A biomarker, a kit and a diagnostic device for diagnosing diabetic nephropathy

Abstract

The present invention relates to the field of biological detection technology, and in particular to a biomarker, a kit and a diagnostic device for diagnosing diabetic nephropathy. The present invention relates to the application of a substance for detecting a biomarker, wherein the biomarker is a PHYHD1 protein in urine exosomes; the application includes: application in the preparation of a product for diagnosing diabetic nephropathy; application in the preparation of a product for screening diabetic nephropathy. The present invention proposes a biomarker PHYHD1 for diagnosing diabetic nephropathy, which can realize non-invasive differential diagnosis of diabetic nephropathy without renal puncture pathological examination.

Description

[0001] The present invention claims priority to a Chinese patent application filed with the National Intellectual Property Administration on July 31, 2023, with application number 202310952639.9, entitled “A biomarker, kit and diagnostic device for the diagnosis of diabetic nephropathy”, the entire contents of which are incorporated by reference into the present invention. Technical Field

[0002] The present invention relates to the field of biological detection technology, and in particular to a biomarker, a kit and a diagnostic device for diagnosing diabetic nephropathy. Background Art

[0003] In recent decades, the incidence of type 2 diabetes mellitus (T2DM) has increased year by year. One of its major complications, diabetic nephropathy (DN), has surpassed primary glomerulonephritis and has become the main cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD). The diagnosis of DN is usually based on the course of chronic diabetes, diabetic retinopathy, proteinuria without macroscopic hematuria, and / or a reduced estimated glomerular filtration rate (eGFR). However, many diabetic patients are diagnosed with non-diabetic renal disease (NDRD) after renal puncture pathological examination. DN and NDRD are different in disease evolution, treatment options, and disease prognosis. Therefore, accurate differential diagnosis is very important. At present, the gold standard for differential diagnosis of DN and NDRD is renal puncture pathological examination.

[0004] Renal puncture pathological examination is a invasive examination with surgical risks, which many patients cannot accept. In addition, this examination requires high technical skills of the operator, and requires the hospital to be equipped with a corresponding pathology laboratory and experienced pathologists. Therefore, it cannot be widely carried out as a routine examination.

[0005] Exosomes are small double-layer spherical membrane vesicles that can be secreted by almost all cells. They carry proteins, amino acids, lipids, metabolites and genetic material from the source cells; they contain physiological and pathological information of the source cells. Exosomes participate in a variety of physiological processes and play a role in the occurrence and development of diseases. Exosomes can be detected in a variety of body fluids such as blood, urine, and cerebrospinal fluid. Urine is produced directly by the kidneys and can be collected in large quantities without trauma. It is an ideal biological sample for studying kidney diseases. Compared with the traditional detection of whole urine, the use of urine exosomes to find non-invasive disease biodiagnostic markers has more advantages.

[0006] Therefore, it was found that by identifying the proteins in urinary exosomes, further establishing a non-invasive, easy-to-operate, highly sensitive and highly specific diabetic nephropathy diagnosis or detection method will help to quickly and accurately diagnose diabetic nephropathy. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a biomarker, a kit and a diagnostic device for diagnosing diabetic nephropathy.

[0008] The present invention provides an application of a substance for detecting a biomarker, wherein the biomarker is PHYHD1 protein in urine exosomes, and the application includes one of the following applications:

[0009] a. Application in the preparation of products for the diagnosis of diabetic nephropathy; the diagnosis includes differential diagnosis of diabetic nephropathy and non-diabetic nephropathy, and differential diagnosis of diabetic nephropathy and type 2 diabetes;

[0010] b. Application in the preparation of products for screening of diabetic nephropathy.

[0011] Optionally, the substance for detecting a biomarker is a reagent for detecting the content of PHYHD1 protein.

[0012] Optionally, the reagents for detecting the content of PHYHD1 protein include reagents for detecting the content of PHYHD1 protein by enzyme-linked immunosorbent assay, immunofluorescence, radioimmunoassay, immunoprecipitation, immunoblotting, immunochemiluminescence, colloidal gold immunoassay, fluorescent immunochromatography or biotin-avidin technology.

[0013] Optionally, the reagent for detecting the content of PHYHD1 protein includes an antibody that binds to the PHYHD1 protein; preferably, the reagent for detecting the content of PHYHD1 protein also includes a secondary antibody that binds to the antibody that binds to the PHYHD1 protein.

[0014] Optionally, in the application of a reagent for detecting the content of PHYHD1 protein by an enzyme-linked immunosorbent assay in the preparation of a product for the diagnosis or screening of diabetic nephropathy, diagnosis or screening is performed based on the detection result of the content of PHYHD1 protein in urine exosomes.

[0015] Optionally, the diagnostic method includes: if the content of PHYHD1 protein is greater than 1.203 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 100% and the specificity is 75%.

[0016] The present invention also provides an application of a kit, which includes the above reagent for detecting the content of PHYHD1 protein, and the application includes one of the following applications:

[0017] a. Application in the preparation of products for the diagnosis of diabetic nephropathy; the diagnosis includes differential diagnosis between diabetic nephropathy and non-diabetic nephropathy, and differential diagnosis between diabetic nephropathy and type 2 diabetes;

[0018] b. Application in the preparation of products for screening of diabetic nephropathy.

