PL237810B1 - Method for detecting kidney damage, occurring in diabetics - Google Patents

Description

The present invention relates to a method of detecting kidney damage appearing in diabetic patients by examining the presence of galectin-3 in urine-derived microbubbles (UEV). The invention should find application in therapy, especially in the early clinical diagnosis of diabetes complications.

Galectin-3 (gal-3) is an animal lectin protein belonging to the family of β-galactoside sugar antenna binding proteins. A characteristic feature of galectins is that they all have a characteristic domain of approx. 130 a.a. recognizing sugars - CRD (Carbohydrate Recognition Domaine) [1]. Currently, 15 different galectins have been identified, which have been classified into 3 groups: tandem (with 2 CRD repeats), which include galectins -4, -6, -8, -9 and -12; prototype (galectin -1, -2, -5, -7, -10, -11, -13, -14 and -15) with one CRD domain and a unique chimeric group having one CRD domain and a long N-terminal domain rich in proline and glycine, which includes gal-3 [2]. In addition, a new group of animal galectins containing 4 CRD domains have recently been discovered. Galectin binds preferentially to glycans that contain N-acetylgalactosamine chains. Gal-3 is composed of 2 domains: the C-terminal domain with the CRD sequence and the N-terminal domain with the phosphorylation site (S6) [3]. The N-terminal domain is composed of 110-130 amino acids, contains a number of homologous 9-amino acid repeats (P, G, T, E), is highly conserved, shows significant homology to heterogeneous nuclear ribonucleoprotein complexes and to the collagen chain (a1) and it is responsible for creating multimeters [4]. In mammals, the N-terminal fragment 1-12 is responsible for the secretion of gal-3 [5]. In the human genome, gal-3 is encoded by the LGALS3 gene, which is located on chromosome 14 in position q21-q22, it consists of 6 exons and 5 introns [6]. LGALS3 expression is variable and is influenced by various internal and external factors. In the cell, it is located both in the cell nucleus and in the cytoplasm, is secreted and can be found on the surface of cells as well as in the intercellular spaces of extracellular vesicles [7]. Gal-3 is expressed in epithelial cells of organs such as lungs, kidneys, epithelium of the small intestine, colon, conjunctiva, cornea and prostate. It is also expressed in myeloid cells, osteoblasts, osteoclasts, keratinocytes, cells of the immune system (neutrophils, eosinophils, basophils, mast cells and dendritic cells, monocytes and macrophages) [2].

The biological activity of gal-3 depends on several factors: localization and phosphorylation on S6 (rarely on S12), which is fundamental for intracellular localization (transport from the nucleus to the cytoplasm) and affinity for ligand (extracellular matrix proteins - ECM) [8.9]. Importantly, gal-3 phosphorylation favors its anti-apoptotic activity [10]. Extracellular gal-3, through its affinity for β-galactosides, binds to numerous ECM proteins, undergoes homodimerization with proteoglycans, and forms pentamers in combination with oligosaccharides [11]. Gal3 does not have a specific receptor or ligand, hence its pleiotropic mechanism of influencing biological processes: both at the extracellular and intracellular levels, which is related to its localization [12]. Fig. 1 presents a diagram showing the participation of galectin-3 in biological processes depending on its localization [12].

Due to the regulation of adhesion processes, the presence of gal-3 affects the migration and polarization of cells, mainly of the epithelium of the urinary system, both in embryonic development and in kidney regeneration [2,8,13,14]. Gal-3 is characterized by differential expression in individual regions of the kidney, normally in the adult kidney, gal-3 expression is restricted to the collecting tubules and the cilia base, and may be temporarily expressed in the proximal tubules during regeneration [15,16]. In an injured kidney, the expression profile of galectin changes. Studies on a rat model showed an increase in gal-3 expression in situ (in kidneys and macrophages) from 2 to 48 hours after toxic acute kidney injury, and a decrease in its expression 28 days after injury [14]. The increase in the expression of gal-3 in macrophages is of key importance for the processes of remodeling and renal fibrosis that occur as a result of kidney damage, on the one hand, gal-3 has a protective anti-apoptotic effect, and on the other, it stimulates proliferation of fibroblasts [17,18].

Accumulating evidence suggests that increased gal-3 expression has a detrimental effect on the cardiovascular system. In patients with chronic heart failure (CHF), Gal-3 concentrations are significantly increased in both saliva and serum, suggesting its usefulness as a marker in the diagnosis of CHF [19, 20].

