KR102065545B1 - Composition for predicting renal fibrosis and the decline in kidney function in patients with diabetic nephropathy - Google Patents

Abstract

The present invention provides a composition for diagnosing renal fibrosis and renal dysfunction comprising an agent capable of amplifying or detecting a marker protein of renal fibrosis and renal dysfunction, a kit comprising the composition and a renal fibrosis and renal dysfunction using the composition or kit. It is about how to diagnose the degree.

Description

Composition for predicting renal fibrosis and the decline in kidney function in patients with diabetic nephropathy}

The present invention relates to a composition for predicting the prognosis of renal fibrosis and renal function lowering in diabetic nephropathy, specifically amplifying or detecting a marker selected from the group consisting of CXCL16 (Chemokine (CXC motif) ligand 16) and endostatin A composition for diagnosing renal fibrosis or degeneration in kidney function, comprising a preparation which can be used, a kit for diagnosing renal fibrosis or hypofunction in diabetic nephropathy patients comprising the composition, renal fibrosis or renal dysfunction The present invention relates to a diagnostic method and a method of providing information for predicting progress.

Kidneys are the excretory organs of vertebrates that filter waste products from the blood and discharge them into the urine. The kidney consists of the outer cortex, the inner medulla and the pyelone inside the medulla. Among them, the medulla is composed of collecting tubes including tubules, and the ends of the collecting tubes are open to the renal pelvis. The tubules of the kidney are composed of different types of cells, depending on the compartment, starting from the area close to the glomerulus, which is called the proximal tubule, the narrow tubule, the ascending tubule, the distal tubule, and the collecting tubule. Since the kidney plays a role without rest, it is required to develop a technology that can quickly and easily diagnose kidney disease.

Among these kidney diseases, diabetic nephropathy is the leading cause of end-stage renal disease worldwide. In general, diabetic nephropathy appears to follow typical clinical results, and proteinuria is known to occur before renal dysfunction occurs. The amount of proteinuria was one of the most important predictors of renal insufficiency, especially in patients with early stages of diabetes mellitus (JASN 2007 vol.18, no.4, 1353-1361, CJASN 2012 vol .7, no. 1, 78-84).

However, recent evidence suggests that the progress of diabetic nephropathy is not so simple. Large epidemiologic studies have shown a decreased incidence of proteinuria in diabetic nephropathy, even with a steady increase in renal impairment without proteinuria (JAMA 2016; 316 (6): 602-10). In addition, several studies have suggested that such proteinuria alone is difficult to accurately predict the clinical course of patients with diabetic nephropathy (J Nephropathol 2, 234-240 (2013), PLoS One. 2012; 7 (5): e36041). Thus, there is a growing need for new markers to accurately predict the course of diabetic nephropathy.

In 2010, the Renal Pathology Society established a new pathological classification system for diabetic nephropathy, including glomerular, tubulointerstitial and vascular components (J Am Soc Nephrol. 2010 Apr; 21 (4): 556-63), three studies have shown that the classification system can be useful for predicting renal function in diabetic nephropathy (Diabetes Res Clin Pract. 2012 Sep; 97 (3): 418 -24, Nephrology (Carlton) .2012 Jan; 17 (1): 68-75, Nephrol Dial Transplant. 2015 Feb; 30 (2): 257-66). Given the rapid increase in the incidence of diabetic nephropathy identified by renal biopsy, this new classification system can help medical staff predict the clinical course of diabetic nephropathy.

 However, patients with a high probability of diabetic nephropathy generally have a problem that clinical renal biopsy is not performed. Therefore, there is a limitation that the classification system is applicable only to diabetic nephropathy patients confirmed through renal biopsy.

In order to overcome the limitations of existing markers for predicting the progression of diabetic nephropathy, various urine markers are being actively researched. Among the various markers, in particular, inflammatory markers are attracting attention because one of the main causes of the development and progression of diabetic nephropathy is low grade inflammation. Various studies have shown that several inflammatory markers are associated with renal decline (Kidney Int 58: 1492-1499, 2000, Diabetes 55: 2993-3003, 2006, Diabetes 51: 3532-3544, 2002, J Am Soc Nephrol 19: 789-797, 2008, Pol Merkur Lekarski 13: 28-32, 2002, Cytokine Growth Factor Rev 17: 441-450, 2006). However, most studies focus on early stage evaluation of diabetic nephropathy. In addition, since diabetic nephropathy is diagnosed based on clinical evidence, previous studies have not investigated the association between urinary inflammatory markers and the pathological characteristics of diabetic nephropathy.

Against this background, the present inventors studied biomarkers in urine that can effectively check the progress, renal function, and renal fibrosis in diabetic nephropathy patients confirmed by renal biopsy. Among the various protein markers that can be detected in urine, The present invention was completed by confirming that the expression levels of CXCL16 and endostatin have high relevance.

One object of the present invention is a kidney comprising an agent for detecting one or more gene mRNAs, or protein levels, selected from the group consisting of CXCL16 (CXC motif ligand 16), endostatin and combinations thereof It is to provide a composition for diagnosing renal fibrosis or renal function (decline in kidney function).

Another object of the present invention is to provide a kit for diagnosing renal fibrosis or renal failure of diabetic nephropathy patients.

Another object of the present invention is to obtain a tissue sample of a subject with diabetic nephropathy; Measuring the expression level of a marker selected from the group consisting of CXCL16, endostatin and combinations thereof from the obtained tissue sample using the composition or kit; And determining the onset or course of renal fibrosis or renal dysfunction of the subject.

Another object of the present invention is to obtain a tissue sample of a subject with diabetic nephropathy; Measuring the expression level of a marker selected from the group consisting of CXCL16, endostatin and combinations thereof from the obtained tissue sample using the composition or kit; And it provides a method of providing information for prognostic prediction of renal fibrosis or renal failure, comprising the step of determining the onset or course of renal fibrosis or renal function decline in the individual.

