HIF-1 Prolyl Hydroxylase Inhibitors Attenuate X-Ray-Induced Radiation Nephropathy through Improving Ferroptosis

Keywords

Radiation; Oxidative Reaction; Lipid Peroxidation; Radiation Nephropathy; Ferroptosis

Abstract

Radiation nephropathy is the damage to the renal parenchyma and blood vessels caused by ionizing radiation, and its mechanism of injury involves oxidative stress, DNA damage, cellular senescence, and other processes, among which oxidative stress plays an obvious role. In the case of excessive accumulation of oxidative stress products, it can lead to lipid peroxidation and iron distribution abnormalities caused by ferroptosis; the latter is involved in a variety of pathophysiological processes, while currently there is no role in radiation nephropathy. The present study sought to investigate the role of ferroptosis in radiation nephropathy, and the mechanism of FG-4592 in relieviating radiation nephropathy, C57BL/6 mice and TCMK-1 cell are separately irradiated with 12GY, 10GY X-ray to construct radiation nephropathy models in vivo and in vitro. Compared with the control group, X-rays promoted the occurrence of lipid peroxidation and ferroptosis, and oxidative stress and lipid peroxidation products were increased and antioxidant products were decreased in the X-rays-induced radionephropathy model; intervention in ferroptosis could alleviate radiation nephropathy, and FG-4592 could regulate lipid metabolism and oxidative reactions to ameliorate feroptosis and attenuate kidney injury through the modulation of HIF activity. In conclusion, in this study, transcriptome analysis was utilized to screen out the pathways involved in the regulation of radiation nephropathy, which provided the basis for the subsequent study of drug intervention in radioactive kidney injury. And FG-4592 had a certain anti-radiation effect in this experiment, which had a protective effect in radiation nephropathy. This study provides new ideas for the development of novel low toxicity, effective radiation-resistant agents.

Abstract

Radiation nephropathy is the damage to the renal parenchyma and blood vessels caused by ionizing radiation, and its mechanism of injury involves oxidative stress, DNA damage, cellular senescence, and other processes, among which oxidative stress plays an obvious role. In the case of excessive accumulation of oxidative stress products, it can lead to lipid peroxidation and iron distribution abnormalities caused by ferroptosis; the latter is involved in a variety of pathophysiological processes, while currently there is no role in radiation nephropathy. The present study sought to investigate the role of ferroptosis in radiation nephropathy, and the mechanism of FG-4592 in relieviating radiation nephropathy, C57BL/6 mice and TCMK-1 cell are separately irradiated with 12GY, 10GY X-ray to construct radiation nephropathy models in vivo and in vitro. Compared with the control group, X-rays promoted the occurrence of lipid peroxidation and ferroptosis, and oxidative stress and lipid peroxidation products were increased and antioxidant products were decreased in the X-rays-induced radionephropathy model; intervention in ferroptosis could alleviate radiation nephropathy, and FG-4592 could regulate lipid metabolism and oxidative reactions to ameliorate feroptosis and attenuate kidney injury through the modulation of HIF activity. In conclusion, in this study, transcriptome analysis was utilized to screen out the pathways involved in the regulation of radiation nephropathy, which provided the basis for the subsequent study of drug intervention in radioactive kidney injury. And FG-4592 had a certain anti-radiation effect in this experiment, which had a protective effect in radiation nephropathy. This study provides new ideas for the development of novel low toxicity, effective radiation-resistant agents.

Keywords

Radiation; Oxidative Reaction; Lipid Peroxidation; Radiation Nephropathy; Ferroptosis

Citation

Wang S, Leng X, Yao P, Deng L, Du Y et al, (2025) HIF-1α Prolyl Hydroxylase Inhibitors Attenuate X-Ray-Induced Radiation Nephropathy through Improving Ferroptosis. J Nephrol Kidney Dis 6(2): 13.

