Biomarker for diagnosing primary membranous nephropathy and application thereof

Abstract

The invention discloses a biomarker for diagnosing primary membranous nephropathy and application of the biomarker, and belongs to the technical field of biological medicine. According to the invention, the content of arginine in the sample urine is detected through high performance liquid chromatography-tandem mass spectrometry, so that whether a subject suffers from the primary membranous nephropathy or not is judged. High performance liquid chromatography-tandem mass spectrometry detection results show that the relative expression level of urine arginine level in patients with primary membranous nephropathy is significantly higher than that of a healthy control group. In addition, the urine arginine level is in significant positive correlation with the PLA2R1 antibody titer of a research object. Experimental results are subjected to subject working curve analysis (ROC), and the results show that the area under the curve is (AUC) 0.802, which indicates that the urine arginine can be used as an effective biomarker to assist in the diagnosis of primary membranous nephropathy and evaluate the severity and disease progression of the primary membranous nephropathy.

Description

Technical Field

[0001] The present invention belongs to the field of biological medicine technology, and particularly relates to a biomarker for diagnosing primary membranous nephropathy and its application. Background Art

[0002] Primary membranous nephropathy (MN) is a chronic glomerular podocyte disease and one of the most common causes of adult nephrotic syndrome, accounting for about 20% - 37% of adult nephrotic syndrome. Currently, the gold standard for diagnosing primary membranous nephropathy is kidney biopsy. However, due to the invasiveness of the biopsy procedure and its complications, it is not suitable for routine clinical monitoring. Therefore, exploring biological markers for non-invasive diagnosis and disease monitoring of primary membranous nephropathy has important clinical value and social significance. Summary of the Invention

[0003] The present invention provides a biomarker for early diagnosis of primary membranous nephropathy and its application. By detecting specific biomarkers, early diagnosis of primary membranous nephropathy is obtained, thereby providing a basis for early screening, intervention treatment, and disease prognosis of primary membranous nephropathy. The biomarker includes urinary arginine. Series of previous studies by our team have shown that substances such as arginine are significantly accumulated in the urine of patients with primary membranous nephropathy and have disease diagnostic value; urinary arginine can be used as a biomarker for predicting progression. However, the study of urinary arginine in patients with primary membranous nephropathy has not been reported.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] In the first aspect, the present invention provides the application of a reagent for detecting the content of urinary arginine in the preparation of a diagnostic product for primary membranous nephropathy.

[0006] Further, the diagnostic product is a reagent or a kit.

[0007] Further, the diagnostic product is used to detect the content of arginine in a urine sample.

[0008] Further, the diagnostic product detects the content of urinary arginine by high performance liquid chromatography-tandem mass spectrometry.

[0009] Further, the above detection method includes: sample pretreatment, chromatographic detection, and mass spectrometric detection.

[0010] Further, the sample pretreatment method is: derivatizing the sample with aminoquinoline-N-hydroxysuccinimidyl carbamate AQC reagent.

[0011] Further, the chromatographic detection conditions are as follows: the chromatographic column is a CORTECS UPLC C18 column, the column temperature is 40°C, the flow rate is 0.3 mL / min, and the injection volume is 20 μL; mobile phase A is an aqueous solution containing 0.1% trifluoroacetic acid, and mobile phase B is an acetonitrile solution. Gradient elution is performed, and the elution program is as follows: 0 - 6.0 min, B 3% - 5%; 9.0 - 10.0 min, B 7% - 12%; 13.8 - 14.5 min, B 12% - 15%; 16.5 - 17.0 min, B 22% - 60%; 17.2 - 20.0 min, B 3% - 3%.

[0012] Further, the mass spectrometry detection conditions are as follows: the capillary voltage of the mass spectrometer is 3.0 KV, the cone voltage is 30 V, the desolvation gas temperature is 500°C, the desolvation gas flow rate is 1000 L / hr, the cone gas flow rate is 150 L / hr, the multiple reaction ion monitoring (MRM) mode is adopted, and detection is performed in the positive ion mode of the electrospray ionization source (ESI).

[0013] In a second aspect, the present invention also provides a diagnostic product for primary membranous nephropathy, including a reagent for detecting the arginine content in urine.

[0014] Further, the reagent further contains one or more pharmaceutically acceptable carriers or excipients.

