Application of uridine in drug development and drugs for preventing and treating diabetic nephropathy

Abstract

The application relates to application of uridine in drug development and drugs for preventing and treating diabetic kidney disease. Through clinical metabolomics identification and animal experiment verification, it is confirmed that uridine is a key active metabolite of DKD, the therapeutic effect of uridine has the characteristics of multi-target and tissue specificity, and a new solution for metabolic drug research and development based on DKD is provided; through pharmacodynamic research and protection mechanism exploration of uridine, various application scenarios of uridine in preventing and / or reducing drug research and development of patients with diabetic kidney disease are further explored, the application of uridine in DKD drugs and drug research and development is more extensive, and a broader prospect is provided for DKD drug research and development.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the application of a clinical diagnostic biomarker—uridine—in the development of drugs for the prevention and treatment of diabetic nephropathy and in those drugs. Background Technology

[0002] A recent report from the International Diabetes Federation indicates that approximately 537 million adults worldwide had diabetes in 2021, a number projected to rise to 783 million by 2045. Diabetic kidney disease (DKD) is the most common complication of type 2 diabetes and a leading cause of end-stage renal disease globally, with an incidence rate now reaching 31.3%. Extensive research confirms that the occurrence and development of DKD involve multiple interdependent risk factors and metabolic pathways, such as metabolic disorders caused by hyperglycemia, hypertension, and hyperlipidemia, and the activation of inflammation, oxidative stress, and apoptosis induced by the renin-angiotensin-aldosterone system (RAAS) and advanced glycation end products (AGEs). Despite the introduction of various therapies and medications in clinical practice, renal function, due to the kidney's "metabolic memory," has not been adequately improved. Therefore, improving diagnosed DKD remains an unmet medical need.

[0003] Metabolomics, by detecting metabolites, is considered the closest representation of an individual, revealing the complex metabolic networks of diseases and providing unique metabolic profiles of disease development. The kidneys, as highly metabolic organs, have complex interactions with circulating metabolites. Numerous metabolomics studies have been used to identify potential biomarkers in various kidney diseases, such as amino acid metabolism, hormonal imbalances, and the TCA cycle. Furthermore, increasing research demonstrates the crucial role of endogenous metabolites in maintaining health, disease progression, and drug activity. Functional metabolites can influence all levels of the omics landscape, mediating physiological and pathological changes in the body; for example, gut microbial metabolites, fatty acids, amino acids, and bile acids. Therefore, diagnosing and treating diseases from a metabolic perspective is becoming an important strategy, simultaneously meeting the needs of drug development and clinical practice. Summary of the Invention

[0004] Based on the above problems and background, this invention provides the application of uridine in the development of drugs for the prevention and treatment of diabetic nephropathy and in these drugs, as detailed below:

[0005] Application of uridine in the development of drugs for the prevention and / or treatment of diabetic nephropathy.

[0006] Preferably, the use of uridine in the preparation of drugs for the prevention and / or treatment of diabetic nephropathy.

[0007] Preferably, the use of uridine in the preparation of drugs for the prevention and / or reduction of kidney damage in patients with diabetic nephropathy.

[0008] Furthermore, the diabetic nephropathy mentioned refers to type II diabetic nephropathy.

[0009] Furthermore, the uridine exerts its effect by influencing the expression of two core genes, EGR1 and COL1A2.

[0010] Furthermore, the uridine can regulate or improve lipid metabolism disorders in patients with diabetic nephropathy.

[0011] Furthermore, the uridine can inhibit renal peroxidation damage and regulate renal cell activity.

[0012] Furthermore, the uridine acts on the mitochondria of renal cortical cells, specifically by inhibiting EGR1-P300-ETS1 and regulating mitochondrial function to treat diabetic nephropathy.

[0013] Furthermore, the renal cortical cells are specifically the proximal convoluted tubule epithelial cells of the human renal cortex.

[0014] Furthermore, the drug for preventing and / or reducing diabetic nephropathy, or the drug development process, also includes pharmaceutically acceptable excipients or drug carriers, and the dosage form of the drug for preventing and / or reducing diabetic nephropathy includes tablets, capsules, soft capsules, granules, powders, pills, gel candies, powders, oral liquids, or drops.

[0015] The advantages of this invention are:

[0016] (1) This invention, through clinical metabolomics identification and animal experiments, confirms that uridine is a key active metabolite of DKD, and its therapeutic effects have the characteristics of multiple targets and tissue specificity, providing a new solution for the development of metabolic drugs based on DKD.

[0017] (2) Through pharmacodynamic studies and investigation of protective mechanisms, this invention further explores multiple application scenarios of uridine in the drug development process for the prevention and / or reduction of diabetic nephropathy, making the application of uridine in DKD drugs and drug development more extensive, and providing a broader prospect for the subsequent DKD drug development.

[0018] (3) The uridine proposed in this study can improve cell morphology, increase cell activity and number, effectively reduce cell apoptosis rate, and significantly weaken inflammatory response. It has a significant regulatory effect in the application of DKD drugs and drug development process, and is especially suitable for early to mid-stage DKD patients, which can delay the progression of the disease to end-stage renal disease. Attached Figure Description

[0019] Figure 1 Metabolomics sample distribution of healthy individuals vs. diabetic nephropathy (DKD) (PCA / OPLS-DA);

[0020] Figure 2 Core differential metabolites in high glucose (HG) and uridine interventions (volcano diagram);

[0021] Figure 3 High glucose-induced differential metabolite pathway enrichment (KEGG).

