Biomarker related to early diagnosis of diabetic nephropathy and application of biomarker

Abstract

The invention relates to the technical field of biological medicine, in particular to a biomarker related to early diagnosis of diabetic nephropathy and application of the biomarker. The invention discloses and verifies the application value of the TRPV2 protein as the biomarker for early diagnosis of diabetic nephropathy for the first time. Clinical queue research proves that compared with a healthy control group, the expression level of TRPV2 protein in proximal renal tubules of kidney tissues of DKD patients is remarkably increased and is continuously increased along with pathological grading progress, TRPV2 protein expression of III-IV-level DKD patients is remarkably higher than that of I-II-level DKD patients, and the TRPV2 protein expression of III-IV-level DKD patients is closely related to the degree of renal function impairment; meanwhile, the AUC of a working characteristic curve of a subject can reach 0.9077 to 0.9291. Therefore, the TRPV2 protein provided by the invention can accurately identify DKD patients and healthy people.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a biomarker related to the early diagnosis of diabetic nephropathy and its application. Background Technology

[0002] Diabetic nephropathy (DKD) is one of the most significant microvascular complications of diabetes and has become the leading cause of chronic kidney disease (CKD) worldwide. With the continued rise in the incidence of diabetes, the prevalence of DKD is also increasing annually, with some patients progressing to end-stage renal disease (ESRD) and requiring renal replacement therapy to sustain life. Furthermore, DKD patients experience a significantly increased incidence of cardiovascular events and mortality, placing a heavy burden on patients' families and the public healthcare system.

[0003] Early diagnosis is a crucial aspect of DKD clinical management, and accurate early diagnosis helps identify high-risk patients. Biomarkers, due to their high specificity, sensitivity, and ability to reflect the pathophysiological processes of the disease, are of significant value in disease diagnosis, prognosis prediction, and treatment monitoring. In recent years, the search for specific biomarkers for the early diagnosis of DKD has become a research hotspot; however, there are currently no clinically validated and widely applicable biomarkers for the early diagnosis of DKD.

[0004] Transient receptor potential vanillic acid 2 (TRPV2) is a non-selective cation channel with high permeability to calcium ions. It is widely expressed in various human tissues and organs, including the glomeruli and tubules of the kidneys, but there are no reports of using it as a biomarker for predicting the prognosis of DKD.

[0005] Given the current lack of accurate and specific biomarkers for early diagnosis of DKD in existing technologies, this invention, through a systematic clinical cohort study, has for the first time discovered that TRPV2 is highly expressed in the renal tissue of DKD patients, and that its expression level is closely related to the degree of pathological damage and the risk of renal function progression. This confirms the application value of TRPV2 as a biomarker for the diagnosis of DKD, thus completing this invention. Summary of the Invention

[0006] The purpose of this invention is to provide a biomarker related to the early diagnosis of diabetic nephropathy and its application, in order to solve the problems existing in the prior art. This invention discovers that TRPV2 protein is highly expressed in the kidney tissue of patients with diabetic nephropathy, and its expression level is positively correlated with the severity of kidney pathological damage, thus serving as a specific biomarker for the early diagnosis of DKD.

[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides a biomarker associated with the early diagnosis of diabetic nephropathy, the biomarker including TRPV2 protein.

[0008] This invention provides the application of reagents for detecting the expression levels of the above-mentioned biomarkers in the preparation of detection products for the early diagnosis of diabetic nephropathy.

[0009] Optionally, the testing product may include a chip or a reagent kit.

[0010] This invention provides a detection product for the early diagnosis of diabetic nephropathy, the product comprising reagents for detecting the expression levels of the aforementioned biomarkers.

[0011] Optionally, the testing product may include a chip or a reagent kit.

[0012] This invention provides the use of inhibitors of the above-mentioned biomarkers in the preparation of medicaments for the prevention and / or treatment of diabetic nephropathy.

[0013] This invention provides the application of the above-mentioned biomarkers as targets in the preparation of drugs for the prevention and / or treatment of diabetic nephropathy.

