Inhibition of Lipid peroxidation by ALR protects the kidney from ischemia-reperfusion injury

Inhibition of Lipid peroxidation by ALR protects the kidney from ischemia-reperfusion injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Inhibition of Lipid peroxidation by ALR protects the kidney from ischemia-reperfusion injury Lili Huang, Ling Zhang, Fangyan Tan, Yixin Ma, Xujia Zeng, Dan Cao, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2649851/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Ferroptosis, characterized by lipid accumulation in intracellular compartments, is related to acute kidney injury (AKI), but the mechanism remains obscure. In our previous study, we reported important roles for augmenter of liver regeneration (ALR) in antioxidant mechanisms. However, the roles of ALR in ferroptosis, especially the morphological changes in mitochondria induced by this type of regulated cell death, remain unclear and warrant further investigation. Methods We subjected Kidney-specific deletion of the ALR gene (ALR-K-KO), as well as HK-2 cells, to ischemia-reperfusion (I/R) induced AKI models. We assessed the kidney function and ferroptosis of proximal tubular epithelial cells. We also examined the level of lipid peroxidation by MS/MS. ALR and Long chain acyl-CoA synthetase 4 (ACSL4) were colocalized and interacting regions were detected by protein docking-analyses. Results Here, we hypothesize that ALR regulates oxylipin accumulation in proximal tubular cells and attenuates ferroptosis induced by ischemia-reperfusion (I/R) injury in AKI. Kidney-specific deletion of the ALR gene (ALR-K-KO) aggravated ferroptosis, accompanied by increased ROS production and mitochondrial damage, whereas overexpression of the ALR gene attenuated lipid accumulation. Moreover, acsl4 loss reduced mostly polyunsaturated fatty acids. In addition, ALR and ACSL4 colocalize in the mitochondria of HK-2 cells and protein docking analysis found the interacting regions. Conclusion We showed for the first time that ALR binds to ACSL4 and regulates ferroptosis in proximal tubular cells by attenuating oxylipin accumulation. Acute kidney injury Augmenter of liver regeneration Ferroptosis Long chain acyl-CoA synthetase 4 Lipid peroxidation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Acute kidney injury (AKI), a common complication associated with high mortality and morbidity in critically ill patients, is characterized by a sharp decline in renal function and extensive proximal tubular cell death in a short period of time (i.e., within 48 h)[ 1 – 3 ]. Ischemia-reperfusion (I/R) injury is the main cause of AKI [ 4 , 5 ]. However, following I/R injury, renal function can recover within a few days of blood flow recovery [ 6 , 7 ]. Unfortunately, if I/R injury is not reversed in a timely manner, then the kidneys are vulnerable to chronic kidney disease, even end-stage renal damage. Presently, the outcomes of interventional treatments, including symptomatic and supportive therapy, as well as renal replacement for chronic AKI, have been disappointing [ 8 , 9 ]. Thus, it is important to define the mechanism of AKI and to identify new therapies for AKI. Recent studies have reported that aberrant biochemical and metabolic processes can trigger a variety of diseases, and they are the causative factors of inflammation [ 10 ]. There is compelling evidence indicating that kidney diseases are closely related to metabolic aberrations [ 11 – 16 ], with kidney diseases causing metabolic aberrations, such as excessive lipid peroxidation, abnormal amino acid metabolism, and aberrant glucose breakdown, and vice versa, especially in I/R injury-induced AKI. Unfortunately, there are few studies addressing lipid peroxidation in AKI. Ferroptosis, a unique iron-dependent form of regulated cell death, is biochemically, morphologically, and physiologically distinct from other forms of cell death, such as apoptosis, necrosis, and autophagy [ 17 – 19 ]. Lipid peroxides, a major source of free radicals, can trigger ferroptosis in the absence of glutathione (GSH)-dependent antioxidant defences [ 18 , 20 ]. As a relatively new type of regulated cell death, the hallmarks of ferroptosis are a series of changes in mitochondria characterized by the appearance of smaller than normal mitochondria, darker‐stained membranes, and disorganization/reduction of mitochondrial crista [ 21 ]. Moreover, emerging evidence suggests that mitochondria play critical roles in ferroptosis [ 22 – 24 ], and ferroptosis is involved in AKI. For instance, the inactivation of the ferroptosis-associated gene, glutathione peroxidase 4 (GPX 4 ), triggers AKI in mice [ 25 , 26 ], whereas the inhibition of ferroptosis by a pharmacological inhibitor can suppress proximal tubular cell death [ 27 ]. Augmenter of liver regeneration (ALR), which is essential for respiration, is a growth-promoting factor in the liver that was initially identified in the rat liver and reported to promote hepatocyte proliferation and liver regeneration [ 28 ]. However, ALR is widely expressed by all mammalian tissues, including the kidneys [ 29 ]. In our previous study, we reported that ALR plays a protective role against oxidative injury by reducing reactive oxygen species (ROS) production and accumulation in renal proximal tubules [ 21 , 30 ]. The accumulation of lipid ROS is another hallmark of ferroptosis, although where and how they are generated during ferroptosis is not clear. Additionally, little is known about the relationship between excessive lipid peroxidation and ALR expression in ferroptosis. In this study, we systematically analyzed the differences in lipid composition after kidney-specific deletion of the ALR gene (ALR-K-KO) in AKI. We also examined the relationship between ACSL4, a ferroptosis-associated maker, and ALR, and concluded that ALR could alleviate lipid accumulation by suppressing the ACSL4 pathway. Materials And Methods Experimental model and subject details All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all procedures and protocols were approved by the Chongqing Medical University Animal Care and Use Committee (approval no. 2018,146). To generate the kidney-specific ALR knockout mice, we conditionally knocked out the ALR gene (also known as GFER) using the Ggt1-Cre/LoxP system. ALR lox/flox mice were used as the control. To generate the proximal tubule-specific ALR knockout mice (PT-ALR KO), we crossed ALR flox/flox mice with ALR Cre expressing mice (Figure S1, Supplemental Material). Establishment of the ischemic AKI model in vivo Bilateral ischemic AKI was induced as previously described [42]. Briefly, mice were anesthetized with 60 mg/kg of sodium pentobarbital in 0.9% NaCl (stock concentration, 5 mg/ml) by intraperitoneal injection. The core body temperature was monitored with a temperature control system with a rectal prob (HP-30, Beijing Cinotech Co., China) and maintained at 36.6–36.8°C (optimal temperature, 36.7°C ± 0.1°C). Bilateral flank incisions were made to expose the kidneys, and renal ischemia was induced for 22 min using micro-clamps and micro-clips (RS-5420/RS-5410, Roboz, UK). The micro-clamps were removed, and the restoration of blood flow was visually confirmed. All mice were euthanized 1, 2, and 3 days after renal ischemia. Blood and kidneys were collected. C ollections of blood, urine, and histological analysis of human kidney tissue This study related to patients has been approved by the Institutional Review Board of The Second Affiliated Hospital, Chongqing Medical University (approval no. 2018,146). Human kidney tissue samples and blood were collected in our experimental studies. The adjacent noncancerous tissues and AKI tissues were respectively used as the normal group(n=6) and AKI group (n=6). The patients from adjacent noncancerous tissues were identified by the Second Affiliated Hospital of Chongqing Medical University by magnetic resonance imaging and pathological examination, all patients provided a written informed consent prior to surgery. The AKI patients were identified by the renal function according to the guideline from Kidney Disease: Improving Global Outcomes. All samples were removed aseptically and frozen in liquid nitrogen until use. Serum creatinine, serum BUN and glomerular filtration rate were collected and measured by the automatic biochemical analyzer. Establishment of the ischemic AKI model in vitro Human kidney proximal tubular HK‐2 cells were cultured in Dulbecco’s Minimum Essential Medium F12 (Gibco, USA) supplemented with 10% fetal bovine serum (Moregate, Australia) and 1% penicillin–streptomycin (Invitrogen, USA) in an atmosphere of 5% CO 2 at 37°C. To induce H/R injury, HK-2 cells were cultured in serum-free medium overnight, followed by serum-free and glucose-free medium in an atmosphere of 94% N 2 , 5% CO 2 , and 1% O 2 for 6 h, as previously described [21, 43]. HK-2 cells were then transferred to an atmosphere of 5% CO 2 and cultured in complete medium for 12 h. Transmission electron microscopy (TEM) Kidneys were immediately collected, minced into 1-mm 3 fragments, and fixed overnight at 4℃. On the following day, the tissues were postfixed in 1% OsO 4 , dehydrated through a graded-alcohol series at room temperature, and embedded in resin. The blocks were sectioned, and 80-nm continuous sections were stained with 2% uranium acetate and observed by TEM (Hitachi, Japan). High-throughput RNA sequencing and informatic analysis Total RNA was isolated with TRIzol Reagent (Invitrogen, China) according to the manufacturer’s instructions. RNA quality, purity, and quantity were assessed by Shanghai NovelBio Bio-Pharm Technology Co., Ltd. The samples were processed by an Illumina HiSeq X Ten Sequencing System. The differentially expressed genes (DEGs) were filtered using the significant threshold value ( p -value), fold change (FC), and false discovery rate (FDR). A total of 5042 DEGs were identified between control and AKI groups (Supplementary Table S1). The DEGs identified between control and ALR KO mice after I/R injury are listed in Supplementary Table S3. Differences in pathways were identified using the Student’s t -test after correcting for multiple hypotheses and accepting pathways with a 5% FDR cutoff. Detection of ROS in cells and tissues For cells, ROS levels were detected by dihydroethidium (BestBio, China). After H/R injury, the cells were harvested and incubated with dihydroethidium (1:1000) for 30 min at 37℃ in the dark. The cells were washed with PBS and analyzed by flow cytometry. For tissues, ROS levels were detected by Cy3 (Sigma, USA). After H/R injury, kidney sections were incubated with Cy3 (1:500) for 30 min at 37℃ in the dark. The cell nuclei were counterstained with DAPI. The cells were viewed by microscopy (Nikon Eclipse C1, Japan). Western blotting The cells were washed, and mitochondrial proteins were extracted using the Mitochondrial Kit (Beyotime Biotech Co., Ltd., China) according to the manufacturer’s instructions. The protein concentration was determined using the BCA Protein Assay (KenGen Biotech, China). The proteins were separated by 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto polyvinylidene membranes. After blocking in 5% nonfat milk in Tris-buffered saline–Tween-20 (TBST) for 2 h at room temperature, the membranes were incubated with specific primary antibodies overnight at 4℃. Subsequently, the membranes were washed three times with TBST and incubated with the indicated secondary antibody for 1 h at room temperature. The ECL (Beyotime Biotech Co., Ltd., China) was used for detecting immunoreactive proteins. The antibodies were as follows: NGAL (1:1000, ab216462m, Abcam, UK), KIM-1 (1:1000, Novus, USA), GPX 4 (1:8000, ab125066, Abcam), ACSL4 (1:5000, ab155282, Abcam), ALR (1:1000, abx129857, Abcam), COX Ⅳ (1:1000, Cell Signaling Technology, USA), and xCT (1:10000, ab175186, Abcam). qPCR analysis RNA was extracted from cells and tissues