Abstract
Background: Renal ischemia reperfusion injury plays a key role in renal transplantation, in which oxidative stress is main injury. Our previous study proved baicalin, a flavonoid glycoside isolated from Scutellaria baicalensis, protects kidney from ischemia reperfusion injury. This study aimed to study the underlying mechanism in vitro. Methods: Human renal proximal tubular epithelial cell line HK-2 cells were stimulated with 500 μmol/L H2O2 for 1 hour and then culture for 6 hours. 100 μmol/L baicalin was added to the culture medium 1 hour prior to the treatment. Morphological change, cell viability, apoptotic rate, and oxidative stress level were evaluated. Western blot and real-time PCR analysis were performed to identify the expression of ER stress hallmarks such as BiP, CHOP, and GRP94. The intranuclear and extranuclear level of Nrf2 were also determined in order to assess the activation of Nrf2 signaling. Results: Hydrogen peroxide produced dramatic injury in HK-2 cells. In H2O2 group, Morphological change of apoptosis was observed. The cell viability was decreased and apoptotic rate was increased. ROS and GSH/GSSG analysis revealed increased oxidative stress. ER stress and Nrf2 signaling were also increased. Baicalin pretreatment ameliorated hydrogen peroxide induced cytotoxicity, reduced oxidative stress and ER stress, and further activated Nrf2 signaling pathway which leads to anti-oxidative response.Figure: No Caption available.Conclusion: This study revealed that baicalin pretreatment serves a protective role against hydrogen peroxide induced cytotoxicity in HK-2 cells, and the mechanism is related to the inhibition of ER stress and the activation of Nrf2 signaling.
Highlights
Renal ischemia-reperfusion injury (IRI) is an inevitable acute injury during renal transplantation, which greatly affects the outcome of allograft [1,2]
In order to observe the change of cell viability along with H 2O2 stimulation time, we compared the Optical density (OD) value by Cell Counting Kit 8 (CCK-8) assay at different time points after H2O2 stimulation (Figure 1C)
The largest difference of cell viability between the H2O2 + Baicalin group and the H2O2 group was observed after 4 h of stimulation, which suggested that 4 h of H2O2 stimulation is most appropriate for our study
Summary
Renal ischemia-reperfusion injury (IRI) is an inevitable acute injury during renal transplantation, which greatly affects the outcome of allograft [1,2]. Renal tubular epithelial cells (TECs) suffer from cell injury and undergo apoptosis in renal IRI, contributing to the loss of graft function. The pathophysiological change and underlying mechanism of TECs apoptosis are quite complex [3], involving the caspase cascade, mitochondria dysfunction, immune injury and endoplasmic reticulum (ER) stress [4]. The prevention of TECs apoptosis is considered to be an effective method to alleviate renal IRI. A serial of conservative and complementary adaptive signaling pathways were activated to cope with the perturbations, which are generally called the unfolded protein response (UPR). The homeostasis in the ER is maintained through the adaptive response of UPR [7]. UPR gradually develops into the apoptosis phase in renal IRI.
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