| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, and
Neurotoxicology Laboratories, Rutgers University College of Pharmacy, Piscataway, NJ 08854
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B activation at the level of IB
and IBß degradation.
In the case of IB, no apparent phosphorylation was detected.
These results suggest that IB proteolysis is triggered during the
renal I/R pro-oxidant state by a still unknown mechanism, which might
be different from other stimuli. A central role of NF-B in CXC
chemokine activation was demonstrated in cell culture anoxia/ATP
repletion experiments as a model of I/R. The data presented indicate
the important role of GP-sensitive signal transduction pathways in the
development of inflammatory response and tissue injury during
I/R. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Neutrophil activation has been gaining attention in the past several years, primarily due to re-evaluation of the role of excessive inflammatory response in kidney I/R injury. In models of renal I/R, depletion of neutrophils, blockade of neutrophil adhesion to the endothelium, and inhibition of the complement system all decrease kidney damage (9). Complementary studies using gene knockouts of the adhesion molecules and chemokines support this conclusion (10). Recent studies have also indicated that ROS can participate in neutrophil recruitment by up-regulation of adhesion molecules and chemotactic factors (11).
We are using transgenic mice overexpressing both types of human glutathione peroxidases, intra- (GPx1) and extracellular (GPxP), as a model system to investigate the influence of increased levels of these antioxidant enzymes on pathways leading to kidney damage. Compared with the two other most abundant antioxidant enzymes, superoxide dismutase and catalase, GP is the most effective in protection of cells against oxidative damage. GP overexpression provides transgenic mice with increased resistance to brain (12) and myocardial I/R injury (13). The data presented here indicate that increased levels of GP activity were able to protect mice against kidney I/R damage. One of the important mechanisms of this effect involves inhibition of activation of neutrophil-attracting chemokines at the level of their transcription.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The generation of transgenic mice with human GPx1 and GPxP genes in the C57BL/6xCBA/J background was described previously (12, 14). Mouse lines GPE23 and GPP17 (containing 200 copies of the human GPx1 gene and two copies of the human GPxP gene, respectively) were used for these studies. As we reported, GPE23 strain express human GPx1 in all tested tissues. Total GP activity was 50% higher in kidney extracts from these transgenic mice compared with that in normal animals. In GPP17 mice, transgene expression was detected in a blood and kidney (50% and 30% higher than in nontransgenic mice, respectively). To obtain nontransgenic and heterozygous transgenic animals for experiments, transgenic founders were bred with (C57BL/6xCBA/J) F1 mice.
I/R of the mouse kidney
The expermental procedure was as described previously (8). These conditions provide reproducible I/R injury in kidneys of normal and transgenic mice. In brief, normal or transgenic males, weighing 25–35 g, were anesthetized with sodium pentobarbital (25 mg/kg) and xylazine (10 mg/kg) and administered heparin (300 USP units/kg of body weight) s.c. before the surgery. Unilateral renal ischemia was induced by occluding the left side renal vein and artery with a microaneurysm clamp. Body temperature was maintained at 37°C during the entire procedure. After 32 min of ischemia, the left kidney was reperfused by declamping the microaneurysm applicator, and right nephrectomy was performed. Sham surgery consisted of a surgical procedure that was identical except that the microaneurysm clamp was not applied. Mice were sacrificed at different time points after surgery. Kidneys were removed immediately after perfusion with cold PBS and rapidly frozen in liquid nitrogen for obtaining extracts and RNA analysis. Blood samples for blood urea nitrogen (BUN) and creatinine determinations were obtained via cardiac puncture. Plasma BUN and creatinine were measured using kits (Sigma, St. Louis, MO).
Mortality study
After the induction of renal ischemia and reperfuson injury or sham operation, 16 animals/transgenic mouse group and 24/nontransgenic group received standard rat chow diet and water ad libitum and were observed for survival up to 12 days.
Histopathology
Kidney tissue was harvested at 24 h after surgery, fixed in a phosphate buffer (pH 7.0) containing 10% formalin, and paraffin embedded. Five-micron sections were stained with hematoxylin and eosin and examined by light microscopy. Damage was assessed by a pathologist and assigned a number from a scale of 0–4 based on qualitative and quantitative criteria. All slides were interpreted blind.
Lipid peroxidation assay
Lipid peroxidation in the kidney was assessed by measurement of malondialdehyde (MDA) and 4-hydroxy-2(E)-nonenal (4-HNE) using a Lipid Peroxidation Kit (Calbiochem, San Diego, CA) according to the manufacturer’s protocol. Lipid peroxidation was normalized to the protein content of the supernatant.
Renal myeloperoxidase (MPO) activity assay
Myeloperoxidase activity in renal tissue was assessed spectrophotometrically at 630 nm, as described previously (15). The assay mixture consisted of 200 µl of heat-inactivated renal supernatant, 100 µl of tetramethylbenzidine dissolved in DMSO (final concentration, 1.6 mM), and 700 µl of 80 mM phosphate buffer, pH 5.4, with H2O2 (final concentration, 3.0 mM). Renal MPO activity was expressed in units, where 1 U represents the amount of enzyme degrading 1 µmol H2O2/min, and was standardized with the protein content of the supernatant (units per milligram of protein).
Detection of apoptosis by DNA fragmentation and ELISA
For DNA fragmentation analysis, kidneys were incubated with extraction buffer containing 50 mM Tris-HCl (pH 8.0), 1% SDS, 100 mM EDTA, and 100 µg/ml of proteinase K at 55°C for 12 h. Following incubation, each sample was treated with 10 µg of RNase for 1 h at 37°C and incubated at 4°C for 2 h with sodium chloride at a final concentration of 1 M. Samples were then centrifuged at 13,000 x g at 4°C for 15 min, and supernatant was gently mixed with 2 vol of phenol/chloroform (1:1). After centrifugation the aqueous phase was collected, and the DNA was precipitated with an equal volume of isopropanol at -20°C overnight. DNA samples were applied on 2.0% agarose gel containing ethidium bromide with m.w. markers. DNA fragmentation was quantitated by measuring cytoplasmic nucleosomes using the Cell Death Detection ELISA Plus kit (Boehringer Mannheim, Indianapolis, IN).
