Method for promoting fatty acid degradation and delaying AKI-CKD conversion

Abstract

The invention relates to the technical field of medical research, in particular to a method for promoting fatty acid degradation and delaying AKI-CKD conversion, which comprises the following steps: a key link of AKI-CKD conversion: peroxisome fatty acid oxidation; the method comprises the following steps of: promoting AKI-CKD transformation by injury of renal tubular epithelial cells (Proximal Tubular Cells, PTCs), and promoting AKI-CKD transformation by injury of the PTCs; the peroxisome FAO affects energy metabolism in PTCs repair; the peroxisome FAO plays a key role in delaying AKI-CKD conversion, and exploration of a regulation and control network mechanism of the peroxisome FAO is very necessary. The application has the advantages that the effect of peroxisome FAO in conversion is focused from the progress characteristic of the AKI-CKD disease, the inhibition of XOR can regulate and control the peroxisome FAO by promoting ABCD3, the regulation and control effect of XOR is preliminarily verified, and a theoretical foundation is laid for subsequently exploring the regulation and control mechanism of XOR; the research preliminarily proves that XOR plays an important role in AKI-CKD disease progression by regulating peroxisome FAO, and provides a new perspective for deeply understanding the pathogenesis of AKI-CKD.

Description

Technical Field

[0001] The present invention relates to the field of medical research technology, and in particular to a method for promoting fatty acid degradation and delaying AKI-CKD transformation. Background Art

[0002] Acute kidney injury (AKI), characterized by a rapid, short-term decline in renal function, is a critical clinical condition with an in-hospital mortality rate as high as 21%. Studies have shown that approximately one-third of hospitalized AKI patients fail to fully recover and eventually develop chronic kidney disease (CKD). Molecular pathological mechanisms include cell cycle arrest, mitochondrial dysfunction, and impaired tubular epithelial cell function. CKD mortality has been increasing annually in recent years and is projected to become the fifth most common cause of death by 2040. Delaying or even blocking the transition from AKI to CKD can improve patients' long-term prognosis and has become a hot topic in the field of nephrology. Therefore, in-depth understanding of the molecular mechanisms of the transition from AKI to CKD and delaying the AKI-CKD transition are of great scientific significance and clinical value. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for promoting fatty acid degradation and delaying AKI-CKD transformation. The method has the advantages of starting from the characteristics of AKI-CKD disease progression, focusing on the role of peroxisomal FAO in transformation, revealing that inhibiting XOR can regulate peroxisomal FAO by promoting ABCD3, and preliminarily confirming the regulatory effect of XOR, laying a theoretical foundation for subsequent exploration of its regulatory mechanism, and solving the problems raised by the above-mentioned background technology.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a method for promoting fatty acid degradation and delaying the transformation of AKI to CKD, the method comprising the following steps:

[0005] S1: A key link in the transition from AKI to CKD: peroxisomal fatty acid oxidation.

[0006] S1.1: Proximal tubular cells (PTCs) damage promotes AKI-CKD transformation.

[0007] S1.2: Peroxisomal FAO affects energy metabolism in PTCs repair.

[0008] S1.3: Peroxisomal FAO plays a key role in delaying the transformation of AKI to CKD, and it is necessary to explore its regulatory network mechanism.

[0009] S2: A key protein regulating peroxisomal FAO: xanthine oxidoreductase.

[0010] S2.1: XOR regulates peroxisomal FAO.

[0011] S2.2: XORI increases ABCD3 protein expression and promotes oxidosomal FAO.

[0012] Furthermore, as a preferred embodiment of the present invention, in step S1.1, the core event of the transition from AKI to CKD is the damage and repair of PTCs. PTCs, as key cells for maintaining renal homeostasis and function, are more susceptible to energy metabolism disorders after AKI injury due to hypoxia, mitochondrial dysfunction, and nutrient sensing pathway disorders. The energy metabolism of PTCs mainly depends on fatty acid β-oxidation (FAO). When AKI occurs, the mitochondrial function in PTCs is severely damaged, inhibiting the FAO pathway. PTCs switch from relying on the FAO mode to relying on the glycolysis mode to make up for the adenosine triphosphate (ATP) required for cell repair. However, the long-term glycolysis mode not only cannot meet the needs of cells, but also produces a large amount of lactic acid, aggravating the lipotoxicity caused by lipid accumulation, forming a cascade damage effect, and further damaging PTCs.

[0013] Furthermore, as a preferred embodiment of the present invention, in step S1.2, FAO mainly occurs in mitochondria and peroxisomes. Peroxisomes are responsible for the initial FAO of very long-chain fatty acids, shortening them to medium-chain fatty acids, which are then transported to mitochondria for further oxidative decomposition, ultimately producing ATP and metabolites, which work together to complete the complete degradation of fatty acids.

[0014] Furthermore, as a preferred embodiment of the present invention, in step S1.3, mitochondrial and peroxisomal FAO are regulated by multiple genes, among which carnitine palmitoyltransferase 1A (CPT1A) is the main translocase of the mitochondrial outer membrane fatty acid shuttle and is the classic mitochondrial FAO rate-limiting enzyme.

