Kinin B1 Receptor Research Articles

Involvement of the Kinin B1 Receptor in Increased Permeability of Cerebral Microvessels in Rats Subjected to Autoimmune Encephalomyelitis.

Kinins are vasoactive peptides that are involved in various cellular mechanisms, including the inflammatory response. Kinins, released in vessel walls, exacerbate inflammation by modulating the production and release of pro-inflammatory factors via two types of G protein-related receptors-B1 and B2 receptors. B1 R is overexpressed during the inflammation that accompanies numerous neurological disorders, including multiple sclerosis (MS), in which loss of BBB integrity is an early pathomechanism of the disease. In this work, we apply pharmacological inhibition of the kinin B1 receptor with DALBK to investigate its effect on blood-brain barrier (BBB) permeability during the course of EAE, an animal model of MS. Functional, ultrastructural and molecular analyses were performed. The expression of selected BBB-associated proteins such as occludin and claudin-5 was assessed, as well as the astrocytic marker GFAP. We show that administration of a specific antagonist attenuates neurological symptoms in EAE rats and recovers the downregulation of TJ proteins and BBB leakage observed during the course of the disease, as well as significantly reducing the disease-specific activation of astroglia. The results show that B1 R-mediated signaling is involved in inducing molecular changes at the level of cerebral microvessels, leading to increased permeability of the BBB following neuroinflammation in EAE.

Abstract P173: Pivotal Role for the Kinin B1R in CB1 Receptor Mediated Neuroinflammation and Pressor Response

The cannabinoid receptors CB1R and CB2R are involved in blood pressure (BP) regulation and CB1R is implicated in the incidence of hypertension. CB1R is highly expressed in the brain but both receptors are also expressed in the heart. Prior research has demonstrated that CB1R mediates significant cardiovascular responses, including elevated BP, heightened plasma norepinephrine levels, and increased sympathetic nerve activity. Furthermore, the activation of the kinin B1 receptor (B1R) mediates similar effects when studied independently. However, the role of B1R in CB1R-mediated BP regulation and the associated signaling mechanisms remain unknown. In this study, we tested the hypothesis that B1R plays a pivotal role in CB1R-mediated pressor response, oxidative stress and neuroinflammation. To test this, we used both in vivo mouse models and in vitro primary hypothalamic neurons. Male C57BL/6NJ wild-type (WT) and B1R knockout mice (B1RKO) underwent surgical carotid artery and jugular vein catheterization to measure BP in conscious state. Following stabilization of BP for at least 90 minutes, the CB1R agonist WIN55,212-2 (300 ug/kg; i.v., WIN) or its vehicle was administered, and recordings continued for 45 minutes. WIN increased BP in WT mice (25±7 mmHg vs baseline), but this effect was blunted in B1RKO mice (n=3, p<0.05). Moreover, administration of WIN led to an augmentation of CB1R expression in the paraventricular nucleus of WT mice, but not in B1RKO mice, thereby indicating the involvement of B1R in CB1R signaling (p<0.05, n=3 mice/group). WT, but not B1RKO, mice treated with WIN displayed a significant increase in oxidative stress levels (p<0.05, n=3 mice/group), as indicated by dihydroethidium staining. To further confirm the role of B1R blockade on CB1R signaling, we cultured primary hypothalamic neurons, as neurons express the highest levels of CB1R in the brain. Neurons were stimulated with WIN (1 uM) both with and without a B1R selective antagonist (SSR240612, 10uM). The results indicated that WIN incubation for 24 hours increased CB1R expression, inflammation, and oxidative stress compared to vehicle treated, and these responses were abrogated by prior B1R blockade (p<0.05, n=5/group), consistent with the in vivo observations. These data provide novel evidence that B1R is implicated in CB1R-mediated proinflammatory responses and serves as a potential therapeutic target to reduce CB1R induced effects.

