Biomarker for detecting kidney fibrosis related diseases and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly discloses a biomarker for detecting kidney fibrosis related diseases and application of the biomarker. The biomarker comprises serum copper cyanin, or the serum copper cyanin and serum creatinine. Compared with other methods for diagnosing renal fibrosis, the serum cupreuloplasmin has the following three advantages: 1, compared with a conventional diagnostic marker (such as serum creatinine), the cupreuloplasmin can detect up-regulation in the serum of a patient suffering from early chronic kidney diseases, and can be combined with an existing mature diagnostic marker for detection; secondly, compared with renal tissue pathologic biopsy, the use of the copper cyanin as the diagnostic marker has non-invasiveness, and only a small amount or even a trace amount of patient serum is needed, so that the harm to the body health of the patient is avoided; and thirdly, compared with a nuclear magnetic resonance method for analyzing the metabolic components of the urine, the method for detecting the markers in the serum is lower in cost, more flexible, high in timeliness and easier to popularize.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a biomarker for detecting diseases related to renal fibrosis and an application thereof. Background Art

[0002] China has the largest number of chronic kidney disease (CKD) and uremia patients in the world, posing a serious threat to national health.

[0003] Renal fibrosis is the pathological basis for the progression of CKD to uremia. Multiple cells, including tubular epithelial cells, interstitial cells, endothelial cells, and inflammatory cells, are involved in tubular-interstitial fibrosis, and the interaction between them initiates the occurrence and progression of renal fibrosis. Among these cells, damaged tubular epithelial cells are considered to be one of the key mechanisms driving renal fibrosis, because the renal tubules are the main components of the kidneys and are susceptible to damage by various factors such as hypoxia, proteinuria, toxins, and metabolic disorders. In general, the renal tubules have a strong regenerative capacity and can be basically completely repaired when the damage is mild; however, when the damage is severe or the continuous damage exceeds the repair capacity, the renal tubules will atrophy, the tubular epithelium will flatten, and various cytokines and other signals will be produced, which will eventually participate in fibrosis or indirectly promote interstitial fibrosis.

[0004] At present, there are many difficulties in the clinical diagnosis of renal fibrosis. ① Renal tissue pathological biopsy is currently the "gold standard" for diagnosing renal fibrosis, but it is an invasive diagnostic method. The puncture process may cause complications such as bleeding, infection, hematuria, and perinephric hematoma, bringing potential safety risks. And because of the invasiveness of renal biopsy, it is difficult to repeat it many times, and it is impossible to continuously and dynamically monitor the progression of renal fibrosis. ② Commonly used renal function indicators such as serum creatinine and urea nitrogen do not change significantly in the early stages of renal fibrosis. Because the kidneys have a strong compensatory function, these indicators will only begin to increase when the glomerular filtration rate drops to a certain level. At this time, renal fibrosis may have progressed to a certain stage, which is not conducive to early diagnosis and intervention. ③ Due to limited resolution and low specificity, imaging examinations are also difficult to accurately identify fibrotic lesions.

[0005] Therefore, finding markers that can be used clinically and effectively diagnose early renal fibrosis is of great significance for the timely detection and treatment of the disease. Summary of the invention

[0006] In order to make up for the bottleneck of existing non-invasive diagnosis technology of renal fibrosis, the purpose of the present invention is to provide a molecular marker that can be used for non-invasive diagnosis of renal fibrosis. Compared with traditional diagnostic methods, the use of serum ceruloplasmin (serum CP) as a non-invasive diagnostic biomarker for renal fibrosis has a higher sensitivity than blood creatinine and can distinguish acute kidney injury from chronic fibrosis, so that patients can know the risk of the disease in the early stage of the disease and take corresponding prevention and treatment measures according to the risk level.

[0007] The purpose of the present invention is to provide the application of serum CP as a non-invasive diagnostic biomarker for renal fibrosis, which confirms that CP is significantly positively correlated with renal fibrosis. CP can be used as a non-invasive diagnostic biomarker for renal fibrosis and as a differential diagnostic marker for acute kidney injury and chronic fibrosis, and has higher sensitivity than blood creatinine.

[0008] To solve the above problems, the present invention adopts the following technical solutions:

[0009] A biomarker for detecting renal fibrosis-related diseases, comprising serum ceruloplasmin, or serum ceruloplasmin and blood creatinine.

