Composition for diagnosing chronic rejection after kidney transplantation

Abstract

The present invention relates to a composition for diagnosing chronic rejection after kidney transplantation and, more specifically, provides a biomarker composition, a diagnostic composition and a kit for diagnosing chronic rejection after kidney transplantation, all of which comprise VSIG4 protein as an active ingredient. The biomarker composition and the like, according to the present invention, can be used to more effectively diagnose chronic rejection after kidney transplantation. Furthermore, a therapeutic drug targeting same can be developed.

Description

Composition for diagnosing chronic rejection after kidney transplantation

[0001] The present invention relates to a composition for diagnosing chronic rejection after kidney transplantation.

[0002] Chronic kidney disease (CKD) is extremely common worldwide, affecting approximately 7-10% of the population. Korea also exhibits a similar pattern. In particular, the number of patients receiving renal replacement therapy (hemodialysis, peritoneal dialysis, or kidney transplantation) due to end-stage renal failure reaches approximately 1,600 per million people. This number is steadily increasing due to the aging population, and the number of patients receiving renal replacement therapy continues to rise. This has led to a rise in medical costs for dialysis. In Korea, dialysis patients, representing just 0.14% of the population, account for 3% of total medical expenses, significantly impacting healthcare finances.

[0003] Treatments for chronic kidney disease include dialysis and transplantation. Transplantation is superior to dialysis in terms of long-term prognosis and quality of life. Transplantation can be divided into living donor transplantation and cadaveric donor transplantation. While living donor transplantation is beneficial for the recipient, it requires a donor, so cadaveric donor transplantation is more common.

[0004] Kidney transplantation involves receiving a kidney from a genetically different donor, so a certain degree of rejection is inevitable. While efforts are made to prevent rejection using immunosuppressants, excessive doses can have adverse effects. Thanks to recent developments in various medications, the incidence of acute rejection in the first year after transplantation has been managed to less than 10%. While there have been significant advances in the diagnosis and treatment of acute rejection, a clear diagnosis and treatment method for chronic rejection, which involves the gradual deterioration of renal function, remains lacking. Therefore, the development of appropriate markers and treatments is urgently needed.

[0005] Currently, markers used in clinical practice include serum creatinine, urine tests, donor-specific antibodies, and renal biopsies. Donor-specific antibody testing is expensive, and renal biopsies have limitations, as they can cause some damage to the transplanted kidney. While various other markers have been introduced, none have yet achieved tangible results. Therefore, the development of markers that can assess the extent of renal damage in transplanted kidneys is urgently needed.

[0006] The purpose of the present invention is to provide a new protein marker, diagnostic composition and diagnostic kit capable of diagnosing chronic rejection after kidney transplantation.

[0007] Another object of the present invention is to provide a method for providing information for diagnosing chronic rejection after kidney transplantation.

[0008] Another object of the present invention is to provide a method for screening a treatment agent for chronic rejection after kidney transplantation.

[0009] To achieve the above purpose, the present invention provides a biomarker composition for diagnosing chronic rejection after kidney transplantation, comprising VSIG4 protein as an active ingredient.

[0010] The present invention provides a composition for diagnosing chronic rejection after kidney transplantation, comprising as an active ingredient a preparation for measuring the expression level of VSIG4 protein.

[0011] The present invention provides a kit for diagnosing chronic rejection after kidney transplantation, comprising the composition for diagnosing chronic rejection after kidney transplantation.

[0012] The present invention provides a method for providing information for diagnosing chronic rejection after kidney transplantation, comprising the steps of: measuring the expression level of VSIG4 protein in a sample isolated from a subject who received a kidney transplant; and comparing the expression level of the VSIG4 protein with at least one indicator selected from the group consisting of a chronicity index, age, blood urea nitrogen (BUN), estimated glomerular filtration rate (eGFR), protein, and albumin.

[0013] In addition, the present invention provides a method for screening a treatment agent for chronic rejection after kidney transplantation, comprising the steps of: treating a biological sample isolated from a subject who received a kidney transplant with a test substance; measuring the expression level of VSIG4 protein in the sample treated with the test substance; and selecting a test substance in which the expression level of the VSIG4 protein is positively correlated with a chronicity index, age, or blood urea nitrogen (BUN), or negatively correlated with estimated glomerular filtration rate (eGFR), protein, or albumin.

