KR101965411B1 - Composition for diagnosing kidney allograft refection and method for detecting a diagnostic marker - Google Patents

Abstract

The present invention relates to a composition for diagnosing kidney transplant rejection and a method for detecting a diagnostic marker. More particularly, the present invention relates to a composition for diagnosing kidney transplant rejection comprising an agent for measuring an expression level of a tetraspanin 1 (TSPAN1) protein or mRNA, and a method for detecting the marker from a biological sample obtained from kidney transplant recipients to provide information necessary for the diagnosis of kidney transplant rejection. As TSPAN1, a biomarker provided in the present invention, is significantly increased in patients exhibiting kidney transplant rejection, it is possible to accurately and quickly diagnose whether the kidney transplant rejection has occurred using the diagnostic composition including the agent for measuring the expression level of TSPAN1or by the TSPAN1 detection method of the present invention.

Description

Technical Field [0001] The present invention relates to a composition for diagnosing renal transplant rejection,

The present invention relates to a composition for diagnosing renal transplant rejection and a diagnostic marker detecting method, and more particularly, to a composition for diagnosing rejection of renal transplantation comprising a preparation for measuring the expression level of protein or mRNA of TSPAN1 (tetraspanin 1) To a method for detecting the marker from a biological sample obtained from a kidney transplant patient to provide information necessary for diagnosis of a transplant rejection reaction.

Kidney transplantation (KT) is the treatment of choice for patients with end-stage renal disease. Successful transplantation improves quality of life and reduces the risk of death as compared to patients receiving chronic dialysis. In addition, the development of more potent and effective immunosuppressants has reduced acute rejection in kidney transplant recipients (KTRs). However, there is no clear improvement in organ transplant survival at present, and there is no reliable diagnostic tool to evaluate it except for allograft biopsy. Kidney allograft biopsy with histopathological examination is the most important criterion for diagnosing acute rejection in KTR, but there are some problems. First, the biopsy is invasive and can lead to serious complications such as bleeding or infection. Second, continuous monitoring is difficult. Third, a lot of money is needed. Finally, histologic examinations generally involve excessive observer differences in interpretation. Early diagnosis of acute rejection is important to improve the survival rate of organ transplantation and organ transplantation. However, early diagnosis may be difficult due to the aforementioned problems.

Conventional indicators of allograft function, such as serum creatinine, proteinuria and color doppler sonography, are noninvasive but not specific for acute rejection. Therefore, non-invasive and sensitive diagnostic tools should be developed to help predict kidney transplant rejection including acute rejection and chronic rejection.

Accordingly, the present inventors have made a diligent effort to detect non-invasive and accurate renal transplantation rejection diagnostic markers, and as a result, the present invention has been completed by identifying a novel biomarker capable of predicting renal transplant rejection through a proteomics approach .

Accordingly, an object of the present invention is to provide a composition for diagnosing rejection of kidney transplantation comprising an agent for measuring the expression level of TSPAN1 (tetraspanin 1) protein or mRNA.

It is another object of the present invention to provide a kit for the diagnosis of rejection of kidney transplantation comprising an agent for measuring the expression level of TSPAN1 (tetraspanin 1) protein or mRNA.

It is another object of the present invention to provide a method for diagnosing renal transplant rejection, comprising the steps of: (a) measuring the expression level of a protein or mRNA of TSPAN1 (tetraspanin 1) in a biological sample obtained from a kidney transplant patient; And (b) comparing the expression level of the TSPAN1 protein or mRNA to that of a normal control to determine that the subject having an increased expression level of the TSPAN1 protein or mRNA is determined to have a rejection of renal transplantation. Of the marker is detected.

Another object of the present invention is to provide a method for screening a therapeutic agent for renal transplant rejection comprising administering to a mammal other than a human a candidate substance for treating renal transplant rejection reaction and measuring the expression level of a protein or mRNA of TSPAN1 .

In order to achieve the above object, the present invention provides a composition for diagnosing rejection of kidney transplantation comprising an agent for measuring the expression level of TSPAN1 (tetraspanin 1) protein or mRNA.

In order to accomplish another object of the present invention, there is provided a diagnostic kit for rejection of kidney transplantation comprising an agent for measuring the expression level of TSPAN1 (tetraspanin 1) protein or mRNA.

In order to accomplish the other object of the present invention, the present invention provides a method for detecting the expression of protein or mRNA of TSPAN1 (tetraspanin 1) in a biological sample obtained from a kidney transplant patient, Measuring a level; And (b) comparing the expression level of the TSPAN1 protein or mRNA to that of a normal control to determine that the subject having an increased expression level of the TSPAN1 protein or mRNA is determined to have a rejection of renal transplantation. Of the marker.

In order to accomplish another object of the present invention, the present invention provides a method for treating renal transplant rejection, comprising administering a candidate substance for treating rejection of kidney transplantation to a mammal other than a human, and measuring the expression level of TSPAN1 protein or mRNA Screening method.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for diagnosing rejection of kidney transplantation comprising an agent for measuring the expression level of TSPAN1 (tetraspanin 1) protein or mRNA.

In the present invention, 'TSPAN1' is an abbreviation of tetraspanin 1 and is a member of transmembrane 4 superfamily. TSPAN1 is known to play an important role in regulating cell development, activation, sexual function and motility.

According to one embodiment of the present invention, the expression of TSPAN1 protein in the urine of a patient exhibiting acute T cell mediated rejection (TCMR) as compared with a stable graft function (STA) showing stable graft function among renal transplantation patients TSPAN1 could be used as a diagnostic marker for acute rejection of kidney transplantation.

In accordance with another embodiment of the present invention, there is provided a method of treating a patient suffering from chronic antibody mediated rejection (TIV) in a urine of a patient having kidney transplantation, wherein the patient has chronic antibody mediated rejection as compared to a long-term graft survival TSPAN1 could be used as a diagnostic marker for the chronic rejection of kidney transplantation.

In the present invention, the term 'expression' means that a protein or a nucleic acid is produced in a cell. "Protein" is used interchangeably with "polypeptide" or "peptide" and refers to a polymer of amino acid residues as commonly found in natural state proteins. A "polynucleotide" or "nucleic acid" refers to deoxyribonucleotides (DNA) or ribonucleotides (RNA) in the form of single- or double-stranded. Unless otherwise constrained, also includes known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. 'mRNA' refers to RNA that transfers genetic information (gene-specific nucleotide sequence) to a ribosome in which an amino acid sequence is specified from a specific gene during protein synthesis.

In the present invention, the term 'renal allograft rejection' refers to a series of immune responses that occur when the recipient's kidney transplanted immune system recognizes the transplantation kidney as an external antigen, Acute rejection, chronic rejection, and mixed rejection, all of which can be classified as mixed rejection. Acute rejection usually occurs within a few days to six months after renal transplantation, with fever, tenderness at the transplantation site, decreased urine, and decreased renal function. Cellular immunity and humoral immunity are involved and cause tissue damage of the transplant kidney. Histologically, renal tubular inflammation and endothelial cell inflammation are seen, and mononuclear cell carcinoma of the kidney and tubular necrosis are observed. Chronic rejection occurs after 6 months after transplantation and is characterized by gradual renal dysfunction accompanied by proteinuria and hypertension. If vascular inflammation and fibrosis occur in the grafted kidney due to the immune rejection of the recipient, the renal function is worsened and the kidney function is progressed as a whole due to overload of the healthy glomeruli.

