All animal experiments were approved by the Institutional Animal Care and Use Committee of Nanfang Hospital, Southern Medical University, and carried out in accordance with the ARRIVE guidelines (McGrath et al., 2010). The study protocol was approved by the Ethics Committee for experimental animals, Southern Medical University.

Male Sprague-Dawley rats (180–200 g) were maintained under a 12/12 h light/dark cycle at 22°C, and fed a standard laboratory chow ad libitum. The animals were randomly assigned to undergo sham operation or five-sixth nephrectomy to establish a renal hypertension model. Briefly, two-thirds of the left kidney was first removed; a week later, the right kidney was totally excised under pentobarbital anesthesia. The sham group only underwent de-capsulation of the kidneys.

Ten weeks after the final surgery, 5/6Nx rats were randomized into 7 groups (n = 6 per group): ➀no treatment; ➁intracerebroventricular injection (ICV) of artificial cerebrospinal fluid (aCSF) as the vehicle; ➂ICV of losartan (Sigma Chemical, 2.29 mmol/l/kg), an angiotensin II subtype 1 receptor (AT1R) antagonist; ➃ICV of farnesylthiosalicylic acid (FTS) (Cayman Chemical, 1 mmol/l/kg), a Ras inhibitor; ➄ICV of 2-(2-Amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059) (Sigma Chemical, 200 μmol/l/kg), a selective MEK inhibitor that effectively inhibits ERK1/2 phosphorylation; ➅ICV of 4-(4-Fluorophenyl)-2-(4-methylsulfinylpheyl)-5-(4-pyridyl)-1H-imidazole (SB203580) (Sigma Chemical, 200 μmol/l/kg), a p38MAP kinase inhibitor; ➆ICV of N-Benzyloxycarbonyl-Asp (OMe)-Glu (OMe)-Val-Asp- (OMe)-fluoro-methylketone (Z-DEVD-FMK) (Calbiochem, 1500 μmol/l/kg), a caspase-3 inhibitor. Sham operated rats (n = 6) with no treatment were used as normal controls.

ICV was performed with a stereotactic frame (David Kopf Instrument Inc., USA) after anesthesia with 3% pentobarbital sodium (0.15 mL/100 g body weight). A brain-infusion cannula (Brain Infusion Kit 2; ALZET Inc., USA) coupled to an osmotic pump (Model 2002; ALZET Inc., USA) was implanted into the cerebral ventricle. The coordinates were −1.0 mm posterior and 1.5 mm lateral from the midline, and 4.5 mm ventral, with respect to the bregma. Osmotic pumps were placed subcutaneously at the back of the neck. Following surgery, the wounds were carefully closed. The implanted osmotic pumps would continuously infuse aCSF or respective drugs into the lateral cerebral ventricle at 0.5 μl/h for 14 days.

Ten weeks after the final nephrectomy or sham operation, rats were weighted; 24-h urine samples were collected and urinary protein excretion was assessed by the Bradford method; blood pressure was determined with a pressure transducer (Gould) placed in the femoral artery and connected to a physiologic recorder (Gilson Medical Electronics) in anesthetized rats (Li et al., 2007). Serum creatinine levels were measured on an automatic biochemical analyzer (AU480, Beckman Coulter). Plasma norepinephrine concentrations were assessed using a competitive ELISA kit using TMB (3, 3, 5, 5-TetraMethyl benzidine solution liquid MeMbrane substrate) as a substrate and finally monitored at 450 nm. Moreover, the standard range and the sensitivity of the kit are 0.2–32 ng/ml and 1.3 pg/ml, respectively (Demeditec Diagnostics, DEE5200).

Two weeks after administration of aCSF or drugs, the above measurements were performed again. Then, all animals were anesthetized with 3% pentobarbital sodium (0.15 mL/100 g body weight) and sacrificed by cervical dislocation.

Some rats were transcardially perfused with 200 ml ice-cold normal saline followed by 400 ml 4% paraformaldehyde. Then, the brains were removed and sectioned, fixed for 6 h, and dehydrated in graded alcohol. Finally, the samples were paraffin embedded and sliced in 5 μm sections for immunochemistry. To identify the position of PVN, the brains were immediately removed and1-mm thick sections were cut using a cryostat. The PVN was defined and excised from 1-mm-sections on dry ice based on an rat brain atlas (Paxinos and Watson, 1998; Figure S1). PVNs were isolated from brains in accordance with the steps above, snap frozen in liquid nitrogen, and stored at −80°C for protein and RNA extraction.

