Renin-angiotensin system acting on reactive oxygen species in paraventricular nucleus induces sympathetic activation via AT1R/PKCγ/Rac1 pathway in salt-induced hypertension

Brain renin-angiotensin system (RAS) could regulate oxidative stress in the paraventricular nucleus (PVN) in the development of hypertension. This study was designed to explore the precise mechanisms of RAS acting on reactive oxygen species (ROS) in salt-induced hypertension. Male Wistar rats were administered with a high-salt diet (HS, 8.0% NaCl) for 8 weeks to induced hypertension. Those rats were received PVN infusion of AT1R antagonist losartan (LOS, 10 μg/h) or microinjection of small interfering RNAs for protein kinase C γ (PKCγ siRNA) once a day for 2 weeks. High salt intake resulted in higher levels of AT1R, PKCγ, Rac1 activity, superoxide and malondialdehyde (MDA) activity, but lower levels of copper/zinc superoxide dismutase (Cu/Zn-SOD), superoxide dismutase (SOD) and glutathione (GSH) in PVN than control animals. PVN infusion of LOS not only attenuated the PVN levels of AT1R, PKCγ, Rac1 activity, superoxide and decreased the arterial pressure, but also increased the PVN antioxidant capacity in hypertension. PVN microinjection of PKCγ siRNA had the same effect on LOS above responses to hypertension but no effect on PVN level of AT1R. These results, for the first time, identified that the precise signaling pathway of RAS regulating ROS in PVN is via AT1R/PKCγ/Rac1 in salt-induced hypertension.

hydroxyl ion, and hydrogen peroxide in the PVN, which contribute to the overactivity of pre-autonomic PVN neurons and result in sympathoexcitation and metabolic disorders in the pathophysiology of hypertension 5,[7][8][9] . Furthermore, data from our laboratory suggest that Ang II increased the PVN levels of ROS, and bilateral PVN infusion of AT1R blocker decreased the production of ROS in PVN, as well as sympathetic nerve activity and blood pressure [10][11][12] . Therefore, the activation of AT1R augments the intracellular level of ROS and sympathetic nerve activity. However, the precise signaling pathway of RAS modulating the oxidative stress in PVN is still unknown in hypertension.
Superoxide generated by NAD(P)H oxidase has emerged as a key intermediary in the central and peripheral effects of Ang II 13 . NAD(P)H oxidase is composed of membrane-bound subunits, gp91 phox and p22 phox , cytoplasmic subunits, p47 phox , p40 phox , p67 phox , and Rac1 and/or Rac2 14 . Rac1 (Ras-related C3 botulinum toxin substrate1) is a small GTPase which is essential for the assembly and activation of NAD(P)H oxidase and the production of superoxide 15 . A growing body of evidence indicate that the increased Ang II in brain induces the NAD(P) H-dependent ROS production by the protein kinase C (PKC) which is a critical step in Rac1 activation and subsequent enzyme assembly 13,16 . PKC is a family of phospholipids serine/threonine protein kinases, which have been classified into conventional PKCs (α , β and γ ), novel PKCs (δ , ε , η and θ ), and atypical PKCs (ζ and λ /ι ) 17 . PKCγ , unlike other PKC isozymes, is expressed only in the central nervous system and eye tissues 17 . Aronowski and colleagues (2000) found that PKCγ is activated in the period of brain ischemia/reperfusion which is closely related to the oxidative stress 18,19 . These findings suggest that the linking of AT1R and NAD(P)H oxidase-derived ROS is involved in the effects of PKCγ /Rac1/NAD(P)H pathway. Furthermore, it is unclear whether AT1R/PKCγ /Rac1/ NAD(P)H pathway in PVN mediates ROS production in the development of hypertension. Therefore, the aim of the present study was to determine whether the precise mechanism of RAS modulating oxidative stress in PVN is through AT1R/PKCγ /Rac1/NAD(P)H signaling pathway during the development of high-salt hypertension.

