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*
Renal Division, Department of Medicine, Montefiore Medical Center, Bronx,
New York
Inotek, Inc., Beverly, Massachusetts.
Correspondence to Dr. Norman Bank, Renal Division, Montefiore Medical Center, 111 East 210 Street, Bronx, NY 10467. Phone: 718-920-4991; Fax: 718-920-6658; E-mail: nbankmd{at}aol.com
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Protein phosphorylation is a common mechanism of transduction of extracellular signals from the cell membrane to cell nuclei, leading to activation of a number of genes (14). Protein kinases and phosphatases modulate the phosphorylation state of the proteins and play a critical role in cell signaling.
In the present study, we examined tyrosine/threonine phosphorylation of enzymes of the three mitogen-activated protein (MAP) kinases (MAPK) in normal and sickle cell mouse kidneys. The enzymes of these three signaling cascades require phosphorylation of specific amino acid residues by ATP for their activation (15,16). Western blots using antibodies directed against specific threonine/tyrosine residues of extracellular signal-regulated kinase (ERK), p38 kinase, and c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK), demonstrated significant reduction of phosphorylation of p38 and increased phosphorylation of JNK. Treatment of the mice with mercaptoethylguanidine (MEG) (17), a scavenger of ONOO- that also inhibits the inducible isoform of NOS (iNOS), restored phosphorylation of p38 toward normal. These observations suggest that ONOO- produced in the kidneys of sickle cell mice interferes with phosphorylation of p38, but does not prevent phosphorylation of JNK.
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Materials and Methods |
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Hßs[ßMDD], the description of which was
published in detail previously
(18,19).
All animal experimentation described in this study was conducted in accord
with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals. The ßs mice manifest a mild degree of
reticulocytosis under ambient room air conditions, but are not anemic. They
have enlarged kidneys and liver, and a normal urine concentrating ability.
When housed under hypoxic conditions (10% O2) for 5 d, however,
they manifest in vivo sickling, vaso-occlusion of numerous medullary
blood vessels, and their ability to concentrate urine is significantly reduced
(19). Control mice used in
this study were of the C57BL/6J strain, purchased from Jackson Laboratories
(Bar Harbor, ME). All animals were allowed free access to a mouse pellet diet. The mice were housed in individual metabolic cages under room air conditions or exposed to hypoxic conditions for 4 to 5 d in a glass environmental chamber (Braintree Scientific, Braintree, MA) filled with constantly flowing 10% O2/0.5% CO2/89.5% N2 gas, as described previously (1). In each experiment, three groups of five mice each were studied (normoxic control, hypoxic control, normoxic ßs, hypoxic ßs, and MEG-treated hypoxic transgenic). MEG, the specific inhibitor of iNOS, was administered to transgenic sickle cell mice exposed to hypoxia, since in this experimental condition there is evidence of nitrosative stress (2). The mice were injected with MEG (10 mg/kg body wt; Inotek Corp., Beverly, MA) intraperitoneally once daily for 5 d. On the day of study, the mice were anesthetized with Inactin-Byk (sodium salt of 5-ethyl-5-(1-methylpropyl)-2-thiobarbiturate; Byk Gulden, Konstanz, Germany; 8 mg/100 g body wt, intraperitoneally). The kidneys were rapidly perfused with ice-cold saline via cardiac puncture, and blood was drained from the severed distal inferior vena cava. Kidneys used for Western blot were homogenized.
Sample Preparation and Protein Extraction
Kidneys were homogenized (Ultra Turrax T25; IKA-Labortechnik, Staufen,
Germany) in ice-cold homogenizing buffer (pH 7.5, Tris 50 mM, NaCl 20 mM,
ethylenediaminetetra-acetic acid 1 mM, Nonidet P-40 0.5%) containing 0.3 ml/g
kidney weight of a commercial protease inhibitor cocktail (Sigma Chemical Co.,
St. Louis, MO), 1 mM sodium orthovanadate, and 30 mM sodium fluoride. The
homogenates were centrifuged at 10,000 rpm at 4°C for 20 min. The
supernatant was collected and was again centrifuged at 10,000 rpm for 2 min.
The protein concentration of the supernatant was determined by the Bradford
method (Bio-Rad). Thirty micrograms of protein was suspended in sample buffer
(62.5 mM Tris-HCl, 10% glycerol, 2% sodium dodecyl sulfate [SDS], 2%
mercaptoethanol, 1% bromphenol blue). Samples were boiled for 3 min and loaded
on 12% SDS polyacrylamide separating gel. Electrophoresis was carried out for
1 h at 200 V. Transfer of proteins to polyvinylidene difluoride (PVDF)
membranes (Immobilon-P, Millipore, Bedford, MA) was performed for 1 h at 100 V
in transfer buffer (25 mM Tris, 0.2 M glycine, 20% methanol).
