USE OF SERUM 2-CYSTEINE PEROXIREDOXINS (2-CYS-PRDX) AS BIOMARKERS OF CHRONIC KIDNEY DISEASES

Abstract

The present invention relates to the use of at least two of 2-Cys-PRDXs selected from a group consisting of PRDX1, PRDX2, PRDX3, PRDX4 and PRDX5 as a biomarker to diagnose, differentiate and/or assess the risk of chronic kidney disease (CKD). The present invention also relates to a method of diagnosis and a method of determining an increased risk of chronic kidney disease (CKD). A kit suitable for diagnosis and differentiation of chronic kidney disease (CKD) selected from the group comprising lupus nephritis (LN), IgA nephropathy (IgAN) and autosomal-dominant polycystic kidney disease (ADPKD) in a subject is also provided.

Claims

1. Use of at least two 2-Cys-PRDX biomarkers selected from a group consisting of PRDX1, PRDX2, PRDX3, PRDX4 and PRDX5, to diagnose, differentiate and/or assess the risk of a chronic kidney disease (CKD), wherein said chronic kidney disease (CKD) is selected from lupus nephritis (LN), IgA nephropathy (IgAN) and/or autosomal-dominant polycystic kidney disease (ADPKD).

2. The use of claim 1, wherein at least two 2-Cys-PRDX biomarkers are selected from a combination of PRDX1+PRDX2, PRDX1+PRDX3, PRDX1+PRDX4, PRDX1+PRDX5, PRDX2+PRDX3, PRDX2+PRDX4, PRDX2+PRDX5, PRDX3+PRDX4, PRDX3+PRDX5 or PRDX4+PRDX5.

3. The use of claim 1 or 2, wherein at least two 2-Cys-PRDX biomarkers are selected from a combination of PRDX1+PRDX4, PRDX2+PRDX4 or PRDX4+PRDX5.

4. The use according to any of claims 1-3, wherein at least three 2-Cys-PRDX biomarkers are used, which are selected from a combination of PRDX1+PRDX2+PRDX3, PRDX 1+PRDX2+PRDX4, PRDX 1+PRDX2+PRDX5, PRDX 1+PRDX3+PRDX4, PRDX1+PRDX3+PRDX5, PRDX1+PRDX4+PRDX5, PRDX2+PRDX3+PRDX4, PRDX2+PRDX3+PRDX5, PRDX2+PRD4+PRDX5, PRDX3+PRDX4+PRDX5.

5. The use according to any of claims 1-4, wherein at least four 2-Cys-PRDX biomarkers are used, which are selected from a combination of PRDX1+PRDX2+PRDX3+PRDX4, PRDX 1+PRDX2+PRDX3+PRDX5, PRDX 1+PRDX2+PRDX4+PRDX5, PRDX 1+PRDX3+PRDX4+PRDX5, PRDX2+PRDX3+PRDX4+PRDX5.

6. The use according to any of claims 1-5, wherein five 2-Cys-PRDX biomarkers are used as a combination PRDX1+PRDX2+PRDX3+PRDX4+PRDX5.

7. A method of diagnosis of a chronic kidney disease (CKD) selected from lupus nephritis (LN), IgA nephropathy (IgAN) and/or autosomal-dominant polycystic kidney disease (ADPKD) in a subject, comprising (a) a step of identification of at least two, three, four or five 2-Cys-PRDX biomarkers selected from a group consisting of PRDX1, PRDX2, PRDX3, PRDX4 and PRDX5 in a serum sample from said subject, and (b) a step of identification of at least two or three or four or five 2-Cys-PRDX biomarkers selected from a group consisting of PRDX1, PRDX2, PRDX3, PRDX4 and PRDX5 in a serum sample from a healthy control, wherein said 2-Cys-PRDX biomarkers correspond to the biomarkers identified in step (a), and (c) a step of comparison of the 2-Cys-PRDX biomarkers identified in step (a) in the serum sample from said subject with the corresponding 2-Cys-PRDX biomarkers identified in step (b) in a serum sample from said healthy control.