Results
Effect of losartan on mean arterial pressure (MAP). High salt diet induced a significant increase in mean arterial pressure (MAP) compared with control rats after 8 weeks (139 ± 4.7 mmHg vs. 94.7 ± 3.6 mmHg, P < 0.05) of high salt diet before bilateral PVN infusion of LOS or microinjections of PKCγ siRNA. In Fig. 1 Effect of losartan on sympathetic nerve activity. In Fig. 2, HS + PVN aCSF rats exhibited a 7.1-fold increase in renal sympathetic nerve activity (RSNA, % of max) and 3.3-fold increase in plasma noradrenaline (NE) when compared with control rats (P < 0.05). Bilateral PVN infusion of LOS declined to a 64.5% decrease RSNA and 54.7% plasma NE in high salt-induced hypertensive rats (P < 0.05). NS + PVN LOS did not attenuate RSNA when compared with NS + PVN aCSF.
Effect of losartan on PKCγ and AT1R positive neurons in PVN. In Fig. 3, immunohistochemistry and immunofluorescence results revealed that high salt diet respectively induced a 4.8-fold increase and 2.6-fold increase in the number of AT1R and PKCγ positive neurons compared with control rats (P < 0.05). Bilateral PVN infusion of LOS respectively declined to a 51.2% decrease and 60% decrease in the number of AT1R and PKCγ positive neurons in hypertensive rats (P < 0.05). NS + PVN LOS did not attenuate the number of AT1R and PKCγ positive neurons when compared with NS + PVN aCSF. immunohistochemistry and immunofluorescence results revealed that high salt diet respectively induced a 2.3-fold increase and 3.5-fold increase in the number of p-Rac1 positive neurons and superoxide compared with control rats (P < 0.05). Bilateral PVN infusion of LOS respectively declined to a 56.0% decrease and 53.1% decrease in the number of p-Rac1 positive neurons and superoxide in hypertensive rats (P < 0.05). NS + PVN LOS did not attenuate the number of p-Rac1 positive neurons and superoxide when compared with NS + PVN aCSF.
Effect of PKCγ (PKCγ siRNA) on mean arterial pressure (MAP). In Fig. 6, the MAP of HS + PVN scrambled siRNA group is also much higher than control animals (at Day 14, 160.6 ± 4.9 vs. 95.1 ± 3.1 mmHg, P < 0.05) at the end of experiment. Bilateral PVN microinjection of PKCγ siRNA significantly decreased the MAP compared to HS + PVN scrambled siRNA rat (at Day 14, 132.6 ± 3.4 vs. 163.5 ± 5.6 mmHg, P < 0.05). Normal salt diet groups did not show any significant change in MAP at the end of experiment.
Effect of PKCγ (PKCγ siRNA) on sympathetic nerve activity. In Fig. 7, HS + PVN scrambled siRNA rats exhibited a 4.1-fold increase in RSNA and a 2.5-fold increase in plasma NE when compared with control rats (P < 0.05). Bilateral PVN infusion of PKCγ siRNA declined to a 65.4% RSNA and 72% plasma NE in high salt-induced hypertensive rats (P < 0.05). NS + PVN PKCγ siRNA did not show any significant change in RSNA and plasma NE when compared with NS + PVN scrambled siRNA at the end of experiment. Effect of PKCγ (PKCγ siRNA) on PKCγ and AT1R positive neurons in PVN. In Fig. 8, high salt diet also induced respectively induced a 6.7-fold increase and 3.2-fold increase in the number of AT1R and PKCγ positive neurons compared with control rats (P < 0.05). However, bilateral PVN microinjection of PKCγ siRNA declined to 60% PKCγ positive neurons, and had no effect on the AT1R positive neurons in PVN in hypertensive rats at the end of experiment.

Effect of PKCγ (PKCγ siRNA) on p-Rac1 positive neurons and the level of Superoxide in
PVN. In Fig. 9, high salt diet also induced respectively induced a 1.3-fold increase and 3.7-fold increase in the number of p-Rac1 positive neurons and superoxide compared with control rats (P < 0.05). However, bilateral PVN microinjection of PKCγ siRNA respectively declined to 34% the number of p-Rac1 positive neurons and 70% superoxide in PVN in hypertensive rats at the end of experiment (P < 0.05). Fig. 10A-C, western blot results indicated that high salt-induced rats respectively induced a 9.6-fold (AT1R), 1.8-fold (PKCγ ) and 7.3-fold (GTP-Rac1, Pull-down Assay) increase, but 7.8-fold decrease (Cu/Zn-SOD) in the protein expression level (P < 0.05) in PVN compared with control rats. Bilateral PVN infusion of PKCγ siRNA in PVN respectively declined to 51% (AT1R), 31% (PKCγ ), and 49% (GTP-Rac1, Pull-down Assay) protein expression level, but increased Cu/Zn-SOD protein level to 32% compared with high salt-induced rats (P < 0.05). And administration of PKCγ siRNA in PVN had no effect on PVN levels of AT1R in salt-induced hypertensive rats. Bilateral PVN infusion of PKCγ siRNA in normal rat downregulated a 55% protein expression compared with normal rats with scrambled siRNA.

