Department of Medicine, McGill University Health Centre, Montreal, Quebec, Canada H3A 2B4
Submitted 12 March 2003 ; accepted in final form 21 June 2003
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ABSTRACT |
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proteinuria; passive Heymann nephritis
Mitogenic and stress-related extracellular signals are primarily
transmitted intracellularly through either of the three MAPK pathways:
1) the ERK pathway, typically activated by growth factors;
2) the JNK pathway, also known as the stress-activated protein kinase
pathway; and 3) the p38 pathway
(14,
23). p38 is activated by a
series of cytokines, growth factors, in addition to the stress and
proinflammatory signals and has important roles in stress responses, cell
survival, apoptosis, and inflammation
(15). Activation of p38
involves phosphorylation of threonine and tyrosine residues in a TGY motif,
resulting in increased enzyme activity. p38 lies downstream of various
signaling molecules including the small G proteins Rac and Cdc42 and the
protein kinase transforming growth factor--activated kinase 1 (TAK1).
p38 is directly activated by MAPK kinases (MKKs) MKK3 and 6. Substrates for
p38 include transcription factors, such as ATF-2 or CREB, and protein kinases,
such as MAPK-associated protein kinase (MAPKAPK)-2 and -3. Of interest,
MAPKAPK-2, which is activated by p38, phosphorylates heat shock protein
(HSP27; Ref. 5). Although the
function of HSP27 is not understood completely, it is known to associate with
the actin cytoskeleton and to modulate its organization
(5,
13,
22). Overexpression of HSP27
in fibroblasts increased stress fiber stability during hyperthermia, prevented
cytochalasin D-mediated actin depolymerization, and increased cortical
filamentous actin, ruffling, and pinocytotic activity
(11,
12). Overexpression of
non-phosphorylatable HSP27 inhibited each of these activities
(11,
12).
We reported previously that complement activates two of the MAPK pathways, namely the ERK and JNK pathways in GEC (3, 17, 31). The JNK pathway protects GEC against complement-mediated injury (17) and also contributes to complement-mediated cyclooxygenase (COX)-2 upregulation in GEC (31). In addition, it was recently reported that in GEC, complement-activated ERK might be important in cellular response to complement-induced DNA damage (18). The present study addressed the role of another member of the MAPK family, p38 in GEC. We demonstrated that complement activates p38 in GEC and that p38 activation leads to cytoprotection. We also showed that HSP27 downstream of p38 may contribute to cytoprotection.
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MATERIALS AND METHODS |
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GEC culture and stimulation by complement. Culture,
characterization, and stable transfection of rat GEC were described previously
(2,
29). A subclone of GEC, which
stably overexpress cPLA2 (GEC-cPLA2)
(16,
29), was used unless otherwise
noted. GEC-Neo is a control GEC clone, which was transfected with vector alone
and expresses only a small amount of cPLA2. GEC-cPLA2
shows augmented response (in cytotoxicity and eicosanoid generation) to
complement stimulation, compared with GEC-Neo
(16,
29). Because GEC in vivo
express a significant amount of cPLA2, GEC-cPLA2 better
represent GEC in vivo than GEC-Neo
(4). Inducible-TAK1 clones were
generated using Ecdysone-Inducible Mammalian Expression System
(Invitrogen-Life Technologies). First, GEC were stably transfected with the
plasmid pVgRXR and selected by zeomycin. Second, HA-TAK1 subcloned into the
pIND/Hygro vector was stably transfected and selected by hygromycin.
Expression of HA-TAK1 was induced with an insect hormone, ponasterone A (4
µM). Rabbit antiserum to GEC
(2) was used to activate
complement on GEC membranes. Briefly, GEC were incubated with antiserum (5%
vol/vol) for 40 min at 22°C. GEC were then incubated with normal human
serum (NS; 2.55.0% vol/vol), or heat-inactivated (decomplemented) human
serum (HIS; 56°C, 30 min, 2.55.0% vol/vol) in controls, for the
indicated times at 37°C. In some experiments, antibody-sensitized GEC were
incubated with C8D (5.0% vol/vol) reconstituted with or without purified human
C8 (80 µg/ml undiluted serum). We generally used heterologous complement to
facilitate studies with complement-deficient sera and to minimize possible
signaling via complement-regulatory proteins; however, in previous studies,
results of several experiments involving arachidonic acid metabolism were
confirmed with homologous (rat) complement
(2). Sublytic concentrations of
complement (5% NS) were established previously
(2). Previous studies showed
that, in GEC, complement is not activated in the absence of antibody.