8. A method of determining an increased risk of a chronic kidney disease (CKD) selected from the group consisting of lupus nephritis (LN), IgA nephropathy (IgAN) and autosomal-dominant polycystic kidney disease (ADPKD) in a subject, comprising identification of at least two, three, four or five biomarkers selected from a group consisting of PRDX1, PRDX2, PRDX3, PRDX4 and PRDX5 in a serum sample from said subject and comparing the presence of these biomarkers with the presence of the corresponding 2-Cys-PRDX biomarkers in a serum sample from a healthy control and based on this comparison assessing the risk of lupus nephritis (LN), IgA nephropathy (IgAN) and autosomal-dominant polycystic kidney disease (ADPKD) in said subject.

9. A kit for diagnosis and differentiation of a chronic kidney disease (CKD), selected from lupus nephritis (LN), IgA nephropathy (IgAN) and/or autosomal-dominant polycystic kidney disease (ADPKD) in a subject, which comprises at least antibodies that specifically bind biomarkers of a CKD and means of identification of such biomarkers that bind to said antibodies, characterized in that said kit comprises as least two 2-Cys-PRDX biomarkers selected from a group consisting of PRDX1, PRDX2, PRDX3, PRDX4 and PRDX5 biomarkers.

10. A method for differentiation of a chronic kidney disease (CKD), selected from lupus nephritis (LN), IgA nephropathy (IgAN) and autosomal-dominant polycystic kidney disease (ADPKD), in a subject comprising: a) detecting the amount of at least two or three or four or five 2-Cys-PRDX biomarkers selected from a group consisting of PRDX1, PRDX2, PRDX3, PRDX4 and PRDX5 in a serum sample from said subject; b) detecting a reference amount of at least two or three or four or five 2-Cys-PRDX biomarkers selected from a group consisting of PRDX1, PRDX2, PRDX3, PRDX4 and PRDX5, which correspond to the biomarkers the amount of which is detected in the step a) in the serum sample from a healthy control; c) comparing the amount of the 2-Cys-PRDX biomarkers detected in the step a) in a serum sample from said subject to the reference amount of the corresponding biomarkers detected in the step b) in a serum sample from a healthy control; and d) identifying the subject as having an increased risk for disease progression if the amount of the biomarkers detected in the step a) in the sample from said subject is greater than the reference amount of the corresponding biomarkers detected in the step b) in a serum sample from a healthy control.

11. The method of claim 10, wherein one of at least two 2-Cys-PRDX biomarkers detected in steps a) and b) is PRDX4.

12. The method of claim 11, wherein the second of at least two 2-Cys-PRDX biomarkers detected in steps a) and b) is PRDX2.

13. The method of claim 11, wherein the second of at least two 2-Cys-PRDX biomarkers detected in steps a) and b) is PRDX3.

14. The method of claim 11, wherein the second of at least two 2-Cys-PRDX biomarkers detected in steps a) and b) is PRDX5.

15. The method of claim 11, wherein the second of at least two 2-Cys-PRDX biomarkers detected in steps a) and b) is PRDX1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Present invention is described herein in relation to the following figures of drawings in which:

[0060] FIG. 1 presents 2-Cys-prdxs (1-5) serum concentration in patients diagnosed with IgAN, LN, ADPKD and healthy, age-, sex-matched control. Panel A (FIG. 1A)reflects control group without any kidney disease or other chronic diseases requiring treatment, the remaining groups have CKD. Bars are representing the mean value of PRDX (1-5) serum concentration (the mark of PRDXs isoforms, changes with progressively darker shades of grey on a plot), the box itself is the standard error (SE) of the results; the whiskers are the upper standard deviation (SD). Panel B and C (FIGS. 1B and 1C, respectively) represent serum levels of 2-cys-prdxs in female (B) and male (C) patients separately, in comparison with healthy sex-matched control. Mann WhitneyU Tests were used to compare PRDX (1-5) concentration values of each group. Values with p<0.05 were considered statistically significant and marked on a graphs with: *statistical significance of all groups comparing to control group; **IgAN patients comparing to LN patients; ***ADPKD patients comparing to LN patients.

