2021 Volume 46 Issue 10 Pages 445-451
The double-stranded RNA-dependent protein kinase (PKR) is involved in inflammatory cytokine expression and disease pathogenesis in many conditions. The aim of this study was to explore the role of PKR in sepsis-induced renal tissue injury. Six-week-old C57BL/6J mice received PKR inhibitor (imoxin) and Endoplasmic reticulum (ER) inducer (tunicamycin) 2 hr prior to induction of inflammation via cecal ligation and puncture (CLP). Renal tissues were collected 24 hr after the CLP treatment and protein expression were assessed. The expression of creatinine (Cre) and blood urea nitrogen (BUN) in serum and inflammation factor in tissues was detected by ELISA, and the apoptosis of renal tissue was detected by TUNEL staining. PKR inhibitors reduce the expression of sepsis-induced ER stress in renal tissue, as well as the pathological changes and renal impairment in renal tissue. PKR inhibitors reduce the expression of sepsis-induced inflammatory response and sepsis-induced apoptosis in renal tissue by ER stress. In conclusion, PKR inhibitor alleviates ER stress and alleviates sepsis-induced renal injury.
Sepsis is a complex clinical syndrome caused by infection and characterized by a systemic inflammatory response (Cecconi et al., 2018). Sepsis usually results in extensive tissue damage and multiple organ dysfunction (Alsharif et al., 2020). Acute renal injury is one of the most common complications of sepsis, which not only increases the complexity of the disease and the cost of care, but also is a high risk factor for sepsis-related death (Chen et al., 2021b). The pathologic mechanism of sepsis-induced acute renal injury is complex, involving multiple factors such as changes of blood flow in the renal, endothelial cell dysfunction, inflammatory cell infiltration of renal parenchymal tissue, renal tubular dysfunction caused by glomerular thrombosis and necrotic cells and debris (Li et al., 2019; Ferrè et al., 2019).
Double-stranded RNA (dsRNA)-activated protein kinase R (PKR), a widely expressed serine/threonine kinase, is a key inducer of inflammation, insulin resistance and glucose homeostasis in obese patients (Lee et al., 1994). Studies have shown that the PKR inhibitor imoxin can prevent in vivo lipopolysaccharide (LPS)-induced skeletal muscle inflammation (Valentine et al., 2018), and PKR inhibitor can reduce LPS-induced lung injury (Li et al., 2017; Zeng et al., 2019). In sepsis response, PKR inhibition can reduce the induced macrophage endotoxin injury by inactivating PI3K/AKT signal (Xu et al., 2018). However, the role of PKR in sepsis-induced renal tissue injury has not been studied, so this study aims to explore the role of PKR inhibitor in sepsis-induced renal tissue injury.
Male C57BL/6 mice (SPF grade, 5 weeks of age) were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, China). All mice were housed on a 12–12 hr light-dark cycle in controlled temperature (20–24°C) and relative humidity (40%–60%) conditions. After an acclimatization period of 1 week, the mice were used in experiments. All experimental protocols in mice were approved by the Ethics Committee of Kunming Medical University Affiliated Yan’an Hospital.
The mice were randomly assigned to five groups: control group (Control, n = 5); sham group (Sham, n = 5); cecal ligation and puncture (CLP) (n = 5), CLP+ imoxin (imoxin, n = 5) and CLP+ imoxin+ tunicamycin group (imoxin+ tunicamycin, n = 5). The mice in the CLP, CLP+ imoxin and CLP+ imoxin+ tunicamycin groups were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (35 mg/kg). A small incision was made on the abdominal endothelium with sterile scissors, and ligation and perforation were performed at 1 cm from the end of the cecum. After the cecal ligation and perforation, the cecum was put back into the abdominal cavity. The wound was sutured and sepsis was induced in the mice. The mice in the CLP+ imoxin group were administered a subcutaneous injection of PKR inhibitor imoxin (0.5 mg/kg) (Calbiochem, San Diego, CA, USA) 2 hr before CLP administration. Tunicamycin induces the accumulation of unfolded proteins in the cellular endoplasmic reticulum (ER) and induces ER stress. The mice in the CLP+ imoxin+ tunicamycin group were administered a subcutaneous injection of ER inductor tunicamycin (0.5 mg/kg) (Calbiochem).
