Non-steroidal mineralocorticoid receptor antagonist finerenone ameliorates Diabetic Nephropathy via suppressing SLC7A11-mediated ferroptosis

Non-steroidal mineralocorticoid receptor antagonist finerenone ameliorates Diabetic Nephropathy via suppressing SLC7A11-mediated ferroptosis | 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 Non-steroidal mineralocorticoid receptor antagonist finerenone ameliorates Diabetic Nephropathy via suppressing SLC7A11-mediated ferroptosis 卢 Yu, 齐汉 zhai, 尤林 王, 刘伟 王, 邱红 利, 燕红 郭, 荣 gou, 林 唐 This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7064578/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 Diabetic tubulopathy (DT) has recently been identified as a critical pathological feature of diabetic nephropathy (DN). Ferroptosis has emerged as an important pathological factor in DT, implicated in various metabolic disorders, including DN. Finerenone (FIN), a novel non-steroidal mineralocorticoid receptor (MR) antagonist, has demonstrated its ability to mitigate kidney inflammation and fibrosis in DN. However, the exact mechanisms underlying these effects remain unclear. SLC7A11 is known for its role in regulating glutathione (GSH) synthesis, which is closely associated with ferroptosis. To investigate how MR modulates SLC7A11-mediated ferroptosis under diabetic and high glucose (HG) conditions, human kidney proximal tubular epithelial (HK-2) cells were exposed to HG treatment. We assessed COL1, TGF-β, ferroptosis-related markers such as GSH and MDA, and proteins linked to ferroptosis, including FTH1, SLC7A11, and GPX4. Additionally, these molecules and proteins were analyzed in the kidneys of diabetic mice treated with FIN. FIN treatment effectively protected the kidneys by inhibiting SLC7A11-mediated ferroptosis in both HG-exposed HK-2 cells and tubular cells from diabetic mice. In summary, our study confirms that the non-steroidal mineralocorticoid receptor antagonist FIN improves diabetic nephropathy by suppressing SLC7A11-mediated ferroptosis, offering potential therapeutic targets and strategies for kidney disease management while providing insights into the mechanisms of clinical drugs. Diabetic Nephropathy、Nonsteroidal mineralocorticoid receptor antagonist、Finerenone、SLC7A11、ferroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction With the development of China’s economy and changes in residents' lifestyles, the incidence of diabetes, particularly type 2 diabetes, has continuously risen, becoming a major public health issue threatening the health of Chinese residents. According to data from the International Diabetes Federation in 2021, approximately 180 million adults in China have diabetes, with a prevalence rate of 12.8%, the highest in the world [ 1 ]. Diabetic nephropathy (DN) is one of the serious complications of diabetes, significantly affecting patients’ quality of life and imposing a substantial economic burden on society, posing a major challenge to China’s medical and healthcare system. Therefore, exploring new pathogenic mechanisms and optimizing treatment strategies to improve the prognosis of DN patients is urgently needed and has significant clinical and social implications. Ferroptosis is an iron-dependent programmed cell death characterized by lipid peroxidation and the accumulation of lipid reactive oxygen species. It is involved in the pathological processes of various kidney diseases, including DN, ultimately leading to kidney injury [ 2 ][ 3 ][ 4 ]. The Xc- system is a non-sodium antiporter that exports intracellular glutamate and imports extracellular cystine in a 1:1 ratio. It consists of two subunits linked by disulfide bonds: the heavy chain subunit solute carrier family 3 member 2 (SLC3A2) and the light chain subunit solute carrier family 7 member 11 (SLC7A11) [ 5 ][ 6 ]. SLC7A11 is a multi-channel transmembrane protein. Since cysteine is a precursor for glutathione (GSH) synthesis, and intracellular cysteine is primarily provided by cystine uptake mediated by SLC7A11, numerous studies have demonstrated the crucial role of SLC7A11-mediated cystine uptake in inhibiting ferroptosis under oxidative stress conditions and maintaining cell survival. Therefore, SLC7A11 is considered a key regulatory protein for ferroptosis [ 7 ]. GPX4, a member of the glutathione peroxidase family, was discovered in 1982 in pig liver and heart tissues [ 8 ]. Its main function is to utilize glutathione as a cofactor to counteract lipid peroxidation, a process triggered by reactive oxygen species (ROS) or free radicals that target cell membrane lipids. ROS-induced oxidation of polyunsaturated fatty acids (PUFAs) generates lipid hydroperoxides and other reactive intermediates, causing structural damage to the cell membrane and potential cell death. Dysregulation of GPX4 leads to ferroptosis [ 9 ]. Non-steroidal mineralocorticoid receptor antagonists (MRAs) can comprehensively block the gene expression of inflammation and fibrosis associated with excessive activation of the mineralocorticoid receptor (MR) [ 10 ][ 11 ]. Finerenone, a third-generation MRA, differs from the steroid structure of spironolactone and eplerenone by its unique non-steroidal structure, which ensures high receptor selectivity while significantly reducing the risk of adverse events associated with steroid MRAs [ 12 ][ 13 ]. Existing research has confirmed that finerenone can effectively block excessive MR activation, exerting anti-inflammatory and anti-fibrotic effects [ 14 ][ 15 ]. It has been shown to reduce the upregulation of inflammatory factors, improve glomerular sclerosis and hypertrophy, alleviate tubulointerstitial fibrosis, reduce macrophage infiltration, and lower endothelial dysfunction, thereby effectively reducing proteinuria and protecting renal function [ 16 ][ 17 ]. FIN and other MRAs have exhibited antioxidant, anti-inflammatory, and anti-fibrotic properties, and have reversed metabolic abnormalities in DN in previous studies [ 18 ]. Insulin resistance, commonly associated with MR activation in DT, is significantly linked to a decline in glomerular filtration rate (GFR). However, in diabetic nephropathy, whether finerenone can alleviate ferroptosis by regulating the SLC7A11 transporter, thereby exerting a protective effect on renal tubular injury, remains underexplored. In this study, we used a diabetic nephropathy mice model and in vitro cultured HK2 cells to explore through in vivo and in vitro experiments how finerenone exerts a protective effect on renal tubules in diabetic nephropathy by regulating the SLC7A11 and alleviating ferroptosis. 2. Methods 2.1 Patients and pathological samples Ten patients diagnosed with DN (aged 18–65 years) from the Department of Nephrology of the First Affiliated Hospital of Zhengzhou University were selected (It conforms to the 2017 edition of the Standards of Medical Care in Diabetes released by the American Diabetes Association, and is in line with the consensus on the diagnosis of DN issued by the American Diabetes Association and the National Kidney Foundation in 2014. It has been confirmed as DN through renal biopsy). Patients with tumors, other liver or kidney diseases, infections, fever, or autoimmune diseases were excluded. After signing the informed consent form, renal tissue samples were collected and clinical laboratory indicators such as blood glucose, serum creatinine, and quantitative urine protein were collected. Normal control renal tissue samples were obtained from the Department of Urology of the First Affiliated Hospital of Zhengzhou University from patients with normal blood glucose who underwent nephrectomy for renal tumors (6 cases). After signing the informed consent form, normal renal tissue 3–5 cm away from the tumor margin of the removed kidney was collected. 2.2 Animal studies db/db mice and db/dm mice were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. [SCXK (Zhe) 2019-0004]. db/db mice are type 2 diabetic nephropathy mice with leptin receptor point mutations and can exhibit obvious diabetic symptoms such as obesity and hyperglycemia at 6 weeks of age. All mice were handled in accordance with the approved protocol by the laboratory and were approved by the Animal Ethics Committee of Zhengzhou University. The purchased mice were 8-week-old male mice, randomly divided into 4 groups, with 8 mice in each group. ① Normal control group: 8-week-old db/dm mice, fed a normal diet, and renal tissue samples were collected at 24 weeks. ②DN model group: 8-week-old db/db mice, fed a high-fat diet until 10 weeks of age, and then received two consecutive intraperitoneal injections of streptozotocin (70 mg/Kg) at 10 weeks of age. Renal tissue samples were collected at 24 weeks.③④Finerenone treatment group: 8-week-old db/db mice, fed until 10 weeks of age and then given finerenone by gavage at doses of 10 mg/Kg/d and 30 mg/Kg/d, divided into 2 groups, with 8 mice in each group.After sacrificing the mice, the kidneys were collected and stored at suitable storage conditions for further experiments。 2.3 Cell culture HK-2 (human renal cortical proximal tubular epithelial cells) culture: Cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher), 100 U/mL penicillin and 100µg/mL streptomycin at 37℃, 5% CO 2 and 95% humidity. All cells were used between passages 5 and 15. The medium was changed every 48 hours, and serum-free medium was used when the cells reached 70% confluence. The cells were treated as follows: 5 mM glucose (Con), 30 mM D-glucose (HG), HG + 1µM finerenone and HG + 3µM finerenone. The SLC7A11 interfering plasmid (shSLC7A11) was transfected into cells 24 hours before HG treatment using lipofectamine 3000 transfection reagent, and then the cells were collected or stained 24 hours later for subsequent experiments. 2.4 Immunohistochemistry, IHC De-waxing, antigen retrieval, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, drop freshly prepared DAB chromogen solution in the immunohistochemistry circle, place under the microscope for observation, quickly rinse after positive reaction to terminate staining. Hematoxylin re-stain the cell nucleus, dehydration, drop neutral gum on the dehydrated section, cover with a coverslip and seal, place in a fume hood to dry. Photography and analysis: Observe and collect pictures under an upright fluorescence microscope. 2.5 Hematoxylin-Eosin Dyeing De-waxing, hematoxylin staining of nuclei, differentiation and bluing, eosin staining, dehydration: Place the slides in an oven at 68℃ for 2 minutes, then immerse them in xylene I and xylene II for 2 minutes each, and air dry. Seal the slides with neutral gum. Observe under an optical microscope and take pictures. 2.6 Masson Dyeing De-waxing, nuclear staining with hematoxylin solution, soaking in Masson's acid fuchsin solution for 10 minutes, washing with 2% glacial acetic acid for 5 minutes, differentiation with 1% phosphomolybdic acid solution for 5 minutes, staining with aniline blue for 5 minutes, and washing with 0.2% glacial acetic acid solution for 5 minutes. Dehydration: successively place the sections inanhydrous ethanol I for 5 minutes-anhydrous ethanol II for 5 minutes-xylene I for 5 minutes - xylene II for 5 minutes, transfer to a fume hood to dry, and seal with neutral gum. Observe and collect images using an upright fluorescence microscope. 2.7 Transient transfection of SLC7A11 shRNA plasmid The shRNA sequence targeting SLC7A11 was synthesized and annealed with the eukaryotic expression vector containing the U6 promoter. Transfection: ①Dissolve 0.8µg of plasmid in 50µL of Opti-MEM serum-free medium ② Dissolve 2µL of lipo3000 in 50µL of Opti-MEM serum-free medium, mix well and let stand at room temperature for 5 minutes③Mix tubes A and B and let stand for 20 minutes. Replace the medium in the 24-well plate with serum-free medium, 400µL/well. Add the mix in tube C to the corresponding wells of the 24-well plate. After 4–6 hours, replace with serum-containing medium. 2.8 Cell viability assay The viability of cells was evaluated using the Cell Counting Kit-8 (CCK-8) assay (Beyotime Biotechnology, China). Following transfection, cells were seeded into 96-well plates at a density of 3000 cells per well. Absorbance values were recorded at various time points ranging from 0 to 96 hours post-transfection. A solution consisting of 100 µL RPMI 1640 medium with 10 µL of CCK-8 reagent was added to each well, followed by a 2-hour incubation period. Subsequently, the absorbance at 450 nm was measured using a microplate reader (Bio-Rad, USA). To ensure reliability, all experiments were performed in three replicate wells and repeated three times. 2.9 GSH assay The measurement of glutathione (GSH) levels was carried out using the DTNB (5,5′-Dithio-bis-(2-nitrobenzoic acid)) method (Solarbio, BC1170), with distinct pre-treatment protocols for tissue and cell samples. For tissue samples, tissues were homogenized on ice at a ratio of 1:10 (grams of tissue to milliliters of reagent) using a pre-chilled homogenizer. The homogenates were centrifuged at 8000 g for 10 minutes at 4°C, and the supernatant was carefully collected and stored at 4°C until analysis. For cell samples, cells were cultured in 100 mm dishes at a density of 3 million cells per dish and treated according to the experimental groups. After treatment, the cells were detached using trypsinization, counted, and 5 million cells were resuspended in 1 mL of assay buffer. To ensure complete lysis, the suspension underwent 2–3 freeze-thaw cycles (freezing in liquid nitrogen and thawing in a 37°C water bath). Following the final thaw, the suspension was centrifuged at 8000 g for 10 minutes, and the supernatant was collected and kept on ice for subsequent steps. After sample collection, proteinase purification was performed. The sample was then mixed with DTNB solution in a 96-well plate, and the reaction was initiated by adding NADPH solution. After incubation for 20 minutes, the GSH concentration was determined at a wavelength of 412 nm using a SpectraMax M2e spectrophotometer (Beyotime Biotechnology, China). 2.10 MDA assay The Lipid Peroxidation MDA Assay was performed using the MDA Assay Kit (Beyotime Biotechnology, China). Cells were cultured in 60 mm culture dishes at a density of 800,000 cells per dish and treated according to their assigned groups. After treatment, the cells were harvested through trypsinization, counted, and a total of 3 million cells were resuspended in 0.3 mL of lysis buffer. Following lysis, the suspension was centrifuged at 10,000–12,000 g for 10 minutes to obtain a clear supernatant. If the supernatant was not sufficiently clear after centrifugation, a 0.2 µm filter was used to further clarify the sample. All steps of sample preparation were carried out on ice or at 4°C. After pre-treatment, the supernatants were processed following the manufacturer's instructions. The samples were heated in a boiling water bath for 15 minutes, cooled to room temperature, and then centrifuged at 1000 g for 10 minutes. The absorbance of the supernatant was measured at 532 nm using an enzyme-linked immunosorbent assay reader. This absorbance value was correlated with the MDA concentration, which was determined based on a standard curve. 