KR102193393B1 - Endocan as a marker for diagnosing or predicting cardiovascular complications and kidney transplant rejection in patients with renal failure, and uses thereof - Google Patents

Abstract

The present invention relates to endocans and their use as biomarkers for diagnosis or prediction of antibody-mediated rejection and cardiovascular complications after kidney transplantation in renal failure patients. According to the present invention, by measuring the level of endocan in a biological sample of a patient with end-stage renal failure, such as plasma and/or urine, antibody-mediated rejection or cardiovascular complications after kidney transplantation can be diagnosed and predicted early in a non-invasive method. F. Measures to suppress the rejection reaction can be taken early after kidney transplantation, and patients with high risk of cardiovascular complications can be selected as high-risk patients early and active treatment can be performed, which will greatly improve the survival rate. It is expected. Furthermore, the present invention may be easily utilized in the development of a new drug using endocan as a new therapeutic target or a control mechanism in terminal renal failure patients.

Description

Endocan as a marker for diagnosing or predicting cardiovascular complications and kidney transplant rejection in patients with renal failure, and uses thereof}

The present invention relates to endocans and their use as biomarkers for diagnosis or prediction of antibody-mediated rejection and cardiovascular complications after kidney transplantation in renal failure patients.

Renal failure, also known as renal failure, refers to a condition in which the kidneys function to filter and discharge waste products from the blood. Because the kidneys are not functioning properly, the concentration of waste products in the blood increases, the discharge of water does not occur, and various complications and high blood pressure occur. Renal failure is classified into acute and chronic, and it is judged by the rate of decline in renal function and the rate of change in the concentration of creatinine in plasma at the time of diagnosis. Acute kidney failure refers to the sudden failure of the kidneys to function properly after undergoing complex surgery, severe conditions, or when blood supply to the kidneys is not smooth. Unlike chronic kidney failure, it can return to normal after treatment. On the other hand, chronic renal failure has no symptoms at an early stage and progresses for months or years, resulting in a slow glomerular filtration rate and various complications. If it gets worse, it can develop into end-stage renal failure. In this case, kidney replacement therapy such as dialysis and kidney transplantation is performed.

Kidney transplantation is currently a selective treatment for patients with end-stage renal failure, and the one-year graft survival rate after transplantation over the past 20 years has gradually increased to 96.5%. However, allograft rejection remains a major cause of early and late allograft dysfunction in kidney transplants despite significant advances in immunosuppressive therapy. Since allograft rejection cannot be sensitively detected by monitoring serum creatinine levels, it is difficult to properly diagnose and immediately manage allograft rejection in clinical practice.

Moreover, the vascular endothelium of the transplanted kidney is a major site for allograft rejection, especially in patients with antibody-mediated immune impairment. Microvascular inflammation (MVI), a histological symptom of glomerulonephritis and capillary vasculitis, is the basis for diagnosing antibody-mediated rejection (ABMR), and several studies have shown that this condition is another factor that generally determines kidney survival. Regardless of this, the prognosis of allografts was poor (Am. J. Transplant. 9, 2520–2531). Currently, invasive renal biopsy is essential for demonstrating MVI with a substantial risk of complications, and many potential biomarkers of microvascular inflammation are being studied, but none are currently available in clinical practice.

In addition, cardiovascular disease is the most common complication that occurs in patients with end-stage renal failure undergoing hemodialysis, and is known to account for 40-50% of deaths in patients with end-stage renal failure, and is also known as a major cause of death after kidney transplantation. . In addition to existing cardiovascular risk factors such as hypertension, diabetes, and dyslipidemia, cardiovascular risk may increase in patients with end-stage renal failure, such as weight overload and impairment of calcium and phosphate disturbances. Therefore, it is most important to predict and appropriately manage high-risk patients who are likely to develop cardiovascular complications. However, biomarkers and related data that can diagnose or predict cardiovascular complications early in patients with end-stage renal failure are still very limited. .

On the other hand, endocan or ESM-1 (Endothelial cell-specific molecule 1) is a water-soluble proteoglycan containing an amino acid polymer (molecular weight 22 kDa) and a single dermatan sulfate chain, and is a human lung or kidney tissue. It is known as a protein that is mainly expressed and secreted in endothelial cells of. According to previous studies, endocan is a specific biomarker of tip cells in the process of angiogenesis, and in the presence of pre-angiogenic growth factors such as vascular endothelial growth factor (VEGF) or fibroblast growth factor-2 (FGF-2). The result of increased expression has been reported, and it has been demonstrated that plasma endocanal levels have potential as endothelial activation markers. However, it is not well known as a specific biomarker for diagnosing antibody-mediated rejection characterized by microvascular inflammation after renal transplantation in patients with end-stage renal failure, early diagnosis of cardiovascular complications, or predicting risk.

As a result of research efforts to discover biomarkers capable of diagnosing or predicting antibody-mediated rejection reactions and cardiovascular complications in patients with end-stage renal failure, endocans in plasma and/or urine as biomarkers. It was found that it can be used, and the present invention was completed based on this.

Accordingly, an object of the present invention is to provide a marker composition for diagnosing antibody-mediated rejection (ABMR) after kidney transplantation, including an endocan gene or a protein encoded by the gene.

In addition, the present invention comprises an agent for measuring the level of the mRNA of the endocan gene or the protein encoded by the gene, a composition for diagnosing antibody-mediated rejection (ABMR) after kidney transplantation, and the composition Another object is to provide a diagnostic kit comprising a.

In addition, the present invention comprises the step of measuring the level of the mRNA of the endocan gene or the protein encoded by the gene in a biological sample derived from a patient undergoing a kidney transplant operation, antibody-mediated rejection (antibody-mediated rejection) ; ABMR) Another purpose is to provide a method of providing information for diagnosis.

In addition, another object of the present invention is to provide a marker composition for diagnosing or predicting cardiovascular complications of renal failure, including an endocan gene or a protein encoded by the gene.

In addition, the present invention includes a composition for diagnosing or predicting cardiovascular complications of renal failure, including an agent for measuring the level of mRNA of the endocan gene or the protein encoded by the gene, and cardiovascular complication diagnosis or Another object is to provide a prediction kit.

In addition, the present invention provides a method for diagnosing or predicting cardiovascular complications, comprising measuring the level of the mRNA of the endocan gene or the protein encoded by the gene in a biological sample derived from a patient with renal failure. It has another purpose.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a marker composition for diagnosing antibody-mediated rejection (ABMR) after kidney transplantation, including an endocan gene or a protein encoded by the gene do.

In one embodiment of the present invention, the endocan gene may be composed of the nucleotide sequence of SEQ ID NO: 1.

In another embodiment of the present invention, the protein encoded by the endocan gene may be composed of the amino acid sequence of SEQ ID NO: 2.

In addition, the present invention provides a composition for diagnosing antibody-mediated rejection (ABMR) after kidney transplantation, including an agent measuring the level of the mRNA of the endocan gene or the protein encoded by the gene. .

In addition, the present invention provides a kit for diagnosing antibody-mediated rejection (ABMR) after kidney transplantation, comprising the composition.

In one embodiment of the present invention, the agent for measuring the mRNA level may be a sense and antisense primer or a probe that complementarily binds to the mRNA of the gene.

In another embodiment of the present invention, the agent for measuring the protein level may be an antibody that specifically binds to the protein encoded by the gene.

In addition, the present invention comprises the step of measuring the level of the mRNA of the endocan gene or the protein encoded by the gene in a biological sample derived from a patient undergoing a kidney transplant operation, antibody-mediated rejection (antibody-mediated rejection) ; ABMR) Provides information provision method for diagnosis.

In one embodiment of the present invention, the biological sample may be tissue, blood, serum, plasma, or urine.

In another embodiment of the present invention, the expression level of the mRNA is polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RNase Protection assay; RPA), microarray (microarray), and may be measured through one or more methods selected from the group consisting of northern blotting (northern blotting).

In another embodiment of the present invention, the protein expression level is Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation method (immunoprecipitation). ), flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, and one or more methods selected from the group consisting of protein chips It may be measured through.

In addition, the present invention provides a marker composition for diagnosing or predicting cardiovascular complications of renal failure, including an endocan gene or a protein encoded by the gene.

