Experimental Physiology

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1 496 Exp Physiol (2015) pp Symposium Report Symposium Report Mechanisms of renal sympathetic activation in renovascular hypertension Ruy R. Campos 1, Elizabeth Barbosa Oliveira-Sales 1,ErikaE.Nishi 1,JulianF.R.Paton 2 and Cassia T. Bergamaschi 1 1 Department of Physiology, Cardiovascular Division, Universidade Federal de São Paulo, São Paulo, Brazil 2 School of Physiology & Pharmacology, Bristol CardioVascular, University of Bristol, Medical Sciences Building, Bristol BS8 1TD, UK Experimental Physiology New findings What is the topic of this review? This review addresses the underlying mechanisms involved in sympathoexcitation during renovascular hypertension, focusing on the importance of increased oxidative stress in the paraventricular nucleus and rostral ventrolateral medulla. What advances does it highlight? Whether renal or autonomic dysfunction is the major contributor to systemic hypertension following a renovascular insult is still a matter of debate. Here, we take an integrative approach by describing the crosstalk between the kidney and brain. We show how changes in the CNS, and in sympathetic premotor neurons in particular, are activated by ischaemic renal disease in an experimental model of renovascular hypertension. This review addresses the underlying mechanisms involved in the sympathoexcitation in renovascular hypertension. We focus on the importance of increased oxidative stress in the paraventricular nucleus of hypothalamus (PVN) and rostral ventrolateral medulla (RVLM) for the autonomic dysfunction associated with renovascular hypertension in the two-kidney, one-clip (2K-1C) model. We found in 2K-1C rats, 6 weeks after clipping, a significant increase in the mrna and protein expression of the angiotensin II type 1 receptor within the RVLM and PVN. In addition, mrna from NADPH oxidase subunits (p47phox and gp91phox) was greater in the RVLM and PVN of 2K-1C rats than in a sham-operated group. However, CuZn superoxide dismutase gene expression in these regions was not changed, suggesting that excessive production of reactive oxygen species overwhelms any endogenous antioxidant system in the RVLM and PVN in renovascular hypertension. In fact, acute administration of tempol or vitamin C (either i.v. or directly into the PVN or RVLM) caused a significant decrease in blood pressure and renal sympathetic nerve activity in 2K-1C rats, but not in control animals. Thus, we suggest that an increase in the activity of RVLM and PVN neurons triggered by angiotensin II and oxidative stress is a major mechanism involved in the maintenance of sympathoexcitation of the cardiovascular system in renovascular hypertension. (Received 24 November 2014; accepted after revision 26 January 2015; first published online 29 January 2015) Corresponding author R. R. Campos: Cardiovascular Division, Department of Physiology, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Botucatu 862, CEP , São Paulo, SP, Brazil. ruy.camposjr@unifesp.br DOI: /expphysiol

2 Exp Physiol (2015) pp Sympathetic activation in hypertension 497 The idea that the sympathetic nervous system plays an important role in initiation or maintenance of hypertension has been considered for decades. In fact, in the 1940 s, surgical lumbar sympathectomy and splanchnic resection were employed for the treatment of hypertension (Smithwick, 1940; Grimson, 1941). However, a major argument against the participation of the sympathetic nervous system in the long-term regulation of arterial pressure and, consequently, in hypertension is the hypothesis that without a chronic and sustained decrease in renal function, an increase in sympathetic tone cannot cause persistent hypertension (Guyton, 1990). Recently, however, the importance of sympathetic nervous system dysfunction in hypertension in humans has been highlighted in the early studies testing selective catheter-based renal sympathetic nerve ablation in patients with resistant hypertension, indicating that chronic activation of the renal nerves (whether afferent and/or efferent) is an important mechanism to trigger and maintain systemic hypertension (Esler, 2010). It is well known that a chronic change in renal sympathetic nerve activity (RSNA) is able to increase renin secretion and sodium reabsorption and induce renal vasoconstriction (DiBona & Kopp, 1997; Dampney et al. 2005). However, the mechanisms