Western Blot
Membranes were blocked in Tris-buffered saline (TBS) Tween 0.1% (pH 7.5,
Tris 20 mM, NaCl 140 mM) (TBS-T) with 5% nonfat dry milk for 1 h at room
temperature. Then, the primary antibody was applied, diluted 1:1000 in 5%
bovine serum albumin/TBS-T, and incubated overnight at 4°C. Two membranes
run in parallel were probed with rabbit polyclonal IgG (affinity-purified)
phosphospecific antibodies that detect only phosphorylated threonine/tyrosine
sites of ERK and p38 kinase. In the case of JNK, a monoclonal antibody was
used (New England Biolabs, Beverly, MA). The second membranes were probed with
rabbit polyclonal IgG antibodies that are specific for the total protein of
each of the three enzymes (New England Biolabs). Activation of the enzymes by
inflammatory cytokines and stress stimuli occurs via dual phosphorylation of
specific threonine and tyrosine sites
(20). The degree of
phosphorylation detected by these phosphospecific antibodies has been shown to
correlate closely with measured enzyme activity
(21,22).
The excess of antibody was removed by three washes in TBS-T and membranes
incubated with horseradish peroxidase (HRP)-conjugated secondary anti-rabbit
antibody, diluted 1:2000 in 5% milk-TBS-T for 1 h at room temperature.
Membranes were washed three times with TBS-T, and the immunoreactive proteins
were detected by enhanced chemiluminescence (Amersham). Biotinylated molecular
weight standards were run with each blot (New England Biolabs).
The same protocol was used for Western blot of MAP kinase phosphatase-1 (MKP-1). Sixty micrograms of kidney proteins was resolved on 7.5% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. Rabbit polyclonal MKP-1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), which does not cross-react with MKP-2, was applied in dilution 1:200 for 2 h. Anti-rabbit HRP-conjugated antibody (Transduction Laboratories, Lexington, KY) diluted 1:2000 was used as a secondary antibody. The band was identified by molecular weight (38 kD) standards.
Nuclear Protein Extraction and Western Blot of Hypoxia-Inducible
Factor-1
Nuclear extracts from normal and transgenic mouse kidneys were probed with
anti-hypoxia-inducible factor-1 (anti-HIF-1) antibody. Kidney
tissues were homogenized with a Dounce homogenizer in 10 mM Tris-HCl (pH 7.6),
1.5 mM MgCl2, 10 mM KCl, 2 mM dithiothreitol, 0.4 mM
phenylmethylsulfonyl fluoride, and 1 mM Na3 VO4. Nuclei
were pelleted by centrifugation at 10,000 rpm for 10 min and then resuspended
in 0.42 M KCl, 20 mM Tris-HCl (pH 7.6), 20% glycerol, 1.5 mM MgCl2,
2 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, and 1 mM
Na3 VO4. The suspension was centrifuged at 10,000 rpm
for 30 min at 4°C to extract nuclear proteins. The protein concentration
was determined by the Bradford method (Bio-Rad). Fifteen micrograms of nuclear
protein extract was resolved on 7.5% SDS polyacrylamide gel and transferred to
PVDF membranes at 100 V for 1 h in transfer buffer (25 mM Tris, 192 mM
glycine, 20% methanol). The membranes were blocked with 5% nonfat dry
milk/TBS-T 0.1% (20 mM Tris, 137 mM NaCl, pH 7.4). The monoclonal
anti-HIF-1 antibody (Novus Biologicals, Littleton, CO) was applied
diluted 1:800 in 5% nonfat dry milk/TBS-T for 1 h. Membranes were washed three
times with TBS-T and then incubated with HRP-conjugated anti-mouse IgG (1:4000
dilution; Transduction Laboratories) for 1 h. The membranes were rinsed three
times with TBS-T and developed with enhanced chemiluminescence reagents
(Amersham). Quantification of the blots was performed using a laser
densitometer and the Image Quant software program.
Immunohistochemistry of formalin-fixed kidney sections from three control
and three ßs mice was also carried out with the
anti-HIF-1 antibody. These methods have been described previously in
detail (2).
