C7ORF41 Alleviated Ferroptosis Through Keap1/Nrf2/HO-1 Axis in Sepsis-Associated Acute Kidney Injury

doi:10.21203/rs.3.rs-4708813/v1

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C7ORF41 Alleviated Ferroptosis Through Keap1/Nrf2/HO-1 Axis in Sepsis-Associated Acute Kidney Injury

https://doi.org/10.21203/rs.3.rs-4708813/v1

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Acute kidney injury (AKI) stemming from sepsis, termed SA-AKI, frequently emerges as a predominant complication among critically ill patients, with over half of intensive care unit (ICU) AKI cases linked to sepsis. Ferroptosis in tubules is implicated in SA-AKI development, yet its regulatory mechanism remains unclear. Recently, C7ORF41, a conserved sequence on chromosome 7, was associated with inflammation and lipid accumulation in palmitic acid. We investigated C7ORF41's role in lipopolysaccharide (LPS) induced AKI models in C57BL mice. Post-LPS treatment, renal tubules showed reduced C7ORF41 expression. C7ORF41 deficiency significantly mitigated LPS induced lipid peroxidation, tissue damage, and renal dysfunction. In vitro experiments showed decreased ferroptotic cell death, lipid ROS, and GPX4 expression in renal tubular cells lacking C7ORF41. From a mechanistic standpoint, ferroptosis is facilitated by C7ORF41 through activating the pathway involving Keap1, Nrf2, and HO-1, known for its cytoprotective and antioxidant properties. Our findings suggest that C7ORF41 promotes ferroptosis in SA-AKI through Keap1/Nrf2/HO-1 Axis, highlighting its potential as a therapeutic target for SA-AKI treatment.

C7ORF41

Acute Kidney Injury

ferroptosis

Keap1

Nrf2/HO-1

LPS

Acute renal failure (ARF) is a prevalent and severe clinical condition, with a rising occurrence rate annually ¹. If not properly managed, ARF can result in the failure of various organs in critically ill individuals, leading to a high mortality rate exceeding 50% ². Despite significant advancements in diagnostic technology and intensive care, the incidence of ARF remains a substantial concern. Sepsis, also known as septicemia or blood poisoning, is a systemic inflammatory response syndrome (SIRS) triggered by severe infection. It is characterized by a dysregulated host response to pathogens, often leading to organ dysfunction and failure. The kidneys are particularly vulnerable during sepsis due to factors such as hypoperfusion, inflammation, and direct toxicity from microbial products. The association between sepsis and AKI is well established in clinical practice and research. Sepsis increases the risk of AKI development, and conversely, AKI significantly worsens the prognosis for septic patients. The mechanisms underlying sepsis-induced AKI are complex and multifactorial, involving hemodynamic changes, immune dysregulation, microvascular dysfunction, and cellular injury. Despite improvements in medical care, the incidence of AKI among sepsis patients in the ICU remains alarmingly high, ranging from 40% to 50%, thereby elevating the likelihood of mortality ³. Sepsis and AKI are closely connected in numerous patients. Sepsis is the primary factor behind AKI, while AKI frequently occurs as a complication of sepsis ⁴. Due to kidney on sepsis. The production of lipopolysaccharide (LPS) is particularly sensitive, contributing to AKI in over 40% of sepsis patients. The occurrence of sepsis will worsen the prognosis of patients and further increase the mortality of patients. At the same time, survival patients are often accompanied by long-term illness, which greatly increases medical expenditure ⁵; ⁶.

C7ORF41, also known as chromosome 7 open reading frame 41, is a conserved sequence located within chromosome 7, reflecting its evolutionary significance. Although research into its energetic and protein composition remains scarce, observations indicate variations in the gene's expression throughout the development of human embryos ⁷. In a recent investigation, the excessive expression of C7ORF41 greatly improved liver inflammation and reduced the buildup of lipids in hepatocytes treated with palmitic acid (PA) through the c-Jun/C7ORF41/NF-κB regulatory network ⁸. Another study found that C7ORF41 is involved in TPA induction by regulating signaling pathways of NF-κB, MAPK/ERK and SAPK/JNK ⁹. The expression of C7ORF41 is upregulated when human CD34+ cells differentiate into megakaryocytes. C7ORF41 activates ERK and JNK signaling pathways and promotes megakaryocytes by upregulating RUNX1 and FLI1 differentiation. Knocking out the C7ORF41 gene in mouse liver cells inhibited megakaryocytogenesis ⁸. The NF-κB can be activated activation of C7ORF41, which in turn inhibits NF-κB signaling. Suggestive evidence points to a possible involvement of C7ORF41 in the advancement of inflammatory conditions in humans.

