Application of reagent for detecting biomarker in preparation of product for noninvasive diagnosis of renal interstitial fibrosis and chronic progressive injury

Abstract

The invention relates to application of a reagent for detecting a biomarker in preparation of a product for noninvasive diagnosis of renal interstitial fibrosis and chronic progressive injury, and belongs to the technical field of biology. The biomarkers are SNX3, VPS4B and SMO derived from urine extracellular vesicles, and the expression level is increased along with the increase of the fibrosis degree of the transplanted kidney. According to the separation method of the urine extracellular vesicles, the urine regulatory proteins wound with the vesicles in the ultracentrifugation process can be effectively removed, and the structural integrity of the extracellular vesicles and the expression of marker proteins are not influenced; the biomarker disclosed by the invention has excellent transplanted kidney fibrosis diagnosis capability, and also has excellent capabilities in diagnosing chronic progressive injury of the transplanted kidney, predicting the risk of dysfunction of the transplanted kidney and renal fibrosis of chronic kidney diseases. The kit has the advantages of good stability, strong specificity and high sensitivity, is simple and convenient to operate and short in detection period, and can be used for non-invasive diagnosis or prognosis monitoring of transplanted kidney or non-transplanted kidney fibrosis.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to the use of reagents for detecting biomarkers in the preparation of products for non-invasive diagnosis of renal interstitial fibrosis and chronic progressive injury. Background Art

[0002] Chronic kidney disease (CKD) has been 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 risen to over 90%, but the 10-year functional survival rate is only approximately 51%.

[0003] Long-term functional decline in renal allografts is primarily attributed to chronic allograft dysfunction (CAD), characterized by interstitial fibrosis and tubular atrophy (IFTA), and is the core cause of long-term allograft failure. Notably, tubulointerstitial fibrosis is the common terminal pathway of nearly all forms of persistent renal injury, and its progression is closely associated with progressive renal function loss and mortality. Fibrosis is not only common in patients undergoing an indicated biopsy but also occurs in patients with partial preservation or recovery of renal function. Clinically, renal function is typically assessed by measuring glomerular filtration rate (GFR). However, GFR does not accurately reflect the extent of renal fibrosis, and the correlation between GFR and fibrosis progression is weak. Therefore, early, noninvasive detection of renal fibrosis after transplantation is not only a key prerequisite for clinical intervention but also an urgent need to improve the long-term prognosis of renal allografts. It is crucial for personalized treatment and improved patient outcomes. However, accurate monitoring and real-time prevention of renal fibrosis in the transplanted kidney remain major clinical challenges.

[0004] A puncture biopsy is the gold standard for diagnosing the type of transplant kidney injury and is the most commonly used clinical procedure. However, because a puncture biopsy is an invasive procedure, it can lead to complications such as bleeding, infection, and damage to surrounding organs. This makes it difficult to continuously monitor the progression of transplant kidney fibrosis and intervene promptly, resulting in difficulties in improving the long-term survival rate of transplanted kidneys. Monitoring the progress of transplant kidney injury through multiple biopsies is painful, expensive, and impractical for patients.

[0005] Currently, clinically used non-invasive markers of allograft renal injury include serum creatinine (Scr), urine protein, and donor-specific antibodies (DSAs). These markers have limitations in sensitivity, specificity, and accuracy, making them ineffective for monitoring the progression of renal fibrosis. With the rapid development of high-throughput omics technologies, urine-derived noninvasive biomarkers have shown promising potential in the diagnosis of renal fibrosis. Urine, an ultrafiltrate produced directly by the kidneys, provides a unique window into the progression of allograft fibrosis. Furthermore, urine is readily accessible and can be collected daily, allowing for continuous, noninvasive monitoring of the pathophysiological status of the transplanted kidney. Studies have identified urinary markers such as cadherin-11 and pigment epithelium-derived factor as potential renal fibrosis markers. However, urine concentration is susceptible to fluctuations in diet, fluid intake, and physiological status, resulting in wide fluctuations in pH, osmolality, and protein concentration, resulting in poor stability.

[0006] Urine extracellular vesicles (uEVs) have been shown to carry markers specific to cells in the renal tubules and various segments of the urinary tract, making them a valuable biomarker source. Their high accessibility in urine makes them a promising target for noninvasive diagnostic research in various kidney diseases, such as autosomal dominant polycystic kidney disease, diabetic nephropathy, and transplant rejection. uEV analysis can detect molecules in urine that are difficult to directly identify due to low concentrations or confinement within vesicles, making them an ideal new liquid biopsy medium.

