Application of reagents for detecting biomarkers in the preparation of products for non-invasive diagnosis of renal interstitial fibrosis and chronic progressive injury.

Abstract

This invention relates to the application of reagents for detecting biomarkers in the preparation of non-invasive diagnostic products for renal interstitial fibrosis and chronic progressive injury, belonging to the field of biotechnology. The biomarkers are SNX3, VPS4B, and SMO, derived from urinary extracellular vesicles, and their expression levels increase with the degree of transplanted kidney fibrosis. The urinary extracellular vesicle separation method of this invention effectively removes urinary glomerulonephrine entangled with vesicles during ultracentrifugation without affecting the structural integrity of the extracellular vesicles or the expression of the marker proteins. The biomarkers of this invention have excellent diagnostic capabilities for transplanted kidney fibrosis and also demonstrate outstanding ability in diagnosing chronic progressive injury of transplanted kidneys, predicting the risk of transplanted kidney failure, and assessing renal fibrosis in chronic kidney disease. They possess advantages such as good stability, high specificity, and high sensitivity, and are simple to operate with a short detection cycle, enabling non-invasive diagnosis or prognostic monitoring of fibrosis in both transplanted and non-transplanted kidneys.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the application of reagents for detecting biomarkers in the preparation of non-invasive diagnostic products for renal interstitial fibrosis and chronic progressive injury. Background Technology

[0002] Chronic kidney disease (CKD) is widely recognized as a major global public health problem. As CKD progresses to end-stage kidney disease (ESKD), patients require renal replacement therapy, including hemodialysis, peritoneal dialysis, and even kidney transplantation. Kidney transplantation is the most ideal renal replacement therapy for ESKD patients, offering significant advantages over lifelong dialysis in terms of treatment costs, quality of life, and long-term survival. Currently, the one-year survival rate of transplanted kidneys has increased to over 90%, but the 10-year functional survival rate is only about 51%.

[0003] Long-term functional decline in transplanted kidneys is primarily attributed to chronic allograftdysfunction (CAD), characterized by interstitial fibrosis and tubular atrophy (IFTA), which is the core cause of long-term transplant failure. Notably, tubulointerstitial fibrosis is a common terminal pathway in almost all persistent kidney injuries, and its progression is closely related to progressive loss of kidney function and patient mortality. Fibrosis is common not only in patients undergoing indicated biopsies but also in those with partially preserved or restored kidney function. Clinically, kidney function is usually assessed by measuring the glomerular filtration rate (GFR). However, GFR does not accurately reflect the degree of kidney fibrosis, and the correlation between GFR and fibrosis progression is not strong. Therefore, early non-invasive detection of post-transplant kidney fibrosis is not only a key prerequisite for clinical intervention but also an urgent need to improve the long-term prognosis of transplanted kidneys, crucial for achieving individualized treatment and improving patient outcomes. However, how to accurately monitor and prevent transplanted kidney fibrosis in real time remains a major challenge in clinical practice.

[0004] Needle biopsy is the gold standard for diagnosing the type of transplant kidney injury and is also the most commonly used method in clinical practice. However, because needle biopsy is an invasive procedure, it may lead to complications such as bleeding, infection, and damage to surrounding organs. It is difficult to continuously monitor the progression of transplant kidney fibrosis and intervene in a timely manner, which makes it difficult to improve the long-term survival rate of transplant kidneys. Furthermore, monitoring the transplant kidney injury process through multiple biopsies is painful and expensive for patients, making it impractical.

[0005] Currently, non-invasive markers of transplant kidney injury used in clinical practice include serum creatinine (Scr), urinary protein, and donor-specific antibodies (DSA). These markers have limitations in sensitivity, specificity, and accuracy, and cannot effectively monitor the progression of renal fibrosis. With the rapid development of high-throughput omics technologies, non-invasive biomarkers derived from urine have shown great potential in the diagnosis of renal fibrosis. Urine, the ultrafiltrate directly produced by the kidneys, provides a unique window for observing the fibrosis process in transplanted kidneys. Furthermore, urine is readily available and can be collected daily, allowing for continuous and non-invasive monitoring of the pathophysiological state of the transplanted kidney. Studies have identified cadherin-11 and pigment epithelium-derived factor in urine as potential markers of renal fibrosis. However, urine concentration is easily affected by fluctuations in diet, water intake, and physiological state; pH, osmolarity, and protein concentration fluctuate significantly, resulting in poor stability.

[0006] As a novel source of biomarkers, urinary extracellular vesicles (uEVs) have been proven to carry cell-specific markers from the renal tubules and various segments of the urinary tract, making them a highly valuable source of biomarkers. Their high accessibility in urine makes them a suitable non-invasive diagnostic research subject for various kidney diseases, such as autosomal dominant polycystic kidney disease, diabetic nephropathy, and transplant rejection. uEV analysis can detect molecules in urine that are too low in concentration or confined within the vesicles to be directly identified, making it an ideal novel liquid biopsy carrier.

[0007] However, the development and application of disease biomarkers in uEVs still face multiple challenges, among which the interference from uromodulin (also known as Tamm-Horsfall protein) is particularly prominent. As the most abundant glycoprotein in urine, uromodulin readily forms polymer networks and precipitates even at low centrifugal forces. However, its polymers may encapsulate uEVs, leading to EV loss during subsequent separation. Furthermore, due to its high abundance, uromodulin may mask the signals of low-abundance EV biomarkers, and co-precipitation of uromodulin can significantly contaminate uEV samples, interfering with proteomics analysis. Although reducing agents (such as DTT or TCEP) can partially depolymerize the uromodulin network to improve EV recovery, they may affect the antigenicity of EV membrane proteins, limiting downstream functional studies. Existing separation methods (such as ultracentrifugation and size exclusion chromatography) are widely used but have limitations. Ultracentrifugation may result in the loss of small EVs due to uromodulin encapsulation, while size exclusion chromatography, although reducing uromodulin contamination, struggles to completely remove protein complexes such as albumin that co-migrate with EVs. In addition, dynamic properties of urine (such as pH, osmotic pressure, and protein concentration) and disease states (such as proteinuria) can further alter sample viscosity, affecting separation efficiency and repeatability.