Immunoblotting. Immunoblotting was performed as described previously (29). Cells or glomeruli were lysed in IP buffer [1% Triton X-100, 125 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA, 2 mM Na3VO4, 10 mM sodium pyrophosphate, 25 mM NaF] containing protease inhibitor cocktail (Roche Diagnostics). After insoluble components were removed by centrifugation (14,000 rpm, 5 min, 4°C), protein concentration of supernatants was quantified using a commercial reagent (Bio-Rad). Equal amounts of protein were separated by 8% SDS-PAGE under reducing conditions. Proteins were electrophoretically transferred to nitrocellulose membrane, blocked with 5% dry milk, and incubated with first antibodies for 16 h at 4°C. After three washes, membranes were incubated with secondary antibodies conjugated with horseradish peroxidase, and horseradish peroxidase activity was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Baie d'Urfé, Quebec).
p38 kinase assay was performed using p38 MAP Kinase Assay Kit (New England BioLabs) as per manufacturer's instructions. In brief, cells or glomeruli were lysed in 1x lysis buffer and phosphorylated (activated) p38 was immunoprecipitated with a phospho-specific monoclonal antibody. Immunoprecipitates were subjected to kinase assay using GST-ATF-2 as substrate, and ATF-2 phosphorylation was then detected by immunoblotting using anti-phospho-ATF-2 (Thr71) antibody.
Cytotoxicity assay was performed as described previously
(2,
30). BCECF is a small molecule
(molecular mass 520.5) that could be loaded into cells as acetoxymethyl ester.
It is released into supernatants when cells are injured; thus it could be used
as a marker of cell injury (2).
After complement stimulation, amounts of BCECF released into supernatants and
associated with cells were quantified using a fluorometer, respectively.
Specific release of BCECF into supernatants was calculated to quantify
cytotoxicity using the following formula
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Lactate dehydrogenase (LDH) is an intracellular enzyme that is substantially larger than BCECF. After complement stimulation, LDH activities in supernatants and cell lysates were quantified as described previously (19). Specific release of LDH was calculated as described previously (19) in an analogous manner to BCECF. We previously employed both BCECF assay and LDH assay to quantify cytotoxicity and obtained parallel results. In general, we use higher concentrations of serum for LDH assay because LDH is substantially larger than BCECF; thus it requires more injury to be released into supernatants.
Quantification of F-actin. Quantification of F-actin was performed as described previously (30). After stimulation, cells were washed with PBS and fixed with 2% paraformaldehyde and 4% sucrose in PBS for 10 min. After incubation in NH4Cl (50 mM) for 5 min, cells were permeabilized with 0.5% Triton X-100 for 10 min and blocked with 3% BSA in PBS for 30 min. Cells were incubated with TRITC-phalloidin (0.1 µg/ml) for 60 min. After three washes with PBS, TRITC-phalloidin was extracted with 2 ml of methanol for 30 min with agitation, and cells were further washed with an additional 1 ml of methanol. Fluorescence of the pooled methanol extracts was quantified in a fluorometer (excitation 542 nm/emission 563 nm). To confirm that there were similar numbers of viable cells in each well, cells were further washed three times with PBS and were incubated with a nucleic acid-binding fluorescent dye, Toto-3 (100 nM, Molecular Probe, Eugene, OR). Cells were scraped with a rubber policeman, and cell suspensions were transferred to test tubes and sonicated. Fluorescence of the samples was quantified in a fluorometer (excitation 642 nm/emission 660 nm), and results of TRITC-phalloidin were normalized to Toto-3 values.
Induction of PHN and treatment with FR-167963. PHN was induced in
male Sprague-Dawley rats (150175 g body wt, Charles River, St.