INTRODUCTION

Ionizing radiation from nuclear industry, nuclear accidents, and other fields threatened the environment and humanity; during that, in medical areas, radiation therapy was an important factor. Such as, radiation therapy is an important part of the treatment regimen for pelvic and abdominal malignant tumours, which usually induces organ damage, including kidney. A clinical study showed that there is a 50% chance of developing radiation nephropathy when they receive radiation doses up to 28Gy (0.25-0.5Gy/dose) in a 5-year period [1]. Radiation nephropathy is defined as the renal parenchymal and vascular damage caused by ionizing radiation to the kidneys and accompanied clinical manifestations such as hypertension, azotemia, and anemia [2]. Therefore, effective measures to relieve radiation damage are necessary. Kidney is a radiation sensitive organ, which may be influenced by radiation mostly. Although it is currently believed that radiation nephropathy was related to oxidative stress, RAS system activation, vascular endothelial cell damage, as well as DNA damage and cell death, the mechanism remained unclear [3,4]. Early and effective intervention may be necessary for radiation nephropathy treatment, but there were no effective anti-radiation drugs for the treatment of radiation nephropathy. Ferroptosis is a nonapoptotic form of cell death induced by lipid peroxidation that is characterized by both iron dependence and ROS accumulation [5,6]; and is associated with a variety of diseases, such as those related to organ ischaemia/reperfusion, renal impairment, and neurological degeneration [6]. Previous studies have shown that drugs can relieve organ injury through antiferroptosis mechanisms; for example, as demonstrated that [7], melanin nanoparticles could alleviate sepsis-induced myocardial injury through antiferroptosis and inhibiting inflammation reactions with the continuous development of research on radiation, the relationship between ferroptosis and radiation has gradually been dissected. Studies have shown that radiation not only damages cellular DNA but also induces ROS production, which ultimately leads to the oxidation of biomolecules, including lipids [8], and lipid peroxidation is a key step in ferroptosis in cells. Radiation-inducedferroptosis has become an important component of current antitumor therapy and an important mechanism of action of radiation-induced damage to normal organs, and tumour cells exhibit a certain degree of radiation resistance when ferroptosis is reduced [9], similarly, it has been reported that inhibiting ferroptosis could attenuate radiation-induced fibrosis in lung tissue [10]. Thus, resistance to ferroptosis may be an effective measure for radiation nephropathy treatment. Hypoxia-inducible factor-1 (HIF-1) has been found to be an endogenous antioxidative stress regulator; HIF-1can activate the nuclear factor erythroid-2-related factor (Nrf2)-mediated antioxidant response element (ARE) pathway and has protective effects on ischaemic heart disease and spinal cord injury [11]; Moreover, HIF-1α pretreatment enhances antioxidant activity to protect against ischaemic heart disease and spinal cord injury [11]. Secondly, in mitochondria, stabilizing the expression of HIF-1 activates pyruvate dehydrogenase kinase 1 (PDK 1) gene expression, the PDK-1 gene expression up-regulation could inactivate the catalytic subunit of pyruvate dehydrogenase (PDH) through phosphorylation, finally preventing the conversion of pyruvate to acetyl coenzyme A in the tricarboxylic acid cycle of mitochondria [12], and decreasing the amount of ROS generated by the electron transport chain. FG-4592 is a small-molecule stabilizer of HIF, studies have shown that FG 4592 could balance oxidative stress by increasing the expression levels of the HIF-1α target genes superoxide dismutase 2 (SOD2), Nrf-2, and haem oxygenase 1 (HO-1); additionally, these factors or catalytic enzymes could mediate ROS deoxygenation [13]. FG-4592 could promote hemopoiesis and ameliorate radiation-induced hemopoetic system damage by mediating the HIF-1α pathway [14], indicating that FG-4592 has not only antioxidant effects but also antiradiation effects. And FG-4592 is an FDA approved drug for improving renal anemia, and the adverse reactions are mainly gastrointestinal reactions, dizziness, and fatigue, with no significant other toxic side effects, which is a high degree of safety. Hence, FG-4592 may be a potential low-toxicity anti-radiation agent. Meanwhile, ferrostatin-1(Fer-1) inhibits ferroptosis through suppressing the accumulation of lipid peroxides and chain carrying peroxyl radicals [15], and fer-1 occupies important roles in tissues or organs protection from radiation injury [16,17]. Therefore, this study aims to explore potential drug relieving radiation-associated kidney injury. In the present study, C57BL/6 mice and TCMK-1 cells were radiated by X-ray, respectively with 12Gy and 10Gy to construct radiation nephropathy models in vitro an in vivo, and divided into 4 groups (control group, irradiation group , irradiation+FG-4592 group, irradiation+ferroptosis inhibitor (ferrostatin-1, Fer-1) group) to investigate the roles of lipid peroxidation and ferroptosis in X-ray induced radiation nephropathy, moreover to further investigate the mechanism of FG-4592 in alleviating radiation nephropathy and provide new ideas for exploring effective and low-toxicity radioresistant agents.

Figure 1: Specific experimental flowchart.

MATERIALS AND METHODS

Animal Model of Radiation Nephropathy

SPF-grade C57BL/6 mice (6-8 weeks-old, male, 20-22 g) were purchased from Chengdu Dashuo Animal Laboratory Co., Ltd. (Chengdu, China) and were housed in a barrier environment in controlled temperature and humidity (20-26 ºC, a relative humidity of 40-70%), according to a 12-hour light/dark cycle, with free access to UV-sterilized water and feed. The mice were randomly divided into a control group (CON group), an irradiation group (IR group), an irradiation+FG-4592 group (IR+FG-4592 group), and an irradiation+ferroptosis inhibitor group (ferrostatin-1, Fer-1) (IR+Fer-1 group), with five mice in each group. After the mice were acclimatized to the environment, the IR+FG-4592 and IR+Fer-1 groups were pretreated, with the IR+FG-4592 group receiving treatment every other day by gavage (25 mg/kg) 5 days prior to irradiation and the IR+Fer-1 group receiving treatment daily by gavage (5 mg/kg) 5 days prior to irradiation. On Day 6, a radiation nephropathy model was constructed by irradiating the peritoneal cavities of mice via X-ray (a single dose of 12 Gy at a dose rate of 235 cGy/min), with all the mice anesthetized intraperitoneally with 10% chloral hydrate before irradiation. After irradiation, changes in the glomerular filtration rate (GFR) of the irradiated mice were dynamically observed by measuring the GFR at 1, 4, 8, and 12 weeks after irradiation, and histopathological changes in the kidneys were recorded at the time points corresponding to the changes in the GFR (Figure 1). This work has received approval for research ethics from the environmental conditions of the Chinese national standard “Experimental animal and environmental facilities” (approval ID: GB14925-2010) for animal experimental facilities, animal feeding management. 

Glomerular Filtration Rate Test

First, the skin of the mice was prepared by removing the hair from the right side of the spine from the scapula to the root of the thigh; then the mice were anesthetized with isoflurane-containing respiratory anesthetics and attached the mini with the battery connected to the dorsal side of the mice and fixing it with adhesive tape. The baseline data were read after 5 minutes, after which a tracer (35 mg/ml, 7 mg/100 g) was injected into the tail vein, and the GFR was then measured by removing the mini after 1.5 hours of recording and reading the data on a computer [18], with MB-Lab2 and MB-Studio3 .

Assay for Renal Function, TGF-β1

Serum was prepared for creatinine and urea nitrogen measurement according to creatinine (Jiangsu Meimian Industrial Co., Ltd., MM 0693M1), urea nitrogen (Jiangsu Meimian Industrial Co., Ltd. MM 0692M1) kit instructions, and serum TGF-β1 levels were detected according to TGF-β1 (Jiangsu Meimian Industrial Co., Ltd., MM-0135M1) kit instruction with Elisa test.

MDA and GSH Assays in the Kidney

MDA and GSH levels in kidney tissues were detected by using an MDA assay kit (Beijing Solebo Technology Co., Ltd., BC0025) and a GSH kit (Beijing Solebo Technology Co., Ltd., BC1175) and following their respective protocols.

Pathological Examination of Kidney

Kidney tissues were fixed with 4% paraformaldehyde, paraffin fixed, cut into thin sections (5-8 µm), and placed on slides, which were subjected to hematoxylin-eosin (HE) and Masson’s trichrome staining.

