Biomarker for predicting curative effect of fenerenone on diabetic nephropathy and application of biomarker

Abstract

The invention discloses a biomarker for predicting the curative effect of fenerenone on treating diabetic nephropathy and application of the biomarker, and four biomarker combinations with clear prediction values are screened and verified on the basis of a core molecular mechanism (mTOR / ULK1 pathway-podocyte autophagy) of fenerenone for treating diabetic nephropathy. And a'baseline + early stage 'dual pre-judgment system and a standardized application process are established. The scheme solves the bottleneck that the curative effect of the fenerenone cannot be pre-judged in the prior art, has the advantages of being clear in mechanism, convenient to detect and accurate in prediction, can be directly applied to clinical individualized treatment guidance, improves the treatment effective rate and reduces the medical cost, and has wide industrialization prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical testing and treatment of diabetic nephropathy, specifically to a set of biomarkers for predicting the efficacy of fenelazol treatment for diabetic nephropathy, along with their detection methods and application procedures, applicable to individualized treatment guidance for patients with diabetic nephropathy in the clinical proteinuria stage. Background Technology

[0002] Diabetic kidney disease (DKD) is one of the most serious microvascular complications of diabetes and the leading cause of end-stage renal disease (ESRD) worldwide, placing a heavy burden on patients' families and healthcare systems. With the continued rise in global diabetes prevalence, the incidence of DKD is also increasing year by year. According to the International Diabetes Federation, by 2045, the number of people with diabetes worldwide will reach 784 million, and 20% to 40% of these patients will also have DKD. The total number of people with diabetes in China has already reached 141.1 million, indicating a large and rapidly growing number of DKD patients. DKD has an insidious onset, low awareness, and few early symptoms. Once it progresses to the clinical proteinuria stage, kidney function damage is often irreversible, ultimately requiring dialysis or kidney transplantation to sustain life. Therefore, there is an urgent need to develop more effective treatment strategies and efficacy assessment methods.

[0003] The pathogenesis of kidney disease (DKD) is extremely complex, resulting from the combined effects of multiple factors and pathways. Core pathological features include podocyte injury, inflammatory response, oxidative stress, and renal interstitial fibrosis. Podocytes, highly differentiated glomerular epithelial cells, line the outer surface of the glomerular basement membrane and are key cells in maintaining the integrity and functional homeostasis of the glomerular filtration barrier. Damage, shedding, and functional abnormalities of podocytes are the core driving factors in the progression of DKD. Under pathological stimuli such as hyperglycemia and advanced glycation end products (AGEs), podocytes exhibit a series of damaging responses, including foot process loss, downregulation of specific protein expression, and increased apoptosis, leading to disruption of the filtration barrier, proteinuria, and ultimately, accelerated deterioration of renal function. Recent studies have confirmed that autophagy dysfunction plays a key role in podocyte injury. Autophagy is an important physiological process by which cells degrade damaged organelles and abnormal protein aggregates through lysosomes to maintain cellular homeostasis. In the case of DKD, podocyte autophagy flux is impaired, leading to the accumulation of harmful substances, which further aggravates podocyte injury and shedding. Therefore, targeted regulation of autophagy and restoration of podocyte function have become important research directions for the treatment of DKD.

[0004] Currently, the main drugs used clinically to treat diabetic kidney disease (DKD) include angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs). While these drugs can reduce proteinuria and slow the decline in renal function to some extent, many patients still experience continued disease progression, and they cannot effectively prevent the disease from developing into end-stage renal disease. Furthermore, they have limitations due to side effects such as dry cough and hyperkalemia, and the clinical need remains far from being met. Finelerone, a novel selective nonsteroidal mineralocorticoid receptor (MR) antagonist, has been approved in many countries for the treatment of chronic kidney disease associated with type 2 diabetes. Clinical studies (such as the FIDELIO-DKD and FIGARO-DKD trials) have confirmed its effectiveness in reducing composite renal endpoint events, reducing proteinuria, and slowing the decline in renal function in DKD patients. Its known renal protective effect is mainly attributed to the inhibition of MR overactivation-mediated inflammatory responses and fibrosis. However, with further research, researchers have found that the mechanism of action of finelerone may be more complex. Whether it exerts its protective effect through the regulation of podocyte autophagy, and the specific molecular targets and signaling pathways, remain unclear.

