Intracerebroventricular Infusion of the (Pro)renin Receptor Antagonist PRO20 Attenuates Deoxycorticosterone Acetate-Salt–Induced Hypertension

Abstract

We previously reported that binding of prorenin to the (pro)renin receptor (PRR) plays a major role in brain angiotensin II formation and the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Here, we designed and developed an antagonistic peptide, PRO20, to block prorenin binding to the PRR. Fluorescently labeled PRO20 bound to both mouse and human brain tissues with dissociation constants of 4.4 and 1.8 nmol/L, respectively. This binding was blocked by coincubation with prorenin and was diminished in brains of neuron-specific PRR-knockout mice, indicating specificity of PRO20 for PRR. In cultured human neuroblastoma cells, PRO20 blocked prorenin-induced calcium influx in a concentration- and AT₁ receptor–dependent manner. Intracerebroventricular infusion of PRO20 dose-dependently inhibited prorenin-induced hypertension in C57Bl6/J mice. Furthermore, acute intracerebroventricular infusion of PRO20 reduced blood pressure in both DOCA-salt and genetically hypertensive mice. Chronic intracerebroventricular infusion of PRO20 attenuated the development of hypertension and the increase in brain hypothalamic angiotensin II levels induced by DOCA-salt. In addition, chronic intracerebroventricular infusion of PRO20 improved autonomic function and spontaneous baroreflex sensitivity in mice treated with DOCA-salt. In summary, PRO20 binds to both mouse and human PRRs and decreases angiotensin II formation and hypertension induced by either prorenin or DOCA-salt. Our findings highlight the value of the novel PRR antagonist, PRO20, as a lead compound for a novel class of antihypertensive agents and as a research tool to establish the validity of brain PRR antagonism as a strategy for treating hypertension.

Hypertension is a risk factor for cardiovascular diseases, including stroke, myocardial infarction, congestive heart failure, and chronic kidney disease, and remains the number one cause of morbidity and mortality worldwide. Significant advances have been made in the treatment of hypertension through the use of diuretics, renin–angiotensin system (RAS) inhibitors, calcium channel blockers, β-blockers, and α-adrenoreceptor antagonists. Despite the availability of numerous antihypertensive medications, the blood pressure (BP) of many patients with hypertension remains uncontrolled. During 2011 to 2012, the estimated proportion of patients with hypertension whose BP was controlled (<140/90 mm Hg) was only 51.9% in the United States.¹ A majority of unresponsive patients exhibited increased sympathetic drive and displayed neurogenic components.^2,3

The brain RAS plays an essential role in neurogenic hypertension.^4,5 Angiotensin (Ang) II, the major bioactive peptide of the RAS, is synthesized locally in the brain and is regulated independently of peripheral Ang II.⁶ The (pro)renin receptor (PRR) is a newly discovered component of the RAS that is highly expressed in the brain.^7,8 We previously reported that the PRR plays a pivotal role in Ang II formation in the brain,⁹ where renin activity is extremely low.¹⁰ PRR knockdown in the subfornical organ of the brain attenuates Ang II–induced hypertension in human renin–angiotensinogen (RA) double-transgenic mice¹¹ and decreases brain Ang II formation.¹² Neuron-specific PRR knockout (PRR-KO) prevents deoxycorticosterone acetate (DOCA)-salt–induced hypertension by inhibiting brain Ang II formation.⁹ Taken together, these findings suggest that PRR might be a promising target in the treatment of neurogenic hypertension.

The renin inhibitor, aliskiren, does not alter renin or prorenin binding to the PRR.^13,14 The peptide, handle region peptide (HRP), derived from the handle region of prorenin, inhibits the conformational change and nonproteolytic activation of prorenin that occurs on binding to PRR.¹⁵ HRP was reported to prevent the development of diabetic nephropathy in rats without affecting hyperglycemia and induce regression of established diabetic nephropathy.¹⁶ HRP also attenuates the development of cardiac fibrosis in spontaneously hypertensive rats.¹⁷ However, the antagonistic effect of HRP on the PRR has not been consistently replicated in other laboratories,^13,14,18,19 casting doubt on the efficacy of this unique decoy peptide. Recent studies have even shown that HRP counteracts the beneficial effects of aliskiren on BP, coronary function, and cardiac hypertrophy in spontaneously hypertensive rats²⁰ and on vascular dysfunction in diabetic hypertensive rats,²¹ suggesting a partial agonistic effect of HRP on the PRR. Indeed, it has been shown that HRP increases phosphorylation of extracellular signal–regulated protein kinase 1 and 2 (ERK-1/2) in the retina.²²

In the present study, we report the development of a novel antagonistic peptide, PRO20, which successfully blocks the binding of prorenin to the PRR in both mouse and human tissues. More importantly, PRO20 reduces prorenin-induced calcium influx and ERK-1/2 phosphorylation in vitro, and attenuates hypertension induced by either prorenin or DOCA-salt, as well as genetic hypertension in RA mice.

