KR20190094967A - Biomarkers for diagnosis or prognosis of fibrosis in kidney allografts - Google Patents

Abstract

The present invention relates to biomarkers for diagnosing or predicting fibrosis after kidney transplantation. More specifically, the present invention relates to compositions, kits, and methods for diagnosing fibrosis or predicting prognosis of transplanted organs after kidney transplantation, the composition including TG2 or IL-6 protein or a substance for measuring mRNA expression level of a gene encoding the protein. The diagnostic marker of the present invention is for predicting the risk of fibrosis or the degree of fibrosis in the transplanted kidney of a patient having undergone kidney transplant surgery. Specifically, by measuring the expression level of the biomarkers of the present invention and calculating the correlation between the biomarkers, it is possible to quickly and accurately predict the fibrosis of transplanted kidney tissue. In addition, it is possible to quantitatively measure and compare individual samples by using the fibrosis progression risk formula derived by combining the biomarkers of the present invention, thereby significantly improving diagnostic specificity and efficiency.

Description

Biomarkers for diagnosis or prognosis of fibrosis in kidney allografts

The present invention relates to a biomarker for diagnosing or predicting fibrosis of transplanted organs after renal transplantation. More specifically, the present invention includes a TG2 or IL-6 protein or a substance measuring mRNA expression level of a gene encoding the protein. The present invention relates to a composition, kit and diagnostic method for diagnosing or predicting fibrosis of a transplanted organ after renal transplantation.

Organ transplants are slowly becoming part of precision medicine. When using current standard immunosuppressive therapy, acute rejection still occurs in 15 to 20% of patients undergoing kidney transplants.To date, tissue biopsies are diagnosed when there is an increase in serum creatine levels or the development of new proteinuria. . Since the renal state at the time of increased serum creatine concentration or the expression of new proteinuria is already well developed, there is a need for a biomarker capable of early detection of subclinical acute rejection.

In identifying and evaluating transplanted organs after transplantation, although needle biopsies are safer and more standardized, there is a risk that bleeding after biopsy can result in the loss of transplant organs. In addition, there is a problem that the interpretation of the biopsy can vary from one observer to another and depends on the error of the sample. To make matters worse, there is a problem that systematic biopsy studies and meta-analysis are not possible because the diagnostic criteria are revised every two years, and therefore the interconversion of tissue classifications is impossible.

Congenital / acquired immune responses are known to appear first at the molecular level before they appear at the histological level. Therefore, the development of non-invasive biomarkers for the prediagnosis and monitoring of clinical conditions after renal transplantation is an essential step in precision medicine.

A single biomarker has been reported to assess interstitial fibrosis & tubular atrophy (IF / TA) of transplanted kidney after kidney transplantation. However, there is no known biomarker combination to assess the severity of interstitial fibrosis and tubular atrophy and distinguish it from tubulointerstitial inflammation.

An object of the present invention is the mRNA expression level of at least one protein selected from the group consisting of TG2 (transglutaminase 2) and IL-6 (interleukin-6, interleukin-6) or a gene encoding the protein It is to provide a composition for diagnosing or predicting fibrosis of transplanted organs after kidney transplant comprising a substance measuring the amount thereof.

Another object of the present invention is to diagnose fibrosis or predict prognosis of transplanted organs after renal transplantation, which comprises a substance measuring mRNA expression level of at least one protein selected from the group consisting of TG2 and IL-6 or a gene encoding the protein. It is to provide a kit for.

Another object of the present invention is to measure the mRNA expression level of at least one protein selected from the group consisting of TG2 and IL6 or a gene encoding the protein in a biological sample obtained from the individual, transplantation using the measured expression level It provides a method for providing information for diagnosing or predicting the prognosis of the transplanted organs after renal transplantation, comprising calculating the risk of progressing fibrosis of the organs and comparing the calculated risks with a reference.

The present invention is to solve the above-described problems, the organ of transplantation after renal transplantation comprising at least one protein selected from the group consisting of TG2 and IL-6 or a substance measuring the mRNA expression level of the gene encoding the protein Provided are compositions for diagnosing or predicting fibrosis.