[0061] FIG. 2 presents 2-Cys-prdxs (1-5) serum concentration depending on the disease duration in female (A) and male (B) patients diagnosed with IgAN (FIGS. 2A and 2B respectively). Bars are representing mean value of PRDX (1-5) concentration, the boxstandard error (SE), the whiskers are upper standard deviation (SD).

[0062] FIG. 3 is a graphical illustration of the correlation analysis of PRDX2 serum concentration and eGFR (calculated according to CKD-EPI equation) in patients diagnosed with IgAN. Panel Areflects both male and female, Bonly female, Conly male IgAN patients. The line parameters such as: rPearsons coefficient, pstatistical significance (p<0.05 are statistical significant) are also presented on a graph.

[0063] FIG. 4 is a graphical illustration of the correlation analysis of PRDX (1, 2 or 4) serum concentration and proteinuria in both male and female (FIG. 4A), only female (FIG. 4B) and only male (FIG. 4C) patients diagnosed with IgAN. Three independent lines are presented, with parameters such as: rPearsons coefficient, pstatistical significance (p<0.05 are statistical significant) are presented on a graph.

[0064] FIG. 5 is a graphical illustration of the correlation analysis of PRDX5 serum concentration and BMI in female (FIG. 5A), male (FIG. 5B) patients diagnosed with IgAN and in female patients diagnosed with LN (FIG. 5C). Parameters such as: rPearsons coefficient and pstatistical significance (p<0.05 are statistical significant) are presented on a graph.

[0065] FIG. 6 is a schematic scheme of the differentiation of LN, ADPKD, IgAN in the correlation with the PRDX (1 and 4) titre. Additional statistical analysis indicates that parameter PRDX1 should help to distinguish patient from a healthy control, as well as to distinguish a patient with LN and with ADPKD from a patient with IgAN (PRDX1>0.08). Moreover, exclusion of ADPKD disease occurs when parameter PRDX4 exceeds value 7.5.

EXAMPLES

Participants

[0066] This study was performed using serum samples collected from 38 patients with biopsy-proven IgAN, 18 patients with biopsy-proven LN, 8 patients diagnosed with ADPKD and 15 healthy volunteers serving as the age- and sex-matched controls. The inclusion criteria for the control group were as follows: age older than 18 years and absence of any kidney disease or other chronic diseases requiring treatment. The exclusion criteria for both groups included: active infection, history of malignancy, previous organ transplantation, or current pregnancy. The blood was drawn once, at different stages of disease activity and duration. The study protocol was approved by the local ethics committee and informed consent was obtained from all participants. The study was performed in accordance with the

[0067] Declaration of Helsinki. Demographic and clinical data of all groups is presented in Table 1 (values are given as meanSD)

TABLE-US-00001 TABLE 1 Charecteristics IgA (n = 38) SLE (n = 17) ADPKD (n = 7) Control (n = 15) Male/female (n) 21/17 2/15 1/6 6/9 Age (years) 40.37 13.70 43.94 12.37 51.86 6.20 38.20 9.79 BMI (kg/m.sup.2) 27.03 4.85 23.43 3.86 26.65 6.17 24.34 3.39 Serum creatinine (mg/dl) 1.31 0.69 0.99 0.46 1.34 0.63 0.83 0.15 eGFR (ml/min/1.73.sup.2) 74.70 32.86 80.96 26.10 57.05 22.70 101.73 14.70 Proteinuria (g/d) 0.73 0.65 0.40 0.60 none none HCT (%) 42.85 4.11 39.64 4.06 43.69 1.43 43.76 3.25 HGB (g/dl) 14.27 1.50 12.78 1.49 14.57 0.75 14.71 1.17

Sample Measurements

[0068] The serum concentration of each of the 2-Cys-PRDXs (1-5) was assessed in five independent ELISA tests (EIAab, Wuhan, China). Tests were proceeded strictly according to manufacturer's instructions.

[0069] In brief summary, the microtiter plate provided with ELISA kit has been pre-coated with an antibody specific to target antigen. The standards and samples were added in a predetermined order in amount of 100 l to the appropriate wells with a biotin-conjugated antibody preparation, specific to target antigen, then avidin conjugated to horseradish peroxidase were added to each microplate well. The enzyme-substrate reaction was terminated by the addition of a sulphuric acid solution. Changes of colour in each well were measured spectrophotometrically at the wavelength of 450 nm. The concentration of targeted antigen in the samples were determined by comparing the O.D. of the samples to the standard curve.

