Complement Activation in Angiotensin II–Induced
Organ Damage
Erdenechimeg Shagdarsuren,* Maren Wellner,* Jan-Hinrich Braesen, Joon-Keun Park, Anette Fiebeler,
Norbert Henke, Ralf Dechend, Petra Gratze, Friedrich C. Luft, Dominik N. Muller
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Abstract—We tested whether or not complement activation participates in angiotensin (Ang) II–induced vasculopathy. We
used double transgenic rats harboring human renin and angiotensinogen genes (dTGR) with or without losartan or the
human renin inhibitor aliskiren. Sprague–Dawley (SD) rats were controls. DTGR had increased blood pressure at week
5 that increased further by week 7. Albuminuria was absent at week 5 but increased markedly in weeks 6 and 7.
C-reactive protein (CRP) elevation, macrophages, T cells, tumor necrosis factor (TNF)-␣, C1q, C3, C3c, and C5b-9
expression preceded albuminuria. C1q, C3, C3c, and C5b-9 were observed in the dTGR vessel media. C5b-9 colocalized
with interleukin (IL)-6. Losartan and aliskiren reduced albuminuria and complement expression. We also studied
vascular smooth muscle cells (VSMC) from dTGR compared VSMC from SD. C3 and IL-6 mRNA were analyzed after
Ang II, TNF-␣, and CRP stimulation. VSMC from dTGR showed increased proliferation and C3 expression compared
with SD. Ang II did not induce C3 mRNA in either VSMC type. However, TNF-␣ and CRP induced C3 mRNA slightly
in SD VSMC but markedly in dTGR VSMC, whereas IL-6 induction was similar in both. Thus, complement activation
and cell infiltration occurred before the onset of albuminuria in Ang II–mediated renal damage. TNF-␣ and CRP played
a major role in C3 activation. VSMC from dTGR are more sensitive for C3 activation. Our data show that, in this Ang
II–induced model, complement activation is a major participant and suggest that TNF-␣ and CRP may play a role in
its induction. (Circ Res. 2005;97:716-724.)
Key Words: angiotensin II 䡲 complement 䡲 immune system 䡲 albuminuria and renal damage
T
he innate complement system eliminates invading pathogens, stimulates opsonization, enhances phagocytosis,
cytolysis, chemotaxis, and solubilizes immune complexes.
Complement forms a bridge between innate and acquired
immunity.1,2 On excessive activation or inappropriate deposition, complement can cause disease.3 The classical alternative and lectin complement pathway merge at the level of C3,
resulting in the generation of C5b-9, the membrane attack
complex. Complement activation has been implicated in the
pathogenesis of numerous proteinuric renal diseases including glomerulonephritis, transplant rejection, and ischemiareperfusion injury.3–7 Pratt et al demonstrated that the absence
of local C3 production modulates renal graft survival and
regulates T-cell priming of donor antigens.4 Very recently,
Lin et al reported that young spontaneous hypertensive rats
(SHR) that have not yet developed hypertension showed
increased C3 expression and increased vascular smooth
muscle cell (VSMC) proliferation. Both were blocked by C3
downregulation.8 Several studies showed that angiotensin
(Ang) II not only is a vasoconstrictor peptide but also
promotes inflammation and renal damage. We showed recently that immunosuppression improved nonimmune Ang
II–mediated renal damage.9 The evidence that Ang II affects
the complement system is indirect. Abbate et al demonstrated
that angiotensin-converting enzyme (ACE) inhibition reduced cell infiltration and C3 immunoreactivity in rats with 5
of 6 nephrectomy.10 To investigate complement in an Ang
II–induced model, we first tested whether or not complement
is activated. Second, we investigated whether or not inhibition of the renin–angiotensin system by 2 independent mechanisms would affect complement activation. Third, we asked
whether or not complement activation occurs before or
subsequent to renal damage. Fourth, we conducted in vitro
signal-transduction experiments to elucidate participants in
complement activation.
Materials and Methods
Experimental Design
We studied male transgenic rats harboring human renin and angiotensinogen genes (dTGR) (RCC Ltd; Basel, Switzerland) and age-
Original received April 26, 2005; revision received July 7, 2005; accepted August 10, 2005.
From the Medical Faculty of the Charité (E.S., J.-H.B., A.F., N.H., R.D., F.C.L., D.N.M.), Franz Volhard Clinic, and HELIOS Klinikum-Berlin; Max
Delbrück Center for Molecular Medicine (M.W., P.G., F.C.L., D.N.M.), Berlin-Buch, Germany; and Medical School of Hannover (J.-K.P.), Hannover,
Germany.