The term "fibrosis of transplanted organs after kidney transplant" refers to fibrosis in the kidney transplanted in a patient who has undergone kidney transplantation, preferably the fibrosis is interstitial fibrosis and tubular atrophy (IFTA) It may be, but is not limited thereto.

As used herein, the term "diagnosis" refers to identifying the presence or characteristics of a pathological condition, and in particular, identifying the presence or symptom of fibrosis in a transplant organ of a patient undergoing a kidney transplant.

As used herein, the term "prediction" relates to the possibility of developing fibrosis in transplanted organs of patients undergoing kidney transplants. Prediction method of the present invention can be used clinically to determine whether the immune rejection response or stability of the transplanted kidney by performing a biomarker test for a patient undergoing a kidney transplant surgery.

The term "biomarker" used in the present invention is a substance that can be determined by discriminating whether fibrosis has occurred or the degree of fibrosis progression of the transplanted kidney, a cell in which fibrosis has occurred compared to a cell or tissue in which fibrosis has not occurred, Include proteins or mRNAs that differ by increasing or decreasing expression in tissues. For the purposes of the present invention, the biomarker for diagnosing or predicting fibrosis of a transplanted organ after renal transplantation is a TG2 or IL-6 protein in a sample, or a gene encoding the same, and includes a set containing one or more of these biomarkers. The diagnostic efficiency can be improved.

As used herein, the term "measurement of expression level of protein" refers to the presence or absence of a fibrosis diagnostic marker (protein) or a gene encoding the same in a biological sample after transplantation in order to diagnose fibrosis of the transplanted organ after kidney transplantation. It means the process of checking the expression level. Expression level measurement or comparative analysis of the protein may include protein chip analysis, immunoassay, ligand binding assay, Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry, matrix-assisted laser desorption / Ionized time-of-flight mass spectrometry), Surface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry, surface-enhanced laser desorption / ionization time-of-flight mass spectrometry, radioimmunoassay, radioimmunoassay radioimmunodiffusiony, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry, complement fixation assay, two-dimensional electrophoresis Liquid chromatography-mass spectrometry (LC-MS), liquid chromatogra (LC-MS / MS) phy-Mass Spectrometry / Mass Spectrometry, Western blotting, and ELISA (enzyme linked immunosorbent assay), but are not limited thereto.

In the composition for diagnosing fibrosis of transplanted organs after renal transplantation of the present invention, the material for measuring the protein expression level may include antibodies, interacting proteins, ligands, oligopeptides, peptide nucleic acid, Peptide nucleic acids), nanoparticles, or aptamers.

As used herein, the term "antibody" refers to a substance that specifically binds to an antigen to produce an antigen-antibody response. For the purposes of the present invention, an antibody refers to an antibody that specifically binds to TG2 protein or IP-6 protein. Antibodies of the invention include all polyclonal antibodies, monoclonal antibodies and recombinant antibodies. Such antibodies can be readily prepared using techniques well known in the art. For example, polyclonal antibodies are produced by methods well known in the art, including the steps of injecting fibrosis marker protein antigens of transplanted organs into the animal and collecting blood from the animal to obtain serum comprising the antibody after the kidney transplant. Can be. Such polyclonal antibodies can be prepared from any animal, such as goats, rabbits, sheep, monkeys, horses, pigs, cattle, dogs, and the like. In addition, monoclonal antibodies are well known in the art by the hybridoma method (see Kohler and Milstein (1976) European Journal of Immunology 6: 511-519), or phage antibody library techniques (Clackson et al, Nature, 352). : 624-628, 1991; Marks et al, J. Mol. Biol., 222: 58, 1-597, 1991). Antibodies prepared by the above method can be isolated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like. In addition, antibodies of the invention include functional fragments of antibody molecules, as well as complete forms with two full length light chains and two full length heavy chains. The functional fragment of an antibody molecule means the fragment which has at least antigen binding function, and includes Fab, F (ab '), F (ab') 2, Fv, etc. In addition, the antibody of the present invention may be commercially obtained.

