Proteomic insights into aprotinin’s immunomodulatory effects in attenuating renal fibrosis in a unilateral ureteral obstruction model

Background Chronic kidney disease involves progressive fibrosis and immune dysregulation. Aprotinin, a broad-spectrum serine protease inhibitor, has shown dose-dependent renal effects, but its role in obstructive nephropathy is unclear. Methods A unilateral ureteral obstruction (UUO) mouse model was used to assess aprotinin at 0.5 mg/day and 1 mg/day delivered via osmotic pumps for 7 days. Renal injury and fibrosis was evaluated by histology, serum markers, and expression of inflammation- and fibrosis-related genes. Mechanistic insights were obtained through LC-MS/MS proteomic profiling, functional enrichment analysis, and CIBERSORT-based immune deconvolution. Key proteins, signaling pathways, and immune cell infiltration were validated by Western blotting, immunohistochemistry, and flow cytometry. Results Aprotinin at 1 mg/day significantly reduced tubular injury and interstitial fibrosis, whereas the 0.5 mg/day dose showed minimal benefit. The higher dose caused mild increases in serum creatinine and blood urea nitrogen. Proteomic analysis identified differentially expressed proteins enriched in immune regulatory pathways. Cathepsin S (CTSS), a protease involved in antigen presentation, was markedly decreased by aprotinin. CIBERSORT revealed reduced follicular helper T cells and partial restoration of naïve CD4 + T cells following treatment. These changes were further confirmed by flow cytometry. Western blotting and immunohistochemistry confirmed reduced CTSS and CD4 expression, along with decreased CD4 + T-cell infiltration and inhibition of ERK signaling. Conclusions Aprotinin attenuates renal fibrosis in UUO in a dose-dependent manner. Its effects are associated with suppression of CTSS, inhibition of ERK signaling, and modulation of CD4 + T-cell subsets, and reduced immune cell infiltration. These findings indicate that aprotinin attenuates renal fibrosis through immunomodulatory mechanisms and support further investigation of serine protease inhibitors in immune-mediated kidney injury.