CN116515986A - Application of UBASH3A protein as biomarker for acute kidney injury - Google Patents

Application of UBASH3A protein as biomarker for acute kidney injury Download PDF

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CN116515986A
CN116515986A CN202310175735.7A CN202310175735A CN116515986A CN 116515986 A CN116515986 A CN 116515986A CN 202310175735 A CN202310175735 A CN 202310175735A CN 116515986 A CN116515986 A CN 116515986A
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ubash3a
protein
kidney injury
acute kidney
seq
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项荣
赵美方
黄皓
张思源
王琴
原壮壮
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Central South University
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Central South University
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to an application of UBASH3A protein in evaluating toxic and side effects of kidney, belonging to the field of biological medicine. The present invention provides the use of UBASH3A protein or mRNA encoding UBASH3A protein or DNA encoding UBASH3A protein for assessing acute kidney injury. The amino acid sequence of the UBASH3A protein is shown as SEQ ID NO. 1. The sequence of the mRNA for encoding UBASH3A protein is shown as SEQ ID NO. 2. The CDS sequence of the DNA encoding UBASH3A protein is shown in SEQ ID NO. 3. By detecting UBASH3A levels in a patient, the likelihood and/or extent of onset of nephrotoxic side effects in the patient after cisplatin administration can be detected/predicted, thereby providing guidance as to whether or not the individual is administered cisplatin.

Description

Application of UBASH3A protein as biomarker for acute kidney injury
Technical Field
The invention relates to an application of UBASH3A protein or mRNA or DNA encoding UBASH3A protein in preparing a biomarker for acute kidney injury, belonging to the field of biological medicine.
Background
Acute kidney injury (Acute kidney injury, AKI) is an acute injury to renal function resulting from infection, drug intake, post-surgical procedures, etc., and is closely related to the prognosis of the patient's life. Acute Kidney Injury (AKI) is a condition in which renal function decreases sharply in a short period of time, from hours to days. Waste in the body cannot be excreted through urine, or overflowed. Dialysis may be required.
Drug-related kidney injury caused by nephrotoxic drugs (e.g., cisplatin, contrast agents, nephrotoxic antibiotics, etc.) accounts for 25% of all AKI cases, and is one of the main causes of AKI. Cisplatin is a common cause of clinical medicine AKI, and can inhibit tumor cell proliferation, and is a powerful chemotherapeutic medicine. However, renal toxicity is the most common side effect of cisplatin and is often manifested as AKI. The mechanism is complex and mainly comprises lipotoxicity, oxidative stress, inflammatory reaction, mitochondrial apoptosis and the like, and the mechanism is still to be studied. It is counted that about 25% -30% of patients experience kidney damage after cisplatin injection.
Diagnostic guidelines for acute Kidney injury have been provided in the past by KDIGO (Kidney Disease: improving Global Outcomes, improving the global prognosis of Kidney Disease). In this KDIGO classification, acute renal failure is diagnosed based on elevated serum creatinine concentration and reduced urine volume. Specifically, (in the kdaigo diagnosis and treatment guideline for acute kidney injury) the stage (stage) classification of AKI is defined by any one of the following:
1. serum creatinine value is raised by more than 0.3mg/dl within 48 hours;
2. serum creatinine values increased by more than 1.5 fold over the previously known or expected basal values within 7 days;
3. the urine volume was reduced to 0.5 ml/kg/hr over 6 hours.
However, with this baseline, the opportunity for therapeutic intervention has mostly been missed.
The AKI early warning markers found at present are mostly specific proteins expressed and secreted when the renal tubule is damaged, wherein neutrophil gelatinase-associated apolipoprotein (NGAL) is reported more, and the marker has better diagnosis effect in experimental animals and clinical patients and has time earlier than that of the traditional marker creatinine, but the detection method is more complex than that of small molecular markers such as creatinine and the like, and the detection is carried out by adopting an enzyme-linked immunosorbent assay. The method has higher sensitivity and specificity, but the operation process is tedious, the detection time is long, and the metabolites in the matrix can cross react with the antibody to interfere the measurement result.
