Additional file 1: Figure S1. of Lipopolysaccharide pretreatment increases protease-activated receptor-2 expression and monocyte chemoattractant protein-1 secretion in vascular endothelial cells

Western blot – Anti-PAR-2 antibody. Lane A: Control cell lysate at 10 μg. Lane B: PAR-2 agonist (AC 55541, 10 μM)-treated cell lysate at 10 μg. Predicted band size: 43~55 kDa. (TIFF 823 kb

Uremic Retention Solute Indoxyl Sulfate Level Is Associated with Prolonged QTc Interval in Early CKD Patients

<div><p>Total mortality and sudden cardiac death is highly prevalent in patients with chronic kidney disease (CKD). In CKD patients, the protein-bound uremic retention solute indoxyl sulfate (IS) is independently associated with cardiovascular disease. However, the underlying mechanisms of this association have yet to be elucidated. The relationship between IS and cardiac electrocardiographic parameters was investigated in a prospective observational study among early CKD patients. IS arrhythmogenic effect was evaluated by in vitro cardiomyocyte electrophysiological study and mathematical computer simulation. In a cohort of 100 early CKD patients, patients with corrected QT (QTc) prolongation had higher IS levels. Furthermore, serum IS level was independently associated with prolonged QTc interval. In vitro, the delay rectifier potassium current (IK) was found to be significantly decreased after the treatment of IS in a dose-dependent manner. The modulation of IS to the IK was through the regulation of the major potassium ion channel protein Kv 2.1 phosphorylation. In a computer simulation, the decrease of IK by IS could prolong the action potential duration (APD) and induce early afterdepolarization, which is known to be a trigger mechanism of lethal ventricular arrhythmias. In conclusion, serum IS level is independently associated with the prolonged QTc interval in early CKD patients. IS down-regulated <i>I_K</i> channel protein phosphorylation and the <i>I_K</i> current activity that in turn increased the cardiomyocyte APD and QTc interval in vitro and in the computer ORd model. These findings suggest that IS may play a role in the development of arrhythmogenesis in CKD patients.</p></div

Ventricular cardiomyocyte action potential (AP) and pseudo-ECG constructed by the O'Hara-Rudy dynamic human ventricular model.

<p>The suppression of inward rectifier potassium current (<i>I</i>_{<b><i>K</i></b>}) mimics the effect of indoxyl sulfate toxicity to ventricular cardiomyocyte AP. The AP duration was gradually increased and the QT interval was also prolonged with the increment of <i>I</i>_{<b><i>K</i></b>} suppression. The early afterdepolarization was noted in the higher suppression of <i>I</i>_{<b><i>K</i></b>} especially in the mid-myocardial cardiomyocyte (arrow). The ventricular arrhythmias like ECG was also noted when the <i>I</i>_{<b><i>K</i></b>} was severely suppressed (empty arrow).</p

Multiple logistic regression analysis with presence of prolonged QTc interval as the dependent variable.

<p>BMI, body mass index; BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein.</p><p>Multiple logistic regression analysis with presence of prolonged QTc interval as the dependent variable.</p

Clinical and biochemical variables associated in univariate analysis with log indoxyl sulfate.

<p>HDL: high-density lipoprotein, LDL: low-density lipoprotein.</p><p>Clinical and biochemical variables associated in univariate analysis with log indoxyl sulfate.</p

The effect of IS on H9c2 cardiomyocyte <i>I</i>_<i>K</i> and potassium channel protein Kv2.1 expression.

<p>(A) The representative current traces for delayed rectifier potassium outward currents (<i>I</i>_{<b><i>k</i></b>}) in H9c2 cells with different indoxyl sulfate (IS) concentration treatment. <i>I</i>_{<b><i>k</i></b>} were elicited by 300 ms depolarizing step pulses from -70 to 50 mV at a holding potential of -60 mV. (B) The average relationships between <i>I</i>_{<b><i>k</i></b>} (pA/pF) and membrane potential in the control, 0.1μM IS, 1 μM IS and 300μM IS groups (n = 6 for each groups) comparing the IS treated group with the control group, <i>I</i>_{<b><i>k</i></b>} was significantly decreased at membrane potentials from 0 mV to 50 mV in a dose-dependent manner. (C and D) The expression of Kv2.1 and phosphorylated Kv2.1 by Western blot in the H9c2 cells treated with different concentration of IS (0.1μM, 1 μM and 300μM). The expressions of Kv2.1 were not significantly different among the control and IS-treated groups (C). However, the phosphorylated Kv2.1 was significantly decreased in the 1 μM IS-and 300 μM-IS treated groups (D). (n = 6 for each groups) *: <i>p</i><0.05 as compared with the control group.</p

Patient clinical laboratory data according to QTc classification.

<p>Data are expressed as mean ± SD, number (%), or median (interquartile range). BMI, body mass index; BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein.</p><p>Patient clinical laboratory data according to QTc classification.</p