Group of urine exosome metabolism markers for identifying lupus nephritis as well as screening method and application of urine exosome metabolism markers

Abstract

The invention discloses a group of urine exosome metabolism markers for identifying lupus nephritis, which is characterized in that the urine exosome metabolism markers comprise one or more of L-tryptophan, L-isoleucine and phenylalanine. The method can be used as a novel noninvasive index for screening and identifying lupus nephritis.

Description

Technical Field

[0001] The present invention belongs to the technical field of metabolomics or lupus nephritis testing and diagnosis, and specifically relates to a group of urine exosome metabolite markers for identifying lupus nephritis. Background Art

[0002] Systemic lupus erythematosus (SLE) is a typical autoimmune disease involving multiple organs and tissues throughout the body, with clinical manifestations of abnormal activation of lymphocytes and abnormal accumulation of autoantibodies. Currently, the diagnosis of systemic lupus erythematosus mainly relies on the detection of autoantibodies, while the diagnosis of lupus nephritis mainly relies on renal biopsy and 24-hour urine protein quantification. These traditional diagnostic methods are limited by defects such as low sensitivity or specificity, invasiveness and delayed diagnosis, which greatly delays the best diagnosis and treatment period for patients. A large amount of evidence shows that exosomes play a key role in the course of various autoimmune diseases (AIDs), and they mainly participate in the pathogenesis of AIDs by affecting intercellular communication and the function of immune cells. The concentration of exosomes themselves and the expression of nucleic acids, proteins and metabolites they encapsulate can reflect the degree of disease progression and are good biomarkers for AIDs. However, current articles on exosome markers mainly focus on the RNA and protein levels, while the development of exosome metabolic markers is often overlooked. Urine collects waste products discharged from blood under homeostatic mechanisms in various forms, and collects a large number of disease-related changes, which can better reflect early minor changes. Urine samples have many advantages, such as being completely non-invasive, convenient for continuous collection, and closely related to kidney disease, making them a good carrier in biomarker research. At present, there are no reports of urinary exosome metabolite markers used in clinical practice or applications. Therefore, this study used non-invasive urinary exosomes as carriers and non-targeted metabolomics technology as a development method to identify for the first time non-invasive urinary exosome metabolite markers specific to lupus nephritis. Purpose of the Invention

[0003] The purpose of the present invention is to identify lupus nephritis-specific urine exosome differential metabolites through metabolomics technology. Based on the above invention purpose, the present invention discloses a group of urine exosome metabolite markers, screening methods and applications for identifying lupus nephritis.

[0004] In a first aspect, the present invention provides a set of urinary exosome metabolite markers for identifying lupus nephritis, wherein the urinary exosome metabolite markers comprise one or more of L-tryptophan, L-isoleucine, and phenylalanine.

[0005] Preferably, the urine exosome metabolic markers are from urine exosome samples of lupus nephritis LN and healthy subjects HC.

[0006] Preferably, the receiver operating characteristic (ROC) curve is used to analyze the diagnostic value of urinary exosome metabolite markers for lupus nephritis.

[0007] In a second aspect, the present invention provides a group of urinary exosome metabolite marker screening methods for identifying lupus nephritis, wherein the screening method process is: S1. Collect urine samples from patients diagnosed with lupus nephritis and healthy patients undergoing physical examinations, and use an exosome extraction kit to extract exosomes from the urine samples as exosome samples; S2. Pipette 20-100 μL of exosome sample, add 120 μL of extraction solution and vortex mix for 30 s, perform ultrasonic extraction at 5 ℃ and 40 kHz for 30 min, let the sample stand at -20 ℃ for 30 min, centrifuge at 4 ℃ and 13000 g for 15 min, remove the supernatant, blow dry the supernatant with nitrogen, add 100-200 μL of re-solution to the supernatant to dissolve the precipitate, perform ultrasonic extraction at 5 ℃ and 40 kHz for 5 min, centrifuge at 4 ℃ and 13000 g for 10 min, remove the supernatant to an injection bottle with an inner tube for LC-MS analysis; during the instrument analysis, insert a quality control sample into every 5-15 samples to examine the repeatability of the entire analysis process.

[0008] Preferably, the extract in step S2 comprises: 50% by volume acetonitrile, 50% by volume methanol, and 0.02 mg / mL L-2-chlorophenylalanine; The complex solution includes: 50% by volume of acetonitrile, the solvent is ultrapure water; Quality control samples included: 20 μL of each sample metabolite was taken and mixed evenly.

