KR20110137278A - Diagnostic biomarker for diabetic nephropathy and identification method thereof - Google Patents

Abstract

PURPOSE: A marker for diabetic nephropathy, isolated from plasma glycoprotein is provided to evaluating prognosis of the disease. CONSTITUTION: A composition for diagnosing diabetic nephropathy contains an antibody which specifically binds to plasma retinol-binding protein(RBP4) and precursor thereof. A composition for diagnosing diabetic nephropathy contains a primer or probe which is specific to RBP4 and the precursor thereof. A kit for diagnosing diabetic nephropathy contains the composition. A microarray for diabetic nephropathy contains the composition.

Description

Biomarker for Diagnosing Diabetic Nephropathy and Its Identification Method {Diagnostic biomarker for Diabetic nephropathy and Identification method}

The present invention relates to diagnostic markers. The present invention also relates to a composition for diagnosing diabetic nephropathy comprising a molecule specific for a protein that is overexpressed or extremely reduced in diabetic nephropathy.

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease (ESRD) and is characterized by Kimmelstiel-Wilson lesions and capillary glomerulonephritis (Ritz, E , Saudi J Kidney Dis Transpl 2006, 17, 481-490. About 20-30% of people with type 1 or type 2 diabetes develop signs of kidney disease. Most ESRD is found in patients with type 2 diabetes because type 2 diabetes is much more frequent than type 1 diabetes. Only some of diabetic nephropathy progress to end stage nephropathy (Molitch, M. E., DeFronzo, R. A., Franz, M. J., Keane, W. F., et al., Diabetes Care 2003, 26 Suppl 1, S 94-98). The development of diabetic nephropathy is thought to be due to the interaction between abnormal metabolic and hemodynamic factors, which increases the development of diabetic nephropathy by activating a common pathway leading to the development of diabetic nephropathy (Parving, HH , Kidney Int 2001, 60, 2041-2055). Diabetic nephropathy is also associated with an increased risk of cardiovascular disease (Caramori, M. L., Mauer, M., Curr Opin Nephrol Hypertens 2003, 12, 273-282).

As diabetic nephropathy develops, the glomeruli continue to injure, developing microalbuminuria, and megaalbuminuria over several years to decades (Otu, HH, Can, H., Spentzos, D., Nelson, RG, et al., Diabetes Care 2007, 30, 638-643). Traditionally, early diabetic nephropathy is defined as microalbuminuria (30-300mg / 24h in urine albumin secretion), and then progresses to megaalbuminuria (> 300mg / 24h), eventually ending with kidney disease (Remuzzi, G., Schieppati, A., Ruggenenti, P., N Engl J Med 2002, 346, 1145-1151). However, microalbuminuria is limited to be used as a marker of diabetic nephropathy because the correlation between microalbuminuria and glomerular damage is very variable. Microalbuminuria is also often more useful as a marker of cardiovascular disease than diabetic nephropathy (Fioretto, P., Mauer, M., Brocco, E., Velussi, M., et al., Diabetologia 1996, 39, 1569-1576 ).

There is currently no selective treatment for diabetic nephropathy. However, in the early stages of the appearance of microalbuminuria in the progression of diabetic nephropathy, it has been reported that strict glycemic control and low-protein diet can reduce or delay the progression of microalbuminuria to the next stage. Therefore, it is necessary to find biomarkers that allow early diagnosis to prevent diabetic nephropathy from progressing to end stage nephropathy.

Recently, proteomic techniques have been used to find biomarkers in samples such as serum and urine of diabetic nephropathy. Plasma samples are limited in finding biomarkers because most of the specific proteins in the plasma are present at very low concentrations.

Glycoprotein enrichment techniques can be used to remove unwanted plasma proteins as well as provide means to enrich the functional subsets of proteins. Glycoproteins also play an important role in cancer progression and immune response, and have the potential as new biomarkers for cancer prognosis, diagnosis, and monitoring. In the past we have successfully found biomarkers for lung cancer using multi-lectin affinity chromatography and liquid chromatography coupled double mass spectrometry (LC-MS / MS) techniques.

High blood sugar levels in diabetes are known to cause abnormal glycation of proteins in the blood, which occur randomly in a non-enzymatic manner. High blood sugar and abnormal glycation damage tissues including the kidneys and directly lead to diabetic nephropathy. In addition, an imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) causes kidney damage. Therefore, it was assumed that glycoproteins would leak into the blood when tissue damage began, or that tissue damage signals would alter the glycoproteome profile in the blood. Finding other plasma glycoproteins in diabetics and diabetic nephropathy may provide a tool for detecting disease early in diabetic nephropathy.

In this regard, the present inventors used a multi-lectin affinity column to examine and study plasma glycoproteome from glycoprotein enriched samples of diabetic nephropathy patients, which can be used as a new biomarker for diagnosing the disease early in diabetic nephropathy. The present invention was completed by identifying proteins.

An object of the present invention is to provide a biomarker for diagnosing diabetic nephropathy identified from plasma glycoproteins.

It is also an object of the present invention to provide a diabetic nephropathy diagnostic composition comprising an antibody that specifically binds to the biomarker.

In order to achieve the above object of the present invention, the present invention provides a composition for diagnosing diabetic nephropathy comprising a molecule specific for glycoprotein that is overexpressed or greatly reduced in serum of diabetic nephropathy patient.

The present invention also provides a method for identifying a marker for diagnosing diabetic nephropathy.

Hereinafter, the present invention will be described in detail.

As used herein, the term "diagnostic" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis is to confirm the transition to nephropathy of diabetic patients.

As used herein, the term "diabetic nephropathy" refers to kidney disease caused by diabetes, and includes all of glomerulosclerosis, vascular lesions and tubulointerstitial disease, but especially diabetes mellitus. Means glomerulosclerosis.

As used herein, the term "diagnostic marker" refers to an organic biomolecule capable of damaging the kidney and progressing to nephropathy with a particularly increasing substance in diabetics. The organic biomolecules include, but are not limited to, polypeptides, proteins, nucleic acids, lipids, glycolipids, glycoproteins, and sugars. In the present invention, preferably, the organic biomolecule refers to a glycoprotein.

To identify new diabetic nephropathy markers, the present inventors identified glycoproteins that are expressed differently between the plasma of diabetic-only patients without nephropathy and those with diabetic nephropathy. To this end, glycoproteins were separated from plasma using a multi-lectin affinity column, glycoproteins were deglycosylated by enzymatic treatment, followed by trypsin treatment to obtain peptides. Peptides secured above were separated by liquid chromatography and analyzed by ion trap mass spectrometry (LC-MS / MS). The LC-MS / MS data was retrieved from the IPI human protein database using a computer program to identify glycoproteins present in the plasma of diabetic nephropathy patients and to compare them with glycoproteins derived from plasma of diabetic patients without kidney complications. By specifically increasing or decreasing in the blood of diabetic nephropathy patients, glycoproteins that can be used as biomarkers for diabetic nephropathy progression were identified.

In an embodiment of the present invention, six blood samples were obtained from diabetic patients without nephropathy and diabetic patients with nephropathy, and centrifuged to obtain plasma samples. From the plasma samples obtained above, the glycoproteins were separated using a multi-lectin affinity column and concentrated by acetone precipitation.

In one embodiment of the present invention, coomassie straining and gelcode (GelCode) sugars were applied to the eluate (concentrated glycoprotein) and the flow-through fraction obtained by the multi-column affinity column. Protein staining was performed to confirm the efficiency of separation of the glycoprotein by the multi-lectin affinity column. As a result, it was confirmed that the glycoprotein in plasma was concentrated by the multi-lectin affinity column.