[0007] However, the development and application of disease biomarkers from uEVs still face multiple challenges, notably the interference of uromodulin (also known as Tamm-Horsfall protein). As the most abundant glycoprotein in urine, uromodulin readily forms polymer networks that precipitate at low centrifugal forces. However, its aggregates may encapsulate uEVs, leading to EV loss during subsequent separation. Furthermore, due to its high abundance, it can mask the signal of less abundant EV markers. Co-precipitation of uromodulin can significantly contaminate uEV samples, interfering with proteomic analysis. Although reducing agents (such as DTT or TCEP) can partially disaggregate uromodulin networks to improve EV recovery, this can affect the antigenicity of EV membrane proteins, limiting downstream functional studies. Existing separation methods (such as ultracentrifugation and size-exclusion chromatography), while widely used, have limitations. Ultracentrifugation can lead to the loss of small EVs due to uromodulin encapsulation, while size-exclusion chromatography can reduce uromodulin contamination but struggles to completely remove protein complexes, such as albumin, that co-migrated with EVs. In addition, the dynamic properties of urine (such as pH, osmotic pressure, protein concentration) and disease states (such as proteinuria) can further change sample viscosity, affecting separation efficiency and reproducibility.

[0008] Therefore, developing efficient and high-purity methods for isolating uEVs is crucial. This requires a balance between removing contaminants such as uromodulin and preserving EV integrity. More standardized methods are needed to ensure the reliability of test results. Examples include combining chemical disaggregation with multi-step gradient centrifugation or developing microfluidic technologies to directly analyze EV subpopulations in unfractionated urine. Standardized isolation procedures will enhance the comparability of data across studies and lay the foundation for disease biomarker discovery and clinical translation.

[0009] Currently, the clinical translation of uEVs has achieved initial success, with biomarkers derived from them now FDA-approved for the diagnosis of prostate cancer, demonstrating their feasibility for clinical application. However, the diagnostic value of uEVs in renal transplantation remains to be further explored. Single-nucleus RNA sequencing studies have revealed that proximal tubular epithelial cells (PTECs), key effector cells of transplanted kidney injury, mediate the fibrotic process by secreting extracellular vesicles (EVs). This suggests that uEVs derived from transplanted kidney TECs may carry key molecular information reflecting the fibrotic state. However, the specific proteomic characteristics of uEVs in the progression of transplanted kidney fibrosis and their regulatory mechanisms have not been systematically elucidated. Therefore, systematic proteomic analysis of uEVs and the discovery of novel biomarkers for the noninvasive diagnosis of transplanted kidney fibrosis have important clinical translational value and are 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 for use in the preparation of a product for non-invasive diagnosis of renal interstitial fibrosis and chronic progressive injury.

[0011] To solve the above technical problems, the technical solution adopted by the present invention is: the use of reagents for detecting biomarkers in the preparation of products for non-invasive diagnosis of renal interstitial fibrosis and chronic progressive injury, wherein the biomarkers are SNX3 (sorting connexin 3), VPS4B (vacuolar protein sorting 4 homolog B) and SMO (smoothened receptor).

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

[0013] Furthermore, the reagent for detecting biomarkers is a reagent that can quantitatively detect SNX3, VPS4B and SMO; the reagent is a substance that can detect 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, and ELISA; the product can be a kit for detecting the content of SNX3, VPS4B and SMO by protein immunoblotting (Western blot) or sandwich enzyme-linked immunosorbent assay (ELISA).

[0014] Furthermore, the method for detecting changes in the expression levels of SNX3, VPS4B, and SMO is specific for uEVs, comprising the following steps: S1. Isolation of uEVs from urine by ultracentrifugation. S2. Detect uEV marker proteins CD9, CD63, Alix, TSG101, and AQP2 by Western blot; characterize the size distribution and morphology of uEVs by DLS and transmission electron microscopy, respectively; S3. Detect the relative levels of SNX3, VPS4B, and SMO in urine before and after ultracentrifugation by Western blot to confirm that SNX3, VPS4B, and SMO are mainly present in uEVs; S4. The relative contents of SNX3, VPS4B, and SMO in uEVs and urine of renal transplant patients with different Banff CI scores were detected by Western blot to confirm that its diagnostic ability is specific for uEVs.

[0015] Furthermore, the method for isolating uEVs comprises 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 in a ratio of 1:4 to the urine, with final concentrations of 20 mM and 8 mM, respectively. Vortex for 90 seconds to disaggregate the uromodulin network to obtain the treated urine. S22, centrifuging the urine obtained in step S11 at 17000 g for 30 min at 4°C to remove large cell debris such as apoptotic bodies to obtain a supernatant; S33, filtering the supernatant obtained in step S22 with a needle filter to remove microvesicles with a diameter greater than 200 nm; S44. Place the supernatant after filtering in step S33 into an ultracentrifuge tube, centrifuge at 4°C, 130,000 g for 120 min, discard the supernatant, add PBS to wash the precipitate, centrifuge again at 4°C, 130,000 g for 120 min, resuspend the precipitate with PBS, and freeze at -80°C for later use.

[0016] Furthermore, the biomarker detection method comprises the following steps: S111, Isolation of uEVs from urine; S222, the expression levels of total SNX3, VPS4B, and SMO were detected by Western blot or sandwich enzyme-linked immunosorbent assay; S333. Urinary creatinine concentration was detected to normalize the expression levels of SNX3, VPS4B, and SMO.

