JP6558729B2 - Inspection method of glomerular injury - Google Patents

Description

  The present invention relates to a glomerular disorder inspection method and the like. More specifically, the present invention relates to a method for examining glomerular disorders, a method for differentiating glomerular disorders from tubular disorders, and a method for diagnosing prognosis of kidney function.

  Fatty acid-binding protein (FABP) is a protein having a molecular weight of about 14 to 15 kDa that binds to a hydrophobic ligand such as a long-chain fatty acid or eicosanoid (Non-patent Document 1). At least nine FABP isoforms are known so far, and FABP4 among them is known to be expressed mainly in adipocytes and macrophages (Non-patent Document 1). In normal renal tissue, it was known that FABP4 expression was observed in interstitial capillaries and vascular endothelial cells (Non-patent Documents 2 and 3). Although sufficient studies have not been made so far regarding the expression of FABP4, it has been reported by some of the present inventors that the expression of FABP4 has been confirmed from the kidney tissues of patients with multiple renal diseases. (Non-Patent Document 4).

On the other hand, FABP4 is associated with chronic inflammation, and in studies using FABP4 knockout mice that play an important role in insulin resistance and arteriosclerosis, it becomes insulin-sensitive due to FABP4 deficiency and suppresses arteriosclerosis (Non-Patent Documents 1 and 5). In addition, since FABP4 does not have a secretory signal peptide, it was considered to be a non-secreted protein. However, recent studies have reported that FABP4 is secreted from adipocytes, and its blood concentration is insulin resistance, diabetes, It is known to be associated with arteriosclerosis (Non-Patent Documents 6 and 7).
However, it has not been known so far that FABP4 is excreted in urine. In addition, a method for diagnosing a metabolic disorder has been reported so far by measuring the concentration of the adipocyte type (A-FABP, FABP4, P2) of the fatty acid binding protein in various body fluids. However, it does not mention that FABP4 is excreted in urine, and does not describe measuring the amount of FABP4 excreted in urine (Patent Document 1).

  By the way, the kidney is composed of a functional unit called nephron, and nephron is composed of a renal body and a tubule composed of a vascular pole and a glomerulus. Kidney dysfunction includes glomerular damage and tubule damage. Urinary excretion of β2-microglobulin (β2-MG), N-acetyl β-D-glucosaminidase (NAG), and liver-type L-FABP (FABP1), which is one of the isoforms of FABP, as an indicator of renal dysfunction Although evaluation of renal interstitial disorder by quantitative measurement has been clinically applied, it mainly evaluates tubular disorders and cannot evaluate glomerular disorders (Non-patent Document 8). In addition, urinary albumin excretion is used as an index for evaluating glomerular damage (Non-patent Document 9), but urinary albumin is not detected unless glomerular damage progresses. Conversely, it is known that secondary urinary albumin excretion causes secondary tubular damage.

  Under such circumstances, a method capable of early diagnosis of the presence, possibility, progression or prognosis of glomerular disorders, and differentiation between glomerular disorders and tubular disorders, and future kidney function It can be said that there is still a need for a method that can determine whether or not there is a possibility of a failure.

Special table 2009-501926

Furuhashi M, Hotamisligil GS., Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets., Nat. Rev. Drug Discov., 7: 489-503, 2008 Elmasri H, et al., Fatty acid binding protein 4 is a target of VEGF and a regulator of cell proliferation in endothelial cells, FASEB J., 23: 3865-3873, 2009 Elmasri H, et al., Endothelial cell-fatty acid binding protein 4 promotes angiogenesis: role of stem cell factor / c-kit pathway, Angiogenesis, 15: 457-468, 2012 The 56th Annual Meeting of the Japanese Nephrological Society Abstract Furuhashi M, Ishimura S, Ota H, Miura T, Lipid chaperones in metabolic inflammation, Int. J. Inflam., 2011: 642612, 2011 Xu A, Wang Y, Xu JY, Stejskal D, Tam S, Zhang J, Wat NM, Wong WK, Lam KS., Adipocyte fatty acid-binding proteinis a plasma biomarker closely associated with obesity and metabolic syndrome., Clin Chem., : 405-413, 2006 Furuhashi M, Saitoh S, Shimamoto K, Miura T. Fatty acid-binding protein 4 (FABP4): pathophysiological insights and potent clinical biomarker of metabolic and cardiovascular diseases. Clin Med Insights Cardiol 8 (S3): 23-33, 2014 Kamijo-Ikemori A, Sugaya T, Kimura K. Urinary fatty acid binding protein in renal disease. Clin Chim Acta 374: 1-7, 2006 Shlipak MG, Day EC. Biomarkers for incident CKD: a new framework for interpreting the literature.Nat Rev Nephrol 9: 478-83, 2013

