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B and AP-1 in the Kidney
From the Laboratory of Vascular and Renal Pathology,*
Fundación Jiménez Díaz, Universidad Autónoma,
Madrid; and the Hospital Clínico,
Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B and AP-1 binding activity that was associated with
inflammatory cell infiltration and tubular damage.
Interestingly, infiltrating cells presented activated NF-B
complexes, suggesting the involvement of AngII in inflammatory
cell activation. When rats were treated with AT1 or
AT2 receptor antagonists different responses were observed.
The AT1 antagonist diminished NF-B activity in
glomerular and tubular cells and abolished AP-1 in renal cells,
improved tubular damage and normalized the arterial blood pressure. The
AT2 antagonist diminished mononuclear cell infiltration and
NF-B activity in glomerular and inflammatory cells, without
any effect on AP-1 and blood pressure. These data suggest that
AT1 mainly mediates tubular injury via
AP-1/NF-B, whereas AT2 receptor participates in
the inflammatory cell infiltration in the kidney by NF-B. Our
results provide novel information on AngII receptor signaling and
support the recent view of Ang II as a proinflammatory
modulator.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transcription factors are important
mediators involved in signal transduction that bind to specific DNA
sequences in gene promoters, and regulate transcriptional activity. In
cultured cells, AngII activates various nuclear transcription factors,
including the activator protein-1 (AP-1),10
STAT family of
transcription factors,11
cyclic adenosine monophosphate
response element binding protein12
and, as we have
previously shown, nuclear factor-B (NF-B).3,13
Emerging attention has been focused on the regulation and function of
transcription factors, such as NF-B and AP-1 during tissue
injury.14,15
NF-B has special interest because it plays
a pivotal role in the control of several genes, including cytokines,
chemokines, adhesion molecules, NO synthase, and angiotensinogen,
involved in the pathogenesis of inflammatory lesions, kidney damage,
and hypertension.14
In several models of renal damage, an
elevated tissular NF-B DNA binding activity that diminished in
response to angiotensin-converting enzyme (ACE) inhibition has been
found.3,16
In other pathological conditions associated
with activated RAS, such as atherosclerosis, the increased tissular
NF-B activity was also found to decrease by ACE
inhibition.13
Double-transgenic rats overexpressing both
renin and angiotensinogen genes exhibited increased NF-B activity in
the heart and kidney. In these animals, the antioxidant pyrrolidine
dithiocarbamate inhibits NF-B, ameliorates inflammation, and
protects against AngII-induced end-organ damage.17
However, the in vivo effect of AngII on NF-B activation,
and the potential receptor subtype involved, have not been elucidated.
Two pharmacologically distinct subclasses of AngII receptors (AT1 and AT2) have been described.18,19 The well-known AngII actions, such as the regulation of blood pressure and water-electrolyte balance, and growth-promoting effects, have been attributed mainly to the activation of various signal-transduction pathways via AT1.18,19 AT1 antagonists are currently used to treat patients with hypertension or heart failure. Treatment with AT1 antagonists causes elevation of plasma AngII, which selectively binds to AT2 and theoretically could exert clinically important, but yet undefined, effects.20 The biological functions and the signal transduction pathway of AT2 are primarily unknown. AT2 regulates cell growth inhibition, blood pressure, diuresis/natriuresis, renal NO production and glomerular monocyte infiltration.9,21,22 The AT2 mRNA is highly expressed in the fetal kidney, in lower levels in the adult, and is re-expressed in pathological situations involving tissue remodeling or inflammation, such as neointima formation, heart failure, and wound healing.21,23,24 Renal AT2 may be activated during sodium depletion or AngII administration in the rat.21,25 Therefore, understanding of AT2-mediated physiopathological actions may have important pharmacological implications.
To elucidate the molecular mechanisms implicated in the AngII-induced
kidney damage we have investigated the renal activity of the
transcription factors NF-B and AP-1, related to the pathological
effects caused by systemic infusion of AngII, such as inflammatory cell
infiltration and tubular damage. We have also determined the receptor
subtype associated with these effects by using the specific receptor
antagonists, losartan for AT1 and PD123319 for
AT2.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The in vivo effect of AngII was evaluated by systemic infusion of AngII (dissolved in saline) into female Wistar rats (subcutaneously by osmotic minipumps; Alza Corp., Palo Alto, CA), at the dose of 50 ng/kg/minute. Animals were sacrificed at 24, 48, and 72 hours (acute study), and at 7 days (chronic study). Then, tissue samples were immediately removed and further processed for histological studies and protein extraction. To determine the role of AngII receptor, a group of rats was treated with the AT1 antagonist losartan (10 mg/kg/day in the drinking water) or the AT2 antagonist PD123319 (30 mg/kg/day, subcutaneously by osmotic minipumps) from 24 hours before AngII infusion, and after an additional 72 hours. Losartan was kindly provided by MSD (Spain), and PD123319 was from Sigma (St. Louis, MO). The doses of losartan and PD123319 have previously demonstrated to cause an effective blockade of AT1 and AT2, respectively.26-29 Control groups of animals of the same age, untreated or treated (AT antagonists and saline-infused), were also studied. Systolic arterial blood pressure was measured in conscious, restrained rats by the tail-cuff sphygmomanometer (NARCO Biosystems, CO). The blood pressure value for each rat was calculated as the average of three separate measurements at each session.