[0022] Figure 4 Pyrimidine / fatty acid metabolic remodeling under uridine intervention (pathway diagram);

[0023] Figure 5 Diagnostic value of differentially metabolites in DKD (ROC analysis);

[0024] Figure 6 The therapeutic effect of uridine on DKD mice (pathology + biochemistry): Figure a is a schematic diagram of the uridine intervention experiment in DKD mice; b is the kidney mass index of each group of mice (n=6-7); ce is the serum urea nitrogen, creatinine and uric acid levels of each group of mice (n=6-7); f is the fasting blood glucose level of each group of mice (n=6-7); gh is the oral glucose tolerance test (OGTT) results of each group of mice (n=6-7); i is the stained micrograph of kidney tissue sections of each group of mice (200×) (n=3); jm is the blood lipid biochemical index (n=6-7); np is the kidney tissue oxidative stress index (n=6-7).

[0025] Figure 7 Molecular mechanisms of uridine (WB + mitochondrial function): Figures ae and e are pathway validation diagrams, showing that HG upregulates EGR1 / P300 / ETS1 (promoting fibrosis), and uridine inhibits P300 / ETS1 by knocking down EGR1 (shEGR1), thus blocking COL1A2 expression; Figures 7f-i are related to mitochondrial protection: uridine is located in mitochondria (Figure 7f), restoring ATP (↑50%), membrane potential (JC-1 red / green ratio ↑), and cristae structure (electron microscopy figure 7i), echoing the fatty acid metabolism repair in Figure 4; Figures j (ROS fluorescence imaging) and k (flow cytometry quantification of ROS) are diagrams of the inhibitory effect of oxidative stress.

[0026] Figure 8Effects of uridine on HK-2 cell apoptosis (Annexin V / PI + apoptosis proteins): Figure a shows the protective effect of different concentrations of uridine on damaged cells; Figure b shows the cell morphology of different groups under a microscope; Figure c shows the relative cell counts of different groups; Figure d shows the flow cytometry analysis of HK-2 cell apoptosis; Figure e shows the detection of nuclear apoptosis morphology by Hoechst 33342 staining, scale bar: 125 µm; Figure f shows the detection and quantitative statistics of apoptosis-related proteins (Casp-3, Bax and Bcl-2) in HK-2 cells by Western blotting.

[0027] Figure 9 Volcano plots of differentially expressed molecules in cell extracts from high glucose / control / uridine treatments and bar charts of differential molecular pathway enrichment analysis based on cell extracts; Figures ab show volcano plots of transcriptomic analysis of uridine protecting HK-2 cells from high glucose-induced damage (n=3); Figure c shows Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes regulated by uridine.

[0028] Figure 10 Volcano plots of differentially expressed molecules in cell lysates treated with high glucose, control, and uridine, and bar charts of differential molecular pathway enrichment analysis based on cell extracts. Figure ab: Volcano plot of proteomics analysis of uridine protecting HK-2 cells from high glucose-induced damage (n=3); Figure c: Kyoto Encyclopedia of Genetics and Genomes (KEGG) pathway analysis of differentially expressed proteins regulated by uridine. Data are expressed as mean ± standard deviation (mean ± SD). Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the content of the present invention is not limited to the following embodiments. Unless otherwise specified, the materials, reagents, etc., used in the embodiments and experimental examples of the present invention can be obtained commercially; unless otherwise specified, the methods used in the embodiments and experimental examples of the present invention are conventional methods.

[0031] Example 1: Clinical metabolomics experiment for diabetic nephropathy

[0032] 1. Inclusion and Exclusion Criteria

[0033] This study complies with the Declaration of Helsinki and has been registered with the Chinese Clinical Trial Registry (http: / / www.chictr.org.cn / ) under registration number ChiCTR2000034438.

[0034] 1.1 Diagnostic criteria

[0035] The diagnostic criteria for diabetic nephropathy (DKD) are a fasting blood glucose level ≥7.0 mmol / L, a urine albumin / creatinine ratio (ACR) ≥30 mg / g, or an estimated glomerular filtration rate (eGFR) <60 mL / min / 1.73 mcg. 2 Lasting for more than 3 months.

[0036] 1.2 Inclusion Criteria

[0037] The participants included in this study were: 1) individuals who met the relevant diagnostic criteria for DKD and were diagnosed; 2) individuals aged between 18 and 80 years, regardless of gender; 3) individuals who were of sound mind, had no intellectual disability, and had normal communication skills; and 4) individuals who voluntarily participated after learning about this study.

[0038] 1.3 Exclusion Criteria

[0039] Patients excluded include: 1) those with primary kidney disease; 2) those with severe cardiovascular, liver, or kidney dysfunction; 3) those infected with HIV, pregnant, with cancer, severe infection, or malignant tumor; 4) those with severe mental illness and cognitive impairment; 5) those diagnosed with type 1 diabetes or type 1 DKD; and 7) those with acute complications of diabetes.

[0040] 2. Sample processing and instrument conditions

[0041] 100 μL of sample was mixed with 400 μL of acetonitrile. The mixture was vortexed for 1 minute and sonicated in an ice-water bath for 10 minutes, then centrifuged at 14,000 rpm for 15 minutes at 4 °C. A UPLC-Q-TOF / MS system (Waters) coupled with a UPLCWaters ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm) was used. Mobile phase A consisted of 0.1% formic acid aqueous solution, and mobile phase B consisted of 0.1% formic acid methanol solution. The elution gradient was set as follows: 0-0.5 min, 3% B; 0.5-7.5 min, 3-80% B; 7.5-8 min, 80-98% B; 8-13 min, 98% B; 13-13.5 min, 98-100% B; 13.5-17 min, 100% B; 17-18 min, 100-3% B; 18-25 min, 3% B. The flow rate was 0.35 mL / min, and the injection volume was 5 μL. Mass spectrometry analysis was performed on a Xevo™ G2 QTof (Waters) with a scan range of 50-1200 m / z.

