Cyclic helix B peptide protects HK-2 cells from oxidative stress by inhibiting ER stress and activating Nrf2 signalling and autophagy

Abstract

Renal ischemia-reperfusion injury (IRI) is present in numerous diseases and is observed following certain treatments, including renal transplantation. Preventing tubular epithelial cells (TECs) from undergoing apoptosis is vital for treatment of renal IRI. Cyclic helix B peptide (CHBP) is a novel agent that has a protective effect on renal IRI in vivo. In the present study, the effect and underlying mechanism of CHBP on TECs was investigated. The HK-2 human renal proximal tubular epithelial cell line was treated with 500 µmol/l H2O2 for 4 h prior to determining the effect of CHBP pretreatment for 1 h on cell viability, caspase 3 activity and expression levels, expression levels of oxidative stress markers, endoplasmic reticulum (ER) stress markers, NF-E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1) and autophagy markers. This was investigated using a Cell Counting kit 8, a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling assay, western blotting, reverse transcription-quantitative polymerase chain reaction and immunocytochemistry. Results revealed that pretreatment with CHBP enhanced HK-2 cell viability, the glutathione/glutathione disulphide ratio, activation of Nrf2 and mRNA expression levels of HO-1 and the expression levels of beclin-1 and light chain 3 A/B-II/I. Conversely, CHBP pretreatment reduced the expression levels of reactive oxygen species, the activity and protein expression levels of capase-3, the mRNA and protein expression levels of C/EBP homologous protein and binding immunoglobulin protein, and the expression levels of phosphorylated (p)-mechanistic target of rapamycin (mTOR) Ser2448 and p62 during oxidative stress. However, the expression of p-mTOR Ser2481 was enhanced after CHBP pretreatment. CHBP pretreatment reduced the expression levels of reactive oxygen species, the activity and protein expression levels of capase-3, the mRNA and protein expression levels of C/EBP homologous protein and binding immunoglobulin protein, and the expression levels of phosphorylated (p)-mechanistic target of rapamycin (mTOR) Ser2481, p62 and p-mTOR Ser 2448 during oxidative stress. In conclusion, CHBP pretreatment protected HK-2 cells from H2O2-induced injury, inhibited ER stress and pro-apoptotic pathways, and activated the Nrf2 signalling pathway and autophagy. These results provide a potential mechanism of how CHBP protects against renal IRI.

Erythropoietin Derived Peptide Improved Endoplasmic Reticulum Stress and Ischemia-Reperfusion Related Cellular and Renal Injury

“…The renoprotection of HBSP and CHBP against IR injury has also been confirmed in our previous research projects (33,34). The pretreatment of CHBP reduced the active level of oxidative damage and ERS induced hydrogen peroxide (H 2 O 2 ) in human proximal tubular cell line, HK-2 cells (35), but its underlying mechanisms have still not been welldefined, especially the impact of CHBP on ERS regulators in TECs, as well as in kidneys post IR injury with extended time to 2 and 8 weeks.…”

“…At 4, 8, 12 h after the exposure of H 2 O 2 , the cells were washed three times with PBS (Ji Nuo, Hangzhou, China) and harvested for further analysis. Twenty ng/ml of CHBP (synthesized by Shanghai Institute of Materia Medica, Chinese Academy of Sciences) was added at the same time upon H 2 O 2 stimulation and cultured for 12 h (35).…”

Erythropoietin and its derivatives: from tissue protection to immune regulation

“…Mitochondria are the energy factories for cells and the main source of reactive oxygen species (ROS); thus, mitochondrial dysfunction underlies many diseases. Through the Nrf2 signaling pathway, CHBP reduced ROS production, alleviated endoplasmic reticulum stress and restored mitochondrial membrane potential and integrity, leading to protection against apoptosis 98,99 . In addition, CHBP suppressed the expression of transient receptor potential melastatin 7 (TRPM7), a membrane Ca 2+ channel and kinase which was upregulated in kidney IRI, and reduced TRPM7like current.…”

Molecular Interactions Between Reactive Oxygen Species and Autophagy in Kidney Disease

“…Schisandrin B, isolated from the fruit of Schisandra chinensis , provided a protective role in cyclosporine (CsA)-induced nephrotocxicity, attenuated CsA-induced nephrotoxicity by activating Nrf2, preventing ROS accumulation and oxidative stress, and allowed recovery of CsA-induced blockade of autophagic flux [168]. Cyclic helix B peptide, a tissue-protective peptide mimicking the 3D structure of erythropoietin, which has been reported to ameliorate IR [169], protected HK-2 cells from H 2 O 2 -induced injury through activation of the Nrf2 signaling pathway and autophagy [170]. However, in this study, the link between Nrf2 and autophagy was not determined.…”