Inonotus obliquus (Chaga) Ameliorates Folic Acid-induced Renal Fibrosis in Mice: the Crosstalk Analysis among PT Cells, Macrophages and T Cells Based on Single-Cell Sequencing

Yueling Peng ¹ Yaling Zhang ¹ Rui Wang ² Xinyu Wang ² Xingwei Liu ¹ Hui Liao ¹ Rongshan Li ^1*

• ¹ Shanxi Provincial People's Hospital, Taiyuan, China
• ² Shanxi Medical University, Taiyuan, Shanxi Province, China

Renal fibrosis, characterized by the abnormal accumulation of extracellular matrix in renal tissue and progressive loss of kidney function, is posing a significant challenge in clinical treatment. While several therapeutic options exist, effective treatments remain limited. Inonotus obliquus (Chaga), a traditional medicinal mushroom, has shown promising effects in chronic kidney disease (CKD), yet its cellular and molecular mechanisms remain largely unexplored.We analysed the chemical composition of Chaga using UPLC-MS and predicted its biological targets using PubChem and Swiss Target Prediction. We used single-cell RNA sequencing to study cellular responses in a mouse model of folic acid-induced renal fibrosis, complemented by spatial transcriptomics to map cellular location patterns. Histological assessment was performed using H&E and Masson trichrome staining.For the first time, we employed single-cell RNA sequencing technology to investigate Chaga treatment in renal fibrosis. Histological analysis revealed that Chaga treatment significantly reduced renal tubular damage scores [from 5.00 (5.00, 5.00) to 2.00 (2.00, 2.00), p < 0.05] and decreased collagen deposition area (from 11.40 ± 3.01% to 4.06 ± 0.45%, p < 0.05) at day 14. Through analysis of 82,496 kidney cells, we identified 30 distinct cell clusters classified into 8 cell types. Key findings include the downregulation of pro-inflammatory M1 macrophages and upregulation of anti-inflammatory M2 macrophages, alongside decreased T cell responses. Single-cell sequencing revealed differential gene expression in proximal tubular subpopulations associated with reduced fibrosis. Pathway and network pharmacology analyses of 60 identified compounds in Chaga and their 675 predicted targets suggested potential effects on immune and fibrotic pathways, particularly affecting Tregs and NKT cells. Cell-to-cell communication analyses revealed potential interactions between proximal tubular cells, macrophages, and T cells, providing insights into possible mechanisms by which Chaga may ameliorate renal fibrosis.Our study provided new insights into the potential therapeutic effects of Chaga in renal fibrosis through single-cell sequencing analysis. Our findings suggest that Chaga may represent a promising candidate for renal fibrosis treatment, though further experimental validation is needed to establish its clinical application.