CN118028453A

CN118028453A - Application of CXCL1 and TNFRSF12A in AKI diagnosis

Info

Publication number: CN118028453A
Application number: CN202410241409.6A
Authority: CN
Inventors: 郭丽丽; 李霄阳; 周向阳; 康慧霞; 马月宏; 张越; 柴启薇; 葛海军; 赵欣; 李荣山
Original assignee: Shanxi Provincial Peoples Hospital
Current assignee: Shanxi Provincial Peoples Hospital
Priority date: 2024-03-04
Filing date: 2024-03-04
Publication date: 2024-05-14

Abstract

The application belongs to the technical field of molecular diagnosis, and particularly relates to application of CXCL1 and TNFRSF12A in AKI prediction, which realizes effective prediction of AKI diseases through joint detection of CXCL1 and TNFRSF12A and has the advantages of remarkable sensitivity and specificity.

Description

Application of CXCL1 and TNFRSF12A in AKI diagnosis

Technical Field

The application belongs to the technical field of biology, and particularly relates to application of CXCL1 and TNFRSF12A in AKI prediction and diagnosis.

Technical Field

AKI (acute kidney injury) is a global public health problem. Kidney disease is usually manifested as asymptomatic, and diagnosis thereof depends mainly on the interpretation of altered renal function or reduced urine output. Current diagnostic criteria for AKI are as follows: (1) urine volume <0.5mL/kg/h for 6 hours; (2) Assuming that impairment of kidney function occurs within 7 days, scr rises to ≡1.5 times the baseline; or (3) a Scr increase of 0.3mg/dL or more within 48 hours. However, changes in urine volume and serum ALB are neither sensitive nor specific for detecting AKI. With researchers having more insight into the underlying molecular mechanisms of AKI, more and more biomarkers have been identified for diagnosing and identifying AKI, such as kidney injury molecule 1 (KIM-1), cystatin-C, and neutrophil gelatinase-associated lipocalin (NGAL), etc., but these indicators are also not sufficiently reliable because they are also insensitive to mild kidney injury. Novel biomarkers of AKI must be developed to enable early diagnosis of mild kidney injury.

Chemokine (CXC motif) ligand 1 is a small molecule cytokine belonging to the CXC chemokine family and expressed by macrophages, neutrophils and epithelial cells and has a cell chemotactic effect on neutrophils. CXCL1 binds to the chemokine receptor CXCR2 and acts as a cell chemotaxis. CXCL1 has main effects including neovascularization, inflammatory reaction, wound healing, tumor formation and the like, and can be used as a gastric cancer progression molecular marker and the like. TNFRSF12A is a TNFSF12/TWEAK receptor, acting as a weak apoptosis inducer in specific cell types, and also promoting angiogenesis, endothelial cell proliferation, and possibly regulating cell adhesion to matrix proteins, TNFRSF12A can serve as a potential immune prognostic marker for gliomas.

Disclosure of Invention

In order to solve the technical problems, the CXCL1 and the TNFRSF12A are found and proved to have obvious disease indication effects in AKI prediction and diagnosis through animal model construction, histology analysis and clinical sample detection, and the application is put forward.

The specific technical scheme of the application is as follows:

the application firstly provides an application of CXCL1 and TNFRSF12A serving as markers in AKI diagnosis.

The application also provides the use of a detector for obtaining CXCL1 and TNFRSF12A levels in a sample for the preparation of a product for diagnosing AKI.

In some aspects, the level comprises a nucleic acid level or a protein level.

In some aspects, the nucleic acid level comprises a DNA level or an RNA level.

In some aspects, the nucleic acid level or protein level includes, but is not limited to, the abundance or concentration of a nucleic acid or protein; mutant abundance or concentration, such as nucleic acid or protein, may also be included in some aspects.

Further, the nucleic acid level is obtained by a sequencing technique, a nucleic acid amplification technique, a nucleic acid hybridization technique, an electrophoresis technique, a biological mass spectrometry technique, or a chromatography technique.

Further, nucleic acid level acquisition methods include, but are not limited to, any of the following: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip, probe hybridization, gel electrophoresis, northern blotting, nucleic acid mass spectrometry, or liquid chromatography.

Further, the protein level is obtained by sequencing techniques, immunological techniques, electrophoretic techniques, biological mass spectrometry techniques, or chromatographic techniques.

Further, protein level acquisition methods include, but are not limited to, any of the following: amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein mass spectrometry or liquid chromatography.

In some aspects, the sample described above comprises one or more of tissue, cells, body fluids, serum, plasma, whole blood, urine, semen, saliva, hydrothorax, ascites, stool, or synovial fluid; preferably, the sample is serum, plasma, whole blood or urine.