This will be described in detail as follows. In addition, each description and embodiment disclosed in this invention is applicable to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention is not to be limited by the specific description described below.

One aspect of the present invention for achieving the above object is an agent for detecting one or more gene mRNA, or protein levels selected from the group consisting of CXCL16 (Chemokine (CXC motif) ligand 16), endostatin and combinations thereof Provided, comprising a composition for diagnosing renal fibrosis or renal function (decline in kidney function).

Another aspect of the present invention for achieving the above object comprises an agent capable of amplifying or detecting a marker selected from the group consisting of CXCL16 (Chemokine (CXC motif) ligand 16), endostatin and combinations thereof Provided is a diagnostic composition for interstitial fibrosis and tubular atrophy (IFTA).

In the present invention, CXCL16 (Chemokine (CXC motif) ligand 16) is a small cytokine belonging to the CXC chemokine lineage, which is found by dendritic cells found in the lymphoid T cell zone and cells found in the red pulp of the spleen. It is known to produce. In some studies blood CXCL16 has been reported to be associated with renal fibrosis (Sci Rep. 2016 Jun 29; 6: 28715, Curr Opin Nephrol Hypertens. 2014 Jan; 23 (1): 93-100, Hypertension. 2013 Dec; 62 (6): 1129-37, J Am Soc Nephrol. 2011 Oct; 22 (10): 1876-86) Urine CXCL16 has not been studied, particularly for its role in diabetic nephropathy and also for diagnosing renal dysfunction. None known, this use was first identified by the inventors.

In the present invention, endostatin is a 20kDa substance derived from type XVIII collagen, and is known to act as an angiogenesis inhibitor similar to angiostatin and thrombospodine. Although studies have reported that blood endostatin reflects renal insufficiency (Am J Nephrol. 2012 May; 35 (4): 335-340), the meaning of endostatin in urine has not been studied and is used for diagnosing renal fibrosis or renal insufficiency. Is not known, this use was first identified by the inventors.

Specific protein information of the CXCL16 (58191) and endostatin (AAF69009.1) markers is known from databases such as NCBI. This invention includes the case where these are respectively used individually or all are used. In addition, even if mutations, deletions, or substitutions occur in a part of the amino acid sequence of the protein, it is included in the scope of the present invention without limitation as long as the diagnosis of renal fibrosis or renal function is possible.

In the present invention, renal fibrosis (renal fibrosis) is a common phenomenon seen in all kidney disease transition to chronic kidney disease with an extracellular matrix accumulated in the kidney tissue. There is no method yet to accurately determine the extent of renal fibrosis other than renal biopsy.

Decline in kidney function in the present invention means the annual reduction rate of renal function represented by eGFR. Patients with diabetic nephropathy are generally known to be able to predict a decrease in renal function of proteinuria but are not known in patients with advanced diabetic nephropathy.

In the present invention, interstitial fibrosis and tubular atrophy (IFTA) refer to an irreversible dysfunction caused by chronic damage of epilepsy and tubules in the kidney. As a common feature of renal fibrosis, patients with severe interstitial fibrosis and tubular atrophy are known to have poor kidney prognosis. However, as with renal fibrosis, there is no method for determining the degree of interstitial fibrosis and tubular atrophy except renal biopsy.

Renal fibrosis or renal insufficiency may be present in individuals with diabetic nephropathy. Diabetic nephropathy in the present invention is also referred to as diabetic kidney disease (diabetic kidney disease), means a disease in which the glomeruli inside the kidney are continuously damaged by diabetes mellitus, hyperglycemia, etc., and kidney function is reduced. If diabetes lasts for a long time, small blood vessels in the body are damaged, and thus the glomeruli responsible for blood filtration are damaged, resulting in proteinuria and reduced kidney function.

In the present invention, the agent for detecting the gene mRNA, or protein level means a means used to amplify or detect the marker, in order to confirm or measure the expression level of the marker protein, the level of the target protein contained in the sample Means the formulation used in the method for measuring. Examples include western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis , Antibodies used in methods such as tissue immunostaining, immunoprecipitation assays, complement fixation assays, FACS and protein chip assays, or aptamers that can bind to such proteins, etc. This can be More specifically, the agent for detecting the mRNA level may be a primer pair or probe specifically binding to the gene, the agent for detecting the protein level is an antibody that specifically binds to CXCL16 or endostatin, the antibody It may be a fragment, or an aptamer, but is not limited thereto.

As used herein, the term "antibody" refers to a proteinaceous molecule capable of specifically binding to the antigenic site of a protein or peptide molecule. The form of the antibody is not particularly limited and, if it is a polyclonal antibody, a monoclonal antibody or antigen-binding, a part thereof may be included in the antibody of the present invention and may include all immunoglobulin antibodies, as well as humanized antibodies. It may also contain a special antibody of. In addition, the antibodies include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains. A functional fragment of an antibody molecule means a fragment having at least antigen binding function and may be Fab, F (ab '), F (ab') 2, Fv, and the like.

As used herein, the term "aptamer" refers to a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) that has a stable tertiary structure and has a property that can bind to a target molecule with high affinity and specificity.

In the present invention, the antibody, fragment or aptamer thereof is not particularly limited in its amino acid sequence or nucleotide sequence as long as the CXCL16 or endostatin marker can be detected, and is particularly limited in the number of amino acids or nucleic acids constituting the sequence. It doesn't work.

The term "diagnosis" of the present invention means confirming the presence, characteristics, progression of the disease, the therapeutic effect of the disease during treatment, and the like. For the purposes of the present invention, the diagnosis means diagnosing renal fibrosis or renal insufficiency by detecting CXCL16 or endostatin in urine, but is not limited thereto.