Immunohistochemical Assay

Brifly, kidney tissues were fixed with 4% paraformaldehyde, paraffin fixed, cut into thin sections (5-8 µm), and placed on slides. After dewaxing and hydration, the slides were boiled in citric acid antigen extract for 15 min at room temperature, cooled, incubated in 3% hydrogen peroxide for 10 min at room temperature Then, the slides were blocked with 10% sheep serum at 4 °C overnight, and after removing the sealer, the HIF 1α mouse monoclonal antibody (Santa Cruz Biotechnology, sc-13515, M, 1:300), 4-HNE mouse monoclonal antibody (Bioss, bs-631R, M, 1:300), and GPX4 rabbit monoclonal antibody (Zen Bio, 381958, R, 1:100) were added and incubated overnight at 4 °C. The sections were washed three times with PBS, and the appropriate amount of secondary antibody was added. Afterwards, the sections were incubated for 30 min at 37 °C in a thermostat and visualized with a DAB kit (ab64238, Shanghai, China) for color development.

Cell Culture and Treatment

TCMK-1 cells were purchased from Guangzhou Gineo Biotechnology Co. and cultured in DMEM containing 10% newborn calf serum, 100 U/ ml penicillin, and 100 µg/ml streptomycin. Incubation conditions were 5%CO2 .,95% relative humidity and 37℃. When cells were grown to 70 80% confluence, cells were cultured in culture dishes, 6-well plates, or 96-well plates. 6 hours before radiation, cells in groups were separately pretreated with FG-4592 (40 µmol/L), Fer-1 (2 µmol/L), DMEM, and then irradiated with 10 Gy at a dose rate of 235 cGy/min. After irradiation, the cells were cultured in a cell incubator for 24 hours for subsequent experiments.

Cellular CCK-8 Assays

TCMK cells (5*103/well) were cultured in 96-well plates 24 hours before radiation, after cells attachment, cell could be irradiated by different doses of X-ray (5Gy-15Gy), and finally, using a CCK8 assay kit (Biyun Tian) to measure cell activity, 100 µl of CCK8 working solution (10 µl of CCK8 reagent + 90 µl of medium) was added an incubated for 1.5-2 hours according to the instructions of the CCK8 kit, and the absorbance was detected at a wavelength of 450 nm.

MDA and GSH Assays in TCMK-1 Cells

Cells (3.0*106/well) were cultured in culture dish 24 hours before treatment, pretreatedt with FG-4592, Fer-1, or without for 6 hours, and then irradiated with 10 Gy at a dose rate of 235 cGy/min. The MDA and GSH contents of the cells were detected by using an MDA assay kit (Beijing Solebo Technology Co., Ltd., BC0025) and a GSH kit (Beijing Solebo Technology Co., Ltd., BC1175), respectively.

Cellular Reactive Oxygen Species Assays

After X-ray irradiation, the adherent TCMK-1 cells were collected into 1.5 ml centrifuge tubes, washed twice with PBS, and centrifuged at 1000 rpm/min for 5 min, after which the cell precipitate was retained for detection. A diluted probe was prepared by adding 10 µM probe to the corresponding volume of serum-free culture medium at a ratio of 1:1200, and the cell suspension was prepared by resuspending the cell pellet. The cells were incubated at 37 ºC for 30 min in the dark, during which time they were mixed every 3-5 min. The probe-labeled single-cell suspension was collected and centrifuged at 1000 rpm/min for 5 min, washed with PBS twice, and then detected according to the FITC fluorescence detection conditions.

Cellular RNA-seq

The total RNA was extracted by TRIzol (Nanjing Novozan Biotechnology Co., Ltd., R401) 24 hours after X-ray irradiation of TCMK-1 cells, and the extracted RNA was frozen at -80 °C for subsequent transcriptome sequencing, which was performed by Henan Huizhiin Biotechnology Co. Before data analysis, data filtering was performed. Cut-adapt was used to remove the junctions at the 3’ ends, allowing 20% base mismatches. Reads with average quality scores lower than Q20 were removed, and the filtered sequences were compared with the reference genes using HISAT2 software (http://geneontology.org/), and expression levels were normalized using FPKM. The DEGs in each group were analyzed using the DESeq package in R. P < 0.05 and |log2 (fold change)|> 1 were considered differentially expressed genes (DEGs), where log2 (fold change) > 1 was considered to indicate upregulated genes and log2 (fold change) < 1 was considered to indicate downregulated genes. The ggplots2 package in R was also used to construct a volcano plot of the DEGs; cluster analyses (http://www.kegg.jp/) and gene set enrichment analysis (GSEA) of KEGG pathway enrichment (https://www.bioinformatics.com.cn) were performed on the DEGs.

Western Blot Analysis

The treated cells were placed on ice, 150 µl of RIPA lysis buffer (RIPA:PMSF 100:1) was added, and the cells were sonicated by an ultrasonic crusher (Shanghai Bilang Instrument Manufacturing Co., Ltd.) for 2 seconds, with an interval of 3 seconds. Afterwards, the mixture was incubated for 1 minute at a power of 10%, sonicated 3 times, and subsequently placed on ice for 30 minutes. The cell protein concentration was detected using BCA reagent, and the cell proteins were then homogenized according to the following formula: loading buffer = 4:1. Afterwards, 5x loading buffer was added, and the mixture was mixed well, boiled for 10 minutes to denature the proteins, and stored at -20 °C. Primary antibodies were diluted according to the corresponding antibody instruction manuals: HIF-1α (Santa Cruz Biotechnology, sc-13515, M, 1:1000), Nrf2 (Bioss bs-1074, R, 1:1000), GPX4 (Zenn BIO, 381958, R, 1:500), SCL7X11 (Affinity Bioscience, DF12509, R, 1:1000), 4-HNE (Bios bs-6313, R, 1:1000), TfR1 (California, USA, 60103175, 1:1000), GAPDH (Zenn BIO, R, 1:5000), and beta-tubulin (Affinity Bioscience, T0023, M, 1:10,000). The secondary antibodies used were goat anti-rabbit IgG (Zen Bio, 511203, R, 1:10,000) and goat anti-rabbit mouse IgG (Zen Bio, 511103, M, 1:10,000).