[0014] The present invention provides a medicament for the prevention and / or treatment of diabetic nephropathy, the medicament comprising inhibitors of the aforementioned biomarkers.

[0015] This invention provides the application of the above-mentioned biomarkers as targets in screening drugs for the treatment of diabetic nephropathy.

[0016] This invention provides a method for screening drugs for the treatment of diabetic nephropathy, including the step of detecting the expression level of TRPV2 protein in subjects before and after drug administration.

[0017] The present invention discloses the following technical effects: This invention is the first to reveal and verify the application value of TRPV2 as a biomarker for the early diagnosis of diabetic nephropathy (DKD). Through clinical cohort studies, this invention confirms that, compared with healthy controls, the expression level of TRPV2 protein in the proximal tubules of renal tissue was significantly increased in DKD patients, and continued to increase with the progression of pathological grading. The TRPV2 protein expression in grade III-IV patients was significantly higher than that in grade I-II patients, and it was closely related to the degree of renal function impairment; simultaneously, the receiver operating characteristic (AUC) curve reached 0.9077-0.9291. Therefore, the TRPV2 protein provided by this invention can accurately distinguish between DKD patients and healthy individuals.

[0018] In summary, this invention, through model construction using a training set and validation using an independent validation set, fully demonstrates the effectiveness and stability of TRPV2 protein as a biomarker, solving the problem of the lack of accurate biomarkers in the early diagnosis of DKD. Its detection method is reproducible and has stable predictive efficacy, providing a reliable basis for clinical translation. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This study compares the TRPV2 protein expression levels in renal tissues of patients with diabetic nephropathy at different pathological stages. A shows the immunohistochemical staining images of TRPV2 protein in the renal tissues of DKD patients and healthy controls; G shows the glomerular field of view; and T shows the tubular and interstitial field of view. B shows the quantitative statistical comparison of TRPV2 immunohistochemical staining results in the renal tissues of DKD patients and healthy controls. C shows the statistical comparison of TRPV2 immunohistochemical staining results after dividing the population into DKD-I / II, DKD-III / IV, and healthy control groups according to renal biopsy pathology. P<0.0001; HC represents the healthy control group, DKD patients (including DKD patients of grades I-II and DKD patients of grades III-IV), DKD patients of grades I-II, and DKD patients of grades III-IV. Figure 2 This study analyzed the correlation between TRPV2 protein expression levels in renal tissue and clinical baseline data in patients with diabetic nephropathy. Specifically, A represents the correlation between TRPV2 expression levels and serum creatinine; B represents the correlation between TRPV2 expression levels and blood urea nitrogen; C represents the correlation between TRPV2 expression levels and eGFR; D represents the correlation between TRPV2 expression levels and hemoglobin levels; E represents the correlation between TRPV2 expression levels and fasting blood glucose levels; F represents the correlation between TRPV2 expression levels and serum albumin; G represents the correlation between TRPV2 expression levels and the urinary albumin / creatinine ratio; H represents the correlation between TRPV2 expression levels and serum triglyceride levels; I represents the correlation between TRPV2 expression levels and very low-density lipoprotein (VLDL); and J represents the correlation between TRPV2 expression levels and serum calcium levels. Figure 3 Receiver operating characteristic (ROC) curves and multivariate logistic regression analysis were used to differentiate TRPV2 protein between DKD patients and healthy individuals; where A is the ROC curve for the training set and B is the ROC curve for the validation set. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Example 1 1. Sample Screening Process 1.1 Source of research subjects The diabetic kidney disease (DKD) patient cohort in this study consisted of patients who visited the Nephrology Center of the First Affiliated Hospital of Zhejiang University School of Medicine between December 2012 and December 2022. The healthy control group consisted of healthy kidney donors who underwent kidney transplantation with a relative at the same hospital during the same period. All participants signed informed consent forms. This study was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine and complies with the principles of the Declaration of Helsinki.

