Low-level tragus stimulation alleviates sepsis-associated acute kidney injury by promoting renal M2 macrophage polarization.

Background: Low-level tragus stimulation (LL-TS) can reduce acute renal injury in sepsis and improve survival rates. However, the underlying molecular mechanisms remains unknown. Renal macrophages that contributed to AKI in sepsis under low-level tragus stimulation were characterized. Methods: Mice were randomly assigned to five groups following diverse treatments: Control, Sham, Sepsis, LL-TS + sepsis, and LL-TS + sepsis + Methyllycaconitine citrate (MLA). Blood creatinine testing and hematoxylin and eosin (HE) staining were used to assess kidney injury. Single-cell RNA sequencing were used to profile renal macrophages from CD45+ cells in mouse kidney. Western blot, immunofluorescence and HE staining were performed to verify the level of the alpha7 nicotinic acetylcholine receptor (α7nAChR) or interleukin-1β expression in kidney. Results: HE staining and blood creatinine testing of renal tissue indicated that LL-TS alleviated renal injury in mice with sepsis-associated acute kidney injury (AKI). A map of the renal cellular landscape was generated to identify macrophages that contributed to AKI in sepsis under LL-TS. Pseudo-timing analysis showed that LL-TS played an anti-inflammatory role by promoting renal monocyte conversion into M2 macrophages. Moreover, LL-TS significantly increased α7nAChR expression and decreased interleukin-1β expression in renal tissues. Conclusions: LL-TS alleviated sepsis-associated AKI by increasing α7nAChR expression and M2 macrophage polarization which promotes the anti-inflammatory process in sepsis. The results suggests that LL-TS may serve as a potential therapeutic strategy for sepsis-induced kidney dysfunction, ultimately improving patient outcomes. Mouse kidneys were collected 24 hours after CLP. In this study, kidney samples were obtained from one mouse in each of the following groups: the control group, the sepsis group, and the LL-TS combined with sepsis group. Each kidney specimen was precisely sectioned into two equal parts, which were subsequently subjected to enzymatic digestion for further analysis. Immune cells were sorted using CD45 magnetic beads. CD45-sorted positive cells were mixed with non-sorted cells after enzymatic digestion of the same tissue block in a 1:2 ratio. The 10X Genomics GemCode technology was used to encapsulate single cells in droplets, which were then processed by OE Biotech. Co., Ltd. (Shanghai, China). A unique molecular identifier was used to barcode every cell and transcript, and a single-cell 30 Reagent Kit v2 (10X Genomics) was used to generate cDNA for Illumina sequencing.