[0017] Furthermore, when the biomarker is used in a non-invasive diagnosis of transplanted kidney interstitial fibrosis product, the method for determining whether a subject suffers from transplanted kidney fibrosis includes: comparing and analyzing the parameters of the SNX3, VPS4B, and SMO protein content or expression level in the subject's uEVs with the sample parameters; if the parameters are higher than the sample parameter threshold, transplanted kidney fibrosis exists; otherwise, transplanted kidney fibrosis does not exist; the sample parameters are selected from the subject's previous test samples and / or specified parameters of the normal content or expression level of SNX3, VPS4B, and SMO proteins in uEVs.

[0018] Furthermore, when the biomarker is used in a non-invasive diagnosis of chronic progressive renal transplant injury, the method for determining whether a subject has chronic progressive renal transplant injury includes comparing and analyzing parameters related to the content or expression level 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 renal transplant injury is present; if the parameters are lower than the sample parameter threshold, acute renal transplant injury or non-specific injury is present. The sample parameters are parameters that indicate normal content or expression level of SNX3, VPS4B, and SMO proteins in uEVs. Chronic progressive renal transplant injury includes allograft glomerulopathy, chronic rejection, polyomavirus nephropathy, and IgA nephropathy.

[0019] Furthermore, when the biomarker is used in a non-invasive diagnosis product for the prognosis of transplanted kidney patients, the risk of transplanted kidney failure in the subject is predicted by detecting the content or expression level of SNX3, VPS4B and SMO proteins in the subject's uEVs.

[0020] Furthermore, when the biomarker is used for non-invasive diagnosis of renal fibrosis in non-transplanted chronic kidney disease patients, the method for determining whether the subject suffers from 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, renal fibrosis is present; 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.

[0021] The beneficial effects of the present invention are: (1) The biomarker of the present invention is derived from urine extracellular vesicles, which can be used to diagnose transplanted kidney fibrosis under non-invasive conditions.

[0022] (2) The biomarkers of the present invention are significantly enriched in uEVs, and their abundance changes are uEV-specific. Their abundance changes in urine have no correlation with the degree of renal fibrosis in patients, indicating that compared with free protein markers in urine, their diagnostic specificity is higher, their sensitivity is stronger, and their diagnosis is more accurate.

[0023] (3) The urine extracellular vesicle separation method of the present invention removes protein particles and large vesicles by depolymerizing uromodulin fibers with Tris-EDTA and filtering with a 0.22 μm needle filter. It can effectively remove uromodulin entangled with vesicles during ultracentrifugation without affecting the structural integrity of extracellular vesicles and the expression of marker proteins.

[0024] (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 rate of 90.6%, and has good clinical application prospects.

[0025] (5) In addition to diagnosing renal allograft fibrosis, the biomarker of the present invention also has excellent ability in diagnosing chronic progressive injury of the renal allograft (AUC = 0.8220).

[0026] (6) The urinary extracellular vesicle proteins SNX3, VPS4B, and SMO proposed in the present invention are used as biomarkers. By detecting the biomarkers, it can be used to predict the risk of patients progressing to transplant renal failure.

[0027] (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 patients with chronic kidney disease. It can be seen that the biomarkers of the present invention have excellent diagnostic ability for transplanted kidney fibrosis, and also have excellent ability in diagnosing chronic progressive damage to transplanted kidneys, predicting the risk of transplanted kidney failure, and renal fibrosis in chronic kidney disease (non-transplant).

[0028] (8) The present invention discloses the use of a urine extracellular vesicle protein marker combination in the preparation of a diagnostic product for renal interstitial fibrosis. By analyzing and screening the extracellular vesicle protein marker combination in urine, accurate diagnosis of renal fibrosis is achieved. This method not only reduces the pain and risk for patients, but also avoids unnecessary biopsies and reduces the possibility of misdiagnosis and missed diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be described in detail below with reference to the accompanying drawings and in combination with examples, and the advantages and implementation modes of the present invention will become more apparent. The contents shown in the accompanying drawings are only used to illustrate the present invention and do not constitute any limitation to the present invention. In the accompanying drawings: Figure 1 This is a graph showing the expression levels of marker proteins in uEVs obtained by ultracentrifugation after urine pretreatment in different ways of the present invention.

[0030] Figure 2 This is an identification diagram of uEVs isolated by the method for isolating urine extracellular vesicles of the present invention.

[0031] Figure 3 This is an analysis diagram of differentially expressed proteins between the CI0, CI1 and CI2 groups of the present invention.

[0032] Figure 4This is a diagram of candidate marker proteins SNX3, VPS4B, and SMO obtained based on protein differential expression levels and verified and screened according to the Nephromine database.

[0033] Figure 5 This is an expression analysis diagram of three marker proteins in Urine and EV-free Urine of the present invention.

[0034] Figure 6 These are immunohistochemical staining images of biopsy tissues of renal transplant patients with different CI scores in the present invention. Scale bar = 100 μm.