The application also provides a product for diagnosing AKI comprising a detector of CXCL1 and TNFRSF12A levels in a sample obtained.

In some aspects, the product is in the form of a kit, including a diagnostic kit.

In some aspects, the level comprises a nucleic acid level or a protein level.

In some aspects, the nucleic acid level comprises a DNA level or an RNA level.

In some aspects, the sample comprises one or more of tissue, cells, body fluids, serum, plasma, whole blood, urine, semen, saliva, hydrothorax, ascites, stool, or synovial fluid; preferably, the sample is serum, plasma, whole blood or urine.

The application also provides a method of diagnosing or prognosticating AKI in vivo or in vitro, comprising the step of detecting CXCL1 and TNFRSF12A levels in a sample of a subject.

Further, the method comprises the steps of:

(i) Obtaining CXCL1 and TNFRSF12A levels in a sample of said subject in the sample;

(ii) Comparison of CXCL1 and TNFRSF12A levels with control samples;

wherein a significant difference in CXCL1 and TNFRSF12A levels in the subject sample and the control sample is an indication that the subject has AKI; or comparing with a set threshold absolute amount; wherein a sample level of the subject above a threshold absolute amount is an indication that the subject has AKI.

There is also provided a method of detecting a marker in a subject suffering from or suspected of suffering from AKI in vivo or in vitro comprising determining or detecting CXCL1 and TNFRSF12A levels in a sample from the subject

The application has at least the following beneficial technical effects:

the application proves that CXCL1 and TNFRSF12A are used as combined markers for the prediction of AKI diseases, and the sensitivity and specificity of the prediction have very remarkable diagnosis advantages, and are more suitable for clinical diagnosis application.

Drawings

Figure 1. Construction and validation results of aki model. Wherein, (a-C) describes a flow chart of IRI, cisplatin or LPS-induced AKI model construction; (D-E) is a representative histological image depicting HE staining of IRI, cisplatin or LPS-induced control mice and AKI model mice; F-G is the kidney injury score of mice of the IRI, cisplatin or LPS-induced AKI model; (H-J) is the Scr level of the IRI, cisplatin or LPS-induced AKI model mice compared to the control group. Compared with control mice, IRI, cisplatin or LPS induced AKI model mice have (K-M) BUN levels ^*p≤0.05,^**p≤0.01,^*** p.ltoreq.0.001.

FIG. 2 shows the results of a functional enrichment analysis of DEPs between the cisplatin-induced AKI group and the control group. Wherein (a) volcanic plot shows the distribution of 92 inflammation-related biomarkers (red, upregulated DEP; gray, no difference; blue, downregulated DEP) in AKI patients after cisplatin treatment compared to control group; (B) a thermal map of significant DEP between cisplatin and control sample; (C) A box scatter plot depicting expression levels ^*p≤0.05,^**p≤0.01,^***p≤0.001,^*** p.ltoreq.0.0001. NC for 12 DEPs between cisplatin-treated samples and control samples, healthy control group; SP, cisplatin-induced AKI group. (D) the first 7 enrichment KEGG enrichment analyses of DEP; (E) Analysis of the enriched DEP resulted in the first 20 enriched GO pathways.

FIG. 3 identification of DEG in IRI-, cisplatin or LPS-induced AKI dataset. Wherein, (a-C) is a difference in major component in samples receiving IRI, cisplatin or LPS treatment compared to control; (D-F) is a volcanic plot depicting DEG identified in IRI-cisplatin or LPS-induced AKI dataset; (G-I) based on their respective P values, a heat map was generated to visualize the first 50 DEG identified in the IRI-cisplatin or LPS-induced AKI dataset.

FIG. 4 enrichment analysis and qPCR validation results of co-DEG. Wherein, (a) Venn plot shows the overlap of four AKI datasets up-regulating DEG; (B) Correlation of CXCL1 and TNFRSF12A in transcriptome and Olink proteome; (C-E) comparing mRNA expression levels of CXCL1 in IRI, cisplatin or LPS-induced AKI mouse kidney tissue with kidney tissue of a control mouse; (F-H) mRNA expression levels of TNFRSF12A in kidney tissue of IRI, cisplatin or LPS-induced AKI mice were compared with expression levels of kidney tissue of control mice at ^*p＜0.05,^** p < 0.01.

FIG. 5 verification of co-DEGs in serum of AKI mice. Wherein, (a-C) serum levels of CXCL1 protein of IRI, cisplatin or LPS-induced AKI mice; (D-F) serum levels ^*p＜0.05^**, p < 0.01 of TNFRSF12A protein from IRI, cisplatin or LPS-induced AKI mice compared to control; (G) Calculating Pearson correlation coefficients to evaluate the relationship between CXCL1 protein levels measured by ELISA (X-axis) and Olink assays (Y-axis); (H) Pearson correlation coefficients were calculated to evaluate the relationship between TNFRSF12A protein levels measured by ELISA (X-axis) and Olink assay (Y-axis).