In one embodiment of the present invention, as a result of analyzing the pathological classification of diabetic nephropathy and the relationship between urine inflammation markers, the expression level of CXCL16 and endostatin was significantly increased in patients with high grade IFTA (Table 8, 12) In patients with high ITFA and / or epilepsy inflammation scores, the rate of reaching end-stage renal disease (ESRD) was found to be higher (FIGS. 4B, C), and CXCL16 and urine in diabetic nephropathy patients. It can be seen that the degree of renal fibrosis and renal function degradation of diabetic nephropathy can be predicted by confirming the expression level of endostatin (or FIG. 6).

Another aspect of the invention provides a kit for diagnosing renal fibrosis or renal failure of diabetic nephropathy patient, comprising the composition.

Another aspect of the invention provides a kit for diagnosing interstitial fibrosis and tubular atrophy of diabetic nephropathy patient, comprising the composition.

The kit of the present invention can be used to diagnose the occurrence or prognosis of renal fibrosis or renal insufficiency by measuring the expression level of CXCL16 or endostatin from a tissue sample of a subject with diabetic nephropathy, but is not particularly limited thereto, but the CXCL16 or endostatin Antibodies, fragments, or aptamers for detecting them, as well as one or more other constituent compositions, solutions or devices suitable for analytical methods may be included.

In order to increase the sensitivity, the urine may be centrifuged or concentrated.

The substrate used in the kit of the present invention may further include a chromophore for confirming contact thereof upon urine contact.

The kit may also include the case of using in combination with the existing kidney disease diagnostic kits, for example in the form of a complex kit to increase the sensitivity and specificity.

In the present invention, the kit for measuring the expression level of the marker may include a substrate, a secondary antibody and a chromophore labeled with a suitable buffer, chromophore or fluorescent substance for immunological detection of the antibody. The substrate may be a nitrocellulose membrane, a 96-well plate synthesized with a polyvinyl resin, a 96-well plate synthesized with a polystyrene resin, a slide glass made of glass, and the like. The chromophore may be a peroxidase, Alkaline phosphatase (alkaline phosphatase) and the like can be used, the fluorescent material may be used FITC, RITC and the like, the color substrate is ABTS (2,2'-azino-bis- (3-ethylbenzothiazoline-6- Sulfonic acid)) or OPD (o-phenylenediamine), TMB (tetramethyl benzidine) can be used.

In addition, a secondary antibody capable of detecting the antibody or aptamer may be further included, and one labeled with a fluorescent substance, a radioisotope, or the like may be used.

Another aspect of the present invention provides a method of treating a subject, comprising the steps of: (a) obtaining a tissue sample of a subject having diabetic nephropathy; (b) measuring the expression level of a marker selected from the group consisting of CXCL16, endostatin and combinations thereof from the tissue samples obtained using the composition of any one of claims 1 to 3 or the kit of claim 4. ; And (c) determining the onset or course of renal fibrosis or renal dysfunction of the subject.

Another aspect of the invention, the present invention comprises the steps of (a) obtaining a tissue sample of a subject with diabetic nephropathy; (b) measuring the expression level of a marker selected from the group consisting of CXCL16, endostatin and combinations thereof from the tissue samples obtained using the composition of any one of claims 1 to 3 or the kit of claim 4. ; And (c) determining the onset or course of renal fibrosis or renal dysfunction of the subject.

Another aspect of the present invention provides a method for diagnosing interstitial fibrosis and tubular atrophy and providing information for prognostic prediction, comprising the steps (a) to (c).

At this time, the composition, kit, CXCL16, endostatin, renal fibrosis or renal impairment are the same as described above, and the sample includes feces, blood, plasma, serum, lymph, cerebrospinal fluid, isolated tissue, isolated cells, saliva, urine Or the like, and more specifically, urine, but is not particularly limited thereto.

The method of the present invention. If the expression level of the marker selected from the group consisting of CXCL16, endostatin and combinations thereof measured in step (b) is higher than that of the control sample, further comprising the step of judging that the prognosis of the subject is poor It may be.

The present invention can easily diagnose and predict the progression of renal fibrosis and renal insufficiency in diabetic nephropathy patients by measuring only the expression level of CXCL16 or endostatin in urine from patients with diabetic nephropathy who have difficulty in renal biopsy.

1 is a diagram showing the number of patients classified by the institution recruiting patients, specific diseases. (Abbreviations: DN, diabetic nephropathy; NDRD, non-diabetic renal disease; KNDP, Korea national diabetes program)
2 is a diagram showing the expression level of urine inflammatory markers in the control group and diabetic nephropathy patients as demonstrated by renal histology. (Abbreviations: DM, diabetes mellitus; DN, diabetic nephropathy; MCP-1, monocyte chemoattractant protein-1; RANTES, Regulated on activation, normal T cell expressed and secreted; IP-10, interferon gamma-induced protein-10; IL- 6, interleukin-6; TNF-α, tumor necrosis factor-α; VEGF, vascular endothelial growth factor)
3 shows the correlation between urine PCR and reduction of eGFR. (Abbreviations: PCR, protein-to-creatinine ratio; eGFR, estimated glomerular filtration rate)
Figure 4 is a diagram showing the kidney results of patients according to the pathological classification of diabetic nephropathy. (A) renal survival in various glomerular lesions, (B) interstitial fibrosis and tubular atrophy, (C) interstitial inflammation, (D) vitreous artery and (E) atherosclerosis.
5 is a diagram showing the renal results of patients according to the expression level of the urine marker. (A) Urine protein-creatinine ratio (PCR), (B) CXCL16, (C) endostatin, and (D) CXCL16 and endostatin combination.
FIG. 6 shows a Receiver operating characteristic (ROC) curve for predicting kidney results using (A) urine protein to creatinine ratio (PCR), (B) CXCL16, (C) endostatin and (D) CXCL16 and endostatin The figure shown.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Experimental Materials and Methods

1-1. Patient Selection and Experimental Design

Patient selection and recruitment proceeded as shown in FIG. 1. Specifically, in previous studies, patients with urine sampling who were diagnosed with diabetic nephropathy by pure kidney biopsy were selected. During the period from January 2012 to December 2016, 220 patients with type 2 diabetes who underwent kidney biopsy at six different hospitals were enrolled. Seventy out of 114 patients diagnosed with purely diabetic nephropathy without developing other kidney disease, agreed to collect urine samples for future biomarker studies.