In the present invention, the rejection of kidney transplantation may include acute rejection, chronic rejection, and mixed rejection. Preferably, acute T cell mediated rejection (TCMR), chronic antibody- In addition, chronic antibody mediated rejection (CAMR), active antibody mediated rejection, chronic active T cell mediated rejection, and mixed rejection, (TCMR), or chronic antibody mediated rejection (CAMR). The present invention also provides a method for preventing or treating an autoimmune disease.

In the present invention, the term " diagnosis " means identifying the presence or characteristic of a pathological condition. The diagnosis in the present invention is to measure the level of TSPAN1 protein or mRNA to confirm the presence or absence of a rejection reaction pathway in kidney transplantation.

Meanwhile, when the diagnostic composition of the present invention is for measuring the expression level of mRNA, the agent for measuring mRNA expression level may be a probe or a primer set that specifically binds to TSPAN1 mRNA.

The mRNA of TSPAN1 may be derived from a mammal including a human, and preferably the mRNA of TSPAN1 may include the nucleotide sequence of SEQ ID NO: 1.

[SEQ ID NO: 1]

tcccggcctc agacacacac accagcagct acacctacac gctgaccatc acaggcacac

agaggcacat ccacctcaca tccacctcat acttgtgtac tctcagggtt cagtctttca

cctatccct ctctgatctg tgcctcccaa taccttccaa gatgtttaca gagacccttc

tccctgtgca gttaggagtg taaggcaaga gagcccctac ttcatggggc agatcaagag

ctgagaccaa agatggtcta tgttgctgac cttgtcctgt cctcctgctg tcttaaacta

tgatccctgc tgcggtcact gaagcctttc cctgtgagca gtggtgtgtg agagccaggc

gtccctctgc ctgcccactc agtggcaaca cccgggagct gttttgtcct ttgtggagcc

tcagcagttc cctctttcag aactcactgc caagagccct gaacaggagc caccatgcag

tgcttcagct tcattaagac catgatgatc ctcttcaatt tgctcatctt tctgtgtggt

gcagccctgt tggcagtggg catctgggtg tcaatcgatg gggcatcctt tctgaagatc

ttcgggccac tgtcgtccag tgccatgcag tttgtcaacg tgggctactt cctcatcgca

gccggcgttg tggtctttgc tcttggtttc ctgggctgct atggtgctaa gactgagagc

aagtgtgccc tcgtgacgtt cttcttcatc ctcctcctca tcttcattgc tgaggttgca

gctgctgtgg tcgccttggt gtacaccaca atggctgagc acttcctgac gttgctggta

gtgcctgcca tcaagaaaga ttatggttcc caggaagact tcactcaagt gtggaacacc

accatgaaag ggctcaagtg ctgtggcttc accaactata cggattttga ggactcaccc

tacttcaaag agaacagtgc ctttccccca ttctgttgca atgacaacgt caccaacaca

gccaatgaaa cctgcaccaa gcaaaaggct cacgaccaaa aagtagaggg ttgcttcaat

cagcttttgt atgacatccg aactaatgca gtcaccgtgg gtggtgtggc agctggaatt

gggggcctcg agctggctgc catgattgtg tccatgtatc tgtactgcaa tctacaataa

gtccacttct gcctctgcca ctactgctgc cacatgggaa ctgtgaagag gcaccctggc

aagcagcagt gattggggga ggggacagga tctaacaatg tcacttgggc cagaatggac

ctgccctttc tgctccagac ttggggctag atagggacca ctccttttag gcgatgcctg

actttccttc cattggtggg tggatgggtg gggggcattc cagagcctct aaggtagcca

gttctgttgc ccattccccc agtctattaa acccttgata tgccccctag gcctagtggt

gatcccagtg ctctactggg ggatgagaga aaggcatttt atagcctggg cataagtgaa

atcagcagag cctctgggtg gatgtgtaga aggcacttca aaatgcataa acctgttaca

atgttgccaa aaaaaaaaa

The diagnostic composition of the present invention comprising the mRNA-specific probe or primer set of the TSPAN1 may further comprise a preparation necessary for a method for detecting known RNA. Methods for detecting known RNA using this composition can be used without limitation to determine the level of mRNA of the markers in a subject.

The 'primer' is a short single strand oligonucleotide that serves as a starting point for DNA synthesis. The primer specifically binds to a polynucleotide which is a template under a temperature condition with a suitable buffer, and a DNA polymerase is added to the primer by addition of a nucleoside triphosphate having a base complementary to the template DNA, Is synthesized. The primer is generally composed of 15 to 30 nucleotide sequences, and the melting temperature (Tm) of the primer varies depending on the base structure and length. The sequence of the primer does not need to have a sequence completely complementary to a part of the nucleotide sequence of the template. It is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, in the present invention, the primer for measuring the expression level of TSPAN1 mRNA does not need to have a perfectly complementary sequence in each gene sequence, and it is possible to amplify a specific region of mRNA or cDNA by DNA synthesis to measure the amount of mRNA Anything that has a length and complementarity suited to the purpose is sufficient. The primer for the amplification reaction is composed of a pair (pair) complementarily binding to a template (or sense) at opposite ends of a specific region of the mRNA to be amplified and an opposite region (antisense), respectively. Primers can be easily designed by those skilled in the art with reference to mRNA or cDNA sequence of KRS or AIMP1. In the present invention, the primer may preferably be a set, a pair, or a combination thereof that specifically binds to the mRNA of TSPAN1 represented by SEQ ID NO: 1.

The 'probe' may be a fragment of a polynucleotide such as RNA or DNA having a base pair length of several to several hundreds, which can specifically bind to a specific gene mRNA or cDNA (complementary DNA) And it is labeled so that the presence or expression level of mRNA or cDNA to be bound can be confirmed. For the purpose of the present invention, a probe complementary to KRS or AIMP1 mRNA can be used for diagnosis of colon cancer by measuring the expression level of KRS or AIMP1 mRNA by performing hybridization with a sample of a subject. The selection and hybridization conditions of the probes can be appropriately selected according to techniques known in the art.

In the present invention, the primer or the probe may be chemically synthesized using a phosphoramidite solid support synthesis method or other well-known methods. The primers or probes may also be modified in various ways according to methods known in the art, so long as they do not interfere with hybridization with KRS or AIMP1 mRNA. Examples of such modifications include, but are not limited to, methylation, capping, substitution with one or more of the natural nucleotide analogs and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, phosphoramidate, carbamate, etc.) ) Or charged conjugates (eg, phosphorothioate, phosphorodithioate, etc.), and binding of labeling materials using fluorescence or enzymes.