FIG. 6 verification of co-DEGs in serum of AKI patients in clinical practice and correlation with clinical features. Wherein (a) the level of CXCL1 protein in serum of an AKI patient in clinical practice; (B) TNFRSF12A protein levels in serum of AKI patients in clinical practice. (C-E) correlation between CXCL1 and serum creatinine (Scr), BUN and Glomerular Filtration Rate (GFR) of hospitalized AKI patients (n=30) and healthy individuals (n=7) detected by ELISA; (E-F) correlation between TNFRSF12A expression and serum Scr and BUN levels in hospitalized AKI patients (n=30) and healthy individuals (n=7) was examined by ELISA. Pearson correlation analysis was used to analyze the correlation.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Partial term definition

Unless defined otherwise hereinafter, all technical and scientific terms used in the detailed description of the application are intended to be identical to what is commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present application.

As used in this application, the indefinite or definite article when used in reference to a singular noun e.g. "a" or "an", "the" includes a plural of that noun.

As used herein, the terms "comprising," "including," "having," "containing," or "involving," are inclusive (inclusive) or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If a certain group is defined below to contain at least a certain number of embodiments, this should also be understood to disclose a group that preferably consists of only these embodiments.

The term "about" in the present application means a range of accuracy that one skilled in the art can understand while still guaranteeing the technical effect of the features in question. The term generally means a deviation of + -10%, preferably + -5%, from the indicated value.

Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the application described herein are capable of operation in other sequences than described or illustrated herein.

The above terms or definitions are provided solely to aid in the understanding of the present application. These definitions should not be construed to have a scope less than understood by those skilled in the art.

Diagnostic use

The CXCL1 and the TNFRSF12A are screened based on the raw information analysis and can serve as potential AKI prediction markers, and clinical samples are further verified to be capable of accurately predicting AKI through PCR and ELISA wet experiments, so that the application provides diagnostic application of the CXCL1 and the TNFRSF12A in AKI.

In particular, in aspects of the present disclosure, diagnostic uses are provided that include CXCL1 and TNFRSF12A as markers for AKI, as well as the use of a detector to detect CXCL1 and TNFRSF12A in a sample in the preparation of a product for diagnosing AKI.

The terms "sample," "specimen," "test sample," "subject sample," and the like as used herein include various sample types obtained from a patient, individual, or subject and useful in diagnostic or monitoring assays. Patient samples may be obtained from healthy subjects, diseased patients, or patients with AKI-related symptoms. Furthermore, a sample obtained from a patient may be divided into parts, and only a part may be used for diagnosis. In addition, the sample or a portion thereof may be stored under conditions that hold the sample for subsequent analysis. This definition specifically includes blood and other liquid samples of biological origin (including but not limited to tissues, cells, body fluids, serum, plasma, whole blood, urine, semen, saliva, hydrothorax, ascites, cerebrospinal fluid, stool, synovial fluid, and the like). In a specific embodiment, the sample comprises a urine sample. In a specific embodiment, the sample comprises a blood sample. In another embodiment, a serum sample is used. The definition also includes samples that are manipulated in any way after sample retrieval, such as by centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washing or enrichment of certain cell populations. These terms also include clinical samples, and also include cells in culture, cell supernatants, tissue samples, organs, and the like. Samples may also include freshly frozen and/or formalin fixed, paraffin embedded tissue blocks, such as blocks prepared from clinical or pathological biopsies, prepared for pathological analysis or study by immunohistochemistry. The sample may be tested immediately after collection, stored at RT, 4 degrees celsius, -20 degrees celsius, or-80 degrees celsius, and tested after 24 hours, 1 week, 1 month, 1 year, 10 years, or up to 30 years of storage.

The terms "individual," "subject," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, in need of diagnosis, treatment, or therapy.

Obtaining a test agent for CXCL1 and TNFRSF12A levels in a sample can be understood to include directly obtaining a test agent for CXCL1 and TNFRSF12A levels in a sample, or indirectly obtaining a test agent for CXCL1 and TNFRSF12A levels in a sample.

According to some embodiments of the application, the marker level indicator may be obtained at any of a nucleic acid level, a protein level, and the like. The detection method is not limited, but any method that can be used to directly or indirectly evaluate the levels of CXCL1 and TNFRSF12A nucleic acids or proteins is suitable for the present application. It is understood that the nucleic acid level may include a DNA level or an RNA level. There are a variety of ways in the art for DNA or RNA level detection, including but not limited to by sequencing techniques, nucleic acid amplification techniques, nucleic acid hybridization techniques, electrophoresis techniques, biological mass spectrometry techniques, or chromatographic techniques, which can be used in the present application. In some embodiments of the application, including but not limited to any of the following specific methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip, probe hybridization, gel electrophoresis, northern blotting, nucleic acid mass spectrometry, or liquid chromatography.

