Biomarker, application and system for detecting renal fibrosis

Abstract

The invention discloses a biomarker for detecting renal fibrosis, application and a system, and relates to the technical field of biological medicines. The invention relates to a biomarker for detecting renal fibrosis. The biomarker comprises urinary copper cyanin. Compared with the conventional diagnostic marker (such as eGFR, RBP, NAG and the like), the urocyanin has the following three advantages: 1, compared with the conventional diagnostic marker (such as eGFR, RBP, NAG and the like), the urocyanin is more sensitive to diagnosis of renal interstitial fibrosis and fibrosis severity; secondly, compared with traumatic renal tissue pathologic biopsy, urine is used as a diagnosis medium, so that true noninvasive effect can be realized, and no harm is caused to the body health of a patient; and thirdly, compared with detection means such as iconography, the method for detecting the urinary cupreocyanin marker is lower in cost, more flexible, high in timeliness and easier to popularize.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a biomarker, application, and system for detecting renal fibrosis. Background Technology

[0002] Chronic kidney disease (CKD) is characterized by high prevalence, poor prognosis, and high medical costs. More seriously, without effective prevention and treatment, when CKD progresses to end-stage renal disease (ESRD), patients can only rely on renal replacement therapy such as dialysis or kidney transplantation to sustain life. However, most kidney diseases have an insidious onset, lacking early warning, non-invasive diagnostic, and precise classification and staging methods, resulting in high incidence, low awareness, delayed medical treatment, and poor treatment outcomes. Therefore, early diagnosis is of significant scientific and clinical value for identifying susceptible populations for CKD, dynamically assessing CKD progression, and enabling timely treatment intervention to reduce the incidence of ESRD and improve prognosis.

[0003] Renal fibrosis is the pathological basis for the progression of CKD to uremia. Currently, the diagnosis and assessment of renal interstitial fibrosis still rely on renal biopsy. Renal tissue biopsy is currently the "gold standard" for diagnosing renal fibrosis, but it is an invasive diagnostic method that cannot be performed frequently or repeatedly. Furthermore, renal fibrosis is a progressive disease with constantly changing conditions, and the biopsy process may cause complications such as bleeding, infection, hematuria, and perirenal hematoma, posing potential safety risks. Due to the invasive nature of renal biopsy, it is difficult to repeat the procedure multiple times, making continuous dynamic monitoring of the progression of renal fibrosis challenging.

[0004] Therefore, there is an urgent need for non-invasive biomarkers that can reflect changes in the progression of renal fibrosis. Currently used clinical indicators for assessing renal fibrosis, such as proteinuria and glomerular filtration rate (GFR), do not show significant changes in the early stages of renal fibrosis. Because the kidneys have a strong compensatory function, these indicators only begin to rise when the glomerular filtration rate drops to a certain level. By this time, renal fibrosis may have already progressed to a certain stage, hindering early diagnosis and intervention. Furthermore, they cannot determine the extent of kidney disease, thus limiting their value in guiding clinical treatment.

[0005] The inventors initially studied serum ceruloplasmin as a non-invasive biomarker for changes in renal fibrosis and applied for corresponding patents. However, as their research progressed, they discovered that serum ceruloplasmin can be secreted by multiple organs, and its levels are easily affected by factors such as infection, trauma, coronary heart disease, and pregnancy. This leads to biases in the sensitivity and specificity of renal fibrosis diagnosis, limiting and impacting its actual clinical application.

[0006] Therefore, there is still an urgent need to find biomarkers that are suitable for clinical application and can effectively diagnose early renal fibrosis in the current technology. Summary of the Invention

[0007] To address the technical problems existing in the prior art, embodiments of the present invention provide a biomarker, application, and system for detecting renal fibrosis. The technical solution is as follows:

[0008] A biomarker for detecting renal fibrosis, the biomarker comprising: urinary ceruloplasmin.

[0009] Optionally, the renal fibrosis is renal interstitial fibrosis.

[0010] Application of reagents for detecting urinary ceruloplasmin in the preparation of products for the early diagnosis, risk assessment, prognosis prediction and / or treatment selection of renal fibrosis.

[0011] Optionally, the renal fibrosis is renal interstitial fibrosis.

