| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
B Activation and Monocyte Chemoattractant Protein-1 Synthesis1
Renal Unit, Fundación Jiménez Díaz, Universidad Autónoma, Madrid, Spain
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. Since NF-
B is involved in the regulation of MCP-1
gene, we explored whether the effects of AngII were mediated through
NF-B activation. Untreated nephritic rats showed increased renal
NF-B activity (3.5-fold) that decreased in response to ACE
inhibition. In mesangial cells, AngII activated NF-B (4.3-fold), and
the NF-B inhibitor pyrrolidine dithiocarbamate abolished the
AngII-induced NF-B activation and MCP-1 gene expression. Our results
suggest that AngII could participate in the recruitment of mononuclear
cells through NF-B activation and MCP-1 expression by renal cells.
This could be a novel mechanism that might further explain the
beneficial effects of ACE inhibitors in progressive renal diseases. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
NF-B is the main factor involved in the transcription of MCP-1 gene
induced by LPS, TNF-, IL-1ß, and phorbol esters (15). MCP-1 gene
expression is regulated mainly by proteins that bind to a B region,
responsible for the induction of transcription after stimulation and to
an Sp1-binding site responsible for the maintenance of basal
transcription (16). The NF-B is a heterodimer of p50 and p65
subunits present in inactive form in the cytoplasm complexed with its
inhibitor IB (17). NF-B activation consists of the dissociation
of IB and the translocation of the heterodimer into the nucleus,
where it binds to a specific DNA sequence and activates the
transcription (18, 19).
Recent data suggest that activation of local renin-angiotensin system participates in the pathogenesis of renal damage (20). Tissue levels of angiotensin II (AngII), the effector peptide of this system, are increased under pathologic conditions. AngII is considered a renal growth factor involved in cell proliferation and extracellular matrix accumulation (20, 21, 22), two features of renal damage. Some evidence suggest that AngII could be involved in cell recruitment. AngII receptors have been demonstrated on human monocytes (23), and AngII stimulates chemotaxis of human mononuclear cells (24, 25). Moreover, in different models of renal damage, angiotensin-converting enzyme (ACE) inhibitors diminished the number of infiltrating cells at glomerular and interstitial level (26, 27, 28).
In this work, we evaluated the hypothesis that AngII may participate in mononuclear cell accumulation during renal injury through the production of MCP-1 by resident renal cells. In an experimental model of immune complex nephritis in rats that present increased renal ACE activity (27), we determined renal MCP-1 expression, inflammatory cell infiltration, and the effect of ACE inhibition.
To determine a potential direct effect of AngII on MCP-1 synthesis we
performed in vitro studies with cultured rat glomerular mesangial
cells. To further investigate this process we evaluated whether the
AngII effects were mediated through NF-B activation.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
All culture reagents were purchased from Life Technologies
(Paisley, U.K.). AngII was obtained from Calbiochem.
[-32P]dCTP (3000 Ci/mmol) and
[
-32P]CTP (3000 Ci/mmol) were from Amersham (Amersham,
Buckinghamshire, U.K.). Primers for RT-PCR studies were obtained from
Genosys Biotechnology (Cambridge, U.K.). The mAbs used in
immunohistochemistry were the following: 0X1 (leukocyte common Ag) and
W3/13 (T lymphocytes) obtained from Seralab (Sussex, U.K.); ED1
(monocytes/macrophages) from Serotec (Oxford, U.K.). Recombinant MCP-1
and goat polyclonal anti-MCP-1 Ab were from Immugenex (Los Angeles,
CA). Horseradish peroxidase-conjugated donkey anti-goat IgG was
from The Binding Site (Birmingham, U.K.), and normal control rabbit IgG
was from Sigma (St. Louis, MO). For supershift assays, we used
anti-p65 and anti-c-Rel Abs from Santa Cruz Biotechnology
(Santa Cruz, CA) and anti-p50 Ab from Chemikon (Temecula, CA).
NF-B consensus oligonucleotide was from Promega (Madison, WI). All
other chemicals were from Sigma. None of the compounds was cytotoxic
for mesangial cells at the concentrations used, as determined by trypan
blue staining (not shown).
Experimental design
Immune complex nephritis was induced in normotensive Wistar rats as previously described (27, 29). Briefly, rats received an initial s.c. injection of 5 mg of OVA in CFA (Difco, Detroit, MI), and 3 wk later, the same dose was given in IFA (Difco). One week later, daily i.p. administration of 10 mg of OVA was started. Proteinuria appears at the 9th week, and when reaching 20 to 50 mg/day animals were randomly distributed into two groups: untreated (proteinuria, 29 ± 11 mg/day), animals with spontaneous development of nephritis; and quinapril-treated (proteinuria, 30 ± 10 mg/day; p = NS vs untreated), animals treated with the ACE inhibitor quinapril (a gift from Parke Davis as powdered hydrochloride) at a concentration of 100 mg/L, added to the drinking water, and replaced every 48 h.
Studies were done 3 wk after the onset of proteinuria when untreated
animals develop full-blown nephrotic syndrome and moderate renal
failure (27, 29). A parallel control group of animals of the same age,
with or without treatment, was also studied. At the time of sacrifice,
animals were fasted overnight and anesthetized with 5 mg/100 g sodium
pentobarbital. The kidneys were perfused in vivo via the abdominal
aorta with 100 ml of normal saline at 4°C. Blood samples and kidneys
were removed immediately and further processed for histologic studies,
RNA extraction, and NF-B activity.
We have previously described the effect of ACE inhibition treatment in this normotensive model of immune complex nephritis (27). In the group of animals studied in the present work, the administration of quinapril during 3 wk caused a significant reduction in proteinuria (80 ± 29 vs 510 ± 49 mg/day in untreated nephritic rats, n = 10, p < 0.01) and in glomerular and tubulointerstitial lesions (not shown). Nephritic rats remained normotensive along the study, and quinapril did not significantly modify systemic blood pressure (not shown). Renal ACE activity was elevated in untreated rats compared with controls and diminished around 60% in response to ACE inhibition, as previously shown (27).
Renal histopathologic studies
Immunohistochemistry. Tissue for immunohistochemistry was embedded in OCT (Tissue-Tek, Miles, Elkhart, IN), snap-frozen in liquid nitrogen, and stored at -80°C until its study. The quantification of the infiltrating glomerular and interstitial cells was performed as previously published (29, 30). The mean numbers of cells per glomerular cross-section were determined by evaluating 50 glomeruli in each renal section. In the interstitium, areas of 0.45 mm2 were counted.
Tissue localization of MCP-1 immunoreactivity. Paraffin-embedded renal tissue was cut at 4 µm and mounted on poly-L-lysine-coated slides, and immunoperoxidase staining was performed. The slides were deparaffinized with graded concentrations of xylene and ethanol. The slides were quenched in methanol containing 3% H2O2/methanol at 25°C for 30 min. They were subsequently incubated in PBS with 6% horse serum in 4% BSA for 1 h at 37°C to reduce nonspecific background staining and then incubated overnight at 4°C with goat polyclonal anti-MCP-1 Ab, 70 µg/ml, in PBS containing 1% horse serum and 4% BSA. After being washed with PBS, the sections were incubated with horseradish peroxidase-conjugated donkey anti-goat IgG, diluted 1:100 in 4% BSA for 30 min, and after washing, they were stained with 0.05% 3,3'-diaminobenzidine (Dako A/S, Glostrup, Denmark) in 0.01% H2O2 for 10 min. The sections were counterstained with Mayer’s hematoxylin and mounted in Pertex (Medite, Burgdorf, Germany). In each experiment, negative controls without the primary Ab or using an unrelated Ab were included to check for nonspecific staining. About 15 glomeruli from each animal were examined, and the immunostaining was graded from 0 to 4+ by a semiquantitative score according to the following criteria: 0, no staining; 1+, minimal staining; 2+, mild staining; 3+, moderate staining; 4+, marked staining.
