RGS19 links autophagy dysfunction to CD8+ T cell-driven renal fibrosis via mTOR signaling

Introduction: Renal fibrosis, a defining feature of chronic kidney disease (CKD), is driven by impaired autophagy and chronic immune activation, but the molecular mechanisms linking these processes remain poorly understood. Regulator of G protein signaling 19 (RGS19), a novel autophagy-related gene, may play a pivotal role in this pathology. This study aimed to explore the role of RGS19 in renal fibrosis and its potential as a therapeutic target. We focused on how RGS19 influences autophagy and immune cell infiltration in fibrotic kidneys.
Methods: We integrated Single-cell transcriptomic sequencing data of kidney biopsy samples from our hospitalized patients and autophagy-related genes from the Human Autophagy Database. Differentially expressed genes (DEGs) were identified using the Limma method, and then multiple machine learning algorithms were employed to optimize candidate genes. CIBERSORT was used to analyze immune cell infiltration in renal fibrosis. Animal models of unilateral ureteral obstruction (UUO) and unilateral renal ischemia-reperfusion (UIRI) were established. Fibrosis markers and autophagy-related proteins were detected using immunohistochemistry, immunofluorescence, and Western blot. Autophagic flux was monitored using RFP-GFP-LC3B lentivirus, and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) was used to measure the expression levels of RGS19 and inflammatory cytokines (Tumor Necrosis Factor-alpha (TNF-α), Interleukin 6 (IL-6), IL-1β). Enzyme-Linked Immunosorbent Assay (ELISA) was performed to measure chemokines (CXCL9, CXCL10), and flow cytometry was used to assess CD8+ T cell proliferation and infiltration. In vitro, a fibrosis model was induced in tubular epithelial cells (TEC) TKPTS using TGF-β1, and CD8+ T cells were isolated from mouse spleens and purified. The effect of RGS19 on autophagic flux and the immune microenvironment was analyzed by co-culturing TGF-β1-induced TKPTS with CD8+ T cells. Furthermore, nanocarriers modified with the fibrotic renal homing peptide RDYH58 Nanoparticles Loaded with siRNA Targeting RGS19 (RDYH58-NPs@si-RGS19) were constructed and characterized to evaluate their efficiency in delivering si-RGS19 and their anti-fibrotic effects both in vitro and in vivo. Experimental results were validated through histological analysis, RT-qPCR, Western blot, and fluorescence microscopy.
Results: Bioinformatics analysis identified RGS19 as a key autophagy-related gene in renal fibrosis, with its high expression strongly correlating with CD8+ T cell infiltration. In the TGF-β1-induced TEC model, RGS19 knockdown significantly reduced p62 and p-Mechanistic Target of Rapamycin (mTOR) levels while restoring LC3B-II expression, indicating that RGS19 regulates autophagic flux in renal fibrosis. Further, co-culture experiments showed that RGS19 knockdown decreased the secretion of CXCL9 and CXCL10, reducing CD8+ T cell proliferation and infiltration. In vivo, RGS19 knockdown significantly alleviated renal fibrosis induced by UUO and UIRI. Additionally, the RDYH58-NPs@si-RGS19 further confirmed its therapeutic potential, demonstrating excellent targeting efficiency and anti-fibrotic effects.
Conclusion: This study reveals for the first time the critical role of RGS19 in renal fibrosis. Knockdown of RGS19 improves the renal fibrosis microenvironment by promoting autophagy, reducing CD8+ T cell infiltration, and slowing disease progression, providing a potential therapeutic target for renal fibrosis.