*Both authors contributed equally to this work.
Correspondence to Dominik N. Muller, Max Delbrück Center and Franz Volhard Clinic, Wiltberg Strasse 50, 13125 Berlin, Germany. E-mail
[email protected]
© 2005 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/01.RES.0000182677.89816.38
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Shagdarsuren et al
matched nontransgenic SD rats (Tierzucht Schönwalde, Germany).9,11 American Physiological Society guidelines for animal care
were followed (permit no. G 237/03). Untreated dTGR (n⫽10 week
5 and 6; n⫽18 week 7) and SD (n⫽8 each group) rats were killed at
age 5, 6, and 7 weeks. Losartan (LOS)-treated and the human renin
inhibitor aliskiren (ALISK)-treated dTGR were killed at week 7.
Four-week-old dTGR were treated with LOS (10 mg/kg in the diet,
n⫽15) or ALISK (3 mg/kg per day SC by minipump infusion, n⫽11)
for 3 weeks. Systolic blood pressure was measured weekly by
tailcuff. Twenty-four-hour urine samples were collected in metabolic
cages from weeks 5 to 7. Serum and plasma were collected at week
7. Urinary rat albumin and serum rat C-reactive protein (CRP) were
determined by ELISA (CellTrend, Germany, and BD Pharmingen,
Germany, respectively). Blood urea nitrogen and creatinine were
determined by an automated clinical method.
Immunohistochemistry, Confocal Microscopy,
and Antibodies
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Ice-cold acetone-fixed cryosections (6 ␮m) were stained by immunofluorescence or alkaline phosphatase–anti-alkaline phosphatase
techniques as described earlier.9,11,12 The sections were incubated
with the following monoclonal antibodies: anti–ED-1 and anti-CD8
(Clone Ox-8) (both from Serotec, Düsseldorf, Germany); anti-CD4
(Clone Ox-38), anti-CD86, anti-Ox62, and anti–major histocompatibility complex (MHC) II (Clone Ox-6) (all BD Pharmingen); and
anti–C5b-9 (2A1) (kindly provided by Dr W. Couser, University of
Washington, Seattle). The following polyclonal antibodies were
used: rabbit anti-rat IgG (DAKO, Hamburg, Germany); antifibronectin (Paesel, Frankfurt, Germany); anti– collagen IV (Southern Biotechnology, Birmingham, Ala); anti-C1q (A 0138; DAKO),
fluorescein isothiocyanate– conjugated anti-C3 (ICN, Eschwege, Germany); fluorescein isothiocyanate–conjugated rabbit anti-C3c complement (Roche Diagnostics, Manheim, Germany); and goat anti–
tumor necrosis factor (TNF)-␣ (Santa Cruz Biotechnologies,
Heidelberg, Germany). Confocal microscopy was performed as
described earlier.13 VSMC were stained with anti–smooth muscle
(SM) ␣-actin (DAKO) and anti-vinculin (BioTrend, Germany) and
analyzed by 2 different investigators (E.S. and J.-K.P.) without
knowledge of the origin of the specimens.
Quantification of Infiltrated Cells and
Complement Expression
Sections were analyzed with a Zeiss Axioplan-2 microscope (Carl
Zeiss) and AxioVision 2 multichannel image processing system
(Carl Zeiss). Semiquantitative scoring of infiltrated (ED-1⫹, CD4⫹,
CD8⫹, Ox62⫹, Ox6⫹, CD86⫹) cells, in 15 different cortical kidney
areas (n⫽6 per group), was performed using computerized cell count
program (KS 300 3.0; Carl Zeiss) with samples examined blind. For
quantification of perivascular macrophage infiltration, all selected
view fields included a small vessel in their analysis. Quantification
of CD4⫹ T cells, MHC II⫹, and CD86⫹ cells was performed
periglomerular, whereas CD8⫹ cells were quantified interstitially.
The number of IgG-positive glomeruli was counted in 50 randomly
selected glomeruli in different cortical areas. For quantitative analysis of complement components C1q, C3, C3c, and C5b-9, we
counted the total number of positive-stained vessels and glomeruli
and positive-stained group of tubules per whole-kidney cross-section
based on the staining intensity.