Preparation of RNA and Northern blot analysis
Total RNA was isolated from kidney samples using TRIzol reagent (Life Technologies, Gaithersburg, MD) according to the manufacturer’s protocol. The RNA concentration was measured using a spectrophotometer. For Northern blot analysis, RNA samples (20 µg) were loaded on 1.4% agarose gels with 2.2 M formaldehyde. After electrophoresis, RNA was transferred from the gels to GeneScreen membranes (New England Nuclear, Boston, MA) using a PosiBlot pressure blotter (Stratagene, La Jolla, CA). After transfer the membranes were fixed and hybridized with labeled DNA fragments at 42°C overnight.
RNase protection assay
RNase protection assay was performed using a Riboquant kit and
an mCK-5 Multiprobe Template Set (PharMingen, San Diego, CA) according
to the manufacturer’s protocol. RNA probes for mouse chemokines
lymphatactin (Ltn), RANTES, eotaxin, MIP-1ß, MIP-1, MIP-2,
IFN-
-inducible-protein(IP-10), MCP-1, and TCA-3) as well as those
for positive controls (L32 and GAPDH) were generated in vitro using a
T7 polymerase transcription kit and
[-32P]UTP as a label. A standard curve was
made using undigested probes as markers, and the identities of
RNase-protected bands were established. To compare RNA amounts in the
protected bands, the films were scanned using an imaging densitometer
(GS-670, Bio-Rad, Hercules, CA), and final values were factored
relative to GAPDH levels.
Immunostaining
Frozen tissue sections (10 µm) were used for immunostaining. Endogenous peroxidase activity was blocked by incubating sections in a blocking buffer (3 M NaN3, 1% H2O2, and 0.1% saponin in Earl’s buffered saline, pH 7.4; Life Technologies) for 60 min in the dark. Endogenous biotin activity was eliminated in two blocking buffers (Avidin/Biotin Blocking kit, Vector, Burlingame, CA) sequentially, for 1 h each. Biotin solution was supplemented with 10% donkey serum. After washing, sections were incubated overnight at room temperature with primary Abs (1/1000 to 1/2500 dilutions). Goat anti-mouse KC and MIP-2 Abs were obtained from R & D Systems (Minneapolis, MN). Sections were then washed three times and incubated for 1 h with biotin-donkey anti-goat IgG as a secondary Abs diluted 1/4000 (Jackson ImmunoResearch Laboratories, West Grove, PA). Slides were washed three times and incubated with Vectastain Elite ABC-peroxidase reagent (Vector) supplemented with 0.1% saponin for 30 min in the dark. Samples were developed with 3,3'-diaminobenzidine tetrahydrochloride (DAB liquid substrate, Sigma) prepared according to the manufacturer’s instructions. For detection of ICAM and VCAM expression, anti-mouse mAbs were purchased from Biodesign (Kennebunkport, ME) and used with a Histomouse-SP kit (Zymed, South San Francisco, CA) according to the manufacturer’s protocols. Slides were coded and interpreted by a pathologist who was blind to treatment. Each experiment was conducted at least twice and interpreted separately. The results of replicate determinations were then compared.
Preparation of the whole cell extract for protein analysis and immunoprecipitations
Kidney tissues (100 mg) were homogenized in 10 vol of RIPA
buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Nonidet P-40, 0.5%
sodium deoxycholate, 0.1% SDS, 1 mM DTT, 0.5 mM PMSF, 10 µg/ml
leupeptin, aprotinin, and pepstatin, and the phosphatase inhibitors (10
mM NaF, 1 mM NaVO4, 1.5 mM
Na2MoO4, 1 mM bensamidine,
20 mM glycerophosphate, and 20 mM p-nitrophenyl phosphate))
using a Tissumizer (Tekmar, Cincinnati, OH). SDS-PAGE (5x) sample
buffer was added to the final concentrations of 50 mM Tris-HCl (pH
6.8), 2% SDS, 100 mM DTT, 0.006% bromophenol blue, and 10% glycerol.
Samples were boiled for 10 min and cooled on ice, and DNA was sheared
by sonication. Lysates were finally centrifuged at 13,000 x
g for 20 min, and supernatants were used for PAGE and
Western blotting. For immunoprecipitation analysis, RIPA homogenates
were centrifuged at 13,000 x g for 20 min. The
supernatants were transferred to new tubes. Six microliters of
anti-phosphotyrosine mAbs (clone PT-66, Sigma) or anti-IB
Abs (sc-371, Santa Cruz Biotechnology, Santa Cruz, CA) were added to
250 µg of protein extract, and the mixture was incubated at 4°C
overnight. Thirty microliters of a 50/50 (w/v) slurry of protein
A-Sepharose (Pharmacia) in RIPA buffer was then added to the samples.
Immunopellets were collected and washed, and protein was eluted by
boiling for 1–2 min in SDS-PAGE sample buffer. Samples were used for
electrophoresis and Western blotting. To prepare total extracts from
cell culture of resident i.p. macrophages, cells were washed with cold
PBS, pH 7.4, and lysed by addition of RIPA buffer. After that,
electrophoresis sample buffer was added, and extracts were prepared as
described above.
Cell culture
Resident i.p. macrophages were obtained from nontransgenic mice
as previously described (16). Macrophages were cultured in
DMEM containing 10% heat-inactivated FCS with 100 U/ml penicillin and
100 µg/ml streptomycin. Cells were collected by centrifugation and
resuspended in complete medium at a density of
107 cells/ml and after 30 min of incubation at
37°C treated for various time periods with 0.5 and 5 mM
H2O2, 100 ng/ml mouse
TNF- (Biodesign), or 100 µM pervanadate. A concentrated solution
of 50 mM pervanadate was prepared just before use from vanadate and
H2O2 according to the
method of Evans et al. (17). After treatment, cells were
recovered by centrifugation, and pellets were used for preparation of
whole cell extracts and Western blotting.