Cell Cultures
The murine tubuloepithelial cells (MCT cell line) were kindly donated by Dr. E. Neilson (University of Pennsylvania). The human monocytic cell line U937 (1593-CRL) was obtained from the American Type Culture Collection (Rockville, MD). U937 cells were grown in suspension and 5 x 106 cells were used in each experiment. These cell lines were cultured in RPMI medium with 10% fetal calf serum (FCS) (Gibco BRL, Paisley, Scotland, UK).
Analysis of Transcription Factor Activity
NF-B and AP-1 activity was evaluated by binding of 10 µg of
protein extracts from renal cortex or cells with an oligoconsensus
NF-B or AP-1 labeled with [
-32P]-ATP and
the complexes formed were analyzed by electrophoretic mobility shift
assay (EMSA). Protein extraction were done as described.3
Growth-arrested MCT were incubated with AngII
(10-7, 10-9 and
10-11 mol/L) for 30, 60, and 120 minutes. As
positive controls, 100 U/mL of tumor necrosis factor-
and
10-7 mol/L phorbol myristate acetate were
used. For in vivo studies, frozen kidney pieces were
pulverized in a metallic chamber and resuspended in a cold extraction
buffer [20 mmol/L HEPES-NaOH (pH 7.6), 20% (vol/vol) glycerol, 0.35
mol/L NaCl, 5 mmol/L MgCl2, 0.1 mmol/L
ethylenediaminetetraacetic acid (EDTA), 1 mmol/L dithiothreitol, 0.5
mmol/L phenylmethyl sulfonyl fluoride (PMSF), 1 mg/L pepstatin A]. The
homogenate was vigorously shaken for 30 minutes, and the insoluble
materials precipitated by centrifugation at 40,000 x g
for 30 minutes at 4°C. For in vitro studies, cells were
resuspended in extraction buffer (10 mmol/L HEPES, pH 7.8, 15 mmol/L
KCl, 2 mmol/L MgCl2, 0.1 mmol/L EDTA, 1 mmol/L
dithiothreitol, 1 mmol/L PMSF) and homogenized. Nuclei and cytosolic
fractions were separated by centrifugation at 1000 x g
for 10 minutes, the nuclei were resuspended in extraction buffer to a
final concentration of 0.39 mol/L KCl, and centrifuged at 100,000
x g for 30 minutes. Supernatants dialyzed overnight against
a binding buffer containing 20 mmol/L HEPES-NaOH (pH 7.6), 20% (v/v)
glycerol, 0.1 mmol/L NaCl, 5 mmol/L MgCl2, 0.1
mmol/L EDTA, 1 mmol/L dithiothreitol, and 0.5 mmol/L PMSF. The
dialysates were cleared by centrifugation at 10,000 x
g for 15 minutes at 4°C and frozen at -80°C. Protein
concentration was quantified by the bicinchoninic acid method
(Pierce, Rockford, IL).
NF-B and AP-1 consensus oligonucleotides
(5'-AGTTGAGGGGACTTTCCCAGGC-3' and 5'-CGCTTGATGAGTCAGCCGGAA-3',
respectively) were end-labeled with
[-32P]ATP (Amersham, Buckinghamshire, UK)
and T4 polynucleotide kinase (Promega). Nuclear extracts were
equilibrated for 10 minutes in a binding buffer [4% glycerol, 1
mmol/L MgCl2, 0.5 mmol/L EDTA, 0.5 mmol/L
dithiothreitol, 50 mmol/L NaCl, 10 mmol/L Tris-HCl, pH 7.5, and 50
µg/ml of poly(dI-dC)] (Pharmacia LKB, Uppsala, Sweden), then the
labeled probe (0.35 pmol) was added and incubated for 20 minutes at
room temperature. Negative controls without cellular extracts, and
competition assays with a 100-fold excess of unlabeled NF-B, mutant
NF-B, and AP-1 oligonucleotides, were performed to establish the
specificity of the reaction. When competition assays were done, the
unlabeled probe was added to this buffer 10 minutes before the addition
of the labeled probe. HeLa cell nuclear extracts were used as a
positive control (not shown). The antibodies to NF-B proteins were
from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and Chemikon
(Temecula, CA). For supershift assays, 1 µg of anti-p50, anti-p65, or
anti-c-Rel antibodies was added to protein extracts from cortex of
AngII-infused rats, and incubated for 1 hour before the addition of the
labeled probe. Supershift assays were also done with HeLa cell nuclear
extracts. The supershift band was competed by a 100-excess of unlabeled
specific (NF-B), but not by mutant NF-B probe (not shown). The
specificity of the antibodies was confirmed by Western blot (not
shown). Oligonucleotides were from Promega Corp. (Madison, WI). The
reaction was stopped by adding gel-loading buffer (250 mmol/L Tris-HCL,
0.2% bromophenol blue, 0.2% xylene cyanol, and 40% glycerol) and
protein-DNA complexes were separated on a nondenaturing, 4% acrylamide
gel in Tris-borate. The gel was dried and exposed to X-ray film.