Cell-Culture Experiments
Aortic VSMC were isolated from dTGR and SD rats as described
previously.13 VSMC from passages 4 and 8 were treated with Ang II
(10 to 7 mol/L; Sigma), TNF-␣ (10 ng/mL; Calbiochem), or human
CRP (50 ␮g/mL; Calbiochem) for 24 hours. Rat tubular epithelial
NRK-52E cells (NRK) were treated with Ang II and TNF-␣. To
analyze whether C3 was regulated in a nuclear factor ␬B (NF-␬B)–
dependent manner, we treated dTGR-VSMC in the absence and
presence of the I-␬B phosphorylation inhibitor BAY 11 to 7085
(20 ␮mol/L, Calbiochem) and I-␬B kinase (IKK)-2 inhibitor sc 514
(100 ␮mol/L; Calbiochem) with TNF-␣ for 3 hours. Cell-
Angiotensin II and Complement
717
proliferation experiments were performed after a method described
by Crouch et al.14 The proliferation was determined after 3 days and
calculated as fold-induction per baseline.
Quantitative TaqMan RT-PCR
RNA isolation and TaqMan RT-PCR were performed as described
previously.15 Each sample was in triplicate. For quantification, the
target sequences were normalized in relation to the 18S product.
Biotez (Berlin, Germany) synthesized the primers. The sequences are
available on request.
Electrophoretic Mobility Shift Assay
VSMC from dTGR and SD were stimulated with TNF-␣ (10 ng/mL)
for 15 minutes. Electrophoretic mobility shift assay was performed
as described previously.16 Total cell extracts (5 ␮g) were incubated
with oligonucleotides, containing the NF-␬B– binding site from the
MHC enhancer (H2K, 5⬘-gatcCAGGGCTGGGGATTCCCCATCTCCACAGG). In competition assays, 50 ng of unlabeled H2K
oligonucleotides were used. For supershift assay, 1 ␮g of anti-p50 or
anti-p65 (both from Santa Cruz Biotechnologies) were added for 20
minutes to the homogenates before addition of the labeled probe. The
shifts (n⫽4) were quantitated with the NIH image program.
Ancillary Experiments
To support our observations, we conducted ancillary experiments
with the methodology described above or reanalyzed specimens from
earlier studies. These protocols and results are given in the online
data supplement available at http://circres.ahajournals.org.
Statistics
Data are presented as means⫾SEM. Statistically significant differences in mean values were tested by ANOVA and blood pressure and
albuminuria by repeated-measures ANOVA and the Scheffé test. A
value of P⬍0.05 was considered statistically significant. The data
were analyzed using Statview statistical software.
Results
Blood Pressure and Renal Damage
Untreated 5-week-old dTGR showed moderately elevated
systolic blood pressure compared with age-matched SD rats
(138⫾4 versus 91⫾5 mm Hg), which progressively increased
over time to week 7 (204⫾5 mm Hg). LOS treatment reduced
blood pressure (141⫾6 mm Hg) partially, whereas ALISK
normalized blood pressure (111⫾2 mm Hg) to SD values
(119⫾6; Figure 1A). In contrast, albuminuria, serum creatinine, and blood urea nitrogen were not different at week 5
between dTGR and SD. Increased albuminuria was first
observed at week 6 and reached a 150-fold increase at week
7 (Figure 1B). LOS and ALISK treatment reduced albuminuria by 85% and 99%, respectively. SD rats showed no
albuminuria. Blood urea nitrogen levels were 13⫾0.5 versus
12⫾0.4 at week 5 and 17⫾1 versus 13⫾1 mg/dL at week 6
but significantly elevated at week 7 (65⫾15 versus 13⫾0.4
mg/dL dTGR versus SD, respectively). LOS- and ALISKtreated dTGR showed normal levels at week 7 (16⫾0.4 and
16⫾0.7 mg/dL, respectively). Serum creatinine was not
changed at weeks 5 and 6 but significantly increased at week
7 (Figure 1C). Both LOS and ALISK reduced serum creatinine significantly (Figure 1C). Renal fibrosis (fibronectin and
collagen IV) was first detected at week 6 and markedly
increased at week 7 in untreated dTGR and was abolished by
LOS and ALISK treatment (data not shown).
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Figure 1. A, Systolic blood pressure, albuminuria, serum creatinine, and CRP are shown in 5-, 6-, 7-week-old untreated dTGR and
LOS- and ALISK-treated dTGR and age-matched SD control rats. Blood pressure increased by week 5 in dTGR and further increased
up to week 7. LOS treatment partially reduced blood pressure, whereas ALISK normalized it. B, Albuminuria was not altered at week 5
among the groups. At week 6, dTGR showed a significant increase in albuminuria and reached 150-fold increase at week 7. Control SD
rats showed no albuminuria. LOS and ALISK treatment normalized it. C, Serum creatinine in dTGR was not changed at weeks 5 and 6
but elevated at week 7 compared with SD. LOS and ALISK reduced it to control level. D, Serum CRP in dTGR was increased ⬇10-fold
at week 5 compared with SD rats. LOS and ALISK reduced it to SD level. Results are mean⫾SEM. *P⬍0.05 vs dTGR, #P⬍0.05 vs
other groups.