[0015] Among patients with primary membranous nephropathy, 70% - 80% of the patients present with nephrotic syndrome, and the incidence of renal vein thrombosis is as high as 40% - 50%. As one of the important causes of nephrotic syndrome, primary membranous nephropathy lacks non-invasive diagnostic methods. This application focuses on exploring the diagnostic efficacy of urinary arginine as a biomarker for primary membranous nephropathy, and realizes non-invasive clinical diagnosis.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] In the present invention, compared with the healthy control group, the abundance of urinary arginine in patients with primary membranous nephropathy is significantly increased (P < 0.05 for all), and quantitative detection indicates a significant increase in the concentration of urinary arginine (P < 0.05 for all). When 98.02 μmol / L is used as the cut-off value for the urinary arginine concentration, the sensitivity for the diagnosis of PMN is 0.619, the specificity is 0.953, the positive predictive value is 0.929, and the negative predictive value is 0.714. This study shows that urinary arginine can be used as an effective biomarker to assist in the diagnosis of primary membranous nephropathy, evaluate its severity, and disease progression. Description of the Drawings

[0018] Figure 1This is the enrichment analysis of different substance pathways in the morning urine samples of the two groups of screened people in Example 1;

[0019] Figure 2 The relative abundance difference of urine arginine in the morning urine samples of the two groups of screened people in Example 1;

[0020] Figure 3 is the correlation between urine arginine abundance and PLA2R1 antibody abundance in Example 1;

[0021] Figure 4 The MRM peak diagram of the arginine standard and internal standard in Example 2;

[0022] Figure 5 is the standard curve for quantitative detection of arginine in Example 2;

[0023] Figure 6 The difference in quantitative concentration of urine arginine in morning urine samples of two groups of validation populations in Example 2;

[0024] Figure 7 It is the ROC curve diagram in Example 2; DETAILED DESCRIPTION

[0025] In order to better illustrate the present invention, the following embodiments are listed. Obviously, the described embodiments are only a part of the present invention, not all embodiments. Based on the embodiments in the present invention, other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0026] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0027] Example 1

[0028] 1. Research subjects and groups

[0029] From September 2022 to March 2024, 30 healthy controls and 30 patients with primary membranous nephropathy were enrolled as research subjects at Tongren Hospital in Shanghai. Inclusion criteria for the enrolled subjects: (1) Kidney biopsy confirmed histopathological damage of the kidney tissue; (2) Age > 18 years old; (3) Signed the informed consent form. Exclusion criteria: (1) Suffering from malignant tumors, lupus or severe liver diseases; (2) Pregnant or lactating patients; (3) Patients who have received kidney transplantation or dialysis treatment; (4) Patients with obvious fibrosis in other organs, such as the liver, heart, lungs and extensive skin scars; (5) Patients suffering from acute or chronic infections. The control group included 30 healthy subjects without proteinuria, hypertension, diabetes, malignant tumors, impaired renal function or acute and chronic inflammatory diseases. This invention has been reviewed and approved by the Ethics Committee of Tongren Hospital in Shanghai (approval number: K2024-068-01), and all research subjects gave informed consent. The data are expressed as mean ± standard deviation. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0030] 2. Extraction of urinary arginine

[0031] Collect 10 mL of urine samples from fasting research subjects in the early morning, filter the samples through a 0.22 μm filter membrane, take 50 μL of the filtered samples, add 10 μL of mixed internal standard and 200 μL of ice-cold LC-MS grade 50% methanol, and centrifuge at 16000 g and 4 °C for 15 min; Take all the supernatant, dry it gently under nitrogen; Dissolve the dried residue in 100 μL of 50% methanol for detection by the RPLC-HRMS / MS untargeted metabolomics platform.

[0032] 3. High performance liquid chromatography tandem mass spectrometry

[0033] Use an ultra-high performance liquid chromatography system (model Ultimate 3000, Thermo Fisher) and an ACQUITY UPLC HSS T3 chromatographic column (2.1 mm × 100 mm, 1.8 μm, Waters) to perform chromatographic separation on the samples. Chromatographic tandem mass spectrometry detection is carried out separately in the positive ion mode and the negative ion mode. Specifically as follows:

[0034] Positive ion mode: Mobile phase A is a methanol solution containing 0.1% formic acid, and mobile phase B is an aqueous solution containing 0.1% formic acid. The metabolites are eluted and separated using a linear gradient mode. The gradient is as follows: 0 - 1 min, 100% B, 10 min, 0% B, 13 min, 0% B, 13.1 min, 100% B, 15 min, 100% B; The column temperature is 50 °C, and the flow rate is 0.35 mL / min;