[0035] Figure 7 This is a correlation analysis chart showing the expression levels of three marker proteins and the degree of fibrosis in the mouse renal fibrosis model induced by unilateral ureteral ligation by immunohistochemical staining in the present invention. Scale bar = 100 μm.

[0036] Figure 8 This is a correlation analysis diagram showing the expression levels of three marker proteins and the degree of fibrosis in the mouse renal fibrosis model induced by folic acid by immunohistochemical staining in the present invention. Scale bar = 100 μm.

[0037] Figure 9 This is a diagram of the present invention showing chronic rejection-induced mouse transplant kidney damage through H&E staining.

[0038] Figure 10 This is a diagram showing the co-localization of biomarkers with EVs markers in chronic rejection-induced fibrotic transplanted kidneys of mice by immunofluorescence co-staining.

[0039] Figure 11 This is a graph showing the expression levels of fibrosis diagnostic markers in uEVs of 59 patients in the cohort identified and validated by Western blot in the present invention.

[0040] Figure 12 This is a receiver operating characteristic curve diagram for distinguishing the presence or absence of fibrosis in transplanted kidneys using markers in the present invention.

[0041] Figure 13 This is a receiver operating characteristic curve diagram for distinguishing whether a transplanted kidney has chronic damage or not by using markers in the present invention.

[0042] Figure 14 This figure shows the expression levels of fibrosis diagnostic markers in uEVs of 32 patients in the validation cohort identified by ELISA in the present invention.

[0043] Figure 15 The cut-off value, receiver operating characteristic curve, and confusion matrix diagram of the logistic regression model constructed for diagnosing transplanted kidney fibrosis based on the ELISA test results are shown in the present invention.

[0044] Figure 16 This is an analysis chart of the present invention for predicting renal function loss in renal transplant patients through marker expression levels.

[0045] 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 the present invention. DETAILED DESCRIPTION

[0046] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below with reference to the accompanying drawings and the best embodiments.

[0047] Experimental Specimens: Subjects enrolled in this study were divided into three groups: a group with no allograft fibrosis (Banff CI score of 0, 41 patients), a group with mild allograft fibrosis (Banff CI score of 1, 41 patients), and a group with moderate to severe allograft fibrosis (Banff CI score of 2, 34 patients). Subject information is shown in Table 1. All participants received a 50 mL second-morning urine sample on the day of biopsy. The sample was immediately centrifuged at 3000 g for 20 minutes at 4°C to remove cells and cell debris, and then frozen at -80°C for later use.

[0048] Table 1 Basic characteristics of the research subjects

[0049] Methods for isolating and detecting urine extracellular vesicles: The method for isolating uEVs comprises the following steps: S1. Urine sample pretreatment: Thaw the frozen urine sample (50 mL) in a 37°C water bath within 1 h. Then, add Tris-EDTA solution with a Tris concentration of 100 mM and EDTA concentrations of 40 mM, 80 mM, and 160 mM to the urine at a ratio of 1:4. Vortex and oscillate for 90 s to disaggregate the uromodulin (THP) network to obtain the treated urine.

[0050] S2. Centrifuge the urine obtained in step S1 at 17,000 g for 30 min at 4°C to remove large cell debris such as apoptotic bodies to obtain a supernatant.

[0051] S3. Filter the supernatant obtained in step S2 using a 0.22 μm needle filter to remove microvesicles with a diameter greater than 200 nm.

[0052] S4. Place the supernatant after filtering in step S3 into an ultracentrifuge tube, centrifuge at 130,000 g at 4°C for 120 min, discard the supernatant, add PBS to wash the precipitate, centrifuge again at 130,000 g at 4°C for 120 min, resuspend the precipitate with an appropriate amount of PBS, and freeze at -80°C for later use.

[0053] like Figure 1 As shown, it shows the effects of different buffers (PBS-EDTA and Tris-EDTA) and EDTA concentrations on the expression levels of various proteins, among which Figure 1 (a) is a Western blot image. Figure 1 (b) Quantitative analysis. Western blot analysis of EV marker expression (CD9, CD63, TSG101, Alix) revealed significantly higher band intensities in the 8 mM EDTA group compared to other concentrations, demonstrating that low-concentration EDTA can effectively release uEVs while maintaining membrane protein integrity. It was determined that final concentrations of 20 mM Tris and 8 mM EDTA, respectively, maximized uEV isolation efficiency and preserved EV structural integrity.

[0054] like Figure 2 As shown, isolation and identification of uEVs from clinical samples: 50 mL of morning urine was collected from patients with Banff CI scores of 0, 1, and 2 on the day of biopsy, and uEVs were purified and subsequently identified using the isolation method described in Example 1.