The term "Peptide Nucleic Acid" (PNA) as used herein refers to a polymer similar to DNA or RNA artificially synthesized. DNA has a phosphate-ribose sugar backbone, whereas PNA has a repeated N- (2-aminoethyl) -glycine backbone linked by peptide bonds, which greatly increases binding and stability to DNA or RNA, leading to molecular biology. It is used in diagnostic analysis and antisense therapies. PNAs are described in Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide". Science 254 (5037): 1497-500.

"Aptamers" in the present invention are oligonucleic acid or peptide molecules, the general contents of which are described in Bock LC et al., Nature 355 (6360): 5646 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78 (8): 42630 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95 (24): 142727 (1998).

The term "measurement of expression level of mRNA" used in the present invention refers to a process of confirming the presence and expression level of mRNA of genes encoding the diagnostic protein in a biological sample in order to diagnose fibrosis of transplanted organs after kidney transplantation. Means to measure. Analytical methods for this purpose include reverse transcriptase (RT-PCR), competitive reverse transcriptase (RT) PCR, real-time reverse transcriptase (Real-time RT-PCR), RNase protection assay (RPA). assays, Northern blotting, DNA chips, etc., but are not limited thereto.

In the composition for diagnosing fibrosis of transplanted organs after renal transplantation according to the present invention, a substance for measuring the expression level of mRNA of the gene encoding TG2 protein or a substance for measuring the expression level of mRNA of the gene encoding IL-6 protein Primers, probes or antisense nucleotides that specifically bind to the mRNA of the gene encoding each protein. Those skilled in the art will be able to easily design primers, probes or antisense nucleotides that specifically bind to mRNA of the gene encoding the protein.

As used herein, the term “primer” refers to a fragment that recognizes a target gene sequence, which includes primer pairs in the forward and reverse directions, but is preferably a primer pair that provides assay results with specificity and sensitivity. High specificity can be imparted when the nucleic acid sequence of the primer is a sequence that is inconsistent with the non-target sequence present in the sample so that only the target gene sequence containing the complementary primer binding site is amplified and does not cause nonspecific amplification. .

As used herein, the term “probe” refers to a substance that can specifically bind to a target substance to be detected in a sample, and means a substance that can specifically confirm the presence of the target substance in the sample through the binding. do. The type of probe is a material commonly used in the art, but there is no limitation, but preferably, PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA And most preferably PNA. More specifically, the probes are biomaterials, including those derived from or similar to organisms or produced in vitro, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, neurons, DNA, and It may be RNA, DNA includes cDNA, genomic DNA, oligonucleotides, RNA includes genomic RNA, mRNA, oligonucleotides, examples of proteins may include antibodies, antigens, enzymes, peptides and the like.

As used herein, the term “antisense” refers to the use of a nucleotide base to allow the antisense oligomer to hybridize with a target sequence in RNA by Watson-Crick base pairing, typically allowing the formation of mRNA and RNA: oligomeric heterodimers within the target sequence. An oligomer having a backbone between the sequence and subunits. The oligomer may have exact sequence complementarity or approximate complementarity to the target sequence.

The present invention also provides a kit for diagnosing or predicting fibrosis of a transplanted organ after kidney transplant comprising the composition. The kit can be prepared by conventional manufacturing methods known in the art. The kit may include, for example, an antibody in freeze-dried form and a buffer, stabilizer, inert protein, and the like.

The kit may further comprise a detectable label. The term “detectable label” means an atom or molecule that enables the specific detection of a molecule comprising a label among molecules of the same kind without a label. The detectable label may be attached to an antibody, interacting protein, ligand, nanoparticle, or aptamer that specifically binds to the protein or fragment thereof. The detectable label may comprise a radionuclide, a fluorophore, an enzyme.

The kit can be used according to various immunoassays or immunostaining methods known in the art. The immunoassay or immunostaining method may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, ELISA, capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining and immunoaffinity purification. Preferably, the kit is a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an enzyme linked immunosorbent assay (ELISA) kit, a protein chip kit, a rapid (rapid) ) Kit or MRM (multiple reaction monitoring).

