Application of targeting iRhom2-ATF6-PANoptosis signaling pathway in prevention and treatment of acute kidney injury

Abstract

This invention belongs to the fields of biomedicine and molecular biology, specifically relating to the application of targeting the iRhom2-ATF6-PANoptosis signaling pathway in the prevention and treatment of acute kidney injury. Specifically, this invention reveals the crucial role of the iRhom2-ATF6-PANoptosis signaling pathway in regulating pan-apoptosis in TECs cells during acute kidney injury (AKI), providing a novel specific target for AKI treatment. Simultaneously, it utilizes two different intervention methods targeting iRhom2: gene silencing (…). siRNA- Rhbdf2 The combined use of α-carotene and a chemical inhibitor (sulforaphane) synergistically inhibits the iRhom2-ATF6-PANoptosis pathway through different mechanisms, achieving significantly better therapeutic effects than monotherapy. This combination therapy holds promise for development into a highly effective and low-toxicity new drug for the prevention and treatment of AKI, thus possessing significant practical application value.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and molecular biology, specifically relating to the application of targeting the iRhom2-ATF6-PANoptosis signaling pathway in the prevention and treatment of acute kidney injury. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Acute kidney injury (AKI) is a serious clinical complication with rapid onset and progression, boasting high morbidity and mortality rates. It is a significant cause of chronic kidney disease and end-stage renal disease. The pathophysiological mechanisms of AKI are complex, and currently, effective treatments are lacking. Treatment primarily involves symptomatic relief and renal replacement therapy, offering limited improvement in renal function. Therefore, actively exploring the pathogenesis of AKI, identifying specific drug targets, and developing kidney-targeted therapies are of great importance for its prevention and treatment.

[0004] Renal tubular epithelial cell death plays a crucial role in the pathophysiology of acute kidney injury (AKI). Multiple cell death pathways, such as apoptosis, pyroptosis, and programmed necrosis, are involved in TEC damage. Panapoptosis is a unique inflammatory cell death pathway that regulates pyroptosis, apoptosis, and programmed necrosis through panapoptotic bodies. Although studies have shown that panapoptosis is involved in AKI progression, the specific regulatory mechanisms of panapoptosis in AKI remain unclear. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the inventors, through long-term technical and practical exploration, have developed an application of targeting the iRhom2-ATF6-PANoptosis signaling pathway in the prevention and treatment of acute kidney injury (AKI). This invention reveals that the iRhom2 (inactive rhomboid protein 2)-ATF6 signaling pathway plays a crucial role in AKI by regulating pan-apoptosis of renal tubular epithelial cells. iRhom2, encoded by the Rhbdf2 gene, assists in the transport of ATF6 from the endoplasmic reticulum to the Golgi apparatus, promoting ATF6 cleavage and nuclear insertion. This, in turn, upregulates the expression of key pan-apoptotic molecules NLRP3, CHOP, and RIPK1, inducing pan-apoptosis of renal tubular epithelial cells and thus exacerbating AKI. Based on this discovery, this invention provides a pharmaceutical composition targeting the iRhom2-ATF6-PANoptosis signaling pathway for the treatment of AKI. This invention is thus completed based on the above research findings.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides siRNA- Rhbdf2 or siRNA coated with biomimetic liposome nanoparticles for neutrophil membranes- Rhbdf2 Application of combined iRhom2 inhibitors in the preparation of drugs for the prevention and treatment of acute kidney injury.

[0007] A second aspect of the present invention provides a pharmaceutical composition having an active ingredient comprising at least (a) and (b): (a) siRNA- Rhbdf2 or siRNA coated with biomimetic liposome nanoparticles for neutrophil membranes- Rhbdf2 ; (b) iRhom2 inhibitor.

[0008] A third aspect of the invention provides the use of the above-described pharmaceutical composition in any one or more of the following: (a) Synergistically inhibits renal tubular epithelial cell damage; (b) Targeting the iRhom2-ATF6-PANoptosis signaling pathway; (c) Prevention and / or treatment of acute kidney injury.

[0009] A fourth aspect of the present invention provides a method for preventing and treating acute kidney injury, the method comprising: administering the above-described pharmaceutical composition to a subject.

[0010] Compared with existing technical solutions, one or more of the above technical solutions have the following beneficial effects: This invention reveals for the first time the crucial role of the iRhom2-ATF6-PANoptosis signaling pathway in the regulation of pan-apoptosis in TECs cells by AKI, providing a novel specific target for AKI treatment. Simultaneously, the combined application of two different interventions targeting iRhom2—gene silencing (siRNA-Rhbdf2) and a chemical inhibitor (sulforaphane)—synergistically inhibits the iRhom2-ATF6-PANoptosis pathway through different mechanisms, achieving significantly better therapeutic effects than single-therapy. Using neutrophil membrane-inspired biomimetic liposome nanoparticles as siRNA delivery carriers, leveraging the natural inflammatory tropism of neutrophils, targeted accumulation of the drug in renal lesions is achieved. This overcomes the shortcomings of free siRNA, such as easy degradation, low transfection efficiency, off-target effects, and immune clearance by traditional nanomaterials, exhibiting advantages such as immune escape, long circulation, good biocompatibility, and easy renal metabolism. The above combined drug regimen holds promise for development into a highly effective and low-toxicity new drug for the prevention and treatment of AKI, thus possessing significant practical application value. Attached Figure Description

[0011] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0012] Figure 1 This invention demonstrates that iRhom2 expression was significantly elevated in the kidneys of mouse AKI models and clinical ATN patients. ab. Expression patterns of Rhomboid family members Rhbdf1 and Rhbdf2 (b) in published single-cell RNA-seq data of the renal IRI model. c. Cluster analysis heatmap showing the expression of the Rhomboid family (14 members) in the kidneys of AKI mice. d. Fold change of the Rhomboid family (14 members) in the kidneys of AKI mice. e. Representative Western blot images and quantification of iRhom2 expression in the kidneys of sham-operated and IRI mice (n=6). f. Representative IHC images and quantitative statistics of iRhom2 in the kidneys of sham-operated and IRI mice (n=6). g. Representative Western blot images and quantitative analysis of iRhom2 expression in the kidneys of cisplatin-induced AKI model mice (n=4). h. Representative IHC images and quantitative analysis of iRhom2 in the kidneys of cisplatin-induced AKI model mice (n=4). i. Dual immunofluorescence staining of iRhom2 and segment-specific markers of renal tubules in sham-operated and IRI mouse kidneys. Segment-specific tubular markers were used as follows: aquaporin-1 (AQP1) for proximal tubules, calcium-binding protein (D28k) for distal tubules, and aquaporin-3 (AQP3) for collecting ducts. j. Representative IHC images and quantitative analysis of iRhom2 in human normal kidneys and kidneys from patients with biopsy-confirmed acute tubular necrosis (ATN). kl represents the correlation analysis (m, n) between iRhom2 expression and serum blood urea nitrogen (BUN) and serum creatinine (Scr) in biopsy-confirmed ATN patients. All data are presented as mean ± standard deviation. Statistical significance was determined by one-way ANOVA using Tukey's multiple comparison test (bd) and two-tailed unpaired Student's t-test (e). P <0.05, P <0.01, P <0.0001.