Hematoxylin and eosin (H&E) stainingAfter the mice were sacrificed, isolated left renals of each group at 24 hr post-CLP administration were fixed in 4% paraformaldehyde (24 hr at room temperature), embedded in paraffin wax, cut into 5-μm sections and stained with H&E. Renal injury was subsequently observed in five randomly selected fields under a light microscope.
ELISAStrictly according to the manufacturer’s instructions, the ELISA kit (Nanjing Jiancheng Bioengineering Institute, Inc., Nanjing, China) was used to analyze the release levels of creatinine (Cre) and blood urea nitrogen (BUN) in serum, tumor necrosis factor-α (TNF-α), Interleukin-1β (IL-1β) and Interleukin-16 (IL-6) in tissues were evaluated using the corresponding detection kits. The serum and cut tissue were centrifuged and the supernatants in the different group were submitted for detection according to the protocols of corresponding manufacturer.
Western blotThe proteins samples in the supernatants were obtained via lysis in RIPA buffer (Beyotime, Jiangsu, China, P0013B) and centrifugation. The separation of proteins was performed on 10% SDA-PAGE. The separated proteins were then wet-transferred on the PVDF membrane. 5% non-fat milk was used for blockage of the membranes and then the membranes were incubated with the primary antibody against Recombinant Eukaryotic Translation Initiation Factor 2 Alpha (p-Eif2α), Recombinant Activating Transcription Factor 4 (ATF4), PKR, Inhibitory Subunit of NF Kappa B Alpha (IKbα), p-IKbα and Cyclooxygenase-2 (COX2), B cell lymphoma (Bcl)2, BCL2-Associated X Protein (Bax) (Cell Signaling Technology, Inc.3 Trask LaneDanvers, MA, USA). Subsequently, the membranes were incubated with goat anti-rabbit HRP-conjugated IgG secondary antibodies (cat. no. AA24142; dilution, 1:5,000; Abcam, Cambridge, UK) at room temperature for 1 hr. The immunoreactive signals were detected using an ECL reagent (Cytiva, Shanghai, China) and ImageJ software (version 146; National Institutes of Health) was employed to analyze the fold-changes of protein levels. All experiments were independently performed three times.
Measurement of cell apoptosisThe cell apoptosis was detected via TUNEL assay. One Step TUNEL Apoptosis Kit (Beyotime) was used herein for detection of the cell apoptosis according to the instructions of the manufacturer. Briefly, the cells after treatment were set on glass slides. After fixed and permeabilized, the cells were incubated with the TUNEL working solution at 4°C, overnight and counterstained with 4,6-diamino-2-phenyl indole (DAPI) (Thermo Fisher Scientific, Waltham, MA, USA) for 2 min. After washing with PBS, the slides were observed under the Olympus FV-500 confocal microscope (Olympus, Pittsburgh, PA, USA).
Statistical analysisAll the data herein were obtained for three repeated experiments and expressed as mean ± SD. The statistical analysis was performed on GraphPad prism. One-way ANOVA followed by Tukey’s post hoc test was used for multiple pairwise comparisons. P < 0.05 was considered to indicate a statistically significant difference.
In order to investigate the effect of PKR inhibitor on the expression level of endoplasmic reticulum (ER) stress in renal tissues induced by sepsis, western blot was used to detect the expression of ER stress in renal tissues. Eukaryotic translation initiation factor2α (eIF2α) is an important molecule in the initiation of protein synthesis. Eif2α is phosphorylated (p-eIF2α) during ERS, thereby inhibiting protein synthesis (Moreno et al., 2012). ATF4 is a signaling molecule downstream of the Endoplasmic reticulum-resident kinase (PERK) pathway in the ER stress signaling pathway (Moulin et al., 2020). ATF4 plays an important role in ontogeny, cellular stress response, amino acid secretion, apoptosis and tumorigenesis (Niccoli et al., 2021; Chen et al., 2021a). As shown in Fig. 1, the expression levels of p-eIF2α, ATF4 and PKR in the CLP group were all up-regulated. After treatment with the inhibitor drug imoxin, levels of p-eIF2α, ATF4 and PKR were decreased compared with those in the CLP group. The results showed that PKR inhibitor alleviates the expression of renal ER stress induced by sepsis.