[0019] The present invention also provides a device for diagnosing diabetic nephropathy, comprising:

[0020] Urine exosome separation module: used to separate urine exosomes from urine samples;

[0021] PHYHD1 protein quantitative detection module: quantitative detection of PHYHD1 protein in urine exosomes by enzyme-linked immunosorbent assay;

[0022] Determination module: used to diagnose based on the PHYHD1 protein content in urine samples, and samples with a content greater than the threshold are determined to be diabetic nephropathy;

[0023] The diagnostic method includes: if the content of PHYHD1 protein is greater than 1.203 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 100% and the specificity is 75%.

[0024] The technical solution provided by the embodiment of the present invention has the following advantages compared with the prior art:

[0025] The present invention proposes a biomarker PHYHD1 for diagnosing diabetic nephropathy, which can realize non-invasive differential diagnosis of diabetic nephropathy without renal puncture pathological examination, and has high sensitivity and high specificity.

[0026] The urine exosome protein PHYHD1 proposed in the present invention can achieve an AUC area of ​​0.8500 in the non-invasive differential diagnosis of DN and NDRD patients through enzyme-linked immunosorbent assay technology, an AUC area of ​​0.9167 in the differential diagnosis of DN and healthy people, and an AUC area of ​​0.8393 in the differential diagnosis of DN and type 2 diabetes patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a flow chart of extracting urine exosomes in Example 1 of the present invention;

[0028] Figure 2 This is a Western Blot protein blotting result of urine exosomes in Example 2;

[0029] Figure 3 This is a transmission electron microscopy characterization image of urine exosomes in Example 2;

[0030] Figure 4 The particle size and concentration distribution curve of urine exosomes in Example 2;

[0031] Figure 5 The number of proteins identified by mass spectrometry of urine exosomes in each group in Example 3;

[0032] Figure 6 This is a Venn diagram for screening significantly differentially expressed proteins between the DN group and the NDRD group in Example 3;

[0033] Figure 7 This is a flow chart for screening significantly differentially expressed proteins between the DN group and the NDRD group in Example 3;

[0034] Figure 8 The volcano plot of the significantly differentially expressed proteins between DN and NDRD in Example 3;

[0035] Fig. 9 This is a heat map of the significantly differentially expressed proteins between DN and NDRD in Example 3;

[0036] Fig.10 This is the screening process of the penalty parameter λ in Example 3;

[0037] Fig.11 The cluster heat map of DN and NDRD of 10 candidate proteins screened by LASSO regression λ.min model in Example 3;

[0038] Fig.12 ROC analysis of significantly differentially expressed proteins in Example 3;

[0039] Fig.13 The ROC curve of the non-invasive differential diagnosis of DN and NDRD patients by urine exosomal protein PHYHD1 in Example 3;

[0040] Fig.14 The ELISA in Example 4 verified the expression level of PHYHD1 in urine exosomes among the groups;

[0041] Fig.15 This is the ROC curve in Example 4. DETAILED DESCRIPTION

[0042] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0043] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0044] The present invention uses label-free quantitative proteomics technology to study the urine exosome proteomics of diabetic nephropathy. The differential proteins in the urine exosomes of patients with diabetic nephropathy (DN) and non-diabetic nephropathy (NDRD) are analyzed. The number of identifications in each group reaches about 3372, and the depth of protein composition identification is good. DEGs screening was used to obtain a series of significantly differentially expressed proteins. Among the 371 differentially expressed proteins, 291 proteins were downregulated and 80 proteins were upregulated in the NDRD group. The present invention further analyzed the top 10 upregulated proteins in the DN group compared with the NDRD group, and the top 10 downregulated proteins in the DN group compared with the NDRD group. The bioinformatics analysis of the significantly differential proteins between DN and NDRD showed that the significantly differential proteins involved the biogenesis of urine exosomes, stress response pathways, immune response pathways and other pathways, suggesting that urine exosomes may be involved in the occurrence and development of DN diseases through the above pathways. The embodiment of the present invention is aimed at the diagnostic efficacy and correlation analysis of differentially expressed proteins in urine exosomes of diabetic nephropathy. 371 significantly differentially expressed proteins were screened out by LASSO regression, and 10 proteins showed a significant clustering trend in DN and NDRD. The LASSO screened proteins were further subjected to logistic regression analysis, and a total of 5 proteins were statistically significant. ROC curve analysis was performed on the above 5 proteins, among which PHYHD1 had the best diagnostic efficacy, and a new application of urine exosome protein PHYHD1 was proposed, which can realize non-invasive diagnosis of NDRD without renal puncture pathological examination; the application includes one of the following applications:

[0045] a. Application in the preparation of products for the diagnosis of diabetic nephropathy; the diagnosis includes differential diagnosis of diabetic nephropathy and non-diabetic nephropathy, and differential diagnosis of diabetic nephropathy and type 2 diabetes;

[0046] b. Use in the preparation of a product for screening diabetic nephropathy, wherein the screening is to obtain samples of diabetic nephropathy from normal samples.