[0010] Optionally, the renal fibrosis-related disease is renal interstitial fibrosis and / or chronic tubulointerstitial damage.

[0011] Use of serum ceruloplasmin as a biomarker in the preparation of a product for early diagnosis, risk assessment, prognosis prediction and / or treatment regimen selection of a subject's renal fibrosis-related disease.

[0012] Optionally, the early diagnosis, risk assessment, prognosis prediction and / or treatment plan selection of the subject's renal fibrosis-related disease includes:

[0013] Determining the serum ceruloplasmin level in a sample from the subject, and comparing the serum ceruloplasmin level in the sample with a reference value, so as to perform early diagnosis, risk assessment, prognosis prediction and / or selection of a treatment regimen for the subject; or

[0014] The serum ceruloplasmin level and the blood creatinine level in the sample from the subject are measured, and the serum ceruloplasmin level×the blood creatinine level in the sample are compared with the reference value to perform early diagnosis, risk assessment, prognosis prediction and / or treatment regimen selection for the subject.

[0015] Optionally, the renal fibrosis-related disease is renal interstitial fibrosis and / or chronic tubulointerstitial damage.

[0016] A product for early diagnosis, risk assessment, prognosis prediction and / or treatment plan selection of a subject with renal fibrosis-related diseases,

[0017] The product comprises a reagent, a kit and / or a detection device for detecting the level of serum ceruloplasmin in a sample from a subject, or

[0018] The product comprises a reagent, a kit and / or a detection device for detecting the serum ceruloplasmin level and the blood creatinine level in a sample from a subject.

[0019] Optionally, the renal fibrosis-related disease is renal interstitial fibrosis and / or chronic tubulointerstitial damage.

[0020] Optionally, the sample is a blood sample from a subject.

[0021] Optionally, the kit comprises:

[0022] (i) a reagent for detecting an effective amount of serum ceruloplasmin in a subject's sample; or a reagent for detecting an effective amount of serum ceruloplasmin and blood creatinine in a subject's sample;

[0023] (ii) Optionally, at least one substance selected from the following group: container packaging, auxiliary agent, solution, buffer, negative control substance, positive control substance or instruction.

[0024] A system for early diagnosis, risk assessment, prognosis prediction and / or treatment plan selection of a subject's renal fibrosis-related disease, the system comprising:

[0025] (1) a first device for collecting and / or receiving data on serum ceruloplasmin levels in a sample from a subject; or a first device for collecting and / or receiving data on serum ceruloplasmin levels and blood creatinine levels in a sample from a subject;

[0026] (2) a second device for analyzing the data to perform early diagnosis, risk assessment, prognosis prediction and / or treatment plan selection for the subject;

[0027] Wherein, the first device comprises the product as described above;

[0028] And / or, the analysis comprises comparing the serum ceruloplasmin level or the serum ceruloplasmin level×blood creatinine level in the sample with a reference value.

[0029] The present invention can achieve the following beneficial effects:

[0030] The inventors have found that compared with other methods for diagnosing renal fibrosis, serum ceruloplasmin has the following three advantages: First, compared with conventional diagnostic markers (such as blood creatinine), ceruloplasmin can be detected to be upregulated in the serum of patients with early renal interstitial fibrosis, and can be detected in conjunction with existing mature diagnostic markers to improve diagnostic efficiency; second, compared with the traditional method of renal tissue pathological biopsy, which is invasive and may cause damage to the patient's body, using ceruloplasmin as a diagnostic marker only requires collecting a small amount or even a trace amount of patient serum, which is completely non-invasive and will not have adverse effects on the patient's health; third, compared with imaging methods such as CT and MRI, the method of detecting markers in serum has lower cost, more flexible detection methods, stronger timeliness, and is easier to promote and popularize in clinical practice, making it convenient for patients to receive diagnosis in a timely and convenient manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings used in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0032] Figure 1 :uIRI fibrosis model construction process;

[0033] Figure 2 :Renal conditions of UUO fibrosis model;

[0034] Figure 3 : Serum creatinine at each time point in uIRI model and UUO model;

[0035] Figure 4 :Masson staining of fibrosis in uIRI model;

[0036] Figure 5 :Masson staining of fibrosis in UUO model;

[0037] Figure 6 : Changes of CP and fibrosis indexes over time in uIRI and UUO models;

[0038] Figure 7 : Serum CP expression in uIRI model and UUO model;

[0039] Figure 8 :Correlation analysis between serum CP and renal fibrosis area;

[0040] Fig. 9 : Serum CP expression levels in patients with different renal fibrosis degrees;

[0041] Fig.10:Correlation analysis between serum CP, serum creatinine and eGFR in patients with renal fibrosis;

[0042] Fig.11 : ROC curve of serum CP and serum creatinine in diagnosing renal interstitial fibrosis in patients with renal fibrosis. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings. The following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.

