WO2020035033A1 - Biomarker calb1 for diagnosing early stages of diabetic nephropathy - Google Patents

Abstract

The present invention provides a biomarker for diagnosing the early stages of diabetic nephropathy and a method for diagnosing early stages of diabetic nephropathy. The biomarker is CALB1 contained in urine extracellularvesicles (urinary EVs) having an expression level in the early stages of diabetic nephropathy significantly higher than that of normal people. The diagnostic method of the invention is non-invasive and has great application prospects.

Description

Biomarker CALB1 for diagnosing early stages of diabetic nephropathy Technical field

The invention relates to the field of protein detection, in particular to the detection of biomarkers in the early stages of diabetic nephropathy.

Diabetes has become a global public health problem. In 2015, the International Diabetes Federation (IDF) announced that China has 109.6 million diabetic patients, most of whom are type 2 diabetes. Impaired fasting blood glucose and / or abnormal glucose tolerance occur before their blood glucose reaches the diagnostic criteria for diabetes, saying It is called pre-diabetes (also known as pre-diabetes). Although these patients do not have a significant increase in blood sugar, organ damage has begun to occur, so it is of great significance for their early diagnosis and can prevent the complications of diabetes more effectively. Diabetic nephropathy and renal failure are common complications of diabetes. At present, the diagnosis of diabetic nephropathy depends on a biopsy of the kidney, but this is a risky and invasive test. Microalbuminuria can diagnose diabetic nephropathy stage 3, but when diabetic nephropathy is still in stage 1,2, microalbumin has not yet appeared in urine. At this time, how to diagnose early diabetic nephropathy is still an international research in the field. The key and difficult points. At present, there is no recognized urine biomarker that can diagnose diabetic nephropathy early before the emergence of microalbuminuria.

Urine extracellular vesicles (EVs) are derived from a variety of cells throughout the urogenital tract, are released into the urine, and can serve as biomarkers for renal dysfunction and structural damage.

Diabetic nephropathy (DN) is a major complication of diabetes (DM) and significantly increases cardiovascular risk and related mortality. Urine extracellular vesicles (EVs) are considered not only as a rich non-invasive source of kidney damage markers, but also as an important marker source of systemic organ or vascular damage. The authors of the present invention sought to verify that certain proteins in urinary EVs may be associated with diabetic nephropathy.

Under physiological conditions, there are a large number of high-abundance proteins in the urine, such as Tamm-Horsfall protein, while the urine proteins that can reflect early information about the disease are often low-abundance. It is difficult to be affected by other high-abundance proteins Detected specifically and sensitively. Significantly, many of these urine proteins that reflect early disease information exist in urine EVs. If we can find a scientific and effective method for enriching urine EVs, then detect the proteins in urine EVs that can reflect early disease information. Can overcome the effects of high abundance protein in urine. In the study of the present invention, we tried to find and verify an important biomarker in urine EVs, which can be finally evaluated or monitored to achieve the purpose of early diagnosis of DN.

Summary of the Invention

The object of the present invention is to provide a protein contained in urine EVs, which is used for early diagnosis of early stages of diabetic nephropathy before microalbumin appears in urine.

We hypothesized that different proteins could be found in the urine EVs of patients with diabetic nephropathy (DN), especially before the emergence of microalbuminuria. In this study, a large number of urine samples used in the study of urinary EVs in patients with DN were obtained in a community molecular epidemiological study supported by the ISN “KHDC” project in the southern community population. In this study, two-dimensional electrophoresis was used to separate urine EVs from patients with DN and healthy subjects, and their protein components were analyzed and quantitatively compared by MALDI TOF / MS analysis.

1980 protein spots were isolated from the urine EVs of patients by electrophoresis, and 40 protein spots were significantly different from the NC group by at least 1.5 times. Mass spectrometry identification of the above 40 protein spots revealed that 22 of them may be closely related to diabetic nephropathy.

