DISCUSSION:
Renal artery stenosis remains a pervasive cause of secondary
hypertension and a condition significantly correlated with high
morbidity and mortality (Textor, 2003), (Kalra, et al., 2005), (de Mast
and Beutler, 2009), (Kalra, et al., 2010). NO plays an important role in
maintaining renal blood flow and glomerular filtration rate following
single renal artery stenosis (Granger, et al., 2002), (Majid and Navar,
1997), (Majid, et al., 1998). In addition, there is emerging
pre-clinical evidence that sGC stimulator drugs which have had notable
anti-fibrotic effects, in conjunction with RAAS blockade confer
resistance to end stage renal disease and chronic kidney disease,
(Beyer, et al., 2015), (Sandner and Stasch, 2017). Such therapies have
demonstrated an ability to elevate blood flow and/or improve cardiac
outcomes as a result of decreased vascular tone and decreased blood
pressure (Stasch, et al., 2001), (Stasch, et al., 2011), (Evgenov, et
al., 2006). Our previous study, in accordance with previous 2K1C models,
showed that the modified 2K1C renal artery stenosis model causes
increased blood pressure without altering body weight or plasma salt
concentrations (DeLalio, et al., 2020). Here we provide the first
evidence that sGC expression unexpectedly increases in renal artery
vascular smooth muscle to preserve renal blood flow.
In this study, we observed a significant increase in vasodilation in the
contralateral renal arteries of renal clipped animals, which was not
observed in other arterial beds. This increase in vasodilation was
likely due to the measurable increase in sGC expression observed in
unobstructed renal artery smooth muscle from clipped animals compared to
their sham controls. We also observed a significant increase in
ACh-dependent vasodilation of renal clip animals over the sham controls,
suggesting significant contribution of the endothelium in this response.
This response may indicate that NO signaling, in the endothelium, which
has been established to be a pivotal player in promoting regulation of
renovascular homeostasis of blood pressure and fluid retention (O’Connor
and Cowley, 2010), (Dautzenberg, et al., 2011), is enhanced compared
with other vascular beds following elevated RAAS activity.
Changes in sGC expression in renal smooth muscle were not limited toin vivo animal tissue chronically exposed to Ang II. Following
treatment of cultured renal pre-glomerular smooth muscle cells (RPGSMCs)
with Ang II for 48 hours, the increase observed in sGC mRNA and protein
expression suggests that RPGSMCs respond differently from aortic smooth
muscle. Aortic smooth muscle and endothelial cells exhibit decreased
functional NO signaling with excess Ang II exposure, via pathological
overproduction of reactive oxygen species (ROS) (Griendling, et al.,
1994), (Doughan, et al., 2008). Moreover, aortic sGC protein expression
decreases with Ang II (Mollnau, et al., 2002), (Rippe, et al., 2017),
and Ang II impairs aortic smooth muscle sGC function (Rippe, et al.,
2017), (Crassous, et al., 2012). Furthermore, these processes prevent
sufficient cGMP production, leading to elevated systemic blood pressure
(Durgin, et al., 2019). On the contrary, our studies show that treatment
with Ang II in conjunction with NO-stimulation caused elevated cGMP
signaling in RPGSMCs, as indicated by VASP phosphorylation. This
indicates enhanced sGC-cGMP signaling following Ang II treatment in
renal vascular smooth muscle.
Remarkably, other known responses to Ang II treatment were noted in
RPGSMCs, such as increased protein expression, elevated F-actin
expression, and increased cell size. These patterns have been observed
in aortic smooth muscle both in vivo following infusion with Ang
II and in vitro following Ang II treatment in culture,
(Geisterfer, et al., 1988), (Zhang, et al., 2005). This suggests that
while the increases in sGC expression are unique to renal smooth muscle,
the hypertrophic responses to Ang II conform to the patterns that have
been observed by others.