The present inventors enzymatically treated the concentrated serum glycoprotein obtained above and obtained a peptide mixture by in-gel digestion. The peptide mixture obtained above was analyzed by LC-MS / MS to identify 43 proteins specifically increased or expressed in diabetic nephropathy patients and 40 proteins specifically in diabetic nephropathy patients compared to diabetic patients.

Some of the 43 up-regulated proteins are reported to be able to be used as markers of diabetic nephropathy in the serum or plasma of diabetic nephropathy patients. Examples include albumin, von Willebrand factor precursor (VWF), plasma retinol-binding protein precursor (RBP-4), apolipoprotein C III precursor (APOC3, Apolipoprotein C-) III precursor), vitamin D-binding protein precursor (GC), luminan precursor, and the like. Proteins other than these are not yet known as markers or biomarkers for diabetic nephropathy. therefore Other glycoproteins identified by the inventors can be used as diagnostic markers for new diabetic nephropathy.

Therefore, the present invention provides a myosin-reactive immunoglobulin heavy chain variable region fragment (Table 4), extracellular matrix protein 1 precursor (EMP1), iso of desmoplakin. Isoform DPI of Desmoplakin (DSP), 49 kDa protein (IPI 00180956), Ig heavy chain V III region VH26 precursor (IGHV2, Ig heavy chain V-III region VH26 precursor), serum amyloid A-4 protein precursor (SAA- 4, Serum amyloid A-4 protein precursor), tetranectin precursor (CLEC3B, Tetranectin precursor), Ig kappa chain V IV region STH analog (IGKV4-1, Similar to Ig kappa chain V-IV region STH), C-reactive protein Isoform 1 of C-reactive protein precursor (CRP), binding plakoglobin (JUP), isoform 1 of E3 ubiquitin-protein ligase UBR1 (UBR1, Isoform 1 of E3 ubiquitin-protein ligase UBR1), complement factor H-related Complement factor H-related protein 4 precursor (CFHR4), small proline-rich protein 2G (SPRR2G), IGKC protein, isoform 2 of carboxypeptidase B2 precursor (CPB2, Isoform 2 of Carboxypeptidase B2 precursor), coagulation factor V (F5), SCC-112 protein, collagen alpha-1 (VI) chain precursor, Ipopsorcin ( RP1-14N1.3, Ifapsoriasin), Isoform C of Fibulin-1 precursor, Ficollin-1 precursor, Procollagen C endopeptidase enhancer 1 precursor (PCOLCE, Procollagen C-endopeptidase enhancer 1 precursor) ), Isoform 1 H3 lysine-9 specific 5 (EHMT1, Isoform 1 of Histone-lysine N-methyltransferase, H3 lysine-9 specific 5), IgG Fc-binding protein precursor (FCGBP, IgG Fc-binding protein precursor), Isoform 2 (ABCB9, Isofor) of ATP-binding cassette subfamily B member 9 precursor m 2 of ATP-binding cassette sub-family B member 9 precursor), 36 kDa protein (IPI 00790690), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Glyceraldehyde-3-phosphate dehydrogenase), protein S100-A8 , Isoform 1 of DNA-dependent protein kinase catalytic subunit (PRKDC), Desmoglein-1 precursor (DSG1), Duxidine precursor (DCD, Dermcidin) precursor), Vasorin precursor (VASN, Vasorin precursor), Isoform 1 (QSOX1, Isoform 1 of Sulfhydryl oxidase 1 precursor), IGLV3-25 protein, SNC66 protein (IGHA1), Complement factor H Diabetic nephropathy as a protein selected from the group consisting of Complement factor H-related protein 3 precursor (CFHR3) and beta-Ala-His dipeptidase precursor (CNDP1, Beta-Ala-His dipeptidase precursor) Characterized by increased expression in Relates to diabetic nephropathy diagnostic biomarker proteins.

In addition, 40 down-regulated proteins of the present invention have not yet been reported to specifically decrease in diabetic nephropathy. Therefore, these proteins can be used as diagnostic markers of new diabetic nephropathy.

Therefore, the present invention is shown in Table 5 Mannose-binding protein C precursor (MBL2, Mannose-binding protein C precursor), Actin cytoplasmic 1 (ACTB, Actin cytoplasmic 1), Hornerine (HRNR, Hornerin), Biotinida Biotinidase precursor (BTD), Dopamine beta-hydroxylase precursor (DBH), Potassium voltage-gated channel subfamily H member 4 (KCNH4), Met binding Isoform 1 of lectin serine protease 2 precursor (MASP2, Isoform 1 of Mannan-binding lectin serine protease 2 precursor), gamma-glutamyl hydrolase precursor (GGH), isoform 1 of melanotransferrin precursor (MFI2, Isoform 1 of Melanotransferrin precursor), fetuin-B precursor (FETUB, Fetuin-B precursor), properdin precursor (CFP, Properdin precusor), Ig kappa chain VI region AG, Immunoglobulin heavy chain variable Reverse (IPI00477804), growth-inhibiting protein 12 (LTF), WW domain binding protein 11 (WBP11, WW domain-binding protein 11), amyloid lambda 6 light chain variable region SAR fragment (Amyloid lambda 6 light chain variable) region SAR Fragment), Isoform 1 of Protein HEG homolog 1 precursor (HEG1), Ig heavy chain V III region TIL, RRP5 protein homolog (PDCD11, RRP5 protein homolog), platelet glycoprotein Ib eggs Platelet glycoprotein Ib alpha chain precursor (GP1BA), Isoform 1 of Filamin-C (FLNC), Vitamin K-dependent protein C precursor (PROC) , Ig heavy chain V III region BRO, Zinc finger protein 291 (ZNF291, Zinc finger protein 291), Centrosome-associated protein 350 (CEP350, Centrosome-associated protein 350), H53_GS1 fragment, Selenoprotein P precursor (SEPP1, Selenoprotein P precursor), dispatched A (DISP1, Dis patched A), Isoform 1 of Gelsolin precursor (GSN), Insulin-like growth factor-binding protein 2 precursor (IGFBP2), Ig kappa chain V III Region HAH precursor (IGKV3-20, Ig kappa chain V-III region HAH precursor), isoform LMW (KNG1, Isoform LMW of Kininogen-1 precursor) of kininogen-1 precursor, myosin reactive immunoglobulin heavy chain variable region fragment ( IPI 00384407), 38 kDa protein (IPI00008669), Ig lambda chain V-III region SH, apolipoprotein (LPA, Apolipoprotein), complement component C8 alpha chain precursor (C8A), complement component C8 beta A protein selected from the group consisting of chain component (C8B, Complement component C8 beta chain precursor), heparin cofactor 2 precursor (SERPIND1) and complement C5 precursor (C5), characterized by reduced expression in diabetic nephropathy, Diabetic nephropathy It relates to a biomarker for protein.

