To utilize transcriptome sequencing datasets for exploring key cell clusters and specific transcriptional regulators during the fibrotic process, aiming to elucidate the mechanism by which sulforaphane ameliorates renal fibrosis in mice.

A single-cell RNA sequencing dataset (GSE175412) derived from a unilateral ureteral obstruction (UUO) renal fibrosis model was utilized. Cell clustering was performed using the Leiden algorithm, followed by manual annotation of the cell clusters. Cell-cell interaction analysis was performed using CellPhoneDB to identify key cell clusters involved in renal fibrosis. Subsequently, the RegDiffusion algorithm combined with AUCell scoring was employed to identify key cell-type-specific transcriptional regulators within each cluster. The bulk RNA-seq dataset of human polycystic kidney disease cells treated with sulforaphane (GSE141740) was utilized. The DESeq2 package was employed to analyze differentially expressed genes between the sulforaphane-treated group and the model group, aiming to identify potential downstream targets of sulforaphane. Subsequently, a UUO model was established by ligating the upper pole of the left ureter in 6 to 8-week-old male C57BL/6J mice to validate the anti-fibrotic effect of sulforaphane and its impact on the expression levels of potential downstream targets. The mice were divided into two groups (n=6 per group): the UUO model group and the UUO plus sulforaphane intervention group. The latter received intraperitoneal injections of sulforaphane [12.5 mg/(kg·d)] starting from the day after surgery. Real-time quantitative PCR was performed to verify the mRNA expression level of sex-determining region Y-related high-mobility-group box transcription factor 4 (Sox4). Masson′s trichrome staining was utilized to evaluate renal interstitial collagen deposition. Additionally, immunohistochemistry and Western blotting were employed to detect fibrotic markers, including vimentin, collagen I, and α-smooth muscle actin. One-way ANOVA was used for comparisons among multiple groups.

The results demonstrated that the proportion of proximal tubular epithelial cells decreased, while the proportion of injured tubular epithelial cells increased in the kidneys of UUO mice. Furthermore, injured tubular epithelial cells were found to interact with fibroblasts and pericytes via multiple pro-fibrotic pathways. Sox4 exhibited high specific activity and significantly elevated mRNA levels in the cluster of injured tubular epithelial cells. Furthermore, sulforaphane treatment significantly downregulated the mRNA expression of Sox4 in human polycystic kidney disease cells. In the UUO plus sulforaphane intervention group, Sox4 mRNA level was significantly downregulated (P<0.05), and renal interstitial collagen deposition was significantly reduced (P<0.05). Besides, the expression of collagen I, α-smooth muscle actin, and vimentin was also significantly decreased (all P<0.05).

Injured tubular epithelial cells served as a key cell cluster in the progression of UUO-induced renal fibrosis, with Sox4 acting as their specific transcriptional regulator. Sulforaphane may exert its anti-fibrotic effects by downregulating Sox4.