In the present invention, the agent for measuring the protein expression level of TSPAN1 may be an antibody that specifically binds to TSPAN1 protein.

In the present invention, the TSPAN1 protein may be derived from a mammal including a human. Preferably, the TSPAN1 protein comprises the amino acid sequence of SEQ ID NO: 2.

[SEQ ID NO: 2]

mqcfsfiktm milfnllifl cgaallavgi wvsidgasfl kifgplsssa mqfvnvgyfl

iaagvvvfal gflgcygakt eskcalvtff filllifiae vaaavvalvy ttmaehfltl

lvvpaikkdy gsqedftqvw nttmkglkcc gftnytdfed spyfkensaf ppfccndnvt

ntanetctkq kahdqkvegc fnqllydirt navtvggvaa gigglelaam ivsmylycnl

q

The term "antibody" refers to an immunoglobulin that specifically binds to an antigenic site. The antibody in the present invention is an antibody that specifically binds only KRS or AIMP1 protein without reacting with other proteins including aminoacyl thiourea synthetase other than KRS or AIMP1. The KRS or AIMP1 antibody can be produced by cloning each gene into an expression vector to obtain a protein encoded by the gene and obtaining the protein from the obtained protein according to a conventional method in the art. KRS or fragments of the AIMP1 protein comprising the AIMP1 antigenic site may be used to produce each protein-specific antibody. The form of the antibody of the present invention is not particularly limited and includes a polyclonal antibody or a monoclonal antibody. Also included in the antibody of the present invention are a portion of whole antibodies as well as any immunoglobulin antibody that binds specifically to KRS or AIMP1 as long as it has antigen-antibody binding properties. F (ab '), F (ab '), < / RTI > which have antigen binding functions, as well as complete forms of antibodies with two full length light chains and two full length heavy chains, 2, and Fv. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies, chimeric antibodies, and recombinant antibodies, as long as they can specifically bind KRS or AIMP1 protein.

The diagnostic composition of the present invention comprising the TSPAN1 protein-specific antibody may further include a preparation necessary for a method for detecting a known protein, and a method for detecting a known protein using the present composition may be used, The level of expression of the TSPAN1 protein can be measured in a sample.

The composition of the present invention may further comprise an agent for measuring the expression level of HPX (hemopexin) protein or mRNA.

According to one embodiment of the present invention, in addition to the TSPAN1 protein in the urine of patients showing acute T cell mediated rejection (TCMR) as compared with the stable graft function (STA) showing stable graft function among renal transplantation patients, HPX The expression level of TSPAN1 and HPX protein was applied to the ROC curve analysis. As a result, AUC of 0.744, sensitivity of 64.0% and specificity of 72.7% Respectively.

In the present invention, the HPX protein and mRNA may be derived from a mammal including a human. Preferably, the HPX protein may be one whose sequence can be confirmed in GenBank: AAA58678.1. HPX mRNA is preferably NCBI Reference Sequence: It may be possible to confirm the sequence in NM_000613.2.

The present invention also provides a kit for the diagnosis of rejection of kidney transplantation comprising an agent for measuring the expression level of TSPAN1 (tetraspanin 1) protein or mRNA.

The diagnostic kit of the present invention may include one or more other component compositions, solutions, or devices suitable for the assay as well as primers, probes, or antibodies recognizing the TSPAN1 protein as a marker or recognizing the TSPAN1 mRNA as a marker.

In a specific embodiment, the diagnostic kit may be a diagnostic kit comprising essential elements necessary for performing a reverse transcription-polymerase reaction. The RT-PCR kit contains the respective primer pairs specific for the marker gene. A primer is a nucleotide having a sequence specific to a nucleic acid sequence of each marker gene, and has a length of about 7 bp to 50 bp, and more preferably about 10 bp to 30 bp. It may also contain a primer specific for the nucleic acid sequence of the control gene. Other reverse transcription polymerase reaction kits may be used in combination with test tubes or other appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC DEPC-water, sterile water, and the like.

Another aspect of the present invention is a diagnostic kit characterized in that it includes an essential element necessary for performing a DNA chip. The DNA chip kit may include a substrate to which a cDNA or oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, preparations, enzymes, and the like for producing a fluorescent-labeled probe. The substrate may also comprise a cDNA or oligonucleotide corresponding to a control gene or fragment thereof.

The sample used for the analysis includes a biological sample capable of identifying a renal transplant rejection-specific protein which can be distinguished from a normal state such as blood, plasma, serum, saliva, saliva, sputum, ascites, vaginal discharge, urine and the like. Preferably from a biological fluid sample, such as blood, serum, plasma or urine. The sample can be prepared to increase the detection sensitivity of the protein marker. For example, serum samples obtained from a patient can be analyzed by anion exchange chromatography, affinity chromatography, size exclusion chromatography, liquid chromatography, Can be pretreated using sequential extraction or gel electrophoresis.

The present invention also relates to a method for diagnosing renal transplant rejection comprising the steps of: (a) determining the level of expression of a protein or mRNA of TSPAN1 (tetraspanin 1) in a biological sample obtained from a kidney transplant patient; And (b) comparing the expression level of the TSPAN1 protein or mRNA to that of a normal control to determine that the subject having an increased expression level of the TSPAN1 protein or mRNA is determined to have a rejection of renal transplantation. Of the marker.

In the present invention, the biological sample can be used without limitation as long as it is collected from a kidney transplant patient who is to be diagnosed as a kidney transplant rejection reaction. For example, the biological sample can be a cell or tissue obtained by biopsy, blood, whole blood, serum, plasma, , Various secretions, urine, feces, and the like. And may be selected from the group consisting of blood, plasma, serum, saliva, saliva, sputum, ascites, vaginal discharge and urine, and most preferably urine.

Expression levels of the TSPAN1 protein can be detected or measured using an antibody that specifically binds to the TSPAN1 protein. The protein-specific antibody is as described for the diagnostic composition of the present invention. Methods for measuring the expression level of a protein can be performed by any method known in the art including, but not limited to, western blotting, dot blotting, enzyme-linked immunosorbent assay Immunohistochemical staining, immunoprecipitation, complement fixation, flow cytometry (FACS), or immunohistochemical staining with protein (s), immunohistochemistry (ELISA), radioimmunoassay (RIA) A chip method, and the like, but the present invention is not limited thereto.

The level of TSPAN1 mRNA is determined by amplifying the mRNA or cDNA of each marker from a sample of a subject using a primer set or a probe that specifically binds to mRNA or by using hybridization with a probe, the presence and expression level of mRNA can be measured. The primers and probes are as described in the diagnostic composition of the present invention. The expression level of mRNA can be measured by any method known in the art without any limitations. For example, reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR PCR), real-time RT-PCR, RNase protection assay (RPA), northern blotting, DNA microarray chip, RNA sequencing RNA sequencing, hybridization using nanostrings, in situ hybridization of tissue sections, but are not limited thereto.