Statistical Analysis

The data were normally distributed, and variation was determined by the chi-square test, calculated as the mean ± standard error of mean (SEM); differences between groups were analyzed using one-way ANOVA in SPASS 22.0 software. Semiquantitative analyses of the integrated optical density or fluorescence intensity in the images were performed using ImageJ software, and the results were considered to be significantly different at p < 0.05.

RESULTS

FG-4592 Improves Radiation-Induced Kidney Injury in Mice

First, in this study, the glomerular filtration rate of mice in the IR group started to decrease after 4 weeks of radiation and then decreased significantly after 12 weeks of radiation (Figure S1). Renal function was assessed by measuring the serum urea nitrogen and serum creatinine levels in the mice. The serum creatinine level of the control mice was 37.872± 1.279 µmol/L, and the serum creatinine level of the mice irradiated with X-rays increased to 41.313±3.501 µmol/L (p=0.0007); however, compared to the radiated mice, the renal function of the mice in the FG-4592 pretreatment group improved, and the serum creatinine level decreased to 35.661±1.547 µmol/L (p<0.0001) (Figure 2A). Furthermore, compared with radiated mice, the serum urea nitrogen concentration was significantly decreased in the control group (13.777±1.186 mmol/L vs. 21.938±1.599 mmol/L, p<0.0001) and FG-4592-pretreated group (14.565±1.722 mmol/L vs. 21.938±1.599 mmol/L, p=0.0002) (Figure 2B). HE staining revealed that in the irradiated group, the renal pathology was characterized by focal segmental sclerosis of the glomeruli, and the impairment in renal tubule was more obvious, including disappearance of normal tubular structure, extensive vacuolization of tubular epithelial cells, and nuclear consolidation in tubular epithelial cells. However, in the control and FG-4592 pretreatment groups, renal tissues of mice had clearly visible renal cystic lumens, and the vascular globules did not show capillary basement membranes or tunica albuginea hyperplasia, with no degenerative necrosis or fibrotic structures (Figure 3). In addition, in the Masson trichrome staining of kidney tissues, the cytoplasm and myofibrils were stained red, while the collagen fibers were stained blue. Obvious fibrosis was observed in the glomeruli and interstitium of irradiated mice, while renal fibrosis was not significant in the control, FG-4592, or Fer 1 pretreatment groups (Figure 3). Masson’s trichrome staining revealed that renal tissue from irradiated mice exhibited obvious fibrosis, while FG-4592 and Fer-1 treatments reduced the degree of renal fibrosis; Fer 1 inhibited renal fibrosis by efficiently capturing oxygen free radicals; TGF-β1 was an important factor in renal fibrosis in radiation nephropathy; and the serum level of TGF-β1 was greater in the irradiated group than in the control group (239.077±27.603 ng/mL vs. 151.740±7.325 ng/ mL, p<0.0001), and the FG-4592-treated group (239.077±27.603 ng/ mL vs. 144.837±5.179 ng/mL, p<0.0001) (Figure 2C). Therefore, FG 4592 could attenuate radiation-associated renal fibrosis. Taken together, these results suggested that FG-4592 plays an important role in relieving radiation nephropathy, which was indicated by changes in renal function and pathology changes.

FG-4592 Protects Against Radiation-Induced TCMK-1 Damage

In the present study, TCMK-1 cells were used to evaluate the effect of the FG-4592 in vitro model of radiation nephropathy. Cellular activity of TCMK-1 cells was measured by using the CCK8 assay. First, to explore the 50% inhibitory concentration of FG-4592 in TCMK-1 cells, the study pretreated TCMK-1 cells with different concentrations of FG 4592 and finally calculated that the 50% inhibitory concentration was  178.5µmol/L, which indicated that FG-4592 had less toxicity to TCMK-1 cells. Additionally, TCMK-1 cells irradiated with different radiation doses (5, 7.5, 10, 12.5, 15, and 17.5 Gy; dose rate: 235 cGy/min), and after 24 hours the cell activity in diferent groups was measured by CCK-8 assay. results showed that FG-4592 had protective effects on TCMK-1 cells, and the activity of cells pretreated with FG-4592 was significantly higher than that of cells in the irradiated group (Figure 4A). To further investigate the protective mechanism of FG-4592 against X-ray-induced cellular damage, cellular reactive oxygen species (ROS), GSH, and MDA were detected, and irradiation significantly increased the levels of cellular oxidation products but decreased the levels of antioxidant products (Figure 4B-D). Thus, FG 4592 exerted a protective effect on TCMK-1 cells through antioxidant effects.

Figure 2: Serum creatinine, blood urea nitrogen and TGF-β1 concentrations in mice (* represents the CON group; #, compared with the IR group). (A) compared to IR group, the level of serum creatine was lower in CON, IR+FG-4592, and IR+Fer-1groups; (B) compared to IR group, the level of serum BUN was lower in CON, IR+FG-4592, and IR+Fer-1groups; (C) compared with that in the CON group, the TGF-β1 content was increased in the IR group, and pretreatment with FG-4592 and Fer-1 decreased this parameter.

Figure S1: Glomerular filtration rate of mice in the IR group started to decrease after 4 weeks of radiation and then decreased significantly after 12 weeks of radiation

