Canagliflozin ameliorates ferroptosis in diabetic podocytes by PCBP1-enriched extracellular vesicles derived from proximal tubular epithelial cells

INTRODUCTION: Podocyte ferroptosis contributes to proteinuria in diabetic kidney disease (DKD). Canagliflozin reduces albuminuria, but the mechanism underlying podocyte protection remains unclear. Because sodium-glucose cotransporter 2 (SGLT2) is expressed predominantly in proximal tubular epithelial cells (PTECs) rather than podocytes, we hypothesized that this effect is mediated indirectly through PTEC-derived signals. OBJECTIVES: To determine whether canagliflozin suppresses podocyte ferroptosis through PTEC-derived extracellular vesicles (EVs) and to define the underlying molecular mechanism. METHODS: A matched subset of 60 patients with DKD (CON, n = 30; CANA, n = 30) from a prospective, multicenter, randomized, open-label trial was analyzed to assess clinical associations. Mechanistic studies were performed in cultured PTECs and podocytes, db/db mice, and renal tubule-specific Foxo1-knockout mice. EV dependence was examined using GW4869. EV proteomics, luciferase reporter assays, chromatin immunoprecipitation, and RNA immunoprecipitation were used to delineate the FOXO1/VPS53-PCBP1/ACSL4 pathway. iRGD-modified PCBP1-enriched EVs were further evaluated in db/db mice. RESULTS: In cultured podocytes, canagliflozin did not directly alter ferroptosis-related changes, whereas EVs from canagliflozin-treated PTECs were protective. In PTECs, canagliflozin promoted FOXO1 nuclear translocation and VPS53 transcription, thereby increasing EV release. The improvement in renal injury and ferroptosis-related markers was attenuated in diabetic mice with renal tubule-specific Foxo1 deletion. EVs derived from canagliflozin-treated PTECs were enriched in PCBP1, which bound ACSL4 mRNA and reduced its stability in podocytes, thereby limiting lipid peroxidation and ferroptosis. iRGD-modified PCBP1-enriched EVs ameliorated renal injury and reduced ferroptosis-related changes in db/db mice. CONCLUSION: The experimental findings support a FOXO1/VPS53-dependent PTEC-EV pathway associated with PCBP1-mediated destabilization of ACSL4 mRNA and protection against ferroptosis in cultured podocytes. These findings identify tubule-to-podocyte EV communication as a mechanism underlying canagliflozin-mediated podocyte protection and support further evaluation of PCBP1-enriched EVs as a potential EV-based therapeutic strategy for DKD.