using the Total RNA Purification Kit (Biyuntian, China). Approximately 1 µg of RNA was reverse transcribed into cDNA using the Reverse Transcription Kit (Takara, Japan). Real-time PCR was performed using the SYBR Premix Kit (TaKaRa, Japan). Primer sequences are listed in Supplementary Table S6 of Supplemental Material. Co-immunoprecipitation HK-2 cells were lysed in lysis buffer, and the protein concentration was determined as previously indicated. The protein lysates (1.5 mg) were incubated with the indicated antibodies at a protein:antibody ratio of 500:1 overnight at 4°C on a rotator. The antibodies were as follows: anti-ALR polyclonal antibody (11293, Proteintech, USA) and anti-ACSL4 polyclonal antibody (ab155282, Abcam). Anti-IgG (3900, Cell Signaling Technology) served as the negative control. On the following day, 100 μL of a 20% slurry of agarose beads (Abcam) was washed three times with lysis buffer and added to the samples for 2 h at 4°C on a rotator. The immunocomplexes were centrifuged at 2500 rpm for 5 min at 4°C, and the supernatants were washed three times with lysis buffer and centrifuged to yield the “input” sample. The immunocomplexes were combined with an equivalent volume of 1× SDS-PAGE sample buffer and heated at 95°C for 5 min. Western blotting was performed as previously described. Cell immunofluorescence To determine whether ALR and ACSL4 colocalize in mitochondria, MitoTracker Red CMXRos (Beyotime Biotech Co., Ltd) was used to label viable mitochondria. HK-2 cells were seeded in confocal dishes and allowed to adhere overnight. After H/R injury, the cells were washed with phosphate-buffered saline three times and stained with trimethylrhodamine, methyl ester, for 20 min, followed by fixation with 4% paraformaldehyde. The cells were washed, permeabilized, blocked with 0.5% BSA, and incubated overnight at 4°C with primary antibodies (ACSL4, 1:100 or ALR, 1:50). On the following day, the cells were incubated with a fluorescein isothiocyanate (FITC)- or a tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary antibody for 60 min in the dark at room temperature. The cell nuclei were counterstained with DAPI. The cells were observed by confocal laser-scanning microscopy. siRNA and plasmids The ACSL4 siRNA target sequence was as follows: 5′-GCAAUAAUCCUGCUAUGGAtt-3′, and the siRNA negative control (NC) sequence was 5′-UUCUCCGAACGUGUCACGUdTdT-3′. The siRNAs were purchased from Shanghai GeneBio (China), and they were used at a concentration of 50 nM. The pReceiver-Lv105 plasmid and empty vector (negative control) were purchased from GeneBio (China). The cells were transiently transfected with Lip2000 Transfection Reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Transfection efficiency was determined by qRT-PCR analysis. The sequences of transient downregulation of ACSL4 are provided in Supplementary Table S6 of Supplemental Material. The sequence of the expression clone, as well as its verification, are provided in Supplementary Figure S4A–C. Lentivirus transfection and stable cell clone establishment The lentiviral-based small hairpin RNA (shRNA) and the ALR overexpression construct were purchased from GeneChem (China). HK-2 cells were infected with lentiviruses with an MOI 6. The full length ALR gene (GFER, NM_005262) was synthesized using a human cDNA library. All the sequences (Supplementary Table 3) were cloned into a lentiviral vector. The order of the elements from the ALR overexpression construct was Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin. At 35% confluence, HK-2 cells were infected with lenti-ALR-EGFP (referred to as ALR overexpression, ALR-OE) or lenti-EGFP (referred to as vector). After 72 h, the transfection efficiency was assessed by fluorescent microscopy, and the cells were selected for 2 weeks using puromycin (3 μg/ml, Sigma‐Aldrich, USA) to produce a stable cell line for subsequent assays. The verification of the lentivirus is presented in Figs S3A–D and S4D. Protein–protein docking The ClusPro server is a widely used tool for protein–protein docking studies and binding affinity analyses [44]. ACSL4 was assigned as the ligand, whereas ALR was assigned as the receptor. The ligand was rotated 70,000 times, and for each rotation, the ligand was translated in x, y, and z planes relative to the receptor on a grid. The translation with the highest score was selected. Of the 70,000 rotations, 1000 rotation–translation combinations with the lowest score were selected, and greedy clustering of these ligand positions with a 9 Å C-alpha RMSD radius was performed to identify the ligand positions with the most “neighbors”, i.e., cluster centers. The top ten cluster centers were retrieved and inspected visually, and the intermolecular contacts from the most probable poses were further evaluated. After analyzing the docked structures and interface residues, molecular images were generated by PyMOL (www.pymol.org). UPLC-QqQ-MS/MS analysis of oxylipins The separation and quantification of oxylipins from HK-2 cells were performed using UPLC coupled with 6460 QqQ-MS/MS (Agilent Technologies, Germany). Briefly, the cells were sonicated twice and stored at -40 ℃ for 1 h. After centrifugation (15 min, 12000 rpm, 4℃), 480μL of the supernatant was transferred to an Eppendorf tube, and 320μL of water was added. After vortexing for 30 s, the sample was further purified with SPE. The SPE cartridges were equilibrated (methanol/water,1:1). After loading the supernatant, the cartridge was washed with 1 mL of 5% MeOH/H 2 O. The flow-through fraction was discarded, and the samples were eluted with 1 mL of MeOH. The eluent was evaporated to dryness under a gentle stream of nitrogen and reconstituted in 100μL of 30% ACN/H 2 O. The reconstituted sample was vortexed for 30 s, homogenized at 60 Hz for 4 min, and sonicated for 5 min in the ice-water bath. After centrifugation (1 min, 12000 rpm, 4℃), the sample was transferred to an EP tube with a filter membrane. After centrifugation (15 min, 12000 rpm, and 4℃), the clear supernatant was subjected to UPLC-MS/MS analysis. The precision of the quantitation was measured as the relative standard deviation (RSD), which was determined by injecting analytical replicates of a QC sample. The accuracy of quantitation was measured as the analytical recovery of the QC sample determined. The percent recovery was calculated as [(mean observed concentration) / (spiked concentration)] × 100%. Statistics and reproducibility Data were presented as the mean ± standard error of the mean (SEM). GraphPad Prism 8.0 Software (GraphPad Software, Inc., USA) was used for statistical analysis. Differences between two groups were assessed by an unpaired Student’s t -test. For multiple group comparisons, one-way ANOVA, followed by Tukey’s post hoc test, was applied. Significant differences were considered at p < 0.05. Results I/R injury up-regulates AKI expression and ferroptosis I/R injury is the main cause of AKI. Thus, the bilateral renal I/R injury model was used [31]. We evaluated pathological changes, as well as renal function, after 25 min of ischemia and different reperfusion times. With prolonged reperfusion time, we observed extensive pathological changes and deteriorating renal function (Fig.1A, C). Compared with wild-type mice, the levels of serum creatine (SCR) and blood urea nitrogen (BUN) were elevated in mice with prolonged reperfusion time (Fig.1A). We observed brush border loss, tubule flattening, and inflammatory cell infiltration (Fig. 1C). In addition, the mRNA and protein levels of biomarkers for proximal tubule injury, namely, kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), were elevated (Fig.1A,B). These results indicate that I/R injury can induce AKI in mice. To explore whether ALR expression is altered and ferroptosis is involved in AKI, we performed RNA sequencing using mouse kidney tissues. RNA-seq data of control mice and mice after I/R injury could be found in Supplement S1, Supplemental Material. In gene set enrichment analysis, several pathways were associated with AKI, with the top three up-regulated pathways related to cell cycle progression, malaria, and amoebiasis and the top down-regulated pathway related to metabolism (Fig.1D). Also, using a volcano plot to compare differentially expressed genes (DEGs) in control mice and mice after I/R injury, we plotted the overall distribution of DEGs (Fig.1E). Several DEGs were associated with ferroptosis. Next, we examined the expression of ALR and the involvement of ferroptosis in I/R injury-induced AKI. Ferroptosis is a form of regulated cell death controlled by glutathione peroxidase 4 (GPX 4 ). Cysteine availability can limit the biosynthesis of glutathione and the import of cystine, which are required by the cystine/glutamate antiporter system x c - [32]. Thus, we examined xCT and GPX4 expression in the kidneys (Fig.2A, B) and observed ferroptosis after I/R injury. After 24 h of reperfusion, GPX4 expression decreased compared with the control, which prompted us to select 24 h as the time point of interest in subsequent experiments. After I/R injury, ALR expression was increased compared with the control (Fig.2A, B). These results were confirmed by immunohistochemistry using clinical specimens, namely, five kidney paracancerous cases (control group) and seven AKI cases from our hospital. Compared with the control group, ALR expression and the levels of SCR and BUN were increased in the AKI group (Fig.2C, D). Patient information is presented in Supplementary Figure S2 and Supplementary table 2. ALR protects against I/R injury-induced ferroptosis in vitro and in vivo To understand the role of ALR in I/R injury-induced AKI, we generated a conditional KO mouse in which the ALR gene was deleted in proximal tubular cells by the Cre-Loxp system (Supplementary Figure S1A). By cross-breeding the ALR floxed mouse with the Ggt1-Cre mouse, we obtained the ALR flox/flox /Ggt1-Cre mouse (PT-KO mouse), with ALR flox/flox mice serving as the control (Supplementary Figure S1B). ALR expression is high in the liver; thus, we examined ALR expression in this organ by q-PCR and western blotting. The protein is abundantly expressed in the liver. In the kidneys, ALR expression in PT-KO mice was decreased compared with control mice (Supplementary Figure S1C,D). Approximately 8 weeks after birth, there were no significant differences in body weight and the kidney/body weight ratio between PT-KO and wild-type mice (Supplementary Figure S1E,F). ALR KO mice were euthanized after 24 h of I/R injury, at which time the proximal tubular epithelial cells were injured. Compared with control mice, HE- and PAS-stained kidney sections demonstrated extensive damage of proximal tubular epithelial cells in PT-KO mice (Fig.3A). Furthermore, after 24 h of reperfusion, we observed brush border loss, tubule flattening, and epithelial cell sloughing in the PT-KO group compared with wild-type mice. Renal function deteriorated in PT-KO mice as evidenced by increased SCR and BUN levels (Fig.3B), but there were no significant differences in these parameters between PT-KO mice and wild-type mice in the absence of I/R injury. These results were confirmed by immunofluorescent staining (Fig.3C). We further evaluated the role of ALR in ferroptosis in vitro and in vivo . After I/R injury, the mRNA and protein levels of ALR in the PT-KO group decreased compared with the control group (Fig.3D, E). To examine the role of ALR in ferroptosis, we transiently overexpressed ALR by lentiviral infection and successfully established the H/R injury model in HK-2 cells (Supplementary Figure S3A-D). Given that ferroptosis associates with mitochondrial changes [23], we examined the levels of mitochondrial proteins in cells associated ferroptosis. After H/R injury, xCT and GPX 4 expression was significantly increased in cells overexpressing ALR compared with those overexpressing the empty vector (Fig.3E) ( p < 0.05). After I/R injury, proximal tubular cells in PT-KO mice had smaller than normal mitochondria with darker-stained membranes and disorganized mitochondrial crista compared with wild-type mice (Fig.3F). These results indicate that cell-specific knockout of ALR aggravates ferroptosis in proximal tubular cells and promotes AKI in mice, whereas overexpression of ALR in HK-2 cells alleviates ferroptosis in proximal tubular cells. We also examined the localization of GPX 4 by confocal laser‐scanning microscopy and observed GPX 4 expression in the nucleus and cytoplasm (Fig.4A). Ferroptosis is promoted by excessive ROS accumulation in cells. Thus, we detected ROS levels in vitro and in vitro . In ALR KO mice, ROS levels in the kidneys were elevated after I/R injury, whereas ALR overexpression reduced ROS levels in HK-2 cells (Fig. 4B, C). We also measured the enzymatic activity of GPX. content. Notably, activity of the antioxidant enzyme GPX activity was significantly reduced in KO ALR mouse after I/R injury compared with the control (Fig.4D). ACSL4 plays a key role in ferroptosis Previous studies have reported that ACSL4 is a biomarker and a regulator of ferroptosis [33, 34]. Thus, we investigated the role of ACSL4 in AKI and employed transcriptome sequencing for this purpose (Fig.5A, B). RNA-seq data of control mice with AKI and ALR-KO mice with AKI could be found in Supplementary Table S3 of supplemental material. Because lipid peroxidation is a critical driver of ferroptosis, so we identified 26 genes related to fatty acid accumulation and ferroptosis (Fig.5C, D) from AKI and ALR KO groups. ACSL4 and ACSL3 were common to both fatty acid accumulation and ferroptosis, and the other genes are listed in Supplementary Table S4. Next, we examined ACSL4 expression in wild-type and ALR KO mice after I/R injury-induced AKI and observed that ACSL4 expression increased significantly in ALR KO mice after I/R injury compared with wild-type mice (Fig. 5E, F). These results indicate that ACSL4 is a biomarker of ferroptosis in AKI. Furthermore, ALR overexpression could further rescue I/R injury in vitro (Fig. 5F). During ferroptosis, in the presence of ferrous iron or lipoxygenase, ALR catalyzes unsaturated fatty acids within the cell membrane, which causes lipid peroxidation and ferroptosis. Thus, we examined the mRNA levels of fatty acid oxidation genes in the kidneys and observed that the levels of enzymes and regulators of fatty acid oxidation were reduced after I/R injury compared with the control, it indicated that ALR plays a key role in ferroptosis by inhibiting fatty acid oxidation (Fig.5G). ALR binds ACSL4 to prevent ferroptosis We used HK-2 cells, a proximal tubular cell line, to determine if ALR binds to ACSL4. ALR and ACSL4 were co-localized in mitochondria in HK-2 cells (Fig.6A). In the kidneys, these two proteins mainly localized in proximal tubules (Fig.6C). These results were confirmed by co-immunoprecipitation (Fig.6B). To further confirm whether ALR and ACSL4 indeed interact, we identified the potential binding sites between these two proteins (Fig.7A-D). ALR is a dimer in its native conformation (Fig.7A). However, interactions between ALR and ACSL4 in publicly available protein–protein interaction databases have not been reported. Thus, molecular docking was used to identify potential interacting residues based on geometric complementarity. Because ALR is a dimer [35], we analyzed the potential sites in the dimer. Interacting regions between ALR (https://www.pdbus.org/, PDB, ID3MBG) and ACSL4 (https://www.uniprot.org/, Uniprot-O60488-F1-model) corroborated available data on the existing domains (Fig.7A-C). The specific sites of contact between ACSL4 with ALR are shown in Fig. 7D. Ferroptosis is driven by fatty acid accumulation Ferroptosis is characterized by the accumulation of ROS and lipid peroxidation products. There are many different classes of lipids in cells, including monounsaturated fatty acids and polyunsaturated fatty acids. However, it was not clear if these fatty acids are oxidized and if they have roles in ferroptosis. To address these questions, we extracted and analyzed lipid metabolites by resolution liquid chromatography (UPLC) coupled with triple quadruple mass spectrometry (QqQ) (UPLC-QqQ-MS/MS). We analyzed the oxylipin ratio between ACSL4-silenced and control specimens to generate the lipidomic profiles. There were 48 lipid metabolites (17 down-regulated and 31 up-regulated) displaying significant changes after ACSL4 knockdown (Fig.8A, Supplementary Table S5). Also, the volcano map show that the down-regulation and up-regulation about oxylipin (Supplementary Figure S4E). As for the qualitative oxylipin profile, of the 48 oxylipins analyzed, there were significant differences in six oxylipins between ACSL4-silenced and control specimens (Fig.8B). To further examine the functions of ALR and ACSL4 in ferroptosis, ALR expression was silenced in HK-2 cells by short hairpin interfering RNA sequences. Next, we performed targeted lipidomics and metabolomics profiling (Fig.8C). We found that the level of 5Z,8Z,11Z,14Z-eicosatetraenoic acid (ARA) increased significantly compared with the control group (Fig.8D). After treatment with ACSL4, consistent with this finding, we examined the ARA level (oxylipin from EPA) and observed a significant increase ( p < 0.05) in the shRNA/ALR group compared with the shRNA/ALR+ACSL4 group (Supplementary Figure S4F). Heatmap showed the detailed concentration from targeted metabolomics profiling (Fig.8E). Taken together, the above results prove that interference ACSL4 and/or ALR reduced mostly polyunsaturated fatty acids. Discussion Ferroptosis, a non-apoptotic form of cell death, is a relatively recently discovered regulated cell death that can be triggered by excessive lipid peroxidation [ 36 ]. An increasing number of studies indicate that Ferroptosis is involved in I/R injury, as well as AKI[ 37 ].I/R injury is the most common cause of AKI, and the pathophysiology of I/R injury-induced AKI can be summarized by hemodynamic alterations, epithelial cell injury, and inflammation [ 38 ]. Furthermore, ROS production has been implicated in the kidneys after I/R injury. ROS are the products of lipid oxidation, and the rapid increase in ROS production can overwhelm antioxidant defenses and further aggravate injury. It is worth mentioning that epithelial cells are susceptible to I/R injury. In our previous study, we reported that ALR was a protective antioxidant molecule in the mitochondrial response to H/R injury-induced oxidative stress in AKI [ 39 ]. Previous research found that defects of ALR leas to intramitochondrial iron accumulation[ 28 ]. In addition, it is worth mentioning that ALR was shown to be responsible for Fe-S cluster transfer and stability of the mitochondrial membrane[ 40 ]. Moreover, ferroptosis is suggested to be iron-dependent and lipid peroxidation accumulations. Here, we highlight the significance of ALR in I/R injury-induced ferroptosis in the kidneys by high-throughput RNA sequencing. Mechanistically, we also demonstrate that decreased ALR expression and excessive oxylipin production synergistically induced ferroptosis after H/R injury. Our study discovered the protective role of ALR against I/R induced ferroptosis. Using a kidney-specific ALR knockout mouse model, we found that knockout ALR aggravated the progression of AKI. On the contrary, overexpression of ALR by transfected with lentivirus protected against ferroptosis in AKI. We further revealed that this protective role of ALR could be enhanced via downregulation of ACSL4, an important regulator of sensitivity of ferroptosis converting and controlling the accessibility of long polyunsaturated fatty acids required for the transduction of ferroptotic cell death metabolic process. ACSL4 is a member of the acy1-CoA synthetase family, and it is a biomarker and regulator of ferroptosis [ 33 ]. ACSL4 is localized in mitochondria, and it prefers polyunsaturated fatty acids [ 41 ]. Here, we identified the common genes between fatty acid oxidation and ferroptosis in the PT-KO mouse, and ACSL4 was the only gene that was common to both processes out of 26 genes. We also observed changes in arachidonic and eicosapentaenoic acids following H/R injury, which resulted in the decrease of ACSL4 expression. Specifically, interference ACSL4 reduced mostly polyunsaturated fatty acids. Similarly, ACSL4 overexpression inhibited oxylipin accumulation. In addition, by docking analysis, we identified the potential binding sites between ALR and ACSL4, and in other studies, we observed that ALR and ACSL4 were colocalized in mitochondria. Given the morphological changes in mitochondria during ferroptosis and the production of oxylipins by mitochondria, these observations highlight the important role of oxylipins, which are triggered by ROS stress, and the critical role of ALR in mediating resistance to oxidative stress. Finally, we discuss how ferroptosis might be targeted in mitochondria. Mitochondria play important roles in I/R injury and ferroptosis, and from the perspective of energy metabolism, proximal tubular cells require a large amount of energy to maintain normal function. Because the energy from fat is more than three times higher than that of glucose, proximal tubular cells prefer fat as the main energy source. There is accumulating evidence suggesting that lipid metabolism may be involved in the pathology of AKI and chronic kidney disease. In addition, lipid peroxidation is a hallmark of ferroptosis, and if we can recognize the mitochondria as a target, ALR could directly bind to ACSL4 to prevent I/R injury-induced ferroptosis. Conclusions In this study, we report for the first time that ALR specifically binds to ACSL4 and regulates ferroptosis in proximal tubular cells by attenuating oxylipin accumulation (Fig. 8F). These results provide new insights on how mitochondria, a key organelle of energy resources for proximal tubular cells, can be targeted to decrease the accumulation of oxidized lipids in I/R injury-induced AKI. Abbreviations AKI: acute kidney injury; ALR: augmenter of liver regeneration; ACSL4:Long chain acyl-CoA synthetase 4 ; BUN: blood urea nitrogen; DEGs :differentially expressed genes; GPX 4 : glutathione peroxidase 4; I/R: Ischemia‐reperfusion;KIM-1: kidney injury molecule-1; NGAL: neutrophil gelatinase-associated lipocalin; PT-ALR KO: proximal tubule-specific ALR knockout; ROS: reactive oxygen species; RSD: relative standard deviation; SCR: serum creatine. Declarations Acknowledgments We thank Prof. Aishun Jin at Chongqing Medical University (Department of Immunology, College of Basic Medicine) for providing the platform for cell culture and the training of cell culture techniques. A uthor contributions LLH, FYT, YXM cultured HK-2 cells. DC and LLD performed the animal models. ZZ and LZ, provided expertise and materials. HS, QL and XHL designed the experiments. LLH and YJZ analyzed the data, and wrote the paper. All authors approved the final version of the manuscript. Funding This work was supported by grants from the National Natural Science Foundation of China (81873604), the Medical Scientific Research Project of the Chongqing Health Commission (2022GDRC005) and Chongqing Science and Technology Agency (CSTB2022NSCQ-MSX0984). Availability of data and materials The datasets generated in this study have been included in this article as figures. Ethics approval and consent to participate The animal study was examined and approved by the Chongqing Medical University Animal Care and Use Committee (approval no. 2018,146). Competing interests The authors report no conflict of interest. References Bellomo R, Kellum JA, Ronco C: Acute kidney injury. Lancet 2012, 380: 756-766. Schrier RW, Wang W, Poole B, Mitra A: Acute renal failure: definitions, diagnosis, pathogenesis, and therapy. Journal of Clinical Investigation 2004, 114: 5-14. Levey AS, James MT: Acute Kidney Injury. Ann Intern Med 2017, 167: ITC66-ITC80. 