[0047] In the above application, the substance for detecting the biomarker is a reagent for detecting the content of PHYHD1 protein, that is, a reagent for detecting the expression level of PHYHD1 protein in urine exosomes.

[0048] In the application of the embodiment of the present invention, the reagents for detecting the content of PHYHD1 protein include reagents for detecting the content of PHYHD1 protein by enzyme-linked immunosorbent assay, immunofluorescence, radioimmunoassay, immunoprecipitation, immunoblotting, immunochemiluminescence, colloidal gold immunoassay, fluorescent immunochromatography or biotin-avidin technique. Specifically, the reagents for detecting the content of PHYHD1 protein include antibodies that bind to PHYHD1 protein, and the reagents for detecting the content of PHYHD1 protein also include secondary antibodies that bind to antibodies that bind to PHYHD1 protein.

[0049] Specifically, in the application of a reagent for detecting the content of PHYHD1 protein by a linked immunosorbent assay in the preparation of a product for diagnosing diabetic nephropathy, diagnosis is performed based on the detection result of the content of PHYHD1 protein in urine exosomes; the diagnostic method includes: if the content of PHYHD1 protein is greater than 1.203 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 100% and the specificity is 75%.

[0050] Further preferably, the diagnostic method further comprises:

[0051] If the content of PHYHD1 protein is greater than 1.180ng / mL, the sensitivity of diagnosing diabetic nephropathy is 20% and the specificity is 100%; if the content of PHYHD1 protein is greater than 1.193ng / mL, the sensitivity of diagnosing diabetic nephropathy is 40% and the specificity is 100%; if the content of PHYHD1 protein is greater than 1.194ng / mL, the sensitivity of diagnosing diabetic nephropathy is 40% and the specificity is 87.5%; if the content of PHYHD1 protein is greater than 1.196ng / mL, the sensitivity of diagnosing diabetic nephropathy is 40% and the specificity is 75%; if the content of PHYHD1 protein is greater than 1.198ng / mL, the sensitivity of diagnosing diabetic nephropathy is 60% and the specificity is 75%; if the content of PHYHD1 protein is greater than 1.201ng / mL, the sensitivity of diagnosing diabetic nephropathy is 40% and the specificity is 87.5%; if the content of PHYHD1 protein is greater than 1.206ng / mL, the sensitivity of diagnosing diabetic nephropathy is 40% and the specificity is 87.5% The sensitivity of diabetic nephropathy is 80% and the specificity is 75%; if the content of PHYHD1 protein is greater than 1.206ng / mL, the sensitivity of diagnosing diabetic nephropathy is 100% and the specificity is 62.5%; if the content of PHYHD1 protein is greater than 1.210ng / mL, the sensitivity of diagnosing diabetic nephropathy is 100% and the specificity is 50%; if the content of PHYHD1 protein is greater than 1.215ng / mL, the sensitivity of diagnosing diabetic nephropathy is 100% and the specificity is 37.5%; if the content of PHYHD1 protein is greater than 1.220ng / mL, the sensitivity of diagnosing diabetic nephropathy is 100% and the specificity is 25%; if the content of PHYHD1 protein is greater than 1.230ng / mL, the sensitivity of diagnosing diabetic nephropathy is 100% and the specificity is 12.5%. The diagnosis is made using the following method: Fig.13 The ROC curve is shown.

[0052] As a preferred embodiment of the present invention, urine exosomes are extracted by excluding uromodulin and albumin from urine samples and co-precipitating with urine exosomes. Since proteinuria specimens contain a large amount of uromodulin interference and albumin interfere with the purification of exosomes, the present invention improves the extraction method and proposes a method for extracting urine exosomes, which can effectively reduce the interference of uromodulin and albumin, while ensuring the extraction effect of urine exosomes, and specifically includes the following steps:

[0053] S1. Take a urine sample, centrifuge it for the first time at 1-5°C, and freeze it at -75°C to -85°C; thaw the frozen sample at 20-28°C until it becomes liquid;

[0054] S2, centrifuge the thawed liquid for the second time at 1-5°C and collect the supernatant;

[0055] S3, taking the supernatant and filtering it with a filter membrane with a pore size of 0.22 μm, and collecting the filtrate;

[0056] S4, centrifuge the filtrate for the third time at 1-5°C and discard the supernatant;

[0057] S5, resuspend the precipitate in buffer solution, centrifuge for the fourth time at 1-5°C, and discard the supernatant;

[0058] S6, resuspend the precipitate with a buffer solution, perform a fifth centrifugation at 1-5°C, discard the supernatant, and resuspend the precipitate to obtain urinary exosomes;

[0059] The rotation speed of the first centrifugation and the second centrifugation is 4000-5000 rpm, and the time of each centrifugation is 30-60 minutes; the centrifugal force of the third centrifugation, the fourth centrifugation and the fifth centrifugation is 180,000×g-220,000×g, and the time of each centrifugation is 1-3 hours.