DTGR Show Increased Serum C-Reactive Protein,
Renal TNF-␣, and Cell Infiltration
Serum CRP in dTGR was increased 10-fold at week 5
compared with SD (51⫾5 versus 4⫾1 mg/mL); CRP reached
66.0⫾5.7 mg/mL at week 7. LOS and ALISK treatment
reduced CRP by 90% and 88% to SD levels (Figure 1D). We
next analyzed the proinflammatory cytokine TNF-␣ in the
kidney. We detected elevated immunoreactivity of TNF-␣ in
untreated dTGR at week 5 that progressively increased over
time. TNF-␣ was detected in glomeruli, in the media of vessel
walls, and in tubular epithelial cells (brush borders). Agematched SD showed no staining. TNF-␣ was partially reduced in LOS-treated dTGR and completely reduced in
ALISK-treated dTGR (Figure 2). Because TNF-␣ promotes
inflammation, we next focused on renal-cell infiltration.
Monocytes/macrophages (ED-1⫹ cells) infiltrated predominantly around the damaged vessels; CD4 T-helper, CD86⫹,
and MHC II⫹ (OX-62⫹) cells showed a perivascular and
interstitial location; cytotoxic CD8 T cells and dendritic cells
were at interstitial, periglomerular, and glomerular locations.
Semiquantification revealed that all cell types, except dendritic cells, were increased in untreated dTGR already at
week 5, before the onset of albuminuria. LOS and ALISK
treatment prevented the infiltration of all cell types (Figure 3),
with 1 exception. CD4⫹ T cells were only partially reduced
by LOS.
Renal Expression of Complement Components
Precedes the Onset of Albuminuria
In contrast to cell infiltration, the role of Ang II on complement components C1q, C3, C3c, and the membrane attack
complex C5b-9 is not well analyzed. Figure 4A shows
increased C1q immunostaining in the media of 5-week-old
dTGR compared with age-matched SD. There were only a
few C1q-positive glomeruli at week 7 by dTGR (data not
shown). Age-matched SD control and LOS- and ALISK-
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SD. LOS and ALISK both reduced renal C5b-9 immunoreactivity (Figure 5A and 5B). C5b-9 is able to induce interleukin (IL)-6. We, thus, performed costainings. The media of
small untreated dTGR vessels showed expression of both
C5b-9 and IL-6 (Figure 5C). However, IL-6 was also expressed in the neointima of damaged dTGR vessels.
VSMC From dTGR Are More Sensitive for
C3 Activation
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VSMC isolated from dTGR showed increased cell proliferation, decreased SM ␣-actin and vinculin immunostaining, and
increased C3 mRNA expression compared with SD VSMC,
indicating a switch from the contractile to a synthetic phenotype (Figure 6A). Ang II treatment did not induce C3 mRNA
in either SD or dTGR VSMC (Figure 6B). TNF-␣ (Figure
6B) and CRP (Figure 6C) induced C3 mRNA slightly above
baseline in the SD VSMC but massively in the dTGR VSMC.
In contrast, IL-6 was induced to a similar extent in both
VSMC types (Figure 6B and 6C). We also investigated
whether inhibition of the IKK–NF-␬B pathway would prevent TNF-␣–induced C3 activation. The I␬B␣ phosphorylation inhibitor BAY 11-8702 and the IKK-2 inhibitor sc 514
inhibited TNF-␣–mediated C3 induction in dTGR VSMC
(Figure 6D). Finally, we found that TNF-␣ induced NF-␬B
DNA-binding activity to a greater extent in VSMC from
dTGR compared with SD (Figure 6E).
We next analyzed the role of Ang II and TNF-␣ on C3 in
renal tubular epithelial cells (NRK). We found only C3 and
IL-6 induction after TNF-␣ (1144⫾215 and 3.1⫾0.4 arbitrary units for C3 and IL-6, respectively), but not after Ang II
(7⫾1 and 2.2⫾0.2 arbitrary units for C3 and IL-6, respectively), compared with unstimulated cells (10⫾2 and 1.7⫾0.3
arbitrary units for C3 and IL-6, respectively).