[0042] The collected data were imported into Masslynx software using QI and Markerlynx XS for peak detection and comparison. The obtained metabolites were then subjected to literature searches and HMDB database searches. To visualize the diagnostic efficacy of the metabolites, we further plotted heatmaps, used hierarchical cluster analysis, and generated receiver operating characteristic (ROC) curves. Furthermore, Pearson correlation coefficient analysis was employed to reveal the intrinsic link between metabolites and DKD. Figure 1 Distribution of metabolomics samples for healthy individuals vs. diabetic nephropathy (DKD).

[0043] 3. Result Characterization

[0044] (1) The clinical characteristics of healthy controls and DKD patients in this study are shown in Table 1. Data were analyzed using independent samples t-test or Mann-Whitney U test and expressed as mean ± standard deviation. Age, weight (BW), body mass index (BMI), blood pressure, eGFR, serum creatinine (CREA), urea concentration, proteinuria, and urine protein / creatinine ratio were significantly different from those of the control group.

[0045] Table 1 Clinical characteristics of the subjects

[0046]

[0047] Data are mean ± SD of continuous measurements, and n is the categorical measure. *: compared with HC (*: p < 0.05, **: p < 0.01); N / A, not applicable; HC, healthy control group; DKD, diabetic nephropathy group.

[0048] (2) We used UPLC-Q-TOF / MS in negative ion mode to detect 57,240 variables in 228 subjects. Subsequently, 35 metabolites with VIP>1 and P<0.05 were screened, of which 12 were downregulated and 23 were upregulated (see Table 2). Correlation analysis and pathway analysis showed that differentially expressed metabolites were involved in TCA cycle, amino acid metabolism, lipid metabolism and nucleotide metabolism ( Figures 2-4 As shown in the figure). ROC curve results show that arachidonic acid, deoxyuridine monophosphate (dUMP), fumaric acid, inosinic acid, stearic acid, and uridine all have high clinical diagnostic efficacy. Figure 5 (As shown). Combined with the results of the previous meta-analysis (Clinical metabolomics characteristics of diabetic kidney disease: A meta-analysis of 1875 cases with diabetic kidney disease and 4503 controls. Diabetes Metab Res Rev. 2024;40(3):e3789), it was found that pyrimidine metabolism is closely related to the occurrence and development of diabetic kidney disease (DKD). Urate is a key metabolite in pyrimidine metabolism and may be a potential functional metabolite. Supplementation with uridine has a protective effect on the kidneys.

[0049] Table 2.35 Information on Differential Metabolites

[0050] Metabolites RT(min) m / z Molecular formula Multiple change <![CDATA[L-Iditol]] # > 0.428 181.07187 <![CDATA[C6H 14 O6]]> 0.59 <![CDATA[D-Fructose # > 0.437 179.05686 <![CDATA[C6H 12 O6]]> 1.94 <![CDATA[Xanthine # > 0.554 151.02534 <![CDATA[C5H4N4O2]]> 1.46 <![CDATA[Uric acid # > 0.575 167.021 <![CDATA[C5H4N4O3]]> 2.22 <![CDATA[uridine # > 0.575 243.06225 <![CDATA[C9H 12 N2O6]]> 0.77 <![CDATA[Phenylalanine # > 1.384 164.07097 <![CDATA[C9H 11 NO2]]> 1.12 <![CDATA[L-Malic acid # > 1.391 133.01491 <![CDATA[C4H6O5]]> 1.34 <![CDATA[Tryptophan]]> # > 1.842 203.08247 <![CDATA[C 11 H 12 N2O2]]> 0.67 <![CDATA[High uric acid Hippuric acid # > 1.907 178.0505 <![CDATA[C6H5CONHCH2COOH]]> 1.66 <![CDATA[Glutamine # > 2.33 145.06193 <![CDATA[C5H 10 N2O3]]> 2.12 <![CDATA[p-Cresol # > 2.466 107.0496 <![CDATA[C7H8O]]> 1.23 <![CDATA[Inosine # > 2.97 267.07153 <![CDATA[C 10 H 12 N4O5]]> 1.43 <![CDATA[Fumaric acid # > 3.317 115.0032 <![CDATA[C4H4O4]]> 1.53 <![CDATA[citric acid # > 3.335 191.01807 <![CDATA[C6H8O7]]> 0.66 <![CDATA[Ascorbic acid # > 5.114 175.0244 <![CDATA[C6H8O6]]> 3.29 <![CDATA[Thymidine # > 6.741 241.0827 <![CDATA[C 10 H 14 N2O5]]> 0.50 <![CDATA[Cholic acid * > 6.773 407.27817 <![CDATA[C 24 H 40 O5]]> 0.76 <![CDATA[Glycoursodeoxycholic acid * > 7.05 448.306 <![CDATA[C 26 H 43 NO5]]> 0.76 <![CDATA[Lysophosphatidylethanolamine LysoPE(22:6) # > 9.07 524.2773 <![CDATA[C 27 H 44 NO7P]]> 0.68 <![CDATA[Linoleic acid * > 9.15 279.2322 <![CDATA[C 18 H 32 O2]]> 0.76 <![CDATA[Lysophosphatidylethanolamine LysoPE(16:0) # > 9.53 452.2778 <![CDATA[C 21 H 44 NO7P]]> 0.69 <![CDATA[Palmitic acid # > 9.65 255.2324 <![CDATA[C 16 H 32 O2]]> 1.69 <![CDATA[Arachidonic acid metabolism (eicosanoids) 12(R)-HETE # > 9.782 319.22761 <![CDATA[C 20 H 32 O3]]> 1.80 <![CDATA[Lysophosphatidylethanolamine LysoPE(18:1) # > 9.89 478.2935 <![CDATA[C 23 H 46 NO7P]]> 0.76 <![CDATA[Lysophosphatidylethanolamine LysoPE(20:1) # > 9.94 506.3245 <![CDATA[C 25 H 50 NO7P]]> 1.48 <![CDATA[Glycerophospholipid metabolism (lysophospholipid) LysoPC(15:0) # > 10.9 480.3089 <![CDATA[C 23 H 48 NO7P]]> 1.34 <![CDATA[Stearic acid # > 10.98 283.2638 <![CDATA[C 18 H 36 O2]]> 0.60 <![CDATA[LysoPE(20:0) (Lysophosphatidylethanolamine) # > 10.99 508.3403 <![CDATA[C 25 H 52 NO7P]]> 1.42 <![CDATA[docosahexaenoic acid # > 12.022 327.23269 <![CDATA[C 22 H 32 O2]]> 0.58 <![CDATA[Uridine 5'-diphosphate # > 13.588 565.047 <![CDATA[C 15 H 24 N2O 17 P2]]> 0.77 <![CDATA[Pyrimidine deoxynucleotide metabolism dUMP # > 14 307.0328 <![CDATA[C9H 13 N2O8P]]> 0.60 <![CDATA[Arachidonic acid # > 14.48 303.23242 <![CDATA[C 20 H 32 O2]]> 2.23 <![CDATA[Inosinic acid # > 14.93 347.03921 <![CDATA[C 10 H 13 N4O8P]]> 0.64 <![CDATA[Fructose 6-phosphate # > 15.477 259.02192 <![CDATA[C6H 13 O9P]]> 1.42 <![CDATA[Glycerophospholipid metabolism (phospholipid) PC(35:4) # > 17.88 766.5376 <![CDATA[C 43 H 78 NO8P]]> 1.36