[0055] in, Figure 2 (a) Western blot analysis of extracellular vesicle marker proteins. Western blot analysis of exosome marker proteins Alix and CD63: Exosome proteins were lysed and loaded onto a 10% polyacrylamide gel. After electrophoresis, the membranes were transferred to PVDF membranes and incubated overnight with 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) diluted in blocking buffer. After incubation with HRP-conjugated secondary antibodies, luminescence detection was performed using chemiluminescent detection reagent (ECL).

[0056] in, Figure 2 (b) DLS-derived uEV size distribution. Extracellular vesicle size was measured using a dynamic light scattering particle size analyzer: extracted extracellular vesicles were diluted with double-distilled water and added to the sample cell of a Malvern nanometer potentiometer for analysis. As can be seen, there was no significant difference in the size distribution of extracellular vesicles from urine collected from patients with different degrees of transplanted kidney fibrosis.

[0057] in, Figure 2 (c) is a cryo-TEM image of uEVs, with a scale of 200 nm. Extracellular vesicle morphology was determined using transmission electron microscopy: the extracellular vesicles extracted in Example 1 were dropped onto a 200-mesh copper grid, allowed to stand at room temperature for 2 minutes, and excess liquid was removed with filter paper. A 20 mg / mL uranyl acetate solution was added to the grid and allowed to stand at room temperature for 1 minute to negatively stain the sample. Excess liquid was removed with filter paper, and the grid was air-dried. The prepared sample was observed under a transmission electron microscope, and photographs were taken. The morphology and diameter of the extracellular vesicles remained unchanged, with the vesicles appearing as cup-shaped structures with a diameter of approximately 80 to 120 nm.

[0058] It can be seen that the uEVs separated according to the method provided by the present invention have a complete morphological structure and excellent quality.

[0059] 4D-DIA proteomics analysis of uEVs protein expression profiles in patients with transplanted renal fibrosis: Protein digestion, desalting and library construction: S10. Reduce 100 µg of protein extracted from each sample. Add 200 mM dithiothreitol (DTT) solution and incubate at 37°C for 1 hour. Then add trypsin (trypsin:protein = 1:50) and incubate at 37°C overnight.

[0060] S20. The next day, add 50 μL of 0.1% FA to terminate the digestion. Wash the C18 column with 100 μL of 100% ACN and centrifuge at 1200 rpm for 3 minutes. Wash the column once with 100 μL of 0.1% FA and centrifuge at 1200 rpm for 3 minutes. Replace the EP tube, add the sample, and centrifuge at 1200 rpm for 3 minutes. Wash the column twice with 100 μL of 0.1% FA and centrifuge at 1200 rpm for 3 minutes. Wash the column once with 100 μL of pH 10 water and replace the EP tube with 70% ACN. Combine the eluates from each sample, lyophilize, and store at -80°C until loading.

[0061] S30. Sample peptides were fractionated using a C18 column on a Rigol L3000 HPLC system at a flow rate of 1 mL / min and a column temperature of 50°C. Gradient elution was performed using mobile phases A [2% acetonitrile (ACN), adjusted to pH 10.0 with ammonium hydroxide] and B (98% ACN, adjusted to pH 10.0 with ammonium hydroxide). The solvent gradient was 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; and 95-5% B, 4 min. The eluent was monitored at a UV wavelength of 214 nm. One fraction was collected every minute and pooled into six fractions. The peptides from these six fractions were then dried in vacuo.

[0062] S40. The sample peptide and the six fraction peptides were reconstituted in 0.1% (v / v) formic acid (FA) in water, and then 0.2 µL of standard peptide (iRTkit, Biognosys) was added to the peptide sample for subsequent analysis.

[0063] S50. To construct the 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.

[0064] LC-MS / MS mass spectrometry analysis: S100, prepare mobile phase A (100% water, 0.1% formic acid) and mobile phase B (80% acetonitrile, 0.1% formic acid).

[0065] Dissolve the lyophilized powder in 10 µL of S200 lyophilized liquid and centrifuge at 14,000 g for 20 min at 4°C. Take a 1 µg sample of the supernatant and inject it into the liquid chromatography-mass spectrometry (LC-MS / MS) analysis. LC elution conditions are shown in Table 2.

[0066] Table 2 Chromatographic gradient

[0067] An Orbitrap Exploris™ 480 mass spectrometer was used with an S300 and FAIMS Pro™ Interface. The compensation voltage (CV) was switched between -45 and -65 every 1 s. A Nanospray Flex™ (NSI) ion source was used. The ion spray voltage was set to 2.0 kV, the ion transfer tube temperature was set to 320°C, and the mass spectrometer was acquired in data-dependent acquisition mode. The full scan range was 350–1500 m / z. The primary mass spectrometer resolution was set to 120,000 (at 200 m / z), the AGC was 300%, and the maximum C-trap injection time was 50 ms. The secondary mass spectrometer detection adopted the "Top Speed" mode with the secondary mass spectrometer resolution set to 15,000 (at 200 m / z), the AGC was 75%, the maximum injection time was 22 ms, and the peptide fragmentation collision energy was set to 33% to generate raw mass spectrometry data.

