A molecular marker for early screening of renal fibrosis and application thereof

Abstract

This invention provides risk biomarkers for renal fibrosis and their applications. Specifically, this invention provides the use of genes, mRNAs, cDNAs, proteins, or detection reagents for biomarkers of renal fibrosis risk, for the preparation of detection reagents or kits for assessing the risk of renal fibrosis. Studies have shown that CCN5 can be used as a biomarker for assessing the occurrence of renal fibrosis with high sensitivity and specificity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to CCN5 as a biomarker for screening renal fibrosis. Background Technology

[0002] Chronic kidney disease (CKD) is a major threat to human health worldwide. CKD is characterized by renal fibrosis, leading to a time-dependent decline in kidney function. The number of CKD patients progressing to end-stage renal disease (ESRD) is increasing at a rate of 5-6% annually. ESRD patients require long-term dialysis or kidney transplantation to sustain life, placing a significant burden on individuals, families, and society. Early diagnosis is of great scientific and clinical value for identifying susceptible populations of CKD, dynamically assessing CKD progression, and thus enabling timely treatment interventions, reducing the incidence of ESRD, and improving prognosis.

[0003] Clinically, urinary protein and serum creatinine levels are often used as indicators to assess the progression of CKD. However, due to the kidney's strong reserve function, by the time patients develop proteinuria and elevated serum creatinine levels, the kidney disease is often already quite severe. Traditional pathological diagnosis relies on renal biopsy, which is an invasive procedure and difficult to repeat multiple times. Finding early, non-invasive biomarkers associated with CKD progression is a pressing challenge in current nephrology research.

[0004] Therefore, it is necessary to develop efficient markers for detecting chronic kidney disease or drugs for treating it. Summary of the Invention

[0005] The purpose of this invention is to provide CCN5, a target for the diagnosis and treatment of renal fibrosis, and its application.

[0006] In a first aspect of the invention, there is provided the use of a gene, mRNA, cDNA, protein, or a detection reagent thereof for a risk marker of renal fibrosis, for preparing a detection reagent or kit, said detection reagent or kit for diagnosing or assisting in the diagnosis of the risk of developing renal fibrosis and for determining the severity of renal fibrosis.

[0007] The renal fibrosis markers include CCN5.

[0008] In another preferred embodiment, the detection reagent or kit is used to detect the expression level of the risk marker in the sample to be tested.

[0009] In another preferred embodiment, the expression level of the risk marker is the expression level in a biological sample.

[0010] In another preferred embodiment, the biological sample is selected from the group consisting of urine, blood, plasma, serum, or combinations thereof.

[0011] In another preferred embodiment, the expression level includes protein expression level or mRNA expression level.

[0012] In another preferred embodiment, the CCN5 gene includes a wild-type CCN5 gene and a mutant CCN5 gene.

[0013] In another preferred embodiment, the renal fibrosis is selected from: unilateral ureteral ligation and folic acid-induced renal fibrosis.

[0014] In a second aspect of the invention, a kit is provided comprising a detection reagent for detecting genes, mRNAs, cDNAs, or proteins, or combinations thereof, of risk markers for renal fibrosis.

[0015] The renal fibrosis risk markers mentioned include CCN5.

[0016] In another preferred embodiment, the detection reagent includes antibodies, primers, probes, sequencing libraries, nucleic acid chips (such as DNA chips) or protein chips.

[0017] In another preferred embodiment, the detection reagent includes a nucleic acid reagent for detecting CCN5 mRNA or cDNA.

[0018] In another preferred embodiment, the protein comprises a full-length protein or a protein fragment.

[0019] In another preferred embodiment, the diagnostic reagent or kit contains one or more detection reagents selected from the group consisting of:

[0020] (a) Specific antibodies against CCN5, specific binding molecules of CCN5; and / or

[0021] (b) Primers or primer pairs, probes or chips for specific amplification of CCN5 mRNA or CCN5 cDNA.

[0022] In another preferred embodiment, the detection reagent is coupled with or carries a detectable marker.

[0023] In another preferred embodiment, the detectable marker is selected from the group consisting of chromophores, chemiluminescent groups, fluorophores, isotopes, or enzymes.