In addition, the present invention comprises a composition for diagnosing or predicting cardiovascular complications of renal failure, comprising an agent for measuring the level of mRNA of the endocan gene or the protein encoded by the gene, and cardiovascular complications of renal failure comprising the composition Kits for diagnosis or prediction are provided.

In addition, the present invention provides a method for diagnosing or predicting cardiovascular complications, comprising measuring the level of the mRNA of the endocan gene or the protein encoded by the gene in a biological sample derived from a patient with renal failure. .

In one embodiment of the present invention, when the endocan protein level is 3.2 log pg/mL or more, it can be predicted that the risk of cardiovascular complications is high.

In another embodiment of the present invention, the biological sample may be blood, serum or plasma.

According to the present invention, by measuring the level of endocan in a biological sample of a patient with end-stage renal failure, such as plasma and/or urine, antibody-mediated rejection or cardiovascular complications after kidney transplantation can be diagnosed and predicted early in a non-invasive method. F. Measures to suppress the rejection reaction can be taken early after kidney transplantation, and patients with high risk of cardiovascular complications can be selected as high-risk patients early and active treatment can be performed, which will greatly improve the survival rate. It is expected. Furthermore, the present invention may be easily utilized in the development of a new drug using endocan as a new therapeutic target or a control mechanism in terminal renal failure patients.

1 is a result of comparative analysis of endocan levels according to renal allograft status, respectively, as a result of normal lesion (NP), acute tubular necrosis (ATN), acute pyelonephritis (APN), BK virus-related nephropathy (BKVN), acute T cells. Plasma endocans and urinary endocans in patients with mediated rejection (TCMR), acute antibody-mediated rejection (aABMR), organ transplant survival (LTGS), and chronic active antibody-mediated rejection (cABMR) Urinary endocan) levels were compared.
FIG. 2 is a result of analyzing the correlation between various Banff pathological scores and levels of endocans in plasma (FIG. 2a) or endocans in urine (FIG. 2b ).
3 shows immunohistochemical staining for transparent cell renal cell carcinoma tissue, normal lesion (NP) patient-derived tissue, acute T cell mediated rejection (TCMR) patient-derived tissue, and acute antibody-mediated rejection patient-derived tissue, respectively. This is the result of observing the expression of endocans in tissues.
Figure 4 shows the differentiated ability of endocan levels in plasma and urine in distinguishing acute antibody-mediated rejection from acute tubular necrosis (ATN), BK virus-related nephropathy (BKVN) and acute T cell mediated rejection (TCMR). This is the result showing the ROC curve for evaluation.
Figure 5 is a kidney survival rate according to the type of diagnosis (renal survival), and acute T-cell mediated rejection (T-cell mediated rejection), acute antibody-mediated rejection (Acute antibody-mediated rejection), chronic active antibody-mediated, respectively. This is a result showing the survival rate according to the level of endocan in plasma and urine in the patient group of chronic active antibody-mediated rejection.
6 shows the comparison of log levels of plasma endocans according to the onset of cardiovascular complications in patients with end-stage renal failure.
7 is a result showing the risk of cardiovascular complications according to plasma endocan levels in patients with end-stage renal failure during the follow-up period.

As a result of research efforts to discover biomarkers capable of diagnosing or predicting antibody-mediated rejection reactions and cardiovascular complications in patients with end-stage renal failure, endocans in plasma and/or urine as biomarkers. It was found that it can be used, and the present invention was completed based on this.

Accordingly, the present invention provides a marker composition for diagnosing antibody-mediated rejection (ABMR) after kidney transplantation, including an endocan gene or a protein encoded by the gene.

In addition, the present invention comprises an agent for measuring the level of the mRNA of the endocan gene or the protein encoded by the gene, a composition for diagnosing antibody-mediated rejection (ABMR) after kidney transplantation, and the composition It provides a kit for diagnosing antibody-mediated rejection (ABMR) after kidney transplant, including.

In the present invention, the endocan gene may be composed of a nucleotide sequence of SEQ ID NO: 1 (NCBI Reference sequence: BC011989.1), and the protein encoding the endocan gene is SEQ ID NO: 2 (NCBI Reference sequence: AAH11989. It may be composed of the amino acid sequence of 1). At this time, the sequence of SEQ ID NO: 1 or 2 and 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95%, 96%, 97%, 98%, 99% or more of the sequence It may include a nucleotide sequence or amino acid sequence having homology.

The term "diagnosis" used in the present invention means in a broad sense to judge the condition of a patient's disease in all aspects. The content of the judgment is the name of the disease, etiology, disease type, severity, detailed mode of the bed, and the presence or absence of complications. In the present invention, the diagnosis is to determine the risk of onset, whether or not, and the level of progression of an antibody-mediated rejection reaction characterized by vascular inflammation after kidney transplantation, and more preferably, the antibody-mediated rejection is acute antibody-mediated rejection. Response or chronically active antibody-mediated rejection.

The present inventors have identified endocans as biomarkers capable of specifically diagnosing or predicting antibody-mediated rejection after kidney transplantation in patients with end-stage renal failure through specific examples.

In one embodiment of the present invention, for a total of 203 kidney transplant patients, 8 groups, namely, normal lesions (NP, n=29), acute tubular necrosis (ATN, n=17), acute pyelonephritis ( APN, n=7), BK virus-related nephropathy (BKVN, n=22), acute T-cell mediated rejection (TCMR, n=46), acute antibody-mediated rejection (acute ABMR n=39), organ transplant survival ( LTGS, n=26), chronically active antibody-mediated rejection (hereinafter, chronic active ABMR, n=17), and the groups were divided into a set of short-term transplant periods (NP, ATN, APN, BKVN, TCMR and acute ABMR) and organ transplant period sets (LTGS and chronic active ABMR). Next, the basic clinical characteristics and data of the patients in each group were analyzed.As a result, patients with APN, BKVN, TCMR and acute ABMR had significantly lower glomerular filtration rates (eGFR), and higher than those of NP and ATN patients. Urine protein to creatinine ratio was shown, and in the organ transplant period set, it was confirmed that LTGS patients showed higher age, longer post-transplantation period and higher glomerular clearance and less proteinuria than chronic active ABMR patients (see Example 2). ).

In another embodiment of the present invention, plasma and urine endocan levels of each group of patients were measured and compared. In the short transplant period set, both plasma and urine endocan levels were significant in acute ABMR patients compared to other groups. It was found that the plasma and urine endocan levels were significantly increased in the chronic active ABMR group compared to the LTGS group in the organ transplant period set. In addition, clinical parameters such as age, post-transplantation period, number of HLA discrepancies, glomerular filtration rate, and urine PCR results were examined for correlations between endocan levels. As a result, the glomerular filtration rate was negative with plasma and urine endocan levels. It was confirmed that it had a correlation of (see Example 3).

In another embodiment of the present invention, as a result of analyzing the association between the Banff pathology score and the endocan levels in plasma and urine, the microvascular inflammation score calculated as the sum of the glomerulonephritis, capillaryitis, and glomerulonephritis and capillary vasculitis scores is It was confirmed that the endocan quartile in plasma and urine was significantly high in patients with high (see Example 4).

In another embodiment of the present invention, immunohistochemical staining was performed on tissues derived from clear cell renal cell carcinoma tissues, NP, TCMR, and acute ABMR patients as positive controls. As a result, endocans were performed on tissues derived from NP and TCMR patients. While this was not detected, it was confirmed that endocans were expressed in proximal tubular epithelial cells of acute ABMR patient-derived tissue (see Example 5).

In another embodiment of the present invention, it was attempted to evaluate the usefulness of plasma and urine endocan levels for distinguishing acute ABMR from ATN, BKVN, and TCMR using AUC determined through ROC curve analysis. As a result, in plasma and urine The AUC of the endocan level was 0.796 and 0.788, respectively, and it was confirmed that the AUC value increased to 0.893 after integrating the endocan levels in plasma and urine (see Example 6).

In another embodiment of the present invention, as a result of analyzing the kidney transplant results according to the type of diagnosis and the level of endocan after kidney transplantation, patients diagnosed with any type of rejection had significantly lower renal allograft survival rates than other groups. As a result of analyzing the survival rate according to the endocan level in the TCMR, acute ABMR, and chronic active ABMR groups, the survival rate was significantly lower in the group with high endocan levels in the TCMR and acute ABMR groups, and significantly in the chronic active ABMR group. No, but similar results were confirmed (see Example 7).