[0008] Therefore, developing efficient and high-purity methods for separating uEVs is crucial. It is necessary to balance the removal of contaminants such as urinary globulins with the preservation of EV integrity, and more standardized methods are needed to ensure the reliability of test results. For example, combining chemical depolymerization with multi-step gradient centrifugation, or developing microfluidic technologies to directly analyze EV subpopulations in unseparated urine. Standardized separation procedures will improve the comparability of data across studies, laying the foundation for disease biomarker discovery and clinical translation.

[0009] Currently, the clinical translation of uEVs has yielded initial results, with its derived biomarkers already approved by the FDA for prostate cancer diagnosis, confirming its clinical feasibility. However, in the field of kidney transplantation, the diagnostic value of uEVs still needs further exploration. Single-nuclear RNA sequencing studies have revealed that proximal tubular epithelial cells (PTECs)—key effector cells in transplanted kidney injury—mediate the fibrosis process by secreting extracellular vesicles (EVs). This suggests that uEVs derived from transplanted kidney TECs may carry key molecular information reflecting the fibrosis status. However, the specific proteomic characteristics and regulatory mechanisms of uEVs in the transplanted kidney fibrosis process have not yet been systematically elucidated. Therefore, systematically analyzing the proteomic characteristics of uEVs and discovering novel biomarkers for non-invasive diagnosis of transplanted kidney fibrosis has significant clinical translational value and is urgently needed. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to provide a reagent for detecting biomarkers and its application in the preparation of products for non-invasive diagnosis of renal interstitial fibrosis and chronic progressive injury.

[0011] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: the application of reagents for detecting biomarkers in the preparation of non-invasive diagnostic products for renal interstitial fibrosis and chronic progressive damage, wherein the biomarkers are SNX3 (sorting connexin 3), VPS4B (vacuole-sorting 4 homologue B) and SMO (smooth receptor).

[0012] Furthermore, the SNX3, VPS4B, and SMO are derived from uEVs, and the expression level changes of the SNX3, VPS4B, and SMO are uEVs-specific.

[0013] Furthermore, the reagents for detecting biomarkers are those capable of quantitatively detecting SNX3, VPS4B, and SMO; these reagents are substances capable of detecting the above three molecules at the protein level, including antibodies, protein expression chips, and reagents and / or chips used for protein detection by means of mass spectrometry protein sequencing, Western blot, or ELISA; the product can be a kit for detecting the content of SNX3, VPS4B, and SMO by Western blot or sandwich enzyme-linked immunosorbent assay (ELISA).

[0014] Furthermore, the method for detecting uEVs-specific changes in the expression levels of SNX3, VPS4B, and SMO includes the following steps:

[0015] S1. UEVs are separated from urine by ultracentrifugation;

[0016] S2. The marker proteins CD9, CD63, Alix, TSG101, and AQP2 of uEVs were detected by Western blot; the particle size distribution and morphology of uEVs were characterized by DLS and transmission electron microscopy, respectively.

[0017] S3. The relative contents of SNX3, VPS4B and SMO in urine before and after ultracentrifugation were determined by Western blot to clarify that SNX3, VPS4B and SMO are mainly present in uEVs.

[0018] S4. Western blot was used to detect the relative levels of uEVs and SNX3, VPS4B, and SMO in urine of kidney transplant patients with different Banff ci scores to determine whether the diagnostic ability is uEVs specific.

[0019] Furthermore, the method for separating the uEVs includes the following steps:

[0020] S11. Urine sample pretreatment: Thaw the frozen urine sample in a 37°C water bath within 1 hour, then add Tris-EDTA solution to the urine at a ratio of 1:4, with final Tris / EDTA concentrations of 20 mM and 8 mM, respectively. Vortex for 90 seconds to depolymerize the uromodulin network and obtain the treated urine.

[0021] S22. Centrifuge the urine obtained in step S11 at 4°C and 17000g for 30 minutes to remove cell debris such as apoptotic bodies and obtain the supernatant.

[0022] S33. Filter the supernatant obtained in step S22 using a needle filter to remove microvesicles with a diameter greater than 200 nm.

[0023] S44. Place the supernatant filtered in step S33 into an ultracentrifuge tube, centrifuge at 130,000g for 120 min at 4°C, discard the supernatant, add PBS to wash the precipitate, centrifuge again at 130,000g for 120 min at 4°C, resuspend the precipitate with PBS, and freeze at -80°C for later use.

[0024] Furthermore, the method for detecting the biomarker includes the following steps:

[0025] S111, Separating uEVs from urine;

[0026] S222. The total expression levels of SNX3, VPS4B, and SMO were detected by Western blot or sandwich enzyme-linked immunosorbent assay.

[0027] S333, the expression levels of SNX3, VPS4B, and SMO were standardized by detecting urinary creatinine concentration.

[0028] Furthermore, when the biomarker is applied to a non-invasive diagnostic product for transplant kidney interstitial fibrosis, the method for determining whether a subject has transplant kidney fibrosis includes: comparing and analyzing the parameters of SNX3, VPS4B, and SMO protein content or expression levels in the subject's uEVs with sample parameters. If the parameters are higher than the sample parameter threshold, transplant kidney fibrosis exists; otherwise, transplant kidney fibrosis does not exist. The sample parameters are selected from the subject's previous test samples and / or parameters of normal SNX3, VPS4B, and SMO protein content or expression levels in specified uEVs.