[0027] 1.2 Inclusion Criteria (1) DKD patient group: ① A confirmed diagnosis of type 2 diabetes (meeting the WHO 2019 diagnostic criteria for diabetes), with a disease duration of ≥3 months; ② The patient was diagnosed with diabetic nephropathy via renal biopsy (the pathological diagnosis met the diagnostic criteria of the "Expert Consensus on the Prevention and Treatment of Diabetic Nephropathy (2023 Edition)"). ③ Baseline estimated glomerular filtration rate (eGFR) ≥ 30 ml / min / 1.73 m 2 (Calculated using the CKD-EPI formula); ④ Age 18-75, gender not limited; ⑤ Voluntarily participate in and cooperate in completing follow-up visits.

[0028] (2) Healthy control group: ① No history of chronic diseases such as diabetes, hypertension, or chronic kidney disease; ② Renal biopsy pathology confirmed that the kidney structure and function were normal (no abnormal lesions in the glomeruli and renal tubules). ③ Frequency matching was performed between age and DKD patient group, with an age matching tolerance of ±5 years. The baseline characteristics of gender and age of the two groups were found to be comparable (P>0.05). ④ Laboratory tests: Serum creatinine (Scr), blood urea nitrogen (BUN), and urine albumin / creatinine ratio (ACR) were all within the normal reference range; ⑤ Voluntary participation and signing of informed consent form.

[0029] 1.3 Exclusion Criteria (1) Comorbid other primary glomerular diseases (such as IgA nephropathy, membranous nephropathy) and secondary kidney diseases (such as lupus nephritis, hypertensive nephropathy); (2) The presence of active infection (such as pneumonia, urinary tract infection), malignant tumor, or autoimmune disease; (3) Pregnant or breastfeeding women; (4) Severe heart and liver dysfunction (such as heart failure, decompensated cirrhosis); (5) History of major surgery or trauma within the past 3 months; (6) Those who refuse renal biopsy or are unable to cooperate in completing sample collection and follow-up; (7) Incomplete clinical data affects the diagnostic analysis.

[0030] 1.4 Sample Screening Steps ① Preliminary screening: Potential study subjects who meet the above inclusion criteria are searched through the hospital's electronic medical record system, and those who obviously do not meet the exclusion criteria are excluded; ② Data verification: Review the patient's inpatient medical records, pathology reports, and laboratory test results (serum creatinine, blood urea nitrogen, eGFR, triglycerides, serum white blood cells, urine albumin / creatinine ratio, and other clinical indicators) to confirm the diagnosis and the completeness of baseline data; ③ Informed consent: Explain the research purpose, process and risks in detail to eligible research subjects, and obtain their signed informed consent form; ④ Final Determination: After joint review by two attending physicians, a total of 110 DKD patients and 29 healthy controls were ultimately included. The training set included 84 DKD patients (details shown in Tables 1-4) and 23 healthy controls (details shown in Table 5). The validation set included 26 DKD patients (details shown in Table 6) and 6 healthy controls (details shown in Table 7).

[0031] Table 1. Specific information of DKD patients in the training set Table 2. Specific information on some DKD patients in the training set (see Table 1) Table 3. Specific information on DKD patients in the training set (see Table 2) Table 4. Specific information on DKD patients in the training set (see Table 3) Table 5. Detailed information on the healthy control group in the training set. Table 6. Specific information of DKD patients in the validation set Table 7. Specific information on the healthy control group in the validation set. 2. Procedure for detecting TRPV2 protein expression level (accession number: Q9Y5S1) 2.1 Sample Collection and Processing (1) Kidney tissue samples: After kidney biopsy, a portion of the tissue was fixed with 4% (w / v) paraformaldehyde for 24 h, dehydrated in a gradient (75% (v / v) alcohol → 85% (v / v) alcohol → 95% (v / v) alcohol → anhydrous ethanol) for 15 min each, cleared twice with xylene (15 min each time), embedded in paraffin and made into 4 μm thick paraffin sections, which were stored at room temperature for later use.

