CARD group (n = 37) - patients with at least one history of a cardiovascular event, admitted to hospital for elective angiography, without any signs of impaired kidney function.

The studies in this group were conducted to check the changes that occur as a result of cardiovascular disease (CVD) but without kidney disease.

CKD1-2 (n=29) (stage G1-G2 CKD) with mild decrease in eGFR (eGFR >90-60 ml/min/1.73 m^2)

The studies in this group were performed to disclose the changes that occur as a consequence of the beginning of kidney function deterioration.

HV (n = 32) - this group was composed of healthy people, with no evidence of impairment in renal function and cardiovascular disorders in the history and at the time of enrollment in the study.

body mass index (BMI) [kg/m^2] was calculated by dividing a person's weight (post-HD weight in HD group) [kg] by the squared their body height [m]

Serum concentration of:

• advanced glycation ends products (AGE) [µg/mg protein];
• 3-nitrotyrosine (3-NT) [µmol/mg protein];
• advanced oxidation protein products (AOPP) [µmol/mg protein];
• carboxymethyle(lysine) (CML) [µg/mg protein]

were determined with the enzyme immunoassay methods (ELISA) using Shanghai Sunred Biological Technology Co kits, China.

metalloproteinases in the serum [ng/ml]:

• metalloproteinase 9 (MMP-9) in the serum was determined by the ELISA method using the Quantikine Human MMP-9 (total) kit, by R&D Systems, Canada;
• tissue inhibitor of metalloproteinase 1 (TIMP-1) in the serum - was determined by the ELISA method using the Quantikine Human TIMP-1 kit, manufactured by R&D Systems, Canada;
• the MMP-9/TIMP-1 ratio was calculated by the quotient of the MMP-9 and the TIMP-1 concentration.

• total cholesterol (T-C) [mg/dl] - was assessed by routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA;
• low-density lipoprotein cholesterol (LDL-C) [mg/dl] - was assessed by routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA;
• high-density lipoprotein cholesterol (HDL-C) [mg/dl] - was assessed by routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA;
• triglycerides (TG) [mg/dl] - were assessed by routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA;
• the concentration of low-density lipoprotein (LDL-C) cholesterol - was determined from Friedewalds' equation (LDL-C [mg/dl] = total cholesterol (T-C) [mg/dl]- HDL-C [mg/dl]- TG[mg/dl]/5).

• iron concentration [mg/dl] - was analyzed with the Cobas Integra 400 plus biochemical analyzer from Roche Diagnostics, USA;
• total iron-binding capacity (TIBC) [mg/dl] - was analyzed with the Cobas Integra 400 plus biochemical analyzer from Roche Diagnostics, USA;
• the unsaturated iron-binding capacity (UIBC) [mg/dl] was determined by an equation in which iron concentration in plasma is subtracted from TIBC [mg/dl];
• ferritin [ng/ml] concentration was determined with the Modular E-170 biochemical analyzer from Roche Diagnostics, USA.

• high-sensitivity C-reactive protein (hsCRP) [mg/l] was measured using DADE Behring, USA and the DADE nephelometer Behring Analyzer II;
• neopterin [nmol/l] was determined by using the Neopterin ELISA kit, DRG International, Inc., USA;
• interleukin 18 (IL-18) [pg/ml] concentration was determined by Colorimetric Sandwich ELISA, Quantikine Human IL-18 R&D Inc., USA.

carotid intima-media thickness (IMT) [mm] was measured by The Accuson CV 70 system (Siemens) with a 10 megahertz (Mhz) transducer.

Two longitudinal projections were assessed (anterolateral and posterolateral). The distal 1 cm of the common carotid artery just proximal to the bulb was measured by means of a computer analysis system (Medical Imaging Applications, LLC).

For non-invasive cardiological examinations, the Portapres device (Finapres Medical Systems (FMS), the Netherlands), the SphygmoCor tonometer (AtCor Medical), the Colin blood pressure monitor (BMP)-7000 (Japan) were used.

Main assessed variables: heart rate (HR) [beats per minute [bpm]]; ejection duration (ED) [millisecons]; peripheral systolic (pSBP) and diastolic blood pressure (pDBP) [mm Hg]; peripheral mean arterial pressure (pMAP) [mm Hg]; peripheral end-systolic pressure (pESP) [mm Hg]; central systolic (cSBP) and diastolic blood pressure (cDBP) [mm Hg]; central mean arterial pressure (cMAP) [mm Hg]; central augmented pressure (cAP) [mmHg]; central mean pressure of diastole (cMPD)[mm Hg]; central mean pressure of systole (cMPS) [mm Hg]; central end-systolic pressure (cESP) [mm Hg].

