The effect of shear stress and urea on endothelial CAT-1 expression
Date
2016
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Chronic Kidney Disease (CKD) affects over 26 million people in the United States and has an estimated prevalence of 11.5%. CKD is associated with an elevated risk of cardiovascular disease (CVD), even if traditional cardiovascular risk factors are not present. This may be due in part to a reduction in the bioavailability of the vasodilator nitric oxide (NO) and an impairment in the transport of the NO substrate L-arginine into the endothelium. Cell culture studies have shown that uremic toxins such as urea play a role in the inhibition of L-arginine transport into endothelial cells, ultimately leading to endothelial dysfunction. L-arginine is transported into the endothelium through the cationic amino acid transporter CAT-1, which is regulated by PKCα. Evidence from previous studies suggests that PKCα phosphorylates CAT-1, directly altering and decreasing its catalytic activity, or that PKCα causes internalization of CAT-1 to the inside of the cell where it can no longer participate in L-arginine transport. Increased endothelial shear stress, which occurs during physical activity as a result of increased blood flow, has been shown to improve L-arginine uptake into endothelial cells. Interestingly, previous studies have shown that in the 5/6 ablation/infarction (A/I) rat model of CKD, 4 weeks of voluntary wheel running improved L-arginine uptake, however, this did not occur through an increase in CAT-1 expression. However, in sedentary CKD animals, PKCα expression was increased compared groups that participated in voluntary wheel running. Additionally, in uremic rats, treatment with rosiglitazone, a PPARγ agonist and anti-diabetic drug that has been shown to be beneficial in CKD, was associated with a decrease in PKCα, as well as a decrease in phosphorylated CAT-1 compared to untreated rats. The benefits of physical activity to potentially decrease the expression of PKCα thereby improving L-arginine uptake through a decrease in phosphorylated CAT-1 makes exercise a potential treatment to reduce the risk of cardiovascular disease in CKD. Our overall hypothesis was that shear stress alters CAT-1 expression and the expression of its phosphorylated state. In our first set of experiments, we studied the effect of different shear stress levels on CAT-1, PKCα, and phosphorylated CAT-1 expression. As shear stress increased, CAT-1 expression also increased, with expression at 20 dyn/cm2 being significantly greater than 0 dyn/cm 2 (p < 0.05). PKCα and phosphorylated CAT-1 expression decreased as shear levels increased starting at 10 dyn/cm2. In the second set of experiments, we explored the effect of different shear stress levels on CAT-1, PKCα, and phosphorylated CAT-1 expression in the presence of 25mM of the uremic toxin, urea. CAT-1 expression remained unchanged with all shear stress levels, suggesting that shear stress may result in a post translational effect of CAT-1. Both PKCα and phosphorylated CAT-1 expression were significantly decreased at all shear levels compared to 0 dyn/cm2 (p < 0.05). This decrease in phosphorylation of CAT-1 by PKCα could in turn lead to enhanced L-arginine transport and vascular function. These results suggest that activities that increase shear stress, such as exercise, may be a beneficial therapy in improving vascular function in patients suffering from CKD.