Ling Zhang

Ischemia–reperfusion (I/R) is the most common cause of acute kidney injury (AKI) and can induce apoptosis in renal epithelial tubule cells. Mitochondrial dysfunction is one of the main reasons for I/R-induced apoptosis. Accumulating evidence suggests that PINK1/Parkin-mediated mitophagy possibly plays a renoprotective role in kidney disease by removing impaired mitochondria and preserving a healthy population of mitochondria. Our previous study showed that augmenter of liver regeneration (ALR) alleviates tubular epithelial cells apoptosis in rats with AKI, although the specific mechanism remains unclear. In this study, we investigated the role of ALR in I/R-induced mitochondrial pathway of apoptosis. We knocked down ALR with short hairpin RNA lentiviral and established an I/R model in human kidney proximal tubular (HK-2) cells in vitro. We observed that the knockdown of ALR aggravated mitochondrial dysfunction and increased the mitochondrial reactive oxygen species (ROS) levels, leading to an increase in cell apoptosis via inhibition of mitophagy. We also found that the PINK1/Parkin pathway was activated by I/R via confocal microscopy and Western blot. Furthermore, the knockdown of ALR suppressed the activation of PINK1 and Parkin. These findings collectively indicate that ALR may protect HK-2 cells from I/R injury by promoting mitophagy, and the mechanism by which ALR regulates mitophagy seems to be related to PINK1 and Parkin. Consequently, ALR may be used as a potential therapeutic agent for AKI in the future.

Acute kidney injury (AKI) is a common and severe clinical condition with high morbidity and mortality. Ischaemia‐reperfusion (I/R) injury remains the major cause of AKI in the clinic. Ferroptosis is a recently discovered form of programmed cell death (PCD) that is characterized by iron‐dependent accumulation of reactive oxygen species (ROS). Compelling evidence has shown that renal tubular cell death involves ferroptosis, although the underlying mechanisms remain unclear. Augmenter of liver regeneration (ALR) is a widely distributed multifunctional protein that is expressed in many tissues. Our previous study demonstrated that ALR possesses an anti‐oxidant function. However, the modulatory mechanism of ALR remains unclear and warrants further investigation. Here, to elucidate the role of ALR in ferroptosis, ALR expression was inhibited using short hairpin RNA lentivirals (shRNA) in vitro model of I/R‐induced AKI. The results suggest that the level of ferroptosis is increased, particularly in the shRNA/ALR group, accompanied by increased ROS and mitochondrial damage. Furthermore, inhibition of system xc‐ with erastin aggravates ferroptosis, particularly silencing of the expression of ALR. Unexpectedly, we demonstrate a novel signalling pathway of ferroptosis. In summary, we show for the first time that silencing ALR aggravates ferroptosis in an in vitro model of I/R. Notably, we show that I/R induced kidney ferroptosis is mediated by ALR, which is linked to the glutathione‐glutathione peroxidase (GSH‐GPx) system.

Ischemia/reperfusion is a major cause of acute kidney injury and can induce apoptosis in renal epithelial tubule (HK-2) cells. Accumulating evidence indicates that endoplasmic reticulum (ER) stress is a major contributor to apoptosis in acute kidney injury. We previously reported that augmenter of liver regeneration (ALR) functions as an anti-apoptotic factor in H₂O₂-treated HK-2 cells although the precise mechanism underlying this action remains unclear. In the present study, we investigate the role of ALR in H₂O₂-induced ER stress-mediated apoptosis. We overexpressed ALR and established a H₂O₂-induced ER stress model in HK-2 cells. Overexpression of ALR reduced the level of reactive oxygen species and the rate of apoptosis in H₂O₂-treated HK-2 cells. Using confocal microscopy and western blot, we observed that ALR colocalized with the ER and mitochondria compartment. Moreover, ALR suppressed ER stress by maintaining the morphology of the ER and reducing the levels of the ER-related proteins, glucose-regulated protein 78 (GRP78), phospho-protein kinase-like ER kinase (p-PERK), phospho-eukaryotic initiation factor 2α (p-eIF2α) and C/EBP-homologous protein (CHOP) significantly (p < 0.05). Mechanistically, ALR promoted Bcl-2 expression and suppressed Bax and cleaved-caspase-3 expression significantly during ER-stress induced apoptosis (p < 0.05). Furthermore, ALR attenuated calcium release from the ER, and transfer to mitochondria, under ER stress. To conclude, ALR alleviates H₂O₂-induced ER stress-mediated apoptosis in HK-2 cells by suppressing ER stress response and by maintaining calcium homeostasis. Consequently, ALR may protect renal tubule epithelial cells from ischemia/reperfusion induced acute kidney injury.

Autophagy may have protective effects in renal ischemia–reperfusion (I/R) injury, although the underlying mechanisms remain unclear. Augmenter of liver regeneration (ALR), a widely distributed multifunctional protein that is originally identified as a hepatic growth factor, may participate in the process of autophagy. To investigate the role of ALR in autophagy, ALR expression is knocked-down in human kidney 2 (HK-2) cells with short hairpin RNA lentivirals. Then, the level of autophagy is measured in the shRNA/ALR group and the shRNA/control group in an in vitro model of ischemia–reperfusion (I/R). The results indicate that the level of autophagy in two groups increase, accompanied by increased reactive oxygen species production, especially in the shRNA/ALR group. The AMPK/mTOR signaling pathway is hyperactive in the shRNA/ALR group. Inhibition of autophagy with the AMPK inhibitor compound C induce apoptosis, especially in the shRNA/ALR group. These findings collectively indicate that ALR negatively regulates the autophagy process through an association with the AMPK/mTOR signaling pathway. Autophagy inhibit apoptosis and play a protective role under conditions of oxidative stress.