  An object of the present invention is to provide a novel method capable of predicting and early diagnosis of glomerular injury. Another object of the present invention is to provide a method capable of easily differentiating glomerular damage and tubule damage. Another object of the present invention is to provide a method capable of determining the possibility of future renal dysfunction.

  In addition to the findings obtained so far that expression of FABP4 was observed in the kidney tissues of a plurality of kidney disease patients, FABP4 is now excreted in urine, and the amount of FABP4 excreted in urine Was found to be related to urinary albumin excretion. Furthermore, the present inventors have found that glomerular disorder can be predicted and diagnosed early based on the detection result of urinary FABP4. Furthermore, the present inventors have found that, based on the detection result of FABP4 in urine, it is possible to differentiate between glomerular disorders and tubule disorders and to determine the possibility of future renal dysfunction. The present invention is based on such knowledge.

According to the present invention, the following inventions are provided.
(1) A method for examining glomerular injury, comprising a step of detecting fatty acid binding protein 4 (FABP4) in urine of a subject.
(2) The test method according to (1), wherein the step of detecting FABP4 is performed by an immunoassay using an antibody against FABP4.
(3) The test method according to (1) or (2), wherein the immunoassay is an ELISA method.
(4) The inspection method according to any one of (1) to (3), wherein the glomerular injury is primary or secondary.
(5) The examination method according to any one of (1) to (4), which is an examination method for diagnosing the presence / absence, possibility of morbidity, progression or prognosis of glomerular disorder.
(6) A method for differentiating glomerular injury and tubular injury based on the result obtained by the inspection method according to any one of (1) to (5).
(7) A method for diagnosing prognosis of kidney function based on the result obtained by the test method according to any one of (1) to (5).
(8) A method for monitoring glomerular injury in a subject,
Measuring the amount of FABP4 in the urine of the subject,
A method wherein glomerular injury has progressed when the amount of FABP4 increases over time.
(9) A test kit for glomerular injury, comprising an antibody against FABP4.
(10) Use of FABP4 as a test marker for glomerular injury.

  According to the present invention, by the simple method of detecting fatty acid binding protein 4 (FABP4) in urine, the presence / absence, morbidity, progress or prognosis of primary or secondary glomerular disorder is diagnosed at an early stage. it can. Furthermore, according to the present invention, it is possible to differentiate glomerular disorders and tubular disorders based on the results obtained by the method, and to determine whether there is a possibility of future renal dysfunction. Can do.

It is a graph which shows the correlation of U-FABP4 excretion amount and UACR in a U-FABP4 detectable group. It is a graph which shows the relationship between the amount of FABP4 excretion in urine, and the change of eGFR.

Detailed description of the invention

[Inspection method]
The test method of the present invention is characterized by including a step of detecting fatty acid binding protein 4 (hereinafter also referred to as “FABP4”) (FABP4) in the urine of a subject. Hereinafter, the inspection method of the present invention will be specifically described.

  FABP4 is also called A-FABP and aP2, and is known as a factor that regulates insulin resistance and lipid and sugar metabolism. Normally, FABP4 is not expressed in glomeruli. In addition, so far, no examination has been made on the excretion of FABP4 in the urine. In the test method of the present invention, FABP4 (U-FABP4) excreted in urine is detected and / or measured.

  In this specification, the term “subject's urine” includes urine obtained (collected) from the subject.

  The urine used in the examination of the present invention can be collected according to a conventional method. Concentrated wake-up urine is preferred. The collected urine is preferably stored at −20 ° C. or lower until the time of examination and is not repeatedly frozen and thawed.