From the results of specific examples of the present invention, by measuring the level of endocans in plasma or urine, antibody-mediated rejection, characterized by microvascular inflammation, was specifically differentiated from other types of rejection after kidney transplantation in patients with terminal renal failure. It was confirmed that it can be diagnosed or predicted early.

In the present invention, the agent for measuring the mRNA level may be a sense and antisense primer or probe that complementarily binds to the mRNA of a gene.

The term “primer” used in the present invention refers to a short gene sequence that serves as a starting point for DNA synthesis, and refers to an oligonucleotide synthesized for use in diagnosis or DNA sequencing. The primers are usually synthesized and used in a length of 15 to 30 base pairs, but may vary depending on the purpose of use, and may be modified by methylation or capping by a known method.

The term "probe" as used herein refers to a nucleic acid capable of specifically binding to an mRNA having a length of several to several hundreds of bases produced through enzymatic chemical separation or purification or synthesis. The presence or absence of mRNA can be confirmed by labeling radioactive isotopes or enzymes, and it can be designed and modified by a known method.

In the present invention, the agent for measuring the protein level may be an antibody that specifically binds to a protein encoded by a gene.

The term “antibody” used in the present invention includes immunoglobulin molecules immunologically reactive with a specific antigen, and includes both monoclonal and polyclonal antibodies. In addition, the antibodies include forms produced by genetic engineering such as chimeric antibodies (eg, humanized murine antibodies) and heterologous antibodies (eg, bispecific antibodies).

The diagnostic kit of the present invention is composed of a composition, solution, or device containing one or more other components suitable for an analysis method.

For example, in order to perform PCR, the kit of the present invention uses genomic DNA derived from a sample to be analyzed, a primer set specific for the marker gene of the present invention, an appropriate amount of DNA polymerase, dNTP mixture, PCR buffer solution, and water. It may be a kit including. The PCR buffer solution may contain KCl, Tris-HCl and MgCl ₂ . In addition, constituents necessary for performing electrophoresis capable of confirming the amplification of PCR products may be additionally included in the kit of the present invention.

In addition, the kit of the present invention may be a kit including essential elements necessary for performing RT-PCR. RT-PCR kits include test tubes or other suitable containers, reaction buffers, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC, in addition to each primer pair specific for the marker gene. -May include DEPC-water, sterilized water, etc. In addition, a primer pair specific to a gene used as a quantitative control may be included.

In addition, the kit of the present invention may be a kit including essential elements necessary to perform a DNA chip. The DNA chip kit includes a substrate to which cDNA corresponding to a gene or a fragment thereof is attached by a probe, and the substrate may include a cDNA corresponding to a quantitative structural gene or a fragment thereof. In addition, the kit of the present invention may be in the form of a microarray having a substrate on which the marker gene of the present invention is immobilized.

In addition, the kit of the present invention may be a kit characterized in that it contains essential elements necessary for performing ELISA. The ELISA kit includes an antibody specific for a marker protein, and includes an agent for measuring the protein level. The ELISA kit may include a reagent capable of detecting an antibody forming an “antigen-antibody complex”, for example, a labeled secondary antibody, chromopores, enzymes, and a substrate thereof. In addition, an antibody specific for the quantitative control protein may be included.

The term "antigen-antibody complex" as used herein refers to a combination of a protein encoded by a gene and an antibody specific thereto. The amount of antigen-antibody complex formation can be quantitatively measured through the signal level of a detection label. The detection label may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioactive isotopes, but is not limited thereto.

In addition, the present invention comprises the step of measuring the level of the mRNA of the endocan gene or the protein encoded by the gene in a biological sample derived from a patient undergoing a kidney transplant operation, antibody-mediated rejection (antibody-mediated rejection) ; ABMR) Provides information provision method for diagnosis.

In the present invention, the biological sample may include tissue, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid and urine, and more preferably tissue, blood, serum, plasma, or urine, but is limited thereto. It does not become.

The expression level of the mRNA is determined by conventional methods known in the art: polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RNase). Protection assay; RPA), microarray (microarray), and may be measured by one or more methods selected from the group consisting of northern blotting (northern blotting), but is not limited thereto.

The protein expression level is determined by conventional methods known in the art, Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), and immunoprecipitation. , Flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, and one or more methods selected from the group consisting of protein chips. It can be measured through, but is not limited thereto.

On the other hand, the present inventors have confirmed through specific examples that endocan can be used as a useful biomarker for early diagnosis or prediction of cardiovascular complications in patients with terminal renal failure undergoing dialysis.

In one embodiment of the present invention, as a result of analyzing basic clinical characteristics and endocan levels according to the occurrence of cardiovascular complications in patients with end-stage renal failure, 52 patients with cardiovascular complications out of 349 patients had diabetes, acute coronary artery. Cardiovascular risk factors such as syndrome, arrhythmia, and heart failure were more common, and increased white blood cell count, hemoglobin, serum albumin, total cholesterol and HDL levels were confirmed to be decreased, and plasma endocan levels were significantly higher. It was found (see Example 8).

In another embodiment of the present invention, as a result of analyzing the correlation between plasma endocan levels and basic clinical characteristics in the two patient groups, arrhythmia, chronic liver disease, HDL, dialysis period, spKt/ through univariate linear regression curve analysis. It was confirmed that V and TNF-α showed a positive correlation with log endocan levels, and also showed an inverse correlation with body mass index, diabetes, WBC, platelets, serum albumin, and LDL. In multiple linear regression analysis, log endocan levels were positively correlated with acute coronary syndrome (ACS), stable angina, TNF-α and predialysis systolic blood pressure (SBP), and were independent of BMI and albumin. It was confirmed that there was an inverse correlation (see Example 9).

In another embodiment of the present invention, a multivariate Cox proportional analysis was performed to evaluate the risk of cardiovascular complications in patients with end-stage renal failure. As a result, log endocan levels were 7.88 (95% CI, 1.69 to 36.88) and onset of cardiovascular complications. , p = 0.01). Furthermore, as a result of the maximum chi-square test, it was confirmed that the optimal cutoff value of the plasma endocan level when predicting cardiovascular complications was 3.2 log pg/mL.From this, the risk ratio for patients with plasma endocan levels of 3.2 log pg/mL or higher was 7.6 (95 % CI, 3.0 ~ 19.2) (p <0.001), it was found that the cumulative incidence of cardiovascular complications was significantly higher (see Example 10).

Accordingly, as another aspect of the present invention, the present invention provides a marker composition for diagnosing or predicting cardiovascular complications of renal failure, including an endocan gene or a protein encoded by the gene.

In addition, the present invention comprises a composition for diagnosing or predicting cardiovascular complications of renal failure, comprising an agent for measuring the level of mRNA of the endocan gene or the protein encoded by the gene, and cardiovascular complications of renal failure comprising the composition Kits for diagnosis or prediction are provided.

The term "cardiovascular complications" used in the present invention may include all known types of cardiovascular diseases that may develop in patients with end-stage renal failure, more preferably myocardial infarction and unstable angina, stable angina, heart failure, Acute coronary syndrome (ACS) including arrhythmia, sudden cardiac arrest, cerebral infarction, spontaneous cerebral hemorrhage, and peripheral vascular obstructive disease may be included, but is not limited thereto.

The term "diagnosis" used in the present invention means in a broad sense to judge the condition of a patient's disease in all aspects. The content of the judgment is the name of the disease, etiology, disease type, severity, detailed mode of the bed, and the presence or absence of complications. In the present invention, the diagnosis is to determine the risk of onset of cardiovascular complications, the onset or not, and the level of progression.

In addition, the present invention provides a method for diagnosing or predicting cardiovascular complications, comprising measuring the level of the mRNA of the endocan gene or the protein encoded by the gene in a biological sample derived from a patient with renal failure. .

In the present invention, when the endocan protein level is 3.2 log pg/mL or more, it can be predicted that the risk of cardiovascular complications is high.

