Renal nitric oxide synthase and nitric oxide production in renal ischemic reperfusion : effects of angiotensin system

Abstract

This study was conducted to investigate renal nitric oxide synthase (NOS) protein expression and nitric oxide (NO) production in unilateral renal ischemic reperfusion (IR) as well as the role of angiotensin system. The male Wistar rats were divided into two main groups; sham operation (S) and IR (30-minute occlusion). In IR groups, the animals were further divided into 3 subgroups treated with: 1) water, 2) angiotensin converting enzyme inhibitor (ACEI; Enalapril(R); 200 mg/L), and 3) angiotensin II receptor type 1 antagonist (ARA; Losartan(R); 500 mg/L). The treatment was performed one day before the operation (S or IR) and continuously for 1 day or 7 days after the operation. On each experimental due date, 24-hr urine and blood samples were collected. The serum were stored at -80 ํC until use for NO production (nitrite), electrolytes, blood urea nitrogen (BUN), creatinine (Cr), and Cr clearance (CCr). The kidneys were removed and fixed for eNOS protein expression and histological study. In addition, the regional renal blood flow (rBF) was measured before, during and after ischemia as well as before sacrificed by a laser doppler flowmeter. By immunohistochemistry, the expression of renal eNOS protein showed more staining in glomerulus as well as in renal tubular epithelial cells in cortex than in medulla. One day after IR caused a marked decrease of eNOS protein expression, especially in cortex. The treatements with ACEI or ARA could ameliorate the loss of renal eNOS protein expression caused by IR in 1-day group. However, this alteration was less observed in 7-day duration after IR, while only the ACEI-treated rats, for 7 days after IR, reduced the expression. One day after IR, serum nitrite concentration was significantly increased (p < 0.01). The treatment with ACEI could normalize the heightened nitrite level induced by IR to be that of sham animal. The ARA-treated rats had a greater extent less concentration. However, 7 days after IR, serum nitrite concentration in all groups was comparable and not significant different from 1-day sham animals. The kidney exposed to IR showed mild to moderate dilatation of cortical tubule with few cast formation and mild brush border membrane loss in 1-day post IR group. The treatment with ACEI or ARA could attenuate structural damages. All 7-day groups studied showed normal structure of renal tissue. During left renal artery occlusion, the rBF decreased by 80 to 85% (p < 0.001) and restored to be 50% and 75 to 80% of baseline levels at 5[superscript th] and 10[superscript th] minute after release, respectively. The values of rBF were completely returned to baseline in either 1-day or 7-day post IR groups. No significant changes of rBF were observed in the right (non-ischemic) kidney. Neither ACEI nor ARA influenced on rBF of both. The fractional excretion (FE) of sodium (FE[subscript Na]+) was markedly diminished (p < 0.001) in 1 day after IR. Only ARA treatment could restore the value to be near that of the sham animals. However, after 7 days of IR, these FE values in all groups were comparable. In addition, both 1-day and 7-day post IR groups demonstrated similar levels of serum Na+, K+, Cl- as well as BUN, Cr, and CCr. These values are in normal range. The present data are the first evidence of IR model in that the angiotensin system plays a crucial role in regulation of renal eNOS protein expression, NO production as well as electrolyes excretion.