When the expression level of the TSPAN1 protein or mRNA is increased in the kidney transplantation patients as measured by the method of step (a), when the expression level of the TSPAN1 protein or mRNA is increased as compared with the normal control, It can be judged that the reaction exists. The normal control includes a normal kidney transplant recipient or a kidney transplant recipient who has undergone a kidney transplantation but has a stable renal function and does not show acute rejection, chronic rejection, or mixed rejection.

The method of the present invention further comprises measuring the expression level of protein or mRNA of HPX (hemopexin) in the step (a) and comparing the expression level of the protein or mRNA of TSPAN1 and HPX in the step (b) And judging that the increased body is present in the rejection reaction of kidney transplantation.

The present invention also provides a screening method for treating renal transplant rejection, comprising the step of measuring the level of expression of the protein or mRNA of TSPAN1 after administration of a candidate substance for treating renal transplant rejection in a mammal other than human.

Specifically, the method can be useful for screening a therapeutic agent for renal transplant rejection by comparing the increase or decrease of TSPAN1 mRNA or protein expression under the presence and absence of a candidate kidney transplant rejection response candidate. Substances that indirectly or directly reduce the level of expression of TSPAN1 mRNA or protein can be selected as therapeutic agents for renal transplant rejection.

That is, the expression level of the marker of the present invention is measured in a biological sample obtained from a mammal other than a human under the absence of a candidate kidney transplant rejection response candidate, and the expression level of the marker of the present invention The expression level is measured to compare the two, and then a substance that reduces the expression level of the marker of the present invention in the absence of the candidate kidney transplantation rejection candidate candidate is lower than the marker expression level in the absence of the kidney transplant rejection reaction therapeutic agent, .

Since the biomarker TSPAN1 provided by the present invention is significantly increased in patients exhibiting a renal transplant rejection reaction, the diagnostic composition or the TSPAN1 detection method comprising the agent for measuring the expression level of TSPAN1 can be used to determine whether or not a kidney transplant rejection Can be accurately and quickly diagnosed.

FIG. 1 is a heat map showing the expression levels of general proteins in patients with stable graft function (STA) and patients with acute TCMR (T cell mediated rejection) that have undergone renal transplantation.
FIG. 2 is a graph showing the results of detection of 5 types of renal transplant rejection response markers selected in the present invention by Western blot in urine of patients with STA (stable graft function) and acute TCMR (T cell mediated rejection) The results are shown as graphs.
FIG. 3 shows the results of ROC curve analysis by analyzing the expression levels of TSPAN1 and HPX in the urine of STA patients and acute TCMR patients.
Figure 4 shows a protein-protein interaction network of TSPAN1 (a) or HPX (b).
FIG. 5 shows Western blot analysis of the expression level of TSPAN1 protein in exosomes extracted from kidney transplanted patients with long-term graft survival (LGS) and chronic antibody medated rejection (CAMR) patients (a) (B) ("L" in FIG. 5A means urine sample of LGS patient and "C" means urine sample of CAMR patient).
FIG. 6 shows the results of ROC curve analysis by analyzing the expression level of TSPAN1 in the urine of LGS patients and CAMR patients.

Hereinafter, the present invention will be described in detail.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Recent developments in omics technologies can provide additional information about disease and can help identify new biomarkers. There are a number of studies to identify diagnostic biomarkers for acute rejection in KTR based on these techniques. However, there is no confirmed positive result yet.

Thus, the present invention seeks to identify novel biomarkers that can non-invasively predict acute T cell mediated rejection (TCMR) using the proteomics approach.

<Material and Experimental Method>

1. Research population and data collection

(1) T cell mediated rejection (TCMR) patients

A total of 84 transplantation biopsies from 385 KTRs were enrolled in the cross-sectional multicenter study ARTKT-1 (Assessment of Immunologic Risk and Tolerance in Kidney Transplantation) at six university hospitals in Korea . Kidney allograft biopsy was performed in patients with acute clinical graft dysfunction or with a protocol. Twenty - two patients were proven to have stable graft function (STA) and 25 patients had acute TCMR demonstrated by biopsy. All blood and urine samples were collected at the time of the biopsy using the same protocol from August 2013 to July 2015. Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. The study protocol was registered in the Clinical Research Information Service (CRIS Registration Number: KCT0001010), and the participating hospitals (Kyungpook National University Hospital (2013-10-010), Kyunghee University Hospital in Kangdong; And the Institutional Review Board of Inje University Pusan Paik Hospital). The study of the present invention was conducted in accordance with the Helsinki Declaration in 2000 and the Helsinki Declaration in 2008.

(2) patients with chronic antibody-mediated rejection (CAMR)

All of the patients studied were selected for ARTKT-1 studies, which included kidney transplant patients (KTR) with transplantation biopsy or stable renal function (estimated glomerular filtration rate (eGFR) ≥ 50ml / min / 1.73m ² ) (LGS, Kyungpook National University Hospital, Kyungpook National University Hospital, Kyungpook National University Hospital, Samsung Medical Center, St. Mary's Hospital of Catholic University, and St. Mary's Hospital).

Nine of them were CAMR renal transplant patients and 33 were LGS kidney transplant patients.

In addition, urine samples of 17 CAMR kidney transplant patients and 24 LGS kidney transplant patients were obtained and analyzed for the verification of marker proteins in addition to the kidney transplant patients.

2. Definition of Patient Status

(1) Definition of STA and acute TCMR

STA was defined as a patient without significant damage to stable serum creatinine and graft biopsy. Acute TCMR was diagnosed according to the Banff '07 classification, which showed interstitial infiltration, tubulitis, and intimal arteritis.

(2) Definition of LGS and CAMR

Patients with ≥50 mL / min / 1.73 m ² of eGFR calculated using the Modification of Dienes in Renal Disease equation for more than 10 years after renal transplantation were defined as LGS. All LGS patients previously had no rejection, proteinuria, or de novo donor-specific antibodies (DSA). CAMR was diagnosed as a renal allograft biopsy according to the Banff 2013 classification. Systemic infection, allograft infection and other inflammatory processes were excluded clinically.

3. Isolation of urine exosomes from LC-MS analysis

Urine from 22 STAs, 25 TCMR patients, 33 LGS and 9 CAMR patients was first centrifuged at 3,000 g for 15 minutes at 4 ° C and centrifuged at 17,000 g for 15 minutes at 4 ° C to remove cells and cell debris . The supernatant was then centrifuged at 200,000 xg for 2 hours at 4 ° C. The pellet of each tube was resuspended in 50 μl of the isolation solution (250 mM sucrose / 10 mM triethanolamine / 0.5 mM PMSF / 1 μM leupeptin) and the zona pellucidase domain (zona) in Tam-Horsfall protein pellucia domains were denatured to inhibit aggregation and incubated at 60 ° C for 10 minutes with 60 mg / ml DTT so that THP could be removed from the supernatant. Crude exosomes were washed by centrifugation at 200,000 xg for 2 hours at 4 ° C in PBS and resuspended in RIPA buffer.

4. Isolation of urine exosomes for Western blot analysis

The exosomes of the 500 μl urine samples were concentrated by centrifugation at 14,000 g at 4 ° C for 10 minutes using Amicon Ultra-15 (MWCO: 100 kDa, Millipore).