The concept of ferroptosis was initially introduced in 2012 by Dixon, who characterized it as a buildup of reactive oxygen species (ROS) within cells ¹⁰. Ferroptosis involves the depletion of cellular antioxidants and elevated intracellular iron levels. A number of studies have investigated ferroptosis in various diseases. Nevertheless, the extent to which ferroptosis is implicated in SA-AKI has yet to be clearly established. In their study, Cao and colleagues discovered that GPX4 plays a crucial role as a downstream mediator in the HDAC3 abnormality and renal ferroptosis while transitioning from AKI to CKD ¹¹. Additionally, Xiao et al reported that renal tubular epithelial cells in SA-AKI mice experienced ferroptosis, and the Nrf2 signaling pathway is utilized by MCTR1 to inhibit ferroptosis in SA-AKI ¹². In another research, it was noted that irisin suppressed the buildup of ROS and the generation of iron in HK-2 cells ¹³. These findings suggest that irisin may have the potential to prevent SA-AKI by suppressing ferroptosis through the SIRT1/Nrf2 signaling pathway. The existing data collectively indicate that ferroptosis significantly impacts SA-AKI. Further investigations are warranted to fully elucidate the molecular mechanisms underlying ferroptosis in SA-AKI and explore potential therapeutic interventions targeting this pathway.

The present research investigated the role of C7ORF41 in SA-AKI and assessed its connection with ferroptosis using both in vivo and in vitro approaches. To accomplish this, we generated C7ORF41 knockout mice to examine whether reduced C7ORF41 expression exacerbated SA-AKI. Additionally, we utilized HK-2 cells with different levels of C7ORF41 expression to study its involvement in ferroptosis.

C7ORF41 expression was decreased in the kidney of septic mice and in HK2 cells exposed to LPS. Our research initially discovered that in a mouse model of septic kidney injury induced by LPS, there was a notable increase in levels of blood Cr and BNU (Figure.1A/1B). Additionally, the kidney tissue exhibited substantial structural damage as evidenced by significant findings in HE tests (Figure.1C/1D). To establish the connection between kidney injury and C7ORF41, we collected kidney tissues to analyze the corresponding RNA and protein(Figure.1E/1F). Our findings indicate a gradual decrease in both the RNA and protein levels of C7ORF41. To further clarify the role of C7ORF41 in SA-AKI, we investigated the role of C7ORF41 in human renal tubular epithelial cells HK2. LPS-treated HK2 cells also showed reduced RNA and protein levels of C7ORF41 a timedependent manner (Figure.1G/1H). These data suggest that C7ORF41 expression is decreased during septic kidney injury and that C7ORF41 may have a nephroprotective role.

Ferroptosis was involved in LPS induced AKI. More and more studies are now finding that ferroptosis is linked to inflammation. To examine the presence of ferroptosis in SA-AKI, we conducted tail vein injections on SA-AKI mice with Fer-1, a compound that inhibits ferroptosis, and RSL3, a substance that induces ferroptosis. The results showed a decrease or increase in blood creatinine and serum urea nitrogen in the inhibitor Fer-1 or agonist RSL3-treated groups, respectively, compared to the experimental groups alone (Figure.2A/2B). The HE staining of kidney tissue revealed that tubular injury, which primarily consisted of widespread tubular cell death, severe cell loss, interstitial edema, and disruption of the brush border in the renal cortex, was either alleviated or exacerbated in the groups treated with Fer-1 or the agonist RSL3 (Figure.2C/2D).

GPX4, a phospholipid hydroperoxidase, serves as the primary indicator of ferroptosis. It hinders lipid peroxidation and can be suppressed by GSH, an antioxidant compound. Depletion of GSH directly activates lipoxygenase, which inhibits GPX4 function and triggers lipid peroxidation. Through the analysis of these biomarkers, it was observed that the experimental-only group exhibited notably elevated levels of reactive oxygen species (ROS), malondialdehyde (MDA), and ferrous ions (Fe²⁺), comparison to the control group. Additionally, this group displayed significantly decreased levels of glutathione (GSH). In contrast, the inhibitor Fer-1 or agonist RSL3-treated group exhibited decreased or increased levels of ROS, MDA, and Fe²⁺, as well as reduced levels of GSH (Figure.2E/2F/2G/2H). Furthermore, the expression of ferroptosis related proteins were also diminished or enhanced relative to the control group (Figure.2I). The data indicate that ferroptosis has a significant impact on the development of kidney damage during sepsis.