[0012] Optionally, the early diagnosis, risk assessment, prognostic prediction, and / or treatment selection for renal fibrosis include:

[0013] The urinary ceruloplasmin level in the sample is measured and compared with a reference value to enable early diagnosis, risk assessment, prognosis prediction, and / or treatment selection for renal fibrosis.

[0014] A system for early diagnosis, risk assessment, prognostic prediction, and / or treatment selection of renal fibrosis, the system comprising:

[0015] (1) A first device for collecting and / or receiving data on the level of urinary ceruloplasmin in a sample;

[0016] (2) A second device for analyzing the data to perform early diagnosis, risk assessment, prognosis prediction and / or treatment selection for renal fibrosis;

[0017] The first device includes: reagents, kits and / or detection devices for detecting the level of urinary ceruloplasmin in samples from the subject;

[0018] The analysis includes comparing the urinary ceruloplasmin level in the sample with a reference value.

[0019] Optionally, the sample is derived from a urine sample of the subject.

[0020] Optionally, the renal fibrosis is renal interstitial fibrosis.

[0021] The beneficial effects of the technical solutions provided in the embodiments of the present invention include at least the following:

[0022] This invention provides the application of urinary ceruloplasmin as a non-invasive diagnostic biomarker for renal fibrosis. The inventors have discovered that urinary ceruloplasmin offers three major advantages compared to other methods for diagnosing renal fibrosis: First, compared to conventional diagnostic markers (such as eGFR, RBP, NAG, etc.), urinary ceruloplasmin is more sensitive in diagnosing the presence and severity of renal interstitial fibrosis; second, compared to invasive renal tissue biopsy, using urine as the diagnostic medium is truly non-invasive and poses no harm to the patient's health; third, compared to imaging and other detection methods, the method for detecting urinary ceruloplasmin is lower in cost, more flexible, more timely, and easier to popularize.

[0023] Compared to serum ceruloplasmin, urinary ceruloplasmin, as a non-invasive diagnostic marker for renal fibrosis, has significant advantages in clinical application value, diagnostic specificity, convenience of detection, and ability to monitor disease dynamics: ① It directly reflects local renal lesions, avoiding systemic interference. Serum ceruloplasmin is mainly synthesized by the liver and widely distributed in the circulatory system. Its level changes are affected by various non-renal factors such as liver function (e.g., hepatitis, cirrhosis), systemic inflammatory responses (e.g., infection, autoimmune diseases), and nutritional status (e.g., diseases related to copper metabolism abnormalities), which can easily lead to false positive or false negative results. For example, when serum ceruloplasmin levels are abnormally elevated in patients with liver disease, it may mask early signs of renal fibrosis or mistakenly identify fluctuations in serum indicators caused by non-renal factors as renal lesions, making it difficult to accurately pinpoint the local fibrotic process in the kidney. Urinary ceruloplasmin has a kidney-specific origin. During renal fibrosis, damaged kidney cells abnormally synthesize ceruloplasmin and secrete it into the urine. Its level changes are directly related to the pathological degree of local renal fibrosis (such as the extent of collagen deposition and the degree of renal tubular atrophy), and are not affected by external factors such as liver function or systemic metabolic status. This allows for a more accurate reflection of internal renal fibrosis and significantly improves diagnostic specificity. Secondly, the detection method is truly non-invasive, improving patient compliance and making clinical application more convenient for dynamic monitoring. Serum ceruloplasmin testing requires venous blood collection, which is an invasive procedure with risks of local infection, bleeding, and pain. For chronic kidney disease patients requiring long-term dynamic monitoring, repeated blood draws can easily cause patient resistance, reducing compliance with follow-up monitoring and making it difficult to continuously track the progression of renal fibrosis. Urinary ceruloplasmin testing only requires collecting a midstream urine sample, is entirely non-invasive, simple to operate, and has no invasive risks. Patient acceptance is high, making it particularly suitable for elderly patients, children, and those requiring long-term follow-up for chronic diseases. Meanwhile, urine sample collection does not require professional medical personnel; patients can easily complete sample collection at home or in an outpatient clinic, greatly reducing the time cost and operational threshold of testing, and providing feasibility for large-scale screening and long-term dynamic monitoring. ③ Earlier detection of early renal fibrosis signals improves the timeliness of diagnosis. The pathological process of renal fibrosis is "insidious." In the early stages, the structural and functional damage to the kidneys is relatively mild, and serological indicators (including serum ceruloplasmin) often show no significant changes due to renal compensatory mechanisms or systemic regulatory effects, making it difficult to detect in the early stages of the disease. As a result, most patients are diagnosed when the disease has progressed to the middle or late stages of fibrosis, missing the best intervention opportunity.As a direct product of kidney metabolism, changes in urine composition can reflect early damage to the kidney's microstructure in real time. When renal fibrosis is in its early stages (such as mild inflammation of the renal tubules and interstitium with a small amount of collagen deposition), the secretion of ceruloplasmin by the intrinsic cells of the kidney has become abnormal, and this abnormality will be rapidly excreted through urine. This causes a significant increase in urinary ceruloplasmin levels even when serum levels are still within the normal range. Urinary ceruloplasmin can detect early signs of renal fibrosis earlier than serum ceruloplasmin, thus gaining a critical time window for early clinical intervention and delaying disease progression. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a graph showing the correlation between ceruloplasmin in mouse kidneys and the area of ​​fibrosis in the uIRI model provided in Example 1 of this invention.