Figure 2. Renal TNF-␣ expression. DTGR kidneys show
increased TNF-␣ immunoreactivity (red) in glomeruli, in the
media of vessel wall, and in tubular epithelial cells dominantly
on tubular brush borders. TNF-␣ immunostaining was detected
already at week 5 in dTGR and increased over time. SD showed
no TNF-␣ staining. LOS and ALISK treatment reduced it. The
autofluorescence (green) was used to demonstrate the kidney
structure.
treated rats showed no staining for C1q. Semiquantification
of C1q demonstrated that the number of C1q-positive vessels
in dTGR increased over time and was higher compared with
SD at all time points. Already at week 5, dTGR showed C3
immunoreactivity in tubular epithelial cells, in the media of
small vessels, and granular staining in glomeruli (Figure 4B).
The intensity and the accumulation of C3 staining increased
over time. We also analyzed the active cleavage product C3c
(Figure 4C). Renal C3c expression was observed in the media
of small vessels in 5-week-old dTGR, whereas C3c expression on the brush borders of tubular epithelial cells was first
detected at week 6. Semiquantification confirmed a higher
number of C3 and C3c staining in dTGR compared with other
groups (Figure 4D). The immunostaining for the membrane
attack complex C5b-9 resembled the pattern of C3. The
number of C5b-9 –positive vessels, tubules, and glomeruli in
untreated dTGR was significantly increased compared with
Discussion
We provide the first evidence that complement components
are activated in a model of Ang II–induced renal damage.
There were 4 major findings. First, complement C1q, C3,
C3c, and C5b-9, as well as increased CRP, renal TNF-␣, and
cell infiltration, occurred before the onset of albuminuria and
were reduced by Ang II type 1 receptor blockade or by the
human renin inhibitor ALISK. Second, our in vitro experiments in VSMC and in renal tubular epithelial cells showed
that Ang II did not directly induce C3. Third, CRP and
TNF-␣ both induced a more pronounced C3 activation in
VSMC from dTGR compared with SD VSMC. NF-␬B
DNA-binding activity in dTGR VSMC was higher compared
with SD VSMC after TNF-␣ induction. Finally, TNF-␣–
induced C3 stimulation was blocked by IKK–NF-␬B inhibition. Taken together, CRP and TNF-␣ play an important role
in C3 activation in vitro and in vivo.
The complement activation in immune-mediated renal
diseases has been appreciated for decades. Nonetheless, not
much information is known about the role of Ang II in
complement activation. Abbate et al provided indirect evidence that Ang II leads to complement activation.10 They
showed that chronic ACE inhibitor treatment reduced complement C3 deposition in the remnant kidney model. From
their study, whether or not the reduction of C3 was attribut-
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Figure 3. Semiquantitative analysis of infiltrated cells in the kidney. Infiltration of all
cell types (except dendritic cells) was
increased already at week 5 in dTGR compared with SD. Cell infiltration further
increased over time. LOS and ALISK treatment reduced the influx of cells to SD
level, except for CD4 T-cell infiltration,
which was only partially reduced by LOS.
Results are mean⫾SEM. *P⬍0.05 vs
dTGR, #P⬍0.05 vs other groups.
able to decreased Ang II levels or to the improved renal
function resulting from the ACE inhibitor treatment is not
clear. We, therefore, addressed the question of whether
complement activation precedes or is the consequence of
albuminuria in Ang II–induced renal damage. Our data
provide clear evidence that the complement components C1q,
C3, C3c, and C5b-9 are activated and that infiltration of
macrophages and T cells occurs before the albuminuria
commences. Both inflammation and complement activation
probably contribute to the renal disease progression. We
observed complement C3 and C3c immunostaining in the
vessel wall and tubules. Complement activation is mainly
initiated by cleavage of C3 to C3b. C3b then binds to cell
membranes, leading to the generation of C3 and C5 convertase, unless C3b is cleaved to inactive C3bi. Thereafter, C3bi
is cleaved by factor I to C3c and C3d. Numerous findings
indicate that glomerular C3c deposition reflects very recent
complement activation. In IgA nephropathy, glomerular C3c
deposition disappeared to normal values in parallel with the
return of elevated urinary C5b-9 excretion.17 We investigated
whether complement activation was a specific finding in our
dTGR model or whether complement activation is a general
feature of Ang II–induced renal damage. We infused SD rats
with Ang II. Furthermore, we analyzed a completely different
transgenic model, namely rats overexpressing the mouse
renin-2 gene. Both models showed elevated blood pressure,
albuminuria, CRP, C1q, C3, C3c, and C5b-9, indicating that
our findings were not limited to the dTGR model (online data
supplement).
