JP2018179669A - Diagnostic marker of diabetic nephropathy - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a diagnostic marker of sensitively diagnosing diabetic nephropathy.SOLUTION: A diagnostic marker of diabetic nephropathy includes pyruvate kinase muscle type 2 (PKM2) in the urine of a subject or fragment thereof. It is diagnosed that the subject is suffered from diabetic nephropathy when PKM2 or the fragment is equal to or more than 1.0 ng/40μL. It is found out that glomerular epithelial cells cultured under high sugar are shifted to a glycolytic-dependent energy production mechanism, that is, metabolic remodeling (MR) is induced.SELECTED DRAWING: Figure 2

Description

  The present invention relates to diagnostic markers and diagnostic kits for diagnosing diabetic nephropathy.

  Chronic kidney disease (CKD) is defined as a condition in which structural or functional abnormalities of the kidney last more than 3 months. It is known that when CKD progresses and it leads to end-stage renal failure, dialysis and transplantation are required, but the risk of developing cardiovascular disease increases as the stage progresses. It is clear from the fact that the representative of CKD is diabetic nephropathy, which occupies the top of dialysis-induced original diseases in Japan.

  Hyperglycemia dilates the import arterioles and increases intraglomerular hydrostatic pressure, resulting in damage to the glomerulus, resulting in hypertension, urinary protein excretion, or enhancement of the renal tissue RA system associated with hyperglycemia, and various metabolic abnormalities. It is the mechanism of diabetic nephropathy that the modification is added and the progression of renal injury is accelerated.

  In order to prevent the progression of diabetic nephropathy to renal failure, it is essential to have an early treatment intervention based on early diagnosis, or an intensifying therapy for cases expected to become severe.

  In the conventional diagnosis of diabetic nephropathy, in addition to the history of diabetes, changes in urinary findings (appearance of proteinuria and microalbuminuria) and eGFR (glomerular filtration rate: serum creatine concentration and age, an index representing renal function) Judgment was made on the basis of, for example, a decrease in gender) (Non-Patent Document 1). However, this prior art may not be able to reliably distinguish diabetic nephropathy from other renal diseases.

  Non-Patent Document 2 describes the diagnosis of diabetic nephropathy using L-type fatty acid binding protein (L-FABP) in urine as a diagnostic marker. In patients with type 2 diabetes, urinary L-FABP becomes significantly higher as the stage progresses, and even normal albuminuria that does not exhibit microalbuminuria also has lower urinary L- compared to the non-diabetic control group. FABP shows significantly higher price.

Evidence-based CKD practice guidelines 2009, p 87-p 104, chapter 8 diabetic nephropathy Kamijo Ikemori A, et al. Diabetes Care 34: 691-696, 2011

  However, in the case of the diagnostic marker of Non-Patent Document 2, in order to diagnose with high accuracy, it is necessary to also monitor the amount of albumin in urine.

  The present invention has been made in view of such problems, and it is an object of the present invention to provide a diagnostic marker capable of sensitively diagnosing diabetic nephropathy.

  The diagnostic marker for diabetic nephropathy according to the present invention is characterized in that it is pyruvate kinase muscle type 2 (PKM2) or a fragment thereof.

  According to the present invention, the progression from diabetic nephropathy to renal failure can be monitored with high accuracy.

FIG. 1 is a photographic view showing expression of PKM2 in glomerular epithelial cells, the left side of FIG. 1 is a photographic view of normal kidney, and the right side of FIG. 1 is a photographic view of diabetic nephropathy. It is the figure which measured the PKM2 density | concentration in urine using ELISA kit. FIG. 1 is a photograph of electrophoresis (SDS-PAGE) analysis showing detection of PKM2 and its fragments in the urine of diabetic nephropathy patients.

  Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings, which are for the purpose of facilitating the understanding of the principle of the present invention, and the scope of the present invention is as follows: The present invention is not limited to the embodiment, and other embodiments in which a person skilled in the art substitutes the configuration of the following embodiments as appropriate are also included in the scope of the present invention.