[0015] Furthermore, as a preferred embodiment of the present invention, in step S2.1, in order to explore the regulatory mechanism of peroxisome FAO after renal injury, the chip data of renal injury patients in the GEO database were screened, and 6 markers (XOR, KOX-1, ROMO1, NOA1, NF-κB and COX-2) were obtained after merging and removing the differences. It was verified in PTCs that the expression of XOR, KOX-1 and ROMO1 was elevated, among which the inter-group difference of XOR was significant and the intra-group difference was minimal. XOR is a classic purine metabolism rate-limiting enzyme. Under physiological conditions, its expression in renal tissue is very low. It mainly catalyzes the hydroxylation of hypoxanthine to xanthine, and xanthine to uric acid. After renal injury, XOR accelerates the progression of renal tissue damage by promoting the deposition of insoluble uric acid crystals and activating the renin-angiotensin-aldosterone system.

[0016] Furthermore, as a preferred embodiment of the present invention, in step S2.1, XORI was given to mice with UIR-induced AKI-CKD model, and it was found that: no change in blood uric acid levels occurred at 14 days of UIR, and XORI did not change the mRNA levels of purine synthesis and salvage pathway regulatory genes; but renal tissue damage was significantly improved. Enrichment analysis found that the correlation between differentially expressed genes and peroxisomal FAO was higher than that with purine metabolism. Studies have shown that XOR can be activated by FAO and is closely related to liver fatty degeneration, which is consistent in principle with the results of preliminary experiments, but has not yet been revealed in kidney-related diseases. This proves that XORI can play a protective role in AKI-CKD by regulating oxisomal FAO.

[0017] Furthermore, as a preferred embodiment of the present invention, in step S2.2, in order to explore the mechanism by which XORI regulates peroxisomal FAO, studies have found that XORI can increase the peroxisomal FAO regulatory gene ABCD3. ATP-binding cassette sub-family D member 3 (ABCD3) is a key transport protein for maintaining peroxisomal homeostasis and is involved in the transport of multiple fatty acids. ABCD3 gene knockout mice exhibit a metabolic phenotype with increased total cholesterol (CHOL) but unchanged triglycerides. This metabolic feature has a similar pathophysiological mechanism to the clinical indicators of XORI patients. Based on this, it is inferred that XORI can promote peroxisomal FAO by increasing the expression of ABCD proteins.

[0018] Furthermore, as a preferred embodiment of the present invention, although the clinically used XORI drug has shown significant efficacy in lowering serum uric acid levels and treating gout, its effect on blood lipids has not yet been reported. Previously, it was found in the UIR model that XORI can effectively delay the transformation of AKI to CKD, but its mechanism of action is not yet clear. Therefore, it is of great significance to further clarify the molecular mechanism of XOR in the pathophysiological process of the kidney.

[0019] Beneficial effects: The technical solution of the present application has the following technical effects: The present invention has the advantages of starting from the characteristics of AKI-CKD disease progression, focusing on the role of peroxisomal FAO in transformation, revealing that inhibiting XOR can regulate peroxisomal FAO by promoting ABCD3, and preliminarily confirming the regulatory role of XOR, laying a theoretical foundation for subsequent exploration of its regulatory mechanism. This study not only preliminarily confirmed that XOR plays an important role in the progression of AKI-CKD disease by regulating peroxisomal FAO, but also provided a new perspective for a deeper understanding of the pathogenesis of AKI-CKD. This discovery laid a solid theoretical foundation for further exploration of the regulatory mechanism of XOR. Subsequent research can be carried out around the specific signaling pathway of XOR regulating peroxisomal FAO, clarifying the molecular regulatory network between XOR and ABCD3, and the differences in this regulatory process in different renal cell types. At the same time, based on the results of this study, targeted intervention strategies for XOR can also be explored, providing potential targets for the development of new AKI-CKD prevention and treatment drugs, which is expected to bring new hope for improving patients' prognosis and quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0021] Figure 1 This is a hypothetical model diagram of the present invention;

[0022] Figure 2 This is a data graph of the present invention;

[0023] Figure 3 This is the characteristic diagram of AKI-CKD conversion in the present invention;

[0024] Figure 4 This is a diagram showing that the present invention inhibits XOR to delay the progression of kidney disease;

[0025] Figure 5 This is a diagram of XOR regulating peroxisomal FAO in the present invention. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. In order to better understand the technical content of the present invention, specific embodiments are cited and explained in conjunction with the drawings as follows. Various aspects of the present invention are described in this disclosure with reference to the drawings, which show many illustrative embodiments. It should be understood that the various concepts and embodiments introduced above, as well as those described in more detail below, can be implemented in any of many ways. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] As attached Figure 1 To the attached Figure 5 As shown: This embodiment provides a method for promoting fatty acid degradation and delaying the transformation of AKI to CKD, the method comprising the following steps:

[0028] S1: A key link in the transition from AKI to CKD: peroxisomal fatty acid oxidation.

[0029] S1.1: Proximal tubular cells (PTCs) damage promotes AKI-CKD transformation.

[0030] S1.2: Peroxisomal FAO affects energy metabolism in PTCs repair.