[0014] The biomarker composition according to the present invention confirms that the VSIG4 protein derived from the serum and urine of kidney transplant patients is related to various indicators including chronic indicators of chronic renal damage after kidney transplantation, especially chronic rejection, and thus, by using this, chronic rejection after kidney transplantation can be diagnosed more effectively and, furthermore, therapeutic drugs targeting this can be developed.

[0015] Figure 1 shows the correlation between estimated glomerular filtration rate (eGFR) and VSIG4 protein according to an experimental example of the present invention, where (a) shows the correlation with chronic indicators, (b) shows the correlation with urine VSIG4, and (c) shows the correlation with serum VSIG4.

[0016] Figure 2 shows the correlation between chronic indicators and serum VSIG4 protein. (a) shows a positive correlation between chronic indicators and serum VSIG4 levels, and (b) shows the change between groups when chronic indicators are divided into three groups.

[0017] Figure 3 shows the correlation between chronic indicators and urine VSIG4 protein. (a) shows a positive correlation between chronic indicators and urine VSIG4 levels, and (b) shows the change between groups when chronic indicators are divided into three groups.

[0018] Hereinafter, the present invention will be described in detail.

[0019]

[0020] The present inventors have completed the present invention by confirming that there is a correlation between VSIG4 protein activity in the serum and urine of kidney transplant patients and chronic indicators of renal damage, and further that VSIG4 can be used as an indicator that reflects the degree of chronic renal damage in kidney transplant patients, and that a therapeutic drug targeting it can be developed.

[0021]

[0022] The present invention provides a biomarker composition for diagnosing chronic rejection after kidney transplantation, comprising VSIG4 protein as an active ingredient.

[0023] In this specification, "VSIG4 (V-set and immunoglobulin domain containing 4; VSIG4 gene ID: 11326)" is a B7 family-related protein that is mainly expressed in macrophages and suppresses T cells by binding to CR1 / CR3 of T cells. In addition to macrophages, VSIG4 has been reported to be expressed in the liver, lungs, heart, and abdominal cavity. Although VSIG4 is known to be involved in various inflammatory responses, its functions other than suppressing T cell function are not yet clear.

[0024] As used herein, "diagnosis" refers to confirming the presence or characteristics of a pathological condition. For the purposes of the present invention, diagnosis may refer to confirming the onset of chronic rejection or chronic renal damage after kidney transplantation.

[0025] In this specification, a "biomarker" is an indicator that can detect changes in the body, and is a substance that can confirm the normal or pathological state of a living organism, whether there is a change therein, etc., and may include organic biomolecules such as polypeptides, nucleic acids, lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, oligosaccharides, etc.), and can be used to diagnose chronic rejection after kidney transplantation, as in the present invention.

[0026]

[0027] The VSIG4 protein according to the present invention may be derived from whole blood, plasma, serum, blood or urine, and preferably may be derived from serum or urine, but is not limited thereto.

[0028] The expression or activity level of VSIG4 protein derived from the above urine may be a value corrected with urine creatinine.

[0029] The expression or activity level of the above VSIG4 protein may have a positive or negative correlation with one or more indicators selected from the group consisting of chronicity index, age, blood urea nitrogen (BUN), estimated glomerular filtration rate (eGFR), protein, and albumin in chronic rejection after kidney transplantation.

[0030] Preferably, the expression or activity level of the VSIG4 protein may be positively correlated with chronic markers, age or blood urea nitrogen, or negatively correlated with glomerular filtration rate, protein or albumin.

[0031] The above chronicity index may be a value calculated and interpreted based on the chronicity index of the Banff score.

[0032] In general, chronic renal damage such as chronic rejection after kidney transplantation is accompanied by an increase in chronic indicators, age, or blood urea nitrogen values, or a decrease in glomerular filtration rate, etc. If the expression or activity level of the VSIG4 protein according to the present invention is shown to increase as the chronic indicators, age, or blood urea nitrogen increase, or as the glomerular filtration rate, protein, or albumin decreases, it can be diagnosed as chronic rejection or chronic renal damage after kidney transplantation.

