Introduction

Sepsis caused by infections of bacteria, fungi, or virus is an immensely severe and life-threatening syndrome. It leads to dysfunctioning of multiple organs and has been the leading cause of death in intensive care units (ICUs).^1,2 Acute kidney injury (AKI) is one of the most common and severe complications that emerges during the progression of sepsis, accounting for more than half of cases of AKI in Intensive care units.³ Specifically, sepsis-induced AKI leads a six- to eight-fold increased risk of deaths among sepsis patients,^3,4 and also an increased incidence of progression of chronic kidney disease in sepsis survivors.⁵ The development and progression of sepsis-induced AKI is an intricate crosstalk of multiple mechanisms, such as inflammatory response, oxidative stress, apoptosis, and ferroptosis.^6–9 Thus, despite advances in treatments, such as renal replacement, blood purification, and pharmacologic therapy,^10,11 high mortality and down-the-line outcomes still continue.¹² Therefore, exploring the potential molecular mechanisms of sepsis-induced AKI and seeking underlying targets could contribute to the development and improvement of therapies for sepsis-induced AKI.

Cation transport regulator-like protein 1 (CHAC1), situated in cytosol, has been identified in mammalian cells as a novel component of the unfolded protein response (UPR) pathway.¹³ CHAC1 is under the modulation of the Activating Transcription Factor 4 (ATF4) arm, ATF3, and C/EBP homologous protein (CHOP), thus it is the downstream of ATF4–ATF3–CHOP pathway.¹³ Notably, CHAC1 can promote apoptosis cascade via apoptosis-inducing factor (AIF) and poly(ADP-ribose) polymerase (PARP); so, it is generally recognized as a mammalian pro-apoptic factor.¹³ In addition, CHAC1 can degrade glutathione (GSH) in the cytosol of mammalian cells through its gamma(γ)-glutamylcyclotransferase activity.¹⁴ GSH is the most abundant non-protein thiol present in all mammalian tissues resistant to oxidative stress.¹⁵ Moreover, depletion of GSH is demonstrated as an early hallmark of apoptosis.¹⁶ Furthermore, GSH is the reducing substrate of glutathione peroxidase 4 (GPx4).¹⁷ The insufficient activity or missing of GPx4 can lead to ferroptosis, indicating that GSH is also essential for ferroptosis.¹⁷ In this study, we speculate that CHAC1 regulates the progress of sepsis-induced AKI through the modulation of apoptosis, oxidative stress, and ferroptosis.

In the current study, an in vitro model of sepsis-induced AKI was established in the human renal proximal tubular epithelial cells, HK-2 cells, with lipopolysaccharide (LPS) challenge. Then the role of CHAC1 in apoptosis, oxidative stress, and ferroptosis was investigated in LPS-induced HK-2 cells.

Materials and methods

Data extraction and analysis of differentially expressed genes (DEGs)

The GSE60088 microarray dataset was accessed from the Gene Expression Omnibus (GEO) database, which developed from the Affymetrix GPL1261 platform. It contained five lung cases of mice with sepsis-induced multiple organ dysfunction syndrome (MODS) challenged by a combination of mechanical ventilation and S. aureus pneumonia (MV+SA), four normal tissues from the lung, five liver cases of mice induced by MV+SA, three normal tissues from the liver, five kidney cases of mice induced by MV+SA (GSM1464844, GSM1464845, GSM1464846, GSM1464847, and GSM1464848), and five normal tissues from the kidney (GSM1464839, GSM1464840, GSM1464841, GSM1464842, and GSM1464843). The DEGs in kidney tissues and normal tissues were analyzed by using the LIMMA package in R language,¹⁸ and the heat map, including the top 30 up-regulated and the top 30 down-regulated genes, was also imaged with the thresholds of logFC (fold change) of ˃1.0 and P ˂ 0.01.

Functional enrichment identification

The functional enrichment identification was determined by the Metascape platform.¹⁹ It integrated the analysis of GO biological processes, KEGG pathway, Reactome gene sets, CORUM, Wiki pathways, and PANTHER pathway20.

Cell culture

HK-2 cells (CL-0109) were acquired from Procell (Wuhan, China) and hatched in minimum essential medium (MEM, PM150410; Procell) with 10% fetal bovine serum (FBS, 164210-50; Procell) and 1% penicillin–streptomycin (PB180120; Procell) at 37°C in an incubator with 5% carbon dioxide (CO₂).

