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Office for Medical Research Administration, National Taiwan University
Hospital, Taipei, Taiwan.
Department of Surgery, National Taiwan University College of Medicine,
Taipei, Taiwan.
Department of Physiology, National Taiwan University College of Medicine,
Taipei, Taiwan.
Department of Urology, National Taiwan University College of Medicine,
Taipei, Taiwan.
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Department of Pathology, National Taiwan University College of Medicine,
Taipei, Taiwan.
Correspondence to Dr. Su-Ming Hsu, Department of Pathology, National Taiwan University College of Medicine, 1-1 Jen-Ai Road, Taipei, Taiwan. Phone: 886-2-23123456 ext. 2980; Fax: 886-2-2394-7927; E-mail: smhsu{at}ha.mc.ntu.edu.tw or Dr. Ming-Kuen Lai, Department of Urology, National Taiwan University College of Medicine, 1-1 Jen-Ai Road, Taipei, Taiwan. Phone: 886-2-23123456 ext. 2135; Fax: 886-2-2321-9145; E-mail: mingkuen{at}ha.mc.ntu.edu.tw
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apoptosis, or programmed cell death, which can be distinguished on the basis of morphologic and biochemical criteria, is different from necrosis (11,12,13). The mechanism responsible for post-I/R apoptosis is attributed to the increasing activity of endonuclease by elevation of calcium entry into cells (12), or the release of reactive oxygen species (ROS) (1,2,3,14,15,16). ROS induce apoptosis by causing DNA damage, oxidation of lipid membranes, and/or direct activation and expression of the genes/proteins responsible for apoptosis (17). For example, increases in the Bax/Bcl-2 ratio (18), expression of caspase and its activity (19,20), and caspase-mediated cleavage of poly-(ADP-ribose)-polymerase (PARP) (21) have been found in organs subjected to I/R injury or in cells after a cytotoxic insult.
The sources of ROS generated after I/R may be circulatory macrophages/neutrophils (22) or the resident cells (23). In the reperfused heart, infiltrating neutrophils are the primary source of ROS (1). In brains subjected to I/R, superoxide detected in the meninges was located primarily in the extracellular space and occasionally in endothelial cells and vascular smooth muscle cells (2). In the liver, the sources of ROS seem to be Kupffer cells, sinusoidal cells, and infiltrating leukocytes (24). In the kidney, demonstration of increased renal venous ROS after I/R by electron spin resonance and the efficacy of antioxidants or free-radical scavengers in minimizing I/R injury (7,14,16) suggest a role of ROS in I/R injury. However, the precise origin of ROS in the I/R kidney has yet to be defined.
In this study, we adapted a modified chemiluminescence (CL) method (25,26,27) for direct measurement of the amount of ROS produced from the kidney surface and from renal veins in I/R kidneys. In addition, we determined the cellular location of de novo ROS synthesis in the I/R kidney by using an in situ vascular perfusion technique, whereby nitro blue tetrazolium (NBT) was perfused through the kidney for depicting cells in which superoxide was formed (28). The onset of apoptosis and its severity in the I/R kidney were determined by examination of the presence of DNA fragmentation and an increase in cellular proteins associated with apoptosis. Our study showed that prolonged ischemia resulted in severe apoptosis in the reperfused kidney in a time-dependent dependent manner. Cells in both proximal tubules (PT) and distal tubules (DT) were subjected to I/R injury, whereas only PT cells produced measurable ROS by the NBT method. Not unexpected, the apoptosis in vivo and in vitro induced by I/R and hypoxia/reoxygenation (H/R) in PT cells, but not in DT cells, can be prevented by superoxide dismutase (SOD) or a novel free-radical scavenger, hexa(sulfobutyl)fullerene (FC4S) (14).
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Materials and Methods |
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Induction of Unilateral Renal Ischemia
For induction of total ischemia in the kidney, the left renal artery was
clamped with a small vascular clamp. All rats were randomly divided into four
groups, depending on the duration of ischemia: 0 min (control), 15 min (I15),
45 min (I45), and 60 min (I60). Shamoperated (control) animals underwent
similar operative procedures without occlusion of the renal artery.