  Glomeruli is the most important micro-organ for renal function, and glomerular epithelial cells (podocytes) play a central role in glomerular function. The present inventors conducted a comprehensive proteome analysis of energy metabolism abnormalities in glomerular epithelial cells placed under high sugar, and as a result, glomerular epithelial cells cultured under high sugar were glycolytic system-dependent energy production We have found a shift in mechanism (ie, that metabolic remodeling (MR) is triggered).

  The present inventor is a complex of a glycolytic enzyme or oxidative phosphorylation pathway in which there is a 2-fold or more change in expression between glomerular epithelial cells in high sugar culture and glomerular epithelial cells in normal sugar concentration culture. We focused on proteins and searched for glomerular epithelial cell specific expression in glomeruli, and determined pyruvate kinase as the final candidate. Pyruvate kinase is an enzyme that catalyzes a reaction of producing pyruvate from ADP and phosphoenolpyruvate.

  As a result of the present inventor's investigation using renal biopsy cases of human diabetic nephropathy, in diabetic nephropathy patients, the expression of PKM2 or a fragment thereof in glomerular epithelial cells is significantly increased as compared to normal. We have acquired what we are doing as new findings, and completed the present invention based on such facts. That is, the diagnostic marker for diabetic nephropathy according to the present invention is PKM2 or a fragment thereof. MR is a change that occurs early as a functional change in glomerular epithelium exposed to hyperglycemia, and the change is considered to lead to inefficiency of energy production, which is in turn linked to pathological changes of cells. That is, MR is likely to be just before the development of diabetic nephropathy tissue damage. Therefore, the present invention is a diagnostic marker that can be detected earlier than existing markers focusing on the detection of MR.

PKM2 is represented by the sequence consisting of SEQ ID NO: 1 below.
MSKPHSEAGT AFIQTQQLHA AMADTFLEHM CRLDIDSPPI TARNTGIICT IGPASRSVET LKEMIKSGMN VARLNFSHGT HEYHAETIKN VRTATESFAS DPILYRPVAV ALDTKGPEIR TGLIKGSGTA EVELKKGATL KITLDNAYME KCDENILWLD YKNICKVVEV GSKIYVDDGL ISLQVKQKGA DFLVTEVENG GSLGSKKGVN LPGAAVDLPA VSEKDIQDLK FGVEQDVDMV FASFIRKASD VHEVRKVLGE KGKNIKIISK IENHEGVRRF DEILEASDGI MVARGDLGIE IPAEKVFLAQ KMMIGRCNRA GKPVICATQM LESMIKKPRP TRAEGSDVAN AVLDGADCIM LSGETAKGDY PLEAVRMQHL IAREAEAAIY HLQLFEELRR LAPITSDPTE ATAVGAVEAS FKCCSGAIIV LTKSGRSAHQ VARYRPRAPI IAVTRNPQTA RQAHLYRGIF PVLCKDPVQE AWAEDVDLRV NFAMNVGKAR GFFKKGDVVI VLTGWRPGSG FTNTMRVVPP P
Although the fragment of PKM2 is not particularly limited, it is shown, for example, by the sequence consisting of SEQ ID NO: 2 below.
IRKASD VHEVRKVLGE KGKNIKIISK IENHEGVRRF DEILEASDGI MVARGDLGIE IPAEKVFLAQ KMMIGRCNRA GKPVICATQM LESMIKKPRP TRAEGSDVAN AVLDGADCIM LSGETAKGDY PLEAVRMQHL IAREAEAAIY HLQLFEELRR LAPITSDPTE ATAVGAVEAS FKCCSGAIIV LTKSGRSAHQ VARYRPRAPI IAVTRNPQTA RQAHLYRGIF PVLCKDPVQE AWAEDVDLRV NFAMNVGKAR GFFKKGDVVI VLTGWRPGSG FTNTMRVVPV P
The present invention can also diagnose the stage of diabetic nephropathy by comparing the degree of expression of PKM2 or a fragment thereof. In addition, the present invention can also predict the aggravation of diabetic nephropathy by comparing the degree of expression of PKM2 or a fragment thereof.

