Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6827—Total protein determination, e.g. albumin in urine
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/04—Endocrine or metabolic disorders
  • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/34—Genitourinary disorders
  • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/56—Staging of a disease; Further complications associated with the disease
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
  • G01N33/5432—Liposomes or microcapsules

Definitions

• the subject of the invention is a method of determination of the degree of renal damage occurring in subjects with diabetes, based on assessing galectin-3 concentration in urine-derived microvesicles (UEV).
• the present invention could be applicable in therapy, in particular in clinical diagnostics of diabetic complications.
• Galectin-3 (gal-3) is a protein with properties of animal lectin, a member of b-galactoside sugar antennae binding protein family.
• a characteristic feature of galectins is the fact that they all have a distinctive carbohydrate recognizing domain comprised of about 130 aminoacids- CRD (Carbohydrate Recognition Domain) [1].
• galectins 15 different galectins have been identified, these being classified into 3 groups: tandem (with 2 CRD repeats), including galectins -4, -6, -8, -9 and -12; prototypical (galectin-1 , -2, -5, -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, where galectin-3 belongs [2] Moreover, recently a new group of animal galectins has been found, comprising 4 CRD domains. Galectins preferentially bind glycans that contain N-acetylgalactosamine chains.
• Gal-3 is composed of 2 domains: C-terminal, wherein the CRD sequence is found and N-terminal containing a phosphorylation site (S6) [3]
• the N-terminal domain is composed of 110-130 amino acids, contains a series of homolo’gous repeats of 9 amino acids (P, G, T, E), it is strongly conserved, shows a significant homology to heterogenous nuclear ribonucleoprotein complexes and to collagen (a1) chain and it is responsible for multimer formation [4].
• the 1-12 fragment is responsible for gal-3 secretion [5]
• Microvesicles or extracellular vesicles are small, spherical membranous structures with a diameter of 20-1000 nm, released into extracellular space [6]
• the term relates both to microvesicles budding directly from the cell membrane during the cell life cycle - the ectosomes (microvesicles) and during programmed cell death - apoptotic blebs, as well as to vesicles of extracellular origin - exosomes, released from multivesicular bodies [7]
• EVs demonstrate numerous common features such as: bilayer lipid membrane and proteins and nucleic acids found within these structures [6, 8, 9].
• the load (cargo) transported by EVs, reflecting the secreting cell condition, is transferred to a target cells - playing an active role in intercellular
• the subject of the invention is a method of determination of the degree of renal damage occurring in subjects with diabetes, characterized in that gal-3 protein concentration is determined in a urine sample from a subject suffering from diabetes, wherein:
• gal-3 protein concentration is determined in microvesicles found in urine (UEV) and elevated values thereof in comparison to the one observed in healthy subjects confirms renal damage.
• microvesicles are isolated from urine by filtration with the HFD method, followed by ultracentrifugation at speed of 150 000 x g for 1.5 h in 4°C.
• gal-3 protein concentration is determined by quantitative ELISA using antibodies specific against gal-3 protein.
• the method of the invention allows to obtain, regardless of the state of diabetes treatment, a reliable diagnostic test allowing a determination of the degree of renal damage occurring in subjects with diabetes.
• Example 1 Determination of the degree of renal damage occurring in subjects with diabetes.
• Chronic Kidney Disease is according to the KDIGO 2012 definition, abnormalities of kidney structure or function, present for > 3 months, with implications for health.
• the severity of CKD is determined based on the GFR value (G category) and the level of albuminuria (A category) [10].
• G category G category
• a category albuminuria
• the most common causes of CKD is diabetic nephropathy (about 30% at the end stage), glomerulonephritis, hypertensive nephropathy, acute renal damage, tubulointerstitial kidney disease, polycystic kidney disease, ischemic nephropathy
• eGFR glomerular filtration rate
• K- coefficient for women 0,7, for men 0,9, or optionally, value of 1
• the above formula is better than the most commonly used variants recommended for evaluation and classification of chronic kidney failure, such as the MDRD formula (Modification of Diet in Renal Disease Study), due to a smaller systematic error, especially for the higher eGFR values, which allows a higher sensitivity of diagnosing renal damage at an early stage, namely stage II [11].
• MDRD formula Modification of Diet in Renal Disease Study
• 60 mL/min/1.73 cm2 is considered to be the threshold value, with values beneath it indicating renal failure [10, 11 , 14].
• the threshold value separating the 2 groups of patients with moderate renal damage into 2 groups with mild to moderate (G3a stage, group 2) and moderate to severe (G3b stage, group 3) renal damage was adopted to be 44 mL/min/1.73 cm2.
• the full clinical picture of CKD dependent on GFR category is comprised in the review nterna. Choroby. Choroby nerek i drag moczowych. Przewlekta choroba nerek” [1 1].