[0009] Preferably, the LC-MS analysis instrument is an ultra high performance liquid chromatography tandem Fourier transform mass spectrometry UHPLC-Exploris240 system.

[0010] In a third aspect, the present invention provides a kit for identifying lupus nephritis, wherein the kit comprises the urine exosome metabolite marker or a detection reagent thereof.

[0011] Beneficial Effects

[0012] 1. The present invention provides three highly specific and sensitive biomarkers, namely L-tryptophan, L-isoleucine and phenylalanine, for diagnosing lupus nephritis, which fills the gap in this field, further improves the accuracy of diagnosis, and helps establish new clinical diagnostic standards for lupus nephritis.

[0013] 2. The diagnostic indicators provided by the present invention are metabolic markers of urinary exosomes. Compared with other peripheral indicators, urine can more directly reflect the pathological changes occurring in the kidneys. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0015] Figure 1 The following are analysis diagrams of the metabolomics sequencing results of urine exosomes in the lupus nephritis group and the healthy physical examination group; A is the result of non-target metabolomics analysis, B is the prediction result of the OPLS-DA model, C is the result of metabolite screening based on metabolomics, and D is the Venn diagram of metabolite screening.

[0016] Figure 2 The following are the screening results of differential urinary exosome metabolic markers; A is the protein abundance detection result of L-tryptophan, B is the protein abundance detection result of phenylalanine, and C is the protein abundance detection result of L-isoleucine.

[0017] Figure 3 Diagnostic performance analysis of differential exosome metabolites in urine; A is the area under the AUC curve of L-tryptophan, B is the area under the AUC curve of phenylalanine, and C is the area under the AUC curve of L-isoleucine. DETAILED DESCRIPTION

[0018] The technical scheme of the present invention is described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in the embodiments are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0019] Example 1 Metabolomics Sequencing Screening of Urine Exosomes 1.1 Collection of urine samples: 29 patients diagnosed with lupus nephritis by the Department of Rheumatology and Immunology of Drum Tower Hospital Affiliated to Nanjing University Medical School and 20 healthy patients were collected, and their midstream urine specimens were collected during routine testing. The inclusion criteria for lupus nephritis required meeting all of the following conditions at the same time: 1) At least 4 of the 11 systemic lupus erythematosus classification criteria established by the American College of Rheumatology (ACR) in 1997; and clear renal puncture pathology or in accordance with the "2019 Chinese Guidelines for the Diagnosis and Treatment of Lupus Nephritis". 2) Age range 18-60 years old; 3) Signed informed consent and willing to provide urine samples.

[0020] Exclusion criteria for lupus nephritis: Patients who meet any of the following criteria are excluded: 1) Patients with other autoimmune diseases other than systemic lupus erythematosus: including dermatomyositis / polymyositis, mixed connective tissue disease, systemic sclerosis, rheumatoid arthritis, etc.; 2) Patients with various infections. The disease activity index score of patients with systemic lupus erythematosus is based on the systemic lupus erythematosus disease activity index-2000 (SLEDAI-2K) scale, and the SLEDAI-2K score is calculated. This study was approved by the Ethics Committee of Drum Tower Hospital Affiliated to Nanjing University Medical School (Project No.: 2023-568-01).

[0021] 1.2 Extraction of urine exosomes: Exosomes were obtained using the Exo-Quick-TC™, Total Exosome Isolation (Invitrogen) kit. The following steps were performed strictly according to the kit instructions: The urine sample was centrifuged at 2000 × g for 30 min at 4 °C to remove cells and debris. The urine was mixed with the sedimentation reagent in a 1:1 ratio and the sample was incubated at room temperature for 1 hour. After incubation, the sample was centrifuged at 10,000 × g for 1 hour at 4 °C, the sample was aspirated with a pipette, and the supernatant was discarded. The exosomes were deposited in a small ball at the bottom of the test tube.