[0026] Figure 2 This is a correlation diagram of ceruloplasmin in mouse kidneys and fibrosis area in the AAN model provided in Example 1 of this invention;

[0027] Figure 3 This is a graph showing the urinary ceruloplasmin level in the uIRI model provided in Embodiment 1 of the present invention;

[0028] Figure 4 This is a graph showing the urinary ceruloplasmin level in the AAN model provided in Embodiment 1 of the present invention;

[0029] Figure 5 This is a graph showing the urinary ceruloplasmin levels in different renal fibrosis groups of CKD patients, as provided in Example 2 of this invention.

[0030] Figure 6 This is a graph showing the correlation analysis between urinary ceruloplasmin and renal fibrosis area in CKD patients provided in Embodiment 2 of the present invention;

[0031] Figure 7 This is the ROC curve diagram of urinary ceruloplasmin and other indicators in CKD patients for diagnosing mild to moderate-severe renal interstitial fibrosis, provided in Embodiment 2 of the present invention. Detailed Implementation

[0032] The technical solution of the present invention will now be described with reference to the accompanying drawings.

[0033] Ceruloplasmin (CP), also known as copper oxidase, is a copper-containing α2 glycoprotein with a molecular weight of approximately 120,000-160,000. It is a single-chain polypeptide containing 6-7 copper atoms per molecule. Its blue color is due to the copper content. It contains about 10% sugar, with sialic acid attached to the terminal polypeptide chain. It exhibits genetic polymorphism. Its functions include regulating the distribution of copper in various parts of the body, synthesizing copper-containing enzyme proteins, acting as an antioxidant, and possessing oxidase activity, catalyzing the oxidation of polyphenols and polyamines. Ceruloplasmin is generally believed to be synthesized by the liver, with a portion excreted via the bile duct, and present in trace amounts in urine.

[0034] To overcome the limitations of existing non-invasive diagnostic techniques for renal fibrosis, the present invention aims to provide a molecular marker that can be used for the non-invasive diagnosis of renal fibrosis.

[0035] The kidneys are the organs that excrete urine, and changes in urine composition are considered a "window" into kidney disease. Urine is rich in biological information, including proteins, extracellular vesicles, and nucleic acids, which can directly indicate glomerular or tubular damage. Furthermore, urine collection is simple, non-invasive, and repeatable, with a daily urine volume generally at least 500 ml, making it a prime source for biomarker research. Therefore, analyzing changes in urine composition can provide an effective humoral approach for finding non-invasive diagnostic biomarkers reflecting the occurrence and progression of renal fibrosis. In addition, repairing damaged renal tubular epithelial cells is a key factor driving the initiation and progression of fibrosis, directly or indirectly participating in the fibrotic process by producing various pro-inflammatory and pro-fibrotic cytokines and signals. Therefore, identifying biomarkers derived from repairing damaged renal tubular epithelial cells is an important approach for the non-invasive diagnosis of renal fibrosis.