The kit can also be used for mass spectrometry. In this case, the specific amino acid residues of the protein are myristoylation, isoprenylation, prenylation, prenylation, glypiation, lipolation, acylation, alkylation alkylation, methylation, demethylation, amidation, ubiquitination, phosphorylation, deamidation, deamidation, glycosylation, oxidation, Or a modification such as acetylation.

In addition, the present invention comprises the steps of measuring the mRNA expression level of at least one protein selected from the group consisting of TG2 and IL-6 or a gene encoding the protein in a biological sample obtained from the individual; Calculating the risk of fibrosis progression of the transplanted organ using the measured expression level; And it provides a method for providing information for diagnosing or prognostic fibrosis of the transplanted organ after kidney transplant comprising the step of comparing the calculated risk with a reference (reference).

In this method, a "biological sample" is a tissue, cell, blood, serum, plasma, saliva, cerebrospinal fluid or urine that differs in protein or gene expression levels due to the degree of fibrosis of transplanted organs in patients undergoing kidney transplants. It means a sample such as.

The protein level can be measured by the immunoassay or immunostaining described above. The method may also be carried out in the form of a microchip or automated microarray system capable of detecting the biomarker protein or fragment thereof in a sample.

The protein levels include multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), sequential windowed data independent acquisition of the total high-resolution (SWATH), and selected reaction monitoring (SRM). ) Or immuno multiple reaction monitoring (iMRM). MRM is a method of determining the exact fraction of a substance, breaking it out on a mass spectrometer, then selecting a particular ion from a broken ion once more and obtaining the number using a continuously connected detector. Using the MRM method, mass spectrometry can be used to quantify the protein or fragment thereof in blood samples from individuals with high or low fibrosis risk of transplanted kidney.

The mRNA level can be measured by RT-PCR, competitive RT-PCR, quantitative RT-PCR, RNase protection assay, Northern blot, or DNA chip.

According to one aspect of the invention, the step of calculating the risk of fibrosis progression, calculating the time difference (KT to BX) between the time of kidney transplantation and biopsy; And calculating the risk of progression of fibrosis of the transplanted organ using the protein level and the time difference between the time of renal transplantation and the time of biopsy.

According to another aspect of the present invention, the risk of fibrosis progression has a positive correlation with the TG2 expression level or the time difference between the time of renal transplantation and biopsy, and is negative with IL-6 expression level. It may have a correlation of.

According to one preferred aspect of the invention, the step of calculating the risk of progression of fibrosis of the transplanted organ may be calculated using the following relationship the protein level measured in each sample:

Risk of progression of fibrosis = 0.373 x log _e (TG2 / Cr)-0.111 x log _e (IL-6 / Cr) + 0.007 x log _e (KT to BX) + 2.455

In this case, the TG2 and IL-6 means the respective protein expression level value, the Cr means the creatinine concentration measurement value of each sample, the KT to BX is the time difference between the time of kidney transplantation and biopsy it means.

According to one aspect of the invention, the fibrosis may be interstitial fibrosis and tubular atrophy (IFTA).

The diagnostic marker of the present invention is for predicting the risk of fibrosis or the degree of fibrosis in the transplanted kidney of a patient who has undergone kidney transplant surgery. Specifically, the diagnostic marker measures the expression level of the biomarker of the present invention and correlates the relationship between the biomarkers. By calculating, one can quickly and accurately predict whether fibrosis in the transplanted kidney tissue has occurred. In addition, by using the fibrosis progress risk calculation formula derived by combining the biomarker of the present invention, it is possible to quantitatively measure and compare individual samples, thereby significantly improving diagnostic specificity and efficiency.

1 is a diagram showing the classification results of the test participants according to an embodiment of the present invention.
Figure 2 shows the results of confirming the concentration of TG2 (a), SDC4 (b) and A1M (c) by ELISA (Donor, kidney donor group, n = 50; Bx, biopsy group, n = 115) No Bx, no biopsy group, n = 53).
Figure 3 shows the results of confirming the concentration of IP-10 (a), IL-6 (b) and MCP-1 (c) using a flow cytometry bead array (CBA) (Donor, Kidney donor group, n = 50; Bx, biopsy group, n = 115; No Bx, biopsy group, n = 53).
FIG. 4 shows biopsy samples of transplanted kidney tissue divided into three groups according to IFTA score (ct + ci), biomarkers TG2 (a), SDC4 (b), A1M (c), IP-10 of each group. (d), the result of quantitative comparison of IL-6 (e) and MCP1 (f) concentration is shown.