Indications for renal biopsy include a period of diabetes less than 5 years; The incidence of proteinuria without diabetic retinopathy; Frequent occurrence of urinary sediment; And / or a rapid decrease in renal function due to hematuria or an unknown cause.

As controls, an additional six high-risk diabetes patients (defined as having impaired fasting glucose and / or impaired glucose tolerance) with age and gender matched, and 10 diabetic patients without proteinuria Recruitment was made as a comparative group of urine inflammation markers (right side of FIG. 1).

Age, sex, height, weight, duration of diabetes, and presence of hypertension were recorded, and general tests including HbA1c, hemoglobin, serum albumin, creatinine, cholesterol, etc., were also performed at kidney biopsy. If possible, the amount of proteinuria was measured by 24-hour urine collection or calculated by spot urine protein-to-creatinine ratio (PCR, g / gCr). Patients diagnosed with diabetic nephropathy were conservatively administered using angiotensin-converting enzymes or angiotensin receptor blockers, unless otherwise contraindicated. Renal function was assessed according to progression to end-stage renal disease (ESRD) requiring dialysis or kidney transplantation.

1-2. Measurement of Urine Inflammation Markers Using Sampling and Multiple Enzyme Immunoassay (ELISA)

Additional urine samples were collected on the morning of the patient's kidney biopsy to measure urine inflammation markers. Samples were centrifuged at 2,000 x g for 20 minutes at room temperature, then the supernatant was placed in a tube and stored in a freezer at -80 ° C until analysis. All samples were processed and stored within 1 hour after collection.

According to the manufacturer's instructions (R & D Systems, MN), various urine inflammatory markers were simultaneously measured using a customized Magnetic Luminex Screening Assay. Based on the results, a total of 10 candidate markers were selected: monocyte chemoattractant protein-1 (MCP-1), regulated on activation, normal T cell expressed and secreted (RANTES), interferon gamma-induced protein-10 ( IP-10), CXCL16, endostatin, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), CXCL9 and growth / differentiation factor-15 (GDF-15) .

Each inflammatory marker was detected by Luminex 200 ^™ (Luminex Corp., Austin, TX) and the concentration was calculated based on standard curves generated from reference samples. Finally, all urinary inflammatory markers were expressed in proportion to urine creatinine concentration (pg / mg Cr).

1-3. Pathological Analysis and Classification of Registered Patients

All biopsy specimens were processed by standard methods for general examination using light microscopy (LM), immunofluorescence and electron microscopy (EM), and then referred to a pathology specialist at each hospital. The pathological diagnosis of diabetic nephropathy was based on the following criteria (J Am Soc Nephrol. 2010; 21: 556-63): diffuse thickening of glomerular basement membranes (395 nm or more in women, 430 nm or more in men) and / Or swelling of glomerular mesialial with or without nodular glomerulosclerosis. Patients with confirmed kidney disease non-diabetic were excluded from the experiment.

Patients were further classified according to the pathological classification of the Renal Pathology Society. To classify enrolled patients, five different pathological parameters were used: interstitial fibrosis and tubular atrophy, interstitial inflammation, arterial artery Arteriolar hyalinosis and arteriosclerosis.

Glomerular lesions were scored as follows.

class I, thickening of glomerular basement membrane confirmed by electron microscopy, no epileptic changes;

class II, interstitial dilatation confirmed by light microscopy;

class III, at least one nodular glomerulosclerosis; And

class IV, glomerulosclerosis in more than 50% of observed glomeruli.

Interstitial fibrosis and tubular atrophy (IFTA) were scored as follows.

0, no sign of IFTA;

1, at least 25% of the total area;

2, 26-50% of the total area;

3, more than 50% of the total area

Epilepsy inflammation was scored as follows.

0, no trace;

1, infiltration of inflammatory cells associated with IFTA;

2, inflammatory cell infiltration in the region without IFTA

The vitreous artery of the arterioles was scored as follows.

0, no trace;

1, development of arteriosclerosis in at least one area;

2, arteriosclerosis occurs in more than one area

Finally, atherosclerosis was scored as follows.

0, no intima thickening;

1, intima thickening thinner than the thickness of the media;

2, thicker lining thicker than the thickness of the media

1-4. Statistical analysis

All statistical analyzes were performed with SPSS for Windows, version 20.0 (SPSS, Chicago, IL). Clinical characteristics and clinical indicators were expressed as mean ± standard deviation (SD) or number and percentage of patients. ANOVA and chi-square test were used to compare baseline characteristics and test findings. Urine inflammation markers were described as median (1, 3 quartile range [IQR]), and Kruskal-Wallis analysis was performed considering that these data correspond to an abnormal distribution. Pearson correlation analysis was used to compare urine inflammation markers, urine protein-to-creatinine ratio (PCR), and eGFR reduction. Time-to-event analysis of renal results was performed by log-rank test. Finally, to distinguish patients with rapidly progressing diabetic nephropathy, the receiver operating characteristic (ROC) curve and the area under the curve (AUC) for evaluating the predictive power of urinary inflammatory markers were identified. P values below 0.05 were considered statistically significant.

Example 2: Experimental Results

2-1. Identifying clinical features and markers of urinary inflammation in registered patients

The basic clinical characteristics of diabetic nephropathy confirmed by biopsy are summarized in Table 1.