[0036] Based on previous research on serum ceruloplasmin as a non-invasive biomarker for changes in renal fibrosis, the inventors have further creatively proposed the concept of urinary ceruloplasmin as a non-invasive biomarker for changes in renal fibrosis.

[0037] However, in current clinical diagnostic techniques, there are significant barriers to the conversion between urinary biomarkers (biomarkers mainly detected in urine samples) and blood biomarkers (biomarkers detected in blood samples). The core reason lies in the fundamental differences in their biological origins, environments, and metabolic mechanisms. From the perspective of the inherent properties of the samples, blood, as the core fluid environment of the body's systemic circulation, plays a crucial role in substance transport, signal transduction, and homeostasis maintenance. Its composition is synergistically regulated by multiple systemic factors. On the one hand, the levels of biomarkers in blood are influenced by multiple factors, including systemic metabolic status (such as glucose and lipid metabolism, amino acid metabolism), multi-organ function (liver synthesis, kidney excretion, spleen clearance), blood dilution effects (such as water intake, vascular osmotic pressure), and systemic regulatory mechanisms (neuro-humoral regulation). These systemic factors collectively lead to blood biomarker levels reflecting more the overall physiological and pathological state of the body than local lesions in a single organ. In contrast, urine is the final metabolic product formed after blood is filtered by the kidneys, reabsorbed by the renal tubules, and secreted by the collecting ducts. Its composition exhibits strict local selective regulation by the kidneys. The presence and concentration of biomarkers in urine are not simply determined by their original concentration in the blood, but rather by a series of screening processes inherent to kidney function. These processes include the permeability of the glomerular filtration barrier, the reabsorption efficiency of the renal tubules, the secretory function of the renal tubular epithelial cells, and local renal factors such as the physicochemical environment of urine (pH, osmotic pressure, protease activity). This means that changes in urine biomarkers directly indicate local structural and functional abnormalities in the kidneys, rather than the systemic state. Furthermore, the clinical significance of biomarkers in blood and urine differs fundamentally: blood biomarkers often reflect "systemic pathological states" (e.g., elevated inflammatory factors indicate systemic inflammation, and abnormal liver function indicators reflect liver damage), while urine biomarkers focus more on "local renal lesions and excretory function" (e.g., urinary protein reflects glomerular barrier damage, and elevated urinary enzymes indicate renal tubular damage). This difference in significance leads to completely different clinical interpretation logics for blood biomarkers related to kidney disease (e.g., serum creatinine) and their urinary counterparts (urinary creatinine) (the former reflects renal function, while the latter is often used as a urine dilution correction indicator). Therefore, not all blood biomarkers are applicable to urine. The conversion from blood biomarkers to urine biomarkers requires independent verification and cannot be directly extrapolated by those skilled in the art.

[0038] Therefore, the inventors further conducted screening and verification experiments to determine whether urinary ceruloplasmin could serve as a non-invasive biomarker for changes in renal fibrosis.

[0039] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0040] Example 1: Animal Research Section

[0041] 1.1 Clarify the correlation between ceruloplasmin in mouse kidneys and the area of ​​fibrosis.

[0042] Two renal fibrosis models, unilateral renal ischemia-reperfusion injury (uIRI) and aristolochic acid nephropathy (AAN), were constructed using male C57 mice (purchased from Spiford Biotechnology Co., Ltd.) to simulate the most common clinical renal interstitial fibrosis caused by ischemia or drug toxicity.

[0043] The steps for constructing a unilateral renal ischemia-reperfusion injury (uIRI) renal fibrosis model are as follows: Mice are anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution at a dose of 30-40 mg / kg. After anesthesia, the back is shaved and disinfected with iodine. A longitudinal incision of approximately 1.5-2 cm is made along the left costal margin, and the left kidney is bluntly dissected, carefully freeing the renal pedicle. The left renal pedicle is clamped with atraumatic micro-arterial clamps for 28 minutes, during which time the animal is placed on a 37°C constant-temperature plate. After the clamping time is completed, the arterial clamps are quickly removed. The left kidney changes from purplish-black to bright red within a short time, indicating successful reperfusion. The kidney and other abdominal organs are repositioned, and the abdominal cavity is closed by suturing layer by layer.