Methods

An expanded Methods section is available in the online-only Data Supplement.

Generation of the PRR Peptide Ligand and Fluorescent Labeling

The first 20 amino acids of the mouse prorenin prosegment (L1PTRTATFERIPLKKMPSVR²⁰), termed PRO20, was synthesized by the Neo Peptide Company (Cambridge, MA). A separate PRO20 peptide fluorescently labeled at the N terminus with fluorescein isothiocyanate (PRO20-FITC) was synthesized by China Peptides Company (Shanghai, China).

Results

Novel PRR Antagonist Peptide Binds to the PRR in Mouse and Human Brain

Fluorescence microscopy analysis showed that PRO20-FITC bound to mouse brain paraventricular nucleus of hypothalamus (PVN; Figure 1A). PRO20-FITC (5 nmol/L) binding to mouse brain tissue was completely blocked by coincubation with unlabeled mouse prorenin (1 μmol/L; Figure 1B). Quantification and plotting of PRO20-FITC binding, expressed as relative fluorescence units (RFUs), for a series of PRO20-FITC concentrations yielded dissociation constants (K_d) of 4.6±2.2 nmol/L and maximum binding (B_max) of 24.0±4.1 RFU for mouse brain (Figure 1C). Similarly, PRO20-FITC bound to human PVN (Figure 1D) and coincubation with unlabeled human prorenin (1 μmol/L) eliminated PRO20-FITC binding to human brain tissue (Figure 1E). The K_d and B_max were 1.8±0.5 nmol/L and 119.5±15.3 RFU for human brain (Figure 1F). Thus, PRO20-FITC bound specifically and saturably to both mouse and human brain sections. In addition, PRO20-FITC binding was decreased in the PVN from neuron-specific PRR-KO mice (Figure S1 in the online-only Data Supplement). We noted that there is 30% to 40% of residual binding in PRR-KO mice. This is possibly because other cell types in the brain also express PRR,⁷ whereas PRR deletion is only in the neurons. The ependymal cells surrounding the third ventricle exhibited high PRO20 binding, reflecting the fact that these cells express high levels of the PRR.⁹ Importantly, PRO20 binding to non-neuronal ependymal cells was retained in PRR-KO mice further supporting the specificity of PRO20 for the PRR. PRO20-FITC binding was also detected in other brain regions involved in the central regulation of BP, including the supraoptic nucleus, rostral ventrolateral medulla, and nucleus of the solitary tract (Figure S2).

Figure 1.

Binding of fluorescently labeled PRO20 to mouse and human brain tissues. A and B, Representative images show the binding of PRO20-fluorescein isothiocyanate (PRO20-FITC; 5 nmol/L; green) in the mouse paraventricular nucleus of hypothalamus (PVN), with or without coincubation with unlabeled mouse prorenin (1 μmol/L). Cell nuclei are indicated by blue DAPI (4′,6-diamidino-2-phenylindole) staining. C, Fluorescence in the mouse PVN was quantified for a series of concentrations (1, 2, 5, 10, and 20 nmol/L) of PRO20-FITC (n=5–6/concentration). D and E, Representative images show the binding of PRO20-FITC (1 nmol/L) in the human PVN, with or without coincubation with unlabeled human prorenin (1 μmol/L). F, Fluorescence in the human PVN was quantified for a series of concentrations (0.2, 0.5, 1, 2, and 5 nmol/L) of PRO20-FITC (n=5–6/concentration). Relative fluorescence unit (RFU) was calculated by subtracting the background from the total fluorescence density in each image quantified by Image J (Version 1.48).