Immunohistochemical assessment of RAS, p-ERK1/2, and Bax levels in PVN samples was performed with the avidin–biotin-peroxidase complex method. Primary antibodies were mouse anti-AGT monoclonal antibodies (1:500, Swant, Switzerland), rabbit polyclonal antibodies raised againstangiotensin II (1:400, Peninsula laboratories, USA), AT1R (1:100, Millipore, USA), and Bax (1:500, Santa Cruz, USA), and rabbit monoclonal antibodies targeting phospho-ERK1/2 (1:100, Cell Signaling Technology, USA), respectively. Biotinylated goat anti-rabbit/mouse secondary antibodies (1:200) were used for detection. Finally, sections were incubated with avidin–biotin–peroxidase complex (VECTASTAIN ABC Kit, USA) and peroxidase substrate solution (Vector Laboratories, USA) until optimal signal intensity developed. After dewaxing and hydration with graded ethanol, photographs were acquired under a microscope and analyzed using the Image-Pro Plus software. Finally, positive cells were counted in 10 low-power field at least three slices per group. Data were presented as positive cells per 10³ μm².

Immunoflourescent TUNEL staining was performed by following steps: The sections were rehydrated and treated with 0.1% Triton-100 for 30 min at room temperature, then applied 100 uL TUNEL reaction mix from the In Situ Cell Death Detection Kit (Roche 11684817910) in a dark humidified chamber at 37°C for 60 min prior to counterstaining tissue with 4′,6-diamidino-2-phenylindole (DAPI) nuclear dye.

PVN samples were homogenized in a lysis buffer and Western blot was performed as previously described (Su Z. et al., 2014). Specifically, Ras active protein (Ras-GTP) was extracted from total tissue lysates by adding 80 ug GST-Raf1-RBD per 500 ug sample and using Active Ras Pull-Down and Detection Kit (Thermo scientific, USA) and assessed by western blot immediately or stored at −20°C.

Primary antibodies used included the following: anti-AT1-R (1:200, Millipore, USA), anti-p-ERK1/2 (1:300, Cell Signaling Technology, USA), anti-p-p38 (1:500, Cell Signaling Technology, USA), anti-p38 (1:1000, Cell Signaling Technology, USA), anti-Bax (1:500, Santa Cruz Biotechnology), anti-Bcl-2 (1:200, Santa Cruz Biotechnology), anti-Cleaved caspase-3 (1:600, Cell Signaling Technology), anti-ERK1/2 (1:1000, Cell Signaling Technology, USA), anti-Ras (1:200, Abcam Cambridge, UK), anti-β-actin (1:5000, Cell Signaling Technology), anti-GAPDH (1:1000, Cell Signaling Technology). The levels of the above proteins were determined as previously described (Su Z. et al., 2014). AT1-R protein content was normalized to GAPDH amounts. Ras, p-ERK1/2, and p-p38 levels were normalized to total Ras, ERK1/2 and p38, respectively. Bax, Bcl-2 and cleaved caspase-3 protein levels were normalized to β-actin.

Total RNA from PVN samples was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA concentrations were measured on a Nanodrop ND1000 spectrophotometer (NanoDrop technologies, Wilmington, DE, USA). Reverse transcription was performed with a One-Step RT-PCR kit (TaKaRa, Kyoto, Japan) according to the manufacturer's protocol. Quantitative real-time PCR was performed in a total volume of 25 μl in duplicate on a Fast Real-Time PCR system 7500 (Applied Biosystems, CA, USA) with the SYBR Premix Ex Taq kit (TaKaRa, Kyoto, Japan). Thermal cycling conditions comprised 30 s at 95°C, followed by 95°C for 5 s, 60°C for 30 s, and 72°C for 60 s for 40 cycles. The relative expression levels of targeted mRNAs were calculated after normalization to GAPDH mRNA using the 2^−ΔΔCT method. The primer sets used are summarized in Table 1.

All analyses were performed with SPSS 19.0 for Windows. Data are mean ± standard error of the mean (SEM). Continuous variables between groups were compared by one-way analysis of variance, followed by Least Significant Difference (LSD test). The level of statistical significance was set at 0.05.