To collect the concentrated urine, the filter device was transferred to a clean centrifuge tube and centrifuged at 1,000 g for 2 minutes at 4 ° C.

5. LC-MS sample preparation

The isolated urine exosomal protein was resuspended in 2 mM SDS and 6 M Urea, 5 mM EDTA in 100 mM triethylammonium bicarbonate (TEABC), pH 8.0 buffer. Protein was chemically denatured with 10 mM DTT for 20 min at 60 [deg.] C and alkylated with 50 mM iodoacetamide at 25 [deg.] C for 20 min. The alkylated protein solution was dissolved in an acrylamide / bisacrylamide solution (30% v / v, 29: 1), 10% (w / v) ammonium persulfate and tetramethylethylenediamine (tetramethylethylenediamine). The gel was cut into small pieces and washed three times with 25 mM TEABC containing 50% acetonitrile (ACN). Proteolytic digestion was carried out overnight at 37 ° C using 25 mM TEABC trypsin (protein: trypsin = 50: 1, w / w). Digested peptides were extracted from the gel by exchange with two buffers consisting of 0.1% formic acid (FA) in 25 mM TEABC or 0.1% FA in 50% ACN. The eluate was concentrated on a SpeedVac and desalted using an HLB cartridge (Waters, Milford, MA, USA).

6. LC-MS analysis

1 [mu] g of the resulting peptide was analyzed by tandem mass spectrometry using nano-UPLC (Waters, Manchester, UK) and Q-Tof Premier (Waters). The peptides were injected into a 2 cm x 180 mu m trap column and analyzed with a 25 cm x 75 mu m nanoACQUITY C18 column (Waters) using an LC system. Mobile phase A consisted of water containing 0.1% FA and mobile phase B consisted of ACN with 0.1% FA. The peptides were analyzed with a gradient of 3% to 45% mobile phase B over 160 minutes at a flow rate of 300 nL / min. This method was applied to five MS / MS scans (m / z) for the five most intense ions present in the full sequential MS scan (m / z 150-1,600, 0.6 sec) and the full scan mass spectrum 100 to 1,990, 0.6 s / sec).

7. Identification and quantification of proteins

For protein identification, MS raw data was converted to a peak list using MASCOT Distiller version 2.1 (Matrix Science, London, UK). All MS / MS raw data were analyzed using MASCOT version 2.2.1 (Matrix Science). MASCOT is configured to search for the IPI (International Protein Index) human database version 3.78 (86,392 entries). The database search for MASCOT was performed with a 0.5Da fragment ion mass tolerance and a parent ion tolerance of 0.2 Da. A missed cleavage was allowed for trypsin digestion. Cysteine carbamidomethylation and methionine oxidation were considered to be various variants. To evaluate the false-discovery rate (FDR) of protein identification, analysis using the same search parameters and validation criteria for a random decoy database generated by MASCOT . Peptide identities were assigned when the MASCOT ion score was P> 0.05. The identity of proteins with two or more peptides has been confirmed. Label-free protein quantification was performed using PEAKS 7 (Bioinformatics Solutions Inc., Waterloo, Canada) in triplicate. Ion chromatography extraction was used to determine the amount of peptide present, and the protein ratio was calculated using the average abundance among the corresponding peptides. Protein ratios were considered acceptable when the identified protein contained one or more unique peptides.

8. Candidate proteomic biomarker validation

The exosomal protein (20 μg) was separated by SDS-PAGE, transferred to nitrocellulose membrane, probed with each primary antibody and incubated with secondary antibody conjugated with HRP (horseradish peroxidase) Respectively. The blot was visualized as ECL (enhanced chemiluminescence) detection reagent and quantitated using ECL hyperfilm. Band volume was measured by densitometry in at least three different experiments.

The primary antibodies are as follows: tetraspanin-1 (TSPAN1, H00010103, 1: 500; Abnova), hemopexin (HPX, ab124935, 1: 500; Abcam), multiple immunoglobulin receptor (TAC, ab75815, 1: 400, abcam), apolipoprotein AI (APOA1, ab52945, 1: 500, Abcam), transthyretin Alpha-2-lycoprotein, AZGP1, ab117275, 1: 500; abcam), ceruloplasmin (CP, ab48614, 1: 500; abcam), and lectin galactoside Lectin galactoside-binding soluble 3 binding protein, LGALS3BP, ab123921, 1: 500; Abcam).

9. Functional Protein Associated Network Analysis

Protein-protein interactions can be detected using the Interacting Genes / Proteins (STRING) database v10.5 ( http://www.string-db.org/) , a search tool for known and predicted protein interactions based on physical and functional associations . Using a search tool. Identified candidate proteomic biomarkers were used for the analysis.

For the search parameters, the active prediction method was set up as an experiment showing a list of protein interaction data sets collected from several databases; The required confidence score was set to 0.4 (medium confidence); The number of displayed interac- tors was set to 50 or less. The organism was set as human. The network view is represented by a mutual mode that allows the node to move freely, and images of the network are exported with a confidence setting that represents the strength that the line thickness is supported by the data.

10. Statistical Analysis

Data were summarized as mean ± standard deviation, median (interquartile range), or number and percentage (%) according to the characterization distribution of variables. Student's t-test or Mann-Whitney U test was used to assess differences between successive variables.

Pearson chi-square tests were applied to compare categorical variables. Receiver Operating Characteristic (ROC) analysis was performed to evaluate the potential of TSPAN1 and HPX to distinguish between STA and acute TCMR. Performance was evaluated by area under the curve (AUC). Statistical analysis was performed using SPSS 20.0 for Windows (SPSS Inc, Chicago, IL, USA). All P values were two-tailed and P <0.05 was considered statistically significant.

<Experimental Results>

1. STA and TCMR Patient Characteristics

Table 1 describes the characteristics of the baseline demographic characteristics of registered KTRs (22 STAs and 25 acute TCMRs).

(1.13 ± 0.35 mg / dL vs. 2.92 ± 1.90 mg / dL, P <0.001, and 74.9 ± 24.2 ml / min / 1.73 m2 vs. TCMR at the time of biopsy, including serum creatinine levels and eGFR ( P <0.001, respectively) and there was a significant difference in the period after KT [STA vs TCMR: 85.5 (16.0-175.0) day vs. 32.9 ± 18.9 ml / min / 1.73 m2, P <0.001, respectively). 259-0 (84.0-696.0) days, P = 0.001]. However, there were no significant differences in sex, age, and HLA mismatches between the two groups. The donor age was similar, and live donors were more common in STA than in acute TCMR (77.3% vs. 44.0%, P = 0.02).

