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.