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Supplementary Files SupplementaryFig.1.tif SupplementaryFig.2.tif SupplementaryFig.3.tif SupplementaryFig.4.tif SupplementaryTab.1.xlsx Supplementarytab.2.docx SupplementaryTab.3.xlsx Supplementarytab.4.docx SupplementaryTab.5.xlsx SupplementaryTab.6.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2649851","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":180492794,"identity":"8b8f3da5-f673-4c17-88eb-b815245985ba","order_by":0,"name":"Lili Huang","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Huang","suffix":""},{"id":180492795,"identity":"e23dee27-7774-47cc-8d3a-b3efeec25a17","order_by":1,"name":"Ling Zhang","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Zhang","suffix":""},{"id":180492796,"identity":"9bb44539-89c9-4cd4-a9fd-169284c159b4","order_by":2,"name":"Fangyan Tan","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fangyan","middleName":"","lastName":"Tan","suffix":""},{"id":180492797,"identity":"bd3cf255-f81c-4df3-a718-802e24b5ab5b","order_by":3,"name":"Yixin Ma","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yixin","middleName":"","lastName":"Ma","suffix":""},{"id":180492798,"identity":"736ab674-324d-4b19-8350-9022dde06171","order_by":4,"name":"Xujia Zeng","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xujia","middleName":"","lastName":"Zeng","suffix":""},{"id":180492799,"identity":"9855fcf5-4f83-487f-8e8b-7bb9ca54b258","order_by":5,"name":"Dan Cao","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Cao","suffix":""},{"id":180492800,"identity":"4566adb5-c72b-4f01-86c5-69b4573d3374","order_by":6,"name":"Lili Deng","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lili","middleName":"","lastName":"Deng","suffix":""},{"id":180492801,"identity":"0f8bdde9-af8f-4612-bd5a-c0aa3712beae","order_by":7,"name":"Qi Liu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Liu","suffix":""},{"id":180492802,"identity":"52b9afe6-499a-4e4b-9c15-6763a56729ee","order_by":8,"name":"Hang Sun","email":"","orcid":"","institution":"The Second Affiliated Hospital of Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hang","middleName":"","lastName":"Sun","suffix":""},{"id":180492803,"identity":"2aa36c17-a8fe-4ab9-8ba4-6c6c4bf74001","order_by":9,"name":"Zheng Zhang","email":"","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zheng","middleName":"","lastName":"Zhang","suffix":""},{"id":180492804,"identity":"2cf5584f-ef42-4291-a1be-faa2f46a05db","order_by":10,"name":"Xiaohui Liao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYBACPgYexscgBhsDA+MBmKgEPi1sDDzMxmAGUA/RWtikoSxitUjkHqsuqKjL45NvfnCAocY62uAA88HbPAx2eTi18JxLuz3jzOFiNjY2gwMMx9JzNxxgS7bmYUguxqmFvcfsNm/bgcQ2NgaDA4wNh4FaeMykeRgOJDbg0sLMY1bM21YH1ML+AaqF/xt+LUBbmHnbmIFaeOC2sOHXwnPGWJoH7JecggMJQL/MPMxmbDnHIBmnFn6JHMPPPMAQk28+vvHBhxrr3L7jzQ9vvKmww6kFBhKgJDMDCDEwGBBQD9cCVT8KRsEoGAWjAAUAAI27T7+LniBRAAAAAElFTkSuQmCC","orcid":"","institution":"Chongqing Medical University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xiaohui","middleName":"","lastName":"Liao","suffix":""}],"badges":[],"createdAt":"2023-03-03 05:29:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2649851/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2649851/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":33912322,"identity":"46e20087-e871-446f-ba9b-78f83a6b5ba9","added_by":"auto","created_at":"2023-03-07 15:44:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":502494,"visible":true,"origin":"","legend":"\u003cp\u003eIschemia-reperfusion injury-induced AKI involves an ALR associated metabolic pathway. Blood and kidneys were collected at 24, 48, and 72 h after I/R injury. \u003cstrong\u003eA\u003c/strong\u003e Baseline serum creatinine and BUN levels. (n = 6). \u003cstrong\u003eB\u003c/strong\u003e KIM-1 and NGAL expression in kidneys as determined by q-PCR and Western blotting. \u003cstrong\u003eC\u003c/strong\u003e Representative HE- and PAS-stained kidney sections (magnification, 20×), and KIM-1 expression in kidneys after I/R injury. \u003cstrong\u003eD\u003c/strong\u003e Gene set enrichment analysis of up-regulated (red) and down-regulated (blue) differentially expressed pathways in mice after 24 h of I/R injury and control mice (n = 4). \u003cstrong\u003eE\u003c/strong\u003e Volcano plot of all genes. Red dots represent up-regulated genes, and blue dots represent down-regulated genes that exceeded the FDR cutoff. The y-axis represents −log\u003csub\u003e10\u003c/sub\u003e of FDR, and the x-axis represents –log\u003csub\u003e2 \u003c/sub\u003efold change. Data represent mean ±SD of at least three independent experiments, ∗\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, compared versus control, ∗∗\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ∗∗∗ \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 compared versus the indicated groups.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/73d206d9c5ff6863b878a17d.png"},{"id":33912670,"identity":"7ef99f37-8772-4d25-b892-05b046087464","added_by":"auto","created_at":"2023-03-07 15:52:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":186440,"visible":true,"origin":"","legend":"\u003cp\u003eIschemia-reperfusion injury-induced AKI involves increased ALR expression and ferroptosis. \u003cstrong\u003eA\u003c/strong\u003e xCT, GPX\u003csub\u003e4\u003c/sub\u003e, and ALR expression in kidneys, as determined by q-PCR (n = 6). \u003cstrong\u003eB\u003c/strong\u003e xCT, GPX\u003csub\u003e4\u003c/sub\u003e, and ALR expression in kidneys, as determined by western blotting (n = 5). \u003cstrong\u003eC\u003c/strong\u003e Representative ALR-stained kidney sections (n = 6). \u003cstrong\u003eD\u003c/strong\u003e Serum creatinine and BUN levels in mice after I/R injury and control mice (n = 6). ∗\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, compared versus control, ∗∗\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ∗∗∗ \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 compared versus the indicated groups.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/2c778906c5ec9d879506399a.png"},{"id":33911542,"identity":"2144cc14-fb76-449f-a97e-91f3487bbcc0","added_by":"auto","created_at":"2023-03-07 15:36:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":761432,"visible":true,"origin":"","legend":"\u003cp\u003eALR protects against I/R injury-induced ferroptosis \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. \u003cstrong\u003eA\u003c/strong\u003e HE- and PAS-stained kidney sections of ALR KO mice (n = 6). Magnification, 20×. \u003cstrong\u003eB\u003c/strong\u003e Biochemical analysis of renal function in ALR KO and wild-type mice. KIM-1 expression in ALR KO and wild-type mice as determined by q-PCR. \u003cstrong\u003eC\u003c/strong\u003e Representative KIM-1- and NGAL-stained kidney sections. Scale bar = 20 μm. \u003cstrong\u003eD\u003c/strong\u003e xCT, GPX\u003csub\u003e4\u003c/sub\u003e, and ALR expression in ALR KO and wild-type mice as determined by q-PCR (n = 6). \u003cstrong\u003eE\u003c/strong\u003e xCT, GPX\u003csub\u003e4\u003c/sub\u003e, and ALR expression in HK-2 cells and the kidneys. \u003cstrong\u003eF\u003c/strong\u003e Representative transmission electron microscopy images of ALR KO and wild-type mice after I/R injury. Magnification, 10,000× or 30,000×. Scale bars = 2 μm or 500 nm, respectively. ∗\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, compared versus control, ∗∗\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ∗∗∗ \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 compared versus the indicated groups.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/6fa0bc7dfb81e1b8ef5ba430.png"},{"id":33912672,"identity":"06f6afa5-5c4e-40bb-8e1d-573afcc450d2","added_by":"auto","created_at":"2023-03-07 15:52:47","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":425369,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of \u003cem\u003eALR \u003c/em\u003eoverexpression on ferroptosis \u003cem\u003ein\u003c/em\u003e \u003cem\u003evitro \u003c/em\u003eand \u003cem\u003ein vivo\u003c/em\u003e. \u003cstrong\u003eA\u003c/strong\u003e GPX\u003csub\u003e4 \u003c/sub\u003eexpression and localization after ALR overexpression in HK-2 cells exposed to H/R injury. GPX\u003csub\u003e4\u003c/sub\u003e localization was examined by confocal laser‐scanning microscopy. Scale bar = 20 μm. \u003cstrong\u003eB\u003c/strong\u003e ROS levels in the kidneys were measured by hydro-Cy3 staining. Stained cells were viewed by confocal laser-scanning microscopy. \u003cstrong\u003eC\u003c/strong\u003e ROS levels in HK-2 cells were measured by dihydroethidium. Stained cells were analyzed by flow cytometry (n = 3). \u003cstrong\u003eD\u003c/strong\u003e Relative GPX content. ∗\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, compared versus control, ∗∗\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, ∗∗∗ \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001 compared versus the indicated groups.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/8f34e1fbb80daf30036b34ba.png"},{"id":33913123,"identity":"3ce69f06-7f9c-4f9c-a18e-0fd7bd1d8e31","added_by":"auto","created_at":"2023-03-07 16:00:47","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":176800,"visible":true,"origin":"","legend":"\u003cp\u003eALR knockout drives expression of fatty acid-associated genes. \u003cstrong\u003eA\u003c/strong\u003e Supervised hierarchical clustering identifies two groups after deep sequencing of RNA. The heatmap was prepared using a diverging scale, in which up-regulated genes are colored red and down-regulated genes are colored green. The first group represents AKI, and the second group represents PT-KO-AKI. These clusters were further classified into AKI (light green) and PT-KO-AKI (orange) (n = 4). \u003cstrong\u003eB\u003c/strong\u003ePathway analysis of differentially expressed genes from (\u003cstrong\u003eA\u003c/strong\u003e) was performed. \u003cstrong\u003eC\u003c/strong\u003e Heatmap of differentially expressed genes related to fatty acid synthesis and ferroptosis between AKI and PT-KO-ALR groups. \u003cstrong\u003eD\u003c/strong\u003e Venn diagram of 20 genes related to fatty acid synthesis and 8 genes related to ferroptosis, with two genes (ACSL4 and ACSL3) common to both fatty acid synthesis and ferroptosis. \u003cstrong\u003eE-F\u003c/strong\u003e ACSL4 expression \u003cem\u003ein vivo\u003c/em\u003e (n = 6) and \u003cem\u003ein vitro\u003c/em\u003e (n = 3) as determined by q-PCR and Western blotting. \u003cstrong\u003eG\u003c/strong\u003eExpression of the fatty acid oxidation enzyme. ∗\u003cem\u003ep\u003c/em\u003e \u0026lt;0.05, compared versus control, ∗∗\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01 compared versus the indicated groups.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/6429d9c4ef53e96484c7b6e8.png"},{"id":33912323,"identity":"76000c7c-d067-445a-b370-f55acfb13690","added_by":"auto","created_at":"2023-03-07 15:44:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":349863,"visible":true,"origin":"","legend":"\u003cp\u003eALR interacts with ACSL4 to prevent ferroptosis. \u003cstrong\u003eA\u003c/strong\u003e ALR and ACSL4 colocalize in the mitochondria of HK-2 cells by confocal laser‐scanning microscopy. Scale bar = 50 μm. \u003cstrong\u003eB\u003c/strong\u003e Co-immunoprecipitation of endogenous ALR and ACSL4 in human proximal tubular cells. \u003cstrong\u003eC\u003c/strong\u003e The sections were co-stained with anti-laminin to outline the tubular area. Scale bar = 20μm (n = 3).