Hypertension remains the leading cause of death globally, accounting for 10.4 million deaths per year. Recent evidence has shown that the kallikrein-kinin system may play a vital role in the neural control of hypertension, through interactions with key regulatory systems such as the renin-angiotensin system (RAS). We previously showed that kinin B1 receptor (B1R) activation promotes the development of DOCA-salt-induced neurogenic hypertension. However, accumulated evidence has shown that elevated levels of angiotensin II (Ang II) in the brain or upregulated neuronal angiotensin II type I receptor (AT1R) expression, results in increased neuroinflammation and the development of neurogenic hypertension. Recently, we presented data supporting B1R blockade prevents Ang II induced inflammation and oxidative stress in primary hypothalamic neurons. Yet, the interactions between the B1R and AT1R have not been fully studied. In this study, we hypothesize that Ang II treatment will increase blood pressure through B1R mediated mechanisms. Ang II infusion (600 ng/kg/min, SC, 2 weeks) increased blood pressure (radiotelemetry) in wild-type (WT) male mice (145 ±13 mmHg vs. 103 ±6, n=6, plt;0.01). This increase in blood pressure was attenuated in B1R knockout (B1RKO) mice with Ang II infusion (118 ±12 mmHg, n=6, plt;0.01). Interestingly, our data showed no significant differences in B1RKO male and female mice with Ang II-infusion. Additionally, B1RKO mice show reduced Ang II-induced AT1R expression in the brain. Using ultra-sensitive mass spectrometry detection, we found that Ang II-induced an increase in B1R specific agonist Lys-Des-Arg9-Bradykinin (LDABK) within the hypothalamus in WT mice (plt;0.01, n=5). More specifically, immunofluorescence staining showed that B1R is upregulated within neurons in the paraventricular nucleus (PVN) of the hypothalamus, a central regulatory center for cardiovascular and autonomic control. Ang II-treated mice had increased inflammation (qRT-PCR), as shown by elevated gene and protein expression of TNFα, IL-1β, and IL-6 (6-, 5- and 8-fold respectively, n=5, plt;0.01 vs. control) in the PVN, which was attenuated in B1RKO mice. To further determine interactions between AT1R and B1R, we used proximity ligation assay (PLA), to elucidate close proximity (lt;40 nM) receptor-receptor interactions indicated by fluorescent PLA signal. Our results showed that AT1R-B1R close proximity interactions are detected within the PVN, and the PLA signals are significantly increased by Ang II treatment in WT mice (plt;0.01, n=5). Furthermore, to supplement our findings we isolated cultured primary hypothalamic neurons from WT and B1RKO mice and investigated the interaction between Ang II and B1R activation. Primary WT neurons treated with Ang II (300 nM, 24 hr) showed increased B1R expression, inflammation, and AT1R-B1R interaction. However, B1RKO neurons or WT neurons pretreated with R715 (a B1R specific peptide antagonist) showed an attenuation of these effects. These results indicate B1R blockade attenuates Ang II-induced hypertension through novel B1R and AT1R interactions. Collectively, our data provide distinct evidence suggesting B1R blockade could serve as a therapeutic target in the treatment of hypertension.
This study was supported by the NHLBI/NIH 5R01HL153115 (S. Sriramula).