For example, in particular embodiments, the polymerase chain reaction method includes primers for CXCL1 and TNFRSF12A amplification, wherein the primers refer to primers capable of specifically amplifying CXCL1 and TNFRSF12A genes, respectively, such as polynucleotides of a certain length, e.g., about 35 nucleotides or longer, that are capable of hybridizing to at least a portion of a template sequence and serve as starting sites for synthesizing primer extension products. In some embodiments, the probe hybridization method includes probes of CXCL1 and TNFRSF12A, wherein a probe refers to a probe capable of specifically recognizing the CXCL1 and TNFRSF12A genes or transcripts of the genes, respectively, which is a molecule capable of binding to a specific sequence or subsequence or other portion of another molecule, typically a nucleic acid probe that binds to another nucleic acid (i.e., the specific sequences of CXCL1 and TNFRSF12A as target nucleotides) by complementary base pairing. In some embodiments, the gene chip refers to a composite structure formed by immobilizing an array of the aforementioned probes on a substrate material (including, but not limited to, polymers such as nylon membrane, nitrocellulose membrane, glass, etc.). In some embodiments, reverse transcription is required to obtain cDNA when RNA detection is desired, and detection is performed by CXCL1 and TNFRSF12A amplified primers.

It will be appreciated that there are a variety of protein detection means in the art including, but not limited to, sequencing techniques, immunological techniques, electrophoretic techniques, biological mass spectrometry techniques or chromatographic techniques, which can be used in the present application; in some embodiments of the application, including but not limited to any of the following specific methods: amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein mass spectrometry or liquid chromatography.

For example, in some embodiments, specific antibodies are included in the method of quantitatively detecting CXCL1 and TNFRSF12A at the protein level. Wherein specific antibodies refer to antibodies capable of specifically recognizing CXCL1 and TNFRSF 12A-encoded proteins, respectively, and specifically include, but are not limited to, at least one of monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies having a desired biological activity), nanobodies, or including certain antibody fragments. It is understood that the antibody may be a human, humanized and/or affinity matured antibody.

It will be appreciated that in order to further enhance the diagnostic effect on AKI, the biomarkers detected by the reagents may also be other diagnostic nucleic acid fragments, proteins, metabolites etc. known to those skilled in the art, which may be used as markers, in such a way that the combined use of multiple biomarkers is achieved, in combination with each other, to achieve a better diagnostic effect on AKI patients, and thus a more efficient diagnostic assessment.

In some embodiments of the application, a test sample according to the application may be selected from the group consisting of tissue, cells, body fluid serum, plasma, whole blood (peripheral blood), urine, semen, saliva, hydrothorax, ascites, cerebrospinal fluid, stool, and synovial fluid; in a preferred embodiment, the test sample is selected from any one of serum, plasma, whole blood or urine.

Diagnostic method

The levels of CXCL1 and TNFRSF12A in AKI patients were found to be higher than those of CXCL1 and TNFRSF12A in healthy persons, so that diagnosis of AKI can be predicted more accurately based on the levels (such as expression levels) of CXCL1 and TNFRSF 12A.

The core of the diagnostic method comprises the step of detecting or determining CXCL1 and TNFRSF12A in a sample of a subject;

in some embodiments, the method comprises the steps of:

(i) Detecting or determining the level of CXCL1 and TNFRSF12A in the test sample;

(ii) Comparison of CXCL1 and TNFRSF12A levels with control samples; wherein the presence of a significant difference in the levels of CXCL1 and TNFRSF12A in the test sample and the control sample is an indication that the subject has AKI;

Or alternatively

(Ii) Comparing with a set threshold absolute amount; wherein a sample level of the subject above a threshold absolute amount is an indication that the subject has AKI.

In some embodiments, the "level of CXCL1 and TNFRSF 12A" or "CXCL1 and TNFRSF12A level" herein includes, but is not limited to, the abundance or concentration of CXCL1 and TNFRSF12A nucleic acid or protein, such as in some specific embodiments, the expression abundance or concentration of the corresponding protein.

It will be appreciated that the control sample may be selected according to actual needs, for example, in disease diagnosis, the control sample is a normal population sample, and when used for prognosis evaluation, the control sample may be a control sample of different prognosis.