Blood Vessels

Abstract: One of the primary target tissues for inflammatory and allergy mediators is the blood vessel, and cytokines have a variety of impacts on it. We describe the usefulness of isolated blood vessels immersed in organ baths as a valuable source of pharmacological data. Contractility assays provide strong potency and selectivity data on agonists, partial agonists, and competitive or noncompetitive antagonists; however, their use in the bioassay of vasoactive drugs is gradually being replaced by more advanced analytical techniques. The human umbilical vein, for instance, has been widely utilized to identify bradykinin B2 receptor ligands. Isolated segments of vascular tissue are highly reactive living tissues, particularly in terms of regulating gene products associated with inflammation (e.g., kinin B1 receptor). The venule’s extremely thin walls have the potential to burst due to their high volume. Venules allow blood to move into larger veins. Both veins and arteries have three layers in their walls. On the other hand, venous pressure is modest. Veins are less elastic and have thin walls, allowing them to maintain a high blood circulation rate. The venous system’s high capacitance enables it to store a significant amount of blood at comparatively low pressures, containing around 75% of the circulating blood at any given time. The veins’ one-way valves permit blood to move forward toward the heart through muscle contractions.

Kinin B1 receptor deficiency promotes enhanced adipose tissue thermogenic response to β3-adrenergic stimulation.

Kinin B1 receptor (B1R) has a key role in adipocytes to protect against obesity and glycemic metabolism, thus becoming a potential target for regulation of energy metabolism and adipose tissue thermogenesis. Kinin B1 knockout mice (B1KO) were subjected to acute induction with CL 316,243 and chronic cold exposure. Metabolic and histological analyses, gene and protein expression and RNA-seq were performed on interscapular brown adipose tissue (iBAT) and inguinal white adipose tissue (iWAT) of mice. B1KO mice, under acute effect of CL 316,243, exhibited increased energy expenditure and upregulated thermogenic genes in iWAT. They were also protected from chronic cold, showing enhanced non-shivering thermogenesis with increased iBAT mass (~ 90%) and recruitment of beige adipocytes in iWAT (~ 50%). Positive modulation of thermogenic and electron transport chain genes, reaching a 14.5-fold increase for Ucp1 in iWAT. RNA-seq revealed activation of the insulin signaling pathways for iBAT and oxidative phosphorylation, tricarboxylic acid cycle, and browning pathways for iWAT. B1R deficiency induced metabolic and gene expression alterations in adipose tissue, activating thermogenic pathways and increasing energy metabolism. B1R antagonists emerge as promising therapeutic targets for regulating obesity and associated metabolic disorders, such as inflammation and diabetes.

Kinin B1 receptor and TLR4 interaction in inflammatory response.

We aimed to broaden our understanding of a potential interaction between B1R and TLR4, considering earlier studies suggesting that lipopolysaccharide (LPS) may trigger B1R stimulation. We assessed the impact of DBK and LPS on the membrane potential of thoracic aortas from C57BL/6, B1R, or TLR4 knockout mice. Additionally, we examined the staining patterns of these receptors in the thoracic aortas of C57BL/6 and in endothelial cells (HBMEC). DBK does not affect the resting membrane potential of aortic rings in C57BL/6 mice, but it hyperpolarizes preparations in B1KO and TLR4KO mice. The hyperpolarization mechanism in B1KO mice involves B2R, and the TLR4KO response is independent of cytoplasmic calcium influx but relies on potassium channels. Conversely, LPS hyperpolarizes thoracic aorta rings in both C57BL/6 and B1KO mice, with the response unaffected by a B1R antagonist. Interestingly, the absence of B1R alters the LPS response to potassium channels. These activities are independent of nitric oxide synthase (NOS). While exposure to DBK and LPS does not alter B1R and TLR4 mRNA expression, treatment with these agonists increases B1R staining in endothelial cells of thoracic aortic rings and modifies the staining pattern of B1R and TLR4 in endothelial cells. Proximity ligation assay suggests a interaction between the receptors. Our findings provide additional support for a putative connection between B1R and TLR4 signaling. Given the involvement of these receptors and their agonists in inflammation, it suggests that drugs and therapies targeting their effects could be promising therapeutic avenues worth exploring.