Reperfusion was initiated by removal of the clamp. The rats in each group were
divided further into subgroups and were subjected to the following time frames
of reperfusion: for 5 min (R5m), 30 min (R30m), 1 h (R1h), 2 h (R2h), 4 h
(R4h), 10 h (R10h), or 24 h (R24 h).
SOD Treatment
A set experimental rats (n = 4 for each ischemic group) was
treated with intravenous SOD (500 U per rat; Sigma, St. Louis, MO) after the
ischemic period (before reperfusion). In this experiment, all ischemic kidneys
were reperfused for 4 h. The experiment was intended for confirmation of the
role of ROS in apoptosis formation in the I/R kidney.
Tissue Preparation
For evaluation of the effect of ischemic intervals on apoptosis formation
and apoptosis-related protein expression, 12 rats in each ischemic group (I15,
I45, and I60) were used. Four rats were killed with an overdose of anesthetics
at the end of ischemia (i.e., no reperfusion), and at 1 h or 4 h
after reperfusion. For comparison, three control rats were used. The kidney
was resected and divided into three parts. One part was stored in 10% neutral
buffered formalin for routine histology and in situ apoptotic assay,
another was prepared for DNA fragmentation electrophoresis, and the third was
quickly frozen in liquid nitrogen and stored at -70°C for protein
isolation.
In Situ Apoptotic Assay
The method for in situ apoptotic assay (i.e., terminal
deoxynucleotidyl transferase-mediated nick-end labeling [TUNEL] method) was
performed according to the method of Gavrieli et al.
(29) with minor modifications
(30). Briefly, 4- to
6-µm-thick sections of the kidney were prepared, deparaffinized, and
stained by the TUNEL—avidin-biotin-peroxidase complex method
(10,30).
Twenty high-power (x400) fields of the outer medulla were randomly
selected in each section, and the number of apoptotic cells was counted. PT
were distinguished from other segments by their characteristic brush border
and their relatively large epithelial cell bodies. DT were recognized by their
smaller (the nuclei are close together) and more regular cells
(31). The number of apoptotic
cells was expressed per 100 of the PT or DT cells in each section.
DNA Extraction and Electrophoresis
The kidneys were homogenized and lysed with 20 ml of buffer containing 10
mM Tris-HCl (pH 7.5), 1 mM ethylenediaminetetraacetate, 0.1 M NaCl, and 1 ml
of 10% sodium dodecyl sulfate (SDS). The lysates were incubated with 200 µl
of 10 mg/ml proteinase K at 50°C overnight. DNA was extracted twice with
phenol and twice with phenol/chloroform (1/1); the upper layers were
collected, and 5 M NaCl was added in a 1:50 (vol/vol) ratio. The sample was
then precipitated overnight at -20°C in absolute ethanol. DNA was pelleted
by centrifugation at 3000 x g at 4°C for 10 min, washed
with 70% ethanol, and dried. The DNA obtained was resuspended in TE (10 mM
Tris-HCl, 1 mM ethylenediaminetetraacetate) at 1 µg/ml and incubated with
0.1 mg/ml DNAse-free RNAse at 37°C for 30 min. Four µ1 of 0.25%
bromophenol blue and 0.25% xylene cyanol in 40% sucrose were added to DNA
samples at a 1:5 (vol/vol) ratio. Electrophoresis was performed at 50 V in
1.6% agarose gels. DNA was visualized with ethidium bromide.