Below, Through the examples will be described in more detail the present invention. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Experimental Method

1. Experimental subject

For quantitative analysis of candidate biomarkers, urine samples were collected from 260 patients aged 17-77 years, who were at least 6 months old, at the time of routine examination or before indication biopsy. In addition, urine samples were collected from 50 surviving kidney donors just prior to resection. Among 260 patients, those without symptomatic biopsies had an average serum creatinine concentration of 2.0 mg / dL (180 umol / L) or less at the time of participation, no history of acute rejection treatment, or cytomegalovirus (CMV) ) Or BK polyomavirus (polyomavirus type BK, BKV) infection was found to be stable after kidney transplantation.

2. Urine Sample Collection and Storage

Approximately 35-50 mL of urine samples were collected from each participant, and 0.5 mL protease inhibitor mixture (5 mM 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride [4- [4] was collected in each urine sample. (2-aminoethyl) benzensulfonyl fluoride hydrolchloride, 2 μM Leupeptin-hemisulfate, and 3.3 mM sodium azide were added whole cell, large membrane particles. urine samples were centrifuged at 4,000 rpm, 4 ° C. for 15 minutes to remove urinary sediments, including particles), and other debris, and the supernatant was stored at −80 ° C. for use in the experiment.

3. Creatinine normalization Creatinine  normalization)

Urine molecule concentration depends on various biologic variables such as liquid intake, body composition, liver function, and kidney function. Urinary creatinine concentration was measured as urine creatinine concentration as an indicator of urine dilution (R & D system. Inc.).

50 μl of 20-fold diluted urine samples and 100 μl of alkaline picrate were mixed and reacted in the dark for 30 minutes. The absorbance of each well was measured at 490 nm using a microplate reader (Sunrise, Tecan Trading AG, Switzerland), using Magellan software [Magellan ^™ data analysis software, Tecan Trading AG]. And analyzed. Creatinine normalization was performed by mathematically calculating the concentration of each molecule according to the creatinine concentration of each sample.

4. ELISA assay-TG2, SDC4  And A1M

ELG kits were used to quantify TG2 (transglutaminase 2; Mybiosource, San Diego, Calif., USA), SDC4 (syndecan 4; R & D system), and A1M (α-1 microglobulin; abcam).

In order to detect TG2, 100 μl of the urine sample diluted 60-fold and the standard sample were placed in the bottom of the micro ELISA plate well and reacted at 37 ° C. for 90 minutes. Next, a detection antibody with biotin was added to each well, and reacted at 37 ° C. for 1 hour. After washing the plate three times with a washing buffer, the solution was added to each well HRP bound for 30 minutes at 37 ℃. After washing, 90 μl of substrate solution was added and reacted at 37 ° C. for 15 minutes. Each well was measured for absorbance at 450 nm using a Microplate reader (Sunrise).

To detect A1M, 50 μl of a 20,000-fold diluted urine sample and a standard sample were placed in a well and reacted at room temperature for 120 minutes. To detect SDC4, 100 μl of an undiluted urine sample was placed in a well for 120 minutes at room temperature. The reaction was carried out by the same method as the TG2 ELISA. Each candidate was normalized to creatinine values.

5. Cytometric  bead array- MCP -1, IP-10 and IL-6

Monocyte chemoattractant protein 1 (MCP-1), interferon-gamma-induced protein 10 using a cytoplasmic bead array (CBA) method with a CBA flexset kit (BD Life Science, CA, USA) concentrations of interferon gamma-inducible protein 10 (IP-10) and interleukin-6 (IL-6) were measured.

Briefly, 50 μl of mixed capture beads were mixed with the same volume of urine or standard sample and reacted for 1 hour while shaking in a dark room at room temperature so that cytokines could adhere well to the capture beads. Next, the PE detection reagent was added to each tube and reacted for 2 hours while shaking in a dark room at room temperature.