To differentiate whether C3 activation is directly mediated
by Ang II or is the consequence of hypertension and/or renal
damage, we analyzed sections from earlier studies on dTGR.
Dexamethasone-treated dTGR were severely hypertensive
with blood pressure levels ⬎200 mm Hg; however, dexamethasone treatment reduced mortality to 0 and markedly
lowered albuminuria.9 The newly stained histological sec-
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Figure 4. Renal expression of complement C1q and C3. A, Photomicrograph
showing C1q immunoreactivity in the
media of small vessel of 5-week-old
dTGR compared with age-matched SD.
Semiquantification of C1q demonstrates
a higher number of C1q⫹ vessels in
dTGR compared with other groups. B,
Kidney cortex sections of dTGR show
C3 immunoreactivity in tubular epithelial
cells (T), in the media of small vessels,
and granular staining in glomeruli (G) at
all time points. SD and LOS- and ALISKtreated dTGR showed some immunoreactivity at the basal site of tubules. C,
Renal C3c expression was observed in
dTGR sections on brush borders of
tubular epithelial cells and in the media
of small vessels. D, C3 semiquantification confirmed the immunohistochemical
results. Results are mean⫾SEM.
*P⬍0.05 vs dTGR, #P⬍0.05 vs other
groups.
tions from the dexamethasone-treated dTGR showed no
complement. None of the complement components were
identified (online data supplement). We also had available
specimens from an earlier study in which we treated dTGR
with a triple therapy of non–renin–angiotensin system inhibitors (reserpine, hydralazine, and hydrochlorothiazide).18 The
rats from that study developed albuminuria and renal complement component expression in a similar fashion as untreated dTGR, despite normal blood pressure values (online
data supplement).
We also performed cell-culture experiments in the current
study. We found no evidence of direct Ang II–mediated C3
induction in the cell types at the time points (3, 6, 24, 48, and
72 hours) tested. One limitation of our in vitro experiments
was the fact that the VSMC were isolated from aorta and not
from small renal vessels. However, the results were nonethe-
less striking. Ang II exposure did little to C3 mRNA but
induced IL-6 in SD VSMC. Because in dTGR, the VSMC are
chronically exposed to Ang II, we suspected that in vivo Ang
II induces local mediators like TNF-␣. In contrast to Ang II,
CRP and TNF-␣ activated C3, both in VSMC and tubular
epithelial cells. Our in vitro findings and data from Lin et al,
who demonstrated C3 activation in VSMC from prehypertensive SHR rats, suggest that high blood pressure is not the
main reason for complement activation.8 Lin et al termed
VSMC from control rats as contractile and VSMC from SHR
as synthetic based on differences in growth rates and gene
expression.8 We observed that dTGR VSMC showed typical
features of the synthetic phenotype with increased cell proliferation, decreased SM ␣-actin and vinculin expression, and
a 4-fold C3 mRNA expression. Lin et al also demonstrated
that exogenous C3 leads to the synthetic VSMC phenotype.8
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Figure 5. A, Renal expression of complement C5b-9 and IL-6. C5b-9 immunoreactivity shows a similar staining pattern as C3. B, Semiquantification confirms the higher number of C5b-9 positivity in dTGR compared with other groups. Results are mean⫾SEM. *P⬍0.05
vs dTGR, #P⬍0.05 vs other groups. C, C5b-9 and IL-6 are colocalized in the media of damaged small dTGR vessels (week 7). Only
IL-6 immunostaining was also observed in the neointima of damaged dTGR vessels, indicated by the white arrows.
In our study, the 4-fold higher C3 levels might have contributed to the VSMC phenotype switch. The dTGR VSMC were
far-more sensitive to CRP and TNF-␣ compared with SD
VSMC, which agrees with the findings of Lin et al.8 These
investigators also provided an interesting new C3 function by
demonstrating that C3 inhibition resulted in decreased cell
proliferation.8 Our VSMC data are the first indicating that
IKK–NF-␬B inhibition prevents C3 activation. In addition,
we found that the synthetic phenotype is more susceptible to
TNF-␣–induced NF-␬B DNA-binding activity induction.
Nevertheless, CRP and TNF-␣ stimulation induced IL-6 to a
similar extent in the synthetic and contractile VSMC, which
speaks for the involvement of alternative regulatory pathways
for C3 and IL-6.
In vitro, TNF-␣ appeared to play a major role in C3
activation. We reported recently that TNF-␣ blockade ame-
liorates renal damage in our transgenic model.9 To clarify
whether or not in vivo TNF-␣ blockade affects renal C3
expression, we reexamined the histological sections from that
study. We found that TNF-␣ blockade markedly reduces
complement C1q and C3 activation (online data supplement).