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/7a0f692b1a0c6b727f0439a4.png"},{"id":33911543,"identity":"0d990ad4-11b8-4c18-a813-970ae432fbcf","added_by":"auto","created_at":"2023-03-07 15:36:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":255350,"visible":true,"origin":"","legend":"\u003cp\u003eALR interacts with ACSL4. \u003cstrong\u003eA\u003c/strong\u003e The structures of ALR and ACSL4. \u003cstrong\u003eB \u003c/strong\u003eThe surface binding model of ALR with ACSL4. \u003cstrong\u003eC\u003c/strong\u003e The interaction between ALR and ACSL4. ALR is colored cyan (chain A) and orange (chain B), and ACSL4 is colored green. The red dashes represent hydrogen bonds. The yellow dashes represent salt bridges. \u003cstrong\u003eD\u003c/strong\u003e The specific sites of contact between ALR and ACSL4.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/90299736bfe021534c2a74ce.png"},{"id":34021523,"identity":"e62e3ffc-ec67-4dec-a449-a0ec2463fafc","added_by":"auto","created_at":"2023-03-09 15:22:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":445729,"visible":true,"origin":"","legend":"\u003cp\u003eThe detail information about oxylipin by UPLC-QqQ-MS/MS. \u003cstrong\u003eA\u003c/strong\u003e Targeted metabolomics profiling assays oxidized lipids 48 h after plasmid transfection of ACSL4. \u003cstrong\u003eB\u003c/strong\u003e The concentration about oxylipin, only statistically significant changes were displayed. \u003cstrong\u003eC\u003c/strong\u003e Targeted metabolomics profiling assays oxylipin after lentivirus-mediated shRNA interfering ALR expression. \u003cstrong\u003eD\u003c/strong\u003e The concentration of 4-HDoH4 and ARA, only statistically significant changes were displayed from (\u003cstrong\u003eC\u003c/strong\u003e). \u003cstrong\u003eE\u003c/strong\u003eTargeted metabolomics profiling assays oxidized lipids after inhibit ALR expression through short hairpin RNA (shRNA) interference and/or transfected with ACSL4 overexpression plasmid. \u003cstrong\u003eF\u003c/strong\u003e ALR interacts with ACSL4. Working model of ALR mediates the function of mitochondrial intermembrane assembly by binding to ACSL4, thereby decreasing ferroptosis. ∗\u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, compared versus control, ∗∗\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01compared versus the indicated groups.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/346b3fc7a659b1cb4d8ee72b.png"},{"id":34021714,"identity":"3dcc7330-5422-4965-b190-6bd18392d4fd","added_by":"auto","created_at":"2023-03-09 15:22:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4640775,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/b54a3aa8-9a54-4673-80ec-6eac77e1009c.pdf"},{"id":33911540,"identity":"8bff6a1d-2159-447d-8b09-38f30ca4faa4","added_by":"auto","created_at":"2023-03-07 15:36:47","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":121660,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.1.tif","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/94dc10900b519088eaf7d043.tif"},{"id":33913549,"identity":"c10ff96f-27fd-4b79-ad70-0320c6dcf980","added_by":"auto","created_at":"2023-03-07 16:08:47","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1185410,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.2.tif","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/0e3cc381386454dab95c0084.tif"},{"id":34021525,"identity":"34dad666-0ded-4070-bc75-492e728c0c8a","added_by":"auto","created_at":"2023-03-09 15:22:10","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":280762,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.3.tif","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/6678695dc7e2c648e505f0c2.tif"},{"id":34021520,"identity":"ce6231ab-b2b9-4e92-8890-6e7f443205c3","added_by":"auto","created_at":"2023-03-09 15:22:06","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":137290,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.4.tif","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/0cec40c1c118f1acbeb6cbf5.tif"},{"id":33913124,"identity":"73ecb685-22e4-482c-935f-5a132cd204d9","added_by":"auto","created_at":"2023-03-07 16:00:47","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":594710,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTab.1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/2e31ee281b06f41580687027.xlsx"},{"id":33911557,"identity":"e711e578-6b09-468a-b2e4-ce96d7b247a8","added_by":"auto","created_at":"2023-03-07 15:36:47","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":45199,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytab.2.docx","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/456f2697873da5ba245ac937.docx"},{"id":34021526,"identity":"2c4a6dff-9363-4eaf-9a61-f7ab75b7de2c","added_by":"auto","created_at":"2023-03-09 15:22:10","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":24103,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTab.3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/0cb5932cfc3c7d020d1314d8.xlsx"},{"id":33911548,"identity":"9be32310-245d-4434-bcc3-f9a5555e75e9","added_by":"auto","created_at":"2023-03-07 15:36:47","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":27390,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarytab.4.docx","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/6e8494dd29867b11566e7197.docx"},{"id":33912673,"identity":"7d5149e6-a76f-4468-8b9a-15ee789a477f","added_by":"auto","created_at":"2023-03-07 15:52:47","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":17100,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTab.5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/53be366ba2b6a076d8ff3c33.xlsx"},{"id":34021521,"identity":"bb44b0ef-ddef-4fcc-87dc-793f42505a90","added_by":"auto","created_at":"2023-03-09 15:22:06","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":21062,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTab.6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2649851/v1/c2a0295ec2554ba54d1faeeb.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Inhibition of Lipid peroxidation by ALR protects the kidney from ischemia-reperfusion injury","fulltext":[{"header":"Background","content":"\u003cp\u003eAcute kidney injury (AKI), a common complication associated with high mortality and morbidity in critically ill patients, is characterized by a sharp decline in renal function and extensive proximal tubular cell death in a short period of time (i.e., within 48 h)[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Ischemia-reperfusion (I/R) injury is the main cause of AKI [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, following I/R injury, renal function can recover within a few days of blood flow recovery [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Unfortunately, if I/R injury is not reversed in a timely manner, then the kidneys are vulnerable to chronic kidney disease, even end-stage renal damage. Presently, the outcomes of interventional treatments, including symptomatic and supportive therapy, as well as renal replacement for chronic AKI, have been disappointing [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Thus, it is important to define the mechanism of AKI and to identify new therapies for AKI. Recent studies have reported that aberrant biochemical and metabolic processes can trigger a variety of diseases, and they are the causative factors of inflammation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. There is compelling evidence indicating that kidney diseases are closely related to metabolic aberrations [\u003cspan additionalcitationids=\"CR12 CR13 CR14 CR15\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], with kidney diseases causing metabolic aberrations, such as excessive lipid peroxidation, abnormal amino acid metabolism, and aberrant glucose breakdown, and vice versa, especially in I/R injury-induced AKI. Unfortunately, there are few studies addressing lipid peroxidation in AKI.\u003c/p\u003e \u003cp\u003eFerroptosis, a unique iron-dependent form of regulated cell death, is biochemically, morphologically, and physiologically distinct from other forms of cell death, such as apoptosis, necrosis, and autophagy [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Lipid peroxides, a major source of free radicals, can trigger ferroptosis in the absence of glutathione (GSH)-dependent antioxidant defences [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. As a relatively new type of regulated cell death, the hallmarks of ferroptosis are a series of changes in mitochondria characterized by the appearance of smaller than normal mitochondria, darker‐stained membranes, and disorganization/reduction of mitochondrial crista [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Moreover, emerging evidence suggests that mitochondria play critical roles in ferroptosis [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and ferroptosis is involved in AKI. For instance, the inactivation of the ferroptosis-associated gene, glutathione peroxidase 4 (GPX\u003csub\u003e4\u003c/sub\u003e), triggers AKI in mice [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], whereas the inhibition of ferroptosis by a pharmacological inhibitor can suppress proximal tubular cell death [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAugmenter of liver regeneration (ALR), which is essential for respiration, is a growth-promoting factor in the liver that was initially identified in the rat liver and reported to promote hepatocyte proliferation and liver regeneration [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, ALR is widely expressed by all mammalian tissues, including the kidneys [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In our previous study, we reported that ALR plays a protective role against oxidative injury by reducing reactive oxygen species (ROS) production and accumulation in renal proximal tubules [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The accumulation of lipid ROS is another hallmark of ferroptosis, although where and how they are generated during ferroptosis is not clear. Additionally, little is known about the relationship between excessive lipid peroxidation and ALR expression in ferroptosis.\u003c/p\u003e \u003cp\u003eIn this study, we systematically analyzed the differences in lipid composition after kidney-specific deletion of the ALR gene (ALR-K-KO) in AKI. We also examined the relationship between ACSL4, a ferroptosis-associated maker, and ALR, and concluded that ALR could alleviate lipid accumulation by suppressing the ACSL4 pathway.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eExperimental model and subject details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all procedures and protocols were approved by the Chongqing Medical University Animal Care and Use Committee (approval no. 2018,146). To generate the kidney-specific ALR knockout mice, we conditionally knocked out the ALR\u003cem\u003e\u0026nbsp;\u003c/em\u003egene (also known as GFER) using the Ggt1-Cre/LoxP system. ALR\u003csup\u003elox/flox\u003c/sup\u003e mice were used as the control. To generate the proximal tubule-specific ALR knockout mice (PT-ALR KO), we crossed ALR\u003csup\u003eflox/flox\u003c/sup\u003e mice with ALR Cre expressing mice (Figure S1, Supplemental Material).