Fig 2: Validation of three key DEGs with RT-qPCR, ELISA, and IHC staining. (A–C) STXBP3, GOT2, and MAP4K5 expression was measured in 10 healthy controls, four patients with without AR episodes (NAR), and 10 patients with acute rejection (AR). (A) The expression of STXBP3 was compared between healthy controls, patients with NAR, and patients with AR (n = 24). (B) The expression of GOT2 was compared between healthy controls, patients with NAR, and patients with AR (n = 24). (C) The expression of MAP4K5 was compared between healthy controls, patients with NAR, and patients with AR (n = 24). (D–F) ELISA validation of STXBP3 and GOT2 expression in four patients with NAR and 10 patients with AR. (G, H) ROC curve was constructed to estimate the diagnostic power of STXBP3 and GOT2 for early AR. STXBP3: AUC = 0.989 (p < 0.0001), cut-off value = 7.840, sensitivity: 0.929, specificity: 0.944; GOT2: AUC = 0.966 (p < 0.0001), cut-off value = 13.147, sensitivity: 0.929, specificity: 0.889; and the combination of STXBP3 and GOT2: AUC = 1.000(p < 0.0001). (I, J) Immunohistochemical staining of kidney tissues showed that both STXBP3 and GOT2 were increased in AR group compared with that in NAR group. The scale bars in i–j = 100 µm.

Fig 3: Construction of Venn diagrams for key DEGs identification in AR and NAR group. First method for identifying 28 co-expressed genes. The intersection of GSE112927 and GSE120396 yielded the first group of 198 co-expressed genes. The 357 co-expressed genes at the intersection of our center and GSE120649, 758 co-expressed genes at the intersection of our center and GSE131179, and 257 co-expressed genes at the intersection of our center and GSE145503 were obtained. The following three groups of genes were then intersected with the first group of 198 co-expressed genes. Three groups of co-expressed genes were merged to obtain 28 co-expressed genes (FH, TKT, SMARCD1, CLIC3, XRCC6, KHSRP, PPP1R3B, TOE1, ZFAND6, ITGAL, KIF3B, EIF4G1, FGFRL1, PRPF19, CD58, NABP1, PPM1G, TBCD, CCDC92, MDH2, PDIA4, SMARCAL1, GOT2, DNAJC11, MED24, BCL6, IFRD1, and STXBP3).

Fig 4: Bioinformatics analysis of our center RNA sequencing dataset. (A) The expression levels of STXBP3, GOT2, and MAP4K5 illustrated using a heatmap. (B) GO analysis of DEGs in our center dataset. (C) KEGG pathway analysis of DEGs in our center dataset.