[0005] Subsequent studies further revealed that the renal protective effect of fenelazol is closely related to the regulation of the mTOR / ULK1 signaling pathway and the activation of podocyte autophagy—a key regulatory axis for autophagy initiation. mTOR kinase inhibits autophagy by phosphorylating ULK1, while when mTOR activity is inhibited, ULK1 is activated and initiates the autophagy process. Fenelazol can alleviate high glucose-induced podocyte damage and apoptosis by inhibiting mTOR phosphorylation, promoting ULK1 phosphorylation, and upregulating the expression of key autophagy proteins LC3B and Beclin1. However, despite the promising prospects of fenelazol in the treatment of disseminated kidney disease (DKD), significant challenges remain in its clinical application: First, there are large individual differences in efficacy, with some patients responding poorly to treatment, yet effective predictive methods are lacking; second, current technologies have not revealed that mTOR / ULK1 pathway-related proteins (p-mTOR, p-ULK1, LC3B, Beclin1) can serve as biomarkers for predicting fenelazol efficacy, making it impossible to screen for high-responder patients in advance; third, clinical medication often follows a "one-size-fits-all" approach, leading to the risk of ineffective treatment for low-responder patients, delaying treatment and wasting medical resources. Therefore, establishing an efficacy prediction system based on the core mechanism of action of fenelazol and screening for specific biomarkers to provide a scientific basis for personalized clinical medication has become a critical issue that urgently needs to be addressed in the current field of DKD treatment.

[0006] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0007] Based on the aforementioned discovery that fenelazol works through the mTOR / ULK1 pathway, this invention provides a biomarker for predicting the efficacy of fenelazol in treating diabetic nephropathy and its application.

[0008] This invention, through in vitro and in vivo experiments, reveals that fenelazol can promote podocyte autophagy by activating the mTOR / ULK1 signaling pathway, thereby exerting a therapeutic effect on diabetic nephropathy. Specifically, fenelazol inhibits mTOR phosphorylation, thereby activating ULK1, ultimately upregulating the expression of key autophagy proteins LC3B and Beclin1, enhancing autophagy activity, and protecting podocytes from high glucose-induced damage and apoptosis.

[0009] The structural formula of fenelone is as follows:

[0010]

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] A biomarker combination for predicting the efficacy of fenelazol treatment for diabetic nephropathy includes four proteins: phosphorylated mTOR (p-mTOR), phosphorylated ULK1 (p-ULK1), LC3B, and Beclin1.

[0013] The disclosed biomarker combination for predicting the efficacy of fenelazol in treating diabetic nephropathy (DKD) using the above-mentioned technical solution comprises the following four proteins: phosphorylated mTOR (p-mTOR), phosphorylated ULK1 (p-ULK1), the autophagy key protein LC3B, and Beclin1. This combination is screened based on the core molecular mechanism of fenelazol treatment for DKD (mTOR / ULK1 pathway - podocyte autophagy), and has been validated through in vitro and in vivo experiments, enabling accurate prediction of patient response to fenelazol treatment.

[0014] Preferably, the diabetic nephropathy is clinical proteinuria-stage diabetic nephropathy, and the patient's estimated glomerular filtration rate (eGFR) is ≥25 ml・min⁻¹・(1.73m²)⁻¹.

[0015] The present invention also provides the application of the aforementioned biomarker combination in the preparation of a detection reagent or kit for predicting the efficacy of fenelazol in the treatment of diabetic nephropathy.

[0016] Specifically, the screening phase of the application process includes: including patients with diabetic nephropathy in the clinical proteinuria stage (eGFR≥25 ml・min⁻¹・(1.73m²)⁻¹), collecting blood / urine samples before treatment, and detecting the combined levels of biomarkers;

[0017] Prediction phase: Based on the above prediction criteria, patients are determined to be "high-response," "intermediate-response," or "low-response." Medication guidance phase: High-response: Administer fennitone at the standard dose (eGFR ≥ 25 and < 60 ml・min⁻¹・(1.73m²)⁻¹ starting at 10 mg / day; eGFR ≥ 60 ml・min⁻¹・(1.73m²)⁻¹ starting at 20 mg / day).