  In the present specification, “detecting” a molecule in urine includes not only examining the presence / absence of the molecule in urine but also measuring the amount of the molecule contained in urine. In addition, since the concentration of urine changes depending on the collection conditions (the amount of water in the body at the time of urine collection, etc.), when detecting and measuring molecules in the urine at any time, it is preferable to perform correction with urine creatinine (creatinine correction).

A method for measuring a molecule to be detected (for example, FABP4) in urine can be performed using any method for detecting and measuring a specific protein in a liquid, such as an immunoassay, an agglutination method, a turbidimetric method, Examples include, but are not limited to, western blotting and surface plasmon resonance (SPR). Alternatively, the amount of protein may be indirectly detected / measured using a method for detecting / measuring mainly DNA or mRNA, such as in situ hybridization.
An immunoassay that measures the amount of the molecule to be detected using an antigen-antibody reaction between the antibody against the molecule to be detected and the molecule to be detected in the urine sample is particularly simple and preferable.

In the immunoassay, an antibody against a detection target molecule that is detectably labeled or an antibody (secondary antibody) against an antibody against the detection target molecule that is detectably labeled is used. Depending on the labeling method of the antibody, it is classified into enzyme immunoassay (EIA or ELISA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), fluorescence polarization immunoassay (FPIA), chemiluminescent immunoassay (CLIA), etc. This method can be used.
In the ELISA method, enzymes such as peroxidase and alkaline phosphatase, in the RIA method, radioactive substances such as ¹²⁵ I, ¹³¹ I, ³⁵ S, and ³ H, in the FPIA method, fluorescein isothiocyanate, rhodamine, dansyl chloride, phycoerythrin, tetramethylrhodamine isotope In the CLIA method, an antibody labeled with a luminescent substance such as luciferase, luciferin, or aequorin is used. In addition, antibodies labeled with nanoparticles such as colloidal gold and quantum dots can also be detected.
In an immunoassay, an antibody against a molecule to be detected can be labeled with biotin and detected by binding avidin or streptavidin labeled with an enzyme or the like.

Among immunoassays, the ELISA method using an enzyme label is preferable because it can easily and rapidly measure an antigen.
The ELISA method includes a competitive method and a sandwich method. In the competitive method, an antibody against a detection target molecule is immobilized on a solid phase carrier such as a microplate, and a urine sample and an enzyme-labeled detection target molecule are added to cause an antigen-antibody reaction. Once washed, it reacts with the enzyme substrate, develops color, and the absorbance is measured. If there are many molecules to be detected in the urine sample, the color development becomes weak. If there are few molecules to be detected in the urine sample, the color development becomes strong. Therefore, the amount of the molecule to be detected can be obtained using a calibration curve.

  In the sandwich method, an antibody against a molecule to be detected is immobilized on a solid phase carrier, a urine sample is added and reacted, and then an antibody against another molecule that recognizes another epitope labeled with an enzyme is added and reacted. . After washing, it reacts with the enzyme substrate, develops color, and measures the absorbance, whereby the amount of the molecule to be detected can be determined. In the sandwich method, an antibody immobilized on a solid phase carrier is reacted with a molecule to be detected in a urine sample, an unlabeled antibody (primary antibody) is added, and an antibody against the unlabeled antibody (secondary antibody) is labeled with an enzyme. Further, it may be added.

  In the present specification, the “solid phase carrier” is not particularly limited as long as it is a carrier capable of immobilizing an antibody, glass, metal, resin-made microtiter plate, substrate, beads, nitrocellulose membrane, nylon membrane, A PVDF membrane etc. are mentioned.

  When the enzyme is peroxidase, the enzyme substrate for detecting the enzyme label is 3,3'-diaminobenzidine (DAB), 3,3 ', 5,5'-tetramethylbenzidine (TMB), o-phenylenediamine (OPD), etc. In the case of alkaline phosphatase, p-nitrophenylphosphate (NPP) or the like can be used.

In the immunoassay, an agglutination method is also preferable as a method for easily detecting a trace amount of protein. Examples of the aggregation method include a latex aggregation method in which latex particles are bound to an antibody.
When the antibody against the molecule to be detected is bound to the latex particle and mixed with the urine sample, the antibody-bound latex particle is aggregated if the molecule to be detected is present. Thus, the concentration of the antigen can be determined by irradiating the sample with near-infrared light and quantifying the aggregate by measuring the absorbance (turbidimetric method) or the scattered light (Hipple method).