In the case of IL-6 analysis, a reagent for further improving the reaction signal was added and reacted for 1 hour. Fluorescently labeled cytokine specific beads were analyzed by flow cytometry and analyzed by FCAP array software (BD). Each candidate was normalized to creatinine values.

6. Signs Allograft Biopsy (indication allograft  biopsy)

123 patients received indication biopsy at the time of the study and urine samples were collected 2-3 hours prior to biopsy. Eight of the biopsies diagnosed with BKVN were excluded to minimize the effect of viral infection on tubulointerstitial inflammation. All biopsy samples were scored according to Banff 2013 classification.

7. Statistical Analysis

The experimental entry group was divided into three subgroups: patients undergoing symptomatic biopsy (n = 115), patients whose transplanted tissue exhibited stable function (n = 53), and living kidney donors (n = 50).

Variables by category are expressed as absolute and relative frequencies. Quantitative variables are expressed as mean and standard deviation (SD). Differences between means were analyzed using analysis of variance of continuous variables (ANOVA). Quantitative values of urine biomarker candidates were adjusted to urine creatinine and then converted to log values (log _e ) prior to analysis. Multivariate linear regression was used to analyze the correlation between the concentrations of each biomarker candidate and the pathological scores (ci + ct, t + i, g + ptc, and cg + cv) indicated by each allograft state.

All statistical analyzes were performed with SPSS version 21.0 (SPSS Inc., Chicago, IL) and R software version 3.1.2 (R Foundation for Statistical Computing, Vienna, Austria) and assume statistical significance when the P value is less than 0.05. It was.

<Experiment Result>

1. Experimental subject

Of the 260 patients who received a kidney transplant, 87 did not have symptomatic biopsies, 34 of whom did not meet stable graft function criteria (21 patients with an average serum creatinine concentration of 2.0 mg / dL or higher; 8 patients with at least one treatment history; 5 patients with CMV or BKV infection detected at the time of the study.

123 of 260 patients underwent symptomatic biopsies and urine samples were taken 2-3 hours prior to biopsy. In these biopsies, eight samples identified with BKVN (polyomavirus type BK virus nephropathy) infection were excluded to minimize the effect of viral infection on tubulointerstitial inflammation.

As a result, the concentration of candidate biomarkers for symptomatic biopsy patients (n = 115) was compared with patients with stable function of transplanted tissue (n = 53) and kidney donors immediately before renal transplantation (n = 50).

The classification results of the experiment participants are shown in FIG. 1, and the characteristics of the experiment participants are shown in Table 1 below.

variable
All participants
(n = 168) symptom Biopsy  patient
(n = 115) Graft
Stable patient
(n = 53) Organ donor
(n = 50) Organ transplanter  Characteristic Mean age, years (SD) 50.9 (11.0) 49.6 (11.7) 53.8 (8.4) 43.7 (13.4) Female gender, n (%) 71 (42.3) 42 (36.5) 29 (54.7) 26 (53.1) Body mass index, kg / m ² (SD) 22.9 (3.6) 22.9 (3.7) 22.9 (3.4) 25.3 (3.3) Previous transplant, n (%) 11 (6.5) 10 (8.7) 1 (1.9) Cause of ESRD, n (%)   Glomerular 31 (18.6) 17 (14.8) 14 (26.9)   Diabetes 27 (16.2) 17 (14.8) 10 (19.2)   Hypertension 21 (12.6) 12 (10.4) 9 (17.3)   PKD 6 (3.6) 4 (3.5) 2 (3.8)   FSGS 2 (1.2) 2 (1.7) 0   Other 51 (30.5) 43 (37.4) 8 (15.4)   Unknown 29 (17.4) 20 (17.4) 9 (17.3) Serum creatinine at the time of enrollment, mg / dL (SD) 1.74 (1.44) 2.51 (1.62) 0.98 (0.25) 0.77 (0.16) Peak PRA,% (SD) 17.2 (30.7) 14.2 (29.2) 23.7 (33.0) Pre-transplant DSA, n (%) 16 (15.4%) 9 (13.8) 7 (17.9) ABO incompatible KT 22 (13.1) 18 (15.7) 4 (7.5) CNI at the time of enrollment, n (%)   Cyclosporine 38 (22.6) 25 (21.7) 13 (24.5)   Tacrolimus 130 (77.4) 90 (78.3) 40 (75.5) Induction regimen, n (%)   No induction 45 (26.8) 37 (32.2) 8 (15.1)   Basiliximab 110 (65.5) 68 (59.1) 42 (79.2)   Anti-thymocyte globulin 13 (7.7) 10 (8.7) 3 (5.7) Time to enrollment, months (SD) 82.5 (79.1) 87.0 (79.2) 72.8 (78.8) Characteristics of Organ Donors Deceased donor, n (%) 44 (26.3) 27 (23.5) 17 (32.7) Mean age, years (SD) 41.7 (14.0) 42.7 (13.1) 39.2 (16.2) Female gender, n (%) 72 (42.9) 56 (48.7) 16 (30.2) Serum creatinine at the time of donation, mg / dL (SD) 0.93 (0.57) 0.88 (0.44) 1.06 (0.78)