In Vivo CL Recording
The method for detection of CL from the organ surface after intrarenal
arterial 2-Methyl-6-[4-methoxyphenyl]-3,7-dihydroimidazo-[1,2-a]-pyrazin-3-one
hydrochloride (MCLA) (TCI-Ace; Tokyo Kasei Kogyo Co. Ltd., Tokyo, Japan)
injection
(2,26)
was adapted for demonstration of ROS production in the I/R kidney. The rat was
maintained on a respirator (tidal volume, 1.0 to 1.5 ml; rate, 80 to 90
cycles/min; inspiratory pressure, 20 to 30 cm H2O) and a
circulating water pad at 37°C during photon detection. For excluding
photon emission from sources other than the kidney, the animal was housed in a
dark box with a shielded plate. Only the renal window was left unshielded and
was positioned under a reflector, which reflected the photons from the exposed
kidney surface onto the detector area.
A single dose (1 mM in 0.1 ml) of N,N'-dimethyldiacridium (lucigenin; Sigma) or a continuous infusion (0.2 mg/ml per h) of MCLA was administered into the control or I/R kidney via an intra-renal arterial catheter. The lucigenin- or MCLA-enhanced CL signal from the kidney surface was measured continuously during administration by use of a Chemiluminescence Analyzing System (CLD-110, Tohoku Electronic Industrial Co., Sendai, Japan). Eight rats (five for lucigenin and three for MCLA treatment) in each ischemic group (I15, I45, and I60) were used in this part of the experiment.
A separate experiment on I45 rats (n = 3) treated with intravenous SOD (cumulative dose to 500 U per rat) before lucigenin injection at 4 h reperfusion was performed. This experiment was intended for confirmation of the enhanced CL induced by I/R that originated from ROS.
Preparation of Whole-Blood Sample for Lucigenin-Enhanced CL
Determination
Four groups of rats (I15, I45, I60, and control; five rats in each group)
were used for measurement of ROS in whole-blood samples. A series of blood
samples (0.2 ml) from the left renal vein or carotid artery were obtained
immediately after reperfusion for various times. The blood samples were
immediately wrapped in aluminum foil and kept on ice until CL measurement,
usually done within 2 h
(32).
Immediately before CL measurement, 0.1 ml of phosphate-buffered saline (pH 7.4) was added to 0.2 ml of blood sample. The CL was measured in a completely dark chamber of the Chemiluminescence Analyzing System. After 100-s background level determination, 1.0 ml of 0.1 mM lucigenin in phosphate-buffered saline (pH 7.4) was injected into the sample. The CL was monitored continuously for an additional 600 s. The total amount of CL was calculated by integrating of the area under the curve and subtracting it from the background level. The assay was performed in duplicate for each sample and was expressed as CL counts/10 s for blood CL. The mean ± SEM of the CL level of each sample was calculated.
In Situ Demonstration of Superoxide Formation by NBT
Three rats in each ischemic group were killed at the end of ischemia, at 1
h and 4 h after reperfusion. An NBT perfusion method was used for localizing
de novo ROS generation in the I/R kidney
(28). An 18-gauge needle
connected to an infusion pump (Infors AG, CH-4103, Bottmingen, Switzerland)
was inserted in the upper abdominal aorta just above both kidneys, whereas the
lower abdominal aorta below both kidneys was ligated. After the bilateral
renal veins were cut, the kidneys were perfused with 37°C Hanks' balanced
salt solution (HBSS; flow rate, 10 ml/min [pH 7.4]). Once blood had been
removed, NBT (1 mg/ml) was added to the HBSS, and the kidneys were perfused
for an additional 10 min at a flow rate of 5 ml/min. All unreacted NBT was
removed from the kidneys by postperfusion with HBSS. The NBT-perfused kidneys
were cut and fixed in zinc/formalin for histologic examination for formazan
deposits, and some samples were prepared for localization of apoptotic cells.
In separate experiments, SOD was included in the perfusate (500 µg/ml) for
confirmation of the specificity of the NBT deposition as the product of
ROS.