[0051] *: p <0.05,#: p <0.01.

[0052] Example 2: Clinical bioinformatics analysis experiment of DKD

[0053] 1. Dataset download and processing

[0054] Microarray data were downloaded from the Gene Expression Comprehensive Database (http: / / www.ncbi.nlm.nih.gov / geo): GSE104954, GSE47185, GSE96804, and GSE142025. Detailed information for these datasets is shown in Table 3. All gene expression profiles were normalized and standardized using the limma R package, and differentially expressed genes (DEGs) were screened using p < 0.05 and |log2FC| > 1.5. The analysis results were visualized using the ggplot and heatmap packages in R.

[0055] Table 3 Information of 4 microarray datasets

[0056]

[0057] 2. Data processing results: DEGs were screened by the "limma" package (p < 0.05 and |log FC| > 1). The GSE104954 dataset contains 201 DEGs (121 up-regulated and 80 down-regulated); the GSE47185 dataset contains 325 DEGs (94 up-regulated and 231 down-regulated); the GSE96804 dataset contains 621 DEGs (282 up-regulated and 339 down-regulated); the GSE142025 dataset contains 1858 DEGs (1039 up-regulated and 819 down-regulated). Among them, 19 DEGs that were expressed in all datasets were screened out. The STRING online database was used to construct a PPI network (confidence 0.4) for the 19 DEGs, and the results were downloaded using Cytoscape software for further analysis. The top 5 hub genes of each algorithm were identified using the cytoHubba plugin of Cytoscape, and a total of 2 hub genes were identified: EGR1 and COL1A2.

[0058] Example 3 Experiment on the protective effect of uridine on the kidney

[0059] 1. Experimental animals

[0060] 6-week-old male C57BL / 6J mice were purchased from Huafukang in Beijing, with the quality certificate number: SCXK (Beijing) 2019-0008.

[0061] 2. Drugs and reagents, as shown in Table 4;

[0062] Table 4 Drugs and reagents

[0063]

[0064] 3. Establishment of the experimental model and administration method

[0065] Mice were randomly divided into a control group (n=8) and a model group (n=16), fed a normal diet and a high-fat diet, respectively, for 4 weeks. Subsequently, mice in the model group received a low-dose STZ (streptozotocin 50 mg / kg) intraperitoneally, while mice in the control group received an equal dose of citrate buffer intraperitoneally. Injections continued for 5 to 7 days, and fasting blood glucose levels were measured. Mice with a fasting blood glucose level ≥11.1 mmol / L for 3 consecutive days were considered diabetic mice. Afterward, the mice were fed for another 4 weeks, and a DKD model was confirmed when urinary albumin was ≥30 mg / 24 h. DKD mice were then randomly divided into two groups (n=8): a DKD group and a uridine group (30 mg / kg / day, ip). Both groups received the same volume of 0.5% Na-CMC for 4 weeks. During treatment, water intake, food consumption, body weight, and FBG levels were monitored weekly.

[0066] 4. Biochemical parameters, kidney index, and histopathological examination

[0067] Oral glucose tolerance was measured using a blood glucose meter. Blood samples were collected and centrifuged at 3000 rpm for 10 min at 4 °C. The supernatant was used to analyze biochemical parameters, including blood urea nitrogen, creatinine, uric acid, total cholesterol, triglycerides, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol. To monitor renal function, the renal index (renal index = kidney weight (mg) / body weight (g)) and the levels of MDA (malondialdehyde) and SOD (superoxide dismutase) were also measured.