Cell treatment and transfection

In order to explore the optimal treatment time of LPS on HK-2 cells, cells were challenged with 10-μg/mL LPS (Escherichia coli 055:B5, L8880; Solarbio, Beijing, China) for 2, 4, 8, and 12 h.²⁰ Meanwhile, two small interfering (si)RNAs targeting CHAC1 (siCHAC1#1 and siCHAC1#2) and the corresponding negative control (siNC) were obtained from GenePharma (Shanghai, China) and transfected in HK-2 cells with lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) for 6 h. Then HK-2 cells were challenged with 10-μg/mL LPS for 8 h. In addition, to further verify the relation between oxidative stress and apoptosis or ferroptosis, HK-2 cells were transfected with plasmid cloning (pc)DNA vector plasmids harboring the sequences of CHAC1 (CHAC1) or the empty pcDNA vector plasmids (vector) with lipofectamine 3000; these were consecutively treated with 5-mM N-acetyl cysteine (NAC) for 1 h as well as 10-μg/mL LPS for 8 h.

Western blot analysis

As described by Fu et al.,²¹ total proteins were extracted from cells with radioimmunoprecipitation assay (RIPA) buffer (R0010, Solarbio) and quantified with the bicinchoninic acid (BCA) protein assay kit (PC0020, Solarbio) by following the direction for use. Protein samples, 20 µg, were separated and electrically transferred onto a polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Billerica, MA, USA). After sealing with 5% skimmed milk (Anchor, Switzerland) at room temperature for 1 h, the membranes were incubated overnight at 4°C with the following primary antibodies targeted to diverse proteins: CHAC1 (1:500, ab217808; Abcam, Cambridge, UK), PARP (1:10,000, ab227244; Abcam), cleaved PARP (1:1000, ab4830; Abcam), caspase-3 (1:500, ab13847; Abcam), cleaved caspase-3 (1:100, ab2302; Abcam), GPx4 (1:1000, ab231174; Abcam), ACSL4 (1:10,000, 22401-1-AP; Proteintech, Wuhan, China) and β-actin (1:5000, ab8227; Abcam). The membranes were then hatched with goat anti-rabbit IgG H&L (HRP) (1:20,000; Abcam) at room temperature for 2 h and visualized by ECL Western blotting substrate (PE0010, Solarbio). The gray value was quantified by QUANTITY ONE software (Bio-Rad, Hercules, CA, USA).

Cell counting kit-8 (CCK-8) assay

The cell viability of HK-2 cells was assessed by CCK-8 assays (Dojindo Laboratories, Kumamoto, Japan) as described previously.²² The optical density (OD) at 450 nm was detected by using a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Flow cytometry assay

The level of reactive oxygen species (ROS) in HK-2 cells was evaluated with the flow cytometry assay after cells were incubated with 5-(and-6)-chloromethyl-2’,7’-dichlorofluorescin diacetate (CM-H2DCFH-DA) in the dark at 37°C for 30 min. For determining cell apoptosis, HK-2 cells were collected, rinsed with phosphate buffer saline (PBS) (P1010, Solarbio), resuspended by 0.5- mL bind buffer, and stained with 5-μL Annexin V/FITC (Thermo Fisher Scientific) and 5-μL propidium iodide (PI; Thermo Fisher Scientific) for 15 min at room temperature. Then, the relative fluorescence intensities and apoptosis of HK-2 cells were examined on a FACScan flow cytometer with the CellQuest software (BD Biosciences, NJ, USA).

Measurement of malonaldehyde (MDA) and GSH levels

The concentrations of MDA and GSH were detected with the MDA test kit (A003-1-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) at 532 nm and the total glutathione/oxidized glutathione assay kit (A061-2-1; Nanjing Jiancheng Bioengineering Institute) at 405 nm according to the operating manual under a microplate reader (Thermo Fisher Scientific).

Iron examination

The level of iron was examined using an iron assay kit (MAK025; Sigma, St. Louis, MO, USA) following the operating instruction manual.

Statistical analysis

Results were expressed as mean ± standard deviation (SD). Data were examined by normal distribution, and the difference was tested with the Student’s t-test between two groups or the one-way analysis of variance (ANOVA) between more than two groups, followed by post hoc Bonferroni test by the SPSS 26.0 software (IBM, Armonk, New York, USA). P ˂ 0.05 was considered as statistically significant.

Results

CHAC1 was overexpressed in the kidneys of septic mice

Through the GEO database, 192 up-regulated genes and 78 down-regulated genes were identified in the mice with sepsis-induced MODS challenged by a combination of MV+SA (Supplementary Tables S1 and S2). The top 30 up--regulated and 30 down-regulated genes are listed in Figures 1A and 1B. Meanwhile, results in Figures 1A and 1C showed that the expression of CHAC1 was observably increased in the kidney tissues of mice induced with MV+SA with logFC = 3.568785 and FDR (P-value adjusted for multiple tests) = 2.65E-07, compared with the kidney tissues of normal mice. Moreover, the functional enrichment analysis of both 192 up-regulated and 78 down-regulated genes was executed through the Metascape platform. As displayed in Figures 1D and 1E, the up-regulated genes were mainly enriched in several pathways, including response to extracellular stimulus, positive regulation of cell death, regulation of DNA-templated transcription in response to stress, and apoptotic signaling pathway. The down-regulated genes were mainly enriched in metabolism of lipids, cholesterol biosynthesis, and glucose homeostasis.