Interaction of Angiotensin-(1−7) with kinins in the kidney circulation: Role of B1 receptors

Changes in renal hemodynamics impact renal function during physiological and pathological conditions. In this context, renal vascular resistance (RVR) is regulated by components of the Renin-Angiotensin System (RAS) and the Kallikrein-Kinin System (KKS). However, the interaction between these vasoactive peptides on RVR is still poorly understood. Here, we studied the crosstalk between angiotensin-(1−7) and kinins on RVR. The right kidneys of Wistar rats were isolated and perfused in a closed-circuit system. The perfusion pressure and renal perfusate flow were continuously monitored. Ang-(1−7) (1.0–25.0 nM) caused a sustained, dose-dependent reduction of relative RVR (rRVR). This phenomenon was sensitive to 10 nM A-779, a specific Mas receptor (MasR) antagonist. Bradykinin (BK) promoted a sustained and transient reduction in rRVR at 1.25 nM and 125 nM, respectively. The transient effect was abolished by 4 μM des-Arg9-Leu8-bradykinin (DALBK), a specific kinin B1 receptor (B1R) antagonist. Accordingly, des-Arg9-bradykinin (DABK) 1 μM (a B1R agonist) increased rRVR. Interestingly, pre-perfusion of Ang-(1−7) changed the sustained reduction of rRVR triggered by 1.25 nM BK into a transient effect. On the other hand, pre-perfusion of Ang-(1−7) primed and potentiated the DABK response, this mechanism being sensitive to A-779 and DALBK. Binding studies performed with CHO cells stably transfected with MasR, B1R, and kinin B2 receptor (B2R) showed no direct interaction between Ang-(1−7) with B1R or B2R. In conclusion, our findings suggest that Ang-(1−7) differentially modulates kinin's effect on RVR in isolated rat kidneys. These results help to expand the current knowledge regarding the crosstalk between the RAS and KKS complex network in RVR.

Microglia Mediate Hypertension and Autonomic Dysfunction through Kinin B1 Receptor Activation

Reactive oxygen species and proinflammatory cytokines in the central nervous system can induce or exacerbate hypertension (HTN) and result in autonomic dysfunction. During HTN, activated microglia modulates autonomic neural activity in the paraventricular nucleus of the hypothalamus (PVN) by regulating neuroinflammation and blood pressure. We previously reported that enhanced kinin B1 receptor (B1R) expression in the brain is associated with increased neuroinflammation and autonomic dysfunction leading to the development of HTN. However, the functional role of B1R within microglia and signaling mechanisms during HTN have yet to be investigated. In this study, we tested the hypothesis that B1R blockade will reduce oxidative stress and mitochondrial dysfunction in microglia, resulting in an attenuation of hypertension and improved autonomic function. Male and female C57BL/6NJ wild-type (WT) and B1R global gene deficient mice (B1RKO) were administered angiotensin II (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. Ang II induced HTN resulted in dysautonomia as indicated by significantly increased plasma norepinephrine levels (p<0.05, n=6) and decreased spontaneous baroreceptor reflex sensitivity (p<0.05, n=6). This further suggests that B1R contributes to impaired autonomic and baroreflex functions in HTN. In addition, CX3CR1 expression in the PVN revealed that microglia-neuron interactions may be increased during HTN. TMEM119 immunofluorescence revealed significant morphological changes in microglia in the PVN of Ang II infused mice compared to saline controls (p<0.05, n=3). To further define the role of microglia, we cultured primary microglia from neonatal mice and found that Ang II induces mitochondrial oxidative stress (MitoSOX) and decreases mitochondrial membrane potential (TMRE) (p<0.01, n=4), however use of B1R specific antagonist SSR240612 was able to reduce these responses. This data coincides with previous data published in primary hypothalamic neurons. To then explore the role of neuron-microglia interactions following Ang II stimulation, we established a direct co-culturing method and through immunocytochemistry, confirmed that levels of oxidative stress and inflammation were significantly increased in our co-culture model compared to when cultured individually (p<0.05, n=3). Pretreatment with B1R antagonist was able to attenuate these effects. This data indicates that autonomic and mitochondrial dysfunction may be regulated, in part, through the kinin B1 receptor in microglia and that neuron-microglia interactions can enhance these effects. This allows us to believe that B1R blockade may serve as a potential therapeutic target for HTN induced mitochondrial and autonomic dysfunction. This study was supported by the NHLBI/NIH 5R01HL153115 (S. Sriramula). This is the full abstract presented at the American Physiology Summit 2024 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.

