AbstractObjects: A multicenter, controlled trial has revealed that early blockade of the renin-angiotensin system in patients with type 1 diabetes and normoalbuminuria did not retard the progression of nephropathy, suggesting that other mechanism(s) are involved in the pathogenesis of early diabetic nephropathy (DN). We have previously demonstrated that endothelial-mesenchymal-transition (EndoMT) contributes to the early development of renal interstitial fibrosis in streptozotocin (STZ)-induced diabetic mice independently of microalbuminuria. In the present study we hypothesized that blocking EndoMT reduces the early development of DN. Research Design and Methods: EndoMT was induced in a mouse pancreatic microvascular endothelial cell line (MMEC) in the presence of advanced glycation end-products (AGEs) and in an endothelial lineage-traceble mouse line Tie2-Cre;Loxp-EGFP by administration of AGEs, with non-glycated mouse albumin serving as a control. Phosphorylated Smad3 was detected by immunoprecipitation/western blotting and confocal microscopy. Blocking studies using RAGE siRNA and a specific inhibitor of Smad3 (SIS3) were performed in MMECs and in STZ -induced diabetic nephropathy in Tie2-Cre;Loxp-EGFP mice. Results: Confocal microscopy and real-time PCR demonstrated that AGEs induced EndoMT in MMECs and in Tie2-Cre;Loxp-EGFP mice. Immunoprecipitation/western blotting showed that Smad3 was activated by AGEs but was inhibited by SIS3 in MMECs and in STZ-induced DN. Confocal microscopy and real-time PCR further demonstrated that SIS3 abrogated EndoMT, reduced renal fibrosis and retarded progression of nephropathy. Conclusions: EndoMT is a novel pathway leading to early development of DN. Blockade of EndoMT by SIS3 may provide a new strategy to retard the progression of DN and other diabetic complications.