In addition, the present invention can be utilized in the diagnosis of diabetic nephropathy, the 40 proteins down-regulated in diabetic nephropathy, specifically myosin-reactive immunoglobulin heavy chain variable region fragment, Extracellular matrix protein 1 precursor (EMP1), Isoform DPI of Desmoplakin (DSP), 49 kDa protein (IPI 00180956), Ig heavy chain V-III region VH26 precursor ( IGHV2, Ig heavy chain V-III region VH26 precursor), serum amyloid A-4 protein precursor (SAA-4, Serum amyloid A-4 protein precursor), tetranectin precursor (CLEC3B, Tetranectin precursor), Ig kappa chain V-IV Region STH analog (IGKV4-1, Similar to Ig kappa chain V-IV region STH), Isoform 1 of C-reactive protein precursor (CRP), binding flacoglobin (JUP, Junction) plakoglobin), E3 ubiquit Isoform 1 of protein ligase UBR1 (UBR1, Isoform 1 of E3 ubiquitin-protein ligase UBR1), complement factor H-related protein 4 precursor (CFHR4), bovine proline-rich protein 2G (SPRR2G, Small proline-rich protein 2G), IGKC protein, isoform 2 (CPB2, Carboxypeptidase B2 precursor) of carboxypeptidase B2 precursor, coagulation factor V (F5), SCC-112 protein , Collagen alpha-1 (VI) chain precursor (COL6A1, Collagen alpha-1 (VI) chain precursor), ipopsoriacin (RP1-14N1.3, Ifapsoriasin), isoform C of fibulin-1 precursor (FBLN1, Isoform C of Fibulin-1 precursor), Procollagen C-endopeptidase enhancer 1 precursor (PCOLCE), isoform 1 H3 lysine-9 specific 5 of histone-lysine N-methyltransferase ( EHMT1, Isoform 1 of Histone-lysine N-methyltransferase, H3 lysine-9 specific 5), IgG Fc-binding protein precursors (FCGBP, IgG Fc-binding protein precursor), Isoform 2 of ATP-binding cassette sub-family B member 9 precursor), 36 kDa protein (IPI 00790690), Glyceral Isoform 1 of DNA-dependent protein kinase catalytic subunit of dehydro-3-phosphate dehydrogenase (GAPDH), protein S100-A8, DNA-dependent protein kinase catalytic subunit ), Desmoglein-1 precursor (DSG1), Dermcidin precursor (DCD, Dermcidin precursor), Vasorin precursor (VASN, Vasorin precursor), Isoform 1 of sulfhydryl oxidase 1 precursor (QSOX1) , Isoform 1 of Sulfhydryl oxidase 1 precursor), IGLV3-25 protein, SNC66 protein (IGHA1), Complement factor H-related protein 3 precursor (CFHR3) and beta-Ala-His dipeptidase Made of precursor (CNDP1, Beta-Ala-His dipeptidase precursor) It provides a diabetic nephropathy diagnostic composition comprising a molecule that specifically binds to a protein selected from the group of Gin.

In addition, 40 down-regulated proteins of the present invention have not yet been reported to specifically decrease in diabetic nephropathy. Therefore, these proteins can be used as diagnostic markers of new diabetic nephropathy. Therefore, the present invention is shown in Table 5 Mannose-binding protein C precursor (MBL2, Mannose-binding protein C precursor), Actin cytoplasmic 1 (ACTB, Actin cytoplasmic 1), Hornerine (HRNR, Hornerin), Biotinida Biotinidase precursor (BTD), Dopamine beta-hydroxylase precursor (DBH), Potassium voltage-gated channel subfamily H member 4 (KCNH4), Met binding Isoform 1 (MASP2), Mano-binding lectin serine protease 2 precursor (MASP2), Gamma-glutamyl hydrolase precursor (GGH), Isoform 1 of melanotransferrin precursor (MFI2, Isoform 1 of Melanotransferrin precursor), fetuin-B precursor (FETUB, Fetuin-B precursor), properdin precursor (CFP, Properdin precusor), Ig kappa chain VI region AG, Immunoglobulin heavy chain Region (IPI 00477804), growth-inhibiting protein 12 (LTF), WW domain binding protein 11 (WBP11, WW domain-binding protein 11), amyloid lambda 6 light chain variable region SAR fragment (Amyloid lambda 6 light chain) variable region SAR Fragment), Isoform 1 of Protein HEG homolog 1 precursor (HEG1), Ig heavy chain V-III region TIL, RRP5 protein homolog (PDCD11, RRP5 protein homolog), platelet glycoprotein Platelet glycoprotein Ib alpha chain precursor (GP1BA), Isoform 1 of Filamin-C (FLNC), Vitamin K-dependent protein C (PROC) precursor), Ig heavy chain V-III region BRO, Zinc finger protein 291 (ZNF291, Zinc finger protein 291), centrosome-binding protein 350 (CEP350, Centrosome-associated protein 350), H53_GS1 fragment, selenoprotein P precursor (SEPP1 , Selenoprotein P precursor), dispatched A (DIS P1, Dispatched A), Isoform 1 of Gelsolin precursor (GSN), Insulin-like growth factor-binding protein 2 precursor (IGFBP2), Ig kappa chain V -III region HAH precursor (IGKV3-20, Ig kappa chain V-III region HAH precursor), isoform LMW (KNG1, Isoform LMW of Kininogen-1 precursor) of kininogen-1 precursor, myosin reactive immunoglobulin heavy chain variable region Fragment (IPI 00384407), 38 kDa protein (IPI 00008669), Ig lambda chain V-III region SH, apolipoprotein (LPA, Apolipoprotein), complement component C8 alpha chain precursor (C8A), complement Diabetic comprising a molecule that specifically binds to a protein selected from the group consisting of component C8 beta chain precursor (C8B), heparin cofactor 2 precursor (SERPIND1) and complement C5 precursor (C5) Preparation of nephrotic diagnostic composition The.

On the other hand, the presence of a specific protein in the biological sample can be confirmed by measuring the formation of the antigen-antibody complex by contacting the sample with an antibody that specifically binds to the protein. As used herein, the term "antigen-antibody complex" refers to a combination of a specific protein in a biological sample with an antibody that specifically recognizes it.

As used herein, the term "biological sample" or "sample" refers to blood and other liquid samples of biological origin. Preferably, the biological sample or sample may be whole blood, plasma, serum or tissue fluid. The sample can be obtained from an animal, preferably a mammal, most preferably a human. The sample may be pretreated before use for detection. For example, it can include filtration, distillation, extraction, concentration, inactivation of disturbing components, addition of reagents, and the like. In addition, nucleic acids and proteins can be separated from the sample and used for detection.

By "antibody" is meant herein a specific protein molecule directed against the antigenic site. Antibodies used in the present invention include monoclonal or polyclonal antibodies, immunologically active fragments (eg, Fab or (Fab) ₂ fragments), antibody heavy chains, humanized antibodies, antibody light chains, genetically engineered single chain F molecules, Chimeric antibodies and the like.

Since the proteins shown in Tables 4 and 5 are known proteins, the antibodies used in the present invention can be prepared by conventional methods well known in the field of immunology using the known proteins as antigens. Proteins used as antigens of antibodies according to the invention can be extracted or synthesized in nature and can be prepared by recombinant methods based on DNA sequences. When using a recombinant technique, a nucleic acid encoding a protein is inserted into an appropriate expression vector, the host cell is cultivated so that the target protein is expressed in the transformant transformed with the recombinant expression vector, and then It can be obtained by recovering the protein.

For example, polyclonal antibodies can be produced by the method of injecting a protein antigen into an animal and collecting blood from the animal to obtain a serum comprising the antibody. Such antibodies can be prepared using various warm-blooded animals such as horses, cattle, goats, sheep, dogs, chickens, turkeys, rabbits, mice or rats.

Monoclonal antibodies are also known as fusion methods (Kohler and Milstein, European J. Immnunol . 6: 511-519 1976), recombinant DNA methods (US Pat. No. 4816567), and phage antibody libraries (Clackson et al., Nature , 352, 624-628, 1991; Marks et al., J. Mol . Biol . 222, 58: 1-597, 1991).

The diagnostic composition of the present invention may further comprise reagents known in the art for use in immunological assays in addition to antibodies specific for the protein. Immunological analysis in the above may include any method that can measure the binding of the antigen and the antibody. Such methods are known in the art and include, for example, immunocytochemistry and immunohistochemistry, radioimmunoassays, enzyme linked immunabsorbent assay (ELISA), immunoblotting, and farar assays. Farr assay), immunoprecipitation, latex aggregation, erythrocyte aggregation, non-turbidity, immunodiffusion, counter-current electrophoresis, single radical immunodiffusion, immunochromatography, protein chips and immunofluorescence.