[0044] As used herein, ceruloplasmin, also known as copper oxidase, is a copper-containing α2 glycoprotein with a molecular weight of about 120,000-160,000. It is a single-chain polypeptide with 6-7 copper atoms per molecule. It is blue due to the copper content, contains about 10% sugar, and the terminal sialic acid is connected to the polypeptide chain. It has genetic gene polymorphism. Its function is to regulate the distribution of copper in various parts of the body, synthesize copper-containing enzyme proteins, act as an antioxidant, and have oxidase activity. It has the ability to catalyze the oxidation of polyphenols and polyamine substrates. It is generally believed that ceruloplasmin is synthesized by the liver, part of which is excreted by the bile duct, and the content in urine is very small.

[0045] 1. Animal Research

[0046] 1.1 Establishment of mouse renal fibrosis model

[0047] Two renal fibrosis models, unilateral renal ischemia-reperfusion injury (uIRI) and unilateral ureteral obstruction (UUO), were established using male C57 mice aged 8-10 weeks.

[0048] uIRI model construction process: After the mouse is anesthetized, the back skin is prepared and the surgical area is disinfected with iodine. A 2 cm small incision is made obliquely on the left back 1.5 cm away from the spine. The skin and lateral abdominal muscles are cut layer by layer to expose the kidney. The connective tissue and fat around the renal pedicle are carefully separated using pointed forceps. After the renal pedicle is fully exposed, the renal pedicle is clamped with a non-invasive micro-vascular clamp at the renal artery and vein closest to the renal hilum. When the color of the kidney changes from bright red to dark red, the kidney is returned to the abdominal cavity. The mouse was placed on a 37-degree constant temperature table and timed for 28 minutes. After the timing ends, the vascular clamp is removed, and the color of the kidney changes from dark red back to bright red. The abdominal cavity is then sutured closed layer by layer. Figure 1 The sham group was only treated with laparotomy and then closed. Serum and renal tissue were collected from the uIRI model 1, 3, 7, 14, and 28 days after modeling.

[0049] UUO model construction process: After the mouse is anesthetized, the hair on the left back is shaved and the skin is prepared and disinfected. A 2 cm small incision is made longitudinally on the left back 1.5 cm away from the spine. The incision should avoid blood vessels and nerves as much as possible to reduce surgical bleeding and nerve damage. Blunt separation of muscle tissue to expose the kidney and upper ureter. Use small curved forceps to clamp a section of sterile surgical suture, pass it through the ureter from below, and ligate it at 1 / 3 of the ureter with suture. After removing the thread head, the incision is sutured layer by layer. The sham group only underwent laparotomy and then closed the abdomen. The UUO model was collected 1 day, 3 days, 7 days, 10 days, and 14 days after surgery. Figure 2 shown.

[0050] 1.2 The serum creatinine of the above mouse models was tested using the creatinine assay kit of Nanjing Jiancheng Bioengineering Institute. The results showed that due to the presence of contralateral healthy kidneys, the serum creatinine levels of uIRI model mice and UUO model mice at each time point were not significantly different from those of the Sham group, and the P value was not statistically significant. Figure 3 shown.

[0051] 1.3 Masson staining was used to evaluate renal fibrosis in uIRI and UUO models at different time points.

[0052] The specific steps are as follows: paraffin sections are dewaxed to water; stained with prepared Weigert iron hematoxylin staining solution for 5 minutes; differentiated with hydrochloric acid ethanol differentiation solution for 2 seconds, washed with distilled water; ammonia water blueing for 5 seconds, washed with distilled water; dyed with Ponceau fuchsin staining solution for 10 minutes, washed with weak acid working solution for 3-5 seconds. Differentiate with phosphomolybdic acid solution for 3 minutes, then directly put into aniline blue staining solution for 20 minutes, and wash with weak acid working solution for 1 minute. Rapidly dehydrate with 95% ethanol. Dehydrate with anhydrous ethanol 3 times, 2-3 seconds each time. Transparent with xylene for 2 minutes. Seal the slides with neutral gum.