The renal failure patient more preferably means a patient with end-stage renal failure undergoing hemodialysis, and the biological sample derived from the patient may include tissue, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid and urine, and more preferably It may be blood, serum, or plasma, but is not limited thereto.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1. Experiment preparation and experiment method

1-1. Recruitment of kidney transplant patients

In Examples 2 to 7, kidney transplants registered in Gangdong Kyunghee University Hospital, Kyunghee University Medical Center, Seoul St. Mary's Hospital, Kyungpook National University Hospital, Samsung Hospital, Busan Paik Hospital, and Seoul National University Hospital between August 2012 and December 2016 The study was conducted on patients, and patients were recruited at the time of graft biopsy, except for the long-term graft survival (LTGS) group. Graft biopsy was performed to assess the cause of graft dysfunction, including increased serum creatinine levels and/or proteinuria (biopsy for cause), or renal allograft status (protocol biopsy), in patients with written consent. Protocol biopsies were performed only at two centers (3 weeks and 6 months after kidney transplantation at Kyunghee University Hospital in Gangdong and 3 months after kidney transplantation at St. Mary's Hospital, Catholic University). As a result, LTGS patients had stable graft function and did not show proteinuria for more than 10 years after kidney transplantation (eGFR> 50 ml/min/1.73 m ² , urine PCR <300 mg/gCr). Proceeded.

Information on age, sex, diabetes and hypertension, immunological status, serum creatinine levels, and urine PCR was obtained from each patient at the time of graft biopsy in the biopsy cohort or at the outpatient visit of the LTGS group. Kidney function was assessed by the estimated glomerular fltration rate (eGFR) calculated using the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) formula. This study (IRB No 2012-01-030) was approved by the institutional review committee of each hospital, and informed consent was obtained from all patients, and all methods were performed according to relevant guidelines and regulations.

1-2. Pathological diagnosis and classification according to kidney allograft status

Patients who underwent graft biopsy were classified according to the Banff classification, and patients with normal pathology (NP) had serum creatinine levels of less than 1.2 mg/dL, histologic rejection, calcineurin inhibitor toxicity, and acute coronary events. Patients without necrosis, tubulointerstitial inflammation, vascular damage or glomerulonephritis were defined. Acute tubular necrosis (ATN) was diagnosed when the graft biopsy was characterized by moderate or severe ATN without other graft damage. In acute pyelonephritis (APN), pyuria was >10 ⁵ CFU/mL when cultured in urine, and was diagnosed as neutrophil infiltration in tubulointerstium. Diagnosed according to the criteria previously described. Finally, T-cell-mediated rejection (TCMR), acute antibody-mediated rejection (ABMR) and chronic active ABMR were diagnosed according to the Banff 2007 criteria. Patients with TCMR and ABMR characteristics, subclinical rejection, calcineurin inhibitor toxicity, and glomerulonephritis were excluded from this study.

All scores and pathologic diagnoses were tubulitis (t), total interstitial inflammation (ti), intimal arteritis (v), glomerulitis (g), and coronary. In the score category, including tubular atrophy (ct), interstitial fibrosis (ci), chronic transplant glomerulopathy (cg), and peritubular capillaritis (ptc). The MVI score was defined as the sum of the g score and ptc score.

1-3. Collection of plasma and urine samples and determination of endocan levels

Plasma and urine samples were collected early in the morning at the time of patient's graft biopsy, outpatients were followed up regularly, and samples were randomly collected from patients in the LTGS group. Peripheral blood samples were placed in an EDTA-treated tube, centrifuged at 2000 g for 30 minutes at room temperature to remove cells from the sample, and then the plasma of the supernatant was divided by 1 ml and stored at -80°C until analysis. Urine samples were collected in a 50 ml sterilized conical tube, centrifuged at 2000 g for 20 minutes at room temperature, and then the supernatant was added to the tube and stored at -80°C until analysis. All samples were processed and stored within 1 hour after collection.

Plasma and urine endocan concentrations were measured at 450 nm absorbance by ELISA analysis using a commercial kit (Boster Biological Technology, Pleasanton, CA) according to the manufacturer's instructions. The concentration of creatinine in urine was also measured through ELISA, and since the concentration of endocan in urine is adjusted by the level of creatinine in urine, it was expressed as the ratio of urinary endocan/urine creatinine (pg/mgCr).

1-4. Immunohistochemical staining

Immunohistochemical staining was performed using a 3 μm tissue section using the Bond Polymer Intense Detection System (Vision BioSystems, Hingham, MA). Briefly, a 3 μm tissue section fixed with formalin and embedded in paraffin was deparaffinized with Bond Dewax solution (Vision BioSystems), and an antigen recovery process was performed at 100°C for 30 minutes using Bond ER solution (Vision BioSystems). . Subsequently, the tissue sections were incubated with hydrogen peroxide for 5 minutes to inhibit endogenous peroxidase, and biotin-free polymer horseradish peroxidase-binding in Bond-maX automatic slide staining machine (Vision BioSystems). The antibody conjugate system was used to incubate for 15 minutes at room temperature with a primary polyclonal antibody against endocan (Abcam, catalog number ab224591, CABMRidge, MA). As a negative control, rabbit IgG isotype control (Abcam, catalog number ab37415) was stained, and cell nuclei were counter-stained with hematoxylin.

1-5. Immunofluorescence staining

Immunofluorescence staining was performed using formalin-fixed and paraffin-embedded 4 μm tissue sections. Subsequently, the tissue sections were subjected to deparaffinization, antigen recovery, and inhibition of endogenous peroxidase activity in the same manner as in Examples 1-4. Next, the sample was treated with 1% bovine serum albumin (BSA) to perform a blocking process, and incubated with an endocan-specific primary antibody (Abcam, catalog number ab103590) for 2 hours. After washing with PBS, the sample was incubated with a secondary antibody conjugated with Alexa Fluor 488 for 1 hour. On the other hand, in order to observe the cell nucleus, contrast staining was performed with DAPI, and the tissue sections were observed with a confocal microscope (LSM-700, Carl Zeiss, Jena, Thuringia, Germany).

1-6. Statistical analysis

All statistical analyzes were performed using SPSS for Windows, version 20.0 (IBM Corp, Armonk, NY). Statistical and clinical data were expressed as mean ± standard deviation or number and percentage of patients. Bonferroni post hoc analysis, independent t-test, and analysis of variance (ANOVA) using the chi-square test were appropriately used to compare the reference characteristics and experimental results. Intergroup analysis of serum and urine endocan levels was performed after log transformation, and Pearson's simple correlation analysis was performed to evaluate the correlation between endocan levels and other parameters. Banff pathological scores were expressed as the mean ± standard error of the mean using a bar graph, and comparisons between these scores were evaluated by ANOVA.

Meanwhile, a ROC curve was created and AUC was calculated to compare the difference between plasma and urine endocan levels in order to distinguish acute ABMR from ATN and TCMR. In addition, to evaluate the kidney outcome, Kaplan-Meier survival curves were analyzed, and when generating ROC and survival curves, covariate adjustment for logistic regression analysis was performed to remove the influence of other variables. On the other hand, a p value less than 0.05 (p <0.05) was considered statistically significant.

1-7. Recruitment and experimental design of patients with terminal renal failure

In order to evaluate the mortality and incidence of end-stage renal disease (ESRD) patients since 2016, the present inventors have established a K-cohort for patients with end-stage renal failure attending six hemodialysis centers at tertiary hospitals in Korea. I did. The cohort recruited patients with end-stage renal failure from June 2016 to September 2018 (CRIS number KCT0003281), and this study was conducted on patients 18 years of age or older who are receiving hemodialysis and who have received written consent. This study was carried out in compliance with the Declaration of Helsinki, and was carried out after obtaining approval for the research protocol (KHNMC IRB No. 2016-04-039) from the institutional review committee of each participating center.

At the beginning of the study, including clinical characteristics, hemodialysis start date, body measurements (height, dry weight, systolic and diastolic blood pressure), various laboratory tests including lipid profile, and hemodialysis efficacy (spKt/V and urea reduction ratio). Medical information about the patient was collected comprehensively. Subsequently, the patients were followed up every 3 months and the onset of cardiovascular complications was monitored.