[0068] The tissue was embedded in embedding gel and sectioned. According to the standard protocol, periodic acid-Schiff (PAS) staining was performed to observe renal glycogen deposition, Masson staining was performed to observe collagen fibers, and Tunel staining was performed to detect renal cell apoptosis.

[0069] 5. DESI-MSI analysis of kidney tissue

[0070] Kidney sections were prepared at -20°C using a Leica CM1950 cryostat, with a thickness of 10 μm. DESI-MSI experiments were performed using a Q Exactive mass spectrometer coupled with 2D Omni spray to analyze and image the tissues at a spatial resolution of 200 μm. The nominal pixel size was set to 50 × 50 μm. Imaging regions were selected, and the resulting data were processed in high-resolution imaging software (version 1.4). A tissue-compatible solvent system, acetonitrile:water (8:2, v:v), was used for analysis at a flow rate of 3 μL / min. Mass spectrometry was acquired in negative ion mode, with a scan range of 100–1000 Da. Tissue samples analyzed by DESI-MSI were subjected to H&E staining for histopathological evaluation.

[0071] 6. Statistical methods

[0072] Data are expressed as mean ± standard deviation and statistical analysis was performed using GraphPad Prism 8.0 software. One-way ANOVA was used to compare differences between groups; ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant.

[0073] 7. Experimental Results

[0074] (1) General state observation: Figure 6 Figure 'a' shows a schematic diagram of the uridine intervention experiment in DKD mice. Two weeks after STZ injection, compared with the control group, the DKD mice showed decreased body weight and food intake, but increased water consumption. After four weeks of uridine treatment, all parameters except food intake showed significant differences.

[0075] (2) Effects of uridine on blood glucose in DKD mice: Compared with the normal group, the fasting blood glucose levels in the model group rats were significantly increased at weeks 4, 8, 12, and 16 (P < 0.01). Compared with the model group, the fasting blood glucose levels were significantly decreased after uridine administration (P < 0.01). Figure 6 f), and oral glucose tolerance is reduced ( Figure 6 g and 6h) (P < 0.05).

[0076] (3) Effects of uridine on lipid metabolism in DKD mice: Compared with the normal group, the model group mice showed increased serum cholesterol, triglyceride, and low-density lipoprotein levels (P < 0.05), and decreased high-density lipoprotein levels (P < 0.05); after uridine treatment, total cholesterol, triglyceride, and low-density lipoprotein levels decreased (P < 0.05), while high-density lipoprotein levels increased (P < 0.05). Figure 6 j- Figure 6 m).

[0077] (4) Effects of uridine on MDA, SOD, and GSH-Px levels in DKD mice: Compared with the normal group, the MDA content in the kidney tissue of the model group mice was significantly increased (P < 0.05), while the SOD and GSH-Px contents were significantly decreased (P < 0.05); after uridine treatment, the MDA content decreased significantly (P < 0.05), while the SOD and GSH-Px contents increased significantly (P < 0.05). Figure 6 n- Figure 6 p).

[0078] (5) Effects of uridine on renal function in DKD mice

[0079] Compared with the normal group, the serum uric acid, blood urea nitrogen, and creatinine levels in the model group mice were significantly increased (P < 0.05); after treatment with uridine, the uric acid, blood urea nitrogen, and creatinine levels were significantly decreased (P < 0.05). Figure 6 b- Figure 6 e).

[0080] (6) Pathological morphological observation of uridine on kidney tissue of DKD mice

[0081] Compared with the normal group, DKD mice showed abnormal overall renal tissue structure, with moderate dilation of glomeruli and renal tubular interstitium, brown apoptotic spots, and glycogen deposition accumulation in the glomerular capsule and mesangium; uridine could improve renal histology and reduce glycogen deposition, renal cell apoptosis, collagen accumulation, and fibrosis in DKD renal tissue. Figure 6 i).

[0082] (7) Effects of uridine on metabolic characteristics of kidney tissue in DKD mice

[0083] Furthermore, previous studies have confirmed that systemic and tissue-specific changes in lipid metabolism can cause glomerular damage. DESI-MS analysis was performed on kidney sections of mice in each group to more comprehensively study the spatially resolved metabolic changes in DKD. Histological features were distinguished based on H&E staining results, and the molecular characteristics of metabolites were obtained in negative ion mode on adjacent tissue sections. Mass spectrometry imaging results showed that uridine supplementation can regulate vital processes such as glucose homeostasis, lipid metabolism, and amino acid metabolism.

[0084] Example 4: Experiment on the protective effect of uridine on renal cells

[0085] 1. Experimental reagents and instruments are shown in Tables 5-7.

[0086] Table 5 Experimental Reagents

[0087] reagents factory MEM medium (containing NEAA) Wuhan Pronosei Life Science Technology Co., Ltd. fetal bovine serum Shanghai Xiaopeng Biotechnology Co., Ltd. Penicillin and streptomycin Gibco pancreatic enzymes Gibco CCK-8 reagent kit White Shark Biotechnology Co., Ltd. Dimethyl sulfoxide Beijing Solarbio Co., Ltd. Natural Product Compound Library L6000 MCE Company Annexin V-FITC Apoptosis Detection Kit Beijing Solarbio Co., Ltd. BCA Quantitative Reagent Kit Beijing Solarbio Co., Ltd. Monoclonal antibody β-Actin Cell Signaling Technology Bcl-2, Bax and Casp-3 primary antibodies ABclonal 15% Gel Preparation Kit Yamei Bio RIPA pyrolysis fluid Beijing Solarbio Company

