Increased Apoptosis in the Paraventricular Nucleus Mediated by AT1R/Ras/ERK1/2 Signaling Results in Sympathetic Hyperactivity and Renovascular Hypertension in Rats after Kidney Injury

Abstract

Background: The central nervous system plays a vital role in the development of hypertension, but the molecular regulatory mechanisms are not fully understood. This study aimed to explore signaling in the paraventricular nucleus (PVN) which might contribute to renal hypertension. Methods: Renal hypertension model was established by five-sixth nephrectomy operation (5/6Nx) in male Sprague Dawley rats. Ten weeks afterwards, they were random assigned to no treatment, or intracerebroventricular injection (ICV) with artificial cerebrospinal fluid, losartan [angiotensin II receptor type 1 (AT1R) antagonist], farnesylthiosalicylic acid (Ras inhibitor), PD98059 (MEK inhibitor), or SB203580 (p38 inhibitor) and Z-DEVD-FMK (caspase-3 inhibitor). Before and after treatment, physiological and biochemical indices were measured. Immunohistochemistry, western blot and RT-PCR were applied to quantify key components of renin-angiotensin system, apoptosis-related proteins, Ras-GTP, and MAPKs in the PVN samples. TUNEL assay was used to measure the situ apoptosis in PVN. Results: The 5/6Nx rats showed significantly elevated systolic blood pressure, urinary protein excretion, serum creatinine, and plasma norepinephrine (p < 0.05) compared to sham rats. The expression of angiotensinogen, Ang II, AT1R, p-ERK1/2, or apoptosis-promoting protein Bax were 1.08-, 2.10-, 0.74-, 0.82-, 0.83-fold higher in the PVN of 5/6Nx rats, than that of sham rats, as indicated by immunohistochemistry. Western blot confirmed the increased levels of AT1R, p-ERK1/2 and Bax; meanwhile, Ras-GTP and p-p38 were also found higher in the PVN of 5/6Nx rats, as well as the apoptosis marker cleaved caspase-3 and TUNEL staining. In 5/6Nx rats, ICV infusion of AT1R antagonist, Ras inhibitor, MEK inhibitor or caspase-3 inhibitor could lower systolic blood pressure (20.8-, 20.8-, 18.9-, 14.3%-fold) together with plasma norepinephrine (53.9-, 57.8-,63.3-, 52.3%-fold). Western blot revealed that blocking the signaling of AT1R, Ras, or MEK/ERK1/2 would significantly reduce PVN apoptosis as indicated by changes of apoptosis-related proteins (p < 0.05). AT1R inhibition would cause reduction in Ras-GTP and p-ERK1/2, but not vice versa; such intervention with corresponding inhibitors also suggested the unidirectional regulation of Ras to ERK1/2. Conclusion: These findings demonstrated that the activation of renin-angiotensin system in PVN could induce apoptosis through Ras/ERK1/2 pathway, which then led to increased sympathetic nerve activity and renal hypertension in 5/6Nx rats.

Keywords: AT1R/Ras/ERK1/2; apotosis; paraventricular nucleus; renal hypertension; sympathetic activity.

Figure 1

Schematic diagram illustrates the apoptotic pathway being examined and where each drug acts. Losartan (angiotensin II receptor type 1 (AT1R) antagonist), FTS (farnesyl thiosalicylic acid, Ras inhibitor), PD98059 (MEK inhibitor), SB203580 (p38 inhibitor), Z-DEVD-FMK (caspase-3 inhibitor).

Figure 2

Expression levels of AGT, AngII, AT1R, Ras-GTP, p-ERK, and p-p38 in PVN of sham-operated and 5/6Nx rats. (A) Representative immunochemistry micrographs of samples from sham and 5/6Nx groups for AGT, AngII, and AT1R respectively (PVN nucleus in this picture was circled by dotted line). (B) Representative quantitative statistics of samples of sham and 5/6Nx groups for AGT, AngII, and AT1R, respectively. (C,D) Representative protein expression and mRNA level of AT1R were assessed in sham and 5/6Nx groups, by RT-PCR and western blot, with GADH used for normalization. (E,F) Ras-GTP and total Ras protein expression levels assessed by western blot and immunohisochemistry technique in sham and 5/6Nx groups, respectively. (G,H) p-ERK 1/2, ERK 1/2, p-P38, and P38 protein expression levels in sham and 5/6Nx groups. Data are mean ± SEM (n = 6 per group). ^*p < 0.05 vs. sham group. 5/6 Nx: five-sixth nephrectomy.

Figure 3

Apoptosis markers' change in PVN samples from rats with renal hypertension and sham operation. (A) C-caspase-3 protein levels in sham and 5/6Nx rats. (B) Bax and Bcl-2 protein levels in sham and 5/6Nx rats. (C) Representative immunochemistry micrographs and quantitative statistics of Bax in sham and 5/6Nx groups. (PVN nucleus in this picture was circled by dotted line). (D) Bax and bcl-2 mRNA levels in sham and 5/6Nx groups, by real-time PCR, with GADH used for normalization. (E) TUNEL staining in sham and 5/6Nx groups (PVN nucleus in this picture was circled by dotted line). Data are mean ± SEM (n = 6 per group). ^*p < 0.05 vs. sham group. 5/6 Nx: five-sixth nephrectomy. C-caspase-3, cleaved-caspase-3.

Figure 4

Protein levels RAS, Ras, ERK, and apoptosis markers in PVN samples after 14 days of blockade with various approaches in renal hypertension and sham rats. (A) AT1R protein levels in sham and 5/6Nx rats treated with intracerebroventricular injection (ICV) various inhibitors. (B) Ras-GTP and total Ras protein levels in sham and 5/6Nx rats treated with intracerebroventricular injection (ICV) various inhibitors. (C) p-ERK1/2 and ERK 1/2 protein levels in sham and 5/6Nx rats treated with intracerebroventricular injection (ICV) various inhibitors. (D) Cleaved caspase-3 protein levels in sham and 5/6Nx rats treated with intracerebroventricular injection (ICV) various inhibitors. (E) Bax protein levels in sham and 5/6Nx rats treated with intracerebroventricular injection (ICV) various inhibitors. (F) Bcl-2 protein levels in sham and 5/6Nx rats treated with intracerebroventricular injection (ICV) various inhibitors. Data are mean ± SEM (n = 6 per group). ^*p < 0.05 vs. sham group; ^#p < 0.05 vs. 5/6Nx+aCSF group. Losartan (Angiotensin II receptor type 1 (AT1R) antagonist), FTS (farnesyl thiosalicylic acid, Ras inhibitor), PD98059 (MEK inhibitor), SB203580 (p38 inhibitor), Z-DEVD-FMK (caspase-3 inhibitor).