[0068] Data processing: The database used in this study was Homo sapiens SP (number of proteins: 20,407, database: Uniprot). The search parameters for the Spectronaut software are shown in Table 3: Table 3 Retrieval parameter settings

[0069] The original data were normalized to eliminate the error caused by the experiment. The data with more than 50% null values ​​in the sample were filtered out. The KNN method was used to fill the missing value data. The T-test method was used for difference analysis. P Value 0.05, fold change 1.2 times, and the analysis results of differential proteins were obtained.

[0070] Screening and verification of differential uEV protein expression: like Figure 3 As shown in Figure 2, differential proteomic analysis of uEVs was performed on patients with different CI scores. There were 382 and 346 differentially expressed proteins in uEVs of patients with CI = 1 and CI = 2, respectively, compared with patients with CI = 0, of which 144 were differentially expressed proteins in common. Figure 4 As shown, combined with the expression of differentially expressed proteins in other fibrotic kidney diseases in the Nephromine public database and ROC analysis, SNX3, VPS4B and SMO were identified as the main differentially expressed proteins. Figure 4(a) is a heat map showing the expression patterns of multiple genes (e.g., SNX3, VPS4B, SMO, etc.) in different groups (ci = 0, ci = 1, ci = 2). Color coding indicates expression value, with red representing high expression and blue representing low expression, visually demonstrating the expression differences of genes in different groups. Figure 4 (b) 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 between different disease groups; Figure 4 (c) is a scatter plot analyzing the correlation between gene expression values ​​and log2 eGFR (glomerular filtration rate). The expression of SNX3 was significantly negatively correlated with eGFR; the expression of VPS4B was also significantly negatively correlated with eGFR, while the expression of SMO was weakly correlated with eGFR.

[0071] like Figure 5 As shown in Figure 2, the sample load was normalized using urine creatinine concentration, and the expression levels of three differentially expressed proteins in urine before and after ultracentrifugation were detected by Western blot. Figure 5 (a) Western blot detection of protein expression in Urine (urine) and EV-free Urine (urine with extracellular vesicles removed), and quantification of the relative expression levels of the above proteins through statistical charts. The results showed that the band intensity of these proteins in Urine was generally higher than that in EV-free Urine, proving that the three marker proteins SNX3, VPS4B and SMO were significantly enriched in uEVs. Figure 5 (b) Western blot analysis shows the expression levels of the three proteins in uEVs and whole urine of patients with different Banff CI scores. The results show that the content of the three marker proteins in uEVs gradually increases with the improvement of the patient's CI score, and there is no significant change in whole urine, indicating that the abundance changes of the three proteins are specific to uEVs.

[0072] like Figure 6 As shown, immunohistochemical staining confirmed the upregulation of the three markers in transplanted kidney biopsy tissue, showing that the expression of the three markers increased with increasing CI score and their renal tubular localization. This provides direct evidence for the renal origin of marker-carrying uEVs.

[0073] The expression of uEVs biomarkers in a mouse renal fibrosis model was significantly correlated with the degree of fibrosis: The present invention established unilateral ureteral ligation (UUO) and folic acid-induced (FA) renal fibrosis mouse models to further clarify the correlation between the expression levels of the three markers in fibrotic kidneys and the progression of fibrosis. Figure 7and Figure 8 As shown in the figure, the expression levels of the three biomarkers were detected by immunohistochemistry in renal tissues at D0, D3, D7, and D14 after UUO modeling and D0, D3, D14, and D28 after FA modeling. The level of collagen deposition was determined by Sirius red staining. Linear regression showed that the expression levels of the three biomarkers in mouse fibrotic kidneys were significantly positively correlated with the degree of fibrosis.

[0074] Increased expression of uEV biomarkers in renal allografts of mice with chronic rejection-induced fibrosis: The present invention established a mouse model of renal allograft fibrosis to further clarify the expression characteristics of three uEV markers in fibrotic allograft kidneys. One month after Balb / c mouse kidneys were transplanted into C57BL / 6 recipients, the transplanted kidneys were removed for histopathological analysis.

[0075] like Figure 9 As shown, H&E staining showed severe 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 graft interstitial fibrosis.

[0076] like Figure 10 As shown, a mouse model of renal allograft fibrosis induced by chronic rejection was successfully established. Figure 10 (a) shows Western blot results. The expression of candidate biomarkers in fibrotic kidney transplants was assessed in both the sham (blank) and allograft (transplant) groups. The intensity of the bands provides a preliminary indication of differential expression of different proteins between the two groups. Darker bands indicate higher protein expression. SNX3, VPS4B, and SMO were significantly upregulated in fibrotic kidney transplants. To further verify whether these markers were carried by tubular-derived EVs, immunofluorescence co-staining was used to examine their colocalization with the late endosomal marker CD63. Figure 10 (b) Bar graph of relative protein expression. SNX3, VPS4B, and SMO were primarily localized in EVs in fibrotic kidney transplants, providing biological evidence for their secretion from renal tubules into urine via EVs. Figure 10 (c) Immunofluorescence staining image showing colocalization of a fibrosis diagnostic marker and the EV marker CD63 in a mouse fibrotic kidney transplant. Scale bar = 20 μm. Fluorescence microscopy reveals the intracellular localization of the proteins. White arrows indicate areas of protein colocalization, while overlapping areas of different fluorescent colors indicate colocalization of the corresponding proteins. Figure 10(d) is the mean fluorescence intensity (MFI) bar graph, indicating that there are significant differences in the fluorescence intensity (i.e., expression levels) of these proteins between the transplantation group and the blank group.