Determination of mRNA Expression of AT2
In kidney samples and in 48-hour serum-deprived MCT cells, AT2 mRNA expression was analyzed by reverse transcriptase-polymerase chin reaction (RT-PCR),23 with specific primers 5'-CTGACCCTGAACATGTTTGCA-3' (sense) and 5'-GGTGTCCATTTCTCTAAGAG-3' (antisense), yielding a 710-bp product. PCR amplification (1 minute at 58°C, 1 minute at 68°C, and 1 minute at 94°C) was linear up to 50 cycles, and data corresponding to cycle 35 were used for calculations. Control experiments were done with RNA samples, but without avian myeloblastosis virus reverse transcriptase. The DNA products were analyzed on 1.5% agarose gel and ethidium bromide staining.
Western Blot of AngII Receptors
Total proteins were obtained from homogenized cells in lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L EGTA, 0.2% Triton X-100, 0.3% Nonidet P-40, 0.1 mmol/L PMSF, and 1 µg/ml pepstatin A) and then separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. After electrophoresis, samples were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked in 0.01 mmol/L Tris, pH 7.5, and 0.4 mol/L NaCl containing 0.1% Tween-20, 1% bovine serum albumin (BSA) and 5% dry skimmed milk for 30 minutes at 37°C, and then incubated in the same buffer with a specific AT2 or AT1 antibody for 18 hours at 4°C. After washing, detection was made by incubation with peroxidase-conjugated secondary antibody, and developed using an ECL chemiluminescence kit (Amersham). Samples from rat adrenal gland were used as positive control for AT2 expression (not shown). The specificity of AT1 and AT2 antibodies was also determined by incubation with blocking peptides (not shown). The antibodies to AT1 and AT2 were from Santa Cruz, secondary horseradish peroxidase-conjugatedantibodies were from The Binding Site (Birmingham, UK), and control rabbit IgG from Sigma.
Renal Histopathological Studies
The kidney samples were studied by staining with hematoxylin/eosin and Masson’s tricrome technique, and examined by light microscopy. The inflammatory cell infiltration was evaluated by immunohistochemistry in formalin-fixed paraffin-embedded sections with an anti-rat CD43 antibody (Pharmingen). This monoclonal antibody reacts with an epitope of CD43 expressed in monocytes, macrophages, natural killer cells, and T cells. A specific monocyte/macrophage marker, an anti-rat ED1 antibody (Serotec) was also used. The presence of AT2 in renal tissue was determined by immunohistochemistry with a specific goat anti-AT2 antibody recognizing rat and human AT2 (Santa Cruz; tested by Western blot). To localize DNA-binding activity of transcription factors Southwestern histochemistry was used.30 The quantification of the infiltrating cells was performed as previously reported.31 The mean number of cells per glomerular cross-section was determined by evaluating 15 glomeruli and the whole interstitium from each animal. Semiquantitative determination was done for morphology, immunohistochemistry, and Southwestern, using the following score: 0, no staining; 1+, mild staining; 2+, moderate staining; 3+, marked staining. Histological studies were quantified by two independent observers in a blinded manner and the mean value was calculated for each rat.
Tissue Localization of CD43, ED-1, and AT2 Immunoreactivity
Paraffin-embedded renal tissue sections (4 µm) were mounted on poly-L-lysine-coated slides. The slides were deparaffinized with xylene and graded concentrations of ethanol and then rehydrated. The endogenous peroxidase was blocked by incubating in 3% H2O2/methanol (1:1) at 25°C for 30 minutes. The slides were subsequently incubated in phosphate-buffered saline (PBS) with 6% horse serum in 4% BSA for 1 hour at 37°C to reduce nonspecific background staining, and then incubated overnight at 4°C with anti-CD43, anti-ED1, or anti-AT2 antibodies in PBS containing 1% serum and 4% BSA. After being washed with PBS, the sections were incubated with secondary anti-IgG HRPO-conjugated antibody diluted 1:100 in 4% BSA/PBS for 30 minutes, and after washing, they were stained with 0.05% 3,3'-diaminobenzidine (DAKO, Glostrup, Denmark) in 0.3% H2O2 for 10 minutes. The sections were counterstained with Mayer’s hematoxylin and mounted in Pertex (Medite). In each experiment, negative controls without the primary antibody, or using an unrelated antibody, were included to check for nonspecific staining.