As used herein, “antibody” refers to an immunoglobulin that specifically binds to an antigen, and may be any subclass of IgM, IgG, IgA, IgE, and IgD. Moreover, as long as it couple | bonds specifically with an antigen, fragments, such as Fab, F (ab ') ₂ , Fab', scFv, di-scFv, may be sufficient.
Both monoclonal and polyclonal antibodies can be produced according to known methods. Monoclonal antibodies can be obtained by, for example, isolating antibody-producing cells from a non-human mammal immunized with a molecule to be detected or a fragment thereof, and fusing them with myeloma cells to produce a hybridoma and purifying the antibody produced by this hybridoma. Can be obtained. A polyclonal antibody can be obtained from the serum of an animal immunized with a molecule to be detected or a fragment thereof.

  An existing antibody may be used as an antibody against FABP4. Examples of the anti-FABP4 antibody include an anti-FABP4 antibody (manufactured by BioVendor) and an anti-FABP4 antibody (manufactured by Abcam).

  Moreover, the immunoassay in this invention can also be performed using a commercially available immunoassay kit.

As used herein, “glomerular disorder” is used in its broadest sense and refers to a pathological condition in which the glomerulus is damaged regardless of the cause. Glomerular disorders include primary due to disorders of the glomerulus itself and secondary due to other systemic diseases.
Primary glomerular disorders include primary nephrotic syndrome, primary mild proteinuria in which the amount of urinary protein does not meet the diagnostic criteria for nephrotic syndrome, and primary glomerulonephritis.
Secondary glomerular disorders include secondary nephrotic syndrome, secondary mild proteinuria in which the amount of urinary protein does not meet the diagnostic criteria for nephrotic syndrome, and secondary glomerulonephritis.

  In this specification, “nephrotic syndrome” is used in the broadest sense, and includes proteinuria (a state in which the amount of urinary protein per day is 3.5 g or more), hypoproteinemia (serum total protein amount) As long as it exhibits 6.0 g / 100 ml or less (in the case of hypoalbuminemia, the amount of serum albumin is 3.0 g / 100 ml or less), primary and secondary nephrotic syndrome are included regardless of the cause.

Examples of primary glomerular disorders include minimal change nephrotic syndrome (MCNS), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN), membrane Membranoproliferative glomerulonephritis (MPGN), acute glomerulonephritis, rapidly progressive glomerulonephritis and IgA nephropathy.
Secondary glomerular disorders include, for example, diabetic nephropathy due to diabetes, lupus nephritis due to collagen disease, renal amyloidosis due to amyloidosis, kidney due to infections such as HIV, HBV, leishmania Symptoms and the like.

  As used herein, “kidney dysfunction” refers to, but is not limited to, the disorders described above and disorders resulting therefrom. In addition, “prognosis of kidney function” means “renal dysfunction” that may occur in the future.

In the present specification, “examination” means examination of a sample collected from a subject in order to obtain information necessary for diagnosis, and the examination method of the present invention can be implemented by, for example, an examination company.
The test includes, but is not limited to, a test for examining the presence or absence, a test for examining the possibility of morbidity, a test for examining the progress or prognosis of the disease, and the like.
In addition, it is also possible to use for the diagnosis the result test | inspected using the test | inspection method concerning this invention. Specifically, a method of diagnosing the prognosis of kidney function based on the result of examination using the examination method according to the present invention can be mentioned.

  One aspect of the test method of the present invention includes a step of comparing the concentration (excretion amount) of the detection target molecule in the urine of the subject with the concentration (excretion amount) of the detection target molecule in the urine of the healthy subject. . When the concentration (excretion) of the molecule to be detected in the urine of the subject is significantly higher than the urine of the healthy subject, the subject may have glomerular disorder or may come in the future High nature.

  As one aspect of the test method of the present invention, when the amount of FABP4 in the urine of the subject is higher than a preset threshold with reference to the amount of FABP4 in the urine of the healthy subject, the subject It is determined that a glomerular disorder in is detected.

