A biomarker for detecting kidney fibrosis-related diseases and its applications

Abstract

The present invention belongs to the field of pharmaceutical technology, and specifically discloses a biomarker for detecting kidney fibrosis-related diseases and its application. The biomarker includes ceruloplasmin in serum, or ceruloplasmin in serum and creatinine. Compared with other methods for diagnosing renal fibrosis, ceruloplasmin in serum has three major advantages: First, compared with conventional diagnostic markers (such as creatinine), ceruloplasmin can be detected to be up-regulated in the serum of patients with early chronic kidney diseases, and can be jointly detected with existing mature diagnostic markers; Second, compared with renal tissue pathological biopsy, using ceruloplasmin as a diagnostic marker is non-invasive, only a small amount or even a trace amount of patient serum is required, and it will not cause harm to the patient's physical health; Third, compared with the method of analyzing the metabolic components of urine by nuclear magnetic resonance, the method of detecting the biomarker in serum has a lower cost, a more flexible detection method, high timeliness and is easier to popularize.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a biomarker for detecting kidney fibrosis-related diseases and its application. Background Art

[0002] Chronic kidney disease (CKD) and uremia seriously threaten the health of the people.

[0003] Kidney fibrosis is the pathological basis for the progression of CKD to uremia. Multiple cells such as renal tubular epithelial cells, interstitial cells, endothelial cells, and inflammatory cells are involved in tubulointerstitial fibrosis, and their interactions initiate the occurrence and progression of renal fibrosis. Among these cells, damaged renal tubular epithelial cells are considered to be one of the key mechanisms driving kidney fibrosis because the renal tubules are the main components of the kidney and are vulnerable to various factors such as hypoxia, proteinuria, toxins, and metabolic disorders. Generally, the renal tubules have strong regenerative ability and can be basically completely repaired when the damage is mild; however, when the damage is severe or the continuous damage exceeds the repair ability, the renal tubules will atrophy, the renal tubular epithelium will flatten, produce various cytokines and other signals, and ultimately participate in fibrosis or indirectly promote interstitial fibrosis.

[0004] Currently, there are many dilemmas in the clinical diagnosis of renal fibrosis. ① Renal tissue pathological biopsy is the "gold standard" for diagnosing renal fibrosis at present, but it is an invasive diagnostic method. The puncture process may cause complications such as bleeding, infection, hematuria, and perirenal hematoma, bringing potential safety risks. And due to the invasiveness of renal biopsy, it is difficult to repeat it multiple times and cannot 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 stage of renal fibrosis. Because the kidney has a strong compensatory function, when the glomerular filtration rate drops to a certain extent, these indicators will begin to increase, and at this time, renal fibrosis may have progressed to a certain stage, which is not conducive to early diagnosis and intervention. ③ Imaging examinations are also difficult to accurately identify fibrotic lesions due to limited resolution and low specificity.

[0005] Therefore, finding biomarkers that can be applied clinically and can effectively diagnose early renal fibrosis is of great significance for the timely discovery and treatment of the disease. Summary of the Invention

[0006] To overcome the bottleneck of existing non-invasive diagnostic techniques for renal fibrosis, the present invention aims to provide a molecular biomarker for non-invasive diagnosis of renal fibrosis. Compared with traditional diagnostic methods, using serum ceruloplasmin (serum CP) as a non-invasive diagnostic biomarker for renal fibrosis has higher sensitivity than serum creatinine and can distinguish acute kidney injury from chronic fibrosis, enabling patients to be aware of the disease risk at an early stage and take corresponding preventive and treatment measures according to the risk level.

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

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

[0009] A biomarker for detecting kidney fibrosis-related diseases, which includes serum ceruloplasmin, or serum ceruloplasmin and serum creatinine.

[0010] Optionally, the kidney fibrosis-related diseases are interstitial renal 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 plan selection of kidney fibrosis-related diseases in an object.

[0012] Optionally, the early diagnosis, risk assessment, prognosis prediction, and / or treatment plan selection of the kidney fibrosis-related diseases in the object include:

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

[0014] Determining the serum ceruloplasmin level and serum creatinine level in a sample from the object and comparing the product of the serum ceruloplasmin level and serum creatinine level in the sample with a reference value to perform early diagnosis, risk assessment, prognosis prediction, and / or treatment plan selection for the object.

[0015] Optionally, the kidney fibrosis-related diseases are interstitial renal fibrosis and / or chronic tubulointerstitial damage.

[0016] A product for early diagnosis, risk assessment, prognosis prediction, and / or treatment plan selection of kidney fibrosis-related diseases in an object,

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

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

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

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

[0021] Optionally, the kit includes:

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

[0023] (ii) Optionally, at least one substance selected from the following group: a container package, an adjuvant, a solution, a buffer, a negative control, a positive control, or an instruction manual.