Knockdown of CHAC1 reduced LPS-induced oxidative stress in HK-2 cells

In order to explore the role of CHAC1 in the sepsis-induced AKI, an in vitro model was established in HK-2 cells with LPS treatment. Following the exposure to LPS for 2, 4, 8, and 12 h, the level of CHAC1 was notably enhanced, with the CHAC1 expression being highest at 8 h of LPS challenge (Figure 2A). As the expression of CHAC1 was up-regulated in tissues of both kidneys of mice with sepsis-induced MODS and LPS-induced HK-2 cells, two siRNAs targeting CHAC1 (siCHAC1#1 and siCHAC1#2) were transfected into HK-2 cells to down-regulate the level of CHAC1 (Figure 2B). The cell viability was significantly reduced with LPS treatment, which was at least partly restored by the treatment of siCHAC1#1 and siCHAC1#2 but not siNC (Figure 2C). Additionally, treatment of siCHAC1#1 and siCHAC1#2 prominently decreased the LPS-induced ROS level and the MDA concentration, while opposite tendency was indicated in the GSH concentration (Figures 2D–F). Thus, the data indicated that silencing of CHAC1 attenuated LPS-induced oxidative stress in HK-2 cells.

Silencing of CHAC1 alleviated LPS-induced apoptosis in HK-2 cells

Furthermore, the apoptosis rate of HK-2 cells was prominently enhanced with LPS treatment, which was notably antagonized with the transfection of siCHAC1#2 (Figure 3A). Consistently, transfection of siCHAC1#2 significantly decreased the LPS-induced relative protein level of Cleaved PARP–PARP and cleaved caspase-3–caspase-3 (Figure 3B). Therefore, knockdown of CHAC1 relieved LPS-evoked apoptosis in HK-2 cells.

Interference of CHAC1 ameliorated LPS-induced ferroptosis in HK-2 cells

Moreover, the role of CHAC1 in ferroptosis was also dissected with knockdown of CHAC1 in HK-2 cells. Treatment of siCHAC1#2 observably increased the LPS-induced relative protein level of GPx4 whereas markedly reduced the LPS-induced relative level expression of ACSL4 (Figure 4A). In addition, administration of siCHAC1#2 significantly neutralized LPS-induced level of iron (Figure 4B). Hence, knockdown of CHAC1 improved LPS-induced ferroptosis in HK-2 cells.

CHAC1 exacerbated ferroptosis and apoptosis by promoting oxidative stress

In order to define relation between oxidative stress and apoptosis or ferroptosis, NAC, an antagonist of oxidative stress, was incubated with HK-2 cells. Transfection of CHAC1 overexpression plasmid markedly accelerated the reduction of the relative protein expression of GPx4 induced by LPS, which was notably improved with the application of NAC (Figure 5A). Meanwhile, an opposite result was observed in the relative protein expression of ACSL4 (Figure 5A). In addition, administration of NAC counteracted the up-regulation of CHAC1-induced MDA concentration, while it markedly recovered the up-regulation CHAC1-induced GSH concentration in LPS-treated HK-2 cells (Figure 5B). Moreover, co-transfection of CHAC1 overexpression plasmid prominently promoted both increased level of iron and rate of apoptosis induced by LPS, which were observably reversed with the use of NAC (Figures 5C and D). Taken together, CHAC1 aggravated ferroptosis and apoptosis by enhancing oxidative stress in LPS-induced HK-2 cells.

Discussion

Sepsis-induced AKI is a singularly grievous complication during the progression of sepsis, which has caused tremendous load on both patients and the society.^3,4 It has been demonstrated that the pathogenesis of sepsis-induced AKI is closely associated with inflammatory response, apoptosis, oxidative stress, and ferroptosis.^6–8 CHAC1 is identified as a mammalian pro-apoptotic factor,¹³ which may be involved in apoptosis, oxidative stress, and ferroptosis. In the present study, the level of CHAC1 was found to be up-regulated in the kidney tissues of mice with sepsis--induced MODS according to bioinformatic analysis. The HK-2 cells were challenged with LPS to construct an in vitro model of sepsis-induced AKI. The CHAC1 level was consistently up-regulated in the LPS-induced HK-2 cells. Knockdown of CHAC1 dampened LPS-induced oxidative stress, apoptosis, and ferroptosis in HK-2 cells. Moreover, the application of NAC reversed the effects of CHAC1 on the apoptosis and ferroptosis of LPS-induced HK-2 cells. Taken together, CHAC1 aggravated LPS-induced ferroptosis and apoptosis in HK-2 cells by promoting oxidative stress.

