Clinical Outcomes of Chronic Kidney Disease Patients on Hemodialysis in Brazil: A Prospective Cohort Study

Evaluation of HRV. HRV measurement was performed using an electrocardiographic device (Wincardio@, Micromed). The registry was acquired for 20 min before, in the second hour, and immediately after a midweek HD session. Patients were in a supine position and breathed normally during the recording. Patients with unstable patterns of breath had their HRV recording postponed. The electrocardiographic device transmitted the R-R interval record to digital storage, which was subsequently analyzed in the time and frequency domains of HRV through CardioSeries@ 2.4 software. In the time domain, RMSSD and SDNN were acquired. In the frequency domain analysis, a tachogram was interpolated in a frequency of 4 Hertz and divided into periods of 256 points with 50% overlap. These signals were submitted to spectral analysis by fast Fourier transformation to obtain the total power of the spectrum. The LF band, representing sympathetic modulation, was between 0.02 and 0.15 Hz. The HF band, attributed to parasympathetic modulation, was between 0.15 and 0.40 Hz.

ACE gene typing. Blood samples were drawn and centrifugated. The leukocyte supernatant pellet was extracted and stored. ACE polymorphism was typed by PCR for DD, ID, and II genotypes. It used the following specific primers for DNA amplification: sense 5=-CTGGAGACCACTCCCATCCTTTCT-3= and antisense 5= GATGTGGCCATCACATTCGTAGA- 3=. Through these primers, it was possible to detect allele deletion and insertion. A confirmatory PCR was done for all samples with the DD genotype to increase the specificity of the genotyping using a pair of specific primers for insertions, i.e., an allele I amplifier. These primers were as follows: sense 5=-TGGGACCACAGCGCCCGCCACTAC-3= and antisense 5=-TCGCCAGCCCTACCATGCCCATAA-3=. The temperature protocol was programed in the thermocycler beginning at 95°C for 5 min and then 40 cycles of denaturation at 95°C for 45 s, annealing at 54°C for 1 min, and extension at 72°C for 30 s each and then at 5 min at 72°C to finish uncompleted chains.

Hydration status assessment. The patient's hydration status was evaluated before and after (up to 20 min) the studied HD session using a Body Composition Monitor (Fresenius), which uses multifrequency bioimpedance spectroscopy to determine the electrical resistances of total body water and extracellular water. Electrodes were attached to the hand and feet opposite to vascular access, with the patient in a supine position, and the measurement was done and stored in a digital format and analyzed with the Fluid Management Tool. The results were provided as overhydration (in liters), which represents the excess of water above reference levels according to the patient's sex and age.

Muscle thickness and echo intensity by ultrasound Patients were evaluated after one midweek dialysis session. They initially were instructed to stand with the legs and ~20 cm apart. The length of right thigh was measured from the superior iliac spine to the proximal border of the patella. Right and left circumferences were assessed at the midpoint of the thighs. At the same level on the anterior face of the thighs, skin was marked with a water-resistant pen. The quadriceps thickness was measured with the probe over these points, perpendicular to the long axis of the limb (cross-sectional image) and captured after proper visualization of the bone surface. The image was stored in device's hard disk for each of the two sites.

The images were analyzed later using the software ImageJ (National Institute of Health, USA, version 1.37) for both muscle thickness and echo intensity (EI). The thickness was measured as the perpendicular distance between the point just above the apex of the femur surface and the point just below the rectus femoris' superficial fascia obtained using the same software with the length function. The measurements were registered in millimeters and posteriorly converted to centimeters.