Southwestern Histochemistry
Sections of renal tissue were dehydrated, rehydrated, fixed with
0.5% paraformaldehyde and treated with 5 mmol/L levamisole for 30
minutes. Then, sections were digested with 0.5% pepsin in 1 N HCl for
30 minutes at 37°C, washed twice with buffer HEPES-BSA (10 mmol/L
HEPES, 40 mmol/L NaCl, 10 mmol/L MgCl2, 1 mmol/L
dithiothreitol, 1 mmol/L EDTA, 0.25% BSA), and treated with 0.1 mg/ml
DNAsa I for 20 minutes. After that, samples were incubated
overnight at 37°C with the NF-B and AP-1 oligonucleotide
digoxigenin-labeled at a final concentration of 100 pmol/ml in
HEPES-BSA with 0.5 µg/ml of poly (dI-dC). After washing, samples were
incubated with an anti-digoxigenin antibody conjugated with alkaline
phosphatase (Boehringer Mannheim, Mannheim, Germany) for 1 hour at
37°C and were revealed with 0.4% 4-nitroblue tetrazolium
chloride and 0.32% X-phosphate. The reaction was stopped with
10 mmol/L Tris, pH 8, 1 mmol/L EDTA. Preparations were mounted with
glycerol. Synthetic consensus sense (NF-B:
5'-AGTTGAGGGGACTTTCCCAGGC-3'; AP-1: 5'-CGCTTGATGAGTCAGCCGGAA-3'),
and mutant sense (NF-B:
5'-AGTTAGCGCTCCTTTCCCAGGC-3'; AP-1:
5'-CGCTTGATAAATCAGCCGGAA-3') probes (Genosys
Biotechnology,London, UK). Double-stranded DNA probes were labeled
with digoxigenin (DIG oligonucleotide 3'end labeling, Boehringer).
Preparations without probe were used as negative controls, and mutant
probes were used to test the specificity of the technique.
Statistical Analysis
Results are expressed as the mean ± SEM. Significance was established using t-test and analysis of variance when appropriate. Differences were considered significant if P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Systemic infusion of AngII (50 ng/kg/min, subcutaneously by
osmotic minipumps) caused a slight increase in systolic blood pressure
after 72 hours (121 ± 9 versus 100 ± 2 mmHg in
controls, n = 10, P < 0.05), but
within the normotensive range. Morphological lesions were examined by
light microscopy. After 72 hours of AngII infusion, the majority of the
glomeruli had a normal appearance. Interstitial inflammatory cell
infiltration and mild tubular lesions were observed (Figure 1)
. The inflammatory cell
infiltration was further examined by immunohistochemistry with a
specific anti-CD43 antibody (Figure 2)
.
In control animals, only few cells with positive anti-CD43
immunostaining were observed (Figure 2; A, C, E, and I
).
AngII-infused animals had some inflammatory cells in the glomeruli
(Figure 2; B, F, and M
). An important increase in the mean number of
inflammatory cells expressing CD43 antigen was observed in the
interstitium, distributed in a focal manner (Figure 2; D, J, and M
).
Some of these cells were monocytes/macrophages, as determined with an
anti-rat ED1 antibody (data not shown). Our findings are similar to
that observed with low doses of AngII infusion, that also showed a
slight increase in blood pressure, glomerular monocyte infiltration,
and fibrosis.6,9
The animals treated with AngII (50
ng/kg/minute) for 7 days showed marked tubular injury (data not shown).
In this sense, AngII infusion for 14 days, although at higher doses
(200 ng/kg/min), resulted in moderate systolic hypertension associated
with cell proliferation, fibrosis, and tubulointerstitial
damage.5,6
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, whereas the
AT2 antagonist diminished inflammatory cell
infiltration (Figure 1D)
, presenting a marked reduction in
CD43-positive cells both in glomerular and interstitial areas (Figure 2, H, L, and M)
. PD123319 also diminished glomerular ED-1-positive
cells (monocytes/macrophages) (data not shown), as previously
described.9
AngII Infusion Activates NF-B in the Kidney
AngII infusion increased renal NF-B DNA-binding activity,
beginning at 24 hours, peaking at 72 hours (6.5-fold versus
control, n = 10, P < 0.05), and
declining at 7 days, but without reaching control values (3.7-fold,
n = 8, P < 0.05 versus
control) (Figure 3)
. Renal NF-B
activity in saline-infused rats showed no difference versus
that in healthy control rats (n = 6,
P = ns, Figure 3A
). The signal of the retarded bands
was only decreased with an excess of unlabeled NF-B, but not mutant
NF-B or AP-1 (unrelated nuclear protein binding), showing the
specificity of the binding (Figure 3B)
. To characterize the nuclear
protein binding to the NF-B motif, protein extracts from
AngII-infused rats were preincubated with antibodies against the
NF-B subunits, p50, p65, and c-rel.14
In the presence
of anti-p50 and anti-p65 antibodies a supershifted band appeared,
whereas there was no changes in the intensity of the complexes (Figure 3, C and D)
. None of the complexes were inhibited by the antibody
against c-Rel (Figure 3, C and D)
, thus this subunit does not seem to
be present in AngII-induced NF-B complexes, as observed in mesangial
cells.3
These results suggest that in AngII-infused rats
the activated NF-B complexes contain p50 and p65 subunits.