[0013] Figure 2 For the renal tubule specificity in the embodiments of the present invention Rhbdf2Construction and identification of knockout (CKO) mice. a. Generation of tubule-specific mice using the Cre-loxP recombinant system. Rhbdf2 Knockout mice ( KSP -Cre + / Rhbdf2 fl / fl a. The experimental protocol. b. Genotyping was confirmed at 2 weeks of age using PCR and agarose gel electrophoresis. c. Rhbdf2 fl / fl and KSP Cre + / Rhbdf2 fl / fl Relative mRNA levels of Rhbdf2 in the kidneys of the group (n=6). d. Representative protein blots and statistical data, showing... Rhbdf2 fl / fl and KSP -Cre + / Rhbdf2 fl / fl Protein levels of iRhom2 in the kidneys of group (n=4). e. Double immunofluorescence staining of iRhom2 and segment-specific markers of renal tubules after renal IRI. The segment-specific renal tubular markers used are as follows: aquaporin-1 (AQP1) for proximal tubules, calcium-binding protein (D28k) for distal tubules, and aquaporin-3 (AQP3) for collecting ducts. P<0.0001.

[0014] Figure 3 For the renal tubule specificity in the embodiments of the present invention Rhbdf2 Knockout alleviates ischemia-reperfusion-induced AKI. a. Schematic diagram showing the experimental procedure. b. SCr levels in different groups of mice (n=6). c. BUN levels in different groups of mice (n=6). d. Representative images of kidney H&E staining and quantitative assessment of renal tubular injury in different groups of mice (n=6). e. Representative images and IHC staining quantification of KIM-1 in the kidneys of different groups of mice (n=6). f. In situ TUNEL assay and quantification to assess renal cell death in the kidneys of different groups of mice (n=6). g. Neutrophil (Ly6B) staining and data analysis (number of cells per high-power field [HPF]) in representative kidney sections from different groups of mice. h. Macrophage (CD68) staining and data analysis (number of cells per high-power field [HPF]) in representative kidney sections from different groups of mice. i. mRNA levels of pro-inflammatory mediators (including TNF-α, IL-18, IL-6, monocyte chemoattractant protein-1 (MCP-1), and IL-1β) in the kidneys of mice from different groups. All data are presented as mean ± standard deviation. Statistical significance was determined by two-way ANOVA (bi). P <0.01, P <0.001, P <0.0001.

[0015] Figure 4 Renal tubule-specific knockout in this embodiment of the invention Rhbdf2 To alleviate cisplatin-induced acute kidney injury. a. SCr levels in mice from different groups (n=6). b. BUN levels in mice from different groups (n=6). c. Statistical graphs of H&E staining and quantitative assessment of renal tubular injury in mice from different groups (n=6). d. Representative images and quantitative statistical graphs of KIM-1 IHC staining in the kidneys of mice from different groups (n=6). e. Representative images and quantitative statistical graphs of in situ TUNEL assays in mice from different groups to assess renal cell death in the kidneys of mice from different groups. All data are presented as mean ± standard deviation. Statistical significance was determined by two-way ANOVA (ah) (n=6). P <0.001, P <0.0001.

[0016] Figure 5 As described in the embodiments of the present invention Rhbdf2 Renal tubule-specific knockout improves AKI by inhibiting panapoptosis. a. Volcano plot analysis shows KSP-cre after renal IRI. + / Rhbdf2 fl / fl and Rhbdf2 fl / fla. Differentially expressed genes (DEGs) in the kidneys of different groups. b. Top enrichment pathway in GO analysis. c. Representative Western blots of PANoptosis-related molecules in the kidneys of different groups, including apoptosis (Caspase-3, Caspase-7, and Caspase-8), pyroptosis (Caspase-1, GSDMD-FL, GSDMD-N, GSDME-FL, and GSDME-N), and necrosis (MLKL, p-MLKL). d. Representative TEM images of kidneys from different mouse groups. e. Representative IHC staining images of Caspase-3, GSDMD-N, and p-MLKL in kidney biopsies from healthy controls and patients with biopsy-confirmed ATN. Correlation analysis of iRhom2 expression with GSDMD-N (f), Caspase-3 (g), and p-MLKL (h) in patients with biopsy-confirmed ATN. One-way ANOVA of two-tailed unpaired Student t-test (c). P <0.05. P <0.01, P <0.001.

[0017] Figure 6 In this embodiment of the invention, H / R treatment promoted pan-apoptosis and inflammation in HK-2 cells. a. Western blot and quantification of representative proteins expressing iRhom2 in different groups of HK-2 cells (n=6). b. PI staining of different groups of HK-2 cells. c. LDH release in different groups of HK-2 cells (n=6). d. Western blot and quantification of representative proteins expressing Caspase-7, Caspase-8, Caspase-3, Caspase-1, GSDMD, GSDMD-N, GSDME, GSDME-N, p-MLKL, and MLKL in different groups of HK-2 cells. e. Real-time PCR detection of mRNA levels of IL-1β, IL-18, IL-6, TNF-α, and MCP-1 in different groups of HK-2 cells. Statistical significance was determined by one-way ANOVA using Tukey's multiple comparison test (a, b) and two-way ANOVA test (d, e, k). P <0.05, P <0.01, P <0.0001.

[0018] Figure 7 In this embodiment of the invention, ATF6 is a novel iRhom2 interacting protein. a. Schematic diagram of immunoprecipitation and mass spectrometry analysis using iRhom2 antibody after H / R treatment of HK-2 cells. b. Schematic diagram of GO enrichment analysis pathway. c. Network analysis of iRhom2-regulated genes. d. Analysis of iRhom2-ATF6 interaction predicted by αfold3. e. Interactive 2D plot of prediction alignment error (PAE) for iRhom2 and ATF6. f. Immunofluorescence staining showing co-localization of iRhom2 and ATF6 proteins in H / R treated HK-2 cells. iRhom2 (green), ATF6 (red), and DAPI (blue). g. Co-IP assay showing the interaction between iRhom2 and ATF6 in different groups of HK-2 cells.

[0019] Figure 8 In this embodiment of the invention, iRhom2 assists in the transport of ATF6 from the endoplasmic reticulum to the Golgi apparatus, promoting ATF6 cleavage activation. a. Western blot detection of ATF6(P) and ATF6(N) expression in the kidneys of different groups of mice (N=6). b. Western blot detection of ATF6(P) and ATF6(N) expression in HK-2 cells of different groups. c. Immunofluorescence staining of ATF6, Golgi apparatus, endoplasmic reticulum, and nuclei in HK-2 cells of different groups. ATF6 (purple), Golgin-97 (red), Calnexin (green), and DAPI (blue). d. Western blot and quantification of representative proteins expressing ATF6(N) in HK-2 cells of different groups. Statistical significance was determined by two-way ANOVA (a, b, d). P <0.05, P <0.01, P <0.001, P <0.0001.

