PKR inhibition reduces high glucose and H2O2-induced injury in rat renal epithelial cells by suppressing the sonic hedgehog pathway.

Double-stranded RNA-dependent protein kinase (PKR) is involved in signaling pathways that mediate responses to oxidative and endoplasmic reticulum (ER) stress and has been linked to various cardiovascular diseases. However, the role of PKR in renal cells under chronic oxidative stress and the direct impact of PKR inhibitor treatment on such stress-induced damage remain unexplored. This study aimed to investigate the effect of PKR inhibition on oxidative stress-induced injury in renal epithelial cells using an in vitro model. Oxidative stress was induced in NRK-52E renal epithelial cells using a combination of high glucose (HG) and hydrogen peroxide (H2O2). Cell viability, immunofluorescence, and gene expression were analyzed using MTT assay, confocal microscopy, and RT-PCR respectively. Moreover, ROS levels were assessed by FACS. Exposure to HG and H2O2 significantly increased PKR expression in renal epithelial cells compared to control cells. Co-treatment with the PKR inhibitor C16 and gene silencing reduced elevated levels of reactive oxygen species (ROS), malondialdehyde (MDA), nitric oxide, and myeloperoxidase (MPO) in NRK-52E cells. Additionally, HG and H2O2 treatment upregulated Shh, Smo, and Gli1 mRNA while downregulated Ptch1 mRNA compared to control cells. All these altered mRNA was reversed by C16 co-treatment and PKR gene silencing. Furthermore, C16 co-treatment supressed fibrosis and EMT related markers, including TGF-β, α-SMA, fibronectin, vimentin, and type-1 collagen while, increased E-cadherin level in NRK-52E cells. In summary, PKR expression was increased in renal epithelial cells under stress-induced conditions. C16 co-treatment under stress-induced conditions protects the renal epithelial cells via inhibition of oxidative stress, fibrosis, EMT, and modulating Sonic hedgehog signaling pathway.