PRO20 Prevents Prorenin-Induced Calcium Influx in SH-SY5Y Cells

ATP (1 μmol/L) increased intracellular calcium in SH-SY5Y cells (Figure 2A and 2B). Ang II (10 nmol/L) also increased intracellular calcium levels, an effect that was blocked by the angiotensin receptor antagonist, losartan (1 μmol/L; Figure 2A and 2B). Application of human prorenin (4 nmol/L) increased neuronal intracellular calcium. This effect was abolished by removal of extracellular calcium but was unaffected by depletion of intracellular calcium stores with cyclopiazonic acid (10 μmol/L; Figure S3). These data indicate that prorenin induced calcium influx in neurons. A previous study showed that prorenin at a concentration of 4 nmol/L increased Ang II–dependent plasminogen-activator inhibitor 1 release.²³ In our study, prorenin at this concentration induced calcium influx that was blocked by losartan (1 μmol/L) and captopril (1 μmol/L; Figure 2C and 2D), demonstrating involvement of the Ang II type 1 receptor (AT₁R) and Ang II formation in prorenin-induced neuronal calcium influx. Scrambled peptide (1 μmol/L) had no effect on prorenin-induced calcium influx (Figure 2E). PRO20 at a concentration of 0.12 μmol/L partially inhibited prorenin-induced calcium influx and of 1.2 μmol/L completely blocked the response (Figure 2E). Concentration-response curves showed that the half-maximal inhibitory concentration (IC₅₀) for PRO20 inhibition of prorenin-induced (4 nmol/L) calcium influx was 81 nmol/L (Figure 2F). Small inhibitory RNA–mediated knockdown of PRR expression in SH-SY5Y cells eliminated the prorenin-induced calcium influx (Figure S4). PRO20 had no effect on human renin or ATP-induced calcium influx in SH-SY5Y cells (Figure S5). Taken together, these data suggest that the novel PRR inhibitor, PRO20, inhibits prorenin-induced calcium influx in human neurons by blocking prorenin binding to the PRR thereby preventing its enzymatic activation.

Figure 2.

PRO20 prevents human prorenin–induced calcium influx in SH-SY5Y cells. A, Representative traces of changes in fluorescence (ΔRFU) compared with time for SH-SY5Y cells loaded with the calcium-indicator Fluo-4-AM in response to application of ATP (1 μmol/L), angiotensin II (Ang II; 10 nmol/L), or Ang II + losartan (1 μmol/L). The arrow indicates drug injection time. B, Summary data for experiments shown in A (n=7–11/group; *P<0.05 vs control; #P<0.05 vs Ang II). C, Representative traces of changes in fluorescence induced by human prorenin (4 nmol/L), prorenin + captopril (1 μmol/L), or prorenin + losartan (1 μmol/L). D, Summary data for experiments shown in C (n=7–11/group; *P<0.05 vs control, #P<0.05 vs prorenin). E, Representative traces of changes in fluorescence induced by human prorenin + scrambled peptide (1 μmol/L), and prorenin with or without PRO20 (0.12 and 1.2 μmol/L). F, PRO20 inhibited prorenin (4 nmol/L)-induced calcium influx in a concentration-dependent manner.

Antihypertensive Effect of PRO20 on Hypertension Induced by Acute Intracerebroventricular Infusion of Prorenin

We previously reported that intracerebroventricular infusion of mouse prorenin at a concentration of 2.4 μmol/L increases BP in conscious wild-type C57Bl/6J mice.⁹ The concentrations of prorenin in brain were ≈2 to 25 ng/g of tissue at the end of the 10-minute infusion. Under these experimental conditions, prorenin induced an Ang II–dependent increase in BP by binding to the PRR. Although the prorenin concentrations after intracerebroventricular infusion were higher than physiological levels (1–2 ng/g of tissue) in the brain, they approached levels seen in DOCA-salt hypertension (2–5 ng/g of tissue).⁹ Thus, intracerebroventricular infusion of prorenin induces acute hypertension, which can be used to assess the antagonistic effect of PRO20 on prorenin binding to PRR. As shown in Figure 3A, intracerebroventricular infusion of mouse prorenin (2.4 μmol/L, 3 μL/min for 10 minutes) together with scrambled peptide (100 μmol/L) induced a rapid increase in BP. PRO20 (100 μmol/L) prevented this prorenin-induced hypertension (Figure 3B). The antihypertensive effect of intracerebroventricular-infused PRO20 was dose-dependent (Figure 3C), with dose-response curves showing that IC₅₀ for PRO20 inhibition of prorenin-induced (2.4 μmol/L) hypertension was 7.9 μmol/L (Figure 3D). In addition, intracerebroventricular infusion of PRO20 had no effect on Ang II–induced pressor responses (Figure S7).

Figure 3.

PRO20 attenuates pressor responses induced by intracerebroventricular infusion of prorenin. A, Real-time blood pressure (BP) trace recorded before and during intracerebroventricular infusion of mouse prorenin (2.4 μmol/L) + scrambled peptide (100 μmol/L). Arrows indicate the beginning of the 10-minute intracerebroventricular infusion. B, Real-time BP trace recorded before and during intracerebroventricular infusion of mouse prorenin (2.4 μmol/L) + PRO20 (100 μmol/L). Arrows indicate the beginning of the 10-minute intracerebroventricular infusion. C, Changes in mean arterial pressure (ΔMAP) induced by intracerebroventricular infusion of mouse prorenin (2.4 μmol/L) or prorenin + PRO20 (1.8, 5.3, 16, 48, and 144 μmol/L; *P<0.05 vs prorenin). D, PRO20 inhibited mouse prorenin (2.4 μmol/L)-induced increases in MAP in a dose-dependent manner (n=5/concentration).