[0060] In the research process, the embodiment of the present invention found that freezing the urine sample and then thawing it at room temperature can help reduce the uromodulin in the exosomes, and filtering through a 0.22μm filter membrane can further reduce the interference of proteins such as uromodulin and albumin, and through three ultracentrifugations with a centrifugal force of 180,000×g to 220,000×g, the interference of proteins such as uromodulin and albumin can be significantly reduced. The obtained exosomes have a clear background, no obvious high-abundance protein co-precipitation, and the abundance is concentrated in 40 to 150nm. Thus, a standard method for the diagnosis of diabetic nephropathy is obtained. The present invention solves the technical problem of interference of a large amount of uromodulin and albumin in the urine of patients with diabetic nephropathy or non-diabetic nephropathy by improving the extraction method, and solves the technical problem of difficulty in extracting urine exosomes in patients with diabetic nephropathy or non-diabetic nephropathy.

[0061] The present invention also relates to the application of the kit, including one of the following applications:

[0062] a. Application in the preparation of products for the diagnosis of diabetic nephropathy; the diagnosis includes differential diagnosis of diabetic nephropathy and non-diabetic nephropathy, and differential diagnosis of diabetic nephropathy and type 2 diabetes;

[0063] b. Application in the preparation of products for screening of diabetic nephropathy;

[0064] The kit includes the above reagents for detecting the content of PHYHD1 protein.

[0065] The embodiment of the present invention also relates to a diagnostic device for diabetic nephropathy, comprising:

[0066] Urine exosome separation module: used to separate urine exosomes from urine samples;

[0067] PHYHD1 protein quantitative detection module: quantitative detection of PHYHD1 protein in urine exosomes by linked immunosorbent assay;

[0068] Determination module: used to diagnose based on the PHYHD1 protein content in urine samples, and samples with a content greater than the threshold are determined to be diabetic nephropathy;

[0069] The diagnostic method includes: if the PHYHD1 protein level is greater than 1.203 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 100% and the specificity is 75%;

[0070] Preferably, the diagnostic method further comprises: if the content of PHYHD1 protein is greater than 1.180 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 20% and the specificity is 100%; if the content of PHYHD1 protein is greater than 1.193 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 40% and the specificity is 100%; if the content of PHYHD1 protein is greater than 1.194 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 40% and the specificity is 87.5%; if the content of PHYHD1 protein is greater than 1.196 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 40% and the specificity is 75%; if the content of PHYHD1 protein is greater than 1.198 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 60% and the specificity is 75%; if the content of PHYHD1 protein is greater than 1.201 ng / mL, If the PHYHD1 protein content is greater than 1.206 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 100%, and the specificity is 62.5%; if the PHYHD1 protein content is greater than 1.210 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 100%, and the specificity is 50%; if the PHYHD1 protein content is greater than 1.215 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 100%, and the specificity is 37.5%; if the PHYHD1 protein content is greater than 1.220 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 100%, and the specificity is 25%; if the PHYHD1 protein content is greater than 1.230 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 100%, and the specificity is 12.5%. The diagnosis is made by Fig.13 The ROC curve is shown.

[0071] Example 1

[0072] This example is used to illustrate the method for extracting urine exosomes, which specifically includes the following steps:

[0073] S1. Take 50 mL of midstream urine sample in the morning, centrifuge it at 4,500 rpm for 45 min at 4°C, and freeze it at -80°C.

[0074] S2. Thaw the frozen sample at 25°C until it becomes liquid;

[0075] S3, centrifuge the thawed liquid for a second time at 4,500 rpm for 45 minutes at 4°C, discard the precipitate and collect the supernatant;

[0076] S4, filtering with a 0.22 μm polyethersulfone sterile filtration device and collecting the filtrate;

[0077] S5, centrifuge the filtrate for the third time at 200,000×g for 2 h at 4°C and discard the supernatant;

[0078] S6. Resuspend the precipitate in 50 mL of 0.01 M PBS buffer solution, centrifuge for the fourth time at 200,000 × g for 2 h at 4°C, and discard the supernatant.

[0079] S7. Resuspend the precipitate with 1 mL of pre-cooled 0.01 M PBS, centrifuge for the fifth time at 200,000 × g for 2 h at 4 °C, discard the supernatant, and use 0.01 M PBS buffer solution to adjust the volume of the precipitate to 60 μL to obtain urine exosomes. The extraction flow chart is as follows Figure 1 shown.

[0080] Example 2

[0081] This example is used to illustrate the extraction effect of urine exosomes:

[0082] 50 mL of midstream urine samples were collected from patients with diabetic nephropathy (DN), patients with non-diabetic renal disease (NDRD), patients with type 2 diabetes (T2DM), and healthy controls (HC), and exosomes were extracted according to the method of Example 1.

[0083] Western Blot was used to identify urine exosomes. The experimental results are as follows: Figure 2 shown.

[0084] Depend on Figure 2 It can be seen that the number of exosome marker proteins (ALIX, TSG101, Syntenin-1, CD9) is greater than or equal to 2 positive, indicating that the extract is exosomes.

[0085] Two samples (sample A and sample B) were randomly selected and the morphology of urine exosomes was identified using transmission electron microscopy. The experimental results are as follows: Figure 3 As shown (magnification is 68000 times).

[0086] Depend on Figure 3It can be seen that exosomes are vesicle-like bodies with a diameter of 40 to 150 nm, with a clear background and no obvious high-abundance protein co-precipitation.