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment of the ischemic AKI model in vivo\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBilateral ischemic AKI was induced as previously described\u0026nbsp;[42]. Briefly, mice were anesthetized with 60 mg/kg of sodium pentobarbital in 0.9% NaCl (stock concentration, 5 mg/ml) by intraperitoneal injection. The core body temperature was monitored with a temperature control system with a rectal prob (HP-30, Beijing Cinotech Co., China) and maintained at 36.6\u0026ndash;36.8\u0026deg;C (optimal temperature, 36.7\u0026deg;C \u0026plusmn; 0.1\u0026deg;C). Bilateral flank incisions were made to expose the kidneys, and renal ischemia was induced for 22 min using micro-clamps and micro-clips (RS-5420/RS-5410, Roboz, UK). The micro-clamps were removed, and the restoration of blood flow was visually confirmed. All mice were euthanized 1, 2, and 3 days after renal ischemia. Blood and kidneys were collected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e\u003cstrong\u003eollections of blood, urine, and histological analysis of human kidney tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study related to patients has been approved by the Institutional Review Board of The Second Affiliated Hospital, Chongqing Medical University (approval no. 2018,146). Human kidney tissue samples and blood were collected in our experimental studies. The adjacent noncancerous tissues and AKI tissues were respectively used as the normal group(n=6) and AKI group (n=6). The patients from adjacent noncancerous tissues were identified by the Second Affiliated Hospital of Chongqing Medical University by magnetic resonance imaging and pathological examination, all patients provided a written informed consent prior to surgery. The AKI patients were identified by the renal function according to the guideline from Kidney Disease: Improving Global Outcomes. All samples were removed aseptically and frozen in liquid nitrogen until use. Serum creatinine, serum BUN and glomerular filtration rate were collected and measured by the automatic biochemical analyzer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEstablishment of the ischemic AKI model in vitro\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman kidney proximal tubular HK‐2 cells were cultured in Dulbecco\u0026rsquo;s Minimum Essential Medium F12 (Gibco, USA) supplemented with 10% fetal bovine serum (Moregate, Australia) and 1% penicillin\u0026ndash;streptomycin (Invitrogen, USA) in an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. To induce H/R injury, HK-2 cells were cultured in serum-free medium overnight, followed by serum-free and glucose-free medium in an atmosphere of 94% N\u003csub\u003e2\u003c/sub\u003e, 5% CO\u003csub\u003e2\u003c/sub\u003e, and 1% O\u003csub\u003e2\u003c/sub\u003e for 6 h, as previously described\u0026nbsp;[21, 43]. HK-2 cells were then transferred to an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e and cultured in complete medium for 12 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission electron microscopy (TEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eKidneys were immediately collected, minced into 1-mm\u003csup\u003e3\u003c/sup\u003e fragments, and fixed overnight at 4℃. On the following day, the tissues were postfixed in 1% OsO\u003csub\u003e4\u003c/sub\u003e, dehydrated through a graded-alcohol series at room temperature, and embedded in resin. The blocks were sectioned, and 80-nm continuous sections were stained with 2% uranium acetate and observed by TEM (Hitachi, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh-throughput RNA sequencing and informatic analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated with TRIzol Reagent (Invitrogen, China) according to the manufacturer\u0026rsquo;s instructions. RNA quality, purity, and quantity were assessed by Shanghai NovelBio Bio-Pharm Technology Co., Ltd. The samples were processed by an Illumina HiSeq X Ten Sequencing System. The differentially expressed genes (DEGs) were filtered using the significant threshold value (\u003cem\u003ep\u003c/em\u003e-value), fold change (FC), and false discovery rate (FDR). A total of 5042 DEGs were identified between control and AKI groups (Supplementary Table S1). The DEGs identified between control and ALR KO mice after I/R injury are listed in Supplementary Table S3. Differences in pathways were identified using the Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test after correcting for multiple hypotheses and accepting pathways with a 5% FDR cutoff.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetection of ROS in cells and tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor cells, ROS levels were detected by dihydroethidium (BestBio, China). After H/R injury, the cells were harvested and incubated with dihydroethidium (1:1000) for 30 min at 37℃ in the dark. The cells were washed with PBS and analyzed by flow cytometry. For tissues, ROS levels were detected by Cy3 (Sigma, USA). After H/R injury, kidney sections were incubated with Cy3 (1:500) for 30 min at 37℃ in the dark. The cell nuclei were counterstained with DAPI. The cells were viewed by microscopy (Nikon Eclipse C1, Japan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cells were washed, and mitochondrial proteins were extracted using the Mitochondrial Kit (Beyotime Biotech Co., Ltd., China) according to the manufacturer\u0026rsquo;s instructions. The protein concentration was determined using the BCA Protein Assay (KenGen Biotech, China). The proteins were separated by 15% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto polyvinylidene membranes. After blocking in 5% nonfat milk in Tris-buffered saline\u0026ndash;Tween-20 (TBST) for 2 h at room temperature, the membranes were incubated with specific primary antibodies overnight at 4℃. Subsequently, the membranes were washed three times with TBST and incubated with the indicated secondary antibody for 1 h at room temperature. The ECL (Beyotime Biotech Co., Ltd., China) was used for detecting immunoreactive proteins. The antibodies were as follows: NGAL (1:1000, ab216462m, Abcam, UK), KIM-1 (1:1000, Novus, USA), GPX\u003csub\u003e4\u003c/sub\u003e (1:8000, ab125066, Abcam), ACSL4 (1:5000, ab155282, Abcam), ALR (1:1000, abx129857, Abcam), COX Ⅳ (1:1000, Cell Signaling Technology, USA), and xCT (1:10000, ab175186, Abcam).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqPCR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was extracted from cells and tissues using the Total RNA Purification Kit (Biyuntian, China). Approximately 1 \u0026micro;g of RNA was reverse transcribed into cDNA using the Reverse Transcription Kit (Takara, Japan). Real-time PCR was performed using the SYBR Premix Kit (TaKaRa, Japan). Primer sequences are listed in Supplementary Table S6 of Supplemental Material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-immunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHK-2 cells were lysed in lysis buffer, and the protein concentration was determined as previously indicated. The protein lysates (1.5 mg) were incubated with the indicated antibodies at a protein:antibody ratio of 500:1 overnight at 4\u0026deg;C on a rotator. The antibodies were as follows: anti-ALR polyclonal antibody (11293, Proteintech, USA) and anti-ACSL4 polyclonal antibody (ab155282, Abcam). Anti-IgG (3900, Cell Signaling Technology) served as the negative control. On the following day, 100 \u0026mu;L of a 20% slurry of agarose beads (Abcam) was washed three times with lysis buffer and added to the samples for 2 h at 4\u0026deg;C on a rotator. The immunocomplexes were centrifuged at 2500 rpm for 5 min at 4\u0026deg;C, and the supernatants were washed three times with lysis buffer and centrifuged to yield the \u0026ldquo;input\u0026rdquo; sample. The immunocomplexes were combined with an equivalent volume of 1\u0026times; SDS-PAGE sample buffer and heated at 95\u0026deg;C for 5 min. Western blotting was performed as previously described.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell immunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether ALR and ACSL4 colocalize in mitochondria, MitoTracker Red CMXRos (Beyotime Biotech Co., Ltd) was used to label viable mitochondria. HK-2 cells were seeded in confocal dishes and allowed to adhere overnight. After H/R injury, the cells were washed with phosphate-buffered saline three times and stained with trimethylrhodamine, methyl ester, for 20 min, followed by fixation with 4% paraformaldehyde. The cells were washed, permeabilized, blocked with 0.5% BSA, and incubated overnight at 4\u0026deg;C with primary antibodies (ACSL4, 1:100 or ALR, 1:50). On the following day, the cells were incubated with a fluorescein isothiocyanate (FITC)- or a tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary antibody for 60 min in the dark at room temperature. The cell nuclei were counterstained with DAPI. The cells were observed by confocal laser-scanning microscopy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003esiRNA and plasmids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ACSL4 siRNA target sequence was as follows: 5\u0026prime;-GCAAUAAUCCUGCUAUGGAtt-3\u0026prime;, and the siRNA negative control (NC) sequence was 5\u0026prime;-UUCUCCGAACGUGUCACGUdTdT-3\u0026prime;. The siRNAs were purchased from Shanghai GeneBio (China), and they were used at a concentration of 50 nM. The pReceiver-Lv105 plasmid and empty vector (negative control) were purchased from GeneBio (China). The cells were transiently transfected with Lip2000 Transfection Reagent (Thermo Fisher Scientific, USA) according to the manufacturer\u0026rsquo;s instructions. Transfection efficiency was determined by qRT-PCR analysis. The sequences of transient downregulation of ACSL4 are provided in Supplementary Table S6 of Supplemental Material. \u0026nbsp;The sequence of the expression clone, as well as its verification, are provided in Supplementary Figure S4A\u0026ndash;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLentivirus transfection and stable cell clone establishment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe lentiviral-based small hairpin RNA (shRNA) and the ALR overexpression construct were purchased from GeneChem (China). HK-2 cells were infected with lentiviruses with an MOI 6. The full length ALR gene (GFER, NM_005262) was synthesized using a human cDNA library. All the sequences (Supplementary Table 3) were cloned into a lentiviral vector. The order of the elements from the ALR overexpression construct was Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin. At 35% confluence, HK-2 cells were infected with lenti-ALR-EGFP (referred to as ALR overexpression, ALR-OE) or lenti-EGFP (referred to as vector). After 72 h, the transfection efficiency was assessed by fluorescent microscopy, and the cells were selected for 2 weeks using puromycin (3 \u0026mu;g/ml, Sigma‐Aldrich, USA) to produce a stable cell line for subsequent assays. The verification of the lentivirus is presented in Figs S3A\u0026ndash;D and S4D.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein\u0026ndash;protein docking\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ClusPro server is a widely used tool for protein\u0026ndash;protein docking studies and binding affinity analyses [44]. ACSL4 was assigned as the ligand, whereas ALR was assigned as the receptor. The ligand was rotated 70,000 times, and for each rotation, the ligand was translated in x, y, and z planes relative to the receptor on a grid. The translation with the highest score was selected. Of the 70,000 rotations, 1000 rotation\u0026ndash;translation combinations with the lowest score were selected, and greedy clustering of these ligand positions with a 9 \u0026Aring; C-alpha RMSD radius was performed to identify the ligand positions with the most \u0026ldquo;neighbors\u0026rdquo;, i.e., cluster centers. The top ten cluster centers were retrieved and inspected visually, and the intermolecular contacts from the most probable poses were further evaluated. After analyzing the docked structures and interface residues, molecular images were generated by PyMOL (www.pymol.org).