Clinical characteristics and laboratory parameters in patients with diabetic nephropathy proven by biopsy Patients with biopsy-proven diabetic nephropathy (n = 70) Age (years) 57.3 ± 11.4 Sex (Male,%) 45 (64.3) BMI (kg / m ² ) 25.2 ± 3.4 Duration of diabetes (years) 11.4 ± 8.3 Hypertension (n,%) 62 (88.6) HbA1c (%) 7.8 ± 2.0 Hemoglobin (g / dL) 10.4 ± 2.0 eGFR (ml / min) 36.1 ± 23.7 Albumin (g / dL) 3.3 ± 0.7 Total cholesterol (mg / dL) 195.8 ± 64.7 Urine PCR (g / gCr) 7.77 ± 6.80

(BMI, body mass index; eGFR, estimated glomerular filtration rate; PCR, protein-to-creatinine ratio)

The average age was 57.3 years, males 64.3% (45/70), and the average duration of diabetes was 11.4 years. Most patients showed moderate to more severe renal dysfunction at renal biopsy, with an average eGFR of 36.1 ml / min per 1.73 m ² and an average urine PCR of 7.77 g / gCr.

Baseline demographics of diabetic patients and high-risk patients without proteinuria are shown in FIG. 1. Age and sex did not differ between diabetic nephropathy patients, and eGFR and proteinuria were in the normal range in the control group.

Expression levels of urine inflammatory markers in the control group and patients with diabetic nephropathy demonstrated in biopsy are shown in FIG. 2. Most urinary inflammatory markers except CXCL9 were found to be significantly higher in diabetic nephropathy patients as demonstrated by biopsy compared to the control. On the other hand, there was no difference in the expression level of urine inflammatory markers between patients at high risk of diabetes and diabetic patients without proteinuria. The level of GDF-15 in urine was above the range in more than 50% of all patients, and no analysis was made.

2-2. Identifying clinical features and markers of urinary inflammation in patients according to pathological classification

As described in Examples 1-3 above, patients with diabetic nephropathy identified by biopsy were classified according to five pathological classification criteria, and their clinical characteristics are shown in Tables 2-6.

Glomerular lesions Class II (n = 14) Class III (n = 32) Class IV (n = 24) p value Age (years) 63.9 ± 11.1 54.3 ± 11.6 57.5 ± 9.9 0.030 Sex (male,%) 7 (50.0) 23 (71.9) 15 (62.5) 0.353 BMI (kg / m ² ) 25.3 ± 3.2 24.5 ± 3.1 26.0 ± 3.9 0.262 Duration of diabetes (year) 15.3 ± 10.5 11.0 ± 8.1 9.6 ± 6.6 0.116 Hypertension (n,%) 13 (92.9) 28 (87.5) 21 (87.5) 0.853 HbA1c (%) 7.7 ± 1.9 7.8 ± 2.0 7.7 ± 2.1 0.941 Hemoglobin (g / dL) 10.7 ± 2.3 10.3 ± 1.6 10.4 ± 2.3 0.758 eGFR (ml / min / 1.73m ² ) 38.5 ± 21.7 39.0 ± 24.6 30.8 ± 23.5 0.408 Albumin (g / dL) 3.7 ± 0.5 3.1 ± 0.6 3.2 ± 0.8 0.032 Total cholesterol (mg / dL) 189 ± 27 213 ± 76 182 ± 61 0.145 Urine PCR (g / gCr) 5.46 ± 5.12 9.55 ± 7.98 6.77 ± 5.42 0.114

Interstitial fibrosis and tubular atrophy 0 and 1
(n = 43) 2
(n = 17) 3
(n = 10) p value Age (years) 58.4 ± 10.7 55.9 ± 11.5 55.0 ± 14.6 0.599 Sex (male,%) 28 (65.1) 9 (52.9) 8 (80.0) 0.360 BMI (kg / m ² ) 25.1 ± 3.6 25.3 ± 3.3 25.4 ± 2.7 0.966 Duration of diabetes (year) 11.8 ± 8.4 9.1 ± 6.6 13.5 ± 10.3 0.371 Hypertension (n,%) 36 (83.7) 16 (94.1) 10 (100) 0.100 HbA1c (%) 7.7 ± 1.7 7.9 ± 2.5 7.9 ± 2.6 0.928 Hemoglobin (g / dL) 10.9 ± 2.1 9.7 ± 1.3 9.4 ± 2.2 0.019 eGFR (ml / min / 1.73m ² ) 45.1 ± 23.2 26.2 ± 18.7 14.3 ± 8.1 <0.001 Albumin (g / dL) 3.4 ± 0.7 3.0 ± 0.5 3.0 ± 0.6 0.051 Total cholesterol (mg / dL) 198 ± 64 181 ± 62 209 ± 75 0.519 Urine PCR (g / gCr) 5.58 ± 3.90 12.66 ± 10.26 8.92 ± 5.19 0.123

Interstitial inflammation 0
(n = 12) One
(n = 42) 2
(n = 16) p value Age (years) 57.2 ± 12.1 57.4 ± 10.8 57.3 ± 13.1 0.999 Sex (male,%) 5 (41.7) 30 (71.4) 10 (62.5) 0.163 BMI (kg / m ² ) 25.7 ± 3.3 25.2 ± 3.7 24.8 ± 2.8 0.779 Duration of diabetes (year) 10.7 ± 8.1 10.5 ± 7.0 14.2 ± 11.1 0.306 Hypertension (n,%) 10 (83.3) 38 (90.5) 14 (87.5) 0.787 HbA1c (%) 8.2 ± 2.3 7.8 ± 2.0 7.4 ± 2.8 0.656 Hemoglobin (g / dL) 10.6 ± 2.1 10.5 ± 2.0 9.9 ± 2.0 0.606 eGFR (ml / min / 1.73m ² ) 42.2 ± 29.4 38.9 ± 23.6 24.0 ± 14.4 0.059 Albumin (g / dL) 3.3 ± 0.8 3.3 ± 0.7 3.2 ± 0.5 0.775 Total cholesterol (mg / dL) 213 ± 79 181 ± 55 224 ± 69 0.044 Urine PCR (g / gCr) 6.15 ± 5.34 7.91 ± 7.59 8.64 ± 5.58 0.624