[0087] Two samples (sample A and sample B) were randomly selected, and the particle size of the separated and purified products was measured using nanoparticle size tracking technology. The particle size and concentration distribution curves are shown in the figure below. Figure 4 As shown. Figure 4 It can be seen that the abundance of exosomes is concentrated in the range of 40 to 150 nm.

[0088] Example 3

[0089] Urinary exosomal protein PHYHD1 for differential diagnosis of DN and NDRD:

[0090] Twelve DN patients (6 males and 6 females) and 15 NDRD patients (12 males and 3 females) were included, and urine exosomes were extracted from urine samples according to the method in Example 1.

[0091] Then the following analysis is performed:

[0092] 1. Mass spectrometry identification results of urine exosome proteins

[0093] The urine exosomes obtained after separation and purification were hydrolyzed with trypsin solution and desalted with C18, and the proteomic spectrum of urine exosomes was obtained using label-free quantitative proteomics technology (DIA).

[0094] A total of 17867±5807 peptides, 2858±668.7 protein groups, and 2936±680.9 proteins were identified in the DN group; a total of 24300±7869 peptides, 3476±625.9 protein groups, and 3561±643.0 proteins were identified in the NDRD group; a total of 27155±9143 peptides, 3584±695.2 protein groups were identified in the T2DM group. , 3674±708.2 proteins; a total of 26680±10593 peptides, 3595±733.0 protein groups, and 3681±752.4 proteins were identified in the healthy control group. On average, 23822±8954 peptides, 3372±722.1 protein groups, and 3456±737.7 proteins were identified in each exosome sample, indicating that the proteomic identification depth was good, as shown in Table 1.

[0095] Table 1: Proteomic mass spectrometry identification results among groups

[0096] DN NDRD T2DM HC Average number of identifications Number of peptides 17867±5807 24300±7869 27155±9143 26680±10593 23822±8954 Protein number 2936±680.9 3561±643.0 3674±708.2 3681±752.4 3372±722.1 Number of protein groups 2858±668.7 3476±625.9 3584±695.2 3595±733.0 3456±737.7

[0097] There was no significant statistical difference in the number of identified peptides, protein groups and proteins among the groups (p>0.05), indicating that the quality of mass spectrometry data was stable, and the level of albumin in urine did not affect the number of urinary exosome protein identifications, suggesting that the optimized urinary exosome separation and purification method can reduce the impact of high-abundance proteins on mass spectrometry data. There was no statistical difference in the number of protein identifications among the groups, such as Figure 5 shown.

[0098] 2. Number of significantly differentially expressed proteins in urine exosomes

[0099] There were 1263 significantly differentially expressed proteins between the DN group and the healthy control group (497 up-regulated and 766 down-regulated proteins in the DN group), 1177 significantly differentially expressed proteins between the NDRD group and the healthy control group (508 up-regulated and 669 down-regulated proteins in the NDRD group), 632 significantly differentially expressed proteins between the T2DM group and the healthy control group (462 up-regulated and 170 down-regulated proteins in the T2DM group), 1200 significantly differentially expressed proteins between the DN group and the T2DM group (337 up-regulated and 863 down-regulated proteins in the DN group), 1260 significantly differentially expressed proteins between the NDRD group and the T2DM group (347 up-regulated and 913 down-regulated proteins in the NDRD group), and 557 significantly differentially expressed proteins between the NDRD group and the DN group (156 up-regulated and 401 down-regulated proteins in the DN group), as shown in Table 2.

[0100] Table 2: Number of significantly differentially expressed proteins among groups

[0101] Total number of differentially expressed proteins Number of upregulated proteins Number of down-regulated proteins HC group vs. DN group 1263 497 766 HC group vs. NDRD group 1177 508 669 HC group vs. T2DM group 632 462 170 T2DM group vs. DN group 1200 337 863 T2DM group vs. NDRD group 1260 347 913 NDRD group vs. DN group 557 156 401

[0102] 3. DEGs method to screen significantly differentially expressed proteins

[0103] There are no differences in the co-expressed proteins in the four groups of urine exosome proteins. In order to screen out the differentially expressed proteins of the disease, it is first necessary to eliminate these co-expressed proteins. Therefore, after the healthy control group was compared with the DN group, NDRD group, and T2DM group, the proteins with no significant difference in expression in the three groups were defined as the co-expressed proteins in the four groups. After eliminating the co-expressed proteins with no significant difference between the four groups, 2029 significantly differentially expressed proteins remained.

[0104] To further screen out the differentially expressed proteins between the DN group and the NDRD group, the common T2DM disease background of the two groups had to be eliminated. There were 1,682 proteins that were significantly differentially expressed in the comparison between the DN group and the T2DM group, and between the NDRD group and the T2DM group. Among them, the proteins that were also significantly differentially expressed in the 2,029 significantly differentially expressed proteins after eliminating the co-expressed proteins were defined as significantly differentially expressed proteins after eliminating the T2DM disease background, totaling 1,308 proteins. Among these 1,308 significantly differentially expressed proteins, there were 371 proteins that were also significantly differentially expressed between the DN group and the NDRD group, such as Wayne Figure 6 As shown: A total of 371 significantly differentially expressed proteins were screened, and 1682 significantly differentially expressed proteins remained after excluding the T2DM background; 2029 proteins remained after excluding the proteins with no significant difference in expression that were co-expressed among the four groups; and 557 significantly differentially expressed proteins were found between the DN group and the NDRD group.