2.11 Lipid peroxidation assay For the lipid peroxidation analysis, cells were seeded into 12-well plates at a density of 80,000 cells per well and treated according to their designated groups. After treatment, the cells were detached using trypsinization, collected by centrifugation at 300g for 5 minutes, and washed three times with PBS. The cell pellet was then incubated with BODIPY™ 581/591C11 dye at a final concentration of 5 µM in a 37°C incubator with 5% CO 2 for 30 minutes. Following incubation, any unbound fluorescent dye was removed by gently washing the cells three times with PBS. The cell suspension was subsequently transferred to flow cytometry tubes for further analysis. Samples were analyzed using a flow cytometer, and the resulting data were processed using FlowJo software. Cell subpopulations were distinguished based on forward scatter (FSC) and side scatter (SSC) characteristics, while the percentage of the positive subpopulation was determined through a single-peak histogram of FL1 fluorescence intensity. 2.12 Abcam Iron assay Take the standard protection agent and add 20 mL of buffer solution, mix well and store at 2–8℃. Take 20µL of 10 mmol/L iron standard solution and mix it evenly with 1980µL of standard protection solution. Determination of ferrous ion concentration: ①Standard wells: Take 200µL of different concentration standards and add them to the corresponding wells of the microplate. Measuring wells: Take 200µL of sample and add it to the corresponding wells of the microplate. ② Add 100µL of chromogenic solution to each well, mix well, and incubate at 37℃ for 10 minutes. Measure the OD value of each well at 593 nm on the microplate reader. Calculate the ferrous ion concentration in the cells according to the standard curve formula. 2.13 EdU-555 cell proliferation assay Under the fluorescence microscope, red fluorescence (DNA molecules in the replication state) and blue fluorescence (cell nuclei) can be observed. When the two are combined, they present purple spot fluorescence. Photos are taken and the images are saved. The Image J software is used tocalculate the proportion of proliferating cells (red fluorescence) to the total cells (blue fluorescence), and statistical analysis is conducted. 2.14 Western blotting analysis Murine renal tissue or cultured cells was homogenized and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with phosphatase inhibitor (Proteintech, China) and protease inhibitor (Proteintech, China). The samples were blotted and tagged with relevant antibodies as previously reported. The antibodies against COL1 and TGF-β,FTH1, SLC7A11and GPX4 were purchased from Proteintech.The protein band was quantified using Image J software. 2.15 Immunofluorescence The HK-2 cells were treated with adenovirus expressing GFP-LC3.The samples were fixed with 4% paraformaldehyde, permeated with methanol/acetone, and infected with TOM20 antibody (1:200, Abcam, ab186734, United States). The samples were then observed under a fluorescence confocal microscope (LSM510 META, Karl Zeiss, Germany). 2.16 Real-time quantitative PCR (RT‒qPCR) After RNA extraction, the concentration and purity of RNA were determined using an ultramicro UV spectrophotometer and recorded. The RNA solution was stored at -80℃. RNA was reverse transcribed into cDNA and amplified using a real-time fluorescence quantitative PCR instrument. Result analysis: GAPDH was used as the internal reference, and the 2 -ΔΔCT method was used for data calculation. Three replicate wells were set for each gene, and each group was repeated at least three times. 2.17 Statistical analysis Statistical analysis was performed using GraphPad Prism 8.0 software. Data were obtained from three or more independent repeated experiments. Two-tailed unpaired Student's t-test was used for comparison between two groups, and one-way ANOVA and Bonferroni test were used for comparison among three or more groups. p < 0.05 was considered statistically significant. 3. Results 3.1 Investigate the expression levels of fibrosis-related markers in the renal tubules of patients with DN and healthy controls To explore the changes of fibrosis-related indicators under the high-glucose environment, we conducted immunohistochemical staining on renal tissue specimens from healthy individuals and DN patients, and detected the indicators related to renal tubules. The results showed that compared with normal renal tissue, the expressions of indicators reflecting fibrosis such as N-cadherin, Fibronectin and α-SMA increased in renal tubules of DN patients, while the expression of E-cadherin decreased. This suggested that the degree of renal tubule fibrosis in DN patients was aggravated (Fig. 1 ). As demonstrated by these outcomes, compared with healthy renal tissue, the degree of renal tubule fibrosis in DN patients is aggravated. 3.2 Evaluating the effects of finerenone on renal tubular atrophy, interstitial inflammatory cell infiltration, and fibrosis in db/db mice. In order to observe the effects of finerenone on renal tubular injury, interstitial inflammatory cell infiltration and fibrosis in the kidneys of db/db mice, we performed HE and Masson staining on the db/dm, db/db and db/db་finerenone groups. Compared with the db/dm group, the renal tubules of db/db mice were atrophied, and the interstitial inflammatory cell infiltration and fibrosis were aggravated; compared with the db/db group, the renal tubules of the db/db་10mg/Kg/d finerenone group and the db/db་30mg/Kg/d finerenone group were atrophied, and the interstitial fibrosis and inflammatory cell infiltration were alleviated (Fig. 2 A).The results of Western Blot experiment showed that compared with the db/dm group, the expression of COL1 and TGF-βrelated to fibrosis in the db/db group was upregulated, suggesting an aggravated degree of fibrosis; compared with the db/db group, the expression of COL1 and TGF-βin the db/db་10mg/Kg/d finerenone group and the db/db་30mg/Kg/d finerenone group was downregulated, suggesting a reduced degree of fibrosis (Fig. 2 B), suggesting that finerenone can alleviate renal tubular atrophy, interstitial inflammatory cell infiltration and fibrosis in db/db mice. 3.3 Exploring the role of finerenone in ferroptosis of renal tissue in db/db mice To further explore the effect of finerenone on the degree of ferroptosis in the kidneys of db/db mice, we detected the expression levels of ferroptosis-related protein SLC7A11 and the ferroptosis-related indicators GSH and MDA in the renal tissues of db/dm, db/db and db/db + finerenone groups of mice.The Western Blot results showed that compared with the db/db group of mice, the expression of ferroptosis-related protein SLC7A11 was increased in the db/db + 10mg/kg/d finerenone group and the db/db + 30mg/kg/d finerenone group of mice, suggesting a reduction in the degree of ferroptosis (Figs. 3 A and 3 B).The levels of GSH and MDA were detected by ELISA. Compared with the db/db group of mice, the levels of GSH, a ferroptosis-related indicator, were increased and the levels of MDA were decreased in the db/db + 10mg/kg/d finerenone group and the db/db + 30mg/kg/d finerenone group of mice, suggesting a reduction in the degree of ferroptosis (Figs. 3 C and 3 D).The above results indicate that finerenone can reduce ferroptosis in the renal tissues of db/db mice. 3.4 Finerenone alleviate high glucose-induced injury to renal tubular epithelial cells. To observe the protective effect of finerenone on renal tubular epithelial cells in a high glucose environment, human proximal tubular epithelial cells HK2 were cultured in vitro. The groups were set as NG group, HG group and HG + finerenone groups with different concentrations (0.1µM, 0.3 µM, 1µM, 3µM, 10µM and 30µM). HK2 cells were treated for 48 hours, and the cell damage and the effect of finerenone on the fibrosis indicators of HK2 cells were detected.The CCK-8 results showed that compared with the NG group, the cell viability of HK2 cells in the HG group was weakened and the number of EdU-labeled positive cells decreased. When different concentrations of finerenone were given to the HG group, the cell viability of HK2 gradually recovered and the number ofEdU-labeled positive cells gradually increased with the increase of finerenone concentration. After reaching a certain concentration, the ability to restore cell viability decreased. The concentrations of 1µM and 3µM were most conducive to cell growth (Fig. 4 A).Laser confocal microscopy showed that compared with the NG group, the intensity of EdU-labeled and DAPI-stained HK2 cells in the HG group decreased, indicating a decline in cell viability. Compared with the HG group, the number of EdU-labeled positive cells in the HG + finerenone groups with different concentrations (1µM and 3µM) gradually increased, and the cell proliferation ability also gradually enhanced (Fig. 4 B, Fig. 4 C). The Western Blot results showed that compared with the NG group, the expression levels of COL1 and TGF-β in HK-2 cells in the HG group significantly increased, indicating an aggravation of fibrosis. Compared with the HG group, the expression levels of COL1 and TGF-βin the HG + finerenone groups with different concentrations (1µM and 3µM) significantly decreased, and the degree of fibrosis was alleviated (Fig. 4 D, Fig. 4 E).QRT-PCR showed that compared with the NG group, the expression of COL1 mRNA and TGF-βmRNA in HK-2 cells in the HG group was significantly upregulated, indicating an aggravation of fibrosis. Compared with the HG group, the expression of COL1mRNA and TGF-βmRNA in the HG + finerenone groups with different concentrations (1µM and 3µM) decreased, and the degree of fibrosis was alleviated (Fig. 4 F, Fig. 4 G).The above results indicate that finerenone can enhance the viability of HK2 cells, promote cell proliferation, and alleviate the degree of fibrosis and cell damage in a high glucose environment. 3.5 Exploring the effect of finerenone on ferroptosis of renal tubular epithelial cells induced by high glucose The NG group, HG group and HG + finerenone groups at different concentrations (1µM and 3 µM) were respectively set up to treat HK2 cells for 48 hours, and the effects of finerenone on the level of HIF-1α and the degree of ferroptosis in HK2 cells under high glucose conditions were observed. The BODIPY 581/591 C11 lipid peroxidation probe was used to detect the intensity of lipid peroxidation related to ferroptosis. Compared with the NG group, the lipid peroxidation in the HG group was significantly enhanced, suggesting an increased degree of ferroptosis. Compared with the HG group, the lipid peroxidation intensity in the HG + finerenone groups at different concentrations (1µM and 3µM) decreased, indicating a reduced degree of ferroptosis (Figs. 5 A and 5 B). The levels of ferroptosis-related indicators GSH and MDA in each group were detected by ELISA. Compared with the NG group, the GSH level in the HG group decreased while the MDA level increased, suggesting an aggravated degree of ferroptosis; compared with the HG group, the GSH level in the HG + finerenone groups at different concentrations (1µM and 3µM) increased while the MDA level decreased, indicating a reduced degree of ferroptosis (Figs. 5 C and 5 D). The Western Blot results showed that compared with the NG group, the levels of ferroptosis-related proteins FTH1, SLC7A11, and GPX4 in the HG group decreased, suggesting an aggravated degree of ferroptosis; compared with the HG group, the levels of FTH1, SLC7A11, and GPX4 in the HG + finerenone groups at different concentrations (1µM and 3µM) increased, indicating a reduced degree of ferroptosis (Figs. 5 E and 5 G). The Fe 2+ concentration in each group was detected by colorimetry. The results showed that compared with the NG group, the Fe 2+ level in the HG group increased; compared with the HG group, the Fe 2+ level in the HG + finerenone groups at different concentrations (1µM and 3µM) decreased, indicating a reduced degree of ferroptosis (Fig. 5 F). The above results indicate that finerenone can alleviate ferroptosis in renal tubular epithelial cells under high glucose conditions. 3.6 Silencing SLC7A11 of HK2 cells in high-glucose environment exacerbating renal tubular epithelial cell injury and counteract the protective effect of finerenone SLC7A11 is considered a key regulatory protein in ferroptosis and plays an important role in regulating ferroptosis. To investigate the role of SLC7A11 in nonenalone-protected HK-2 cells under high glucose conditions, we silenced SLC7A11 in HK-2 cells and divided them into shNC + vehicle group, shSLC7A11 + vehicle group, shNC + Fine group, and shSLC7A11 + Fine group. We then examined the cell count, cell viability, and cell damage in each group. The CCK-8 results showed that compared with the shNC + vehicle group, the cell viability in the shSLC7A11 + vehicle group decreased; compared with the shNC + Fine group, the cell viability in the shSLC7A11 + Fine group decreased, and the differences were statistically significant. This suggests that after nonenalone treatment, cell damage was reduced, but after silencing SLC7A11, cell viability decreased, and the protective effect of nonenalone on cells was also counteracted (Fig. 6 A).The QRT-PCR results indicated that compared with the shNC + vehicle group, the expression of COL1 mRNA and TGF-β mRNA in the shSLC7A11 + vehicle group increased; compared with the shNC + Fine group, the expression of COL1mRNA and TGF-βmRNA in the shSLC7A11 + Fine group increased. This suggests that after silencing SLC7A11 in HK-2 cells, the indicators of cell fibrosis increased, and cell damage worsened (Fig. 6 B).Laser confocal microscopy showed that compared with the shNC + vehicle group, the intensity of EdU-labeled and DAPI-stained cells in the shSLC7A11 + vehicle group decreased, indicating a decrease in cell viability; compared with the shNC + Fine group, the intensity of EdU-labeled and DAPI-stained cells in the shSLC7A11 + Fine group decreased, indicating a decrease in cell viability and a reduction in cell proliferation ability (Fig. 6 C).The Western Blot results showed that compared with the shNC + vehicle group, the expression levels of COL1 and TGF-β in the shSLC7A11 + vehicle group significantly increased, suggesting an aggravation of fibrosis; compared with the shNC + Fine group, the expression levels of COL1 and TGF-β in the shSLC7A11 + Fine group significantly increased, suggesting an aggravation of fibrosis (Fig. 6 D).These results suggest that under high glucose conditions, regardless of whether nonenalone intervention is given or not, silencing the SLC7A11 leads to a decrease in HK2 cell viability, weakened cell proliferation, and enhanced expression of fibrosis-related genes. 