[0035] Negative ion mode: Mobile phase A is a methanol solution containing 6.5 mM ammonium bicarbonate, and mobile phase B is an aqueous solution containing 6.5 mM ammonium bicarbonate. The metabolites were eluted and separated using a linear gradient mode. The gradient is as follows: 0 - 0.5 min, 100% B; 9 min, 0% B; 10.4 min, 0% B; 10.5 min, 100% B; 12 min, 100% B; column temperature is 50 °C, and the flow rate is 0.4 mL / min;

[0036] The metabolites were ionized and scanned in positive ion mode and negative ion mode respectively using the heated electrospray ionization source (HESI) of the Thermo Fisher Scientific quadrupole-orbitrap tandem mass spectrometer (Q Exactive). The main parameters of the ion source were set as follows: the spray voltage was 3.5 kV, the capillary temperature and the auxiliary gas temperature were both 350 °C, the first-stage mass spectrometry scanning range was mass-to-charge ratio 60 - 900, and the data-dependent acquisition (DDA) mode was used to collect the second-stage mass spectrometry fragment information of 8 precursor ions in each scanning cycle (the HCD collision energy was set to 15, 30, and 45 eV).

[0037] 4. Statistical methods

[0038] Thermo Fisher's Compound Discoverer (version 3.3) was used to process the non-targeted metabolomics data. The following three parameters were used for substance identification, including (1) the accurate mass-to-charge ratio of the first-stage mass spectrometry (precursor ion), denoted as MS1; (2) the fragment ion mass spectrometry of MS1, denoted as the second-stage mass spectrometry (MS2); (3) the chromatographic retention time (RT). The output data was processed using the QC normalization method. Based on these three parameters, it was used to match and compare with the locally built database, and the difference in substance abundance between the two groups of data was calculated by fold change (Log2FC). After the data passed the normality test, an independent samples t-test was used to compare the differences between the two groups of data, and the Pearson correlation coefficient was used to calculate the correlation between the relative abundance of arginine and the PLA2R1 antibody titer. All P-values were two-tailed values, and P < 0.05 was considered statistically significant for the difference.

[0039] 5. Result verification

[0040] 1) The difference in relative abundance of urinary arginine detection between the two groups of people was significant:

[0041] Based on the RPLC-HRMS / MS non-targeted metabolomics platform of the present invention, it was found that the relative abundance of urinary arginine detection in the population with primary membranous nephropathy was significantly increased.

[0042] 2) Correlation analysis of urinary arginine levels and PLA2R1 antibody titers in each group:

[0043] The results of high performance liquid chromatography tandem mass spectrometry showed that the relative expression of urine arginine level in primary membranous nephropathy patients (n=30) was significantly higher than that in the healthy control group (n=30) (P<0.05). In addition, urine arginine level was significantly positively correlated with the PLA2R1 antibody titer of the study subjects (30 primary membranous nephropathy patients and 30 healthy controls) (r=-0.3079 and r=-0.4734, both P<0.05) ( Figure 2 , Figure 3 ).

[0044] The detection of arginine in different groups in the present invention shows that compared with healthy controls, the urine arginine concentration and marker expression of primary membranous nephropathy patients are significantly increased. Therefore, the increased expression of urine arginine in the disease group in the present invention can be considered as a manifestation of kidney damage in primary membranous nephropathy.

[0045] Example 2

[0046] Another batch of clinical samples was selected for independent biological experiments. The validation set included 21 healthy controls and 21 patients with primary membranous nephropathy as research subjects. Accurately weigh the arginine standard and prepare a series of calibration solutions (500, 400, 200, 100, 50, 10, 5 mmol / L). Add 10 ul of arginine internal standard solution (Arginine- 13C-D4), vortex oscillation, add 200 μL of acetonitrile, centrifuge at 16000 g and 4 °C for 15 min; take all the supernatant, dry it with nitrogen, add 200 μL of ultrapure water to the above sample to dissolve it, filter it through a 0.22 μm filter membrane filter head, take 10 μL of the filtrate, add 70 μL of borate buffer solution, vortex and mix well, then add 20 μL of AQC derivatization reagent solution, place it at room temperature for 1 min and then heat it at 55 °C for 10 min, take it out and cool it to room temperature, and then it can be detected by the Waters TQD quantitative detection platform. Chromatographic column: CORTECS UPLC C18 (1.6 μm, 2.1×150 mm), column temperature: 40 °C, flow rate: 0.3 mL / min, injection volume: 20 μL, mobile phase A: aqueous solution containing 0.1% trifluoroacetic acid, mobile phase B: acetonitrile; gradient conditions: 0 - 6.0 min, B 3% - 5%, 9.0 - 10.0 min, B 7% - 12%, 13.8 - 14.5 min, B 12% - 15%, 16.5 - 17.0 min, B 22% - 60%, 17.2 - 20.0 min, B 3% - 3%. Set the capillary voltage of the mass spectrometer to 3.0 KV, the cone voltage to 30 V, the desolvation gas temperature to 500 °C, the desolvation gas flow rate to 1000 L / hr, the cone gas flow rate to 150 L / hr, adopt the multiple reaction ion monitoring (MRM) mode, and detect the ion peak of arginine (345.2→171.1) in the positive ion mode with an electrospray ionization source (ESI).