Mouse proximal tubular and mesangial cells (MCT and MMC, respectively) were provided by Dr. E. Neilson (University of Pennsylvania, Philadelphia, PA) (18). Cells were maintained in DMEM plus 10% FCS. Before stimulation, cell cultures were serum deprived for 24 h to decrease the basal level of chemokine activation. Cells were reversibly depleted of ATP as described by Poombe et al. (19) by exposure to cyanide (0.5 mM) and 2-deoxyglucose (5 mM) in the absence of glucose for 40 min, followed by re-exposure to glucose to allow cellular ATP levels to recover. At different time points during repletion, cells were collected and used for analysis. To measure cell ATP content, cell monolayers were snap-frozen with slurry of isopentane and dry ice, followed by extraction with 4% perchloric acid. ATP content was measured using a luciferase kit with luciferin (Promega).
To test the role of NF-B binding to the B site of the MIP-2
promoter region, a fragment spanning -573 to +39 nt of this gene was
amplified by PCR using genomic DNA and cloned into the luciferase
reporter vector PGL3-Basic (Promega). The plasmid was designated pLUC.
To introduce mutations in the B site, oligonucleotide with GG to CC
substitutions (5'-ACCCTGAGCTCAGCCAATTTCCCTGGTCCCG-3') and
the QuickChange site-directed mutagenesis kit (Stratagene) were used
according to the manufacturer’s protocol. The plasmid was designated
pLUC/mut. Expression plasmids were cotransfected with pTL-TK (Promega)
as an internal control reporter into the MCT cells using Lipofectamine
Plus Reagent (Life Technologies) 48 h before anoxia. After the
anoxia/ATP repletion protocol, cell lysates were obtained, and
luciferase activity was measured using the Dual-Luciferase reporter
assay system (Promega). Firefly luciferase activity was normalized to
Renilla luciferase activity from the same sample. To test
the effects of GPx1 overexpression on activation of the MIP-2 promoter
in cell culture, MCT cells were cotransfected with pLUC and pcDNA3.1
(Invitrogen) expression vector containing GPx1 cDNA (pcDNA/GP) in a 1/4
ratio. As a control, cells were cotransfected with pLUC and pcDNA3.1
vector alone. pTL-TK was added as an internal control for the
efficiency of transfection in all experiments. After the anoxia/ATP
repletion protocol, cell lysates were obtained, and luciferase activity
was measured as described above.
Protein electrophoresis and Western blotting
After measuring the protein concentration, protein samples were
denatured in a boiling bath in a sample buffer containing 250 mM
Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 2% 2-ME and were
separated in minigels (7 cm; Bio-Rad, Hercules, CA) by 10% SDS-PAGE.
To obtain a higher resolution of proteins, they were separated in 20-cm
gels, allowing a 32.6-kDa band of a prestained marker (Bio-Rad) to
reach the bottom of the gel. After electrophoresis, gels were
electroblotted onto a polyvinylidene difluoride membrane (Immobilon,
Millipore, Bedford, MA). Membranes were blocked in a Blotto solution
containing 1x TBS (10 mM Tris-HCl (pH 8.0) and 150 mM NaCl), 5% milk,
and 0.1% Tween-20 for 1 h at room temperature. Membranes were
then incubated with primary Abs diluted in Blotto for 12 h at
4°C. Rabbit polyclonal Abs against p65 (sc-372), IB (sc-371),
and IBß (sc-969) were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). For detection of the phosphorylated form of IB,
rabbit anti-phosphospecific IB (Ser32)
Abs (kit 9240, BioLabs, Beverly, CA) were used. Phospho- and
nonphospho-IB cell extracts (kit 9243) from the same company were
applied on the same gels as the controls. After extensive washings in
1x TBS with 0.1% Tween-20, proteins were detected with Phototope-HRP
Western Blot Detection Kit and LumiGlo reagent (both from BioLabs).
Autoradiographs were quantitated by densitometry as described
above.
Preparation of the nuclear extracts and EMSA
Nuclear extracts were prepared as previously described
(20). Aliquots of the extract were stored at -80°C.
Protein content was assayed using the Bio-Rad protein reagent.
Oligonucleotides used in the EMSAs were the following:
5'-AGCTCAGGGAATTTCCCTGGTCC-3' containing the mouse MIP-2 NF-B
binding site, and 5'-GGCCAGGGAATTTCCCGGAGTA-3' and
5'-TTGCAGGGAAACACCCTGTACT-3' containing B1 and B2 sites of the
mouse KC promoter region, respectively (21). For each
oligonucleotide, the two complementary strands were synthesized using
an Applied Biosystems (Foster City, CA) Automated DNA synthesizer.
These oligonucleotides were annealed, end-labeled using polynucleotide
kinase and [-32P]ATP (New Life Science
Products), and purified by PAGE. For EMSA, 5–10 µg of extract was
incubated in a reaction mixture containing 20 mM HEPES (pH 7.9), 60 mM
KCl, 5 mM MgCl2, 1 mM DTT, 0.2 mM EDTA, 0.5%
Nonidet, 1 µg poly(dI-dC), and 8% glycerol in final volume of 20
µl for 20 min at 4°C. After preincubation,
105 cpm of radiolabeled DNA probe was added, and
incubation was continued for 15 min at room temperature. The
DNA-protein complexes were separated on native 5% polyacrylamide gels
in 0.25x Tris-borate-EDTA buffer. Supershift assay was conducted after
incubation of the nuclear extracts with Abs (0.5 µg, the same as used
for Western analysis, and anti-mouse p50 Abs, sc-1192, from Santa
Cruz Biotechnology) for 20 min at 4°C followed by EMSA. For
competition assay, cold double-stranded oligonucleotides were added to
the reaction mixtures at a 10- or 25-fold molar excess upon
radiolabeled probes.
Statistical analyses
The mortality between nontransgenic and transgenic mice was compared using the Kaplan-Meyer method. All other data were analyzed by ANOVA with the StatView program (SAS Institute, San Francisco, CA). The null hypothesis was rejected at the 0.05 level. All data are expressed as the mean ± SEM.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The survival curves of control and transgenic animals after 32 min
of renal ischemia and subsequent reperfusion are shown in Fig. 1
. None of the sham-operated animals died
in this experiment. After 10 days, renal I/R injury resulted in 46, 88,
and 91% survival in nontransgenic, GPx1, and GPxP mice, respectively.