Effect of PKCγ (PKCγ siRNA) on protein expression levels of AT1R, PKCγ, p-Rac1 and Cu/ Zn-SOD in PVN. In
Effect o PKCγ (PKCγ siRNA) on mRNA levels of AT1R, PKCγ, and Cu/Zn-SOD in PVN. In Fig. 10D, RT-PCR results indicated that high salt-induced rats respectively induced a 10.1-fold (AT1R) and 8.4-fold (PKCγ ), but 7.2-fold decrease (Cu/Zn-SOD) in the protein expression level (P < 0.05) in PVN compared with control rats. Bilateral PVN infusion of PKCγ siRNA in PVN respectively declined to 31% (PKCγ ) mRNA level, but increased Cu/Zn-SOD mRNA level to 36% compared with high salt-induced rats (P < 0.05). And administration of PKCγ siRNA in PVN had no effect on PVN mRNA levels of AT1R in salt-induced hypertensive rats. Bilateral PVN infusion of PKCγ siRNA in normal rat downregulated a 52% mRNA level compared with normal rats with scrambled siRNA. Effect of losartan and PKCγ (PKCγ siRNA) on arterial pressure and heart rate (HR). The mean arterial pressure (MAP) and heart rate (HR) were measured with a pressure transducer (MLT0380, AD Instruments, Australia) via a catheter in the right carotid artery. The MAP and HR in high salt-induced hypertensive rats were significantly higher than that in normal salt diet rats. There was no significant difference in the body weight between hypertensive rats and control rats. Chronic PVN infusion of LOS and PKCγ siRNA attenuated MAP and HR in hypertensive rats, but not in normal salt diet rats ( Table 1).

Effect of losartan and PKCγ (PKCγ siRNA) on oxidative stress in PVN. Compared with NS rats,
HS + PVN aCSF and HS + PVN scrambled siRNA groups had higher level of malondialdehyde (MAD) and NAD(P)H oxidase activity, but lower levels of superoxide dismutase (SOD) and glutathione (GSH) in PVN. Infusion of LOS or microinjection of PKCγ siRNA into the PVN attenuated the increased levels of MAD and NAD(P)H oxidase activity, but decreased levels of SOD activity and GSH in PVN of HS groups (Table 2).

Discussion
The novel finding of this study is that both AT1R and PKCγ play an important role in modulating the salt-induced central RAS activation and oxidative stress responses. And these results, for the first time, identified that the precise signaling pathway of RAS regulating ROS in PVN may be through AT1R/PKCγ /Rac1 pathway, which induces overproduction of ROS from Rac1-dependent NAD(P)H and finally increase sympathetic nerve activity and blood pressure. Therefore, PKCγ signaling pathway contributes to RAS modulating oxidative stress in PVN in the development of salt-induced hypertension.
PVN is a central integration site for the regulation of cardiovascular functions 2 . Substantial findings have confirmed high salt intake is a significant environmental factor, which strongly associated with RAS and ROS in the PVN 3,20 . Our study also showed that the activated RAS could regulate the reactive oxygen species overproduction in PVN in the progression of hypertension. As the primary effector peptide of the RAS, Ang II binding to AT1R contributes to the regulation of blood pressure and the development and/or the maintenance of metabolic syndrome. There is also increasing evidence showed that the augmented Ang II in brain induces the NAD(P)   stress in the cardiovascular and neurodegenerative diseases 22 . And PKCγ is a unique isoform of PKC that is found in neurons and eye tissues. Rac1 (Ras-related C3 botulinum toxin substrate1) is a small GTPase which is essential for the assembly and activation of NAD(P)H oxidase 23 . Growing evidence indicate that NOX/Rac1 activation is a main pathway in cardiovascular and cerebrovascular disorders such as myocardial infarction, hypertension, atherosclerosis and stroke 14 . Zimmerman M. C. (2004) had, for the first time, identified a Rac1-dependent NAD(P) H oxidase as the source of central Ang II-induced O 2 production, and implicated this oxidase in cardiovascular diseases associated with dysregulation of brain Ang II signaling, including hypertension 14 . Moreover, increasing evidences demonstrate that increased Ang II in brain induces the NAD(P)H-dependent ROS production by PKC is a critical step in Rac1 activation and subsequent enzyme assembly.
In this study, PVN microinjection of PKCγ siRNA could decrease the sympathetic nervous activity and mean arterial pressure, which indicates that PKCγ plays a significant role in the brain Ang II regulation of oxidative stress in the pathophysiology of high salt-induced hypertension. And our results also show that high salt diet not only elevated the PVN level of AT1R, PKCγ , Rac1, and NADPH oxidase-dependent superoxide in PVN, but also decreased the antioxidant capacity in PVN, therefore, increased the sympathetic nervous activity and mean arterial pressure. Chronic infusion of AT1R antagonist (LOS) into PVN decreased the PVN expression of AT1R, PKCγ , Rac1 and the level of superoxide in hypertensive rats, but increased the antioxidant capacity in PVN. Campese VM (2000) found LOS could stimulate the production of nitric oxide in PVN, and the nitric oxide could regulate the sympathetic outflow and reduces blood pressure levels 24 . This response is also consistent with our results. However, PVN microinjection of PKCγ siRNA suppressed NAD(P)H oxidase-dependent oxidative stress in PVN, decreased the PVN levels of PKCγ , Rac1 and NAD(P)H oxidase subunits but did not affect AT1R. So we would only speculate that AT1R could act on PKCγ and Rac1-dependent NAD(P)H oxidase and regulates the NAD(P)H oxidase-dependent oxidative stress in PVN, which implicated RAS regulating ROS in PVN may be through the AT1R/PKCγ /Rac1 signaling pathway and increase the sympathetic nervous activity and mean arterial pressure in the progression of salt-induced hypertension, which is presented in Fig. 11.  General experimental protocol. Rats were fed on the normal salt diet containing 0.3% NaCl (NS) or high salt diet containing 8% NaCl (HS) for 8 weeks to induce hypertension. After 8 weeks, animals from NS group and HS group are received bilateral PVN infusion of AT1R anantagonist losartan (LOS, 10 μ g/h) or artificial cerebrospinal fluid (aCSF), and bilateral PVN microinjection of PKCγ small interfering RNA (PKCγ siRNA), or scrambled siRNA for 2 weeks with high salt diet respectively 25 .