[0047] Result verification:

[0048] The correlation between the arginine concentration in the morning urine samples of the validation set and the clinical indicators is as follows:

[0049] 1) The concentration of arginine detected by high performance liquid chromatography tandem mass spectrometry in the samples of the validation set was significantly higher in patients with primary membranous nephropathy (n = 21) than in the healthy control group (n = 21) ( Figure 4 、 Figure 5 , P < 0.0001 for both).

[0050] 2) Analysis of urinary arginine as a biomarker for primary membranous nephropathy and its diagnostic performance:

[0051] Use the urinary arginine variable to establish a logistic regression, and then calculate the probability given by the logistic regression, and then the ROC curve of the combined diagnosis of multiple indicators can be drawn.

[0052] The experimental results in Example 2 were analyzed by receiver operating characteristic curve (ROC), and the result showed that the area under the curve (AUC) was 0.802. When 98.02 umol / L was used as the cut-off value of urine arginine concentration, the sensitivity for the diagnosis of PMN was 0.619, the specificity was 0.953, the positive predictive value was 0.929, and the negative predictive value was 0.714. This indicates that urine arginine concentration has good diagnostic value in primary membranous nephropathy.

[0053] In summary, sensitivity is the "true positive rate", which is the probability of correctly diagnosing patients; specificity is the "true negative rate", which is the probability of correctly diagnosing non-patients; the closer the area under the curve AUC is to 1, the better the diagnostic efficacy. In conclusion, urine arginine can be used as an effective biomarker for primary membranous nephropathy, and the diagnostic efficacy of primary membranous nephropathy is high.

[0054] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of a reagent for detecting urine arginine content in the preparation of a diagnostic product for primary membranous nephropathy.

2. The use according to claim 1, characterized in that: The diagnostic product is a reagent or a kit.

3. The use according to claim 1, characterized in that: The diagnostic product is used to detect the arginine content in a urine sample.

4. The use according to claim 3, characterized in that: The diagnostic product detects the arginine content in urine by high performance liquid chromatography tandem mass spectrometry.

5. The use according to claim 4, characterized in that: The above detection method includes: sample pretreatment, chromatographic detection, and mass spectrometry detection.

6. The use according to claim 5, characterized in that: The sample pretreatment method is: using aminoquinoline-N-hydroxysuccinylcarbamate AQC reagent to derivatize the sample.

7. The use according to claim 5, characterized in that: The chromatographic detection conditions are as follows: the chromatographic column is a CORTECS UPLC C18 column, the column temperature is 40° C., the flow rate is 0.3 mL / min, and the injection volume is 20 μL; the mobile phase A is an aqueous solution containing 0.1% trifluoroacetic acid, the mobile phase B is an acetonitrile solution, and the elution is performed under gradient conditions, and the elution program is as follows: 0-6.0 min, B 3%-5%, 9.0-10.0 min, B 7%-12%, 13.8-14.5 min, B 12%-15%, 16.5-17.0 min, B 22%-60%, 17.2-20.0 min, B 3%-3%.

8. The use according to claim 5, characterized in that: The mass spectrometry detection conditions are as follows: the mass spectrometer capillary voltage is 3.0 KV, the cone voltage is 30 V, the desolvation gas temperature is 500° C., the desolvation gas flow rate is 1000 L / hr, the cone gas flow rate is 150 L / hr, and the multiple reaction ion monitoring (MRM) mode is adopted, and the electrospray ion source (ESI) positive ion mode is used for detection.

9. A diagnostic product for primary membranous nephropathy, characterized in that: Includes a reagent for measuring the amount of arginine in urine.

10. The diagnostic product according to claim 9, characterized in that The reagent also includes one or more pharmaceutically acceptable carriers or excipients.

Images