Basic information of study subjects Clinical features Stable renal function normal (STA) Acute T-cell mediated rejection (TCMR) Significance level P value Number of patients 22 25 Average age (years) 44.2 ± 13.6 47.2 ± 11.8 0.177 Gender (Male%) 12 (54.5) 16 (64.0) 0.510 After transplantation (days) 85.5 (16.0, 175.0) 259.0 (84.0, 696.0) 0.001 Serum creatinine (mg / dL) 1.13 + 0.35 2.92 ± 1.90 <0.001 Glomerular filtration rate (ml / min / 1.73 m ² ) 74.9 ± 24.2 32.9 ± 18.9 <0.001 HLA mismatches 3.6 ± 1.7 3.5 ± 1.5 0.812 Donor age (years) 46.9 ± 11.3 44.8 ± 16.9 0.623 Donor gender (male,%) 11 (50.0) 16 (66.7) 0.510 Biological Donor Ratio (%) 17 (77.3) 11 (44.0) 0.020

2. Identification and selection of STA and TCMR proteomic biomarkers

Proteomic analysis was performed with nano-UPLC-MS / MS to identify exosomal proteins in accumulated urine. The entire exosome protein pool was assessed using MASCOT in the IPI human sequence database. 138 and 100 proteins were identified in urine exosomes of STA and acute TCMR, respectively, using high confidence peptide sequences with an error rate of less than 5%. Of these, 69 proteins were common to STA and acute TCMR. Excepting general proteins, 69 proteins were identified in STA, and 31 proteins were identified only in acute TCMR. Protein quantification was performed using the sum of normalized ion intensities, expressed as relative values, to identify the proportion of proteins commonly identified in each group. The quantitative analysis results are shown in Figure 1 as a heatmap showing 46 proteins up-regulated in STA and 17 proteins up-regulated in acute TCMR. These proteins are listed in Table 2 below, with sample profile ratios between the two groups.

Protein lists showing differences between STA group and TCMR group (63 species)
Registration Number valence
(-10lg P ) Stable Renal Function Normal Area Acute T-cell mediated rejection group area Sample Profile (Non) Explanation IPI00021841 11.12 0 6.28E + 02 0: 1.00 APOA1, Apolipoprotein A1 IPI00783987 11.04 0 3.90E + 02 0: 1.00 C3, Complement C3 (Fragment) IPI00247063 10.91 1.03E + 03 0 1.00: 0 NEP, Neprilysin IPI00022488 10.88 0 1.16E + 03 0: 1.00 HPX, Hemopexin IPI00004573 10.88 0 1.29E + 03 0: 1.00 PIGR, Polymeric immunoglobulin receptor IPI00925712 10.8 8.36E + 02 0 1.00: 0 PROM1, Isoform 6 of Prominin-1 IPI00024292 10.68 9.91E + 02 0 1.00: 0 LRP2, low-density lipoprotein receptor-related protein 2 IPI00845263 10.43 2.75E + 02 0 1.00: 0 FN1, fibronectin isoform 4 preproprotein IPI00014055 10.28 1.65E + 03 0 1.00: 0 NAPSA, Napsin-A IPI00023673 10.26 3.66E + 03 0 1.00: 0 LGALS3BP, Lectin galactoside-binding soluble 3-binding protein IPI00221224 10.25 1.72E + 03 0 1.00: 0 ANPEP, Aminopeptidase N IPI00304273 10.24 0 9.73E + 01 0: 1.00 APOA4, Apolipoprotein A-IV IPI00215997 10.14 1.46E + 03 0 1.00: 0 CD9, CD9 antigen IPI01009677 10.12 2.18E + 02 0 1.00: 0 ABCB1 IPI00009865 9.97 0 1.22E + 03 0: 1.00 KRT10, Keratin type I cytoskeletal 10 IPI00947307 9.97 0 3.15E + 02 0: 1.00 CP, cDNA FLJ58075 highly similar to Ceruloplasmin IPI00847635 9.94 4.68E + 02 0 1.00: 0 SERPINA3, Isoform 1 of Alpha-1-antichymotrypsin IPI01010887 9.79 7.45E + 02 0 1.00: 0 STOM, cDNA FLJ52062 highly similar to Erythrocyte band 7 integral membrane protein IPI00014375 9.77 4.52E + 02 0 1.00: 0 ENPEP, Glutamyl aminopeptidase IPI00844536 9.62 0 6.85E + 02 0: 1.00 RBP4 IPI00400826 9.61 1.42E + 03 0 1.00: 0 CLU, Isoform 2 of Clusterin IPI00022426 9.36 1.32E + 03 0 1.00: 0 AMBP IPI00914893 9.24 6.44E + 02 0 1.00: 0 SLC12A1, Isoform F of Solute carrier family 12 member 1 IPI00019359 8.86 0 6.13E + 02 0: 1.00 KRT9, Keratin type I cytoskeletal 9 IPI00924574 8.66 1.45E + 03 0 1.00: 0 APOD IPI00299086 8.57 2.11E + 03 0 1.00: 0 SDCBP, Isoform 1 of Syntenin-1 IPI00843975 8.51 3.74E + 02 0 1.00: 0 EZR, Ezrin IPI00007221 8.37 3.25E + 02 0 1.00: 0 SERPINA5, Plasma serine protease inhibitor IPI00796919 8.36 2.84E + 02 0 1.00: 0 GLB1, Beta-galactosidase IPI00022974 8.36 7.90E + 02 0 1.00: 0 PIP, Prolactin-inducible protein IPI00479018 8.31 7.59E + 02 0 1.00: 0 SDCBP, Isoform 2 of Syntenin-1 IPI00918002 8.24 7.08E + 02 0 1.00: 0 MUC5B, Mucin 5AC oligomeric mucus / gel-forming IPI00018901 8.24 5.37E + 02 0 1.00: 0 GGT1, Glutathione hydrolase 1 proenzyme IPI00022624 8.19 1.07E + 03 0 1.00: 0 GPRC5A, Retinoic acid-induced protein 3 IPI00219018 8.06 4.39E + 02 0 1.00: 0 GAPDH, Glyceraldehyde-3-phosphate dehydrogenase IPI00246058 7.94 3.40E + 02 9.08E + 01 1.00: 0.27 PDCD6IP, Programmed cell death 6-interacting protein IPI00909703 7.81 3.20E + 02 8.58E + 01 1.00: 0.27 ANXA11 IPI00166729 7.71 6.57E + 02 9.87E + 01 1.00: 0.15 AZGP1, Zinc-alpha-2-glycoprotein IPI00922787 7.39 1.82E + 02 6.18E + 01 1.00: 0.34 BROX, cDNA FLJ78829 IPI00980890 7.32 8.40E + 02 3.32E + 02 1.00: 0.40 LAMP2, cDNA FLJ58780 IPI01021535 7.25 3.54E + 02 5.75E + 01 1.00: 0.16 SLC44A4, Choline transporter-like protein 4 isoform 3 IPI00943265 7.1 6.73E + 01 3.58E + 02 1.00: 5.32 IGKV4-1, Similar to Ig kappa chain V-IV region precursor IPI00013446 6.35 8.78E + 02 3.83E + 02 1.00: 0.44 PSCA, Prostate stem cell antigen IPI00553177 6.29 1.80E + 02 8.32E + 02 1.00: 4.63 SERPINA1, Alpha-1-antitrypsin IPI00022463 6.26 7.85E + 02 1.87E + 03 1.00: 2.38 TF, Serotransferrin IPI00220327 6.26 4.04E + 02 1.21E + 03 1.00: 3.01 KRT1, Keratin type II cytoskeletal 1 IPI00030936 5.92 1.55E + 03 3.68E + 02 1.00: 0.24 TSPAN1, tetraspanin-1 IPI00646907 5.72 1.54E + 02 0 1.00: 0 SLC12A3, Isoform 2 of Solute carrier family 12 member 3 IPI00418169 5.7 1.42E + 02 0 1.00: 0 ANXA2, Isoform 2 of Annexin A2 IPI00884105 5.61 2.63E + 02 0 1.00: 0 LAMP1, Lysosome-associated membrane glycoprotein 1 IPI00003765 5.58 1.11E + 02 0 1.00: 0 CAPN7, Calpain-7 IPI00946636 5.54 1.40E + 02 0 1.00: 0 ATP6V1A, cDNA FLJ51804 IPI00020091 5.52 0 1.12E + 02 0: 1.00 ORM2, Alpha-1-acid glycoprotein 2 IPI00940393 5.51 3.85E + 02 0 1.00: 0 EEF1A1 IPI00013179 5.49 7.22E + 02 0 1.00: 0 PTGDS, Prostaglandin-H2 D-isomerase IPI00160130 5.43 7.75E + 00 0 1.00: 0 CUBN, Cubilin IPI00217232 5.42 0 3.40E + 02 0: 1.00 SUCLA2, Isoform 2 of Succinyl-CoA ligase [ADP-forming] subunit beta mitochondrial IPI00294713 5.4 8.29E + 02 0 1.00: 0 MASP2, Isoform 1 of Mannan-binding lectin serine protease 2 IPI00930226 5.39 0 2.86E + 02 0: 1.00 ACTG1, cDNA FLJ57283 IPI00414684 5.38 2.71E + 02 0 1.00: 0 SEMG1, Isoform 2 of Semenogelin-1 IPI00037070 5.37 2.88E + 02 0 1.00: 0 HSPA8 IPI00022371 5.31 0 5.59E + 02 0: 1.00 HRG, Histidine-rich glycoprotein IPI00299116 5.29 1.75E + 03 0 1.00: 0 PODXL, Podocalyxin-like isoform 2 precursor