UBASH3A belongs to the UBASH3 family, UBASH3 is a family of ubiquitin-like proteins, and it has now been found that there are mainly two members in humans that have highly conserved N-terminal ubiquitin-related UBA domains, SH3 domains, C-terminal phosphatase domains and dimerization domains, which can form homo-and heterodimers. The research shows that UBASH3 protein is important tyrosine phosphatase in organism, its SH3 structural domain can combine closely with receptor tyrosine kinase phosphorylating residue, form protein complex to signal transduction, participate in basic physiological activities such as cell proliferation, differentiation, etc., regulate and control pathological processes such as cancer cell migration, inflammation, etc. The human UBASH3A gene is located on chromosome 21q22.3, encodes a protein containing 661 amino acids, and is located in both cytoplasm and nucleus; UBASH3A is expressed in spleen, peripheral blood leukocytes, thymus, while it is highly expressed in embryonic kidneys, but hardly expressed in normal adult kidneys. Currently UBASH3A is mainly reported to be associated with primary sclerosing cholecystitis, vitiligo, breast cancer and type 1 diabetes, but there is no study associated with kidney damage, particularly AKI.
Disclosure of Invention
The invention aims to provide a novel biomarker for acute kidney injury.
In order to solve the technical problems, the technical scheme of the invention is as follows:
use of UBASH3A protein or mRNA encoding UBASH3A protein or DNA encoding UBASH3A protein as biomarker for acute kidney injury, wherein the amino acid sequence of UBASH3A protein is shown in SEQ ID No. 1.
Wherein, the cDNA sequence of UBASH3A gene encoding protein can be obtained by reverse transcription of the mRNA sequence of UBASH3A protein and can be directly translated into the protein amino acid sequence of UBASH3A, thus, the cDNA sequence of UBASH3A gene encoding protein can also be used as a detection marker of acute kidney injury.
Preferably, the sequence of the mRNA encoding UBASH3A protein is shown as SEQ ID NO. 2.
The Ensembl number of the gene encoding UBASH3A protein is ENSG000160185, and the CDS sequence of the gene is shown in SEQ ID NO. 3.
Preferably, the acute kidney injury is caused by cisplatin, a contrast agent, or a nephrotoxic antibiotic.
Preferably, the biomarker is a serum diagnostic marker, a plasma diagnostic marker or a histiocyte diagnostic marker.
The invention also claims a kit for acute kidney injury detection comprising a reagent for specifically detecting UBASH3A protein expression level, a reagent for specifically detecting UBASH3A protein-encoding mRNA level, or a reagent for specifically detecting UBASH3A protein-encoding DNA level.
Preferably, the detecting comprises: colloidal gold immunochromatography, immunoblotting, immunohistochemistry, immunofluorescence, flow cytometry, enzyme-linked immunosorbent assay, nucleic acid probe assay or qPCR method. For example, [ Huang M, wang J, torre, et al SAVER: gene expression recovery for single-cell RNA sequencing [ J ]. Nature methods,2018,15 (7): 539-542.], [ Brich S, bozzi F, perrone F, et al Fluorescence In Situ Hybridization (FISH) provides estimates of minute and interstitial BAP1, CDKN2A, and NF2gene deletions in peritoneal mesothelioma [ J ]. Modern Pathology,2019:1-11.], [ Chen C Y, logan R W, ma T, et al effects of aging on circadian patterns of gene expression in the human prefrontal cortex [ J ]. Proceedings of the National Academy of Sciences,2016,113 (1): 206-211.], and the like.
Preferably, the kit comprises one or more of a specific antibody for UBA 3A protein, a specific primer for amplifying mRNA or DNA encoding UBA 3A protein, and a probe or chip for detecting mRNA or DNA encoding UBA 3A protein.
For example, the commercial antibody Cat No. GTX116432 against UBASH3A protein.
For example, a primer specific for UBACH 3A gene is designed to detect UBACH 3A gene using polymerase chain reaction, and a primer specific for UBACH 3A gene is designed to detect mRNA of UBACH 3A using reverse transcription polymerase chain reaction.
Preferably, the sequences of the specific primers for amplifying mRNA or gene encoding UBASH3A protein are shown as SEQ ID NO.4 and SEQ ID NO. 5.
Preferably, the specific primer for amplifying mRNA or gene encoding UBASH3A protein further comprises a PCR amplification specific primer of internal reference gene GAPDH, and the sequences are shown as SEQ ID NO.6 and SEQ ID NO. 7.