Histologic studies were quantified by two independent observers without knowing to which group the sample belonged, and the mean value was calculated for each rat.
Mesangial cell culture
Rat mesangial cells were cultured from isolated glomeruli of healthy rats by sieving techniques and differential centrifugation (31). Mesangial cells were grown in RPMI 1640 medium buffered with 25 mM HEPES at pH 7.4 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine in the presence of 10% FCS and cultured at 37°C in 5% CO2 atmosphere. At confluence, cells grown in 75-cm2 flasks were made quiescent for 48 h in 0.5% FCS medium, and then different studies were performed. Glomerular mesangial cells were characterized by phase contrast microscopy, positive staining for desmin and vimentin, and negative staining for keratin and factor VIII Ag, excluding epithelial and endothelial cell contamination, respectively (32).
Molecular techniques
cDNA probes.
The cDNA probes for human MCP-1 (JE*/pGEM-hJE34) and
glyceraldehyde-3'-phosphate dehydrogenase (G3PDH) were obtained from
the American Type Culture Collection, Manassas, VA. Probes were
radiolabeled by nick translation (Boehringer Mannheim, Mannheim,
Germany) with [-32P]dCTP.
Northern blot assay.
Total RNA from cells and renal tissue was extracted by the method of
Chomczynski and Sacchi (33) and quantitated by absorbance at 260 nm.
Equal amounts of RNA (10–30 µg) were denatured, electrophoresed in a
l% agarose-formaldehyde gel, and transferred to nylon membranes
(Genescreen, New England Nuclear, Boston, MA). The quality of RNA was
determined by ethidium bromide staining (not shown). RNA was fixed to
the nylon membrane by baking for 90 min at 80°C. The membranes were
prehybridized for 4 h at 42°C in hybridization solution (50%
formamide, 1% SDS, 5x SSC, 1x Denhardt’s, 0.1 mg/ml denatured
salmon sperm DNA, and 50 mM sodium phosphate buffer, pH 6.5), and
hybridization was conducted at 42°C for 16 to 18 h in fresh
hybridization solution with 20% dextran sulfate and
-32P-denatured probe. The membranes were washed
using 2x SSC, 0.1% SDS, for 30 min at room temperature and then twice
with 0.2x SSC, 0.1% SDS, at 55°C for 15 min. Autoradiographic
signals obtained with the G3PDH cDNA probe served as a control for
equal loading of the gel. The ratio of mRNA vs G3PDH was set at unity
for basals, and other lanes on the same gel were expressed as
n-fold increases over this value.
Reverse transcription and semiquantitative PCR analysis.
To obtain cDNA for the PCR, 1 µg of RNA from each sample was
transcribed in a final volume of 20 µl which contained 5 mM
MgCl2, RT buffer (10 mM Tris-HCl, 50 mM KCl, and 0.1%
Triton X-100), 1 mM deoxynucleotide mixture, 20 U of RNasin (an RNase
inhibitor), 15 U of reverse transcriptase of the avian Moloney virus,
and 50 ng of random primer. The reaction mixture was incubated at
42°C for 45 min. At the end of the incubation, samples were heated at
95°C to eliminate transcriptase activity and to denaturalize the
RNA-cDNA hybrids. PCR was conducted in the presence of
[-32P]dCTP for 25, 30, 35, and 40 cycles under the
same conditions as for MCP-1 and glyceraldehyde 3'-phosphate
dehydrogenase (G3PDH), used as internal control (1 min at 54°C to
allow annealing of the primers, 3 min at 72°C for primer extension,
and 1 min at 94°C to denature the double-stranded DNA). The following
primers were used for rat MCP-1 (34): (sense)
5'-TTCTGGGCCTGTTGTTCACA-3' and (antisense)
5'-GGTCACTTCTACAGAAGTCC-3', that yielded a product of 409 bp. G3PDH
was used as internal control and the following primers were used (35):
(sense) 5'-AATGCATCCTGCACCACCAA-3' and (antisense)
5'-GTAGCCATATTCATTGTCATA-3' that yielded products of 515 bp. The
amplification of PCR was linear up to 35 cycles, both for MCP-1 and for
G3PDH, and data for cycle 25 were used for calculations. In all
experiments, the presence of possible contaminants was checked by
control reactions in which amplification was conducted in complete
reaction mixture lacking template DNA or with RNA samples from RT
reactions done in the absence of avian Moloney virus reverse
transcriptase. The DNA products from the PCR reactions were analyzed on
a 4% polyacrylamide-urea gel in Tris-borate, EDTA buffer (45 mM
Tris-HCl, 45 mM boric acid, 1 mM EDTA). The polyacrylamide gels were
dried, exposed to x-ray films, and scanned using the IQ densitometer
(Image Quant Densitometer, Molecular Dynamics, Sunnyvale, CA).
Protein extraction
From tissue. For protein extraction from tissue samples, the method of Negoro et al. (36) was used with some modifications. Briefly, frozen kidney cortex sections were pulverized in a metallic chamber and resuspended in 1 ml of cold extraction buffer containing 20 mM HEPES-NaOH (pH 7.6), 20% (v/v) glycerol, 0.35 M NaCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, and 1 µg/ml pepstatin A. The homogenate was vigorously shaken, insoluble materials were precipitated by centrifugation at 40,000 x g for 30 min at 4°C, and the supernatant was dialyzed overnight against a binding buffer containing 20 mM HEPES-NaOH (pH 7.6), 20% (v/v) glycerol, 0.1 M NaCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, and 0.5 mM PMSF. The dialysate was cleared by centrifugation at 10,000 x g for 15 min at 4°C and frozen at -80°C in aliquots until use. Protein concentration was quantified by the BCA method (Pierce, Rockford, IL).
From cells. Quiescent mesangial cells were stimulated for different periods of time. Then, cells were trypsinized and resuspended in 5 cell pellet vol of buffer A (10 mM HEPES (pH 7.8), 15 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 1 mM PMSF). After 10 min on ice, the cells were pelleted, resuspended in 2 cell pellet vol of buffer A, and homogenized. Nuclei were centrifuged at 1000 x g for 10 min, washed twice in buffer A, and resuspended in 2 vol of this buffer. Then 3 M KCl was added drop by drop to reach 0.39 M KCl. Nuclei were extracted for 1 h at 4°C and centrifuged at 100,000 x g for 30 min. Supernatant was dialyzed in buffer C (50 mM HEPES (pH 7.8), 50 mM KCl, 10% glycerol, 1 mM PMSF, 0.1 mM EDTA, and 1 mM DTT) and then cleared by centrifugation and stored at -80°C. Protein concentration was determined by the BCA method.
Electrophoretic mobility shift assays (EMSA)
Gel shift assays were performed with a commercial kit following
the instructions of the manufacturer (Promega). Briefly, NF-B
consensus oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3') was
32P-end-labeled by incubation for 10 min at 37°C
with 10 U of T4 polynucleotide kinase (Promega) in a reaction
containing 10 µCi of [-32P]ATP, 70 mM Tris-HCl, 10
mM MgCl2, and 5 mM DTT. The reaction was stopped by the
addition of EDTA to a final concentration of 0.05 M. Nuclear or
cellular protein (10 µg) was equilibrated for 10 min in a binding
buffer containing 4% glycerol, 1 mM MgCl2, 0.5 mM EDTA,
0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5), and 50 µg/ml of
poly(dI-dC) (Pharmacia, Uppsala, Sweden). NF-B activity was
quantified in 10 µg of total protein from each pool, and experiments
were done in duplicate. HeLa cell nuclear extract was used as a known
positive control because it has been shown to contain NF-B (37). To
assess the specificity of the reaction, the following controls were
done: negative assay without cellular extracts; and competition assays
with a 100-fold excess of unlabeled NF-B, mutant NF-B, and
unspecific (activating protein 1 (AP-1)) oligonucleotides. When
competition assays were done, the unlabeled probe was added to this
buffer 10 min before the addition of the labeled probe. The labeled
probe (0.35 pmol) was added to the reaction and incubated for 20 min at
room temperature. For supershift assays, 1 µg of anti-p50,
anti-p65, or anti-c-Rel Abs was added and incubated for 1
h before the addition of the labeled probe. The specificity of
anti-p50 and anti-p65 Abs was tested by Western blot. Briefly,
nuclear extracts were obtained from cells treated with TNF- and
AngII, analyzed by PAGE-SDS. After incubation with anti-p50 and
anti-p65 Abs, a band of the expected size was found (not shown).
