L-arginine transport and endothelial dysfunction in chronic kidney disease: the role of exercise

Martens, Christopher
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University of Delaware
Chronic kidney disease (CKD) affects more than 26 million people in the United States and is associated with an elevated risk of cardiovascular disease (CVD) that persists even in the absence of traditional cardiovascular risk factors. Impairments to the vascular endothelium precede the development of atherosclerosis and have been extensively studied as a potential therapeutic target to treat CVD, however few studies have been performed in CKD. A reduction in the bioavailability of the vasodilator nitric oxide (NO) contributes to CVD-related mortality in CKD and is likely related to a reduced availability of the NO substrate L-arginine. Interestingly, the use of L-arginine in studies of endothelial dysfunction in late-stage CKD has produced mixed results unlike other diseases where it has been largely effective. Evidence from cell culture studies suggests that urea and other uremic toxins inhibit L-arginine uptake into endothelial cells and may provide a unique explanation for why L-arginine treatment has been ineffective in late-stage CKD. Additionally, competition for L-arginine from the enzyme arginase as well as increased oxidative stress are possible mechanisms by which NO production and bioavailability are reduced. Physical activity has been shown improve vascular function in patients with CVD through reductions in oxidative stress and improvements in NO synthesis, and may be an effective therapy to reverse vascular dysfunction in CKD. Increased physical activity has also been shown to augment L-arginine uptake in the forearm vasculature of heart failure patients. The known cardiovascular benefits of aerobic exercise combined with its potential to improve L-arginine uptake make increased physical activity an attractive therapy to reduce cardiovascular risk in CKD. Our overall hypothesis was that a deficiency in L-arginine availability contributes to endothelial dysfunction in rats with chronic kidney disease and that this impairment would be restored by increased physical activity. In our first series of experiments, we explored the mechanisms of impaired endothelium-dependent relaxation (EDR) in a rat model of moderate to severe CKD. The enzyme arginase has been implicated as a potential contributor to vascular dysfunction by contributing to the consumption of L-arginine, reducing its availability for NO production. The purpose of this study was to determine if arginase contributes to endothelial dysfunction in the 5/6 ablation/infarction (AI) rat model of CKD. 12 week old male Sprague Dawley rats underwent AI surgery to induce CKD, or a SHAM surgery to serve as a control. EDR of aortic rings to acetylcholine was significantly impaired in AI animals vs. SHAM after 8 weeks (Emax; 56.17 ± 7.82 vs 100.85 ± 5.55, p<0.05; LogEC50; -5.67 ± 0.14 vs -7.74 ± 0.25, p<0.05), and was not improved by arginase inhibition (BEC) alone or in combination with L-arginine. Additionally, scavenging of superoxide (Tempol, Tempol + L-arginine, Tempol + L-arginine + BEC) was not effective suggesting that a mechanism independent of oxidative stress contributes to EDR in moderate to severe CKD. Neither arginase enzyme activity (0.012 ± 0.004 vs. 0.014 ± 0.006) or protein expression of either isoform of arginase (arg1: 0.79 ± 0.03 vs. 1.00 ± 0.11; arg2: 0.85 ± 0.18 vs. 1.00 ± 0.25) was increased in AI animals vs. SHAM, confirming that arginase does not contribute to impaired EDR in CKD. Aortic uptake of radio-labeled L-arginine was attenuated in AI animals vs. SHAM (47.06 ± 12.2 vs. 100 ± 16.3, p <0.05) and was associated with a reduced protein expression of the L-arginine transporter CAT-1 (0.37 ± 0.06 vs. 1.0 ± 0.17, p<0.05). These data suggest that arginase does not contribute to endothelial dysfunction in CKD; however, impaired L-arginine transport may play an important role in diminishing substrate availability for NO production leading to endothelial dysfunction. Our second set of experiments was designed to reverse the impairment in endothelial function using a voluntary wheel running intervention. We tested the effect of 4 weeks of voluntary wheel running (RUN) and/or ARG supplementation on vascular function in rats with CKD. We hypothesized that ARG intervention alone would be ineffective due to impaired L-arginine transport and that RUN would improve EDR, in part due to improved utilization of L-arginine. Based on this hypothesis, we also predicted that the greatest improvement in EDR would be observed when animals were treated with ARG and RUN in combination. 12 week old male Sprague Dawley rats underwent either AI surgery to induce CKD, or a SHAM surgery as a control. CKD animals either remained sedentary (SED) or received one of the following interventions: supplemental ARG (1.25 g/L in drinking water), RUN, or combined RUN+ARG. Interventions began 4 weeks after surgery to allow time for disease progression. Animals were sacrificed 8 weeks after surgery and EDR was assessed by dose response to acetylcholine in aortic rings. EDR was significantly impaired in SED vs. SHAM animals after 8 weeks, demonstrated by an attenuated area under the curve (AUC; 44.56 ± 9.01 vs. 100 ± 4.58, p<0.05) and reduced maximal response (Emax; 59.9% ± 9.67 vs. 94.31% ± 1.27, p <0.05). AUC was not improved by ARG treatment but was significantly improved above SED animals in both RUN and RUN+ARG treated animals. Emax was elevated above SED in RUN+ARG animals only. CAT-1 protein expression was impaired in SED animals and was not improved by any of the interventions. Aortic uptake of radio-labeled L-arginine was attenuated in SED and ARG-treated animals and was significantly improved in both RUN and RUN+ARG animals. PKCα has been implicated as a contributor to reduced L-arginine transport and was shown to be significantly elevated in SED animals, but returned to similar levels as SHAM animals in both RUN and RUN+ARG treated animals. The results indicate that voluntary wheel running is an effective therapy to improve vascular function in CKD, in part through improvements in L-arginine uptake and suggest that exercise may be a beneficial adjunct therapy to address cardiovascular disease in patients with CKD.