Preferably, the test sample is a human serum sample, whole blood sample, and tissue cell sample, as well as other human cell lines.
The invention also provides application of the kit in preparation of an acute kidney injury detection reagent.
The invention uses the expression or content level of any one of the following detection objects (1), (2) and (3) as a marker of acute kidney injury:
(1) UBASH3A protein;
(2) mRNA encoding said UBASH3A protein;
(3) DNA encoding the UBASH3A protein.
The specific method comprises the following steps: and collecting a sample of the tested person and comparing the sample with the expression level of the UBASH3A protein or the mRNA or the gene in a normal control person or a standard sample. According to the comparison result, detecting/predicting whether the sample to be detected has cisplatin nephrotoxic side effect reaction according to the following method: if the expression level of the UBASH3A protein or the mRNA or the gene in the sample to be tested is higher than the normal expression level of the UBASH3A protein or the mRNA or the gene in the normal control or the standard sample, the sample to be tested is more likely to generate cisplatin nephrotoxic side effect reaction; conversely, when the expression level of the UBASH3A protein or the mRNA or the gene in the test sample is lower than or equal to the normal expression level of the UBASH3A protein or the mRNA or the gene in the normal control or the standard sample, the test sample is free of cisplatin nephrotoxic side effect or the cisplatin nephrotoxic side effect may have a lower degree of reaction; the control is a healthy human or a normal cell line of human origin; preferably, the sample to be tested is serum or normal cell lysate. Wherein the sample of the tester includes, but is not limited to, a serum sample of a patient, a whole blood sample, a tissue cell sample and a relevant cell lysate.
According to the prior research, UBASH3A participates in a plurality of processes of acute kidney injury reaction caused by medicaments such as cisplatin and the like, plays an important role in oxidative stress, inflammatory reaction and apoptosis necrosis, and can be used as an important marker for occurrence and development of cisplatin kidney toxic side effect reaction. By detecting UBASH3A levels in a patient, the likelihood and/or extent of onset of nephrotoxic side effects in the patient after cisplatin administration can be detected/predicted, thereby providing guidance as to whether or not the individual is administered cisplatin.
Drawings
FIG. 1-1 shows the measurement of serum creatinine in normal adult and clinical AKI patients according to the present invention.
FIGS. 1-2 are graphs showing the detection of UBASH3A content in serum of normal adult and clinical AKI patients using ELISA in the present invention.
FIG. 2 shows the results of detection of UBASH3A expression in kidney tissue of normal adult and clinical AKI patients using immunohistochemistry.
FIG. 3 shows the results of detection of UBASH3A high expression and kidney injury in UBASH3A deficient mice induced by cisplatin.
Detailed Description
The present invention will be described in detail with reference to examples. The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The amino acid sequence of UBASH3A protein in the following examples is shown as SEQ ID NO.1 in the sequence table, and the coding gene sequence is shown as SEQ ID NO.3 in the sequence table.
Example 1 detection of UBASH3A protein expression in peripheral blood of Normal person in patients with acute kidney injury
The test person: clinically definite acute kidney injury patient and control group normal person
(1) 3-5ml of peripheral venous blood of a patient is extracted from the disposable blood collection tube.
(2) The blood sample was transferred to a 15mL centrifuge tube and centrifuged at 2000rpm/min for 20 minutes to separate serum from the whole blood sample.
(3) After centrifugation, taking the supernatant as a serum sample, and storing the serum sample in an ultralow temperature refrigerator at-80 ℃ for later use.
(4) At the time of use, the solution was removed and centrifuged briefly.
(5) The expression level of UBASH3A was detected using an ELISA kit (vacyveromyces, F9970-a), the specific experimental procedure being as follows:
(5.1) the kit provided stock was diluted to a gradient of 480pg/mL, 240pg/mL, 120pg/mL, 60pg/mL, 30pg/mL, respectively.
And (5.2) respectively arranging blank holes, standard substance holes and sample holes to be tested. On the enzyme-labeled coating plate, 50 mu L of standard substance is accurately added, 40 mu L of sample diluent is firstly added into a sample hole to be detected, then 10 mu L of sample to be detected (the final dilution of the sample is 5 times) is added, and the mixture is gently shaken and mixed uniformly.