[0031] S1.3: Peroxisomal FAO plays a key role in delaying the transformation of AKI to CKD, and it is necessary to explore its regulatory network mechanism.

[0032] S2: A key protein regulating peroxisomal FAO: xanthine oxidoreductase.

[0033] S2.1: XOR regulates peroxisomal FAO.

[0034] S2.2: XORI increases ABCD3 protein expression and promotes oxidosomal FAO.

[0035] Specifically, in step S1.1, the core event in the transition from AKI to CKD lies in the damage and repair of PTCs. As key cells for maintaining renal homeostasis and function, PTCs are more susceptible to energy metabolism disorders after AKI injury due to hypoxia, mitochondrial dysfunction, and nutrient sensing pathway disorders. The energy metabolism of PTCs mainly depends on fatty acid β-oxidation (FAO). When AKI occurs, mitochondrial function in PTCs is severely damaged, inhibiting the FAO pathway. PTCs switch from relying on FAO to relying on glycolysis to make up for the adenosine triphosphate (ATP) required for cell repair. However, long-term glycolysis not only fails to meet cellular needs, but also produces a large amount of lactic acid, exacerbating the lipotoxicity caused by lipid accumulation, forming a cascade damage effect, and further damaging PTCs. Therefore, restoring FAO is a key link in promoting the repair of PTCs damage and rebuilding their physiological functions.

[0036] Specifically, in step S1.2, FAO mainly occurs in mitochondria and peroxisomes. Peroxisomes are responsible for the initial FAO of very long-chain fatty acids, shortening them to medium-chain fatty acids, which are then transported to mitochondria for further oxidative decomposition, ultimately producing ATP and metabolites. The two work together to complete the complete degradation of fatty acids. It was previously believed that mitochondrial FAO was irreplaceable in energy metabolism, but in the study of kidney disease, it was found that treatments that promote mitochondrial function recovery can aggravate renal tissue fibrosis and increase the risk of AKI biomarkers. Some studies have reported that FAO changes occur early after AKI injury, but they did not track the metabolic characteristics of the transition from AKI to CKD. Based on this, the damage and repair mechanisms of FAO need to be further clarified after kidney injury. In order to reveal the metabolic characteristics of the transition from AKI to CKD, a unilateral ischemia-reperfusion model was constructed. The researchers studied the AKI-CKD transition induced by uninjury (UIR) and collected renal tissues at different time points for transcriptome sequencing (RNA-seq). The results showed that FAO was inhibited and ATP decreased significantly after UIR injury. Enrichment analysis found that the changes in differentially expressed genes were closely related to FAO during the 7-14 day UIR period. Localization analysis of the differentially expressed genes revealed that they were highly concentrated in peroxisomes (see Research Basis 1.1), suggesting that peroxisomal FAO plays an important role after 7 days of UIR (i.e., the AKI-CKD transition stage).

[0037] Specifically, in step S1.3, mitochondrial and peroxisomal FAO are regulated by multiple genes, among which carnitine palmitoyltransferase 1A (CPT1A), as the main translocase of the mitochondrial outer membrane fatty acid shuttle, is the classic mitochondrial FAO rate-limiting enzyme. Recent studies have found that in the CKD model, the loss of CPT1A should inhibit mitochondrial FAO, leading to lipid accumulation and aggravating renal tissue damage, but the peroxisomal compensatory increase in FAO did not aggravate renal fibrosis. Peroxisomes are dynamic organelles whose number, morphology, and activity change according to tissue status. Metabolism reported in 2025 that targeted repair of peroxisomal dysfunction is one of the potential therapeutic strategies to slow the progression of renal fibrosis, and JASN reported that oral suberic acid can prevent AKI by increasing peroxisomal FAO. In view of this, the expression of FAO regulatory genes 1 / 3 / 7 / 14 days after UIR was further detected, and the results showed that: UIR At 14 days, peroxisomal FAO regulatory genes (ACOX1 and ABCD3) decreased further compared to 7 days after UIR, but mitochondrial FAO regulatory genes (CPT1A and TFAM) did not show a similar trend (see Research Basis 1.1). This preliminarily confirms that peroxisomal FAO plays a key role in delaying the transformation of AKI to CKD, and it is necessary to explore its regulatory network mechanism.

[0038] Specifically, in step S2.1, in order to explore the regulatory mechanism of peroxisomal FAO after renal injury, the chip data of renal injury patients in the GEO database were screened, and 6 markers (XOR, KOX-1, ROMO1, NOA1, NF-κB and COX-2) were obtained after merging and removing differences. It was verified that the expression of XOR, KOX-1 and ROMO1 was increased in PTCs. Among them, the inter-group difference of XOR was obvious and the intra-group difference was the smallest. XOR is a classic purine metabolism rate-limiting enzyme. Under physiological conditions, its expression in renal tissue is very low. It mainly catalyzes the hydroxylation of hypoxanthine to xanthine, and xanthine hydroxylation to uric acid. After renal injury, XOR accelerates the progression of renal tissue damage by promoting the deposition of insoluble uric acid crystals and activating the renin-angiotensin-aldosterone system. The study found that: 1. In CKD patients with normal uric acid, XOR activity is negatively correlated with renal function; 2. The use of XOR inhibitors (XORI) significantly delayed the decline of renal function in patients (see Research Basis 2.1). Some of the results are in ASN. KidneyWeek presented a poster suggesting that XOR may have renal protective effects independent of purine metabolism, but the mechanism by which it exerts this protective effect is unclear.