[0088] Table 6 Experimental Instruments

[0089] Instruments and Consumables factory HK-2 cells Wuhan Pronosei Life Science Technology Co., Ltd. Explorer G3 Automated Drug Screening System PerkinElmer Multifunctional ELISA reader Thermo Chemiluminescence scanner LI-COR Flow cytometer Beckman Coulter

[0090] Table 7 Database Information

[0091] Databases and software factory Pubchem https: / / pubchem.ncbi.nlm.nih.gov / PDB https: / / www.rcsb.org Autodock https: / / autodock.scripps.edu Pymol https: / / pymol.org / 2 / Obgui https: / / openbabel.org / docs / GUI / GUI.html

[0092] 2. Cell Culture

[0093] HK-2 cells were cultured in MEM (5.5mM) complete medium: 90% MEM medium, 10% fetal bovine serum and 1% penicillin-drug antibody, 37 ℃, 5% CO2.

[0094] 3. Effects of uridine on the morphology, number, and viability of HK-2 cells

[0095] HK-2 cells were seeded in 96-well and 6-well plates. After cell adhesion the next day, the cells were treated with 500 μmol / L uridine for 48 h. Morphology, trypsin digestion counts, and CCK-8 assays were then observed under a microscope.

[0096] 4. Effects of uridine on HK-2 cell apoptosis

[0097] HK-2 cells were seeded into 6-well plates and allowed to adhere overnight. They were then exposed to a high-glucose medium (50 mM) and / or 500 μM uridine for 48 h. Hoechst staining was performed according to the manufacturer's instructions. Furthermore, flow cytometry analysis was performed using the Annexin V-FITC kit.

[0098] After uridine treatment, HK-2 cells underwent protein extraction, BCA quantification, electrophoresis, membrane transfer, and color development: 15% SDS-PAGE separating gels were prepared based on the molecular weights of Bax, Bcl-2, and Casp-3 proteins.

[0099] 7. Results

[0100] (1) Effects of uridine on the morphology, number and activity of HK-2 cells

[0101] Phenotypic results demonstrated that uridine had a protective effect against high glucose damage in HK-2 cells. High glucose at 50 mM caused HK-2 cells to change from a cobblestone-like morphology to a fibrous morphology, and the cell number also decreased significantly. However, HK-2 cells treated with uridine did not show significant changes in cell morphology, and the cell number significantly recovered. Figure 8 a- Figure 8 c).

[0102] (2) Effect of uridine on HK-2 cell apoptosis

[0103] Hoechst staining in the high-glucose group showed typical morphological features of apoptosis, such as nuclear pyknosis, crescent-shaped nuclei, nuclear fission, and apoptotic bodies, suggesting that high glucose can induce nuclear fragmentation and autolysis in the late apoptotic phase. Administration of uridine significantly alleviated apoptosis. Figure 8 d). Figure 8d is a flow cytometry plot for apoptosis detection. The horizontal axis (Comp-FL1-A: AnnexinV FITC-A) reflects early apoptosis (extravasation of phosphatidylserine on the cell membrane); the vertical axis (Comp-FL2-A: PI PIEA) reflects late apoptosis or necrosis (disruption of cell membrane integrity, PI entering the cell to stain nuclear DNA). Quadrants and cell states correspond as follows: Q1 (Annexin V⁻ / PI⁺): Necrotic cells (cell membrane ruptured, PI positive, Annexin V negative); Q2 (Annexin V⁺ / PI⁺): Late apoptotic cells (cell membrane integrity disrupted, phosphatidylserine eversion occurs); Q3 (Annexin V⁺ / PI⁻): Early apoptotic cells (only phosphatidylserine eversion occurs, cell membrane remains intact, PI cannot enter); Q4 (Annexin V⁻ / PI⁻): Live cells (cell membrane intact, neither phosphatidylserine eversion nor PI staining occurs).

[0104] The three sets of images from left to right represent different treatment groups (e.g., control group, high glucose group, and uridine group). The results show that Annexin V / PI staining was used to assess HK-2 cell apoptosis. Compared with the control group, high glucose significantly induced HK-2 cell apoptosis, and uridine treatment reduced the apoptosis rate of HK-2 cells from 30.71% to 15.51%, significantly inhibiting HK-2 cell apoptosis. Figure 8 d).

[0105] Western blot analysis showed that uridine rescued the high glucose-induced increase in Bax and Casp-3 expression and the decrease in Bcl-2 expression. Figure 8 e- Figure 8 f).

[0106] Example 4: Experiment on the protective mechanism of uridine on the kidneys

[0107] 1. Transcriptomics experiments

[0108] Nine replicates were set up for each group, with three replicates randomly pooled into one sample. Total RNA was extracted from HK-2 cells in the control group, high glucose group, and uridine intervention group using TRIzol reagent. After the samples passed the tests, mRNA was purified using magnetic beads with Oligo(dT) beads, and fragmentation buffer was added to fragment the mRNA. Using mRNA as a template, first-strand cDNA was synthesized using randomized hexanucleotide primers, and second-strand cDNA was synthesized with buffer, dNTPs, and DNA polymerase I. AMPure XP beads were used to further purify the double-stranded cDNA, and suitable fragments were selected as templates for PCR enrichment to obtain the final cDNA library. After purification and quantification, the library was sequenced on the Illunima NovaSeq 6000 platform.

[0109] The Cutadapt software was used to filter and screen the raw data. The BWT algorithm compared the valid data with human reference genes, and the R package DESeq2 was used for differential gene analysis. To eliminate the problem of high false positive rates, the threshold criteria for screening differentially expressed genes were set as |log2FC|≥1 and padj < 0.05. After data screening, Fisher's exact test was used to determine the functional categories of all differentially expressed genes enriched.