Knockdown of C7ORF41 aggravated renal injury and increased ferroptosis during SA-AKI in vivo. In order to evaluate the impact of C7ORF41 on ferroptosis in SA-AKI, we employed C7ORF41 knockout mice (hereinafter called C7ORF41KO) and littermate control C7ORF41WT mice. The findings showed that deletion of C7ORF41 exacerbated renal injury in septic mice, including significantly elevated sCr and BUN (Figure.3A/3B), and histological section staining revealed more severe tissue destruction (Figure.3C/3D) in the C7ORF41KO group, with increased nuclear lysis and cytoplasmic vacuolization in the renal tubules. Electron microscopic observations suggested that kidney tissue in the C7ORF41KO group showed more mitochondrial swelling, a reduced number of cristae and ruptured outer membranes compared to the control group (Figure.3E). Further detection of ferroptosis-related biomarkers showed that ROS, MDA and Fe²⁺ levels were significantly higher and GSH levels were significantly lower in the C7ORF41KO group (Figure.3F/3G/3H/3I). Simultaneously, the inclusion of Fer-1, a ferroptosis inhibitor, has the potential to counteract these occurrences. Further detection of ferroptosis-related protein expression by WB revealed that, in agreement with the previous results, FTH1, XCT, and GPX4 protein expression was reduced in the C7ORF41KO group. Similarly FTH1, XCT, and GPX4 protein expression increased after the addition of Fer-1, an ferroptosis inhibitor (Figure.3J). These data suggest that C7ORF41 knockdown increases SA-AKI ferroptosis and exacerbates renal injury.

Downregulation of C7ORF41 increased ferroptosis in HK2 cells exposed to LPS in vitro. In order to further explore the potential exacerbation of ferroptosis in renal tubular epithelial cells caused by C7ORF41 deficiency, we induced LPS stimulation in HK2 cells.Initially, we introduced control shRNA or C7ORF41-shRNA into HK2 cells and evaluated the levels of RNA and protein, indicating the successful establishment of C7ORF41 knockdown and control stable transfer cell lines (Figure.4A/4B). The analysis of markers associated with ferroptosis revealed a notable rise in levels of ROS, MDA, and Fe²⁺, while levels of reduced GSH exhibited a significant decrease in the group with down-regulated C7ORF41. Meanwhile, the addition of Fer-1, an ferroptosis inhibitor, reversed these indices accordingly (Figure.4C/4D/4E/4F). WB measurements showed a decrease in the levels of FTH1, XCT, and GPX4 proteins in the group with down-regulated C7ORF41 expression. Similarly, the expression of FTH1, XCT, and GPX4 proteins increased after the addition of Fer-1, an ferroptosis inhibitor (Figure.4G). These data suggest that C7ORF41 down-regulation increases LPS induced ferroptosis in HK2 cells.

The functioning of C7ORF41 occurs through the Keap1-Nrf2/HO-1 pathway. A multitude of research points to the Keap1/Nrf2/HO-1 signaling axis as a pivotal element in the modulation of both inflammation and oxidative stress ^14–16. Building on this foundation, we propose that Keap1/Nrf2/HO-1 may play a significant role in how C7ORF41 influences ferroptosis. Experimental observations across various living models and controlled lab environments have revealed a notable elevation of Keap1 proteins following LPS stimulation when benchmarked against a control set. Conversely, the elimination of C7ORF41 led to an upregulated Keap1 expression, while the levels of Nrf2 and HO-1 proteins diminished in comparison with samples that were exclusively exposed to LPS (Figure.5A).