Acute Intracerebroventricular Infusion of PRO20 Lowers BP in Established Ang II–Dependent and DOCA-Salt–Induced Hypertension

Brain RAS overactivity is an important mechanism underlying both Ang II–dependent (RA mice)²⁴ and DOCA-salt–induced hypertensions.^24,25 For example, intracerebroventricular infusion of losartan totally prevented the development of DOCA-salt hypertension.⁹ Interestingly, although RA mice have higher Ang II levels in the circulation, systemic administration of the angiotensin-converting enzyme inhibitor enalapril did not normalize BP in these mice.²⁶ However, intracerebroventricular-injected AT₁R blocker to the RA mice reduced BP in these animals to nearly baseline values of control mice, suggesting activation of the brain RAS plays a major role in the maintenance of elevated BP in this model.²⁴ To test whether PRR antagonism blocks hypertension, we intracerebroventricularly infused PRO20, a scrambled peptide, or losartan in genetically hypertensive RA mice and in mice with established DOCA-salt hypertension. Intracerebroventricular infusion of a scrambled peptide had no effect on BP in either RA mice (Figure 4A) or DOCA-salt hypertensive mice (Figure 4B). However, intracerebroventricular infusion of PRO20 rapidly reduced BP in both RA mice (Figure 4C and 4E) and DOCA-salt hypertensive mice (Figure 4D and 4F). This anti-hypertensive effect of PRO20 lasted ≈10 minutes after intracerebroventricular infusion. Acute intracerebroventricular infusion of losartan also reduced mean arterial BP (MAP) in established hypertension in both RA (Figure 4E) and DOCA-salt–treated mice (Figure 4F). Intracerebroventricular infusion of PRO20 or losartan did not alter MAP in normotensive, SHAM mice (Figure 4F). The short duration of the effect suggests that PRO20 is rapidly metabolized in the mouse brain. This might partially explain why the in vivo IC₅₀ of PRO20 is much higher than the in vitro K_d.

Figure 4.

Intracerebroventricular infusion of PRO20 lowers blood pressure (BP) in mice with established angiotensin (Ang II)-dependent and deoxycorticosterone acetate (DOCA)-salt–induced hypertension. Real-time BP trace recorded before, during, and after intracerebroventricular infusion of (A) scrambled peptide (1 mmol/L) in renin–angiotensinogen (RA) double-transgenic mice, (B) scrambled peptide (1 mmol/L) in DOCA-salt hypertensive mice, (C) PRO20 (1 mmol/L) in RA mice, and (D) PRO20 (1 mmol/L) in DOCA-salt hypertensive mice. Arrows indicate the beginning of the 10-minute intracerebroventricular infusion. E, Summary data showing changes in mean arterial pressure (ΔMAP) in response to a 10-minute intracerebroventricular infusion of scrambled peptide (1 mmol/L), PRO20 (1 mmol/L), or losartan (3 mmol/L) in hypertensive RA mice (*P<0.05 vs RA + scrambled; n=4). F, ΔMAP during intracerebroventricular infusion of scrambled peptide (1 mmol/L), PRO20 (1 mmol/L), or losartan (3 mmol/L) in DOCA-salt hypertensive mice (*P<0.05 vs DOCA + scrambled; n=4 in the SHAM group, n=5 in the DOCA group).

Chronic Intracerebroventricular Infusion of PRO20 Attenuates the Development of DOCA-Salt Hypertension

DOCA-salt treatment gradually increased MAP in mice receiving intracerebroventricular infusion of artificial cerebro-spinal fluid (aCSF) plateauing at 134±3.6 mm Hg (Figure 5A). Chronic intracerebroventricular infusion of PRO20 attenuated DOCA-salt–induced hypertension (113±3.3 mm Hg), whereas subcutaneous infusion of PRO20 had no effect on DOCA-salt–induced hypertension. Heart rate (HR) did not differ significantly between intracerebroventricular PRO20- and aCSF-treated groups (Figure 5B).

Figure 5.