[0024] In another preferred embodiment, the specific antibody for CCN5 is a monoclonal antibody or a polyclonal antibody.

[0025] In another preferred embodiment, the expression level of CCN5 protein in the sample is detected by RT-PCR or immunohistochemistry.

[0026] In another preferred embodiment, the gene, mRNA, cDNA, or protein of CCN5 is derived from a subject suffering from renal fibrosis or chronic kidney disease.

[0027] In another preferred embodiment, the subject is a human or a non-human mammal.

[0028] In another preferred embodiment, the detection is for an ex vivo sample; preferably, the ex vivo sample is selected from the group consisting of urine, blood, plasma, serum, or combinations thereof.

[0029] In another preferred embodiment, the kit contains genes, mRNAs, cDNAs, and / or proteins of renal fibrosis risk markers as controls or quality control products.

[0030] In another preferred embodiment, the kit further includes a label or instructions indicating that the kit is used for (a) assessing the risk of developing renal fibrosis and / or (b) evaluating the effectiveness of treatment for renal fibrosis.

[0031] In another preferred embodiment, the detection of the renal fibrosis risk markers can be quantitative.

[0032] In another preferred embodiment, the renal fibrosis is selected from: unilateral ureteral ligation and folic acid-induced renal fibrosis.

[0033] In a third aspect of the present invention, a detection method is provided, comprising the steps of:

[0034] (a) Provide a test sample, said test sample being selected from a urine or blood sample;

[0035] (b) The expression level of CCN5 in the test sample was detected and denoted as C1; and

[0036] (c) Compare the CCN5 concentration C1 with the control reference value C0;

[0037] When C1 / C0 > 1, the test subject is judged to have a high risk of developing renal fibrosis.

[0038] In another preferred embodiment, when C1 / C0 ≥ 1.5, preferably ≥ 2, and preferably ≥ 3, the subject of the test is judged to have a high risk of developing renal fibrosis.

[0039] In another preferred embodiment, the sample to be tested is urine. If the test result for the risk of renal fibrosis or chronic kidney disease in the subject meets the following condition, it indicates that the subject has a high risk of developing renal fibrosis or chronic kidney disease: C1 ≥ 2 ng / ml, preferably ≥ 2.5 ng / ml, more preferably ≥ 3 ng / ml.

[0040] In another preferred embodiment, the method is an in vitro method.

[0041] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0042] In a fourth aspect of the invention, a diagnostic device for renal fibrosis is provided, the device comprising:

[0043] (a) An input module configured to input CCN5 data of a biological sample of an object;

[0044] (b) A processing module configured to process renal fibrosis risk marker data and provide an assessment of the risk of occurrence; wherein the processing includes comparing the expression level C1 of input CCN5 with a control reference value C0;

[0045] Among them, when C1 is significantly higher than C0, it indicates a high risk of renal fibrosis in the subject; when C1 is not significantly higher than C0, it indicates a low risk of renal fibrosis in the subject; and

[0046] (c) Output module, which is configured to output the evaluation results.

[0047] In another preferred embodiment, the device further includes a detection module configured to detect the transcriptional or expression level of CCN5.

[0048] In another preferred embodiment, the device further includes a storage module configured to store pre-set threshold values ​​for markers and comparison result values.

[0049] In another preferred embodiment, the device further includes a control module configured to control the operation of the modules.

[0050] In another preferred embodiment, the detection module is selected from the group consisting of: ELISA analyzer, PCR sequencer, sequencer, or combinations thereof.

[0051] In a fifth aspect of the invention, there is provided the use of a CCN5 inhibitor for preparing a composition or formulation for use selected from the group consisting of:

[0052] (i) Inhibit the expression levels of fibronectin, COL1A1 (COL I), vimentin, and α-SMA proteins;

[0053] (ii) Inhibit the levels of Fibronectin, COL1A1, Vimentin, and α-SMA mRNA;

[0054] (iii) To alleviate or treat renal fibrosis or chronic kidney disease in the subject.

[0055] In another preferred embodiment, the CCN5 inhibitor is an inhibitor of the CCN5 gene, RNA, or its encoded protein.

