Abstract 522: Contribution of Reactive Oxygen Species and Rho Kinase in Sphingosine-1-phosphate Mediated Afferent Arteriolar Vasoconstriction of Ischemia-Reperfusion Rats

Abstract

Our recent studies indicate that sphingosine-1-phosphate (S1P) is a potent vasoconstrictor of afferent arterioles (AA) and is enhanced in renal ischemia-reperfusion (IR) injury. We hypothesized that renal IR-induced increases in reactive oxygen species (ROS) and activation of the rho kinase pathway contribute to the enhanced S1P sensitivity of AA in IR rats. IR injury was induced by bilateral renal artery occlusion for 60 min followed by 24 hrs reperfusion. Renal function was assessed by plasma creatinine (Cr) or glomerular filtration rate (GFR). The AA response to S1P was assessed using the in vitro blood-perfused juxtamedullary nephron preparation with and without superfusion with the ROS scavenger, Tempol, or the rho kinase inhibitor, Y27623. Plasma Cr was markedly increased in IR (3.86±0.20 vs. 0.93±0.07 mg/L in sham, n=5-7, P < 0.05). GFR, calculated from the half time (T 1/2 ) of sinistrin clearance, was significantly reduced in IR rats. The T 1/2 averaged 166±28 min in IR vs. 25±4 min in sham rats corresponding to GFRs of 0.20±0.02 and 1.33±0.23 ml/min (P < 0.05, n=3 each), respectively. The basal AA diameter was significantly decreased in IR compared to sham rats (10.2±0.5 vs. 15.0±0.4 μm, P < 0.05, n=9 each). Superfusion of Tempol (10 -4 M) did not alter the basal AA diameter in sham but increased diameter in IR by 8±1% (P < 0.05, n=6 each). S1P superfusion evoked concentration-dependent AA vasoconstriction in both groups, however, Tempol significantly shifted the S1P response curve to the right in IR. Increasing concentrations of S1P (10 -10 -10 -5 M) reduced AA diameter to 98±2, 96±2, 98±2, 94±3, 81±9 and 59±5% of control in IR rats similar to the response in Tempol-treated sham rats (99±1, 99±2, 96±2, 88±5, 65±6 and 48±4%, respectively, P > 0.05). In contrast, superfusion of Y27632 (10 -5 M) markedly increased the basal AA diameter in both groups. AA diameter increased by 81±17% from 10.6±1.3 to 19.2±3.3 μm in IR vs. a 53±21% increase in sham rats (P > 0.05, n=3 each). Y27632 also significantly blunted S1P-mediated AA vasoconstriction similarly in both groups. These results indicate a significant contribution of ROS and rho kinase to both the elevated afferent arteriolar tone and the enhanced S1P sensitivity of AA in IR induced acute kidney injury.