These results underscore the importance of TNF-␣ in the
innate immunity that occurs in this model.
The C5b-9 membrane attack complex forms pores in cells,
resulting in cell activation or, at higher concentration, leading
to cell death by lysis. Experiments in complement-deficient
mice provided evidence that C5b-9 is involved in deoxycorticosterone acetate salt–induced hypertension-mediated renal
damage,19 nonimmune remnant kidney model,20 and renal
ischemia reperfusion injury.21 In vitro experiments showed
that C5b-9 induces IL-6 and TNF-␣ synthesis in tubular
epithelial cells.22 Viedt et al showed that C5b-9 induced IL-6
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Figure 6. Synthetic VSMC phenotype is more susceptible for C3 induction. A, VSMC from dTGR showed a higher proliferation rate
(left), decreased SM ␣-actin and vinculin expression (middle), and 4-fold increased C3 mRNA levels (right). B and C, TNF-␣ and CRP
induced C3 mRNA slightly in the contractile VSMC phenotype but more pronounced in the synthetic VSMC phenotype. IL-6 was
induced to a similar extent in both VSMC phenotypes. Ang II did not induce C3 mRNA in either phenotype but induced IL-6 in the contractile VSMC phenotype. D, I␬B phosphorylation and IKK inhibitor prevented TNF-␣–induced NF-␬B DNA-binding activity. E, Electrophoretic mobility shift assay showed that TNF-␣–induced NF-␬B DNA-binding activity was significantly more increased in the synthetic
compared with the contractile VSMC phenotype. Arrows indicate p50 and p65 supershift. Quantification is shown in F. Results are
mean⫾SEM. *P⬍0.05 vs unstimulated control, #P⬍0.05 vs unstimulated SD VSMC, ‡P⬍0.05 vs TNF-␣ or CRP-stimulated SD VSMC.
in VSMC through the activation of NF-␬B and activator
protein-1.23 We found that C5b-9 and IL-6 colocalized in
damaged small dTGR vessels in VSMC. Interestingly, we
observed only IL-6 immunoreactivity, but no C5b-9, in the
neointima of these vessels.
Whether CRP is only a biomarker for inflammation or
participates adversely in the pathogenesis of cardiovascular
disease is a matter of debate. CRP is a predictor for myocardial infarction, stroke, metabolic syndrome, peripheral vascular disease, and vascular mortality among individuals with
no known cardiovascular disease.24 In rodent models, CRP is
involved in the pathogenesis of myocardial infarction,25
causes a higher rate of thrombotic occlusions,26 and mediates
atherosclerosis in apolipoprotein E gene deficient mice.27 In
isolated VSMC, CRP is able to activate NF-␬B and activator
protein-128 and increase Ang II type 1–receptor expression
and binding-site, migration and proliferation.29 We focused
on CRP-mediated C3 and IL-6 activation in the synthetic and
contractile VSMC phenotypes. CRP activated IL-6 in both
phenotypes to a similar extent, whereas CRP markedly
induced C3 in the synthetic VSMC phenotype. In human
coronary endothelial cells, CRP activated membrane cofactor
protein-1, vascular cell adhesion molecule-1, and intercellular
adhesion molecule-1.30 In our model, circulating CRP was
elevated long before any evidence for vascular injury. Possibly, Ang II sensitized the vasculature to effects of CRP,
TNF-␣, and other mediators. We believe that the role of CRP
and complement might have been underestimated in Ang
II–related hypertension. We showed the utility of renin–
angiotensin system blockade in these experiments. However,
additional mechanistic experiments are necessary. Examples
include use of complement component (C5A inhibitors are
available) inhibitors or studies in gene-deficient mice. Nevertheless, our observations are novel and suggest additional
avenues to ameliorate Ang II–induced target-organ damage.
Acknowledgments
The studies were supported by grants-in-aid from the Nationales
Genom Netzwerk (KGCV1 01GS0416) and the European Union
(EuReGene). D.N.M. holds a Helmholtz fellowship. The Deutsche
Forschungsgemeinschaft supported D.N.M. and F.C.L. D.N.M. and
724
Circulation Research
September 16, 2005
F.C.L. received a grant from Novartis to study Aliskiren. F.C.L. has
served as an advisor for Novartis. We are grateful to Dr W. Couser
(University of Washington, Seattle) for providing the antibodies
against C5b-9. We thank A. Schiche, J. Meisel, P. Quass, M.-B.
Köhler, G. N⬘diaye, M. Schmidt, R. Langanki, U. Gerhard, and J.