[0056] In another preferred embodiment, the CCN5 inhibitor is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, antisense oligonucleotides (ASO), nucleic acid aptamers, gene editors, or combinations thereof.

[0057] In another preferred embodiment, the CCN5 inhibitor is an antibody.

[0058] In another preferred embodiment, the composition is a pharmaceutical composition.

[0059] In another preferred embodiment, the pharmaceutical composition is administered to a subject with high CCN5 expression.

[0060] In a sixth aspect of the invention, a method for preventing and / or treating renal fibrosis or chronic kidney disease is provided, comprising the step of administering a CCN5 inhibitor to a subject in need.

[0061] In another preferred embodiment, the object includes a human or a non-human mammal.

[0062] In another preferred embodiment, the subject is a patient with renal fibrosis or chronic kidney disease.

[0063] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0064] Figure 1 The study showed the concentration of CCN5 in urine under two different renal fibrosis modeling methods (unilateral ureteral ligation and intraperitoneal injection of hyperuricemic acid) by enzyme-linked immunosorbent assay (ELISA).

[0065] Figure 2The study showed that the CCN5 content in the urine of a mouse model of renal fibrosis (UUO) was detected by enzyme-linked immunosorbent assay (ELISA).

[0066] Figure 3 The study showed that overexpression of CCN5 in renal fibroblasts can exacerbate renal fibrosis in mice induced by ureteral ligation.

[0067] Figure 4 The study showed that knocking out CCN5 in renal fibroblasts can alleviate renal fibrosis in mice induced by ureteral ligation. Detailed Implementation

[0068] Through extensive and in-depth research, the inventors unexpectedly discovered CCN5, a biomarker in renal fibrosis samples, for the first time. The increase in CCN5 levels is accompanied by renal fibrosis, and inhibiting CCN5 expression significantly improves the progression of renal fibrosis and chronic kidney disease. This invention was completed based on this discovery.

[0069] the term

[0070] As used herein, the terms "transcriptional marker" or "transcriptional biomarker" are used interchangeably, both referring to a type of biomarker that marks a specific disease state by detecting the level of gene transcriptional expression in tissues or cells. Biomarkers are biological indicators that can objectively measure physiological or pathological states, including changes at the physiological, biochemical, immune, cellular, and molecular levels. Blood transcriptional markers, specifically, detect the transcriptional expression level of specific genes in blood samples to mark a particular disease state.

[0071] Transcriptional biomarkers, such as those based on blood samples, detect the transcriptional expression levels of single genes or multiple genes in the blood and calculate a biomarker score using specific algorithms. This score often differs significantly between patients and healthy individuals, thus enabling the identification of disease states. Currently, transcriptional biomarkers are widely used in the diagnosis and screening of various diseases, such as various tumors and infectious diseases. As used herein, the terms "RNA sequencing" or "RNA-seq" are used interchangeably, both referring to a technology capable of high-throughput sequencing of all RNA within cells or tissues. This involves randomly fragmenting cDNA obtained from reverse transcription of mRNA, constructing a sequencing library, and then performing high-throughput sequencing on the cDNA fragments, thereby enabling the simultaneous detection of the relative amounts of mRNA transcribed from all genes.

[0072] As used herein, the term "sample" or "sample" refers to material specifically associated with a subject from which specific information relating to the subject can be determined, calculated, or inferred. A sample may consist wholly or partially of biological material derived from the subject.

[0073] As used herein, the term "expression" includes the production of mRNA from a gene or a gene segment, and includes the production of proteins encoded by the RNA or gene segment, as well as the presence of detection substances associated with expression. For example, the binding of cDNA, binding ligands (such as antibodies) to genes or other oligonucleotides, proteins, or protein fragments, and the chromogenic portion of the binding ligand are all included within the scope of the term "expression." Therefore, an increase in the density of half-points on an immunoblot such as a Western blot also falls within the scope of the biologically molecular-based term "expression."

[0074] As used herein, the term "reference value" or "control reference value" refers to a value that is statistically relevant to a particular outcome when compared with the results of an analysis. In a preferred embodiment, the reference value is determined based on a statistical analysis of the mRNA expression and / or protein expression of comparative renal fibrosis markers. Some such studies are shown in the Examples section of this document. However, studies from the literature and user experience with the methods disclosed herein can also be used to produce or adjust reference values. Reference values ​​can also be determined by considering circumstances and outcomes that are particularly relevant to the patient's ethnicity, medical history, genetics, age, and other factors.