In order to further verify the Keap1/Nrf2/HO-1 signaling pathway in the promotion of ferroptosis by C7ORF41, we introduced the overexpression of Nrf2 to shC7ORF41 HK2 cells and analyzed the levels of ferroptosis-related biomarkers and related proteins (Figure.5B). WB measurements showed a increase in the levels of FTH1, XCT, and GPX4 proteins in the group of pcDNA-Nrf2 HK2 cells (Figure.5C). Further detection of ferroptosis-related biomarkers showed that ROS, MDA and Fe²⁺ levels were decrease and GSH levels were increase in the pcDNA-Nrf2 group (Figure.5D/5E/5F/5G). These suggested that overexpression of Nrf2 significantly reversed the LPS-induced ferroptosis in HK2 cells. Consistent with our expectations, the addition of pcDNA-Nrf2 reversed ferroptosis, further confirming that C7ORF41 exacerbates septic kidney injury through Keap1/Nrf2/HO-1 signalling.

This study focused on the potential mechanism by which the gene C7ORF41 influences the condition known as SA-AKI. Our initial findings revealed that the renal expression of C7ORF41 increased in response to LPS stimulation, prompting us to explore its role in SA-AKI using C7ORF41 knock-out mice. In our in vivo experiments, we observed that C7ORF41 deficiency exacerbated renal dysfunction in LPS-induced mice, likely due to the knockout of C7ORF41 increasing ferroptosis and thus aggravating SA-AKI. We further demonstrated in vitro that C7ORF41 facilitates the initiation of the Keap1/Nrf2/HO-1 signaling cascade. Interestingly, we observed that the downregulation of C7ORF41 led to a decrease in Nrf2 levels, an essential regulator of ferroptosis, resulting in the suppression of ferroptosis markers such as FTH1, XCT, and GPX4. These findings suggest that Nrf2 plays a role in counteracting the impact of C7ORF41 on ferroptosis, indicating that Nrf2 is involved in the inhibitory effects of C7ORF41 on ferroptosis in the context of SA-AKI.

ARF encompasses a range of clinical conditions characterized by a sudden decline in kidney function. The mechanism underlying sepsis-induced AKI remains unclear, contributing to a limited understanding of strategies for preventing and managing septic AKI in patients. In recent years, several studies have demonstrated that genes and proteins can effectively relieve SA-AKI through various mechanisms ^17–19. The human chromosome 7 (7p14.3) contains C7ORF41, which consists of four exons and encodes a protein that is 131 amino acids long. At the time of writing, there are 246 species that have homologous genes to C7ORF41. The genetic material contains a protein that is widely preserved in animals with backbones, although its function and arrangement of sections are not well understood; it is abundantly present in the brain, fatty tissues, kidneys, ovaries, and various other body tissues ⁵; ⁶; ²⁰. The role of MTURN, the xenopus laevis counterpart of human C7ORF41, in primary neurogenesis and its involvement in the regulation of neural differentiation signaling pathways have been established ²¹. However, the role of C7ORF41 in SA-AKI remains largely unknown. In this study, we found that lost of C7ORF41 significantely aggravated LPS induced renal pathological changes in vivo. In addition, we have identified a role for C7ORF41 in ferroptosis. There have been several reports suggesting that ferroptosis can occur in AKI due to LPS ^22–24, that renal ferroptosis is significantly increased when SA-AKI occurs, and that inhibition of ferroptosis can achieve a reduction in renal injury to improve renal function. Our research aligns with these reports and discovered that there were ferroptosis related alterations in SA-AKI. Additionally, the utilization of ferroptosis inhibitors and agonists revealed a corresponding decrease and increase in ferroptosis, indicating that C7ORF41 mitigated renal damage by suppressing the onset of ferroptosis.

Iron is a vital micronutrient in the human body that plays various roles in biological processes, such as generating ATP and synthesizing DNA and hemoglobin ²⁵; ²⁶. High amounts of iron ions within cells, particularly ferrous ions, have the potential to trigger lipid peroxidation. Dixon et al conducted a study in the year 2012 ¹⁰. They coined the term ferroptosis to describe this form of cell death, which is triggered by an overabundance of lipid peroxidation and relies on iron. Ferroptosis is characterized by the accumulation of ROS in lipids, reduced levels of GSH, and the retention of iron within cells. Excessive accumulation of substantial quantities of ROS can trigger oxidative stress within cells, causing harm to proteins, nucleic acids, and lipids, and ultimately resulting in the occurrence of ferroptosis. Ferroptosis, in terms of its morphology, genetics, and mechanisms, distinguishes itself from previously recognized forms of cell death like apoptosis, necrosis, pyroptosis, and autophagy.

