Di Ianni, Alisha2017-01-272017-01-272016http://udspace.udel.edu/handle/19716/20329Chronic 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.Chronic renal failure.Strains and stresses.Carrier proteins.Vascular endothelial cells.Urea.Protein kinase C.Arginine.Phosphorylation.Gene expression.Thesis970393576