[0077] The expression levels of SNX3, VPS4B, and SMO in uEVs can realize non-invasive diagnosis of transplanted renal fibrosis: In the present invention, morning urine was collected from 59 renal transplant patients on the day of biopsy. Urinary extracellular vesicles were purified according to the method for isolating urinary extracellular vesicles of the present invention. The expression levels of SNX3, SMO and VPS4B in uEVs of patients with different CI scores were quantified and compared by Western Blot.

[0078] like Figure 11 As shown in Figure 3, the expression of three uEVs proteins was significantly increased in patients with transplanted renal fibrosis.

[0079] like Figure 12 As shown, in order to clarify whether these three uEVs proteins can be used for non-invasive diagnosis of transplant fibrosis, the present invention constructed the protein ROC curve based on the Western Blot quantitative results, showing the diagnostic ability of uEVs proteins SNX3, VPS4B and SMO for fibrotic transplant kidney.

[0080] The present invention found that all three can effectively distinguish patients with fibrotic transplanted kidneys from patients with other types of renal injury. 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.

[0081] The expression levels of SNX3, VPS4B and SMO in uEVs can realize the non-invasive diagnosis of chronic progressive renal transplant injury: To clarify the clinical diagnostic value of candidate markers in addition to diagnosing fibrosis, the present invention divided patients into chronic progressive graft injury group (including antibody-mediated rejection, transplant glomerulopathy and mixed rejection) and other pathology groups according to the biopsy diagnosis results, such as Figure 13 As shown, receiver operating characteristic (ROC) analysis validated the diagnostic efficacy of candidate biomarkers for chronic allograft injury, demonstrating the diagnostic potential of uEV proteins SNX3, VPS4B, and SMO for chronic progressive allograft nephropathy. The ROC-AUCs for SNX3, VPS4B, and SMO were 0.7210, 0.6649, and 0.7527, respectively. These results demonstrate that the candidate biomarkers exhibit good discriminatory power in distinguishing chronic from acute allograft injury, providing a basis for early clinical intervention to delay the progression of chronic allograft nephropathy.

[0082] To further verify the diagnostic capabilities of these three uEVs proteins in clinical applications, the present invention collected morning urine from 32 renal transplant patients undergoing biopsy on the day of their biopsy and detected the content of the three proteins in uEVs by ELISA. Figure 14 As shown in the data, after normalization to urine creatinine concentration, the contents of SNX3, VPS4B, and SMO in uEVs gradually increased with the increase of the patient's CI score, and showed a good linear correlation with the collagen deposition level shown by Masson staining.

[0083] like Figure 15 As shown, based on the ELISA quantitative results, the present invention constructed the ROC curve of the protein, and 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, showing the diagnostic ability of uEVs proteins SNX3, VPS4B, and SMO for fibrotic transplanted kidney.

[0084] After separately plotting the ROC curves for SNX3, VPS4B, and SMO in uEVs of all study subjects, a logistic regression was established using these three variables. Logistic regression provides the coefficients for joint analysis, generating a joint calculation formula. Substituting the SNX3, VPS4B, and SMO values ​​of different individuals into the formula, the probability of an individual having the disease can be calculated. Based on the probabilities given by the logistic regression, the ROC curve for multi-indicator joint diagnosis can be drawn. The calculation formula for the joint diagnosis model is as follows: Logit(P)=6206.64×[SNX3]-10.06×[VPS4B]+1165.64×[SMO]-8.23 The AUC of the joint model can be improved to 0.9909 ( P <0.0001), with a cut-off value of 50%. The confusion matrix showed that the combined model achieved a sensitivity of 95.2%, a specificity of 81.8%, and a diagnostic accuracy of 90.6%, outperforming either indicator alone.

[0085] To clarify the prognostic ability of the markers, the marker levels in uEVs of renal transplant patients were detected by ELISA and divided into low-risk and high-risk groups according to the cut-off value (50%) of the combined diagnostic model, such as Figure 16 As shown in Figure 2, high-risk patients had significantly lower eGFR than low-risk patients at the time of biopsy, and the eGFR decreased significantly after three months, with all patients developing allograft failure (eGFR less than 30 mL / min / 1.73 m 2 ).