Constant, Quebec) by intravenous injection (400 µl/rat) of sheep anti-Fx1A
antiserum as described previously
(29). Preparation of anti-Fx1A
antiserum was described previously
(20). Rats did not show
significant proteinuria up to 7 days after injection. However, significant
proteinuria was observed 14 days after injection (160 mg/day; normal rats
excrete less than 10 mg of protein per day). FR-167963 was suspended in 0.5%
methylcellulose solution, and 32 mg · kg1
· day1 were given subcutaneously from
day 7 through 14. On day 14, rats were killed after
24-h urine collection in metabolic cages and glomeruli were isolated by
differential sieving as described previously
(29).
Quantification of glomerular rat C3 deposition. Quantification of glomerular rat C3 deposition was performed as described previously with a minor modification (17). Briefly, 4-µm cryostat kidney sections were stained with fluorescein-conjugated rabbit anti-rat C3 (Cappel, Scarborough, Ontario). The immunofluorescence images were captured using a Nikon Diaphoto immunofluorescence microscope and a Nikon Coolpix995 digital camera with a fixed exposure time. Images were transferred to the Adobe Photoshop program, and fluorescence intensity of glomeruli was quantified by histogram analysis. At least eight glomeruli were quantified for each group.
Statistics. Data are presented as means ± SE. The t-statistic was used to determine significant differences between two groups. One-way ANOVA was used to determine significant differences among groups. Where significant differences were found, individual comparisons were made between groups using the t-statistic.
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RESULTS |
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To assess if complement-mediated p38 activation occurs in vivo, we next
studied p38 activation in glomeruli from rats with PHN. PHN was induced by a
single injection of anti-Fx1A antiserum (MATERIALS AND METHODS),
and glomeruli were isolated 14 days later, when rats developed significant
proteinuria (160 mg/day, control rat: <10 mg/day). p38 was activated
by approximately sixfold in glomeruli from rats with PHN, compared with
control (control 13 ± 1, PHN 80 ± 22, P < 0.05,
n = 3 and 6; Fig.
2A). Phosphorylation of p38 was also higher in PHN (2.9
± 0.2-fold), compared with control (control 1 ± 0.1, PHN 2.9
± 0.2, P < 0.01, n = 3 and 6;
Fig. 2B). These
results indicate that p38 is activated by complement in GEC in vitro and in
vivo.
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Arachidonic acid contributes to complement-induced p38 activation. We next addressed the mechanisms of complement-induced p38 activation. It was reported previously that complement activates cPLA2 in a [Ca2+]i- and protein kinase C-dependent manner, leading to a liberation of arachidonic acid from the membrane phospholipid pool (16). We also reported that complement-induced JNK activation was, at least in part, mediated by arachidonic acid release (17). We first studied if liberation of arachidonic acid contributes to complement-induced p38 activation. GEC-cPLA2 were stimulated with antibody and complement, and p38 activation was compared with control GEC, which were transfected with vector only (GEC-Neo). p38 was activated by complement 1.4 ± 0.1 times in GEC-Neo, whereas the stimulation was 2.0 ± 0.1 times in GEC-cPLA2 (n = 3, P < 0.02), suggesting that liberation of arachidonic acid, at least partially, contributes to complement-induced p38 activation (Fig. 3A). When GEC-Neo were stimulated with exogenous arachidonic acid (30 µM), p38 was clearly activated (2.8 ± 0.6-fold of control, P < 0.05, n = 5), supporting the role of arachidonic acid in p38 activation (Fig. 3B).
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Unstimulated cultured rat GEC express COX-1, whereas COX-2 expression is
induced by complement. Arachidonic acid liberated by cPLA2 is
further converted by COX-1 and -2 into biologically active eicosanoids such as
PGE2, PGF2, and TXA2
(29). These eicosanoids act
via respective specific cell-surface receptors in an autocrine fashion and are
known to activate p38 in some systems. To study if eicosanoids contribute to
complement-induced p38 activation, we stimulated GEC with PGE2,
PGF2
, and U-46619 (stable analog of
TXA2). When individually tested, these three eicosanoids failed to
activate p38 consistently. Only when three eicosanoids were tested
simultaneously was there a modest increase in p38 phosphorylation (
20%,
not shown). Furthermore, a nonselective COX inhibitor, indomethacin, failed to
inhibit complement-induced p38 activation (not shown). Taken together, these
results indicate that arachidonic acid contributes to complement-induced p38
activation but not through conversion into COX metabolites. Instead,
arachidonic acid itself and/or other mediators stimulated by arachidonic acid,
such as reactive oxygen species (ROS) (see Role of ROS in the activation
of p38 by complement), may be responsible for p38 activation.