PL 237 810 B1

Undoubtedly, gal-3 plays an important role in diabetes. It has the properties of binding advanced glycation endproducts (AGEs) that arise in hyperglycemia, participates in the internalization of AGEs and their degradation [21]. Moreover, it participates in the regulation of the expression of AGE receptors (RAGE) and the internalization of oxidized lipids. Therefore, gal-3 deficiency leads to accelerated aging induced by the accumulation of AGEs and oxidized lipids, thereby increasing the glomerular susceptibility to diabetes-induced damage [22].

Gal-3, along with cardiac troponins (cTn) and brain natriuretic peptide (BNP), has been recognized as a marker of heart disease, mainly as a prognostic marker of post-infarction heart failure [23, 24]. The indication for gal-3 determination was included in the American cardiological guidelines (ACCF / AHA) for risk stratification in patients with acute and chronic heart failure (CHF) [25].

There are few reports on the use of gal-3 as a potential marker of renal failure and the conclusions are inconclusive.

A child study for the Framingham study showed that increased levels of gal-3 in plasma are associated with an increased risk of a sharp decrease in the renal filtration index (GFR) and the development of chronic kidney disease (CKD) [26].

It has been shown that serum gal-3 level correlates inversely with kidney function expressed as GFR in both CHF and non-HF patients [27].

A case-control study showed that urinary gal-3 concentrations are not increased in patients with HF, despite a significant increase in plasma gal-3 concentrations. The authors conclude that impaired renal function in patients with heart failure may explain the described relationship between renal function and gal-3 concentrations [28].

An independent relationship between serum gal-3 concentrations and microalbuminuria, which is an early marker of altered renal function, suggests a possible role of gal-3 in the development of cardiorenal syndrome in CHF patients [29].

Negative study showing that there is no association with serum gal-3 concentrations and urinary albumin concentrations in patients with type 2 diabetes [30].

Increased concentration of gal-3 is associated with a decrease in eGFR and positively correlates with the concentrations of neuropeptides: NT-proBNP, proANP, chromogranin A and copeptin [31].

Microbubbles or extracellular vesicles (EVs) are tiny, spherical membrane structures with a diameter of 20-1000 nm, released into the intercellular space [32]. The term refers to both microbubbles that bud directly from the cell membrane during the life cycle of the cell - ectosomes (microvesicles) or during programmed cell death - apoptotic blebs, as well as intracellular vesicles - exosomes released from multivesicular bodies [33]. Despite the different size characteristics (exosomes: 30-100 nm, ectosomes: 100-1000 nm, apoptotic vesicles: 500-1000 nm), microbubbles have many common features, such as: lipid bilayer, proteins and nucleic acids inside these structures [32, 34, 35]. The content (cargo) transported by microbubbles, which reflects the status of the secreting cell, is transferred to the target cell - it actively participates in intercellular signaling [36]. EVs can be obtained from all body fluids (e.g. blood, urine, saliva, cerebrospinal fluid) and their analysis may replace invasive and expensive traditional biopsy in the future. In the case of diseases of the genitourinary system, diagnostics may also be carried out on the basis of the analysis of urine extracellular microvesicles (UEVs) [37, 38, 39].

In view of the above-described prior art, there is still a need to provide a method allowing the early diagnosis of kidney damage arising in diabetic patients.

Surprisingly, such a method was achieved in the present invention.

The subject of the invention is a method of detecting kidney damage appearing in diabetic patients, characterized in that a urine sample from a diabetic patient is tested for the presence of gal-3 protein in urine microbubbles (UEV), and the presence of this protein indicates kidney damage. , where: a) UEV microbubbles are isolated from the urine sample, b) proteins are isolated from the obtained UEV microbubbles, c) the composition of the proteins obtained is analyzed to determine the presence of gal-3 protein,

The kidney damage is manifested by an abnormal renal glomerular filtration rate (GFR) below 60 mL / min / 1.73 m ² .

Preferably, in step a) the urine sample is subjected to ultracentrifugation, preferably at 150,000 xg for about 90 minutes. It is also possible to use another technique for isolating the MVs. For example, it is preferable to pre-concentrate the urine by filtering. Other ultracentrifugation conditions are also possible, for example the sample may be ultracentrifuged at 150,000 xg for 60 min or 100,000 xg for 90 min. Preferably, in step b) the resulting microbubbles are treated with a solution containing 10% SDS, 1M TRIS-HCl at pH = 8.0 and deionized water, and after determining the protein concentration, DTT is added to the solution to obtain a final concentration of 0.1 M and denatures samples at 99 ° C for 5 minutes.