Immunoblot Analysis for Bax, Bcl-2, CPP32, and PARP
We measured the amounts of Bax/Bcl-2, caspase 3, and PARP
(18,19,21)
in renal tissues of I/R rats. For protein analysis, left kidney samples were
homogenized with a prechilled mortar and pestle in extraction buffer, which
consisted of 10 mM Tris-HCl (pH 7.6), 140 mM NaCl, 1 mM phenylmethylsulfonyl
fluoride, 1% NP-40, 0.5% deoxycholate, 2% ß-mercaptoethanol, 10 µg/ml
pepstatin A, and 10 µg/ml aprotinin. The mixtures were homogenized
completely by vortexing and kept at 4°C for 30 min. The homogenate was
centrifuged at 12,000 x g for 12 min at 4°C, the
supernatant was collected, and the protein concentrations were determined by
BioRad Protein Assay (BioRad Laboratories, Hercules, CA). Antibodies raised
against Bax (Catalog # AB2916, human Bax synthetic peptide, amino acids 44 to
62; Chemicon, Temecula, CA), Bcl-2 (Catalog # B46620, human Bcl-2 peptide,
amino acids 49 to 179; Transduction, Bluegrass-Lexington, KY), the activation
fragments (32 kD of proenzyme and 17 kD of cleaved product) of caspase 3
(CPP32/Yama/Apopain; Catalog # 06-735, human full-length caspase 3 fusion
protein containing a histidine-6 tag; Upstate Biotechnology, Lake Placid, NY),
PARP (Catalog # G3741, N-terminal peptide from the p85 fragment; Promega,
Madison, WI), and ß-actin (Catalog # A5316, Clone AC-74; Sigma) were
used. All of these antibodies cross-react with respective rat antigens.
SDS-polyacrylamide gel electrophoresis (PAGE) was performed on 12.5% separation gels in the absence of urea and was stained with Coomassie brilliant blue. Proteins on the SDS-PAGE gels, each lane containing 30 µg of total protein, were transferred to nitrocellulose filters. The immunoreactive bands were detected by incubation with the antibody described above, followed by secondary antibody-alkaline phosphatase, and finally with NBT and 5-bromo-4chloro-3-indolyl phosphate, toluidine salt (Roche Diagnostic GmbH, Mannheim, Germany) stock solution for 30 min at room temperature.
Isolation of Renal PT and DT
We also designed experiments to study the role of ROS in the formation of
apoptosis in different portions of the renal tubules. Female Wistar rats
(n = 4) were used. Animals were anesthetized with sodium
pentobarbital (60 mg/kg, intraperitoneally). Kidneys were flushed with 20 ml
of ice-cold Krebs-Henseleit-saline buffer (KHS) via an aortal catheter.
Specific isolation of PT and DT was performed as described previously
(33). After the kidneys were
flushed with KHS, they were perfused with 10 ml of 0.1% Type IV collagenase in
KHS (Sigma). Minced renal cortices were incubated for 25 min at 37°C in 30
ml of 0.1% collagenase-KHS, continuously gassed with 95% O2/5%
CO2, and gently agitated. PT and DT cells were isolated in
pregassed 45% Percoll-KHS solution (Sigma) by centrifugation at 20,000 x
g in a fixed-angle rotor (SS34 rotor of Sorvall RC5C centrifuge,
Newtown, CT) for 30 min at 4°C. The band enriched in PT or DT was
withdrawn and washed two times with ice-cold KHS. Each of the two types of
cells was cultured. Alkaline phosphatase activity as a marker enzyme for PT
and hexokinase activity as a marker enzyme for DT were measured
spectrophotometrically with p-nitrophenylphosphate and a pyridine
nucleotide-linked assay, respectively
(34). The PT and DT cultures
were also processed for cytologic examination after formal fixation and
coloration with periodic acid-Schiff. Identification of PT by their granular
yellow cytoplasm and pink brush border membrane and DT by their clear
cytoplasm and the absence of a brush border membrane is made possible by this
procedure (34).