[0039] Specifically, in step S2.1, XORI was given to mice with UIR-induced AKI-CKD model, and it was found that: there was no change in blood uric acid levels at 14 days of UIR, and XORI did not change the mRNA levels of purine synthesis and salvage pathway regulatory genes; but it significantly improved renal tissue damage. Enrichment analysis found that the correlation between differentially expressed genes and peroxisomal FAO was higher than that with purine metabolism (see Research Basis 2.1). Studies have shown that XOR can be activated by FAO and is closely related to liver fatty degeneration, which is consistent with the results of preliminary experiments in principle, but has not yet been revealed in kidney-related diseases. This proves that XORI can play a protective role in AKI-CKD by regulating oxisomal FAO.

[0040] Specifically, in step S2.2, to explore the mechanism by which XORI regulates peroxisomal FAO, the study found that XORI can elevate the peroxisomal FAO regulatory gene ABCD3 (see Research Basis 2.2). ATP-binding cassette sub-family D member 3 (ABCD3) is a key transport protein for maintaining peroxisomal homeostasis and is involved in the transport of multiple fatty acids. ABCD3 gene knockout mice exhibit a metabolic phenotype with increased total cholesterol (CHOL) but unchanged triglycerides. This metabolic feature has a similar pathophysiological mechanism to the clinical indicators of XORI patients (see Research Basis 2.1). Based on this, it is inferred that XORI can promote peroxisomal FAO by increasing ABCD protein expression.

[0041] Research content 1: Demonstrate the effect of XOR on AKI-CKD transformation

[0042] (1) XOR participates in AKI-CKD transformation by regulating peroxisomal FAO

[0043] In animal and cell experiments, an AKI-CKD model was constructed after interfering with XOR expression to clarify the impact of XOR on AKI-CKD transformation. Animal experiments used an existing XORI and administered it orally once daily (15 mg / kg) 24 hours after UIR surgery. Mice were sacrificed at different time points and serum, kidney tissue, and urine were collected to detect renal tissue damage, peroxisome function, mitochondrial homeostasis, and changes in tissue metabolites. A hypoxia / reoxygenation (H / R) model was constructed by interfering with XOR protein expression in a human PTCs cell line (HK2) cultured in vitro. Cells were harvested at different time points during reoxygenation to examine the degree of cellular fibrosis, oxisome structure and function, mitochondrial homeostasis, and changes in metabolites.

[0044] (2) XOR regulates ABCD3 protein expression and regulates oxidase FAO

[0045] In animal and cell experiments, XOR was intervened to clarify its relationship with ABCD3, such as the effect on protein expression and colocalization. After intervening XOR expression in PTCs, an AKI-CKD model was established, and ABCD1 / 2 / 3 proteins were detected at different time points to exclude the influence of other homologous transporters. XOR, ABCD3, and peroxisome markers were fluorescently colocalized in cells and tissues. ABCD3 overexpression plasmids and knockdown small interfering RNA (siRNA) were constructed, and the effects of intervening ABCD3 expression on XOR function were examined.

[0046] Key scientific issues to be addressed

[0047] Is XOR regulation of peroxisomal FAO the key to AKI-CKD transformation?

[0048] Previous studies have suggested that XOR promotes disease progression after renal injury by regulating purine metabolism, but it was found that XOR activity is closely related to renal function in CKD patients with normal uric acid levels. Inhibiting XOR expression in the mouse UIR model did not change blood uric acid levels but significantly improved tissue damage, demonstrating that XOR has other ways of affecting kidney injury. Previous studies have found that inhibiting XOR expression can activate peroxisomal FAO by promoting ABCD3 protein expression, promoting fatty acid degradation, and delaying the transformation from AKI to CKD. This study will further clarify the regulatory effect of XOR on peroxisomal FAO based on existing research.

[0049] Example 1: Demonstration of the effect of XOR on AKI-CKD conversion

[0050] This section will examine the effects of intervening in XOR expression on AKI-CKD transformation at the cellular and animal levels.

[0051] Animal experiment: 70 8-week-old, male, SPF-grade C57 / B6J mice were randomly divided into 7 groups. The UIR model was established and XORI 15 μg / mL (MCE, HY-14268) was gavage-treated 24 hours later. The mice were killed on the 1st, 3rd, 7th and 14th days respectively. The urine of the mice was collected in a metabolic cage 1 day in advance after fasting for 8 hours. Serum was collected from the orbital vein when the mice were killed. The renal tissue was collected to make frozen sections for immunofluorescence and other related experiments. Some tissues were fixed with 4% paraformaldehyde to make paraffin sections for pathological evaluation. The ultrastructure of renal tubular peroxisomes was observed by transmission electron microscopy. Some tissues were subjected to Western Blot and qPCR to detect related gene expression.

