Table 2 Protocol for the study procedures

FMD

- use of Philips CX50 Ultrasound System with a 12-Mhz probe;

- the vasoactive medications removal for 24 h before the procedure;

- patient in supine position for at least 15 min before the examination;

- the right arm will be immobilized in the extended position for the brachial artery 2–4 cm above the antecubital fossa recording; the contralateral arm will be used if an arteriovenous fistula is present;

- 3 adjacent measurements of end-diastolic brachial artery diameter will be made from single 2D frames;

- the cuff will be inflated up to 50 mmHg higher value of systolic blood pressure and kept this way for 5 minutes;

- the maximum FMD diameters will be analysed from the average of the 3 consecutive maximum diameter measurements, and 1 minute after the cuff will be deflated;

- the FMD will be calculated as the percentage change in diameter compared with baseline resting diameters, in agreement with the criteria set by the International Brachial Artery Reactivity Task Force.

Arterial stiffness

- applanation tonometry using SphygmoCorTM; PWV Inc., Westmead, Sydney, Australia;

- the patient will be recumbent 10 minutes before the measures;

- measurements: sequentially pulse of the carotid and femoral, the transit time from the R-wave of the simultaneously acquired electrocardiogram to the foot of the carotid and femoral pulse, and the difference acquired electrocardiogram to the foot of the carotid and femoral pulse;

- the carotid-femoral PWV will be calculated from the difference between these 2 transit times divided by distances measured from the body surface (arterial path length).

IMT

- Philips CX50 Ultrasound System with a 12 MHz probe;

- high-resolution B-mode ultrasound of the common carotid arteries with scanning of the longitudinal axis until the bifurcation and of the transversal axis will be performed;

- two longitudinal measurements will be obtained for each carotid artery, by rotating the vessels at 180^o increments along their axis;

- IMT will be measured at 1 cm proximal to the bifurcation on each side.

Echocardiography

- according to the American Society of Echocardiography recommendations by an observer unaware of the LUS and bioimpedance results;

- measurements: cardiac anatomy (e.g. volumes, geometry, mass) and function (e.g. left ventricular function, valvular function, right ventricular function, and pulmonary artery pressure);

- in patients with adequate acoustic windows, both left ventricular global longitudinal strain (LVGLS) and right ventricular free wall strain (RVFWS) will be calculated.

LUS

- patient in the supine position;

- measurements of the anterior and lateral chest on both sides of the chest, from the second to the fourth (on the right side to the fifth) intercostal spaces, at parasternal to mid-axillary lines;

- B-lines will be recorded in each intercostal space and were defined as a hyperechoic, coherent US bundle at narrow basis going from the transducer to the limit of the screen; − B-lines starting from the pleural line can be either localized or scattered to the whole lung and be present as isolated or multiple artifacts;

- the sum of B-lines produces a score reflecting the extent of lung water accumulation (0 being no detectable B-line).

BIS

- portable whole-body multi-frequency bioimpedance analysis device (BCM®Body Composition Monitor – Fresenius Medical Care D GmbH);

- electrodes will be attached to the patient’s forearm and ipsilateral ankle, with the patient in a supine position;

- measurements: body resistance and reactance to electrical currents of 50 discrete frequencies, ranging between 5 and 1000 kHz;

- calculations: ECW, ICW and TBW, to determine the amount of fluid overload;

- AFO is defined as the difference between the expected patient’s ECW under normal physiological conditions and the actual ECW, whereas RFO is defined as the absolute fluid overload AFO to ECW ratio.

Blood collection for biomarker evaluation

- whole blood will be collected in gel & clot activator tubes (minimum 3.5 mL tube) at each intervention visit for each patient;

- serum will be collected by centrifugation and frozen in aliquots at −80 ° C until further analysis;

- IL1, IL6, VCAM-1, endoglin, NO and ADMA in serum samples will analysed by specific enzyme linked immunosorbent assay (ELISA) kits on a Sunrise Basic Tecan microplate reader.