RESULTS:
To determine the effects of RAAS activation on the renal vasculature, we
used a two-kidney-one-clip (2K1C) model (Figure 1A), which involves
surgical stenosis of one of the renal arteries. To assess the
vasoreactivity responses of the unclipped contralateral (right) renal
arteries, wire myography was performed on the right (unclipped) renal
arteries of clipped and sham control animals. We observed that
contralateral clipped arteries contracted significantly less to 10 μM
phenylephrine than their sham counterparts (Figure 1B). We also observed
a significant improvement in endothelium-dependent vasodilation in
response to ACh (Figure 1C). The calculated values with respect to sham
control and renal clip animals is 1.6x10-7M ±
4.0x10-8M and 1.1x10-7M ±
3.2x10-8M, respectively for EC50, and
we observed 55.9 ± 5.2% and 76.8 ± 5.1%, respectively for
Emax values. Similarly, a significant improvement in
NO-dependent vasodilation in response to the NO-donor, sodium
nitroprusside (SNP), was observed (Figure 1D) with calculated
EC50 values of 3.99x10-5 M ±
3.95x10-5 M and 1.22x10-5 M ±
7.83x10-6 M for sham and renal clip groups,
respectively. These data also show a calculated Emax of
44.5 ± 4.5% versus 66.3 ± 4.2% for sham control and clipped animals,
respectively. These findings indicate a significant improvement in the
vasorelaxation responses of the contralateral clipped arteries over sham
controls to both an endothelium-dependent vasodilator and a NO-donor.
To determine if the increased NO-dependent vasodilatory response in
contralateral clipped arteries were due to changes in sGC expression, we
quantified protein expression within renal arteries. We found that the
expression of the β subunit of sGC (sGCβ) was significantly elevated in
contralateral renal arteries of clipped animals (Figure 2B, F) compared
to sham controls. No significant differences were seen in nuclei
staining (Figure 2A, E), smooth muscle α-actin (ACTA2) expression
(Figure 2C, G), or the endothelial cell marker, von Willebrand Factor
(vWF, Figure 2D, H), between groups.
Next, we sought to determine what drives sGC expression changes in renal
artery smooth muscle. It is well established that reduced renal blood
flow increases angiotensin II (Ang II) in models of 2K1C (Murphy, et
al., 1984), (Sadjadi, et al., 2002). Therefore, we treated rat renal
preglomerular smooth muscle cells (RPGSMCs) with vehicle or
10-6 M Ang II to test if Ang II increased sGC
expression. Ang II treatment led to increased RPGSMC cell area and
augmented filamentous (F)-actin expression (Figure 3B, E), indicating
RPGSMC hypertrophy (Stephenson, et al., 1998). Additionally, Ang-II
resulted in increased sGCβ protein expression by 1.7-fold via
immunofluorescence and 4-fold via western blot analysis (Figure 3C, F,
G). Consistent with increased sGC protein expression, we found that Ang
II treatment also caused a 3.6-fold increase in sGCα mRNA (Figure 4A)
and a 4.4-fold increase in sGCβ mRNA (Figure 4B). To test if increased
sGC expression impacted cGMP production and PKG activity, RPGSMCs
treated with Ang II or vehicle, and subjected to treatment with the NO
donor, DEA-NONOate, for 15 minutes prior to harvest to induce
sGC-mediated cGMP production. Quantification of vasodilator stimulated
protein (VASP) phosphorylated at the serine 239 position, a surrogate
indicator of cGMP-dependent protein kinase activity (Smolenski, et al.,
1998), showed an 8-fold increase in pVASP in Ang II-treated cells
stimulated with DEA-NONOate compared to vehicle controls (Figure 3H).
Taken together, these data show that Ang II in vitro augments sGC
expression and cGMP signaling, indicating that elevated RAAS activity
increases sGC expression and downstream signaling in vivo .
We next tested which Ang II receptor subtype- either the
AT1R or the Angiotensin Type 2 Receptor
(AT2R) -was responsible for increasing sGC mRNA and
protein expression. RPGSMCs co-treated Ang II and Losartan, an
AT1R antagonist
(Timmermans, et al., 1995), caused inhibition of Ang II – induced
increases in sGCα mRNA (Figure 4A), sGCβ mRNA (Figure 4B), and sGCβ
protein expression (Figure 4C & D). Conversely, RPGSMCs co-treated with
Ang II and PD123319, an AT2R antagonist (Blankley, et
al., 1991), showed no significant impact on the Ang II – induced
increases in sGCα mRNA (Figure 4A), sGCβ mRNA (Figure 4B), or sGCβ
protein expression (Figure 4C & D).
Recently, we published evidence that the FoxO family of transcription
factors regulate the mRNA expression of sGC in aortic smooth muscle
(Galley, et al., 2019). To determine whether the FoxO family of
transcription factors also influence the function of renal smooth
muscle, we treated RPGSMCs with 10-6 M AS1842856, a
small molecule Fox O transcription factor inhibitor (Nagashima, et al.,
2010), alone and in conjunction with Ang II. When AS1842856 was
administered alone to RPGSMCs, a significant reduction in sGCα mRNA
(Figure 5A), sGCβ mRNA (Figure 5B), and sGCβ protein expression (Figure
5C) was observed. When administered with Ang II, AS1842856 produced no
effect on either sGCα mRNA (Figure 5A), sGCβ mRNA (Figure 5B), or sGCβ
protein expression (Figure 5C) when compared to vehicle controls. These
data indicate the FoxO transcription factors are necessary for the Ang
II-mediated sGC expression increases in renal smooth muscle.
Next, we tested Ang II receptor antagonists and FoxO inhibitors on cGMP
production, determined by pVASP expression following NO-stimulation with
DEA-NONOate. At baseline, where no DEA-NONOate-stimulation occurred, no
significant differences were observed between the treatment groups.
Similar to the observed effect in sGC expression, co-treatment with Ang
II and PD123319 produced significant increases in downstream sGC
function via VASP phosphorylation following DEA-NONOate stimulation
compared to controls treated with DEA-NONOate. AS1842856 or Losartan
showed no significant differences from control-treated cells stimulated
with DEA-NONOate (Figure 6A & B). These data show that PD123319 has no
significant effect and that the blunting effect of Losartan or AS1842856
on the Ang II-mediated responses also inhibited downstream cGMP
signaling following NO-dependent stimulation.