Preservation of renal blood flow by the antioxidant EUK-134 in LPS-treated pigs

Abstract

Sepsis is associated with an increase in reactive oxygen species (ROS), however, the precise role of ROS in the septic process remains unknown. We hypothesized that treatment with EUK-134 (manganese-3-methoxy N,N'-bis(salicyclidene)ethylene-diamine chloride), a compound with superoxide dismutase and catalase activity, attenuates the vascular manifestations of sepsis in vivo. Pigs were instrumented to measure cardiac output and blood flow in renal, superior mesenteric and femoral arteries, and portal vein. Animals were treated with saline (control), lipopolysaccharide (LPS; 10 µg·kg-1·h-1), EUK-134, or EUK-134 plus LPS. Results show that an LPS-induced increase in pulmonary artery pressure (PAP) as well as a trend towards lower blood pressure (BP) were both attenuated by EUK-134. Renal blood flow decreased with LPS whereas superior mesenteric, portal and femoral flows did not change. Importantly, EUK-134 decreased the LPS-induced fall in renal blood flow and this was associated with a corresponding decrease in LPS-induced protein nitrotyrosinylation in the kidney. PO2, pH, base excess and systemic vascular resistance fell with LPS and were unaltered by EUK-134. EUK-134 also had no effect on LPS-associated increase in CO. Interestingly, EUK-134 alone resulted in higher CO, BP, PAP, mean circulatory filling pressure, and portal flow than controls. Taken together, these data support a protective role for EUK-134 in the renal circulation in sepsis.

Figure 1

Mean arterial blood gases over time for the four conditions (mean ± SEM). Upper left, PO₂ fell in the LPS and LPS plus EUK-134 (LPS + EUK) groups and did not change in the control or EUK-134 alone (EUK) groups; Lower left, pH fell in the LPS and LPS + EUK groups but did not change in the control or EUK groups; Upper right, PCO₂ was comparable across all the groups (by design it was controlled by adjusting the ventilator); and Lower right, base excess (BE) fell in both the LPS and LPS + EUK groups, but not in control or EUK groups. * p < 0.001.

Figure 1

Mean arterial blood gases over time for the four conditions (mean ± SEM). Upper left, PO₂ fell in the LPS and LPS plus EUK-134 (LPS + EUK) groups and did not change in the control or EUK-134 alone (EUK) groups; Lower left, pH fell in the LPS and LPS + EUK groups but did not change in the control or EUK groups; Upper right, PCO₂ was comparable across all the groups (by design it was controlled by adjusting the ventilator); and Lower right, base excess (BE) fell in both the LPS and LPS + EUK groups, but not in control or EUK groups. * p < 0.001.

Figure 2

Cardiac output and blood pressure over time for the four conditions (mean ± SEM). Left, cardiac output (L/min) was higher in the LPS group and LPS plus EUK-134 groups than the control group (p < 0.02 and p < 0.005, respectively). Cardiac output in EUK-134 alone was also higher than control (p < 0.001) and LPS alone (p < 0.005); Right, blood pressure (BP) was lowest in the LPS group but was not significantly different from control (p < 0.068); This drop was rescued in the LPS plus EUK-134 group, which was superimposable with the control group. BP was best maintained or slightly rose in the EUK-134 group; This group was significantly greater than the LPS (p < 0.001), control (p < 0.001), and LPS plus EUK-134 group (p < 0.001). * p < 0.02, 0.005 or 0.001.

Figure 3

Pulmonary artery pressure (PAP) and mean circulatory filling pressure (MCFP) over time for the four conditions (mean ± SEM). Left, PAP in the LPS group was higher than control (p < 0.001). LPS plus EUK-134 (p < 0.001) and EUK alone (p < 0.02) were higher than the control but were not significantly different from each other. LPS plus EUK-134 was lower than LPS alone; Right, MCFP in LPS was not different from control although there appeared to be a “spike” in the pressure that was also seen with LPS plus EUK-134 and had the same timing as the rise in PAP. MCFP was greater in the EUK-134 alone group than in the control (p < 0.003) and LPS plus EUK-134 (p < 0.003). MCFP of the LPS plus EUK-134 group was greater than that of LPS (p < 0.014). * p < 0.02, 0.005 or 0.001.

Figure 4

Systemic vascular resistance (SVR) and resistance to venous return (RVR) over time for the four conditions (mean ± SEM). Left, SVR fell in both LPS and LPS plus EUK-134 groups (p < 0.001). There were no differences between LPS and LPS plus EUK-134 nor between Control and EUK alone; Right, RVR was not different among the four groups. * p < 0.001.

Figure 5

Mean blood flows in different vascular beds as a percent of baseline over time for the four conditions (mean ± SEM). Upper left, renal arterial flow; Renal artery flow was lower in the LPS group than the other three groups (p < 0.001 for each condition); Lower left, portal venous flow; Portal flow was higher in the EUK-134 alone group than in the other three conditions (p < 0.001); Upper right, femoral arterial flow; Femoral flow in the LPS plus EUK-134 group was lower than the other three groups (p < 0.001); and Lower right, superior mesenteric flow; There was no difference among the four conditions. * p < 0.001; NS: not statistically significant.

Figure 6

Western blots with anti-nitrotyrosine antibody of homogenates of renal tissue. There was an increase density at a band at 32 kDa (marked by arrow) with LPS and this was not apparent when animals were pretreated with EUK-134. Bar graph represents relative band optical density (OD) quantification. C = Control, L = LPS, L + E = LPS plus EUK-134 and E = EUK-134; NT = Nitrotyrosine. * p < 0.05 vs. C; ** p < 0.05 vs. L.