Statistical Analyses
Statistical analyses were performed by one-way ANOVA. Results are expressed
as means ± SEM of three independent experiments. In each experiment, a
group of five animals was analyzed. P values <0.05 were considered
significant.
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Results |
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Phosphorylation of ERK and Effect of MEG
Figure 2 shows Western blots
of ERK (p42/44 MAP kinase), using phosphospecific
(Figure 2A) and
non-phosphospecific antibodies (Figure
2B). Phosphorylation of threonine and tyrosine tended to be
slightly reduced in transgenic ßs mice under room air and
hypoxic conditions (as a percentage of control ± SEM:
ßs room air, 78 ± 9; ßs hypoxia, 69
± 11, P = 0.07) (Figure 1,
A and C), as well as normal mice under hypoxia (80 ± 12,
P = 0.2), but the differences were not statistically significant by
ANOVA. The nonphosphorylated ERK antibody detects the total ERK and the p42
and p44 isoforms, but because of their close molecular weights, they are
presented as one band. Phospho-ERK antibody, however, binds the two isoforms
separately, and gives ERK a double-band appearance. Densitometry was therefore
carried out on the summation of the two bands, and showed no statistical
differences among the groups of mice. MEG administration increased
phosphorylation toward normal. Figure
1B demonstrates that the amount of protein loaded onto the gel was
equal in all lanes.
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Phosphorylation of SAPK/JNK and Effect of MEG
In contrast to p38, phosphorylation of SAPK/JNK
(Figure 3) was found to be
markedly increased in transgenic ßs mice
(Figure 3A) (as a percentage of
control ± SEM: ßs room air, 300 ± 20;
ßs hypoxia 309 ± 26, P < 0.001) for both
(Figure 3C). As shown in
Figure 3A, the bulk of the
signal in the sickle cell mice appears higher in the gel than in the control
mice. Because the phosphospecific antibody used in this study detects three
isoforms of JNK, the higher location may represent phosphorylation of several
isoforms. MEG had no significant effect on the increased JNK phosphorylation
(268 ± 16, P = 0.5). As shown in
Figure 3B, abundance of total
JNK was equal in all lanes. The results summarized in
Figure 3C are from three groups
of five mice each.
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Western blot analysis of MKP-1, an enzyme that can dephosphorylate threonine/tyrosine of MAP kinases and thereby modulate their activity, is shown in Figure 4. The bands were identified by molecular weight standards. The results of three groups of four mice each showed no significant differences (laser densitometry) in abundance of this protein among the various animals (P < 0.05).
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Immunohistochemistry and Western blots with anti-HIF-1 antibody are
shown in Figures 5 and
6. A marked increase in the
expression of HIF-1 in transgenic ßs mice housed under
either room air or hypoxic conditions was found on Western blot
(Figure 5). Densitometry
measurements (data not shown) indicated a statistically significant increase
in abundance of HIF-1 in the normoxic sickle cell mice, compared with
normal mice, and a further statistically significant increase in the sickle
cell mice exposed to chronic hypoxia (P < 0.05). As seen in
Figure 6A, there was no
staining in the renal medulla of a normal mouse.
Figure 6B demonstrates
scattered areas of HIF-1-positive immunostaining in collecting duct
cells of the medulla of a sickle cell mouse kidney. Similar results were
obtained in two other groups of normal and ßs mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, shown in
Figures 5 and
6, provide further evidence
that in vivo hypoxia is sufficient to induce this protein in the
renal medulla of sickle cell mice. HIF-1 regulates the transcriptional
function of several hypoxia-regulated genes, including iNOS
(24), therefore serving as a
general mechanism of oxygen-sensing and response
(14). It has been found that
desferrioxamine, an iron chelator, activates iNOS gene expression in
macrophages, and this occurs via DNA binding to the HIF-1 consensus
sequence of the iNOS promoter
(24). The increased expression
of HIF-1 in the sickle cell mice suggests therefore that
hypoxia-induced HIF-1 may be the critical activator of iNOS in these
kidneys, leading to high output of NO and formation of ONOO- in the
hypoxic environment of the sickle cell renal medulla. Because ONOO- has been reported to inactivate a number of enzymes by nitrating tyrosine residues, we examined phosphorylation of the three subfamilies of MAPK. The MAPK superfamily of enzymes plays a number of important roles in transduction of signals from the cell surface to the nuclei. The ERK, JNK, and p38 enzymes studied here are the terminal enzymes of three subfamilies, each of which consists of a cascade of kinases where each kinase phosphorylates and thereby activates the next member in the sequence. Phosphorylation of both threonine and tyrosine residues in a specific Thr-X-Tyr sequence within the catalytic core of the enzyme is required for activation, and dephosphorylation of either residue causes inactivation. Antibodies directed against specific phosphorylated threonine and tyrosine residues in ERK, JNK, and p38 have been useful tools in studying these signal transduction pathways in a variety of different experimental conditions.