Reagents used in immunological analysis include labels, solubilizers, and detergents capable of producing a detectable signal. In addition, when the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminator.

The label capable of generating a detectable signal enables qualitatively or quantitatively measuring the formation of an antigen-antibody complex, examples of which include enzymes, fluorescent materials, ligands, luminescent materials, microparticles, and redox molecules. And radioisotopes can be used. Enzymes include β-glucuronidase, β-D-glucosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glycosidase, hexokinase, malate dehydrogenase, and glucose-6 Phosphate dehydrogenase, invertase and the like can be used. As the fluorescent substance, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, fluorine isothiocyanate and the like can be used. Ligands include biotin derivatives, and luminescent materials include acridinium esters, luciferin, and luciferase. The microparticles include colloidal gold and colored latex, and the redox molecules include ferrocene, ruthenium complex, biologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone and hydroquinone. Radioisotopes include ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, and the like. However, any of those that can be used in immunological assays other than those exemplified above may be used.

In addition, as a method for confirming the presence of the marker protein of the present invention, a peptide that specifically binds to the marker protein of the present invention may be used.

Therefore, the present invention provides a composition for diagnosing diabetic nephropathy comprising a peptide specifically binding to the proteins shown in Tables 4 and 5. The present invention also provides a diabetic nephropathy diagnosis method using the diabetic nephropathy composition.

In addition, the diagnostic composition of the present invention may be provided in an immobilized state using various methods known on a suitable carrier or support to increase the speed and convenience of diagnosis ( Antibodies : A Labotory Manual, Harlow & Lane). Cold Spring Harbor, 1988). Examples of suitable carriers or supports include agarose, cellulose, nitrocellulose, dextran, Sephadex, Sepharose, liposomes, carboxymethyl cellulose, polyacrylamides, polyesters, companion rocks, filter papers, ion exchange resins, plastic films, plastic tubes , Glass, polyamine-methyl vinyl-ether-maleic acid copolymers, amino acid copolymers, ethylene-maleic acid copolymers, nylons, cups, flat packs and the like. Other solid substrates include cell culture plates, ELISA plates, tubes and polymeric membranes. The support may have any possible form, for example spherical (bead), cylindrical (test tube or inner surface of the well), planar (sheet, test strip).

Preferably, the diabetic nephropathy diagnostic composition of the present invention may be provided in a diagnostic kit or microarray form.

The diagnostic kit may be provided, for example, in the form of a lateral flow assay kit based on immunochromatography for detecting a specific protein in a sample. Lateral flow assay kits include a sample pad to which a sample is applied, a release pad coated with a detection antibody, a developing membrane (e.g., nitro) in which the sample is moved and separated and an antigen-antibody reaction occurs. Cellulose) or strip, and an absorption pad.

The microarray is generally intended to attach an antibody onto a slide glass surface treated with a specific reagent to detect a protein that specifically attaches to the antibody by an antigen-antibody reaction.

The present invention is shown in Table 4 myosin-reactive immunoglobulin heavy chain variable region fragments, extracellular matrix protein 1 precursor (EMP1), desmoplakin isoform DPI (DSP, Isoform DPI of Desmoplakin), 49 kDa protein (IPI 00180956), Ig heavy chain V-III region VH26 precursor (IGHV2, Ig heavy chain V-III region VH26 precursor), serum amyloid A-4 protein precursor (SAA- 4, Serum amyloid A-4 protein precursor), tetranectin precursor (CLEC3B, Tetranectin precursor), Ig kappa chain V-IV region STH analog (IGKV4-1, Similar to Ig kappa chain V-IV region STH), C-reactive Isoform 1 of C-reactive protein precursor (CRP), binding plakoglobin (JUP), isoform 1 of E3 ubiquitin-protein ligase UBR1 (UBR1, Isoform 1 of E3 ubiquitin- protein ligase UBR1), the complement factor H-related protein Complement factor H-related protein 4 precursor (CFHR4), small proline-rich protein 2G (SPRR2G), IGKC protein, isoform 2 of carboxypeptidase B2 precursor (CPB2, Isoform 2 of Carboxypeptidase B2 precursor), coagulation factor V (F5), SCC-112 protein, collagen alpha-1 (VI) chain precursor, Ipopsorcin (RP1 -14N1.3, Ifapsoriasin), Isoform C of Fibulin-1 precursor (FBLN1), Procollagen C endopeptidase enhancer 1 precursor (PCOLCE, Procollagen C-endopeptidase enhancer 1 precursor) Isoform 1 of Histone-lysine N-methyltransferase, H3 lysine-9 specific 5, IgG Fc-binding protein precursors (FCGBP, IgG) Fc-binding protein precursor), isoform 2 of ATP-binding cassette subfamily B member 9 precursor -binding cassette sub-family B member 9 precursor), 36 kDa protein (IPI 00790690), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Glyceraldehyde-3-phosphate dehydrogenase), protein S100-A8, DNA-dependent Isoform 1 of DNA-dependent protein kinase catalytic subunit (PRKDC), desmoglein-1 precursor (DSG1), dermcidin precursor (DCD), bar of protein kinase catalytic subunit Vasorin precursor (VASN), isoform 1 of sulfhydryl oxidase 1 precursor (QSOX1, Isoform 1 of Sulfhydryl oxidase 1 precursor), IGLV3-25 protein, SNC66 protein (IGHA1), complement factor H-related protein 3 Primers specific for nucleic acids encoding proteins selected from the group consisting of precursor (CFHR3, Complement factor H-related protein 3 precursor) and beta-Ala-His dipeptidase precursor (CNDP1, Beta-Ala-His dipeptidase precursor) With probe It provides a diagnostic composition for diabetic nephropathy.

In addition, the present invention is shown in Table 5 Mannose binding protein C precursor (MBL2, Mannose-binding protein C precursor), Actin cytoplasmic 1 (ACTB, Actin cytoplasmic 1), Hornerine (HRNR, Hornerin), biotinida Biotinidase precursor (BTD), Dopamine beta-hydroxylase precursor (DBH), Potassium voltage-gated channel subfamily H member 4 (KCNH4), Met binding Isoform 1 (MASP2), Mano-binding lectin serine protease 2 precursor (MASP2), Gamma-glutamyl hydrolase precursor (GGH), Isoform 1 of melanotransferrin precursor (MFI2, Isoform 1 of Melanotransferrin precursor), fetuin-B precursor (FETUB, Fetuin-B precursor), properdin precursor (CFP, Properdin precusor), Ig kappa chain VI region AG, Immunoglobulin heavy chain variable Reverse (IPI00477804), growth-inhibiting protein 12 (LTF), WW domain binding protein 11 (WBP11, WW domain-binding protein 11), amyloid lambda 6 light chain variable region SAR fragment (Amyloid lambda 6 light chain variable) region SAR Fragment), Isoform 1 of Protein HEG homolog 1 precursor (HEG1), Ig heavy chain V-III region TIL, RRP5 protein homolog (PDCD11, RRP5 protein homolog), platelet glycoprotein Ib Platelet glycoprotein Ib alpha chain precursor (GP1BA), Isoform 1 of Filamin-C (FLNC), Vitamin K-dependent protein C precursor (PROC) ), Ig heavy chain V III region BRO, Zinc finger protein 291 (ZNF291, Zinc finger protein 291), Centrosome-binding protein 350 (CEP350, Centrosome-associated protein 350), H53_GS1 fragment, Selenoprotein P precursor (SEPP1, Selenoprotein) P precursor), dispatched A (DISP1, Dispatched A), Isoform 1 of Gelsolin precursor (GSN), Insulin-like growth factor-binding protein 2 precursor (IGFBP2), Ig kappa chain V- III region HAH precursor (IGKV3-20, Ig kappa chain V-III region HAH precursor), isoform LMW (KNG1, Isoform LMW of Kininogen-1 precursor) of kininogen-1 precursor, myosin reactive immunoglobulin heavy chain variable region fragment (IPI 00384407), 38 kDa protein (IPI00008669), Ig lambda chain V-III region SH, apolipoprotein (LPA, Apolipoprotein), complement component C8 alpha chain precursor (C8A, complement component C8 alpha chain precursor) Diabetes comprising a primer or probe specific for a nucleic acid encoding a protein selected from the group consisting of beta chain precursor (C8B, Complement component C8 beta chain precursor), heparin cofactor 2 precursor (SERPIND1) and complement C5 precursor (C5) Castle god It provides a diagnostic composition.