3.7 Silencing SLC7A11 in a high-glucose environment exacerbating ferroptosis in renal tubular epithelial cells and counteract the protective effect of finerenone To explore the effect of SLC7A11 on ferroptosis of renal tubular epithelial cells under high glucose conditions, we silenced the SLC7A11 in HK-2 cells and divided them into shNC + vehicle group, shSLC7A11 + vehicle group, shNC + Fine group, and shSLC7A11 + Fine group, and then detected the ferroptosis-related indicators of each group. The Fe 2+ concentration of each group was detected by colorimetry. The results showed that compared with the shNC + vehicle group, the Fe 2+ concentration in the shSLC7A11 + vehicle group increased; compared with the shNC + Fine group, the Fe 2+ concentration in the shSLC7A11 + Fine group increased, suggesting that the intracellular ferrous ion concentration was significantly reduced after finerenone treatment, but the knockdown of SLC7A11 in cells cancelled this protective effect (Fig. 7 A). The intensity of ferroptosis-related lipid peroxidation was detected by BODIPY 581/591 C11 lipid peroxidation probe. The results showed that compared with the shNC + vehicle group, lipid peroxidation in the shSLC7A11 + vehicle group was significantly enhanced, suggesting that the degree of ferroptosis was aggravated; compared with the shNC + Fine group, lipid peroxidation in the shSLC7A11 + Fine group was significantly enhanced, suggesting that the degree of ferroptosis was aggravated (Fig. 7 B). The levels of ferroptosis-related indicators GSH and MDA in each group were detected by ELISA. The results showed that compared with the shNC + vehicle group, the GSH level in the shSLC7A11 + vehicle group decreased, while the MDA level increased, suggesting that ferroptosis was aggravated; compared with the shNC + Fine group, the GSH level in the shSLC7A11 + Fine group decreased, while the MDA level increased, suggesting that ferroptosis was aggravated (Fig. 7 C, Fig. 7 D).These results suggest that under high glucose conditions, Silencing SLC7A11 can exacerbate ferroptosis in renal tubular epithelial cells and counteract the protective effect of finerenone. 4. Discussion DN is a chronic disease affecting millions of diabetic patients worldwide. Early diagnosis and prevention are crucial for treating DN, as renal damage becomes difficult to reverse once patients reach the clinical proteinuria stage. Therefore, the development of new treatment methods is of vital importance [ 19 ]. The disease mechanism of DN is complex, with hyperglycemia considered the initiating factor for diabetes-related nephropathy. As the disease progresses, metabolic disorders, hemodynamic abnormalities, inflammation, and fibrosis contribute to the pathological process, leading to structural and functional damage of the kidneys and ultimately resulting in adverse cardiovascular and renal outcomes. Among these, inflammation and fibrosis play a key role in the occurrence and progression of diabetes-related renal injury [ 20 ]. Ultrastructural glomerular damage and proteinuria have long been central to research on the pathogenesis of DN [ 21 ]. However, contemporary studies have increasingly focused on renal tubular changes in DN, as clinical signs of renal impairment are closely linked to tubulointerstitial fibrosis and tubular atrophy [ 22 ][ 23 ]. Developing efficient therapies targeting the specific pathophysiological changes in renal tubules remains a significant challenge [ 24 ]. The mineralocorticoid receptor (MR) is a transcription factor that binds to ligands and activates signaling cascades, regulating processes such as water and salt balance, inflammation, fibrosis, apoptosis, and endothelial function [ 25 ]. Current evidence suggests that MR activation may slow the progression of renal dysfunction in patients with chronic kidney disease (CKD) and type 2 diabetes, as well as effectively reduce proteinuria in individuals with non-diabetic CKD [ 26 ][ 27 ][ 28 ]. A study confirmed that finerenone could be an effective treatment option for immunoglobulin A nephropathy (IgAN) patients without diabetes who are receiving conventional treatment, maintaining stable renal function and serum potassium levels [ 29 ]. MR activation can negatively affect various cell types, including renal tubular epithelial cells, vascular smooth muscle cells, endothelial cells, adipocytes, and inflammatory cells, leading to serious consequences for both the kidneys and cardiovascular system. A recent single-cell sequencing study found that MR expression in the proximal convoluted tubular epithelial cells of diabetic patients was increased compared to the control group [ 30 ]. In addition to inducing changes in gene expression, aldosterone has non-genomic effects in renal tubular epithelial cells, activating protein kinases and second messenger signals. The binding of aldosterone to its receptor also induces oxidative stress, inflammation, and fibrosis. A study explored the mechanism of action of finerenone in HK-2 cells and the kidneys of DN mice under high glucose conditions [ 31 ]. The research found that finerenone restored mitochondrial autophagy in HK-2 cells and renal tubular cells of DN mice under high glucose conditions through the PI3K/Akt/eNOS signaling pathway, while reducing oxidative stress, mitochondrial fragmentation, and apoptosis [ 32 ]. These findings highlight the key role of finerenone in improving renal tubular cell injury. Ferroptosis, caused by the excessive accumulation of lipid hydroperoxides in an iron-dependent manner, was also explored [ 33 ]. xCT/SLC7A11 is a 12-pass transmembrane protein responsible for the primary transport activity, highly specific for cystine and glutamate, while SLC3A2 is a single-pass transmembrane protein with a glycosylated extracellular domain at its C-terminus. SLC3A2 functions mainly as a chaperone protein and is crucial for regulating the stability and transport function of xCT/SLC7A11 [ 34 ]. xCT/SLC7A11 promotes the production of reduced GSH and acts as a cofactor for ROS detoxification enzymes (such as GPX4), converting accumulated lipid hydroperoxides into lipid alcohols. This reduces peroxide-related products and protects cells from ROS-induced damage, inhibiting ferroptosis. Through the GPX4-mediated reaction, GSH is oxidized to its oxidized form (GSSG) and is recycled back to GSH at the expense of NADPH by glutathione reductase (GR) [ 35 ]. Iron-dependent programmed cell death, known as ferroptosis, is marked by the accumulation of lipid peroxides [ 36 ]. This phenomenon is regulated by System Xc −, a cystine/glutamate antiporter in the cell membrane, consisting of two components: SLC7A11, which transports cystine and glutamate, and SLC3A2, which ensures the stability of SLC7A11 [ 37 ]. Acting as the central component of System Xc −, SLC7A11 supports cellular redox equilibrium by enabling cystine uptake for the synthesis of glutathione (GSH), an essential antioxidant that inhibits lipid peroxidation. This action decreases lipid peroxide concentrations and safeguards cells against ferroptosis [ 38 ]. The activity and regulation of SLC7A11, primarily located in the plasma membrane, are shaped by its distinct membrane positioning [ 39 ]. In other diseases, such as hepatocellular carcinoma, Parkinson's disease, and endometriosis, regulating SLC7A11-mediated ferroptosis has shown therapeutic potential [ 40 ][ 41 ][ 42 ]. However, the exact processes governing its movement and placement on the cell membrane still require further investigation. Since ferroptosis plays a role in the inflammatory and oxidative pathological processes of various diseases, many studies have focused on its involvement in diabetic complications, including DN. It has been found that maintaining iron metabolism balance in renal cells is crucial for normal renal function [ 43 ]. Wu et al. [ 44 ] observed enhanced ferroptosis in DN, as indicated by elevated expression of ACSL4, PTGS2, and NOX1, and decreased expression of GPX4. Feng et al. [ 45 ] reported that compared with db/m mice, MDA levels were elevated, while those of SOD, CAT, and GSH were decreased in the kidneys of db/db mice. After administering Ferrostatin-1, MDA levels were reduced, SOD levels decreased, CAT levels were lowered, and GSH levels were increased in db/db mice. Renal injury, fibrosis, and lipid protein levels in the urine of db/db mice were also alleviated. The results of our experiments in mice showed that compared with db/db mice, the degree of renal tubular atrophy, interstitial fibrosis and inflammatory cell infiltration was reduced in db/db mice treated with 10 mg/kg/d finerenone and db/db mice treated with 30 mg/kg/d finerenone. The levels of ferroptosis-related protein SLC7A11 increased, GSH levels increased and MDA levels decreased, suggesting that the degree of ferroptosis was reduced. After finerenone intervention in HK2 cells under high glucose conditions, the levels of ferroptosis-related proteins FTH1, SLC7A11 and GPX4 increased, the degree of lipid peroxidation decreased, GSH increased and MDA decreased, suggesting that the degree of ferroptosis was reduced. After silencing the SLC7A11, regardless of whether finerenone intervention was given or not, the viability of HK2 cells decreased, cell proliferation weakened, the expression of fibrosis-related genes increased, and the indicators of ferroptosis also worsened(Fig. 8 ). In conclusion, our study identified finerenone ameliorates Diabetic Nephropathy via suppressing SLC7A11-mediated ferroptosis.This finding can deepen our understanding of the pathogenesis of renal tubular injury in diabetic nephropathy and provide a certain theoretical basis for future exploration of new treatment methods. This could provide deeper insights into the complexity of inflammation and help develop more targeted therapeutic strategies. Future studies should focus on integrating these pathways for a better understanding of finereone treatment in DN. Declarations Ethics approval and consent to participate The research study was authorized by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University and adhered to the principles of the Declaration of Helsinki. Consent for publication Not applicable. Competing interests The authors declare no conflict of interest. Funding This study was funded by National Natural Science Foundation of China(82370727) Author Contribution Authors' contributionsLu Yu: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Project administration, Methodology, Investigation, Formal analysis, Data curation. Zihan Zhai: Validation, Resources, Project administration, Methodology, Funding acquisition. Yulin Wang: Validation, Project administration, Methodology, Formalanalysis, Data curation, Conceptualization. Liuwei Wang: Validation, Project administration, Investigation, Formal analysis, Data curation. Qiuhong Li: Validation, Project administration, Methodology. Yanhong Guo: Validation, Project administration, Methodology. Rong Gou: Project administration, Methodology. Lin Tang: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization Acknowledgement Acknowledgements The authors thank The First Affiliated Hospital of Zhengzhou University and Research Institute of Nephrology, Zhengzhou University for their assistance and support. Availability of data and materials All data will be made available upon request. References Wang L, Peng W, Zhao Z, Zhang M, Shi Z, Song Z, Zhang X, Li C, Huang Z, Sun X et al (2021) Prevalence and Treatment of Diabetes in China, 2013–2018. JAMA 326:2498–2506 [CrossRef] [PubMed] Tang D, Chen X, Kang R et al (2021) Ferroptosis: molecular mechanisms and health implications. Cell Res 31(2):107–125 Lei G, Zhuang L, Gan B (2022) Targeting ferroptosis as a vulnerability in cancer. Nat Rev Cancer 22(7):381–396 Jiang X, Stockwell BR, Conrad M (2021) Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol 22(4):266–282 Chen X, Kang R, Kroemer G et al (2021) Broadening horizons: the role of ferroptosis in cancer. Nat Rev Clin Oncol 18(5):280–296 Koppula P, Zhuang L, Gan B (2021) Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy. Protein Cell 12(8):599–620 Koppula P, Zhuang L, Gan B (2021) Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy. Protein Cell 12(8):599–620 doi: 10. 1007/s13238 -020-00789-5. Epub 2020 Oct 1. PMID: 33000412 Jiao L, Daolin T (2024) Rui, Kang,Targeting GPX4 in ferroptosis and cancer: chemical strategies and challenges[. J] Trends Pharmacol Sci 45:0 Liang Z, Guangyu L, Tao Z et al (2025) Palmitoylation of GPX4 via the targetable ZDHHC8 determines ferroptosis sensitivity and antitumor immunity[. J] Nat Cancer 0:0 Barrera-Chimal J, Lima-Posada I, Bakris GL, Jaisser F (2022) Mineralocorticoid receptor antagonists in diabetic kidney disease - mechanistic and therapeutic effects. Nat Rev Nephrol 18:56–70. https://doi.org/10.1038/s41581-021-00490-8 Barrera-Chimal J, Girerd S, Jaisser F (2019) Mineralocorticoid receptor antagonists and kidney diseases: pathophysiological basis. Kidney Int 96:302–319. https://doi.org/10.1016/j.kint.2019.02.030 Nakagawa T (2010) Diabetic nephropathy: aldosterone breakthrough in patients on an ACEI. Nat Rev Nephrol 6:194–196. https://doi.org/10.1038/nrneph.2010.32 Kawanami D et al (2021) Mineralocorticoid receptor antagonists in diabetic kidney disease. Front Pharmacol 12:754239. https://doi.org/10.3389/fphar.2021.754239 Bakris GL et al (2020) Effect of finerenone on chronic kidney disease outcomes in type 2 diabetes. N Engl J Med 383:2219–2229. https://doi.org/10.1056/NEJMoa2025845 Filippatos G et al (2021) Finerenone and cardiovascular outcomes in patients with chronic kidney disease and type 2 diabetes. Circulation ygh 143:540–552. https://doi.org/10.1161/circulationaha.120.051898 Rossing P et al (2022) Finerenone in Patients With Chronic Kidney Disease and Type 2 Diabetes According to Baseline HbA1c and Insulin Use: An Analysis From the FIDELIO-DN Study. Diabetes Care 45(4):888–897 Pitt B, Filippatos G, Agarwal R, Anker SD, Bakris GL, Rossing P, Joseph A, Kolkhof P, Nowack C, Schloemer P, Ruilope LM (2021) FIGARO-DKD Investigators. Cardiovascular Events with Finerenone in Kidney Disease and Type 2 Diabetes. N Engl J Med 385(24):2252–2263. 