Chronic intracerebroventricular infusion of PRO20 attenuates the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Daily mean arterial pressure (MAP; A) and heart rate (HR; B) after chronic intracerebroventricular (ICV) infusion of artificial cerebrospinal fluid (aCSF; n=9) or PRO20 (n=9), and subcutaneous (SC) infusion of PRO20 (n=5) at baseline and after DOCA-salt treatment for 21 days. C, Maximum changes in HR in response to intraperitoneal propranolol (5 mg/kg; cardiac sympathetic tone). D, Maximum changes in MAP in response to intraperitoneal chlorisondamine (6 mg/kg; vasomotor sympathetic tone). E, Maximum changes in HR in response to intraperitoneal methylatropine (1 mg/kg; cardiac parasympathetic tone). F, Spontaneous baroreflex sensitivity (SBRS) in mice with intracerebroventricular-administered aCSF or PRO20 at baseline and 21 days after DOCA-salt treatment. G, Intrinsic HR recorded after injection of propranolol (5 mg/kg, IP) or methylatropine (1 mg/kg, IP). H, Ang II levels in the cerebral cortex (Cor), hypothalamus (Hypo), and brain stem (BS) of mice in SHAM, DOCA-salt + intracerebroventricular aCSF, and DOCA-salt + intracerebroventricular PRO20 groups (*P<0.05 vs baseline aCSF intracerebroventricular and #P<0.05 vs DOCA + aCSF intracerebroventricular in C, D, E, and F; *P<0.05 vs SHAM and #P<0.05 vs DOCA + aCSF intracerebroventricular in H).

At baseline, cardiac sympathetic tone (ΔHR_symp), vasomotor sympathetic tone (ΔBP_symp), cardiac parasympathetic tone (ΔHR_para), and spontaneous baroreflex sensitivity were similar between aCSF- and PRO20-treated groups: ΔHR_symp, −52.4±5.8 versus −57.2±5.4 bpm (Figure 5C); ΔBP_symp, −32.6±4.7 versus −31.4±2.4 mm Hg (Figure 5D); ΔHR_para, 142.8±9.8 versus 144.2±12 bpm (Figure 5E); spontaneous baroreflex sensitivity, 2.9±0.2 versus 3.1±0.2 ms/mm Hg (Figure 5F). DOCA-salt treatment significantly increased ΔHR_symp (−115.8±15.8 bpm) and ΔBP_symp (−71.6±4.6 mm Hg), and decreased ΔHR_para (62.9±6.5 bpm) and spontaneous baroreflex sensitivity (1.4±0.3 ms/mm Hg) in aCSF-treated mice compared with baseline. Chronic intracerebroventricular infusion of PRO20 attenuated the increase in ΔHR_symp (−77.8±14.8 bpm) and ΔBP_symp (−44.2±6.6 mm Hg), and improved ΔHR_para (119.3±9 bpm) and spontaneous baroreflex sensitivity (2.6±0.3 ms/mm Hg) during DOCA-salt treatment, without affecting intrinsic HR (Figure 5G).

DOCA-salt treatment for 3 weeks significantly increased brain Ang II levels in the cortex (421±80 versus 150±32 pg/g), hypothalamus (1372±88 versus 380±60 pg/g), and brain stem (1308±288 versus 456±33 pg/g) compared with sham treatment (Figure 5H). PRO20 treatment for 3 weeks significantly reduced Ang II levels in DOCA-salt–treated mice in the cortex (322±88 versus 421±80 pg/g) and hypothalamus (904±47 versus 1372±88 pg/g) compared with aCSF-infused DOCA-salt–treated mice. Ang II levels in the brain stem (1226±197 versus 1308±288 pg/g) were not affected by treatment with PRO20.

PRO20 Prevents Direct Prorenin-Induced ERK-1/2 Phosphorylation in Neuro-2A Cells

Neuro-2A cells were cultured in serum-free medium overnight before treatment. Incubation of cells with prorenin alone markedly increased ERK-1/2 phosphorylation, which was partially blocked by losartan (Figure S8), suggesting that prorenin-induced ERK-1/2 phosphorylation in Neuro-2A cells was both Ang II dependent and independent. To exclude Ang II–mediated signaling effects and examine the Ang II–independent direct prorenin/PRR signaling, we pretreated cells with 10 μmol/L losartan for 30 minutes before treatment for 15 minutes with prorenin (4 nmol/L), HRP (1 μmol/L), or PRO20 (1 μmol/L). Total ERK-1/2 and phosphorylated ERK-1/2 were determined by Western blotting. The effect of prorenin on ERK-1/2 phosphorylation in cells with PRR knockdown was also assessed in cells transfected with adeno-associated virusΔPRR-shRNA for 3 days. As shown in Figure 6, prorenin increased ERK-1/2 phosphorylation in the presence of losartan. This effect was prevented by PRO20 treatment or PRR knockdown, but not by HRP. Phosphorylated ERK-1/2 levels were unaffected by treatment with PRO20 or HRP alone.

Figure 6.