Preferably, in step c) the gal-3 protein is identified by means of a nano-liquid chromatograph coupled to a time-of-flight mass spectrometer with a matrix-assisted laser desorption and ionization technique of the sample. The Gal-3 protein can also be identified in other ways: e.g., preliminary separation of the protein by electrophoresis, not only by nano-liquid chromatography; identification by various mass spectrometry techniques (MS, quadrupol etc); or identification using specific antibodies, especially enzyme immunoassay: ELISA and Western Blot.

Surprisingly, the work leading to the present invention has established that the presence of gal-3 containing microbubbles in the urine may result from damage to the kidney cells, especially as a result of diabetes.

The method according to the invention makes it possible to obtain, irrespective of the state of diabetes treatment, a reliable diagnostic indicator allowing the determination of kidney damage, which is probably a diabetic complication.

P r z k ł a d 1

Diagnosing kidney damage appearing in diabetic patients

Description of the study group.

The study group consisted of patients with regular (CD) and unregulated (UD) type 2 diabetes, in whom renal failure (RF) occurred, 6 people in total, and 1 healthy person from the control group (C). The criterion for inclusion in the CD group was diagnosed and treated diabetes with the level of glycated hemoglobin (HbA1C) <7%, in the UD group the level of HbA1C> 7%, according to the 2016 PTD guidelines [40]. RF was defined as the value of the glomerular filtration rate (GRF) calculated according to the MDRD2 formula below 60 mL / min / 1.73 m ² .

In addition, a medical history was prepared, which included information on demographic data, blood pressure, BMI (body mass index), eating habits and addictions (smoking, alcohol consumption), as well as the type of antidiabetic treatment and medications (diuretics: potassium-sparing, loop, thiazide and thiazide-like, β-blockers, ACEI, Ca antagonists, Na / K ATPase inhibitors, vasodilators, clopidogrel, acetylsalicylic acid, statins), information on surgeries and procedures, incl. Coronary artery bypass graft (CABG) and percutaneous coronary intervention (PCI), the presence of comorbidities and cancers. Patients with the following findings during the examination or interview were excluded from the study: - presence or suspicion of a fresh bacterial or viral infection, - neoplastic diseases,

- previous cardiovascular events or cardiac surgery in the 6 months preceding the interview,

- features of liver damage,

- steroid and non-steroidal anti-inflammatory therapy,

- surgical treatment of obesity,

- the use of hormone replacement therapy in the case of women,

- autoimmune diseases (i.e. chronic arthritis, antiphospholipid syndrome). Concentration and isolation of UEVs microbubbles from urine samples.

The morning urine in the amount of approx. 250 ml was collected from the patients and the control group. Initial urine was centrifuged at 2,000 g 30 min to remove epithelial cells, bacteria and pellet. Approximately 5 mL of urine was collected from the supernatant and subjected to ultracentrifugation in an Optima ™ MAX-XP centrifuge (Beckman Coulter Life Sciences, Indianapolis, USA) using a horizontal rotor (MLS-50 Rotor, Swinging Bucket, 4 x 5.0 mL) 150 000 xg at 4 ° C for 90 min. The supernatant after ultracentrifugation was decanted and added to the pellet

5 mL of urine again. Centrifugation was repeated. The resulting pellet was washed with 5 mL of phosphate buffered saline without Ca ²⁺ and Mg ²⁺ (PBS without Ca ^{2+ /} Mg ²⁺ ).

Assessment of the presence of UEVs in transmission electron microscopy (TEM).

The pellet samples were centrifuged in an Eppendorf tube and fixed with 2.5% glutaraldehyde (Cat # G5882, Sigma-Aldrich, St. Louis, USA) in 0.1 M cacodyl buffer (Cat # C4945, Aldrich, St. Louis, USA) for 2 hours at room temperature and then in 1% osmium tetroxide (OsO4) for 1 hour. Samples were dehydrated in ethanol and embedded in PolyBed 812 at 68 ° C. Sections for analysis were placed on a grid (300 mesh grids). The sections were then contrasted with uranyl acetate and lead citrate. A JEOL JEM 2100HT electron microscope (JEOL Ltd., Tokyo, Japan) with a voltage acceleration of 80kV was used for the observation.

Protein isolation from UEVs pellet.