[0032] 2.2 Immunohistochemical detection procedure (kidney tissue sample) (1) Dewaxing: Dewaxing the paraffin sections of kidney tissue in xylene I and xylene II for 15 min each; (2) Hydration: Add anhydrous ethanol → 95% (v / v) alcohol → 85% (v / v) alcohol → 75% (v / v) alcohol for 5 min each time, and finally rinse with PBS buffer (pH=7.4) 3 times for 5 min each time; (3) Antigen retrieval: The slides were placed in citrate buffer (Solepro, catalog number: C1010, 0.01mol / L, pH=6.0), autoclaved for 10 min, and then naturally cooled to room temperature. After that, the slides were rinsed with PBS 3 times for 5 min each time. (4) Blocking: Add 3wt% hydrogen peroxide solution and incubate at room temperature for 10 min to block endogenous peroxidase activity. Rinse with PBS 3 times for 5 min each time. Then add 5% bovine serum albumin (BSA) blocking solution and incubate at 37°C for 30 min. Pour off the blocking solution and do not rinse. (5) Primary antibody incubation: Add diluted rabbit anti-human TRPV2 monoclonal antibody (Novus, catalog number: NBP1-92576, dilution ratio 1:200) and incubate overnight (12-16h) in a humidified chamber at 4°C. (6) Secondary antibody incubation: The next day, the slides were taken out and warmed to room temperature for 30 min. They were washed with PBS 3 times for 5 min each time. Horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (manufacturer: Gene Tech, catalog number: GK500710, dilution ratio 1:1000) was added and incubated at 37℃ for 30 min. They were washed with PBS 3 times for 5 min each time. (7) Color development: Add DAB color development solution (manufacturer: Gene Tech, product number: GK500710, ready to use), incubate at room temperature in the dark for 3-5 minutes, observe the degree of color development under a microscope, and stop the color development with distilled water after the positive signal is clear; (8) Counterstaining: Counterstain with hematoxylin solution for 5 min, differentiate with hydrochloric acid alcohol for 30 s, rinse with running tap water for 10 min to return to blue; (9) Dehydration and transparency: Place 75% (v / v) alcohol → 85% (v / v) alcohol → 95% (v / v) alcohol → anhydrous ethanol in sequence for 5 min each, and xylene I and xylene II for 10 min each; (10) Sealing: Use neutral resin to seal the film, and let it dry before use.

[0033] 2.3 Quantitative Analysis Procedure (1) Image acquisition: A Leica microscope was used with a 40x objective lens and a 10x eyepiece under bright field. Five non-overlapping proximal tubule fields of view were randomly selected (avoiding the glomerulus and necrotic areas). Images were taken for each field of view (resolution: 1024×768 pixels). (2) Quantitative calculation: Fiji ImageJ software (version 1.8.0) was used for analysis. First, a uniform optical density threshold was set (to exclude background interference). The integrated optical density value (IOD) and positive staining area (Area) of each field of view were measured respectively. The average optical density value (Mean OD=IOD / Area) was calculated. The average value of 5 fields of view was taken as the final expression level of TRPV2 protein in the sample.

[0034] 2.4 Quality Control (1) Antibody quality control: For each batch of tests, a positive control (kidney tissue sections with known high expression of TRPV2 protein) and a negative control (using PBS instead of primary antibody) are set up to ensure antibody specificity; (2) Repeatability verification: Randomly select 20 samples for repeated testing (the same sample is tested 3 times consecutively), calculate the coefficient of variation (CV), and CV < 10% is considered reliable test results; (3) Observer consistency: The images were analyzed independently by two experienced pathologists in a blinded manner, and the consistency was assessed by the Kappa test. A Kappa value > 0.8 was considered to be good consistency.