[0075] As used herein, the term "risk marker of the present invention" refers to the CCN5 marker, and also to the "transcription marker" of the present invention.

[0076] Specific antibodies

[0077] In this invention, the terms "specific antibody" and "CCN5 specific antibody" are used interchangeably to refer to antibodies that can be used to specifically bind to and detect the renal fibrosis risk markers of this invention.

[0078] The antibodies of the present invention targeting the renal fibrosis risk marker CCN5 include specific polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies.

[0079] This invention includes not only complete monoclonal or polyclonal antibodies, but also antibody fragments with immunological activity, such as Fab' or (Fab)2 fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules (Ladner et al., U.S. Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have mouse antibody binding specificity but still retain the antibody portion derived from humans.

[0080] The antibodies of this invention can be prepared using various techniques known to those skilled in the art. For example, purified gene products of human renal fibrosis risk markers or their antigenic fragments can be administered to animals to induce the production of polyclonal antibodies. Similarly, cells expressing human renal fibrosis risk marker proteins or their antigenic fragments can be used to immunize animals to produce antibodies. The antibodies of this invention can also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology.

[0081] Antibodies against human renal fibrosis risk marker proteins can be used in immunohistochemistry to detect these proteins in specimens, especially tissue or blood samples. Since these renal fibrosis risk marker proteins are present in blood or tissue samples, their expression levels can be a target for detection.

[0082] CCN5

[0083] Cell communication network factor 5 (CCN5, also known as WISP2) is a member of the stromal cell protein CCN family. It is a WNT1-induced signaling pathway protein containing tandem modules: 1) an insulin-like growth factor binding protein module (IGFBP); 2) a von Willebrand factor C repeat module (VWC); and 3) a platelet-reactive protein type 1 repeat module (TSP-1). Unlike other CCN family members, it lacks a module containing a cysteine ​​carboxyl terminus (CT). The amino acid sequence of the CCN5 protein used in this invention is shown in SEQ ID NO. 1 or 2, where SEQ ID NO. 1 is the human CCN5 protein and SEQ ID NO. 2 is the mouse CCN5 protein.

[0084] The proteins of this invention can be recombinant proteins, natural proteins, or synthetic proteins. They can be naturally purified products, chemically synthesized products, or produced using recombinant technology from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the proteins of this invention can be glycosylated or non-glycosylated. The proteins of this invention may or may not include an initial methionine residue.

[0085] Kidney fibrosis

[0086] Renal fibrosis is a pathophysiological change, a progressive process in which kidney function deteriorates from healthy to damaged, then to completely lost. Stimulated by various pathogenic factors such as trauma, infection, inflammation, circulatory disorders, and immune responses, the intrinsic cells of the kidneys are damaged. In later stages, large amounts of collagen are deposited and accumulated, causing the renal parenchyma to gradually harden, forming scars, until the kidneys completely lose their organ function. The process of fibrosis and hardening of the intrinsic cells of the kidney is the process of renal fibrosis. Renal fibrosis is characterized by abnormal deposition of the extracellular matrix (ECM).

[0087] Chronic kidney disease

[0088] Chronic kidney disease (CKD) is characterized by renal fibrosis, which leads to a time-dependent decline in kidney function.

[0089] Detection methods

[0090] Based on the differential expression of renal fibrosis risk markers in urine or blood samples, this invention also provides a corresponding method for assessing the risk of renal fibrosis.

[0091] This invention relates to diagnostic test methods for quantitatively and locally detecting protein or mRNA levels of risk markers for renal fibrosis. These tests are well known in the art. The protein or mRNA levels of risk markers for renal fibrosis detected in these tests can be used to determine (including as a supplementary determination) whether a person is at risk of renal fibrosis.

[0092] A preferred method is to perform quantitative detection of mRNA or cDNA by PCR / qPCR / RT-PCR.

[0093] A preferred method is to perform quantitative detection by sequencing mRNA or cDNA.