[0020] Figure 9In this embodiment of the invention, iRhom2 promotes pan-death cell death via ATF6. a. PI staining of HK-2 cells in different groups. b. LDH release in HK-2 cells in different groups (n=6). c. Western blot detection of the expression of Caspase-7, Caspase-8, Caspase-3, Caspase-1, GSDMD, GSDMD-N, GSDME, GSDME-N, p-MLKL, and MLKL in HK-2 cells in different groups. d. TEM images of different HK-2 cell groups. e. qpcr detection of Xbp1, Chop, and HSP90 mRNA levels in different HK-2 cell groups. f. Western blot and quantification of representative proteins expressing NLRP3, Ripk1, and Hsp90 in different HK-2 cell groups. Statistical significance was determined by one-way ANOVA using Tukey's multiple comparison test (a, b) and two-way ANOVA test (h, i). P <0.05, P <0.01, P <0.05.001, P <0.0001.

[0021] Figure 10 To illustrate how sulforaphane alleviates renal tubular injury (IRI) in mice in this embodiment of the invention. a. Schematic diagram of animal experiments (n=8). b. SCr levels in different groups of mice (n=8). c. BUN levels in different groups of mice (n=8). d. Representative images of H&E staining and quantitative assessment of renal tubular injury in different groups of mice (n=8). e. Representative images of KIM-1 in the kidneys of different groups of mice and quantitative analysis of IHC staining. f. In situ TUNEL assay and statistical analysis (n=8).

[0022] Figure 11 The neutrophil membrane biomimetic nanoparticles in this embodiment of the invention encapsulate siRNA-Rhbdf2 (MLip-NPs-siRNA- Rhbdf2 This reduces IRI damage in mice by inhibiting iRhom2. a. Neutrophil membrane biomimetic nanoparticles encapsulate siRNA- Rhbdf2 Schematic diagram. b. Representative images of particle size, hydrodynamic dimensions (c), and surface zeta potential (d) detected by transmission electron microscopy. e. Examination of MLip-NP-siRNA using in vivo imaging instruments. Rhbdf2f. Scr levels in different groups of mice (n=6). g. BUN levels in different groups of mice (n=6). h. Representative images of H&E staining and quantitative assessment of renal tubular injury in different groups of mice (n=6). i. Representative images and quantification of Cleaved caspase-3, GSDMD-N, and p-MLKL IF staining in the kidneys of different groups of mice (n=6). All data are presented as mean ± standard deviation. Statistical significance was determined by two-way ANOVA (gl). P <0.01, P <0.001, P <0.0001.

[0023] Figure 12 The siRNA in the embodiments of the present invention- Rhbdf2 Combined use with sulforaphane reduced renal tubular epithelial cell damage. a. Flow cytometry analysis of HK-2 cells in different groups (n=3). b. PI staining of HK-2 cells in different groups (n=3).

[0024] Figure 13 In this embodiment of the invention, iRhom2 induces panapoptosis of renal tubular cells through the ATF6 signaling pathway, which aggravates AKI damage. siRNA-Rhbdf2, sulforaphane, and their combined application can prevent and treat AKI by inhibiting this pathway. Detailed Implementation

[0025] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0026] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0027] In a typical embodiment of the present invention, siRNA- Rhbdf2 or siRNA coated with biomimetic liposome nanoparticles for neutrophil membranes- Rhbdf2 Application of combined iRhom2 inhibitors in the preparation of drugs for the prevention and treatment of acute kidney injury.

[0028] The iRhom2 inhibitor can be sulforaphane.

[0029] In this invention, the siRNA- Rhbdf2 The nucleotide sequence is: 5'-GATGCCCAAGATTGTGGAT-3' (SEQ ID NO.1).

[0030] In this invention, the siRNA-coated neutrophil membrane biomimetic liposome nanoparticles are... Rhbdf2 Its preparation method includes: Protamine and siRNA- Rhbdf2 The mixture is mixed to form core nanoparticles; DOTAP and CHOL are prepared into liposomes; after the liposomes are mixed and incubated with the core nanoparticles, neutrophil membranes are added, and the mixture is extruded to obtain the final product.

[0031] The nanoparticles have a particle size of less than 200 nm.

[0032] This invention demonstrates through experiments that the application of the iRhom2 inhibitor sulforaphane inhibits the iRhom2-ATF6 signaling pathway, and that the combined application of sulforaphane and siRNA-Rhbdf2 can achieve a synergistic effect.

[0033] In another specific embodiment of the present invention, a pharmaceutical composition is provided, wherein the active ingredients of the pharmaceutical composition include at least (a) and (b): (c) siRNA- Rhbdf2 or siRNA coated with biomimetic liposome nanoparticles for neutrophil membranes- Rhbdf2 ; (d) iRhom2 inhibitor.

[0034] The iRhom2 inhibitor is sulforaphane.

[0035] The siRNA- Rhbdf2 The mass molar ratio of the iRhom2 inhibitor to the inhibitor is 5-50:1 (preferably 25:1, g / mol).

[0036] The pharmaceutical composition may also include at least one inactive pharmaceutical ingredient.

[0037] The inactive components of the drug can be pharmaceutically commonly used carriers, excipients, and diluents. Furthermore, according to conventional methods, it can be formulated into oral, topical, suppository, and sterile injectable solutions such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and sprays.

[0038] The non-pharmaceutical active ingredients that may be included, such as carriers, excipients, and diluents, are well known in the art, and those skilled in the art can determine that they meet clinical standards.

[0039] In another specific embodiment of the invention, the drug of the invention can be administered into the body by known means. For example, it can be delivered to the tissue of interest via intravenous systemic delivery or local injection. Alternatively, it can be administered via intravenous, percutaneous, intranasal, mucosal, or other delivery methods. Such administration can be performed via a single dose or multiple doses. Those skilled in the art will understand that the actual dose to be administered in the invention can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0040] In another specific embodiment of the present invention, the use of the above-described pharmaceutical composition in any one or more of the following: (a) Synergistically inhibits renal tubular epithelial cell damage; (b) Targeting the iRhom2-ATF6-PANoptosis signaling pathway; (c) Prevention and / or treatment of acute kidney injury.

[0041] Specifically, the synergistic inhibition of renal tubular epithelial cell damage is manifested in the synergistic reduction of renal tubular epithelial cell death mediated by hypoxia / reoxygenation.

[0042] In another specific embodiment of the present invention, a method for preventing and treating acute kidney injury is provided, the method comprising: administering the above-mentioned pharmaceutical composition to a subject.

[0043] The present invention will be further illustrated below with specific examples. These examples are for illustrative purposes only and do not limit the scope of the invention. Experimental conditions not specifically specified in the examples are generally performed under conventional conditions or as recommended by the sales company; unless otherwise specified in the present invention, these conditions are commercially available.

