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190 Hypertonie 2011 - Poster Hypertonie 2011 - Poster 191
genetic variants, which were represented in
Fabry patients, are located within transcrip-
tionally active regions, possibly altering tran-
scription factor binding sites and therefore, af-
fecting GLA expression. In further experiments
the promoter and the involved transcription
factors will be analyzed in more detail.
Signaltransduktion und molekulare
Mechanismen
PS 66
Angiotensin II-Induced Activation of NADPH
Oxidase Activity Depends on Rho Kinase in
Isolated Vascular Smooth Muscle Cells
Schlüter T. 1 , Reimer N. 1 , Steinbach A. 1 , Rettig
R. 1 , Grisk O. 1
1Universitätsmedizin Greifswald, Physiologie,
Karlsburg, Germany
Objective: NADPH oxidase (Nox) and Rho kinase
(ROCK) contribute to the development of hypertension.
We tested the hypotheses that angiotensin
II-induced activation of Nox depends on
ROCK and that Nox-dependent superoxide formation
and ROCK activity are increased in renal
resistance arteries from rats with angiotensin
II-dependent hypertension.
Methods: Rat vascular smooth muscle cells
(VSMC) were stimulated with angiotensin II
in the absence and presence of ROCK inhibition.
Nox-dependent superoxide formation was
measured by lucigenin-enhanced chemiluminescence.
Renal resistance arteries were isolated
from cyp1a1ren-2 transgenic rats without
or with 3 and 14 days of ren-2 transgene
induction. Nox activity, mRNA expression and
MYPT1 phosphorylation were measured and in-
tact vessels were investigated by small vessel
myography.
Results: Angiotensin II stimulation caused
a 2.5-fold increase in Nox activity in primary
VSMC that was abolished by ROCK inhibition.
Three days after ren-2 transgene induction,
Nox activity was increased by 50% (p < 0.05) accompanied
by increased Nox1 and Nox2 mRNA
expression in renal resistance arteries from
cyp1a1ren-2 transgenic rats. These effects disappeared
14 days after transgene induction.
ROCK mRNA expression and MYPT1 phosphorylation
analyses did not reveal ROCK activation
in renal resistance arteries. Agonist-induced
constriction of renal resistance arteries did not
show enhanced sensitivity to superoxide scavenging
and ROCK inhibition 3 days after transgene
induction.
Conclusion: Angiotensin II-induced activation
of Nox activity is ROCK-dependent in isolated
VSMC while Nox activation in renal resistance
arteries from rats with developing angiotensin
II-dependent hypertension is not accompanied
by ROCK activation. This suggests different
mechanisms of Nox activation in isolated VSMC
and renal resistance arteries in vivo.
PS 67
Syndecan4 Selectively Modulates TRPC6-
Mediated Calcium Signaling in Podocytes
Liu Y. 1 , Thilo F. 2 , Echtermeyer F. 3 , Jensen B.L. 4 ,
Gollasch M. 5 , Tepel M. 6
1Odense University Hospital, Odense, Denmark,
2Charité - Campus Benjamin Franklin, Berlin,
Germany, 3Med Hochschule Hannover, Hannover,
Germany, 4University of Southern Denmark,
Institute of Molecular Medicine, Odense, Denmark,
5Charite, Campus Virchow-Klinikum, Berlin,
Germany, 6Odense University Hospital, Department
of Nephrology, Odense, Denmark
Background: Transient receptor potential cation
channel 6 (TRPC6)-mediated calcium signaling
plays a key role in the vascular dysfunction associated
with arterial hypertension and kidney
disease. Syndecan4 is a transmembrane heparan
sulfate proteoglycan that modulates signal
transduction and regulates localization and
activity of proteins and channels. The present
study investigated whether Syndecan4 modulates
TRPC6-mediated calcium signaling in
podocytes.
Materials and Methods: Conditionally immortalized
mouse podocytes were cultivated at 33°C
for propagation and at 38°C for differentiation.
Knockdown of Syndecan4 was performed using a
silencer siRNA transfection kit. For Syndecan4
overexpression podocytes were transfected either
with the full-length Syndecan4 sequence (Sdc4FL)
or a cytoplasmic deletion construct
(Sdc4CY). Protein expression was investigated
by immunoblotting. TRPC6-mediated calcium
influx into fluo-4-loaded podocytes was measured
using confocal laser scanning microscopy.
Calcium influx was induced by the known
TRPC6 agonist flufenamic acid (FFA).
Results: Knockdown of Syndecan4 in podocytes
selectively reduced TRPC6 protein expression by
30% (p< 0.05), but did not affect TRPC3 protein
assessed by immunoblotting. Control scrambled
siRNA showed no effects on TRPC6 protein expression
(101 ± 2% of control). Consecutively,
siRNA against Syndecan4 reduced the TRPC6mediated
and FFA-induced peak calcium influx
by 58% (p< 0.01).
Syndecan4 overexpression in podocytes after
transfection with Sdc4FL selectively increased
TRPC6 protein expression by 40% (p< 0.01),
whereas TRPC3 protein was not affected.
In addition, increased TRPC6 channel density
after transfection with Sdc4FL enhanced calcium
influx by 111% (p< 0.01).
After Syndecan4 knockdown the 1-oleoyl-
2-acetyl-sn-glycerol-induced calcium influx
was reduced by 49% (p< 0.05). This reduction
by siRNA was prevented in the presence of
nonspecific TRPC blockers, 2-aminoethoxydiphenylborane
or SKF-96365.
Conclusion: Syndecan4 knockdown reduces,
whereas Syndecan4 overexpression increases
TRPC6 protein expression and TRPC6-mediated
calcium flux in podocytes. Syndecan4 may affect
calcium signaling and may be implicated in the
pathogenesis of hypertension.
PS 68
Monocyte Chemoattractant Protein-1 Is
Induced by Serum Amyloid A in Vascular
Smooth Muscle Cells
Schuchardt M. 1 , Tölle M. 1 , Huang T. 1 , Zidek W. 1 ,
van der Giet M. 1
1Charité - Universitätsmedizin Berlin, CC13 -
Schwerpunkt Nephrologie, Berlin, Germany
Objective: Serum amyloid A (SAA) has apolipoprotein
properties and accumulates in HDL.
SAA levels are elevated in patients with endstage
renal disease (ESRD). We could previously
show that HDL from ESRD patients has reduced
anti-inflammatory capacity by inhibiting
MCP-1 production. In this study we investigate
the signaling pathway of SAA-induced MCP-1
production in VSMCs.
Methods: MCP-1 formation was detected by
real-time PCR and by Luminex technology.
Determination of MAPK phosphorylation was