There was a significant difference in mortality between control and
both groups of transgenic animals (p < 0.03).
Kidney damage was also evaluated by measurement of blood BUN and
creatinine. Levels of BUN at 24 h after ischemic injury in
sham-operated, nontransgenic, GPx1, and GPxP were 20 ± 4,
131 ± 2, 72 ± 7, and 70 ± 5 mg/dl, respectively,
whereas levels of creatinine in the same animal groups were 0.1 ±
0.05, 2.04 ± 0.08, 0.65 ± 0.10, and 0.51 ± 0.06
mg/dl, respectively. Data obtained indicate that the nontransgenic
group had approximately 2.0-fold higher levels of BUN and 3.0-fold
higher levels of creatinine compared with both transgenic groups
(p < 0.001), suggesting a significantly
greater decline in renal function in normal animals compared with mice
overproducing human GPs. Light microscopic analysis supports this
conclusion (Fig. 2). After 32 min of
ischemia followed by 24-h reperfusion, the outer medulla of kidney in
control group showed extensive tubular epithelial necrosis and
neutrophil infiltration. Proteinaceous casts filled the lumen of the
tubules. In contrast, sections from both GPx1 and GPxP kidneys
demonstrate significantly less tubular necrosis and fewer PMNs. Tubular
casts were present in fewer nephrons. The damage score in control mice
was approximately 3.5 (of 4), representing a >50% area of medullary
tubular epithelial necrosis or filled with necrotic debris; in
contrast, GPx1 and GPxP groups had significantly smaller damage scores,
1.5 and 1.6 respectively (p < 0.01;
n = 5).
|
|
The majority of injured tubular cells during I/R undergo necrosis,
although pathological evidence of apoptosis in kidney has been
demonstrated following brief periods of ischemia and subsequent
reperfusion in animals (22) and clinical acute renal
failure in humans (23). To evaluate the level of
apoptosis, DNA was extracted from kidneys after I/R and analyzed by
electrophoresis (Fig. 3A). A
180-bp ladder pattern of fragmentation was detected in samples from
nontransgenic mice, as has been reported previously (22).
In contrast, laddering was not observed in samples from either GPx1 or
GPxP mice. Since DNA fragmentation can also occur in cellular necrosis,
the more specific quantitative ELISA assay, which detects histone
proteins, associated with the cytoplasmic fragmented DNA in mono- and
oligonucleosomes, was used. The level of apoptosis detected by this
method, in kidneys of GPx1 and GPxP mice, was approximately 6 times
lower than its level in the nontransgenic mouse group (Fig. 3B). These data indicate that overexpression of GPx1 and
GPxP interferes with the apoptotic cell death pathway during renal I/R
injury.
|
To evaluate the degree of lipid peroxidation in injured kidney, as
an indication of oxidative stress, we determined levels of MDA and
4-HNE. Several studies have demonstrated, through measurement of
hydroperoxide-initiated chemiluminescence, that oxidative stress starts
to develop in kidneys during the early period of reperfusion (5–30
min) (24), whereas the more permanently increased level of
lipid peroxidation may be reliably measured at a later period of time
(up to 48 h) (1). Fig. 4A shows an elevated level of
lipid peroxidation in the nontransgenic mouse group compared with that
in GPx1 and GPxP by 20 min after reperfusion (
40% higher then in
both transgenic mouse groups; p < 0.005). At 24 h
of reperfusion lipid peroxidation increased in all three groups of mice
after I/R. Nevertheless, the levels of MDA and 4-HNE were 2.6-, 1.8-,
and 1.7-fold higher in nontransgenic, GPx1, and GPxP mice than in the
sham-operated group, respectively. Overexpression of both types of GPs
decreased the level of oxidative stress in the kidney of mice
immediately after the beginning of reperfusion as well as later, at
24 h, when it might reflect a state of already developed tissue
dysfunction.
|
B). These data indicate that overexpression of GPx1 and
GPxP can decrease oxidative stress as well as neutrophil retention
after I/R injury, which might be one reason for the improvement in
renal function and survival rate in transgenic animals. Expression of chemokines during kidney I/R
PMN immigration from the vasculature to the kidney parenchyma
during I/R is considered to be a complex process that includes
leukocyte rolling followed by endothelial adhesion and transendothelial
migration. The leukocyte recruitment is driven by the actions of
complement, cytokines, adhesion receptors, and chemokines. Despite data
in the literature regarding antioxidant-sensitive mechanisms for ICAM-1
and VCAM-1 expression (25, 26), we were not able to
demonstrate any difference in the activation of these proteins by
immunohistochemistry or mRNA analysis between kidneys of normal and
transgenic mice after I/R. ICAM-1 mRNA expression is shown in Fig. 5A. This might indicate that
the increased level of GPs in our transgenic mice was unable to
influence ROS that are important for activation of these adhesion
receptors. It also may be due to the differences between sites of ROS
generation and location of hGP in the transgenic mice.
|
A. The
level of mRNA was 10 and 4.5 times higher in normal mice compared with
those in GPx1 and GPxP animals mice at 6 h after reperfusion,
respectively. We also took advantage of the Riboquant kit and mCK-5
Multiprobe Template Set (PharMingen), which allow the measurement of
mRNA expression of nine mouse chemokines as well as two housekeeping
genes, to normalize the quantity of mRNA in each sample. Total RNA was
purified from kidneys of normal and transgenic mice after different
reperfusion times and was analyzed by RNase protection assay using a
kit. The results are shown in Fig. 5B. Comparison of the
levels of activation of different chemokines indicates that they might
be divided into several groups. Eotaxin was induced at 2 and 6 h
reperfusion in similar quantities in all three groups. MIP-1ß, IP-10,
MCP-1, and TCA-3 were equally activated at 2 h in normal, GPx1,
and GPxP mice, whereas at 6 h a high level of expression persisted
only in the first group. The most significant difference, however, was
observed in the level of expression of MIP-2. This chemokine was highly
inducible in normal mice. At 6 h after ischemia, its level was 7.5
and 4.3 times higher in these animals compared with those in GPx1 and
GPxP mice, respectively (Fig. 5B). Importantly, by this time
point only a few PMNs were detected in kidneys of normal mice,
indicating that induction of this chemokine precedes massive migration
of leukocytes. KC and MIP-2 belong to the family of CXC chemokines,
which have been shown to cause PMN activation and mediate chemotaxis
and induction of the respiratory burst (28). To access the
level of KC and MIP-2 protein expression, we performed immunohistologic
analysis of the tissue sections using rabbit anti-mKC and
anti-mMIP Abs.
