## 2 Results
### Gene Ontology Analysis  
Gene ontology analysis of the phosphoproteins indicated that the phosphorylated protein upregulated in the DN group belonged to \"microtubule\" and \"microtubule-based movement\" in the cell component clustering, \"microtubule-based movement\" in the biological process clustering, \"microtubule binding\" and \"tubulin binding\" in the molecular function clustering (Figure 4). Microtubules play a key role in regulating the secretion of insulin particles through their transport to the cell membrane. Glucose regulates microtubule function and promotes microtubule formation in the Golgi apparatus, thereby promoting insulin granule formation. The upregulation of microtubule clustering in DN may be due to prolonged hyperglycemia, resulting in compensatory upregulation of microtubule clustering and increased insulin secretion [22]. Figure 3: The volcano plots represent the quantitative analysis of the proteomes and phosphoproteomes of uEVs from patients with DN, DM, and NC subjects (DN = Diabetic nephropathy; DM = diabetes; NC = normal control). The colored circles indicates the phosphopeptides detected significantly changed among patients with DN, DM, and NC subjects. The red and blue circles indicates the phosphopeptides detected significantly upregulated and downregulated, respectively. Protein-Protein Interaction Prediction of the Phosphoproteins in uEVs from Diabetic Patients Compared to Patients with DN  
To gain a further understanding of the functional interactions among the significantly altered phosphoproteins between patients with DN and DM, a protein-protein interaction network was analyzed using STRING, setting the minimum interaction coefficient to an intermediate confidence level of 0.7. The connections are depicted in Figure 5, with different colors representing various biological processes in the clustering. The network demonstrated that several phosphoproteins are involved in cellular component organization and cellular processes. AQP2 interacts with the calcium channel protein TRPV5 and the sodium phosphate channel protein SLC34A1, both of which belong to the same channel protein interaction network. GSK3\(\beta\) and BAIAP2, which are associated with glycometabolism, shared the same interaction networks. GSK3\(\beta\) and AQP2 are key proteins in the interaction network. Figure 4: Gene Ontology (GO) enrichment analysis of significantly changed phosphoprotein in uEVs among patients with DN, DM, and NC subjects. (**a**) GO enrichment analysis of significantly upregulated phosphoproteins of uEVs from DN patients compared to DM patients. (**b**) GO enrichment analysis of significantly upregulated phosphoproteins of uEVs from DM patients compared to NC subjects. (**c**) GO enrichment analysis of significantly upregulated phosphoproteins of uEVs from DM patients compared to NC subjects. (**d**) GO enrichment analysis of significantly downregulated phosphoproteins of uEVs from DM patients compared to NC subjects. (BP: biological process; CC: cell component; MF: molecular function). Validation of Identified Phosphorylation Specific to DN Patients by Phos-Tag Western Blotting and Immunohistochemistry  
The heterogeneity of DN renders it unlikely that a single diagnostic biomarker can be identified. However, multiple candidate markers that reflect the onset and progression  
Figure 5: The interaction network of aberrant phosphoproteins in uEVs among patients with DN, DM, and NC subjects was constructed by STRING at confidence score >0.7. (**a**) The STRING network analysis of aberrant phosphoproteins in EVs from DN patients compared to DM patients. (**b**) The STRING network analysis of aberrant phosphoproteins in EVs from DN patients compared to NC subjects. (**c**) The STRING network analysis of aberrant phosphoproteins in uEVs from DN patients compared to NC subjects. of DN may offer improved diagnostic and prognostic values.