FG-4592 Improves Ferroptosis in Vivo and In Vitro in Radiation Nephropathy

Results of Animal Experiments

1. FG-4592 Upregulates HIF-1α Expression and Alleviates Lipid Peroxidation Responses in the Kidney: To investigate the role of FG 4592 in radiation nephropathy in vivo, the present study used MDA and GSH assays to detect MDA and GSH levels in mouse kidney tissues. As expected, X-ray radiation significantly decreased GSH levels in kidney tissues (1629.412±74.381 µg/mg prot vs. 2143.892±187.67 µg/mg prot, p=0.0063), while lipid peroxidation products such as MDA were elevated (21.656±3.867 nmol/mg prot vs. 16.044±2.184 nmol/mg prot, p=0.1329) (Figure 5A-B). However, pretreatment with FG-4592 alleviated the above radiation-associated changes, the GSH contents significantly increased (2318.485±281.637 µg/mg prt vs. 1629.412±74.381 µg/ mg prt, p=0.0004), while the MDA content significantly decreased (10.941±2.507 nmol/mg prot vs. 21.656±3.867 nmol/mg prot, p=0.0021) (Figure 5A-B). Both MDA and GSH play important roles in the process of ferroptosis. MDA is the main metabolism of lipid peroxidation, and its level indirectly indicates the level of lipid peroxidation. GSH is also an important component of defence against ferroptosis, i.e., the SLC7A11 GSH–GPX4 axis, which represents key components of antiferroptosis. Ionizing radiation could lead to a decrease in GSH, thus weakening GPX4-mediated defense against ferroptosis and promoting cell injury and death. In contrast, pretreatment with FG-4592 inhibited the GSH depletion induced by X-rays, increased in vivo GSH levels, and suppressed ferroptosis. Immunohistochemical analysis of renal tissues revealed that the expression of 4-HNE was significantly increased in the renal tissues of X-ray-irradiated mice and decreased in the FG-4592 and Fer-1 pretreatment groups (Figure 6C); conversely, the expression of GPX4 was decreased in the renal tissues of the X-ray-irradiated mice and increased in the FG-4592 and Fer-1 pretreatment groups (Figure 6B). Moreover, immunohistochemistry (IHC) of mouse kidney tissues revealed that FG 4592 enhanced HIF-1α expression in mouse kidney tissues. Thus, FG 4592 can increase the level of HIF-1α and inhibit radiation-induced lipid peroxidation.

In Vitro Experimental Results

1. FG-4592 Mediates HIF-1α Expression and Regulates Lipid Metabolism in TCMK-1 cells: To explore the specific mechanism of FG 4592 in radiation nephropathy, transcriptome sequencing of TCMK-1 cells in CON, IR, IR+FG-4592, and IR+Fer-1 groups was performed. Compared to cells in the CON group, there were a total of 360/605/530 upregulated differentially expressed genes (DEGs) and 33/161/50 downregulated DEGs in IR, IR+FG-4592, and IR+Fer-1 groups separately. And compared to cells in the IR group, there were a total of 161/132 upregulated DEGs,197/512 downregulated DEGs in the IR+FG-4592 and IR+Fer-1 groups (Figure S2). Meanwhile, the KEGG pathway enrichment was determined by GSEA analysis. The GSEA analysis between the control group and the irradiation group results revealed that significant difference in lipid metabolism including arachidonic acid metabolism, butanoate metabolism, linoleic acid metabolism and pyruvate metabolism, and some signaling pathways,eg. cAMP signaling pathway, MAPK signaling pathway, PI3K-Akt signaling pathway, etc. (Figure 7A-B). The GSEA analysis between the FG-4592 pretreatment group and the irradiation group showed that significance difference in lipid metabolism (such as arachidonic acid metabolism, pyruvate metabolism) , AMPK signaling pathway, FoxO signaling pathway, HIF-1 signaling pathway and JAK-STAT signaling pathway (Figure 7C-D), and during that FG-4592 negatively regulated arachidonic metabolism and JAK-STAT signaling pathway, positively regulated pyruvate metabolism, HIF-1 signaling pathway, AMPK signaling pathway, and FoxO signaling pathway. Thus, it can be hypothesized that FG-4592 pretreatment can exert protective effects on TCMK-1 cells through mediating HIF-1 stablization and regulating lipid metabolism.

Figure 4: Cell activity and determination of ROS, MDA, and GSH levels after irradiation and following treatment with FG-4592 and Fer-1 (* represents the CON group; #, compared with the IR group). (A) The activity of TCMK-1 cells was negatively related to the X-ray dose, and FG-4592 could protect TCMK-1 cells from radiation damage; (B) the content of MDA was increased in the IR group compared to that in the CON group, while FG-4592 and Fer-1 decreased the level of MDA in TCMK-1 cells; (C) IR decreased the content of GSH in TCMK-1 cells, but the effect was altered by pretreatment with FG-4592 and Fer-1; and (D) pretreatment with ROS in different groups and IR increased the ROS in TCMK-1 cells;(E-F) the results ROS of 4 groups in Flow cytometry detection.

Figure 5: Determination of MDA and GSH levels in mouse kidney tissue (* represents the CON group; #, compared with the IR group). (A) the MDA content in kidneys was increased by irradiation in the IR group compared with the CON group, while the MDA content was decreased by FG-4592 and Fer-1 treatment; (B) GSH levels in kidneys responded oppositely to MDA, and the MDA content in the IR group was much higher than those in the CON, IR+FG-4592/Fer-1 groups.

Figure 6: Immunohistochemistry analysis of HIF-1α, GPX4, and 4-HNE in mouse kidney tissues and semiquantitative analysis (N=5). The arrows indicate the positions of HIF-1α, GPX4, and 4-HNE; * indicates the IR group compared with the CON group; and # indicates the IR+FG-4592/Fer 1 group compared with the IR group. (A) Semiquantitative analysis of HIF-α; the HIF-α level in mice in the IR group was lower than in the other groups. (B) Semiquantitative analysis of GPX4; and GPX4 level in mice in the IR group was lower than in the other groups. (C) Semiquantitative analysis of 4-HNE; the 4-HNE level in mice in the IR group was higher than in the other groups.

Figure S2: 161/132 upregulated DEGs, 197/512 downregulated DEGs in the IR+FG-4592 and IR+Fer-1 groups.