Staining of KC, shown in Fig. 6, was
significantly less intense in GPx1 and GPxP kidneys than in
nontransgenic kidneys, which is in agreement with our RNA analysis. In
nontransgenic mice dark staining was seen throughout the cortex, mostly
in tubules. Similar data were obtained with MIP-2 Abs (data not shown).
Therefore, an analysis of chemokine expression in transgenic mice
overexpressing GPs demonstrates the ability of these enzymes to
significantly affect the level of MIP-2 and KC RNA and proteins. These
findings correlate with our data regarding MPO measurements as well as
the histology of kidneys from different animals (see Figs. 2 and 4). No
reports regarding the cellular distribution of these chemokine
expressions during kidney I/R have been published to date.
|
B binding activity in kidney by GPs
To investigate the mechanism of inhibition of chemokine expression
by GPs, we analyzed activation of NF-B. This transcription factor is
known to be responsible for the activation of many genes mediating the
inflammatory response in general (29) as well as during
I/R in particular (30). It was shown that KC gene contains
two structurally distinct sites, B1 and B2, which cooperatively
mediate KC activation by LPS in RAW 264.7 cells and by TNF- in
NIH-3T3 cells (21), whereas MIP-2 contains only one B
site, very similar to B1 site of the KC. Although there are binding
sites for other transcription activators in the promoter regions of KC
and MIP-2, no significant homology has been observed between
them.
We studied DNA binding activities in kidney nuclear extracts from
normal and transgenic mice after I/R using the oligonucleotides, which
contain B MIP-2 site and KC B1 and B2 sites. Since the binding
sequences of the first two sites are identical, very similar results
were obtained. At 6 h of reperfusion NF-B binding activity was
significantly higher in kidneys from normal mice than in those from
both GPx1 and GPxP transgenic animals (Fig. 7, A and B). In
both cases, binding was specific, and the composition of binding
complexes from p65/p50 proteins was proven by supershift assay. We did
not observe any presence of c-Rel in the complexes. When an
oligonucleotide containing the KC B2 site for DNA binding was used,
one predominant band was observed in nuclear extracts of animals after
I/R (Fig. 7C). Surprisingly, the position of the band was
significantly higher than those in the B MIP-2 and KC B1 cases.
We were not able to compete the binding with cold B1 oligonucleotide
and supershift this band with anti-p65 and p50 Abs. According to
our preliminary analysis by UV cross-linking of
bromodexoxyuridine-labeled oligonucleotide, this complex consists of
protein with Mr approximately 100 kDa.
These data are in contrast to those mentioned before, which demonstrate
that binding to KC B2 site in macrophages activated with LPS is
mediated by the p65/p50 complex (21). Identification of
this novel protein is currently underway. Interestingly, the binding
site in this region of KC is very different from the canonical NF-B
sites and does not contain any T residues in the second half of the
site. As in the case of the other tested oligonucleotides, binding to
the KC B2 site was significantly higher in normal animals at 2 and
6 h than in GP mice (Fig. 7C). the data obtained
indicate that kidneys from GPx1 and GPxP transgenic mice during I/R
modulate DNA binding in the regulatory regions of at least two
chemokines, thereby mediating activation and migration of PMNs. The
ability of ROS to induce DNA binding by NF-B as well as the ability
of antioxidants to inhibit activation of this binding were demonstrated
in several studies (31, 32), although the biological
consequences of these effects in in vivo setting has not been studied
before.
|
B and IBß degradation
NF-B activation is regulated by a family of inhibitory IB
proteins that are coupled to NF-B and block its transport to the
nucleus (29). Activation of cells by various stimuli leads
to the rapid depletion of these proteins by proteolytic degradation.
Studies indicate that the majority of p65/p50 complexes are regulated
by IB and IBß. We measured the presence of both
inhibitory proteins in kidney extracts from normal and transgenic mice
at different time points after I/R (Fig. 8). At 30 and 45 min after I/R, fast
degradation of both IB and IBß was detected, although the
level of depletion was significantly higher in normal mice (57% for
IB and 70% for IBß in nontransgenic mice in comparison to 0
and 4% in GPx1 and 22 and 23% in GPxP mice at 45 min of reperfusion,
respectively). Interestingly, the level of IB at 6 h after
I/R in both types of transgenic mice was increased by 30–50% compared
with that in sham-operated or nonoperated mice. The degradation of
IB and IBß correlates very well with the level of nuclear
p65. These data suggest that after 30 min of kidney I/R, a significant
influx of NF-B into the nucleus in normal mice most likely leads to
the activation of genes that are regulated by this
transcription factor, including KC and MIP-2 chemokines. Overexpression
of GPs seems to inhibit this process in transgenic mice very
efficiently.
|
B phosphorylation during kidney I/R
Since inducible phosphorylation of IB followed by
proteasome-mediated degradation is a well-documented fact
(33) and has even been questioned in relation to the
IBß in a recent report (34), we studied IB
phosphorylation during kidney I/R injury. No bands, other than
nonactivated hypophosphorylated IB, were seen using
anti-IB Abs in kidney extracts at any time during I/R;
furthermore, no staining was visible using anti-phosphospecific
IB (Ser32) Abs (data not shown).
Nevertheless, clear degradation of IB was visible during
reperfusion. Similar data were obtained with kidney extracts from
transgenic animals. Moreover, to exclude the possibility of incomplete
inhibition of phosphatase activity in kidney extracts, we mixed
pellets of the cells treated with
H2O2 with pieces of kidneys
after I/R and homogenated the samples, as was done with kidneys
alone.