underlying the initiation and maintenance of sympathoexcitation in hypertension are not fully understood. In the present short review, we highlight the changes in signalling within sympathetic premotor neurons in a rat model of renovascular hypertension. In particular, we consider those spinally projecting neurons in the paraventricular nucleus (PVN) of the hypothalamus and in the rostral ventrolateral medulla (RVLM), two major regions of the CNS involved in the control of sympathetic vasomotor tone (Guyenet, 2006). Considering the intimate and reciprocal communication between the kidney and the brain through both hormonal and neural pathways and the introduction of renal denervation as a possible treatment for drugresistant hypertension (Esler et al. 2010; Bhatt et al. 2014; Krum et al. 2014), a discussion of some of the mechanisms linking sympathoexcitation and hypertension seems most timely. Our review is based on data obtained from the two-kidney one-clip (2K-1C) rat model of hypertension developed by Goldblatt et al. (1934). This classical model of renovascular hypertension is induced by a partial occlusion of one renal artery, leading to increased renin secretion and, consequently, hyperactivation of the renin angiotensin aldosterone system and arterial hypertension. Interestingly, between 3 and 4 weeks after clipping, the hypertension has a strong neurogenic component. Indeed, both the low-frequency power of systolic blood pressure and the RSNA are significantly increased in 2K-1C rats (de Oliveira-Sales et al. 2010; Oliveira-Sales et al. 2014). In addition, either ganglionic blockade or inhibition of the RVLM leads to normalization and a larger reduction in blood pressure in 2K-1C compared with control rats, indicating that sympathoexcitation is an important factor in the maintenance of experimental renovascular induced hypertension (Bergamaschi et al. 1995). Furthermore, in human renovascular hypertension, there is a significant increase in muscle sympathetic nerve activity and in the total body spillover of noradrenaline (Johansson et al. 1999). These observations emphasize the importance and major contribution of sympathetic activation in the pathophysiology of renovascular hypertension. Next, we consider what changes occur in the signalling in both the RVLM and the PVN that could bring about raised sympathetic discharge. Our attention focuses on the roles of central angiotensin II (Ang II) and oxidative stress as key mechanisms underpinning the sympathoexcitation in renovascular hypertension. We found in 2K-1C rats, 6 weeks after clipping, a significant increase in the mrna and protein expression of the Ang II type 1 (AT 1 ) receptor within the RVLM (de Oliveira-Sales et al. 2010). Losartan injection into this region significantly reduced blood pressure and RSNA in renovascular hypertensive rats but not in control animals. These data suggest that in renovascular hypertension, the upregulated AT 1 receptor in the RVLM maintains sympathoexcitation and high blood pressure. Interestingly, the circulating level of Ang II in this phase of hypertension is only slightly increased, but tissue AT 1 receptor expression was significantly upregulated in the RVLM. Whether Ang II peptide levels are also increased in the RVLM remains to be determined. Furthermore, not only in renovascular hypertensive rats, but also in rabbits with chronic heart failure, the sympathoexcitation was associated with increased expression of the AT 1 receptor in the RVLM, suggesting that the sympathoexcitation in heart failure is, in part, dependent on increased actions of Ang II within the RVLM (Zucker et al. 2014). Moreover, intracerebroventricular infusion of AT 1 receptor antisense to inhibit receptor synthesis prevents 2K-1C hypertension (Galli & Phillips, 2001). An interesting hypothesis proposed by Xue et al. (2012) was that S.C. or intracerebroventricular administration of low doses of Ang II sensitized the brain to produce an enhanced hypertensive response to Ang II associated with increased mrna expression of renin angiotensin aldosterone system components in regions of the brain related to sympathetic activity control, including the lamina terminalis and PVN. Interestingly, we found a significant upregulation of mrna expression of the AT 1 receptor in the PVN in the 2K-1C rat and showed that increased actions of the AT 1 receptor in such regions are essential to maintain the sympathoexcitation and hypertension (de Oliveira-Sales et al. 2010). Considering the PVN-RVLM pathway (Ferguson & Washburn, 1998), an intriguing possibility is that the PVN may be a source