1-8. Definition of occurrence of cardiovascular complications

The occurrence of cardiovascular complications in patients with end-stage renal failure was defined as at least one of the following: myocardial infarction and unstable angina, stable angina, heart failure, arrhythmia, sudden cardiac arrest, cerebral infarction, excluding blood vessel damage caused by hemodialysis. Acute coronary syndrome (ACS), including spontaneous cerebral hemorrhage and peripheral vascular obstructive disease. Disease-free survival was measured at enrollment until the onset of complex cardiovascular disease.

1-9. Laboratory analysis

General biochemistry laboratory parameters were determined by standard methods. Plasma samples were collected in tubes treated with ethylene diaminetetraacetic acid on the study registration day. Thereafter, centrifugation was performed at room temperature at 1,000 g for 15 minutes, and then stored at -80°C until analysis. ELISA was performed using the Molecular Luminex® Screening Assay multiplex kit (R & D Systems, Inc., Minneapolis, MN, USA).

1-10. Statistical analysis

Continuous variables were expressed as mean±standard deviation (SD) and analyzed by Student's t-test or Mann-Whitney U test. Categorical variables were expressed in terms of frequency and percentage, and analysis of categorical data was performed using a Chi-square test or Fisher's exact test. We used a logarithmic value for the endocan level because the data were distributed in a biased manner. Linear regression analysis was used to evaluate the correlation between the baseline clinical features and the log level of endocans. Multivariate Cox regression analysis was performed with all univariates of p <0.1 to find a predictive model for cardiovascular risk.

The maximum chi-square test was used to find the optimal cut-off level of plasma endocans to predict cardiovascular risk. Kaplan-Meier calculated the hourly event end time. In the test, the two groups were compared using a log rank test that is stratified according to the cutoff value of the log endocan. To develop predictive markers of cardiovascular complications, a stratified Cox proportional-risk regression was used to estimate the risk ratio and 95% confidence interval, and the analysis was software statistical package for Windows (SPSS), software version 20.0 (SPSS Inc., Chicago, IL, USA), and P <0.05 was considered statistically significant.

Example 2. Analysis of basic statistical and clinical characteristics in kidney transplant patients

In this Example, a study was conducted to discover markers capable of early diagnosis or predicting kidney transplant rejection in patients undergoing kidney transplant surgery, and experiments and results for this were disclosed in Examples 2 to 7.

First, a total of 203 kidney transplant patients were recruited for this study, and the basic clinical characteristics and experimental data for the patients were analyzed and shown in Table 1 below. The patients were classified into 8 different diagnostic groups as follows: normal lesions (hereinafter, NP, n=29), acute tubular necrosis (hereinafter, ATN, n=17), acute pyelonephritis (hereinafter, APN, n=7), BK virus-related nephropathy (hereinafter BKVN, n=22), acute T-cell mediated rejection (hereinafter TCMR, n=46), acute antibody-mediated rejection (hereinafter acute ABMR n=39), organ transplant survival (hereinafter, LTGS, n =26), chronically active antibody-mediated rejection (hereinafter chronic active ABMR, n=17). In addition, the groups were further classified into two sets according to the transplant vintage, and analyzed separately for each set; The short transplant vintage set included NP, ATN, APN, BKVN, TCMR and acute ABMR patient groups, and the long transplant vintage set included LTGS and chronically active ABMR patient groups. .

As a result of the analysis, as shown in Table 1 below, there was no difference in age or sex between each group in the short-term transplant period set, but the elapsed period after transplantation was more in TCMR and acute ABMR patients than in NP, ATN, APN, and BKVN patients. Appeared long. In the protocol biopsy, 34 (21.3%) samples were collected, including 23, 9, and 2 samples of NP, ATN, and APN patients, respectively. In contrast, BKVN and BKVN, respectively, to assess graft dysfunction during graft biopsy. All samples were collected from patients with TCMR, acute ABMR and chronic active ABMR. The ratio between the number of human leukocyte antigen (HLA) mismatches with kidney transplant patients incompatible with ABO did not differ between groups. On the other hand, patients with APN, BKVN, TCMR, and acute ABMR showed remarkably low glomerular filtration rate (eGFR) (72.0±35.1 vs. 57.8±22.0 vs. 35.0±15.9 vs. 36.0±13.3 vs. 33.4±14.9 vs. 26.5±14.9 vs. 26.5±) 12.0 ml/min/1.73 m ² , NP vs. ATN vs. APN vs. BKVN vs. TCMR vs. acute ABMR; p<0.001), higher urine protein to creatinine ratio (PCR) compared to NP and ATN patients (102 ±163 vs. 81±97 vs. 392±351 vs. 264±166 vs. 835±1308 vs. 1157±1208 mg/gCr, NP vs. ATN vs. APN vs. BKVN vs. TCMR vs. acute ABMR; p =0.002). In the organ transplant period set, patients with LTGS were older than those with chronically active ABMR and had a longer post-transplantation period. Finally, patients with LTGS had higher glomerular filtration rates and less proteinuria than those with chronic active ABMR (73±17 vs. 30±18 ml/min/1.73 m ^{2, respectively} ; p<0.001 and 145±148 vs. 2456±) 2192 mg/gCr; p<0.001; LTGS vs. chronic active ABMR).

[Table 1]

Example 3. Comparative analysis of endocan levels in plasma and urine

As a result of measuring and comparing endocan levels in the patient's plasma and urine according to the state of the kidney allograft, as shown in FIG. 1, plasma endocan levels were compared to NP, ATN, APN, BKVN, and TCMR patients in the short-term transplant period set. It was found to be significantly higher in patients with acute ABMR (271.5±134.6 vs. 314.7±228.9 vs. 207.2±86.0 vs. 309.0±204.6 vs. 311.2±184.8 vs. 605.4±306.5 pg/ml, NP vs. ATN vs. APN vs. BKVN vs. TCMR vs. acute ABMR; p<0.001). On the other hand, there was no significant difference in plasma endocan levels between NP, ATN, APN, BKVN, and TCMR groups. In the organ transplant period set, plasma endocan levels were significantly higher in chronically active ABMR patients than in LTGS patients (219.0±133.8 vs. 406.5±171.7 pg/ml, LTGS vs. chronic active ABMR; p<0.001). .

On the other hand, as a result of comparative analysis of the levels of endocans in the urine, the level of endocans was also significantly increased in patients with acute ABMR compared to patients in other groups in the short-term transplant period set (45.1±76.3 vs. 79.2±87.3 vs. 79.2±87.3 vs. 113.1±86.0 vs. 102.9±101.1 vs. 258.6 ± 374.5 vs. 791.8 ± 1284.0 pg/mgCr, NP vs. ATN vs. APN vs. BKVN vs. TCMR vs. acute ABMR; p<0.001), compared to NP patients In TCMR patients, urinary endocan levels were high (P=0.003). In addition, it was confirmed that urinary endocan levels were higher in patients with chronic active ABMR than in patients with LTGS (174.5±228.8 vs. 731.2±1097.1 pg/mgCr, LTGS vs. chronic active ABMR; p=0.006).

In addition, as a result of investigating the correlation between the clinical parameters mentioned above and the endocan level, as shown in Table 2 below, age, transplant vintage, and the number of HLA mismatching Was not associated with plasma or urine endocan levels, glomerular filtration rate (eGFR) was found to be negatively correlated with plasma and urine endocan levels, and the correlation coefficient was much higher at urine endocan levels (plasma Endocane: r=-0.195, p=0.007 and urine endocan: r=-0.425, p<0.001). Urine PCR results showed a positive correlation with the level of endocans in urine, but plasma endocan levels did not show a correlation (urine endocan: r=0.578, p<0.001 and plasma endocan: r=0.183, p. =0.126), the presence of donor-specific antibodies was not shown to be related to plasma or urine endocan levels.

Plasma endocan (log ₁₀ ) Urinary endocan (log ₁₀ ) r p r p Age (years) -0.051 0.489 0.061 0.416 Transplant vintage (week) -0.081 0.261 0.104 0.155 HLA mismatching (n) -0.031 0.665 -0.109 0.137 eGFR (ml/min/1.73 m ² ) -0.195 0.007 -0.425 <0.001 Urine PCR (log ₁₀ , mg/gCr) 0.183 0.126 0.578 <0.001

Example 4. Analysis of association between Banff score and plasma and endocan quartile in urine

Banff pathology scores according to Banff2007 classification of each diagnostic group are summarized in Table 3 below. TCMR patients had pronounced tubulitis (median 2) and interstitial inflammation (median 2), whereas patients with acute ABMR had prominent glomerulonephritis (median 2) and capillary vasculitis (median 2). In addition, microvascular damage was most pronounced in chronically active ABMR patients than in other groups of patients (median g 3 and median ptc 3). Moreover, chronic tubulointerstitial changes were also observed in chronically active ABMR patients (median ct 2 and median ci 2).