[0018] Moderate response: Standard dose of fenestrone combined with intensive basic therapy (such as strict blood sugar and blood pressure control);

[0019] Low responsiveness: Avoid fenestrone monotherapy; combination therapy with other kidney-protective drugs (such as traditional Chinese medicine combinations) is recommended. Efficacy monitoring phase:

[0020] Biomarkers were retested two weeks after treatment, and the treatment plan was dynamically adjusted.

[0021] Preferably, the test samples are patient blood, serum, plasma or urine sediment. These samples are non-invasive and readily available in clinical settings, avoiding invasive kidney tissue biopsy.

[0022] Preferably, the detection method is ELISA, Western Blot, or immunofluorescence technique.

[0023] The technology used is mature and reproducible, including: ELISA (Enzyme-Linked Immunosorbent Assay): suitable for batch clinical testing, simple to operate and cost-effective;

[0024] Western Blot: Used for precise quantification in the laboratory to detect the p-mTOR / mTOR and p-ULK1 / ULK1 ratios;

[0025] Immunofluorescence technique: used to detect the localization and expression intensity of LC3B and Beclin1 in tissue or cell samples.

[0026] Preferably, the efficacy prediction criteria are: p-mTOR / mTOR ratio ≤1.2, p-ULK1 / ULK1 ratio ≥0.8, LC3B expression level ≥60% of the normal control group, and Beclin1 expression level ≥60% of the normal control group before treatment. If two or more criteria are met, the total effective rate of treatment is predicted to be ≥90%.

[0027] Specifically, based on in vitro and in vivo experimental data (SD rat DKD model + MPC-5 podocyte cell line validation), the following predictive criteria were established:

[0028] Pre-treatment (baseline) prediction: If the patient sample meets two or more of the following criteria, the overall response rate after 3 months of fenelazol treatment is predicted to be ≥90%: p-mTOR / mTOR ratio ≤1.2 (compared to the normal control group); p-ULK1 / ULK1 ratio ≥0.8 (compared to the normal control group); LC3B expression level ≥60% of the normal control group; Beclin1 expression level ≥60% of the normal control group.

[0029] Early treatment (2 weeks) prediction: If the sample meets one or more of the following conditions after 2 weeks of treatment, the probability of predicting the final treatment effect as "short-term remission" (UACR reduction ≥50%) is ≥70%: p-mTOR / mTOR ratio decreases by ≥20% from baseline; p-ULK1 / ULK1 ratio increases by ≥20% from baseline; LC3B or Beclin1 expression level increases by ≥30% from baseline.

[0030] Non-response prediction: If the pre-treatment p-mTOR / mTOR ratio is >1.5 and the LC3B expression level is <40% of that in the normal control group, the predicted efficacy of fenelazol monotherapy is <40%, and it is recommended to adjust the treatment regimen.

[0031] The beneficial effects of this invention are:

[0032] 1) It solves the problem of the "one-size-fits-all" approach to existing fenestrone treatment by using biomarkers to predict and screen high-response patients, avoiding ineffective medication for low-response patients, improving treatment efficacy, and achieving precise individualized medication;

[0033] 2) The biomarker is directly associated with the core pathway of fenelazol treatment for DKD (mTOR / ULK1-podocyte autophagy), which has been confirmed by in vitro and in vivo experiments (SD rat model + MPC-5 podocytes) and inhibitor reversal experiments (3-MA). The predictive results are reproducible and traceable, thus the mechanism is clear and highly reliable.

[0034] 3) Samples are easy to obtain (blood / urine), the testing technology is mature, the operation process is simple, it is suitable for promotion and application in medical institutions at all levels, and it has strong clinical applicability;

[0035] 4) It can also take into account the safety of highly responsive patients, because patients with this condition also show a low risk of hyperkalemia (≤3.2%), which can indirectly provide a reference for medication safety.