[0094] Polynucleotides that serve as risk markers for renal fibrosis can be used for the diagnosis of renal fibrosis risk. Some or all of the polynucleotides of this invention can be immobilized as probes on microarrays or DNA chips for differential gene expression analysis and gene diagnosis.

[0095] Furthermore, this invention can also perform detection at the protein level. For example, antibodies against renal fibrosis risk markers can be immobilized on a protein chip for detecting renal fibrosis risk proteins in a sample.

[0096] Test kit

[0097] Based on the correlation between CCN5 and the risk of renal fibrosis, CCN5 can be used as a marker for assessing the risk of renal fibrosis.

[0098] This invention also provides a kit for assessing the risk of renal fibrosis, the kit containing a detection reagent for detecting the CCN5 gene, mRNA, cDNA, protein, or combinations thereof. Preferably, the kit contains an anti-CCN5 antibody or immunoconjugate of this invention, or an active fragment thereof; or contains primers or primer pairs, probes, or chips that specifically amplify the CCN5 mRNA or cDNA.

[0099] In another preferred embodiment, the kit also includes a label or instructions.

[0100] The main advantages of this invention are:

[0101] (a) The present invention uses urine samples, which is more suitable for early screening and diagnosis, and has the characteristics of being faster, more convenient and lower cost.

[0102] (b) Compared with existing methods for screening renal fibrosis, the biomarkers of the present invention have higher specificity, are non-invasive, and provide more accurate and stable results.

[0103] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0104] Example 1

[0105] Twenty wild-type C57 mice were selected and divided into four groups of five. Two methods were used to establish the renal fibrosis model. The first method involved unilateral ureteral ligation for 7 days to establish the renal fibrosis model (UUO), with the other group being a sham operation group. The second method involved intraperitoneal administration of a high dose of folic acid (FA), with the control group receiving a fused media control reagent; renal fibrosis was established 30 days later. Urine was collected from both types of mice at their respective experimental endpoints, and the CCN5 content in the urine was detected using a mouse CCN5 enzyme-linked immunosorbent assay kit.

[0106] The results are as follows Figure 1 As shown, CCN5 levels were increased in the urine of two mouse models of renal fibrosis.

[0107] Example 2

[0108] Clinical CKD patients and corresponding healthy adults were included according to the clinical diagnostic criteria for chronic CKD. Urine samples were collected from the included subjects, and the CCN5 content in mouse blood was detected using a mouse CCN5 enzyme-linked immunosorbent assay kit.

[0109] The results are as follows Figure 2 As shown, the level of CCN5 in the urine of CKD patients is increased.

[0110] Example 3

[0111] Tool mice using ColCreERT2 and CCN5 flox / flox Rosa26 mice were hybridized to obtain mice that overexpressed CCN5 specific to kidney fibroblasts (ColCreCCN5). TG The two groups (WT:ColCreCCN5) were respectively... WT TG: ColCreCCN5 TG Seven days after unilateral ureteral ligation, renal fibrosis modeling was established in mice. Seven days later, kidney tissue from the ligated and affected sides of the mice was collected, paraffin-embedded, dehydrated, and sectioned. Finally, Masson collagen staining was performed to assess the severity of renal fibrosis in the two groups of mice.

[0112] The results are as follows Figure 3 As shown in B, in vivo experiments using Masson staining revealed that fibroblast-specific CCN5 overexpression can exacerbate renal fibrosis caused by ureteral ligation.

[0113] like Figure 3 As shown in Figure C, Western blotting experiments showed that fibroblast-specific CCN5 overexpression can promote the expression of fibrosis-related proteins such as Fibronectin, COL1A1, Vimentin, and α-SMA.

[0114] like Figure 3 As shown in DF, real-time quantitative PCR was used to detect that fibroblast-specific CCN5 overexpression can promote the expression of fibrosis-related mRNAs such as Fibronectin (D), COL1A1 (E), Vimentin (F), and α-SMA (F).