[0052] Alternative plan: If the mouse UIR model fails or multiple models are verified, the aristolochic acid-induced AKI-CKD model is selected. 5 mg / kg aristolochic acid is injected intraperitoneally for 5 consecutive days, and XORI is administered by gavage on the 7th day. The relevant data of the mice on days 10 and 20 are collected and analyzed.

[0053] Evaluation and testing indicators:

[0054] ①Observe the weight, hair, and mental behavior of mice every day, including food intake, water intake, and feces.

[0055] ② Detect the activities of Scr, BUN, UA, XOR in blood and NGAL, KIM-1, XOR in urine of mice.

[0056] ③Observe tissue damage and fibrosis under an optical microscope (H&E, Masson and Sirius Red staining, etc.).

[0057] ④XOR expression and changes: Western Blot and qPCR were used to detect XOR expression changes, and immunofluorescence was used to detect the co-localization of XOR and the proximal renal tubule marker - Lotus tefouronolobus lectin (LTL).

[0058] ⑤ Peroxisome function assessment: Observe the ultrastructure of the oxisome under transmission electron microscopy to determine whether it is a round or oval organelle with a single membrane structure; detect the number of peroxisomal marker proteins (such as ABCD3 and ACOX2) by immunofluorescence; detect peroxisomal membrane proteins (PMP22) and transport-related proteins ABCD1 / 2 / 3 by Western blot and qPCR; and detect catalase activity.

[0059] ⑥ Mitochondrial function assessment: Western Blot and qPCR detection of mitochondrial CPT1A, CPT1C, etc. expression; tissue detection of total ATP changes, mitochondrial DNA (mtDNA) copy number, mitochondrial membrane potential, etc.

[0060] ⑦ Changes in tissue metabolites: Gas chromatography-mass spectrometry (GC-MS) was used to analyze changes in tissue metabolites, particularly levels of peroxisome-associated metabolites (such as very-long-chain fatty acids, phytanic acid, pristanic acid, and pipecolic acid). Tissue Oil Red O staining was performed to detect changes in tissue lipid metabolism.

[0061] Cell experiments

[0062] XOR overexpression plasmid and control vector plasmid were constructed and verified in advance. HK2 cells were used as the research object. After cell counting, they were plated in six-well plates. After 8 hours of transfection with XOR plasmid, they were hypoxic in a three-gas incubator (1% O2, 5% CO2, 37°C) for 24 hours, then transferred to a normal incubator (21% O2, 5% CO2, 37°C) and reoxygenated for 0 / 12 / 24 hours before the cells were collected. XOR knockdown siRNA and control were constructed. After cell counting, they were plated in six-well plates. After 8 hours of transfection with XOR knockdown siRNA using lipo2000, they were hypoxic for 24 hours, then transferred to a normal incubator for 0 / 12 / 24 hours, and the cells were collected for testing.

[0063] Alternative approach: If the H / R-induced cellular AKI-CKD model fails, HK2 cells can be treated with 200 μM CoCl2 to simulate the effects of hypoxia.

[0064] Evaluation and testing indicators:

[0065] ①Observe the changes in cell morphology and activity under an optical microscope;

[0066] ②PTCs cell function: Western blot, qPCR, and ELISA were used to detect changes in XOR activity and expression; cell damage markers NGAL and KIM-1; inflammatory markers IL-6 and TNF-α; and fibrosis-related markers α-SMA and Fibronectin. Oil Red and Nile Red staining were used to observe changes in intracellular lipid accumulation.

[0067] ③ Peroxisome function assessment: Observe the ultrastructure of oxisomes under transmission electron microscopy; measure the number of oxisomes; detect peroxisomal membrane proteins (PMP22) and transport-related proteins ABCD1 / 2 / 3 by Western blot and qPCR; detect catalase activity; construct a vector (Vector-mCherry-PTS1) containing the fusion of red fluorescent mCherry and peroxisome target signaling protein 1 (PTS1) in advance, transfect it into cells simultaneously with the interference plasmid, and detect the number of peroxisomes by immunofluorescence (see Key Laboratory Techniques for details).

[0068] ④ Mitochondrial function assessment: Western Blot and qPCR were used to detect the expression of mitochondrial CPT1A, CPT1C, etc.; mitochondrial DNA (mtDNA) copy number, mitochondrial membrane potential and cellular oxygen consumption, etc.

[0069] ⑤ Changes in cellular metabolites: Gas chromatography-mass spectrometry (GC-MS) was used to analyze changes in metabolites, especially the levels of peroxisome-related metabolites (such as very long-chain fatty acids, phytanic acid, pristanic acid, and pipecolic acid).

[0070] XOR regulates oxidosomal FAO by regulating ABCD3 protein expression

[0071] ABCD3 overexpression plasmid and knockdown siRNA were constructed in advance, the HK2 cell line was cultured, and the H / R-induced AKI-CKD time point model was constructed after intervening in ABCD3 expression, and the cells were collected for testing.

[0072] HK2 cell lines were cultured and H / R-induced AKI-CKD models were constructed at different time points after XOR intervention. Cells were collected and related indicators were detected.