[0044] The steps for constructing an aristolochic acid-induced nephropathy (AAN) model of renal fibrosis are as follows: Eight-week-old mice were given a single intraperitoneal injection of aristolochic acid at a dose of 5 mg / kg.

[0045] Urine and kidney tissue were collected at various time points in the model (1 day, 3 days, 7 days, 14 days, and 28 days). The kidneys were fixed, dehydrated, embedded, and sectioned for subsequent experiments.

[0046] The above-mentioned mouse kidney tissue was subjected to fibrosis staining (Masson staining). The specific steps are as follows:

[0047] ① Dewaxing of paraffin sections: Immerse the sections in xylene, anhydrous ethanol, and 95% ethanol in sequence, and finally rinse with running water.

[0048] ② Hematoxylin staining of nuclei: Immerse in hematoxylin staining solution and incubate at room temperature for 5 minutes. Then remove non-specific staining of the cytoplasm with hydrochloric acid-ethanol, and neutralize the acidic differentiation solution with ammonia water to enhance the stability of the blue staining in the nucleus.

[0049] ③ Ponceau Sin-Fuchsin staining: Incubate in Ponceau Sin-Fuchsin staining solution at room temperature for 10 minutes to ensure that the cytoplasm of renal tubular epithelial cells and the myofibrous fibers of the glomerular mesangial area are fully stained and appear bright red.

[0050] ④ Differentiation with phosphomolybdic acid solution: Incubate in 1% phosphomolybdic acid solution at room temperature for about 10 minutes until the bright red color of the renal tubular epithelial cells fades and the glomerular structure becomes clear.

[0051] ⑤ Aniline blue staining of collagen fibers: Incubate in aniline blue staining solution for 5-20 minutes until the collagen fibers in the fibrotic area of ​​the mouse kidney turn dark blue, while the non-fibrotic area remains largely unstained.

[0052] This method allows the fibrotic areas of the kidney to be colored blue, and the proportion of the fibrotic area to the total area of ​​the kidney (i.e., the proportion of the blue positive area) can be calculated.

[0053] The expression level of ceruloplasmin in the kidney was further detected by immunohistochemistry. The steps of immunohistochemistry are as follows:

[0054] ① Dewaxing to water: Immerse the slices in xylene, anhydrous ethanol, and 95% ethanol in sequence, and finally rinse with running water.

[0055] ② Antigen retrieval: Place the slides in citrate retrieval solution (pH 6.0), heat in a pressure cooker until the water boils and continue for 15 minutes, then cool naturally to room temperature, and then wash with PBS 3 times, 5 minutes each time.

[0056] ③ Endogenous peroxidase blockade: Add 3% hydrogen peroxide (H2O2) solution to the tissue section and incubate for 10 minutes; wash 3 times with PBS buffer for 5 minutes each time to completely remove H2O2.

[0057] ④ Blocking: Add 10% sheep serum to the sample and incubate at room temperature for 1 hour to block non-specific binding sites.

[0058] ⑤ Primary antibody incubation: Remove the blocking solution, add 30 μL of diluted ceruloplasmin primary antibody (purchased from Abclonal) to each tissue spot, place in a humidified chamber, and incubate overnight at 4°C. Then wash the samples three times with PBS for 5 minutes each time to remove unbound primary antibody.

[0059] ⑥ Secondary antibody incubation: Add an appropriate amount of horseradish peroxidase-labeled secondary antibody (purchased from Beyotime Biotechnology Co., Ltd.) and incubate at 37°C for 1 hour. Then wash the sample three times with PBS for 5 minutes each time to remove unbound secondary antibody.

[0060] ⑦ DAB staining: Add DAB staining solution to the tissue section and observe the staining process in real time under a microscope at room temperature. When the positive signal is clear and the background is not obviously stained, immediately wash twice with distilled water for 3 minutes each time to terminate the staining reaction.

[0061] ⑧ Hematoxylin staining of nuclei, hydrochloric acid and ethanol differentiation, and ammonia water to turn blue.

[0062] ⑨ Dehydration, clearing and sealing.

[0063] Areas positive for ceruloplasmin are brown; the area of ​​these areas was calculated. Correlation analysis was used to evaluate the correlation between ceruloplasmin expression in mouse kidneys and the area of ​​fibrosis.