Mounting evidence suggests that ferroptosis plays a crucial role in the progression of AKI. Tan et al discovered that the absence of legumain reduced the severity of acute tubular injury, inflammation, and ferroptosis in a model of kidney injury caused by ischemia-reperfusion ²⁷. This indicates that legumain plays a role in facilitating chaperonin-mediated GPX4 autophagy, ultimately promoting ferroptosis in the neural tubules during AKI. Zhang et al demonstrated that the presence of the ferroptosis inhibitor ferrostatin-1 decreased blood urea nitrogen, blood creatinine, and tissue harm in a mouse model of AKI. Additionally, the VDR agonist paricalcitol mitigated cisplatin-induced kidney injury in AKI by reducing lipid peroxidation ²⁸. According to research, it is recommended that ferroptosis has a significant impact on cisplatin-induced AKI. The activation of VDR may hinder ferroptosis through the regulation of GPX4 expression, ultimately reducing the severity of kidney injury caused by cisplatin. Gao et al found that Quercetin has the ability to hinder ferroptosis in renal tubular epithelial cells during AKI ²⁹. This is achieved by elevating GSH levels through MDA and ROS. Moreover, the researchers identified ATF3 as a crucial transcription factor in this process, offering a novel approach for AKI treatment. In an AS-AKI model, we examined the correlation between inflammation and ferroptosis. The results suggest that inflammation can cause ferroptosis in kidney tissue and is more severe in C7ORF41 knockout mice. This confirmed that ferroptosis regulates the inflammatory response after LPS stimulation of HK2, while C7ORF41 could protect the kidney by inhibiting ferroptosis to attenuate the inflammation caused by LPS.

We further investigated the mechanism by which C7ORF41 inhibits ferroptosis attenuating inflammation caused by LPS. Numerous researches have indicated that the Keap1/Nrf2/HO-1 signaling pathway plays a role in controlling oxidative stress. Han et al showed that Panaxydol had a notable positive effect on the pulmonary pathological alterations, lessened pulmonary edema and inflammation, and suppressed ferroptosis and inflammatory reactions in bronchial epithelial cells through the Keap1/Nrf2/HO-1 pathway. As a result, it offered defense against acute lung injury induced by LPS ³⁰. A study on acetaminophen-induced acute liver injury discovered that salvianolic acid C safeguarded liver cells from harm by blocking the Keap1/Nrf2/HO-1 signaling pathway, reducing mitochondrial oxidative stress, suppressing inflammatory response, and preventing caspase mediated apoptosis ³¹. In another study on Methotrexate-induced acute kidney injury, Maged E Mohamed confirmed that geraniol reduced Keap1 levels, increased Nrf2 and HO-1 expression, raised antioxidant markers GSH, SOD, CAT, and GSHPx, decreased MDA and NO, mitigated apoptosis regulators Bax, caspase-3 and − 9, enhanced Bcl2 expression, and ultimately alleviated pathological changes in kidney tissue ⁷. Hence, it is speculated that the involvement of Keap1/Nrf2/HO-1 in the inhibition of ferroptosis by C7ORF41. Consistent with the hypothesis, in C7ORF41 decreased the expression of Keap1 while elevating the expression of Nrf2 and HO-1. Additionally, by utilizing pcDNA to elevate Nrf2 expression, we observed a substantial reduction in the protective effects of C7ORF41 against ferroptosis and inflammation. This implies that Nrf2 plays a crucial part in the mechanism through which C7ORF41 inhibits ferroptosis.

Antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase) and nuclear factor erythroid 2-related factor (Nrf2)/heme oxygenase (HO-1) have the potential to act as antioxidants, thereby contributing significantly to AKI by mitigating oxidative stress-induced damage. Below, the description of how these substances impact the plant's antioxidant activity is provided. Nrf2 is a protein that can be activated and has protective and antioxidant properties. Under normal conditions, Nrf2 binds to the Keap1 protein in the cytoplasm and the activity of Nrf2 is precisely regulated by the negative regulatory protein Keap1. Keap1 is thought to confine Nrf2 to the cytoplasm by binding to the actin cytoskeleton and Nrf2, respectively. Upon exposure to oxidative stress, cells undergo dissociation of Nrf2 from Keap1, followed by translocation to the nucleus and subsequent binding to M af in order to create a heterodimer. This heterodimer then activates downstream gene expression mediated by ARE, as stated in reference ³². Nrf2 activation and HO-1 expression can be induced simultaneously by oxidative stress and pro-inflammatory elements, and Nrf2 can also directly control the activity of the HO-1 promoter. HO-1, also known as Heme Oxygenase-1, plays a crucial role in breaking down heme into ferrous iron, carbon monoxide, and biliverdin. This essential antioxidant enzyme effectively prevents cytotoxicity caused by oxidative stress and inflammatory responses from different sources ³³. On one side, the deterioration of the heme unit aids in impeding its pro-oxidant impacts. Conversely, the by-product biliverdin and its diminished bilirubin exhibit a potent ability to eliminate reactive oxygen species, including peroxides, peroxynitrite, hydroxyl, and superoxide radicals ³⁴. In sepsis models, the presence of LPS greatly amplifies the generation of ROS, triggers the initiation of inflammatory reactions, reduces the activity of antioxidant enzymes (catalase, SOD, and GPx), and elevates MPO activity linked to the infiltration of neutrophils. However, an excessive amount of ROS leads to cellular harm and oxidative stress. The findings of this research suggest that C7ORF41 reduces the production of ROS and activates the liberation of Nrf2 from Keap1. The upregulated Nrf2 presence within the nucleus plays a role in triggering the production of HO-1 and GPX4, which aid in the removal of ROS via oxidative harm.

The results of the present study suggest that the renoprotective effect of C7ORF41 on AKI is achieved through the Keap1/Nrf2/HO-1 axis by inhibiting oxidative stress and reducing ROS production.

In conclusion, our study provides evidences that C7ORF41 protects AKI via inhibiting ferroptosis. The study indicates that (1) C7ORF41 effectively prevents kidney damage in SA-AKI; (2) In HK2 cells treated with LPS, C7ORF41 can alleviate inflammation by inhibiting ferroptosis; (3) The mechanisms by which C7ORF41 regulates ferroptosis and inflammation in HK2 cells involve the Keap1/Nrf2/HO-1 pathway (Figure.6). We consider this research as an initial step towards developing therapies for AKI that target ferroptosis. C7ORF41 could potentially serve as a new therapeutic target for treating SA-AKI.

Cell culture. In these studies, HK-2 cells, acquired from the Cell Bank of the Chinese Academy of Sciences in Shanghai, China, were employed. These cells were cultivated in an incubator designated for cell culture, maintained at a temperature of 37°C and an atmosphere containing 5% CO2. The growth medium used was Dulbecco’s Modified Eagle Medium (DMEM)/F-12, enriched with 10% fetal bovine serum (FBS) and 1% penicillin, to nourish the cells. To assess the extent of cellular impairment in SA-AKI, the HK-2 cells underwent treatment with 10 µg/mL LPS obtained from Sigma (Product code L3129).

Cell lines. HK-2 cells underwent transfection with lentiviral particles carrying C7ORF41 short hairpin RNA (shC7ORF41) or with control viral particles (C7ORF41-vector) obtained from Shanghai Shenggong Bioengineering Co., Ltd., China. The infection of HK-2 cells was executed at a multiplicity of infection (MOI) of 50, using 5 µg/mL of polybrene over a duration of 18 hours. Following this, the HK-2 cells were seeded into 6-well plates and subjected to 5 µg/mL of puromycin treatment for 72 hours to ensure cell selection. The efficiency of the transfection was evaluated by quantitative Polymerase Chain Reaction (qPCR) and Western blot analyses.

Total RNA extraction and qRT-PCR. To extract total RNA from HK-2 cells, the RNAiso Plus reagent from TaKaRa (Product code 9108Q) was used. This RNA was then reverse-transcribed into complementary DNA (cDNA) using reverse transcription kits provided by Invitrogen (Kit K1621). The resultant cDNA was subsequently stored at a temperature of -20°C. Real-time PCR assays were conducted utilizing a qPCR kit supplied by Novoprotein (Catalogue #E096-01A). To normalize the expression levels of the target genes, the housekeeping gene GAPDH was used as an internal control.

The PCR protocol entailed an initial pre-denaturation step at 95°C for 30 seconds, followed by 40 cycles of PCR reaction, which consisted of denaturation at 95°C for 5 seconds and annealing/extension at 60°C for 30 seconds, and concluded with a melt curve analysis stage. Gene expression levels were quantified as fold changes relative to the control by applying the 2-AACT method. The specific primers used in the qPCR are detailed subsequently:

Nrf2: 5`-GGAUGGAUUUCUACGCCGACC-3` .