|
B
activation in vivo by a digoxygenin-labeled NF-B
oligonucleotide that binds to nuclear active NF-B
complexes.30
Control rats showed no nuclear staining for
NF-B (Figure 4; A, B, and C
). In
AngII-infused rats, there was an increase in positive nuclear staining,
with activated NF-B complexes located mainly in the glomeruli
(mesangial, endothelial, and epithelial cells; Figure 4, D and E
), and
in tubulointerstitial areas (tubuloepithelial and mononuclear
infiltrating cells; Figure 4F
). Moreover, there was an increase in
NF-B staining in renal arteries, located in endothelial and vascular
smooth muscle cells (Figure 4, H and I)
. The semiquantitative score of
NF-B staining of the different groups is shown in Figure 4J
.
Competition of binding by an excess of 100-fold unlabeled NF-B
oligonucleotide, incubation with a mutant digoxigenin-labeled
NF-B (100 pmol/L), or the absence of digoxigenin-labeled NF-B,
did not show any nuclear signal, (Figure 5)
demonstrating the specificity of the
binding. These controls were done in each experiment.
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B Activation in AngII-Infused Animals
We determined the effect of AT1 and
AT2 antagonists in renal NF-B DNA binding
activity, in rats infused with AngII for 72 hours, by EMSA and
Southwestern histochemistry. Both treatments partially diminished renal
NF-B activity in AngII-infused rats [losartan, 44% (inhibition
versus AngII-infusion, n = 6,
P < 0.05); and PD123319, 53%
(n = 4, P < 0.05; EMSA)]
(Figure 6)
. Neither losartan nor PD123319
alone affected renal NF-B activity (not shown). By Southwestern
histochemistry we observed that both treatments diminished nuclear
NF-B staining in the glomeruli (Figure 7, C and D)
. The effect of the
AT1 blocker was mainly observed in
tubuloepithelial cells (Figure 7G)
, whereas AT2
antagonist presented a marked effect on infiltrating cells (Figure 7H)
.
The semiquantitative score of NF-B staining in the glomeruli,
infiltrating and tubular cells of the different groups is shown in
Figure 7I
.
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In renal cortex from AngII-infused rats there was an increase in
AP-1 DNA-binding activity at 72 hours (2.6-fold versus
control, n = 8, P < 0.05) decreasing
after 7 days (Figure 8, A and B)
. By
Southwestern histochemistry we observed that AngII-infused rats for 72
hours presented positive nuclear staining for AP-1 in glomeruli and
tubulointerstitial areas (Figure 8C)
. The specificity of the binding
was evaluated by competition experiments (not shown). Treatment with
AT1 antagonist blocked the AngII effect on renal
AP-1 binding activity (90% inhibition versus
AngII-Infusion, n = 4, P < 0.05,
EMSA), whereas AT2 antagonist did not have any
significant effect (Figure 9)
.
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Many investigators have demonstrated the presence of
AT1 in the adult kidney and in cultured renal
cells.1,21,32
However, there is still some controversial
data about renal AT2.9,21,24,33,34
For this reason, we investigated the presence of
AT2 in the kidney, and in cultured renal cells
by immunohistochemistry and Western blot (protein levels) and
RT-PCR (gene expression). In renal sections of control rats, there was
a positive immunostaining for AT2 located mainly
in tubuloepithelial cells and, to a lesser extent, in resident
glomerular cells (Figure 10A, a and b)
.
Moreover, in rats with immune complex nephritis, which presented an
activation of the local RAS,31
a strong glomerular
staining with the anti-AT2 antibody was observed
(Figure 10A, c)
. Sections were incubated in the absence of primary
antibody, as a negative control of the technique (Figure 10A, d)
. When
total RNA from renal cortex was analyzed by RT-PCR, a band
corresponding to the AT2 mRNA was observed
(Figure 10B)
. AT2 was also detected in cultured
mesangial cells from normotensive Wistar rats, but not from
hypertensive rats,34
in rat glomerular endothelial
cells9
and rabbit tubuloepithelial cells.33
Murine tubuloepithelial cells (MCT) were incubated for 48 hours in RPMI
without serum. Total proteins were analyzed by Western blot and a band
of 44 kd of apparent molecular weight corresponding to the predicted
size was detected (Figure 10C)
. These cells also expressed
AT2 mRNA (Figure 10B)
. All these data are in
agreement with previous studies showing detectable levels of
AT2 in the kidney in normal and pathological
conditions.
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B in Cultured Renal Cells
We performed in vitro studies to further demonstrate
that the in vivo AngII effect on NF-B could be because of
a direct, nonhemodynamic, action. We have previously demonstrated that
AngII activates NF-B in cultured glomerular mesangial
cells.3
However, whether AngII regulated NF-B activity
in tubuloepithelial cells has not been evaluated. For this purpose, we
used MCT cells that possess AT12
and
AT2 (Figure 10)
.
