Use of a reagent for detecting the ratio of urinary syndecan-4 to urinary creatinine in the preparation of a product for diagnosing proteinuria

Abstract

The application provides application of a reagent for detecting a urine Syndecan-4 / urine creatinine ratio in preparation of a product for diagnosing proteinuria, and belongs to the technical field of biological detection.The application provides a biomarker for proteinuria diagnosis, and the biomarker is a urine Syndecan-4 / urine creatinine ratio.It is found by the application that the urine Syndecan-4 / urine creatinine ratio of a proteinuria patient is significantly higher than that of a healthy volunteer, so that the urine Syndecan-4 / urine creatinine ratio can be used as a biomarker for clinical diagnosis of proteinuria.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and more particularly to the application of a reagent for detecting the urine Syndecan-4 / urine creatinine ratio in the preparation of products for diagnosing proteinuria. Background Technology

[0002] Proteinuria is a core phenotype of chronic kidney disease and various glomerular diseases (such as diabetic nephropathy, hypertension-related kidney damage, and immune glomerulonephropathy). Clinically, the urinary microalbumin / creatinine ratio (ACR) is commonly used for screening and follow-up. Compared to 24-hour urine protein quantification, ACR sampling is more convenient and has higher compliance, but it is easily affected by renal hemodynamics and short-term physiological states. In reality, factors such as exercise, fever and infection, changes in body position, fluctuations in blood pressure and volume, high-protein diets, and drug treatment can all cause short-term increases or decreases in albuminuria, resulting in significant biological variability in the same patient at different time points. This weakens its ability to indicate the true extent of disease activity and structural damage, leading to biases in disease assessment, treatment evaluation, and risk stratification.

[0003] In the pathophysiology of glomerular diseases, damage to the glomerular filtration barrier (mainly composed of the endothelium / glycocalyx, glomerular basement membrane, and podocyte slit membrane structure) is a core element in the development of proteinuria. Among these, podocytes, as key cells maintaining the selectivity and structural stability of the filtration barrier, are considered closely related to the occurrence and development of proteinuria due to damage and detachment. Podocyte injury can lead to slit membrane structural disorder and weakened cell-basement membrane adhesion, further causing podocyte detachment from the basement membrane or a decrease in their number, thereby weakening the integrity of the filtration barrier and exacerbating protein leakage. Therefore, podocyte-related molecular events have the potential value of informing mechanistic aspects of structural damage to the glomerular filtration barrier.

[0004] Syndecan-4 (SDC4) is a transmembrane heparan sulfate proteoglycan involved in cell-matrix adhesion, cytoskeleton remodeling, and mechanical signal transduction. It works with adhesion complexes such as integrins to maintain stable cell-extracellular matrix connections. Under glomerular pathological stress (e.g., abnormal mechanical load, inflammatory mediators, metabolic abnormalities), cell surface proteoglycans can undergo shedding mediated by related proteases, with their extracellular domains entering body fluids in soluble form and being excreted in urine. This "membrane protein shearing-shedding" event may not only be associated with decreased adhesion capacity and cell detachment tendency but may also reflect the process of damage to local glycocalyx and adhesion structures. Therefore, the presence and elevation of soluble SDC4 in urine may serve as a mechanistic signal associated with filtration barrier structural damage, adhesion complex disruption, and membrane protein shedding, providing a supplementary assessment dimension for proteinuria-related kidney injury.

[0005] Unlike albuminuria, which tends to exhibit a phenotype of "increased permeability leading to albumin leakage," urinary soluble SDC4 is more likely to directly reflect pathological processes such as damage to the glycocalyx-adhesion complex on the surface of filtration barrier cells, shedding of membrane proteins, and activation of related proteases, possessing the potential to approach the origin of the disease at a mechanistic level. Therefore, creatinine correction of urinary SDC4 (SDC4 / Cr) can, to some extent, offset differences in urine volume dilution, improving the comparability of samples from different individuals and at different time points; and it is suitable for testing in morning urine or random urine, providing supplementary and corrective information for "true disease activity / structural damage load" in the context of physiological fluctuations or proteinuria-reducing treatment, applicable to scenarios such as screening, stratified assessment, efficacy monitoring, and prognostic judgment.