[0035] 3 Results Immunohistochemical staining revealed that, in the training set, compared with the healthy control group, the expression level of TRPV2 protein in the proximal tubules of the kidneys of DKD patients was significantly increased (Mean OD: 0.33±0.03 vs 0.28±0.01, P<0.001). Figure 1 Further analysis based on renal biopsy pathological grading showed that TRPV2 protein expression levels were correlated with the severity of DKD pathological damage. Specifically, the TRPV2 protein expression levels in the renal tissues of patients with grade III-IV DKD were significantly higher than those in patients with grade I-II DKD (0.34±0.03 vs 0.32±0.03, P=0.034). Figure 1 ).

[0036] Correlation analysis showed that in the DKD patient cohort of the training set, the expression level of TRPV2 protein in renal tissue was positively correlated with renal function indicators Scr (r=0.7158, P<0.0001) and BUN (r=0.4805, P<0.0001), negatively correlated with eGFR (r=-0.8639, P<0.0001), negatively correlated with hemoglobin (Hb) (r=-0.5246, P<0.0001), positively correlated with glucose level (Glucose) (r=0.2505, P=0.0093), and negatively correlated with serum albumin level (Alb) (r=-0.2841, P=0.0033). In addition, TRPV2 protein expression levels were positively correlated with the urinary albumin / creatinine ratio (ACR) (r=0.5858, P<0.0001), and positively correlated with triglyceride levels (r=0.3253, P=0.0006) and very low-density lipoprotein (VLDL-C) levels (r=0.3444, P=0.0003). Furthermore, TRPV2 protein expression was negatively correlated with serum calcium levels (r=-0.3230, P=0.0007). Figure 2 ).

[0037] ROC curve analysis results show: 1. In the training set, TRPV2 protein accurately distinguished between DKD patients and healthy individuals. The receiver operating characteristic (ROC) curve showed an AUC of 0.9291 (95% confidence interval, CI: 0.8738–0.9844), an optimal cutoff value of 0.3150, a sensitivity of 0.8434 (95% confidence interval, CI: 0.7502–0.9061), and a specificity of 0.8636 (95% confidence interval, CI: 0.6667–0.9525). P < 0.0001, indicating that the TRPV2 protein in the training set has good distinguishing efficacy between DKD patients and healthy individuals. Figure 3 (A in GraphPad Prism 10.0).

[0038] 2. In the validation set, TRPV2 protein accurately distinguished between DKD patients and healthy individuals. The receiver operating characteristic (ROC) curve showed an AUC of 0.9520 (95% confidence interval, CI: 0.8676–1.000), an optimal cutoff of 0.2950, ​​a sensitivity of 0.9600 (95% confidence interval, CI: 0.8046–0.9979), and a specificity of 0.8000 (95% confidence interval, CI: 0.3755–0.9897), with a P=0.0017. This suggests that the TRPV2 protein in the validation set has good efficacy in distinguishing between DKD patients and healthy individuals. Figure 3 (B in GraphPadPrism 10.0).

[0039] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A biomarker associated with the early diagnosis of diabetic nephropathy, characterized in that, The biomarker includes the TRPV2 protein.

2. The use of the reagent for detecting the expression level of the biomarker as described in claim 1 in the preparation of a detection product for the early diagnosis of diabetic nephropathy.

3. The application according to claim 2, characterized in that, The testing products include chips or reagent kits.

4. A detection product for the early diagnosis of diabetic nephropathy, characterized in that, The product includes a reagent for detecting the expression level of the biomarker as described in claim 1.

5. The testing product according to claim 4, characterized in that, The testing products include chips or reagent kits.

6. The use of the inhibitor of the biomarker according to claim 1 in the preparation of a medicament for the prevention and / or treatment of diabetic nephropathy.

7. The use of the biomarker of claim 1 as a target in the preparation of a medicament for the prevention and / or treatment of diabetic nephropathy.

8. A drug for the prevention and / or treatment of diabetic nephropathy, characterized in that, The drug includes an inhibitor of the biomarker as described in claim 1.

9. The use of the biomarker of claim 1 as a target for screening drugs for the treatment of diabetic nephropathy.

10. A method for screening drugs for treating diabetic nephropathy, characterized in that, This includes steps to detect the expression level of TRPV2 protein in subjects before and after drug administration.

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