[0064] Experimental results:

[0065] Table 1. Area of ​​ceruloplasmin-positive region and renal fibrosis area in the kidneys of the uIRI model.

[0066]

[0067] Table 2. Immunization and renal fibrosis area of ​​ceruloplasmin-positive areas in the kidneys of AAN models.

[0068]

[0069] See the experimental results. Figures 1 to 2 .from Figure 1 As can be seen from the results, in the uIRI model, as the degree of fibrosis increases and the fibrosis area increases, the area of ​​ceruloplasmin-positive area in the kidney also gradually increases. The two are significantly correlated, with a correlation coefficient of 0.8625 and a P value of <0.001.

[0070] from Figure 2 As can be seen from the AAN model, as the area of ​​fibrosis increases, the area of ​​ceruloplasmin-positive area in the kidney also gradually increases, and the two are significantly correlated, with a correlation coefficient of 0.775 and a P value of <0.001.

[0071] 1.2 Urinary ceruloplasmin detection

[0072] The content of ceruloplasmin in the urine of fibrotic model mice was detected using a commercially available ceruloplasmin activity assay kit (purchased from Solarbio Science & Technology Co., Ltd.), and the urinary creatinine (Cr) level was also measured (creatinine kit purchased from Nanjing Jiancheng Biotechnology Co., Ltd.). The ratio of these two levels was used to eliminate the interference of urine concentration or dilution on the urinary ceruloplasmin detection results, thus more accurately reflecting the true level of ceruloplasmin excretion by the mouse kidneys. Specific detection procedures are detailed in the kit instructions.

[0073] Further quantitative analysis of collagen in Masson staining of fibrotic mouse kidneys was performed, following the experimental procedures outlined in 1.1. The correlation between urinary ceruloplasmin and renal fibrosis area was analyzed using the Speraman method.

[0074] Experimental results:

[0075] See the experimental results. Figures 3 to 4 .from Figure 3 and Figure 4 It can be seen that in both unilateral renal ischemia-reperfusion injury (uIRI) and aristolochic acid nephropathy (AAN) renal fibrosis models, the level of ceruloplasmin in mouse urine gradually increases with the extension of fibrosis model time.

[0076] The correlation analysis between urinary ceruloplasmin and renal fibrosis area showed that the level of urinary ceruloplasmin and fibrosis area were significantly correlated in the model, with correlation coefficients of 0.8779 and 0.6229, respectively, and P value < 0.01.

[0077] Table 3. Experimental results of urinary ceruloplasmin activity and renal fibrosis area in the uIRI model.

[0078]

[0079] Table 4. Experimental results of urinary ceruloplasmin activity and renal fibrosis area in the AAN model.

[0080]

[0081] The results of experiments 1.1-1.2 above indicate that during the process of renal fibrosis, the kidneys produce large amounts of ceruloplasmin, which is secreted into the urine. The levels of ceruloplasmin derived from the kidneys and in urine are positively correlated with the area of ​​renal fibrosis, suggesting that urinary ceruloplasmin may serve as a non-invasive diagnostic biomarker for renal fibrosis.

[0082] Example 2: Clinical Research Section

[0083] 2.1 This study included 220 CKD patients who underwent renal biopsy due to their condition between January and June 2025. Thirty healthy volunteers were also included. Inclusion criteria were: renal biopsy required due to the patient's condition; eGFR < 90 ml / minper 1.73 m 2 Exclusion criteria: urine protein greater than 3.5g / 24h; accompanied by massive hematuria; unable to provide a urine sample; failure to sign an informed consent form.

[0084] Clinical information of 220 CKD patients is as follows:

[0085] Table 5 Clinical information of 220 CKD patients

[0086]

[0087] 2.2 The levels of ceruloplasmin in the urine of healthy controls and patients with nephropathy were detected using a commercially available ceruloplasmin kit (Human CP (Ceruloplasmin) ELISA Kit) (purchased from eLabscience). Urinary creatinine (Cr) levels were also measured (creatinine kit purchased from Chengdu Boshitai Biotechnology Co., Ltd.). The ratio of these two levels was used to eliminate the interference of urine concentration or dilution on the urinary ceruloplasmin detection results, thus more accurately reflecting the true level of ceruloplasmin excretion by the kidneys of CKD patients. The unit for urinary ceruloplasmin is mg / g Cr. Patients were further grouped according to the severity of renal interstitial fibrosis (fibrosis area <25%; 25%≤fibrosis area≤50%; 50%<fibrosis area).