  The threshold value in the present invention can be set in advance according to race, age, and the like. Further, the threshold value can be set in advance by measuring the amount of FABP4 in the urine of a healthy person by the above-described method for detecting and measuring urinary FABP4 and referring to the measured value.

  Thus, according to the present invention, glomerular damage can be detected by using FABP4 in urine as a marker of glomerular damage.

  In the present specification, the “subject” may be a person who may or may have a glomerular disorder. In a medical examination or the like, a healthy person may be the subject. The subject is not limited to humans, and may be other mammals (mouse, rat, rabbit, dog, cat, cow, horse, pig, monkey, etc.).

[Method for differentiating glomerular and tubular disorders]
The present invention includes a method for differentiating between glomerular disorders and tubular disorders, comprising a step of detecting a molecule to be detected in urine of a subject. That is, it includes a method for differentiating between glomerular disorders and tubular disorders based on the results obtained by the examination method of the present invention.
In the present specification, “differentiation” means that the amount of FABP4 contained in the urine of a subject having urine protein is the amount of FABP4 in the urine of a healthy subject based on the result obtained by the test method of the present invention. If it is equal to or greater than a preset threshold value, it is determined that the patient is suffering from glomerular disorder, and if it is less than the threshold value, it is determined that the patient is suffering from tubular disorder.

[Diagnosis of renal function prognosis]
The present invention includes a method for diagnosing prognosis of kidney function, comprising a step of detecting a molecule to be detected in urine of a subject. That is, it includes a method for diagnosing prognosis of kidney function based on the results obtained by the test method of the present invention.
As used herein, “diagnosis” refers to a method by which a doctor examines whether a patient has or is likely to suffer from a glomerular disorder based on the results obtained by the test method of the present invention, The method or the like of examining the progress or prognosis of, but not limited to.

[Monitoring method of glomerular injury]
The present invention includes a method for monitoring glomerular damage in a subject, comprising measuring the amount of FABP4 in the urine of the subject and monitoring the amount of FABP4 over time. In the monitoring method of the present invention, when the amount of FABP4 in the urine of the subject increases with time, it is determined that the glomerular disorder of the subject has progressed. One aspect of the glomerular disorder monitoring method of the present invention includes a method including the following steps.
First step: The amount of FABP4 in the urine of the subject is measured.
Second step: After the first step, the amount of FABP4 in the urine of the subject is measured continuously or discontinuously over time.
Third step: The difference between the measurement value in the second step and the measurement value in the first step ((measurement value in the second step) − (measurement value in the first step)) is calculated.
Fourth step: If the difference value calculated in the third step is negative, it is determined that the glomerular disorder is maintained or improved, and if the value is positive, the glomerular disorder is worsened. Judge that you are doing.
Further, in the above monitoring method, if the absolute value of the calculated difference value is large, it is determined that the degree of improvement or deterioration is large, and if the value is small, the degree of improvement or deterioration is small. to decide.
Such monitoring methods can be used to accurately grasp the progression of glomerular disorders and thus renal dysfunction, and to administer more appropriate drugs and appropriate treatments at more appropriate times. it can.

[How to improve]
In addition, according to another aspect of the present invention, in any one of the above methods, the thread further comprises a step of administering an effective amount of a glomerular disorder ameliorating agent to a subject having glomerular disorder. A method of improving sphere disorders is provided. Here, “improvement” includes not only the meaning of “treatment” of an established pathological condition, but also the meaning of “prevention” that is prepared in advance for an expected deterioration and prevents the occurrence of a disease in advance. .

  Although it does not specifically limit as a glomerular disorder improving drug, For example, a chemotherapeutic agent (such as a new quinolone antibacterial agent), an adrenal steroid (such as methylprednidolone), an immunosuppressant (such as cyclophosphamide, azathioprine, cyclosporin A) ), Anticoagulants (such as heparin, warfarin), antiplatelet drugs (such as dipyridamole, dilazep hydrochloride), fibrinolytic drugs (such as urokinase), anti-inflammatory drugs (such as non-steroidal anti-inflammatory drugs such as indomethacin), Examples include diuretics (such as furosemide and other loop treatments) or antihypertensive drugs. The effective amount and dosing schedule of the glomerular disorder ameliorating drug is appropriately determined by those skilled in the art in consideration of the sex, age, weight, symptom, etc. of the subject.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[Measurement of various parameters]
The subjects of the present example were 392 participants in the physical measurement and follow-up survey conducted by the inventors every year for residents in Hokkaido's Tanno Town and Sobetsu Town.