Genetic Deletion of Carboxypeptidase N Attenuates Angiotensin II-Induced Hypertension and Target Organ Damage

Objectives: Kininase I transforms bradykinin and kallidin, into kinin B1 receptor (B1R) agonists des-Arg9-BK and des-Arg10-kallidin, respectively. Carboxypeptidase M (CPM) is the membrane bound isoform of kininase I and carboxypeptidase N (CPN) is the isoform located in the plasma. Activation of the B1R has been implicated in the development of hypertension, diabetes and atherosclerosis. Pharmacologic inhibition of kininase I is associated with reductions in B1R expression, inflammation, oxidative stress, and improvements in insulin resistance. B1R can interact with aminopeptidase N (CD13) or high-mobility group box 1 (HMGB1) to mediate an inflammatory response. However, the exact role of kininase I in the development of hypertension and the regulation of these B1R-mediated signaling mechanisms remains unknown. In this study, we hypothesize that gene deletion of CPN will prevent B1R activation, reduce inflammation via interfering with CD13 and HMGB1 signaling, and ultimately attenuate the progression of hypertension and target organ damage. Methods and Results: To test our hypothesis we utilized 12-week-old male and female C57BL/6NJ wild-type (WT) and CPN gene knockout (CPNKO) mice. These mice were infused angiotensin II (Ang II, 600 ng/kg/min) or saline (vehicle) via osmotic minipumps for 2 weeks. Vehicle treated CPNKO and WT mice showed no significant difference in mean arterial blood pressure (MAP) (104 ±7 vs 102 ±5 mmHg, n=5, p≤0.05). Ang II-induced elevations in MAP were significantly reduced in CPNKO mice compared to WT mice infused with Ang II (129 ±8 vs 150 ±6 mmHg, n=5, p≤0.05). Interestingly, we did not find any significant sex differences in MAP in CPNKO groups. Additionally, Ang II treatment significantly increased cardiomyocyte hypertrophy in male and female WT mice, but this effect was blunted in CPNKO mice (n=5, p≤0.05; wheat germ agglutinin staining). Ang II infusion promoted collagen deposition in the aorta of WT male and female mice, but not in CPNKO mice (n=5, p≤0.05; picrosirius red/Masson’s trichrome). In male WT mice, Ang II significantly increased the immunoreactivity of CPM in the heart, but this was abrogated in CPNKO mice infused with Ang II (n=4, p≤0.05). Similarly, B1R was also upregulated in male WT mice hearts when compared to CPNKO mice after Ang II infusions (n=3, p≤0.05). We also found that Ang II significantly increased HMGB1 and CD13 in male WT hearts and brains, but not in CPNKO mice (n=4, p≤0.05). In addition, oxidative stress levels in the brain were significantly increased by Ang II in WT mice and was attenuated in CPNKO mice (n=4, p≤0.05; dihydroethidium staining). Conclusions: Genetic deletion of CPN attenuates Ang II-induced hypertension by reducing B1R mediated reductions in CD13 and HMGB1-mediated inflammatory responses and oxidative stress. Our data suggests that targeting kininase I may be a novel therapeutic target for hypertension and target organ damage. Funding: This study was supported by NHLBI/NIH 5R01HL153115 (S. Sriramula). This is the full abstract presented at the American Physiology Summit 2024 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.

Role of kinin receptors in skin pigmentation

Previous studies have shown that all kinin system is constitutively expressed in the normal and inflamed skin, with a potential role in both physiological and pathological processes. However, the understanding regarding the involvement of the kinin system in skin pigmentation and pigmentation disorders remains incomplete. In this context, the present study was designed to determine the role of kinins in the Monobenzone (MBZ)-induced vitiligo-like model. Our findings showed that MBZ induces higher local skin depigmentation in kinin receptors knockout mice (KOB1R, KOB2R and KOB1B2R) than in wild type (WT). Remarkably, lower levels of melanin content and reduced ROS generation were detected in KOB1R and KOB2R mice treated with MBZ. In addition, both KOB1R and KOB2R show increased dermal cell infiltrate in vitiligo-like skin, when compared to WT-MBZ. Additionally, lack of B1R was associated with greater skin accumulation of IL-4, IL-6, and IL-17 by MBZ, while KOB1B2R presented lower levels of TNF and IL-1. Of note, the absence of both kinin B1 and B2 receptors demonstrates a protective effect by preventing the increase in polymorphonuclear and mononuclear cell infiltrations, as well as inflammatory cytokine levels induced by MBZ. In addition, in vitro assays confirm that B1R and B2R agonists increase intracellular melanin synthesis, while bradykinin significantly enhanced extracellular melanin levels and proliferation of B16F10 cells. Our findings highlight that the lack of kinin receptors caused more severe depigmentation in the skin, as well as genetic deletion of both B1/B2 receptors seems to be linked with changes in levels of constitutive melanin levels, suggesting the involvement of kinin system in crucial skin pigmentation pathways.