[0036] 5) In summary, this invention is the first to elucidate a novel mechanism by which fenelazol treats diabetic nephropathy (DKD) through a specific pathway: "inhibition of mTOR phosphorylation → activation of ULK1 phosphorylation → upregulation of LC3B / Beclin1 → promotion of podocyte autophagy." This discovery not only provides new theoretical basis for the renal protective effect of fenelazol but also provides new scientific support for its application in the treatment of DKD. Compared with existing technologies, this invention clarifies the specific targets and signaling pathways of fenelazol in the regulation of podocyte autophagy, providing new targets and ideas for the development of autophagy-based therapeutic drugs for DKD. Attached Figure Description

[0037] Figure 1 A logic diagram for finelenone treatment of diabetic nephropathy based on network pharmacology and bioinformatics (A. Protein-protein interaction (PPI) network of finelenone-related targets; B. PPI network of diabetic nephropathy (DKD)-related targets; C. Construction of PPI network of common targets of finelenone and DKD; D. Terminology of biological processes (BP), cellular components (CC), and molecular functions (MF); E. GO analysis of overlapping genes of finelenone and DKD; F. Enrichment analysis of the first 9 KEGG pathways of overlapping genes of finelenone and DKD).

[0038] Figure 2 Metabolic characteristics of each experimental group (A. Drug timeline in rats; B. Blood glucose level; C. Body weight; D. 24-hour urinary protein; E. Serum creatinine; F. Blood urea nitrogen; G. Electrolytes).

[0039] Figure 3 Pathological changes in kidney problems (A, representative images of kidney tissue stained with HE, Masson, and PAS, scale bar: 50 µm; B, representative transmission electron micrographs of glomeruli, scale bar: 1.0 µm; C, relative area of ​​collagen fiber deposition (n=5); D, relative area of ​​glycogen deposition (n=5); E, relative thickness of glomerular basement membrane (n=3)).

[0040] Figure 4 The diagram shows the effect of fenestrone in alleviating podocyte damage caused by a high-glucose environment (A, cell viability evaluation using CCK-8 assay; B, representative Western blot results of nephrin and podocin; C, relative protein levels of nephrin and podocin; D, representative flow cytometry results of podocyte apoptosis).

[0041] Figure 5Schematic diagram of the regulation of podocyte autophagy by finerenone in vivo and in vitro (A, Representative transmission electron microscopy images of autolysosomes, scale bar: 1.0 µm; B, Representative in vivo immunofluorescence images of LC3B, scale bar: 50 µm; C, Relative fluorescence intensity of LC3B expression; D, Relative fluorescence intensity of Beclin-1 and LC3B in vitro; E, Representative immunofluorescence images of Beclin-1 and LC3B in vitro);

[0042] Figure 6 Schematic diagram of the molecular docking and regulatory effects of finerenone on the mTOR / ULK1 pathway (A, Molecular docking map of finerenone and ULK1; B, Molecular docking map of finerenone and mTOR; C, Representative image of Western blot; D, Quantitative results of Western blot for the expression of p-mTOR / mTOR and p-ULK1 / ULK1);

[0043] Figure 7 Results graph of the regulation of autophagic flux by finerenone in vitro (A, Representative image of Western blot; B, Quantitative Western blot results for the expression of p-mTOR / mTOR and p-ULK1 / ULK1; C, Representative fluorescence images of Beclin-1 and LC3B; Note, scale bar: 100 µm. (German) Representative immunofluorescence images of Beclin-1 and LC3B). Detailed implementation manners

[0044] The present invention will be further described below through specific examples. To make the invention object, technical solution and beneficial technical effects of the present invention clearer, the present invention will be further described in detail below in combination with the examples. It should be understood that the examples described in this specification are only for explaining the present invention and not for limiting the present invention.

[0045] Unless otherwise stated, all instruments and reagents used in the examples are commercially available or can be synthesized according to conventional methods, and can be directly used without further treatment, and the instruments used in the examples are all commercially available.

