Kinin B1 receptor antagonism prevents the transactivation of brain TLR4 in angiotensin II-induced hypertension
Authors: Theobald Drew , Sriramula Srinivas 
Published: May 2023

Background and Hypothesis: Toll like receptor 4 (TLR4) is a component of the innate immune system and is associated with hypertension due to its role in inflammation and oxidative stress. TLR4 inhibition in the brain hypothalamic paraventricular nucleus (PVN) has been found to attenuate inflammation and hypertension. Previously, we reported that kinin B1 receptor (B1R) expression is increased in neurons of mice with angiotensin II-induced hypertension. However, the interaction between TLR4 and B1R in hypertension is unknown. In this present study, we hypothesize that B1R blockade will prevent transactivation of TLR4 receptor, thereby, attenuates Ang II-induced hypertension. Methods and Results: Male and female C57BL/6NJ wild-type (WT) and B1R global gene deficient mice (B1RKO) were administered Ang II (600 ng/kg/min; SC, 2 weeks) or saline (vehicle) via osmotic minipumps. Ang II infusion significantly increased mean arterial pressure (radiotelemetry) in WT mice (145 ±13 mmHg vs. 103 ±6, n=6, p<0.01), which was attenuated in B1RKO mice. Interestingly, our data did not show significant differences in blood pressure in male and female B1RKO mice with Ang II-infusion. Fluorescence staining revealed that TLR4 is upregulated in neurons of the PVN of Ang II-infused WT mice and was blunted in B1RKO mice. In addition, B1R and TLR4 are co-localized in PVN neurons in hypertensive mice. To determine the direct interactions between TLR4 and B1R, we used proximity litigation assay (PLA) to examine the close proximity (<40nm) of receptor-receptor expression and interaction. Our results indicate there are B1R-TLR4 receptor interactions in the PVN as indicated by increased fluorescent PLA signal in Ang II infused WT mice (p<0.05, n=5). To further understand the association between B1R and TLR4, we cultured neonatal primary hypothalamic neurons and microglia from WT mice and treated them with Ang II (300 nM, 24 hr) and found an increase in TLR4 and B1R protein expression. Pretreatment with a B1R specific antagonist R715 (10 uM) prevented these receptor expressions in both cell types. To supplement these findings, we used PLA on primary microglia and neurons to determine the direct receptor-receptor interaction of B1R and TLR4. We identified that PLA signal is significantly increased in neurons (p<0.05, n=3) and microglia (p<0.05, n=3) treated with Ang II, further confirming that there is a direct interaction between TLR4 and B1R. Furthermore, pretreatment with R715 attenuated this B1R-TLR4 interaction in both cell types, indicating B1R antagonism may prevent the transactivation of TLR4 by Ang II treatment. Conclusions: Our data provides novel evidence that blocking B1R can attenuate Ang II-induced hypertension in the brain, possibly through reduction in B1R-TLR4 interaction mediated signaling, suggesting that B1R blockade may serve as a potential therapeutic agent in treating hypertension. Funding: This study was supported by NHLBI/NIH 5R01HL153115 (S. Sriramula). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.