The detection of a specific nucleic acid using a primer may be performed by amplifying the sequence of the gene of interest using an amplification method such as PCR, and then checking whether the gene is amplified by a method known in the art. In addition, the detection of a particular nucleic acid using a probe may be performed by contacting a sample nucleic acid with a probe under suitable conditions and confirming the presence of the hybridizing nucleic acid.

The "primer" refers to a nucleic acid sequence having a short free hydroxyl group, which forms a complementary template and base pair, and serves as a starting point for template strand copying. Primers of the invention can be chemically synthesized using methods known in the art, such as, for example, phosphoramidite solid support methods.

The "probe" refers to a nucleic acid fragment such as RNA or DNA consisting of several to several hundred bases that can specifically bind to mRNA and is labeled to confirm the presence or absence of a specific mRNA. Probes can be prepared in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, RNA probes, and the like, and can be labeled with biotin, FITC, rhodamine, DIG, or the like, or radioisotopes.

The probe may also be labeled with a detectable substance, for example, a radiolabel that provides a suitable signal and has sufficient half-life. Labeled probes are described in Sam et al., Molecular Cloning , A Laboratory Mannual, 1989) can be hybridized to a nucleic acid on a solid support.

As a method for detecting a specific nucleic acid using the probe or primer, for example, but not limited to, polymerase chain reaction (PCR), DNA sequencing, RT-PCR, primer extension method (Nikiforeov et al., Nucl Acids Res 22, 4167-4175, 1994), oligonucleotide extension assays (Nickerson et al., Pro Nat Acad Sci USA, 87, 8923-8927, 1990), allele specific PCR (Rust et al., Nucl Acids Res , 6, 3623-3629, 1993), RNase mismatch cleavage (Myers et al., Science , 230, 1242-1246, 1985), single strand conformation lymorphism, Orita et al., Pro Nat Acad Sci USA, 86, 2766-2770, 1989), simultaneous SSCP and heteroduplex analysis (Lee et al., Mol Cells , 5: 668-672, 1995), modified gradient gel electrophoresis (DGGE, Cariello et al., Am j hum Genet , 42, 726-734, 1988), modified high pressure liquid chromatography (Underhill et al., Genome Res , 7, 996-1005, 1997), hybridization reactions, DNA chips and the like. Examples of such hybridization reactions include Northern hybridization (Maniatis T. et al., Molecular Cloning , Cold Spring Habor Laboratory, NY, 1982), in situ hybridization (Jacquemier et al., Bull Cancer , 90: 31-8, 2003) and microarrays (Macgregor, Expert Rev Mol Diagn 3: 185-200, 2003) have methods.

The diabetic nephropathy diagnostic composition of the present invention may further include a reagent generally used in the method for detecting the above-described nucleic acid. For example, metal ion salts such as deoxynulceotide triphosphate (dNTP), a heat resistant polymerase, and magnesium chloride required for a PCR reaction may be included, and include dNTP, a sequencease, etc. required for sequencing. Can be.

Preferably, the composition for diagnosing diabetic nephropathy of the present invention, for example, but not limited to RT-PCR kit containing each primer pair specific for the marker gene of the present invention, cDNA of the marker gene of the present invention Or it may be provided in the form of a diagnostic kit or microarray such as a DNA chip including a substrate to which the oligonucleotide is attached.

Moreover, as another viewpoint, this invention

(a) isolating glycoproteins in a bodily fluid sample using a multi-lectin affinity column;

(b) concentrating the isolated glycoprotein of step (a) by acetone precipitation;

(c) diglycosylating the enzyme by processing the glycoprotein concentrated in step (b);

(d) SDS-PAGEing the diglycosylated protein in step (c);

(e) obtaining a peptide by in-gel trypsin digestion;

(f) analyzing the peptide of step (e) by LC-MS / MS to identify a diabetic nephropathy diagnostic marker comprising identifying a protein expressed or reduced in diabetic nephropathy patients compared to diabetic patients do. Preferably the body fluid is blood or tissue fluid, more preferably plasma.

Preferably, the step (f) is performed by applying a multiple reaction monitoring (MRM) or selective reaction monitoring (SRM) method in a mass spectrometer to determine precursor ions and transition ions of the peptide. Provided is a method for identifying a diabetic nephropathy diagnostic marker characterized by quantitatively analyzing and identifying a protein expressed or reduced in diabetic nephropathy patients using a peak on a mass spectrometer.

As described above, the marker for diagnosing diabetic nephropathy of the present invention is particularly useful for evaluating progression, prognosis, and treatment status to diabetic nephropathy because it is specifically overexpressed or reduced in blood of diabetic nephropathy patients.

1 is a protein profile of urine samples analyzed via SDS-PAGE and Coomassie staining. Protein profiles were used for sample selection for plasma sample analysis.
DC, urine DN of diabetic, urine of diabetic nephropathy
Figure 2 shows the separation of glycoproteins from human plasma. Proteins obtained from the multi-lectin affinity column eluate and the perfusion fraction were separated by SDS-PAGE and stained with Coomassie Blue (CBB) (A) and Gelcode Glycoprotein Staining (B).
3 shows the distribution and classification of proteins associated with diabetic nephropathy. (A) cell distribution profile, (B) biological process profile, (C) molecular function profile.
4 shows a comparison of biological processes and molecular functions between upregulated and downregulated proteins in diabetic nephropathy. (A) comparison of biological process profiles, (B) comparison of molecular function profiles.
FIG. 5 shows the validation of Lumican, Basorin and RBP-4 expression levels by Western blot and densitometry analysis. (A) Separate hematopoietic plasma samples from control group (9) and nephropathy group (9) were treated with anti-rumican antibody (1: 1000 dilution), anti-basorin antibody (1: 1000 dilution), and anti-RBP-4 The antibody (1: 1000 dilution) was used for western blot analysis. (B) The thickness of the band of the western blot was measured using Scion Image. The points in the data represent each patient and the line points to the median.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

Example 1 Stepwise Selection of Plasma Samples from Diabetic Control and Diabetic Nephropathy Patients

Blood and urine samples were taken from type 2 diabetic patients (control, 23 males) without nephropathy and type 2 diabetic patients (diabetic nephropathy, 14 males) with nephropathy. Samples were taken overnight after fasting overnight. The protocol for this study was accredited by the Kyungpook National University Hospital Biosafety Ethics Committee and received informed consent from all 37 patients. Heparinized whole blood samples were centrifuged at 4 to 2,000 rpm for 15 minutes to separate plasma samples and stored at -70 until use. Urine samples were also centrifuged to remove cell residue and salts were removed by dialysis (molecular-porous membrane tubing, 6-8,000 Da cutoff, Spectrumlabs USA) for 4 to 12 hours. These samples were placed in plastic urine containers and stored at -70 until use.

