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]. Presently, 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]. In mammals, the 1-12 fragment is responsible for gal-3 secretion [5],

Microvesicles or extracellular vesicles (EVs) 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] Despite the different characteristics in terms of size (exosomes: 30-100 nm, ectosomes: 100-1000 nm, apoptotic blebs: 500-1000 nm), 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 signalization.

In light of the prior art described above there is still a need for providing a method allowing determination of renal damage occurring in subjects with diabetes.

Surprisingly, such a method was found in the present invention. 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:

- concentration of gal-3 protein in urine below 1 1.06 ng/mL indicates the subject having a degree of renal damage corresponding to a moderately severe or severe decrease in glomerular filtration rate eGFR below 44 mL/min/1.73 cm2,

- concentration of gal-3 protein in urine above 1 1.06 ng/mL indicates the subject having a degree of renal damage corresponding to a moderate, small or mild decrease in glomerular filtration rate eGFR above 44 mL/min/1.73 cm2.

In a preferred embodiment of the method of the invention, additionally 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. Preferably, 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.

Preferably, gal-3 protein concentration is determined by quantitative ELISA using antibodies specific against gal-3 protein.

Surprisingly, the studies that that led to the present invention established that there is a correlation for gal-3 content levels in complete urine and the gal-3 containing microvesicle levels, with the degree of kidney cell damage, in particular due to diabetes.

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.

Herein below, examples of embodiments of the invention are shown.

Example 1. Determination of the degree of renal damage occurring in subjects with diabetes.

Description of the study group. 109 patients with type 2 diabetes were recruited to the study, as well as 7 healthy subjects (control). A sample of first morning urine, from middle stream was obtained. Patients were recruited at the Nephrology Department at the University Hospital in Krakow (Katedra Nefrologii Szpitala Uniwersyteckiego w Krakowie) and at the Clinical Provincial Hospital no. 2 in Rzeszow (Kliniczny Szpital Wojewodzki Nr 2 w Rzeszowie).

Patients were divided into 5 groups based on the severity of renal damage during the course of chronic kidney disease caused by diabetes. Chronic Kidney Disease (CKD) 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]. 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

[11]·

Classification criteria for the degree of renal damage in the course of CKD. The criterion for including a patient in a particular group was the estimated value for glomerular filtration rate eGFR (estimated Glomerular Filtration Rate). The eGFR factor was estimated based on blood serum creatinine levels, age, sex and ethnicity of the patient, and the CKD EPI formula was used for its calculation [12, 13], wherein: eGFR = 141 1,018 [female] - 1,159 [black ethinicity ] Skr- serum creatinine concentration in units of [mg/dL]

K- coefficient, for women 0,7, for men 0,9, or optionally, value of 1

a - coefficient, for women -0,329, for men -0.411

mm - minimal

Skr

max - maximal— -

Kor 1

age - age in years

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].

Dividing patients into groups hased on degree of renal damage

Based on the degree of renal damage expressed on the basis of eGFR, patients with diabetic CKD were classified into 5 groups [10, 14]:

1 above 90 and between 60 - 89 mL/min/1.73 cm2, G1 stage - normal or increased GFR, G2 stage - renal damage with mild decrease in eGFR, (n=79)

2 45 - 59 mL/min/1 73cm2, G3a stage - renal damage with decrease in eGFR between mild and moderate; (n=5)

3 30 - 44 mL/min/1 ,73cm2, G3b stage - renal damage with decrease in eGFR between moderate and severe; (n=3)

4 15 - 29 mL/min/1 73cm2, G4 stage - renal damage with severe decrease in eGFR; (n=11),

5 <15 mL/min/1 73cm2, G5 stage - end-stage renal failure (n=11)

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:

A. > 44 mL/min/1.73 cm2 (groups 1 , 2; e. i. G1 , G2, G3a)

B. =< 44 mL/min/1.73 cm2 (groups 3, 4, 5; e.i. G3b, G4, G5)

Ethics. The study had the required approval of the Bioethics Committee of the Jagiellonian University Collegium Medicum (Komisja Bioetyczna Uniwersytetu Jagiellohskiego Collegium Medicum) (no. KBET/206/B/2013). The approval is valid until 31 st December 2017. Participation in the study was possible only after the patient gave consent confirmed by their own signature, with the recruitment for the study being conducted by specialist physicians.