[0006] However, current routine clinical testing systems are centered on albuminuria, and there is still a relative lack of standardized, scalable in vitro diagnostic products for urine SDC4. At the same time, the wide range of protein concentrations in urine samples, the complex matrix composition, and the high requirements for batch-to-batch consistency and calibration systems mean that it is still necessary to establish a sensitive, specific, and reproducible testing protocol that is compatible with routine urine sample collection and pretreatment procedures, along with calibrators, quality control products, and a systematic kit for combined calibration with urine creatinine, to meet the needs of clinical translation and industrialization. Summary of the Invention

[0007] The purpose of this invention is to provide a reagent for detecting the urine Syndecan-4 / urine creatinine ratio in the preparation of products for diagnosing proteinuria.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a biomarker for the diagnosis of proteinuria, wherein the biomarker is the urine Syndecan-4 / urine creatinine ratio.

[0009] This invention provides the application of a reagent for detecting the urine Syndecan-4 / urine creatinine ratio in the preparation of products for diagnosing proteinuria.

[0010] The present invention provides a kit for diagnosing proteinuria, the kit comprising a reagent for detecting the ratio of urinary Syndecan-4 to urinary creatinine.

[0011] Preferably, the reagent for detecting the ratio of urinary Syndecan-4 to urinary creatinine is a reagent for detecting the content of Syndecan-4 and urinary creatinine in urine.

[0012] Preferably, the reagent used to detect the Syndecan-4 content in urine is an enzyme-linked immunosorbent assay (ELISA) reagent.

[0013] Preferably, the enzyme-linked immunosorbent assay (ELISA) reagent comprises: a capture antibody against Syndecan-4, Syndecan-4 standard, biotinylated assay reagent, streptavidin-HRP antibody, and substrate chromogenic solution.

[0014] Preferably, the reagent for detecting urinary creatinine content is the modified Jaffe creatinine detection reagent.

[0015] Preferably, the modified Jaffe creatinine detection reagent includes: an alkaline reagent, picric acid, and a creatinine standard solution.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention found that the urine Syndecan-4 / urine creatinine ratio in patients with proteinuria was significantly higher than that in healthy volunteers, indicating that the urine Syndecan-4 / urine creatinine ratio can be used as a biomarker for the clinical diagnosis of proteinuria.

[0017] This invention also found that the urine Syndecan-4 / urine creatinine ratio is significantly positively correlated with the commonly used clinical quantitative proteinuria index ACR, indicating that the urine Syndecan-4 / urine creatinine ratio can be used as a supplementary index to ACR for earlier or mechanism-related risk identification and stratified management. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 This is the standard curve of SDC4 concentration versus OD value in Example 1; Figure 2 This is the standard curve of creatinine concentration versus OD value in Example 1; Figure 3 This is a comparison of urinary SDC4 / Cr levels between proteinuria patients and healthy controls in Example 3; Figure 4 The ROC curve for diagnosing proteinuria using SDC4 / Cr in Example 3; Figure 5 The results of the full-sample correlation analysis between the SDC4 / Cr ratio and clinical proteinuria indicators in Example 3; Figure 6 The results of the intra-group correlation analysis between the SDC4 / Cr ratio and clinical proteinuria indicators in Example 3 are shown. Detailed Implementation

[0020] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0021] Example 1

[0022] Eight patients diagnosed with proteinuria and ten healthy volunteers without proteinuria from the Second Affiliated Hospital of Nanjing Medical University were selected as subjects to detect multiligand proteoglycan 4 (SDC4) and urinary creatinine in urine.

[0023] Sample collection and processing: Collect the subjects' first morning urine, mix well, centrifuge at 5000 rpm for 20 min to remove precipitate and impurities, and use the supernatant as the urine sample to be tested. Store the sample at 4℃ and test as soon as possible (for long-term storage, store at -80℃, with ≤2 freeze-thaw cycles).

[0024] 1. Quantitative detection of SDC4 in urine

[0025] To ensure that the test results fall within the linear quantification range, the urine sample to be tested can be pre-diluted in a gradient (if using a commercial ELISA kit, the dilution factor can be set according to the quantification range given in the instructions; if you are building your own system, you can build a standard curve and determine the linear interval by adding standards).