Induction of H/R of the renal tubule cells was performed as described previously (10). The cultures first were placed in an atmosphere of 95% O2/5% CO2 at 37°C for 30 min. Hypoxia was achieved by gassing with 95% N2/5% CO2 for 15 min, whereas reoxygenation was performed by reintroduction of 95% O2/5% CO2 for 30 min. To study the role of ROS in the formation of apoptosis, we added 100 U of SOD and 50 µM FC4S (provided by Dr. L. Y. Chiang, Center for Condensed Matter Sciences, National Taiwan University) (14), a novel water-soluble fullerene with free-radical scavenging activity, to the cell medium. For determination of the number of apoptotic cells in culture, the cells were fixed with 70% ethanol, stained with propidium, and counted with FACSCalibur (Becton Dickinson, San Jose, CA). Cell viability was counted with a Trypan blue dye exclusion test (33). The amounts of ROS in PT and DT cell cultures (106 cells/ml) were detected by the lucigenin-enhanced CL test described above.
For immunoblotting analysis of PT and DT, 1x106 cells were used per sample; these were centrifuged and washed in KHS. Next, 40 µl of sample buffer (containing 50 mM Tris [pH 6.8], 6 M urea, 6% ß-mercaptoethanol, 0.03% bromophenol blue, and 3% SDS) was added, and the samples were sonicated. These were then heated at 80°C for 5 min, and samples were subsequently loaded and run on a 10% SDS-PAGE gel for 1 h at 140 V. After electrophoretic transfer of the separated polypeptides to nitrocellulose, the membrane was probed with the apoptosis-related antibodies as described above.
Statistical Analyses
All values were expressed as mean ± SEM. Differences within groups
were evaluated by paired t test. One-way ANOVA was used for
establishing differences among groups. Intergroup comparisons were made by
Duncan's multiple-range test. Differences were regarded as significant if
P < 0.05 was attained.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the I15 kidneys, a few apoptotic cells were observed in the specimens at 1 to 4 h after reperfusion (Figure 1, Table 1), and the apoptotic cells occurred mainly in the PT. In I45 and I60 kidneys, increased numbers of apoptotic cells were detected in both PT and DT after reperfusion. The numbers of apoptotic cells reached a plateau when the kidneys were reperfused for 4 to 6 h. There were no apparent apoptotic cells detected in the kidneys subjected to 10 h and 24 h of reperfusion, probably because of the removal of apoptotic cells by a tissue repair mechanism.
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SOD treatment (500 U/rat) before reperfusion reduced the number of apoptotic cells in renal tubules, especially in the PT (Figure 1). The number of apoptotic cells in PT in I15/R4 h kidneys was 3.0 ± 0.6% and was reduced to 0.2 ± 0.1% with SOD treatment. Similar results were obtained in I45/R4 h and I60/R4 h kidneys; the percentages of apoptotic cells were 8.9 ± 1.9% and 13.8 ± 2.7%, respectively, without SOD treatment and were reduced to 0.3 ± 0.1% and 3.2 ± 0.7% with SOD treatment. However, the number of apoptotic cells remained unchanged in DT in both nontreated and SOD-treated rats (4.1 ± 1.2% versus 3.8 ± 0.9% in I45 kidneys and 7.2 ± 1.2% versus 6.5 ± 1.4% in I60 kidneys; P > 0.01).
Effect of Ischemic Interval or I/R on Renal DNA Fragmentation
Patterns
By gel electrophoresis, no apparent DNA fragmentation was detected from
ischemic kidneys without reperfusion. An apoptotic DNA laddering pattern was
clearly evident when ischemic kidneys were subjected to 1 to 4 h of
reperfusion. In all three ischemic groups of animals, there was an increasing
"smear" DNA pattern after prolonged reperfusion, i.e.,
> 10 to 24 h (Figure 2).
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Kidney CL In Vivo
Intrarenal arterial injection of lucigenin into the I/R kidney led to a
rapid rise in CL counts to a peak at 2 min postinjection
(Figure 3). The peak value of
lucigenin-enhanced CL from the I/R kidney surface was then rapidly decreased
to a basal level as the renal circulation resumed (this usually took 4 min).