Another mechanism of NF-B activation that might have a direct
relevance to I/R damage is tyrosine phosphorylation (35, 36). We analyzed tyrosine phosphorylation in kidney during I/R,
theorizing that it might be modulated by overexpression of antioxidant
enzymes under stress conditions, as has been shown previously
(16). To detect tyrosine phosphorylation, extracts from
kidneys were first immunoprecipitated with anti-phosphotyrosine Abs
and then probed with anti-IB Abs (Fig. 9, A and B). As a
positive control, extracts from mouse macrophages were treated with
pervanadate, an efficient inhibitor of tyrosine phosphatases and known
to induce of IB tyrosine phosphorylation (37). As
expected, in pervanadate-treated macrophage extracts, IB was
readily phosphorylated at the tyrosine residue (Fig. 9C).
Nevertheless, no tyrosine phosphorylation was detected in kidney
extracts from normal mice during I/R or from any other group of
animals. The data obtained suggest that the IB protein is not
phosphorylated during kidney I/R, although it clearly undergoes
proteolytic degradation. Of course we cannot completely exclude a small
degree of phosphorylation, which we were not able to detect, or the
possibility that this phosphorylated IB is degraded extremely
fast. Future work will be devoted to a search for the mechanisms that
signal degradation of IB during kidney I/R. Experiments with
specific protease inhibitors should help to clarify this issue with
IB phosphorylation as well. Importantly, it does not appear to be
unique for the I/R process. Recently, Kretz-Remy et al.
(38) demonstrated that amino acid analogues activate
NF-B in T47D cells through redox-dependent IB degradation
without detectable IB phosphorylation; however, the authors did
not specifically analyze the status of tyrosine phosphorylation in
their conditions.
|
Although the final goal in studying the inflammatory response
during kidney I/R injury is to understand in vivo phenomenon, work with
animal models poses difficulties in defining molecular events in a very
complex tissue environment. Therefore, we tested the expression of KC
and MIP-2 in mouse tubular and mesangial cell lines, MCT and MMC. We
used published protocols that involve depletion of ATP by exposure to
cyanide (0.5 mM) and 2-deoxyglucose (5 mM) in the absence of glucose
for 40 min followed by re-exposure to glucose to allow cellular ATP to
recover (19). We measured intracellular ATP content during
the procedure and observed an approximately 90% decrease in ATP in
both MCT and MMC cells with almost 100% recovery to initial levels at
60 min of re-exposure to glucose. These cells, collected at different
time points, were used for Northern analysis as well as RNase
protection assay with chemokine probes and for analysis of IB
degradation as a mechanism of NF-B activation. Results are shown in
Fig. 10, A and B.
These results indicate significant induction of KC and MIP-2 mRNA in
MCT cells during the ATP repletion phase, similar to the expression of
those chemokines during reperfusion in kidney (see Fig. 5).
Interestingly, MMC cells reveal only slight induction of KC mRNA during
anoxia. As already noted, our immunocytochemical analysis showed that
CXC chemokine expression was mainly observed in tubular cells. Results
obtained with cell culture lines are in agreement with those data.
Chemokine expression in animals correlated with NF-B activation and
IB degradation. We tested both the DNA binding activity of this
transcription factor and IB degradation in the cell culture model as
well. At 20 min of ATP repletion, DNA binding activity (not shown) and
IB degradation were induced at an early phase of re-exposure to
glucose in MCT cells (see Fig. 10C), but not in MMC cells.
Similar results were obtained with IBß, although the extent of
degradation in this case was significantly less (not shown). No
induction of phosphorylation using Western blotting with
anti-phosphospecific IB Abs was observed at any tested time
point. The data allow us to conclude that chemical anoxia/ATP repletion
might be used as a model system to study the mechanism of chemokine
activation induced after anoxia/ischemia.
|
B activation in chemokine
induction in the cell culture model, we used expression plasmids
containing wild-type as well as mutated MIP-2 promoter region fused to
the luciferase reporter gene. Mutations were introduced in the NF-B
binding site. Comparison of anoxia/ATP repletion-induced reporter gene
expression after transient transfection of wild-type and mutant
plasmids is shown in Fig. 11. ATP
repletion leads to the activation of relative luciferase expression in
MCT cells. Mutations in the NF-B DNA binding site markedly reduced
luciferase activation. The ability of an increased level of GP to
inhibit activation of MIP-2 expression in tubular cells was also
examined As shown, MCT cells cotransfected with MIP-2
promoter-luciferase and GPx1 expression plasmids showed no activation
of luciferase activity during the ATP repletion phase. This is in
contrast to MCT cells cotransfected with MIP-2 promoter-luciferase and
vector plasmids. The level of luciferase activity in these cells was
even slightly decreased compared with the basic level. The data
presented directly prove that NF-B is an indispensable transcription
factor, mediating MIP-2 activation in a cell culture model of
ischemia/reperfusion.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
As apoptosis is a hallmark of cellular injury well known to be induced by oxidative stress and is inhibited by antioxidants (39), we tested the level of apoptosis in kidneys of nontransgenic and GP mice under conditions of I/R. This process was significantly reduced by overexpression of both types of GP in our animals. It has previously been shown only in vitro that cells transfected with the GPx1 gene are more resistant to induced apoptosis (40) and that selenium-deficient MDBK cells, which have low levels of GP, are more prone to programmed cell death by H2O2 treatment (41).