[0033] The biomarker according to the present invention can be regarded as a highly reliable marker because it shows the same results in repeated experiments and changes in its expression level show significant results, and thus the predicted results can be reasonably trusted.

[0034]

[0035] The present invention provides a composition for diagnosing chronic rejection after kidney transplantation, comprising as an active ingredient an agent for measuring the expression or activity level of VSIG4 protein.

[0036] The above formulation may be, but is not limited to, an antibody, peptide, peptide mimetic, aptamer or compound that specifically binds to the VSIG4 protein.

[0037] As used herein, "antibody" is a term known in the art, and refers to a specific immunoglobulin directed against an antigenic site. The antibody in the present invention refers to an antibody that specifically binds to the protein, and the antibody can be produced according to a conventional method in the art. The form of the antibody includes a polyclonal antibody or a monoclonal antibody, and all immunoglobulin antibodies can be included. The antibody refers to a complete form having two full-length light chains and two full-length heavy chains. In addition, the antibody may also include specialized antibodies such as humanized antibodies.

[0038] The term "peptide" in this specification has the advantage of high binding affinity to target substances and resists denaturation even during heat and chemical treatments. Furthermore, due to its small molecular size, it can be attached to other proteins to form fusion proteins. Specifically, it can be attached to polymer protein chains, making it suitable for use as a diagnostic kit and drug delivery material.

[0039] As used herein, the term "aptamer" refers to a type of polynucleotide composed of a special type of single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure in itself and the characteristic of being able to bind to a target molecule with high affinity and specificity. As described above, an aptamer is composed of a polynucleotide that can specifically bind to an antigenic substance in the same way as an antibody, but is more stable than a protein, has a simpler structure, and is easy to synthesize, and thus can be used as a substitute for an antibody.

[0040]

[0041] The present invention provides a kit for diagnosing chronic rejection after kidney transplantation, comprising the composition for diagnosing chronic rejection after kidney transplantation.

[0042] The above kit can be used to diagnose the development of chronic rejection after kidney transplantation by measuring the expression or activity level of VSIG4 protein from a sample isolated from an individual suspected of having chronic rejection after kidney transplantation.

[0043] The above kit for diagnosing chronic rejection after kidney transplantation may further include one or more other component compositions, solutions or devices suitable for the analysis method, and the kit may be, but is not limited to, a protein chip kit.

[0044] The kit may further include a substrate, a buffer solution, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, a chromogenic substrate, etc. The substrate may include a nitrocellulose membrane, a 96-well plate synthesized with a polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, and a glass slide glass. The chromogenic enzyme may include peroxidase, alkaline phosphatase, etc., and the fluorescent substance may include FITC (Fluorescein isothiocyanate), RITC (rhodamine B isothiocyanate), etc. In addition, ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), OPD (O-phenylene diamine), or TMB (tetramethylbenzidin) may be used as the chromogenic substrate solution.

[0045]

[0046] The present invention provides a method for providing information for diagnosing chronic rejection after kidney transplantation, comprising the steps of: measuring the expression level of VSIG4 protein in a sample isolated from a subject who received a kidney transplant; and comparing the expression level of the VSIG4 protein with at least one indicator selected from the group consisting of a chronicity index, age, blood urea nitrogen (BUN), estimated glomerular filtration rate (eGFR), protein, and albumin.

[0047] Through the above information provision method, when the expression or activity level of the VSIG4 protein of the sample has a positive or negative correlation with the above indicators, it can be determined as chronic rejection after kidney transplantation, and by providing this information, the future treatment direction according to the onset of chronic rejection after kidney transplantation can be set, so that treatment can be performed more quickly and effectively.

[0048] As used herein, “subject” means any animal, including humans, that has received a kidney transplant and has developed or may develop chronic rejection.

[0049] The sample isolated from the above object may include a sample such as tissue, cell, whole blood, plasma, serum, blood, saliva or urine, which can determine the level of expression or activity of the VSIG4 protein, and is preferably serum or urine, but is not limited thereto.