The following parameters of vessel stiffness were assessed by Pulse Trace 2000 (Micro Medical Ltd., Rochester, Kent, United Kingdom):

• reflection index (RI) [in percentages [%]];
• vascular stiffness index (SI) [m/s];
• peripheral pulse pressure (pPP) [mm Hg];
• central puls pressure (cPP) [mm Hg]
• peripheral pulse pressure/central pulse pressure (pPP/cPP ratio).

During a 2-year follow-up from the enrollment to this study, CV-related fatal incidents history has been recorded for each subject separately. The primary endpoint was fatal acute myocardial infarction (AMI) or acute ischemic stroke or any unexpected or sudden death only if autopsy proved CV-related. If there was doubt about the cause of death or there was no contact with the patient during the two years from study enrollment, that patient was excluded and not considered further.

glucose (Glu) [mg/dl] was assessed in the serum by a routine technique using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

klotho [ng/ml] - was analyzed in the serum by Human KL(Klotho) [ng/ml] ELISA Kit, Shanghai Sunred Biological Technology Co kit, China.

FGF-23 [pg/ml] - was analyzed in rhe serum using Human FGF-23 ELISA Kit, Sigma-Aldrich, USA.

• total and ionized calcium [mg/dl],
• phosphate [mg/dl],
• intact parathormone (iPTH) [mg/dl]

were assessed by routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

activity of:

• alanine transaminase (ALT) [U/l];
• aspartate transaminase (AST) [U/l];
• alkaline phosphatase (ALP) [U/l]

were assessed by routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

• total protein (TP) [g/dl];
• albumin (ALB) [g/dl]

were assessed in the serum by routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

• creatinine in the serum [mg/dl] - the assay is based on the reaction of creatinine with sodium picrate as described by Jaffe (Jaffes' colorimetric method) - was assessed by routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA;
• urea [mg/dl] in the serum - was assessed by routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

myeloperoxidase (MPO) [ng/ml] in the serum- was determined by the ELISA method using the Quantikine Human MPO test by R&D Systems kit, Canada.

soluble receptor for advanced glycation end products (sRAGE) [µg/mg protein] in the serum was tested with enzymatic immunoassay (Quantikine ELISA) using R&D Systems kit, Canada.

• methylglyoxal (MG) [µg/mg protein];
• carboxyethyle(lysine) (CEL) [µg/mg protein];
• carbamyl protein groups [µg/mg protein] were assessed in the serum by competitive enzyme immunoassay (competitive ELISA) using kits from Cell Biolabs Inc, USA.

• potassium (K) [mmol/l];
• sodium (Na) [mmol/l];
• magnesium (Mg) [mg/dL]

were assessed in the serum by routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

NT-proBNP [fmol/ml] - was analyzed in the serum by enzyme immunoassay using the Nt-proBNP kit from Biomedica, Slovakia.

eGFR [ml/min/1.73m^2] - according to the Kidney Disease: Improving Global Outcomes (KDIGO) 2012 recommendations was calculated based on the Modification of Diet in Renal Disease (MDRD) formula: eGFR = 186 x [creatinine concentration in mg/dl] - 1.154 x [age in years] - 0.203 x [0.724] for the female gender.

creatinine [mg/dl] concentration in the serum was assessed by routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA (based on Jaffes' colorimetric method - the assay is based on the reaction of creatinine with sodium picrate as described by Jaffe).

eGFR - according to the Kidney Disease: Improving Global Outcomes (KDIGO) 2012 recommendations was calculated based on the Modification of Diet in Renal Disease (MDRD) formula: eGFR = 186 x [creatinine concentration in mg/dl] - 1.154 x [age in years] - 0.203 x [0.724] for the female gender.