NP
(n=29) ATN
(n=17) APN
(n=7) BKVN
(n=22) TCMR
(n=46) Acute
ABMR
(n=39) Chronic
active ABMR
(n=17) t 0 (0, 0) 0 (0, 0) 0 (0, 1) 1 (1, 2) 2 (1, 2) 0 (0, 2) 0 (0, 1) v 0 (0, 0) 0 (0, 0) 0 (0, 0) 0 (0, 0) 0 (0, 0) 0 (0, 0) 0 (0, 0) ti 0 (0, 0) 0 (0, 0) 1 (0, 2) 1 (0, 2) 2 (2, 3) 0 (0, 2) 2 (1, 2) g 0 (0, 0) 0 (0, 0) 0 (0, 0) 0 (0, 0) 0 (0, 1) 2 (1, 3) 3 (2, 3) ct 0 (0, 0) 0 (0, 0) 0 (0, 1) 0 (0, 1) 1 (0, 1) 1 (0, 2) 2 (1, 2) cv 0 (0, 0) 0 (0, 0) 0 (0, 1) 0 (0, 1) 0 (0, 0) 0 (0, 1) 2 (1, 2) ci 0 (0, 0) 0 (0, 0) 0 (0, 1) 0 (0, 1) 1 (0, 1) 1 (0, 1) 2 (1, 2) cg 0 (0, 0) 0 (0, 0) 0 (0, 0) 0 (0, 0) 0 (0, 0) 0 (0, 0) 3 (2, 3) ptc 0 (0, 0) 0 (0, 0) 0 (0, 0) 0 (0, 0) 0 (0, 0) 2 (2, 3) 3 (2, 3) MVI 0 (0, 0) 0 (0, 0) 0 (0, 0) 0 (0, 0) 0 (0, 1) 4 (3, 5) 5 (4, 6)

Enrolled patients were organized into quartiles according to plasma and urine endocan levels, and various Banff pathology scores were compared according to endocan quartiles (plasma endocan levels: 1Q, <189.0; 2Q, ≥189.0 and <280.3. ; 3Q,≥280.3 and <450.8; 4Q,≥450.8 pg/ml and urine endocan levels: 1Q,<18.2; 2Q,≥18.2 and <94.7; 3Q,≥94.7 and <243.6; 4Q,≥243.6 pg/mgCr ). 2A and 2B, the scores of Tubulitis or Interstitial inflammation were not related to the plasma or urinary endocan quartile, whereas, in contrast, glomerulitis and capillary vasculitis ( Peritubular capillaritis), and microvascular inglammation (MVI) scores calculated as the sum of glomerulonephritis and capillaritis scores were significantly higher in patients with high endocan quartiles in plasma and urine.

Example 5. Analysis of immunohistochemical staining and immunofluorescence staining for endocan in kidney allograft tissue

Endocane levels were analyzed by immunohistochemical methods in kidney tissue, and the results are shown in FIG. 3. As a positive control (A), tissues of patients with clear cell renal cell carcinoma were stained with an endocan antibody, and an immune response was observed in the vascular endothelium of the tumor. As a result of observation, it was confirmed that endocans were not detected in tissues of NP(B) and TCMR(C) patients, whereas endocans were expressed only in proximal tubular epithelial cells of tissues derived from acute ABMR patients (D). However, in these patients, endocan expression was not observed in large or small blood vessels including glomerular or peritubular capillaries. On the other hand, it was confirmed that endocan expression was not a false positive result through staining using an isotype control group.

Since the immunohistochemical staining results were not consistent with the immunofluorescence staining results in the previous study, the present inventors further performed immunofluorescence staining for endocans. As a result, the expression pattern of endocans shown in immunofluorescence staining was similar to that observed in the immunohistochemical staining results. That is, the results of endocan immunofluorescence staining in NP and TCMR patient-derived tissues were negative, endocans were observed in proximal renal tubular cells of acute ABMR patient tissues, and endocans were not stained in glomeruli and blood vessels.

Example 6. ROC curve analysis for evaluation of plasma and urine endocan levels for differentiation of ATN, BKVN, TCMR and acute ABMR

AUC determined through ROC curve analysis was used to evaluate the diagnostic utility of plasma and urine endocan levels to distinguish acute ABMR from ATN, BKVN and TCMR. These four conditions cannot be distinguished by clinical symptoms, and a final diagnosis cannot be made without graft biopsy. The ROC curve was analyzed by adjusting age, sex, duration of transplantation, presence of diabetes and hypertension, ABO incompatibility, number of HLA mismatches, and eGFR to eliminate confounding effects by other variables. As a result, as can be seen in FIG. 4, the AUC of the plasma and urine endocan levels were 0.796 and 0.788, respectively, and the AUC value increased to 0.893 after integrating the plasma and urine endocan levels.

Example 7. Comparative analysis of patient treatment methods and kidney transplant results

After the diagnosis of rejection was confirmed, the patients received immunosuppressive treatment in various combinations. The majority of patients diagnosed with rejection (78.3%, 84.6%, and 76.5% of patients with TCMR, acute ABMR and chronic active ABMR, respectively) received high-dose intravenous steroid therapy, and a significant number of ABMR patients received alloreactive antibodies. In addition, intravenous immunoglobulins (79.5% and 58.8% of patients with acute ABMR and chronic active ABMR, respectively) and rituximab (74.4% and 70.6% of patients with acute ABMR and chronic active ABMR, respectively) were administered in order to eliminate them. In addition, therapeutic plasma exchange was frequently performed in the group of patients with acute ABMR, and the prescription pattern of immunosuppressants did not change for the diagnosis of NP and ATN.

Finally, kidney transplant results according to diagnosis and endocan levels were analyzed, and survival curves were generated after the multivariate adjustment described above to avoid possible disturbance effects. As shown in FIG. 5, patients diagnosed with rejection of any type had a significantly lower renal allograft survival rate compared to other groups (P<0.001) (Renal survival). Interestingly, the combination of plasma and urine endocan levels was associated with allograft survival in the TCMR and ABMR groups, and patients with higher endocan levels in the TCMR and acute ABMR groups had significantly higher renal survival rates than those with lower endocan levels. It was found to be low (p=0.004 and p=0.007), and similar results were found in chronic active ABMR patients, but there was no significant difference (p=0.089).

Example 8. Analysis of basic clinical characteristics and endocan levels according to cardiovascular complications in terminal renal failure patients

From this example, a study was conducted to find a marker capable of early diagnosis or predicting the onset of cardiovascular complications in patients with end-stage renal failure, and experiments and results for this were disclosed in Examples 8 to 10.

First, 349 end-stage renal failure patients who received hemodialysis registered at 6 tertiary hospitals were followed up for an average of 15.7 months, and the basic clinical characteristics of the patients and the results of various cytokines are shown in Table 4 below. As a result of observation, cardiovascular complications occurred in 52 (15%) of 349 patients, and cardiovascular risk such as diabetes (diabetes), acute coronary syndrome (ACS), arrhythmia, and heart failure. Factors were more common in patients with cardiovascular disease. In addition, patients with cardiovascular complications showed higher white blood cell count (WBC) than those who did not, and lower hemoglobin, serum albumin (Albumin), total cholesterol and high-density lipoprotein (HDL). Showed the level. Plasma endocan levels were significantly higher in patients with cardiovascular complications (3.1 ± 0.2 log pg/mL) compared to patients without cardiovascular complications (3.0 ± 0.2 log pg/mL). Confirmed (p = 0.02). On the other hand, other cytokines such as IL-6 and TNF-α were not significantly different between the two groups. In addition, the plasma endocan levels according to the occurrence of cardiovascular complications are compared and shown in FIG. 6.