[0110] 2. Proteomics experiments

[0111] SDT (4% SDS, 100 mM Tris-HCl, pH 7.6) was used for sample lysis and protein extraction. Protein amounts were quantified using the BCA Protein Assay Kit (Bio-Rad, USA). Protein digestion was performed with trypsin according to the Filter-Assisted Sample Preparation (FASP) procedure. Digested peptides from each sample were desalted on an MCX, concentrated by vacuum centrifugation, and reconstituted in 40 µL of 0.1% (v / v) formic acid. 100 μg peptide mixtures from each sample were labeled with TMT reagents according to the manufacturer's instructions (Thermo Scientific). The labeled peptides were homogenized, fractionated using a high-pH reverse-phase peptide fractionation kit (Thermo Scientific), and lyophilized in 12 μL of 0.1% formic acid. LC-MS / MS analysis was performed on a Q Exactive mass spectrometer coupled with Easy nLC. The chromatographic column used was packed with nanoViper C18. The linear gradient of buffer A (0.1% formic acid buffer) and buffer B (84% acetonitrile and 0.1% formic acid aqueous solution) was separated at a flow rate of 0.3 ml / min.

[0112] Raw LC / MS data were retrieved from the UniProt-reviewed human protein database, and differentially expressed proteins were screened according to the criteria of upregulation >1.2-fold or downregulation <0.83 and p <0.05. All identified proteins were further subjected to GO and KEGG functional enrichment analyses.

[0113] 3. Discovery and verification of the protective mechanism of uridine

[0114] Transcriptomic experiments revealed that 59 differentially expressed genes (DEGs) were regulated after uridine supplementation, such as EGR1, EGR3, DUSP2, and EIF4A1. Figure 9 a and 9b). KEGG analysis showed that ( Figure 9 c) These genes were significantly enriched in the Apelin, GnRH, AGE-RAGE, and MAPK signaling pathways. Further GO enrichment analysis revealed that these differentially expressed DEGS genes are involved in key biological processes such as DNA binding, ATP binding, glomerular cell proliferation, apoptosis, lipid biosynthesis, protein modification, cell adhesion, and cell metabolism, processes closely related to the pathogenesis of DKD. It is speculated that these genes may be involved in their regulation, possibly related to subcellular functions such as mitochondria and the nucleus, which is significant for understanding the molecular mechanisms of uridine treatment for DKD. Notably, EGR1, as a clinical pivot gene and a differentially expressed gene for uridine protection against diabetic nephropathy, will be a focus of future research.

[0115] A total of 7093 proteins were identified by proteomics analysis. Figure 10 a and Figure 10 (b) Compared to the control group, the high glucose group showed significant upregulation of 108 proteins and significant downregulation of 62 proteins. The volcano plot showed the results for 511 differentially expressed proteins (242 upregulated, 269 downregulated). Compared to the high glucose group, the uridine intervention group identified 511 differently expressed proteins (242 downregulated, 269 upregulated), all of which are potential important targets for DKD treatment. These proteins are mainly involved in glycerophospholipid metabolism, the RAS signaling pathway, the MAPK signaling pathway, and the Apelin signaling pathway. Figure 10 c). GO enrichment analysis showed that these differentially expressed proteins are associated with apoptosis, ATP synthesis, metal ion binding, and the activity of transmembrane ion transporters, which are crucial for the balance between cell survival and apoptosis.

[0116] Through comprehensive multi-omics analysis, differentially expressed genes and proteins involved in key pathways (Apelin and MAPK), along with EGR1, were imported into the STRING website for protein-protein interaction (PPI) network analysis. With a minimum interaction score set to 0.9, the results showed a certain correlation between EGR1, P300, and ETS1. Based on literature research, it is hypothesized that uridine may exert a therapeutic effect on DKD through the EGR1-P300-ETS1 signaling axis. Figure 7 a). To investigate whether uridine exerts its effect by inhibiting the EGR1-P300-ETS1 signaling axis, we first performed Western blot analysis. The results showed that, compared with the control group, the levels of EGR1, P300, and ETS1 proteins were significantly increased in the HG group, and uridine supplementation significantly inhibited the expression of these proteins. Figure 7 b). To verify the upstream regulatory role of EGR1 in the signaling pathway, we successfully established an HK-2 cell model with EGR1 silencing (shEGR1) using lentivirus-mediated RNA interference. Subsequent Western blot analysis showed that, compared with normal HK-2 cells, the expression level of P300 protein was simultaneously downregulated in the shEGR1 group (b). Figure 7 c); Simultaneously, treatment with the P300 activator cholera toxin B (CTB) led to a simultaneous increase in ETS1 expression levels ( Figure 7 (d) This finding highlights the role of P300 in regulating ETS1 levels and further supports the involvement of the EGR1-P300-ETS1 signaling axis in the renal protective effect of uridine. Sustained uridine supplementation significantly inhibited the activation of the EGR1-P300-ETS1 signaling cascade in renal tissue. Figure 7 e). Overall, these results confirm that uridine exerts a nephroprotective effect by inhibiting the EGR1-P300-ETS1 signaling cascade.

[0117] 4. Subcellular localization experimental protocol

[0118] (1) Synthesis of CY3-uridine

[0119] Weigh 7 mg of CY3-COOH and dissolve it in 3 mL of DMF. Add uridine, DIC, HOBt and DMAP and dissolve completely. Stir the reaction under nitrogen protection at 50 °C for 12 h. Remove the solvent by rotary evaporation under reduced pressure. Purify by column chromatography with dichloromethane:methanol = 10:1 as the eluent. Dry under vacuum to obtain the CY3-uridine product.