In growth-arrested MCT AngII augmented NF-B DNA-binding activity,
being maximal with 10-9 mol/L after 30 minutes
(3.1-fold versus control, n = 7,
P < 0.05) and declining at 1 hour (Figure 11B)
. AngII also increased AP-1 DNA
binding in MCT (Figure 11B)
, showing a similar response to that in
mesangial cells.32
To investigate the receptor subtype
involved in AngII-induced NF-B and AP-1 activation growth-arrested
MCT were preincubated for 30 minutes with losartan or PD123319 (range
10-5 to 10-7 mol/L), and
then stimulated with 10-9 mol/L AngII for an
additional 30 minutes. The AT1 antagonist
diminished in a dose-dependent manner the AngII-induced NF-B binding
activity, with a maximal inhibitory effect at
10-5 mol/L (79% versus AngII alone,
n = 6, P < 0.05) (Figure 11A)
, whereas
the AT2 antagonist only caused a slight
diminution at 10-5 mol/L (35% versus
AngII alone, n = 6) (Figure 11A)
. By contrast, only
losartan blocked AngII-induced AP-1 binding activity (Figure 11B)
. All
these data suggest that in cultured tubuloepithelial cells AngII
activated NF-B and AP-1 DNA binding mainly by
AT1, similar to that observed in vivo
in tubulointerstitial areas.
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B DNA
binding activity in human mononuclear cells.13,35
These
cells possess all of the components of the RAS, including
AT1 and AT2
receptors.36
In human U937 mononuclear cells, pretreatment
with AT1 or AT2 antagonists
(10-5 to 10-7 mol/L)
diminished in a dose-dependent manner the AngII-induced NF-B DNA
binding activity (Figure 11C)
, suggesting that both receptors are
involved in the NF-B activation caused by AngII in mononuclear
cells. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and AP-1 in
the kidney being related to inflammatory cell infiltration and tubular
damage. Elevated tissular NF-B and AP-1 has been observed in certain
pathological conditions, associated to activated RAS, including
experimental and human glomerulonephritis.3,14-17,37-39
Blockade of NF-B reduced renal damage.17
ACE inhibitors
also diminished renal NF-B activity in experimental models of renal
injury, associated or not with hypertension,3,16
suggesting that NF-B regulation maybe be a therapeutical target in
kidney diseases. Our data provide the first in vivo evidence
of NF-B activation by AngII, and identify the cell-specific
localization of NF-B DNA binding. Thus, in AngII-infused rats a
positive NF-B staining was observed in both resident renal cells and
infiltrating mononuclear cells. Activated NF-B complexes were
located mainly in the glomeruli (mesangial, endothelial, and epithelial
cells), tubuloepithelial cells, and renal arteries (endothelial and
vascular smooth muscle cells). Previous studies, besides ours, have
shown that AngII activated NF-B in cultured cells, including
glomerular mesangial cells.3,13,35,40
In the present
study, we have also found that in cultured tubuloepithelial cells,
AngII caused a rapid and transient, dose-dependent increase in NF-B
DNA binding activity, with a similar potency to that of inflammatory
cytokines. Although in AngII-infused rats there was a slight increase
in systolic blood pressure (around 20 mmHg), the in vitro
studies further demonstrate a direct, nonpressure-related AngII effect
on the regulation of NF-B activity in the kidney.
The potential role of AngII in the inflammatory response is a new and
active field of investigation. AngII is involved in the pathogenesis of
immune-mediated renal diseases.3,41,42
We have found that
AngII infusion causes inflammatory cell infiltration in the glomeruli
and markedly in the interstitium associated with areas of focal
inflammation, as previously reported.5,9
Several data
support the idea of an important role of AngII in the inflammatory
process. AngII causes monocyte chemotaxis and adhesion to mesangial and
endothelial cells43-45
and NF-B
activation.3,35
We have observed in AngII-infused rats
that the infiltrating inflammatory cells exhibited activated NF-B
complexes. Resident renal cells also respond to AngII stimulation with
activation of NF-B and overexpression of related genes, such as
MCP-1 and RANTES,3,9
that could be responsible
for monocyte infiltration in the kidney. All these data suggest that
the effect of AngII on inflammatory cell recruitment could be direct,
through activation of mononuclear cells, and indirect, via chemokine
production by renal cells, both processes mediated by NF-B
activation. A common feature of all stages of atherosclerosis is
inflammation of the vessel wall. Activation of NF-B has been
observed in atherosclerotic lesions,46
and in cultured
vascular smooth muscle and endothelial cells stimulated with
AngII.13,40
We have found that AngII-infusion activates
NF-B in renal arteries, suggesting a more general feature not only
reduced to renal parenchyma. On the whole, our in vivo data
support the new view of AngII as a proinflammatory mediator.