Variables All
(n = 349) ESRD Patients with cardiovascular composite outcomes
(n = 52) ESRD Patients without cardiovascular composite outcomes
(n = 297) P value Age, mean years ± SD 62 ± 13 64 ± 13 62 ± 13 0.60 Male gender 233 (67) 34 (65) 199 (67) 0.78 BMI ± SD 23 ± 3.9 23.5 ± 3.5 22.9 ± 3.9 0.34 Follow up duration, months± SD 15.7 ± 7.9 9.1 ± 6.43 16.8 ± 7.6 <0.001 Underlying disease Diabetes 194 (56) 38 (73) 156 (53) 0.01 HbA1c,% ± SD 6.6 ± 1.4 6.6 ± 1.1 6.6 ± 1.4 0.76 Hypertension 299 (86) 46 (88) 253 (85) 0.75 Heart disease 110 (32) 31 (60) 79 (26) <0.001 Previous history of ACS 48 (14) 18 (34) 30 (10) <0.001 Valvular heart diseases 2 (0.6) 0 (0) 2 (0.7) 1.00 Heart failure 19 (6) 6 (12) 13 (5) 0.08 Arrhythmia 36 (10) 11 (22) 25 (8) <0.001 Peripheral vascular diseases 6 (2) 1 (2) 5 (2) 0.94 Cerebrovascular diseases 75 (21) 13 (25) 61 (21) 0.49 Solid tumor or hematologic malignancy 41 (12) 7 (13) 34 (11) 0.75 Chronic liver disease 34 (10) 4 (8) 30 (10) 0.81 Chronic pulmonary disease 10 (3) 1 (2) 9 (3) 0.97 Rheumatoid diseases 8 (2) 1 (2) 7 (2) 0.95 Cytokines Log endocan/ESM-1,
log pg/mL ± SD
(Endocan/ESM-1,
pg/mL ± SD) 3.02 ± 0.19
(1164 ± 599) 3.11 ± 0.23
(1480 ± 932) 3.01 ± 0.19
(1109 ± 501) 0.02 IL-6, pg/mL ± SD 4.65 ± 7.08 4.64 ± 3.08 4.66 ± 7.54 0.99 TNF-alpha, pg/mL ± SD 9.67 ± 5.39 10.76 ± 5.55 9.48 ± 5.35 0.12 Laboratory findings WBC, mean /
mm ³ ± SD 5.92 ± 2.01 6.45 ± 2.49 5.83 ± 1.92 0.02 Platelet, x 10 ³ /mm ³ ± SD 175 ± 55 181 ± 56 174 ± 55 0.39 Hemoglobin, g/dL ± SD 10.43 ± 1.22 10.26 ± 1.03 10.46 ± 1.26 0.29 Albumin, g/dL ± SD 3.81 ± 0.34 3.72 ± 0.37 3.82 ± 0.33 0.06 Ca x P, mg ² /dL ² ± SD 40.54 ± 12.68 43.71 ± 10.97 40.96 ± 12.50 0.10 Total cholesterol, mg/dL ± SD 136.86 ± 31.21 126.66 ± 29.37 138.43 ± 31.23 0.03 LDL, mg/dL ± SD 77.71 ± 26.81 76.74 ± 28.42 77.87 ± 26.60 0.78 HDL, mg/dL ± SD 45.34 ± 13.45 40.55 ± 10.73 46.08 ± 13.69 0.01 Hemodialysis informations Dialysis duration, mean months ± SD 58 ± 65 53 ± 52 58 ± 66 0.57 Pre-dialysis SBP, mmHg ± SD 144 ± 20 145 ± 17 144 ± 21 0.55 spKt/V ± SD 1.57 ± 0.30 1.51 ± 0.30 1.58 ± 0.30 0.13

Example 9. Analysis of correlation between plasma endocan levels and basic clinical characteristics

The correlation between plasma endocan levels and various clinical features in patients with end-stage renal failure undergoing hemodialysis is shown in Table 5 below. More specifically, in the univariate linear regression curve, arrhythmia ( β = 0.12, p <0.001), chronic liver disease ( β = 0.09, p = 0.01), high density lipoprotein ( β = 0.003, p = 0.001), and duration of dialysis ( β = 0.002, p = 0.001), spKt/V ( β = 0.08, p = 0.03) and TNF-α ( β = 0.006, p = 0.003) showed positive correlations with log endokan levels. Log endocan levels are also known as body mass index (BMI) ( β = -0.02, p <0.001), diabetes ( β = -0.07, p <0.001), WBC ( β = -0.02, p = 0.004), platelets ( β = -0.001, p <0.001), serum albumin ( β = -0.08, p = 0.01) and LDL ( β = -0.001, p = 0.003) showed an inverse correlation. In multiple linear regression analysis, log-endocan levels were found in acute coronary syndrome (ACS) ( β = 0.08, p = 0.03), stable angina ( β = 0.13, p = 0.004), and TNF-α ( β = 0.005, p = 0.01). ) And predialysis systolic blood pressure (SBP) ( β = 0.001, p = 0.03), and BMI ( β = -0.10, p = 0.01) and albumin ( β = -0.10, p = It was confirmed that there was an independent inverse correlation with 0.04).

Variables Simple linear regression analysis Multiple linear regression analysis

SE p value

SE p value Age 0.001 0.001 0.12 Male gender 0.03 0.02 0.17 BMI -0.02 0.003 <0.001 -0.01 0.004 0.01 Underlying disease Diabetes -0.07 0.02 <0.001 -0.03 0.04 0.47 HbA1c -0.02 0.01 0.03 -0.01 0.01 0.35 Hypertension 0.01 0.03 0.78 Previous history of ACS 0.05 0.03 0.09 0.08 0.04 0.03 Angina pectoris 0.07 1.94 0.05 0.13 0.04 0.004 Valvular heart diseases 0.02 0.14 0.86 Heart failure 0.07 0.04 0.12 Arrhythmia 0.12 0.03 <0.001 0.05 0.04 0.24 Peripheral vascular diseases 0.06 0.07 0.45 Cerebrovascular diseases -0.04 0.03 0.15 Solid tumor or hematologic malignancy 0.03 0.03 0.38 Chronic liver disease 0.09 0.03 0.01 0.06 0.04 0.20 Chronic pulmonary disease -0.03 0.06 0.65 Rheumatoid diseases 0.09 0.07 0.19 Cytokines IL-6 <0.001 0.002 0.82 TNF-alpha 0.006 0.002 0.003 0.005 0.002 0.01 Laboratory findings WBC -0.02 0.005 0.004 -0.001 0.01 0.83 Platelet -0.001 <0.001 <0.001 <-0.001 <0.001 0.13 Hemoglobin -0.02 0.01 0.08 -0.02 0.01 0.16 hs-CRP <-0.001 0.001 0.87 Albumin -0.08 0.03 0.01 -0.10 0.04 0.01 Ca x P -0.001 0.001 0.20 Total cholesterol -0.001 <0.001 <0.001 -0.001 0.001 0.10 LDL -0.001 <0.001 0.003 <-0.001 <0.001 0.68 HDL 0.003 0.001 0.001 <-0.001 0.001 0.68 Hemodialysis informations Dialysis duration 0.001 <0.001 0.001 <0.001 <0.001 0.51 Pre-dialysis SBP 0.001 0.001 0.07 0.001 0.001 0.03 spKt/V 0.08 0.03 0.03 -0.02 0.05 0.72

Example 10. Evaluation of the risk of cardiovascular complications in patients with terminal renal failure

As a result of the multivariate Cox proportional analysis, the log endocan level was found to be independently associated with the onset of cardiovascular complications and the risk ratio of 7.88 (95% CI, 1.69 ~ 36.88, p = 0.01) as shown in Table 6 below. In the univariate Cox proportional-risk model, diabetes, ACS, heart failure, arrhythmia, log endocan levels, WBC, albumin and HDL were associated with the incidence of cardiovascular complications. Multivariate Cox proportional analysis showed that diabetes, ACS, arrhythmia, and low levels of HDL were significantly associated with cardiovascular complications: diabetes risk ratio was 1.98 (95% CI, 1.03-3.81, p = 0.04), ACS. Is 2.48 (95% CI, 1.31-4.68, p = 0.01), arrhythmia is 2.24 (95% CI, 1.10-4.73, p = 0.04), and HDL is 0.97 (95% CI, 0.94-0.99, p = 0.02) Was.