Specifically, our data shows that the AT1R, but not the
AT2R, is responsible for the elevated expression of sGC
observed in renal smooth muscle in response to Ang II. Indeed,
co-treatment with Losartan and Ang II was sufficient to reverse all of
the Ang II-induced phenotypes we observed in RPGSMCs, while Ang II
co-treatment with PD123319 did not impact any of the phenotypes
facilitated by Ang II treatment alone. This response may be due to the
high density of AT1Rs that have been observed in the
adventitia of the renal vasculature (Doughan, et al., 2008),
(Harrison-Bernard, et al., 1997), and the increased constriction of
renal vasculature and, to a smaller extent, gut vasculature following
acute Ang II infusion (Jackson and Herzer, 2001). Curiously, this
contrasts with the role for the AT2R in cardiac
function, which has been shown to improve outcomes following treatment
with AT2R-specific agonists following myocardial
infarction (Kaschina, et al., 2008). These findings suggest that the
observed effect on sGC expression and function are largely independent
of AT2R activation. Truncation products such as
angiotensin 1-7 or angiotensin IV may also play a role (Savergnini, et
al., 2010),(Esteban, et al., 2005), albeit minor. Taken together, these
findings suggest that renal smooth muscle responds uniquely to Ang II
via the AT1R to promote increased sGC expression and
sGC-cGMP induced vasodilation while maintaining the canonical
hypertrophic responses associated with elevated Ang II exposure.
Consistent with our previous work in aortic SMCs (Galley, et al., 2019),
Ang II studies in RPGSMCs showed that inhibition of the forkhead box
subclass O (FoxO) transcription factors significantly impairs sGC
expression. This finding indicates FoxO regulation of sGC expression
applies to multiple vascular beds, thus regulating dilatory function in
multiple branches of the vascular tree. The FoxO protein(s) responsible
are not yet known and the specific role of the FoxO transcription
factors in the development and pathology of renal artery stenosis
requires further study to assess their diverse functions in vascular
physiology. It is nevertheless clear that the Ang II-mediated increases
in sGC function cannot occur without functional FoxO transcription
factor activity. Ang II can activate Akt (Li and Malik, 2005), and
Akt-mediated phosphorylation is a common regulatory mechanism known to
modulate FoxO transcriptional activity (Biggs, et al., 1999), (Brunet,
at al., 1999), (Kops, et al., 1999). In addition, Ang II has been shown
to cause increases in ROS , and oxidative stress is known to impact FoxO
transcription factor activity through acetylation/deacetylation
(Salminen, et al., 2013), (Ichiki, et al., 2003), (Motta, et al., 2004),
(van der Horst, et al., 2004). These findings suggest that there could
be an indirect regulatory mechanism between the AT1R and
FoxO transcription factors. Future research in this area should
investigate the potential mechanistic links between agonism of the
AT1R and activation of the FoxO transcription factors.
Moreover, our research has shown that oxidation or loss of sGC heme iron
leads to NO insensitivity, making the protein more responsive to sGC
activating compounds which target oxidized or heme-deficient sGC to
produce cGMP (Rahaman, et al., 2017), (Durgin, et al., 2019).
Investigation of Ang II-mediated ROS production may reveal a novel
therapeutic target for sGC activating drugs under conditions where high
RAAS activity promotes oxidative stress.
Collectively, we show for the first time that in response to elevated
RAAS activity, renal smooth muscle responds through an
AT1R and FoxO transcription factor-dependent mechanism
to increase sGC expression and cGMP signaling. These responses likely
constitute a compensatory response to allow for maintenance of
homeostatic blood volume and salt balance to counteract Ang II-dependent
increases in systemic blood pressure, and a means of preserving normal
body weight and plasma sodium concentration. Combined, this study marks
an important discovery of how the renal vasculature responds to elevated
circulating plasma Ang II, advancing our understanding of renal vascular
hypertension and the regulation of cGMP signaling within the renal
vascular wall.