The basal CL level detected from the normal kidney surface was approximately
1437 to 1566 counts/10 s. The sham-operated kidneys and the ischemic kidney
without reperfusion did not show increased CL after administration of
lucigenin (Table 2), suggesting
that the enhanced CL resulted from reperfusion injury.
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A minimal 5-min reperfusion was required for detection of increases in CL, and the observed peak level (maximal value) of CL, i.e., the amount of ROS product, seemed to be correlated with the duration of ischemia. The peak levels of CL from the kidney surface were increased 3.5-fold (5811 counts/10 s), 7-fold (9666 counts/10 s), and 9-fold (13,924 counts/10 s) in the I15, I45, and I60 groups followed by 5-min reperfusion, respectively. Lucigenin-enhanced CL was not demonstrated in kidneys after 10 to 24 h of reperfusion (Table 2).
The CL detected from a single dose of lucigenin and that from continuous infusion of MCLA were similar. Continuous infusion of MCLA into a kidney without I/R insult displayed a basal level of CL at approximately 1150 to 1500 counts/10 s. In I/R kidneys, an abrupt increase in MCLA-enhanced CL was observed and was maintained at the increased level for 4 to 5 h (Figure 3B).
In I45 rats treated with SOD before lucigenin injection at 4 h of reperfusion, the peak value of CL from the kidneys was depressed significantly (Figure 3C), suggesting that the increased CL counts were derived from ROS after I/R insult. When we correlated the apoptotic cell number with the renal CL in the sham-operated rats and the three ischemic groups, a positive linear regression curve (r2 = 0.9758, P < 0.01) was obtained (Figure 4).
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Effect of Ischemic Interval on Renal Venous and Systemic
Arterial-Blood CL
We also adapted the CL method to examine the amounts of ROS in the renal
venous and carotid arterial blood samples. The renal venous CL was increased
45-fold (47,864 counts/10 s), 85-fold (85,230 counts/10 s), and 110-fold
(117,410 counts/10 s) in I15, I45, and I60 rats (with 5-min reperfusion),
respectively (Figure 5,
Table 1). A similar result was
obtained with the carotid arterial CL, with 40-fold, 75-fold, and 95-fold
increases in I15/R5m, I45/R5m, and I160/R5m rats, respectively. The increases
in renal venous and carotid arterial CL persisted for 4 h after reperfusion
and returned nearly to basal levels after 10 to 24 h of reperfusion.
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In Situ Localization of ROS Formation by NBT Perfusion
We used an NBT staining method for localization of ROS production in renal
tissue. NBT is a dye that is reduced to an insoluble formazan derivative upon
exposure to superoxide (28).
The blue-colored formazan is readily detectable in tissue by light microscopy.
Figure 1A shows that superoxide
generation was not seen in control kidneys not subjected to ischemia. There
was significant superoxide production (blue formazan deposits) in ischemic
kidneys after 4 h of reperfusion (Figure
1). An increased ischemic interval in I45 kidneys resulted in a
higher density of blue formazan deposits as compared with that in I15 kidneys.
The NBT deposits were located mainly in the PT and very sparsely in DT
(Figure 1, B and C). In rats
treated with SOD, significant decreases in NBT deposits in PT cells were
observed.
We performed double staining with the use of the NBT and TUNEL methods for evaluation of the spatial relationship of ROS production and apoptotic nucleus formation in renal tissues. Both NBT cellular deposits and apoptotic nuclei could be detected readily in tissue sections. As shown in Figure 1, the apoptotic nuclei appeared in either PT or DT epithelial cells but rarely in glomerular cells. SOD treatment reduced the NBT deposit, as did the formation of apoptotic nuclei in PT. The apoptotic nuclei remained in DT in the SOD-treated rats, indicating that formation of apoptosis in DT was not influenced by the small amounts of ROS produced (see data presented in the Effects of H/R on Tubular Cells in Culture section).