Role of ROS in the activation of p38 by complement. We previously showed that complement stimulates ROS generation in GEC in an NADPH oxidase-dependent manner, which contributes to JNK activation (17). We also showed that arachidonic acid stimulates ROS generation in GEC (17). To test whether ROS contribute to complement-mediated p38 activation, we examined complement-mediated p38 activation in GEC treated with anti-oxidants, GSH, and NAC. As shown in Fig. 4, GSH (10 mM) and NAC (10 mM) completely abolished complement-induced p38 phosphorylation [control 3.7 ± 0.3-fold of HIS, GSH 0.9 ± 0.03-fold of HIS (P < 0.01 vs. control), NAC 1.0 ± 0.07-fold of HIS (P < 0.01 vs. control), n = 3]. Thus ROS are likely to be responsible for complement-induced p38 activation. One mechanism of ROS generation might be arachidonic acid released by cPLA2 (17).
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Impact of p38 activation on GEC function. We next studied the impact of p38 activation on complement-mediated GEC injury. We initially hypothesized that p38 activation contributes to complement-mediated cell injury. However, to our surprise, when GEC were pretreated with p38 inhibitors (PD-169316 and FR-167653, 10 µM each), complement-induced cytotoxicity quantified by specific BCECF release (see MATERIALS AND METHODS) was significantly increased, compared with vehicle-treated cells [Fig. 5A: DMSO: NS 2.5 13 ± 3%, NS 2.75 24 ± 5%, NS 3 38 ± 5%, PD-169316: NS 2.5 20 ± 3% (P < 0.05 vs. DMSO), NS 2.75 29 ± 4% (P < 0.05 vs. DMSO), NS 3 46 ± 4% (P < 0.05 vs. DMSO), n = 34; Fig. 5B: DMSO: NS 2.5 18 ± 5%, NS 2.75 36 ± 7%, NS 3 49 ± 9%, FR-167653: NS 2.5 26 ± 7% (P < 0.05 vs. DMSO), NS 2.75 45 ± 7%, NS 3 58 ± 8% (P < 0.05 vs. DMSO), n = 34]. The concentration of FR-167653 used (10 µM) inhibited complement-induced p38 activity by 90% in GEC (data not shown). Similar results were obtained when LDH release was used to quantify cytotoxicity [DMSO: NS 5 25 ± 9%, NS 7.5 36 ± 10%, FR-167653: NS 5 29 ± 11%, NS 7.5 52 ± 10% (P < 0.05 vs. DMSO), n = 6]. These results suggest that p38 activation may protect cells from complement-mediated injury.
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We and others previously reported that complement C5b-9 disrupts actin microfilaments in cultured GEC (30, 33). One possible mechanism for p38-mediated cytoprotection could be via modulation of the actin cytoskeleton. Thus we next studied the impact of FR-167653 on complement-induced actin depolymerization. When GEC-cPLA2 were incubated with antibody and complement for 3 h, F-actin content decreased by 9 ± 3%. FR-167653 (10 µM) significantly augmented the reduction to 18 ± 4% (P < 0.05, n = 5 each). Thus p38 activation, at least partly, prevents complement-induced actin depolymerization.
To further validate these results, we next generated subclones of GEC that inducibly express a constitutively active mutant of TAK1, a kinase upstream of p38 (MATERIALS AND METHODS). When one such clone (Fig. 6A, #1) was stimulated with an insect hormone, ponasterone A, expression of TAK1 was induced after 2 h, peaking at 6 h. Phosphorylation of p38 was also observed and peaked at 6 h (Fig. 6A). Ponasterone A has no known impacts on mammalian cells. When this inducible clone was stimulated with ponasterone A for 6 h and exposed to antibody and increasing concentrations of complement (NS), complement-mediated cytotoxicity was attenuated, compared with controls (incubated with ethanol in the place of ponasterone A). Conversely, when cells were preincubated with the p38 inhibitor FR-167653 (10 µM, without stimulation with ponasterone A), cytotoxicity was augmented, compared with control, consistent with the previous results (Fig. 6B). Of interest, when cells were stimulated with ponasterone A in the presence of FR-167653, the impact of ponasterone A (TAK1 induction) was neutralized, but cytotoxicity did not reach the level of FR-167653 treatment alone [control 47 ± 1%, ponasterone A 33 ± 3% (P < 0.02 vs. control), ponasterone A plus FR-167653 43 ± 3%, FR-167653 alone 57 ± 2% (P < 0.05 vs. control); Fig. 6C]. These results indicate that the cytoprotective effect of TAK1 induction is, at least in part, mediated by p38 activation. However, there might be additional pathways downstream of TAK1, which contribute to cytoprotection.