Kinin B1 receptor controls maternal adiponectin levels and influences offspring weight gain

Given the importance of the kinin B1 receptor in insulin and leptin hormonal regulation, which in turn is crucial in maternal adaptations to ensure nutrient supply to the fetus, we investigated the role of this receptor in maternal metabolism and fetoplacental development. Wild-type and kinin B1 receptor-deficient (B1KO) female mice were mated with male mice of the opposite genotype. Consequently, the entire litter was heterozygous for kinin B1 receptor, ensuring that there would be no influence of offspring genotype on the maternal phenotype. Maternal kinin B1 receptor blockade reduces adiponectin secretion by adipose tissue exvivo, consistent with lower adiponectin levels in pregnant B1KO mice. Furthermore, fasting insulinemia also increased, which was associated with placental insulin resistance, reduced placental glycogen accumulation, and heavier offspring. Therefore, we propose the combination of chronic hyperinsulinemia and reduced adiponectin secretion in B1KO female mice create a maternal obesogenic environment that results in heavier pups.

Kinin B1 receptor modulates glucose homeostasis and physical exercise capacity by altering adrenal catecholamine synthesis and secretion

Our group has shown in several papers that kinin B1 receptor (B1R) is involved in metabolic adaptations, mediating glucose homeostasis and interfering in leptin and insulin signaling. Since catecholamines are involved with metabolism management, we sought to evaluate B1R role in catecholamine synthesis/secretion. Using B1R global knockout mice, we observed increased basal epinephrine content, accompanied by decreased hepatic glycogen content and increased glucosuria. When these mice were challenged with maximal intensity exercise, they showed decreased epinephrine and norepinephrine response, accompanied by disturbed glycemic responses to effort and poor performance. This phenotype was related to alterations in adrenal catecholamine synthesis: increased basal epinephrine concentration and reduced norepinephrine content in response to exercise, as well decreased gene expression and protein content of tyrosine hydroxylase and decreased gene expression of dopamine beta hydroxylase and kinin B2 receptor. We conclude that the global absence of B1R impairs catecholamine synthesis, interfering with glucose metabolism at rest and during maximal exercise.

Prevention of Inflammation, Neovascularization, and Retinal Dysfunction by Kinin B1 Receptor Antagonism in a Mouse Model of Age-Related Macular Degeneration.

The kallikrein-kinin system (KKS) contributes to vascular inflammation and neovascularization in age-related macular degeneration (AMD), particularly via the kinin B1 receptor (B1R). The aim of the present study was to determine the protective effects of the topical administration of the B1R antagonist (R-954) on inflammation, neovascularization, and retinal dysfunction in a murine model of neovascular AMD. Choroidal neovascularization (CNV) was induced in C57BL6 mice using an argon laser. A treatment with ocular drops of R-954 (100 μg/15 μL, twice daily in both eyes), or vehicle, was started immediately on day 0, for 7, 14, or 21 days. CNV, invasive microglia, and B1R immunoreactive glial cells, as well as electroretinography alterations, were observed within the retina and choroid of the CNV group but not in the control group. The staining of B1R was abolished by R-954 treatment as well as the proliferation of microglia. R-954 treatment prevented the CNV development (volume: 20 ± 2 vs. 152 ± 5 × 104 µm3 in R-954 vs. saline treatment). R-954 also significantly decreased photoreceptor and bipolar cell dysfunction (a-wave amplitude: -47 ± 20 vs. -34 ± 14 µV and b-wave amplitude: 101 ± 27 vs. 64 ± 17 µV in R-954 vs. saline treatment, day 7) as well as angiogenesis tufts in the retina. These results suggest that self-administration of R-954 by eye-drop treatment could be a promising therapy in AMD to preserve retinal health and vision.

TRPV4 Activation and its Intracellular Modulation Mediated by Kinin Receptors Contribute to Painful Symptoms Induced by Anastrozole.