Arteriolar hyalinosis of the arterioles 0
(n = 7) One
(n = 58) 2
(n = 5) p value Age (years) 61.3 ± 11.1 56.5 ± 11.4 61.0 ± 12.6 0.442 Sex (male,%) 3 (42.9) 21 (36.2) 5 (100) 0.049 BMI (kg / m ² ) 26.5 ± 3.4 24.9 ± 3.3 27.4 ± 4.3 0.154 Duration of diabetes (year) 7.3 ± 9.4 11.5 ± 7.8 16.0 ± 11.4 0.197 Hypertension (n,%) 7 (100) 50 (86.2) 5 (100) 0.836 HbA1c (%) 7.6 ± 1.9 7.8 ± 2.1 7.5 ± 1.0 0.922 Hemoglobin (g / dL) 10.4 ± 2.3 10.3 ± 2.0 11.7 ± 2.0 0.325 eGFR (ml / min / 1.73m ² ) 37.8 ± 31.1 36.2 ± 23.4 32.4 ± 20.0 0.925 Albumin (g / dL) 3.2 ± 0.7 3.3 ± 0.7 3.2 ± 0.6 0.991 Total cholesterol (mg / dL) 194 ± 53 193 ± 66 225 ± 70 0.588 Urine PCR (g / gCr) 3.86 ± 1.47 8.01 ± 7.01 10.51 ± 6.72 0.203

Arteriosclerosis 0
(n = 33) One
(n = 31) 2
(n = 6) p value Age (years) 56.5 ± 9.5 55.8 ± 12.1 69.8 ± 10.8 0.016 Sex (male,%) 24 (72.7) 14 (45.2) 4 (66.7) 0.306 BMI (kg / m ² ) 25.1 ± 2.9 25.5 ± 3.7 24.4 ± 4.5 0.778 Duration of diabetes (year) 10.8 ± 8.1 10.9 ± 8.0 16.8 ± 10.3 0.243 Hypertension (n,%) 29 (87.9) 27 87.1) 6 (100) 0.594 HbA1c (%) 7.6 ± 2.0 8.2 ± 2.0 6.6 ± 0.4 0.216 Hemoglobin (g / dL) 10.7 ± 2.3 10.1 ± 1.7 10.4 ± 2.0 0.533 eGFR (ml / min / 1.73m ² ) 35.5 ± 23.8 34.9 ± 22.3 45.4 ± 31.3 0.604 Albumin (g / dL) 3.3 ± 0.7 3.3 ± 0.6 3.3 ± 0.4 1.000 Total cholesterol (mg / dL) 205 ± 70 193 ± 61 160 ± 44 0.302 Urine PCR (g / gCr) 6.76 ± 4.63 8.64 ± 8.61 8.91 ± 6.57 0.502

(Abbreviations: BMI, body mass index; eGFR, estimated glomerular filtration rate; PCR, protein-to-creatinine ratio)

For glomerular lesions, there were no patients in class I, one patient in 14 patients (20.0%), class II, 32 patients (45.7%), class III, 24 patients (34.3%). Was class IV. eGFR levels did not differ between groups.

Two patients (2.9%), 41 (58.8%), 17 (24.3%) and 10 (14.3%) had an ITFA score of 0, 1, 2 and 3. Since there were too few patients with an IFTA score of 0, statistical analysis was performed after adding those with IFTA scores of 0 and 1. Patients with high IFTA scores showed significantly lower eGFR and lower hemoglobin levels (p <0.001 and 0.019, respectively).

Twelve patients (17.1%), 42 patients (60.0%) and 16 patients (22.9%) were included in the interstitial inflammation scores 0, 1 and 2. Although not statistically significant (p = 0.059), patients with high levels of epileptic inflammation tended to have low eGFR levels.

Seven patients (10.0%), 58 patients (82.9%), and 5 patients (7.1%) had vitreous disease scores of 0, 1, and 2, respectively, and atherosclerosis scores of 0, 1, and 2 The patients were 33 patients (47.1%), 31 patients (44.3%) and 6 patients (8.6%). There were no differences in clinical picture among patients with different grades of vitreous arteriosclerosis or arteriosclerosis.

In summary, among the various pathological features of diabetic nephropathy, in particular, IFTA was a pathological finding that was significantly associated with renal function. On the other hand, glomerular lesions, interstitial inflammation and other vascular lesions were found not to be directly related to clinical parameters.

In addition, it was evaluated whether the urine markers could reflect intrarenal pathological findings. Expression levels of urine inflammatory markers according to different pathological classifications are shown in Tables 7-11.