[0046] In addition, all experiments were carried out using male SD rats (body weight 210 g ± 20%) provided by the Animal Experiment Center of Xinjiang Medical University (license number: SCXK(Xin)2023 - 0002). This study was carried out in accordance with the protocol reviewed and approved by the Experimental Animal Ethics Committee of the First Affiliated Hospital of Shihezi University (approval number: 2023 - 178 - 01)

[0047] Example 1: Regulation of biomarker expression by finerenone through the mTOR / ULK1 pathway

[0048] The experimental materials and operating procedures are as follows:

[0049] 1.1 Cell Culture and Grouping: Mouse podocyte cell line MPC-5 was used and cultured in RPMI 1640 complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. After proliferation at 33℃, the cells were transferred to 37℃ for induced differentiation. The experiment was divided into 8 groups: normal control group, high glucose group (HG, 30 mmol / L), high glucose + 2.5 μmol / L fenelone group, high glucose + 5 μmol / L fenelone group, high glucose + 10 μmol / L fenelone group, high glucose + 10 μmol / L fenelone + 10 mmol / L 3-methyladenine (3-MA) ​​group, high glucose + 10 mmol / L 3-MA group, and 10 mmol / L 3-MA group.

[0050] 1.2 Drug treatment: Cells in each group were cultured for 24 hours under corresponding conditions. Fennellone and 3-MA were added simultaneously after high glucose stimulation.

[0051] 1.3 Detection Method:

[0052] Western Blot analysis: Total protein was extracted from cells, and protein concentration was determined by BCA method. After separation by SDS-PAGE electrophoresis, the protein was transferred to a PVDF membrane, blocked with 5% skim milk for 1 hour, and incubated overnight at 4°C with p-mTOR, mTOR, p-ULK1, ULK1, and β-actin primary antibodies. After washing with TBST, the membrane was incubated with HRP-labeled secondary antibody at room temperature for 1 hour. Chemiluminescence imaging and quantification were then performed.

[0053] Immunofluorescence assay: Cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.3% Triton X-100, blocked with 5% bovine serum albumin, incubated overnight with LC3B and Beclin1 primary antibodies at 4°C, incubated with Cy3-labeled secondary antibody at room temperature for 2 hours, and then counterstained with DAPI. Images were acquired using a fluorescence microscope and the average fluorescence intensity was measured.

[0054] Results: Consistent with high glucose exposure, 3-MA treatment significantly increased mTOR phosphorylation and decreased ULK1 phosphorylation (P < 0.0001); fenelazol (10 μmol / L) treatment significantly ameliorated these high glucose-induced phosphorylation changes, decreasing the p-mTOR / mTOR ratio and increasing the p-ULK1 / ULK1 ratio (P < 0.0001; Figures 7A and 7B). Immunofluorescence results showed that the fluorescence intensity of LC3B and Beclin1 in the high glucose group was significantly lower than that in the normal control group. The expression levels of both were significantly upregulated after fenelazol treatment, while the combined 3-MA treatment completely reversed the fenelazol-mediated increase in these markers (P < 0.0001; Figures 7C, 7D, and 7E). Molecular docking analysis confirmed that fenelone has binding energies of -7.7 kcal / mol with mTOR and -7.9 kcal / mol with ULK1, respectively, indicating strong binding affinity (Figures 6A and 6B).

[0055] These results indicate that fenelitonee regulates the expression of four biomarkers—p-mTOR, p-ULK1, LC3B, and Beclin1—through the mTOR / ULK1 axis, thereby activating podocyte autophagy.

[0056] Example 2: Association between biomarkers and podocyte protection under high glucose conditions in vitro

[0057] The experimental materials and operating procedures are as follows:

[0058] 2.1 Cell Culture and Grouping: MPC-5 podocytes were cultured and differentiated according to the above method and divided into normal control group, high glucose group (HG, 30 mmol / L), high glucose + 2.5 μmol / L fenelone group, high glucose + 5 μmol / L fenelone group, and high glucose + 10 μmol / L fenelone group.

[0059] 2.2 Cell viability assay: Cells were sputtered at a concentration of 1×10⁻⁶. 4 Each well was seeded with 10 μL of CCK-8 reagent and incubated for 2 hours. The absorbance was then measured at 450 nm using a microplate reader.

[0060] 2.3 Detection of podocyte protein expression: The expression of renin and podocyte protein was detected by Western blotting, and the operation procedure was the same as in Example 1.