3. Candidate STA and TCMR proteomic biomarker validation

We selected candidate proteins based on proteomic analysis to discover specific exosome proteins in each group. A total of 63 proteins were screened by protein quantification of urine exosomes using label-free LC-MS / MS. STA or TCMR, and selected candidate biomarkers among the identified proteins in the ExoCarta database. Finally, we selected five candidate proteins that are expected to differ between STA and acute TCMR. The identified proteins were TSPAN1, HPX, PIGR, APOA1 and LGALS3BP.

To verify the expression of the selected candidate biomarkers, western blot analysis of the collected urine was performed. TSPAN1 and HPX expression levels were significantly higher in patients with acute TCMR than those in STA (STA: acute TCMR ratio (1: 1.81, P = 0.009, 1: 3.48, P = 0.046, PIGR (ratio = 1: 1.27, P = 0.50), APOA1 (ratio = 1: 1.35, P = 0.54), and LGALS3BP (ratio = 1: 0.96, P = 0.91).

The potential of TSPAN1 and HPX, which can distinguish STA from acute TCMR through ROC analysis, is then evaluated and shown in FIG. As a result, the AUC was 0.744, and the sensitivity and specificity were 64.0% and 72.7%, respectively.

4. Functional interaction network analysis of verified proteomic biomarkers

After validation, the STRING database was used to predict functional protein-protein interactions. Two verified proteins, TSPAN1 and HPX, were used for interaction analysis and experimentally induced interactions were used to depict each interaction (Figure 4).

As a result, integrin alpha, beta subunits, and other transmembrane 4 superfamily proteins interacted with TSPAN1 in the TSPAN1 network map.

In addition, HPX network maps showed that albumin, growth factors, matrix metalloproteinases (MMPs) and tissue inhibitors of TIMPs (metalloproteinases) interact with HPX.

5. Verification of LGS and CAMR biomarkers

For the discovery of specific exosome proteins in a group of 33 LGS kidney transplant patients and 9 urine samples of 9 CAMR patients, the inventors selected candidate proteins based on proteomic analysis. A total of 50 proteins were detected in protein quantification of urine exosomes. In the dual ExoCarta database, we selected proteins that were significantly up-regulated and identified exosome proteins in each group. Finally, seven proteins (APOA1, TSPAN1, TTR, PIGR, HPX, AZGP1 and CP) were selected between LGS and CAMR to be selected for different expression patterns (results not shown).

In order to verify the expression of seven proteins expressing the difference in expression between LGS and CAMR, urine samples obtained from 24 patients with LGS kidney transplantation and 17 patients with CAMR were analyzed by Western blotting. As a result, the level of expression of TSPAN1 in urine exoemia was LGS: CAMR = 1: 1.5 (p <0.0001), indicating that TSPAN1 could be a marker for diagnosing CAMR (FIG.

 In addition, the potential of TSPAN1 to distinguish LGS from CAMR through ROC analysis is evaluated and shown in Fig. As a result, it was confirmed that AUC was 0.882, sensitivity was 76.5%, and specificity was 83.3%.

Since the biomarker TSPAN1 provided by the present invention is significantly increased in patients exhibiting a renal transplant rejection reaction, the diagnostic composition or the TSPAN1 detection method comprising the agent for measuring the expression level of TSPAN1 can be used to determine whether or not a kidney transplant rejection Can be diagnosed accurately and quickly, and thus the useability is very good in industry.

