Kininase I Mediated Signaling Evidenced in Hypertension and Target Organ Damage

Abstract

Carboxypeptidase N (CPN) and Carboxypeptidase M (CPM) are isoforms of kininase I, with CPN restricted to the plasma and CPM located on the plasma membrane. Kininase I is a critical pleiotropic regulator of inflammation, as it cleaves several inflammatory mediators such as anaphylatoxins, creatine kinase MM, stromal cell-derived factor-1alpha and kinins. Kinins are important vasoactive peptides which activate the kinin B2 receptor (B2R), and cause vasodilation. However, kininase I converts these kinins into agonists for the kinin B1 receptor (B1R), a receptor associated with vasoconstriction and hypertension. However, the role of kininase I in hypertension has not been identified. Male and female carboxypeptidase N1 (Cpn1) whole-body gene knockout (CPNKO) mice were utilized to investigate the role of CPN in the development of angiotensin II (Ang II)-induced hypertension and target organ damage. CPNKO and wild-type (WT) mice were implanted with radiotelemetry probes to measure blood pressure, and later with mini osmotic pumps to infuse mice with either saline or angiotensin II (Ang II; 600/ng/kg/min -1) for 4 weeks.

In a separate experiment, we used tail cuff plethysmography to measure blood pressure in WT male and female mice infused with either saline or a combined dosage of the non-selective kininase I pharmacological inhibitor (Mergetpa; 10 mg/day) and Ang II (600/ng/kg/min -1) over 4 weeks. Aortic segments isolated from these mice were subjected to wire myography, to assess the effect of CPN gene deletion on vascular function. The activity and expression of kininase I and B1R were analyzed using various protein detection techniques in the brain, heart, and aorta. Proximity ligation assays were utilized to identify alterations in protein-protein interactions between B1R and other protein partners, such as CPM and aminopeptidase N (CD13), in the hypothalamic paraventricular nucleus (PVN) of WT and CPNKO mice. CPN activity assays revealed that overstimulation with Ang II in WT mice promoted CPN activity in plasma. Interestingly, Ang II infusions did not cause a significant modulation in CPM expression specifically in the PVN, heart, and aorta of CPNKO mice, compared to saline treated CPNKO and WT mice. Alternatively, CPM expression was robustly increased following Ang II infusions in WT mouse PVN, heart and aortic tissues. Mean arterial pressure (MAP), measured using radiotelemetry, was not significantly different in saline treated CPNKO and WT mice in both sexes. However, MAP was significantly elevated in WT mice infused with Ang II, but this effect was attenuated in Ang II infused CPNKO mice. Furthermore, the expression of B1R in the brain, heart and aorta was significantly enhanced by Ang II in WT mice compared to CPNKO counterparts. Based on the extent of hypertrophy and picrosirius red staining observed in Ang II infused WT and CPNKO mice, the genetic deletion of CPN protects against cardiac hypertrophy and fibrosis caused by Ang II in both sexes. Interestingly, markers associated with inflammation (IL-1β, IL-6, MCP-1/CCL2, and TNF) and oxidative stress (superoxide, 3-nitrotyrosine) were also found elevated in the heart,

aorta, and PVN of Ang II infused WT mice, and these outcomes were mitigated in Ang II infused CPNKO counterparts. Furthermore, the expression CD13 in the PVN was enhanced following Ang II infusion in WT mice, while CPNKO exhibited significantly lower levels of CD13 expression in the PVN. Astonishingly, proximity ligation assays revealed that protein-protein interactions between both CPM and B1R, as well as CD13 and B1R were heightened in WT mice infused with Ang II but diminished in CPN deficient mice. Mergetpa, the non-selective kininase I inhibitor, was associated with a reduction in MAP and the expression of CPM and B1R in the PVN of WT mice, compared to WT mice infused with Ang II alone. Lastly, vascular function was assessed using wire myography revealed that male Ang II infused CPNKO and Mergetpa treated mice have preserved endothelial dependent relaxation mechanisms, compared to WT mice overstimulated with Ang II. Based on our results, kininase I blockade prevented Ang II induced hypertension and targeted end-organ damage by mitigating B1R activation. B1R mediates inflammation and oxidation and CPM/B1R or CD13/B1R protein-protein interactions may promote Ang II-induced hypertension. Specifically, the pro-inflammatory effects of kininase I initiate B1R activation, leading to the generation of reactive oxygen species (ROS) and subsequent immune cell infiltration/activation and endothelial dysfunction. Alternatively, we suggest that B1R activation mediated by CD13 signaling may contribute to Ang II-induced inflammation and fibrosis, to a lesser extent. Within the context of this study, we did not identify any clear potential sex differences associated with kininase I blockade, in WT and CPNKO mice. We have evidenced for the first time that kininase I mediated B1R activation is correlated with pathogenesis of Ang II and may serve as a potential therapeutic target for the treatment of an overactive RAAS in hypertension.