  In order to obtain the clinical background information of the subjects, each subject was subjected to physical measurements after 6-9 hours of fasting at night. Specifically, blood pressure was measured twice consecutively using an automatic sphygmomanometer (HEM-907, manufactured by OMRON), and the average was used for the analysis of this example. Also, the height and weight were measured, and the body weight (kg) was divided by the square of the height (m) to calculate the body mass index (BMI value). After physical measurement, peripheral venous blood and urine were collected from each subject and used as samples of this example. Biochemical analysis was performed on serum, plasma and urine samples immediately after collection or after storage at −80 ° C. The results of the clinical background of the subjects are shown in Table 1, and the results of biochemical analysis of the collected samples are shown in Table 2.

  In this example, the concentrations of FABP4 (S-FABP4) in serum and FABP4 (U-FABP4) in serum were measured using a commercially available enzyme immunoassay kit for FABP4 (RD191036200, manufactured by Biovendor R & D). Measured. The concentration of FABP1 (U-FABP1) in urine was measured using a commercially available enzyme immunoassay kit (CMIC001, manufactured by CIMIC Co.) of FABP1 (also known as L-FABP).

Moreover, the excretion amount of U-FABP4 and U-FABP1 was corrected (normalized) by the urinary creatinine level (μg / gCr).
Fasting plasma insulin concentrations were measured by radioimmunoassay. The concentration of creatinine and the concentration of lipids including total cholesterol, HDL cholesterol and triglycerides were determined using a general method using enzymes. The LDL cholesterol concentration was calculated using the Friedewald formula. The concentration of hemoglobin A1c (HbA1c) was determined using a latex agglutination method and expressed as an international standard value (NGSP value). High sensitivity CRP (hsCRP) was measured using a turbidimetric method. HOMA-R, which is an index of insulin resistance, has a known formula:
Insulin (μU / ml) x glucose (mg / dl) / 405
Was calculated using. The ratio of albumin to creatinine in urine (UACR; mg / gCr) was used as a marker for microalbuminuria. Estimated glomerular filtration rate (eGFR) is the equation for Japanese:
eGFR (mL / mn / 1.73m ² ) = 194 × Cr ^(-1.094) × age ^(-0.287) (for men)
eGFR (mL / mn / 1.73m ² ) = 194 x Cr ^(-1.094) x age ^(-0.287) x 0.739 (for women)
Was calculated using. In order to evaluate the decrease in eGFR after one year, the degree of change in eGFR was calculated by subtracting the eGFR value for one year from the eGFR value for the next year.

  Each calculated p was expressed as mean ± standard deviation or median (interquartile range). The distribution of each parameter was tested for normality by the Shapiro-Wilk test, and the parameters of the non-normal distribution were logarithmically transformed. Parameter differences between the two groups were tested by unpaired t-test. The correlation between the two parameters was evaluated using Pearson's correlation coefficient. One-way analysis of variance and Tukey-Kramer post hoc test were used to detect significant differences in the three groups of data.

  Multiple regression analyzes were performed to identify independent determinants (explanatory variables) for U-FABP4, UACR and the degree of change in eGFR after one year. A p-value of less than 0.05 was considered statistically significant.

  Of the 392 subjects, 166 were male and 226 were female, with gender differences in girth and prevalence of dyslipidemia and stroke. In addition, 123 subjects did not receive any medication.

  Biochemical analysis showed gender differences in total cholesterol level, HDL cholesterol level, LDL cholesterol level, glucose (blood glucose) level and creatinine level. Moreover, the serum FABP4 (S-FABP4) concentration was significantly higher in women than in men. Moreover, about the amount of urinary FABP4 (U-FABP4) excretion, the significant difference by gender was not recognized. Furthermore, in this measurement, U-FABP4 was below the detection limit in 23.7% of subjects.

  Next, about 93 subjects who were below the detection limit for U-FABP4 (U-FABP4 undetectable group) and 299 subjects who were above the detection limit (U-FABP4 detectable group) Each clinical index was compared. The results are shown in Table 3.