[0022] 1.3 Urine exosome metabolome sequencing: 1.3.1 Chromatographic conditions: 10 μL of sample was separated on an HSS T3 column (100 mm × 2.1 mm, 1.8 μm) and then entered the mass spectrometer detection. Mobile phase A was a water / acetonitrile (95 / 5, volume / volume) solution containing 0.1% formic acid, and mobile phase B was an acetonitrile / isopropanol / water (47.5 / 47.5 / 5, volume / volume / volume) solution containing 0.1% formic acid. Positive ion mode separation gradient: 0-3 min, mobile phase B increased from 0% to 20%; 3-4.5 min, mobile phase B increased from 20% to 35%; 4.5-5 min, mobile phase B increased from 35% to 100%; 5-6.3 min, mobile phase B maintained at 100%; 6.3-6.4 min, mobile phase B decreased from 100% to 0%; 6.4-8 min, mobile phase B maintained at 0%. Negative ion mode separation gradient: 0-1.5min, mobile phase B increased from 0 to 5%; 1.5-2min, mobile phase B increased from 5% to 10%; 2-4.5min, mobile phase B increased from 10% to 30%; 4.5-5min, mobile phase B increased from 30% to 100%; 5-6.3min, mobile phase B maintained linearly at 100%; 6.3-6.4min, mobile phase B decreased from 100% to 0%; 6.4-8min, mobile phase B maintained linearly at 0%. The flow rate was 0.40 mL / min, and the column temperature was 40℃.

[0023] 1.3.2 Mass spectrometry conditions: The sample mass spectrometry signal was collected in positive and negative ion scanning mode, with a mass scanning range of 70-1050m / z. The sheath gas flow rate was 60 psi, the auxiliary gas flow rate was 20 psi, the auxiliary gas heating temperature was 350 °C, the capillary temperature was 320 °C, the positive mode ion spray voltage was set to 3400 V, the negative mode ion spray voltage was set to -3000 V, and the normalized collision energy was 20-40-60eV cyclic collision energy. The primary mass spectrometry resolution was 60000, the secondary mass spectrometry resolution was 15000, and the data were collected in DDA mode.

[0024] 1.3.3 Material identification and analysis: 20-100 μL of exosome sample was aspirated, 120 μL of extraction solution was added and vortexed for 30 s, low-temperature ultrasonic extraction was performed at 5 ℃ and 40 kHz for 30 min, the sample was allowed to stand at -20 ℃ for 30 min, centrifuged at 4 ℃ and 13000 g for 15 min, the supernatant was removed, the supernatant was blown dry with nitrogen, 100-200 μL of re-solution was added to the supernatant to re-dissolve the precipitate, low-temperature ultrasonic extraction was performed at 5 ℃ and 40 kHz for 5 min, centrifuged at 4 ℃ and 13000 g for 10 min, the supernatant was removed to an injection vial with an inner insert for LC-MS analysis; during the instrument analysis, a quality control sample was inserted into every 5-15 samples to examine the repeatability of the entire analysis process.

[0025] After the computer is completed, the LC-MS raw data are imported into the metabolomics processing software Progenesis QI (Waters Corporation, Milford, USA) for baseline filtering, peak identification, integration, retention time correction, and peak alignment. Finally, a data matrix of retention time, mass-to-charge ratio and peak intensity is obtained. At the same time, the MS and MSMS mass spectrometry information is compared with the metabolic public database HMDB (http: / / www.hmdb.ca / ) and Metlin (https: / / metlin.scripps.edu / ) to obtain metabolite information.

[0026] Example 2 Clinical application of differential exosome metabolic markers in urine of lupus nephritis 2.1 Identification and analysis of urine exosome metabolomics sequencing results: The present invention performed non-targeted metabolomics analysis on urine exosomes extracted from 29 patients with lupus nephritis and 20 normal patients. Figure 1 As shown in A, RL represents the lupus nephritis patient group, and RH represents the healthy physical examination group. The metabolite distribution dispersion between the two groups is very large, that is, the classification effect is very significant.

[0027] In the OPLS-DA model, 200 random permutations were used for permutation test for cross validation. R2Y represents the explanatory rate of the constructed model for the Y matrix, and Q2 represents the predictive ability of the model. The closer R2Y and Q2 are to 1, the more stable and reliable the model is. Q2>0.5 represents a good predictive ability of the model, and Q2<0.5 represents a poor predictive ability of the model. The results are shown in Figure 1 As shown in Figure B, the R2Y value is 0.977 and the Q2 value is 0.9, indicating that the resulting model is stable and reliable without overfitting.