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.

Measurement of galectin-3 concentration in urine and in isolated vesicles using ELISA. The concentration of gal-3 in urine samples and in UEV using the Human LGALS3/ Galectin-3 ELISA Kit (Sigma Aldrich, cat. no. RAB0661), according to the manufacturer’s instructions.

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). In the next step, 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. After incubation, the plate was again washed 4 times. In the next step, TMB substrate (3,3’,5,5’-tetramethylbenzidine) for the HRP enzyme was added, triggering a color reaction. Finally, the addition of the Stop Solution causes inhibition of the reaction. The reading of absorbance was performed for the wavelength of 450 nm.

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).

Statistical analysis. Median for gal-3 was calculated for individual groups, as well as interquartile range (Q1-Q3). The p value was calculated using the Mann Whitney U test, in the Statistica 13 software. To assess the correctness of the classifier, which is the new biomarker galectin-3 (gal- 3), a ROC (Receiver Operating Characteristic) curve analysis was performed using tools available with the Statistica 13 software with add-on for medical analysis. Moreover, for goodness of fit and accuracy analysis of the selected model, area under the curve (AUC) for the ROC curve was determined. The value of the AUC index is within the range of [0, 1], the higher it is the better the model [15].

Results:

Concentration of the gal-3 protein in urine. The results of measurements and comparison of gal-3 concentration in urine between individual groups are shown on Fig. 1. Results are shown as obtained for controls (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 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).

Table 1. Median values for gal-3 concentration in urine for individual groups.

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 2. Median values for gal-3 concentration in vesicles for individual groups.

Identification of gal-3 with semi-quantitative methods (polyacrylamide gel electrophoresis and Western Blot). On the basis of values for relative band intensity for gal-3 obtained using Western Blot, Fig. 3 was prepared, which shows values obtained for individual studied groups. A square in the middle of a box shows the mean value while a horizontal line indicates the median.

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).

Table 3. Median values for gal-3 concentration in vesicles for individual groups.

Receiver Operating Curve (ROC) analysis. In order to determine the cut-off value for gal-3 concentration in patients with diabetic CKD, a ROC analysis was performed (see Table 4). Based on the sensitivity and specificity analysis, the cut-off value was determined for differentiating the degree of renal damage G3a/G3b (eGFR below 44 ml/min/1.73cm2), for gal-3 concentration values, in all groups of patients with diabetic CKD. The significant p value (error rate) was obtained for gal-3 at the concentration of 11.06 ng/mL.

Table 4. Results of ROC analysis for gal-3 concentrations in complete urine from patients with diabetic CKD.

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. The determined cutoff is 11 .06 ng/mL with p=0.05. 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.

Table 4. AUC values with assessment of goodness of fit for the test

Conclusions:

1. Gal-3 protein presence was confirmed in UEVs from urine and in complete urine in patients with an early stage of diabetic CKD.

2. It was shown that at an early stage of diabetic CKD (G1 , G2, G3a) there is an increase in the gal-3 protein concentration in urine in patients with diabetic CKD, whereas there is a decrease in gal-3 concentration in patients with moderately severe and severe decrease in eGFR (G3b, G4 and G5)

3. It was shown that with an increase of the degree of severity of renal damage according to the KDIGO 2012 definition and scale, the gal-3 protein concentration in urine of patients with diabetic CKD falls.

4. 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).

5. Low gal-3 concentrations in urine in patients with diabetic CKD and a high gal-3 protein content in UEV are biomarkers for developing a diagnostic test for discrimination between degrees of renal damage in diabetic CKD.

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