  In the U-FABP4 detectable group, the age was significantly higher than in the undetectable group.

  Next, biochemical analysis was performed on the U-FABP4 detectable group and the undetectable group. The results are shown in Table 4.

In the U-FABP4 detectable group, eGFR was significantly lower than that in the undetectable group. This suggested a decrease in kidney function in subjects with high urinary FABP4 excretion. In the U-FABP4 detectable group, the S-FABP4 concentration was significantly higher than that in the undetectable group. From this, it was suggested that the amount of FABP4 excreted in urine reflects the concentration of FABP4 in serum.
Furthermore, in the urinalysis, the ratio of albumin to creatinine in urine (UACR) was significantly higher in the U-FABP4 detectable group than in the undetectable group. That is, it was shown that more protein is excreted in urine in subjects with high urinary FABP4 excretion. This suggested a decrease in glomerular function in subjects with a high urinary FABP4 excretion.

  Next, in the U-FABP4 detectable group, the relationship between urinary FABP4 excretion and each clinical index was analyzed by simple regression analysis and multiple regression analysis using the stepwise method. The results are shown in Table 5.

In the U-FABP4 detectable group, U-FABP4 excretion showed a significant negative correlation with blood insulin concentration. On the other hand, U-FABP4 excretion showed a significant positive correlation with age, systolic blood pressure, neutral fat, HbA1c, UACR, S-FABP4 and U-FABP1. In the multiple regression analysis using the stepwise method, the absolute value of the t value exceeded 2 and the p value fell below 0.05 for age, S-FABP4, UACR and U-FABP1. Therefore, these parameters were shown to have a strong positive correlation with the amount of U-FABP4 excretion, and it became clear that they could be adopted as independent explanatory variables for U-FABP4 excretion.
In addition, by subsequent multiple regression analysis, age, S-FABP4 concentration, UACR and U-FABP1 excretion amount independently correlated with U-FABP4 excretion amount, and 40.2% explanation (R ² = 0. 402). Of particular note is that U-FABP4 excretion independently has a strong positive correlation with UACR. That is, it was shown that more protein is excreted in urine in subjects with high urinary FABP4 excretion. This strongly suggested a decrease in glomerular function in subjects with high urinary FABP4 excretion.

  In the U-FABP4 detectable group, simple regression analysis and multiple regression analysis using the stepwise method were performed for the relationship between the ratio of albumin to creatinine in urine (UACR) and each clinical index. The results are shown in Table 6.

In the U-FABP4 detectable group, the ratio of albumin to creatinine (UACR) in urine was determined by age, systolic blood pressure, diastolic blood pressure, triglyceride (neutral fat), glucose (blood sugar), HbA1c, blood urea nitrogen (BUN). ), EGFR, S-FABP4 concentration, U-FABP1 excretion and U-FABP4 excretion were significantly positively correlated. Moreover, in the multiple regression analysis using the stepwise method, the absolute value of t value exceeded 2 and systolic blood pressure, HbA1c, and U-FABP4 excretion amount, and p value was less than 0.05. Therefore, these parameters were shown to be strongly correlated with UACR, and it became clear that they could be adopted as independent explanatory variables for UACR. However, U-FABP1 excretion was not adopted. Furthermore, the adopted systolic blood pressure, HbA1c and U-FABP4 excretion parameters were shown to explain UACR 21.2% (R ² = 0.212). That is, it became clear that systolic blood pressure, HbA1c and U-FABP4 excretion reflect the albumin ratio (UACR) to creatinine in urine.
Furthermore, U-FABP4 excretion was an independent explanatory variable for UACR, even after correcting for the effects of age, sex, systolic blood pressure, HbA1c, eGFR, S-FABP4 and U-FABP1. Of particular note is that U-FABP4 excretion can be adopted as an independent explanatory variable for UACR even after such correction. That is, it was shown that U-FABP4 excretion has a strong positive correlation with UACR. This means that more protein is excreted in the urine in the subject having a high amount of FABP4 excreted in the urine. Therefore, in subjects with high urinary FABP4 excretion, the decrease in glomerular function was strongly suggested.