[0028] Based on the metabolomics results, a total of 963 metabolites were screened between the two groups. Figure 1 As shown in Figure C, compared with the healthy subjects, 408 metabolites were significantly upregulated in lupus nephritis patients, 555 metabolites were significantly downregulated in lupus nephritis patients, and 1798 metabolites showed no change in trend. Figure 1 As shown in D, the Venn diagram showed that 222 metabolites were unique to the lupus nephritis group, 186 metabolites were unique to the healthy subjects group, and 2674 metabolites were shared by the two groups.

[0029] 2.2 Analysis of the diagnostic efficacy of differential metabolites in urine of lupus nephritis: VIP (Variable important in projection) is the variable weight value of the PLS-DA model variable, which can be used to measure the influence and explanatory power of the difference in the accumulation of each metabolite on the classification and discrimination of each group of samples. VIP ≥ 1 is a common screening standard for differential metabolites. Based on metabolomics data, the present invention screened out a total of 963 metabolites between the two groups. According to VIP> 1 and the one-tailed T test, p <0.05 and the difference multiple were set, and finally three differential metabolites were screened, namely L-tryptophan, phenylalanine and L-isoleucine. Figure 2 As shown in Figure 2, these three metabolites showed a significant up-regulation trend compared with the control group, and the one-tailed T test showed p <0.01.

[0030] The area under the ROC curve (AUC) has been widely recognized as an inherent accuracy indicator for evaluating the authenticity of diagnostic tests. The AUC of a completely worthless diagnostic test is 0.5, and the AUC of an ideal diagnostic test is 1. It is generally believed that an AUC between 0.7 and 0.9 has a certain diagnostic value. Figure 3 As shown, the areas under the AUC curves of the three differential urinary exosome metabolic markers of L-tryptophan, phenylalanine and L-isoleucine of these proteins listed in the present invention are all above 0.7, indicating that the above markers can better screen out lupus nephritis, and the areas under the AUC curves are 0.7741, 0.7828 and 0.7888, respectively.

[0031] The embodiments of the present invention are only to introduce the specific implementation methods thereof, and are not intended to limit the protection scope thereof. The technicians in the industry may make some modifications inspired by the embodiments, so any equivalent changes or modifications made according to the scope of the present invention are within the scope of the patent claims of the present invention.

Claims

1. A set of urinary exosome metabolite markers for identifying lupus nephritis, characterized in that: The urine exosome metabolic markers include one or more of L-tryptophan, L-isoleucine, and phenylalanine.

2. A set of urinary exosome metabolic markers for identifying lupus nephritis according to claim 1, characterized in that: The urinary exosome metabolic markers are from the urinary exosome samples of lupus nephritis LN and healthy subjects HC.

3. The use according to claim 1, characterized in that: The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of urinary exosome metabolic markers for lupus nephritis.

4. A method for screening urine exosome metabolite markers for identifying lupus nephritis, characterized in that: The screening method process is: S1. Collect urine samples from patients diagnosed with lupus nephritis and healthy patients undergoing physical examinations, and use an exosome extraction kit to extract exosomes from the urine samples as exosome samples; S2. Pipette 20-100 μL of exosome sample, add 120 μL of extraction solution and vortex mix for 30 seconds, perform ultrasonic extraction at 5°C and 40 kHz for 30 minutes, let the sample stand at -20°C for 30 minutes, centrifuge at 4°C and 13000 g for 15 minutes, remove the supernatant, blow dry the supernatant with nitrogen, add 100-200 μL of re-solution to the supernatant to dissolve the precipitate, perform ultrasonic extraction at 5°C and 40 kHz for 5 minutes, centrifuge at 4°C and 13000 g for 10 minutes, remove the supernatant to an injection bottle with an inner tube for LC-MS analysis; during the instrument analysis, insert a quality control sample into every 5-15 samples to examine the repeatability of the entire analysis process.

5. The method for screening a group of urine exosome metabolite markers for identifying lupus nephritis according to claim 4, characterized in that: The extract in step S2 comprises: 50% by volume of acetonitrile, 50% by volume of methanol, and 0.02 mg / mL of L-2-chlorophenylalanine; The complex solution includes: 50% by volume of acetonitrile, the solvent is ultrapure water; Quality control samples included: 20 μL of each sample metabolite was taken and mixed evenly.

6. The method for screening a group of urine exosome metabolite markers for identifying lupus nephritis according to claim 4, characterized in that: The LC-MS analysis instrument is an ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry UHPLC-Exploris240 system.

7. A kit for identifying lupus nephritis, characterized in that: The kit comprises the urine exosome metabolite marker or a detection reagent thereof according to claim 1.

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