[0073] HK2 cell lines were cultured and simultaneously overexpressed with XOR and ABCD3 plasmids. H / R-induced AKI-CKD models at different time points were then constructed and the cells were collected for testing.

[0074] Evaluation and testing indicators:

[0075] ① Detect the expression of ABCD1 / ABCD2 / ABCD3 and peroxisome function;

[0076] ② ABCD3 was co-localized with peroxisome fluorescence staining to spatially prove its location.

[0077] For other research indicators, please refer to Section 1.2 Cell Experiment in [Research Plan 1].

[0078] Example 2: Clinical verification of the effect of XOR on renal injury

[0079] Effects of XOR inhibition on peroxidase function in patients

[0080] Patients who used XORIs clinically and met the criteria for the diagnosis of AKI-CKD transformation were screened. Controls were patients who did not use XORI inhibitors, including AKI patients who did not use XORIs and those who used XORIs. Discarded serum and clinical biochemical data of the patients were collected.

[0081] The renal biopsy pathology database was screened, and the control was the normal renal tissue distal to the renal mass removed by surgery. The condition was traced, and the patients were divided into the XORI treatment group and the non-treatment group according to whether they used XORI.

[0082] Evaluation and testing indicators:

[0083] ① Record the patient's baseline characteristics and randomly divide them into groups based on the balance of the patient's baseline characteristics.

[0084] ② Detect the concentrations of very long-chain fatty acids, phytanic acid, pristanic acid and pipecolic acid in serum.

[0085] ③ Immunohistochemistry or immunofluorescence detection of tissue ABCD3 and XOR protein expression changes and their relationship with renal injury.

[0086] Key laboratory technologies

[0087] Peroxisome structure and function detection: 1. Fluorescence localization: A vector fused with m-Cherry (red label) and peroxisome target signaling protein 1 (PTS1) was constructed in advance (Shanghai Sangon Biotechnology Co., Ltd.) and co-transfected with XOR plasmid into PTCs cells (six-well plate, 1.5 μg / plasmid, 3 μg per well, Polyjet 9ul / well), the medium was changed 6 hours after transfection and the cell AKI-CKD model was established. After the cells were harvested, the number of peroxisomes was detected by immunofluorescence (PTS1 protein can be recognized by the cytoplasmic receptor protein PEX5, and then PEX5 interacts with proteins on the peroxisomal membrane); 2. Catalase (CAT) detection: Catalase is a marker enzyme of peroxisomes, and a commercial detection kit is available (Biyuntian, S0051); 3. Peroxisome ultrastructure observation: Renal cortical or renal tubular epithelial cells were fixed with 2.5% glutaraldehyde and 1% osmium acid, and ultrathin sections were cut. The number and structure of peroxisomes in the field of view were directly observed by transmission electron microscopy to determine whether they were round or elliptical. Each sample had more than 5 fields of view, and the number of polyoxisomes and structural damage were counted one by one; 4. Peroxisome FAO function detection in clinical patients: Biochemical detection of plasma very long chain fatty acid concentrations, phytanic acid, pristanic acid, and pipecolic acid concentrations. When peroxisomal FAO is inhibited, the expression of these fatty acids will increase.

[0088] 10x Visium HD spatial transcriptome tissue submission and data analysis: The fresh kidney tissue submitted for inspection needs to be frozen and embedded in OCT in an embedding cassette (Japan Sakura, PN-4583). The tissue section size should be less than 6.5×6.5mm (each embedding cassette can be used for N=3 samples) and then sent to Novogene for testing. The applicant has previously reached a long-term cooperation with Novogene. The subsequent spatial transcriptome data analysis involves complex bioinformatics technologies, mainly including spatial gene expression matrix construction, spatial normalization and dimensionality reduction, spatial clustering and annotation, differential expression analysis and functional enrichment, and spatial pattern visualization.

[0089] Please refer to Figure 2 Figure 1: A: Venn diagram showing the number of overlapping genes between the LASSO logistic regression algorithm and the random forest algorithm (left), followed by mRNA level measurement in TGF-β-induced HK2 cells. B: Correlation study between serum XOR levels and patient eGFR. C: HK2 metabolomics results. **** indicates P < 0.0001, ** indicates P < 0.01.

[0090] 1. A key link in the progression of AKI-CKD: peroxisomal FAO.

[0091] Research basis 1.1: Peroxisomal FAO is suppressed in AKI-CKD

[0092] This part uses an animal AKI-CKD model to find that peroxisomal FAO is further suppressed as UIR progresses from 7 days to 14 days. After establishing a UIR-induced AKI-CKD model, mouse kidney tissue was collected on days 1, 3, 7, and 14 for RNA sequencing. The results showed that compared with the control group, disease progression at day 3 of UIR was closely related to peroxisomes and FAO. During the progression from day 7 of UIR to day 14 of UIR, peroxisomes and FAO played an even more important role, and differentially expressed genes were mainly located in peroxisomes.

[0093] Further testing of the peroxidase FAO-related genes ACOX1 and ABCD3 revealed that peroxisomal FAO-related genes were expressed in the disease state and further decreased from 7 to 14 days after UIR. However, no similar trend was observed for the mitochondrial FAO-related genes CPT1a and TFAM. At the protein level, ABCD3 expression continued to decrease under disease conditions, with a statistically significant difference. At 14 days after UIR, tissue energy was significantly reduced, with a statistically significant difference.