As shown in Table 1 above, the kidney donor was younger and showed a higher BMI index as compared to the group receiving the kidney transplant. In addition, serum creatinine levels in patients with symptomatic biopsies were significantly higher than in the other groups. The baseline characteristics between the symptomatic biopsy group and the stably implanted patient group were not significantly different except that serum creatinine was high in the symptomatic biopsy patient.

2. Biomarkers  compare

Results of confirming the concentration of TG2, SDC4 and A1M using ELISA are shown in Figures 2a to 2c, and the results of confirming the concentration of MCP-1, IP-10 and IL-6 using a cytometric bead array (CBA) 3a to 3c. As a result, log _e TG2 / Cr (Fig. 2a, Bx = 0.63 ± 0.88; No Bx = -0.15 ± 0.58; Donor = -0.36 ± 0.34, p <0.001), log _e in the biopsy (Bx) group compared to the other groups. A1M / Cr (FIG. 2c, Bx = 2.86 ± 0.55; No Bx = 2.41 ± 0.61; Donor = 2.01 ± 0.51, p <0.001), log _e IL-6 / Cr (FIG. 3b, Bx = 1.79 ± 0.82; No Bx = 1.36 ± 0.85; Donor = 1.44 ± 0.60, p <0.001), and log _e MCP-1 / Cr (FIG. 3c, Bx = 0.40 ± 0.49; No Bx = −0.03 ± 0.54; Donor = 0.05 ± 0.34, p < 0.001) was found to increase significantly.

3. Signs Biopsy  According to classification Biomarkers  Compare (ct + ci)

In the symptomatic biopsy group, interstitial fibrosis and tubular atrophy (IFTA) symptoms were scored and classified into three groups according to IFTA scores (ct + ci) to compare biomarker concentrations in each group (Fig. 4a to 4f). As a result, as the IFTA score increases, log _e TG2 / Cr value significantly increased, while it was confirmed that the log _e IL-6 / Cr value significantly decreased (Fig. 4a, Fig. 4e).

Data were calibrated according to the patient's age, sex, body mass index (BMI), diabetes, hypertension, cardiovascular disease, and renal transplantation and biopsy, and the correlations between each biomarker were identified. . As a result, as shown in Table 2 below, interstitial fibrosis and tubular atrophy (IFTA) have a positive correlation with TG2 concentration and the time between KT to biopsy and negative IL-6 concentration. It can be confirmed that the correlation.

Multivariable (R-square = 0.312) variable Coefficient Lower Upper P value Coefficient Lower Upper P value age -0.003 -0.032 0.027 0.864 gender -0.885 -1.589 -0.181 0.014 BMI -0.052 -0.146 0.041 0.270 diabetes 0.552 -0.238 1.342 0.169 High blood pressure -0.497 -1.405 0.411 0.281 CVD -0.273 -1.559 1.013 0.675 KTtoBX 0.009 0.005 0.013 <0.001 0.007 0.003 0.011 <0.001 log ( TG2cr ) 0.424 0.274 0.574 <0.001 0.373 0.225 0.521 <0.001 log ( SDCcr ) 0.451 0.048 0.854 0.029 log ( A1Mcr ) 0.469 0.212 0.725 <0.001 log ( IP10cr ) 0.104 -0.136 0.345 0.391 log ( IL6cr ) -0.061 -0.254 0.131 0.529 -0.111 -0.274 0.051 0.178 log ( MCP1cr ) 0.399 0.093 0.705 0.011 A constant term 2.455 1.663 3.247 <0.001

From the above, a functional formula for evaluating the degree of interstitial fibrosis and tubular atrophy (IFTA) of the kidney after transplantation could be expressed as follows.