[0026] This embodiment uses a human multiligand proteoglycan 4 (SDC4) quantitative detection kit (Upin Biotechnology Co., Ltd., catalog number: SYP-H1355) for detection. The detection was performed according to the instructions, and the specific steps are as follows: A solid-phase carrier coated with an antibody targeting SDC4 (96-well high-binding ELISA plate) was used. Standards, sample diluent, and diluted urine samples (5-fold dilution) were added to the wells, each in 50 μL volume. The plates were incubated at 37°C for 60 min to allow SDC4 to bind to the capture antibody. Blank wells were also prepared without any sample. After washing the plate (5 times, 3 minutes each) to remove unbound components, biotinylated detection antibody was added to form an immune complex; blank wells were left untreated. After another wash, streptavidin-HRP antibody was added and incubated, followed by another wash; blank wells were left untreated. Substrate chromogenic buffer (TMB) was added to all wells, and stop solution was added after 15 min to terminate the reaction. The absorbance (OD) was then read at 450 nm. The OD values ​​of all wells were subtracted from the blank well values ​​to correct for background and obtain the true OD values. The results were based on a standard curve (Table 1). Figure 1 (In the figure, X value represents SDC4 concentration and Y value represents absorbance OD value) The sample concentration is calculated and multiplied by the dilution factor to obtain the original urine concentration (Table 2).

[0027] Table 1 Standard Curve of SDC4 Concentration and OD Value

[0028] Table 2. SDC4 concentration in original urine samples

[0029] 2. Urine creatinine detection - biochemical rate method

[0030] Detection principle: Under alkaline conditions, creatinine and picrate form a colored complex, and its absorbance varies around 510 nm; kinetic readings (fixed time difference) are used to reduce matrix interference.

[0031] Reagent: BioAssay Systems Creatinine Assay Kit (catalog number DICT-500), containing Reagent A / Reagent B (the two solutions are mixed to form a working solution).

[0032] Instruments: Microplate reader, 96-well plate with transparent flat bottom.

[0033] The urine sample to be tested was diluted 10 times with deionized water so that the calculated creatinine concentration fell within the linear range of the standard curve.

[0034] After preparing the working solution according to the kit instructions, add standard wells, blank wells, and sample wells to a 96-well plate. Add an equal volume of working solution to each well, mix well, and react at room temperature for 5 min. Measure the absorbance at 510 nm. Plot a standard curve with the creatinine standard concentration (mg / dL) on the x-axis and the corresponding absorbance signal value on the y-axis, and fit it using a linear regression equation. Let the standard curve equation be y = ax + b, where y is the absorbance signal value and x is the creatinine concentration. Then, the formula for calculating the creatinine concentration of the diluted sample is: x = (yb) / a; the formula for calculating the creatinine concentration of the original urine is: Coriginalurine = [(yb) / a] × 10. If the sample concentration exceeds the linear range of the standard curve, further dilute and retest, multiplying by the corresponding dilution factor.

[0035] The obtained standard curves are shown in Table 3 and Figure 2 As shown in the figure, the X value represents the creatinine concentration, and the Y value represents the absorbance (OD) value. The results of the creatinine concentration measurement in the original urine sample are shown in Table 4.

[0036] Table 3 Standard Curve of Creatinine Concentration vs. OD Value

[0037] Table 4. Urine creatinine concentration

[0038] 3. SDC4 / Cr ratio

[0039] The urinary SDC4 concentration obtained above is corrected for urinary creatinine concentration to obtain the SDC4 / Cr ratio. The SDC4 / Cr ratio can be expressed in any equivalent unit such as pg / mg or ng / mg.

[0040] The results are shown in Table 5. It can be seen that the SDC4 / Cr ratio is significantly higher in patients with proteinuria compared with healthy individuals, suggesting that SDC4 / Cr can be used as a biomarker for diagnosing proteinuria.

[0041] Table 5. Calculation results of urinary SDC4 / Cr ratio in patients with proteinuria and healthy individuals.