Supershift assays were also done with HeLa cell nuclear extracts. The
supershift band was competed by a 100-fold excess of unlabeled specific
(NF-B), but not by mutant NF-B probe (not shown). The reaction
was stopped by adding gel loading buffer (250 mM Tris-HCl, 0.2%
bromphenol blue, 0.2% xylene cyanol, and 40% glycerol) and run on a
nondenaturing, 4% acrylamide gel at 100 V at room temperature in TBE.
The gel was dried and exposed to x-ray film and scanned using the IQ
densitometer.
Western blot analysis
Confluent resting mesangial cells were incubated for 24 h
with 10-7 M AngII and 100 U/ml TNF-, used as
positive control. Then, the conditioned medium was isolated,
concentrated 10 times by centrifugation using ultrafree MC filters
(Millipore, Bedford, MA), and kept at -20°C until analysis. Protein
content was determined by the BCA method. The samples were separated by
SDS-PAGE and transferred to polyvinylidene difluoride membranes.
Membranes were blocked by a 30-min incubation in 0.01 mM Tris, pH 7.5,
and 0.1 mM NaCl containing 0.1% Tween 20, 1% BSA, and 5% milk and
then incubated in the same buffer with an anti-MCP-1 Ab for 18
h at 4°C. After washing, detection was made by incubation with
peroxidase-conjugated anti-rabbit IgG and developed using an ECL
chemiluminescence kit (Amersham). The films were scanned using the IQ
densitometer. Recombinant human MCP-1 was used as positive control.
Statistical analysis
Autoradiographs were scanned by densitometry. Results are expressed as n-fold increase over control in densitometric arbitrary units and expressed as mean of the experiments made. Results of immunohistochemistry are expressed as the mean ± SEM. Comparisons between means of multiple groups were analyzed by ANOVA and Student’s t test. Significance was established using the GraphPAD Instat (GraphPAD Software, San Diego, CA), and differences were considered significant if the p value was <0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We have previously described that administration of the ACE inhibitor quinapril to rats with immune complex nephritis caused a significant decrease in glomerular and interstitial cell infiltration (27). By means of mAbs, we characterized the phenotype of the inflammatory cells in the animals used in the present work. Compared with the untreated nephritic rats, animals receiving quinapril for 3 wk had significantly less glomerular cells expressing the leukocyte common Ag (3.0 ± 0.5 vs 8.0 ± 0.4 untreated rats, cells/glomerulus), T lymphocytes (3.2 ± 0.7 vs 7.5 ± 1.1), and monocytes/macrophages (5.0 ± 1.9 vs 7.5 ± 0.1) (n = 9 for all groups studied; p < 0.05 for all parameters). More marked effects were noted on the interstitial cell infiltrates, with an important reduction in the number of cells expressing the leukocyte common Ag (91 ± 9 vs 283 ± 57 untreated rats, cells/mm2), T lymphocytes (78 ± 12 vs 201 ± 60), and macrophages (37 ± 10 vs 148 ± 14), reaching values similar to those of healthy control rats (n = 9; p < 0.01). The presence of infiltrating cells in renal tissue has been associated with proteinuria in several human and experimental nephritis (2, 3, 4, 5, 6). In quinapril-treated rats, proteinuria was significantly reduced vs untreated rats (80 ± 29 vs 510 ± 49 mg/day, n = 10, p < 0.05). In this model, we have observed that a good correlation existed between infiltrating cells and proteinuria (r2 = 0.99; p < 0.01).
Renal MCP-1 mRNA and protein expression are up-regulated in nephritic rats and are diminished by ACE inhibition
The recruitment of monocyte-macrophages in renal tissue, through the release of chemotactic factors by resident and infiltrating cells, is an important fact in the induction and progression of renal injury (2, 3, 4). MCP-1 expression has been detected in nephritic glomeruli (14). For this reason, in nephritic rats, we have studied renal MCP-1 mRNA levels and protein localization as well as its modulation by ACE inhibition.
MCP-1 gene expression was studied with a semiquantitative PCR technique
by amplifying a fragment of MCP-1 gene in the same conditions as a
fragment of the housekeeping gene G3PDH (see Materials and
Methods). Samples of total RNA from renal cortex of four
different animals of each group were pooled and analyzed in two RT-PCR
experiments. In nephritic rats, there was a fourfold increase in MCP-1
mRNA levels with respect to healthy controls (Fig. 1
). Quinapril treatment induced a
dramatic reduction in MCP-1 gene expression (Fig. 1).
|
, A and B).
In untreated nephritic rats, an intense MCP-1 staining was seen in
glomerular capillaries, mesangial area, proximal tubules, and
interstitial mononuclear cell infiltrates (Fig. 2C).
In nephritic rats treated with quinapril, there was a marked diminution
in MCP-1 staining (Fig. 2D). No immunoperoxidase
staining was observed when a nonimmune serum was used (negative
controls; Fig. 2E). The semiquantification of
immunostaining for MCP-1, done in a double-blinded manner, is shown in
Figure 3.
|
|
Renal NF-B is activated in nephritic rats and is diminished by
ACE inhibition
Recent studies have shown the activation of transcription factors
during tissue damage (38). Cellular extracts were obtained from samples
of the renal cortex of three animals from each group and then pooled.
NF-B activity was quantified in 10 µg of total protein from each
pool, and EMSA experiments were done in duplicate. In relation to
controls, untreated nephritis showed a 3.5-fold increase in NF-B
activity that decreased to control values in quinapril-treated animals
(Fig. 4A).
The reaction was proved to be specific since an excess of unlabeled
NF-B, but not mutant NF-B or AP-1 (unrelated nuclear protein
binding), decreased the signal of the retarded bands (Fig. 4A).
|
B motif,
cellular extracts were preincubated with Abs to the protein subunits of
the NF-B p50, p65, and c-Rel (39). Incubation of the extracts with
anti-p50 and anti-p65 Abs yielded a supershifted band. In the
presence of anti-p65 Ab, a slight reduction in the intensity of the
bands was also noted. No effect was observed with anti-c-Rel Ab
(Fig. 4
B). Angiotensin II induces MCP-1 mRNA and protein expression in cultured mesangial cells
The above-mentioned experiments suggested that in situations of renal injury, associated to increased local AngII generation, this peptide could participate in cell recruitment. However, we cannot discard that other factors involved in cell recruitment such as cytokines and immune complexes, present in the cell microenvironment during renal damage (29, 40), could be responsible for the increase in MCP-1 expression and inflammatory cell accumulation. To further investigate this point, we studied whether AngII regulates MCP-1 production in cultured glomerular mesangial cells. These cells produce MCP-1 in response to several stimuli, like cytokines or immune complexes (10, 11, 41), and may play a key role in inflammatory reactions in different glomerular diseases (42).