[0029] Furthermore, when the biomarker is applied to a non-invasive diagnostic product for chronic progressive injury of transplanted kidneys, the method for determining whether a subject has chronic progressive injury of the transplanted kidney includes: comparing and analyzing relevant parameters such as the content or expression levels of SNX3, VPS4B, and SMO proteins in the subject's uEVs with sample parameters. If the relevant parameters are higher than the sample parameter threshold, chronic progressive injury of the transplanted kidney is present; if the parameters are lower than the sample parameter threshold, it is acute injury or nonspecific injury of the transplanted kidney. The sample parameters are defined as parameters indicating normal content or expression levels of SNX3, VPS4B, and SMO proteins in uEVs. Chronic progressive injury of the transplanted kidney includes transplanted kidney glomerulonephropathy, chronic rejection, polyomavirus nephropathy, and IgA nephropathy.

[0030] Furthermore, when the biomarkers are applied to non-invasive diagnostic products for the prognosis of transplanted kidney patients, the risk of transplant failure in the subject can be predicted by detecting the content or expression level of SNX3, VPS4B and SMO proteins in the subject's uEVs.

[0031] Furthermore, when the biomarker is applied to a non-invasive diagnostic product for renal fibrosis in non-transplanted chronic kidney disease patients, the method for determining whether a subject has renal fibrosis includes: comparing and analyzing the parameters of the content or expression level of SNX3, VPS4B, and SMO proteins in the subject's uEVs with the sample parameters; if the parameters are higher than the sample parameter threshold, then renal fibrosis exists; otherwise, renal fibrosis does not exist; the sample parameters are the specified parameters of the content or expression level of SNX3, VPS4B, and SMO proteins in uEVs.

[0032] The beneficial effects of this invention are as follows:

[0033] (1) The biomarker of the present invention is derived from extracellular vesicles in urine, which can be used to diagnose fibrosis of transplanted kidneys under non-invasive conditions.

[0034] (2) The biomarkers of the present invention are significantly enriched in uEVs, and their abundance changes are uEVs specific. Their abundance changes in urine are not correlated with the degree of renal fibrosis in patients, indicating that compared with free protein biomarkers in urine, they have higher diagnostic specificity, stronger sensitivity, and more accurate diagnosis.

[0035] (3) The urine extracellular vesicle separation method of the present invention removes protein particles and large vesicles by depolymerizing uromolysin fibers with Tris-EDTA and filtering with a 0.22μm needle filter. It can effectively remove uromolysin entangled with vesicles during ultracentrifugation, without affecting the structural integrity of extracellular vesicles and the expression of marker proteins.

[0036] (4) The biomarker of the present invention has excellent diagnostic ability for transplanted kidney fibrosis. Its expression level in uEVs is significantly positively correlated with the degree of interstitial fibrosis. The ROC-AUC for diagnosing transplanted kidney fibrosis can reach 0.9906, with an accuracy of 90.6%, and has good clinical application prospects.

[0037] (5) In addition to diagnosing transplanted kidney fibrosis, the biomarkers of the present invention also have excellent ability in diagnosing chronic progressive damage to transplanted kidneys (AUC=0.8220).

[0038] (6) The extracellular vesicle proteins SNX3, VPS4B and SMO in urine proposed in this invention can be used as biomarkers to predict the risk of patients progressing to transplant kidney failure.

[0039] (7) The ability of the biomarkers of the present invention to diagnose renal fibrosis is not limited to the kidney transplant population, but also shows the ability to diagnose renal fibrosis in uEVs of chronic kidney disease patients. It can be seen that the biomarkers of the present invention have excellent diagnostic ability for transplant kidney fibrosis, and also have excellent ability in diagnosing chronic progressive damage to transplant kidneys, predicting the risk of transplant kidney failure, and renal fibrosis (non-transplant) in chronic kidney disease.

[0040] (8) This invention discloses the application of a combination of extracellular vesicle protein markers in urine in the preparation of diagnostic products for renal interstitial fibrosis. By analyzing and screening the combination of extracellular vesicle protein markers in urine, accurate diagnosis of renal fibrosis is achieved. This method not only reduces patient suffering and risks but also avoids unnecessary biopsies, reducing the possibility of misdiagnosis and missed diagnosis. Attached Figure Description

[0041] The present invention will be described in detail below with reference to the accompanying drawings and examples. The advantages and implementation methods of the present invention will become more apparent from this description. The accompanying drawings are for illustrative purposes only and do not constitute any limitation on the present invention. In the accompanying drawings:

[0042] Figure 1 This is a graph showing the expression levels of biomarker proteins in uEVs obtained by superposition after different methods of urine pretreatment according to the present invention.

[0043] Figure 2 This is an identification diagram of uEVs isolated by the urine extracellular vesicle separation method of the present invention.

[0044] Figure 3 This is a diagram showing the differentially expressed proteins between the ci0, ci1, and ci2 groups in this invention.

[0045] Figure 4The image shows candidate biomarker proteins SNX3, VPS4B, and SMO obtained by the present invention based on differential protein expression levels and verified by the Nephromine database.

[0046] Figure 5 This is a diagram showing the expression analysis of three marker proteins in Urine and EV-free Urine of this invention.

[0047] Figure 6 Immunohistochemical staining images of biopsy tissues from kidney transplant patients with different CI scores according to this invention, scale bar = 100 μm.