In the present study, we used these antibodies to examine the state of phosphorylation of ERK, JNK, and p38 in the kidneys of normal and sickle cell mice. We found significant reduction in the phosphorylated state of p38, and a trend toward reduced phosphorylation of ERK. In sharp contrast, phosphorylation of JNK was markedly increased. Immunoblotting with non-phosphospecific ERK, p38, and JNK antibodies showed that equal amounts of protein were loaded onto each lane, thus ruling out differences in protein abundance and significant alteration in protein antigenicity. Studies by other investigators demonstrated a close correlation between enzyme activity of these transduction enzymes on the one hand and phosphorylation state on the other, measured by phosphospecific antibodies similar to those used in the present study (21,22,25,26). The mechanism(s) of activation may have varied in these previous studies, since certain stimuli are highly selective for a given pathway, whereas others activate two or three pathways. Inactivation can also result from various causes.
The markedly increased phosphorylation of JNK (Figure 3) suggests activation of the SAPK cascade, presumably by extracellular signals such as hypoxia, reactive oxygen species, transforming growth factor-ß, or inflammatory cytokines (16). Sustained JNK activation has been identified as a pathway leading to accelerated apoptosis via induction of c-Jun in rat mesangial cells (27) and whole organ DNA extracts of kidney and heart (28). Although the p38 pathway is also activated in response to extracellular stress stimuli, JNK and p38 MAP kinase represent two independent parallel pathways. Regulation is due in part to upstream SAPK activators and kinases, which are different for the two pathways. For example, in cardiac muscle, a specific upstream activator exists that activates JNK during hypertrophy without affecting ERK or p38 (29). Disparate activation of MAP kinases during the developmental stages of experimental glomerulonephritis has also been observed (25,26). Thus, selective phosphorylation of JNK without increased phosphorylation of p38 (Figure 1) or ERK (Figure 2) is not an unusual finding. As mentioned above, sustained activation of JNK has been associated with apoptosis (27). In a previous study (2), we found evidence of apoptosis in the medullary collecting duct cells of sickle cell mouse kidneys. It seems possible based on the present findings that activation of JNK played a role in the apoptosis.
In sharp contrast to the increased phosphorylation of JNK, we observed significant decreases in p38 phosphorylation in sickle cell mouse kidneys, under room air and especially under hypoxic conditions (Figure 1). We examined two possible mechanisms for this impaired phosphorylation. It is well established that phosphorylation of MAPK is tightly regulated by protein phosphatases that can dephosphorylate them. MAP kinase phosphatase-1 (MKP-1) has been shown to dephosphorylate and inactivate MAPK p42/p44, JNK, and p38 by an inhibitory feedback loop serving to modulate activity of these enzymes (27,30,31,32). Upregulation of MKP-1 in the sickle cell mouse kidneys might therefore account for reduced p38 phosphorylation. To examine this possibility, we carried out Western blots of MKP-1. As shown in Figure 4, equal abundance of MKP-1 was found in normal versus sickle cell mice. These results suggest that changes in MPK-1 protein are an unlikely explanation for the observed decrease in p38 phosphorylation.
A second hypothesis we investigated is that ONOO- nitrosylated tyrosine residues of p38, thereby preventing phosphorylation. Peroxynitrite has been found to inactivate a number of enzymes, by highly selective nitration of specific tyrosine and threonine residues. The nitration is not random, but rather depends on the configuration of Thr-X-Tyr presenting sequences, as dual phosphorylation of threonine and tyrosine is required for full activation (33). Interestingly, only a small number of the total tyrosine residues become nitrated on exposure to ONOO-, but this can be sufficient to completely inactivate certain enzymes (10,13,33). With regard to the use of phosphospecific antibodies, it has been shown that tyrosine, but not phosphotyrosine, can be nitrated by ONOO- (34). Moreover, Gow et al. (12) showed that ONOO- reduces tyrosine phosphorylation of endothelial cell lysates by quantitatively nitrating tyrosine residues. These considerations suggest that the reduced abundance of phosphospecific p38 detected by the antibody (Figure 3) could be due to altered antigenicity resulting from nitrosylation of tyrosine. Nevertheless, the reduced signal on Western blot would still reflect impaired phosphorylation of Thr180/Tyr182 because only unphosphorylated tyrosine is nitrated by ONOO- (34).
