The diabetic microenvironment causes mitochondrial oxidative stress in glomerular endothelial cells and pathological crosstalk with podocytes

In the setting of diabetes mellitus, mitochondrial dysfunction and oxidative stress are important pathogenic mechanisms causing end organ damage, including diabetic kidney disease (DKD), but mechanistic understanding at a cellular level remains obscure. In mouse models of DKD, glomerular endothelial cell (GEC) dysfunction precedes albuminuria and contributes to neighboring podocyte dysfunction, implicating GECs in breakdown of the glomerular filtration barrier. In the following studies we wished to explore the cellular mechanisms by which GECs become dysfunctional in the diabetic milieu, and the impact to neighboring podocytes.

Dissecting the impact of the diabetic environment on individual cell-types from the kidney glomerulus indicates that GECs become dysfunctional and pathological to neighboring podocytes by increased levels of mitochondrial superoxide in GEC. These studies indicate that GEC-signaling to podocytes contributes to the loss of the glomerular filtration barrier in DKD. Video abstract.

Mouse GECs were exposed to high glucose media (HG) or 2.5% v/v serum from diabetic mice or serum from non-diabetic controls, and evaluated for mitochondrial function (oxygen consumption), structure (electron microscopy), morphology (mitotracker), mitochondrial superoxide (mitoSOX), as well as accumulation of oxidized products (DNA lesion frequency (8-oxoG, endo-G), double strand breaks (γ-H2AX), endothelial function (NOS activity), autophagy (LC3) and apoptotic cell death (Annexin/PI; caspase 3). Supernatant transfer experiments from GECs to podocytes were performed to establish the effects on podocyte survival and transwell experiments were performed to determine the effects in co-culture.

Diabetic serum specifically causes mitochondrial dysfunction and mitochondrial superoxide release in GECs. There is a rapid oxidation of mitochondrial DNA and loss of mitochondrial biogenesis without cell death. Many of these effects are blocked by mitoTEMPO a selective mitochondrial anti-oxidant. Secreted factors from dysfunctional GECs were sufficient to cause podocyte apoptosis in supernatant transfer experiments, or in co-culture but this did not occur when GECs had been previously treated with mitoTEMPO.