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

[0025] (1) A first device for collecting and / or receiving data on the ceruloplasmin level in a sample of a subject; or a first device for collecting and / or receiving data on the ceruloplasmin level and the serum creatinine level in a sample of a subject;

[0026] (2) A second device for analyzing the data to perform early diagnosis, risk assessment, prognosis prediction, and / or treatment plan selection of a kidney fibrosis-related disease in the subject;

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

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

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

[0030] The inventor of the present invention has found that compared with other methods for diagnosing renal fibrosis, ceruloplasmin has the following three major advantages: First, compared with conventional diagnostic markers (such as serum creatinine), ceruloplasmin can be detected to be up-regulated in the serum of patients with early renal interstitial fibrosis, and it can be combined with existing mature diagnostic markers for detection to improve the 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 the patient's serum, which is completely non-invasive and will not have an adverse impact on the patient's physical health; Third, compared with imaging methods such as CT and magnetic resonance, the method of detecting markers in serum has a lower cost, a more flexible detection method, stronger timeliness, and is also easier to promote and popularize in clinical practice, facilitating the vast number of 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 drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0032] Figure 1 : Process of constructing the uIRI fibrosis model;

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

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

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

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

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

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

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

[0040] Figure 9 : Serum CP expression levels in different groups of patients with different degrees of renal fibrosis;

[0041] Figure 10: Correlation analysis of serum ceruloplasmin (CP) with serum creatinine and estimated glomerular filtration rate (eGFR) in patients with renal fibrosis;

[0042] Figure 11 : Receiver operating characteristic (ROC) curve of serum CP and serum creatinine for diagnosing renal interstitial fibrosis in patients with renal fibrosis. Detailed implementation manners

[0043] To make the objectives, technical solutions and advantages of the present invention more clear and understandable, the following further describes the present invention in detail with reference to specific embodiments and the accompanying drawings. The following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0044] As used herein, ceruloplasmin, also known as ferroxidase, is a copper-containing α2 glycoprotein with a molecular weight of about 120,000 - 160,000. It is a single-chain polypeptide, containing 6 - 7 copper atoms per molecule. It is blue due to the presence of copper, contains about 10% sugar, and the terminal sialic acid is linked to the polypeptide chain, with 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 having oxidase activity, with the ability to catalyze the oxidation of polyphenol and polyamine substrates. It is generally considered that ceruloplasmin is synthesized by the liver, and a part is excreted through the biliary tract, and its content in urine is very low.

[0045] I. Animal research part

[0046] 1.1 Construction of mouse renal fibrosis models

[0047] Two renal fibrosis models, namely unilateral kidney ischemia-reperfusion injury (uIRI) and unilateral ureteral obstruction (UUO), were constructed using 8 - 10-week-old male C57 mice.

[0048] Procedure for constructing the uIRI model: After anesthetizing the mice, the back was shaved and the surgical area was disinfected with iodophor. A 2-cm small oblique incision was made 1.5 cm away from the spine on the left back, and the skin and lateral abdominal muscles were incised layer by layer to expose the kidney. The connective tissue and fat around the renal pedicle were carefully separated using sharp forceps. After fully exposing the renal pedicle, the renal pedicle was clamped with a non-invasive microvascular clip at the renal artery and vein closest to the renal hilum. After the kidney color changed from bright red to dark red, the kidney was retracted into the abdominal cavity. The mice were placed on a 37-degree constant temperature table and timed for 28 minutes. After the timing ended, the vascular clip was removed. After the kidney color changed from dark red back to bright red, the abdominal cavity was sutured layer by layer as Figure 1 shown. The sham group in the sham operation group only underwent laparotomy and then abdominal closure. Serum and renal tissues were collected from the uIRI model at 1 day, 3 days, 7 days, 14 days, and 28 days after model establishment.

[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 tissues and perform ultrasonic lysis; after ice-bathing for 30 minutes, centrifuge at 12,000 rpm at 4°C, and aspirate the supernatant protein; detect the protein concentration by the BCA method; adjust the protein sample concentration of each group to be the same to ensure the same loading amount; heat at 95°C for 10 minutes to denature the protein; prepare an 8% - 12% corresponding PAGE-SDS polyacrylamide gel according to the molecular weight of the target protein; perform electrophoresis after loading 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, develop the image by chemiluminescence method.

[0057] The expression levels of fibrosis indicators Fibronectin and α-SMA in the tissues of mouse renal fibrosis models (uIRI and UUO) at different time points were detected by the WB method. It can be seen that the expressions of Fibronectin and α-SMA were significantly up-regulated in a gradient 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, as Figure 6 shown.