First, urine samples were collected from 37 male type 2 diabetic patients to test their albumin levels. According to the albumin / creatinine ratio (mg / g) of urine, patients were divided into type 2 diabetic control (hereinafter referred to as control) and type 2 diabetic nephropathy (hereinafter referred to as nephrotic). Of the 37 patients, 22 patients (13 controls and 9 nephropathy) were selected for further analysis, except those with other complications such as hypertension or retinopathy. After testing a total of 22 urine proteins by 1D-SDS-PAGE and Coomassie staining, plasma samples of 6 patients from each group were finally selected (FIG. 1).

Clinical data for patients with sample selection (Control 6, Nephropathy 6) are shown in Table 1. The urinary albumin / creatinine ratio was significantly higher in the nephrotic group than in the control group (P <0.05). HbA1c values were higher than the normal range in both groups, but values in the nephropathy group were much higher than in the control group. Serum creatinine values also tended to be higher in the nephropathy group than in the control group, but the difference was not significant. Serum albumin, serum total cholesterol, HDL cholesterol, LDL cholesterol, triglycerides and hemoglobin levels were not significantly different. After fasting, blood glucose was higher than normal, but there was no significant difference between the two groups.

Clinical Data of Patients with Type 2 Diabetes and Diabetic Nephropathy Normal range DC DN Average age (years) 59.8 ± 6.1 64.7 ± 3.3 Gender Male Female) south south Blood glucose after fasting (mg / dL) 70-110 128.8 ± 41.7 126.5 ± 80.1 HbA1c (%) 4 ~ 6.5 6.7 ± 0.7 8.6 ± 0.9 * Serum Albumin (g / dL) 3.2 ~ 4.8 4.4 ± 0.2 4.2 ± 0.5 Serum creatinine (mg / dL) 0.9-1.5 0.9 ± 0.2 1.5 ± 0.8 Serum Total Cholesterol (mg / dL) 125-200 157.3 ± 16.3 136.8 ± 23.2 HDL cholesterol (mg / dL) > 35 47.5 ± 15.3 40.5 ± 8.9 LDL cholesterol (mg / dL) <130 105.7 ± 10.2 85.2 ± 23.2 Triglycerides (mg / dL) <200 84.5 ± 27.9 118.2 ± 55.9 Hemoglobin (g / dL) 13-18 14.4 ± 0.7 13.0 ± 1.9 Urine albumin / creatinine ratio (mg / 24 h) <30 11.4 ± 4.9 451.2 ± 493.9 *

The data are mean ± SD unless otherwise indicated. * P <0.05

<Example 2> Isolation, Concentration and Proteomic Analysis of Plasma Glycoproteins

Plasma samples were prepared from whole blood samples (control group 6, nephrotic group 6). Plasma glycoproteins were extracted by multi-lectin affinity chromatography and analyzed via SDS-PAGE / Coomassie staining (FIG. 2 (A)).

2.1 Isolation and Concentration of Glycoproteins from Plasma

Jackalin (AIL), Concanavalin A (Con), Snowdrop Lectin (GNA), Lentil Lectin (LCH), Maackia amurensis lectin (MAL), Peanut Aggle Multi-lectin affinity columns (Qiagen, USA) filled with rutinin (peanut agglutinin (PNA), elderberry lectin (SNA) and malt agglutinin (WGA) were used. ConA, GNA, and LCH bind to N-glycosylated proteins, MAL, SNA, and WGA bind to proteins including sialic acid, and bind to AIL and PNA O-glycosylated proteins. The binding buffer was poured into the column to equilibrate, and then the plasma 50 prepared in Example 1 was diluted in the binding buffer 500 to which the protease antagonist was added, placed in the column, and centrifuged at 500 rpm for 2 minutes to obtain a perfusion fraction. Elution buffer was added and centrifuged to yield an eluate which was then precipitated and desalted with acetone.

2.2 Kumasi blue  And Gel cord  Glycoprotein Staining

The samples were separated by electrophoresis. The gel was washed three times with H ₂ O for 5 minutes and gently shaken for 1 hour at room temperature and stained with Bio-Safe Coomassie staining solution (Coomassie G250 Stain; Bio-Rad). The gel was placed in secondary distilled water and incubated for 30 minutes, and then washed three times with secondary distilled water for 10 minutes. Glycoprotein was stained using gelcode kit (PIERCE) to test the efficiency of multilectin chromatography. The gel was incubated for 30 minutes in 50% methanol, washed for 10 minutes with 3% acetic acid solution, incubated for 15 minutes in an oxidized solution, and then washed for 5 minutes with 3% acetic acid. The gel was gently shaken for 15 minutes by addition of a gel-code glycoprotein staining reagent, and then washed with 3% acetic acid and distilled water.

2. 3 persons -Gel trypsin cutting

Known methods (Heo, SH, Lee, SJ, Ryoo, HM, Park, JY, Cho, JY, Proteomics 2007, 7 , 4292-4302) was performed in-gel trypsin digestion (digestion).

Protein bands were removed from Coomassie stained gels and bleached by incubation in 75 mM ammonium bicarbonate / 40% ethanol (1: 1) solution. Desulfurization was performed with 5 mM DTT / 25 mM ammonium bicarbonate for 60 to 30 minutes. It was then alkylated with 55 mM iodoacetoamide at room temperature for 30 minutes. The gel pieces thus treated were dried with 100% ACN. Gel pieces were then rehydrated with 10 25 mM ammonium bicarbonate buffer containing 20 / ml of modified sequencing grade trypsin (Roche Applied Science) and incubated overnight at 37. Trypsin peptide mixture was eluted from the gel using 0.1% formic acid.

When gelcoat glycoprotein staining was used, staining of the protein in the elution fraction was more pronounced than in the perfusion fraction (FIG. 2B). Trypsin agonist peptides of Coomassie stained gel bands were analyzed by LC-MS / MS.

2.4 LC - ESI - MS Of MS  analysis

Thermo Finnigan's ProteomeX workstation LTQ linear ion trap MS (Thermo Electron, San Jose, CA, USA) equipped with NSI source (San Jose, CA) uses conventionally known methods (Heo, SH, Lee, SJ, Ryoo, HM, Park, JY, Cho, JY, Proteomics 2007, 7 , 4292-4302). LC-MS / MS analysis was performed.

12 ml of peptide samples obtained after the in-gel digestion were injected and loaded into peptide trap cartridges (Agilent, Palo Alto, CA). Peptides loaded on a 10 cm reversed phase PicoFrit column filled with C18 with pore size 300 mm 3 were eluted and the gradient eluate was separated. The mobile phase consisted of H ₂ O (A) and ACN (B) and both contained 0.1% v / v formic acid. The flow rate was maintained at 200 nL / min. The concentration gradient started at 2% B and reached 60% B after 50 minutes, then reached 80% B for 5 minutes and 100% A for the last 15 minutes. After performing data-dependent acquisition (m / z 400-1800) and performing a survey MS scan respectively, five MS / MS scans were performed for 30 seconds with the power drain option turned on. The spray voltage was 1.9 kV and the temperature of the ion transfer tube was set to 195. Standardized collision energy was set at 35%.

< Example  3>

3-1. Data Analysis

The dual mass spectra were analyzed with the Sorcerer program using the SEQUEST algorithm, followed by the Scaffold program. The final data set was prepared using orthogonal filtering criteria (peptide probability greater than 95.0% and at least two identified peptides would be correct for each protein).

Extract dual mass spectra, deconvolution, deisotope using Sorcerer 3.4 beta 2 (Sorcerer software 3.10.4, Sorcerer Web interface 2.2.0 r334 and Trans-Proteomic Pipeline 2.9.5) ) did. All MS / MS samples were analyzed using SEQUEST (ThermoFinnigan, SanJose, CA; version v.27, rev. 11). SEQUEST was set up to search the ipiHuman 3.29 database (IPI ver. 3.29, 69,965 entries), considering semiTrypsin as a digestive enzyme. SEQUEST search variables were set to fractional ion mass error of 1.00 Da and parent ion error of 1.5 Da. Oxidation of methionine and iodoacetamide modification of cysteine were designated as fixed modifications. To improve false-positive statistics, the decoy option was selected during the data retrieval process in the Sorcerer program, thereby improving quality by reducing noise effects.