[0105] The overall screening process is as follows Figure 7 shown.

[0106] 4. Significantly differentially expressed proteins between DN and NDRD

[0107] After the above screening process, 371 proteins were considered to be significantly differentially expressed between DN patients and NDRD patients; among them, 291 proteins were downregulated and 80 proteins were upregulated in the NDRD group, such as volcano Figure 8 As shown in Figure 3, 371 proteins were defined as significantly differentially expressed between DN patients and NDRD patients, 291 proteins were downregulated in the NDRD group, and 80 proteins were upregulated in the NDRD group. The 371 significantly differentially expressed proteins showed a distinct clustering trend between DN and NDRD patients, such as Fig. 9 shown.

[0108] The protein names, protein descriptions, coding genes, average expression abundance multiples (AVG Log2 Ratio) between the DN group and the NDRD group, and significance (Qvalue) of the top 10 up-regulated and down-regulated non-contaminating proteins are shown in Tables 3 and 4.

[0109] Table 3: Top 10 upregulated proteins in DN group compared with NDRD group

[0110]

[0111]

[0112] Table 4: Top 10 down-regulated proteins in DN group compared with NDRD group

[0113]

[0114] 5. LASSO regression analysis of significantly differentially expressed proteins

[0115] After removing the co-expressed proteins of each group and T2DM background proteins, there were 371 significantly differentially expressed proteins in the urine exosomes of DN and NDRD patients. To further clarify the significantly differentially expressed proteins, LASSO regression was used to construct a penalty function, and the regression coefficients of the independent variables were compressed to achieve the contraction of high-dimensional data.

[0116] The selection process of penalty parameter λ is as follows: Fig.10 As shown, the two dotted lines indicate λ.min and λ.1se respectively. λ between these two values ​​is considered appropriate. The model constructed by λ.1se is the simplest and uses a small number of proteins, while the model constructed by λ.min has a higher accuracy and uses a larger number of proteins. The obtained λ.1se is 0.2828 and λ.min is 0.093. The model constructed by λ.1se includes 1 protein (PHYHD1), and the model constructed by λ.min includes 12 proteins, among which KRT33B is a microfibrous keratin related to the reproductive system; PRSS1 is a trypsin protein used for solution enzyme cleavage. These two proteins are considered to be contaminating proteins and are therefore excluded from the subsequent analysis. The clustering heat map of the 10 candidate proteins between DN and NDRD patients is shown below. Fig.11 shown.

[0117] 6. Perform logistic regression analysis on candidate proteins

[0118] After removing the contaminating proteins from the 12 proteins included in the LASSO regression λ.min model, the remaining 10 candidate proteins were first subjected to univariate logistic regression analysis, and proteins with p values ​​< 0.05 were selected for multivariate logistic regression analysis. The results of the univariate logistic regression analysis are shown in Table 5.

[0119] Table 5: Results of LASSO regression λ.min model incorporating protein logistic regression analysis

[0120] Protein name Beta value OR 95% CI P-value APOB -.069 0.933(0.883，0.987) 0.015* BPIFB1 0.332 1.394(0.906，2.144) 0.131 C12orf4 3.810 45.164(1.123，1816.743) 0.043* COMP -0.416 0.660(0.459，0.949) 0.025* C18orf63 -0.054 0.948(0.896，1.003) 0.062 MFSD10 -0.408 0.665(0.438，1.009) 0.550 PPP1R12A 0.223 NA 0.565 PHYHD1 2.451 11.604(1.830，73.574) 0.009* RETN -0.008 0.992(0.978，1.007) 0.291 SIDT1 -0.069 0.933(0.883，0.987) 0.015*

[0121] Significance is marked with * (p<0.05).

[0122] The results of multivariate logistic regression analysis showed that the omnibus test was significant < 0.01, and the Holmes-Lemeshow test was significant 0.516, indicating that the model fit was good, among which only PHYHD1 protein was significant, with a β value of 2.451, an OR value of 11.604 (1.830, 73.574), and a p value of 0.009. This is consistent with the results of the λ.1se model constructed in the LASSO regression analysis, in which PHYHD1 was included. Therefore, PHYHD1 was considered to be a significantly differentially expressed protein.

[0123] 7. ROC analysis of significantly differentially expressed proteins

[0124] After LASSO regression analysis and Logistic regression analysis, the significantly differentially expressed protein PHYHD1 was screened out as a diagnostic marker. The ROC curve is shown in Fig.12 shown.

[0125] When the cutoff value was 8880.323, the area under the curve (AUC) was 0.919, p < 0.001, the sensitivity was 93.3%, and the specificity was 91.7%, indicating that it had good diagnostic efficacy.