[0058] 1.5 Detection of CP in serum.

[0059] The activity of ceruloplasmin in the serum of fibrosis model mice was detected using a commercial CP activity kit from Solarbio. It can be seen that as the time of the fibrosis model extended, the activity of CP in the serum of mice gradually increased, as Figure 7 shown.

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

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

[0062]

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

[0064]

[0065] Part Two: Clinical research part

[0066] In this embodiment, 80 patients with chronic tubulointerstitial damage (IFTA) confirmed by renal biopsy pathology, without heart, liver, and lung insufficiency, tumors, diabetes, infections, or pregnancy, were collected in this experiment. And 42 healthy volunteers were included.

[0067] There were no statistically significant differences in age and gender between the two groups (P > 0.10), and they were comparable, as shown in Table 3.

[0068] Clinical indicators of traditional renal fibrosis were detected, including serum creatinine and urea nitrogen, and eGFR was calculated. The expression level 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 5.76 mmol / l, P < 0.01), and serum ceruloplasmin (24.7 vs 21.1 mg / dl, P < 0.01) in the IFTA group were all higher than those in 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 grouped 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, as Figure 9 shown. The results of the correlation study showed that the expression of serum CP was negatively correlated with the serum creatinine level (R 2 = 0.471, P < 0.01) and positively correlated with eGFR (R 2 = 0.383, P < 0.01), as Figure 10 shown.

[0072] To verify the diagnostic value of serum CP for chronic tubulointerstitial fibrosis, ROC curves were plotted for 122 study subjects, and the AUC values were calculated respectively. The results are shown in Tables 4 - 5 and Figure 11 shown. The AUC value of serum for the diagnosis of mild and severe renal interstitial fibrosis reached 0.941, higher than the diagnostic value of the traditional fibrosis index serum creatinine (Cr) (AUC = 0.909), showing good test performance.

[0073] Table 4: Figure 11 The concentrations of serum ceruloplasmin and serum creatinine in

[0074]

[0075]

[0076]

[0077]

[0078] Table 5: Area under the ROC curve

[0079] 。

[0080] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a reagent for detecting ceruloplasmin or a reagent for detecting ceruloplasmin × serum creatinine in the preparation of a product for early diagnosis, risk assessment, prognosis prediction, and / or treatment option selection of a disease related to renal fibrosis in a subject, wherein, The kidney fibrosis-related disease is renal interstitial fibrosis and / or chronic tubulointerstitial damage.

2. The use according to claim 1, characterized in that, The early diagnosis, risk assessment, prognosis prediction, and / or treatment plan selection for the kidney fibrosis-related disease of the subject 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 to perform early diagnosis, risk assessment, prognosis prediction, and / or treatment plan selection for the subject; or determining the serum ceruloplasmin level and serum creatinine level in a sample from the subject, and comparing the serum ceruloplasmin level × serum creatinine level in the sample with a reference value to perform early diagnosis, risk assessment, prognosis prediction, and / or treatment plan selection for the subject.

3. A system for the early diagnosis, risk assessment, prognosis prediction, and / or treatment plan selection of diseases related to renal fibrosis of an object, characterized in that, The system includes: (1) A first device for collecting and / or receiving data on the serum ceruloplasmin level in a sample of a subject; or a first device for collecting and / or receiving data on the serum ceruloplasmin level and serum creatinine level in a sample of 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 with a kidney fibrosis-related disease; wherein, the first device includes: a product for early diagnosis, risk assessment, prognosis prediction, and / or treatment plan selection of a kidney fibrosis-related disease of a subject, wherein the product includes a reagent, a kit, and / or a detection device for detecting the serum ceruloplasmin level in a sample from the subject, or the product includes a reagent, a kit, and / or a detection device for detecting the serum ceruloplasmin level and serum creatinine level in a sample from the subject, wherein the kidney fibrosis-related disease is renal interstitial fibrosis and / or chronic tubulointerstitial damage; The analysis includes comparing the serum ceruloplasmin level or the serum ceruloplasmin level × serum creatinine level in the sample with a reference value.

4. The system according to claim 3, wherein The sample is from a blood sample of the subject.

5. The system according to claim 3, wherein The kit includes: (i) A reagent in an effective amount for detecting serum ceruloplasmin in a sample of a subject; or a reagent in an effective amount for detecting serum ceruloplasmin and serum creatinine in a sample of a subject; (ii) At least one substance selected from the following group: container packaging, adjuvant, solution, buffer, negative control, positive control, or instruction manual.

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