[0120] CY3-uridine was analyzed using an SPD-20A detector and an API3200 mass spectrometer. 5 μl of sample solution was injected into a Shim-pack GIST-HP C18 at a flow rate of 0.2 ml / min and a detection wavelength of 550 nm. The mobile phase consisted of (phase A) 0.1% formic acid in methanol and (phase B) 0.1% formic acid in aqueous solution. Specific conditions are shown in Tables 8 and 9.

[0121] Table 8 Gradient elution conditions

[0122] T(min) Phase A Phase B 0 70% 30% 10 70% 30%

[0123] Table 9 Mass Spectrometry Parameter Information

[0124] Air curtain gas (psi) Spray voltage (V) Atomization temperature (°C) Nebulized gas (psi) Auxiliary gas (psi) Injection voltage (V) Positive ion mode 15 5500 400 45 45 10

[0125] (2) Subcellular localization of CY3-uridine

[0126] Before addition to cells, the non-toxicity dose of CY3-uridine in cells was determined. 1×10⁻⁶ 5 HK-2 cells were seeded in 6-well plates pre-placed with coverslips. After overnight cell adhesion, a non-toxic dose of CY3-uridine was added. After incubation for 24 h and 48 h, the cells were immobilized with mounting medium containing DAPI dye. Fluorescence imaging was performed using single-channel mode at 550 nm excitation. The results showed that uridine could be localized to the mitochondria of HK-2 cells and accumulated over time (see...). Figure 7 (f) suggests that the renal protective effect of uridine may be closely related to its ability to improve mitochondrial dysfunction. To comprehensively assess the effects of uridine on mitochondrial function, the effects of uridine on ATP production, mitochondrial membrane potential, mitochondrial morphology, and reactive oxygen species (ROS) levels in HG-damaged HK-2 cells were systematically investigated. The results showed that, compared with the HG group, uridine treatment significantly increased ATP production in cells (f). Figure 7 g), mitochondrial membrane potential significantly increased ( Figure 7 h). Transmission electron microscopy observation of mitochondrial morphology showed that uridine treatment significantly reduced mitochondrial ultrastructural damage, such as reduced mitochondrial fragmentation and cristae disappearance (h). Figure 7 i). Flow cytometry and fluorescence analysis after DCFH-DA staining showed that uridine significantly reduced ROS release in HG-damaged HK-2 cells (i). Figure 7 j and Figure 7 These results confirm that uridine can effectively repair mitochondrial dysfunction and reduce mitochondrial damage.

[0127] In summary, pyrimidine metabolism disorders play a crucial role in diabetic kidney disease (DKD) patients. Uridine, a core intermediate and key functional molecule in the pyrimidine metabolic pathway, has significant implications for DKD research. A series of experiments have demonstrated that uridine is a key bioactive metabolite in DKD. Uridine supplementation can improve the general condition of DKD mice, regulate glucose and lipid metabolism disorders, reduce oxidative damage to kidney tissue, and improve renal function indicators, thus protecting the kidneys. Furthermore, uridine can significantly improve the pathological changes in kidney tissue of DKD mice, alleviate kidney pathological damage to a certain extent, and regulate specific metabolic changes in kidney tissue, involving glucose metabolism, the TCA cycle, nucleotide metabolism, and lipid metabolism. At the mechanistic level, this study reveals for the first time that uridine exerts its renal protective effect through a dual pathway: on the one hand, uridine significantly improves mitochondrial function and enhances cellular energy metabolism; on the other hand, uridine effectively reduces renal fibrosis and inflammatory response by inhibiting the EGR1-P300-ETS1 signaling axis.

[0128] For those skilled in the art, this invention is not limited to the details of the exemplary embodiments described above, and can be implemented in other specific forms without departing from the spirit or essential characteristics of this invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of this invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within this invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0129] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Any minor modifications, equivalent substitutions, and improvements made to the above embodiments based on the technical essence of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of uridine in the preparation of drugs for the prevention and / or treatment of diabetic nephropathy.

2. The use of uridine in the preparation of drugs for the prevention and / or reduction of kidney damage in patients with diabetic nephropathy.

3. The application according to claim 1 or 2, wherein the diabetic nephropathy refers to type II diabetic nephropathy.

4. The application according to claim 3, characterized in that, The uridine exerts its effect by influencing the expression of two core genes, EGR1 and COL1A2.

5. The application according to claim 3, characterized in that, The uridine can regulate or improve lipid metabolism disorders in patients with diabetic nephropathy.

6. The application according to claim 3, characterized in that, The uridine can inhibit oxidative damage to the kidneys and regulate renal cell activity.

7. The application according to claim 6, characterized in that, The uridine acts on the mitochondria of renal cortical cells, specifically by inhibiting EGR1-P300-ETS1 and promoting NADP+ / NADPH-SIRT1 to regulate mitochondrial function, thereby playing a therapeutic role in diabetic nephropathy.

8. The application according to claim 7, characterized in that, The renal cortical cells specifically refer to the proximal convoluted tubule epithelial cells of the human renal cortex.

9. The application according to claim 3, characterized in that, The drugs for preventing and / or reducing diabetic nephropathy also include pharmaceutically acceptable excipients or drug carriers, and the dosage forms of the drugs for preventing and / or reducing diabetic nephropathy include tablets, capsules, granules, powders, pills, gel candies, powders, oral liquids, or drops.

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