Gapdh: 5`-GTCTCCTCTGACTTCAACAGCG-3`.

Then cDNA was used as the template for SYBR qRT-PCR.

Western blot analysis. To extract total proteins from HK-2 cells and mouse kidney tissues, the process adhered to the protocol provided by the manufacturer. Each sample contributed approximately 60 micrograms of proteins, which were then separated via SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) and subsequently transferred onto PVDF (Polyvinylidene Fluoride) membranes. The membranes, once prepared, were blocked with 5% non-fat milk for one hour to prevent non-specific binding. Following the blocking step, they were incubated overnight with the primary antibody tailored to the protein of interest.

On the subsequent day, the membranes underwent incubation with the secondary antibody, specific to the primary antibody, for one hour. This was done to enable the detection of the protein of interest by enhancing the signal. The detection of protein bands was facilitated by a protein development instrument, indicating the successful binding of the antibodies to their corresponding proteins.

Cell viability assay. Around 3,000 cells were placed into each well of a 96-well plate and were incubated for 24 hours in the presence of various concentrations of LPS (Lipopolysaccharide). After each treatment, 10 microliters of CCK-8 solution (Product Number CK04; obtained from Dojindo Molecular Technologies, Inc.) was added to each well. The cells were then incubated for an additional hour with the CCK-8 solution, which facilitates the assessment of cell viability based on metabolic activity.

Following the incubation with the CCK-8 solution, the absorbance was measured at a wavelength of 450 nm to determine cell viability. To ensure the reliability and reproducibility of the results, the entire procedure was repeated three times.

Transmission electron microscopy. After the necessary steps, the Kidney tissues were trimmed to a dimension of 1 mm3 and then fixed in a 2% glutaraldehyde solution for 1 hour at room temperature. Subsequently, the samples were exposed to a dimly illuminated setting and treated with 2% uranium acetate for a period of 10 minutes at room temperature. Afterwards, they were washed for a duration of 20 seconds. Subsequently, the samples underwent a 10-minute treatment with lead citrate at room temperature, followed by a 20-second rinse. In the end, the samples were recognized at the electron microscopy center located in Renmin Hospital, which is associated with Wuhan University. Acquisition of images was done using the HITACHI Transmission Electron Microscope (model H7650B). Representative images are displayed for each structure of interest, with a minimum of 10 images acquired for each.

ROS analysis. The assessment of ROS levels in HK2 cells was conducted using a ROS detection kit (from Beyotime Institute of Biotechnology, catalog# S0033M), adhering strictly to the guidelines provided by the manufacturer. The HK2 cells were initially seeded into six-well plates with a cell density of 600,000 cells per well. They were then exposed to LPS at a concentration of 10 µg/mL for a 24-hour period for the purpose of ROS induction. To further investigate the role of ferroptosis in ROS levels, HK2 cells were also treated with RSL3 (a ferroptosis inducer) and ferrostatin-1 (a ferroptosis inhibitor) at 37°C for 2 hours. Subsequently, post this pre-treatment, cells were again subjected to LPS (10 µg/mL) for another 24 hours at the same temperature of 37°C. For the detection of ROS, after the second LPS treatment, cells were incubated with 10 µM of the fluorescent probe 2,7-dichlorofluorescein diacetate (H2DCF-DA), at 37°C for one hour. This dye is utilized for the intracellular detection of ROS due to its oxidation by ROS into a highly fluorescent compound. Post-incubation, the cells were meticulously washed twice with PBS to remove any residual dye and then resuspended in 200 µL of PBS. The quantification of ROS was done through Flow Cytometry, using equipment from BD Biosciences, and data analysis was conducted with FlowJo 7.6 software. For each treatment condition, a minimum of 10,000 cells were analyzed to ensure statistical relevance and accuracy.

The levels of MDA and GSH in samples of cells and kidney tissues. The concentrations of MDA and GSH within both cell and renal tissues were quantified utilizing respective assay kits for MDA and GSH, adhering to the guidelines set by the providers. These substances' levels were detected at wavelengths of 450 nm and 405 nm, correspondingly, via a microplate fluorometer. Moreover, to determine the total protein content in the samples, the Bradford assay was employed, a method provided by the Beyotime Institute of Biotechnology in Haimen, China.