Phosphorylated extracellular signal-regulated protein kinase (ERK) 1 and 2 (P-ERK-1/2) and total ERK (T-ERK-1/2) in Neuro-2A cells. Representative Western blots showing P-ERK-1/2 (upper blot) and T-ERK-1/2 (lower blot) following the indicated treatments. Quantification of the blots indicates that prorenin increased ERK-1/2 phosphorylation. PRO20 or prorenin receptor knockdown (PRR-KO) prevented prorenin-induced ERK-1/2 phosphorylation; however, handle region peptide (HRP) had no effect (n=4/group; *P<0.05 vs losartan treatment; #P<0.05 vs losartan + prorenin treatment).

Discussion

Although genetic knockout studies have shown the importance of the brain PRR in the regulation of BP and the local generation of Ang II,^9,11,12 no PRR antagonists have been developed to assess the physiological and pathophysiological significance of the PRR until now. In this study, we describe a specific PRR ligand (PRO20) that blocks activation of prorenin by binding to the PRR. The main findings of this study can be summarized as follows: (1) PRO20 blocks the binding of prorenin to the PRR in both the mouse and human brain; (2) PRO20 prevents human prorenin–induced calcium influx in human neuronal cells; (3) acute intracerebroventricular infusion of PRO20 attenuates prorenin-induced hypertension and reduces MAP in hypertensive mice; (4) chronic intracerebroventricular infusion of PRO20 attenuates the development of DOCA-salt–induced hypertension and decreases brain Ang II formation; and (5) PRO20 prevents prorenin-induced ERK-1/2 phosphorylation in mouse Neuro-2A cells.

The design of PRO20 (first 20 amino acids of the prorenin prosegment) was based on previous findings.^15,27 Studies to determine the physical structure of prorenin have identified multiple possible binding sites to the PRR but to our knowledge, only HRP has been developed for in vivo tests. Previous reports have suggested that HRP is capable of acting as an antagonist of the PRR, preventing diabetic nephropathy¹⁶ and cardiac fibrosis in stroke-prone, spontaneously hypertensive rats.¹⁷ However, HRP has no direct inhibitory effect on the binding of prorenin to the PRR.¹⁴ PRO20 contains most of the previously reported PRR binding sites in prorenin.²⁷ In addition, the N terminus of PRO20 is in close proximity to a previously identified PRR binding domain in (pro)renin (S149QGVLKEDVF158).²⁸ We hypothesized that a 3D conformation of prorenin reveals the PRR binding site. PRO20, as part of this 3D conformation proposed binding site, would thus act as a competitive antagonist of prorenin binding to the PRR. Using PRO20-FITC, we tested the binding affinity and specificity of PRO20 to the PRR in both the mouse and human brain, where the PRR is highly expressed.^11,29 PRO20 binding was completely blocked by coincubation with unlabeled mouse or human prorenin, suggesting that the binding of PRO20 to the PRR is specific. In addition, brain sections from neuron-specific PRR-KO mice bound less PRO20-FITC in the PVN, but not in ependymal cells which further confirms the specificity of PRO20 for the PRR.

It is known that the modulatory action of Ang II on intra-cellular calcium is critical for its intracellular signaling pathways in neurons.^30,31 Consistent with these previous reports, we found that Ang II increased intracellular calcium in a human neuronal cell line (SH-SY5Y). More importantly, we found that human prorenin induced calcium influx to a degree similar to that of Ang II. This increase was completely blocked by the AT₁R blocker, losartan, or the angiotensin-converting enzyme inhibitor, captopril, suggesting that human prorenin induces calcium influx through generation of Ang II. Prorenin can only be converted to active renin in the juxtaglomerular cells of the kidney³²; therefore, it is unlikely that the effects of intracerebroventricular-infused prorenin are attributable to its conversion to renin. Instead, we propose that the effects of prorenin arise from nonproteolytic activation of prorenin via binding to the PRR.¹³ This conclusion is supported by our previous finding that binding of prorenin to the PRR is the major pathway of Ang II formation by neurons.⁹ In the present study, PRO20 blocked prorenin-induced calcium influx in SH-SY5Y cells in a concentration-dependent manner, suggesting that PRO20 competes with prorenin for binding to the PRR. Renin-induced calcium influx (Figure S4) was not inhibited by PRO20, consistent with the fact that PRO20 was designed to specifically block the binding of prorenin, but not renin to PRR. In addition, renin can metabolize angiotensinogen independently of the PRR.