[0050] Preferably, the expression or activity level of the VSIG4 protein can be positively correlated with chronic indicators, age, or blood urea nitrogen, or negatively correlated with glomerular filtration rate, protein, or albumin, thereby being judged as chronic rejection after kidney transplantation.

[0051] That is, chronic renal damage such as chronic rejection after kidney transplantation is accompanied by an increase in chronic indicators, age, or blood urea nitrogen values, or a decrease in glomerular filtration rate, etc., so if the expression or activity level of the VSIG4 protein according to the present invention is shown to increase as the chronic indicators, age, or blood urea nitrogen increase, or as the glomerular filtration rate, protein, or albumin decreases, it can be determined to be chronic rejection or chronic renal damage after kidney transplantation.

[0052]

[0053] The present invention provides a method for screening a treatment agent for chronic rejection after kidney transplantation, comprising the steps of: treating a biological sample isolated from a subject who received a kidney transplant with a test substance; measuring the expression level of VSIG4 protein in the sample treated with the test substance; and selecting a test substance in which the expression level of the VSIG4 protein is positively correlated with a chronicity index, age, or blood urea nitrogen (BUN), or negatively correlated with estimated glomerular filtration rate (eGFR), protein, or albumin.

[0054] The above screening method is a method of comparing the expression or activity of the protein and the above indicators in the presence and absence of a candidate substance for chronic rejection treatment after kidney transplantation, and can be usefully used for screening chronic rejection treatment after kidney transplantation, chronic renal damage treatment, etc., and a substance whose expression or activity level of the VSIG4 protein is highly correlated with the above indicators can be selected as a treatment thereof.

[0055]

[0056] In the present invention, the protein expression or activity level may be measured by at least one method selected from the group consisting of Western blot, Enzymelinked immunosorbent assay (ELISA), Radioimmunoassay (RIA), Radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorter (FACS), and protein chip, but is not limited thereto.

[0057] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0058]

[0059] <Experimental Method>

[0060] 1. Target selection

[0061] A retrospective study was conducted on 64 patients who underwent renal biopsies at Inje University Ilsan Paik Hospital from June 2013 to May 2021 and consented to research on human-derived materials at the time of biopsy. Renal biopsies were performed if the patient had evidence of worsening renal function or proteinuria. This study was approved by the Inje University Ilsan Paik Hospital Medical Ethics Committee.

[0062]

[0063] 2. Clinical indicators

[0064] All subjects were identified for age, sex, degree of human leukocyte antigen (HLA) mismatch, body mass index, and time from transplantation to renal biopsy. Blood urea nitrogen (BUN), creatinine (Cr), estimated glomerular filtration rate (eGFR (chronic kidney disease-epidemiology collaboration; CKD-EPI formula)), tCO2, hemoglobin, white blood cells, fasting glucose, uric acid, protein, albumin, total cholesterol, triglyceride, and low density lipid cholesterol. The amount of proteinuria was calculated using protein and creatinine in urine.

[0065]

[0066] 3. Indicators of chronic renal damage

[0067] The degree of chronic renal damage in renal biopsy findings was assessed using H&E-stained slides obtained at the time of renal biopsy, and the indices were interpreted based on the 2019 revised Banff score (Loupy, Am J Transpl 2020). The chronic Banff score is classified into CI, CT, CV, CG, and PTCML, and their respective meanings are shown in Table 1.

[0068] Table 1 below shows the individual Banff scores for acute, chronic, and acute / chronic scores, which are used to grade acute and chronic active antibody-mediated rejection (ABMR) and T-cell mediated rejection (TCMR) using the Banff standardization system.

[0069]

[0070] [ci: interstitial fibrosis, ct: tubular atrophy, cv: arterial fibrointimal thickening, cg: transplant glomerulopathy]

[0071]

[0072] Among each index, ci, ct, cv, and cg were scored from 0 to 3 points, and the total chronicity index was calculated as ci+ct+cv+ (cgx2) for a total of 15 points based on existing literature. Among the subjects, 44 cases were analyzed in the final analysis, excluding 20 cases in which it was difficult to measure all chronic phase findings in renal biopsies. A renal pathology specialist interpreted the renal biopsies.