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Inhibition of p38 activation augments proteinuria in PHN. The
above results suggest that p38 activation is cytoprotective for GEC against
complement-mediated cell injury in vitro. We next addressed whether p38
activation is also cytoprotective in vivo using the PHN rat model of
membranous nephropathy. In PHN, it is known that complement C5b-9 causes GEC
injury, which leads to proteinuria
(21,
32). As shown in
Fig. 2, p38 was significantly
activated in glomeruli from rats with PHN, compared with control rats. We
anticipated that if p38 is cytoprotective for GEC, inhibition of p38 would
lead to augmented proteinuria in PHN. Thus we examined the impact of a
specific p38 inhibitor, FR-167653, on proteinuria. First, to confirm the
effect of FR-167653, rats were treated with FR-167653 from day 7 to
14 (MATERIALS AND METHODS) and p38 activity in glomeruli
was studied on day 14. In FR-167653-treated rats, p38 activity in
glomeruli was markedly inhibited to a level comparable to that of control rats
[control 13 ± 1, PHN 80 ± 22 (P < 0.05 vs. control),
PHN-FR-167653 12 ± 5 (not significant from control), n =
36; Fig. 7A].
Phosphorylation of p38 was not affected by this inhibitor in a consistent
manner, in agreement with a previous report
(27)
(Fig. 7A). In rats
treated with vehicle, urinary protein excretion on day 14 was 161
± 33 mg/day (n = 7), significantly higher than normal rats
(10 mg/day). Consistent with the in vitro cytoprotective effect of p38
activation, rats treated with FR-167653 showed augmented proteinuria (288
± 54 mg/day, P < 0.05 vs. vehicle, n = 9;
Fig. 7B). To verify
whether complement activation was influenced by FR-167653, we quantified C3
deposition in glomeruli (see MATERIALS AND METHODS). Glomerular C3
deposition was 91 ± 4 in rats with PHN and 93 ± 3 in rats with
PHN treated with FR-167653. Thus FR-167653 did not affect complement
activation in glomeruli. These results support a cytoprotective role for p38
in complement-mediated GEC injury in vivo.
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HSP27 overexpression protects GEC from injury. The above results
indicate cytoprotective effects of the p38 MAPK pathway in GEC. HSP27 is one
of the molecules downstream of p38 MAPK. It is known that when the p38 MAPK
pathway is activated, MAPKAPK-2 is phosphorylated/activated and in turn
phosphorylates HSP27 (5). It
was reported previously that in the rat puromycin aminonucleoside (PAN)
nephrosis model, glomerular expression and phosphorylation of HSP27 were
increased (24). Moreover,
overexpression of HSP27 in mouse podocytes provided protection against PAN
(25). We also demonstrated
that glomerular expression of HSP27 is increased by 1.6-fold in rats with
PHN, compared with control rats, similar to the increase seen in PAN nephrosis
(24)
(Fig. 8A). When
treated with FR-167653, glomerular expression of HSP27 showed an upward trend,
although the difference was not statistically significant [control 52 ±
2, PHN 84 ± 10 (P < 0.01 vs. control), FR-167653 117
± 11 (P < 0.01 vs. control), n = 58 rats;
Fig. 8A]. We also
studied phosphorylation of HSP27 using phospho-HSP27-specific antibody.
Phosphorylation of HSP27 in glomeruli was significantly increased in PHN
(Fig. 8B). In contrast
to protein expression, phosphorylation of HSP27 was markedly inhibited by
FR-167653 [control 15 ± 2, PHN 64 ± 11 (P < 0.01 vs.
control), FR-167653 34 ± 11, n = 6 rats each;
Fig. 8B]. These
results suggest that glomerular phosphorylation of HSP27 in PHN is, at least
in part, mediated by p38 MAPK.