  Diabetic nephropathy is based on clinical characteristics (urine protein, creatinine clearance), and is stage I (pre-nephropathy), stage II (early nephropathy), stage III early (overt nephropathy prepro), stage III It is classified into late stage (late nephropathy late), stage IV (renal failure stage), and stage V (dialysis therapy stage). According to the present invention, diabetic nephropathy in all stages I to V can be diagnosed.

  Glomerular epithelial cells are in direct contact with the urinary cavity, and it is possible to measure PKM2 or a fragment thereof that has leaked into urine. By measuring PKM2 or a fragment thereof in the urine of a subject, it is possible to easily diagnose diabetic nephropathy. The sample of the subject is not limited to urine, and may be blood (whole blood, serum, plasma), saliva, semen, etc., for example.

  The diagnostic kit for diagnosing diabetic nephropathy according to the present invention comprises means for detecting or quantifying PKM2 or a fragment thereof in a sample obtained from a subject.

  The detection or quantification of PKM2 or a fragment thereof is not particularly limited, and may simply detect the presence or absence of PKM2 or a fragment thereof, and the expression amount of PKM2 or a fragment thereof may be relative or absolute. It may be determined.

  Measurement of the expression level of PKM2 or a fragment thereof can be performed by an immunological method, for example, combining electrophoresis separation with detection or quantification by fluorescence, enzyme, radioisotope etc. (Western blot method, Fluorescent two-dimensional electrophoresis, including immunostaining (including fluorescent antibody method, enzyme antibody method, heavy metal labeled antibody method, radioactive isotope labeled antibody method), enzyme-linked immunosorbent assay (ELISA), dot blotting method Etc., preferably ELISA.

  In the present invention, for example, (1) measuring PKM2 or a fragment thereof in a urine sample, and (2) based on the measurement result, diagnosing diabetic nephropathy based on the step of diagnosing the presence or absence of onset of diabetic nephropathy. Diagnosis of nephropathy is possible.

  In step (1), a urine sample derived from a subject is prepared, and the content (concentration) of PKM2 or a fragment thereof present therein is measured. The subject is a diabetic patient or a potentially diabetic patient (a person who may suffer from diabetes). As described above, the sample derived from the subject is not limited to the urine sample, and may be, for example, blood, saliva, semen or the like.

  In step (2), the presence or absence of onset of diabetic nephropathy is diagnosed based on the measurement result. The reference value and the cut-off value used for the diagnosis can be appropriately determined in consideration of the state of the sample to be used, the required accuracy, and the like. For example, the amount (reference value) of PKM2 or a fragment thereof in urine of a person who does not develop diabetic nephropathy (a symptomatic patient) is previously measured, and the amount of PKM2 or a fragment thereof in the urine of a subject When it shows a significant difference from the reference value, it can be diagnosed as developing diabetic nephropathy.

  For example, when the amount of PKM2 or a fragment thereof in urine of a person who does not develop diabetic nephropathy is 0 ng / 40 μL, the amount of PKM2 or a fragment thereof in urine is 0.5 ng / 40 μL, preferably 0.8 ng / It can be diagnosed as having diabetic nephropathy when it is 40 μL, more preferably 1.0 ng / 40 μL or more.

The diagnosis is not limited to the qualitative one, and can be performed semiquantitatively or quantitatively. For example, as shown below, it is possible to set the possibility of developing diabetic nephropathy for each range of measurement values.
x <Measured value: probability of developing diabetic nephropathy> 70%
y ≦ measured value <x: possibility of developing diabetic nephropathy is 30% to 70%
Measured value <y: The possibility of developing diabetic nephropathy is less than 30% According to the diagnosis of this step (2), it can be performed mechanically without a doctor's diagnosis.