[0044] Example Test methods 1.1 Construct a mouse AKI pathological model and detect changes in iRhom2 expression. 1.1.1 Establishment of an animal model of renal ischemia-reperfusion injury and tissue collection (1) All experimental protocols for animal research were approved by the Ethics Committee of Shandong University (ECSBMSSDU2017-2-005).

[0045] (2) Construction of an animal model of renal ischemia-reperfusion injury Eight-week-old male C57BL / 6J wild-type mice were randomly assigned to groups. Mice were anesthetized with 0.3% sodium pentobarbital and placed on a 37°C heated blanket. The abdominal cavity was opened along the linea alba to expose both kidneys, and the renal pedicles were clamped with microarterial clamps for 30 minutes. At the end of the ischemic period, the microarterial clamps were removed (reperfusion), and the kidneys were observed for 5 minutes to ensure the reperfusion process. Levofloxacin hydrochloride was instilled to prevent infection, and the incision was sutured. Renal cortex was harvested at 6, 12, 24, and 48 hours after reperfusion, and again at 72 hours to establish an animal model of renal ischemia-reperfusion injury.

[0046] (3) Methods for obtaining renal cortex Mice were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital. After the anesthesia took effect, the retinal venous plexus was severed, and blood was collected using a 1.5 ml EP tube. After standing at room temperature for 2 hours, the blood was centrifuged at 3000 rpm for 10 minutes to collect serum. The heart was perfused with pre-cooled physiological saline using a perfusion apparatus, while the mouse kidneys were gently massaged with fingers until both the kidneys and liver turned yellowish-white. The kidneys were harvested and cut in half along the medial center. One half was frozen at -80°C, and the other half was immersed in 4% paraformaldehyde.

[0047] 1.1.2 Detection of iRhom2 expression in a renal ischemia-reperfusion injury model (1) Single-cell RNA-seq (scRNA-seq) data analysis: scRNA-seq data of mouse IRI were obtained from GSE139107.

[0048] (2) Analysis of whole-genome microarray results mRNA was extracted from kidney tissues of the sham group and I / R 24h in the renal ischemia-reperfusion injury model and sent to Sinotech Genomics Corporation. After quality control, second-generation transcriptome sequencing analysis was performed, and heatmaps were generated.

[0049] (3) RT-qPCR detection Rhbdf1 and Rhbdf2 mRNA expression at different time points after ischemia-reperfusion.

[0050] Renal cortex from the same location in the kidneys of mice in each group was collected in 2.0 ml grinding tubes. mRNA was extracted from the kidneys using the Trizol method, and the mRNA was reverse transcribed into cDNA using the Evo M-MLV reverse transcription kit. RT-qPCR was then used for detection. Rhbdf1 and Rhbdf2 β-Actin was used as an internal control to measure mRNA expression at different time points after ischemia-reperfusion.

[0051] (4) Western blot analysis of iRhom2 protein expression at different time points after ischemia-reperfusion. Renal cortex from the same location in the kidneys of mice in each group was collected into 2.0 ml grinding tubes. A mixture of RIPA (strong) protein lysis buffer and PMSF (100:1) was added, and the mixture was homogenized. After lysis on ice for 30 min, the mixture was centrifuged at 4°C, 12000 rpm / min for 15 min. The supernatant was collected, and protein concentration was determined by BCA method for protein quantification. Western blotting was used to detect the protein expression of iRhom2 at different time points after ischemia-reperfusion.

[0052] (5) Immunohistochemistry was used to detect the protein expression distribution of iRhom2 at different time points after ischemia-reperfusion. Kidney tissue that had been soaked in 4% paraformaldehyde for 24 hours was embedded in paraffin, cut into 2μm paraffin sections, and immunohistochemistry was used to detect the protein expression distribution of iRhom2 at different time points after ischemia-reperfusion.

[0053] (6) Immunofluorescence assay to detect tissue localization of iRhom2 at different time points after ischemia-reperfusion. The paraffin sections were subjected to a fluorescence double labeling experiment with iRhom2 and the proximal convoluted tubule (AQP1), distal convoluted tubule (Calbindin D28K), and collecting duct (AQP3).

[0054] 1.1.3 In vitro hypoxia injury model to detect iRhom2 expression 1.1.3.1 In vitro simulation of renal ischemia-reperfusion injury stimulation (1) H / R stimulation HK-2 cells were cultured in six-well cell culture plates. When the cell density was about 60%, the cells were washed with PBS and replaced with EBSS medium. The cells were then placed in a hypoxic incubator with 1% O2 and 5% CO2 for 2 hours. The control group was placed in a normal cell culture incubator. After the time was up, the cells were replaced with DMEM-F12 medium with 10% FBS and placed back in a normal incubator for 12 hours, 24 hours, and 48 hours of further culture.

[0055] (2) RT-qPCR detection Rhbdf1 and Rhbdf2 mRNA expression under in vitro stimulation Remove the six-well cell culture plate from the 37°C incubator, discard the culture medium, wash the cells with PBS, extract mRNA using the Trizol method, perform reverse transcription, and detect by RT-qPCR. Rhbdf1 and Rhbdf2 The expression of mRNA under in vitro stimulation.

[0056] (3) RT-qPCR detection of mRNA expression of inflammatory factors in vitro mRNA was extracted from each H / R stimulation group using the Trizol method, reverse transcribed, and then RT-qPCR was used to detect the mRNA expression of inflammatory factors such as TNF-α, MCP-1, IL-6, IL-1β, and IL-18.

[0057] (4) Western blot analysis of iRhom2 protein expression under in vitro stimulation Remove the six-well cell culture plate from the 37°C incubator, discard the culture medium, wash the cells with PBS, add a mixture of RIPA (weak) protein lysis buffer and PMSF (100:1), scrape off the cells, and extract the protein supernatant. Protein concentration was measured and quantified using the BCA method, and Western blotting was used to detect iRhom2 protein expression under in vitro stimulation.

[0058] 1.1.4 Construct a cisplatin-induced acute kidney injury model and detect iRhom2 protein expression. (1) Methods for establishing an animal model of cisplatin-induced acute kidney injury Ten-week-old male C57BL / 6J mice were randomly divided into two groups. The model group was injected intraperitoneally with cisplatin at a dose of 25 mg / kg, while the control group was injected with physiological saline. The acute kidney injury model was established on days 1, 2, and 3 after injection.

[0059] (2) Detection of iRhom2 expression in a cisplatin-induced acute kidney injury model RT-qPCR, WB, and IHC were used to detect changes in iRhom2 expression and tissue distribution at different time points after cisplatin injection.

[0060] 1.1.5 Detection of iRhom2 expression changes in clinical sections of patients with acute tubular necrosis Renal biopsy pathological section samples were collected with the approval of the Ethics Committee of Shandong University (document number ECSBMSSDU2017-1-002), and were obtained from the Department of Pathology, School of Basic Medical Sciences, Shandong University. Histological examination and biochemical analysis confirmed acute tubular injury, and no other features of kidney disease were found (specimen information is shown in Table 1). Control pathological section samples were obtained from individuals who underwent tumor nephrectomy and had no AKI or other kidney disease. IHC was used to detect the expression of iRhom2 in clinical sections of acute tubular necrosis.