Anastrozole, an aromatase inhibitor, induces painful musculoskeletal symptoms, which affect patients' quality of life and lead to therapy discontinuation. Efforts have been made to understand the mechanisms involved in these painful symptoms to manage them better. In this context, we explored the role of the Transient Receptor Potential Vanilloid 4 (TRPV4), a potential transducer of several nociceptive mechanisms, in anastrozole-induced musculoskeletal pain in mice. Besides, we evaluated the possible sensibilization of TRPV4 by signalling pathways downstream, PLC, PKC and PKCε from kinin B2 (B2R) and B1 (B1R) receptors activation in anastrozole-induced pain. Anastrozole caused mechanical allodynia and muscle strength loss in mice. HC067047, TRPV4 antagonist, reduced the anastrozole-induced mechanical allodynia and muscle strength loss. In animals previously treated with anastrozole, the local administration of sub-nociceptive doses of the TRPV4 (4α-PDD or hypotonic solution), B2R (Bradykinin) or B1R (DABk) agonists enhanced the anastrozole-induced pain behaviours. The sensitizing effects induced by local injection of the TRPV4, B2R and B1R agonists in animals previously treated with anastrozole were reduced by pre-treatment with TRPV4 antagonist. Furthermore, inhibition of PLC, PKC or PKCε attenuated the mechanical allodynia and muscle strength loss induced by TRPV4, B2R and B1R agonists. The generation of painful conditions caused by anastrozole depends on direct TRPV4 activation or indirect, e.g., PLC, PKC and PKCε pathways downstream from B2R and B1R activation. Thus, the TRPV4 channels act as sensors of extracellular and intracellular changes, making them potential therapeutic targets for alleviating pain related to aromatase inhibitors use, such as anastrozole.

Abstract 081: Transactivation Of Brain ADAM17 In Angiotensin II-Induced Hypertension Can Be Prevented By Kinin B1 Receptor Blockade

A disintegrin and metalloprotease 17 (ADAM17) has been implicated in mediating inflammation in several cardiovascular diseases. Targeting brain ADAM17 using siRNA revealed a reduction in blood pressure, confirming its role in hypertension. Previously, we reported that kinin B1 receptor (B1R) activation increases ADAM17 activity in primary hypothalamic neurons, while pretreatment with a B1R antagonist mitigated this effect. However, whether there is a direct interaction between ADAM17 and B1R in hypertension is unknown. In this study, we tested the hypothesis that B1R blockade will prevent the transactivation of ADAM17, thereby, preventing the development of angiotensin II-induced hypertension. Male and female C57BL/6NJ wild-type (WT) and B1R knockout mice (B1RKO) were administered Ang II (600 ng/kg/min; SC, 2 weeks) or saline 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. Both male and female B1RKO mice showed similar attenuation of Ang II-induced hypertension. Ang II-infusion led to a significant upregulation of ADAM17 in the PVN of WT mice and was blunted in B1RKO mice (p<0.05, n=3). Proximity ligation assay (PLA) was used to examine close proximity (<40nm) interactions between two receptors. ADAM17-B1R interactions in the PVN of hypertensive mice were significantly increased (p<0.05, n=3). To further understand this correlation, primary neurons and microglia from the hypothalamus of neonatal WT mice were cultured and treated with Ang II (300 nM) for 24 hours. Ang II increased B1R and ADAM17 protein expression in both cell types and was prevented by a B1R antagonist SSR240612 (10 uM) (p<0.05, n=5). To supplement these findings, PLA was used on primary neurons and microglia and revealed an increase in ADAM17-B1R interactions following Ang II. Furthermore, B1R antagonism attenuated this ADAM17-B1R interaction in both neurons and microglia (p<0.05, n=5). Our data provides novel evidence that B1R blockade can attenuate Ang II-induced hypertension in the brain, possibly through a reduction in B1R-ADAM17 interactions, suggesting that B1R blockade may serve as a potential antihypertensive therapeutic agent.

Emerging Role of Kinin B1 Receptor in Persistent Neuroinflammation and Neuropsychiatric Symptoms in Mice Following Recovery from SARS-CoV-2 Infection.