The resulting pellet was dissolved in 60 μL 10% SDS (Cat # L3771, Sigma-Aldrich, St. Louis, USA) 10 μL 1M Tris-HCl pH 8.0 (Cat # T1503 Sigma-Aldrich, St. Louis, USA) and 30 μL of deionized water [41]. Protein concentration was determined by the BCA method (Cat # 23227, Pierce Biotechnology, Thermo Scientific, USA). The mean protein concentration was 1.14 ± 1.04 mg / mL, the total amount of protein used for LC / MALDI was 40 µg. Then denaturation was performed (5 min at 99 ° C) in the presence of 0.1 M DTT.

Protein identification - LC / MALDI-TOF / TOF-MS method.

Proteomic analysis was performed using a nano-liquid chromatograph (EASY-nLC II ™, Bruker Daltonics, Germany) coupled with a time-of-flight mass spectrometer using a matrix assisted laser desorption and ionization (MALDI) technique: MALDI- TOF / TOF-MS ultrafleXtreme ™ (Bruker Daltonics, Germany) using WARP-LC software (ver. 1.2, Bruker) for data collection and analysis. A detailed description of the methodology has been published previously [42]. The precision tolerance was 100 ppm for the masses of the peptides and 0.7 Da for the masses of the ionic fragments. Individual peptide alignment was considered statistically significant for scores> 28. Protein identification performed by hand, based on two unique peptides with a probability of less than 0.05.

PL 237 810 Β1

Results

Table 1. Results of biochemical studies of patients

Parameter Control (C) n = 1 Controlled diabetes (CD) n = 1 Uncontrolled diabetes (UD) n = 1 Renal failure (RF) n = 4 Sample code 29 8 3 4.28.44.61 Age [years] 45 61 40 61.3 + 16.3 Sex woman man man man BMI [kg / m ² ] 22.6 27.8 33.8 30.6 6.8 Serum glucose [mM] 5.3 7.1 22.2 13.6.2 + 6.8 HbA1C [%] n 6.9 13.1 9.6 + 4.1 Serum creatinine [μΜ] 57 117 86 137 ± 21 GFR [mL / min / 1.73m ² ] 99 55 85 46 ± 6.14 Albumin in urine [mg / L] 5.85 102 821 387 + 359 Urine creatinine [mM] 57 11.5 5.0 9.3 ± 6.2

Fig. 2 presents micrograph of microbubbles of urine sediment (UEV) obtained by ultracentrifugation from a healthy person (C) and a person with unregulated type 2 diabetes (UD).

Moreover, the obtained results of the proteomic analysis performed by the LC / MALDI-TOF / TOF-MS method show the presence of gal-3 protein in UEV from urine of patients with controlled diabetes (CD). Gal-3 protein was not detected in a healthy person (Fig. 3A), gal-3 protein was detected in patients with advanced renal failure (RF) as well as in controlled diabetes (CD) - see Fig. 3B. In uncontrolled diabetes (UD) and GFR> 60, no gal-3 was detected in UMVs (see Fig. 3C), suggesting that urinary galectin3 in microbubbles (microbubbles) of diabetic-damaged kidney cells (UEVs) may be used as an early marker of kidney damage in diabetic patients.

Claims (4)

1. A method of detecting kidney damage occurring in diabetic patients, characterized in that a urine sample from a diabetic patient is tested for the presence of gal 3 protein in microbubbles present in the urine (UEV), and the presence of this protein indicates kidney damage, with what: a) UEV microbubbles are isolated from the urine sample, b) proteins are isolated from the obtained UEV microbubbles, (c) the composition of the proteins obtained is analyzed to determine the presence of gal-3 protein, kidney damage being manifested by an abnormal renal glomerular filtration rate (GFR) below 60 mL / min / 1.73 m ² . 2. The method according to p. A method as claimed in claim 1, characterized in that in step a) the urine sample is subjected to ultracentrifugation, preferably at 150,000 xg for 90 minutes. 3. The method according to p. The method of claim 1, characterized in that in step b) the obtained microbubbles are treated with a solution containing 10% SDS, 1M TRIS-HCl at pH = 8.0 and deionized water, and after determining the protein concentration, DTT is added to the solution to obtain a final concentration of 0 , 1 M and denature samples at 99 ° C for 5 minutes. 4. The method according to p. A method according to claim 1, characterized in that in step c) the gal-3 protein is identified by means of a nano-liquid chromatograph coupled to a time-of-flight mass spectrometer using a matrix-assisted laser desorption and ionization technique.