(5.3) sealing the plate with a sealing plate, and then placing the plate in a 37 ℃ oven for 30 minutes for incubation.
(5.4) diluting the concentrated washing solution with distilled water for later use.
(5.5) carefully removing the sealing plate film, discarding the liquid, spin-drying, filling each hole with the washing liquid, standing for 30 seconds, discarding, repeating the above steps for 5 times, and drying.
(5.6) 50. Mu.l of enzyme-labeled reagent was added to each well, except for blank wells.
(5.7) the incubation step of step 5.3 was repeated.
(5.8) the washing step of step 5.5 is repeated.
(5.9) 50. Mu.l of the color-developing agent A and 50. Mu.l of the color-developing agent B are added into each hole, mixed by gentle shaking, and developed for 10 minutes at 37 ℃ in a dark place.
(5.10) 50. Mu.L of stop solution was added to each well to terminate the reaction (at this time, blue turned to yellow).
(6) After the reaction, absorbance (OD value) of each well was measured sequentially with a multifunctional microplate reader (Synergy) at a wavelength of 50nm and a blank Kong Diaoling. The measurement should be performed within 15 minutes after the addition of the stop solution.
(7) Drawing a standard curve on a coordinate paper by taking the concentration of a standard substance as an abscissa and the OD value as an ordinate, and finding out the corresponding concentration from the standard curve according to the OD value of a sample; multiplying by the dilution factor; or calculating a linear regression equation of the standard curve by using the concentration and the OD value of the standard substance, substituting the OD value of the sample into the equation, calculating the concentration of the sample, and multiplying the sample by the dilution multiple to obtain the actual concentration of UBASH3A protein in the sample.
The result shows that: as shown in fig. 1-1, the acute kidney injury marker creatinine content was significantly elevated in patients with acute kidney injury. Meanwhile, the data in the figures 1-2 show that compared with normal people in a control group, the expression level of UBASH3A protein in peripheral blood of patients with acute kidney injury is obviously increased, and the increase of UBASH3A is suggested to be closely related to the occurrence and development of acute kidney injury. I.e. as the level of UBASH3A expression in a patient increases, the extent of acute kidney injury response in the body is also enhanced. Thus, it was demonstrated that the expression level of UBASH3A can be used as a marker for the detection of acute kidney injury, that is, UBASH3A protein or mRNA encoding UBASH3A protein or gene encoding UBASH3A protein can be used as a marker for the detection of acute kidney injury.
Example 2 detection of UBASH3A protein expression in immunohistochemistry on kidney tissue sections of patients with acute kidney injury and Normal human
The test person: clinically definite acute kidney injury patient and control group normal person
(1) AKI kidney tissue was from a kidney-piercing patient and normal control tissue was from a kidney-contusion specimen donation without AKI.
(2) Kidney tissue was paraffin and frozen for sectioning.
(3) After slicing, 4% paraformaldehyde was fixed and incubated with UBASH3A protein antibody (primary antibody) overnight at 4 ℃.
(4) Rinsing with 1 x pbs, after incubation with the corresponding secondary antibody, rinsing three more times.
(5) 40uL DAB color developing agent is added into 1mL color developing buffer solution, after being uniformly mixed, the mixture is dripped onto a slice, and the slice is covered, photographed and stored after 5-10 min.
The result shows that: as shown in FIG. 2, immunohistochemical results showed lower expression of UBASH3A in normal adult kidney tissues and significantly increased kidney tissue expression in AKI patients (scale, 100 μm). The UBASH3A expression level is shown to be closely related to the kidney injury degree of a patient.
Example 3, UBASH3A high expression and UBASH3A deleted mice following cisplatin-induced renal injury assay test results test subjects: UBASH3A high expression and UBASH3A deletion mice, littermates wild type mice
UBASH3A knockout mice were purchased from the tsu racing biotechnology limited company. Construction of UBASH3A highly expressed mouse recombinant adenovirus vector by Hemsl Biotechnology (Shanghai) Inc
(1) The recombinant adenovirus (adenovirus-UBASH 3A) is obtained by constructing a UBASH3A high-expression adenovirus shuttle vector and an over-expression control by the Heng-Shanghai biotechnology (Shanghai) limited company and testing the virus concentration to be 1.4 x 10-12 vg/mL.