Mesangial cells were stimulated with several concentrations of AngII
(10-7 to 10-11 M) for 3, 6, and 24
h. After incubation, RNA was extracted, and MCP-1 mRNA expression was
determined by Northern blot. Under basal conditions, a MCP-1 mRNA band
(0.8 kb) was apparent (Fig. 5A), as described
(8, 9, 10, 11). In response to AngII stimulation, an increase in MCP-1 mRNA
expression was found, being maximal at 6 h and returning to basal
levels after 24 h (Fig. 5A). This result showed
a similar kinetics to that induced by 1 µg/ml LPS (Fig. 5A) and TNF- (not shown), as previously described
(8, 11, 43). This effect was dose dependent with maximal responses at
10-7 to 10-9 M AngII (
2.7-fold increase vs
basal, p < 0.05) (Fig. 5B).
|
80%) (Fig. 5C), suggesting a PKC-dependent mechanism. None of
the two inhibitors had any effect on the MCP-1 gene expression in
control cells, and 100 mM PMA, a known PKC activator, was used as a
positive control (Fig. 5C).
We also evaluated the role of the de novo protein synthesis in this
process. Preincubation of mesangial cells with 1 µg/ml cycloheximide
for 1 h increased MCP-1 mRNA in unstimulated and AngII-treated
cells (Fig. 5C), indicating that its induction occurs
in the absence of new protein synthesis and may be under the control of
a labile repressor protein.
To determine whether the increase in MCP-1 expression in response to
AngII led to the release of protein to the extracellular medium,
mesangial cells were incubated for 24 h with 10-7 M
AngII and 100 U/ml TNF- (positive control), and MCP-1 synthesis was
determined by Western blot. Under control conditions, mesangial cells
secreted small amounts of MCP-1, but after treatment with AngII or
TNF-, a marked increase was observed (threefold) (Fig. 6). The MCP-1 was detected as a band of
12 to 14 kDa, similar to that of the recombinant MCP-1, used as
standard, and similar to that demonstrated in murine and human
mesangial cells after treatment with IgG and IgA aggregates (11, 41).
|
B in mesangial cells
NF-B has been identified as an important DNA-binding protein
that initiates the transcription (17, 18). Growth-arrested mesangial
cells were incubated with AngII (10-7,
10-9, and 10-11 M) for 30, 60, and 120 min.
Nuclear extracts were obtained, and NF-B activity was determined by
binding assay of nuclear proteins to an oligoconsensus labeled with
-32P. Optimal induction was found after 30 min of
stimulation and declined over the next 2 h (Fig. 7A). All subsequent
experiments were done at 30 min. AngII caused induction of NF-B
DNA-binding activity, being maximal at 10-9 M (4.3-fold
over basal, n = 5, p < 0.05) (Fig. 7B). Two strong inducers of NF-B activation, LPS
(1 µg/ml) and TNF- (100 U/ml) (15), were used as positive controls
in all experiments. Different cell culture preparations of nuclear
extracts yielded mainly identical data, with some differences in the
relative intensity of basal and positive control. The NF-B activated
by AngII contains several DNA-protein complexes with different
electrophoretic mobilities, shown by two bands (Fig. 7). All these
complexes were specific because an excess of the same unlabeled
oligonucleotide efficiently competed for the formation of the
complexes, whereas an oligonucleotide containing mutations, or
nonrelated, did not (Fig. 7, A and C).
|
B-Rel family could potentially interact
with the B motif, and therefore we used specific Abs to determine
the composition of the NF-B. When AngII-treated nuclear extracts
were preincubated for 1 h with 1 µg of anti-p50 and
anti-p65 Abs, a supershifted band appeared, and a reduction in the
intensity of the complexes was noted with the anti-p65 Ab (Fig. 7D). None of the complexes were inhibited by the Ab
against c-Rel (Fig. 7D); thus, this subunit does not
seem to be present in mesangial cells as is the case in human vascular
smooth muscle cells (44). These results suggest that in mesangial
cells, AngII-activated NF-B complexes contain p50 and p65
subunits.
AngII increases MCP-1 mRNA expression through NF-B activation in
mesangial cells
The nuclear factor NF-B is involved in the MCP-1 transcription
induced by IL-1ß, TNF-, and phorbol esters (16, 45). One potential
mechanism of inhibiting NF-B activation is by reducing oxidant
stress (46). The antioxidant PDTC has been shown to block cytokine- and
phorbol ester-induced NF-B activation in several cell lines (46, 47). The preincubation of mesangial cells with 200 µM PDTC for 1
h abolished the AngII-induced NF-B activation (Fig. 8C), which correlated
with the inhibition of AngII-induced MCP-1 mRNA levels (Fig. 8A). These data suggest that the increase in MCP-1
mRNA expression induced by AngII in mesangial cells is at least, in
part, NF-B mediated.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B activity. In addition, in cultured glomerular mesangial
cells, AngII elicited an up-regulation of MCP-1 gene expression and
synthesis in part due to the activation of NF-B. Several studies have shown that activation of tissue renin-angiotensin system is involved in the pathogenesis of kidney diseases (20, 26). The fact that the administration of ACE inhibitors reduced the number of inflammatory cells in various models of renal injury (25, 26, 27), as well as the accumulation of mononuclear cells in the kidney of normal rats after 7 to 14 days of systemic AngII infusion (47, 48), suggests that this peptide may be involved in the recruitment of mononuclear cells. In this regard, AngII may participate at many points in the onset and progression of inflammation. AngII induces adhesion molecule expression in human endothelial cells (49) and activates human monocytes leading to increased adhesion to endothelial cells (50). AngII is a chemotactic factor for mononuclear cells (24, 25). Since monocyte-macrophages play an important role in the induction of renal injury (2, 3, 4), we wondered whether AngII may regulate the synthesis of chemoattractant proteins in the kidney. Recent studies have demonstrated that several renal cells can produce MCP-1 after appropriate stimulation (8, 9, 10, 11) and that MCP-1 is overexpressed in the kidney in experimental and human glomerulonephritis (7). Moreover, the administration of anti-MCP-1 Abs to rats with nephrotoxic nephritis decreased glomerular monocyte-macrophage infiltration (51).
In this article, we observe that rats with immune complex nephritis presented increased MCP-1 gene and protein expression. This up-regulation was chiefly located in resident renal cells (glomerular and tubular epithelial cells) and in infiltrating mononuclear cells. The administration of the ACE inhibitor quinapril to rats with established nephritis decreased MCP-1 gene expression and protein levels and the number of infiltrating cells in the glomerular and interstitial areas. In this normotensive model of renal injury, we have recently demonstrated an increase in ACE activity (27) and an up-regulation and redistribution of angiotensinogen, ACE, and AT1 receptor gene expression in renal tissue (52). In addition, in this article we demonstrate that in cultured glomerular mesangial cells AngII can trigger the expression and synthesis of MCP-1, in a manner similar to that of inflammatory cytokines. Therefore, all these results strongly suggest that local AngII through MCP-1 production by resident renal cells may play a central role in the regulation of monocyte recruitment in renal pathology.
We further investigated the signal transduction pathways in
response to AngII stimulation in cultured mesangial cells. AngII
increases protein tyrosine phosphorylation and activates several
protein kinases, including protein kinase C (32, 53, 54). AngII also
phosphorylates the STAT family of transcription factors (55), and in
vascular smooth muscle cells AngII activates nuclear factors that bind
to AP-1 and NF-B sequences, promoters found in several genes such as
TGF-ß and MCP-1 (56, 57). The intracellular mechanisms leading to
mesangial MCP-1 gene activation in response to different stimuli have
not been fully characterized, although this effect appears to be
transcriptionally regulated (39). In this paper, we note that
preincubation of mesangial cells with PKC inhibitors blocked the MCP-1
gene expression elicited by AngII, suggesting that PKC activation is
involved in this process. One feature of primary response genes,
including MCP-1, is that in the presence of translation blockers, such
as cycloheximide, cytokines cause mRNA superinduction (5), as we have
observed in response to AngII. This effect could be due to an increase
in mRNA stability or, as recently suggested, to enhanced transcription
due to NF-B superinduction induced by cycloheximide treatment
(58).