All data are expressed as mean SE. Mean values were analyzed by ANOVA and post-hoc Tukey's test. Statistical significance was set to P <0.05.

3-2. Standards of Protein Identification

MS / MS based peptide and protein identification was confirmed using Scaffold (version Scaffold-02_04_00, Proteome Software Inc., Portland, OR). Protein Prophet algorithm (Keller, A., Nesvizhskii, AI, Kolker, E., Aebersold, R., Anal Chem 2002, 74 , 5383-5392) recognized protein identification when established with a probability of at least 95.0% and comprising at least two identified peptides. Protein probability was determined by protein prediction algorithm. Proteins containing similar peptides and cannot be differentiated solely based on MS / MS analysis were grouped to satisfy the principles of parsimony. Peptide false positive rate (FPR) was calculated using the scaffold software. For each charge state, a table of incorrect assignments was made to calculate FPRi, = [((incorrectly specified number at 95% probability) / (total incorrectly specified number)] * 100, where i is the charge It is a state. Protein recognition was considered accurate when the protein prediction algorithm was associated with a 95% probability and at least two peptides matched the protein sequence and each had a 95% probability based on the protein prediction algorithm. FPR is the sum of the values for each charge state. After protein identification, each data set was subtracted with ProtAn X. NetNGlyc 1.0 and NetOGlyc 3.1 were used to statistically analyze potential N- and O-linked glycosylation sites.

Table 2 shows the number of proteins identified from plasma samples from each person and the peptide false positive rate (FPR) measured using the Decoy database. FPR was calculated using algorithm-specific thresholds and the number of correct peptide identifications assumed. The calculated protein in each group was 164 in the control group (DC) and 174 in the nephropathy group (DN). Approximately 89% of the proteins identified using the NetNGlyc 1.0 and NetOGlyc 3.1 programs were predicted to have N- or O-linked glycosylation sites (Table 3). This result shows that multi-lectin chromatography and LC-MS / MS analysis are well suited for glycoprotein analysis of plasma samples.

Number of Proteins Identified by LC-MS / MS Analysis of Multi-Lectin Affinity Chromatography Concentrated Plasma Glycoprotein Samples DC1 DC2 DC3 DC4 DC5 DC6 DN1 DN2 DN3 DN4 DN5 DN6 Identified  Number of proteins ^a 111 101 114 127 108 109 97 124 127 111 110 100 Peptide  False positive rate (%) ^b 1.22 1.23 1.33 1.07 1.18 1.13 1.50 1.35 1.26 1.05 1.07 1.08

a, at least two peptides per protein are required to be positive for protein identification (95% confidence).

b, calculation of false positive rate was described in the Example.

Total number and percentage of expected glycosylated protein Identified Protein Expected N-linked glycosylation site Expected O-linked Glycosylation Location Total estimated glycoproteins N.D. ^* . Percentage of expected glycoproteins among the total identified proteins DC 164 432 582 146 18 89 DN 174 456 530 154 20 89

* ND: glycosylation site not detected.

3.3. Compared to control Nephropathy In the patient  Differences in Plasma Glycoprotein Expression

Plasma glycoproteins identified from samples from control and nephrotic patients were compared and analyzed. There were 139 proteins common to both groups of plasma. Peptide hit counts in a total of 43 proteins were two-fold higher in nephropathy than in controls (Table 4). In contrast, 40 proteins had twice the number of peptide hits in the control group than in the nephropathy group (Table 5). Up-regulated or down-regulated proteins were classified as extracellular, membrane or nuclear proteins depending on their expected cell location (FIG. 3A). These proteins were also classified according to biological process (FIG. 3B) and molecular function (FIG. 3C). During biological processes, inflammatory response proteins associated with cell adhesion, acute phase response, and blood coagulation were higher in diabetic nephropathy patients, while immune response proteins mediating complement activity and cellular defense responses were reduced compared to the overall (FIG. 4A). . In molecular function, most up- or down-regulated proteins of diabetic nephropathy patients had binding functions, including antigen, ATP, mineral and lipid binding functions (FIG. 4B).

Upregulated Proteins in Diabetic Nephropathy IPI  number Genetic symbol Protein Name DN
Average
range
(%) DC
Peptide
(hit)
Average DN
Peptide
(hit)
Average * DC / DN multiple IPI00783024 -  Myosin reactive immunoglobulin heavy chain variable region (fragment) 14.5 0 6.7 - IPI00003351 ECM1  Extracellular Matrix Protein 1 Precursor 12.8 0 2.2 - IPI00013933 DSP  Desmoplakin's Isoform DPI 5.0 0 2.2 - IPI00180956 -  49 kDa Protein 11.1 0 1.8 - IPI00220120 IGHV2  Ig heavy chain V-III region VH26 precursor 18.8 0 1.5 - IPI00022420 RBP4  Plasma retinol-binding protein precursors 9.2 0 1.3 - IPI00019399 SAA4  Serum Amyloid A-4 Protein Precursor 15.4 0 1.3 - IPI00009028 CLEC3B  Tetranectin precursor 10.4 0 1.0 - IPI00026197 IGKV4-1  Ig kappa chain V-IV region STH analogs 20.0 0 0.8 - IPI00555812 GC  Vitamin D-binding Protein Precursors 8.9 0 0.8 - IPI00022389 CRP  Isoform 1 of C-reactive protein precursor 12.1 0 0.8 - IPI00554711 JUP  Binding Flacoglobin 5.9 0 0.8 - IPI00217405 UBR1  Isoform of E3 Ubiquitin Protein Ligase UBR1 1 0.7 0 0.7 - IPI00021578 CFHR4  Complement factor H-associated protein 4 precursor 11.8 0 0.7 - IPI00021857 APOC3  Apolipoprotein C-III Precursor 19.2 0 0.7 - IPI00000849 SPRR2G  Bovine Proline-Rich Protein 2G 30.1 0 0.7 - IPI00845354 IGKC  IGKC Protein 14.5 0 0.5 - IPI00293057 CPB2  Isoform 2 of Carboxypeptidase B2 Precursor 8.6 0 0.5 - IPI00022937 F5  Coagulation Factor V 1.1 0 0.5 - IPI00303063 SCC-112  SCC-112 Protein 1.7 0 0.5 - IPI00291136 COL6A1  Collagen Alpha-1 (VI) Chain Precursor 2.4 0 0.5 - IPI00397801 RP1-14N1.3  Ipropsoriacin 2.1 0 0.5 - IPI00296537 FBLN1  Isoform C of Fibulin-1 Precursor 2.8 0 0.5 - IPI00299738 PCOLCE  Procollagen C-Endopeptidase Enhancer 1 Precursor 7.1 0 0.5 - IPI00015526 EHMT1  Isoform 1, H3 lysine-9 specific 5 of histone-lysine N-methyltransferase 3.4 0 0.3 - IPI00242956 FCGBP  IgGFc-binding protein precursor 0.2 0 0.3 - IPI00216589 ABCB9  Isoform 2 of ATP-binding cassette subfamily B member 9 precursor 8.7 0 0.3 - IPI00790690 -  36 kDa Protein 17.6 0 0.3 - IPI00219018 GAPDH  Glyceraldehyde-3-phosphate dehydrogenase 7.5 0 0.3 - IPI00007047 S100A8  Protein S100-A8 11.8 0 0.3 - IPI00296337 PRKDC  Isoform 1 of DNA-dependent protein kinase catalytic subunit 1.0 0 0.3 - IPI00025753 DSG1  Desmoglin-1 precursor 1.5 0 0.3 - IPI00027547 DCD  Duxidine Precursor 10.0 0 0.3 - IPI00216773 ALB  ALB Protein 6.3 0.8 9.0 10.8 IPI00023014 VWF  Von Willebrand factor precursor 2.1 0.5 2.2 4.3 IPI00395488 VASN  Basorin precursor 4.3 0.5 1.5 3.0 IPI00003590 QSOX1  Isoform 1 of sulfhydryl oxidase 1 precursor 5.8 0.5 1.3 2.7 IPI00550162 IGLV3-25  IGLV3-25 Protein 12.0 0.3 0.8 2.5 IPI00020986 LUM  Lumican Precursor 7.1 0.7 1.7 2.5 IPI00383164 IGHA1  SNC66 Protein 6.4 1.0 2.5 2.5 IPI00027507 CFHR3  Complement factor H-related protein 3 precursor 8.0 1.5 3.3 2.2 IPI00022434 ALB Serum albumin 47.7 122.5 252.5 2.1 IPI00064667 CNDP1  Beta-Ala-His Dipeptidase Precursor 8.9 0.5 1.0 2.0