The importance of AngII receptors in vivo has been
demonstrated by treatment with specific receptor antagonists. For
example, AT1 participates in AngII-induced
hypertension, cardiac hypertrophy, and extracellular matrix
accumulation;27-29,47
whereas AT2
mediates endothelium-dependent vasodilatation,21
renal NO
production48
and its role in trophic effects on vascular
smooth muscle cells is controversial.27-29
The
receptor subtype involved in renal injury is not completely defined.
AT1 antagonists are currently used in the
treatment of hypertension and proteinuria in patients with diabetic
nephropathy.49
In models of renal injury,
AT1 antagonists decrease proteinuria, matrix
accumulation, and production of growth factors, such as
TGF-ß.21,50,51
The information provided by
AT2 blockers and AT2
knockout mice supports an important role for the
AT2 in the physiopathology of the
kidney.21,52
AT2 might participate
in blood pressure control, cGMP production in response to sodium
depletion, in natriuretic response via bradykinin/NO, and in renal
prostanoid production and metabolism.21,48,52,53
Unilateral obstruction in the knockout mice for the
AT2 gene caused accelerated renal interstitial
fibrosis and collagen deposition.54
In AngII-infused rats
we have found that treatment with the AT1
antagonist losartan restored blood pressure and improved tubular
damage, whereas administration of the AT2 blocker
PD123319 decreased inflammatory cell infiltration. To further
investigate the molecular mechanism of AngII-induced renal injury
associated to each receptor activation, we evaluated tissue levels and
distribution of transcription factors. In AngII-infused animals, both
AT1 and AT2 antagonists
decreased AngII-increased renal NF-B activity, whereas only
AT1 blocker decreased renal AP-1 activity. Some
divergences in cellular distribution of NF-B activated cells were
observed in response to each receptor antagonist. Both antagonists
decreased NF-B-positive immunostaining in the glomeruli. Losartan
markedly diminished NF-B in tubular cells, and PD123319 did so
mainly in inflammatory cells. In addition, AngII increased NF-B and
AP-1 DNA binding activity mainly by AT1 in
cultured tubuloepithelial cells, and activated NF-B via both
AT1 and AT2 in mononuclear
cells.
One important finding of the present study is that the potential role
of AT2 in the renal inflammatory process may be
mediated by NF-B activation. Thus, in AngII-infused rats, the
AT2 blocker diminished both mononuclear
infiltration and NF-B DNA binding activity in inflammatory cells.
Several experimental data support our hypothesis. In anti-thymocyte
serum-induced nephritis two AT1 antagonists
caused a significant, but not a total reduction, in MCP-1, a chemokine
under NF-B control, and reduced glomerular macrophages/monocytes
infiltration only around 30 to 50%.55
In the model of
ureteral obstruction nephropathy, monocyte/macrophage infiltration was
reduced by ACE inhibitors, but not by AT1
antagonists.50,56,57
In this model, both
AT1 and AT2 antagonists
partially diminished NF-B activity.58
ACE inhibitors
diminished the renal activity of NF-B and the expression of
NF-B-regulated genes, such as MCP-1 and the adhesion molecule
VCAM-1,3,16,50
associated with the decrease in renal
monocyte infiltration. In AngII-infused rats AT2,
but not AT1 antagonists blocked glomerular
monocyte infiltration and the expression of
RANTES,9
another NF-B controlled chemokine.
From these data, we could hypothesize that differences in renal
monocyte recruitment between ACE inhibitors and
AT1 blockers could be because of the action of
AngII via AT2/NF-B pathway, and show a
potential clinically important unresolved question, that needs to be
addressed in human diseases. In cultured cells, AngII via
AT1/NF-B pathway up-regulates some genes, such
as interleukin-6, VCAM-1, MCP-1, and
angiotensinogen.59-62
In vascular smooth muscle cells,
AngII increased gene and transactivated a NF-B-driven VCAM-1
promoter through AT1.59
Both
AT1 and AT2 mediate
AngII-induced NF-B DNA binding and transcription of a NF-B
reporter gene.62
Recently, we have unraveled some of the
intracellular signals elicited by AT1 and
AT2. The potential mechanism of
AT1/NF-B/gene regulation could depend on
redox-sensitive signals and activation of protein tyrosine kinases and
mitogen-activated protein, as occurs with proinflammatory cytokines,
whereas the AT2/NF-B pathway seems to be
mediated by ceramide production.62
All these data suggest
that AngII could regulate a great variety of pathological genes through
NF-B activation via AT1 and/or
AT2, although the gene and receptor involved
could be specific for each tissue or pathological condition.
Another important finding is the effect of AT1
antagonist in AngII-induced renal damage. Losartan restored blood
pressure, improved tubular damage, and decreased renal AP-1 activity.