In some specific embodiments, a set value of CXCL1 and TNFRSF12A expression levels may be given, which may be determined based on the expression levels of CXCL1 and TNFRSF12A in normal samples of normal humans and/or non-AKI patients, e.g., selecting an average value of CXCL1 and TNFRSF12A expression levels in normal samples of an appropriate number of samples, or setting a reasonable multiple, such as 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, etc., based on the average value, when the expression levels of CXCL1 and TNFRSF12A in a subject are higher than the set value, the AKI is determined. It will be appreciated that the need for a set point determined based on the mean, or a multiple of the mean, has a good classification meaning, and that known samples can be tested by conventional statistical testing methods based on the classification of the set point, and that the set point can be used as a criterion when the result has a statistical meaning. Where the levels of CXCL1 and TNFRSF12A are values indicating that this biomarker is derived for the subject either directly or further indirectly from direct measurements, which typically is derived at least in part from the abundance or concentration of the biomarker in the sample of the subject. Wherein the indirectly derived values are derived by applying a function to the measured value of this biomarker. Direct measurements include, but are not limited to, values of biomarkers determined by at least one of sequencing, hybridization, mass spectrometry, immunoassays, immunofluorescence, flow cytometry, and the like.

Product(s)

According to the core diagnostic use of the present application, the procedure for detecting CXCL1 and TNFRSF12A levels can be configured in the form of corresponding products for diagnosis or prediction of AKI. The product includes reagents for detecting CXCL1 and TNFRSF12A biomarkers, respectively. It will be appreciated that such product formats are diverse, including but not limited to kit formats.

Kit form

In some embodiments of the application, kits for detecting or analyzing CXCL1 and TNFRSF12A levels to predict AKI disease are also disclosed herein. Such a kit may comprise reagents for detecting the levels of both markers and instructions for predicting AKI disease based on the detected levels.

The kit may comprise a set of reagents for generating a data set by at least one assay. This set of reagents was able to detect quantitative CXCL1 and TNFRSF12A levels, respectively. The set of reagents may also further detect the level of one or more other markers. In certain aspects, the agent is detected at the level of a nucleic acid or protein.

When the detection agent is detected at the nucleic acid level, it is understood that there are a variety of nucleic acid detection means in the art, including but not limited to by sequencing techniques, nucleic acid amplification techniques, nucleic acid hybridization techniques, electrophoresis techniques, biological mass spectrometry techniques, or chromatographic techniques, which can be used in the present application. In some embodiments of the application, including but not limited to any of the following specific methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip, probe hybridization, gel electrophoresis, northern blotting, nucleic acid mass spectrometry, or liquid chromatography.

Thus, for example, in some embodiments, the reagents may be sequencing reagents, such as a first generation Sanger sequencing reagent, a second generation sequencing ("NGS") reagent, a third generation sequencing reagent, and the like. In other embodiments, the reagents are PCR primer reagents, wherein a primer refers to a primer capable of specifically amplifying CXCL1 and TNFRSF12A genes, respectively, such as a polynucleotide of a certain length, e.g., a primer of about 35 nucleotides or more, capable of hybridizing to at least a portion of a template sequence and serving as a starting site for the synthesis of a primer extension product. In other embodiments, the reagent is a probe reagent, wherein the probe is a probe capable of specifically recognizing the CXCL1 and TNFRSF12A genes or transcripts of the genes, respectively, which is a molecule capable of binding to a specific sequence or subsequence or other portion of another molecule, typically a nucleic acid probe that binds to another nucleic acid (i.e., the specific sequence of CXCL1 or TNFRSF12A as target nucleotide) by complementary base pairing.

When the detection agent is detected at the protein level, it is understood that there are a variety of protein detection means in the art including, but not limited to, sequencing techniques, immunological techniques, electrophoretic techniques, biological mass spectrometry techniques, or chromatographic techniques, which can be used in the present application; in some embodiments of the application, including but not limited to any of the following specific methods: amino acid sequencing, enzyme linked immunosorbent assay, radioimmunoassay, immunohistochemical method, immunoblotting, flow cytometry, gel electrophoresis, protein mass spectrometry or liquid chromatography.

Thus, for example, in some embodiments, the agent is a specific antibody, wherein specific antibody refers to an antibody capable of specifically recognizing CXCL1 and TNFRSF 12A-encoded proteins, respectively, specifically including but not limited to at least one of a monoclonal antibody (e.g., full length or intact monoclonal antibody), a polyclonal antibody, a multivalent antibody, a multispecific antibody (e.g., bispecific antibody having a desired biological activity), or including certain antibody fragments. It is understood that the antibody may be a human, humanized and/or affinity matured antibody.

In some embodiments, such kits may include a carrier, package, or container that is compartmentalized to receive one or more containers, such as vials, tubes, and the like, each of the containers comprising one of the individual elements to be used in the method. Kits of the application may include a container as described above as well as one or more other containers containing materials required from a commercial end-user standpoint, including buffers, diluents, filters, and package insert with instructions for use.

