To establish a novel mouse model of chronic renal allograft rejection.

C57BL/6 and Balb/c mice were selected as experimental animals to establish three groups: syngeneic control group, conventional allograft group and novel allograft group. Post-operative serum creatinine and blood urea nitrogen levels were monitored regularly. At 16 weeks post-transplantation, graft tissues were harvested for pathological assessment and deep phenotypic analysis. The Banff scoring system was used to evaluate renal allograft pathology. Raw sequencing data were processed using CellRanger 3.1 software. Major cell types were identified and annotated by comparing gene expression profiles with publicly available cell-type-specific gene databases. Cell cycle scoring was performed using the CellCycleScoring function in the Seurat package. Gene Ontology (GO) functional enrichment analysis was conducted using the ClusterProfiler package in R. Comparisons of normally distributed quantitative data were performed using one-way analysis of variance, while non-normally distributed data were analyzed using the Kruskal-Wallis H test. Categorical data were compared using the Chi-square test. Survival curve was generated using the Kaplan-Meier method and compared using the Log-rank test. A P-value <0.05 was considered statistically significant.

Serum creatinine (except at 8 weeks post-transplantation) and blood urea nitrogen levels in the novel allograft group were significantly higher than those in the syngeneic control and conventional allograft group at various post-operative time points (all P<0.05). Pathological analysis showed statistically significant differences in Banff scores for interstitial fibrosis, tubular atrophy, vascular fibrous intimal thickening, and transplant glomerulopathy among the syngeneic control, novel allograft, and conventional allograft groups (H=24.385, 24.120, 23.550 and 26.840, respectively, all P<0.05). Significant differences were observed in the fibrosis area and lymphocyte infiltration rate among the novel allograft, conventional allograft, and syngeneic control group (F=129.071 and 9.543, respectively, all P<0.05). Semi-quantitative analysis of immunohistochemical staining revealed statistically significant differences in the positive areas of Collagen-Ⅲ, alpha-smooth muscle actin, CD3 and CD19 among the three groups (F=740.142, 223.663, 106.611 and 1 026.355, respectively, all P<0.05). Immunofluorescence staining results indicated stronger infiltration of monocytes and macrophages in the graft tissues of the novel allograft group compared to the syngeneic control group. Cell type identification and annotation showed that epithelial cells dominated in the syngeneic control group, whereas the novel allograft group exhibited a higher proportion of immune cells. Single-cell sequencing analysis demonstrated a significant increase in the proportion of immune cells in the novel allograft group; cell cycle analysis suggested that cells in this group were predominantly in the active proliferative G2M and S phases. Gene set variation analysis and GO analysis showed significant upregulation of Hallmark gene sets related to immune activation in the novel allograft group.

The novel chronic renal allograft rejection mouse model effectively recapitulates the processes of chronic allograft rejection and renal interstitial fibrosis.