[0073]

[0074] 4. VSIG4 measurement

[0075] VSIG4 levels in blood and urine were measured using an ELISA kit (LSbio, Shirley, USA), and the average value was used after two measurements. Urine VSIG4 levels were corrected for urine creatinine.

[0076]

[0077] 5. Statistical processing

[0078] Subjects were divided into three groups (0-4, 5-8, and 9 or higher) based on chronic index values, and comparisons were made between groups. All experimental results were analyzed using the statistical program SPSS 25.0, and a P < 0.05 value was defined as statistically significant.

[0079]

[0080] <Experimental Results>

[0081] 1. Analysis of subject characteristics

[0082] The general characteristics of the 44 patients are shown in Table 2. Twenty-four men and 20 women were included, and the mean age was 50.9 ± 12.8 years. At the time of transplantation, 3.44 ± 1.40 out of 6 HLA mismatches were present, and renal biopsies were performed an average of 4.86 ± 3.97 years after transplantation.

[0083]

[0084]

[0085] 2. Correlation between chronic indicators and other indicators

[0086] We analyzed the correlation between chronic markers and VSIG4 with other indicators (Table 3). Serum VSIG4 showed a higher correlation than urine VSIG4 with eGFR and chronic markers. Serum VSIG4 showed a positive correlation with age and blood urea nitrogen (BUN), and a negative correlation with eGFR, protein, and albumin (Figure 1). In particular, serum VSIG4 showed a higher correlation with eGFR than with chronic markers.

[0087]

[0088]

[0089] 3. Chronic markers and serum VSIG4

[0090] As shown in Figure 2, serum VSIG4 levels showed a positive correlation with chronic index values ​​(a), and when chronic indexes were divided into three groups, significant differences were observed between the groups (b).

[0091]

[0092] 4. Chronic markers and urine VSIG4

[0093] As shown in Figure 3, the urine VSIG4 level showed a positive correlation with the chronic index value (a), and when the chronic index was divided into three groups, a significant difference was shown between the groups (b).

[0094]

[0095] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. In other words, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A biomarker composition for diagnosing chronic rejection after kidney transplantation, comprising VSIG4 protein as an active ingredient.

2. In paragraph 1, The above VSIG4 protein is, A biomarker composition characterized in that it is derived from whole blood, plasma, serum, blood or urine.

3. A composition for diagnosing chronic rejection after kidney transplantation, comprising as an active ingredient a preparation for measuring the expression level of VSIG4 protein.

4. In paragraph 3, The above formulation is, A diagnostic composition characterized in that it is an antibody, peptide, peptide mimetic, aptamer or compound that specifically binds to the VSIG4 protein.

5. A kit for diagnosing chronic rejection after kidney transplantation, comprising a composition according to claim 3 or 4.

6. A step of measuring the expression level of VSIG4 protein in a sample isolated from a subject who received a kidney transplant; and A method for providing information for diagnosing chronic rejection after kidney transplantation, comprising a step of comparing the expression level of the VSIG4 protein with at least one indicator selected from the group consisting of a chronicity index, age, blood urea nitrogen (BUN), estimated glomerular filtration rate (eGFR), protein, and albumin.

7. In paragraph 6, The above sample, A method of providing information, characterized in that the information is whole blood, plasma, serum, blood or urine.

8. In paragraph 6, The above chronicity index is, A method of providing information, characterized in that the values ​​are calculated and interpreted based on the chronic indicators of the Banff score.

9. A step of treating a test substance with a biological sample isolated from an individual who received a kidney transplant; A step of measuring the expression level of VSIG4 protein in a sample treated with the above test substance; and A method for screening a treatment agent for chronic rejection after kidney transplantation, comprising the step of selecting a test substance in which the expression level of the VSIG4 protein has a positive correlation with a chronicity index, age, or blood urea nitrogen (BUN), or a negative correlation with estimated glomerular filtration rate (eGFR), protein, or albumin.

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