Impaired Vascular Function Contributes to Exercise Intolerance in Chronic Kidney Disease

Amaryllis H. Van Craenenbroeck; Emeline M. Van Craenenbroeck; Katrijn Van Ackeren; Vicky Y. Hoymans; Gert A. Verpooten; Christiaan J. Vrints; Marie M. Couttenye

Disclosures

Nephrol Dial Transplant. 2016;31(12):2064-2072. 

In This Article

Materials and Methods

Subjects

Ambulatory patients with a diagnosis of CKD according to the Kidney Disease Outcomes Quality Initiative (KDOQI) guidelines, regardless of severity or cause, were systematically screened for eligibility at the outpatient clinic of Antwerp University Hospital. Of the 966 patients assessed in the period between April 2012 and July 2014, 63 patients were included in the study (Figure 1). None were enrolled in a formal exercise trial. Diagnosis of CKD was based on eGFR using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula[18] and/or the presence of kidney injury, as recommended by the National Kidney Foundation's KDOQI guidelines.[19] Patients with a history of overt cardiovascular disease, including coronary artery disease, peripheral vascular disease or cerebrovascular disease, were excluded. Other exclusion criteria were renal replacement therapy, pregnancy, age <18 years, treatment with immunosuppressive or oral anticoagulation therapy and active malignant disease.

Figure 1.

Patient flow. Of 966 patients assessed in the period between April 2012 and July 2014, 63 patients were included in the study. Eight hundred and forty-three subjects were excluded for not meeting the inclusion criteria, with overt cardiovascular disease accounting for 78% of the exclusions. Of the 123 eligible patients, 40 refused to participate and 63 were included in the study after obtaining informed consent.

Eighteen healthy subjects without a relevant medical history or pharmacological treatment and with no abnormalities on exercise testing, were asked to participate. The study was approved by the ethics committee of the Antwerp University Hospital and conformed to the principles outlined in the Declaration of Helsinki. All participants gave written informed consent.

Study Design

All study participants underwent initial blood and urine sampling, vascular assessment, electrocardiogram (ECG) and cardiopulmonary exercise tests (CPETs). Participants were asked to refrain from food, caffeine and excessive physical exertion for 12 h prior to the study.

In CKD patients, the absence of structural heart disease was confirmed by transthoracic echocardiography. Systolic and diastolic function was assessed by measurement of left ventricular ejection fraction (LVEF, modified Simpson rule), left ventricular end-diastolic diameter (LVEDD) and diastolic function (E/e').

CPET

A symptom-limited CPET was performed on a bicycle ergometer (Cardiovit CS-200 Ergo-Spiro, Schiller AG, Baar, Switzerland). A ramp protocol was used, starting with an equivalent of 20 or 40 watts (W) and increasing by incremental steps equivalent of 10 or 20 W/min. Twelve-lead ECG was recorded continuously and blood pressure was measured at baseline and every 2 min. Peak oxygen consumption (VO2peak) was expressed as the highest attained VO2 during the final 30 s of exercise. Maximal work economy was defined as maximal workload at VO2peak (Wmax/VO2peak). Online analysis of VE/VO2 and VE/VCO2 curves permitted encouragement of patients to exercise until exhaustion, confirmed by a respiratory exchange ratio (RER) >1.10.

Blood Sampling and Analysis

Serum creatinine, total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides, iron, total iron binding capacity (TIBC), transferrin saturation and high-sensitivity C-reactive protein (hs-CRP) were quantified using routine laboratory techniques (Dimension Vista 1500 System, Siemens). Complete blood count was measured on an Advia haematology analyzer (Advia 2120, Siemens Healthcare Diagnostics). Plasma samples [ethylenediaminetetraacetic acid (EDTA)] were immediately stored at −80°C for later quantification of miRNA (batch analysis). In all patients, the urine protein:creatinine ratio was evaluated in a spot morning urine sample.

Vascular Assessments

All measurements were performed between 8:00 and 11:00 a.m. in a quiet room with a stable temperature of 21–24°C.

Endothelial-dependent Vasodilation. Endothelial-dependent vasodilation was assessed by flow-mediated dilation (FMD) of the brachial artery using ultrasound (10 MHz ultrasound Doppler probe, AU5 ultrasound system, Esaote, Biomedica, Genova, Italy) according to current guidelines.[20,21] After measuring baseline internal diameter, a pneumatic tourniquet, placed 1–2 cm distal to the elbow, was inflated to 200 mmHg or at least 50 mmHg above peak systolic pressure for 5 min. After cuff release, the diameter was recorded continuously for 4 min and FMD was expressed as the per cent dilation from baseline to maximal post-occlusion diameter. Endothelial-independent vasodilation was measured after sublingual administration of 0.4 mg glyceryl trinitrate (GTN-MD). Offline analyses were performed by a single trained investigator using FMD-I software.

Arterial Stiffness. Carotid-femoral pulse wave velocity (PWV) was measured using the SphygmoCor device (AtCor Medical, West Ryde, Australia) according to current guidelines.[22,23] The system uses a single high-fidelity applanation tonometer (Millar Instruments, Houston, TX, USA) to obtain a proximal (i.e. carotid) and distal pulse (i.e. femoral), recorded consecutively, and calculates PWV from the transit time between the two arterial sites as PWV = distance (m)/transit time (s). As such, higher PWV values represent stiffer arteries. All measurements were performed in triplicate and were repeated if they did not meet the quality control guidelines, as defined by the manufacturer.

Targeted Quantification of miRNA

A panel of five miRNAs, reported in the literature to be involved in vascular homeostasis, was designed. This included miR-21, miR-126, miR-146a, miR-150 and miR-210.[24] Stored plasma samples were thawed on ice and centrifuged at 4°C for 10 min (16 000 g). Total RNA, including miRNA, was isolated from 200 μL EDTA plasma with the miRNeasy serum/plasma kit (Qiagen, Venlo, The Netherlands). To test for sample-to-sample variation in RNA isolation, a fixed amount of synthetic Cel-miR-39 was added to the sample immediately after lysis with Qiazol. Total RNA was extracted using chloroform, ethanol and a spin column and eluted in 15 μL RNAse-free water. An aliquot of the isolated RNA was used for multiplexed reverse transcription of mature miR-21, miR-126, miR-146a, miR-150 and miR-210 into cDNA using specific stem-loop primers (Applied Biosystems). Levels of selected miR were quantified using real-time PCR via TaqMan probes (Applied Biosystems) in a Biorad CFX96 real-time PCR system. Exogenously added synthetic miR-Cel-39 was used as a spike-in normalization control. All reactions and analyses were performed in duplicate. The threshold coefficient of variation (CV) accepted for intra-assay replicates was set at 4%. Ct values were used for relative miRNA quantification using the ΔCt method. Relative miRNA levels were expressed as log (2−ΔCT*100).

Statistical Analysis

Data are expressed as mean ± standard deviation (SD). Normality was assessed using one-sample Kolmogorov–Smirnov and logarithmic transformation was applied where necessary. Differences between the three groups were analysed using the χ 2 test or by one-way ANOVA followed by the Sidak post hoc test for multiple comparison corrections. Where applicable, comparisons between the two CKD groups were performed using the χ 2 test or independent samples t-test. Bivariate correlations were measured by Pearson's correlation analysis. Stepwise multiple linear regression analysis was used to evaluate independent determinants of VO2peak, adjusting for all significant determinants on bivariate correlation analysis.

All tests were two-sided and a P-value <0.05 was considered statistically significant. All analyses were performed using PASW Statistics 22.0 (SPSS, Chicago, IL, USA).

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