Binding of prorenin to the PRR also initiates Ang II–independent intracellular signaling via mitogen-activated protein kinases, ERK-1/2, P38, and c-Jun N-terminal kinase, increasing the synthesis of profibrotic molecules, such as plasminogen activator inhibitor-1, fibronectin, collagen, and transforming growth factor-β.³³ Transgenic rats overexpressing human PRR develop proteinuria and a progressive nephropathy.³⁴ The glomeruli of these transgenic rats show an increase in ERK-1/2, P38, and c-Jun N-terminal kinase activity, despite normal renal Ang II levels, suggesting a direct pathological role of the PRR in renal damage. Increased plasma prorenin levels in human diabetic patients precede the onset of microalbuminuria and predict microvascular complications.³⁵ Increased prorenin may directly trigger end organ damage by activating the PRR through Ang II–independent signaling pathways. In the present study, we found that prorenin increased Ang II–independent ERK-1/2 phosphorylation in Neuro-2A cells and showed that PRO20 prevented this increase, suggesting that blocking activation of the PRR by PRO20 may exert beneficial effects by reducing damage in end organs where prorenin is increased. The data suggest that PRO20 is a competitive inhibitor of prorenin binding to the PRR, devoid of any agonism of PRR signaling in contrast to HRP.^21,22

The DOCA-salt hypertension model is commonly used to mimic hypertension associated with low circulating renin levels, accounting for 27% of human cases of essential hypertension.³⁶ The combination of DOCA and high salt markedly suppresses the systemic RAS,^37,38 whereas brain RAS signaling is elevated in this model.^39,40 We previously reported that neuron-specific PRR-KO prevents the development of DOCA-salt hypertension by decreasing brain Ang II formation.⁹ Chronic intracerebroventricular infusion of PRO20 also attenuated the development of DOCA-salt hypertension and reduced the increase in Ang II in the hypothalamus and cerebral cortex. However, PRO20 had no effect on the increased Ang II formation in the brain stem of DOCA-salt–treated mice. The brain stem contains autonomic nuclei, including the solitary tract nucleus, rostral ventrolateral medulla, and area postrema, which play important roles in the regulation of autonomic function and BP. Activation of AT₁R by Ang II in these regions contributes to the regulation of BP.^41,42 In our previous report, neuron-specific PRR-KO prevented the development of DOCA-salt hypertension and reduced the increase in Ang II in the brain stem, as well as the cortex, and hypothalamus. In the present study, PRO20 did not completely prevent DOCA-salt hypertension; and Ang II levels in the brain stem were not significantly reduced. These data support a role for angiotensinergic neurons in the brain stem for maintenance of BP in DOCA-salt hypertension. The inability of PRO20 to inhibit Ang II formation in the brain stem may reflect a low level of PRO20 reaching the brain stem from intracerebroventricular infusion. In addition, inhibition of Ang II formation in the PVN attenuates the development of hypertension by reducing proinflammatory cytokines in the PVN and restoring the balance of excitatory and inhibitory neurotransmitters.⁴³ In agreement with this report, we found that intracerebroventricular-infused PRO20 decreased Ang II formation in the hypothalamus along with attenuation of DOCA-salt hypertension. Our findings point to the notion that Ang II signaling from the hypothalamic regions or forebrain regulates presympathetic neurons in the brain stem, affecting angiotensinergic and non-angiotensinergic pathways as proposed in Figure 7. Taken together, these data suggest that PRO20 inhibits activation of the brain RAS during DOCA-salt hypertension. Notably, infusion of PRO20 subcutaneously had no effect on DOCA-salt hypertension, reflecting the central nervous system (CNS) locus of this hypertensive model. This interpretation is supported by a previous study showing that systemic RAS blockade has no effect on DOCA-salt hypertension.⁴⁴

Figure 7.

Proposed pathways for angiotensinergic neurons in the regulation of sympathetic activity. In presympathetic neurons, prorenin (PR) binds intracellular (pro)renin receptor (PRR), leading to intracellular formation of angiotensin II (Ang II), which is subsequently secreted into the extracellular space. Alternatively, extracellular prorenin binds to the PRR on the neuronal membrane and metabolizes extracellular angiotensinogen (AGT) secreted by astrocytes or neurons to generate Ang I. Angiotensin-converting enzyme (ACE), located on the external surface of cell membranes or in the interstitial fluid, converts Ang I to Ang II. The intracellular Ang II can be transported to axon terminals to act as a neurotransmitter. Extracellular Ang II binds to the angiotensin II type 1 receptor (AT₁R) to modulate neuronal activity and neurotransmitter release at the synapse. PVN indicates paraventricular nucleus of hypothalamus; and RVLM, rostral ventrolateral medulla nucleus.