10.1056/NEJMoa2110956 Epub 2021 Aug 28. PMID: 34449181 Zachariah T, Radhakrishnan J (2024) Potential Role of Mineralocorticoid Receptor Antagonists in Nondiabetic Chronic Kidney Disease and Glomerular Disease. Clinical journal of the American Society of Nephrology: CJASN, Wheeler DC et al (2021) Effects of dapagliflozin on major adverse kidney and cardiovascular events in patients with diabetic and non-diabetic chronic kidney disease: a prespecified analysis from the DAPA-CKD trial. The lancet. Diabetes Endocrinol 9(1):22–31 Sawaf H, Thomas G, Taliercio JJN, GeorgesVachharajani, Tushar JM, Ali (2022) Therapeutic Adv Diabet Nephrop J Clin Med, 11(2) Stefansson VTN et al (2022) Molecular programs associated with glomerular hyperfiltration in early diabetic kidney disease. Kidney Int 102:1345–1358 Yao L et al (2022) Mitochondrial dysfunction in diabetic tubulopathy. Metab Clin Exp 131:155195 Ma Z et al (2020) p53/microRNA-214/ULK1 axis impairs renal tubular autophagy in diabetic kidney disease. J Clin Invest 130:5011–5026 Li A et al (2022) Vitamin D-VDR (vitamin D receptor) regulates defective autophagy in renal tubular epithelial cell in streptozotocin-induced diabetic mice via the AMPK pathway. Autophagy 18:877–890 Barrera-Chimal J, Girerd S, Jaisser F (2019) Mineralocorticoid receptor antagonists and kidney diseases: pathophysiological basis. Kidney Int 96:302–319. https:// doi.org/10.1016/j.kint.2019.02.030 Maryam A, Nadeem MS, Fatima A, Asmat KN (2024) Exploring the potential of fnerenone in non-diabetic chronic kidney disease: a promising frontier. Int Urol Nephrol Mårup FH, Thomsen MB, Birn H (2024) Additive efects of dapaglifozin and fnerenone on albuminuria in non-diabetic CKD: an open-label randomized clinical trial. Clin Kidney J Bakris GL, Agarwal R, Anker SD, Pitt B, Ruilope LM, Rossing P, Kolkhof P, Nowack C, Schloemer P, Joseph A, Filippatos G, FIDELIO-DKD Investigators (2020) Efect of fnerenone on chronic kidney disease outcomes in type 2 diabetes. N Engl J Med 383:2219–2229 Chen TF-L, Pei S (2025) Effectiveness and safety of finerenone in non-diabetic patients with IgA nephropathy[. J] J Nephrol 0:0 Wang P (2023) Non-Steroidal Mineralocorticoid Receptor Antagonists (MRAs)-Finerenone Ameliorates Mitochondrial Dysfunction via PI3K/Akt Signaling Pathway in Diabetic Tubulopathy. J Am Soc Nephrol 34(11S):840–841 Zi-Han LZ-J, Sun,Sydney CW, Tang et al (2025) Finerenone Alleviates Over-Activation of Complement C5a-C5aR1 Axis of Macrophages by Regulating G Protein Subunit Alpha i2 to. Improve Diabet Nephrop [J] Cells 14:0 Zhixi L, Yue B, Cheng W et al (2025) Extracellular vesicle-packaged GBP2 from macrophages aggravates sepsis-induced acute lung injury by promoting ferroptosis in pulmonary vascular endothelial cells[. J] Redox Biol 82:0 Dixon SJ et al (2012) Ferroptosis: An iron-dependent form of nonapoptotic cell death. Cell 149:1060–1072 Yang WS et al (2014) Regulation of ferroptotic cancer cell death by GPX4. Cell 156:317–331 Jiang X, Stockwell BR, Conrad M (2021) Ferroptosis: Mechanisms, biology and role in disease. Nat Rev Mol Cell Biol 22:266–282 Lei G, Zhuang L, Gan B (2022) Targeting ferroptosis as a vulnerability in cancer. Nat Rev Cancer 22(7):381–396 Luo Y, Xiang W, Liu Z et al (2022) Functional role of the SLC7A11-AS1/xCT axis in the development of gastric cancer cisplatin-resistance by a GSH-dependent mechanism, Free Radic. Biol Med 184:53–65 Ishimoto T, Nagano O, Yae T et al (2011) CD44 variant regulates redox status in cancer cells by stabilizing the xCT subunit of system xc(-) and thereby promotes tumor growth. Cancer Cell 19(3):387–400 Tsuchihashi K, Okazaki S, Ohmura M et al (2016) The EGF receptor promotes the malignant potential of Glioma by regulating amino acid transport system xc(-). Cancer Res 76(10):2954–2963 Qiong W, Zongwen L, Jing J et al (2025) Correction: Macrophages originated IL-33/ST2 inhibits ferroptosis in endometriosis via the ATF3/SLC7A11 axis.[J]. Cell Death Dis 16:0 Zhe Y, Jing L, Wen A et al (2025) EOGT knockdown promotes ferroptosis and inhibits hepatocellular carcinoma proliferation by regulating SLC7A11 via HEY1.[J]. Cell Signal 0:0 Jinghui X, Xiaofei, He, Lili, Li et al (2025) Voluntary exercise alleviates neural functional deficits in Parkinson's disease mice by inhibiting microglial ferroptosis via SLC7A11/ALOX12 axis.[J].NPJ Parkinsons Dis. 11:0 Swelm RPLV, Wetzels JFM, Swinkels DW The multifaceted role of iron in renal health and disease. Nature Reviews Nephrology Wu Y et al (2021) HMGB1 regulates ferroptosis through Nrf2 pathway in mesangial cells in response to high glucose. Biosci Rep, 41(2) Tiwari B et al (2013) Markers of Oxidative Stress during Diabetes Mellitus. Journal of biomarkers, 2013: p. 378790 Additional Declarations No competing interests reported. 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-7064578","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":481732881,"identity":"1c9ad696-18c5-4431-9c13-97133d823606","order_by":0,"name":"卢 Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACfv7+h49/GPyXY2NvIFKL5IwzzMYMFczG/DwHiNRicCCHTZrhDHOi5IwEYm1pOHtMurCNLcHg5uONNxhqbKIJauFn7ku2ntnGk2dwO63YguFYWm4DYVsOGN7gbZMoNridYybB2HCYsBaDAwkGErxtBokbbp4hWkuOkTTPmYTEmTN4iNQiOeNYsuGMigPAQAb6JYEYv/DzNx988MHgADAqD2+88aHGhrAWFEdKJJCiHKKFVB2jYBSMglEwMgAA06hDS711duUAAAAASUVORK5CYII=","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":true,"prefix":"","firstName":"卢","middleName":"","lastName":"Yu","suffix":""},{"id":481732882,"identity":"41172500-393c-4936-9e1a-e9a13115319d","order_by":1,"name":"齐汉 zhai","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"齐汉","middleName":"","lastName":"zhai","suffix":""},{"id":481732883,"identity":"a8a63c0c-cf9d-47f9-ad6b-138af3f6f7af","order_by":2,"name":"尤林 王","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"尤林","middleName":"","lastName":"王","suffix":""},{"id":481732884,"identity":"63b238b9-4882-4c69-b9b1-ea05b15bf933","order_by":3,"name":"刘伟 王","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"刘伟","middleName":"","lastName":"王","suffix":""},{"id":481732887,"identity":"6c60fe8d-1bb7-4207-a0ef-b3ae2ff23a1c","order_by":4,"name":"邱红 利","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"邱红","middleName":"","lastName":"利","suffix":""},{"id":481732889,"identity":"e07e43fc-fd43-4f5c-bc5b-431ea41f197a","order_by":5,"name":"燕红 郭","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"燕红","middleName":"","lastName":"郭","suffix":""},{"id":481732893,"identity":"ef2ceb4e-cf0a-474c-be8f-85f6be6ed6fa","order_by":6,"name":"荣 gou","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"荣","middleName":"","lastName":"gou","suffix":""},{"id":481732896,"identity":"6e4f1ad2-3387-4ad4-ad36-6546ee7b3e91","order_by":7,"name":"林 唐","email":"","orcid":"","institution":"The First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"林","middleName":"","lastName":"唐","suffix":""}],"badges":[],"createdAt":"2025-07-07 10:53:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7064578/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7064578/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87802347,"identity":"bd257722-a690-43a8-ab64-f14043faedc7","added_by":"auto","created_at":"2025-07-29 08:02:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":462027,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemical staining of renal tissues from normal individuals and DN patients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical staining was used to detect the expression levels of α-SMA, N-cadherin, E-cadherin and Fibronectin in normal renal tissues and renal tubules of DN patients. \u003cem\u003e***p\u0026lt;0.001 \u003c/em\u003e(n=10).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7064578/v1/35da40b5b8a386bbb8a4d24a.png"},{"id":87805584,"identity":"590b4da5-1bbc-41db-b155-b5faf29e1cae","added_by":"auto","created_at":"2025-07-29 08:26:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1243359,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFinerenone can alleviate renal tubular atrophy, interstitial inflammatory cell infiltration and fibrosis in db/db mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Conventional HE and Masson staining were used to observe the degree of renal tubular atrophy, interstitialinflammatory cell infiltration and fibrosis in the db/db, db/db＋10 mg/kg/d finerenone and db/db + 30 mg/kg/d finerenone groups. B. Western Blot was used to detect the levels of COL1 and TGF-βin the renal tissues of the four groups of mice. *, control vs. db/db. #, db/db vs. db/db + finerenone. \u003cem\u003e#p \u0026lt; 0.05, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001, ***p \u0026lt; 0.001\u003c/em\u003e (n = 8).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7064578/v1/2b188934afd8ae5de2bbbe68.png"},{"id":87803812,"identity":"7ee0d936-f981-410b-8009-9956b569e2c7","added_by":"auto","created_at":"2025-07-29 08:10:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":81112,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFinerenone can alleviate ferroptosis-related indicators in the renal tissue of db/db mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. B. Western Blot was used to detect the SLC7A11 levels in the four groups of mice: db/dm, db/db, db/db + 10 mg/kg/d finerenone, and db/db + 30 mg/kg/d finerenone; C. D. The levels of GSH and MDA in the renal tissues of the four groups of mice were detected by ELISA. *, control vs. DN. #, db/db vs. db/db + Fine. \u003cem\u003e#p \u0026lt; 0.05, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001, ***p \u0026lt; 0.001\u003c/em\u003e (n = 3).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7064578/v1/947a7c747e35628f90b1d664.png"},{"id":87803813,"identity":"e6c85a29-16b4-4aa0-8012-27489442810c","added_by":"auto","created_at":"2025-07-29 08:10:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":748611,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFinerenone can alleviate renal tubular epithelial cell injury under high glucose conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. CCK-8 assay was used to detect the viability of HK2 cells in each group; B. C. Laser confocal microscopy was used to detect the number of EdU-labeled positive cells in each group of HK2 cells; D. E. Western Blot was used to detect the expression levels of COL1 and TGF- β in each group; F. G. QRT-PCR was used to detect the expression levels of COL1 mRNA and TGF-βmRNA in each group of HK-2 cells. *, NG vs. HG. #, HG vs. HG + Fine. \u003cem\u003e#p \u0026lt; 0.05, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001, ***p \u0026lt; 0.001\u003c/em\u003e(n = 3).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7064578/v1/0cc8c519bc1adf4657b9a034.png"},{"id":87802354,"identity":"0ad99c71-0a86-45c9-bae3-c9147a89a04f","added_by":"auto","created_at":"2025-07-29 08:02:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":140290,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFinerenone can alleviate ferroptosis of renal tubular epithelial cells under high glucose conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.B. The lipid peroxidation intensity of each group was detected by A.B. BODIPY 581/591 C11 lipid peroxidation probe; C.D. The concentrations of GSH and MDA in each group were detected by ELISA method; E. The Fe\u003csup\u003e2+\u003c/sup\u003e concentration in each group of HK2 cells was detected by E. colorimetric method; F.G. The levels offerroptosis-related proteins FTH1, SLC7A11, and GPX4 in each group were detected by Western Blot. *, NG vs. HG. #, HG vs. HG+Fine. \u003cem\u003e##p\u0026lt;0.01, ###p\u0026lt;0.001, ***p\u0026lt;0.001\u003c/em\u003e (n=3).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7064578/v1/e7d1e66ebe27614cebd84527.png"},{"id":87803815,"identity":"a07f6d97-8e90-450b-97c4-59f39904a09b","added_by":"auto","created_at":"2025-07-29 08:10:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":183191,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSilencing SLC7A11 in a high-glucose environment exacerbating renal tubular epithelial cell injury and\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ecounteract the protective effect of finerenone.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. CCK8 assay was used to detect the effect of knockdown of SLC7A11, a member of the SLC family, on the cytoprotective effect of finerenone under high glucose conditions; B. QRT-PCR was employed to examine the impact of SLC7A11 knockdown on the expression of fibrosis-related genes in HK2 cells under high glucose conditions; C. EdU assay was conducted to assess the effect of SLC7A11 knockdown on finerenone-induced cell proliferation under high glucose conditions; D. Western Blot was utilized to investigate the influence of SLC7A11 knockdown on the protective effect of finerenone against high glucose-induced fibrosis-related gene expression; *, NG vs. HG. \u003cem\u003e**p\u0026lt;0.01, ***p\u0026lt;0.001\u003c/em\u003e (n=3).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-7064578/v1/59e8f0dfc4a96e6fea5e61ce.png"},{"id":87804552,"identity":"19335f86-89a6-40ac-9ac7-987ec007e8fc","added_by":"auto","created_at":"2025-07-29 08:18:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":665259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSilencing SLC7A11 in a high-glucose environment exacerbating ferroptosis in renal tubular epithelial\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ecells and counteract the protective effect of finerenone\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Colorimetric assay was used to detect the effect of SLC7A11 knockdown on ferrous ion concentration in HK2 cells under high glucose conditions; B. BODIPY 581/591 C11 lipid peroxidation probe was employed to examine the impact of SLC7A11 knockdown on lipid peroxidation in HK2 cells under high glucose conditions; C-D. ELISA was utilized to measure the influence of SLC7A11 knockdown on GSH and MDA levels in HK2 cells. *, NG vs. HG. \u003cem\u003e**p\u0026lt;0.01, ***p\u0026lt;0.001\u003c/em\u003e (n=3).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7064578/v1/d94577590f5b152667b3325e.png"},{"id":87803817,"identity":"9b0304ed-d953-47a2-b279-b471e86dd9cd","added_by":"auto","created_at":"2025-07-29 08:10:08","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":136203,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProposed model for the signaling pathway of finerenone ameliorates DN via suppressing SLC7A11-mediated ferroptosis\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7064578/v1/6f52ba2d9238e5b970f4f278.png"},{"id":88505217,"identity":"35eed5d9-8ace-4674-9996-f7dece2f3e0d","added_by":"auto","created_at":"2025-08-07 07:21:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4665016,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7064578/v1/4b81e1f9-d82d-43ce-b268-d6ccf71d54ea.