2. FG-4592 Improves Radiation-Induced Ferroptosis in TCMK 1 cells: The results also showed that X-ray irradiation promotes lipid catabolism, and lipid peroxidation is the key to ferroptosis. In this study, cellular transcriptome sequencing analysis has revealed that the expression levels of genes related to lipid catabolism and peroxidation were significantly upregulated in the IR group, whereas the expression levels of genes related to lipid synthesis and antioxidant production were significantly increased in the FG-4592 group; furthermore, X-ray irradiation also increased cellular ROS and MDA levels but decreased the GSH content in TCMK-1 cells (Figure 4-B-D), while FG-4592 pretreatment reversed the above changes, consistent with the changes observed in the Fer-1 pretreatment group. The Western blot results showed that X-ray irradiation downregulated the expression of Nrf-2, GPX4, and SCL7A11 (Figure 8A-C, 8G-K), but upregulated the expression of 4-HNE and TfR1 (Figure 8D-F), however, pretreatment with FG-4592 and Fer 1 could upregulate the expression of antioxidant-related proteins, which played an important role in against ferroptosis; more importantly, using FG-4592 and Fer-1 downregulated the expression of 4-HNE and TfR1, which is a biomarker of ferroptosis. Thus, FG-4592 pretreatment inhibited ferroptosis by inhibiting lipid peroxidation, increasing cellular antioxidant products, and decreasing oxidized products.

DISCUSSION

Ferroptosis is an iron-dependent, lipid superoxide-driven form of cell death, involving to several diseases, and ferroptosis a key factor in X-ray injury to the kidney in the present study, and FG-4592 was found to attenuate radiation-induced kidney injury by modulating ferroptosis through inhibition of lipid peroxidation. Usually, the kidney is involved in the regulation of lipid metabolism. On the one hand, free fatty acids (FFA) enter renal cells through the differentiation cluster 36 (CD36), fatty acid transport proteins (FATP), and hepatic fatty acid binding proteins (FABP), and synthesize triglycerides (TG) in the cytoplasm, which are deposited on the endoplasmic reticulum and gradually fuse to form lipid droplets, which interact with mitochondria and participate in metabolism [19]. On the other hand, lipids are one of the energy-producing substrates of the kidney, and are not only the main energy-producing substrates for the renal tubule but also an alternative energy-producing substrate when glucose levels are low [20]. However, in pathological conditions, lipids exert lipotoxicity through multiple signaling pathways, oxidative stress, and inflammatory responses, damaging renal tissue cells [21]. Studies have shown that lipid deposition in renal tissue can occur in patients with chronic kidney disease [21], and ectopic lipid deposition has been associated with renal cell injury, glomerulosclerosis, interstitial fibrosis, and proteinuria [22]. In conclusion, under normal conditions, the kidney is involved in the regulation of lipid metabolism; under pathological conditions, lipid metabolism may promote renal tissue damage and accelerate the deterioration of renal function, and the maintenance of a relatively balanced lipid metabolism may reduce renal damage.

Figure 7: The GSEA analysis of KEGG between different groups. (A) GSEA ananlysis revealed significance in enrichment of lipid metabolism between IR and CON group; (B) the enrichment of arachidonic acid metabolism between IR and CON group; (C) GSEA analysis of signaling pathway; (D) GSEA ananlysis revealed significance in enrichment of lipid metabolism between IR+FG-4592 and IR group; (E) the enrichment of arachidonic acid metabolism between IR+FG-4592 and IR group; (F) GSEA analysis revealed significance in enrichment of signaling pathway.

Figure 8: Western blot analysis of HIF-1α, Nrf-2, GPX4, SCL7A11, HO-1,4-HNE, and TfR-1 levels and the results of semiquantitative analysis (* represents the CON group; #, compared with the IR group). (A) Western blot results for GPX 4 and SCL7A11. (B)(C) Semiquantitative analysis of GPX 4 and SCL7A11; GPX 4 and SCL7A11 expression levels were significantly decreased in the IR group compared with the CON group, and IR+FG-4592 and IR+Fer-1 increased GPX 4 and SCL7A11 expression compared with the IR group. (D) Western blot results for 4-HNE and TfR-1. (E) (F) Semiquantitative analysis of 4-HNE and TfR-1; 4-HNE and TfR-1 expression levels were significantly increased in the IR group compared with the CON group, and IR+FG-4592 and IR+Fer-1 decreased 4-HNE and TfR-1 expression compared with the IR group. (G) Western blot results for HIF 1 α and Nrf-2. (H)(I) Semiquantitative analysis of HIF-1 α and Nrf-2; HIF-1 α and Nrf-2 expression levels were significantly decreased in the IR group compared with the CON group, and IR+FG-4592 and IR+Fer-1 increased HIF-1 α and Nrf-2 expression compared with the IR group. (J) Western blot results for HO-1. (K) Semiquantitative analysis of HO-1; HO-1 expression was much higher in the IR group than in the CON, IR+FG-4592, and IR+Fer-1 groups.