  In addition, the correlation between U-FABP4 excretion and UACR was examined for 299 people in the U-FABP4 detectable group. The results are shown in FIG.

  The result of FIG. 1 also showed that the amount of U-FABP4 excretion and UACR have a positive correlation. That is, it means that more protein is excreted in urine in a subject having a high amount of FABP4 excreted in urine. This also strongly suggests that the function of the glomeruli is reduced in subjects with high urinary FABP4 excretion.

From the above results, it can be seen that the higher the amount of urinary FABP4 excreted, the higher the amount of protein (albumin) excreted in urine. That is, it is suggested that the higher the amount of FABP4 excreted in urine, the higher the possibility that glomerular damage has occurred. Therefore, by detecting and / or measuring the amount of FABP4 excreted in urine, it becomes possible to diagnose the presence or absence of glomerular disorder and the possibility of morbidity. Similarly, it is possible to differentiate between glomerular disorders and tubule disorders.
Since glomerular disorders are directly or indirectly linked to renal dysfunction, detection and / or measurement of urinary FABP4 may indicate whether or not kidney dysfunction is affected Can also be diagnosed.

[Tracking survey]
Of the 392 subjects who had undergone the first examination, 325 persons were subjected to a second examination (re-examination) one year later. Depending on the measurement value (baseline) at the time of the first examination of the U-FABP4 excretion amount, the re-examination subjects are classified into low U-FABP4 (T1), medium U-FABP4 (T2) and high U-FABP4. Divided into three groups (T3). The subjects who did not detect U-FABP4 at the first examination were classified as T1.

The composition of each group was as follows. About U-FABP4
Low U-FABP: T1 <0.04 (μg / gCr) 108 out of 108 U-FABP: 0.04 (μg / gCr) ≦ T2 <0.30 (μg / gCr) 108 High U-FABP: T3 ≧ 0.30 (μg / gCr) 109 people.

The eGFR in each group of U-FABP4 was measured, and the change from the eGFR at the first examination was evaluated based on the following formula.
(Change in eGFR) = (eGFR value at the time of reexamination) − (first eGFR value)
The results are shown in FIG.

  As shown in FIG. 2, the decrease in eGFR was significantly greater in the high group (T3) than in the group (T1) where the baseline U-FABP4 excretion was low.

  The degree of change in eGFR after 1 year and the baseline excretion of U-FABP4 had a negative correlation (r = −0.140, p = 0.011). That is, it became clear that the decrease in eGFR was larger as the baseline excretion amount of U-FABP4 was larger.

  From this result, it can be seen that the higher the amount of FABP4 excreted in urine, the greater the decrease in eGFR after 1 year. That is, it is suggested that the higher the amount of FABP4 excreted in urine, the more the glomerular damage progresses with time and the worse the prognosis. Therefore, by detecting and / or measuring the amount of FABP4 excreted in urine, it is possible to diagnose the progress of glomerular injury and its prognosis. Since glomerular damage is directly or indirectly linked to renal dysfunction, the prognosis of renal function can be diagnosed by detecting and / or measuring the amount of FABP4 excreted in urine. It becomes possible.

Claims (10)

A method for the inspection of glomerular disorder, comprising the step of detecting a fatty acid binding protein 4 in the urine of a subject (FABP4), Methods. Detecting said FABP4 is performed by immunoassay using an antibody against FABP4, methods who claim 1. Wherein the immunoassay is an ELISA method, method who claim 2. The glomerular disorder is primary or secondary, Method person according to any one of claims 1 to 3. Whether diseased glomerular disorders, morbidity, a way to diagnose the progress or prognosis method towards according to any one of claims 1-4. A method for differentiating between glomerular injury and renal tubular disorder results obtained by way of any one of claims 1 to 5 is used, METHODS. A method for diagnosis of prognosis of kidney function, the results obtained by way of any one of claims 1 to 5 is used, METHODS. A method for determining the progression of glomerular injury in a subject , comprising measuring the amount of FABP4 in the urine of the subject. A test kit of glomerular injury ing contains antibodies to FABP4, said antibody is used for detection of FABP4 in urine of a subject, the kit for testing. Use of FABP4 in the urine of a subject as a test marker for glomerular injury.