In this present study, we examined the antihypertensive effect of PRO20 in 3 different hypertensive models namely, intracerebroventricular prorenin-induced, DOCA-salt–induced, and RA mice. Intracerebroventricular infusion of losartan abolishes prorenin-induced hypertension, and totally prevents the development of DOCA-salt hypertension.⁹ Intracerebroventricular-injected AT₁R blocker reduced BP in RA mice to nearly baseline values in control mice.²⁴ Taken together, RAS activation and Ang II–dependent signaling in the brain are the major mechanisms in these 3 hypertensive models. Because prorenin activation is the first step to form Ang II inside the brain, the antihypertensive effect of intracerebroventricular infusion of PRO20 to block prorenin binding to the PRR is likely attributable to the blockade of Ang II–dependent signals. We show in this report that PRO20 blocks Ang II–independent ERK-1/2 phosphorylation in vitro; however, to our knowledge, it is not clear whether this pathway in the CNS is responsible for the development of hypertension. Future studies will investigate the significance of Ang II–independent PRR signaling in the CNS in BP regulation.

All RAS components are found in the CNS, including the PRR,¹¹ (pro)renin,⁹ angiotensinogen,⁴⁵ angiotensin-converting enzyme,⁴⁶ Ang II,⁴⁷ and AT₁R,⁴⁸ and at low levels, renin.⁴⁹ We previously showed that the neuronal PRR is present in the cell membrane, as well as in the cytoplasm.⁹ Accordingly, we proposed 2 pathways by which activation of the PRR initiates RAS activation and Ang II formation in the neurons. In the first, prorenin binds and activates intracellular PRR, leading to intracellular formation of Ang II, which is subsequently secreted into the extracellular space. In the second, extracellular prorenin binds to the PRR on the cell surface using extracellular angiotensinogen secreted by astrocytes or neurons leading to Ang II formation. In both cases, Ang II is present in the extracellular fluid and binds to AT₁R to modulate neuronal activity (Figure 7).

In conclusion, we have generated a new PRR ligand that blocks the binding of prorenin to PRR in both human and mouse brain tissues. PRO20 attenuates prorenin and DOCA-salt–induced hypertension, as well as transgenic (RA mouse) Ang II–dependent hypertension. PRO20 inhibits direct activation of PRR by prorenin in cultured neuronal cells. Blockade of prorenin binding to the PRR in the CNS may represent a novel approach for treating hypertension and other cardiovascular diseases involving brain RAS activation.

Perspectives

Previous studies have shown that binding of prorenin to the PRR is the main pathway for Ang II formation in the brain, and knockdown of the brain PRR attenuates the development of hypertension in different hypertensive models. However, no effective pharmacological PRR antagonists have been available for studying the physiological and pathophysiological significance of the PRR beyond genetic knockdown studies. In the present study, we developed a specific PRR antagonist (PRO20) that prevents the binding of prorenin to the PRR. We demonstrated that PRO20 prevents human prorenin–induced calcium influx in human neuronal cells and mouse prorenin-induced ERK-1/2 phosphorylation in mouse neuronal cells. Acute intracerebroventricular infusion of PRO20 attenuates prorenin-induced hypertension and reduces MAP in mice with established hypertension. Chronic intracerebroventricular infusion of PRO20 attenuates the development of DOCA-salt–induced hypertension, diminishes Ang II generation, and improves autonomic function. These findings support the potential of PRR antagonists as a novel means for treating neurogenic hypertension.

Novelty and Significance.

What Is New?

• PRO20 blocks the binding of prorenin to (pro)renin receptor (PRR) in both mouse and human brain tissues.

• PRO20 prevents human prorenin–induced calcium influx in human neuronal cells.

• Acute intracerebroventricular infusion of PRO20 attenuates prorenin-induced hypertension and reduces mean arterial pressure in hypertensive mice.

• Chronic intracerebroventricular infusion of PRO20 attenuates the development of deoxycorticosterone acetate-salt–induced hypertension and decreases brain Ang II formation in mice.

• PRO20 prevents prorenin-induced extracellular signal-regulated protein kinase 1 and 2 phosphorylation.

What Is Relevant?

• Our data suggest that PRO20 is a novel PRR antagonist that prevents the binding of prorenin to the PRR and lowers blood pressure in different hypertensive models.

• PRO20 may be a novel tool for studying the physiological and pathophysiological importance of PRR during hypertension by virtue of its complete lack of agonism for PRR signaling, as well as its ability to block PR binding to the PRR. PRO20 presents a novel mechanism for the treatment of essential hypertension.

Summary

PRO20 blocks the binding of prorenin to the PRR in both mouse and human tissues. PRO20 dose-dependently reduces prorenin-induced calcium influx and extracellular signal-regulated protein kinase 1 and 2 phosphorylation in vitro and attenuates hypertension induced by either prorenin or deoxycorticosterone acetate-salt, as well as genetic hypertension.