Ionizing radiation generates large amounts of ROS by mediating oxidative stress, leading to oxidation of cellular proteins, DNA, and lipid peroxidation, which ultimately leads to cell death and tissue damage [23]. lipid peroxidation is a key factor in cellular ferroptosis. Studies have shown [24], that ionizing radiation induces cellular ferroptosis to damage the intestine, while ferroptosis inhibitors attenuate intestinal damage. Thus, ionizing radiation may damage tissues by inducing cellular ferroptosis, and intervening in this process attenuates the extent of tissue damage. Radiation-induced kidney injury is the damage of renal parenchyma and vasculature caused by ionizing radiation to the kidney [2], and the mechanism of injury involves multiple processes such as oxidative stress, DNA damage, activation of the RAS system, and cellular senescence, during that oxidative stress plays a significant role. In a model of radiation nephropathy, ionizing radiation increases renal tissue oxidation products, such as increased lipid peroxide MDA, while antioxidant (e.g., GSH) content decreases [25]. Combined with the results of this study, the content of lipid peroxidation products such as MDA, 4-HNE, and ROS increased in the X-ray irradiated group, while the content of antioxidants such as GSH and GPX 4 decreased. Therefore, the lipid peroxidation reaction is an important factor in the X-ray kidney injury,and the intervention of the lipid peroxidation reaction and ferroptosis is a potential effective measure to intervene in the radiation-induced kidney injury. Fer-1 was one of the first synthetic radical-trapping antioxidants (RTAs) reported to block ferroptosis, and it is widely used as a reference compound [15-26]. Fer-1 was shown to downregulate ferroptosis to relieve LPS-induced acute lung injury [16], and could also alleviate kidney injury induced by cisplatin or other drugs [27,28]. More importantly, Fer 1 plays a sexual role in radiation-induced tissue-organ damage, Zhang et al. found that Fer-1 exerted a protective role in mouse models of hematopoietic acute radiation syndrome by inhibiting changing processes caused by ionizing radiation [29] (e. g. downregulating ASCL 4, GPX 4; increasing unstable iron pool content), While alleviating the radiation related intestinal injury by targeting to the STAT 1-IRF1-ACSL4 signaling axis [17]. Thus, Fer-1 may play an important role in ionizing radiation related tissue damage. In our study, Fer-1 was also found to reduce the lipid peroxidation reaction and the production of peroxidation products (e. g., reduce MDA, 4-HNE, ROS), and to alleviate the X-ray-induced kidney injury. However, some studies have found that the effect of Fer-1 in vivo is weak due to the instability of plasma and metabolism [30,31]. Therefore, developing a more stable and effective drug is particularly important. FG-4592 is a novel small-molecule stabilizer of HIF already marketed to correct renal anemia. In this context, FG-4592 plays a role in maintaining HIF levels in vivo by inhibiting PHD hydroxylation and reducing HIF degradation [32]. Studies have shown that FG-4592 can delay or alleviate disease-related injuries by regulating mitochondrial oxidative stress and energy metabolism, such as antisepsis, attenuating adriamycin-related cardiotoxicity, or kidney injury [33,34]. Moreover, FG-4592 has some anti radiation effects in the blood systems [14], and radiation enteritis. For example, it was reported that FG-4592 could alleviate radiation-induced intestinal injuries by promoting intestinal stem cell recovery and reducing the damage incurred by intestinal epithelial cells [35,36]. However, its application in radiation nephropathy has not been evaluated. Therefore, this study investigated the effects of FG-4592 on radiation nephropathy and the underlying mechanisms in vivo and in vitro. The results of this study showed that the GSH content in the renal tissues of mice in the FG-4592 pretreatment group was greater than that in the IR group, while the MDA content was lower. Moreover, in the FG-4592 pretreatment group, the renal function of mice was not injured and the serum creatinine and urea nitrogen levels were lower than those in the IR group. The same conclusion was also reached from the in vitro experiments: compared with those in the IR group, the GSH content in TCMK-1 cells in the FG-4592 pretreatment group was elevated, whereas the levels of MDA and ROS were decreased. Thus, FG-4592 exerts certain antioxidant and antiradiation effects. Previously, it was reported that pretreatment with amifostine (a strong antioxidant) could reduce radiation-induced kidney injury [37]; additionally, coenzyme Q10 (CoQ10) also could significantly ameliorate radiation-induced renal injury and reduce fibrosis in renal tissue [38]. Consistent with the results of the present study, antioxidants are potential targets for the mitigation of radiation nephropathy. In fact, oxidative stress plays an important role in the mechanism of injury in radiation nephropathy. Radiation causes a redox imbalance by ionizing water molecules, generating oxygen free radicals, and damaging DNA strands. However, when redox imbalance occurs, it can cause peroxidation of unsaturated fatty acids to lead to ferroptosis. And previous studies have shown that inhibition of ferroptosis can alleviate cellular damage, promote the recovery of organ function [7]. Therefore, does FG-4592 have an effect on preventing ferroptosis? In the in vivo experiments, the GSH and MDA levels and tissue immunohistochemistry (IHC) results (for GPX4 and 4-HNE) of renal tissues from mice in each group were analyzed, and the expression levels of GSH and GPX4 in the FG-4592 and Fer-1 pretreatment groups were greater than those in the IR group, while MDA and 4-HNE expression levels were downregulated. In vitro experiments also revealed that the GSH levels in TCMK-1 cells in the FG-4592 and Fer-1 pretreatment groups were significantly greater than that in the IR group, while the MDA and ROS levels were significantly lower. Therefore, FG-4592 has an antiferroptotic effect, and this anti ferroptosis agent can alleviate radiation-induced tissue and organ damage. Previous studies have shown that ferroptosis is associated with tumor radiosensitivity [39], and as is reported that the ferroptosis inducer (Erastin) could promote ferroptosis and increase the radiosensitivity of tumour cells by inhibiting the cystine–glutamate transport system [40]. In contrast, resistance to ferroptosis may inhibit irradiation-induced damage, as demonstrated that the inhibition of ferroptosis increased radiation resistance in lung cancer cells [41]. By analogy, anti-ferroptosis agents are potential targets for intervention in radiation nephropathy. To further investigate the mechanism of action of FG-4592 in radiation nephropathy, transcriptome sequencing of TCMK-1 cells and heatmap analysis of DEGs were performed, which revealed that the expression levels of genes mediating cellular fatty acid catabolism and oxidative product generation were upregulated in cells of the IR group (e.g., Cyp4a12a; Cyp4a12b; Ncf1), while the expression levels of genes mediating fatty acid synthesis or antioxidant product generation were downregulated compared to those in the control and drug pretreatment groups (e.g., Far2; Gstk1).The results of this study suggest that X-ray radiation damages TCMK-1 cells by promoting fatty acid catabolism and oxidative product production, while FG-4592 mitigates radiation induced cellular damage by inhibiting radiation-induced changes. Moreover, the pathways associated with the DEGs were significantly enriched according to GSEA analysis. GSEA analysis between the control group and the irradiation group results revealed a significant difference in lipid metabolism including arachidonic acid metabolism, butanoate metabolism, linoleic acid metabolism, and pyruvate metabolism, and some signaling pathways, eg. cAMP signaling pathway, MAPK signaling pathway, amd PI3K-Akt signaling pathway. And irradiation positively regulated arachidonic acid metabolism and linoleic acid metabolism, negatively regulated butanoate metabolism and pyruvate metabolism. And the above signaling pathways involve in mediating lipid metabolism, which is known to be a key factor in radiation-induced ferroptosis in cells. In that, arachidonic acid and linoleic acid are main polyunsaturated fatty acids (PUFAs), which would be peroxidized by radiation leading to ferroptosis [42]. In tumor radiation therapy, radiation therapy can promote lipid oxidation and ferroptosis in tumour cells by inhibiting SLC7A11 expression [43]. And one study found that a water-soluble formulation of arachidonic acid could induce the death of cancer cells by inducing ferroptosis [44]. In addition, pyruvate metabolism also regulated the feeroptosis process. Song et al., proved that pyruvate dehydrogenase kinase 4 (PDK4) inhibited ferroptosis through suppressing pyruvate oxidation [45], and fatty acid synthesis, the role of pyruvate metabolism in ferroptosis was related to the balance of oxidation-antioxidant balance. In a sense, lipid metabolism was closely related to ferroptosis, signaling pathway involving regulating lipid metabolism might affect ferroptosis. For example, AMPK signaling pathway regulated lipid accumulation and lipid oxidation, and a study showed that the AMPK/ FoxO3a activation inhibited ferroptosis induced by erastin [46]. While, the GSEA analysis between the FG-4592 pretreatment group and the irradiation group showed a significant difference in lipid metabolism (such as arachidonic acid metabolism, pyruvate metabolism), AMPK signaling pathway, FoxO signaling pathway, HIF-1 signaling pathway, and JAK-STAT signaling pathway. And obviously, FG-4592 negatively regulated arachidonic acid metabolism and JAK-STAT signaling pathway, positively regulated pyruvate metabolism, HIF-1 signaling pathway, AMPK signaling pathway and FoxO signaling pathway. In summary, FG-4592 mediated the metabolism of lipids in different ways. Among these signaling pathways, HIF-1α regulates the expression of iron metabolism-related genes and inhibits ferroptosis to promote tumor cell growth through the hypoxia response element (HRE) [47,48]. The HIF signaling pathway inhibits the occurrence of ferroptosis in acute kidney injury by reducing mitochondrial oxidative stress damage [49]. The FG-4592 used in this study is a novel proline hydroxylase inhibitor; i.e., it exerts its biological effects by stabilizing HIF expression. In vitro and in vivo experiments revealed that renal tissue and cellular HIF-1α expression levels were upregulated in the FG-4592 pretreatment group compared to the IR group, and Nrf-2, GPX4, and SCL7A11 expression levels were upregulated, inhibiting lipid peroxidation and promoting GSH synthesis and transport. In a study, FG-4592 was shown to inhibit ferroptosis to alleviate renal injury by activating the Akt/GSK-3β/Nrf-2 signaling pathway [33], and FG-4592 not only reduced fatty acid peroxidation products in renal tissues but also promoted GSH synthesis and transport, which was consistent with the present results on the regulation of lipid metabolisms [33]. However, in the present study, cellular protein Western blot analysis revealed that haem oxygenase-1 (HO-1) expression was upregulated in the IR group. HO-1 catalyzes the degradation of haem to bilirubin, carbon monoxide, and free iron, which has an antioxidant effect [50,51], and most related studies suggest that Nrf-2 mediates the activation of HO-1 to play antioxidant and antiferroptotic roles [52]. However, in the present study, the difference in Nrf-2 expression was inconsistent with that in HO-1 expression, and HO-1 was highly expressed only in the IR group. Taken together with the results of the current research, the role of HO-1 in ferroptosis is still controversial. On the one hand, HO-1 has an antioxidant effect that can mediate an antiferroptosis effect; on the other hand, HO-1 can promote the release of iron from haemocatabolism and metabolism, and iron overload is another key factor in the occurrence of ferroptosis. HO-1 not only inhibits ferroptosis but also promotes ferroptosis [53,54], and its specific mechanism of action needs to be further investigated. In summary, this study evaluated the therapeutic effect and mechanism of FG-4592 in a model of radiation nephropathy and demonstrated that HIF stabilizers can treat radiation nephropathy by regulating lipid metabolism and redox balance to inhibit the process of ferroptosis, providing a basis for the subsequent development of novel, low-toxicity, and effective radioresistant agents. However, there are deficiencies in this study, the specific molecular mechanism of FG-4592 remains to be investigated.