[0086] To further verify the diagnostic ability of these three uEVs proteins for renal fibrosis in CKD patients (non-transplantation), the present invention collected urine from 10 healthy volunteers and 20 CKD patients, and detected the content of the three proteins in uEVs by ELISA. Figure 17 As shown, after normalization for urine creatinine concentration, the levels of SNX3, VPS4B, and SMO in uEVs from patients with end-stage renal disease were significantly higher than those in patients without renal fibrosis (CKD stage I / II) and healthy volunteers. The expression levels of these three proteins, as determined by ELISA, were incorporated into a diagnostic model constructed using logistic regression to estimate the probability of renal fibrosis in CKD patients. A threshold of 50% was effective in distinguishing patients with renal fibrosis, demonstrating the feasibility of this method for the clinical diagnosis of renal fibrosis in noninvasive patients.

[0087] The embodiments of the present invention are described in detail above, but the contents are only preferred embodiments of the present invention and should not be considered to limit 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. Use of a reagent for detecting biomarkers in the preparation of a product for non-invasive diagnosis of renal interstitial fibrosis and chronic progressive renal injury, characterized by: The biomarkers are SNX3, VPS4B and SMO.

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

3. Use of the reagent for detecting biomarkers according to claim 2 in the preparation of a product for non-invasive diagnosis of renal interstitial fibrosis and chronic progressive renal injury, characterized in that: The method for detecting changes in the expression levels of SNX3, VPS4B, and SMO is specific for uEVs, comprising the following steps: S1. Isolation of uEVs from urine by ultracentrifugation. S2. Detect uEV marker proteins CD9, CD63, Alix, TSG101, and AQP2 by Western blot; characterize the size distribution and morphology of uEVs by DLS and transmission electron microscopy, respectively; S3. Detect the relative levels of SNX3, VPS4B, and SMO in urine before and after ultracentrifugation by Western blot to confirm that SNX3, VPS4B, and SMO are mainly present in uEVs; S4. The relative contents of SNX3, VPS4B, and SMO in uEVs and urine of renal transplant patients with different Banff CI scores were detected by Western blot to confirm that its diagnostic ability is specific for uEVs.

4. Use of the reagent for detecting biomarkers according to claim 2 in the preparation of a product for non-invasive diagnosis of renal interstitial fibrosis and chronic progressive injury, characterized in that: The method for isolating uEVs comprises 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 in a ratio of 1:4 to the urine, with final concentrations of 20 mM and 8 mM, respectively. Vortex for 90 seconds to disaggregate the uromodulin network to obtain the treated urine. S22, centrifuging the urine obtained in step S11 at 17000 g for 30 min at 4°C to remove large cell debris such as apoptotic bodies to obtain a supernatant; S33, filtering the supernatant obtained in step S22 with a needle filter to remove microvesicles with a diameter greater than 200 nm; S44. Place the supernatant after filtering in step S33 into an ultracentrifuge tube, centrifuge at 4°C, 130,000 g for 120 min, discard the supernatant, add PBS to wash the precipitate, centrifuge again at 4°C, 130,000 g for 120 min, resuspend the precipitate with PBS, and freeze at -80°C for later use.

5. Use of the reagent for detecting biomarkers according to claim 1 in the preparation of a product for non-invasive diagnosis of renal interstitial fibrosis and chronic progressive renal injury, characterized in that: The biomarker detection method comprises the following steps: S111, Isolation of uEVs from urine; S222, the expression levels of total SNX3, VPS4B, and SMO in urine were detected by Western blot or sandwich enzyme-linked immunosorbent assay; S333. Urinary creatinine concentration was detected to normalize the expression levels of SNX3, VPS4B, and SMO.

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

7. Use of the reagent for detecting biomarkers according to claim 1 in the preparation of a product for non-invasive diagnosis of renal interstitial fibrosis and chronic progressive renal injury, characterized in that: When the biomarker is used in a non-invasive diagnosis product for chronic progressive injury of transplanted kidney, the method for determining whether a subject has chronic progressive injury of 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 transplanted kidney exists; if the parameters are lower than the sample parameter threshold, it is acute injury to the transplanted kidney or non-specific injury; the sample parameters are parameters for normal content or expression level of SNX3, VPS4B and SMO proteins in specified uEVs.

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

9. Use of the reagent for detecting biomarkers according to claim 7 in the preparation of a product for non-invasive diagnosis of renal interstitial fibrosis and chronic progressive renal injury, characterized in that: When the biomarker is used in a non-invasive diagnosis product for the prognosis of transplanted kidney patients, the risk of transplanted kidney failure in the subject is predicted by detecting the content or expression level of SNX3, VPS4B and SMO proteins in the subject's uEVs.

10. Use of the reagent for detecting biomarkers according to claim 7 in the preparation of a product for non-invasive diagnosis of renal interstitial fibrosis and chronic progressive renal injury, characterized in that: When the biomarker is used for non-invasive diagnosis of renal fibrosis in non-transplanted chronic kidney disease patients, the method for determining whether the subject suffers from 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, renal fibrosis is present; 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.

Images