[0048] Figure 7 This is a graph showing the correlation between the expression levels of three biomarker proteins and the degree of fibrosis in a mouse model of renal fibrosis induced by unilateral ureteral ligation, as presented in this invention via immunohistochemical staining. Scale bar = 100 μm.

[0049] Figure 8 This is a graph showing the correlation between the expression levels of three biomarker proteins and the degree of fibrosis in a folic acid-induced mouse renal fibrosis model, as demonstrated by immunohistochemical staining in this invention. Scale bar = 100 μm.

[0050] Figure 9 This invention presents an image showing chronic rejection-induced transplanted kidney injury in mice using H&E staining.

[0051] Figure 10 This is a map showing the colocalization of biomarkers with EVs biomarkers in chronic rejection-induced fibrotic transplanted kidneys in mice, as presented in this invention, using immunofluorescence co-staining.

[0052] Figure 11 This is a graph showing the expression levels of fibrosis diagnostic markers in 59 patients with uEVs identified and validated by Western blot in this invention.

[0053] Figure 12 This is a subject operating characteristic curve for distinguishing between transplanted kidneys with and without fibrosis using biomarkers, as presented in this invention.

[0054] Figure 13 This is a subject operating characteristic curve for the present invention, which uses biomarkers to distinguish between transplanted kidneys with and without chronic damage.

[0055] Figure 14 This is a graph showing the expression levels of fibrosis diagnostic markers in 32 patients with uEVs identified and validated by ELISA in this invention.

[0056] Figure 15 This invention provides cut-off values, receiver operating characteristic curves, and confusion matrix diagrams for diagnosing transplanted kidney fibrosis using a logistic regression model constructed based on ELISA test results.

[0057] Figure 16 This is an analysis diagram illustrating the prediction of renal function loss in kidney transplant patients based on biomarker expression levels, as presented in this invention.

[0058] Figure 17 This is a graph showing the expression levels of fibrosis diagnostic markers in uEVs of healthy volunteers and CKD patients identified by ELISA in this invention. Detailed Implementation

[0059] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and preferred embodiments.

[0060] Experimental specimens: The subjects included in this study were divided into three groups: transplanted kidney without fibrosis (Banff Ci score of 0, 41 cases), transplanted kidney with mild fibrosis (Banff Ci score of 1, 41 cases), and transplanted kidney with moderate to severe fibrosis (Banff Ci score of 2, 34 cases). Subject data are shown in Table 1. For each participant, 50 mL of the second morning urine sample was collected on the day of biopsy and immediately centrifuged at 3000g for 20 min at 4℃ to remove cells and cell debris. The sample was then frozen at -80℃ for later use.

[0061] Table 1 Basic characteristics of the research subjects

[0062]

[0063] Methods for isolating and detecting extracellular vesicles in urine:

[0064] The method for separating uEVs includes the following steps:

[0065] S1. Urine sample pretreatment: The frozen urine sample (50 mL) was thawed in a 37°C water bath within 1 hour. Then, Tris-EDTA solutions with Tris concentration of 100 mM, EDTA concentrations of 40 mM, 80 mM, and 160 mM were added to the urine at a ratio of 1:4. The mixture was vortexed for 90 seconds to depolymerize the uromodulin (THP) network and obtain the treated urine.

[0066] S2. Centrifuge the urine obtained in step S1 at 4°C and 17000g for 30 minutes to remove large cell debris such as apoptotic bodies and obtain the supernatant.

[0067] S3. Filter the supernatant obtained in step S2 using a 0.22μm needle filter to remove microvesicles with a diameter greater than 200nm.

[0068] S4. Place the supernatant filtered in step S3 into an ultracentrifuge tube, centrifuge at 130,000g for 120 min at 4°C, discard the supernatant, add PBS to wash the precipitate, centrifuge again at 130,000g for 120 min at 4°C, resuspend the precipitate with an appropriate amount of PBS, and freeze at -80°C for later use.

[0069] like Figure 1 As shown, the effects of different buffers (PBS-EDTA and Tris-EDTA) and EDTA concentrations on the expression levels of various proteins are illustrated. Figure 1 (a) is a Western blot plot. Figure 1 (b) is a quantitative analysis diagram. Western blot analysis of EV markers (CD9, CD63, TSG101, Alix) showed that the band intensity of the 8 mM EDTA (final concentration) group was significantly higher than that of other concentrations, indicating that low concentrations of EDTA can effectively release uEVs and maintain membrane protein integrity. Finally, it was determined that final concentrations of Tris and EDTA of 20 mM and 8 mM, respectively, maximized the efficiency of uEV separation and ensured the structural integrity of EVs.

[0070] like Figure 2 As shown, the isolation and identification of uEVs from clinical samples:

[0071] 50 mL of morning urine from patients with Banff ci scores of 0, 1, and 2 on the day of biopsy was collected. uEVs were purified using the separation method described in Example 1 and then identified.

[0072] in, Figure 2 (a) Western blot diagram of extracellular vesicle marker proteins. Western blot detection of exosome marker proteins Alix and CD63: Exosome proteins were lysed and added to a 10% polyacrylamide gel. After electrophoresis, they were transferred to a PVDF membrane. Primary antibodies (CD9, 1:1000 dilution; CD63, 1:1000 dilution; TSG101, 1:1000 dilution; Alix, 1:1500 dilution; Calnexin, 1:1000 dilution; AQP2, 1:1000 dilution) were diluted with blocking buffer and incubated overnight. After incubation with HRP-labeled secondary antibody, chemiluminescence detection was performed using ECL (extracorporeal photoluminescence assay).

[0073] in, Figure 2 (b) The particle size distribution of uEVs measured by DLS. Extracellular vesicle particle size was detected using a dynamic light scattering particle size analyzer: the extracted extracellular vesicles were diluted with double-distilled water and added to the sample cell of a Malvern nanoparticle size potentiometer for detection. It can be seen that there was no significant difference in the particle size distribution of extracellular vesicles in urine from patients with different degrees of transplant kidney fibrosis.

