Advanced glycation end-products (AGEs) are important mediators of diabetic complications via incompletely understood pathways. AGEs bind to intracellular ERM proteins (ezrin, radixin and moesin) that modulate cell shape, motility, adhesion and signal transduction. AGEs bind to the N-terminal domain of ezrin but not full-length ezrin. The AGE binding site may be made accessible either by proteolysis releasing an N-terminal fragment or ezrin activation by phosphorylation. Increased intracellular calcium is a primary event in cell activation by high glucose or AGEs. Calpain activity is increased concomitantly, and ezrin is a calpain substrate. The present study assessed whether glycated proteins affect ezrin cleavage and activation in renal tubule epithelial cells. After 7 days, AGE-BSA decreased ezrin levels in MDCK renal tubular cells to 66 ± 4% of control. AGE-RNAse, ribosylated fetal bovine serum and methylglyoxal-BSA all had similar effects. The AGE-BSA-induced decrease in ezrin was abolished by calpastatin peptide, a specific calpain inhibitor, and 1,2-bis-aminophenoxyethanetetraacetic acid acetoxymethyl ester (BAPTA-AM), a calcium chelator. Ezrin breakdown products were increased in AGE-BSA-treated cells, with a main fragment of 43 kDa. In vitro, calpain 1 cleaved recombinant human ezrin, generating breakdown fragments including an N-terminal fragment of 43 kDa. Studies with ezrin mutants showed that non-phosphorylated ezrin was more susceptible to calpain cleavage. AGE-BSA decreased phosphorylated ERM levels to 31 ± 12% in MDCK cells. Thus, AGE-BSA promotes calpain-mediated proteolysis of ezrin in MDCK cells by both increasing calpain activity and reducing phosphorylation. Therapies targeting both glycated proteins and calpain may provide protection against diabetic complications. Structured digital abstractl Calpain-1 cleaves Ezrin by protease assay (View Interaction: 1, 2)