[0126] PHYHD1 (AUC=0.919, 95% CI=0.805-1.000),

[0127] APOB (AUC=0.878, 95% CI=0.736-1.000),

[0128] C12orf4 (AUC=0.828, 95% CI=0.668-0.987),

[0129] MFSD10 (AUC=0.814, 95% CI=0.639-0.989),

[0130] COMP (AUC=0.800, 95% CI=0.628-0.972).

[0131] 8. Calculate the ROC curve of urine exosomal protein PHYHD1 for non-invasive differential diagnosis of DN and NDRD patients. Fig.13 shown.

[0132] Depend on Fig.13 It can be seen that the AUC area for distinguishing DN and NDRD patients can reach 0.919, p < 0.001, the sensitivity is 93.3%, and the specificity is 91.7%.

[0133] Example 4

[0134] Validation of the efficiency of PHYHD1 as a differential diagnostic biomarker.

[0135] According to the method of Example 1, urine exosomes were isolated from 8 DN patients, 5 NDRD patients, 7 T2DM patients and 9 healthy subjects. The characteristics of the 29 subjects are shown in Table 6.

[0136] Table 6: Basic demographic and clinical characteristics of the validation cohort for PHYHD1 as a biomarker

[0137] Variable DN(n=8) NDRD (n=5) T2DM (n=7) HC (n=9) Age (years) 57.50±7.71 58.00±5.00 54.29±11.64 47.67±6.00 Gender (male / female) (5 / 3) (3 / 2) (4 / 3) (5 / 4) Serum albumin (g / L) <![CDATA[34.20±7.47 a ]]> 34.92±12.43 42.49±4.20 45.03±2.55 HbA1c (%) <![CDATA[7.39±1.60 a ]]> 6.62±0.99 <![CDATA[9.57±2.32 a ]]> 5.43±0.24 Blood glucose (mmol / L) 6.99±2.21 6.50±2.88 <![CDATA[11.21±5.24 a ]]> 5.09±0.36 Urine protein (g / 24h) <![CDATA[2.71±1.98 ab ]]> <![CDATA[2.83±4.11 a ]]> 0.02±0.05 Serum creatinine (μmol / L) <![CDATA[150.44±53.27 ab ]]> 114.40±35.06 71.94±23.20 65.89±10.67 Urea nitrogen (mmol / L) <![CDATA[9.51±2.53 ab ]]> 7.97±1.89 4.90±1.80 4.67±0.96 <h2 style=";text-align:left;direction:ltr"><![CDATA[eGFR(mL min <h2 style=";text-align:left;direction:ltr"> -1 <h2 style=";text-align:left;direction:ltr"> 1.73m<h2 style=";text-align:left;direction:ltr"> -2 <h2 style=";text-align:left;direction:ltr"> )]]><h2 style=";text-align:left;direction:ltr"> <![CDATA[39.85±17.76 b ]]> 50.48±17.91 97.55±25.17

[0138] All data are expressed as mean ± standard deviation. a p<0.05vs.HC; b p<0.05 vs. T2DM; c p<0.05vs.NDRD. HbA1c: glycosylated hemoglobin; eGFR: estimated glomerular filtration rate.

[0139] Urine exosomes were extracted from urine samples according to the method of Example 1. The biomarker PHYHD1 in urine exosomes was detected using an ELISA kit (Abbexa Ltd., abx538764). The results were as follows: Fig.14 As shown. Fig.14 The results showed that there were statistical differences in the expression of PHYHD1 between DN and NDRD, DN and T2DM, and DN and healthy subjects.

[0140] Draw the ROC curve, such as Fig.15 shown.

[0141] Depend on Fig.15 The ROC curve analysis showed that PHYHD1 was the best (highest sum of sensitivity and specificity) for determining the concentration of urinary exosomes in the differential diagnosis of DN and NDRD. When the concentration of DN was higher than 1.203 μg / L, the sensitivity was 100% and the specificity was 75% (AUC = 0.850).

[0142] The above description is only a specific embodiment of the present invention, so that those skilled in the art can understand or implement the present invention. Various modifications to these embodiments will be 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 present invention. Therefore, the present invention will not be limited to the embodiments described herein, but should conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of a substance for detecting a biomarker, characterized in that: The biomarker is PHYHD1 protein in urine exosomes; The application includes one of the following applications: a. Application in the preparation of a product for the diagnosis of diabetic nephropathy; the diagnosis includes differential diagnosis of diabetic nephropathy and non-diabetic nephropathy; b. Application in the preparation of products for screening of diabetic nephropathy; the screening includes screening of diabetic nephropathy and non-diabetic nephropathy.

2. The use according to claim 1, characterized in that: The substance for detecting biomarkers is a reagent for detecting the content of PHYHD1 protein.

3. The use according to claim 2, characterized in that: The reagents for detecting the content of PHYHD1 protein include reagents for detecting the content of PHYHD1 protein by enzyme-linked immunosorbent assay, immunofluorescence assay, radioimmunoassay, immunoprecipitation, immunoblotting, immunochemiluminescence, colloidal gold immunoassay, fluorescent immunochromatography or biotin-avidin technique.