Deletion analysis of the 5'-flanking region of MCP-1 transfected into
tumor cell lines showed that an NF-B element was required for the
cytokine-mediated reporter gene activity (16). We have observed that
NF-B activation preceded up-regulation of MCP-1 gene expression
yielded by AngII. The rapid rise of MCP-1 mRNA levels induced by AngII
and the kinetics of NF-B activation are consistent with a role for
NF-B in transcriptional activation of MCP-1 gene. Moreover, such
kinetics has been described for MCP-1 in response to cytokines (5), and
activation of NF-B was well correlated with MCP-1 expression in
mesangial cells (45). In this sense, we have found that in cultured
mesangial cells, the blockade of NF-B activation with the
antioxidant PDTC inhibited AngII-induced up-regulation of MCP-1 mRNA
expression, suggesting that this phenomenon may be at least partially
NF-B mediated.
Recently, it has been demonstrated that in murine mesangial cells AngII
increases IL-6 synthesis (59) and that in renal interstitial
fibroblasts it up-regulates angiotensinogen gene expression (22), these
two proteins being regulated by NF-B. We have observed that in
nephritic animals, coinciding with the up-regulation of MCP-1, there
was an increase in NF-B activity in renal cortex that was normalized
in response to ACE inhibition. Our results strongly suggest that in
vivo activation of transcription factors can be important during kidney
damage. Since NF-B regulates the expression of several genes,
including those of chemokines and cytokines, it is possible that AngII
could participate in the regulation of the inflammatory process during
renal injury through the triggering of other inflammatory genes,
besides MCP-1.
Several lines of evidence indicate that reactive oxygen intermediates
serve as messengers in NF-B activation (60). In fact, PDTC decreased
NF-B activation and gene expression of several inflammatory genes
(45, 46). In this work, we have observed that in cultured mesangial
cells reactive oxygen intermediates may be involved in AngII response
since the antioxidant PDTC blocked NF-B activation. Recent data have
shown that oxygen radicals participate in intracellular transduction
signals elicited by AngII in myogenic cells, regulating the induction
of early genes (61), that can lead to cell proliferation or hypertrophy
depending on cell environment. Moreover, hypertension caused by AngII
is in part mediated by free radical generation (62).
Several studies have demonstrated that the vasoactive peptide AngII
elicits cell proliferation and matrix production, two key events in the
progression of renal damage (20, 21, 22). The results reported in this
article suggest a novel mechanism by which AngII could participate in
this process. AngII could be responsible for the recruitment of
mononuclear cells into kidney tissue through the activation of NF-B
and the synthesis of MCP-1. This mechanism might further explain the
beneficial effects of ACE inhibitors in progressive renal diseases.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Jesús Egido, Renal Research Laboratory, Fundación Jiménez Díaz, Avenida Reyes Católicos, 2, 28040 Madrid, Spain. E-mail address:
3 Abbreviations used in this paper: MCP-1, monocyte chemotactic protein-1; AngII, angiotensin II; ACE, angiotensin-converting enzyme; EMSA, electrophoretic mobility assay; PDTC, pyrrolidine dithiocarbamate; PKC, protein kinase C; G3PDH, glyceraldehyde-3'-phosphate dehydrogenase; AP-1, activating protein 1.
Received for publication May 27, 1997. Accepted for publication February 26, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
induce gene production of leukocyte chemotactic factors, colony-simulating factors and interleukin-6 in human mesangial cells. Am. J. Pathol. 138:991.[Abstract]
, tumor necrosis factor-, IgG aggregates and cAMP in mouse mesangial cells. J. Immunol. 150:1971.[Abstract]
regulation of human renal cortical epithelial cell-derived monocyte chemotactic peptide-1. Kidney Int. 44:43.[Medline]
B in mouse glomerular mesangial cells in response to tumor necrosis factor-, immunoglobulin G, and adenosine 3':5'-cyclic monophosphate. J. Clin. Invest. 94:1629.
B and SP1 regulate transcription of the human monocyte chemoattractant protein 1 gene. J. Immunol. 153:2052.[Abstract]
B: structure-function relationship of its protein subunits. Biochem. J. 290:297.
B: a specific inhibitor of the NF kappa B transcription factor. Science 242:540. B- is necessary for activation of the transcription factor NF-B. Nature 365:182.[Medline]
and IL-6, and proliferation. J. Immunol. 153:5247.[Abstract]
B-like transcription factor activation in response to atherogenic diet in mice. J. Clin. Invest. 91:2572.
B: regulation by distinct protein subunits. Biochim. Biophys. Acta 1072:63.[Medline]
, platelet-activating factor and fibronectin in proliferative glomerulonephritis. Clin. Exp. Immunol. 101:334.[Medline]
receptors of human mesangial cells activates transcription factor nuclear factor-B and induces expression and synthesis of monocyte chemoattractant protein-1, IL-8, and IFN-inducible protein 10. J. Immunol. 159:3474.[Abstract]
B signaling pathway participates in dysregulation of vascular smooth muscle cells in vitro and in human atherosclerosis. J. Biol. Chem. 272:15817.B correlates with MCP-1 expression by human mesangial cells. Kidney Int. 48:1263.[Medline]
B activation, MCP-1 expression and macrophage infiltration in a rabbit model of early accelerated atherosclerosis. Circulation 95:1532.B by actinomycin D and cycloheximide in epithelial cells. Biochem. Biophys. Res. Commun. 218:518.[Medline]
B activation: proposed location of antioxidant-inhibitable step. J. Immunol. 153:5008.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  R. A. Irani, Y. Zhang, S. C. Blackwell, C. C. Zhou, S. M. Ramin, R. E. Kellems, and Y. Xia The detrimental role of angiotensin receptor agonistic autoantibodies in intrauterine growth restriction seen in preeclampsia J. Exp. Med., November 23, 2009; 206(12): 2809 - 2822. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Stegbauer, D.-H. Lee, S. Seubert, G. Ellrichmann, A. Manzel, H. Kvakan, D. N. Muller, S. Gaupp, L. C. Rump, R. Gold, et al. Role of the renin-angiotensin system in autoimmune inflammation of the central nervous system PNAS, September 1, 2009; 106(35): 14942 - 14947. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 U. Panzer, O. M. Steinmetz, J.-E. Turner, C. Meyer-Schwesinger, C. von Ruffer, T. N. Meyer, G. Zahner, C. Gomez-Guerrero, R. M. Schmid, U. Helmchen, et al. Resolution of renal inflammation: a new role for NF-{kappa}B1 (p50) in inflammatory kidney diseases Am J Physiol Renal Physiol, August 1, 2009; 297(2): F429 - F439. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Meyer-Schwesinger, S. Dehde, C. von Ruffer, S. Gatzemeier, P. Klug, U. O. Wenzel, R. A. K. Stahl, F. Thaiss, and T. N. Meyer Rho kinase inhibition attenuates LPS-induced renal failure in mice in part by attenuation of NF-{kappa}B p65 signaling Am J Physiol Renal Physiol, May 1, 2009; 296(5): F1088 - F1099. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. H. Lee, J. H. Woo, S. U. Woo, K. S. Kim, S. M. Park, E.-h. Joe, and I. Jou The 15-Deoxy-{Delta}12,14-Prostaglandin J2 Suppresses Monocyte Chemoattractant Protein-1 Expression in IFN-{gamma}-Stimulated Astrocytes through Induction of MAPK Phosphatase-1 J. Immunol., December 15, 2008; 181(12): 8642 - 8649. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Park, D.-R. Ryu, J. J. Li, D.-S. Jung, S.-J. Kwak, S. H. Lee, T.-H. Yoo, S. H. Han, J. E. Lee, D. K. Kim, et al. MCP-1/CCR2 system is involved in high glucose-induced fibronectin and type IV collagen expression in cultured mesangial cells Am J Physiol Renal Physiol, September 1, 2008; 295(3): F749 - F757. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Ootaki, M. Yamashita, Y. Ootaki, K. Kamohara, S. Weber, R. S. Klatte, W. A. Smith, A. L. Massiello, S. N. Emancipator, L. A.R. Golding, et al. Reduced Pulsatility Induces Periarteritis in Kidney: Role of the Local Renin-Angiotensin System J. Thorac. Cardiovasc. Surg., July 1, 2008; 136(1): 150 - 158. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Lorz, A. Benito-Martin, A. Boucherot, A. C. Ucero, M. P. Rastaldi, A. Henger, S. Armelloni, B. Santamaria, C. C. Berthier, M. Kretzler, et al. The Death Ligand TRAIL in Diabetic Nephropathy J. Am. Soc. Nephrol., May 1, 2008; 19(5): 904 - 914. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. B. Sanz, P. Justo, M. D. Sanchez-Nino, L. M. Blanco-Colio, J. A. Winkles, M. Kreztler, A. Jakubowski, J. Blanco, J. Egido, M. Ruiz-Ortega, et al. The Cytokine TWEAK Modulates Renal Tubulointerstitial Inflammation J. Am. Soc. Nephrol., April 1, 2008; 19(4): 695 - 703. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Sanchez-Lopez, J. Rodriguez-Vita, C. Cartier, M. Ruperez, V. Esteban, G. Carvajal, R. Rodrigues-Diez, J. J. Plaza, J. Egido, and M. Ruiz-Ortega Inhibitory effect of interleukin-1 on angiotensin II-induced connective tissue growth factor and type IV collagen production in cultured mesangial cells Am J Physiol Renal Physiol, January 1, 2008; 294(1): F149 - F160. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Socha, M. Manhiani, N. Said, J. D. Imig, and K. Motamed Secreted Protein Acidic and Rich in Cysteine Deficiency Ameliorates Renal Inflammation and Fibrosis in Angiotensin Hypertension Am. J. Pathol., October 1, 2007; 171(4): 1104 - 1112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Xu, O. A. Carretero, C.-X. Lin, M. A. Cavasin, E. G. Shesely, J. J. Yang, T. L. Reudelhuber, and X.-P. Yang Role of cardiac overexpression of ANG II in the regulation of cardiac function and remodeling postmyocardial infarction Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1900 - H1907. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Jurewicz, D. H. McDermott, J. M. Sechler, K. Tinckam, A. Takakura, C. B. Carpenter, E. Milford, and R. Abdi Human T and Natural Killer Cells Possess a Functional Renin-Angiotensin System: Further Mechanisms of Angiotensin II-Induced Inflammation J. Am. Soc. Nephrol., April 1, 2007; 18(4): 1093 - 1102. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. K. Fujihara, G. R. Antunes, A. L. Mattar, D. M. A. C. Malheiros, J. M. Vieira Jr., and R. Zatz Chronic inhibition of nuclear factor-{kappa}B attenuates renal injury in the 5/6 renal ablation model Am J Physiol Renal Physiol, January 1, 2007; 292(1): F92 - F99. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Tsuchiya, T. Yoshimoto, Y. Hirono, T. Tateno, T. Sugiyama, and Y. Hirata Angiotensin II induces monocyte chemoattractant protein-1 expression via a nuclear factor-{kappa}B-dependent pathway in rat preadipocytes Am J Physiol Endocrinol Metab, October 1, 2006; 291(4): E771 - E778. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Tedgui and Z. Mallat Cytokines in Atherosclerosis: Pathogenic and Regulatory Pathways Physiol Rev, April 1, 2006; 86(2): 515 - 581. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. K. Fujihara, C. R. Sena, D. M. A. C. Malheiros, A. L. Mattar, and R. Zatz Short-term nitric oxide inhibition induces progressive nephropathy after regression of initial renal injury Am J Physiol Renal Physiol, March 1, 2006; 290(3): F632 - F640. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. C. Hall and N. Dalbeth Disease modification and cardiovascular risk reduction: two sides of the same coin? Rheumatology, December 1, 2005; 44(12): 1473 - 1482. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y Inokuchi, T Morohashi, I Kawana, Y Nagashima, M Kihara, and S Umemura Amelioration of 2,4,6-trinitrobenzene sulphonic acid induced colitis in angiotensinogen gene knockout mice Gut, March 1, 2005; 54(3): 349 - 356. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Gruden, G. Setti, A. Hayward, D. Sugden, S. Duggan, D. Burt, R. E. Buckingham, L. Gnudi, and G. Viberti Mechanical Stretch Induces Monocyte Chemoattractant Activity via an NF-{kappa}B-Dependent Monocyte Chemoattractant Protein-1-Mediated Pathway in Human Mesangial Cells: Inhibition by Rosiglitazone J. Am. Soc. Nephrol., March 1, 2005; 16(3): 688 - 696. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Dalbeth, J. Edwards, S. Fairchild, M. Callan, and F. C. Hall The non-thiol angiotensin-converting enzyme inhibitor quinapril suppresses inflammatory arthritis Rheumatology, January 1, 2005; 44(1): 24 - 31. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. N. A. Nabah, T. Mateo, R. Estelles, M. Mata, J. Zagorski, H. Sarau, J. Cortijo, E. J. Morcillo, P. J. Jose, and M.-J. Sanz Angiotensin II Induces Neutrophil Accumulation In Vivo Through Generation and Release of CXC Chemokines Circulation, December 7, 2004; 110(23): 3581 - 3586. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Sironi, P. Gelosa, U. Guerrini, C. Banfi, V. Crippa, M. Brioschi, E. Gianazza, E. Nobili, A. Gianella, M. de Gasparo, et al. Anti-Inflammatory Effects of AT1 Receptor Blockade Provide End-Organ Protection in Stroke-Prone Rats Independently from Blood Pressure Fall J. Pharmacol. Exp. Ther., December 1, 2004; 311(3): 989 - 995. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Benigni, D. Corna, C. Zoja, L. Longaretti, E. Gagliardini, N. Perico, T. M. Coffman, and G. Remuzzi Targeted Deletion of Angiotensin II Type 1A Receptor Does not Protect Mice from Progressive Nephropathy of Overload Proteinuria J. Am. Soc. Nephrol., October 1, 2004; 15(10): 2666 - 2674. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Sekiguchi, X. Li, M. Coker, M. Flesch, P. M Barger, N. Sivasubramanian, and D. L Mann Cross-regulation between the renin-angiotensin system and inflammatory mediators in cardiac hypertrophy and failure Cardiovasc Res, August 15, 2004; 63(3): 433 - 442. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. T.H. Lee, Z. Cao, D. M. Long, S. Panagiotopoulos, G. Jerums, M. E. Cooper, and J. M. Forbes Interactions between Angiotensin II and NF-{kappa}B-Dependent Pathways in Modulating Macrophage Infiltration in Experimental Diabetic Nephropathy J. Am. Soc. Nephrol., August 1, 2004; 15(8): 2139 - 2151. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Alvarez, M. Cerda-Nicolas, Y. Naim Abu Nabah, M. Mata, A. C. Issekutz, J. Panes, R. R. Lobb, and M.