Reactive oxygen species-evoked endoplasmic reticulum stress mediates albumin load-induced epithelial-mesenchymal transition in podocytes.

BACKGROUND

Albuminuria is associated with the progression of renal disease. Epithelial-mesenchymal transition (EMT) plays a significant role in cell migration and fibrosis. Podocytes may undergo EMT after injury, leading to podocyte migration and detachment, which results ultimately in defective glomerular filtration. Endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) play major roles in EMT. The pathophysiology linking albuminuria to the progression of renal disease is complex and not yet fully understood. In this study, we evaluate whether podocytes can undergo EMT through ROS/ER stress pathways after albumin overload.

METHODS

Podocytes were exposed to medium alone or to high concentrations of delipidated, endotoxin-free human serum albumin (HSA, 10 mg/ml) with or without antioxidant (N-acetylcysteine, NAC) and ER stress inhibitors (salubrinal, Sal; 4-phenylbutyrate, 4-PBA). Intracellular ROS generation was measured using the fluorescent indicator 20, 70-dichlorofluorescin diacetate (DCF-DA). Both mRNA and protein expressions of the EMT biomarker (α-smooth muscle actin, α-SMA) and ER stress biomarkers (GRP78 and CHOP) were assessed by real-time PCR and Western blotting.

RESULTS

After HSA treatment, endocytosis of HSA by podocytes was observed, intracellular ROS generation and ER stress biomarkers (GRP78 and CHOP) were increased, NAC down-regulated ROS generation and ER stress biomarkers (GRP78 and CHOP), and the ER stress inhibitor (4-PBA) also down-regulated ROS generation. The α-SMA mRNA and protein expressions were up-regulated after HSA treatment but down-regulated by the NAC and ER stress inhibitors (Sal and 4-PBA).

CONCLUSION

The intracellular albumin load can induce ROS and ER stress in podocytes. ER stress, in turn, induces EMT through crosstalk between ROS and ER stress.