Crosstalk Between RAGE and the Kallikrein-Kinin System in Hyperglycemia and Traumatic Brain Injury

Abstract

Background: Diabetes is characterized by chronic hyperglycemia that promotes metabolic stress, oxidative damage, and dysregulation of multiple molecular pathways. These metabolic disturbances have been known to exacerbate neuro-inflammation since persistent hyperglycemia drives the accumulation of advanced glycation end products (AGEs), increasing oxidative stress and making the brain more vulnerable to chronic inflammation. When an individual begins to experience vision loss due to hyperglycemia, reduced peripheral sensation, and cognitive decline, their risk of falling and injuring their heads increases. In the context of Traumatic Brain Injury (TBI), chronic hyperglycemia is known to exacerbate secondary injury outcomes by amplifying oxidative stress and inflammation. However, the underlying mechanisms linking Type 1 Diabetes to TBI remain incompletely understood, especially regarding the involvement of the plasma kallikrein-kinin system (PKKS), the Receptor for Advanced Glycation End-products (RAGE), and downstream necroptosis. RAGE is emerging as a key mediator of these effects, yet its involvement in regulated cell-death pathways and its relationship with the PKKS remains unclear. We hypothesize that diabetic conditions enhance neuro-inflammatory responses following TBI by further activating RAGE signaling and promoting necroptotic activity, reflected by shifts in markers such as receptor-interacting serine/threonine-protein kinase 1 and 3: RIPK1, RIPK3; and MLKL, with changes in Plasma Kallikrein (PKall) activity and oxidative stress. Aims: This study aims to characterize the expression profile of PKKS, S100B, RAGE, and downstream pro-inflammatory, anti-inflammatory, oxidative stress, and necroptotic markers in the brains of diabetic and non-diabetic mice subjected to TBI. It also aims to examine the impact of high-glucose on microglial cell activation, evaluating the specific role of RAGE and its crosstalk with PKKS in regulating downstream necroptotic and inflammatory signaling Methods: For the in vivo experiments, male C57BL/6 mice were divided into streptozotocin-induced diabetic groups and non-diabetic control groups. The mice were subjected to TBI on the right parietal cortex and sacrificed at 3- or 7-days post-injury. For the in vitro experiments, murine N9 microglial cells were cultured in high-glucose conditions and treated with a RAGE antagonist (FPS-ZM1). To test PKKS mechanisms, cells were also treated with PKall in the presence and absence of bradykinin 2 receptor (B2R, HOE140) and protease activator receptor 2 (PAR2, GB83) antagonists. Total RNA was extracted and gene expression was evaluated across all models using RT-qPCR Results: In vivo, diabetes alone significantly upregulated baseline RAGE, S100B, and IL-1β expression. Furthermore, we demonstrated that TBI triggers a coordinated upregulation of RAGE, PKKS, necroptotic, pro-inflammatory, and oxidative stress related genes, early on at day 3 post-TBI, with a shift toward anti-inflammatory and pro-fibrotic signaling by day 7 post-TBI. Critically, preexisting diabetes dysregulates this response, producing a rapid but unstable gene expression across pro-inflammatory, KKS, and necroptotic pathways. In vitro experiments confirmed that high-glucose conditions directly activate RAGE-dependent inflammation and necroptosis, which was effectively reversed by RAGE inhibition with FPS-ZM1. Furthermore, PKall treatment significantly increased RAGE and necroptotic mediator’s expression, which was successfully blocked using B2R and PAR2 antagonists Conclusion: We demonstrated that pre-existing diabetes dysregulates the brain’s response to trauma, resulting in a rapid but unstable activation of inflammatory, necroptotic and KKS signaling pathways. Most importantly, we uncovered a previously unreported reciprocal positive feedback loop in which plasma kallikrein conditionally activates RAGE and downstream necroptotic markers, which establish RAGE–KKS-necroptosis signaling as a novel interconnected axis in hyperglycemia and TBI, presenting promising targets for therapeutic intervention.