[0088] Experimental results:

[0089] Table 6. Urinary ceruloplasmin levels in healthy controls and at different degrees of renal fibrosis (IFTA)

[0090]

[0091] See the experimental results. Figures 5 to 6 .

[0092] from Figure 5 As can be seen, the level of ceruloplasmin in urine gradually increases with the aggravation of renal interstitial fibrosis.

[0093] from Figure 6 As can be seen from the correlation study results, the urinary ceruloplasmin level is significantly positively correlated with the area of ​​renal interstitial fibrosis, with a correlation coefficient R=0.8 and P<0.001, indicating that urinary ceruloplasmin can well reflect the severity of renal fibrosis.

[0094] 2.3 To verify the diagnostic value of urinary ceruloplasmin for renal interstitial fibrosis, ROC curves were plotted for 220 CKD subjects, and AUC values ​​were calculated. The method for plotting the ROC curves for urinary ceruloplasmin in mild and severe renal interstitial fibrosis is as follows:

[0095] ① Determine the variable type: Clarify that "urinary ceruloplasmin" is the test indicator variable (continuous variable) and "degree of renal interstitial fibrosis" is the outcome variable (binary variable, "mild" and "severe" need to be assigned values ​​first, such as "mild = 0, severe = 1").

[0096] ② The plotting was performed using the R language (pROC package). The core principle is to iterate through all possible cutoff values ​​for urinary ceruloplasmin, calculate the sensitivity (true positive rate, TPR) and 1-specificity (false positive rate, FPR) for each cutoff value, and plot a curve with FPR on the horizontal axis and TPR on the vertical axis. The area under the curve (AUC) is used to quantify the diagnostic efficacy of urinary ceruloplasmin for mild and severe renal interstitial fibrosis (the closer the AUC is to 1, the stronger the diagnostic efficacy).

[0097] Experimental results:

[0098] The experimental results are shown in Table 7 and Figure 7 .

[0099] Table 7 Area under the ROC curve

[0100]

[0101] like Figure 7 As shown, the AUC value of urinary ceruloplasmin reached 0.938 in the diagnosis of mild and severe renal interstitial fibrosis, which is significantly higher than the diagnostic value of traditional fibrosis indicators and also higher than the diagnostic value of serum ceruloplasmin, demonstrating good test efficacy.

[0102] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A biomarker for detecting renal fibrosis, characterized in that, The biomarkers include: urinary ceruloplasmin.

2. The biomarker according to claim 1, characterized in that, The renal fibrosis is renal interstitial fibrosis.

3. Application of reagents for detecting urinary ceruloplasmin in the preparation of products for the early diagnosis, risk assessment, prognosis prediction and / or treatment selection of renal fibrosis.

4. The application according to claim 3, characterized in that, The renal fibrosis is renal interstitial fibrosis.

5. The application according to claim 3, characterized in that, The early diagnosis, risk assessment, prognostic prediction, and / or treatment selection for renal fibrosis include: The urinary ceruloplasmin level in the sample is measured and compared with a reference value to enable early diagnosis, risk assessment, prognosis prediction, and / or treatment selection for renal fibrosis.

6. A system for early diagnosis, risk assessment, prognostic prediction, and / or treatment selection of renal fibrosis, characterized in that, The system includes: (1) A first device for collecting and / or receiving data on the level of urinary ceruloplasmin in a sample; (2) A second device for analyzing the data to perform early diagnosis, risk assessment, prognosis prediction and / or treatment selection for renal fibrosis; The first device includes: reagents, kits and / or detection devices for detecting the level of urinary ceruloplasmin in samples from the subject; The analysis includes comparing the urinary ceruloplasmin level in the sample with a reference value.

7. The system according to claim 6, characterized in that, The sample was obtained from the subject's urine sample.

8. The system according to claim 6, characterized in that, The renal fibrosis is renal interstitial fibrosis.

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