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUPLC-QqQ-MS/MS analysis of oxylipins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe separation and quantification of oxylipins from HK-2 cells were performed using UPLC coupled with 6460 QqQ-MS/MS (Agilent Technologies, Germany). Briefly, the cells were sonicated twice and stored at -40 ℃ for 1 h. After centrifugation (15 min, 12000 rpm, 4℃), 480\u0026mu;L of the supernatant was transferred to an Eppendorf tube, and 320\u0026mu;L of water was added. After vortexing for 30 s, the sample was further purified with SPE. The SPE cartridges were equilibrated (methanol/water,1:1). After loading the supernatant, the cartridge was washed with 1 mL of 5% MeOH/H\u003csub\u003e2\u003c/sub\u003eO. The flow-through fraction was discarded, and the samples were eluted with 1 mL of MeOH. The eluent was evaporated to dryness under a gentle stream of nitrogen and reconstituted in 100\u0026mu;L of 30% ACN/H\u003csub\u003e2\u003c/sub\u003eO. The reconstituted sample was vortexed for 30 s, homogenized at 60 Hz for 4 min, and sonicated for 5 min in the ice-water bath. After centrifugation (1 min, 12000 rpm, 4℃), the sample was transferred to an EP tube with a filter membrane. After centrifugation (15 min, 12000 rpm, and 4℃), the clear supernatant was subjected to UPLC-MS/MS analysis. The precision of the quantitation was measured as the relative standard deviation (RSD), which was determined by injecting analytical replicates of a QC sample. The accuracy of quantitation was measured as the analytical recovery of the QC sample determined. The percent recovery was calculated as [(mean observed concentration) / (spiked concentration)] \u0026times; 100%.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics and reproducibility\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData were presented as the mean \u0026plusmn; standard error of the mean (SEM). GraphPad Prism 8.0 Software (GraphPad Software, Inc., USA) was used for statistical analysis. Differences between two groups were assessed by an unpaired Student\u0026rsquo;s\u003cem\u003e\u0026nbsp;t\u003c/em\u003e-test. For multiple group comparisons, one-way ANOVA, followed by Tukey\u0026rsquo;s post hoc test, was applied. Significant differences were considered at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eI/R injury up-regulates AKI expression and ferroptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI/R injury is the main cause of AKI. Thus, the bilateral renal I/R injury model was used\u0026nbsp;[31]. We evaluated pathological changes, as well as renal function, after 25 min of ischemia and different reperfusion times. With prolonged reperfusion time, we observed extensive pathological changes and deteriorating renal function (Fig.1A, C). Compared with wild-type mice, the levels of serum creatine (SCR) and blood urea nitrogen (BUN) were elevated in mice with prolonged reperfusion time (Fig.1A). We observed brush border loss, tubule flattening, and inflammatory cell infiltration (Fig. 1C). In addition, the mRNA and protein levels of biomarkers for proximal tubule injury, namely, kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), were elevated (Fig.1A,B). These results indicate that I/R injury can induce AKI in mice.\u003c/p\u003e\n\u003cp\u003eTo explore whether ALR expression is altered and ferroptosis is involved in AKI, we performed RNA sequencing using mouse kidney tissues. RNA-seq data of control mice and mice after I/R injury could be found in Supplement S1, Supplemental Material. In gene set enrichment analysis, several pathways were associated with AKI, with the top three up-regulated pathways related to cell cycle progression, malaria, and amoebiasis and the top down-regulated pathway related to metabolism (Fig.1D). Also, using a volcano plot to compare differentially expressed genes (DEGs) in control mice and mice after I/R injury, we plotted the overall distribution of DEGs (Fig.1E). Several DEGs were associated with ferroptosis.\u003c/p\u003e\n\u003cp\u003eNext, we examined the expression of ALR and the involvement of ferroptosis in I/R injury-induced AKI. Ferroptosis is a form of regulated cell death controlled by glutathione peroxidase 4 (GPX\u003csub\u003e4\u003c/sub\u003e). Cysteine availability can limit the biosynthesis of glutathione and the import of cystine, which are required by the cystine/glutamate antiporter system x\u003csub\u003ec\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e[32]. Thus, we examined xCT and GPX4 expression in the kidneys (Fig.2A, B) and observed ferroptosis after I/R injury. After 24 h of reperfusion, GPX4 expression decreased compared with the control, which prompted us to select 24 h as the time point of interest in subsequent experiments. After I/R injury, ALR expression was increased compared with the control (Fig.2A, B). These results were confirmed by immunohistochemistry using clinical specimens, namely, five kidney paracancerous cases (control group) and seven AKI cases from our hospital. Compared with the control group, ALR expression and the levels of SCR and BUN were increased in the AKI group (Fig.2C, D). Patient information is presented in Supplementary Figure S2 and Supplementary table 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eALR protects against I/R injury-induced ferroptosis in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003evitro\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eand in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003evivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo understand the role of ALR in I/R injury-induced AKI, we generated a conditional KO mouse in which the ALR gene was deleted in proximal tubular cells by the Cre-Loxp system (Supplementary Figure S1A). By cross-breeding the ALR floxed mouse with the Ggt1-Cre mouse, we obtained the ALR\u003csup\u003eflox/flox\u003c/sup\u003e/Ggt1-Cre mouse (PT-KO mouse), with ALR\u003csup\u003eflox/flox\u003c/sup\u003e mice serving as the control (Supplementary Figure S1B). ALR expression is high in the liver; thus, we examined ALR expression in this organ by q-PCR and western blotting. The protein is abundantly expressed in the liver. In the kidneys, ALR expression in PT-KO mice was decreased compared with control mice (Supplementary Figure S1C,D). Approximately 8 weeks after birth, there were no significant differences in body weight and the kidney/body weight ratio between PT-KO and wild-type mice (Supplementary Figure S1E,F). ALR KO mice were euthanized after 24 h of I/R injury, at which time the proximal tubular epithelial cells were injured. Compared with control mice, HE- and PAS-stained kidney sections demonstrated extensive damage of proximal tubular epithelial cells in PT-KO mice (Fig.3A). Furthermore, after 24 h of reperfusion, we observed brush border loss, tubule flattening, and epithelial cell sloughing in the PT-KO group compared with wild-type mice. Renal function deteriorated in PT-KO mice as evidenced by increased SCR and BUN levels (Fig.3B), but there were no significant differences in these parameters between PT-KO mice and wild-type mice in the absence of I/R injury. These results were confirmed by immunofluorescent staining (Fig.3C). We further evaluated the role of ALR in ferroptosis \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. After I/R injury, the mRNA and protein levels of ALR in the PT-KO group decreased compared with the control group (Fig.3D, E). To examine the role of ALR in ferroptosis, we transiently overexpressed ALR by lentiviral infection and successfully established the H/R injury model in HK-2 cells (Supplementary Figure S3A-D). Given that ferroptosis associates with mitochondrial changes\u0026nbsp;[23], we examined the levels of mitochondrial proteins in cells associated ferroptosis. After H/R injury, xCT and GPX\u003csub\u003e4\u003c/sub\u003e expression was significantly increased in cells overexpressing ALR compared with those overexpressing the empty vector (Fig.3E) (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). After I/R injury, proximal tubular cells in PT-KO mice had smaller than normal mitochondria with darker-stained membranes and disorganized mitochondrial crista compared with wild-type mice (Fig.3F). These results indicate that cell-specific knockout of ALR aggravates ferroptosis in proximal tubular cells and promotes AKI in mice, whereas overexpression of ALR in HK-2 cells alleviates ferroptosis in proximal tubular cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe also examined the localization of GPX\u003csub\u003e4\u003c/sub\u003e by confocal laser‐scanning microscopy and observed GPX\u003csub\u003e4\u003c/sub\u003e expression in the nucleus and cytoplasm (Fig.4A). Ferroptosis is promoted by excessive ROS accumulation in cells. Thus, we detected ROS levels \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. In ALR KO mice, ROS levels in the kidneys were elevated after I/R injury, whereas ALR overexpression reduced ROS levels in HK-2 cells (Fig. 4B, C). We also measured the enzymatic activity of GPX. content.\u0026nbsp;Notably, activity of the antioxidant enzyme GPX activity was significantly reduced in KO ALR\u003cem\u003e\u0026nbsp;\u003c/em\u003emouse after I/R injury compared with the control (Fig.4D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACSL4 plays a key role in ferroptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies have reported that ACSL4 is a biomarker and a regulator of ferroptosis [33, 34]. Thus, we investigated the role of ACSL4 in AKI and employed transcriptome sequencing for this purpose (Fig.5A, B). RNA-seq data of control mice with AKI and ALR-KO mice with AKI could be found in Supplementary Table S3 of supplemental material. Because lipid peroxidation is a critical driver of ferroptosis, so we identified 26 genes related to fatty acid accumulation and ferroptosis (Fig.5C, D) from AKI and ALR KO groups. ACSL4 and ACSL3 were common to both fatty acid accumulation and ferroptosis, and the other genes are listed in Supplementary Table S4. Next, we examined ACSL4 expression in wild-type and ALR KO mice after I/R injury-induced AKI and observed that ACSL4 expression increased significantly in ALR KO mice after I/R injury compared with wild-type mice (Fig. 5E, F). These results indicate that ACSL4 is a biomarker of ferroptosis in AKI. Furthermore, ALR overexpression could further rescue I/R injury \u003cem\u003ein vitro\u003c/em\u003e (Fig. 5F). During ferroptosis, in the presence of ferrous iron or lipoxygenase, ALR catalyzes unsaturated fatty acids within the cell membrane, which causes lipid peroxidation and ferroptosis. Thus, we examined the mRNA levels of fatty acid oxidation genes in the kidneys and observed that the levels of enzymes and regulators of fatty acid oxidation were reduced after I/R injury compared with the control, it indicated that ALR plays a key role in ferroptosis by inhibiting fatty acid oxidation (Fig.5G).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eALR binds ACSL4 to prevent ferroptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe used HK-2 cells, a proximal tubular cell line, to determine if ALR binds to ACSL4. ALR and ACSL4 were co-localized in mitochondria in HK-2 cells (Fig.6A). In the kidneys, these two proteins mainly localized in proximal tubules (Fig.6C). These results were confirmed by co-immunoprecipitation (Fig.6B). To further confirm whether ALR and ACSL4 indeed interact, we identified the potential binding sites between these two proteins (Fig.7A-D). ALR is a dimer in its native conformation (Fig.7A). However, interactions between ALR and ACSL4 in publicly available protein\u0026ndash;protein interaction databases have not been reported. Thus, molecular docking was used to identify potential interacting residues based on geometric complementarity. Because ALR is a dimer [35], we analyzed the potential sites in the dimer. Interacting regions between ALR (https://www.pdbus.org/, PDB, ID3MBG) and ACSL4 (https://www.uniprot.org/, Uniprot-O60488-F1-model) corroborated available data on the existing domains (Fig.7A-C). The specific sites of contact between ACSL4 with ALR are shown in Fig. 7D.