[0061] 1.2 Tubular-specific knockout of Rhbdf2 reduces AKI-induced renal damage through ATF6-induced pan-apoptosis. 1.2.1 Renal tubular epithelial cell-specific knockout Rhbdf2 Mouse construction Build using Cre-loxp system cdh16-cre + / - / Rhbdf2fl / fl ( KSP-cre + / Rhbdf2 fl / fl Mice carrying the loxp site Rhbdf2 homozygous female mice ( Rhbdf2 fl / fl ， Purchased from Shanghai Southern Model Center) and cdh16-cre + / - Male mice (purchased from the Shanghai Southern Modeling Center) were mated to produce the first generation F1, whose genotype was identified as follows: cdh16-cre + / - / Rhbdf2 fl / + The mice continued to be with Rhbdf2 fl / fl Mouse breeding to obtain renal tubular epithelial cell-specific knockout Rhbdf2 mice, i.e. cdh16-cre + / - / Rhbdf2 fl / fl .and cdh16-cre + / - / Rhbdf2 fl / fl The later breeding of mice uses cdh16-cre + / - / Rhbdf2 fl / fl and Rhbdf2 fl / fl Mating pattern, offspring from the same litter cdh16-cre - / - / Rhbdf2 fl / fl It can be used as a control group to extract mouse tail DNA for mouse genotyping.

[0062] 1.2.2 In vivo validation of the role of iRhom2 in ischemia-reperfusion-induced acute kidney injury (1) Construction of mouse model and material collection 8-week-old males Rhbdf2 fl / fl Rhbdf2 - / - and KSP-cre / Rhbdf2 fl / fl Mice were randomly divided into groups, and kidney cortex was collected 24 hours after ischemia-reperfusion. The modeling method was the same as above.

[0063] (2) Measure the serum creatinine and blood urea nitrogen levels. The levels of serum creatinine and blood urea nitrogen in a renal ischemia-reperfusion model were detected using a fully automated biochemical analyzer.

[0064] (3) HE staining detection Rhbdf2 fl / fl Rhbdf2 - / - and KSP-cre / Rhbdf2 fl / fl Morphological damage to mouse kidney tissue Mouse kidney paraffin sections were baked, dewaxed and hydrated, stained with hematoxylin for nuclei, differentiated with 1% hydrochloric acid alcohol, blued with 1% ammonia water, stained with eosin for cell morphology, dehydrated, and mounted with neutral resin.

[0065] (4) PAS staining detection Rhbdf2 fl / fl Rhbdf2 - / - and KSP-cre / Rhbdf2 fl / fl Morphological damage to mouse kidney tissue Mouse kidney paraffin sections were baked, dewaxed and hydrated, oxidized with an oxidant at room temperature in the dark, reacted with Schiff base at room temperature in the dark, stained with hematoxylin, dehydrated in a gradient, and mounted with neutral resin.

[0066] (5) IHC detection of Kim-1 expression in each group of mice Mouse kidney paraffin sections were baked, dewaxed and hydrated, antigen retrieval was performed, catalase was blocked, sheep serum was blocked at 37 degrees Celsius, Kim-1 (1:50) primary antibody was added and incubated overnight at 4 degrees Celsius, reaction enhancement solution was used to enhance the reaction, universal histochemical secondary antibody was incubated at 37 degrees Celsius, DAB staining was performed, hematoxylin was stained, gradient dehydration was performed, and neutral resin was used for mounting.

[0067] (6) TUNEL staining was used to detect renal tubular apoptosis in mice in each group. Mouse kidney paraffin sections were baked, dewaxed and hydrated, proteinase K antigen was removed, TUNEL staining solution was added, and the sections were incubated at 37 degrees Celsius in the dark for 1 hour. The nuclei were stained with DAPI, mounted with mounting medium, and fixed with nail polish.

[0068] (7) IHC detection of renal inflammatory infiltration Paraffin-embedded tissue sections were subjected to IHC to detect renal infiltration by macrophages and neutrophils. Macrophages were labeled with CD68, and neutrophils were labeled with ly6B.

[0069] (8) RT-qPCR detection of mRNA expression of inflammatory factors in vivo mRNA was extracted from the kidneys of mice in each group using the Trizol method, reverse transcribed, and then RT-qPCR was used to detect the mRNA expression of inflammatory factors such as TNF-α, MCP-1, IL-6, IL-1β, and IL-18.

[0070] 1.2.3 Elucidate the role of iRhom2 in cisplatin-induced acute kidney injury. (1) Construction of mouse model and material collection Select 10-week-old males Rhbdf2 fl / fl and Rhbdf2 - / - Mice were randomly divided into two groups: the model group was injected intraperitoneally with cisplatin at a dose of 25 mg / kg, and the control group was injected with physiological saline. Samples were collected 2 days later.

[0071] (2) Measure the serum creatinine and blood urea nitrogen levels. The levels of serum creatinine and blood urea nitrogen in a cisplatin-induced acute kidney injury model were detected using a fully automated biochemical analyzer.

[0072] (3) 2.3.3 HE staining detection of cisplatin-stimulated groups Rhbdf2 fl / fl Rhbdf2 - / - Morphological damage to mouse kidney tissue The staining method is the same as above.

[0073] (4) PAS staining detection of cisplatin-stimulated groups Rhbdf2 fl / fl Rhbdf2 - / - Morphological damage to mouse kidney tissue The staining method is the same as above.

[0074] (5) IHC detection of Kim-1 in cisplatin-stimulated groups Rhbdf2 fl / fl Rhbdf2 - / - Expression in mice The staining method is the same as above.

[0075] 1.2.4 iRhom2 regulates the transport of ATF6 from the endoplasmic reticulum to the Golgi apparatus, promoting its cleavage and nuclear translocation, upregulating the expression of panapoptotic molecules RIP1 and NLRP3, inducing panapoptosis of renal tubular epithelial cells, and exacerbating AKI. (1) In vivo and in vitro experiments to detect the regulation of ATF6 activation from endoplasmic reticulum to Golgi translocation by iRhom2. In vivo experiments used Western blotting (WB) to detect changes in ATF6 signaling pathway molecules induced by ischemia-reperfusion injury after tubular-specific knockout of iRhom2. In in vitro experiments, after silencing iRhom2, WB was used to detect changes in endoplasmic reticulum signaling molecules under H / R stimulation. Simultaneously, co-immunoprecipitation (CO-IP) was used to detect the presence of an interaction between iRhom2 and ATF6.

[0076] (2) Immunofluorescence colocalization experiment to investigate the process by which iRhom2 assists ATF6 in translocation from the endoplasmic reticulum to the Golgi apparatus under H / R stimulation in vitro.