<110> Kyungpook National University Industry-Academic Cooperation Foundation          KYUNGPOOK NATIONAL UNIVERSITY HOSPITAL <120> Composition for diagnosing kidney allograft refection and method          for detecting a diagnostic marker <130> NP18-0046 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 1638 <212> DNA <213> Artificial Sequence <220> <223> Human tetraspanin 1 <400> 1 tcccggcctc agacacacac accagcagct acacctacac gctgaccatc acaggcacac 60 agaggcacat ccacctcaca tccacctcat acttgtgtac tctcagggtt cagtctttca 120 cctatccctc tctgatctgt gcctcccaat accttccaag atgtttacag agacccttct 180 ccctgtgcag ttaggagtgt aaggcaagag agcccctact tcatggggca gatcaagagc 240 tgagaccaaa gatggtctat gttgctgacc ttgtcctgtc ctcctgctgt cttaaactat 300 gatccctgct gcggtcactg aagcctttcc ctgtgagcag tggtgtgtga gagccaggcg 360 tccctctgcc tgcccactca gtggcaacac ccgggagctg ttttgtcctt tgtggagcct 420 cagcagttcc ctctttcaga actcactgcc aagagccctg aacaggagcc accatgcagt 480 gcttcagctt cattaagacc atgatgatcc tcttcaattt gctcatcttt ctgtgtggtg 540 cagccctgtt ggcagtgggc atctgggtgt caatcgatgg ggcatccttt ctgaagatct 600 tcgggccact gtcgtccagt gccatgcagt ttgtcaacgt gggctacttc ctcatcgcag 660 ccggcgttgt ggtctttgct cttggtttcc tgggctgcta tggtgctaag actgagagca 720 agtgtgccct cgtgacgttc ttcttcatcc tcctcctcat cttcattgct gaggttgcag 780 ctgctgtggt cgccttggtg tacaccacaa tggctgagca cttcctgacg ttgctggtag 840 tgcctgccat caagaaagat tatggttccc aggaagactt cactcaagtg tggaacacca 900 ccatgaaagg gctcaagtgc tgtggcttca ccaactatac ggattttgag gactcaccct 960 acttcaaaga gaacagtgcc tttcccccat tctgttgcaa tgacaacgtc accaacacag 1020 ccaatgaaac ctgcaccaag caaaaggctc acgaccaaaa agtagagggt tgcttcaatc 1080 agcttttgta tgacatccga actaatgcag tcaccgtggg tggtgtggca gctggaattg 1140 ggggcctcga gctggctgcc atgattgtgt ccatgtatct gtactgcaat ctacaataag 1200 tccacttctg cctctgccac tactgctgcc acatgggaac tgtgaagagg caccctggca 1260 agcagcagtg attgggggag gggacaggat ctaacaatgt cacttgggcc agaatggacc 1320 tgccctttct gctccagact tggggctaga tagggaccac tccttttagg cgatgcctga 1380 ctttccttcc attggtgggt ggatgggtgg ggggcattcc agagcctcta aggtagccag 1440 ttctgttgcc cattccccca gtctattaaa cccttgatat gccccctagg cctagtggtg 1500 atcccagtgc tctactgggg gatgagagaa aggcatttta tagcctgggc ataagtgaaa 1560 tcagcagagc ctctgggtgg atgtgtagaa ggcacttcaa aatgcataaa cctgttacaa 1620 tgttgccaaa aaaaaaaa 1638 <210> 2 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> Human tetraspanin 1 <400> 2 Met Gln Cys Phe Ser Phe Ile Lys Thr Met Met Ile Leu Phe Asn Leu   1 5 10 15 Leu Ile Phe Leu Cys Gly Ala Leu Leu Ala Val Gly Ile Trp Val              20 25 30 Ser Ile Asp Gly Ala Ser Phe Leu Lys Ile Phe Gly Pro Leu Ser Ser          35 40 45 Ser Ala Met Gln Phe Val Asn Val Gly Tyr Phe Leu Ile Ala Ala Gly      50 55 60 Val Val Val Phe Ala Leu Gly Phe Leu Gly Cys Tyr Gly Ala Lys Thr  65 70 75 80 Glu Ser Lys Cys Ala Leu Val Thr Phe Phe Ile Leu Leu Leu Ile                  85 90 95 Phe Ile Ala Glu Val Ala Ala Val Val Ala Leu Val Tyr Thr Thr             100 105 110 Met Ala Glu His Phe Leu Thr Leu Leu Val Val Pro Ala Ile Lys Lys         115 120 125 Asp Tyr Gly Ser Gln Glu Asp Phe Thr Gln Val Trp Asn Thr Thr Met     130 135 140 Lys Gly Leu Lys Cys Cys Gly Phe Thr Asn Tyr Thr Asp Phe Glu Asp 145 150 155 160 Ser Pro Tyr Phe Lys Glu Asn Ser Ala Phe Pro Pro Phe Cys Cys Asn                 165 170 175 Asp Asn Val Thr Asn Thr Ala Asn Glu Thr Cys Thr Lys Gln Lys Ala             180 185 190 His Asp Gln Lys Val Glu Gly Cys Phe Asn Gln Leu Leu Tyr Asp Ile         195 200 205 Arg Thr Asn Ala Val Thr Val Gly Gly Val Ala Gly Ile Gly Gly     210 215 220 Leu Glu Leu Ala Ala Met Ile Val Ser Met Tyr Leu Tyr Cys Asn Leu 225 230 235 240 Gln    

Claims (14)

A composition for diagnosing rejection of kidney transplantation comprising an agent for measuring the expression level of protein or mRNA of urinary TSPAN1 (tetraspanin 1).
2. The composition according to claim 1, wherein the agent for measuring the expression level of the mRNA is a probe or a primer that specifically binds to mRNA of TSPAN1.
2. The composition of claim 1, wherein the agent that measures the level of expression of the protein is an antibody specific for a protein of TSPAN1.
2. The composition of claim 1, wherein the mRNA of TSPAN1 comprises the nucleotide sequence of SEQ ID NO: 1.
2. The composition of claim 1, wherein the protein of TSPAN1 comprises the amino acid sequence of SEQ ID NO: 2.
The composition of claim 1, wherein said composition further comprises an agent that measures the level of expression of HPX (hemopexin) protein or mRNA.
The method according to claim 1, wherein the rejection of the kidney transplant is selected from the group consisting of acute T cell mediated rejection (TCMR), chronic antibody mediated rejection (CAMR) Wherein the antibody is selected from the group consisting of active antibody mediated rejection, chronic active T cell mediated rejection, and mixed rejection.
A kit for the diagnosis of rejection of kidney transplantation comprising an agent for measuring the expression level of protein or mRNA of urinary TSPAN1 (tetraspanin 1).
In order to provide information necessary for diagnosis of rejection of renal transplantation,
(a) measuring the expression level of TSPAN1 (tetraspanin 1) protein or mRNA in the urine obtained from a kidney transplant patient; And
(b) comparing the expression level of the TSPAN1 protein or mRNA with that of a normal control, and determining that the test subject whose expression level of the TSPAN1 protein or mRNA is increased has a rejection reaction of the kidney transplantation, A method for detecting a marker.
delete 10. The method according to claim 9, wherein the expression level of the mRNA is measured in a group consisting of a reverse transcriptase polymerase, a competitive reverse transcriptase polymerase, a real-time reverse transcriptase polymerase, an RNase protection assay, a Northern blotting and a DNA chip &Lt; / RTI &gt;
10. The method according to claim 9, wherein the method for measuring the protein expression level is selected from the group consisting of Western blotting, ELISA, radioimmunoassay, radial immunodiffusion, Oucheroni immunodiffusion, rocket immunoelectrophoresis, Wherein the method is one or more selected from the group consisting of a fixed assay method, a FACS and a protein chip method.
The method according to claim 9, wherein the expression level of HPX (hemopexin) protein or mRNA is further measured in step (a), and the expression level of protein or mRNA of TSPAN1 and HPX is compared with a normal control in step (b) And determining that the increased body is present in the rejection reaction of kidney transplantation.
A method for screening a therapeutic agent for renal transplant rejection comprising administering to a mammal other than a human a renal transplant rejection therapeutic candidate substance, the level of expression of the protein or mRNA of urinary TSPAN1.

Images