Czychi for excellent technical assistance.
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Complement Activation in Angiotensin II−Induced Organ Damage
Erdenechimeg Shagdarsuren, Maren Wellner, Jan-Hinrich Braesen, Joon-Keun Park, Anette
Fiebeler, Norbert Henke, Ralf Dechend, Petra Gratze, Friedrich C. Luft and Dominik N. Muller
Downloaded from http://circres.ahajournals.org/ by guest on June 18, 2017
Circ Res. 2005;97:716-724; originally published online August 18, 2005;
doi: 10.1161/01.RES.0000182677.89816.38
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2005 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/97/7/716
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2005/08/18/01.RES.0000182677.89816.38.DC1
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Online Data Supplement
Shagdarsuren et al.
Results on-line supplement
We performed additional studies to separate Ang II-related effects from blood
pressure-related actions in our model. We stained kidney sections from dTGR treated with
triple therapy (hydralazine, hydrochlorothiazide and reserpine) or with dexamethasone
(DEXA) from our earlier studies on dTGR.1,2 DEXA-treated dTGR were severely
hypertensive (blood pressure levels >200 mm Hg).2 However, DEXA reduced mortality to
zero in this model and markedly lowered albuminuria. The histological sections from the
DEXA-treated dTGR showed no complement. None of the complement components were
identified. We also had available specimens from an earlier study in which we treated dTGR
with a “triple therapy” of non-renin-angiotensin system inhibitors. The rats from that study
developed albuminuria and expressed renal complement components in a similar fashion as
untreated dTGR, despite normal blood pressure values.1 Renal complement C3 expression is
shown in figure 1 supplement. Therefore, we conclude that hypertension is not the major
cause for complement activation in this model.
We suspected that TNF-α might have played an important role in complement
activation. Therefore, we stained kidney sections from dTGR treated with the TNF-α blocker,
eternacept, for C1q and C3.2 Both complement components were significantly reduced,
compared to controls as shown in figure 2 supplement.
We also investigated whether complement activation was a specific finding in our
dTGR model or whether complement activation is a general feature of Ang II-induced renal
damage. We investigated SD rats infused with Ang II as well as transgenic rats over
expressing the mouse renin-2 gene (TGR(mREN-2)27), a transgenic model that also depends
on Ang II. Similar to our dTGR model, we found elevated blood pressure, albuminuria, renal
cell infiltration, CRP release, and the activation of complement components, as shown in
figure 3 supplement. Systolic blood pressure and albuminuria were similar in TGR(mREN)27
and dTGR, but sightly lower in Ang II-infused SD rats. In contrast, CRP levels were similar
in all three models. All three models showed C1q, C3, and C5b-9 complement activation.
Nevertheless, there were some minor differences between the models. Glomerular C5b-9 was
more highly expressed in TGR(mREN)27 compared to dTGR and Ang II-infused SD. In
contrast, TGR(mREN)27 and Ang II-infused SD rats showed fewer C1q and C3 positive
vessels compared to dTGR. Tubular and glomerular C3 was similar in all three models.
Online Data Supplement
Shagdarsuren et al.
Figure legends
Fig. 1 supplement. Untreated and triple therapy (TRIPLE)-treated dTGR showed renal
complement C3 expression. In contrast, dexamethasone (DEXA)-treated dTGR were C3
negative.
Fig. 2 supplement. Semi-quantification of C1q and C3 inuntreated and etanercept-treated
dTGR is shown. Both complement components were significantly reduced by etanercept
treatment.
Fig. 3 supplement. A. Systolic blood pressure, albuminuria and C-reactive protein (CRP) are
elevated in TGR(mREN)27 and Ang II-infused SD rats compared to SD controls.
B.
Complement C1q, C3 and C5b-9 were significantly more expressed in TGR(mREN)27 and
Ang II-infused SD rats compared to SD controls.
Supplement References
1.
Mervaala E, Muller DN, Schmidt F, Park JK, Gross V, Bader M, Breu V, Ganten D,
Haller H, Luft FC. Blood pressure-independent effects in rats with human renin and
angiotensinogen genes. Hypertension. 2000;35:587-594.
2.
Muller DN, Shagdarsuren E, Park JK, Dechend R, Mervaala E, Hampich F, Fiebeler
A, Ju X, Finckenberg P, Theuer J, Viedt C, Kreuzer J, Heidecke H, Haller H, Zenke M, Luft
FC. Immunosuppressive treatment protects against angiotensin II-induced renal damage. Am J
Pathol. 2002;161:1679-1693.