[0074] in, Figure 2 (c) is a cryo-transmission electron microscopy (TEM) image of uEVs, with a scale bar of 200 nm. Extracellular vesicle morphology was identified using TEM: Extracellular vesicles extracted in Example 1 were dropped onto a 200-mesh copper mesh sample grid, allowed to stand at room temperature for 2 min, and excess liquid was blotted with filter paper; 20 mg / mL uranium acetate solution was added to the sample grid, allowed to stand at room temperature for 1 min, and the sample was negatively stained. Excess liquid was blotted with filter paper, and the sample grid was dried; the prepared sample was observed under a TEM and images were captured. It can be seen that the morphology and diameter of the extracellular vesicles did not change; the morphology was a cup-shaped vesicle-like structure, with a diameter of approximately 80-120 nm.

[0075] It is evident that the uEV obtained by the method provided by the present invention has a complete morphological structure and excellent quality.

[0076] 4D-DIA proteomics analysis of uEV protein expression profiles in transplant kidney fibrosis patients:

[0077] Protein digestion, desalting, and library construction:

[0078] S10. Reduce 100µg of protein extracted from each sample. Add 200mM dithiothreitol (DTT) solution and incubate at 37°C for 1 hour. Then add trypsin (trypsin:protein = 1:50) and incubate overnight at 37°C.

[0079] S20. On the second day, add 50 μL of 0.1% FA to terminate digestion. Wash the C18 column with 100 μL of 100% ACN and centrifuge at 1200 rpm for 3 min; wash the column once with 100 μL of 0.1% FA and centrifuge at 1200 rpm for 3 min. Replace the EP tube, add the sample, and centrifuge at 1200 rpm for 3 min; wash the column twice with 100 μL of 0.1% FA and centrifuge at 1200 rpm for 3 min. Wash once with 100 μL of pH=10 water; replace the EP tube and elute with 70% ACN. Combine the eluents from each sample and lyophilize. Store at -80°C before loading.

[0080] S30. Fractionation of the sample peptides was performed on a Rigol L3000 HPLC using a C18 column at a flow rate of 1 mL / min and a column temperature of 50 °C. Mobile phases A [2% acetonitrile (ACN), pH adjusted to 10.0 with ammonium hydroxide] and B [98% ACN, pH adjusted to 10.0 with ammonium hydroxide] were used for gradient elution. The solvent gradient was set as follows: 5% B, 0 min; 5-8% B, 5 min; 8-18% B, 35 min; 18-32% B, 22 min; 32-95% B, 2 min; 90% B, 4 min; 95-5% B, 4 min. The eluent was monitored at 214 nm UV wavelength, and one tube was collected every minute. The eluents were combined into six fractions, which were then dried under vacuum.

[0081] S40. Reconstruct the sample peptide and 6 fractionated peptides in 0.1% (v / v) formic acid (FA) water, and then add 0.2 µL of standard peptide (iRTkit, Biognosys) to the peptide sample for subsequent analysis.

[0082] S50. To construct a transition library, shotgun proteomics analysis was performed using an EASY-nLC™ 1200 ultra-high performance liquid chromatography system and an Orbitrap QExactive HF-X mass spectrometer (Thermo Fisher Scientific) in data-dependent acquisition (DDA) mode.

[0083] LC-MS / MS mass spectrometry analysis:

[0084] S100, prepare mobile phase A (100% water, 0.1% formic acid) and mobile phase B (80% acetonitrile, 0.1% formic acid).

[0085] S200: Dissolve the lyophilized powder in 10 µL of liquid solution, centrifuge at 14000 g for 20 min at 4 °C, and inject 1 µg of the supernatant as sample for liquid chromatography-mass spectrometry (LC-MS). The LC elution conditions are shown in Table 2.

[0086] Table 2 Chromatographic gradient

[0087]

[0088] The S300 uses an Orbitrap Exploris™ 480 mass spectrometer with the optional FAIMS Pro™ Interface. The compensation voltage (CV) switches between -45 and -65 every 1 second. A Nanospray Flex™ (NSI) ion source is used, with the ion spray voltage set to 2.0 kV and the ion transfer tube temperature set to 320°C. Mass spectrometry employs a data-dependent acquisition mode, with a full scan range of 350–1500 m / z. The primary mass spectrometry resolution is set to 120,000 (200 m / z), AGC to 300%, and the maximum C-trap injection time to 50 ms. Secondary mass spectrometry detection uses the "Top Speed" mode, with a resolution of 15,000 (200 m / z), AGC to 75%, a maximum injection time to 22 ms, and the peptide fragmentation collision energy set to 33%. Raw mass spectrometry data is generated.

[0089] Data processing:

[0090] The database used in this study was Homo sapiens SP (protein count: 20,407, database: uniprot). The database search parameters using Spectronaut software are shown in Table 3.

[0091] Table 3 Search Parameter Settings

[0092]

[0093] The original data were normalized to eliminate experimental errors. Data with more than 50% missing values ​​were filtered out. Missing values ​​were filled using the KNN imputation method. A t-test was used for difference analysis. P Value 0.05, Fold change 1.2-fold, yielding the analysis results of differentially expressed proteins.

