Kininase I inhibition reduces B1R mediated signaling and attenuates angiotensin II-induced hypertension

Abstract

Objectives: Kininase I, or carboxypeptidase M and N (CPM/CPN), metabolizes bradykinin and kallidin into endogenous kinin B1 receptor (B1R) agonists. B1R expression in the brain is linked to neuroinflammation and autonomic dysfunction, and the development of hypertension. The metalloprotease aminopeptidase N (CD13) can interact with B1R to enhance proinflammatory cytokine production. However, the exact role of kininase I in the development of hypertension has not been yet investigated. In the present study, we tested the hypothesis that inhibition of kininase I will prevent the activation of the B1R, and reduce CD13 signaling, thus ultimately attenuating the progression of hypertension. Methods and Results: To inhibit the kininase I activity, we have utilized carboxypeptidase N gene deleted knockout (CPNKO) mice. Twelve-week-old male and female C57BL/6NJ wild-type (WT) and CPNKO mice, were administered angiotensin II (Ang II, 600 ng/kg/min; 2 weeks) or saline (vehicle) via osmotic minipumps. The mean arterial blood pressure (MAP) was measured using radio telemetry probes for 2 weeks. CPNKO mice did not show any significant differences in MAP at baseline. Ang II-infusion significantly increased the MAP in WT mice compared to saline infused control mice (147 ±5 vs 102 ±2 mmHg, n=5, p<0.05), which was attenuated in CPNKO mice (125 ±4 mmHg, n=5, p<0.01). Interestingly, there were no significant differences in MAP following Ang II-infusion in male and female CPNKO mice. Ang II treatment resulted in sympathoexcitation in WT mice, as indicated by increased urinary norepinephrine levels (446 ±38 ng/ml, p<0.05 vs. control), but this increase was blunted in CPNKO mice infused with Ang II (246 ±14 ng/ml, p<0.05 vs. WT+Ang II). WT mice treated with Ang II had increased inflammation, as shown by elevated gene expression (measured by qRT-PCR) of TNF, IL-1β and IL-6 (5-, 3.6-, and 4-fold, respectively, n=6, p<0.01 vs. WT) in the hypothalamic paraventricular nucleus (PVN). The expression of these inflammatory genes was attenuated in CPNKO mice with Ang II infusion. Immunofluorescence staining showed that Ang II-infusion also significantly increased the expression of B1R, CPM, and CD13 in the PVN, a central regulatory center for cardiovascular and autonomic control. CPM has been shown to interact directly with B1R. To further determine interactions between B1R and CPM, we used proximity ligation assay (PLA) to elucidate close (<40 nM) protein-protein interactions, as indicated by fluorescent PLA signals. Our data suggest that direct B1R-CPM interactions occurring within the PVN as the PLA signals are significantly increased by Ang II treatment in WT mice (p<0.05, n=3), and blunted in CPNKO mice. Conclusions: Kininase I inhibition by CPN gene deletion attenuates Ang II-induced hypertension by reducing B1R and CD13 acti vation, sympathoexcitation, and inflammation. Our data suggest that targeting kininase I may be a novel therapeutic target for 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.