In some embodiments, the kit further includes a sample processing reagent, which may include at least one of a sample lysing reagent, a sample purifying reagent, and a sample extracting reagent.

In some embodiments, the product further comprises at least one of a standard, a calibrator, a control, and a buffer. Wherein the reference substance is a control substance for checking the validity of the experiment and is used as a comparison of the judging result. The buffer may be any solution known in the art that provides suitable buffer conditions during the detection process.

The kit may include instructions for use of the set of reagents. For example, the kit may comprise instructions for performing at least one assay, such as an immunoassay, a protein binding assay, an antibody-based assay, an antigen binding protein-based assay, a protein-based array, an enzyme-linked immunosorbent assay (ELISA), flow cytometry, a protein array, blotting, western blotting, nephelometry, chromatography, mass spectrometry, enzymatic activity, and an immunoassay selected from RIA, immunofluorescence, immunochemiluminescence, immunoelectrochemiluminescence, immunoelectrophoresis, competitive immunoassay, and immunoprecipitation.

In addition to the above, the kit will further comprise instructions for carrying out the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is that they are printed information on a suitable medium or substrate, for example, one or more sheets of paper on which the information is printed, in the packaging of the kit, in the form of package inserts, and the like.

The application is illustrated below in connection with specific embodiments.

Example 1 construction of AKI animal model and injury evaluation

1. Mouse model construction

C57BL/6 mice (female, 6-8w,20-25 g) were supplied by the Shanxi province people hospital animal service center and kept in pathogen-free environment. All animal experiments were approved by the institutional animal care and use committee of the national hospitals affiliated with the university of mountain western medicine. Cisplatin-induced AKI mice were given cisplatin by a single intraperitoneal injection (20 mg/kg; HY-17394, medChemExpress). After 72 hours, the mice were anesthetized and sacrificed. Lipopolysaccharide (LPS) -induced AKI mice were intraperitoneally injected with LPS (L2630, sigma-Aldrich) at a dose of 10mg/kg body weight and animals were sacrificed 24 hours after LPS injection. As a negative control, physiological saline was used as a comparison with cisplatin or LPS group.

Kidney IRI mice were anesthetized by intraperitoneal administration of sodium pentobarbital at a dose of 50 mg/kg. A micro vascular clamp was used to block bilateral renal blood supply for 30 minutes to induce complete renal ischemia. Mice were euthanized 24 hours after the subsequent infusion. The negative surgery group received the same surgery, except that the renal pedicles were clamped. Blood samples were collected, serum was isolated to determine creatinine levels, urea nitrogen detection, and Olink sequencing. Kidneys were collected for paraffin embedding and RNA extraction.

Three AKI mouse models, i.e., LPS, cisplatin, and IRI-induced AKI mice, were established by the methods described above, with specific results shown in figures 1A-1C. HE staining showed pathological changes in the AKI group, such as significant swelling of tubular epithelial cells, brush border elimination, and renal cavity expansion (fig. 1D and 1E).

2. Tubular injury degree scoring

Kidney tissue was fixed using 4% paraformaldehyde solution and subsequently embedded in paraffin. Tissue sections with a thickness of 4 μm were histologically analyzed after staining with Hematoxylin and Eosin (HE). The extent of tubule injury is assessed by scoring the percentage of tubules that characterize tubule necrosis, mold formation, and tubule expansion.

The results are shown in figures 1F and 1G, with significantly higher tubular injury scores for the AKI group.

3. Measurement of renal injury

The level of Scr was measured using a creatinine (Cr) measurement kit (C011-2-1; nanjing Jiangcheng bioengineering institute), and the level of BUN was measured using a urea measurement kit (C013-2-1; nanjing Jiangcheng bioengineering institute) to evaluate kidney damage.

The results are shown in figures 1H-1M, with significantly elevated BUN and Scr levels in serum of AKI mice compared to control group, demonstrating successful establishment of AKI model.

Example 2 screening and analysis of AKI biomarkers

1. Olink-based differential protein level analysis

1) Olink detection: plasma samples from cisplatin-induced AKI or control mice were analyzed using Olink inflammatory panels containing 92 inflammatory biomarkers to evaluate and compare the protein expression levels of the 92 proteins. Briefly, each target protein is bound to a pair of antibodies by a unique nucleotide sequence probe, and the correct and orthotopic probes are bound by complementary pairs of 5-bp base pairs at the ends to form a double stranded template under the action of an elongase, followed by qPCR or NGS detection. The final test values are expressed as normalized protein expression values after log ² transformation.