DC / DN multiples: Number of average DC peptide hits divided by average DN peptide hits.

Downregulated Proteins in Diabetic Nephropathy IPI  number Genetic symbol Protein Name DC  Average
range
(%) DC
Peptide
(hit)
Average DN
Peptide
(hit)
Average DN Of
DC  Drainage IPI00004373 MBL2  Mannose Binding Protein C Precursor 40.4 2.2 0 - IPI00021439 ACTB  Actin Cytoplasmic 1 6.1 1.5 0 - IPI00398625 HRNR Homerin 1.3 1.5 0 - IPI00218413 BTD  Biotinidase precursor 6.5 1.2 0 - IPI00171678 DBH  Dopamine Beta-hydroxylase Precursor 4.3 1.0 0 - IPI00017522 KCNH4  Potassium Channel Subfamily H Member 4 1.6 0.8 0 - IPI00294713 MASP2  Isoform of Met-bound lectin serine protease 2 precursor 1 2.9 0.7 0 - IPI00023728 GGH  Gamma-glutamyl hydrolase precursors 8.5 0.7 0 - IPI00029275 MFI2  Isoform of Melanotransferin precursor 1 1.8 0.7 0 - IPI00005439 FETUB  Fetuin-B Precursor 5.5 0.7 0 - IPI00021364 CFP  Properdine Precursor 5.7 0.5 0 - IPI00387022 -  Ig kappa fragment V-I region AG 25 0.5 0 - IPI00477804 -  Immunoglobulin Heavy Chain Variable Regions 14.3 0.5 0 - IPI00298860 LTF  Growth Inhibitor Protein 12 1.3 0.5 0 - IPI00170786 WBP11  WW domain binding protein 11 10.0 0.5 0 - IPI00386839 -  Amyloid Lambda 6 Light Chain Variable Region SAR (Short) 32.8 0.5 0 - IPI00297263 HEG1  Isoform 1 of protein HEG homolog 1 precursor 3.8 0.3 0 - IPI00382478 -  Ig heavy chain V-III region TIL 16.5 0.3 0 - IPI00400922 PDCD11  RRP5 protein homologue 1.8 0.3 0 - IPI00011255 GP1BA  Platelet Glycoprotein Ib Alpha Chain Precursor 4.2 0.3 0 - IPI00178352 FLNC  Isoform of Philamine-C 1 1.4 0.3 0 - IPI00021817 PROC  Vitamin K-dependent protein C precursor 2.6 0.3 0 - IPI00382480 -  Ig heavy chain V-III region BRO 35 0.3 0 - IPI00307114 ZNF291  Zinc Finger Protein291 1.6 0.3 0 - IPI00103595 CEP350  Centrosome-Related Protein 350 0.2 0.3 0 - IPI00307702 -  H53_GS1 (short) 7.8 0.3 0 - IPI00029061 SEPP1  Selenoprotein P Precursor 11.0 0.3 0 - IPI00172479 DISP1  Dispatched A 3.2 0.3 0 - IPI00026314 GSN  Isoform of gelsorine precursor 1 2.4 0.3 0 - IPI00297284 IGFBP2  Insulin-like growth factor-binding protein 2 precursor 3.7 0.3 0 - IPI00030205 IGKV3-20  Ig kappa chain V-III region HAH precursor 15.8 5.7 0.5 11.3 IPI00215894 KNG1  Isoform LMW of Kininogen-1 Precursor 7.0 7.3 0.8 8.8 IPI00384407 -  Myosin-reactive immunoglobulin heavy chain variable region (fragment) 15.9 1.7 0.5 3.3 IPI00008669 -  38 kDa Protein 11.7 3.2 1.0 3.2 IPI00382436 -  Ig lambda chain V-III region SH 13.9 1.0 0.3 3.0 IPI00029168 LPA  Apolipoprotein 1.3 4.0 1.5 2.7 IPI00011252 C8A  Complement C8 Alpha Chain Precursor 6.9 11.5 4.3 2.7 IPI00294395 C8B  Complement Component C8 Beta Chain Precursor 7.6 4.8 2.0 2.4 IPI00292950 SERPIND1  Heparin Cofactor 2 Precursor 10.0 15.0 7.2 2.1 IPI00032291 C5  Complement C5 precursor 7.1 29.7 15.0 2.0

* DN / DC multiple: The average DN peptide hit divided by the average DC peptide hit.

< Example  4> LC - MS Of MS  Data validation

To verify LC-MS / MS data, nine samples from each of nephrotic and control groups were western blotting to analyze the values of lumincan, basorin and RBP-4 (FIG. 5A).

20 μg of crude plasma was isolated by 12% SDS-PAGE. After electrophoresis, the proteins were transferred to nitrocellulose membrane (Whatman, Germany) and lumican, vasorin and RBP-4 were detected. The membrane was overnight at 4 with anti-lumincan antibody, anti-human vasorine antibody (R & D, MN, USA, 1: 1000 dilution), or anti RBP-4 antibody (AbFRONTIER, Seoul, Korea, 1: 1000 dilution) Incubated. 1 hour at room temperature with horseradish peroxidase bound anti-goat IgG (Santa Cruz Biotechnology, CA, USA, 1: 1,500 dilution) or anti-mouse IgG (Stressgen, MI, USA, 1: 1000 dilution) Incubated. Immune response proteins were detected using an ECL PLUS Western Blot Analysis System (Amersham Biosciences, UK). The thickness of the band was measured by photographing with Scion Image (Scion, Frederick, MD) (FIG. 5B). Graticule slides were measured using the Scion Image, and the distance of 250 μm was calculated equal to 733 pixels.

In nephropathy group, it can be seen that leucan, basorin and RBP-4 are expressed at a higher level than the control group. Compared with the control group, the mean concentrations of lumincan, basorin and RBP-4 bands in the nephropathy group were 3.3, 1.8 and 1.7 fold, respectively. These results show that lumincan, basorin and RBP-4 are suitable markers for predicting diabetic nephropathy and monitoring the progress of the disease.

Claims (4)

Diabetic nephropathy diagnostic composition comprising an antibody that specifically binds to plasma retinol-binding protein (RBP4) and precursors thereof. A composition for diagnosing diabetic nephropathy comprising a primer or probe specific for a nucleic acid encoding a plasma retinol-binding protein (RBP4) and a precursor thereof. Kit for diagnosing diabetic nephropathy comprising the composition of claim 1. A microarray for diagnosing diabetic nephropathy comprising the composition of claim 1.

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