4. The use according to claim 2, characterized in that: The reagent for detecting the content of PHYHD1 protein includes an antibody that binds to PHYHD1 protein; And / or, the reagent for detecting the content of PHYHD1 protein further comprises a secondary antibody that binds to the antibody that binds to the PHYHD1 protein.

5. The use according to claim 3, characterized in that: In the use of a reagent for detecting the content of PHYHD1 protein by an enzyme-linked immunosorbent assay in the preparation of a product for diagnosing or screening diabetic nephropathy, diagnosis or screening is performed based on the detection result of the content of PHYHD1 protein in urine exosomes.

6. The use according to claim 5, characterized in that: The diagnostic method includes: if the content of PHYHD1 protein is greater than 1.203 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 100% and the specificity is 75%; and / or, if the level of PHYHD1 protein is greater than 1.180 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 20% and the specificity is 100%; If the PHYHD1 protein level is greater than 1.193 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 40% and the specificity is 100%; If the PHYHD1 protein level is greater than 1.194 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 40% and the specificity is 87.5%; If the PHYHD1 protein level is greater than 1.196 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 40% and the specificity is 75%; If the PHYHD1 protein level is greater than 1.198 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 60% and the specificity is 75%; If the PHYHD1 protein level is greater than 1.201 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 80% and the specificity is 75%; If the PHYHD1 protein level is greater than 1.206 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 100% and the specificity is 62.5%; If the PHYHD1 protein level is greater than 1.210 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 100% and the specificity is 50%; If the PHYHD1 protein level is greater than 1.215 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 100% and the specificity is 37.5%; If the PHYHD1 protein level is greater than 1.220 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 100% and the specificity is 25%; If the PHYHD1 protein content is greater than 1.230 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 100% and the specificity is 12.5%.

7. The use according to claim 1, characterized in that: The method for extracting urine exosomes comprises the following steps: S1. Take a urine sample, centrifuge it for the first time at 1-5°C, and freeze it at -75°C to -85°C; thaw the frozen sample at 20-28°C until it becomes liquid; S2, centrifuge the thawed liquid for the second time at 1-5°C and collect the supernatant; S3, taking the supernatant and filtering it with a filter membrane with a pore size of 0.22 μm, and collecting the filtrate; S4, centrifuging the filtrate for the third time at 1-5°C, and discarding the supernatant; S5, resuspend the precipitate in buffer solution, centrifuge for the fourth time at 1-5°C, and discard the supernatant; S6. Resuspend the precipitate with buffer solution, perform the fifth centrifugation at 1-5°C, discard the supernatant, and resuspend the precipitate to obtain urinary exosomes.

8. The use according to claim 7, characterized in that: The rotation speed of the first centrifugation and the second centrifugation is 4000-5000 rpm, and the time of each centrifugation is 30-60 minutes; the centrifugal force of the third centrifugation, the fourth centrifugation and the fifth centrifugation is 180,000×g-220,000×g, and the time of each centrifugation is 1-3 hours.

9. An application of a kit, characterized in that: The application includes one of the following applications: a. Application in the preparation of a product for the diagnosis of diabetic nephropathy; the diagnosis includes differential diagnosis of diabetic nephropathy and non-diabetic nephropathy; b. Application in the preparation of a product for screening of diabetic nephropathy, wherein the screening includes screening of diabetic nephropathy and non-diabetic nephropathy; The kit comprises the reagent for detecting the content of PHYHD1 protein as described in any one of claims 2 to 6.

10. A diagnostic device for diabetic nephropathy, the diagnosis comprising differential diagnosis between diabetic nephropathy and non-diabetic nephropathy, the diagnostic device comprising: Urine exosome separation module: used to separate urine exosomes from urine samples; PHYHD1 protein quantitative detection module: quantitatively detecting the PHYHD1 protein in the urine exosomes by enzyme-linked immunosorbent assay; Determination module: used for making a diagnosis based on the PHYHD1 protein content in the urine sample, and determining the sample as having diabetic nephropathy if the content is greater than a threshold; The diagnostic method includes: if the content of PHYHD1 protein is greater than 1.203 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 100% and the specificity is 75%; And / or, the diagnostic method further comprises: If the PHYHD1 protein level is greater than 1.180 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 20% and the specificity is 100%; If the PHYHD1 protein level is greater than 1.193 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 40% and the specificity is 100%; If the PHYHD1 protein level is greater than 1.194 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 40% and the specificity is 87.5%; If the PHYHD1 protein level is greater than 1.196 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 40% and the specificity is 75%; If the PHYHD1 protein level is greater than 1.198 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 60% and the specificity is 75%; If the PHYHD1 protein level is greater than 1.201 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 80% and the specificity is 75%; If the PHYHD1 protein level is greater than 1.206 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 100% and the specificity is 62.5%; If the PHYHD1 protein level is greater than 1.210 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 100% and the specificity is 50%; If the PHYHD1 protein level is greater than 1.215 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 100% and the specificity is 37.5%; If the PHYHD1 protein level is greater than 1.220 ng / mL, the sensitivity for diagnosing diabetic nephropathy is 100% and the specificity is 25%; If the PHYHD1 protein content is greater than 1.230 ng / mL, the sensitivity of diagnosing diabetic nephropathy is 100% and the specificity is 12.5%.

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