Ten weeks after the 5/6 nephrectomy (5/6 Nx), body weight, 24 h urinary protein excretion, systolic blood pressure, serum creatinine, and plasma norepinephrine in model rats were evaluated, in comparison with sham-operated controls (Table S1, p < 0.05). Plasma norepinephrine concentrations were used as an indicator of sympathetic nerve activity (Zoccali et al., 2002). As shown in Table S1, 5/6Nx rats showed similar body weights, but significantly higher 24 h urine protein excretion as well as systolic blood pressure, serum creatinine, and plasma norepinephrine (p < 0.05). These findings indicated a successful establishment of a rat model of chronic renal hypertension.

There were no differences among rats with renal hypertension administered aCSF and various agents, in body weight and serum creatinine (Table 2, p < 0.05). Compared with the aCSF group, intracerebroventricular injection with losartan, FTS, PD98059, and Z-DEVD-FMKICV resulted in significantly decreased urinary protein, systolic blood pressure and plasma norepinephrine levels (Table 2, p < 0.05). However, the p38 inhibitor SB203580 showed no effects on the above parameters.

Immunochemistry, western blot and real-time PCR were used to assess the expression of AGT, Ang II, and AT1R. Compared with the values obtained for sham-operated rats, AGT, Ang II, and AT1R amounts in PVN from rats with renal hypertension were significantly higher (Figures 2A–D, p < 0.05). Western blot and immunochemistry were used to detect Ras-GTP, p-ERK1/2, and p-p38. Compared with sham-operated rats, the model animals showed significantly higher Ras-GTP, p-ERK1/2 and p-p38 protein levels in PVN samples (Figures 2E–H, p < 0.05).

Immunochemistry, western blot and real-time PCR showed that cleaved caspase-3 and Bax amounts in PVN from model rats were significantly higher compared with the values obtained for sham-operated rats Bcl-2 expression was reduced in the model rats, both at the gene and protein levels, although the latter did not reach statistical significance (Figures 3A–D, p < 0.05). TUNEL staining is a well-defined method for detecting apoptotic DNA fragmentation in tissue. Figure 3E demonstrated that TUNEL-positive cells apparently increased in the hypothalamus PVN of 5/6Nx rats compared to sham rats.

The observations after nephrectomy and subsequent drug treatments suggested that increased apoptosis of central nervous system neurons may play a crucial role in elevating blood pressure in case of kidney damage, possibly by relieving the constraints on the sympathetic nervous system; upregulated AT1R, Ras, ERK1/2, and caspase-3 signaling pathways in the CNS may constitute the underlying mechanisms at the molecular level. However, p38 MAPK activation appeared to play no decisive role in hypertension.

To determine the possible regulation mechanism of AT1R on blood pressure and sympathetic nerve activity, AT1R protein expression in PVN of rats with renal hypertension was assessed after intracerebroventricular injection with aCSF, losartan, FTS, PD98059, and Z-DEVD-FMK, by western blot. Interestingly, high AT1R levels in PVN of model rats were decreased only by ICV losartan (p < 0.05). Meanwhile, FTS, PD98059, SB203580, and Z-DEVD-FMK administered by ICV showed no effects on AT1R protein expression (Figure 4A).

Meanwhile, Ras activity was significantly lower in samples from model groups treated (ICV) with losartan and FTS (p < 0.05); however, ICV treatment with PD98059, SB203580, and Z-DEVD-FMK yielded no effects on Ras activity (Figure 4B). These findings demonstrated that AT1R regulated Ras activity, with the remaining blocking agents failing, in the PVN of rats with renal hypertension.

Interestingly, ERK1/2 phosphorylation was significantly lower in PVN samples from model rats that received ICV injection of losartan, FTS, and PD98059 (p < 0.05), with SB203580 and Z-DEVD-FMK showing no effects on ERK1/2 activity (Figure 4C). These findings demonstrated that both AT1R and Ras regulated ERK1/2 phosphorylation, unlike the remaining blocking agents, in the PVN of rats with renal hypertension.

The effects of these blocking agents were evaluated on markers of cell apoptosis. As shown in Figure 4, Bax and cleaved caspase-3 levels were significantly higher in PVN samples from model rats compared with Sham-operated animals, while Bcl-2 amounts were significantly lower. Meanwhile ICV injection of losartan, FTS, and PD98059 in rats with renal hypertension significantly reduced Bax amounts, and markedly increased Bcl-2 levels. In addition, ICV injection of losartan, FTS, PD98059, and Z-DEVD-FMK in rats with renal hypertension significantly reduced cleaved caspase-3 amounts (Figures 4D–F, p < 0.05).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.