2) DEP analysis and functional enrichment analysis: DEP between cisplatin group and control group by R software package'Analyze "for identification. The p-value of DEP is less than 0.05. Visualization of DEP is performed by volcanic and thermal maps through the R package "ggpolt 2". To elucidate the functional role of DEPs and to determine DEPs-rich pathways, gene Ontology (GO) and kyoto gene and genome encyclopedia (KEGG) analyses were performed using an R-package "cluster analyzer".

The experimental results are as follows:

The information about DEP is shown in Table 1. The levels of 11 different proteins were significantly increased in the AKI group and decreased in the 1 protein group compared to the control group (fig. 2A). FIG. 2B shows a heat map of the differentially expressed proteins, and FIG. 2C shows the differences in protein expression between the two groups.

Table 1, differences in plasma inflammatory proteins between AKI and control.

^a FC (fold change) is calculated as a ratio of log2 between AKI and NC. The ^b p values calculated using Student t-test. Shows significantly different proteins (p < 0.05).

To further understand DEPs functional features, the results of GO and KEGG pathway enrichment analysis performed showed that the KEGG pathway is rich in a number of annotated proteins that are mostly involved in cytokine-cytokine receptor interactions (fig. 2D). Furthermore, GO enrichment analysis showed that in cisplatin-induced AKI, these protein-enriched pathways were negative regulators of the activin receptor signaling pathway (fig. 2E).

2. PCA-based differential expression gene analysis

Olink data intuitively reflect changes in protein levels, whereas mRNA levels may reflect intermediate states of gene expression, part of which performs differential analysis of mRNA levels in model mice. This section provides a thorough analysis of transcriptome data obtained from mice injected intraperitoneally with cisplatin for 72 hours, with LPS for 24 hours, or with ischemia reperfusion surgery for 24 hours.

1) Microarray data acquisition: the data sets GSE165100, GSE108195 and GSE227623 (https:// www.ncbi.nlm.nih.gov/GEO /) related to AKI in GEO are screened for the public database to obtain the corresponding gene expression matrix, and the detailed information is shown in Table 2.

TABLE 2 information of selected microarray dataset

2) Differential expression gene screening in multiple AKI models

Principal Component Analysis (PCA) was performed to initially evaluate sample conditions under different treatments. The Limma package of R statistical analysis software is used to identify the different sets of DEG. DEG was analyzed from a series of matrices obtained from GSE165100, GSE108195, and GSE227623 using Limma according to the criteria of P <0.05 and |log2 Fold Change (FC) | > 1.2. Volcanic and thermal maps are generated by the R software package "ggplot" for visualizing DEG.

The experimental results are as follows: principal Component Analysis (PCA) was used to determine principal component differences between the different groups (fig. 3A-3C). Cisplatin, LPS or ischemia reperfusion groups showed significant differences compared to the control or sham groups. 1848 DEG were identified in total between LPS treated and control groups, 912 up-regulated and 936 down-regulated DEG were detected (FIG. 3D). A total of 5381 DEG were found in cisplatin treated and control groups; these DEG consist of 3361 upregulated DEG and 2020 downregulated DEG (FIG. 3E). In addition, 672 DEG, including 426 up-regulated and 246 down-regulated DEG, were present in the IRI group and the sham operation group altogether (FIG. 3F). Heat maps of the first 50 genes with statistical significance of low P values are shown (fig. 3G-3I).

3. Combined analysis and qPCR validation of transcriptome and Olink proteome in vivo

1) To study the crossover point between transcriptomes and Olink proteomes, overlapping sets of upregulated DEG in transcriptome analysis and upregulated DEP in Olink proteome analysis were identified and visualized using Venn diagram.

The Venn plot results showed that CXCL1 and TNFRSF12A were present in four data sets simultaneously (fig. 4A). The four data sets were then analyzed for correlation between CXCL1 and TNFRSF 12A. The results showed a positive correlation (R ² =0.59, p= 0.000096) between CXCL1 and TNFRSF12A (fig. 4B).

2) Next, the present application validated the expression levels of CXCL1 and TNFRSF12A in mice. Specifically, kidney tissue was collected at designated time points and total RNA was isolated using TRIzol reagent (TaKaRa). Subsequently, first strand cDNA synthesis was performed using the first strand synthesis system from TransGen (China). Gene expression levels were determined by qRT-PCR performed on the ABI 7500 real-time PCR system using the comparative Cycle Threshold (CT) method. Table 3 lists specific primers for qPCR detection. The data were normalized to the expression level of the endogenous gene β -actin in each sample.

TABLE 3 primers used in quantitative real-time PCR

MRNA analysis results showed that CXCL1 and TNFRSF12A expression was significantly up-regulated in LPS, cisplatin and IRI groups compared to control groups (FIGS. 4C-H).