Imoxin alleviates the expression of ER stress induced by sepsis in renal tissue. The expression levels of p-eIF2α, ATF4 and PKR in the CLP group were detected by western blot. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control; ##P < 0.01 and ###P < 0.001 vs. sham; ΔΔΔP < 0.001 vs. clp.
To further explore the effects of PKR inhibitors on renal tissue and renal function. HE assay was used to detect the pathological level of renal tissues. As shown in Fig. 2, renal tissues of CLP group showed pathological changes, including renal tubular cell necrosis, cell apoptosis, cell exfoliation, vacuole formation and tubular expansion. The pathological changes of renal tissue of mice treated with imoxin were serve. Compared with the imoxin treatment group, the pathologic condition of mice treated with imoxin pretreatment and the addition of ER inducer tunicamycin was increased.
Imoxin alleviates pathological changes of renal tissue. HE assay was used to detect the pathological level of renal tissues.
Serum of mice in each group was detected by ELISA. The expression of Cre and BUN in CLP group was up-regulated, and PKR inhibitor could reduce the expression of Cre and BUN. Compared with the imoxin treatment group, the expression of Cre and BUN was further up-regulated after the addition of ER inducer (Fig. 3A). All the results showed that imoxin could alleviate the pathological changes of renal tissue and renal function injury.
Imoxin alleviates pathological changes of renal function injury and inflammatory responses. (A) The expression of Cre and BUN in each group was detected by ELISA. (B) The expression of inflammatory factors TNF-α, IL-6 and IL-1β was detected by ELISA. (C) The expression trend of IKbα, p-IKbα and COX2 was detected by western blot. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control; #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. sham; ΔP < 0.05, ΔΔP < 0.01 and ΔΔΔP < 0.001 vs. clp. $$ P < 0.01 and $$$ P < 0.001 vs. imoxin.
Further, the expression of inflammatory factors TNF-α, IL-6 and IL-1β were detected by ELISA. The expression of TNF-α, IL-6 and IL-1β was all increased in CLP group, and imoxin attenuated the expression of inflammatory factors. Compared with the imoxin treatment group, the expressions of TNF-α, IL-6 and IL-1β was further up-regulated after the addition of ER inducer (Fig. 3B). The expression of p-IKBα and COX2 in CLP cells was increased, and the expression of p-IKBα and COX2 was decreased after imoxin treatment, which was partially alleviated by tunicamycin treatment. The expression level of IKBα was up-regulated in the imoxin group, which further indicated the anti-inflammatory effect of imoxin. (Fig. 3C). The apoptosis of cells was detected by TUNEL staining and western blot. As shown in Fig. 4A–B, the level of apoptosis was increased in the CLP group, and PKR inhibitors attenuated expression of sepsis-induced apoptosis in renal tissue cells by ER stress.
Imoxin alleviates the expression of sepsis-induced apoptosis through ER stress. The apoptosis of renal tissue cells was detected by TUNEL staining (A) and western blot (B). *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control; #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. sham; ΔΔΔP < 0.001 vs. clp. $P < 0.05 and $$P < 0.01 vs. imoxin.