-J. Sanz Direct evidence of leukocyte adhesion in arterioles by angiotensin II Blood, July 15, 2004; 104(2): 402 - 408. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Esteban, O. Lorenzo, M. Ruperez, Y. Suzuki, S. Mezzano, J. Blanco, M. Kretzler, T. Sugaya, J. Egido, and M. Ruiz-Ortega Angiotensin II, via AT1 and AT2 Receptors and NF-{kappa}B Pathway, Regulates the Inflammatory Response in Unilateral Ureteral Obstruction J. Am. Soc. Nephrol., June 1, 2004; 15(6): 1514 - 1529. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. R. R. Goncalves, C. K. Fujihara, A. L. Mattar, D. M. A. C. Malheiros, I. L. Noronha, G. de Nucci, and R. Zatz Renal expression of COX-2, ANG II, and AT1 receptor in remnant kidney: strong renoprotection by therapy with losartan and a nonsteroidal anti-inflammatory Am J Physiol Renal Physiol, May 1, 2004; 286(5): F945 - F954. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Toko, Y. Zou, T. Minamino, M. Sakamoto, M. Sano, M. Harada, T. Nagai, T. Sugaya, F. Terasaki, Y. Kitaura, et al. Angiotensin II Type 1a Receptor Is Involved in Cell Infiltration, Cytokine Production, and Neovascularization in Infarcted Myocardium Arterioscler Thromb Vasc Biol, April 1, 2004; 24(4): 664 - 670. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Wu, M. Iwai, Z. Li, T. Shiuchi, L.-J. Min, T.-X. Cui, J.-M. Li, M. Okumura, C. Nahmias, and M. Horiuchi Regulation of Inhibitory Protein-{kappa}B and Monocyte Chemoattractant Protein-1 by Angiotensin II Type 2 Receptor-Activated Src Homology Protein Tyrosine Phosphatase-1 in Fetal Vascular Smooth Muscle Cells Mol. Endocrinol., March 1, 2004; 18(3): 666 - 678. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Ni, S. Kitamoto, M. Ishibashi, M. Usui, S. Inoue, K.-i. Hiasa, Q. Zhao, K.-i. Nishida, A. Takeshita, and K. Egashira Monocyte Chemoattractant Protein-1 Is an Essential Inflammatory Mediator in Angiotensin II-Induced Progression of Established Atherosclerosis in Hypercholesterolemic Mice Arterioscler Thromb Vasc Biol, March 1, 2004; 24(3): 534 - 539. [Abstract] [Full Text]
|
|
|
|
|
|
E. A. McPherson, Z. Luo, R. A. Brown, L. S. LeBard, C. C. Corless, R. C. Speth, and S. P. Bagby Chymase-like Angiotensin II-Generating Activity in End-Stage Human Autosomal Dominant Polycystic Kidney Disease J. Am. Soc. Nephrol., February 1, 2004; 15(2): 493 - 500. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Suzuki, T. Yamamoto, N. Ikegaya, and A. Hishida Dietary salt intake modulates progression of antithymocyte serum nephritis through alteration of glomerular angiotensin II receptor expression Am J Physiol Renal Physiol, February 1, 2004; 286(2): F267 - F277. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Perez de Lema, C. de Wit, C. D. Cohen, E. Nieto, A. Molina, B. Banas, B. Luckow, A. B. Vicente, F. Mampaso, and D. Schlondorff Angiotensin Inhibition Reduces Glomerular Damage and Renal Chemokine Expression in MRL/lpr Mice J. Pharmacol. Exp. Ther., October 1, 2003; 307(1): 275 - 281. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Suzuki, M. Ruiz-Ortega, C. Gomez-Guerrero, Y. Tomino, and J. Egido Angiotensin II, the immune system and renal diseases: another road for RAS? Nephrol. Dial. Transplant., August 1, 2003; 18(8): 1423 - 1426. [Full Text] [PDF]
|
|
|
|
|
|
Y. Suzuki, M. Ruiz-Ortega, C. Gomez-Guerrero, Y. Tomino, and J. Egido Angiotensin II, the immune system and renal diseases: another road for RAS? Nephrol. Dial. Transplant., August 1, 2003; 18(88): 1423 - 1426. [Full Text]
|
|
|
|
 |
|
 B. Amann, R. Tinzmann, and B. Angelkort ACE Inhibitors Improve Diabetic Nephropathy Through Suppression of Renal MCP-1 Diabetes Care, August 1, 2003; 26(8): 2421 - 2425. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Wada, H. Yokoyama, K. Matsushima, and K.-i. Kobayashi Monocyte chemoattractant protein-1: does it play a role in diabetic nephropathy? Nephrol. Dial. Transplant., March 1, 2003; 18(3): 457 - 459. [Full Text] [PDF]
|
|
|
|
|
|
S.-L. Lin, Y.-M. Chen, C.-T. Chien, W.-C. Chiang, C.-C. Tsai, and T.-J. Tsai Pentoxifylline Attenuated the Renal Disease Progression in Rats with Remnant Kidney J. Am. Soc. Nephrol., December 1, 2002; 13(12): 2916 - 2929. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Zatz, I. L. Noronha, and C. K. Fujihara Experimental and clinical rationale for use of MMF in nontransplant progressive nephropathies Am J Physiol Renal Physiol, December 1, 2002; 283(6): F1167 - F1175. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Chen, Y. Wei, G. C. Sharp, and H. Braley-Mullen Inhibition of TGF{beta}1 by Anti-TGF{beta}1 Antibody or Lisinopril Reduces Thyroid Fibrosis in Granulomatous Experimental Autoimmune Thyroiditis J. Immunol., December 1, 2002; 169(11): 6530 - 6538. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Suzuki, C. Gomez-Guerrero, I. Shirato, O. Lopez-Franco, P. Hernandez-Vargas, G. Sanjuan, M. Ruiz-Ortega, T. Sugaya, K. Okumura, Y. Tomino, et al. Susceptibility to T Cell-Mediated Injury in Immune Complex Disease Is Linked to Local Activation of Renin-Angiotensin System: The Role of NF-AT Pathway J. Immunol., October 15, 2002; 169(8): 4136 - 4146. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Viedt, R. Dechend, J. Fei, G. M. Hansch, J. Kreuzer, and S. R. Orth MCP-1 Induces Inflammatory Activation of Human Tubular Epithelial Cells: Involvement of the Transcription Factors, Nuclear Factor-{kappa}B and Activating Protein-1 J. Am. Soc. Nephrol., June 1, 2002; 13(6): 1534 - 1547. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Lorenzo, M. Ruiz-Ortega, P. Esbrit, M. Ruperez, A. Ortega, S. Santos, J. Blanco, L. Ortega, and J. Egido Angiotensin II Increases Parathyroid Hormone-Related Protein (PTHrP) and the Type 1 PTH/PTHrP Receptor in the Kidney J. Am. Soc. Nephrol., June 1, 2002; 13(6): 1595 - 1607. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
O. Lorenzo, M. Ruiz-Ortega, Y. Suzuki, M. Ruperez, V. Esteban, T. Sugaya, and J. Egido Angiotensin III Activates Nuclear Transcription Factor-{kappa}B in Cultured Mesangial Cells Mainly via AT2 Receptors: Studies with AT1 Receptor-Knockout Mice J. Am. Soc. Nephrol., May 1, 2002; 13(5): 1162 - 1171. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Smit-van Oosten, R. H Henning, and H. van Goor Strain differences in angiotensin-converting enzyme and angiotensin II type I receptor expression. Possible implications for experimental chronic renal transplant failure Journal of Renin-Angiotensin-Aldosterone System, March 1, 2002; 3(1): 46 - 53. [Abstract] [PDF]
|
|
|
|
 |
|
