Active Vitamin D and Vitamin D Receptor Help Prevent High Glucose Induced Oxidative Stress of Renal Tubular Cells via AKT/UCP2 Signaling Pathway

It has been documented that vitamin D supplementation showed an improvement of symptoms of diabetic nephropathy; however, the underlying mechanisms remain unknown. We here tested the hypothesis that active vitamin D is able to up-regulate AKT/UCP2 signaling to alleviate oxidative stress of renal tubular cell line HK2.

siVDR, AKT inhibitor, and UCP2 inhibitor elevated the ROS and apoptosis of HK2 cells while attenuating the mitochondrial membrane potential, suggesting that vitamin D protects renal tubular cell from high glucose by AKT/UCP2 signaling pathway.

There are eight groups in the present study: normal glucose, osmotic control (5.5 mmol/L D-glucose+24.5 mmol/L D-mannitol), NAC control (30 mmol/L D-glucose + 1.0 mmol/L N-Methylcysteine), high glucose, high glucose+VD, high glucose (HG)+VD+siVDR, HG+VD+AKT inhibitor (AI), and high glucose+VD+UCP2 inhibitor (Gelipin). Concentration of superoxide dismutase (SOD) and malondialdehyde (MDA) was analyzed by ELISA. Reactive oxygen species (ROS), mitochondrial membrane potential and apoptosis were measured by flow cytometry. JC-1 was evaluated by flow cytometry. The presence of VDR, AKT, and UCP2 in HK cells was assessed using RT-PCR and western blot analyses.

VD administration significantly upregulated the SOD activation and downregulated MDA levels compared to HG group. siVDR, AKT inhibitor, and UCP2 inhibitor significantly suppressed the activation of SOD and increased the expression of MDA compared to VD group. ROS generation and apoptosis of HK2 cells in HG+VD group were significantly lower than those in HG, HG+VD+siVDR, HG+VD+AI, and HG+VD+Gelipin group. ΔΨm in HG+VD group was obviously higher than those in HG, HG+VD+siVDR, HG+VD+AI, and HG+VD+Gelipin group. Decreased mRNA and protein levels of VDR, p-AKT, and UCP2 were observed in HG+VD+siVDR, HG+VD+AI, and HG+VD+Gelipin group compared to those in HG+VD group. Conclusions: siVDR, AKT inhibitor, and UCP2 inhibitor elevated the ROS and apoptosis of HK2 cells while attenuating the mitochondrial membrane potential, suggesting that vitamin D protects renal tubular cell from high glucose by AKT/UCP2 signaling pathway.