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Non-steroidal mineralocorticoid receptor antagonist finerenone ameliorates Diabetic Nephropathy via suppressing SLC7A11-mediated ferroptosis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eWith the development of China\u0026rsquo;s economy and changes in residents' lifestyles, the incidence of diabetes, particularly type 2 diabetes, has continuously risen, becoming a major public health issue threatening the health of Chinese residents. According to data from the International Diabetes Federation in 2021, approximately 180\u0026nbsp;million adults in China have diabetes, with a prevalence rate of 12.8%, the highest in the world [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Diabetic nephropathy (DN) is one of the serious complications of diabetes, significantly affecting patients\u0026rsquo; quality of life and imposing a substantial economic burden on society, posing a major challenge to China\u0026rsquo;s medical and healthcare system. Therefore, exploring new pathogenic mechanisms and optimizing treatment strategies to improve the prognosis of DN patients is urgently needed and has significant clinical and social implications.\u003c/p\u003e\u003cp\u003eFerroptosis is an iron-dependent programmed cell death characterized by lipid peroxidation and the accumulation of lipid reactive oxygen species. It is involved in the pathological processes of various kidney diseases, including DN, ultimately leading to kidney injury [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e][\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e][\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The Xc- system is a non-sodium antiporter that exports intracellular glutamate and imports extracellular cystine in a 1:1 ratio. It consists of two subunits linked by disulfide bonds: the heavy chain subunit solute carrier family 3 member 2 (SLC3A2) and the light chain subunit solute carrier family 7 member 11 (SLC7A11) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e][\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. SLC7A11 is a multi-channel transmembrane protein. Since cysteine is a precursor for glutathione (GSH) synthesis, and intracellular cysteine is primarily provided by cystine uptake mediated by SLC7A11, numerous studies have demonstrated the crucial role of SLC7A11-mediated cystine uptake in inhibiting ferroptosis under oxidative stress conditions and maintaining cell survival. Therefore, SLC7A11 is considered a key regulatory protein for ferroptosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. GPX4, a member of the glutathione peroxidase family, was discovered in 1982 in pig liver and heart tissues [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Its main function is to utilize glutathione as a cofactor to counteract lipid peroxidation, a process triggered by reactive oxygen species (ROS) or free radicals that target cell membrane lipids. ROS-induced oxidation of polyunsaturated fatty acids (PUFAs) generates lipid hydroperoxides and other reactive intermediates, causing structural damage to the cell membrane and potential cell death. Dysregulation of GPX4 leads to ferroptosis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNon-steroidal mineralocorticoid receptor antagonists (MRAs) can comprehensively block the gene expression of inflammation and fibrosis associated with excessive activation of the mineralocorticoid receptor (MR) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e10\u003c/span\u003e][\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Finerenone, a third-generation MRA, differs from the steroid structure of spironolactone and eplerenone by its unique non-steroidal structure, which ensures high receptor selectivity while significantly reducing the risk of adverse events associated with steroid MRAs [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e12\u003c/span\u003e][\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Existing research has confirmed that finerenone can effectively block excessive MR activation, exerting anti-inflammatory and anti-fibrotic effects [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e14\u003c/span\u003e][\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. It has been shown to reduce the upregulation of inflammatory factors, improve glomerular sclerosis and hypertrophy, alleviate tubulointerstitial fibrosis, reduce macrophage infiltration, and lower endothelial dysfunction, thereby effectively reducing proteinuria and protecting renal function [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e16\u003c/span\u003e][\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. FIN and other MRAs have exhibited antioxidant, anti-inflammatory, and anti-fibrotic properties, and have reversed metabolic abnormalities in DN in previous studies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Insulin resistance, commonly associated with MR activation in DT, is significantly linked to a decline in glomerular filtration rate (GFR). However, in diabetic nephropathy, whether finerenone can alleviate ferroptosis by regulating the SLC7A11 transporter, thereby exerting a protective effect on renal tubular injury, remains underexplored.\u003c/p\u003e\u003cp\u003eIn this study, we used a diabetic nephropathy mice model and in vitro cultured HK2 cells to explore through in vivo and in vitro experiments how finerenone exerts a protective effect on renal tubules in diabetic nephropathy by regulating the SLC7A11 and alleviating ferroptosis.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003e2.1 Patients and pathological samples\u003c/h2\u003e\n\u003cp\u003eTen patients diagnosed with DN (aged 18\u0026ndash;65 years) from the Department of Nephrology of the First Affiliated Hospital of Zhengzhou University were selected (It conforms to the 2017 edition of the Standards of Medical Care in Diabetes released by the American Diabetes Association, and is in line with the consensus on the diagnosis of DN issued by the American Diabetes Association and the National Kidney Foundation in 2014. It has been confirmed as DN through renal biopsy). Patients with tumors, other liver or kidney diseases, infections, fever, or autoimmune diseases were excluded. After signing the informed consent form, renal tissue samples were collected and clinical laboratory indicators such as blood glucose, serum creatinine, and quantitative urine protein were collected. Normal control renal tissue samples were obtained from the Department of Urology of the First Affiliated Hospital of Zhengzhou University from patients with normal blood glucose who underwent nephrectomy for renal tumors (6 cases). After signing the informed consent form, normal renal tissue 3\u0026ndash;5 cm away from the tumor margin of the removed kidney was collected.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003e2.2 Animal studies\u003c/h2\u003e\n\u003cp\u003edb/db mice and db/dm mice were purchased from Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. [SCXK (Zhe) 2019-0004]. db/db mice are type 2 diabetic nephropathy mice with leptin receptor point mutations and can exhibit obvious diabetic symptoms such as obesity and hyperglycemia at 6 weeks of age. All mice were handled in accordance with the approved protocol by the laboratory and were approved by the Animal Ethics Committee of Zhengzhou University. The purchased mice were 8-week-old male mice, randomly divided into 4 groups, with 8 mice in each group. ① Normal control group: 8-week-old db/dm mice, fed a normal diet, and renal tissue samples were collected at 24 weeks. ②DN model group: 8-week-old db/db mice, fed a high-fat diet until 10 weeks of age, and then received two consecutive intraperitoneal injections of streptozotocin (70 mg/Kg) at 10 weeks of age. Renal tissue samples were collected at 24 weeks.③④Finerenone treatment group: 8-week-old db/db mice, fed until 10 weeks of age and then given finerenone by gavage at doses of 10 mg/Kg/d and 30 mg/Kg/d, divided into 2 groups, with 8 mice in each group.After sacrificing the mice, the kidneys were collected and stored at suitable storage conditions for further experiments。\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003e2.3 Cell culture\u003c/h2\u003e\n\u003cp\u003eHK-2 (human renal cortical proximal tubular epithelial cells) culture: Cells were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher), 100 U/mL penicillin and 100\u0026micro;g/mL streptomycin at 37℃, 5% CO\u003csub\u003e2\u003c/sub\u003e and 95% humidity. All cells were used between passages 5 and 15. The medium was changed every 48 hours, and serum-free medium was used when the cells reached 70% confluence. The cells were treated as follows: 5 mM glucose (Con), 30 mM D-glucose (HG), HG\u0026thinsp;+\u0026thinsp;1\u0026micro;M finerenone and HG\u0026thinsp;+\u0026thinsp;3\u0026micro;M finerenone. The SLC7A11 interfering plasmid (shSLC7A11) was transfected into cells 24 hours before HG treatment using lipofectamine 3000 transfection reagent, and then the cells were collected or stained 24 hours later for subsequent experiments.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003e2.4 Immunohistochemistry, IHC\u003c/h2\u003e\n\u003cp\u003eDe-waxing, antigen retrieval, endogenous peroxidase inactivation, blocking, primary antibody incubation, secondary antibody incubation, drop freshly prepared DAB chromogen solution in the immunohistochemistry circle, place under the microscope for observation, quickly rinse after positive reaction to terminate staining. Hematoxylin re-stain the cell nucleus, dehydration, drop neutral gum on the dehydrated section, cover with a coverslip and seal, place in a fume hood to dry. Photography and analysis: Observe and collect pictures under an upright fluorescence microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003e2.5 Hematoxylin-Eosin Dyeing\u003c/h2\u003e\n\u003cp\u003eDe-waxing, hematoxylin staining of nuclei, differentiation and bluing, eosin staining, dehydration: Place the slides in an oven at 68℃ for 2 minutes, then immerse them in xylene I and xylene II for 2 minutes each, and air dry. Seal the slides with neutral gum. Observe under an optical\u003c/p\u003e\n\u003cp\u003emicroscope and take pictures.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003e2.6 Masson Dyeing\u003c/h2\u003e\n\u003cp\u003eDe-waxing, nuclear staining with hematoxylin solution, soaking in Masson's acid fuchsin\u003c/p\u003e\n\u003cp\u003esolution for 10 minutes, washing with 2% glacial acetic acid for 5 minutes, differentiation with 1%\u003c/p\u003e\n\u003cp\u003ephosphomolybdic acid solution for 5 minutes, staining with aniline blue for 5 minutes, and washing\u003c/p\u003e\n\u003cp\u003ewith 0.2% glacial acetic acid solution for 5 minutes. Dehydration: successively place the sections inanhydrous ethanol I for 5 minutes-anhydrous ethanol II for 5 minutes-xylene I for 5 minutes - xylene II for 5 minutes, transfer to a fume hood to dry, and seal with neutral gum. Observe and collect images using an upright fluorescence microscope.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003e2.7 Transient transfection of SLC7A11 shRNA plasmid\u003c/h2\u003e\n\u003cp\u003eThe shRNA sequence targeting SLC7A11 was synthesized and annealed with the eukaryotic expression vector containing the U6 promoter. Transfection: ①Dissolve 0.8\u0026micro;g of plasmid in 50\u0026micro;L of Opti-MEM serum-free medium ② Dissolve 2\u0026micro;L of lipo3000 in 50\u0026micro;L of Opti-MEM serum-free medium, mix well and let stand at room temperature for 5 minutes③Mix tubes A and B and let stand for 20 minutes. Replace the medium in the 24-well plate with serum-free medium, 400\u0026micro;L/well. Add the mix in tube C to the corresponding wells of the 24-well plate. After 4\u0026ndash;6 hours, replace with serum-containing medium.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch2\u003e2.8 Cell viability assay\u003c/h2\u003e\n\u003cp\u003eThe viability of cells was evaluated using the Cell Counting Kit-8 (CCK-8) assay (Beyotime Biotechnology, China). Following transfection, cells were seeded into 96-well plates at a density of 3000 cells per well. Absorbance values were recorded at various time points ranging from 0 to 96 hours post-transfection. A solution consisting of 100 \u0026micro;L RPMI 1640 medium with 10 \u0026micro;L of CCK-8 reagent was added to each well, followed by a 2-hour incubation period. Subsequently, the absorbance at 450 nm was measured using a microplate reader (Bio-Rad, USA). To ensure reliability, all experiments were performed in three replicate wells and repeated three times.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003e2.9 GSH assay\u003c/h2\u003e\n\u003cp\u003eThe measurement of glutathione (GSH) levels was carried out using the DTNB (5,5\u0026prime;-Dithio-bis-(2-nitrobenzoic acid)) method (Solarbio, BC1170), with distinct pre-treatment protocols for tissue and cell samples. For tissue samples, tissues were homogenized on ice at a ratio of 1:10 (grams of tissue to milliliters of reagent) using a pre-chilled homogenizer. The homogenates were centrifuged at 8000 g for 10 minutes at 4\u0026deg;C, and the supernatant was carefully collected and stored at 4\u0026deg;C until analysis. For cell samples, cells were cultured in 100 mm dishes at a density of 3\u0026nbsp;million cells per dish and treated according to the experimental groups. After treatment, the cells were detached using trypsinization, counted, and 5\u0026nbsp;million cells were resuspended in 1 mL of assay buffer. To ensure complete lysis, the suspension underwent 2\u0026ndash;3 freeze-thaw cycles (freezing in liquid nitrogen and thawing in a 37\u0026deg;C water bath). Following the final thaw, the suspension was centrifuged at 8000 g for 10 minutes, and the supernatant was collected and kept on ice for subsequent steps. After sample collection, proteinase purification was performed. The sample was then mixed with DTNB solution in a 96-well plate, and the reaction was initiated by adding NADPH solution. After incubation for 20 minutes, the GSH concentration was determined at a wavelength of 412 nm using a SpectraMax M2e spectrophotometer (Beyotime Biotechnology, China).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e2.10 MDA assay\u003c/h2\u003e\n\u003cp\u003eThe Lipid Peroxidation MDA Assay was performed using the MDA Assay Kit (Beyotime Biotechnology, China). Cells were cultured in 60 mm culture dishes at a density of 800,000 cells per dish and treated according to their assigned groups. After treatment, the cells were harvested through trypsinization, counted, and a total of 3\u0026nbsp;million cells were resuspended in 0.3 mL of lysis buffer. Following lysis, the suspension was centrifuged at 10,000\u0026ndash;12,000 g for 10 minutes to obtain a clear supernatant. If the supernatant was not sufficiently clear after centrifugation, a 0.2 \u0026micro;m filter was used to further clarify the sample. All steps of sample preparation were carried out on ice or at 4\u0026deg;C. After pre-treatment, the supernatants were processed following the manufacturer's instructions. The samples were heated in a boiling water bath for 15 minutes, cooled to room temperature, and then centrifuged at 1000 g for 10 minutes. The absorbance of the supernatant was measured at 532 nm using an enzyme-linked immunosorbent assay reader. This absorbance value was correlated with the MDA concentration, which was determined based on a standard curve.