Nonneuronal Acetylcholine Signaling in the Kidneys Connects Neuromodulation to Metabolism

Background: Vagus nerve stimulation (VNS) ameliorates ischemic and septic kidney injury via the cholinergic anti-inflammatory pathway (CAP), primarily through acetylcholine (ACh) signaling. While splenic macrophages are well-studied in this context, the identity and function of ACh-secretion renal cells remain unclear. We hypothesized that VNS activates non-neuronal ACh secretion in the kidney. To test this, we utilized single-cell RNA sequencing (scRNA-seq) and ChAT-reporter mice to map ACh-producing cells and assess renal gene expression changes in response to VNS. Methods: Renal ACh content was measured by mass spectrometry at timepoints from 0 to 48 h post-VNS. Kidneys from ChAT-Cre:R26GRR reporter mice and VNS- or sham-treated wild-type mice (N=1 per group) were processed for scRNA-seq using the 10x Genomics Chromium system. ChAT+ cells were enriched by red fluorescence sorting. Data were analyzed using Seurat. Cell types were mapped and annotated using UMAP projections and verified by ChAT immunostaining. Differentially expressed genes (DEGs) between VNS and sham groups were identified in each cell type. The α4nAChR knockout mice were generated via CRISPR/Cas9 targeting exon 5. Results: ChAT-expressing renal cells included proximal tubule cells, loop of Henle, podocytes, and immune cells. Immunofluorescence showed distinct subcellular ChAT localization across these cell types. ACh levels peaked at 4 h post-VNS, supporting non-neuronal secretion. Among VNS-induced DEGs, Pck1 was selectively upregulated in proximal tubules. Its expression pattern resembled the renal ACh peak and was confirmed by qPCR. Notably, Chrna4 (encoding α4 nicotinic acetylcholine receptor, α4nAChR) was uniquely expressed in proximal tubules. In Chrna4-knockout mice, VNS failed to induce Pck1, suggesting α4nAChR-mediated regulation independent of the canonical α7nAChR pathway. Conclusion: VNS induces a delayed increase in renal ACh levels, likely originating from non-neuronal sources. Renal ACh-producing cells, particularly proximal tubule cells, also respond to ACh via an autocrine mechanism involving α4nAChR, leading to upregulation of Pck1. These findings uncover a novel renal cholinergic signaling pathway, distinct from the classical cholinergic anti-inflammatory pathway, and suggest a kidney-intrinsic circuit that links neuromodulation to metabolic regulation. Funding: Government Support – Non-U.S.