[0053] The results showed that mild fibrosis began to appear on the 3rd day of the uIRI model, and the fibrosis was limited to the outer medulla region adjacent to the damaged S3 segment; collagen fibers were deposited in the inner and outer medulla regions on the 7th day. By the 28th day after uIRI, a large amount of collagen fiber deposition could be observed throughout the kidney, such as Figure 4 shown.

[0054] In the UUO model, the left kidney of mice with ureteral ligation for 10 days showed thinning of the renal cortex, significant dilation of the renal pelvis and calyces, atrophy of the renal parenchyma, disappearance of the tubules and interstitial structures, unclear boundaries between the cortex and medulla, and obvious fibrosis compared with the contralateral kidney. Figure 5 shown.

[0055] 1.4 Western blot was used to detect renal fibrosis markers and CP expression.

[0056] The specific steps are as follows: add RIPA and PMSM to mouse kidney tissue for ultrasonic lysis; ice bath for 30 minutes, centrifuge at 12000rpm at 4℃, and absorb supernatant protein; detect protein concentration by BCA method; adjust the concentration of each group of protein samples to be consistent so that the loading amount is consistent; heat at 95℃ for 10 minutes to denature the protein; prepare 8%~12% corresponding PAGE-SDS polyacrylamide gel according to the molecular weight of the target protein; perform electrophoresis after adding the sample; then transfer the protein from the polyacrylamide gel to a nitrocellulose membrane, add the corresponding primary and secondary antibody working solutions for incubation; finally, perform chemical luminescence development.

[0057] The expression of fibrosis indicators Fibronectin and α-SMA in the tissues of mouse renal fibrosis models (uIRI and UUO) at different time points was detected by WB method. It can be seen that the expression of Fibronectin and α-SMA increased significantly with time. Further detection of the expression of CP at different stages of the two fibrosis models showed that as the fibrosis model worsened, the expression of CP gradually increased. Figure 6 shown.

[0058] 1.5 Serum testing for CP.

[0059] The activity of ceruloplasmin in the serum of mice with fibrosis model was detected using the commercial CP activity kit of Solebo Reagent Company. It can be seen that as the fibrosis model time increases, the activity of CP in the serum of mice gradually increases. Figure 7 shown.

[0060] 1.6 Correlation analysis between serum CP and renal fibrosis area. The fibrosis area of ​​the kidneys of uIRI and UUO model mice was quantitatively analyzed by Image J software, and the correlation analysis between serum CP and renal fibrosis area was performed by Speraman method. It can be seen that the activity of serum CP was significantly correlated with the fibrosis area in both uIRI and UUO models, with correlation coefficients of 0.8137 and 0.7265, respectively, and P values ​​<0.001. The results are shown in Tables 1-2 and Figure 8 shown.

[0061] Table 1: Serum ceruloplasmin activity and renal fibrosis area in uIRI model

[0062]

[0063] Table 2: Serum ceruloplasmin activity and renal fibrosis area in UUO model

[0064]

[0065] Part II: Clinical Research

[0066] In this example, 80 patients with chronic tubulointerstitial damage (IFTA) diagnosed by renal biopsy pathology and without heart, liver, lung dysfunction, tumor, diabetes, infection, or pregnancy were collected, and 42 healthy volunteers were included.

[0067] There was no significant difference in age and gender between the two groups (P>0.10), and the two groups were comparable (see Table 3).

[0068] The clinical indicators of traditional renal fibrosis were tested, including serum creatinine and urea nitrogen, and eGFR was calculated. The expression of serum CP was detected. The results showed that the serum creatinine value (147 vs 80 umol / l, P<0.01), blood urea nitrogen value (7.59 vs 5.76mmol / l, P<0.01), and serum ceruloplasmin (24.7 vs 21.1 mg / dl, P<0.01) of the IFTA group were higher than those of the healthy control group, as shown in Table 3.