Interestingly, the effect of losartan on NF-B activity was mainly
located in tubular cells. Many studies have demonstrated that the renal
protection caused by ACE inhibitors or AT1
antagonists are not only because of blood pressure control, but also to
blockade of tissue RAS, in particular to the cellular actions of
AngII.1,2
ACE inhibitors and AT1
antagonists diminished profibrotic genes in experimental nephritis
associated or not to hypertension 1,2,31,50,51
. In
AngII-infused rats, losartan diminished AngII-induced aortic
fibronectin by a nonpressure-mediated mechanism.63
We have
observed that in cultured tubuloepithelial cells AngII, via
AT1, activated NF-B and AP-1. Although a
limitation of our in vivo studies is that we cannot clearly
dissociate between direct/pressure effect, our in vitro data
support the idea that the beneficial effect of
AT1 antagonist could be attributed to a direct
AngII/AT1 action in tubular cells. In cultured
renal cells, AT1 regulates cell growth, matrix
production, and the induction of profibrotic
genes.1,2,8,32
Systemic AngII infusion into rats is
characterized by cell proliferation and fibrosis,4,5
that
are preceded by changes in the expression of some genes that are
regulated by AP-1, such as c-fos, TGF-ß, and
fibronectin.4-8,57
Among these genes, TGF-ß has a
pivotal role in kidney diseases. In cultured renal cells, AngII
increased TGF-ß via AT1.1,2,8,32
In tubuloepithelial cells, AngII causes hypertrophy by TGF-ß
production.2
TGF-ß is also involved in AngII-induced
matrix production in cultured renal cells and in models of renal
injury.1,2,31,32,50,51,64
AP-1 also regulates genes of the
extracellular matrix proteins, such as fibronectin, that are induced by
AngII via AT1,1,51,64
and diminished
by ACE inhibitors and AT1 antagonists in various
models of kidney injury.31,57
Injection of AngII induced
the expression of c-fos/jun via AT1 in the brain
of spontaneously hypertensive rats.65
We have observed
that AngII infusion caused activation of AP-1 via
AT1, suggesting that the beneficial effect of
losartan treatment could be due, at least in part, to the regulation of
AP-1 transcription factor. On the whole, these experimental data
support the idea that losartan blocks
AT1-mediated cellular AngII actions, such as gene
overexpression and activation of transcription factors (AP-1 and
NF-B), and therefore ameliorates renal damage.
Although our studies have described several AngII-mediated effects, as well as the receptors involved, there are many open questions that need future research. AT1 blockade leads to free ligand (AngII) that could theoretically bind to AT2, and then exert some potential beneficial effects, including vasodilatation and antiproliferative/apoptotic responses,21 although there is little evidence of this mechanism in the kidney. On the other hand, AT2 could exert pathological effects through the recruitment of mononuclear cells, although this point needs to be addressed in human diseases. In addition, AT1 blockade stimulates aminopeptidase A activity,66 an enzyme that degrades AngII to AngIII, suggesting a potential increase in AngIII that could bind to AT2.18 AngIII causes proteinuria and increases growth-related, profibrotic and proinflammatory genes.44,67,68 Increased renal AngII production and aminopeptidase A expression have been described in pathological settings associated to tissue RAS activation, such as hypertension, diabetes mellitus, nephritis, and AngII infusion.69,70 These data indicate that although AngII is the main effector peptide of the renin angiotensin system other components of this system, such as AngIII, could play an active role in renal injury.
Our results suggest that the effects of AngII on renal structures take
place through different receptors and transcription factors. AngII
participates in inflammatory cell infiltration mainly mediated by
NF-B activation via AT2, whereas tubular
damage seems to occur through AP-1 by AT1. These
results describe a novel signaling mechanism associated to
AngII-induced renal injury, and may contribute to a better
understanding of the pathological effects of this peptide in several
diseases associated with activated RAS, as renal and cardiovascular
disease.
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Acknowledgements |
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Footnotes |
|---|
Supported by grants from Fondo de Investigación Sanitaria (99/0425), Comunidad de Madrid (97/084/0003 08.4/0017/2000), Ministerio de Educación (SAF 97/0055, PM 97/0085), EU Concerted Action Grant BMH 4-CT98–3631 (DG 12-SSM1), and Fundación Renal Iñigo Alvarez de Toledo. O.L is a fellow of FIS.
M. R.-O. and O. L. contributed equally to this work.
Accepted for publication December 1, 2000.
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B and activator protein-1 in cultured mesangial and mononuclear cells. Kidney Int 2000, 57:2285-2298[Medline]
B through AT1 and AT2 receptors in cultured vascular smooth muscle cells. Molecular mechanisms. Circ Res 2000, 23:1266-1272
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L. Wu, M. Iwai, H. Nakagami, Z. Li, R. Chen, J. Suzuki, M. Akishita, M. de Gasparo, and M. Horiuchi Roles of Angiotensin II Type 2 Receptor Stimulation Associated With Selective Angiotensin II Type 1 Receptor Blockade With Valsartan in the Improvement of Inflammation-Induced Vascular Injury Circulation, November 27, 2001; 104(22): 2716 - 2721. [Abstract] [Full Text] [PDF]
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