[0042] Example 2

[0043] The remaining 20 patients diagnosed with proteinuria at the Second Affiliated Hospital of Nanjing Medical University were tested according to the method in Example 1, and the results are shown in Table 6.

[0044] Table 6 Clinical test results

[0045] The data from Tables 5 and 6 show that the urinary SDC4 / Cr ratio in patients with proteinuria is significantly different from that in healthy individuals, further suggesting that SDC4 / Cr can serve as a biomarker for diagnosing proteinuria.

[0046] Example 3

[0047] This embodiment analyzes the correlation between SDC4 / Cr levels and proteinuria patients based on data from Embodiments 1 and 2.

[0048] First, based on the data from Examples 1 and 2, the urinary SDC4 / Cr levels in the proteinuria patient group and the healthy control group were compared. The results are as follows: Figure 3 As shown, the urinary SDC4 / Cr level was elevated in the proteinuria patient group (patient group), with a median of 27.26 pg / mg and a median of 17.50 pg / mg in the control group (normal individuals), a statistically significant difference (P=0.0296). Further ROC curves were plotted with proteinuria status as the outcome variable, and the results are as follows... Figure 4 As shown, the AUC of urinary SDC4 / Cr was 0.807, indicating that this index has good auxiliary diagnostic efficacy for proteinuria.

[0049] Subsequently, based on the data from Examples 1 and 2, the correlation between the SDC4 / Cr ratio and clinical proteinuria indicators (24-hour urinary protein) was analyzed. The results of the full-sample correlation analysis are as follows: Figure 5 As shown, the correlation analysis results within the patient groups are as follows: Figure 6 As shown in the figure. The results showed that urinary SDC4 / Cr was significantly positively correlated with urinary protein levels (Spearman r = 0.633 for the whole sample, P < 0.001; Spearman r = 0.560 for the patient group, P = 0.0019), suggesting that urinary SDC4 / Cr can be used to reflect the degree of proteinuria-related kidney injury and has stratified assessment value.

[0050] In summary, the urinary SDC4 / Cr level in patients with proteinuria was higher than that in healthy controls and was positively correlated with the urinary protein level. This suggests that this indicator can be used to develop products for the auxiliary diagnosis of proteinuria and the auxiliary assessment of the degree of proteinuria-related kidney damage, and can be further used for screening and stratified assessment of subjects.

[0051] It is important to emphasize that urinary protein mainly reflects the phenotype of "increased permeability" of the filtration barrier; while SDC4 originates from the shedding of glycocalyx-adhesion complexes on the surface of glomerular filtration barrier-related cells, which is closer to pathological processes such as cell structure damage and protease activation. Therefore, it can be used as a supplementary indicator of ACR for earlier or mechanism-related risk identification and stratified management.

[0052] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A biomarker for the diagnosis of proteinuria, characterized in that, The biomarker is the urine Syndecan-4 / urine creatinine ratio.

2. The use of a reagent for detecting the urine Syndecan-4 / urine creatinine ratio in the preparation of products for diagnosing proteinuria.

3. A kit for diagnosing proteinuria, characterized in that, The kit includes reagents for detecting the ratio of urine Syndecan-4 to urine creatinine.

4. The reagent kit according to claim 3, characterized in that, The reagent used to detect the ratio of urinary Syndecan-4 to urinary creatinine is a reagent used to detect the content of Syndecan-4 and urinary creatinine in urine.

5. The reagent kit according to claim 4, characterized in that, The reagent used to detect the Syndecan-4 content in urine is an enzyme-linked immunosorbent assay (ELISA) reagent.

6. The reagent kit according to claim 5, characterized in that, The enzyme-linked immunosorbent assay (ELISA) reagent includes: a capture antibody against Syndecan-4, Syndecan-4 standard, biotinylated assay reagent, streptavidin-HRP antibody, and substrate chromogenic solution.

7. The reagent kit according to claim 4, characterized in that, The reagent used to detect urinary creatinine levels is a modified Jaffe creatinine assay reagent.

8. The reagent kit according to claim 7, characterized in that, The modified Jaffe creatinine detection reagent includes: an alkaline reagent, picric acid, and a creatinine standard solution.

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