Objective Augmenter of liver regeneration (ALR) is a growth factor that is ubiquitously expressed in multiple forms among eukaryotes. The present study focused on the role of endogenous ALR on the hypoxia/reoxygenation (H/R)-induced inflammatory response in human kidney 2 (HK-2) cells, and the underlying molecular mechanisms. Methods To determine the relationship between exogenous and endogenous ALR, exogenous ALR was administrated to HK-2 cells, and endogenous ALR protein and mRNA expression was examined by Western blotting and quantitative real-time polymerase chain reaction (qPCR), respectively. In order to knockdown endogenous ALR expression, HK-2 cells were infected with lentiviral shRNA/ALR, after which cell viability was determined by the MTS cell viability assay. Cells were subjected to hypoxia for 6 h and reoxygenation for 12 h. Levels of monocyte chemotactic protein (MCP-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA) and qPCR. Cells were harvested, and nuclear and phosphorylated protein extracts were prepared from the HK-2 cell lysates. Nuclear factor κB (NF-κB), and phosphorylated extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) were analyzed by Western blotting. The translocation of NF-κB was detected by immunofluorescence. Results Exogenous ALR inhibited the expression of endogenous ALR. Lentiviral shRNA/ALR markedly downregulated endogenous ALR expression, whereas there were no changes in ALR expression in lentiviral shRNA/control HK-2 cells. The results of the MTS assay showed that silencing ALR expression did not influence cell viability. H/R led to increased production of MCP-1, IL-6, and TNF-α. However, knockdown of ALR attenuated the inflammatory response via inhibition of ERK, p38, and JNK phosphorylation. The translocation of NF-κB into the nucleus was also decreased. Conclusions These results suggest that there is a negative feedback loop involving ALR in HK-2 cells. Knockdown of ALR exerts anti-inflammatory actions via suppression of the mitogen-activated protein kinase signaling pathway.

The effects of augmenter of liver regeneration (ALR) on the acute kidney injury (AKI) rats were investigated by measuring the inflammatory response associated with transcription factor nuclear factory (NF-κB) pathway.

Oxidative stress plays an important role in cellular destruction. Augmenter of liver regeneration (ALR) is an anti-apoptotic factor that is expressed in all mammalian cells and functions as an anti-oxidant by stimulating the expression of a secretory isoform of clusterin and inhibiting reactive oxygen species (ROS) generation. Previous work from our group showed that ALR expression is upregulated in acute kidney injury (AKI) rats, and recombinant human ALR reduces tubular injury. In the present study, we used small interfering RNA (siRNA) silencing of ALR to examine its role in H₂O₂ induced mitochondrial injury and apoptosis. Knockdown of ALR increased ROS levels, reduced mitochondrial membrane potential, and increased the release of mitochondrial proteins and the rate of apoptosis in response to H₂O₂. In addition, the ratio of Bax/Bcl-2 was increased in siRNA/ALR groups treated with H₂O₂. These data confirm the protective role of ALR against oxidative stress-induced mitochondrial injury and suggest a potential mechanism underlying the protective role of ALR in AKI.

Elevated plasma low-density lipoprotein (LDL) is associated with systemic inflammation, and is an important factor in the pathogenesis of chronic kidney disease. The aim of the present study was to investigate the effects of endoplasmic reticulum (ER) stress preconditioning on LDL-induced inflammatory responses, in human mesangial cells (HMCs). HMCs were exposed to LDL (200 nm), with or without pretreatment with tunicamycin, an ER stress inducer, and tested for changes to gene expression levels. Small interfering RNA technology was used to knockdown the expression of inositol-requiring enzyme-la (IRE1 alpha) and X-box-binding protein-1 (XBP-1), in order to determine their effects on LDL-treated HMCs. LDL treatment resulted in a significant, and time-dependent, increase in the relative mRNA expression levels of proinflammatory cytokines and CD40, which was coupled with enhanced phosphorylation of IRE1 alpha, I kappa B kinase (IKK), and nuclear factor (NF)-kappa B p65 and p65 nuclear translocation. The LDL-induced inflammatory responses were significantly reduced in the IRE1 alpha-depleted HMCs. Furthermore, pretreatment with tunicamycin significantly attenuated the induction of proinflammatory cytokines and CD40, by LDL. Whereas, silencing XBP1 expression significantly restored the production of proinflammatory cytokines, in the LDL-treated HMCs with ER stress preconditioning. The phosphorylation levels of IRE1 alpha, IKK, and NF-kappa B p65 were markedly increased in the XBP1-depleted HMCs. Conversely, overexpression of XBP1 blocked LDL-induced inflammation in the HMCs. The results of the present study demonstrate that ER stress preconditioning antagonizes LDL-induced inflammatory responses in HMCs, which may be mediated through upregulation of XBP1, and subsequent inactivation of the IRE1 alpha/IKK/NF-kappa B pathway.