Because increased resistance of transgenic animals to I/R might result from a direct effect of antioxidant enzymes on damaging ROS as well as from an indirect influence on ox/redox-sensitive mechanisms, we investigated further one of the critical mediators of I/R injury, neutrophil recruitment. Measurements of PMN by MPO activity, as well as by histopathologic examination indicated reduced infiltration of these leukocytes into renal parenchyma. Recent observations suggest that ROS might play an important role in the regulation of leukocyte migration to the tissues (7, 42). Among the important targets of ROS/antioxidant action is expression of neutrophil chemoattractants-chemokines. It has been shown that I/R induces expression of different chemokines in kidney, liver, lung, brain, and heart (43). Most importantly, immunization of rabbits with IL-8 Abs significantly reduced lung injury and PMN infiltration in an I/R model in vivo (27), suggesting a critical role for this chemokine in I/R damage. Similar data were obtained with another CXC chemokine, ENA-78 (44). During the last 5 yr, several papers have demonstrated increased neutrophil infiltration in transgenic mice overexpressing chemokines and a lower inflammatory response in mice with deletion of the genes encoding a chemokines (45). All these data implicate at least CXC chemokines as important contributors to the pathogenesis of I/R injury. It has also been demonstrated that expression of these chemokines is influenced by ROS/antioxidant levels (46). Analysis of chemokine expression during kidney I/R demonstrates that in our transgenic animals production of several chemokines, belonging to both CXC and CC groups, is affected. The most significant decrease in expression was observed in two CXC chemokines, KC and MIP-2. These chemokines were specifically induced at high levels in tubular cells of nontransgenic mice, which correlates with a predominant role of PMN in this model of I/R injury. Although it might not be the only mechanism of protection in our transgenic mice, the effects of GP overexpression on development of an inflammatory response during renal I/R is sufficient to render transgenic mice resistant to injury.
Expression of chemokines, leading to neutrophil-mediated inflammation,
is activated by a series of transcription factors, among which NF-B
is thought to play a central role. To demonstrate a direct relationship
between NF-B activation and chemokine expression we used cell
culture models of I/R that have been successfully applied to study
signal transduction pathways and mechanisms of cell injury (19, 47). A chemical anoxia/ATP repletion procedure in MCT cells is
well characterized and possesses two major features of the tubular cell
injury in vivo, oxidative stress and energy deprivation with
concomitant resupply of ATP. Importantly, we demonstrated that this
treatment leads to the activation of CXC chemokines and preserves cell
type specificity. By analyzing expression of the reporter gene directed
by MIP-2 promoter region, we showed that NF-B binding is critical
for the activation of this gene in in vitro model of I/R, and GP
overproduction in cell culture leads to the inhibition of this
activation. Of course, these data do not exclude involvement of other
transcription factors in activation of CXC chemokine expression in I/R.
Although NF-B nuclear activity was induced in several models of I/R,
the exact mechanism of this process, especially in vivo, has not been
studied. In our kidney I/R model, we were indeed able to demonstrate
elevated DNA binding activity to the B site of MIP-2 as well as
B1 site of KC in nuclear extracts. Importantly, DNA binding activity
was decreased in transgenic mice, suggesting inhibition of NF-B
activation, probably leading to the decreased production of those
chemokines. It is also possible that GP overexpression might affect
chemokine activation through other mechanisms, taking into account the
ability of ROS/antioxidants to influence many intracellular processes.
Among those, for example, are post-transcriptional mechanisms, such as
a steady state level of chemokine mRNA, known to be an important factor
for expression of chemokines that are sensitive to oxidative stress
(48, 49). These possibilities are currently under
investigation. The regulation of NF-B activation by antioxidant
enzymes might occur on several levels, including upstream signal
transduction pathways. Alternatively, changes in the redox status of
critical cysteine residues may influence the interaction between the
NF-B subunits as well as between subunits and other protein factors
and DNA. In general, as previously demonstrated, reductants decrease
NF-B activity, whereas oxidants greatly activate it (16, 31). We show that during the development of kidney I/R injury,
degradation of IBß along with IB took place and was also
affected by an increased level of GP activity, which correlated with
the appearance of nuclear p65.
The fundamental role and mechanism of phosphorylation in initiation of
IB protein degradation have been an area of intensive investigation
during the past several years (reviewed in Ref. 33). We
did not detect any obvious phosphorylation on Ser and Tyr residues of
IB before degradation during kidney I/R. As already noted,
another group recently demonstrated IB degradation without
phosphorylation (38). Most importantly, this degradation,
leading to the NF-B activation, was redox dependent. Cells
overexpressing GPx1 were able to abolish this degradation. The authors
suggest that GPx1 is able to influence both phosphorylation-dependent
as well as independent proteolysis of IB. The signal for
targeting of IB degradation in the second case is not known. The
present results indicate that this still unexplored mechanism might
have wider implication, for example in I/R.
In experiments involving amino acid analogues (38), their
incorporation probably leads to the appearance of misfolded proteins
that become targets for proteasomes, which might themselves undergo
ox/redox regulation. Importantly, I/R is known to induce formation of
misfolded proteins due to oxidative stress, calcium overload,
activation of proteases, alteration in cytoskeleton, depletion of
energy sources, etc. (50). It is well established that a
family of heat shock proteins that act as chaperons to transport and
salvage denatured proteins is highly induced after the onset of kidney
ischemia and reperfusion (51). Our future work will be
directed toward identification of the signals that lead to IBs
degradation during I/R, and are sensitive to GP overexpression. The
data presented here show that these signals are critical for arranging
an inflammatory response during I/R, specifically by activating
chemotactic molecules. They also might involve upstream signal
transduction pathways, which were not studied in this work, but are
known to be important for the outcome of I/R injury. These include
stress- and mitogen-activated protein kinases (c-Jun N-terminal
kinase/stress-activated protein kinase and mitogen-activated protein
kinase/extracellular signal-related kinase), which are stimulated
during kidney I/R, are sensitive to ox/redox regulation, and are
implied in activation of NF-B (29, 52).
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Abbreviations used in this paper: I/R, ischemia/reperfusion; BUN, blood urea nitrogen; GP, glutathione peroxidase; GPx1, intracellular glutathione peroxidase; GPxP, extracellular glutathione peroxidase; 4-HNE, 4-hydroxy-2(E)-nonenal; MCP, monocyte chemoattractant protein; MDA, malondialdehyde; MPO, myeloperoxidase; PMN, polymorphonuclear leukocytes; ROS, reactive oxygen species; IP-10, IFN--inducible protein-10.
Received for publication March 16, 1999. Accepted for publication September 2, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Bß and abrogation of NFB activity in peritoneal macrophages stimulated with lipopolysaccharide. J. Biol. Chem. 272:23025.B sequence motifs cooperatively control LPS-induced KC gene transcription in mouse macrophages. J. Immunol. 155:3593.[Abstract]
B and Rel proteins: evolutionary conserved mediators of immune responses. Annu. Rev. Immunol. 16:225.[Medline]
B binding site: evidence for a role of NF-B in ischemia-reperfusion injury. Circulation 96:280.
