Over 26 million Americans have chronic kidney disease (CKD) and many more are at risk due to hypertension and diabetes. According to the 2017 Center for Disease Control’s CKD Surveillance Study, 66% of those with CKD also have cardiovascular disease (CVD) compared to 33% of people in the general population. Endothelial dysfunction precedes the development of CVD (15, 30, 75, 84) and has been recognized by the American Heart Association as a key link between CVD development in CKD. Inhibition of L-arginine transport through the cationic amino acid transporter-1 (CAT-1) as a result of elevated urea levels in late stage CKD makes endothelial dysfunction more difficult to treat than in earlier stages (10, 25, 74, 136). Previous studies in our lab have demonstrated in a rat model of CKD that low volume aerobic exercise helped restore L-arginine transport (77). Increased shear stress as a result of wheel running was a likely factor affecting L-arginine transport however it is difficult to isolate the effect of shear stress during exercise in rodents due to other factors in play during exercise . We sought to isolate the effect of shear stress on CAT-1 mediated L-arginine transport in cultured endothelial cells (HUVECs) exposed to urea. Our central hypothesis for this study was that shear stress would increase L-arginine transport and alter the expression of CAT-1 via alterations in its phosphorylated state and impact subcellular colocalization of CAT-1 and eNOS.
First, we investigated the effect of multiple levels of shear stress in the presence of urea or mannitol (osmotic control) on L-arginine transport and NOx production utilizing a previously established radiolabeled L-arginine uptake assay (45, 77) and commercially available NOx assay kits, respectively. Cells were harvested immediately or 6 hours following shear stress based on preliminary studies. We observed that L-arginine transport was increased immediately following shear stress in cells treated with both mannitol and urea (main effect: shear, p=0.005; treatment, p=0.258). Six hours after shear stress, L-arginine transport remained elevated in mannitol samples but transport was blunted by urea (main effect: shear, p=0.005; treatment, p<0.001). The augmented L-arginine transport from shear stress corresponded to an increase in NOx production compared to pre shear stress (p<0.001). We hypothesized that these changes were due to post translational modification of CAT-1 via phosphorylation. Phosphorylated CAT-1 (measured via western blotting) was elevated 6 hours post shear stress (main effect treatment p < 0.001) but not immediately after (main effect treatment p=0.841) indicating a possible shear-based mechanism which reduced phosphorylation following shear stress.
In our second set of experiments, we used a novel imaging method to examine the colocalization of CAT-1 and eNOS at the plasma membrane of HUVECs. Cells were stained for CAT-1 and eNOS following shear stress. Quantitative analysis of the CAT-1 pixel intensity revealed a greater amount of CAT-1 at the membrane immediately post shear stress. This was attenuated in the presence of urea (main effect: shear stress, p<0.001; treatment, p<0.001). We also observed that immediately following shear stress there was greater colocalization of CAT-1 and eNOS as indicated by a Mander’s value <0.5 (62) (main effect treatment: p<0.001; shear, p=0.001; interaction, p=0.001). Colocalization was sustained at 6 hours post (main effect treatment: p=0.762; shear, p=0.014).
In conclusion, these studies provide evidence that shear stress impacts the phosphorylation state of CAT-1 and subcellular location, which improved L-arginine transport and NOx production. Further studies are needed to understand the exact mechanism by which CAT-1 and eNOS translocate to the membrane.
|Advisor:||Edwards, David G.|
|Commitee:||Lennon, Shannon L., Wenner, Megan M., Guers, John J.|
|School:||University of Delaware|
|School Location:||United States -- Delaware|
|Source:||DAI-B 82/3(E), Dissertation Abstracts International|
|Subjects:||Physiology, Cellular biology, Health sciences|
|Keywords:||CAT-1, eNOS, Exercise, Kidney Disease, Shear Stress, Urea|
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