Measurement of BUN, sCr. Kidney tissue and blood were collected for biochemical analysis. Serum levels of BUN and sCr were assessed by employing the Mouse BUN Elisa kit provided by Shanghai Huabang Biotechnology Co. (HB-P9S949X), following the guidelines provided by the manufacturer.

Kidney tissue histopathological, immunohistochemistry (IHC). Harvested recently obtained kidney tissues were promptly preserved in formalin and subsequently encased in paraffin. The tissues were then sliced into sections with a thickness of 5 um, which were utilized for hematoxylin-eosin (H&E) staining, IHC. To evaluate the extent of renal interstitial injury in mice, various factors are considered, including the shape of tubular endothelial cells, the integrity of the brush border, the presence of renal epithelial casts, and the number of necrotic cells in the lumen. Image J software detected the photographs.

Iron measurements. The cells were homogenized using an iron assay kit (Abcam, China) in iron assay buffer, following the provided instructions.Briefly, all cell lysates and standards were added to 96-well plates, and assay buffer was also added.Incubation of all samples took place at a temperature of 37°C for a duration of 30 minutes.Afterwards, the samples were supplemented with iron probes and incubated at a temperature of 37°C for a duration of 1 hour. Finally, the OD value was measured with a colorimetric microplate reader at 593 nm.

Animals and experimental protocol. Cyagen Biotechnology Co., LTD (located in Suzhou, China) provided 40 C7ORF41 conventional knockout mice (KOCMP-10672-Mturn), while Beijing HFK Bioscience Co., Ltd. (based in Beijing, China) supplied 40 C57BL mice. Male mice that were 8 weeks old and weighed between 23 and 25 grams were utilized. The Ethics Committee of Renmin Hospital of Wuhan University reviewed and granted approval for the animal study. In order to create a mouse model of nonfatal kidney injury caused by sepsis, wild-type (WT) and C7ORF41 knockout (KO) mice were given a single injection of 5 mg/kg Escherichia coli LPS (serotype 0111 B4, Sigma) intraperitoneally. The control group was administered an equivalent amount of sterile saline. C7ORF41 knockout mice or wild-type mice were randomly assigned to two groups: control group and LPS group, each consisting of a minimum of 20 mice. After a 24 hours period, the mice were euthanized and their samples were collected for future utilization. The anesthetic drug given was sodium pentobarbital at a dosage of 30 mg/kg. Carbon dioxide induction is the method employed for euthanizing the mice. At the beginning, the flow of air should remain consistent with a carbon dioxide level ranging from 30–70% V/min for approximately one minute. Furthermore, it is crucial to uphold the circulation of air for a minimum duration of 1 minute subsequent to clinical death in order to prevent reversal. A minimum of three replications were conducted for all animal experiments.

Ethics declarations. The Ethics Committee of Renmin Hospital of Wuhan University reviewed and granted approval for the animal study. All experiments were performed in accordance with relevant named guidelines and regulations. The authors complied with the ARRIVE guidelines.

Statistical analysis. To ensure the reliability and precision of the results, each experiment was repeated three times. Such replication increases confidence in the consistency and reproducibility of the experimental outcomes. For the purpose of statistical evaluation, SPSS software (version 22.0) was utilized. Specifically, the Analysis of Variance (ANOVA) statistical test was conducted. ANOVA is a technique used to compare the means of three or more groups to see if at least one group mean is statistically different from the others.

Acknowledgements

We thank all the people and organizations that helped us in this study.

Author contributions statement

Yunzhao Yang and Xuan Peng conceptualized the study. Zhong Wang contributed to the datacuration. Huaxin Wang conducted the formal analysis. Xi Yu and Chenglin Ye investigated the study. Haoren Shao contributed to the methodology. Xi Yu and Chenglin Ye wrote, reviewed, and edited the manuscript. All authors contributed to the article and approved the submitted version. All authors have agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even ones in which the author was not personally involved, are appropriately investigated, resolved, and the resolution documented in the literature.

Data availability statement

Requests for materials should be addressed to X.P.

Additional information

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

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C7ORF41 Alleviated Ferroptosis Through Keap1/Nrf2/HO-1 Axis in Sepsis-Associated Acute Kidney Injury

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