3 498 R. R. Campos and others Exp Physiol (2015) pp of the additional excitatory angiotensinergic drive to the RVLM in Goldblatt hypertension. Considering that Ang II increases the activity of NADPH oxidase, the major source of superoxide anions (O 2 ), and that O 2 increases neuronal excitation, we tested the hypothesis that increased oxidative stress in the PVN and RVLM is important to trigger sympathoexcitation and hypertension in the 2K-1C model. We found that mrna from NADPH oxidase subunits (p47phox and gp91phox) is greater in the RVLM and PVN of 2K-1C rats than in a sham-operated group. In addition, CuZn superoxide dismutase (CuZnSOD) gene expression in these regions was not changed, suggesting that an excessive production of reactive oxygen species overwhelms any endogenous antioxidant system in the RVLM and PVN in renovascular hypertension (Oliveira-Sales et al. 2008, 2009). Additionally, acute administration of tempol or vitamin C (either I.V. or directly into the PVN or RVLM) caused a significant decrease in blood pressure and RSNA in 2K-1C rats, but not in control animals (Oliveira-Sales et al. 2008, 2009). One important aspect is that no changes in the NADPH oxidase subunits were observed in the cerebral cortex, which acted as a control region, suggesting that the increase in oxidative stress is probably selective or preferential to the PVN and RVLM, consistent with the expression patterns of AT 1 receptorsinthebrain(song et al. 1992). The results support the idea that an increase in oxidative stress in sympathetic premotor neurons plays a major role in maintaining high arterial pressure and sympathetic drive in 2K-1C hypertension. To test the role of oxidative stress in the RVLM for the sympathoexcitation in renovascular hypertension chronically, we first administered vitamin C (150 mg kg 1 ) Figure 1. Relative levels of mrna of angiotensin II type 1 (AT 1 ) receptor, NADPH oxidase subunits and CuZn superoxide dismutase (CuZnSOD) compared with mrna of β-actin in the rostral ventrolateral medulla (RVLM) and paraventricular nucleus (PVN) p47phox (A), gp91phox (B), AT 1 (C) and CuZnSOD (D) in the RVLM and PVN of control (CT; n = 4), CT + vitamin C (n = 4), two-kidney, one-clip (2K-1C; n = 6) and 2K-1C + vitamin C animals (n = 5). P < 0.05 versus SHAM. P < 0.05 versus 2K-1C (one-way ANOVA followed by the Newman Keuls post hoc test).

4 Exp Physiol (2015) pp Sympathetic activation in hypertension 499 for 7 days consecutively. This significantly decreased blood pressure and RSNA and improved the sensitivity of arterial baroreceptor control of heart rate and RSNA (Nishi et al. 2010). Interesting, mrna expression of AT 1 receptor and NADPH oxidase subunits within the RVLM and PVN were significantly reduced after vitamin C treatment (Fig. 1), but not in a control region of the cerebral cortex, indicating that the improvement in cardiovascular and autonomic function induced by antioxidant therapy was mediated by reduced actions of Ang II and oxidative signalling in specific regions of the brain. Second, we overexpressed superoxide dismutase (to degrade superoxide) using an adenoviral vector (Ad-CMV-CuZnSOD) that was injected into the RVLM parenchyma either in rats with renovascular hypertension or in sham-operated control animals. Arterial pressure was measured chronically using radio-telemetry. The elevated superoxide levels in the RVLM were normalized by expression of CuZnSOD in RVLM. The hypertension produced in 2K-1C rats was reversed 1 week after virus-mediated expression of CuZnSOD and lasted. The antihypertensive effect was maintained for 3 weeks and was associated with a decrease in the low-frequency power of systolic blood pressure, suggesting reduced sympathetic vasomotor modulation. Finally, the expression of CuZnSOD was localized in tyrosine hydroxylase-positive neurons in the RVLM, the barosenstive C1 neurons of the RVLM related to cardiovascular control. Consistent with these results was the finding that deletion of angiotensin II type 1A receptors from C1 RVLM neurons results in reduced sympathetic nerve activation and fluid and electrolyte retention during angiotensin infusion in mice (Jancovski et al. 2014). One of the possible sources for AT 1 receptor activation in the RVLM is Ang II acting in the subfornical