A literature search was performed on the above-mentioned differential proteins, and a full search of urine CALB1 (Calb1 calbindin 1, NCBI accession number P05937, molecular weight 30291.1, isoelectric point 4.7), which accounted for the majority of kidney diseases (including glomeruli) Nephritis, diabetic nephropathy) No report on urine CALB1 was found. The literature related to urine CALB1 mainly focuses on idiopathic hypercalciuria. The above search results show that in the glomerular diseases that account for the vast majority of kidney diseases, including diabetic nephropathy we studied, there is no report of urine CALB1 in the world.

CALB1 is a vitamin D-dependent calcium-binding protein (CaBP), which is localized in distal tubules and renal tubules. Lee, CT, Lien, YH, Lai, LW, Chen, JB, et al .. Increasing calcium and magnesium transporter abundance instreptozotocin-induced diabetes mellitus. KIDNEY INT 2006; 69: 1786-1791. (STZ) Studies of induced diabetic rats have shown that CALB1 messenger RNA is increased in the renal cortex and medulla. The mechanism of how CALB1 mediates calcium metabolism is unclear. Insulin administration can effectively correct hyperglycemia-related hypercalciuria and reverse the increase in calcium transporter abundance in STZ-induced diabetic rats. Therefore, the increase in CALB1 may be a compensation mechanism that offsets the high calcium load in DN kidneys. Our study for the first time reported an increase in expression of CALB1 in urinary EVs in patients with diabetes and diabetic nephropathy.

Beneficial effect: The present invention provides a urine extracellular vesicle containing a marker CALB1, which can be used for non-invasive diagnosis of early diabetic nephropathy.

Specification attached

Figure 1. Changes in the expression of CALB1 in urine EVs in five groups of test subjects

detailed description

The present invention is further explained below with reference to specific examples, but the scope of the claims of the present invention is not limited. The reagents used in the present invention are commercially available unless otherwise specified.

Example 1. Transmission electron microscope (TEM) and nanoparticle trajectory analysis (NTA) of urine EVS

Participants were divided into 5 groups: healthy control group (NC, n = 15), pre-diabetes group (PreDM, n = 15), diabetes group with normal proteinuria level (DM, n = 15), microalbuminuria diabetes Group (DM-micro, n = 15) and large albuminuria diabetes group (DM-macro, n = 15). Urine was collected from all participants on the first morning. Using our previously established methods (Musante, Saraswat, M, Ravida, A, Byrne, B, et al .. Recovery of northerninovesicles from ultracentrifugation supernatants. Nephrol Dial Transplant 2013; 28: 1425-1433.), Using hydrostatic dialysis methods For urine EVS enrichment in urine.

Specifically, 100 ml of morning urine of a research subject was collected without a protease inhibitor. After low-speed centrifugation, the supernatant was collected and stored in a -80 ° C refrigerator.

HETTICH CENTRIFUGE universal 320 (UK) centrifuge, 2000g RCF, centrifuged at room temperature for 30min to remove T-H protein, cell debris and other sediment. Collect the supernatant.

Nanofiltration ultrafiltration and concentration of samples: A self-made nanomembrane concentrating device was used, which was connected to a 1000kDa nanomembrane, washed with SDS, and washed with miliQ water. Checked that there were no leaks or air leaks. After the device was intact, it began to process samples, processing eight samples each time The urine of all diabetic and normal controls was treated in several batches. During the process of concentrating the sample by the nanometer film, closely observe whether the device is running perfectly. If there is a liquid leakage phenomenon, replace the new nanometer film in time and reprocess the sample. Our nano-membrane concentrator is running perfectly, and this phenomenon has not happened yet. When ultrafiltration reaches 6-8ml, add 10-20ml miliQ water, collect the filtrate, repeatedly concentrate and filter three times, then add 200ml miliQ pure water, and continue to concentrate ultrafiltration. When the urine is concentrated to 3-5ml, collect the sample and save it for later use.

Urine urine EVs from 5 groups were identified by transmission electron microscopy (TEM). Nanoparticle trajectory analysis was performed, and the NanoSight NS300 camera equipped with sCMOS was used to estimate the number and size distribution of urine EVS. Five micrograms of urine EVs proteins were separated using SDS-PAGE in the Bradford protein assay. The gel was transferred to a polyvinylidene fluoride (PVDF) membrane, incubated with specific rabbit anti-human antibody TSG101, and then incubated with a horseradish peroxidase-conjugated secondary antibody. Finally, observe the samples with Kodak IS 4000R image station.