As an intracellular pathogenic receptor molecule, PKR is activated after being stimulated by viruses, bacterial infections and interferons (Reimer et al., 2021; Kalra et al., 2020). It can transmit signals not only to classical substrates, such as EIF2, but also to MAPK, NF-κB, Stat, etc., and is an important node in the interconnection of different regulatory pathways (Chai et al., 2011; Bullido et al., 2008). Researchers believe that it is the complex regulatory role of PKR that makes it possible for PKR to become a key molecule to link different areas of research and even to understand different mechanisms of the same disease (Chang et al., 2021). PKR may be able to explain part of the mechanism of renal injury and provide a possibility for therapeutic exploration of renal injury in the absence of long-term breakthrough in the exploration of renal injury therapy. After successfully establishing the CLP model in this study, PKR inhibitors, imoxin alleviates pathological changes of renal tissue and renal function injury. Also, imoxin alleviates the expression of sepsis-induced inflammatory responses and apoptosis. Therefore, our study demonstrates that PKR may be involved in the pathogenesis of acute renal injury.
ER plays an important role in the pathological state of sepsis (Kozlov et al., 2009; Schildberg et al., 2005). Therefore, it is very important to study new intervention methods to improve endothelium injury caused by ER stress and prevent coagulation dysfunction in patients with severe sepsis. Sepsis is also accompanied by oxidative stress, which induces the formation of reactive oxygen species (ROS) and the lipid peroxidation marker malondialdehyde (MDA) (Prayag, 2000).The acceleration of ER dysfunction is related to oxidative stress. ER stress is a disorder of ER function caused by various physical and chemical factors, such as oxidative stress, nutrient deprivation, toxic substances, and hypoxia (Cook et al., 2016; Chinta et al., 2008). Through the activation of ER receptors, it causes downstream self-protection reactions-unfolded protein reaction, ER overload reaction and steroidal regulatory cascade reaction (Gentz et al., 2013). PERK is a I type transmembrane kinase located on the endoplasmic omentum. After dissociation from BIP, the oligomeric phosphorylation of PERK itself is activated, and ATF4 is selectively translated and encoded mRNA and ATF4 further induce the expression of CHOP gene, inhibit the expression of anti-apoptotic protein Bcl-2, cause the imbalance of Bcl-2/Bax ratio, activate the Caspase protease family, and finally cause the apoptosis of stress cells (Hiramatsu et al., 2015). More and more studies have shown that ER stress and cell apoptosis are involved in ALI induced by various factors (Zeng et al., 2017).
NF Kappa B is an important transcription factor for the expression of inflammatory cytokines, chemokines, tissue factors and adhesion molecules (Dolatshahi et al., 2021). Under resting state, p65 and p50 formed trimer with Inhibitory Kappa B Ikb, and were located in cytoplasm in inactive state (Zozo et al., 2021). NF Kappa B is the first transcription factor that has been shown to act directly on oxidative stress (Dolatshahi et al., 2021). After the dysregulation of various intracellular REDOX effects, the transcriptional activity of NF Kappa B is altered, which leads to the alteration of downstream gene expression. IKB plays an important role in the inflammatory response. Increased degradation and decreased synthesis of IKB will lead to the release of more NF Kappa B. Therefore, an important direction of anti-inflammatory is to reduce the degradation of IKB, such as increasing the expression of IKB (Zozo et al., 2021). In this study, with the addition of ER inducer, we found that imoxin alleviates the expression of ER stress induced by sepsis in renal tissue, and imoxin alleviates the expression of sepsis induced inflammatory responses and apoptosis through ER stress, which is consistent with relevant studies.
In both in vivo and in vitro studies, activation of PKR has been associated with the initiation of several pathological events, such as insulin resistance, glucose intolerance, increased expression of inflammatory and oxidative stress markers in diabetes, and cardiovascular complications (Udumula et al., 2017). Imoxin has been shown to have anti-inflammatory effects in a variety of tissues (Nakamura et al., 2014; Supinski and Callahan, 2011). The role of PKR in inflammation seems to depend on the environment and cell type (Watanabe et al., 2018). With the further study of the mechanism of action of PKR inhibitor, imoxin may have the possibility of clinical application, providing more options for clinical treatment.
In conclusion, the mouse model of septic kidney injury induced by CLP was successfully replicated in this study, which confirmed that imoxin could play a protective role against septic kidney injury by inhibiting ER stress and cell apoptosis, and provided a theoretical basis for the clinical application of imoxin.
Conflict of interestThe authors declare that there is no conflict of interest.