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Thus we next studied whether the cytoprotective effect of the p38 MAPK pathway is mediated via MAPKAPK-2 and HSP27 in rat GEC in culture. To test this hypothesis, we first studied if MAPKAPK-2 is phosphorylated by complement in GEC. When GEC were exposed to antibody and complement, phosphorylation of p38 was observed for 2060 min (Fig. 9A). Phosphorylation of MAPKAPK-2 was observed in a similar time course (Fig. 9A). We next studied the impact of overexpression of HSP27 on complement-mediated GEC injury. The plasmid encoding the wild-type HSP27 was stably transfected into GEC, and its expression was verified by immunoblotting. Two subclones (HSP27WT1 and 2) that overexpressed the wild-type HSP27 were selected for further study (Fig. 9B). Control 1 is a subclone of GEC that was transfected with vector alone and control 2 was transfected with the wild-type HSP27 but the expression level was minimal. It should be noted that control cells also expressed HSP27 when the film was exposed longer (Fig. 9B). When HSP27WT1 and 2 were simulated with antibody and complement, specific LDH release was markedly attenuated, compared with the two control cells [NS 2.5: control 1 30 ± 4%, control 2 24 ± 3%, HSP27WT1 6 ± 1% (P < 0.001 vs. both controls), HSP27WT2 7 ± 1% (P < 0.001 vs. both controls), NS 5: control 1 56 ± 8%, control 2 48 ± 5%, HSP27WT1 10 ± 1% (P < 0.001 vs. both controls), HSP27WT2 16 ± 1% (P < 0.001 vs. both controls), NS 10: control 1 73 ± 6%, control 2 66 ± 2%, HSP27WT1 25 ± 1% (P < 0.001 vs. both controls), HSP27WT2 30 ± 1% (P < 0.001 vs. both controls), n = 612; Fig. 9C]. To study if phosphorylation of HSP27 is important in cytoprotection, we next established subclones of GEC that overexpress a non-phosphorylatable mutant of HSP27. In this mutant, two Ser residues that are known to be phosphorylated by MAPKAPK-2 are mutated to Ala (10). Two subclones (HSP27mut1 and 2) were chosen for further studies (Fig. 9B). Control 3 is a subclone of GEC that was transfected with vector alone, and control 4 was transfected with HSP27mut, but the expression level was minimal. When HSP27mut1 and 2 were stimulated with complement, specific LDH release was slightly attenuated, compared with control cells, but the attenuation was much smaller than in cells overexpressing the wild-type HSP27 [NS 2.5: control 3 25 ± 4%, control 4 29 ± 2%, HSP27mut1 19 ± 2%, HSP27mut2 31 ± 5%, NS 5: control 3 60 ± 3%, control 4 48 ± 2%, HSP27mut1 41 ± 2% (P < 0.05 vs. control 3), HSP27mut2 48 ± 2%, NS 10: control 3 66 ± 2%, control 4 62 ± 1%, HSP27mut1 53 ± 1% (P < 0.05 vs. both controls), HSP27mut2 50 ± 3% (P < 0.05 vs. control 3), n = 412; Fig. 9D]. These results indicate that overexpression of HSP27 induces cytoprotection in GEC and suggest that MAPKAPK-2-mediated phosphorylation of HSP27 is important in this cytoprotection.