[0077] Calnexin-labeled endoplasmic reticulum and Golgin-97-labeled Golgi apparatus were used in an immunofluorescence co-localization experiment with iRhom2 to investigate whether iRhom2 undergoes a translocation process from the endoplasmic reticulum to the Golgi apparatus under in vitro simulated renal ischemia-reperfusion.

[0078] (3) siRNA-mediated gene silencing Small interfering RNA (siRNA) was transfected into HK-2 cells using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA). Targeted Rhbdf2 siRNA sequence: Rhbdf2 siRNA (5'-GATGCCCAAGATTGTGGAT-3-3'). siRNA targeting the ATF6 sequence: ATF6 siRNA (5'-GACCCAAGACUCAAACAATT-3'). A control sequence was used where no known mammalian gene homologous sequences were detected.

[0079] (4) Flow cytometry detection of cell death Cell death was determined by staining for APC-bound annexin V and 7-AAD. Data were analyzed using the CytExpert program (both from Beckman Coulter, Inc.).

[0080] (5) Lactate dehydrogenase (LDH) release test LDH release from the supernatant of HK-2 cells was detected using an LDH cytotoxicity assay kit (Beyotime).

[0081] (6) Real-time imaging of cell death Propidium iodide (PI; Sigma, St. Louis, MO, USA) incorporation was used to detect cell death.

[0082] (7) Western blot and IHC detection of the expression of key molecules of panapoptosis in HK-2 cells, AKI kidney specimens and ATN clinical specimens Pan-apoptotic related proteins: apoptosis (Caspase-3, Caspase-7, and Caspase-8), pyroptosis (Caspase-1, Gsdmd, and Gsdme), and programmed necrosis (p-Mlkl). (8) Electron microscopy was used to observe the morphological changes of AKI kidney and HK2 cells.

[0083] 1.3 Exploration of the role of inhibiting the iRhom2-ATF6 pathway in the treatment of AKI 1.3.1 Study on the therapeutic effect of iRhom2 inhibitor sulforaphane on AKI (1) Sulforaphane (Sigma, S4441), 12.5 mg / kg, was administered to mice via intraperitoneal injection seven times, starting five days before renal ischemia and continuing until 24 hours post-surgery, with injections every 24 hours. (2) The ischemia-reperfusion AKI model was prepared as before. Serum creatinine and blood urea nitrogen (BUN) levels were measured, and HE staining, Kim1 and TUNEL staining were performed. 1.3.2 Neutrophil membrane biomimetic nanoliposome particles encapsulating siRNA- Rhbdf2 Study on the therapeutic effect of AKI (1) Collection and lysis of neutrophils Neutrophils were isolated according to the experimental procedures provided in the Mouse Bone Marrow Neutrophil Extraction Kit (Solabio). Neutrophils cultured in T75 flasks were collected under aseptic conditions by centrifugation (250g, 5min). Each flask of collected cells was washed twice with PBS, then 1 mL of hypotonic cell lysis buffer (containing PMSF) was added, and the cells were lysed on ice for 30 min. The cells were homogenized and centrifuged at 700g for 10 min at 4 °C. The supernatant was collected, followed by centrifugation at 14000g for 30 min at 4 °C. The cell membrane pellet was collected, resuspended in PBS, and stored at -80 °C for later use. Membrane protein concentrations were determined using the BCA method for quantification before use.

[0084] (2) Preparation of biomimetic nanoparticles for neutrophil membranes Take an appropriate amount of protamine (1 mg / ml) and siRNA- Rhbdf2 (20uM) Vortex mixing yields core nanoparticles. DOTAP and CHOL (molar ratio 1:1) are dissolved in chloroform and thoroughly mixed. The organic solvent is removed by rotary evaporation under reduced pressure. The liposomes are obtained by ultrasonic hydration with DEPC water and ultrasonication in a water bath. The liposomes are then mixed with the nanocores and incubated in a 40°C water bath for 30 minutes. A certain mass of neutrophil membrane (liposome:membrane (mass ratio) = 2:1) is added. The liposomes are extruded more than 10 times through a liposome extruder until the extruder syringe is smooth, thus obtaining neutrophil membrane biomimetic nanoparticles.

[0085] (3) Characterization of biomimetic nanoparticles for neutrophil membranes The morphology of the samples prepared in the above process was observed using transmission electron microscopy (TEM). 10 μL each of Lip, Lip-NP, and MLip-NP were dropped onto a copper grid, allowed to stand for one minute, and then excess liquid was absorbed with filter paper. The samples were photographed after the copper grid was completely dry. The particle size distribution and zeta potential of Lip, Lip-NP, and MLip-NP were measured using a particle size potential analyzer (DLS).

[0086] (4) Neutrophil biomimetic nanoparticle-coated siRNA- Rhbdf2 Treatment of AKI Contains 2OD siRNA- Rhbdf2 Neutrophil-inspired nanoparticles were dissolved in 100 μL of physiological saline and administered via tail vein injection 1 hour before ischemia.

[0087] 1.3.3 siRNA- Rhbdf2 Combined use with sulforaphane reduces H / R-induced HK-2 cell death. Small interfering RNA (siRNA-) Rhbdf2 HK-2 cells were transfected with Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA) at a concentration of 500 ng / ml. After 6 h of transfection, the transfection solution was removed, and the cells were treated with sulforaphane (20 μM) H / R for 24 h before being collected for analysis.

[0088] 1.4 Statistical Analysis Statistical analysis was performed using GraphPad Prism 8.0 software. Data are expressed as mean ± standard deviation (Mean ± SD). Spearman's test was used to statistically analyze the correlation between two variables. For normally distributed data, a two-tailed unpaired Student's t-test was used for comparisons between two groups; for non-normally distributed data, the Mann-Whitney rank-sum test was used. One-way ANOVA was used to determine differences between multiple groups with one variable, followed by the Tukey test. Two-way ANOVA and the Tukey test were used to compare multiple groups with multiple variables. For data with a non-Gaussian distribution, nonparametric statistical analysis was performed using the Kruskal-Wallis test, followed by multiple comparisons using the Dunn test. P <0.05、 P <0.01、 P <0.001、 P <0.0001.

[0089] Test results 1) iRhom2 expression was significantly increased in the kidneys of mouse AKI models and clinical ATN patients: Analysis of published single-cell RNA-seq data from mice with renal ischemia / reperfusion injury (IRI) revealed that among the 14 members of the Rhomboid family... Rhbdf2 Specific upregulation in renal tubules ( Figure 1 a, 1b), consistent with the analysis of transcriptome sequencing results of ischemia-reperfusion kidneys (a, 1b). Figure 1 cd). Western blot ( Figure 1 e) and immunohistochemistry ( Figure 1 f) The results also confirmed that iRhom2 expression was upregulated in ischemia-reperfusion-induced AKI. iRhom2 expression was also increased in the kidneys of cisplatin-induced AKI mice, indicating that iRhom2 upregulation is a common feature of AKI. Figure 1 To further pinpoint the sites of increased iRhom2 expression during AKI, immunofluorescence double staining was performed using iRhom2 (green) and various tubule markers (red). The results showed that iRhom2 expression was increased in the proximal tubule, distal tubule, and collecting tube. Figure 1 i). Clinical specimen biopsy confirmed upregulation of iRhom2 expression in the kidneys of patients with acute tubular necrosis (ATN). Figure 1 j), iRhom2 expression and serum urea nitrogen level (R2 = 0.5725, Figure 1 k), serum creatinine level (R2 = 0.6635, Figure 1 The results showed a positive correlation with the expression of iRhom2 in the renal tubules during AKI, suggesting that it may play a key role in AKI.