Statistical Analysis

[0070] The measurements of 80 samples in each test were collected, divided into appropriate groups (depending on type of disease), expressed as meansSD and statistically analysed (Statistica 12.0, StatSoft). The statistical analysis comparing LN, IgAN and ADPKD patients to healthy controls were performed using Mann Whitney U Test (where p<0.05 was considered significant).

Results Discussion

[0071] If not provided otherwise, when a titre of concentration of PRDX is mentioned it should be understood as ng/ml.

[0072] As presented in FIG. 1A, the mean concentrations of PRDX1-5 in overall population of IgAN and ADPKD patients were not significantly different from the controls. In comparison, serum concentrations of all PRDXs were significantly elevated in LN patients, as compared to the control group. Notably, there was a robust intragroup variability in this group, especially in regard to PRDX1 and 2 concentrations, which corresponds to the complex nature of LN as a disease. This results prove that PRDX1-5 are selective biomarkers of LN.

[0073] Moreover, further statistical analysis for IgAN, LN, ADPKD and control, grouped by sex, revealed that more significant differences can be observed in the female (FIG. 1B) than in the male (FIG. 1C) population, which suggests that the oxidative stress management may vary between genders. PRDX 2 serum concentration is significantly correlated to proteinuria in females with IgAN, but not in LN or ADPKD. This fact may be used in differentiation of IgAN from the latter diseases during pregnancy, when renal biopsy is highly unrecommended. It suggests that comparison of serum concentration of at least two of five (1-5) isoforms of PRDXs might be also used as a preliminary tool for CKD diagnosis.

[0074] Furthermore, statistical analysis also shows that PRDX3 (PRDX3>5.03) is an important parameter which is influencing the LN prognosis. Change of PRDX3 parameter by value of 1 ng/ml increases the LN probability by 12.401% (Pr>0.00879).

[0075] As can be seen from FIG. 2 disease duration in IgAN patients has no significant influence on the PRDX 1-5 serum concentrations. Therefore, PRDX 1-5 cannot be used to diagnose and differentiate IgAN from other CKD disease at any time from disease's onset.

[0076] Differently, disease duration in LN patients has a significant influence on the PRDX 1-5 serum concentrations. Therefore, PRDX 1-5 can be used to diagnose and differentiate LN from other CKD disease at any time from disease's onset.

[0077] As can be seen from FIG. 3 the eGFR is inversely correlated with renal function. PRDX 1-5 serum concentrations in IgAN patients have no significant correlation to the eGFR. Therefore, PRDX 1-5 can be used to diagnose and differentiate IgAN from other CKD at any stage of renal impairment. PRDX 1-5 serum concentrations in LN patients have no significant correlation to the eGFR. Therefore, PRDX 1-5 can be used to diagnose and differentiate LN from other CKD at any stage of renal impairment.

[0078] As is evident from FIG. 4 proteinuria depends on the activity of glomerular diseases. PRDX 1 and 4 serum concentrations have significant correlation to the daily urinary protein loss in IgAN patients. Therefore, PRDX 1 and 4 may be used to assess disease activity. PRDX 2 serum concentration is significantly correlated to proteinuria in females with IgAN. Therefore, PRDX 2 may be used to assess disease activity in this subgroup of IgAN patients. Neither PRDX 1, nor 2, nor 4 serum concentrations in males with IgAN correlate significantly to proteinuria. Therefore, PRDX 1, 2 and 4 in males serum can be used to differentiate IgAN from other CKD regardless of the degree of proteinuria.

[0079] PRDX 1-5 serum concentrations have no significant correlations to the daily urinary protein loss in LN patients. Therefore, PRDX 1-5 can be used to diagnose and differentiate LN from other CKD regardless of the degree of proteinuria.