(2) The recombinant adenovirus for UBASH3A was injected into the tail of a wild-type mouse by intravenous injection in an appropriate amount (the dosage design is referred to Nat Med.2004, 10:382-388), and the mouse with UBASH3A high expression was obtained after 14 days.
(3) And (5) mixing cisplatin storage solution on a vortex instrument for a short time, and putting the mixture into a centrifugal machine for short time centrifugation after mixing uniformly. The mice were fed intraperitoneally at a concentration of 20mg/kg for 3 days.
(4) After about 200ul of urine from the mice was collected using a 1.5ml EP tube, the mice were sacrificed by cervical dislocation, and about 1ml of blood from the mice was obtained by taking blood from the eyeballs in a 1.5ml EP tube.
(5) The blood was placed in a centrifuge and centrifuged at 2000rpm/min for 20 minutes to separate serum from the whole blood sample.
(6) The supernatant was taken as a serum sample and the creatinine content in the serum was determined according to the instructions using the creatinine assay kit from Sigma-Aldrich, usa: 100uL of serum is added into 1mL of first extract, after centrifugation for 10min at 4 ℃ and 12000g, 0.8mL of supernatant is taken, then 0.15mL of second extract is added, evenly mixed at 4 ℃ and after centrifugation for 10min, 12000g of supernatant is taken. And adding 20uL of a sample, 20uL of a first reagent, 20uL of a second reagent, 5uL of a third working solution of the reagent and 5uL of a fourth reagent into each measuring tube, fully mixing uniformly, reacting for 10min at 37 ℃, adding five 40uL of the reagent and six 90uL of the reagent, fully mixing uniformly, developing color at 37 ℃ for 30min, and measuring under a fluorescence spectrophotometer.
(6) Taking the supernatant as a serum sample, and determining the urea nitrogen content in the serum by using a urea nitrogen detection kit of Sigma-Aldrich company in the United states: the enzyme-labeled instrument is preheated for 30min, the wavelength is adjusted to 630nm, and the spectrophotometer distilled water is zeroed. 1mg/mL urea nitrogen standard solution was diluted to 25ug/mL with distilled water for use. Adding 30uL of a sample, 60uL of a first reagent and 110uL of a second reagent into each measuring tube, fully and uniformly mixing, reacting for 10min at 37 ℃, adding 120uL of a third reagent, 90uL of a fourth reagent, fully and uniformly mixing, standing for 30min at 37 ℃, adding 90uL of distilled water, fully and uniformly mixing, absorbing 200uL of the reaction solution into a 96-well plate, and measuring under a fluorescence spectrophotometer.
The result shows that: as shown in fig. 3, UBASH3A high expression cisplatin-induced mice had significantly elevated serum creatinine and urea nitrogen compared to littermate wild-type cisplatin-induced mice; UBASH3A knockout resulted in a significant decrease in creatinine and urea nitrogen in mice induced by cisplatin compared to littermate wild-type cisplatin-induced mice. It was shown that UBASH3A expression levels are closely correlated with the extent of kidney injury in animals at the animal level.
Example 4 correlation analysis of UBASH3A protein expression levels in patients with the appearance of acute renal injury following administration of cisplatin or contrast media
The test person: patients in need of cisplatin or contrast agent administration in clinical treatment
(1) Patients requiring cisplatin or contrast agent administration in clinical therapy were collected, peripheral venous blood samples were drawn prior to their administration, serum samples were isolated as in example 1, and UBASH3A expression levels were detected by ELISA.
(2) Average of normal human UBASH3A expression level is about 20pg/mL; the average of UBASH3A expression levels in AKI patients was about 50pg/mL. Selecting, as a < 20pg/mL group, those patients in need of cisplatin or contrast agent administration having UBA 3A expression values below 20pg/mL based on the measured UBA 3A expression levels; patients with UBASH3A expression values above 50pg/mL were selected as > 50pg/mL groups.
(3) The physical condition of patients included in the study group after cisplatin or contrast agent administration was tracked, and statistical analysis was performed according to whether the patients had acute kidney injury, which was classified into "acute kidney injury after administration" and "acute kidney injury did not occur after administration".