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFerroptosis is driven by fatty acid accumulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFerroptosis is characterized by the accumulation of ROS and lipid peroxidation products. There are many different classes of lipids in cells, including monounsaturated fatty acids and polyunsaturated fatty acids. However, it was not clear if these fatty acids are oxidized and if they have roles in ferroptosis. To address these questions, we extracted and analyzed lipid metabolites by resolution liquid chromatography (UPLC) coupled with triple quadruple mass spectrometry (QqQ) (UPLC-QqQ-MS/MS). We analyzed the oxylipin ratio between ACSL4-silenced and control specimens to generate the lipidomic profiles. There were 48 lipid metabolites (17 down-regulated and 31 up-regulated) displaying significant changes after ACSL4 knockdown (Fig.8A, Supplementary Table S5). Also, the volcano map show that the down-regulation and up-regulation about oxylipin (Supplementary Figure S4E). As for the qualitative oxylipin profile, of the 48 oxylipins analyzed, there were significant differences in six oxylipins between ACSL4-silenced and control specimens (Fig.8B).\u0026nbsp;To further examine the functions of ALR and ACSL4 in ferroptosis, ALR expression was silenced in HK-2 cells by short hairpin interfering RNA sequences.\u0026nbsp;Next, we performed targeted lipidomics and metabolomics profiling (Fig.8C). We found that the level of 5Z,8Z,11Z,14Z-eicosatetraenoic acid (ARA) increased significantly compared with the control group (Fig.8D). After treatment with ACSL4, consistent with this finding, we examined the ARA level (oxylipin from EPA) and observed a significant increase (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05) in the shRNA/ALR group compared with the shRNA/ALR+ACSL4 group (Supplementary Figure S4F). Heatmap showed the detailed concentration from targeted metabolomics profiling (Fig.8E). Taken together, the above results prove that interference ACSL4 and/or ALR reduced mostly polyunsaturated fatty acids.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFerroptosis, a non-apoptotic form of cell death, is a relatively recently discovered regulated cell death that can be triggered by excessive lipid peroxidation [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. An increasing number of studies indicate that Ferroptosis is involved in I/R injury, as well as AKI[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].I/R injury is the most common cause of AKI, and the pathophysiology of I/R injury-induced AKI can be summarized by hemodynamic alterations, epithelial cell injury, and inflammation [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Furthermore, ROS production has been implicated in the kidneys after I/R injury. ROS are the products of lipid oxidation, and the rapid increase in ROS production can overwhelm antioxidant defenses and further aggravate injury. It is worth mentioning that epithelial cells are susceptible to I/R injury. In our previous study, we reported that ALR was a protective antioxidant molecule in the mitochondrial response to H/R injury-induced oxidative stress in AKI [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Previous research found that defects of ALR leas to intramitochondrial iron accumulation[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In addition, it is worth mentioning that ALR was shown to be responsible for Fe-S cluster transfer and stability of the mitochondrial membrane[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Moreover, ferroptosis is suggested to be iron-dependent and lipid peroxidation accumulations. Here, we highlight the significance of ALR in I/R injury-induced ferroptosis in the kidneys by high-throughput RNA sequencing. Mechanistically, we also demonstrate that decreased ALR expression and excessive oxylipin production synergistically induced ferroptosis after H/R injury. Our study discovered the protective role of ALR against I/R induced ferroptosis. Using a kidney-specific ALR knockout mouse model, we found that knockout ALR aggravated the progression of AKI. On the contrary, overexpression of ALR by transfected with lentivirus protected against ferroptosis in AKI. We further revealed that this protective role of ALR could be enhanced via downregulation of ACSL4, an important regulator of sensitivity of ferroptosis converting and controlling the accessibility of long polyunsaturated fatty acids required for the transduction of ferroptotic cell death metabolic process.\u003c/p\u003e \u003cp\u003eACSL4 is a member of the acy1-CoA synthetase family, and it is a biomarker and regulator of ferroptosis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. ACSL4 is localized in mitochondria, and it prefers polyunsaturated fatty acids [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Here, we identified the common genes between fatty acid oxidation and ferroptosis in the PT-KO mouse, and ACSL4 was the only gene that was common to both processes out of 26 genes. We also observed changes in arachidonic and eicosapentaenoic acids following H/R injury, which resulted in the decrease of ACSL4 expression. Specifically, interference ACSL4 reduced mostly polyunsaturated fatty acids. Similarly, ACSL4 overexpression inhibited oxylipin accumulation. In addition, by docking analysis, we identified the potential binding sites between ALR and ACSL4, and in other studies, we observed that ALR and ACSL4 were colocalized in mitochondria. Given the morphological changes in mitochondria during ferroptosis and the production of oxylipins by mitochondria, these observations highlight the important role of oxylipins, which are triggered by ROS stress, and the critical role of ALR in mediating resistance to oxidative stress.\u003c/p\u003e \u003cp\u003eFinally, we discuss how ferroptosis might be targeted in mitochondria. Mitochondria play important roles in I/R injury and ferroptosis, and from the perspective of energy metabolism, proximal tubular cells require a large amount of energy to maintain normal function. Because the energy from fat is more than three times higher than that of glucose, proximal tubular cells prefer fat as the main energy source. There is accumulating evidence suggesting that lipid metabolism may be involved in the pathology of AKI and chronic kidney disease. In addition, lipid peroxidation is a hallmark of ferroptosis, and if we can recognize the mitochondria as a target, ALR could directly bind to ACSL4 to prevent I/R injury-induced ferroptosis.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we report for the first time that ALR specifically binds to ACSL4 and regulates ferroptosis in proximal tubular cells by attenuating oxylipin accumulation (Fig.\u0026nbsp;8F). These results provide new insights on how mitochondria, a key organelle of energy resources for proximal tubular cells, can be targeted to decrease the accumulation of oxidized lipids in I/R injury-induced AKI.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAKI:\u0026nbsp;acute kidney injury;\u0026nbsp;ALR: augmenter of liver regeneration; ACSL4:Long chain acyl-CoA synthetase 4 ; BUN: blood urea nitrogen; DEGs :differentially expressed genes; GPX\u003csub\u003e4\u003c/sub\u003e: glutathione peroxidase 4; I/R: Ischemia‐reperfusion;KIM-1: kidney injury molecule-1; NGAL: neutrophil gelatinase-associated lipocalin; PT-ALR KO: proximal tubule-specific ALR knockout; ROS: reactive oxygen species; RSD: relative standard deviation; SCR: serum creatine.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Aishun Jin at Chongqing Medical University (Department of Immunology, College of Basic Medicine) for providing the platform for cell culture and the training of cell culture techniques.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003euthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLLH, FYT, YXM cultured HK-2 cells. DC and LLD performed the animal models. ZZ and LZ, provided expertise and materials. HS, QL and XHL designed the experiments. LLH and YJZ analyzed the data, and wrote the paper. All authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China (81873604), the Medical Scientific Research Project of the Chongqing Health Commission (2022GDRC005) and Chongqing Science and Technology Agency (CSTB2022NSCQ-MSX0984).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated in this study have been included in this article as figures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal study was examined and approved by the Chongqing Medical University Animal Care and Use Committee (approval no. 2018,146).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBellomo R, Kellum JA, Ronco C: \u003cstrong\u003eAcute kidney injury.\u003c/strong\u003e \u003cem\u003eLancet \u003c/em\u003e2012, \u003cstrong\u003e380:\u003c/strong\u003e756-766.\u003c/li\u003e\n\u003cli\u003eSchrier RW, Wang W, Poole B, Mitra A: \u003cstrong\u003eAcute renal failure: definitions, diagnosis, pathogenesis, and therapy.\u003c/strong\u003e \u003cem\u003eJournal of Clinical Investigation \u003c/em\u003e2004, \u003cstrong\u003e114:\u003c/strong\u003e5-14.\u003c/li\u003e\n\u003cli\u003eLevey AS, James MT: \u003cstrong\u003eAcute Kidney Injury.\u003c/strong\u003e \u003cem\u003eAnn Intern Med \u003c/em\u003e2017, \u003cstrong\u003e167:\u003c/strong\u003eITC66-ITC80.\u003c/li\u003e\n\u003cli\u003ePefanis A, Ierino FL, Murphy JM, Cowan PJ: \u003cstrong\u003eRegulated necrosis in kidney 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\u003cstrong\u003e12:\u003c/strong\u003e255-278.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Acute kidney injury, Augmenter of liver regeneration, Ferroptosis, Long chain acyl-CoA synthetase 4, Lipid peroxidation","lastPublishedDoi":"10.21203/rs.3.rs-2649851/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2649851/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eFerroptosis, characterized by lipid accumulation in intracellular compartments, is related to acute kidney injury (AKI), but the mechanism remains obscure. In our previous study, we reported important roles for augmenter of liver regeneration (ALR) in antioxidant mechanisms. However, the roles of ALR in ferroptosis, especially the morphological changes in mitochondria induced by this type of regulated cell death, remain unclear and warrant further investigation.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe subjected Kidney-specific deletion of the ALR gene (ALR-K-KO), as well as HK-2 cells, to ischemia-reperfusion (I/R) induced AKI models. We assessed the kidney function and ferroptosis of proximal tubular epithelial cells. We also examined the level of lipid peroxidation by MS/MS. ALR and Long chain acyl-CoA synthetase 4 (ACSL4) were colocalized and interacting regions were detected by protein docking-analyses.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHere, we hypothesize that ALR regulates oxylipin accumulation in proximal tubular cells and attenuates ferroptosis induced by ischemia-reperfusion (I/R) injury in AKI. Kidney-specific deletion of the ALR gene (ALR-K-KO) aggravated ferroptosis, accompanied by increased ROS production and mitochondrial damage, whereas overexpression of the ALR gene attenuated lipid accumulation. Moreover, acsl4 loss reduced mostly polyunsaturated fatty acids. In addition, ALR and ACSL4 colocalize in the mitochondria of HK-2 cells and protein docking analysis found the interacting regions.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eWe showed for the first time that ALR binds to ACSL4 and regulates ferroptosis in proximal tubular cells by attenuating oxylipin accumulation.\u003c/p\u003e","manuscriptTitle":"Inhibition of Lipid peroxidation by ALR protects the kidney from ischemia-reperfusion injury","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-03-07 15:36:42","doi":"10.21203/rs.3.rs-2649851/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bead447a-e73b-40f5-b573-bbf47a2eee90","owner":[],"postedDate":"March 7th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-03-07T21:44:19+00:00","versionOfRecord":[],"versionCreatedAt":"2023-03-07 15:36:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2649851","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2649851","identity":"rs-2649851","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}