[0080] Since BMI may be correlated with proteinuria and is associated with eGFR, as it can be seen on FIG. 5, PRDX 1-5 serum concentrations have no significant correlations to the BMI in IgAN patients. Therefore, PRDX 1-5 can be used to differentiate IgAN from other CKD regardless of the body weight. On the contrary PRDX 1-4 serum concentrations have no significant correlations to the BMI in LN patients. Therefore, PRDX 1-4 can be used to differentiate LN from other CKD regardless of the body weight.

[0081] Moreover, PRDX 5 serum concentration is significantly correlated to the BMI in LN female patients (FIG. 5C). Therefore, PRDX 5 concentration should be interpreted with caution in obese females.

[0082] As can be seen from FIG. 6 combination of two 2-Cys-PRDXs is useful to differentiate chronic kidney disease (CKD) selected from the group comprising lupus nephritis (LN), IgA nephropathy (IgAN) and autosomal-dominant polycystic kidney disease (ADPKD). In case of combination of PRDX-1 and PRDX4 the titer of PRDX1 greater than 0.08 suggest that the subject does not suffer from IgA. To differentiate unequivocally patients suffering from LN and ADPKD it is sufficient to determine titer of PRDX4.

[0083] Additionally, based on the results as obtained and presented on the Figures the following can/might be concluded: [0084] 1. PRDX1 or PRDX2 distinguishes between healthy controls and patients with CKD; [0085] 2. PRDX2 or PRDX3 distinguishes IgAN from all subjects. Moreover, every PRDX distinguishes IgA from LN, whereas PRDX4 distinguishes IgAN from ADPKD. [0086] 3. PRDX 2 or PRDX3 or PRDX4 or PRDX5 distinguishes LN from all subjects, as well as from IgA and from ADPKD. PRDX 1 or PRDX2 or PRDX3 or PRDX4 or PRDX5 distinguishes LN from IgA and from ADPKD.

[0087] Summarized parameters that differentiate between all three diseases are shown in the Table 2 below:

TABLE-US-00002 TABLE 2 All Control IgAN LN ADPKD All X 1, 2 2, 3 2, 3, 4, 5 1, 4 Control X none 1, 2, 3, 4, 5 4 IgAN X 1, 2, 3, 4, 5 4 LN X 1, 2, 3, 4, 5 ADPKD X

Results Interpretation

[0088] The present study showed that serum levels of all 2-Cys-PRDXs were elevated in LN patients, while in other diseases (IgAN and ADPKD) they were on similar levels as in healthy controls (FIG. 1A). Indeed, the statistical analysis using Mann Whitney U Test, revealed that there were no statistically significant differences between healthy controls and patients with IgAN or ADPKD. However, there was a difference in each PRDXs isoform (1-5) serum concentration in patients with LN (p<0.05).

[0089] Therefore, it was claimed that elevated 2-Cys-PRDXs concentrations in serum could be a useful biomarker of LN versus other CKD-related diseases, in addition to currently used markers such as a range of autoantibodies and other markers in LN. Moreover, PRDX 1-5 can be used to diagnose and differentiate LN from other CKD at any time from disease's onset, at any stage of renal impairment, and regardless of the degree of proteinuria or BMI (except for PRDX 5 in females).Additionally, correlation between PRDX2 serum concentration and estimated glomerular filtration rate (eGFR), which was calculated according to CKD-EPI equation in patients diagnosed with IgAN (FIG. 4), correlation between PRDX (1, 2 or 4) serum concentration and proteinuria (FIG. 5) and correlation between PRDX5 serum concentration and BMI (FIG. 6) shows the association of these parameters with the 2-Cys-PRDXs serum levels. As presented on the FIGS. 4-6 such correlation might also be used to differentiate patients with various types or mechanisms of kidney failure and improve early-diagnosis methods. Therefore, the studies summarized above prove that serum concentrations of 2-Cys-PRDXs are very promising new biomarkers, which can improve current diagnostic methods of CKD and are sufficiently sensitive and accurate to detect early LN and/or to monitor its progression.

[0090] It must be underlined that the present invention is both cost-efficient, as well as, hassle-free for the potential subject, or a patient since it doesn't require use of any equipment such as USG or distress due to a biopsy which are perceived as onerous and harmful for health, especially in pregnant patients.