*2.3. Functional Annotation of Di*ff*erential Protein Expression in the PPG and GPG Groups*

To find the differential abundant proteins (DAPs) from among the 1117 proteins, fold-changes and *p*-values were calculated by Mann–Whitney U tests of the two groups. A volcano plot showing log2-fold-changes against minus log10 *p*-values identified 46 proteins as being upregulated in the PPG and 54 proteins in the GPG (|log2 fold-change| > 0.5; *p*-value < 0.05; Figure 2 and Table S3). These differentially expressed proteins included the six previously described candidate urinary biomarkers (APOE, CO3, COF1, NID1, OSTP-5, and PODXL) of glomerular or tubular injury [11].

**Figure 2.** Volcano plot of urinary proteomic data. Volcano plots are depicted with the fold change of each protein abundance and the *p* value was calculated by performing a Mann–Whitney U-test. The averages of the urinary proteomic abundance data of good prognostic group (*N* = 35) were compared with the averages of the data for poor prognostic group (*N* = 19). Red circles show 54 urinary proteins that have significant increases in PPG. Blue circles show 46 urinary proteins which have significant decreases in PPG. Gray circles are urinary proteins without statistical meaning. Green circles are previously released as urinary protein markers for glomerular injury or tubular injury.

To determine whether urinary DAPs were associated with specific biological processes, up- and downregulated proteins in the PPG were subjected to gene ontology (GO) enrichment analysis. To integrate the three domains of GO and easily visualize the relationship between terms, ClueGO tools were applied with default settings (kappa score 0.4 and group merger of 50% of genes) to functionally organize the GO term networks [12].

Downregulated proteins in PPG were significantly enriched with an FDR < 0.01 (Figure 3A,B). Biological processes associated with these proteins included negative regulation of lipid localization, collagen catabolic process, positive regulation of neural precursor cell proliferation, and neuron projection regeneration. Resulting analysis of molecular function indicated that transforming growth factor beta binding and cargo receptor activity were annotated. The networks between proteins and functional GO terms showed that three proteins were negative regulators of lipid transport, as well as being associated with another GO term (Figure 3C). These included APOE, which is involved in neuron projection regeneration; THBS1, which is involved in transforming growth factor beta binding; and EGF, which is involved in positive regulation of neural precursor cell proliferation.

**Figure 3.** Up-regulated proteome in good prognosis group (GPG) and gene ontology (GO) analysis. (**A**) Functional GO network displaying grouping of GO terms enriched in GPG up-regulated proteins. (**B**) Enriched GO terms in biological process and molecular function. (**C**) The network between GO terms and corresponding proteins represents the relationship between GO terms via the proteins. The abundances of each protein represent the violin plots in two groups. The numbers listed below represent the measured numbers for each group.

Upregulated proteins in PPG were identified in enriched functional GO groups with an FDR < 0.01 (Figure 4A,B). Biological processes associated with these proteins included platelet degranulation, retina homeostasis, and heterotypic cell–cell adhesion. The molecular functional processes related with these proteins contained collagen binding. These urinary proteins were located in the lysosomal lumen and blood microparticles. The networks between proteins and functional GO terms indicated that the proteins in blood microparticles were functionally involved in platelet degranulation (Figure 4C). Platelets in patients with CKD are deficient in reactivity [13]. Leukocytes adhere to and destroy damaged kidney cell walls in patients with CKD [14], accompanied by bone marrow-derived kidney fibrosis, which is highly associated with cell–cell adhesion [15]. CKD is also associated with retinal abnormalities [16] and the possible destruction of retinal homeostasis, as confirmed in this study.

**Figure 4.** Up-regulated proteome in poor-prognostic group (PPG) and GO analysis. (**A**) Functional GO network displaying grouping of GO terms enriched in PPG up-regulated proteins. (**B**) Enriched GO terms in biological process, molecular function, and cellular component. (**C**) The network between GO terms and their contained proteins represents the relationship between GO terms via the proteins. The abundance of proteins represents the violin plots in two sample groups. The numbers listed below represent the measured numbers for each group.