 J.-K. Park, A. Fiebeler, D. N. Muller, E. M.A. Mervaala, R. Dechend, F. Abou-Rebyeh, F. C. Luft, and H. Haller Lacidipine Inhibits Adhesion Molecule and Oxidase Expression Independent of Blood Pressure Reduction in Angiotensin-Induced Vascular Injury Hypertension, February 1, 2002; 39(2): 685 - 689. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ruiz-Ortega, O. Lorenzo, M. Ruperez, V. Esteban, Y. Suzuki, S. Mezzano, J.J. Plaza, and J. Egido Role of the Renin-Angiotensin System in Vascular Diseases: Expanding the Field Hypertension, December 1, 2001; 38(6): 1382 - 1387. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 Y. GORIN, N.-H. KIM, D. FELIERS, B. BHANDARI, G. G. CHOUDHURY, and H. E. ABBOUD Angiotensin II activates Akt/protein kinase B by an arachidonic acid/redox-dependent pathway and independent of phosphoinositide 3-kinase FASEB J, September 1, 2001; 15(11): 1909 - 1920. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. A. Mezzano, M. Ruiz-Ortega, and J. Egido Angiotensin II and Renal Fibrosis Hypertension, September 1, 2001; 38(3): 635 - 638. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Alvarez and M.-J. Sanz Reactive oxygen species mediate angiotensin II-induced leukocyte-endothelial cell interactions in vivo J. Leukoc. Biol., August 1, 2001; 70(2): 199 - 206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Nakayama, A. Furusu, Q. Xu, T. Konta, and M. Kitamura Unexpected Transcriptional Induction of Monocyte Chemoattractant Protein 1 by Proteasome Inhibition: Involvement of the c-Jun N-Terminal Kinase-Activator Protein 1 Pathway J. Immunol., August 1, 2001; 167(3): 1145 - 1150. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. P. Lewington, M. Arici, K. P. G. Harris, N. J. Brunskill, and J. Walls Modulation of the renin-angiotensin system in proteinuric renal disease: are there added benefits? Nephrol. Dial. Transplant., May 1, 2001; 16(5): 885 - 888. [Full Text] [PDF]
|
|
|
|
|
|
A. Zernecke, K. S. C. Weber, L. P. Erwig, D. C. Kluth, B. Schroppel, A. J. Rees, and C. Weber Combinatorial Model of Chemokine Involvement in Glomerular Monocyte Recruitment: Role of CXC Chemokine Receptor 2 in Infiltration During Nephrotoxic Nephritis J. Immunol., May 1, 2001; 166(9): 5755 - 5762. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ruiz-Ortega, O. Lorenzo, M. Ruperez, J. Blanco, and J. Egido Systemic Infusion of Angiotensin II into Normal Rats Activates Nuclear Factor-{{kappa}}B and AP-1 in the Kidney : Role of AT1 and AT2 Receptors Am. J. Pathol., May 1, 2001; 158(5): 1743 - 1756. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Gomez-Garre, R. Largo, N. Tejera, J. Fortes, F. Manzarbeitia, and J. Egido Activation of NF-{{kappa}}B in Tubular Epithelial Cells of Rats With Intense Proteinuria : Role of Angiotensin II and Endothelin-1 Hypertension, April 1, 2001; 37(4): 1171 - 1178. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. C. Paul Glomerular hypertension--an under-appreciated aspect of chronic rejection Nephrol. Dial. Transplant., February 1, 2001; 16(2): 213 - 215. [Full Text] [PDF]
|
|
|
|
|
|
M. SATOH, N. KASHIHARA, Y. YAMASAKI, K. MARUYAMA, K. OKAMOTO, Y. MAESHIMA, H. SUGIYAMA, T. SUGAYA, K. MURAKAMI, and H. MAKINO Renal Interstitial Fibrosis Is Reduced in Angiotensin II Type 1a Receptor-Deficient Mice J. Am. Soc. Nephrol., February 1, 2001; 12(2): 317 - 325. [Abstract] [Full Text]
|
|
|
|
|
|
F. C. Luft Workshop: Mechanisms and Cardiovascular Damage in Hypertension Hypertension, February 1, 2001; 37(2): 594 - 598. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Schwobel, T. Fischer, B. Lanz, and M. Mohaupt Angiotensin II receptor subtypes determine induced NO production in rat glomerular mesangial cells Am J Physiol Renal Physiol, December 1, 2000; 279(6): F1092 - F1100. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Piqueras, P. Kubes, A. Alvarez, E. O'Connor, A. C. Issekutz, J. V. Esplugues, and M.-J. Sanz Angiotensin II Induces Leukocyte-Endothelial Cell Interactions In Vivo Via AT1 and AT2 Receptor-Mediated P-Selectin Upregulation Circulation, October 24, 2000; 102(17): 2118 - 2123. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. M. Behr, X. Wang, N. Aiyar, R. W. Coatney, X. Li, P. Koster, C. E. Angermann, E. Ohlstein, G. Z. Feuerstein, and J. Winaver Monocyte Chemoattractant Protein-1 is Upregulated in Rats With Volume-Overload Congestive Heart Failure Circulation, September 12, 2000; 102(11): 1315 - 1322. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. N. Muller, E. M. A. Mervaala, F. Schmidt, J.-K. Park, R. Dechend, E. Genersch, V. Breu, B.-M. Loffler, D. Ganten, W. Schneider, et al. Effect of Bosentan on NF-{kappa}B, Inflammation, and Tissue Factor in Angiotensin II-Induced End-Organ Damage Hypertension, August 1, 2000; 36(2): 282 - 290. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ruiz-Ortega, O. Lorenzo, M. Ruperez, S. Konig, B. Wittig, and J. Egido Angiotensin II Activates Nuclear Transcription Factor {kappa}B Through AT1 and AT2 in Vascular Smooth Muscle Cells : Molecular Mechanisms Circ. Res., June 23, 2000; 86(12): 1266 - 1272. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ruiz-Ortega, O. Lorenzo, M. Ruperez, and J. Egido ACE inhibitors and AT1 receptor antagonists--beyond the haemodynamic effect Nephrol. Dial. Transplant., May 1, 2000; 15(5): 561 - 565. [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kim and H. Iwao Molecular and Cellular Mechanisms of Angiotensin II-Mediated Cardiovascular and Renal Diseases Pharmacol. Rev., March 1, 2000; 52(1): 11 - 34. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Gomez-Guerrero, N. Duque, M. T. Casado, C. Pastor, J. Blanco, F. Mampaso, F. Vivanco, and J. Egido Administration of IgG Fc Fragments Prevents Glomerular Injury in Experimental Immune Complex Nephritis J. Immunol., February 15, 2000; 164(4): 2092 - 2101. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. SEGERER, P. J. NELSON, and D. SCHLÖNDORFF Chemokines, Chemokine Receptors, and Renal Disease: From Basic ScienceTo Pathophysiologic and Therapeutic Studies J. Am. Soc. Nephrol., January 1, 2000; 11(1): 152 - 176. [Abstract] [Full Text]
|
|
|
|
|
|
D. N. Muller, R. Dechend, E. M. A. Mervaala, J.-K. Park, F. Schmidt, A. Fiebeler, J. Theuer, V. Breu, D. Ganten, H. Haller, et al. NF-{kappa}B Inhibition Ameliorates Angiotensin II-Induced Inflammatory Damage in Rats Hypertension, January 1, 2000; 35(1): 193 - 201. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. E. Donovan, D. A. Mark, H. Z. He, H.-C. Liou, L. Kobzik, Y. Wang, G. T. De Sanctis, D. L. Perkins, and P. W. Finn NF-{kappa}B/Rel Transcription Factors: c-Rel Promotes Airway Hyperresponsiveness and Allergic Pulmonary Inflammation J. Immunol., December 15, 1999; 163(12): 6827 - 6833. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. WANG, G. K. RANGAN, Y.-C. TAY, Y. WANG, and D. C. H. HARRIS Induction of Monocyte Chemoattractant Protein-1 by Albumin Is Mediated by Nuclear Factor {kappa}B in Proximal Tubule Cells J. Am. Soc. Nephrol., June 1, 1999; 10(6): 1204 - 1213. [Abstract] [Full Text]
|
|
|
|
|
|
R. Largo, D. Gomez-Garre, K. Soto, B. Marron, J. Blanco, R. M. Gazapo, J. J. Plaza, and J. Egido Angiotensin-Converting Enzyme Is Upregulated in the Proximal Tubules of Rats With Intense Proteinuria Hypertension, February 1, 1999; 33(2): 732 - 739. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Kalra, N. Sivasubramanian, and D. L. Mann Angiotensin II Induces Tumor Necrosis Factor Biosynthesis in the Adult Mammalian Heart Through a Protein Kinase C-Dependent Pathway Circulation, May 7, 2002; 105(18): 2198 - 2205. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