  As described above, the inventor found that glomerular epithelial cells cultured under high sugar shift to a glycolytic system-dependent energy production mechanism, that is, that MR is triggered, but transcription factor analysis Using the qPCR microarrays described above, it was revealed that the transcription factors that trigger the MR were MEF2C and MYF5. That is, MEF2C and MYF5 can be diagnosed as diabetic nephropathy when their expression is reduced as compared to that of healthy persons, and MEF2C and MYF5 have a lower expression, and so on. It can be judged that the progression of nephropathy is early.

  Therefore, the diagnostic marker for diabetic nephropathy according to the present invention is a diagnostic marker for diabetic nephropathy consisting of PKM2 or a fragment thereof and transcription factor MEF2C, wherein the expression of PKM2 or a fragment thereof is increased and the expression of transcription factor MEF2C is decreased In some cases, it is diagnosed as diabetic nephropathy.

  Further, the diagnostic marker for diabetic nephropathy according to the present invention is a diagnostic marker for diabetic nephropathy consisting of PKM2 or a fragment thereof and transcription factor MYF5, wherein the expression of PKM2 or a fragment thereof is increased and the expression of transcription factor MYF5 is decreased In some cases, it is diagnosed as diabetic nephropathy.

  The diagnostic marker for diabetic nephropathy according to the present invention is a diagnostic marker for diabetic nephropathy consisting of PKM2 or a fragment thereof, transcription factor MYF5 and transcription factor MEF2C, and upregulation of PKM2 or a fragment thereof, transcription factor MYF5 Is diagnosed as diabetic nephropathy in the case of reduced expression of the gene and expression of the transcription factor MEF2C.

(1) Comprehensive proteome analysis Using cultured human glomerular epithelial cells (podocyte: Podo), cultured in high sugar (360 mg / dl) to simulate diabetes conditions Podo cultured under conditions equivalent to normoglycemic (90 mg / dl) As a result of performing comprehensive protein expression analysis or gene expression analysis with Podo, Podo cultured in high sugars has reduced mitochondrial function, and its energy metabolism mechanism is from oxidative phosphorylation to glycolysis It has been found that MR is triggered to increase the dependence on energy, and energy production is inefficient by MR.

Comprehensive renal proteome analysis was performed using renal biopsy tissue of diabetic nephropathy cases, and four proteins involved in MR whose expression was significantly changed (1.5 or more times) were detected. The expression of PKM2 was found to be epithelial cell-specific in glomeruli and the same as proteome results, when the expression in normal kidney and diabetic nephropathy kidney was examined using each antibody. It has been significantly increased in sexual nephropathy. The results are shown in FIG. The left side of FIG. 1 is a photograph of a normal kidney, and the right side of FIG. 1 is a photograph of diabetic nephropathy. On the left side of FIG. 1, positive cells were not found in the glomeruli, but on the right side of FIG. 1, enhanced expression of PKM2 was observed specifically for glomerular epithelial cells.
(2) Measurement in urine A renal biopsy was performed, and five subjects and one healthy subject whose tissue diagnosis was confirmed were subjects. According to renal biopsy tissue diagnosis, in 5 subjects, 3 cases of diabetic nephropathy patients, 1 case of nephrosclerosis patients, and 1 case of diabetics complicated to nephrosclerosis.

In the early morning, 40 mL of fresh urine was collected, and concentration measurement was performed using a commercially available PKM2 measurement ELISA kit (BioVender RD191345200R). The results are shown in FIG. As shown in FIG. 2, in diabetic nephropathy, it was confirmed that the PKM2 concentration (in terms of gCre) was increased. In FIG. 2, DMN indicates diabetic nephropathy, and DM indicates diabetes.
(3) MR-induced transcription factor Using renal biopsy tissue of diabetic nephropathy (DMN, N = 5) and normal kidney (Cont, N = 5), in addition to MEF2C, MYF5, PGC-1α, culture experiment Glomeruli were evaluated blindly after immunostaining of Pyruvate Kinase, Muscle (PKM) which showed the largest expression change in glycolytic enzymes. The results are shown in Table 1 below.

