment. We infer
that IL-1b was also over-activated by ICE in this model.
Specific ICE inhibitor indirectly inhibits activation of IL-18 and
IL-1b through inhibiting the activity of ICE, and thereby
protects against acute ischemic ARF.
Because IL-18 is the strongest IFN-g
inducer[13], we then detected the expression of the
IFN-g gene in renal tissue. We found that renal
IFN-g mRNA expression was up-regulated significantly in model controls, and decreased after the
mice were treated with specific ICE inhibitor. The results
again demonstrate that IFN-g plays an important role in
ischemic ARF, and ICE inhibitor suppresses the expression
of IFN-g mRNA through inhibiting activation of IL-18, thus
further protecting against ischemic ARF.
In summary, the present study demonstrates that
specific ICE inhibitors are promising agents in the treatment of
ischemic ARF. It exerts renal-protective effects by inhibiting
strong ICE activity, thereby regulating the activation of
IL-18 and IL-1b, and further regulating abnormal expression
of IFN-g.
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