[0090] 2) Renal tubule-specific knockout Rhbdf2 Mouse construction Cdh16-cre recombinant system constructs renal tubule-specific Rhbdf2 Knockout mice ( KSP- cre + / Rhbdf2 fl / fl () Figure 2 a) to verify the role of iRhom2 expressed in renal tubules in AKI. This was achieved through gene identification ( Figure 2 b) Rhbdf2 mRNA expression ( Figure 2 c), iRhom2 Western blot ( Figure 2 d) and iRhom2 (green) with various renal tubular markers (red) immunofluorescence double labeling ( Figure 2 e) Confirmation of renal tubule-specific knockout Rhbdf2 Mouse model successfully constructed. Renal tubule specificity. Rhbdf2The knockout mice had a normal phenotype, with no obvious defects in kidney morphology and function.

[0091] 3) Rhbdf2 Renal tubule-specific knockout improves AKI Using an ischemia-reperfusion and cisplatin-induced AKI model, renal tubule-specific... Rhbdf2 Knockout mice were used to verify the role of iRhom2 expressed in renal tubules in acute kidney injury (AKI). (Animal experimental diagram) Figure 3 a) Compared with the control group Rhbdf2 fl / fl compared to KSP- cre + / Rhbdf2 fl / fl mouse serum creatinine ( Figure 3 b) and blood urea nitrogen (BUN) Figure 3 c) Decreased, HE results showed that the morphological damage of renal tubular edema, brush border loss, and nucleus loss caused by renal IRI was significantly improved, and immunohistochemical staining of the renal tubular injury marker KIM-1 showed reduced renal tubular injury. Figure 3 d, e) and TUNEL staining results showed a significant reduction in cell death ( Figure 3 f), The above results suggest that specific renal tubule knockout Rhbdf2 It can significantly improve renal IRI. It can also reduce renal inflammatory response and decrease the infiltration of neutrophils and macrophages. Figure 3 g, h) and inflammatory factors (g, h) Figure 3 i) expression.

[0092] In a cisplatin-induced AKI model, renal tubule specificity Rhbdf2 Knockout can also reduce kidney damage. Figure 4 ae).

[0093] 4) Rhbdf2 Renal tubule-specific knockout improves AKI by inhibiting panapoptosis Analysis of mouse kidney IRI transcriptome sequencing results, IR / Rhbdf2 fl / fl With IR / KSP- cre + / Rhbdf2 fl / fl There were 322 differentially expressed genes between the groups ( P <0.05, |log2(FC)|≥1), during which 63 genes were upregulated and 259 genes were downregulated ( Figure 5 a). IR / KSP -Cre + / Rhbdf2 fl / fl Mice and IR / Rhbdf2fl / fl Comparative GO enrichment pathway analysis of mouse transcriptomes revealed the greatest differences in intracellular protein transport pathways, followed by inflammation and apoptosis pathways, indicating renal tubule-specific knockout. Rhbdf2 It may alleviate AKI by regulating intracellular protein transport, cell death, and inflammation. Figure 5 b). Apoptosis, programmed necrosis, and pyroptosis, collectively known as panapoptosis, represent a novel pro-inflammatory programmed cell death pathway. Western blot analysis detected renal IRI in mice (IR / KSP -Cre + / Rhbdf2 fl / fl Mouse) pan-apoptosis-related proteins (apoptosis (Caspase-3, Caspase-7 and Caspase-8)) Figure 5 c) Pyrodeation (Caspase-1, Gsdmd, and Gsdme) Figure 5 e) and programmed necrosis (p-Mlkl) Figure 5 f) expression was significantly reduced; IR / KSP -Cre + / Rhbdf2 fl / fl Morphological characteristics of panapoptosis in mouse kidney electron microscopy sections (mainly manifested as nuclear pyroptosis, cell swelling (necrosis), membrane pore formation (pyroptosis), and formation of macrovesicles from the plasma membrane (pyroptosis)) compared with IR / Rhbdf2 fl / fl The number of mice decreased significantly ( Figure 5 d). Immunohistochemical results showed that the expression of Caspase-3, GSDMD-N, and p-Mlkl was increased in the kidneys of IRI mice and ATN patients. Figure 5 e), iRhom2 expression in the kidneys of ATN patients is related to Caspase-3 (R 2 = 0.6139, Figure 5 f), GSDMD-N (R) 2 = 0.8268, Figure 5 g) or p-Mlkl (R 2 = 0.5830, Figure 5 There is a positive correlation between h). The above results suggest that iRhom2 promotes pan-apoptosis of renal tubular epithelial cells, exacerbating AKI.

[0094] 5) iRhom2 promotes pan-apoptosis and inflammation in HK-2 cells. In vitro experiments have shown that H / R (hypoxia / reoxygenation) treatment of the human renal tubular epithelial cell line HK-2, Rhbdf2 Increased expression levels of mRNA and iRhom2 protein ( Figure 6 a). PI staining ( Figure 6b) Lactate dehydrogenase (LDH) release detection Figure 6 c) The results showed that siRNA-R hbdf2 Knock down Rhbdf2 Expression significantly reduced HK-2 cell death after H / R treatment. Western blot results showed that siRNA- Rhbdf2 The expression of caspase-7, caspase-8, and caspase-3 (apoptosis), gasdermin D (GSDMD), gasdermin E (GSDME), caspase-1 (pyroptosis), and p-MLKL (programmed necrosis) was reduced in HK-2 cells. Figure 6 d). After H / R treatment, the mRNA expression levels of IL-1β, IL-18, IL-6, TNF-α, and MCP-1 in siRNA-Rhbdf2 HK-2 cells were significantly reduced. Figure 6 e). The above in vitro results suggest that iRhom2 can regulate pan-apoptosis and inflammation in renal tubular epithelial cells.