Univariable Cox regression analysis Multivariable Cox regression analysis Variables Odds ratio (95% CI) P value Variables Odds ratio (95% CI) P value Log endocan 6.24 (1.60-24.36) 0.01 Log endocan/ESM-1 21.39
(4.72-97.01) 0.01 Diabetes 2.02 (1.11-3.67) 0.02 Diabetes 1.98 (1.03-3.81) 0.04 Acute coronary syndrome 4.09 (2.35-7.13) <0.001 Acute coronary syndrome 2.48 (1.31-4.68) 0.01 Heart failure 2.73
(1.23-6.05) 0.01 Heart failure 1.51 (0.61-3.74) 0.37 Arrhythmia 3.37
(1.80-6.31) <0.001 Arrhythmia 2.24 (1.10-4.73) 0.04 White blood cell 1.16 (1.04-1.30) 0.01 White blood cell 1.16 (1.00-1.34) 0.05 Albumin 0.39
(0.19-0.81) 0.01 Albumin 0.65 (0.27-1.59) 0.35 Ca x P 1.02 (0.99-1.04) 0.15 High-density lipoprotein 0.97
(0.94-0.99) 0.01 High-density lipoprotein 0.97 (0.94-0.99) 0.02

In addition, as shown in FIG. 7, the maximum chi-square test result showed that the optimal cutoff value of the plasma endocan level was 3.2 log pg/mL when predicting cardiovascular complications. Patients with endocan levels of 3.2 log pg/mL or more had a significantly higher risk ratio of 7.6 (95% CI, 3.0 ~ 19.2) (p <0.001), and the cumulative incidence of cardiovascular complications was significantly higher.

Through the above results, it was confirmed that the endocan level can be used as an independent marker for predicting the risk of cardiovascular complications in patients with end-stage renal failure.

The above-described description of the present invention is for illustration purposes only, and those of ordinary skill in the art to which the present invention pertains can understand that it is possible to easily transform it into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting.

<110> University-Industry Cooperation Group of Kyung Hee University <120> Endocan as a marker for diagnosing or predicting cardiovascular complications and kidney transplant rejection in patients with renal failure, and uses thereof <130> PD19-081 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 1634 <212> DNA <213> Homo sapiens <400> 1 gctgcttccc accagcaaag accacgactg gagagccgag ccggaggcag ctgggaaaca 60 tgaagagcgt cttgctgctg accacgctcc tcgtgcctgc acacctggtg gccgcctgga 120 gcaataatta tgcggtggac tgccctcaac actgtgacag cagtgagtgc aaaagcagcc 180 cgcgctgcga gaggacagtg ctcgacgact gtggctgctg ccgagtgtgc gctgcagggc 240 ggggagaaac ttgctaccgc acagtctcag gcatggatgg catgaagtgt ggcccggggc 300 tgaggtgtca gccttctaat ggggaggatc cttttggtga agagtttggt atctgcaaag 360 actgtcccta cggcaccttc gggatggatt gcagagagac ctgcaactgc cagtcaggca 420 tctgtgacag ggggacggga aaatgcctga aattcccctt cttccaatat tcagtaacca 480 agtcttccaa cagatttgtt tctctcacgg agcatgacat ggcatctgga gatggcaata 540 ttgtgagaga agaagttgtg aaagagaatg ctgccgggtc tcccgtaatg aggaaatggt 600 taaatccacg ctgatcccgg ctgtgatttc tgagagaagg ctctattttc gtgattgttc 660 aacacacagc caacatttta ggaactttct agattatagc ataaggacat gtaatttttg 720 aagaccaaat gtgatgcatg gtggatccag aaaacaaaaa gtaggatact tacaatccat 780 aacatccata tgactgaaca cttgtatgtg tttgttaaat attcgaatgc atgtagattt 840 gttaaatgtg tgtgtatagt aacactgaag aactaaaaat gcaatttagg taatcttaca 900 tggagacagg tcaaccaaag agggagctag gcaaagctga agaccgcagt gagtcaaatt 960 agttctttga ctttgatgta cattaatgtt gggatatgga atgaagactt aagagcagga 1020 gaagatgggg agggggtggg agtgggaaat aaaatattta gcccttcctt ggtaggtagc 1080 ttctctagaa tttaattgtg cttttttttt ttggctttgg gaaaagtcaa aataaaacaa 1140 ccagaaaacc cctgaaggaa gtaagatgtt tgaagcttat ggaaatttga gtaacaaaca 1200 gctttgaact gagagcaatt tcaaaaggct gctgatgtag ttcccgggtt acctgtatct 1260 gaaggacggt tctggggcat aggaaacaca tacacttcca taaatagctt taacgtatgc 1320 cacctcagag ataaatctaa gaagtatttt acccactggt ggtttgtgtg tgtatgaagg 1380 taaatattta tatattttta taaataaatg tgttagtgca agtcatcttc cctacccata 1440 tttatcatcc tcttgaggaa agaaatctag tattatttgt tgaaaatggt tagaataaaa 1500 ctatgactct ataaggtttt caaacatctg aggcatgata aatttattat ccataattat 1560 agtaataata accttaataa gcataagaaa aacagagtca ctctggattt caaaaatgtc 1620 aaaaaaaaaa aaaa 1634 <210> 2 <211> 184 <212> PRT <213> Homo sapiens <400> 2 Met Lys Ser Val Leu Leu Leu Thr Thr Leu Leu Val Pro Ala His Leu 1 5 10 15 Val Ala Ala Trp Ser Asn Asn Tyr Ala Val Asp Cys Pro Gln His Cys 20 25 30 Asp Ser Ser Glu Cys Lys Ser Ser Pro Arg Cys Glu Arg Thr Val Leu 35 40 45 Asp Asp Cys Gly Cys Cys Arg Val Cys Ala Ala Gly Arg Gly Glu Thr 50 55 60 Cys Tyr Arg Thr Val Ser Gly Met Asp Gly Met Lys Cys Gly Pro Gly 65 70 75 80 Leu Arg Cys Gln Pro Ser Asn Gly Glu Asp Pro Phe Gly Glu Glu Phe 85 90 95 Gly Ile Cys Lys Asp Cys Pro Tyr Gly Thr Phe Gly Met Asp Cys Arg 100 105 110 Glu Thr Cys Asn Cys Gln Ser Gly Ile Cys Asp Arg Gly Thr Gly Lys 115 120 125 Cys Leu Lys Phe Pro Phe Phe Gln Tyr Ser Val Thr Lys Ser Ser Asn 130 135 140 Arg Phe Val Ser Leu Thr Glu His Asp Met Ala Ser Gly Asp Gly Asn 145 150 155 160 Ile Val Arg Glu Glu Val Val Lys Glu Asn Ala Ala Gly Ser Pro Val 165 170 175 Met Arg Lys Trp Leu Asn Pro Arg 180

Claims (19)

An antibody-mediated rejection (ABMR) diagnostic marker composition after kidney transplantation, comprising an endocan gene or a protein encoded by the gene.
A composition for diagnosing antibody-mediated rejection (ABMR) after kidney transplant, comprising an agent measuring the level of the mRNA of the endocan gene or the protein encoded by the gene.
The method of claim 2,
The composition for measuring the mRNA level is a sense and antisense primer or probe that complementarily binds to the mRNA of the gene.
The method of claim 2,
The composition for measuring the protein level is an antibody that specifically binds to a protein encoded by the gene.
A kit for diagnosing antibody-mediated rejection (ABMR) after kidney transplant, comprising the composition of claim 2.
Diagnosis of antibody-mediated rejection (ABMR), comprising measuring the level of the mRNA of the endocan gene or the protein encoded by the gene in a biological sample derived from a patient who has undergone kidney transplant surgery. How to provide information for
The method of claim 6,
The method for providing information, characterized in that the biological sample is tissue, blood, serum, plasma or urine.
The method of claim 6,
The expression level of the mRNA is polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay (RPA), microarray. (microarray), and northern blotting (northern blotting), characterized in that measured through at least one method selected from the group consisting of, information providing method.
The method of claim 6,
The protein expression level is Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation, flow cytometry, Characterized in that it is measured by one or more methods selected from the group consisting of immunofluorescence, ouchterlony, complement fixation assay, and protein chip, How to provide information.
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