organ (SFO), leading to activation of the PVN and, subsequently, the RVLM; this is the well-established SFO PVN RVLM pathway (Zimmerman et al. 2004). In fact, the hypertension and sympathoexcitation induced byangiicanbepreventedorattenuatedbyselective blocking of AT 1 receptors or oxidative stress formation in this pathway, in the SFO, PVN or RVLM (Zimmerman et al. 2002, 2004; Oliveira-Sales et al. 2010). Considering that all components of the renin angiotensin system are synthesized in the brain, we cannot rule out an important role of brain-derived Ang II in the regulation of sympathetic vasomotor tone and blood pressure (Paul et al. 2006). Recently, hypertension-induced changes in the permeability of the blood brain barrier in the RVLM and PVN of spontaneously hypertensive rats and 2K-1C rats were described, suggesting an additional mechanism involved in the sympathoexcitation triggered by circulating Ang II leading to chronic hypertension (Biancardi et al. 2014; Nishi et al. 2014). Figure 2 summarizes the major hypothesis presented in this brief review. Changes in signalling in the PVN and RVLM may contribute to the maintenance of sustained increases in sympathetic activity in renovascular hypertension. Previously, we have described AT 1 receptors Figure 2. Schematic model of possible mechanism leading to the sympathoexcitation in renovascular hypertension Circulating angiotensin II (Ang II) acting in the circumventricular organs (CVOs; outside the blood brain barrier, BBB), changes in the BBB permeability or local formation of Ang II in the brain would increase local oxidative stress in the paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM), triggering autonomic dysfunction. Abbreviations: AT 1, angiotensin II type 1 receptors; IML, intermediolateral column; ROS, reactive oxygen species; and SFO, subfornical organ.

5 500 R. R. Campos and others Exp Physiol (2015) pp on the endothelium of the microcirculation in the nucleus tractus solitarii (NTS; Paton et al. 2001). Indeed, Ang II mediated signalling across the blood brain barrier, where AT 1 receptors were activated by blood-borne Ang II that triggered endothelial nitric oxide synthase activation, and NO signalling within the brain parenchyma was found to depress baroreflex function via a GABAergic mechanism within the NTS (Paton et al. 2002, 2007). Such a mechanism was supported by the finding that systemic Ang II-induced baroreflex depression was reversed by microinjection of candesartan into the NTS (Tan et al. 2007). Whether such a mechanism operates to increase superoxide in the 2K-1C model remains to be addressed. Moreover, given that astrocytes in the RVLM are a target for Ang II (Guo et al. 2010), we also need to consider this cell type in the future as a component of the signalling pathway in Goldblatt hypertension as well as an additional target when considering antihypertensive therapy. Thus, we suggest that an increase in the activity of RVLM and PVN neurons triggered by Ang II and oxidative stress is a major mechanism involved in the maintenance of sympathoexcitation of the cardiovascular system in renovascular hypertension. In Fig. 2, we outline future directions for understanding the role of the CNS in hypertension that would further the development of new therapeutic targets for the treatment of systemic hypertension. In summary, sympathetic nervous system activation in Goldblatt hypertension may be triggered by different pathways. First, circulating Ang II may reach AT 1 receptors in regions of the brain outside the blood brain barrier, such as the SFO, leading to downstream activation of the PVN and RVLM, i.e. the SFO PVN RVLM pathway. In addition, local formation of Ang II in the brain targeting endothelial cells and astrocytes and changes in permeability of the blood brain barrier are important mechanisms leading to increased oxidative stress and activation of the sympathetic nervous system. References Bergamaschi C, Campos RR, Schor N & Lopes OU (1995). Role of the rostral ventrolateral medulla in maintenance of blood pressure in rats with Goldblatt hypertension. Hypertension 26, Bhatt DL, Kandzari DE, O Neill WW, D Agostino R, Flack JM, Katzen BT, Leon MB, Liu M, Mauri L, Negoita M, Cohen SA, Oparil S, Rocha-Singh K, Townsend RR & Bakris GL; SYMPLICITY HTN-3 Investigators (2014). A controlled trial of renal denervation for resistant hypertension. 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