Long-Term Proteomic Changes from Human Urinary Extracellular Vesicles in Diabetic Patients after Administrating SGLT2 Inhibitors

Background: Sodium-glucose cotransporter-2 (SGLT2) inhibitors exert their effect by selectively blocking the SGLT2 transporters in the renal proximal tubules and provide nephroprotective effects. However, the long-term effects of SGLT2 inhibitors on human renal proteins and transporters remain unclear. Methods: We collected urine from eighteen patients with type 2 diabetes treated with SGLT2 inhibitors before SGLT2 inhibitors and at 3, 6, 9 months after SGLT2 inhibitors. Urinary extracellular vesicles (uEVs) were isolated by ultracentrifuge. We carried out large-scale LC-MS/MS-based quantitative proteomics from purified uEVslabeled by tandem mass tags (TMT). Results: Characterization of uEVs was confirmed by nanoparticle tracking analysis, transmission electron microscopy, and immunoblotting. Totally, 1108 quantifiable proteins were identified from uEVs. Functional enrichment analysis from upregulated proteins involved in “inhibition of epithelial cell proliferation” and “cell adhesion remodeling”. Downregulated proteins mainly affected cell membrane such as PODXL, ATP1B1, and AQP1. In addition, we identified the co-expressed differentially expressed proteins (DEPs) including upregulated proteins (GPR110, CHMPA4, APPL2, TPPP3) and downregulated proteins (IGHG1, PODXL, SLC4A4, COBLL1) at different time points. After clustering of DEPs from uEVs based on nephron segments, SGLT2 inhibitors recovered the changed renal tubular proteins identified in diabetic patients compared to healthy controls. Among renal solute carrier groups (SLCs), SGLT2 inhibitors mainly affected the SLCs in proximal tubules (SLC5A1, SLC5A2, and SLC4A4) but not the distal tubules (SLC12A1 and SLC12A3). Moreover, proteomic analysis of uEVs in db/db mice administered with SGLT2 inhibitors also showed the changed proteins in terms of “Focal adhesion” and “Slit diaphragm”. Conclusion: SGLT2 inhibitors affected renal proteins involving changes of membrane proteins and cellular differentiation in diabetic patients. The effects of SGLT2 inhibitors on solute carrier group (SLCs) mainly focused on proximal tubules. This uEVs proteomic study could help us know how long-term effects of SGLT2 inhibitors on human kidney. Funding: Government Support – Non-U.S.