In podocytes cultured under hyperglycemia, the mitochondrial (Mit) oxidative phosphorylation system shifts to glycolytic-dependent inefficient energy production, and upstream of it, the transcription factors MEF2C, MYF5 decrease, and then It was found that PGC-1α reduction was involved.
(4) Fragment of PKM2 measured in urine 40 mL of fresh morning urine of diabetic nephropathy patients are collected and the presence of PKM2 fragment contained in the urine is analyzed by SDS polyacrylamide electrophoresis (SDS-PAGE) analysis It was tested. The polyacrylamide gel used an acrylamide concentration of 14.0%. The migrated polyacrylamide gel was stained with Coomassie blue G-250. The results are shown in FIG. In FIG. 3, Pt # 19 represents urine of diabetic nephropathy patients, Ori represents raw urine, and x33 represents urine that has been concentrated. The 58.6 Kd indicated by the arrow is uncleaved PKM2 and 31.5 Kd is a fragment of PKM2. As shown in FIG. 3, not only PKM2 but also fragments thereof were detected in the urine of diabetic nephropathy patients. This PKM2 fragment had the sequence shown in SEQ ID NO: 2.

  It can be used to diagnose diabetic nephropathy.

Claims (9)

  A diagnostic marker for diabetic nephropathy, characterized in that it is pyruvate kinase muscle type 2 (PKM2) or a fragment thereof. The diagnostic marker for diabetic nephropathy according to claim 1, wherein the fragment comprises the following sequence.
IRKASD VHEVRKVLGE KGKNIKIISK IENHEGVRRF DEILEASDGI MVARGDLGIE IPAEKVFLAQ KMMIGRCNRA GKPVICATQM LESMIKKPRP TRAEGSDVAN AVLDGADCIM LSGETAKGDY PLEAVRMQHL IAREAEAAIY HLQLFEELRR LAPITSDPTE ATAVGAVEAS FKCCSGAIIV LTKSGRSAHQ VARYRPRAPI IAVTRNPQTA RQAHLYRGIF PVLCKDPVQE AWAEDVDLRV NFAMNVGKAR GFFKKGDVVI VLTGWRPGSG FTNTMRVVPV P   The diagnostic marker for diabetic nephropathy according to claim 1 or 2, characterized in that it is pyruvate kinase muscle type 2 (PKM2) or a fragment thereof in the urine of a subject.   4. The method according to any one of claims 1 to 3, wherein when the pyruvate kinase muscle type 2 (PKM2) or a fragment thereof in urine of the subject is 1.0 ng / 40 μL or more, it is diagnosed as developing diabetic nephropathy. The diagnostic marker for diabetic nephropathy according to any one of the preceding claims. It is a diagnostic marker of diabetic nephropathy consisting of pyruvate kinase muscle type 2 (PKM2) or a fragment thereof and transcription factor MEF2C,
The diabetic nephropathy according to any one of claims 1 to 4, characterized in that it is diagnosed as diabetic nephropathy in the case of increased expression of pyruvate kinase muscle type 2 (PKM2) or a fragment thereof and decreased expression of transcription factor MEF2C. Markers for diabetic nephropathy. A diagnostic marker for diabetic nephropathy, which comprises pyruvate kinase muscle type 2 (PKM2) or a fragment thereof and the transcription factor MYF5,
The diabetic according to any one of claims 1 to 4, characterized in that it is diagnosed as diabetic nephropathy in the case where the expression of pyruvate kinase muscle type 2 (PKM2) is increased and the expression of transcription factor MYF5 is decreased. Diagnostic marker for nephropathy.   A diagnostic kit for diagnosing diabetic nephropathy, comprising: means for detecting or quantifying pyruvate kinase muscle type 2 (PKM2) or a fragment thereof in a sample obtained from a subject.   The diagnostic kit according to claim 7, wherein the sample is urine of a subject.   The diagnosis according to claim 7, characterized in that it is diagnosed as developing diabetic nephropathy when pyruvate kinase muscle type 2 (PKM2) or a fragment thereof in urine of a subject is 1.0 ng / 40 μL or more. kit.