The results show that urine EVs have a uniform distribution, exhibit a membrane-encapsulated structure, and are cup-shaped or round with a diameter of 40-100 nm. A more comprehensive urinary EVs size analysis was performed for each group using NTA. The curve shows that the main peak is between 55 and 110 nm.

Example 2. Protein electrophoresis and mass spectrometry identification of urine EVs obtained in Example 1

The internal standard protein (TSG101) in each sample was labeled in equal amounts using Cy2. Proteins from the control group and patients were labeled with cy3 and cy5 dyes, respectively. IPG ImmobilineTM Drystrip (24cm, pH 4-7) was used for isoelectric focusing (IEF) and IPGphor (GE Healthcare) was performed at 1000V (2 hours) gradient; 8000V (2 hours); in the dark The two-dimensional electrophoresis program (2D-DIGE) was performed at 17 W / gel for 5-6 hours. The gel was scanned with a Typhoon 9410 scanner (GE Healthcare). Ettan SpotPicker (GE Healthcare) was used to extract the difference protein points (| ratio |> 1.5) that consistently changed between the two groups (preDM, DM, DM-micro and DM-macro compared to NC) in all of these protein point maps , P <0.05), followed by thorough washing in ultrapure water and in-gel trypsin digestion. Protein identification was performed by ABI 4800 Proteomics Analyzer MALDI-TOF-MS (Applied Biosystems). The GlobalProteome Server Explorer software (Applied BioSystems) was used to search the mass spectrometry identification in the SwissProt database for protein identification.

Comparative data analysis was performed using the ExoCarta database (www.exocarta.org). Gene ontology software (GO) was used to analyze the molecular functions and biological processes of these identified proteins. By searching the references, some potential biomarkers that were not found as diabetes and diabetic nephropathy were finally screened.

The results are shown in Figure 1. Western blotting showed that TSG101 existed in the five groups, but the intensity of TSG101 in the DM, DM-micro and DM-macro groups was significantly stronger than that in the NC and PreDM groups. Perhaps this may be due to the fact that diabetic patients with and without kidney damage secrete more urine EVS than NC and PreDM patients.

And two-dimensional DIGE (2D-DIGE) analysis showed that in 5 groups, compared with the NC group, there were nearly 40 proteins in 1980 protein spots between the PreDM, DM, DM-micro and DM-large groups. A significant 1.5-fold difference, | ratio |> 1.5, p <0.05. The aforementioned differential protein spots were selected for identification by mass spectrometry. According to our analysis, 22 specific proteins were selected for the next data analysis, as shown in Table 1 below.

Table 1: Differential proteins

The data was analyzed by the ExoCarta, Gene Ontology database to reveal proteomics consistent with urinary EVS, especially urine origin and associated with diabetic nephropathy. According to the ExoCarta database, all identified differential proteins have been previously reported in normal urine EVs. As shown in Figure 1, it was found that CALB1 showed increased expression. Calb1calbindin1, NCBI accession number P05937, molecular weight 30291.1, isoelectric point 4.7, a literature search was performed, and no correlation between CALB1 in urine EVs and diabetes / diabetic nephropathy was found. . The newly found urine EVs containing CALB1 can be used to identify "pre-microalbuminuria" in early renal damage in diabetic patients.

Claims (5)

1. A biomarker for diagnosing diabetic nephropathy at an early stage, the biomarker is CALB1 contained in urine EVs, and its NCBI accession number is P05937.
2. Application of CALB1 contained in urinary EVS in the early stage of diagnosing diabetic nephropathy, the CALB1 expression level is significantly increased relative to normal people.
3. The application according to claim 2, wherein the CALB1 expression level is increased by more than 30% relative to a normal person.
4. The application according to claim 3, wherein the CALB1 expression level is increased by more than 50% relative to a normal person.
5. The application according to claim 4, wherein the CALB1 expression level is increased by more than 100% relative to a normal person.

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