Activation of Orexin 1 Receptors in the Paraventricular Nucleus Contributes to the Development of Deoxycorticosterone Acetate-Salt Hypertension Through Regulation of Vasopressin

Abstract

Salt-sensitivity is a major factor in the development of hypertension. The brain orexin system has been observed to play a role in numerous hypertensive animal models. However, orexin's role in the pathology of salt-sensitive hypertension (SSH) remains to be adequately explored. We assessed the impact of orexin hyperactivity in the pathogenesis of the deoxycorticosterone acetate (DOCA) - salt rat model, specifically through modulation of Arginine Vasopressin (AVP). Adult male rats were separated into three groups: vehicle control, DOCA-salt, and DOCA-salt+OX1R-shRNA. DOCA-salt rats received subcutaneous implantation of a 21-day release, 75 mg DOCA pellet in addition to saline drinking water (1% NaCl and 0.2% KCl). DOCA-salt+OX1R-shRNA rats received bilateral microinjection of AAV2-OX1R-shRNA into the paraventricular nucleus (PVN) to knockdown function of the Orexin 1-Receptor (OX1R) within that area. Following 2-week to allow full transgene expression, a DOCA pellet was administered in addition to saline drinking solution. Vehicle controls received sham DOCA implantation but were given normal water. During the 3-week DOCA-salt or sham treatment period, mean arterial pressure (MAP) and heart rate (HR) were monitored utilizing tail-cuff plethysmography. Following the 3-week period, rat brains were collected for either PCR mRNA analysis, as well as immunostaining. Plasma samples were collected and subjected to ELISA analysis. In line with our hypothesis, OX1R expression was elevated in the PVN of DOCA-salt treated rats when compared to controls. Furthermore, following chronic knockdown of OX1R, the hypertension development normally induced by DOCA-salt treatment was significantly diminished in the DOCA-salt+OX1R-shRNA group. A concurrent reduction in PVN OX1R and AVP mRNA was observed in concert with the reduced blood pressure following AAV2-OX1R-shRNA treatment. Similarly, plasma AVP concentrations appeared to be reduced in the DOCA-salt+OX1R-shRNA group when compared to DOCA-salt rats. These results indicate that orexin signaling, specifically through the OX1R in the PVN are critical for the onset and maintenance of hypertension in the DOCA-salt model. This relationship is mediated, at least in part, through orexin activation of AVP producing neurons, and the subsequent release of AVP into the periphery. Our results outline a promising mechanism underlying the development of SSH through interactions with the brain orexin system.

Keywords: blood pressure; deoxycorticosterone acetate; hypertension; orexin; paraventricular nucleus; vasopressin.

Figure 1

Deoxycorticosterone acetate (DOCA)-salt treatment increases orexin 1-receptor (OX1R) expression in the paraventricular nucleus (PVN) of Sprague Dawley (SD) rats. OX1R mRNA (A) and immunofluorescence (B) was compared in the PVN area between DOCA-salt treated (n = 4) and vehicle control (n = 3) rats. DOCA-salt treated rats showed a significant increase in OX1R expression within the PVN area (^*p < 0.05). PVN, paraventricular nucleus; OX1R, orexin 1-receptor.

Figure 2

Deoxycorticosterone acetate-salt treatment significantly increases plasma arginine vasopressin (AVP) concentrations in SD rats. Following 3 weeks of DOCA-salt treatment or vehicle control, ELISA analysis was performed on the plasma of DOCA-salt (n = 8) and vehicle control (n = 4) rats to confirm the effectiveness of DOCA-salt treatment in causing increased plasma AVP. DOCA-salt rats showed a significantly increased plasma AVP concentration (^*p < 0.05). AVP, arginine vasopressin.

Figure 3

Chronic knockdown of OX1R in the PVN attenuates increased mean arterial pressure (MAP) induced by DOCA-salt treatment in SD rats. Rats were bilaterally injected with AAV2-OX1R-shRNA into the PVN 2 weeks prior to DOCA-salt treatment. Following this, blood pressure was monitored for 3 weeks during DOCA-salt treatment. Central knockdown of OX1R (n = 4) within the PVN results in attenuation of mean arterial pressure compared to DOCA-salt treated rats (n = 4; A; ^*p < 0.05), while there was no difference between OX1R knockdown rats and controls (n = 5). OX1R knockdown within the PVN had no impact on heart rate between groups (B; p > 0.05). MAP, mean arterial pressure; HR, heart rate.

Figure 4

Paraventricular nucleus bilateral microinjection of AAV2-OX1R-shRNA significantly reduces OX1R expression within the brain. Following 3 weeks of DOCA-salt treatment, rat PVN areas were collected and subjected to either PCR analysis or immunostaining. mRNA levels of OX1R (A) were significantly reduced following OX1R knockdown (n = 5; ^*p < 0.05) compared to DOCA-salt treated rats (n = 10). There were no significant differences between OX1R knockdown rats and controls (n = 7; p > 0.05). These results were further supported by immunostaining analysis (B). PVN, paraventricular nucleus; OX1R, orexin 1-receptor.

Figure 5

Paraventricular nucleus bilateral microinjection of AAV2-OX1R-shRNA significantly reduces central production and subsequent peripheral release of AVP. Following 3 weeks of DOCA-salt treatment, brain PVN areas as well as plasma were collected to test AVP central expression and peripheral secretion. PVN OX1R knockdown (n = 5) resulted in a significantly reduced PVN AVP mRNA expression (A) compared to DOCA-salt treated rats (n = 9; ^*p < 0.05), but not control rats (n = 8; p > 0.05). Similarly, OX1R knockdown in the PVN (n = 5) appeared to partially reduce plasma AVP levels compared to DOCA-salt rats (n = 6; B), although this did not reach significance (p = 0.077). OX1R knockdown rats showed no differences in plasma AVP levels when compared to control rats (n = 6; p > 0.05). Lastly, immunostaining revealed a reduced protein expression of AVP in the PVN area following OX1R knockdown (C). PVN, paraventricular nucleus; AVP, arginine vasopressin.

Figure 6

The hypothesized relationship mediating the impact of central orexin system functioning on hypertension development in the DOCA-salt rat model. DOCA-salt treatment results in activation of circumventricular organs (CVOs), which send projections to the lateral hypothalamus (LH), stimulating Orexin A (OXA) release. OXA then interacts with OX1R at the PVN. This then facilitates an increased production and secretion of AVP to the periphery, where it causes increased blood pressure through various means including vasoconstriction, fluid reabsorption, and increased blood volume. CVO, circumventricular organs; LH, lateral hypothalamus; OXA, orexin-A; OX1R, orexin 1-receptor; PVN, paraventricular nucleus; and AVP, arginine vasopressin.