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DISCUSSION |
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With the use of the p38 inhibitor FR-167653, Wada et al. (35) elegantly demonstrated that inhibition of p38 MAPK ameliorated histological changes and proteinuria in the nephrotoxic serum nephritis model. The same group also demonstrated that FR-167653 ameliorated histological changes and prolonged survival of MRL-Faslpr mice (6). The current results would appear to contradict these earlier reports. However, it should be noted that both nephrotoxic serum nephritis and MRL-Faslpr mice are models in which glomerular infiltration by leukocytes has a major role. In contrast, infiltrating leukocytes have a minor or negligible role in PHN (32). Because p38 inhibition by FR-167653 inhibits production of inflammatory cytokines by monocyte/macrophages (27), it is highly likely that the effects of FR-167653 observed in nephrotoxic serum nephritis and MRL-Faslpr mice are mediated by inhibition of monocyte/macrophage activation. In fact, in these models, the amelioration of histological changes and proteinuria was paralleled by decreased expression of MCP-1 and glomerular macrophage/lymphocyte infiltration (6, 35). It is therefore possible that the cytoprotective role of p38 in GEC was unmasked in PHN because leukocyte activation has little role in this proteinuric model. Recently, Stambe et al. (26) confirmed the protective effect of a p38 inhibitor (NPC-31145) in rat nephrotoxic serum nephritis. In this report, activated (phosphorylated) p38 was clearly localized in infiltrating neutrophils. Of interest, in glomeruli of normal rat and human, p38 was expressed in podocytes (26). p38 in podocytes was phosphorylated even in the normal kidneys, and phosphorylation was further augmented in nephrotoxic serum nephritis (rat) and postinfectious glomerulonephritis (human) (26). These results support the role of p38 in normal podocyte function as well as in podocyte injury in glomerular disease. In the current study, although it is not possible to determine the site of p38 activation in vivo directly, it is reasonable to assume that GEC are the main site because in PHN, the injury is limited to this cell type (32).
To date, four isoforms of p38 MAPK, i.e., p38, p38
,
p38
, and p38
, have been identified
(15). However, no systematic
study has been conducted regarding which isoforms are expressed in glomeruli
or in GEC. The anti-p38 antibodies used in the current study as well as in the
report by Stambe et el. (26)
are specific for the p38
isoform, confirming the expression of the
p38
isoform in GEC. In contrast, p38
is highly expressed in the
brain and heart and p38
is expressed primarily in skeletal muscle
(15); thus these two isoforms
are not likely to be contributing to cytoprotection in GEC. In addition, the
inhibitor we used in the current study (FR-167653) does not inhibit p38
or p38
(28); thus the
cytoprotective effect we observed is not likely to be mediated by these
isoforms. Therefore, it is most likely that the p38
isoform is
responsible for the cytoprotective effect observed in GEC in the current
study.
Recently, the importance of the actin cytoskeleton in the structure and function of GEC has been highlighted (8). We and others reported that complement induces disruption of the actin cytoskeleton (30, 33). Thus one possible mechanism of the cytoprotective effects of p38 is via interaction with the actin cytoskeleton. Several cytoskeletal proteins are substrates for the p38 pathway, including the microtubule-associated protein tau, the actin-associated protein HSP27, and the intermediate filament proteins h-caldesmon, vimentin, and keratin polypeptides 8, 18 (15). Among them, HSP27 has been most extensively studied. HSP27 is phosphorylated by MAPKAPK-2, which is phosphorylated/activated by p38 MAPK (5). Phosphorylated HSP27 affords resistance to cells against an inhibitor of actin polymerization, cytochalasin D, in a p38-dependent manner (5). Phosphorylation of HSP27 also conferred thermal resistance by interacting with actin filaments (11, 12). In the kidney, Smoyer et al. (24) demonstrated that glomerular HSP27 was upregulated and phosphorylated in the rat model of PAN nephrosis. Overexpression of the wild-type HSP27 in mouse podocytes (GEC) provided protection against PAN-induced microfilament disruption; however, the role of phosphorylation of HSP27 was not addressed in that study (25). It was also reported that HSP27 limits injury in ATP-depleted renal epithelial cells by associating with the actin cytoskeleton (34). In the current study, we demonstrated that MAPKAPK-2 is phosphorylated by complement in a similar time course as p38 (Fig. 9A). Furthermore, GEC that overexpress the wild-type HSP27 showed increased resistance to complement-mediated cell injury (Fig. 9C). However, this protective effect was markedly decreased when phosphorylation sites of HSP27 were mutated (Fig. 9D). Our results are therefore in agreement with previous reports and further demonstrate that phosphorylation of HSP27 is indeed a critical mediator of cytoprotection in GEC. Other cytoskeletal proteins downstream of p38 may also contribute to cytoprotection. Further studies are required to clarify the role of the other cytoskeletal proteins in cytoprotection in GEC.
In summary, p38 MAPK is activated by complement C5b-9 in GEC. Activation of p38 leads to phosphorylation of MAPKAPK-2 and HSP27, which contributes to cytoprotection most likely via interaction with the actin cytoskeleton.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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