[0095] 6) ATF6 is a novel iRhom2 interacting protein. To explore the mechanism by which iRhom2 regulates pan-apoptosis and inflammation in TECs, HK-2 cells were treated with H / R and then subjected to immunoprecipitation and mass spectrometry analysis using iRhom2 antibody. Figure 7 a). GO enrichment analysis revealed that intracellular protein transport, apoptosis, and endoplasmic reticulum stress are important regulatory pathways of iRhom2 ( Figure 7 b). Based on the function of iRhom2 and the results of transcriptome and mass spectrometry analysis, iRhom2 interacting proteins were screened, and ATF6 (activating transcription factor 6) was identified as a novel iRhom2 binding molecule. Figure 7 c). Through alpha fold3 ( Figure 7 d) Using ChimeraX software analysis of the hydrogen bonds between iRhom2 and ATF6, we found that the Rhomboid domain of iRhom2 is mainly bound to the 3′ end or middle region of the entire length of ATF6, consistent with the results of AE analysis. Figure 7 e), ATF6 is an iRhom2 interacting protein. Furthermore, immunofluorescence showed partial co-localization of endogenous iRhom2 and ATF6 in H / R-treated HK-2 cells. Figure 7 f). Co-IP results showed that H / R treatment enhanced the interaction between endogenous ATF6 and iRhom2 in HK-2 cells (f). Figure 7 g).

[0096] 7) iRhom2 assists in the transport of ATF6 from the endoplasmic reticulum to the Golgi apparatus, promoting ATF6 cleavage activation. Whether iRhom2 is involved in ATF6 activation was investigated using Western blot analysis. The results showed that after Rhbdf2 tubule-specific knockout, the expression of cleaved ATF6 (ATF6-p50) was significantly reduced. Figure 8 a). In vitro results showed that Rhbdf2 knockdown significantly inhibited H / R-induced ATF6 cleavage into the nucleus ( Figure 8 b, 8c). Confocal microscopy results showed that in the control group, iRhom2 and ATF6 were mainly co-localized in the endoplasmic reticulum. After H / R stimulation, iRhom2 and ATF6 translocated from the endoplasmic reticulum to the Golgi apparatus. Inhibiting the expression of Rhbdf2 significantly inhibited the translocation of ATF6 from the endoplasmic reticulum to the Golgi apparatus. Figure 8 c). To further determine the mechanism by which iRhom2 mediates ATF6 cleavage activation, Western blot results showed that the addition of the protein transport inhibitor Brefeldin A (BFA) affected iRhom2-induced ATF6 cleavage. Figure 8 d) The above results indicate that iRhom2 assists ATF6 in its transport from the endoplasmic reticulum to the Golgi apparatus, promoting the cleavage activation and nuclear translocation of ATF6.

[0097] 8) iRhom2 promotes pan-apoptosis via ATF6 The study further explored the mechanism by which iRhom2 regulates apoptosis. PI staining ( Figure 9 a) LDH release detection ( Figure 9 b) It was found that ATF6 gene silencing significantly reduced cell death in HK-2 cells induced by Rhbdf2 overexpression; Western blot results showed that the expression of key molecules involved in panapoptosis, including Caspase-7, Caspase-8, Caspase-3 (apoptosis), GSDMD, GSDME, Caspase-1 (pyroptosis), and p-MLKL (programmed necrosis), was significantly reduced. Figure 9 c). Further examination of the expression of Xbp1, Chop, and HSP90 was conducted to explore the mechanism by which iRhom2 promotes the formation of pan-apoptotic bodies. Figure 9 e). Experimental results show that iRhom2 upregulates NLRP3, CHOP, and RIPK1 expression via ATF6 ( Figure 9 i). These results indicate that iRhom2 promotes panapoptotic body formation and induces panapoptosis in renal tubular epithelial cells by regulating the expression of NLRP3, CHOP, and RIPK1 through ATF6.

[0098] 9) Exploration of the inhibitory effect of the iRhom2-ATF6 pathway in the treatment of AKI Sulforaphane is an iRhom2 inhibitor ( Figure 10a) The application of sulforaphane can significantly reduce serum creatinine in ischemia-reperfusion AKI (a) Figure 10 b) and blood urea nitrogen (BUN) Figure 10 c) Levels, reducing kidney damage ( Figure 10 d, e) and cell death ( Figure 10 f). The above studies suggest that sulforaphane may be a potential drug for the treatment of AKI.

[0099] Preparation of neutrophil membrane biomimetic liposome nanoparticles coated with siRNA- Rhbdf2 (M-Lip NPs siRNA- Rhbdf2 () Figure 11 a Scanning electron microscopy and dynamic light scattering analysis results showed that M-Lip-NPs siRNA- Rhbdf2 Average diameter less than 200 nm Figure 11 b and 11c), uniform dispersion ( Figure 11 d). In vivo imaging results showed that, compared with the Lip NPs group, 24 hours after tail vein injection, Cy5-labeled siRNA- Rhbdf2 MLip NPs significantly accumulate in the kidneys ( Figure 11 e), which may be related to the targeting of neutrophil membrane-mediated inflamed tissues. Injection of siRNA- Rhbdf2 Following MLipNP, renal IRI was significantly reduced ( Figure 11 hi).

[0100] After exploring the effects of monotherapy, this invention focuses on siRNA- Rhbdf2 (SEQ ID NO.1) was used in combination with sulforaphane to explore the prevention and treatment of AKI, and flow cytometry was used. Figure 12 a) and PI ( Figure 12 (b) The results showed that the combined use of these two treatments significantly reduced H / R-induced cell death compared to using either treatment alone, producing a synergistic effect. These findings suggest that the combined use of these two treatments could be a potential therapeutic option for AKI.

[0101] The above results indicate that inhibiting the iRhom2-ATF6 pathway may be developed into an effective target for the prevention and treatment of AKI.

[0102] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. (a) siRNA- Rhbdf2 or siRNA coated with biomimetic liposome nanoparticles for neutrophil membranes- Rhbdf2 The application of combination (b) iRhom2 inhibitor in the preparation of drugs for the prevention and treatment of acute kidney injury; wherein the iRhom2 inhibitor is sulforaphane; wherein the siRNA- Rhbdf2 The nucleotide sequence is: 5'-GATGCCCAAGATTGTGGAT-3' (SEQ ID NO.1).

2. The application as described in claim 1, characterized in that, The siRNA-coated neutrophil membrane biomimetic liposome nanoparticles Rhbdf2 Its preparation method includes: Protamine and siRNA- Rhbdf2 The mixture is mixed to form core nanoparticles; DOTAP and CHOL are prepared into liposomes; after the liposomes are mixed and incubated with the core nanoparticles, neutrophil membranes are added, and the mixture is extruded to obtain the final product.

3. The application as described in claim 1, characterized in that, The nanoparticles have a diameter of less than 200 nm.

4. A pharmaceutical composition, characterized in that, The active ingredients of the pharmaceutical composition include at least (a) and (b): (a) siRNA- Rhbdf2 or siRNA coated with biomimetic liposome nanoparticles for neutrophil membranes- Rhbdf2 ; (b) iRhom2 inhibitors; The iRhom2 inhibitor is sulforaphane; the siRNA- Rhbdf2 The nucleotide sequence is: 5'-GATGCCCAAGATTGTGGAT-3' (SEQ ID NO.1).

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition further includes at least one inactive pharmaceutical ingredient.

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