CHAC1 is a novel component of UPR pathway, whose dysregulation has been reported in a variety of disease models. For instance, upregulation of CHAC1 has been discovered in intraocular (uveal tract) melanoma patients, predicting a poor outcome.²³ Similar results are also reported in breast and ovarian cancer patients.²⁴ In line with these findings, we also found that the expression of CHAC1 was enhanced in the kidney tissues of mice with sepsis-induced MODS based on bioinformatic analysis. Moreover, the CHAC1 was consistently expressed highly in LPS-induced HK-2 cells. Therefore, CHAC1 was overexpressed in sepsis-induced AKI.

Molecular mechanisms, such as apoptosis, oxidative stress, and ferroptosis, have been revealed to participate in the progression of a variety of diseases.^25–30 In sepsis--induced AKI, Huang et al. demonstrated that suppressing pannexin-1 inhibited cell apoptosis, which could contribute to the remission of sepsis-induced AKI.³¹ Methyl jasmonate, a bioactive oxylipid, attenuates apoptosis and inflammation in LPS-induced HK-2 cells.³² Similarly, surfactant protein D, an innate immune molecule, mitigates sepsis-induced AKI by reducing apoptosis.³³ Ibrutinib, an inhibitor of Bruton’s tyrosine kinase, attenuates oxidative stress in the kidney of AKI-associated dysfunction.³⁴ Al-Harbi et al. reported that spleen tyrosine kinase signaling relieves sepsis--induced AKI by inhibiting inflammation and oxidative stress.³⁵ In addition, the study conducted by Guo et al. revealed that ginsenoside Rg1, belonging to the class of steroid glycosides, suppressed ferroptosis to improve sepsis-induced AKI.⁶ Moreover, ferroptosis was involved in LPS-induced AKI through mitochondria-derived ROS.³⁶ In this study, our results also demonstrated that LPS treatment enhanced cell apoptosis, oxidative stress, and ferroptosis in HK-2 cells that were notably attenuated by the knockdown of CHAC1. CHAC1 acts as a proapoptotic factor,¹³ and promotes apoptosis in temozolomide-induced glioma cytotoxicity.³⁷ More importantly, CHAC1 mediates the degradation of GSH,¹⁴ which is strongly related to oxidative stress,¹⁵ apoptosis,¹⁶ and ferroptosis.¹⁷ Taken together, these results demonstrated that silencing of CHAC1 attenuated LPS-induced oxidative stress, apoptosis, and ferroptosis in HK-2 cells.

Owing to harmful stimulation of external and internal conditions, oxidative stress generates superfluous ROS, which can assault cell DNA, protein, and lipid, eventually resulting in a series of pathogenies, such as apoptosis and ferroptosis.^38,39 Cui et al. showed that alfalfa saponins, a natural extract of Alfalfa (Medicago sativa), suppressed oxidative stress-mediated apoptosis in piglet cells.⁴⁰ Bardoxolone methyl, a semi-synthetic triterpenoid, mitigates osteoarthritis by preventing oxidative stress-induced apoptosis.⁴¹ Similarly, Zhu et al. summarized the molecular mechanisms of modulating oxidative stress-induced ferroptosis, and its potential therapeutic value in cancer.²⁸ In addition, oxidative stress-induced ferroptosis is also involved in the progression of intervertebral disc degeneration.⁴² Similar these previous findings, NAC, an inhibitor of oxidative stress, reversed the effect of CHAC1 on apoptosis and ferroptosis in LPS-induced HK-2 cells in the current study, indicating that the role of CHAC1 in the pathogenies of LPS-induced HK-2 cells was associated with oxidative stress-induced apoptosis and ferroptosis.

Conclusion

The expression of CHAC1 was up-regulated in the kidney tissues of mice with sepsis-induced MODS and LPS-induced HK-2 cells. Overexpression of CHAC1 promoted the apoptosis, oxidative stress, and ferroptosis of LPS-induced HK-2 cells. Furthermore, the application of NAC reversed the effects of CHAC1 on the apoptosis and ferroptosis of LPS-induced HK-2 cells. Taken together, these findings illustrated that CHAC1 exacerbated ferroptosis and apoptosis by enhancing oxidative stress in LPS-induced HK-2 cells. However, an in vivo validation is essential in the present study. In brief, our study contributes to understand the molecular mechanisms for sepsis-induced AKI, which could further provide a potential target for AKI therapy.