[0094] Screening and validation of differentially expressed uEVs proteins:

[0095] like Figure 3 As shown, differential proteomics analysis of uEVs in patients with different CI scores revealed 382 and 346 differentially expressed proteins in uEVs of patients with CI=1 and CI=2, respectively, compared to patients with CI=0. Among these, 144 proteins were co-expressed differentially. Figure 4 As shown, combining the expression data of differentially expressed proteins in other fibrotic nephropathy patients from the Nephromine public database with ROC analysis, SNX3, VPS4B, and SMO were identified as the main differentially expressed proteins. Figure 4(a) is a heatmap showing the expression patterns of multiple genes (such as SNX3, VPS4B, SMO, etc.) in different groups (ci=0, ci=1, ci=2). The color coding represents the expression value, with red representing high expression and blue representing low expression, visually presenting the differences in gene expression in different groups; Figure 4 (b) is a box plot comparing the log2 expression values ​​of the three genes SNX3, VPS4B, and SMO in the healthy donor (blue), diabetic nephropathy (orange), IgA nephropathy (purple), and lupus nephritis (red) groups, revealing the differences in gene expression among different disease groups; Figure 4 (c) is a scatter plot, analyzing the correlation between gene expression values ​​and log2 eGFR (glomerular filtration rate). SNX3 expression is significantly negatively correlated with eGFR; VPS4B expression is also significantly negatively correlated with eGFR, while SMO expression is less correlated with eGFR.

[0096] like Figure 5 As shown, after normalizing the loading amount with urinary creatinine concentration, the expression levels of three differentially expressed proteins in urine before and after separation were detected by Western blot. Figure 5 (a) Western blot analysis was performed to detect the expression of proteins in Urine and EV-free Urine (urine with extracellular vesicles removed), and the relative expression levels of these proteins were quantified using statistical charts. The results showed that the band intensities of these proteins in Urine were generally higher than those in EV-free Urine, demonstrating that the three marker proteins SNX3, VPS4B, and SMO were significantly enriched in uEVs. Figure 5 (b) The expression levels of the three proteins in uEVs and whole urine of patients with different Banff ci scores were detected by Western blot. The results showed that the content of the three marker proteins in uEVs gradually increased with the increase of the patient's ci score, and there was no significant change in whole urine, indicating that the abundance changes of the three proteins were uEVs specific.

[0097] like Figure 6 As shown, immunohistochemical staining confirmed the upregulation of three biomarkers in transplanted kidney biopsy tissue, demonstrating that the expression of these three biomarkers increased with increasing CI scores and their correlation with renal tubular localization. This provides direct evidence for the kidney origin of uEVs carrying these biomarkers.

[0098] In a mouse model of renal fibrosis, the expression of uEVs biomarkers was significantly correlated with the degree of fibrosis.

[0099] This invention established a mouse model of unilateral ureteral ligation (UUO) and folic acid-induced (FA) renal fibrosis to further clarify the correlation between the expression levels of three biomarkers in fibrotic kidneys and the progression of fibrosis. Figure 7 and Figure 8 As shown, immunohistochemical staining was used to detect the expression levels of three biomarkers in kidney tissues at D0, D3, D7, and D14 after UUO modeling and at D0, D3, D14, and D28 after FA modeling. Collagen deposition levels were measured using Sirius red staining. Linear regression analysis showed a significant positive correlation between the expression levels of the three biomarkers in the fibrotic kidneys of mice and the degree of fibrosis.

[0100] Increased expression of uEV biomarkers in chronic rejection-induced fibrotic transplanted kidneys in mice:

[0101] This invention established a mouse model of transplanted kidney fibrosis to further clarify the expression characteristics of three uEVs biomarkers in fibrotic transplanted kidneys. One month after Balb / c mouse kidneys were transplanted into C57BL / 6 recipients, the transplanted kidneys were harvested for histopathological analysis.

[0102] like Figure 9 As shown, H&E staining revealed severe renal tubular damage and atrophy in the transplanted kidney; Masson staining and Sirius red staining showed the level of collagen deposition in the mouse transplanted kidney, and Masson trichrome staining and Sirius red staining further confirmed significant interstitial fibrosis in the graft.

[0103] like Figure 10 As shown, after successfully constructing a mouse model of transplanted kidney fibrosis induced by chronic rejection, among which, Figure 10 (a) is the Western blot result. The expression of candidate biomarkers in fibrotic transplanted kidneys was detected in two groups: Sham (blank group) and Allograft (transplant group). The intensity of the bands initially revealed the differences in protein expression between the two groups; a darker band indicated higher protein expression. It was observed that SNX3, VPS4B, and SMO were all significantly upregulated in fibrotic transplanted kidneys. To further verify whether these biomarkers were carried by tubular-derived EVs, their co-localization with the late endosomal biomarker CD63 was detected by immunofluorescence co-staining. Figure 10 (b) is a bar chart showing the relative expression levels of proteins. SNX3, VPS4B, and SMO are mainly located within EVs in fibrotic transplanted kidneys, providing biological evidence that they are secreted into the urine from the renal tubules of the transplanted kidney via EVs. Figure 10(c) is an immunofluorescence staining image; immunofluorescence staining shows that the diagnostic marker of fibrosis and the EV marker protein CD63 co-localize in the fibrotic transplanted kidney of mice, scale bar = 20 μm. Fluorescence microscopy reveals the intracellular localization of proteins. The areas indicated by the white arrows are co-localized regions, and overlapping areas of different fluorescent colors indicate the co-localization of the corresponding proteins. Figure 10 (d) is a bar chart of mean fluorescence intensity (MFI). This shows that there are significant differences in the fluorescence intensity (i.e., expression level) of these proteins between the transplantation group and the blank group.

[0104] The expression levels of SNX3, VPS4B, and SMO in uEVs can enable non-invasive diagnosis of transplant kidney fibrosis.

[0105] This invention collected morning urine from 59 kidney transplant patients on the day of biopsy. Urinary extracellular vesicles were purified according to the method of this invention. The expression levels of SNX3, SMO and VPS4B in uEV of patients with different CI scores were quantified and compared by Western blotting.