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003e2.11 Lipid peroxidation assay\u003c/h2\u003e\n\u003cp\u003eFor the lipid peroxidation analysis, cells were seeded into 12-well plates at a density of 80,000 cells per well and treated according to their designated groups. After treatment, the cells were detached using trypsinization, collected by centrifugation at 300g for 5 minutes, and washed three times with PBS. The cell pellet was then incubated with BODIPY\u0026trade; 581/591C11 dye at a final concentration of 5 \u0026micro;M in a 37\u0026deg;C incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e for 30 minutes. Following incubation, any unbound fluorescent dye was removed by gently washing the cells three times with PBS. The cell suspension was subsequently transferred to flow cytometry tubes for further analysis. Samples were analyzed using a flow cytometer, and the resulting data were processed using FlowJo software. Cell subpopulations were distinguished based on forward scatter (FSC) and side scatter (SSC) characteristics, while the percentage of the positive subpopulation was determined through a single-peak histogram of FL1 fluorescence intensity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003e2.12 Abcam Iron assay\u003c/h2\u003e\n\u003cp\u003eTake the standard protection agent and add 20 mL of buffer solution, mix well and store at 2\u0026ndash;8℃. Take 20\u0026micro;L of 10 mmol/L iron standard solution and mix it evenly with 1980\u0026micro;L of standard protection solution. Determination of ferrous ion concentration: ①Standard wells: Take 200\u0026micro;L of different concentration standards and add them to the corresponding wells of the microplate. Measuring wells: Take 200\u0026micro;L of sample and add it to the corresponding wells of the microplate. ② Add 100\u0026micro;L of chromogenic solution to each well, mix well, and incubate at 37℃ for 10 minutes. Measure the OD value of each well at 593 nm on the microplate reader. Calculate the ferrous ion concentration in the cells according to the standard curve formula.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e2.13 EdU-555 cell proliferation assay\u003c/h2\u003e\n\u003cp\u003eUnder the fluorescence microscope, red fluorescence (DNA molecules in the replication state) and blue fluorescence (cell nuclei) can be observed. When the two are combined, they present purple spot fluorescence. Photos are taken and the images are saved. The Image J software is used tocalculate the proportion of proliferating cells (red fluorescence) to the total cells (blue fluorescence), and statistical analysis is conducted.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003e2.14 Western blotting analysis\u003c/h2\u003e\n\u003cp\u003eMurine renal tissue or cultured cells was homogenized and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with phosphatase inhibitor (Proteintech, China) and protease inhibitor (Proteintech, China). The samples were blotted and tagged with relevant antibodies as previously reported. The antibodies against COL1 and TGF-\u0026beta;,FTH1, SLC7A11and GPX4 were purchased from Proteintech.The protein band was quantified using Image J software.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003e2.15 Immunofluorescence\u003c/h2\u003e\n\u003cp\u003eThe HK-2 cells were treated with adenovirus expressing GFP-LC3.The samples were fixed with 4% paraformaldehyde, permeated with methanol/acetone, and infected with TOM20 antibody (1:200, Abcam, ab186734, United States). The samples were then observed under a fluorescence confocal microscope (LSM510 META, Karl Zeiss, Germany).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003e2.16 Real-time quantitative PCR (RT‒qPCR)\u003c/h2\u003e\n\u003cp\u003eAfter RNA extraction, the concentration and purity of RNA were determined using an ultramicro UV spectrophotometer and recorded. The RNA solution was stored at -80℃. RNA was reverse transcribed into cDNA and amplified using a real-time fluorescence quantitative PCR instrument. Result analysis: GAPDH was used as the internal reference, and the 2 -\u0026Delta;\u0026Delta;CT method was used for data calculation. Three replicate wells were set for each gene, and each group was repeated at least three times.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003e2.17 Statistical analysis\u003c/h2\u003e\n\u003cp\u003eStatistical analysis was performed using GraphPad Prism 8.0 software. Data were obtained from three or more independent repeated experiments. Two-tailed unpaired Student's t-test was used for comparison between two groups, and one-way ANOVA and Bonferroni test were used for comparison among three or more groups. p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Investigate the expression levels of fibrosis-related markers in the renal tubules of patients with DN and healthy controls\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the changes of fibrosis-related indicators under the high-glucose environment, we conducted immunohistochemical staining on renal tissue specimens from healthy individuals and DN patients, and detected the indicators related to renal tubules. The results showed that compared with normal renal tissue, the expressions of indicators reflecting fibrosis such as N-cadherin, Fibronectin and \u0026alpha;-SMA increased in renal tubules of DN patients, while the expression of E-cadherin decreased. This suggested that the degree of renal tubule fibrosis in DN patients was aggravated (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eAs demonstrated by these outcomes, compared with healthy renal tissue, the degree of renal tubule fibrosis in DN patients is aggravated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Evaluating the effects of finerenone on renal tubular atrophy, interstitial inflammatory cell infiltration, and fibrosis in db/db mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to observe the effects of finerenone on renal tubular injury, interstitial inflammatory cell infiltration and fibrosis in the kidneys of db/db mice, we performed HE and Masson staining on the db/dm, db/db and db/db་finerenone groups. Compared with the db/dm group, the renal tubules of db/db mice were atrophied, and the interstitial inflammatory cell infiltration and fibrosis were aggravated; compared with the db/db group, the renal tubules of the db/db་10mg/Kg/d finerenone group and the db/db་30mg/Kg/d finerenone group were atrophied, and the interstitial fibrosis and inflammatory cell infiltration were alleviated (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA).The results of Western Blot experiment showed that compared with the db/dm group, the expression of COL1 and TGF-\u0026beta;related to fibrosis in the db/db group was upregulated, suggesting an aggravated degree of fibrosis; compared with the db/db group, the expression of COL1 and TGF-\u0026beta;in the db/db་10mg/Kg/d finerenone group and the db/db་30mg/Kg/d finerenone group was downregulated, suggesting a reduced degree of fibrosis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB), suggesting that finerenone can alleviate renal tubular atrophy, interstitial inflammatory cell infiltration and fibrosis in db/db mice.\u003c/p\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003e3.3 Exploring the role of finerenone in ferroptosis of renal tissue in db/db mice\u003c/h2\u003e\n\u003cp\u003eTo further explore the effect of finerenone on the degree of ferroptosis in the kidneys of db/db mice, we detected the expression levels of ferroptosis-related protein SLC7A11 and the ferroptosis-related indicators GSH and MDA in the renal tissues of db/dm, db/db and db/db\u0026thinsp;+\u0026thinsp;finerenone groups of mice.The Western Blot results showed that compared with the db/db group of mice, the expression of ferroptosis-related protein SLC7A11 was increased in the db/db\u0026thinsp;+\u0026thinsp;10mg/kg/d finerenone group and the db/db\u0026thinsp;+\u0026thinsp;30mg/kg/d finerenone group of mice, suggesting a reduction in the degree of ferroptosis (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB).The levels of GSH and MDA were detected by ELISA.\u003c/p\u003e\n\u003cp\u003eCompared with the db/db group of mice, the levels of GSH, a ferroptosis-related indicator, were increased and the levels of MDA were decreased in the db/db\u0026thinsp;+\u0026thinsp;10mg/kg/d finerenone group and the db/db\u0026thinsp;+\u0026thinsp;30mg/kg/d finerenone group of mice, suggesting a reduction in the degree of ferroptosis (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD).The above results indicate that finerenone can reduce ferroptosis in the renal tissues of db/db mice.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003e3.4 Finerenone alleviate high glucose-induced injury to renal tubular epithelial cells.\u003c/h2\u003e\n\u003cp\u003eTo observe the protective effect of finerenone on renal tubular epithelial cells in a high glucose environment, human proximal tubular epithelial cells HK2 were cultured in vitro. The groups were set as NG group, HG group and HG\u0026thinsp;+\u0026thinsp;finerenone groups with different concentrations (0.1\u0026micro;M, 0.3 \u0026micro;M, 1\u0026micro;M, 3\u0026micro;M, 10\u0026micro;M and 30\u0026micro;M). HK2 cells were treated for 48 hours, and the cell damage and the effect of finerenone on the fibrosis indicators of HK2 cells were detected.The CCK-8 results showed that compared with the NG group, the cell viability of HK2 cells in the HG group was weakened and the number of EdU-labeled positive cells decreased. When different concentrations of finerenone were given to the HG group, the cell viability of HK2 gradually recovered and the number ofEdU-labeled positive cells gradually increased with the increase of finerenone concentration. After reaching a certain concentration, the ability to restore cell viability decreased. The concentrations of 1\u0026micro;M and 3\u0026micro;M were most conducive to cell growth (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA).Laser confocal microscopy showed that compared with the NG group, the intensity of EdU-labeled and DAPI-stained HK2 cells in the HG group decreased, indicating a decline in cell viability. Compared with the HG group, the number of EdU-labeled positive cells in the HG\u0026thinsp;+\u0026thinsp;finerenone groups with different concentrations (1\u0026micro;M and 3\u0026micro;M) gradually increased, and the cell proliferation ability also gradually enhanced (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\n\u003cp\u003eThe Western Blot results showed that compared with the NG group, the expression levels of COL1 and TGF-\u0026beta; in HK-2 cells in the HG group significantly increased, indicating an aggravation of fibrosis. Compared with the HG group, the expression levels of COL1 and TGF-\u0026beta;in the HG\u0026thinsp;+\u0026thinsp;finerenone groups with different concentrations (1\u0026micro;M and 3\u0026micro;M) significantly decreased, and the degree of fibrosis was alleviated (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE).QRT-PCR showed that compared with the NG group, the expression of COL1 mRNA and TGF-\u0026beta;mRNA in HK-2 cells in the HG group was significantly upregulated, indicating an aggravation of fibrosis. Compared with the HG group, the expression of COL1mRNA and TGF-\u0026beta;mRNA in the HG\u0026thinsp;+\u0026thinsp;finerenone groups with different concentrations (1\u0026micro;M and 3\u0026micro;M) decreased, and the degree of fibrosis was alleviated (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG).The above results indicate that finerenone can enhance the viability of HK2 cells, promote cell proliferation, and alleviate the degree of fibrosis and cell damage in a high glucose environment.\u003c/p\u003e\n\u003ch2\u003e3.5 Exploring the effect of finerenone on ferroptosis of renal tubular epithelial cells induced by high glucose\u003c/h2\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\"\u003eThe NG group, HG group and HG\u0026thinsp;+\u0026thinsp;finerenone groups at different concentrations (1\u0026micro;M and 3 \u0026micro;M) were respectively set up to treat HK2 cells for 48 hours, and the effects of finerenone on the level of HIF-1\u0026alpha; and the degree of ferroptosis in HK2 cells under high glucose conditions were observed. The BODIPY 581/591 C11 lipid peroxidation probe was used to detect the intensity of lipid peroxidation related to ferroptosis. Compared with the NG group, the lipid peroxidation in the HG group was significantly enhanced, suggesting an increased degree of ferroptosis. Compared with the HG group, the lipid peroxidation intensity in the HG\u0026thinsp;+\u0026thinsp;finerenone groups at different concentrations (1\u0026micro;M and 3\u0026micro;M) decreased, indicating a reduced degree of ferroptosis (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). The levels of ferroptosis-related indicators GSH and MDA in each group were detected by ELISA. Compared with the NG group, the GSH level in the HG group decreased while the MDA level increased, suggesting an aggravated degree of ferroptosis; compared with the HG group, the GSH level in the HG\u0026thinsp;+\u0026thinsp;finerenone groups at different concentrations (1\u0026micro;M and 3\u0026micro;M) increased while the MDA level decreased, indicating a reduced degree of ferroptosis (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD).\n\u003cp\u003eThe Western Blot results showed that compared with the NG group, the levels of ferroptosis-related proteins FTH1, SLC7A11, and GPX4 in the HG group decreased, suggesting an aggravated degree of ferroptosis; compared with the HG group, the levels of FTH1, SLC7A11, and GPX4 in the HG\u0026thinsp;+\u0026thinsp;finerenone groups at different concentrations (1\u0026micro;M and 3\u0026micro;M) increased, indicating a reduced degree of ferroptosis (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE and \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG). The Fe\u003csup\u003e2+\u003c/sup\u003e concentration in each group was detected by colorimetry. The results showed that compared with the NG group, the Fe\u003csup\u003e2+\u003c/sup\u003e level in the HG group increased; compared with the HG group, the Fe\u003csup\u003e2+\u003c/sup\u003e level in the HG\u0026thinsp;+\u0026thinsp;finerenone groups at different concentrations (1\u0026micro;M and 3\u0026micro;M) decreased, indicating a reduced degree of ferroptosis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e\n\u003cp\u003eThe above results indicate that finerenone can alleviate ferroptosis in renal tubular epithelial cells under high glucose conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Silencing SLC7A11 of HK2 cells in high-glucose environment exacerbating renal tubular epithelial cell injury and counteract the protective effect of finerenone\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSLC7A11 is considered a key regulatory protein in ferroptosis and plays an important role in regulating ferroptosis. To investigate the role of SLC7A11 in nonenalone-protected HK-2 cells under high glucose conditions, we silenced SLC7A11 in HK-2 cells and divided them into shNC\u0026thinsp;+\u0026thinsp;vehicle group, shSLC7A11\u0026thinsp;+\u0026thinsp;vehicle group, shNC\u0026thinsp;+\u0026thinsp;Fine group, and shSLC7A11\u0026thinsp;+\u0026thinsp;Fine group. We then examined the cell count, cell viability, and cell damage in each group. The CCK-8 results showed that compared with the shNC\u0026thinsp;+\u0026thinsp;vehicle group, the cell viability in the shSLC7A11\u0026thinsp;+\u0026thinsp;vehicle group decreased; compared with the shNC\u0026thinsp;+\u0026thinsp;Fine group, the cell viability in the shSLC7A11\u0026thinsp;+\u0026thinsp;Fine group decreased, and the differences were statistically significant. This suggests that after nonenalone treatment, cell damage was reduced, but after silencing SLC7A11, cell viability decreased, and the protective effect of nonenalone on cells was also counteracted (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA).The QRT-PCR results indicated that compared with the shNC\u0026thinsp;+\u0026thinsp;vehicle group, the expression of COL1 mRNA and TGF-\u0026beta; mRNA in the shSLC7A11\u0026thinsp;+\u0026thinsp;vehicle group increased; compared with the shNC\u0026thinsp;+\u0026thinsp;Fine group, the expression of COL1mRNA and TGF-\u0026beta;mRNA in the shSLC7A11\u0026thinsp;+\u0026thinsp;Fine group increased. This suggests that after silencing SLC7A11 in HK-2 cells, the indicators of cell fibrosis increased, and cell damage worsened (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB).Laser confocal microscopy showed that compared with the shNC\u0026thinsp;+\u0026thinsp;vehicle group, the intensity of EdU-labeled and DAPI-stained cells in the shSLC7A11\u0026thinsp;+\u0026thinsp;vehicle group decreased, indicating a decrease in cell viability; compared with the shNC\u0026thinsp;+\u0026thinsp;Fine group, the intensity of EdU-labeled and DAPI-stained cells in the shSLC7A11\u0026thinsp;+\u0026thinsp;Fine group decreased, indicating a decrease in cell viability and a reduction in cell proliferation ability (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC).The Western Blot results showed that compared with the shNC\u0026thinsp;+\u0026thinsp;vehicle group, the expression levels of COL1 and TGF-\u0026beta; in the shSLC7A11\u0026thinsp;+\u0026thinsp;vehicle group significantly increased, suggesting an aggravation of fibrosis; compared with the shNC\u0026thinsp;+\u0026thinsp;Fine group, the expression levels of COL1 and TGF-\u0026beta; in the shSLC7A11\u0026thinsp;+\u0026thinsp;Fine group significantly increased, suggesting an aggravation of fibrosis (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD).These results suggest that under high glucose conditions, regardless of whether nonenalone intervention is given or not, silencing the SLC7A11 leads to a decrease in HK2 cell viability, weakened cell proliferation, and enhanced expression of fibrosis-related genes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n\u003ch2\u003e3.7 Silencing SLC7A11 in a high-glucose environment exacerbating ferroptosis in renal\u0026nbsp;\u003cstrong\u003etubular epithelial cells and counteract the protective effect of finerenone\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eTo explore the effect of SLC7A11 on ferroptosis of renal tubular epithelial cells under high glucose conditions, we silenced the SLC7A11 in HK-2 cells and divided them into shNC\u0026thinsp;+\u0026thinsp;vehicle group, shSLC7A11\u0026thinsp;+\u0026thinsp;vehicle group, shNC\u0026thinsp;+\u0026thinsp;Fine group, and shSLC7A11\u0026thinsp;+\u0026thinsp;Fine group, and then detected the ferroptosis-related indicators of each group. The Fe\u003csup\u003e2+\u003c/sup\u003e concentration of each group was detected by colorimetry. The results showed that compared with the shNC\u0026thinsp;+\u0026thinsp;vehicle group, the Fe\u003csup\u003e2+\u003c/sup\u003econcentration in the shSLC7A11\u0026thinsp;+\u0026thinsp;vehicle group increased; compared with the shNC\u0026thinsp;+\u0026thinsp;Fine group, the Fe\u003csup\u003e2+\u003c/sup\u003e concentration in the shSLC7A11\u0026thinsp;+\u0026thinsp;Fine group increased, suggesting that the intracellular ferrous ion concentration was significantly reduced after finerenone treatment, but the knockdown of SLC7A11 in cells cancelled this protective effect (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA). The intensity of ferroptosis-related lipid peroxidation was detected by BODIPY 581/591 C11 lipid peroxidation probe. The results showed that compared with the shNC\u0026thinsp;+\u0026thinsp;vehicle group, lipid peroxidation in the shSLC7A11\u0026thinsp;+\u0026thinsp;vehicle group was significantly enhanced, suggesting that the degree of ferroptosis was aggravated; compared with the shNC\u0026thinsp;+\u0026thinsp;Fine group, lipid peroxidation in the shSLC7A11\u0026thinsp;+\u0026thinsp;Fine group was significantly enhanced, suggesting that the degree of ferroptosis was aggravated (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eB). The levels of ferroptosis-related indicators GSH and MDA in each group were detected by ELISA. The results showed that compared with the shNC\u0026thinsp;+\u0026thinsp;vehicle group, the GSH level in the shSLC7A11\u0026thinsp;+\u0026thinsp;vehicle group decreased, while the MDA level increased, suggesting that ferroptosis was aggravated; compared with the shNC\u0026thinsp;+\u0026thinsp;Fine group, the GSH level in the shSLC7A11\u0026thinsp;+\u0026thinsp;Fine group decreased, while the MDA level increased, suggesting that ferroptosis was aggravated (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eC, Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eD).These results suggest that under high glucose conditions, Silencing SLC7A11 can exacerbate ferroptosis in renal tubular epithelial cells and counteract the protective effect of finerenone.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eDN is a chronic disease affecting millions of diabetic patients worldwide. Early diagnosis and prevention are crucial for treating DN, as renal damage becomes difficult to reverse once patients reach the clinical proteinuria stage. Therefore, the development of new treatment methods is of vital importance [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The disease mechanism of DN is complex, with hyperglycemia considered the initiating factor for diabetes-related nephropathy. As the disease progresses, metabolic disorders, hemodynamic abnormalities, inflammation, and fibrosis contribute to the pathological process, leading to structural and functional damage of the kidneys and ultimately resulting in adverse cardiovascular and renal outcomes. Among these, inflammation and fibrosis play a key role in the occurrence and progression of diabetes-related renal injury [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eUltrastructural glomerular damage and proteinuria have long been central to research on the pathogenesis of DN [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, contemporary studies have increasingly focused on renal tubular changes in DN, as clinical signs of renal impairment are closely linked to tubulointerstitial fibrosis and tubular atrophy [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e22\u003c/span\u003e][\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Developing efficient therapies targeting the specific pathophysiological changes in renal tubules remains a significant challenge [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The mineralocorticoid receptor (MR) is a transcription factor that binds to ligands and activates signaling cascades, regulating processes such as water and salt balance, inflammation, fibrosis, apoptosis, and endothelial function [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Current evidence suggests that MR activation may slow the progression of renal dysfunction in patients with chronic kidney disease (CKD) and type 2 diabetes, as well as effectively reduce proteinuria in individuals with non-diabetic CKD [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e26\u003c/span\u003e][\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e27\u003c/span\u003e][\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A study confirmed that finerenone could be an effective treatment option for immunoglobulin A nephropathy (IgAN) patients without diabetes who are receiving conventional treatment, maintaining stable renal function and serum potassium levels [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. MR activation can negatively affect various cell types, including renal tubular epithelial cells, vascular smooth muscle cells, endothelial cells, adipocytes, and inflammatory cells, leading to serious consequences for both the kidneys and cardiovascular system. A recent single-cell sequencing study found that MR expression in the proximal convoluted tubular epithelial cells of diabetic patients was increased compared to the control group [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In addition to inducing changes in gene expression, aldosterone has non-genomic effects in renal tubular epithelial cells, activating protein kinases and second messenger signals. The binding of aldosterone to its receptor also induces oxidative stress, inflammation, and fibrosis. A study explored the mechanism of action of finerenone in HK-2 cells and the kidneys of DN mice under high glucose conditions [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe research found that finerenone restored mitochondrial autophagy in HK-2 cells and renal tubular cells of DN mice under high glucose conditions through the PI3K/Akt/eNOS signaling pathway, while reducing oxidative stress, mitochondrial fragmentation, and apoptosis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These findings highlight the key role of finerenone in improving renal tubular cell injury. Ferroptosis, caused by the excessive accumulation of lipid hydroperoxides in an iron-dependent manner, was also explored [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. xCT/SLC7A11 is a 12-pass transmembrane protein responsible for the primary transport activity, highly specific for cystine and glutamate, while SLC3A2 is a single-pass transmembrane protein with a glycosylated extracellular domain at its C-terminus. SLC3A2 functions mainly as a chaperone protein and is crucial for regulating the stability and transport function of xCT/SLC7A11 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. xCT/SLC7A11 promotes the production of reduced GSH and acts as a cofactor for ROS detoxification enzymes (such as GPX4), converting accumulated lipid hydroperoxides into lipid alcohols. This reduces peroxide-related products and protects cells from ROS-induced damage, inhibiting ferroptosis. Through the GPX4-mediated reaction, GSH is oxidized to its oxidized form (GSSG) and is recycled back to GSH at the expense of NADPH by glutathione reductase (GR) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Iron-dependent programmed cell death, known as ferroptosis, is marked by the accumulation of lipid peroxides [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This phenomenon is regulated by System Xc \u0026minus;, a cystine/glutamate antiporter in the cell membrane, consisting of two components: SLC7A11, which transports cystine and glutamate, and SLC3A2, which ensures the stability of SLC7A11 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Acting as the central component of System Xc \u0026minus;, SLC7A11 supports cellular redox equilibrium by enabling cystine uptake for the synthesis of glutathione (GSH), an essential antioxidant that inhibits lipid peroxidation. This action decreases lipid peroxide concentrations and safeguards cells against ferroptosis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The activity and regulation of SLC7A11, primarily located in the plasma membrane, are shaped by its distinct membrane positioning [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In other diseases, such as hepatocellular carcinoma, Parkinson's disease, and endometriosis, regulating SLC7A11-mediated ferroptosis has shown therapeutic potential [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e40\u003c/span\u003e][\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e41\u003c/span\u003e][\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. However, the exact processes governing its movement and placement on the cell membrane still require further investigation. Since ferroptosis plays a role in the inflammatory and oxidative pathological processes of various diseases, many studies have focused on its involvement in diabetic complications, including DN. It has been found that maintaining iron metabolism balance in renal cells is crucial for normal renal function [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Wu et al. [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e44\u003c/span\u003e] observed enhanced ferroptosis in DN, as indicated by elevated expression of ACSL4, PTGS2, and NOX1, and decreased expression of GPX4. Feng et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e45\u003c/span\u003e] reported that compared with db/m mice, MDA levels were elevated, while those of SOD, CAT, and GSH were decreased in the kidneys of db/db mice. After administering Ferrostatin-1, MDA levels were reduced, SOD levels decreased, CAT levels were lowered, and GSH levels were increased in db/db mice. Renal injury, fibrosis, and lipid protein levels in the urine of db/db mice were also alleviated.\u003c/p\u003e\u003cp\u003eThe results of our experiments in mice showed that compared with db/db mice, the degree of renal tubular atrophy, interstitial fibrosis and inflammatory cell infiltration was reduced in db/db mice treated with 10 mg/kg/d finerenone and db/db mice treated with 30 mg/kg/d finerenone. The levels of ferroptosis-related protein SLC7A11 increased, GSH levels increased and MDA levels decreased, suggesting that the degree of ferroptosis was reduced. After finerenone intervention in HK2 cells under high glucose conditions, the levels of ferroptosis-related proteins FTH1, SLC7A11 and GPX4 increased, the degree of lipid peroxidation decreased, GSH increased and MDA decreased, suggesting that the degree of ferroptosis was reduced. After silencing the SLC7A11, regardless of whether finerenone intervention was given or not, the viability of HK2 cells decreased, cell proliferation weakened, the expression of fibrosis-related genes increased, and the indicators of ferroptosis also worsened(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e ).