The iron overload caused by non-enzymatically glycated transferrin triggers oxidative stress and ferroptosis by regulating the PI3/AKT signaling pathway and the expression of Nrf2 to promote renal damage in diabetes.

Non-enzymatic glycation is an important post-translational modification of proteins, ultimately forming advanced glycation end products (AGEs). In a high-glucose environment, the glycation degree of transferrin (Tf) increases, leading to elevated levels of AGE-modified transferrin (AGE-Tf), which reduces the binding capacity of Tf for iron. However, the role of AGE-Tf-associated iron dysregulation in diabetic nephropathy remains unclear. In this study, human podocytes were treated in vitro with AGE-Tf prepared by incubating Tf with 5.6 or 33.3 mM glucose (hereafter termed 5.6-Glc-AGE-Tf and 33.3-Glc-AGE-Tf, respectively) in combination with iron. Additionally, plasmids encoding Tf with mutations at three glycation sites (K206A/K296A/K534A; MT-Tf) or wild-type Tf (WT-Tf) were packaged into adeno-associated viral vectors and administered to diabetic rats. Compared with podocytes treated with 5.6-Glc-AGE-Tf, podocytes treated with 33.3-Glc-AGE-Tf exhibited reduced viability and greater oxidative stress. After ferric-citrate addition, intracellular Fe2+ was also higher in the 33.3-Glc-AGE-Tf group than in the 5.6-Glc-AGE-Tf group. The 33.3-Glc-AGE-Tf group showed decreased GPX4, Nrf-2, and SLC7A11 expression and increased ACSL4 expression. In vivo, diabetic rats receiving an empty vector (DM-empty-vector) or a wild-type Tf vector (DM-WT-Tf-vector) exhibited higher renal AGE-Tf, Fe2+, and oxidative stress levels; lower nephrin and podocin expression; ferroptosis-related alterations; mesangial cell proliferation and matrix expansion; increased interstitial collagen deposition; and focal nodular sclerosis. These alterations were attenuated in diabetic rats receiving the glycation-resistant mutant Tf vector (DM-MT-Tf-vector). Deferoxamine (DFO) or TBHQ (an Nrf-2 activator) ameliorated oxidative stress, iron dysregulation, and ferroptosis in AGE-Tf-treated podocytes and in the kidneys of diabetic rats receiving the WT-Tf vector, whereas LY294002 (a PI3K/AKT pathway inhibitor) primarily alleviated oxidative stress. These findings identify AGE-Tf-associated iron dysregulation as a potential therapeutic target in diabetic kidney disease.