[0069] Table 3. Comparison of baseline data between the IFTA group and the healthy control group

[0070]

[0071] Further grouping according to the severity of renal interstitial fibrosis (fibrosis area ≤ 1% is 0 points; 1% < fibrosis area ≤ 25% is 1 point; 25% < fibrosis area ≤ 50% is 2 points; 50% < fibrosis area is 3 points), it was found that with the aggravation of tubulointerstitial fibrosis, the expression of serum CP gradually increased, such as Fig. 9 The results of the correlation study showed that serum CP expression was negatively correlated with serum creatinine level (R 2 =0.471, P<0.01, and positively correlated with eGFR (R 2 =0.383, P<0.01), such as Fig.10 shown.

[0072] In order to verify the diagnostic value of serum CP for chronic tubulointerstitial fibrosis, ROC curves were drawn for 122 subjects and the AUC values ​​were calculated. The results are shown in Tables 4-5 and Fig.11 As shown, serum The AUC value for the diagnosis of mild and severe renal interstitial fibrosis reached 0.941, which is higher than the diagnostic value of the traditional fibrosis indicator blood creatinine (Cr) (AUC=0.909), showing good test efficiency.

[0073] Table 4: Fig.11 Serum ceruloplasmin and serum creatinine concentrations

[0074]

[0075]

[0076]

[0077]

[0078] Table 5: Area under the ROC curve

[0079] .

[0080] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A biomarker for detecting renal fibrosis-related diseases, characterized in that: Includes serum ceruloplasmin, or, serum ceruloplasmin and serum creatinine.

2. The biomarker according to claim 1, characterized in that The renal fibrosis-related diseases are renal interstitial fibrosis and / or chronic tubulointerstitial damage.

3. Use of serum ceruloplasmin as a biomarker in the preparation of products for early diagnosis, risk assessment, prognosis prediction and / or treatment regimen selection of subjects with renal fibrosis-related diseases.

4. The use according to claim 3, characterized in that The early diagnosis, risk assessment, prognosis prediction and / or treatment plan selection of the subject's renal fibrosis-related diseases include: Determining the serum ceruloplasmin level in a sample from the subject, and comparing the serum ceruloplasmin level in the sample with a reference value, so as to perform early diagnosis, risk assessment, prognosis prediction and / or selection of a treatment regimen for the subject; or The serum ceruloplasmin level and the blood creatinine level in the sample from the subject are measured, and the serum ceruloplasmin level×the blood creatinine level in the sample are compared with the reference value to perform early diagnosis, risk assessment, prognosis prediction and / or treatment regimen selection for the subject.

5. The use according to claim 3, characterized in that: The renal fibrosis-related diseases are renal interstitial fibrosis and / or chronic tubulointerstitial damage.

6. A product for early diagnosis, risk assessment, prognosis prediction and / or treatment regimen selection of a subject's renal fibrosis-related disease, characterized in that: The product comprises a reagent, a kit and / or a detection device for detecting the level of serum ceruloplasmin in a sample from a subject, or The product comprises a reagent, a kit and / or a detection device for detecting the serum ceruloplasmin level and the blood creatinine level in a sample from a subject.

7. The product according to claim 6, characterized in that The renal fibrosis-related diseases are renal interstitial fibrosis and / or chronic tubulointerstitial damage.

8. The product according to claim 6, characterized in that The sample is a blood sample from a subject.

9. The product according to claim 6, characterized in that The kit comprises: (i) a reagent for detecting an effective amount of serum ceruloplasmin in a subject's sample; or a reagent for detecting an effective amount of serum ceruloplasmin and blood creatinine in a subject's sample; (ii) Optionally, at least one substance selected from the following group: container packaging, auxiliary agent, solution, buffer, negative control substance, positive control substance or instruction.

10. A system for early diagnosis, risk assessment, prognosis prediction and / or treatment plan selection of a subject's renal fibrosis-related disease, characterized in that: The system comprises: (1) a first device for collecting and / or receiving data on serum ceruloplasmin levels in a sample from a subject; or a first device for collecting and / or receiving data on serum ceruloplasmin levels and blood creatinine levels in a sample from a subject; (2) a second device for analyzing the data to perform early diagnosis, risk assessment, prognosis prediction and / or treatment plan selection for the subject; Wherein, the first device comprises the product according to any one of claims 6-9; And / or, the analysis comprises comparing the serum ceruloplasmin level or the serum ceruloplasmin level×blood creatinine level in the sample with a reference value.

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