[0106] like Figure 11 As shown, the expression of all three uEVs proteins was significantly increased in patients with transplanted kidney fibrosis.

[0107] like Figure 12 As shown, to clarify whether these three uEVs proteins can be used for non-invasive diagnosis of transplanted fibrosis, the present invention constructed ROC curves of the proteins based on Western Blot quantitative results, demonstrating the diagnostic capabilities of uEVs proteins SNX3, VPS4B, and SMO for fibrotic transplanted kidneys.

[0108] This invention found that all three can effectively distinguish between fibrotic transplanted kidneys and other types of kidney injury patients. The AUCs of SNX3, VPS4B, and SMO were 0.8454 (P<0.0001), 0.7433 (P=0.0028), and 0.7667 (P=0.0001), respectively.

[0109] The expression levels of SNX3, VPS4B, and SMO in uEVs can enable non-invasive diagnosis of chronic progressive graft injury in transplanted kidneys. To clarify the clinical diagnostic value of candidate biomarkers beyond fibrosis, this invention categorizes patients based on biopsy results into a chronic progressive graft injury group (including antibody-mediated rejection, transplanted glomerulonephropathy, and mixed rejection) and other lesion groups, such as... Figure 13As shown, ROC analysis validated the diagnostic efficacy of the candidate biomarkers for chronic graft injury, demonstrating the diagnostic capabilities of uEVs proteins SNX3, VPS4B, and SMO for chronic progressive transplant nephropathy. The ROC-AUC values ​​for SNX3, VPS4B, and SMO were 0.7210, 0.6649, and 0.7527, respectively. These results indicate that the candidate biomarkers exhibit good discriminatory ability in distinguishing between chronic and acute graft injury, providing a basis for early clinical intervention to delay the progression of chronic transplant nephropathy.

[0110] To further verify the diagnostic capabilities of these three uEVs proteins in clinical applications, this invention collected morning urine samples from 32 kidney transplant patients on the day of biopsy and detected the levels of the three proteins in uEVs using ELISA. Figure 14 As shown, after normalization of urinary creatinine concentration, the contents of SNX3, VPS4B and SMO in uEVs gradually increased with the increase of patient ci score, and showed a good linear relationship with the collagen deposition level shown by Masson staining.

[0111] like Figure 15 As shown, based on the ELISA quantitative results, the present invention constructed ROC curves for the proteins. The AUCs of SNX3, VPS4B, and SMO were 0.9174 (P<0.0001), 0.7238 (P=0.0470), and 0.8727 (P=0.0009), respectively, demonstrating the diagnostic ability of uEVs proteins SNX3, VPS4B, and SMO for fibrotic transplanted kidneys.

[0112] After plotting the ROC curves for SNX3, VPS4B, and SMO for all uEVs in the study subjects, a logistic regression was established using these three variables. The logistic regression yielded coefficients for the joint analysis, generating a joint calculation formula. By substituting the SNX3, VPS4B, and SMO measurements from different individuals into the formula, the probability of an individual having the disease could be calculated. Based on the probabilities provided by the logistic regression, the ROC curve for the multi-indicator joint diagnosis could be plotted. The calculation formula for the joint diagnostic model is as follows:

[0113] Logit(P)=6206.64×[SNX3]-10.06×[VPS4B]+1165.64×[SMO]-8.23

[0114] The AUC of the joint model can be improved to 0.9909 ( P <0.0001), cut-off value of 50%. Confusion matrix shows that the combined model has a sensitivity of 95.2%, a specificity of 81.8%, and a diagnostic accuracy of 90.6%, which is better than any single indicator.

[0115] To clarify the prognostic potential of biomarkers, based on biomarker levels in uEVs of kidney transplant patients detected by ELISA, patients were divided into low-risk and high-risk groups according to the cut-off value (50%) of the combined diagnostic model. Figure 16 As shown, high-risk patients had significantly lower eGFR at the time of biopsy than low-risk patients, and this decrease was even more pronounced three months later, with all patients developing transplant kidney failure (eGFR below 30 mL / min / 1.73 m). 2 ).

[0116] To further verify the diagnostic ability of these three uEVs proteins for renal fibrosis (non-transplant) in CKD patients, this invention collected urine samples from 10 healthy volunteers and 20 CKD patients, and detected the content of the three proteins in uEVs using ELISA. Figure 17 As shown, after normalizing urinary creatinine concentration, the levels of SNX3, VPS4B, and SMO in uEVs of patients with end-stage renal disease were significantly higher than those in patients without renal fibrosis (CKD stage I / II) and healthy volunteers. Substituting the expression levels of the three proteins measured by ELISA into a logistic regression-based diagnostic model yielded the predicted probability of renal fibrosis in CKD patients. Using a 50% threshold, this model effectively distinguished patients with renal fibrosis, demonstrating the feasibility of its non-invasive clinical application in diagnosing renal fibrosis.

[0117] The embodiments of the present invention have been described in detail above, but the content described is only a preferred embodiment of the present invention and should not be considered as limiting the scope of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the present invention.

Claims

1. The application of reagents for detecting biomarkers in the preparation of non-invasive diagnostic products for renal interstitial fibrosis and chronic progressive injury, characterized in that: The biomarkers are SNX3, VPS4B, and SMO.

2. The application of the reagent for detecting biomarkers according to claim 1 in the preparation of non-invasive diagnostic products for renal interstitial fibrosis and chronic progressive injury, characterized in that: The SNX3, VPS4B, and SMO are derived from uEVs, and the changes in the expression levels of the SNX3, VPS4B, and SMO are uEVs-specific.