The results are shown in Table 1. The result shows that: the detection by ELISA experiments and selection grouping based on patient UBASH3A protein expression levels, the < 20pg/mL group collected 31 persons, > 50pg/mL group collected 18 persons, and a total of 49 persons were included in the study group and further analyzed.
TABLE 1 correlation analysis of UASH 3A protein expression levels in patients with the occurrence of acute renal injury following administration of cisplatin or contrast media
As shown in table 1, in the 49 study group, the number of people who developed acute kidney injury after administration was 19, and the number of people who did not observe acute kidney injury after administration was 30. Among 31 persons in the group with UBASH3A expression value lower than 20pg/mL, 2 persons have acute kidney injury after administration, and the rest 29 persons have no symptoms of acute kidney injury. In the group 18, in which UBASH3A expression value was higher than 50pg/mL, acute kidney injury occurred after 17 persons had taken the drug, and no symptoms of acute kidney injury were observed after only 1 person had taken the drug. Namely, when the UBASH3A expression value is lower than 20pg/mL, the probability of acute kidney injury after the patient takes the medicine is 6.4%; when UBASH3A expression value is higher than 50pg/mL, the probability of acute kidney injury after administration of the drug to patients is 94.4%. Of the 19 patients with acute kidney injury, 18 patients with UBA 3A expression level higher than 50pg/mL had a 94.7% ratio, and the specificity was strong. Chi-square test of two groups of data, X 2 The value was found to be 22.69,the P value is less than 0.001, the two groups of data are independent and reliable, and the UBASH3A expression level has strong correlation with the occurrence of acute kidney injury after medicine. The data indicate that patients with high levels of UBASH3A protein expression have a high likelihood of developing acute kidney injury following administration of cisplatin or contrast agents, and therefore it is advisable to avoid administration of cisplatin or contrast agents to patients with high levels of UBASH3A protein expression. Thus, UBASH3A protein can be used as a biomarker for acute kidney injury.
The foregoing examples are set forth in order to provide a more thorough description of the present invention, and are not intended to limit the scope of the invention, since modifications of the invention in various equivalent forms will occur to those skilled in the art upon reading the present invention, and are within the scope of the invention as defined in the appended claims.

Claims (10)

  1. Use of UBASH3A protein or mRNA encoding UBASH3A protein or DNA encoding UBASH3A protein as biomarker for acute kidney injury or for the preparation of a reagent for detecting acute kidney injury, characterized in that the amino acid sequence of UBASH3A protein is shown in SEQ ID No. 1.
  2. 2. The use according to claim 1, wherein the mRNA encoding UBASH3A protein has the sequence shown in SEQ ID No. 2.
  3. 3. The use according to claim 1, wherein the acute kidney injury is caused by cisplatin, a contrast agent or a nephrotoxic antibiotic.
  4. 4. The use according to claim 1, wherein the biomarker is a serum diagnostic marker, a plasma diagnostic marker or a histiocyte diagnostic marker.
  5. 5. A kit for the detection of acute kidney injury, comprising reagents for detecting UBASH3A protein or mRNA encoding UBASH3A protein or DNA encoding UBASH3A protein.
  6. 6. The kit of claim 5, wherein the detecting comprises: colloidal gold immunochromatography, immunoblotting, immunohistochemistry, immunofluorescence, flow cytometry, enzyme-linked immunosorbent assay, nucleic acid probe assay or qPCR method.
  7. 7. The kit according to claim 5, wherein the kit comprises one or more of an antibody specific for UBASH3A protein, a specific primer for amplifying mRNA or DNA encoding UBASH3A protein, and a probe or chip for detecting mRNA or DNA encoding UBASH3A protein.
  8. 8. The kit according to claim 5, wherein the sequence of the specific primer for amplifying mRNA or DNA encoding UBASH3A protein is shown in SEQ ID NO.4 and SEQ ID NO. 5.
  9. 9. The kit according to claim 5, wherein the specific primers for amplifying mRNA or DNA encoding UBASH3A protein further comprise PCR amplification specific primers of internal gene GAPDH, the sequences of which are shown in SEQ ID NO.6 and SEQ ID NO. 7.
  10. 10. Use of a kit according to any one of claims 5-9 for the preparation of a reagent for the detection of acute kidney injury.
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