[0115] Example 4

[0116] Tool mice using ColCreERT2 and CCN5 flox / flox Mice with CCN5 knockout specific to kidney fibroblasts (ColCreERT2 CCN5) were obtained through hybridization. fl / fl The two groups (WT:CCN5) mentioned above are respectively... flox / floxKO: ColCreCCN5 flox / flox Seven days after unilateral ureteral ligation, renal fibrosis modeling was established in mice. Seven days later, kidney tissue from the ligated and affected sides of the mice was collected, paraffin-embedded, dehydrated, and sectioned. Finally, Masson collagen staining was performed to assess the severity of renal fibrosis in the two groups of mice.

[0117] The results are as follows Figure 4 As shown in B, in vivo experiments using Masson staining revealed that fibroblast-specific CCN5 knockout can alleviate renal fibrosis caused by ureteral ligation.

[0118] like Figure 4 As shown in Figure C, Western blotting experiments showed that fibroblast-specific CCN5 knockout could inhibit the expression of fibrosis-related proteins such as Fibronectin, COL I, Vimentin, and α-SMA.

[0119] like Figure 4 As shown in DH, real-time quantitative PCR was used to detect that fibroblast-specific CCN5 knockout could inhibit Fibronectin ( Figure 4 D), COL I ( Figure 4 E), Vimentin Figure 4 F), α-SMA ( Figure 4 F) Expression of fibrosis-related mRNAs.

[0120] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of a gene, mRNA, cDNA, protein, or a detection reagent thereof for a risk marker of renal fibrosis, for the preparation of a detection reagent or kit, said detection reagent or kit being used to diagnose or assist in the diagnosis of the risk of developing renal fibrosis and to determine the severity of renal fibrosis; wherein The renal fibrosis markers include: CCN5.

2. Use according to claim 1, characterized in that, The detection reagent or kit is used to detect the expression level of the risk marker in the sample to be tested.

3. A kit comprising a detection reagent for detecting genes, mRNA, cDNA, or proteins, or combinations thereof, of risk markers for renal fibrosis. wherein The aforementioned risk markers for renal fibrosis include: CCN5.

4. The kit of claim 3, wherein The diagnostic reagent or kit contains one or more detection reagents selected from the group consisting of: (a) Specific antibodies against CCN5 and specific binding molecules of CCN5; and / or (b) Primers or primer pairs, probes or chips for specific amplification of CCN5 mRNA or CCN5 cDNA.

5. The kit of claim 3, wherein The detection is for in vitro samples; preferably, the in vitro sample is selected from the group consisting of urine, blood, plasma, serum, or combinations thereof.

6. A method of detection, characterized in that Including the following steps: (a) Provide a test sample, said test sample being selected from a urine or blood sample; (b) The expression level of CCN5 in the test sample was detected and denoted as C1; and (c) Compare the CCN5 concentration C1 with the control reference value C0; When C1 / C0 > 1, the test subject is judged to have a high risk of developing renal fibrosis.

7. The method of claim 6, wherein, The sample for testing is urine. If the test result for the risk of renal fibrosis or chronic kidney disease of the test subject meets the following conditions, it indicates that the subject has a high risk of developing renal fibrosis or chronic kidney disease: C1 ≥ 2 ng / ml, preferably ≥ 2.5 ng / ml, more preferably ≥ 3 ng / ml.

8. A diagnostic device for renal fibrosis, the device comprising: (a) An input module configured to input CCN5 data of a biological sample of an object; (b) A processing module configured to process renal fibrosis risk marker data and provide an assessment of the risk of occurrence; wherein the processing includes comparing the expression level C1 of input CCN5 with a control reference value C0; Among them, when C1 is significantly higher than C0, it indicates a high risk of renal fibrosis in the subject; when C1 is not significantly higher than C0, it indicates a low risk of renal fibrosis in the subject; and (c) Output module, which is configured to output the evaluation results.

9. Use of a CCN5 inhibitor for the preparation of a composition or formulation, said composition or formulation for use selected from the group consisting of: (i) Inhibit the expression levels of Fibronectin, COL1A1, Vimentin, and α-SMA proteins; (ii) Inhibit the levels of Fibronectin, COL1A1, Vimentin, and α-SMA mRNA; (iii) alleviating or treating renal fibrosis or chronic kidney disease in the subject.

10. Use according to claim 9, characterized in that, The pharmaceutical composition is administered to a subject with high expression of CCN5.

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