3. The application of the reagent for detecting biomarkers according to claim 2 in the preparation of non-invasive diagnostic products for renal interstitial fibrosis and chronic progressive injury, characterized in that: The method for detecting changes in the expression levels of SNX3, VPS4B, and SMO that are specific to uEVs includes the following steps: S1. UEVs are separated from urine by ultracentrifugation; S2. The marker proteins CD9, CD63, Alix, TSG101, and AQP2 of uEVs were detected by Western blot; the particle size distribution and morphology of uEVs were characterized by DLS and transmission electron microscopy, respectively. S3. The relative contents of SNX3, VPS4B and SMO in urine before and after ultracentrifugation were determined by Western blot to clarify that SNX3, VPS4B and SMO are mainly present in uEVs. S4. Western blot was used to detect the relative levels of uEVs and SNX3, VPS4B, and SMO in urine of kidney transplant patients with different Banff ci scores to determine whether the diagnostic ability is uEVs specific.

4. The application of the reagent for detecting biomarkers according to claim 2 in the preparation of non-invasive diagnostic products for renal interstitial fibrosis and chronic progressive injury, characterized in that, The method for separating uEVs includes the following steps: S11. Urine sample pretreatment: Thaw the frozen urine sample in a 37°C water bath within 1 hour, then add Tris-EDTA solution to the urine at a ratio of 1:4, with final Tris / EDTA concentrations of 20 mM and 8 mM, respectively. Vortex for 90 seconds to depolymerize the uromodulin network and obtain the treated urine. S22. Centrifuge the urine obtained in step S11 at 4°C and 17000g for 30 minutes to remove cell debris such as apoptotic bodies and obtain the supernatant. S33. Filter the supernatant obtained in step S22 using a needle filter to remove microvesicles with a diameter greater than 200 nm. S44. Place the supernatant filtered in step S33 into an ultracentrifuge tube, centrifuge at 130,000g for 120 min at 4°C, discard the supernatant, add PBS to wash the precipitate, centrifuge again at 130,000g for 120 min at 4°C, resuspend the precipitate with PBS, and freeze at -80°C for later use.

5. The application of the reagent for detecting biomarkers according to claim 1 in the preparation of non-invasive diagnostic products for renal interstitial fibrosis and chronic progressive injury, characterized in that: The method for detecting the biomarker includes the following steps: S111, Separating uEVs from urine; S222. The total expression levels of SNX3, VPS4B, and SMO in urine were detected by Western blot or sandwich enzyme-linked immunosorbent assay. S333, the expression levels of SNX3, VPS4B, and SMO were standardized by detecting urinary creatinine concentration.

6. The application of the reagent for detecting biomarkers according to claim 1 in the preparation of non-invasive diagnostic products for renal interstitial fibrosis and chronic progressive injury, characterized in that: When the biomarker is applied to a non-invasive diagnostic product for transplanted kidney interstitial fibrosis, the method for determining whether a subject has transplanted kidney fibrosis includes: comparing and analyzing the parameters of SNX3, VPS4B, and SMO protein content or expression levels in the subject's uEVs with sample parameters. If the parameters are higher than the sample parameter threshold, transplanted kidney fibrosis is present; otherwise, transplanted kidney fibrosis is not present. The sample parameters are selected from the subject's previous test samples and / or parameters indicating normal SNX3, VPS4B, and SMO protein content or expression levels in specified uEVs.

7. The application of the reagent for detecting biomarkers according to claim 1 in the preparation of non-invasive diagnostic products for renal interstitial fibrosis and chronic progressive injury, characterized in that: When the biomarker is applied to a non-invasive diagnostic product for chronic progressive injury of transplanted kidneys, the method for determining whether a subject has chronic progressive injury of the transplanted kidney includes: comparing and analyzing the relevant parameters of the content or expression level of SNX3, VPS4B, and SMO proteins in the subject's uEVs with the sample parameters. If the relevant parameters are higher than the sample parameter threshold, chronic progressive injury of the transplanted kidney is present; if the parameters are lower than the sample parameter threshold, it is acute injury or nonspecific injury of the transplanted kidney. The sample parameters are those parameters indicating that the content or expression level of SNX3, VPS4B, and SMO proteins in uEVs is normal.

8. The application of the reagent for detecting biomarkers according to claim 7 in the preparation of non-invasive diagnostic products for renal interstitial fibrosis and chronic progressive injury, characterized in that: The chronic progressive damage to the transplanted kidney includes transplanted kidney glomerulonephropathy, chronic rejection, polyomavirus nephropathy, and IgA nephropathy.

9. The application of the reagent for detecting biomarkers according to claim 7 in the preparation of non-invasive diagnostic products for renal interstitial fibrosis and chronic progressive injury, characterized in that: When the biomarkers are applied to non-invasive diagnostic products for the prognosis of transplanted kidney patients, the risk of transplant failure in the subject can be predicted by detecting the content or expression level of SNX3, VPS4B and SMO proteins in the subject's uEVs.

10. The application of the reagent for detecting biomarkers according to claim 7 in the preparation of non-invasive diagnostic products for renal interstitial fibrosis and chronic progressive injury, characterized in that: When the biomarker is applied to a non-invasive diagnostic product for renal fibrosis in non-transplanted chronic kidney disease patients, the method for determining whether a subject has renal fibrosis includes: comparing and analyzing the parameters of the content or expression level of SNX3, VPS4B, and SMO proteins in the subject's uEVs with the sample parameters. If the parameters are higher than the sample parameter threshold, then renal fibrosis exists; otherwise, renal fibrosis does not exist. The sample parameters are the specified parameters of the content or expression level of SNX3, VPS4B, and SMO proteins in uEVs.

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