The fibrinolytic system attenuates vascular tone: effects of tissue plasminogen activator (tPA) and aminocaproic acid on renal microcirculation

Abstract

1. The renal medulla is a major source of plasminogen activators (PA), recently shown to induce vasodilation in vitro. Treatment with PA inhibitors has been associated with renal dysfunction, suggesting compromised renal microvasculature. We investigated the impact of the PA inhibitor epsilon amino-caproic acid (EACA) upon vascular tone in vitro, and studied the effect of both tPA and EACA upon intrarenal hemodynamics in vivo. 2. In vitro experiments were carried out in isolated aortic rings and with cultured vascular smooth muscle cells. Studies of renal microcirculation and morphology were conducted in anesthetized Sprague-Dawley rats. 3. In isolated aortic rings, EACA (but not the other inhibitors of the fibrinolytic system PAI-1 or alpha-2 antiplasmin) reduced the half-maximal effective concentration of phenylephrine (PE) required to induce contraction (from 32 nm in control solution to 2 and 0.1 nm at EACA concentrations of 1 and 10 microm, respectively). Using reteplase (retavase) in the same model, we also provide evidence that the vasoactivity of tPA is in part kringle-dependent. In cultured vascular smooth muscle cells, Ca(2+) internalization following PE was enhanced by EACA, and retarded by tPA. 4. In anesthetized rats, EACA (150 mg x kg(-1)) did not affect systemic blood pressure, total renal or cortical blood flow. However, the outer medullary blood flow declined 12+/-2% below the baseline (P<0.03). By contrast, tPA (2 mg x kg(-1)), transiently increased outer medullary blood flow by 8+/-5% (P<0.02). Fibrin microthrombi were not found within the renal microvasculature in EACA-treated animals. 5. In conclusion, both fibrinolytic and antifibrinolytic agents modulate medullary renal blood flow with reciprocal effects of vasodilation (PA) and vasoconstriction (EACA). In vitro studies suggest that these hemodynamic responses are related to direct modulation of the vascular tone.

Figure 1

Renal hemodynamic response to EACA. (a) Changes in systemic blood pressure (BP), total renal blood flow (RBF) and selective cortical (CF) and outer medullary (MF) flows are displayed as filled circles, while the calculated corresponding total renal (RVR), cortical (CR) and medullary vascular resistance (MR) are presented as empty symbols. Medullary flow significantly declines, with the null hypothesis rejected at a P-value of 0.007. Values at 2′–10′ are significantly lower than baseline measurements (n=8, P<0.03, Newman–Keuls post hoc test). The slope of reciprocal rise in medullary resistance falls short of statistical significance, but values at 2′–10′ are significantly higher than baseline (P<0.05, paired t-test). Blood pressure, total renal and cortical flows (determined with a superficial nonpenetrating probe) are unaffected by EACA. (b) Cortical flow (CF) and resistance (CR), determined with a penetrating needle probe inserted into the superficial cortex, are again unaffected by EACA (n=6).

Figure 2

Renal hemodynamic response to tPA. Symbols and abbreviations are similar to those of Figure 1. While blood pressure, total renal and selective cortical blood flows remain unchanged during the first 7.5′, medullary flow rises and regional resistance falls (n=12, P<0.02 for the 7.5′ time point as compared to baseline values, paired t-test). Subsequently, blood pressure declines with renal microcirculatory deterioration both in the cortex and outer medulla (P<0.01, ANOVA).

Figure 3

Effect of EACA on isometric contraction of vascular smooth muscle. (a) Isometric tension was determined in isolated aortic rings incubated with PE. The PE concentration–contraction curve determined in control Krebs–Henselite solution is shifted to the left by the addition of EACA (1 and 10 μM). Each point along these curves represents the mean of 3–9 experiments. (b) Effect of EACA on half-maximal effective concentration (EC₅₀) of PE in isometrically contracted aortic rings: EC₅₀, defined as the PE concentration that induces 50% of the maximal contraction, extrapolated from (a), significantly falls with the addition of EACA (^*P<0.001 vs controls for both concentrations of EACA).

Figure 4

Effect of tPA and reteplase on half-maximal effective concentration (EC₅₀) of PE in isometrically contracted aortic rings: EC₅₀ is defined as the PE concentration that induces 50% of the maximal contraction. While the full-length wild-type tPA (WT-tPA) exerts opposing pro-vasodilatory and vasoconstrictive effects at low (1 nM) and high (20 nM) concentrations, respectively (increasing and reducing EC₅₀ of PE, respectively), the delta kringle reteplase (DK-tPA), a tPA variant lacking kringle-1 and a finger domain, exerts vasodilation at both concentrations (^*P<0.001 vs control conditions – CTR).

Figure 5

Effect of tPA and EACA on PE-induced Ca²⁺ mobilization in human umbilical vein SMC. SMC were incubated with buffer containing ⁴⁵Ca²⁺. The experimental groups are: control group with no additives (CTR); 100 nM PE; 100 nM PE and 50 nM tPA (PE+tPA); 100 nM PE and 1 μM EACA (PE+EACA); and EACA (1 μM; 10 μM; 50 μM). While PE-mediated Ca²⁺ mobilization in vascular SMC is reduced by tPA, it is enhanced by EACA. Moreover, at high concentrations, EACA enhances Ca²⁺ mobilization in the absence of PE. (^*P<0.01 vs CTR; ^#P<0.01 vs PE).