Glomerular lesions Class II (n = 14) Class III
(n = 32) Class IV (n = 24) p value MCP-1 / Cr 6.4 (2.9, 15.2) 12.7 (5.7, 25.0) 8.9 (4.0, 20.9) 0.180 RANTES / Cr 0.1 (0, 0.3) 0.2 (0.1, 0.3) 0.1 (0, 0.3) 0.310 IP-10 / Cr 0.4 (0.2, 1.1) 0.6 (0.3, 0.9) 0.3 (0.2, 0.5) 0.106 CXCL16 / Cr 3.6 (1.6, 9.3) 4.6 (2.5, 8.6) 7.7 (3.8, 16.6) 0.280 Endostatin / Cr 447.1 (114.9, 1172.0) 694.5 (356.8, 1957.2) 1119.1 (364.1, 3093.7) 0.452 IL-6 / Cr 0.1 (0, 0.2) 0.3 (0.1, 0.8) 0.3 (0, 0.5) 0.154 TNF-α / Cr 0 (0, 0) 0 (0, 0.1) 0 (0, 0.1) 0.100 VEGF / Cr 1.2 (1.0, 1.8) 1.7 (1.2, 2.3) 1.4 (1.0, 1.8) 0.075 CXCL9 / Cr 1.1 (0.8, 2.3) 1.8 (1.1, 3.2) 1.6 (0.9, 2.3) 0.202

Interstitial fibrosis and tubular atrophy 0 and 1
(n = 43) 2
(n = 17) 3
(n = 10) p value MCP-1 / Cr 6.5 (2.7, 14.5) 13.4 (7.8, 34.3) 20.7 (9.0, 35.5) 0.004 RANTES / Cr 0.2 (0.1, 0.3) 0.1 (0, 0.4) 0.2 (0.1, 0.3) 0.773 IP-10 / Cr 0.4 (0.2, 0.8) 0.4 (0.3, 0.8) 0.4 (0.4, 0.7) 0.517 CXCL16 / Cr 3.6 (1.6, 6.3) 8.4 (5.4, 19.8) 16.5 (6.1, 23.0) <0.001 Endostatin / Cr 443.7 (231.4, 947.6) 1905.4 (1014.1, 7489.8) 3967.9 (1403.2, 7827.5) <0.001 IL-6 / Cr 0.1 (0, 0.5) 0.4 (0.1, 0.6) 0.5 (0.1, 0.9) 0.178 TNF-α / Cr 0 (0, 0.1) 0 (0, 0.1) 0.2 (0, 0.3) 0.179 VEGF / Cr 1.4 (1.0, 2.0) 1.5 (1.1, 2.2) 1.5 (1.0, 3.8) 0.670 CXCL9 / Cr 1.3 (0.8, 2.1) 2.9 (1.1, 4.3) 2.1 (1.2, 2.8) 0.402

Interstitial inflammation 0
(n = 12) One
(n = 42) 2
(n = 16) p value MCP-1 / Cr 12.2 (3.6, 21.2) 8.3 (4.0, 18.2) 15.9 (5.7, 27.2) 0.331 RANTES / Cr 0.2 (0.1, 0.3) 0.1 (0, 0.3) 0.2 (0.1, 0.3) 0.949 IP-10 / Cr 0.5 (0.2, 0.9) 0.4 (0.3, 0.7) 0.4 (0.3, 1.1) 0.761 CXCL16 / Cr 6.2 (0.8, 8.4) 4.7 (2.1, 10.1) 7.3 (3.4, 17.0) 0.391 Endostatin / Cr 928.2 (83.0, 1215.8) 548.4 (342.1, 1666.2) 1428.2 (460.0, 8156.2) 0.076 IL-6 / Cr 0.3 (0.1, 0.5) 0.2 (0.1, 0.6) 0.4 (0.1, 0.8) 0.690 TNF-α / Cr 0 (0, 0.1) 0 (0, 0.1) 0 (0, 0.2) 0.702 VEGF / Cr 1.3 (0.9, 2.0) 1.5 (1.1, 2.1) 1.4 (0.8, 2.8) 0.576 CXCL9 / Cr 1.6 (0.5, 3.6) 1.3 (0.9, 2.8) 2.1 (1.3, 2.7) 0.393

Arteriolar hyalinosis of the arterioles 0
(n = 7) One
(n = 58) 2
(n = 5) p value MCP-1 / Cr 4.3 (2.6, 20.1) 9.5 (4.9, 18.4) 32.3 (2.9, 100.2) 0.574 RANTES / Cr 0.2 (0, 0.3) 0.1 (0, 0.3) 0.2 (0.1, 6.2) 0.258 IP-10 / Cr 0.4 (0.3, 0.9) 0.4 (0.3, 0.8) 0.5 (0.3, 1.1) 0.798 CXCL16 / Cr 3.6 (1.8, 17.3) 5.1 (2.2, 10.2) 4.6 (2.1, 31.2) 0.865 Endostatin / Cr 515.6 (264.7, 8320.6) 799.8 (337.8, 1739.0) 515.6 (263.9, 13195.7) 0.878 IL-6 / Cr 0.1 (0, 0.4) 0.2 (0.1, 0.6) 0.9 (0.1, 0.5) 0.453 TNF-α / Cr 0 (0, 0.1) 0 (0, 0.1) 0.2 (0, 0.3) 0.179 VEGF / Cr 1.4 (0.8, 3.3) 1.5 (1.1, 2.1) 1.4 (0.9, 4.0) 0.892 CXCL9 / Cr 2.6 (1.4, 2.9) 1.3 (0.9, 2.5) 2.5 (1.1, 11.9) 0.182

Arteriosclerosis 0
(n = 33) One
(n = 31) 2
(n = 6) p value MCP-1 / Cr 9.5 (3.5, 19.4) 9.2 (4.2, 19.8) 16.0 (5.2, 72.8) 0.574 RANTES / Cr 0.1 (0, 0.3) 0.2 (0, 0.3) 0.3 (0.1, 3.7) 0.210 IP-10 / Cr 0.4 (0.3, 0.7) 0.4 (0.2, 0.7) 0.8 (0.6, 1.4) 0.064 CXCL16 / Cr 4.4 (2.0, 12.5) 9.6 (2.5, 10.7) 4.7 (2.1, 15.2) 0.857 Endostatin / Cr 799.8 (275.0, 1642.7) 721.9 (356.7, 2733.6) 446.1 (341.0, 5210.4) 0.784 IL-6 / Cr 0.2 (0, 0.7) 0.2 (0.1, 0.5) 1.0 (0.1, 1.3) 0.299 TNF-α / Cr 0 (0, 0.1) 0 (0, 0.1) 0 (0, 0.1) 0.536 VEGF / Cr 1.5 (1.0, 2.0) 1.4 (1.2, 2.2) 1.8 (1.0, 3.8) 0.681 CXCL9 / Cr 1.3 (0.9, 2.2) 1.8 (1.1, 3.0) 3.7 (0.9, 8.2) 0.071