CONCLUSION

Our study mainly paid attention to the role of ferroptosis in radiation nephropathy to further explore effective measure for relieving kidney injury. We found that lipid peroxidation induced by radiation is closely related to kidney injury, and FG-4592 could regulate the metabolism of lipid, play an important role in anti-ferropotosis to relieve kidney damage.In a sense, radiation-associated damage is inescapable, searching for effective radioresistant agents is as well essential and promising. While the study initiates the role of ferropotosis in radiation nephropathy, we just focus on the lipid metabolism, but neglect the role of iron metabolism, further study is needed.

ACKNOWLEDGEMENTS

We thank AJE (https://china.aje.com/cn/researcher/rewards) for English language editing during the preparation of this manuscript.

AUTHOR CONTRIBUTION STATEMENT

Xingli Leng: conceptualization, methodology, writing original drafts, review, and editing; Peng Yao: conceptualization, methodology, funding acquisition; Lin Deng: conceptualization, methodology, writing original drafts; Yinyuan Du: writing original drafts, review and editing; Xia Feng: writing - original draft preparation and editing; Minglin Liu: Writing - original draft preparation and editing; Shaoqing Wang: conceptualization, data curation, formal analysis, writing - original draft, writing review, and editing, supervision, funding acquisition.

CONFLICT OF INTEREST

The manuscript is original and has not been previously published elsewhere. All the authors and the institutions where the work was carried out have approved the submission of this manuscript. All animal procedures in this study were approved by the Ethics Committee of Chengdu Medical College, The Second Affiliated Hospital of Chengdu Medical College (China National Corporation 416 Hospital).

DATA AVAILABILITY STATEMENT

No data was used for the research described in the article.

FUNDING

This work was supported by the Key Projects of the Nuclear Medicine Science and Technology Innovation of CNNC in 2021 (grant number ZHYLZD2021005), the Key Project of the Project of Scientific and Technological Innovation and Entrepreneurship in Sichuan Province in 2022 (grant number 2023JDRC0089), the Chengdu Medical College Graduate Innovative Research Fund Project in 2023 (grant number YCX2023-01-18), and the Sichuan Provincial Administration of Traditional Chinese Medicine Special Project on Chinese Medicine Research in 2024 (grant number 2024MS155)

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