[0094] Please refer to Figure 3 As shown, Figure 3 Characteristic of AKI-CKD transformation. AB: RNAseq sequencing results, KEGG enrichment analysis, and GO cellular component analysis of renal tissue at different time points after UIR model; DG: mRNA levels of ACOX1, ABCD3, CPT1a, and TFAM genes were detected at different time points after UIR model; H: Western Blot detection and semi-quantitative analysis of ABCD3 protein expression in the kidney at different time points; I: ATP detection and analysis of renal tissue at 14 days after UIR, **** indicates P < 0.0001, *** indicates P < 0.001, and * indicates P < 0.05.

[0095] 2. Key protein regulating peroxisomal FAO: XOR

[0096] Research basis 2.1: Inhibiting XOR delays kidney disease progression

[0097] This study demonstrated that inhibition of XOR significantly slowed the progression of renal injury, clinically manifested by lower total cholesterol. Patients treated with XORI were followed up for 18 months. A decrease in serum cholesterol (Scr) greater than 20% compared to baseline was considered significant, while an Scr decrease ≤20%, renal replacement therapy, or death were considered ineffective. The results showed that XOR inhibitors significantly delayed the decline in eGFR, with a statistically significant difference. Co-localization staining of LTL and XOR in renal tissue of mice 14 days after UIR demonstrated that XOR was primarily expressed in the proximal renal tubular epithelium. Biochemical markers in patients treated with XORI for more than six months revealed a statistically significant decrease in total cholesterol levels after XORI. XOR did not increase significantly after one day of UIR, but increased significantly after three days, with a statistically significant difference.

[0098] Please refer to Figure 4 As shown, Figure 4 To inhibit XOR and delay the progression of renal disease, A: ROC analysis of patients who (or did not) receive XOR inhibitors; B: Colocalization staining of LTL and XOR in renal tissue of mice 14 days after UIR; C: Total cholesterol levels in patients who received XORI for more than six months; D: Semi-quantitative analysis of renal XOR protein expression by Western blot at different time points. **** indicates P < 0.0001, and ** indicates P < 0.01.

[0099] Research basis 2.2: XOR participates in the transformation of AKI-CKD by regulating peroxisomal FAO

[0100] This part verifies from mouse and cell experiments that inhibiting XOR increases the peroxisomal FAO regulatory gene ABCD3 and delays the transformation from AKI to CKD.

[0101] Eight-week-old SPF-grade C57 / B6J mice were selected, and the UIR model was established 24 hours later. The mice were given 15 mg / kg of XOR inhibitor daily according to their body weight. The mice were killed at different time points and the required tissues and blood were collected for testing. The results showed that the XORI group significantly alleviated renal tubular swelling on day 3 of UIR, and reduced renal tubular atrophy and tissue structure disorder on day 14 of UIR. RNA seq testing of tissues on day 3 of UIR revealed that XORI mainly exerted its effect by regulating FAO, especially peroxisome-related FAO, suggesting that inhibition of XOR may exert a protective effect through the peroxisome-related fatty acid degradation pathway.

[0102] Western Blot experiments were performed on renal tissues at different time points to detect the expression of related proteins. The results showed that at 3 days after UIR, XORI significantly reduced kidney injury molecule 1 (KIM-1) and increased the expression of peroxisomal membrane protein ABCD3. Similarly, at 14 days after UIR, it reduced the fibrosis protein α-SMA and promoted the expression of ABCD3 protein, and the differences were statistically significant. No increase in blood UA was found in mice killed at 14 days after UIR. At the mRNA level, the XORI group significantly increased the peroxisomal FAO-related proteins ABCD3 and Acox3, but did not affect the mitochondrial FAO regulatory enzyme Cpt1a, the purine synthesis pathway catalytic enzymes PPAT and PAICS, and the purine metabolism salvage pathways APRT and HPRT. After expressing the XOR plasmid, TGF-β-induced pericellular lipid droplet aggregation was aggravated.

[0103] Please refer to Figure 5 , Figure 5 To investigate the regulation of peroxisomal FAO by XOR, A: HE staining of renal tissue at different time points after UIR; B: KEGG analysis of differentially expressed FAO pathways after 3 days of UIR; CE: Western blot analysis of Kim-1, ABCD3, and α-SMA protein expression after 3 days of UIR; F: Serum UA levels in mice; GH: qPCR analysis of ABCD3, ACOX1, CPTA1, PPAT, PAICs, APRT, and HPRT mRNA levels; I: Oil red staining to detect TGF-β-induced lipid droplet accumulation after XOR overexpression. **** indicates P < 0.0001, *** indicates P < 0.001, ** indicates P < 0.01, and * indicates P < 0.05.

[0104] It should be noted that, in this document, relational terms such as first and second, etc. are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations.