Data are expressed as median (25 ^th , 75 ^th percentages)

(Abbreviations: MCP-1, monocyte chemoattractant protein-1; RANTES, Regulated on activation, normal T cell expressed and secreted; IP-10, interferon gamma-induced protein-10; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; VEGF, vascular endothelial growth factor)

There was no significant difference in the expression level of urine inflammatory markers when patients were classified according to the grade of glomerular lesion. In addition, the severity of interstitial inflammation, arteriosclerosis, and atherosclerosis was not significantly associated with marker expression.

On the other hand, it was confirmed that the expression levels of the three urine inflammatory markers of MCP-1, CXCL16 and endostatin were significantly elevated in high grade IFTA patients (p = 0.004, <0.001 and <0.001, respectively).

Correlation analysis revealed that most urinary inflammatory markers except RANTES were significantly correlated with urine PCR (Table 12), especially MCP-1, CXCL16, endostatin and IL-6 were the most significant urinary inflammatory markers. High (R = 0.687, 0.490, 0.540, 0.527, respectively). The four markers also showed a significant negative correlation with the annual decline in eGFR (R = -0.496, -0.520, -0.602, -0.476, respectively). Urine PCR itself was associated with a decrease in eGFR, but the correlation coefficient was found to be lower than the urine marker (R = -0.315, Figure 3).

Relationship between Urine Inflammation Markers, Urinary Protein / Creatinine Ratio, and Annual Decline in Renal Function Urine PCR (g / gCr) Decline in eGFR (% / yr) R p R p MCP-1 0.687 <0.001 -0.496 0.001 RANTES 0.228 0.057 -0.255 0.108 IP-10 0.372 0.002 -0.207 0.194 CXCL16 0.490 <0.001 -0.520 <0.001 Endostatin 0.540 <0.001 -0.602 <0.001 IL-6 0.527 <0.001 -0.476 0.002 TNF-α 0.320 0.007 -0.281 0.075 VEGF 0.324 0.006 -0.299 0.057 CXCL9 0.433 <0.001 -0.351 0.024

(Abbreviations: PCR, protein-to-creatinine ratio; eGFR, estimated glomerular filtration rate; MCP-1, monocyte chemoattractant protein-1; IP-10, interferon gamma-induced protein-10; IL-6, interleukin-6; TNF -tumor necrosis factor-α; VEGF, vascular endothelial growth factor)

Confirmation of renal function results in diabetic nephropathy patients confirmed by biopsy according to 2-3 pathological classification and urinary inflammatory markers

Renal results of patients according to pathological classification and urine inflammatory markers are shown in FIGS. 4 and 5. To assess the effects of these markers on renal results, patients were divided into thirds according to urine PCR and inflammatory markers.

The rate of reaching end stage kidney disease (ESRD) was found to be significantly higher in patients with high ITFA and / or interstitial inflammation scores (p = 0.009 and 0.016, respectively, B and C in FIG. 4). On the other hand, glomerular lesions, arteriosclerosis and atherosclerosis did not affect kidney survival (A, D and E in FIG. 4).

Interestingly, urine PCR was found not to be associated with ESRD development (p = 0.241, FIG. 5A). On the other hand, patients with increased expression levels of CXCL16, endostatin, and the combination of the two markers showed higher rates of reaching ESRD (p = 0.015 and p = 0.002 and <0.001; respectively B, C and D in FIG. 5). ).

Finally, the predictive power of progression to ESRD was evaluated through the ROC curve (FIG. 6). Similar to the results described above, urine PCR was found not to be associated with the development of ESRD (AUC value 0.534, A in FIG. 6). On the other hand, urine CXCL16 and endostatin exhibited AUC values of 0.693 and 0.756, respectively, confirming that they could be used to predict the development and progression of ESRD (Figs. 6B and C). (discriminative power) was confirmed to be higher (AUC value 0.824, Figure 6 D).

Thus, in summating these findings, CXCL16 and endostatin expressed in urine are useful as biomarkers for predicting the degree of renal failure and renal fibrosis in patients with advanced diabetic nephropathy with poor predictive prognosis of urine proteinuria and biopsy. It can be seen that it can be used.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (10)

Developing diabetic nephropathy, including one or more gene mRNAs selected from the group consisting of urine-derived CXCL16 (CXC motif ligand 16), endostatin, and combinations thereof, or agents that detect protein levels A diagnostic composition for predicting renal fibrosis or degenerative kidney function in a subject.
delete The composition of claim 1, wherein the agent for detecting mRNA levels comprises primer pairs or probes that specifically bind to the gene.
The composition of claim 1, wherein the agent for detecting protein levels is an antibody, a fragment of the antibody, or an aptamer specifically binding to CXCL16 or endostatin.
Kit for diagnosing renal fibrosis or renal failure of diabetic nephropathy patient, comprising the composition of claim 1.
delete delete (a) obtaining a urine sample of an individual with diabetic nephropathy;
(b) Expression level of a marker selected from the group consisting of CXCL16, endostatin and combinations thereof from the urine sample obtained using the composition of any one of claims 1 and 3-4 or the kit of claim 5 Measuring; And
(c) determining the onset or course of renal fibrosis or renal insufficiency of said individual, wherein the method of providing information for prognostic prediction of renal fibrosis or renal insufficiency.
delete The method of claim 8,
If the expression level of the marker selected from the group consisting of CXCL16, endostatin and combinations thereof measured in step (b) is higher than the control sample, it is determined that the progress of the subject is bad.

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