Body mass index (BMI) - [kg/m^2] was calculated by dividing a person's weight (post-HD weight in HD group) [kg] by the squared their body height [m].

total cholesterol (T-C) [mg/dl] concentration in the serum was assessed by routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

high-density lipoprotein cholesterol (HDL-C) [mg/dl] concentration in the serum was assessed by routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

low-density lipoprotein (LDL-C) cholesterol concentration in the serum was determined from Friedewals' equation (LDL-C [mg/dl] = total cholesterol (T-C) [mg/dl] - HDL-C [mg/dl] - TG[mg/dl]/5).

It was assessed by the routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

triglycerides (TG) [mg/dl] concentration in the serum was assessed by the routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

activity of aspartate transaminase (AST) [U/l]; was assessed in the serum by the routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

activity of alanine transaminase (ALT) [U/l] was assessed in the serum by the routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

activity of alkaline phosphatase (ALP) [U/l] was assessed in the serum by the routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

total protein (TP) [g/dl] concentration in the serum was assessed by routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

albumin (ALB) [g/dl] concentration in the serum was assessed by the routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

iron concentration [mg/dl] in the serum - was assessed with the Cobas Integra 400 plus biochemical analyzer from Roche Diagnostics, USA;

total iron-binding capacity (TIBC) [mg/dl] - was determined with the Cobas Integra 400 plus biochemical analyzer from Roche Diagnostics, USA.

unsaturated iron-binding capacity (UIBC) [mg/dl] was determined by an equation in which iron [mg/dl] concentration in plasma is subtracted from TIBC [mg/dl].

ferritin [ng/ml] concentration in the serum was determined with the Modular E-170 biochemical analyzer from Roche Diagnostics, USA.

total and ionized calcium [mg/dl] serum concentrations were assessed by routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

phosphate [mg/dl] serum concentration was assessed by the routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

intact parathormone (iPTH) [mg/dl] serum concentration was assessed by the routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

klotho (KL) [ng/ml] serum concentration was analyzed by Human KL(Klotho) [ng/ml] ELISA Kit, Shanghai Sunred Biological Technology Co kit, China.

fibroblast growth factor 23 (FGF-23) [pg/ml] serum concentration was analyzed using Human FGF-23 ELISA Kit, Sigma-Aldrich, USA.

Electrolytes: potassium (K) [mmol/l] and sodium (Na) [mmol/l] serum concentrations were assessed by the routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

magnesium (Mg) [mg/dl] serum concentration was assessed by routine techniques using Cobas Integra 400 plus biochemical analyzer by Roche Diagnostics, USA.

Serum concentration of 3-nitrotyrosine (3-NT) [µmol/mg protein] was determined with the enzyme immunoassay method (ELISA) for 3NT using Shanghai Sunred Biological Technology Co kits, China.

Serum concentration of advanced glycation ends products (AGE) [µg/mg protein] was determined with the enzyme immunoassay method (ELISA) for AGE using Shanghai Sunred Biological Technology Co kits, China.

Serum concentration of carboxymethyle(lysine) (CML) [µg/mg protein] was determined with the enzyme immunoassay method (ELISA) for CML using Shanghai Sunred Biological Technology Co kits, China.

Serum concentration of advanced oxidation protein products (AOPP) [µmol/mg protein] was determined with the enzyme immunoassay method (ELISA) for AOPP using Shanghai Sunred Biological Technology Co kits, China.

metalloproteinase 9 (MMP-9) [ng/ml] concentration in the serum was determined by the ELISA method using the Quantikine Human MMP-9 (total) kit, by R&D Systems, Canada.

tissue inhibitor of metalloproteinase 1 (TIMP-1) [ng/ml] concentration in the serum - was determined by the ELISA method using the Quantikine Human TIMP-1 kit, manufactured by R&D Systems, Canada.

the MMP-9/TIMP-1 ratio was calculated by the quotient of the MMP-9 [ng/ml] and the TIMP-1 [ng/ml] concentration.

high-sensitivity C-reactive protein (hsCRP) [mg/l] concentration in the serum was measured using DADE Behring, USA, and the DADE nephelometer Behring Analyzer II.

neopterin [nmol/l] serum concentration was determined by using the Neopterin ELISA kit, DRG International, Inc., USA.

interleukin 18 (IL-18) [pg/ml] concentration in the serum was determined by Colorimetric Sandwich ELISA, Quantikine Human IL-18 R&D Inc., USA.

myeloperoxidase (MPO) [ng/ml] in the serum - was determined by the ELISA method using the Quantikine Human MPO test by R&D Systems kit, Canada.