To test the role of ONOO-, we administered MEG, a potent inhibitor of iNOS. This compound has been shown to have dual actions to scavenge peroxynitrite and to also inhibit iNOS (17,35,36). MEG belongs to a class of S-substituted isothioreas, such as S-aminoethylisothiourea (AEITU). AEITU has been shown to inhibit NOS catalytic activity, and also to decrease the translation of iNOS mRNA and decrease mRNA stability (37). The compound has previously been found to reduce ONOO- formation and the toxic effects of ONOO- in two experimental inflammatory conditions (17,38). As shown in Figures 1 and 2, MEG increased phosphorylation of p38 and ERK in hypoxic ßs mouse kidneys. These observations provide supporting evidence that impaired phosphorylation of these enzymes was due in part to nitration of tyrosine residues by ONOO- and/or a closely related nitrating compound.
The MAP kinase signaling cascades directly phosphorylate and activate certain nuclear transcription factors, thus regulating expression of several genes, including osmolyte transporter genes (p38, ERK) (22), genes responsible for cell growth and proliferation (ERK), and genes involved in cell death and apoptosis (JNK) (39). In the renal medulla, synthesis and transport of protective organic osmolytes is critical for survival of kidney cells exposed to wide variations in ambient osmolality (22,40). Hyperosmotic stress has been shown to induce the three MAP kinase pathways in many cells, including Madin-Darby canine kidney (MDCK) cells (40,41), and in kidneys of intact rats (22). Of the three MAP kinase pathways, p38 kinase activity was found to be essential for induction in MDCK cells of genes involved in synthesis of betaine transporter, an important osmolyte (40). In the renal medulla of sickle cell mice, abnormal conditions exist that might have an impact on ERK, p38, and JNK. First, significant medullary hypoxia has been demonstrated in several mouse models of sickle cell disease, even under room air conditions (23). Hypoxia would be expected to activate JNK via extracellular stress signals (16). In the case of the ßs mice, chronic hypoxia leads to a concentrating defect (19) and, presumably, a corresponding fall in medullary osmolality. Reduced medullary osmolality was shown to decrease the activities of p38, JNK, and ERK in normal rats (22). Thus, the decreases in p38 and ERK phosphorylation in the hypoxic ßs mice, shown in Figures 1 and 2, might have been due to a fall in medullary osmolality. Of additional interest is the finding by Sheikh-Hamad et al. (40) that inhibition of p38 kinase by a specific inhibitor correlated with upregulation of JNK-1 activity in osmotically stressed MDCK cells. They postulated that increased activity of JNK-1 observed during inhibition of p38 kinase is consistent with regulation of JNK-1 by p38 kinase. According to this view, the decrease in p38 phosphorylation found in the present study might have directly influenced the increase in JNK phosphorylation. However, the fact that MEG restored phosphorylation of p38 in the hypoxic ßs mice (Figure 3) supports the hypothesis that ONOO- played an important role in preventing phosphorylation of p38. The impaired phosphorylation of p38 in the sickle cell mouse kidneys could impair survival of cells exposed to osmotic stress, and thereby lead to medullary pathology.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-globins in transgenic mice:
Hemoglobin composition and hematological consequences. Proc Natl
Acad Sci USA 89:12150
-12154, 1992-globins in transgenic mice: Erythrocyte abnormalities, organ damage,
and the effect of hypoxia. Proc Natl Acad Sci USA89
: 12155-12159,1992 in rat mesangial cells. J
Biol Chem 273:4027
-4034, 1998This article has been cited by other articles:
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  N. Kinjo, H. Kawanaka, T. Akahoshi, S. Yamaguchi, D. Yoshida, G. Anegawa, K. Konishi, M. Tomikawa, K. Tanoue, A. Tarnawski, et al. Significance of ERK nitration in portal hypertensive gastropathy and its therapeutic implications Am J Physiol Gastrointest Liver Physiol, November 1, 2008; 295(5): G1016 - G1024. [Abstract] [Full Text] [PDF]
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 M. Bassil, Y. Li, and M. B. Anand-Srivastava Peroxynitrite inhibits the expression of Gi{alpha} protein and adenylyl cyclase signaling in vascular smooth muscle cells Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H775 - H784. [Abstract] [Full Text] [PDF]
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