Implantation of bilateral PVN osmotic minipump for chronic infusion.
The method for PVN minipump has been previously described 8,26,27 . Each rat head was placed into a stereotaxic apparatus after anesthetized with ketamine (90 mg/kg) and xylazine (10 mg/kg) intraperitoneally (i.p). A skin incision was made, and the skull was opened. The location was 1.8 mm caudal to the bregma, 0.4 mm lateral to central line, and 7.9 mm below the skull surface (Chen et al., 2011; Zhu et al., 2004). The skull was then opened, and the minipumps (ALZET Osmotic Pumps, 2002 Model, 0.5 ul/h) connected with losartan (LOS, 10 μ g/h) or artificial cerebrospinal fluid (aCSF) for the continuous infusion were implanted subcutaneously in the back of the neck. The infusion was continued for 2 weeks. Rats received buprenorphine (0.01 mg/kg, sc) immediately following surgery. The success rate of bilateral PVN microinjection was around 68%. Bilateral PVN cannulae implantation for chronic infusion studies. The method for implantation of bilateral PVN cannulae has been described previously. Briefly, under anesthesia, rat head was placed into a stereotaxic apparatus. A stainless steel double cannula (Plastics One, Inc.) with a center-to-center distance of 0.5 mm, was implanted into the PVN using an introducer, according to stereotaxic coordinates (1.8 mm caudal to the bregma, 0.4 mm lateral to central line, and 7.9 mm below the skull surface) 10 . The cannula was fixed to the cranium using dental acrylic and two stainless steel screws. 50 nL of scrambled siRNA or small interfering RNAs for PKCγ (PKCγ siRNA) were microinjected into the bilateral PVN each side, which were completed within 1 min once per day. The success rate of bilateral PVN cannulation is about 65%.
Blood pressure measurement. Arterial pressure was measured noninvasively via tail-cuff instrument and their Recording System. Conscious rats from each group were warmed to an ambient temperature of 30 °C by placing them in a holding device mounted on a thermostatically controlled warming plate. Each rat was allowed to accommodate the cuff for 10 minutes before blood pressure measurement. The rat arterial pressure was measured every week during the 8 weeks and every day after LOS or siRNA infusion. Mean arterial pressure and heart rate data were consisted of 20 times, which were collected for 40 min within the same 2-hour time window each day and then averaged those data until the end of this study 28 . Sympathetic neural recordings. Rats were anaesthetized with a ketamine (90 mg/kg) and xylazine (10 mg/kg) mixture (ip). After retroperitoneal laparotomy, the left renal nerves were isolated via glass microelectrode technique under an inversion microscope. The renal nerve was hung by a platinum electrode which is connected to the recording system. In order to moisturize the nerves and isolate electrical disturbance, the nerve should be covered by paraffin oil tampons. Maximum renal sympathetic nerve activity (RSNA) was detected using an intravenous bolus administration of sodium nitroprusside (SNP, 10 mg). The recordings of rectified and integrated RSNA were analyzed using methods described as previously 8,26 .  Immunohistochemistry and immunofluorescence staining. Rats were anaesthetized and received a thoracotomy and were perfused through the left ventricle first with 300 mL of 0.01 M phosphate-buffered solution (PBS) at pH 7.4 and then with 300 mL of 4% paraformaldehyde. The brains were immediately removed and immersed in 4% paraformaldehyde, and then immersed in 30% sucrose for at least 2 days. Samples were embedded in OCT and microdissection procedures were used to isolate the PVN tissue. The tissues were collected from both sides of the PVN of individual rat and sectioned into several 18 mm transverse sections at about 1.80 mm from bregma and stored at − 80 °C for future use for immunohistochemistry and immunofluorescence staining 5 .