All the above results indicate that CXCL1 and TNFRSF12A are likely to be involved in the pathogenesis of LPS, cisplatin or IRI-induced kidney cell damage in vivo.

Example 3 ELISA validation of CXCL1 and TNFRSF12A in AKI mouse plasma

CXCL1 and TNFRSF12A were identified as biomarker proteins based on Olink and integration of transcriptome data. Thus, the levels of CXCL1 and TNFRSF12A in AKI mice were quantified using an ELISA kit. 24 plasma samples were collected from mice, including control mice and mice induced with IRI, LPS and cisplatin (6 mice per group). The obtained data was analyzed using single factor analysis.

The results showed that the levels of CXCL1 and TNFRSF12A were increased in IRI, LPS and cisplatin-induced AKI mouse serum samples (fig. 5A-F). Furthermore, there was a correlation between multiplex analysis using Olink data and ELISA results (fig. 5G and H).

Example 4 clinical sample testing

To further verify that CXCL1 and TNFRSF12A are potential predictive biomarkers in AKI, this example detects CXCL1 levels and TNFRSF12A levels in patient samples in a clinical practice environment by ELISA kit to determine their correlation with clinical features, the specific method steps are as follows:

1) Patient sample collection: 37 human plasma samples were collected from the hospital laboratory, including 30 pathologically confirmed AKI patients (using only 48 hours of serum creatinine standard ≡0.3mg/dL or ≡1.5 times the baseline) and 7 negative control patients. Plasma was stored at-80 ℃ until laboratory analysis was performed.

2) ELISA detection: all serum samples were removed from storage at-80 ℃ and thawed on ice prior to the experiment. Mouse serum samples were quantified by using ELISA kits (EM 0003 and EM2139, fineTest). For human serum samples, quantification was performed using ELISA kits (EH 0005 and EH2382, fineTest).

3) Statistical analysis: descriptive statistics include mean (SD). The difference between the two groups was analyzed using the two-tailed Student t-test. Statistical analysis was performed on all data collected. P < 0.05 is considered statistically significant.

The results were as follows: both CXCL1 and TNFRSF12A expression were significantly increased in AKI patients (fig. 6A and 6B), and were statistically significant. In addition, the present application also analyzed the correlation between CXCL1 and TNFRSF12A and clinical features (Scr, BUN and GFR). As shown in fig. 6C, there is a significant positive correlation between CXCL1 and BUN (R ² =0.2633, p < 0.05). Likewise, the results of CXCL1 and Scr are also comparable (R ² =0.1152, p < 0.05), as shown in fig. 6D. GFR is significantly inversely correlated with CXCL1 expression (R ² =0.2260, p < 0.05) (fig. 6E). Furthermore, TNFRSF12A correlated positively with BUN (R ² =0.2702, p < 0.05) and Scr (R ² =0.1773, p < 0.05), but correlated negatively with Glomerular Filtration Rate (GFR) (R ² =0.2581, p < 0.05) (fig. 6E-G). The above results indicate that the combination detection of CXCL1 and TNFRSF12A has a significant disease-indicating effect in AKI prediction.

Finally, it should be noted that the above embodiments are merely illustrative of the technical solution of the present application, and not limiting thereof; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.

Claims

1. Use of a detector of CXCL1 and TNFRSF12A levels in a sample obtained for the preparation of a product for diagnosing AKI.

2. A product for diagnosing AKI comprising a detector of CXCL1 and TNFRSF12A levels in a sample obtained.

3. The use of claim 1 or the product of claim 2, wherein the level comprises a nucleic acid level or a protein level.

4. The use or product according to claim 3, wherein the nucleic acid level is obtained by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

5. The use or product according to claim 3, wherein the protein level is obtained by sequencing techniques, immunological techniques, electrophoretic techniques, biological mass spectrometry techniques or chromatographic techniques.

6. The use according to claim 1 or the product according to claim 2, wherein the sample comprises one or more of tissue, cells, body fluid, serum, plasma, whole blood, urine, semen, saliva, hydrothorax, ascites, faeces or synovial fluid; preferably, the sample is serum, plasma, whole blood or urine.

Use of cxcl1 and TNFRSF12A as markers in AKI diagnosis.

8. A method of diagnosing AKI in vivo or in vitro comprising the step of detecting CXCL1 and TNFRSF12A levels in a sample of a subject.

9. The method according to claim 8, characterized in that it comprises the steps of:

(ii) Comparison of CXCL1 and TNFRSF12A levels with control samples;

10. A method of detecting a marker in a subject having or suspected of having AKI in vivo or in vitro, comprising determining or detecting CXCL1 and TNFRSF12A levels in a sample from the subject.