Risk of progression of fibrosis = 0.373 x log _e (TG2 / Cr)-0.111 x log _e (IL-6 / Cr) + 0.007 x log _e (KT to BX) + 2.455

In this case, the TG2 and IL-6 means the respective protein expression level value, the Cr means the creatinine concentration measurement value of each sample, the KT to BX is the time difference between the time of kidney transplantation and biopsy it means.

Claims (10)

Of transplanted organs after renal transplantation comprising a TG2 (transglutaminase 2) or IL-6 (interleukin-6, interleukin-6) protein or a substance measuring the mRNA expression level of the gene encoding the protein Composition for diagnosing or predicting fibrosis.
The method of claim 1,
The fibrosis is tubular interstitial fibrosis and tubular atrophy (interstitial fibrosis and tubular atrophy, IFTA) characterized in that the composition for diagnosing or predicting the fibrosis of the transplanted organ after kidney transplantation.
The method of claim 1,
The substance for measuring the protein level comprises an antibody, an interaction protein, a ligand, an oligopeptide, a peptide nucleic acid (PNA), a nanoparticle or an aptamer specifically binding to the protein or fragment thereof. A composition for diagnosing or prognostic fibrosis of a transplanted organ after kidney transplantation.
The method of claim 1,
The substance for measuring the mRNA level is a composition for diagnosing or predicting fibrosis of the transplanted organ after kidney transplant, characterized in that it comprises a primer, a probe or an antisense nucleotide that specifically binds to the gene.
A kit for diagnosing or predicting fibrosis of a transplanted organ after kidney transplant comprising the composition of claim 1.
Measuring mRNA expression levels of TG2 (transglutaminase 2) or IL-6 (interleukin 6) or a gene encoding the protein in a sample obtained from the individual;
Calculating fibrosis progression risk of the transplanted organ using the measured protein levels; And
A method of providing information for diagnosing or prognostic fibrosis of a transplanted organ following renal transplantation, comprising comparing the calculated risk with a reference.
The method of claim 6,
Calculating the risk of progressing the fibrosis,
Calculating a time difference (KT to BX) between the time of renal transplantation and the time of biopsy; And
Calculating the risk of fibrosis progression of the transplanted organ using the time difference between the protein underwater and the time of renal transplantation and biopsy, providing information for diagnosing or predicting fibrosis of the transplanted organ after renal transplantation Way.
The method of claim 7, wherein
The fibrosis progression risk is positively correlated with the TG2 expression level or time difference between the time of renal transplantation and biopsy, and is characterized by a negative correlation with IL-6 expression level. Method of providing information for diagnosing or prognostic fibrosis of transplanted organs after transplantation.
The method of claim 7, wherein
Computing the risk of progression of fibrosis of the transplanted organ is characterized by calculating the protein level measured in each sample by using the following relationship The method for providing information for diagnosing or predicting the fibrosis of the transplanted organ after kidney transplantation:
Risk of progression of fibrosis = 0.373 x log _e (TG2 / Cr)-0.111 x log _e (IL-6 / Cr) + 0.007 x log _e (KT to BX) + 2.455
In this case, the TG2 and IL-6 means the respective protein expression level value, the Cr means the creatinine concentration measurement value of each sample, and the KT to BX is the time difference between the time of renal transplantation and the biopsy Means).
The method according to any one of claims 6 to 9,
The fibrosis is tubular interstitial fibrosis and tubular atrophy (interstitial fibrosis and tubular atrophy, IFTA) characterized in that the method for providing information for diagnosing or predicting the fibrosis of the transplanted organ after kidney transplantation.

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