Protracted Ischemic Intervals and Apoptosis-Regulated Gene
Expression
The expression of Bax, Bcl-2, CPP32, and PARP in the kidney samples after
I/R was assessed by immunoblotting with antibodies against Bax, Bcl-2, CPP32,
and PARP (Figure 6). The
expression of Bcl-2 was detected in control renal tissues. Bcl-2 was
apparently depressed in the ischemic stage, but gradually returned to its
pre-ischemic level or higher after reperfusion.
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Expression of Bax but not of CPP32 and PARP was detected in control tissues. The expression of all three proteins was increased after 1 h of reperfusion and reached a high level at 4 h of reperfusion. Increased ischemic intervals seemed to potentiate their expression (Figure 6). By densitometry, the Bax/Bcl-2 ratio was increased 1.5-, 1.9-, and 2.1-fold in I15/R4h, I45/R4h, and I60/R4h kidneys, respectively. Expression of CPP32 (32 kD, proenzyme) and its cleaved product (17 kD) was increased 8-, 10-, and 12-fold and 6-, 10-, and 17-fold in I15, I45, and I60 kidneys, respectively. Expression of PARP was increased 3-, 5-, and 8-fold in I15, I45, and I60 kidneys, respectively.
Effects of H/R on Tubular Cells in Culture
PT and DT cells isolated from rat kidney displayed >88% viability by the
Trypan blue exclusion test. The characterization of PT and DT was performed by
measurement of marker enzyme activity. Alkaline phosphatase activity and
hexokinase activity in PT culture (n = 3) were 545 ± 125
nmol/min and 98 ± 27 nmol/min, respectively, whereas these activities
in DT culture (n = 3) were 107 ± 24 nmol/min and 866 ±
189 nmol/min, respectively. The purity of PT and DT cell cultures was 90
± 3% and 86 ± 4%, respectively, as determined by periodic
acid-Schiff staining. When these cells were exposed to H/R, a greater decrease
in cell viability in PT cells (56 ± 7%) was evident as compared with
that of DT cells (69 ± 5%; Figure
7). A 10-fold increase in the number of apoptotic cells was noted
in PT culture (from 2.4 ± 0.5% to 23 ± 5%), as compared with a
3-fold increase in DT culture (from 1.9 ± 0.5% to 6.8 ± 2.1%;
P < 0.05).
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Treatment of cultures with SOD or FC4S, a novel water-soluble fullerene with free-radical scavenging activity (14), protected the PT cells from H/R injury (Figure 7). The viability of PT cells subjected to H/R was 56 ± 7% versus 79 ± 5% or 77 ± 5% in cultures treated with SOD or FC4S. The percentage of apoptotic cells among PT cells subjected to H/R was 23 ± 5% without free-radical scavenger treatment versus 13 ± 4% with SOD treatment or 14 ± 4% with FC4S treatment (P < 0.05). However, treatment with SOD or FC4S did not seem to affect the viability of the reoxygenated DT cells (viability, 69 ± 5% without free-radical scavenger versus 74 ± 6% with SOD or 72 ± 6% with FC4S, and of apoptotic cells from 6.8 ± 2.1% without free-radical scavenger versus 6.2 ± 2.0% with SOD or 6.5 ± 1.5% with FC4S).
The ROS response of PT and DT culture of H/R is displayed in Figure 7. ROS detected in PT and DT culture media were 606 ± 115 counts/10 s and 558 ± 99 counts/10 s respectively. After H/R, ROS in PT and DT culture media were increased to 2732 ± 488 counts/10 s and 1215 ± 205 counts/10 s, respectively. Addition of SOD or FC4S to the culture medium significantly decreased H/R-induced ROS to near-basal levels in both PT and DT cultures.