[0061] 2.4 Apoptosis detection: Cells were injected at a rate of 5 × 10⁻⁶ cells / year. 5Cells were seeded per well in 6-well plates. After 24 hours of treatment, cells were collected, resuspended in Annexin V binding buffer, and incubated with Annexin V-FITC and PI in the dark for 15 minutes. Apoptosis was analyzed by flow cytometry.

[0062] 2.5 Detection of autophagy markers: The number of autolysosomes was observed by transmission electron microscopy, and the expression of LC3B and Beclin1 was detected by immunofluorescence. The operation steps were the same as in Example 1.

[0063] Results: Podocyte viability was significantly reduced in the high glucose group (P < 0.0001), renin and podocyte protein expression were downregulated (P < 0.01), and apoptosis rate was increased. Fennellone treatment reversed these changes in a dose-dependent manner, with a concentration of 5 μmol / L effectively antagonizing high glucose-induced damage (Figs. 4A, 4B, 4C, 4D). Transmission electron microscopy showed that the number of autolysosomes in podocytes in the fenelrenone group was significantly higher than that in the high glucose group (Fig. 5A). Immunofluorescence results showed that the relative fluorescence intensity of LC3B and Beclin1 was significantly reduced in the high glucose group (P < 0.0001), and their expression levels were significantly upregulated after fenelrenone treatment (P < 0.001; Figs. 5B, 5C, 5D, 5E). Simultaneously, Western blot confirmed that fenelrenone could reduce the high glucose-induced p-mTOR / mTOR ratio and increase the p-ULK1 / ULK1 ratio (P < 0.001; Figs. 6C, 6D).

[0064] These results indicate that changes in the expression of core biomarkers are directly related to the podocyte protective efficacy of fenelazol, and can be detected by methods such as ELISA, Western Blot, and immunofluorescence.

[0065] Example 3: Association between biomarkers and therapeutic efficacy in an in vivo model of diabetic nephropathy

[0066] 3.1 Animal Model Establishment and Grouping: Sixty male SD rats were randomly divided into four groups: control group (CON, standard diet), diabetic nephropathy group (DN group, high-fat diet + streptozotocin STZ), low-dose fenelazolone group (FIN-L, 1 mg / kg / day), and high-dose fenelazolone group (FIN-H, 10 mg / kg / day). After 8 weeks of high-fat diet feeding, rats in the DN, FIN-L, and FIN-H groups received a single intraperitoneal injection of STZ (30 mg / kg). Fasting blood glucose was measured via tail vein on days 3, 7, and 10 post-injection. A result ≥11.1 mmol / L was diagnostic of diabetes.

[0067] 3.2 Drug administration and sample collection: After diagnosis, rats were administered the corresponding dose of feneriphenone by gavage daily for 8 weeks. During this period, body weight and fasting blood glucose were monitored every 4 weeks, and 24-hour urine samples were collected to detect urinary albumin. After treatment, rats were anesthetized, and blood samples were collected by cardiac puncture and left kidney tissue was separated.

[0068] 3.3 Detection Method:

[0069] Biochemical tests: Serum tests for Scr, BUN, and potassium levels;

[0070] Pathological examination: Kidney tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, stained with HE, PAS, and Masson staining, and observed under an optical microscope; renal cortex tissue was fixed in 2.5% glutaraldehyde, post-fixed in osmium tetroxide, dehydrated, embedded, and then ultrathinly sectioned for transmission electron microscopy to observe the ultrastructure of podocytes and GBM thickness.

[0071] Biomarker detection: Western blotting was used to detect the expression of p-mTOR, p-ULK1, LC3B, and Beclin1 in kidney tissue. The procedure was the same as in Example 1.