• Criteria for cut-off determination for galectin-3 The threshold value for the eGFR factor for differentiation of patients with CKD in complications of diabetes was adopted to be 44 mL/min/1.73 cm2. Since clinically the G3 group is very heterogenous and the degree of severity for the disease can vary widely, which is reflected in etiopathology of the disease (the division to G3a and G3b) and differing clinical picture [11 , 12] Taking that criteria, the patients for gal-3 concentration assessment in urine were divided into 2 groups:
• UEV isolation from urine sample First morning urine, from middle stream, in a volume of about 100 mL was collected from patients with type II diabetes, with the patients recruited to the study beforehand. Urine was subjected to preliminary centrifugation at the speed of 2000 x g for 30 minutes in room temperature (Hermle Z-300K centrifuge) in order to remove cell debris, bacteria and other contaminants. After centrifugation, the supernatant was removed and was used in the next step of isolation of urine-derived extracellular microvesicles (UEV). The supernatant was filtered using the HFD method (Hydrostatic Filtration Dialysis) in order to concentrate the EVs population. The concentrated EVs population was ultracentrifuged at the speed of 150 000 x g for 1.5 h in 4°C, the obtained vesicle pellet being suspended in 60 ul of demineralized water.
• HFD method Hydrostatic Filtration Dialysis
• This method employed a 96-welled plate coated with anti-galectin-3 antibody.
• the plate was loaded with a protein standard (in varying concentrations in order to plot a calibration curve) and with samples of isolated vesicles or of complete urine.
• the plate was incubated for 2.5 h, room temperature, shaking, and then the plate was washed 4 times with buffer provided with the kit (Wash Buffer).
• biotinylated antibody was added and it was again incubated (1 hour, room temperature, shaking). After incubation, the plate was washed 4 times using Wash Buffer. Then, streptavidin conjugated with horseradish peroxidase (HRP) was added. The plate was incubated for 45 minutes in room temperature with shaking.
• HRP horseradish peroxidase
• the concentrations for individual samples were calculated based on the plotted calibration curve.
• the semi-quantitative analysis of the gal-3 protein in UEV from urine of patients using Western Blot For the assessment of the gal-3 protein levels in UEVs isolated from urine of patients with diabetic CKD and in controls, the Western Blot method as used. For this purpose, proteins were isolated from the isolated UEVs and concentrations thereof were measured using the BCA method. Then, the proteins were separated using gel electrophoresis with the PAGE- SDS method and blots were made using antibodies: primary galectin-3 antibody (cat. no. 60207- 1-lg, CloneNo.: 1C1 B2; manufacturer Prointech Germany) and secondary: HRP-conjugated (Mouse/Rabbit), in the Lumi-LightPLUS Western Blotting Kit (cat. no.12015218001 , Roche).
• primary galectin-3 antibody cat. no. 60207- 1-lg, CloneNo.: 1C1 B2; manufacturer Prointech Germany
• HRP-conjugated Mae/Rabbit
• Table 1 shows the values for the median, the first and third quartile (Q1 -Q3) and the p value calculated using the Mann Whitney U test (control group was compared with every patient group).
• Gal-3 protein levels in UEV isolated from urine with the HFD method Gal-3 levels in isolated vesicles from urine is shown on Fig. 2. Results are shown as obtained for the control group (0) and for individual patient groups with varying degree of renal damage (1 -5). A square in the middle of a box shows the mean value while a horizontal line indicates the median.
• Table 2 shows the values for the median, the first and third quartile (Q1 -Q3) and the p value calculated using the Mann Whitney U test (control group was compared with every patient group).
• Table 3 shows the values for the median, the first and third quartile (Q1 -Q3) and the p value calculated using the Mann Whitney U test (control group was compared with every patient group).
• ROC Receiver Operating Curve
• the cut-off value of 11.06 ng/mL differentiates between patients with diabetic renal damage with normal or increased eGFR, a mild decrease and a moderate decrease in eGFR (G1 , G2 and G3a) and patients with moderately severe and severe decrease in eGFR (G3b, G4 and G5).
• the plot for the ROC curve and determination of AUC with indicated cut-off value is illustrated on Fig. 4, wherein the plot for the ROC curve is shown, for the purpose of determining the gal-3 concentration value for differentiation between patients with diabetic CKD.
• Table 4 provides the values for the area under the ROC curve, the AUC value with the assessment of goodness of fit for the test, the p probability value being determined to be p ⁇ 0.000005.
• the cut-off value of 11.06 ng/mL differentiates patients with diabetic renal damage with normal or increased eGFR, a mild decrease and a moderate decrease in eGFR (G1 , G2 and G3a) from patients with moderately severe and severe decrease in eGFR (G3b, G4 and G5).
• Menon RP Hughes RC: Determinants in the N-terminal domains of galectin-3 for secretion by a novel pathway circumventing the endoplasmic reticulum-Golgi complex. EurJ Biochem 264: 569-576, 1999
• Kidney Function and Disease Report of a Kidney Disease: Improving Global Outcomes (KDIGO) Consensus Conference Levey, Andrew S. et al. Kidney International 2020 DOI: https://doi.Org/10.1016/j.kint.2020.02.010

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• 2019
  • 2019-02-21 PL PL428992A patent/PL240124B1/pl unknown
• 2020
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