[0105] While the present invention has been disclosed above with reference to preferred embodiments, this is not intended to limit the present invention. Persons skilled in the art will readily appreciate that various modifications and variations can be made without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A method for promoting fatty acid degradation and delaying the transformation of AKI to CKD, characterized by: The method comprises the following steps: S1: Key link in the AKI-CKD transition: peroxisomal fatty acid oxidation; S1.1: Proximal tubular cell (PTCs) damage promotes AKI-CKD transformation; S1.2: Peroxisomal FAO affects energy metabolism in PTCs repair; S1.3: Peroxisomal FAO plays a key role in delaying the transition from AKI to CKD, and it is necessary to explore its regulatory network mechanism; S2: Key protein regulating peroxisomal FAO: xanthine oxidoreductase; S2.1: XOR regulates peroxisomal FAO; S2.2: XORI increases ABCD3 protein expression and promotes oxidosomal FAO.

2. The method of claim 1, wherein: In step S1.1, the core event in the transition from AKI to CKD lies in the damage and repair of PTCs. PTCs, as key cells for maintaining renal homeostasis and function, are more susceptible to energy metabolism disorders after AKI injury due to hypoxia, mitochondrial dysfunction, and nutrient sensing pathway disorders. The energy metabolism of PTCs mainly depends on fatty acid β-oxidation (FAO). When AKI occurs, mitochondrial function in PTCs is severely damaged, inhibiting the FAO pathway. PTCs switch from relying on the FAO mode to relying on the glycolysis mode to make up for the adenosine triphosphate (ATP) required for cell repair. However, long-term glycolysis mode not only cannot meet cellular needs, but also produces a large amount of lactic acid, exacerbating the lipotoxicity caused by lipid accumulation, forming a cascade damage effect, and further damaging PTCs.

3. The method of promoting fatty acid degradation and delaying AKI-CKD conversion according to claim 1, characterized in that: In step S1.2, FAO mainly occurs in mitochondria and peroxisomes. Peroxisomes are responsible for the initial FAO of very long-chain fatty acids, shortening them to medium-chain fatty acids, which are then transported to mitochondria for further oxidative decomposition, ultimately producing ATP and metabolites. The two work together to complete the complete degradation of fatty acids.

4. The method of promoting fatty acid degradation and delaying AKI-CKD conversion according to claim 1, characterized in that: In step S1.3, mitochondrial and peroxisomal FAO are regulated by multiple genes, among which carnitine palmitoyltransferase 1A (CPT1A) is the main translocase of the mitochondrial outer membrane fatty acid shuttle and is the classic mitochondrial FAO rate-limiting enzyme.

5. The method for promoting fatty acid degradation and delaying AKI-CKD conversion according to claim 1, characterized in that: In step S2.1, in order to explore the regulatory mechanism of peroxisomal FAO after renal injury, the chip data of renal injury patients in the GEO database were screened, and six markers (XOR, KOX-1, ROMO1, NOA1, NF-κB and COX-2) were obtained after merging and removing differences. It was verified that the expression of XOR, KOX-1 and ROMO1 was elevated in PTCs. Among them, the inter-group difference of XOR was significant and the intra-group difference was minimal. XOR is a classic purine metabolism rate-limiting enzyme. Under physiological conditions, its expression in renal tissue is very low. It mainly catalyzes the hydroxylation of hypoxanthine to xanthine, and the hydroxylation of xanthine to uric acid. After renal injury, XOR accelerates the progression of renal tissue damage by promoting the deposition of insoluble uric acid crystals and activating the renin-angiotensin-aldosterone system.

6. The method of promoting fatty acid degradation and delaying AKI-CKD conversion according to claim 1, characterized in that: In step S2.1, XORI was administered to mice with UIR-induced AKI-CKD model, and it was found that: no change in blood uric acid levels occurred at 14 days of UIR, and XORI did not change the mRNA levels of purine synthesis and salvage pathway regulatory genes; but renal tissue damage was significantly improved. Enrichment analysis found that the correlation between differentially expressed genes and peroxisomal FAO was higher than that with purine metabolism. Studies have shown that XOR can be activated by FAO and is closely related to liver fatty degeneration, which is consistent with the results of preliminary experiments in principle, but has not yet been revealed in kidney-related diseases. This proves that XORI can play a protective role in AKI-CKD by regulating oxisomal FAO.

7. The method of promoting fatty acid degradation and delaying AKI-CKD conversion according to claim 1, characterized in that: In step S2.2, to explore the mechanism by which XORI regulates peroxisomal FAO, studies have found that XORI can increase the peroxisomal FAO regulatory gene ABCD3. ATP-binding cassette sub-family D member 3 (ABCD3) is a key transport protein for maintaining peroxisomal homeostasis and is involved in the transport of multiple fatty acids. ABCD3 gene knockout mice exhibit a metabolic phenotype with increased total cholesterol (CHOL) but unchanged triglycerides. This metabolic feature has a similar pathophysiological mechanism to the clinical indicators of XORI patients. Based on this, it is inferred that XORI can promote peroxisomal FAO by increasing ABCD protein expression.

8. The method of promoting fatty acid degradation and delaying AKI-CKD conversion according to claim 1, characterized in that: Although the clinically used XORI drugs have shown significant efficacy in lowering serum uric acid levels and treating gout, no effect on blood lipids has been found so far.

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