[0072] Results: Compared with the DN group, the fenelazol treatment group showed significantly lower levels of blood glucose, 24-hour urinary protein, serum creatinine (Scr), and blood urea nitrogen (BUN) (P < 0.0001), alleviated weight loss, and showed no significant increase in serum potassium levels (all within the normal range; Figures 2B, 2C, 2D, 2E, 2F, 2G). Pathological staining showed reduced glomerular sclerosis, decreased glycogen deposition, and decreased collagen fiber deposition in the fenelazol group (P < 0.0001; Figures 3A, 3C, 3D); transmission electron microscopy showed improved foot process injury and restored GBM thickness (P < 0.0001; Figures 3B, 3E). Meanwhile, the p-mTOR / mTOR ratio in kidney tissue decreased, while the expression of p-ULK1 / ULK1, LC3B, and Beclin1 increased, with the high-dose group showing a more significant effect (consistent with in vitro experimental results); Figure 1 (KEGG enrichment analysis) validated the rationality of the target, with overlapping genes of fenelazol and DKD significantly enriched in the mTOR signaling pathway and autophagy pathway.

[0073] These results indicate that in a clinical proteinuria-prone diabetic nephropathy model (eGFR ≥ 25 ml・min⁻¹・(1.73 m²)⁻¹), changes in the expression of core biomarkers are directly associated with the renal function protective efficacy of fenelazol.

[0074] Example 4: Study on the molecular mechanism of fenelazol-regulated autophagy and verification of the stability of the biomarker detection method.

[0075] 4.1 Sample preparation: Kidney tissues from 20 rats in Example 3 and podocyte samples treated with high glucose and fenelazol in Example 2 were selected. Tissue protein and total cell protein were extracted from each sample. Serum samples were taken from 20 rats in Example 3.

[0076] 4.2 Detection Method:

[0077] Western Blot detection: p-mTOR, p-ULK1, LC3B, and Beclin1 expression were detected according to the steps described in Example 1, and the band intensity was quantified using Fiji ImageJ software.

[0078] Detection: Using the corresponding commercial ELISA kits and following the kit instructions, the protein concentrations of p-mTOR, p-ULK1, LC3B, and Beclin1 in serum and cell supernatant were detected. The absorbance was read using an ELISA reader and the concentrations were calculated.

[0079] 4.3 Data Comparison: Analyze the correlation coefficient, intra-batch coefficient of variation, and inter-batch coefficient of variation of the two detection methods.

[0080] Results: The correlation coefficients between Western blot and ELISA detection of the p-mTOR / mTOR ratio were 0.89, the p-ULK1 / ULK1 ratio was 0.91, the LC3B expression level was 0.87, and the Beclin1 expression level was 0.88. The intra-assay coefficient of variation for the ELISA method was ≤5%, and the inter-assay coefficient of variation was ≤8%, showing good consistency with the Western blot results. The correlation coefficients between the average fluorescence intensity of LC3B and Beclin1 detected by immunofluorescence and the Western blot quantitative results were 0.86 and 0.85, respectively (corresponding to the results of Examples 1 and 2).

[0081] These results demonstrate that ELISA, Western Blot, and immunofluorescence techniques can all reliably detect the core biomarkers, and the results are consistent.

[0082] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A combination of biomarkers for predicting the efficacy of fenelazol in treating diabetic nephropathy, characterized in that, It contains four proteins: phosphorylated mTOR (p-mTOR), phosphorylated ULK1 (p-ULK1), LC3B, and Beclin1.

2. The biomarker combination according to claim 1, characterized in that, The diabetic nephropathy referred to is clinical proteinuria-stage diabetic nephropathy, with the patient's estimated glomerular filtration rate (eGFR) ≥25 ml・min⁻¹・(1.73m²)⁻¹.

3. The use of the biomarker combination as described in any one of claims 1-2 in the preparation of a detection reagent or kit for predicting the efficacy of fenelazol in the treatment of diabetic nephropathy.

4. The application according to claim 3, characterized in that, The test samples are the patient's blood, serum, plasma, or urine sediment.

5. The application according to claim 3, characterized in that, The detection methods are ELISA, Western Blot, or immunofluorescence.

6. The application according to claim 3, characterized in that, The efficacy prediction criteria are as follows: before treatment, the p-mTOR / mTOR ratio is ≤1.2, the p-ULK1 / ULK1 ratio is ≥0.8, the LC3B expression level is ≥60% of the normal control group, and the Beclin1 expression level is ≥60% of the normal control group. If two or more criteria are met, the total effective rate of treatment is predicted to be ≥90%.

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