Evidence suggests that patients with long COVID can experience neuropsychiatric, neurologic, and cognitive symptoms. However, these clinical data are mostly associational studies complicated by confounding variables, thus the mechanisms responsible for persistent symptoms are unknown. Here we establish an animal model of long-lasting effects on the brain by eliciting mild disease in K18-hACE2 mice. Male and female K18-hACE2 mice were infected with 4 × 103 TCID50 of SARS-CoV-2 and, following recovery from acute infection, were tested in the open field, zero maze, and Y maze, starting 30 days post infection. Following recovery from SARS-CoV-2 infection, K18-hACE2 mice showed the characteristic lung fibrosis associated with SARS-CoV-2 infection, which correlates with increased expression of the pro-inflammatory kinin B1 receptor (B1R). These mice also had elevated expression of B1R and inflammatory markers in the brain and exhibited behavioral alterations such as elevated anxiety and attenuated exploratory behavior. Our data demonstrate that K18-hACE2 mice exhibit persistent effects of SARS-CoV-2 infection on brain tissue, revealing the potential for using this model of high sensitivity to SARS-CoV-2 to investigate mechanisms contributing to long COVID symptoms in at-risk populations. These results further suggest that elevated B1R expression may drive the long-lasting inflammatory response associated with SARS-CoV-2 infection.

Kinin B1 Receptor Antagonism Prevents Acute Kidney Injury to Chronic Kidney Disease Transition in Renal Ischemia-Reperfusion by Increasing the M2 Macrophages Population in C57BL6J Mice.

Chronic kidney disease (CKD) is a multifactorial, world public health problem that often develops as a consequence of acute kidney injury (AKI) and inflammation. Strategies are constantly sought to avoid and mitigate the irreversibility of this disease. One of these strategies is to decrease the inflammation features of AKI and, consequently, the transition to CKD. C57Bl6J mice were anesthetized, and surgery was performed to induce unilateral ischemia/reperfusion as a model of AKI to CKD transition. For acute studies, the animals received the Kinin B1 receptor (B1R) antagonist before the surgery, and for the chronic model, the animals received one additional dose after the surgery. In addition, B1R genetically deficient mice were also challenged with ischemia/reperfusion. The absence and antagonism of B1R improved the kidney function following AKI and prevented CKD transition, as evidenced by the preserved renal function and prevention of fibrosis. The protective effect of B1R antagonism or deficiency was associated with increased levels of macrophage type 2 markers in the kidney. The B1R is pivotal to the evolution of AKI to CKD, and its antagonism shows potential as a therapeutic tool in the prevention of CKD following AKI.

Kinin receptors regulate skeletal muscle regeneration: differential effects for B1 and B2 receptors.

After traumatic skeletal muscle injury, muscle healing is often incomplete and produces extensive fibrosis. Bradykinin (BK) reduces fibrosis in renal and cardiac damage models through the B2 receptor. The B1 receptor expression is induced by damage, and blocking of the kallikrein-kinin system seems to affect the progression of muscular dystrophy. We hypothesized that both kinin B1 and B2 receptors could play a differential role after traumatic muscle injury, and the lack of the B1 receptor could produce more cellular and molecular substrates for myogenesis and fewer substrates for fibrosis, leading to better muscle healing. To test this hypothesis, tibialis anterior muscles of kinin receptor knockout animals were subjected to traumatic injury. Myogenesis, angiogenesis, fibrosis, and muscle functioning were evaluated. Injured B1KO mice showed a faster healing progression of the injured area with a larger amount of central nucleated fiber post-injury when compared to control mice. In addition, they exhibited higher neovasculogenic capacity, maintaining optimal tissue perfusion for the post-injury phase; had higher amounts of myogenic markers with less inflammatory infiltrate and tissue destruction. This was followed by higher amounts of SMAD7 and lower amounts of p-SMAD2/3, which resulted in less fibrosis. In contrast, B2KO and B1B2KO mice showed more severe tissue destruction and excessive fibrosis. B1KO animals had better results in post-injury functional tests compared to control animals. We demonstrate that injured skeletal muscle tissues have a better repair capacity with less fibrosis in the presence of B2 receptor and absence of B1 receptor, including better performances in functional tests.

Kinin B2 Receptor Mediates Cisplatin-Induced Painful Peripheral Neuropathy by Intracellular Kinase Pathways and TRPA1 Channel Sensitisation.