methylglyoxal (MG) [µg/mg protein] concentration in the serum was assessed by competitive enzyme immunoassay (competitive ELISA) using MG kits from Cell Biolabs Inc, USA.

carboxyethyle(lysine) (CEL) [µg/mg protein] concentration in the serum was assessed by competitive enzyme immunoassay (competitive ELISA) using CEL kits from Cell Biolabs Inc, USA.

carbamyl protein groups [µg/mg protein] concentration in the serum were assessed by competitive enzyme immunoassay (competitive ELISA) using carbamyl protein groups kits from Cell Biolabs Inc, USA.

soluble receptor for advanced glycation end products (sRAGE) [µg/mg protein] concentration in the serum was tested with enzymatic immunoassay (Quantikine ELISA) using R&D Systems sRAGE kit, Canada.

Blood pressure was measured using the Colin BPM 7000 on both arms (participants were seated). Next, a piezoelectric tonometer Colin BPM was placed over the radial artery for the acquisition of the radial arterial pressure waveform in a supine position. This signal was sent to the SphygmoCor and after averaging various parameters have been assessed and recorded:

• peripheral systolic blood pressure (sSBP) [mmHg];
• peripheral diastolic blood pressure (pDBP)[mm Hg];
• peripheral mean arterial pressure (pMAP) [mm Hg];
• peripheral end-systolic pressure (pESP) [mm Hg];
• central systolic blood pressure (cSBP) [mm Hg];
• central diastolic blood pressure (cDBP) [mm Hg];
• central mean arterial pressure (cMAP) [mm Hg];
• entral augmented pressure (cAP) [mm Hg];
• central mean pressure of diastole (cMPD) [mm Hg];
• central mean pressure of systole (cMPS)[mm Hg];
• central end-systolic pressure (cESP)[mm Hg]

Blood pressure was measured using the Colin BPM 7000 on both arms (participants were seated). Next, a piezoelectric tonometer Colin BPM was placed over the radial artery for the acquisition of the radial arterial pressure waveform in a supine position. This signal was sent to the SphygmoCor and after averaging various parameters have been assessed and recorded:

- heart rate (HR) in beats per minute [bpm]

Blood pressure was measured using the Colin BPM 7000 on both arms (participants were seated). Next, a piezoelectric tonometer Colin BPM was placed over the radial artery for the acquisition of the radial arterial pressure waveform in a supine position. This signal was sent to the SphygmoCor and after averaging various parameters have been assessed and recorded:

- ejection duration (ED) in milliseconds [msec]

Carotid intima-media thickness (IMT) [mm] was measured by The Accuson CV 70 system (Siemens) with a 10 megahertz (Mhz) transducer.

Two longitudinal projections were assessed (anterolateral and posterolateral). The distal 1 cm of the common carotid artery just proximal to the bulb was measured by means of a computer analysis system (Medical Imaging Applications, LLC).

The following parameter of vessel stiffness was assessed by Pulse Trace 2000 (Micro Medical Ltd., Rochester, Kent, United Kingdom):

- reflection index (RI) in percentages [%].

The following parameter of vessel stiffness was assessed by Pulse Trace 2000 (Micro Medical Ltd., Rochester, Kent, United Kingdom):

-vascular stiffness index (SI) [m/s].

The following parameters of vessel stiffness were assessed by Pulse Trace 2000 (Micro Medical Ltd., Rochester, Kent, United Kingdom):

• peripheral pulse pressure (pPP) [mm Hg];
• central pulse pressure (cPP) [mm Hg]

Peripheral pulse pressure/central pulse pressure (pPP/cPP) ratio was assessed by dividing peripheral pulse pressure (pPP) [mm Hg] by central pulse pressure (cPP) [mm Hg].

During a 2-year follow-up from the enrollment to this study, CV-related fatal incidents history has been recorded for each subject separately. The primary endpoint was fatal acute myocardial infarction (AMI) or acute ischemic stroke or any unexpected or sudden death only if autopsy proved CV-related. If there was doubt about the cause of death or there was no contact with the patient during the two years from study enrollment, that patient was excluded and not considered further.

N-terminal pro-B-type natriuretic peptide (NT-proBNP) [fmol/ml] concentration in the serum was analyzed by enzyme immunoassay using the Nt-proBNP kit from Biomedica, Slovakia.