B phosphorylation and degradation
and subsequent NFB activation by glutathione peroxidase
overexpression. J. Cell Biol. 133:1083.
B. Immunol. Today 19:80.[Medline]
Bß degradation. J. Biol. Chem. 272:9942.B activates NFB without proteolytic degradation of IB. Cell 86:787.[Medline]
B through the phosphorylation of IB on tyrosine residues. Cancer Res. 54:1425.B serine phosphorylation in nuclear factor B activation by tumor necrosis factor. J. Biol. Chem. 270:18881.B through redox-dependent IB degradation by the proteasome without apparent IB phosphorylation. J. Biol. Chem. 273:3180. in endothelial and epithelial cells. J. Biol. Chem. 272:32910.B activities and stability of IL-8 transcripts are implicated in IL-8 mRNA superinduction in lung epithelial H292 cells. Biochem. J. 330:429.
mRNA by oxidative stress. J. Biol. Chem. 271:5878.This article has been cited by other articles:
|
|
 |
|
  H. Zhu, L. Zhang, A. R. Amin, and Y. Li Coordinated Upregulation of a Series of Endogenous Antioxidants and Phase 2 Enzymes as a Novel Strategy for Protecting Renal Tubular Cells from Oxidative and Electrophilic Stress Experimental Biology and Medicine, June 1, 2008; 233(6): 753 - 765. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Matsushima, S. Kinugawa, T. Ide, H. Matsusaka, N. Inoue, Y. Ohta, T. Yokota, K. Sunagawa, and H. Tsutsui Overexpression of glutathione peroxidase attenuates myocardial remodeling and preserves diastolic function in diabetic heart Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2237 - H2245. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Jiang, O. Prokopenko, L. Wong, M. Inouye, and O. Mirochnitchenko IRIP, a New Ischemia/Reperfusion-Inducible Protein That Participates in the Regulation of Transporter Activity Mol. Cell. Biol., August 1, 2005; 25(15): 6496 - 6508. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Mizuno and T. Nakamura Prevention of Neutrophil Extravasation by Hepatocyte Growth Factor Leads to Attenuations of Tubular Apoptosis and Renal Dysfunction in Mouse Ischemic Kidneys Am. J. Pathol., June 1, 2005; 166(6): 1895 - 1905. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. T. Hua, H. Albadawi, F. Entabi, M. Conrad, M. C. Stoner, B. T. Meriam, R. Sroufe, S. Houser, G. M. LaMuraglia, and M. T. Watkins Polyadenosine Diphosphate-Ribose Polymerase Inhibition Modulates Skeletal Muscle Injury Following Ischemia Reperfusion Arch Surg, April 1, 2005; 140(4): 344 - 351. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Shiomi, H. Tsutsui, H. Matsusaka, K. Murakami, S. Hayashidani, M. Ikeuchi, J. Wen, T. Kubota, H. Utsumi, and A. Takeshita Overexpression of Glutathione Peroxidase Prevents Left Ventricular Remodeling and Failure After Myocardial Infarction in Mice Circulation, February 3, 2004; 109(4): 544 - 549. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y.-J. Day, M. A. Marshall, L. Huang, M. J. McDuffie, M. D. Okusa, and J. Linden Protection from ischemic liver injury by activation of A2A adenosine receptors during reperfusion: inhibition of chemokine induction Am J Physiol Gastrointest Liver Physiol, February 1, 2004; 286(2): G285 - G293. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. V. Bonventre and J. M. Weinberg Recent Advances in the Pathophysiology of Ischemic Acute Renal Failure J. Am. Soc. Nephrol., August 1, 2003; 14(8): 2199 - 2210. [Full Text] [PDF]
|
|
|
|
|
|
J. V. Bonventre Dedifferentiation and Proliferation of Surviving Epithelial Cells in Acute Renal Failure J. Am. Soc. Nephrol., June 1, 2003; 14(90001): S55 - 61. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. K. Meldrum, A. L. Burnett, X. Meng, R. Misseri, M. B.K. Shaw, J. P. Gearhart, and D. R. Meldrum Liposomal Delivery of Heat Shock Protein 72 Into Renal Tubular Cells Blocks Nuclear Factor-{kappa}B Activation, Tumor Necrosis Factor-{alpha} Production, and Subsequent Ischemia-Induced Apoptosis Circ. Res., February 21, 2003; 92(3): 293 - 299. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Whitin, S. Bhamre, D. M. Tham, and H. J. Cohen Extracellular glutathione peroxidase is secreted basolaterally by human renal proximal tubule cells Am J Physiol Renal Physiol, July 1, 2002; 283(1): F20 - F28. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Maser, D. Vassmer, B. S. Magenheimer, and J. P. Calvet Oxidant Stress and Reduced Antioxidant Enzyme Protection in Polycystic Kidney Disease J. Am. Soc. Nephrol., April 1, 2002; 13(4): 991 - 999. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Ishibashi, O. Prokopenko, K. R. Reuhl, and O. Mirochnitchenko Inflammatory Response and Glutathione Peroxidase in a Model of Stroke J. Immunol., February 15, 2002; 168(4): 1926 - 1933. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. S. Esworthy, R. Aranda, M. G. Martin, J. H. Doroshow, S. W. Binder, and F.-F. Chu Mice with combined disruption of Gpx1 and Gpx2 genes have colitis Am J Physiol Gastrointest Liver Physiol, September 1, 2001; 281(3): G848 - G855. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Mirochnitchenko, O. Prokopenko, U. Palnitkar, I. Kister, W. S. Powell, and M. Inouye Endotoxemia in Transgenic Mice Overexpressing Human Glutathione Peroxidases Circ. Res., August 18, 2000; 87(4): 289 - 295. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
DNA digested with PstI was used. The level of
apoptosis was also measured by detection of nucleosomes in kidney
cytoplasmic fractions (B). Diluted cytoplasmic extracts
were directly used in ELISA as described in Materials and
Methods. *, p < 0.001.
n = 10.