Chemotherapy-induced peripheral neuropathy is a severe clinical problem frequently associated with cisplatin use. Although its pathophysiology is poorly understood, it is known that kinin receptors and the transient receptor potential ankyrin 1 (TRPA1) channel play a significant role in the peripheral neuropathy induced by cisplatin in rodents. However, the role of signalling pathways downstream from B2 kinin receptors activation and sensitisation of the TRPA1 channel remains unknown in this model. The cisplatin-induced neuropathy model caused mechanical and cold allodynia in male Swiss mice. Antagonists for kinin B2 and B1 receptors and the TRPA1 channel attenuated the painful parameters. Local sub-nociceptive doses of kinin B2 receptor (bradykinin) and TRPA1 channel (allyl isothiocyanate; AITC) agonists enhanced the painful parameters in cisplatin-treated mice, which their respective antagonists attenuated. Furthermore, we demonstrated the interaction between the kinin B2 receptor and the TRPA1 channel in cisplatin-induced peripheral neuropathy since phospholipase C (PLC) and protein kinase C epsilon (PKCε) inhibitors attenuated the increase in mechanical and cold allodynia evoked by bradykinin and AITC in cisplatin-treated mice. Therefore, regulating the activation of signalling pathways downstream from the kinin B2 receptors activation and TRPA1 channel sensitisation can mitigate the painful peripheral neuropathy decurrent of the oncology treatment with cisplatin.

Evaluation of Novel B1R/B2R Agonists Containing TRIOZAN™ Nanoparticles for Targeted Brain Delivery of Antibodies in a Mouse Model of Alzheimer Disease.

The blood-brain barrier (BBB) is a major obstacle to the development of effective therapeutics for central nervous system (CNS) disorders, including Alzheimer's disease (AD). This has been particularly true in the case of monoclonal antibody (mAbs) therapeutic candidates, due to their large size. To tackle this issue, we developed new nanoformulations, comprising bio-based Triozan polymers along with kinin B1 and B2 receptor (B1R and B2R) peptide agonist analogues, as potent BBB-permeabilizers to enhance brain delivery of a new anti-C1q mAb for AD (ANX005). The prepared B1R/B2R-TRIOZAN™ nanoparticles (NPs) displayed aqueous solubility, B1R/B2R binding capacity and uniform sizes (~130-165 nm). The relative biodistribution profiles of the mAb loaded into these NPs versus the naked mAb were assessed in vivo through two routes of administrations (intravenous (IV), intranasal (IN)) in the Tg-SwDI mouse model of AD. At 24 h post-administration, brain levels of the encapsulated mAb were significantly increased (up to 12-fold (IV) and 5-fold (IN), respectively) compared with free mAb in AD brain affected regions, entorhinal cortex and hippocampus of aged mice. Liver uptakes remained relatively low with similar values for the nanoformulations and free mAb. Our findings demonstrate the potential of B1R/B2R-TRIOZAN™ NPs for the targeted delivery of new CNS drugs, which could maximize their therapeutic effectiveness.

Kinins' Contribution to Postoperative Pain in an Experimental Animal Model and Its Implications.

Postoperative pain causes discomfort and disability, besides high medical costs. The search for better treatments for this pain is essential to improve recovery and reduce morbidity and risk of chronic postoperative pain. Kinins and their receptors contribute to different painful conditions and are among the main painful inflammatory mediators. We investigated the kinin's role in a postoperative pain model in mice and reviewed data associating kinins with this painful condition. The postoperative pain model was induced by an incision in the mice's paw's skin and fascia with the underlying muscle's elevation. Kinin levels were evaluated by enzyme immunoassays in sham or operated animals. Kinin's role in surgical procedure-associated mechanical allodynia was investigated using systemic or local administration of antagonists of the kinin B1 receptor (DALBk or SSR240612) or B2 receptor (Icatibant or FR173657) and a kallikrein inhibitor (aprotinin). Kinin levels increased in mice's serum and plantar tissue after the surgical procedure. All kinin B1 or B2 receptor antagonists and aprotinin reduced incision-induced mechanical allodynia. Although controversial, kinins contribute mainly to the initial phase of postoperative pain. The kallikrein-kinin system can be targeted to relieve this pain, but more investigations are necessary, especially associations with other pharmacologic targets.