The expression of Bax, Bcl-2, CPP32, and PARP in PT and DT cultures after H/R was assessed by immunoblotting with antibodies against Bax, Bcl-2, CPP32, and PARP (Figure 8). Weak expression of Bcl-2 was detected in control PT and DT cultures, and its expression was apparently enhanced in both cultures after H/R insult. The enhanced Bcl-2 expression could be suppressed by treatment with free-radical scavengers (especially SOD). Expression of Bax, CPP32, and PARP was readily detected in control PT and DT cultures. The expression of all three of proteins was increased after H/R; however, the increased expression was depressed by SOD and FC4S treatment (Figure 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our study showed that prolonged ischemia resulted in severe apoptosis in the reperfused kidney in a time-dependent manner. This was demonstrated by increases in DNA fragmentation, in apoptotic cell number, and in "proapoptotic" proteins such as Bax, CPP32, and PARP. A minimum of 15 min of ischemia is required for the detection of apoptotic nuclei during the reperfusion phase, whereas reperfusion as short as 5 min was sufficient to allow production of ROS by PT cells in the kidneys. Prolonged ischemia seemed to potentiate the proapoptotic mechanisms.
Indirect evidence has shown that ROS have an important role in I/R injury; however, obtaining direct proof is hampered because of the high reactivity and the short biologic half-life of ROS. A number of techniques have been developed for study of the production and mechanisms of ROS in I/R tissue. These include electron spin resonance spectroscopy (35), the NBT reduction method (15, 28), DAB-Mn2+ cytochemistry (2), and measurement of the induction or depletion of endogenous ROS scavengers (7, 14, 16, 36, 37). In this study, we used an enhanced CL method to study ROS production. This method has been applied to measurement of ROS production in cultured cells (22), in the whole-blood system (32, 38), in isolated perfused organs (3, 39), and in lung and brain in vivo (2, 26). We showed that the level of lucigenin- or MCLA-enhanced CL detected from the renal surface was correlated with that in whole blood from renal venous efflux or from systemic arterial ROS measured by the lucigenin-enhanced CL method. In either method, a significant level of CL was detected only in reperfused ischemic kidneys but not in sham-operated kidneys or in ischemic kidneys without reperfusion. This strongly suggests a role of reperfusion in the induction of ROS generation and, subsequently, apoptosis formation.
By using an in situ vascular NBT perfusion technique, we showed that the cellular source of ROS synthesis was mainly the PT epithelial cells in the I/R kidney. The apoptotic cell death as well as the ROS production in PT cells can be ameliorated by SOD treatment before reperfusion. In contrast, SOD treatment did not seem to minimize apoptosis in DT cells in I/R kidneys. We also noted that, in culture, the number of apoptotic cells and the amounts of ROS formed are more evident in PT cells than in DT cells subjected to H/R insult. Kiyama et al. (31) reported that upon exposure to transient ischemia, the DT of the kidney often escape the severe damage that afflicts the PT. The resistance to I/R injury by DT epithelial cells may be attributed to a relative abundance of the manganese SOD gene in DT, whereas low levels of expression were detected in PT (31). This could also explain the generally low level of ROS formation in DT cells. As expected, addition of SOD or FC4S for scavenging ROS in culture provided significant protection against H/R-induced apoptosis in PT but had only a minor effect in DT cells.
Our results indicate that, as in many organs, the increased Bax, CPP32, and PARP levels could contribute to apoptotic cell death in I/R kidneys. The expression patterns of these proteins in kidneys subjected to I/R as well as in PT and DT cultures subjected to H/R are similar. The level of these proteins seems not to explain the different degree of apoptosis in PT and DT cells in culture. Perhaps multiple and complex proteins, in addition to these four proteins (Bax, Bcl-2, CPP32, and PARP), are involved in this process. Thus, the marked difference between the PT and DT in their susceptibility to ischemic and/or hypoxic injury is attributable to their intrinsic cellular properties, which include endogenous antioxidants, e.g., SOD).
In summary, the present study indicates that ROS are produced in significant amounts in PT epithelium under reperfusion conditions and therefore may be responsible for the apoptotic death of these cells. In DT epithelial cells in I/R kidneys, unlike PT, mechanisms other than formation of ROS may prevail for the induction of apoptosis. Treatment with SOD and other free-radical scavengers as well may be effective for the prevention of ROS-mediated apoptosis in PT.
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