Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC185
Effects of End Stage Chronic Renal Failure on L-arginine-nitric oxide pathway and urea cycle in red blood cells
M. B. Moss1,4, M. A. Siqueira1, M. A. Martins1, N. R. Pereira1, S. F. Santos2, J. R. Lugon3, T. M. Brunini1, A. Mendes-Ribeiro1,4
1. Departamento de Farmacologia e Psicobiologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil. 2. Disciplina de Nefrologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil. 3. Disciplina de Nefrologia, Universidade Federal Fluminense, Rio de Janeiro, Brazil. 4. Departamento de Ci
Chronic renal failure (CRF) is characterized by the presence of endothelial dysfunction, associated with increased bioavailability of nitric oxide (NO) in blood cells (1). L-arginine is a cationic amino acid that is converted into NO by a family of enzymes referred as NO synthase (NOS). It is also converted into L-ornithine and urea by arginase and both enzymes compete for the same substrate (2). Recent evidence has shown that red blood cells (RBC) produce and release NO, which regulates RBC deformability and promotes vasodilation (3). The aim of the present study was to investigate the effects of CRF on the L-arginine-NO pathway, the arginase pathway in RBC and RBC osmotic fragility (OF) as a measure of membrane instability. Were included In this study 13 CRF patients under haemodialysis and 12 healthy controls. L-arginine Influx was determined by measuring the uptake of L-[3H]-arginine (5-100 µM). N-ethylmaleimide was used to isolate transport system y+L. Basal NOS activity was measured by the conversion of L-[3H]-arginine into L-[3H]-citrulline. Expression of arginase I and II was accessed by Western Blotting. Arginase activity was analyzed in RBC lysate through the conversion of [C14]-L-arginine into [C14]-urea. OF was assessed by the incubation of RBC with different concentrations of NaCl. The present study was approved by the Pedro Ernesto Hospital Ethical Committee (process 451-CEP/HUPE), and informed consent was obtained from each participant. Values are means ± S.E.M. compared by the Student’s t-test. L-arginine influx (µmol/L/cells/h) was increased in RBC from CRF patients via system y+ (1908 ± 364 vs. 638 ± 109, p<0.05) and y+L (771 ± 230 vs. 221 ± 64, p<0.05), however, no difference was detected in NOS activity (IRC: 0.031 ± 0.001 vs. 0.037 ± 0.005 nmol/108 cells, controls). RBC expressed only arginase I and CRF patients showed an overexpression of this enzyme (5 replicates of each group) associated with increased enzyme activity (IRC: 34.4 ± 5 vs. 21 ± 2 nmol urea/mg protein/ 2h controls, p<0.05). RBC from CRF patients showed increased OF at 0.7 (IRC: 18.9 ± 5 vs. 5.3 ± 2 % haemolysis controls, p<0.05) and 0.65 % NaCl (IRC: 34.1 ± 12 vs. 7.5 ± 3 % haemolysis controls, p<0.05). Thus the present study showed that L-arginine influx is increased in RBC from CRF patients, which could be explained in part by alterations in the plasmatic membrane of these cells. However, in these patients the increased uptake of L-arginine is probably shifted towards the urea cycle, with activation of arginase I and higher levels of this enzyme in RBC. Even though there is an upregulation of the arginase pathway, NOS activity remained within normal levels. These data suggest that in RBC, L-arginine influx and arginase I are key factors involved in the alterations of erythrocytes detected in CRF.
Where applicable, experiments conform with Society ethical requirements