\u003c/p\u003e\u003cp\u003eIn conclusion, our study identified finerenone ameliorates Diabetic Nephropathy via suppressing SLC7A11-mediated ferroptosis.This finding can deepen our understanding of the pathogenesis of renal tubular injury in diabetic nephropathy and provide a certain theoretical basis for future exploration of new treatment methods. This could provide deeper insights into the complexity of inflammation and help develop more targeted therapeutic strategies. Future studies should focus on integrating these pathways for a better understanding of finereone treatment in DN.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\u003cp\u003eThe research study was authorized by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University and adhered to the principles of the Declaration of Helsinki.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis study was funded by National Natural Science Foundation of China(82370727)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthors'\u0026nbsp;contributionsLu Yu: Writing \u0026ndash; review \u0026amp; editing, Writing \u0026ndash; original draft, Visualization, Validation, Software, Project administration, Methodology, Investigation, Formal analysis, Data curation. Zihan Zhai: Validation, Resources, Project administration, Methodology, Funding acquisition. Yulin Wang: Validation, Project administration, Methodology, Formalanalysis, Data curation, Conceptualization. Liuwei Wang: Validation, Project administration, Investigation, Formal analysis, Data curation. Qiuhong Li: Validation, Project administration, Methodology. Yanhong Guo: Validation, Project administration, Methodology. Rong Gou: Project administration, Methodology. Lin Tang: Writing \u0026ndash; review \u0026amp; editing, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eAcknowledgements The authors thank The First Affiliated Hospital of Zhengzhou University and Research Institute of Nephrology, Zhengzhou University for their assistance and support.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\u003cp\u003eAll data will be made available upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWang L, Peng W, Zhao Z, Zhang M, Shi Z, Song Z, Zhang X, Li C, Huang Z, Sun X et al (2021) Prevalence and Treatment of Diabetes in China, 2013\u0026ndash;2018. JAMA 326:2498\u0026ndash;2506 [CrossRef] [PubMed]\u003c/li\u003e\n\u003cli\u003eTang D, Chen X, Kang R et al (2021) Ferroptosis: molecular mechanisms and health implications. Cell Res 31(2):107\u0026ndash;125\u003c/li\u003e\n\u003cli\u003eLei G, Zhuang L, Gan B (2022) Targeting ferroptosis as a vulnerability in cancer. Nat Rev Cancer 22(7):381\u0026ndash;396\u003c/li\u003e\n\u003cli\u003eJiang X, Stockwell BR, Conrad M (2021) Ferroptosis: mechanisms, biology and role in disease. Nat Rev Mol Cell Biol 22(4):266\u0026ndash;282\u003c/li\u003e\n\u003cli\u003eChen X, Kang R, Kroemer G et al (2021) Broadening horizons: the role of ferroptosis in cancer. Nat Rev Clin Oncol 18(5):280\u0026ndash;296\u003c/li\u003e\n\u003cli\u003eKoppula P, Zhuang L, Gan B (2021) Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy. Protein Cell 12(8):599\u0026ndash;620\u003c/li\u003e\n\u003cli\u003eKoppula P, Zhuang L, Gan B (2021) Cystine transporter SLC7A11/xCT in cancer: ferroptosis, nutrient dependency, and cancer therapy. Protein Cell 12(8):599\u0026ndash;620 doi: 10. 1007/s13238 -020-00789-5. Epub 2020 Oct 1. PMID: 33000412\u003c/li\u003e\n\u003cli\u003eJiao L, Daolin T (2024) Rui, Kang,Targeting GPX4 in ferroptosis and cancer: chemical strategies and challenges[. J] Trends Pharmacol Sci 45:0\u003c/li\u003e\n\u003cli\u003eLiang Z, Guangyu L, Tao Z et al (2025) Palmitoylation of GPX4 via the targetable ZDHHC8 determines ferroptosis sensitivity and antitumor immunity[. J] Nat Cancer 0:0\u003c/li\u003e\n\u003cli\u003eBarrera-Chimal J, Lima-Posada I, Bakris GL, Jaisser F (2022) Mineralocorticoid receptor antagonists in diabetic kidney disease - mechanistic and therapeutic effects. Nat Rev Nephrol 18:56\u0026ndash;70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41581-021-00490-8\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eBarrera-Chimal J, Girerd S, Jaisser F (2019) Mineralocorticoid receptor antagonists and kidney diseases: pathophysiological basis. Kidney Int 96:302\u0026ndash;319. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.kint.2019.02.030\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eNakagawa T (2010) Diabetic nephropathy: aldosterone breakthrough in patients on an ACEI. Nat Rev Nephrol 6:194\u0026ndash;196. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nrneph.2010.32\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eKawanami D et al (2021) Mineralocorticoid receptor antagonists in diabetic kidney disease. Front Pharmacol 12:754239. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphar.2021.754239\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eBakris GL et al (2020) Effect of finerenone on chronic kidney disease outcomes in type 2 diabetes. N Engl J Med 383:2219\u0026ndash;2229. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1056/NEJMoa2025845\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eFilippatos G et al (2021) Finerenone and cardiovascular outcomes in patients with chronic kidney disease and type 2 diabetes. Circulation ygh 143:540\u0026ndash;552. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1161/circulationaha.120.051898\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eRossing P et al (2022) Finerenone in Patients With Chronic Kidney Disease and Type 2 Diabetes According to Baseline HbA1c and Insulin Use: An Analysis From the FIDELIO-DN Study. Diabetes Care 45(4):888\u0026ndash;897\u003c/li\u003e\n\u003cli\u003ePitt B, Filippatos G, Agarwal R, Anker SD, Bakris GL, Rossing P, Joseph A, Kolkhof P, Nowack C, Schloemer P, Ruilope LM (2021) FIGARO-DKD Investigators. Cardiovascular Events with Finerenone in Kidney Disease and Type 2 Diabetes. N Engl J Med 385(24):2252\u0026ndash;2263. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1056/NEJMoa2110956\u003c/span\u003e\u003c/span\u003eEpub 2021 Aug 28. PMID: 34449181\u003c/li\u003e\n\u003cli\u003eZachariah T, Radhakrishnan J (2024) Potential Role of Mineralocorticoid Receptor Antagonists in Nondiabetic Chronic Kidney Disease and Glomerular Disease. Clinical journal of the American Society of Nephrology: CJASN,\u003c/li\u003e\n\u003cli\u003eWheeler DC et al (2021) Effects of dapagliflozin on major adverse kidney and cardiovascular events in patients with diabetic and non-diabetic chronic kidney disease: a prespecified analysis from the DAPA-CKD trial. The lancet. Diabetes Endocrinol 9(1):22\u0026ndash;31\u003c/li\u003e\n\u003cli\u003eSawaf H, Thomas G, Taliercio JJN, GeorgesVachharajani, Tushar JM, Ali (2022) Therapeutic Adv Diabet Nephrop J Clin Med, 11(2)\u003c/li\u003e\n\u003cli\u003eStefansson VTN et al (2022) Molecular programs associated with glomerular hyperfiltration in early diabetic kidney disease. Kidney Int 102:1345\u0026ndash;1358\u003c/li\u003e\n\u003cli\u003eYao L et al (2022) Mitochondrial dysfunction in diabetic tubulopathy. Metab Clin Exp 131:155195\u003c/li\u003e\n\u003cli\u003eMa Z et al (2020) p53/microRNA-214/ULK1 axis impairs renal tubular autophagy in diabetic kidney disease. J Clin Invest 130:5011\u0026ndash;5026\u003c/li\u003e\n\u003cli\u003eLi A et al (2022) Vitamin D-VDR (vitamin D receptor) regulates defective autophagy in renal tubular epithelial cell in streptozotocin-induced diabetic mice via the AMPK pathway. Autophagy 18:877\u0026ndash;890\u003c/li\u003e\n\u003cli\u003eBarrera-Chimal J, Girerd S, Jaisser F (2019) Mineralocorticoid receptor antagonists and kidney diseases: pathophysiological basis. Kidney Int 96:302\u0026ndash;319. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps:// doi.org/10.1016/j.kint.2019.02.030\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003cli\u003eMaryam A, Nadeem MS, Fatima A, Asmat KN (2024) Exploring the potential of fnerenone in non-diabetic chronic kidney disease: a promising frontier. Int Urol Nephrol\u003c/li\u003e\n\u003cli\u003eM\u0026aring;rup FH, Thomsen MB, Birn H (2024) Additive efects of dapaglifozin and fnerenone on albuminuria in non-diabetic CKD: an open-label randomized clinical trial. Clin Kidney J\u003c/li\u003e\n\u003cli\u003eBakris GL, Agarwal R, Anker SD, Pitt B, Ruilope LM, Rossing P, Kolkhof P, Nowack C, Schloemer P, Joseph A, Filippatos G, FIDELIO-DKD Investigators (2020) Efect of fnerenone on chronic kidney disease outcomes in type 2 diabetes. N Engl J Med 383:2219\u0026ndash;2229\u003c/li\u003e\n\u003cli\u003eChen TF-L, Pei S (2025) Effectiveness and safety of finerenone in non-diabetic patients with IgA nephropathy[. J] J Nephrol 0:0\u003c/li\u003e\n\u003cli\u003eWang P (2023) Non-Steroidal Mineralocorticoid Receptor Antagonists (MRAs)-Finerenone Ameliorates Mitochondrial Dysfunction via PI3K/Akt Signaling Pathway in Diabetic Tubulopathy. J Am Soc Nephrol 34(11S):840\u0026ndash;841\u003c/li\u003e\n\u003cli\u003eZi-Han LZ-J, Sun,Sydney CW, Tang et al (2025) Finerenone Alleviates Over-Activation of Complement C5a-C5aR1 Axis of Macrophages by Regulating G Protein Subunit Alpha i2 to. Improve Diabet Nephrop [J] Cells 14:0\u003c/li\u003e\n\u003cli\u003eZhixi L, Yue B, Cheng W et al (2025) Extracellular vesicle-packaged GBP2 from macrophages aggravates sepsis-induced acute lung injury by promoting ferroptosis in pulmonary vascular endothelial cells[. J] Redox Biol 82:0\u003c/li\u003e\n\u003cli\u003eDixon SJ et al (2012) Ferroptosis: An iron-dependent form of nonapoptotic cell death. Cell 149:1060\u0026ndash;1072\u003c/li\u003e\n\u003cli\u003eYang WS et al (2014) Regulation of ferroptotic cancer cell death by GPX4. Cell 156:317\u0026ndash;331\u003c/li\u003e\n\u003cli\u003eJiang X, Stockwell BR, Conrad M (2021) Ferroptosis: Mechanisms, biology and role in disease. Nat Rev Mol Cell Biol 22:266\u0026ndash;282\u003c/li\u003e\n\u003cli\u003eLei G, Zhuang L, Gan B (2022) Targeting ferroptosis as a vulnerability in cancer. Nat Rev Cancer 22(7):381\u0026ndash;396\u003c/li\u003e\n\u003cli\u003eLuo Y, Xiang W, Liu Z et al (2022) Functional role of the SLC7A11-AS1/xCT axis in the development of gastric cancer cisplatin-resistance by a GSH-dependent mechanism, Free Radic. Biol Med 184:53\u0026ndash;65\u003c/li\u003e\n\u003cli\u003eIshimoto T, Nagano O, Yae T et al (2011) CD44 variant regulates redox status in cancer cells by stabilizing the xCT subunit of system xc(-) and thereby promotes tumor growth. Cancer Cell 19(3):387\u0026ndash;400\u003c/li\u003e\n\u003cli\u003eTsuchihashi K, Okazaki S, Ohmura M et al (2016) The EGF receptor promotes the malignant potential of Glioma by regulating amino acid transport system xc(-). Cancer Res 76(10):2954\u0026ndash;2963\u003c/li\u003e\n\u003cli\u003eQiong W, Zongwen L, Jing J et al (2025) Correction: Macrophages originated IL-33/ST2 inhibits ferroptosis in endometriosis via the ATF3/SLC7A11 axis.[J]. Cell Death Dis 16:0\u003c/li\u003e\n\u003cli\u003eZhe Y, Jing L, Wen A et al (2025) EOGT knockdown promotes ferroptosis and inhibits hepatocellular carcinoma proliferation by regulating SLC7A11 via HEY1.[J]. Cell Signal 0:0\u003c/li\u003e\n\u003cli\u003eJinghui X, Xiaofei, He, Lili, Li et al (2025) Voluntary exercise alleviates neural functional deficits in Parkinson's disease mice by inhibiting microglial ferroptosis via SLC7A11/ALOX12 axis.[J].NPJ Parkinsons Dis. 11:0\u003c/li\u003e\n\u003cli\u003eSwelm RPLV, Wetzels JFM, Swinkels DW The multifaceted role of iron in renal health and disease. Nature Reviews Nephrology\u003c/li\u003e\n\u003cli\u003eWu Y et al (2021) HMGB1 regulates ferroptosis through Nrf2 pathway in mesangial cells in response to high glucose. Biosci Rep, 41(2)\u003c/li\u003e\n\u003cli\u003eTiwari B et al (2013) Markers of Oxidative Stress during Diabetes Mellitus. Journal of biomarkers, 2013: p. 378790\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"Diabetic Nephropathy、Nonsteroidal mineralocorticoid receptor antagonist、Finerenone、SLC7A11、ferroptosis","lastPublishedDoi":"10.21203/rs.3.rs-7064578/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7064578/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDiabetic tubulopathy (DT) has recently been identified as a critical pathological feature of diabetic nephropathy (DN). Ferroptosis has emerged as an important pathological factor in DT, implicated in various metabolic disorders, including DN. Finerenone (FIN), a novel non-steroidal mineralocorticoid receptor (MR) antagonist, has demonstrated its ability to mitigate kidney inflammation and fibrosis in DN. However, the exact mechanisms underlying these effects remain unclear. SLC7A11 is known for its role in regulating glutathione (GSH) synthesis, which is closely associated with ferroptosis. To investigate how MR modulates SLC7A11-mediated ferroptosis under diabetic and high glucose (HG) conditions, human kidney proximal tubular epithelial (HK-2) cells were exposed to HG treatment. We assessed COL1, TGF-β, ferroptosis-related markers such as GSH and MDA, and proteins linked to ferroptosis, including FTH1, SLC7A11, and GPX4. Additionally, these molecules and proteins were analyzed in the kidneys of diabetic mice treated with FIN. FIN treatment effectively protected the kidneys by inhibiting SLC7A11-mediated ferroptosis in both HG-exposed HK-2 cells and tubular cells from diabetic mice. In summary, our study confirms that the non-steroidal mineralocorticoid receptor antagonist FIN improves diabetic nephropathy by suppressing SLC7A11-mediated ferroptosis, offering potential therapeutic targets and strategies for kidney disease management while providing insights into the mechanisms of clinical drugs.\u003c/p\u003e","manuscriptTitle":"Non-steroidal mineralocorticoid receptor antagonist finerenone ameliorates Diabetic Nephropathy via suppressing SLC7A11-mediated ferroptosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-29 08:02:03","doi":"10.21203/rs.3.rs-7064578/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":"7ff4c370-0337-4c9e-8237-38e3c6084ee2","owner":[],"postedDate":"July 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-03T13:53:19+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-29 08:02:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7064578","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7064578","identity":"rs-7064578","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}