Introduction
Circulatory failure is the most common and fatal hallmark of sepsis in critically ill patients. The key hemodynamic characteristic of sepsis is arterial vasodilation associated with a decrease in total vascular resistance. Hypotension in septic patients results from both hypovolemia and inadequate vasodilatation [
1]. Typically, septic patients show marked hyporeactivity to both endogenously generated and exogenously administered vasoconstrictors [
1,
2]. The resistance to vasoconstrictors includes otherwise potent factors such as norepinephrine, endothelins, and angiotensin II. Down-regulation of the corresponding receptors, such as adrenergic α-receptors, ET
A receptors and angiotensin AT1 receptors, is involved in the functional resistance to vasopressor hormones. Furthermore, because the concentrations of catecholamines, endothelins and angiotensin II are markedly elevated during sepsis, their corresponding receptors may be desensitized [
3‐
6]. Additionally, the excess presence of vasodilatory substances, such as nitric oxide and prostanoids, may cause the maximum dilatation of resistance vessels and may exceed the regulatory range of vascular smooth muscle cells, which is usually determined by the balanced presence of vasoconstrictors and vasodilators [
7].
Diminished vasoconstrictor function may be related to reduced receptor transcription, translation and/or membrane trafficking [
8‐
13]. For the latter, the involvement of receptor-associated proteins might be relevant, including the recently discovered angiotensin II AT1 receptor-associated proteins [
8,
14]. Several different receptor-associated proteins have been described for the AT1 receptor, which is the most relevant mediator of the renin angiotensin system (RAS). Among these, the AT1-associated protein Arap1 (AT1 receptor-associated protein 1) appears to be most relevant because it is expressed predominantly in the vasculature of many organs, overlapping in location with AT1 receptors [
15‐
18]. Arap1 supports the trafficking of the AT1 receptor to the cell membrane, leading to locally enhanced sensitivity [
17]. Furthermore, the expression of Arap1 is regulated in a manner such that the presence of angiotensin II results in a marked down-regulation of Arap1, establishing a local negative feedback loop by reducing the vasculature's sensitivity when angiotensin II concentrations rise [
18].
In the present study, we used Arap1-deficient mice to address the hypothesis that Arap1 might be involved in the vasculature's loss of sensitivity to angiotensin II during sepsis.
We found that during lipopolysaccharide (LPS)-induced sepsis in mice, Arap1 expression was markedly down-regulated; Arap1 expression was similarly reduced in cultured cells in the presence of pro-inflammatory cytokines. The blood pressure homeostasis during endotoxemia was more severely compromised in Arap1-deficient mice than in wildtypes, suggesting that down-regulation of Arap1 expression during the course of sepsis may contribute to the development of hyporeactivity to angiotensin II.
Discussion
Arap1 is a protein that interacts with the angiotensin II AT1 receptor, and
in vitro studies suggested that Arap1 facilitates the surface expression of the AT1 receptor and, hence, acts as a positive local regulator of AT1 receptor function [
17]. In the present study, we used Arap1-deficient mice to investigate the possible involvement of Arap1 in sepsis-induced hypotension. We hypothesized that a dysregulation of Arap1 expression during sepsis may be involved in the hyporeactivity of vascular AT1 receptors, contributing to a decrease in the total vascular resistance.
Vascular hypo-responsiveness to pressor substances, such as angiotensin II contributes to hypotension in septic patients [
1]. Arap1 was shown to enhance AT1 receptor surface expression in cultured cells [
17], a result that was functionally confirmed at the whole organ level in our study. Therefore, we hypothesized that Arap1-dependent modulation of the AT1 receptor may contribute to angiotensin resistance during sepsis. In fact, the Arap1 expression in several organs declined during the course of the experimental sepsis, reaching levels below 10% of the baseline. This down-regulation of Arap1 expression could be recapitulated
in vitro, when cultured mesangial cells were exposed to pro-inflammatory cytokines, such as TNFα and IFNγ. Thus, the reduced Arap1 expression during sepsis may be mediated by pro-inflammatory cytokines, all of which are known to be markedly elevated during endotoxemia [
23,
24]. Furthermore, Arap1 abundance has been shown to be dependent on angiotensin II levels, with angiotensin II suppressing Arap1 expression; the regulation of Arap1 expression was related to changes in transcriptional activity, because Arap1 mRNA and protein abundances changed in parallel under all conditions [
18]. Plasma angiotensin II concentrations were shown to be 5-fold increased six hours after the induction of endotoxemia by a single LPS injection in rats [
23], and PRA was elevated up to 25-fold in septic human patients [
5]. Thus, angiotensin II in combination with pro-inflammatory cytokines likely accounts for the down-regulation of Arap1 during LPS-induced sepsis. The virtual loss of Arap1 during endotoxemia would reduce the surface expression of the AT1 receptor in the vasculature and would contribute to the hyporesponsiveness to angiotensin II observed during sepsis. With the endotoxemia-triggered down-regulation of Arap1 to levels below 10% of the control level, the wildtype mice approximated the Arap1-deficient mice. Thus, despite the marked blood pressure differences between the Arap1+/+ and -/- mice during the early time course of endotoxemia, blood pressure was indistinguishable between the genotypes when the blood pressure nadir was reached, approximately six hours after the LPS administration. At this particular time, the Arap1 expression in the wildtype mice was as low as 9% of the baseline abundance, similar to the situation in the Arap1-/- mice.
In contrast to the baseline conditions, when the MAP was maintained in the Arap1-/- mice, the fall in blood pressure after the induction of endotoxemia was more pronounced in the Arap1-/- than in the wildtype mice, at least before the minimum values were reached after approximately six hours. Apparently, when the RAS is stimulated, and when increased concentrations of angiotensin II are required to maintain the MAP, the relevance of Arap1 for AT1 receptor surface expression is unmasked. Our experiments with mice pre-treated with enalapril further suggest that blood pressure maintenance is more dependent on intact AT1 receptor activity during sepsis than under normal conditions. Thus, enalapril reduced the baseline MAP from 104.9 to 98 mmHg in the wildtype mice and from 106.8 to 92 mmHg in the Arap1-/- mice before the LPS administration, a difference of 6.9 and 14.8 mmHg, respectively. However, six hours after the induction of endotoxemia, the blood pressure differences caused by enalapril increased to -19 in the Arap1-/- and to -21 mmHg in the wildtype mice, with no difference between the genotypes, suggesting that the relative contribution of angiotensin II to maintaining MAP had increased.
A closer inspection of the time course of the MAP after LPS injection revealed a transient recovery of the blood pressure approximately 2.5 hours after the induction of endotoxemia. This transient recovery has been suggested to be related to an activation of the RAS and an increased endothelin production [
25]. Our data are consistent with this assumption because the transient increase in MAP was blunted in the presence of enalapril in both genotypes, suggesting that an activation of the RAS predominantly accounts for the temporary stabilization of blood pressure. It should be noted that the Arap1 expression at this time point was only slightly reduced when compared with the expression during the later course of endotoxemia.
Although down-regulation of Arap1 during endotoxemia appears to be relevant for the development of hypotension and, by inference, hyporesponsiveness to angiotensin II, it should be noted that other mechanisms, such as inadequate formation of vasodilator agents and resistance to other vasoconstrictors, are relevant in the pathogenesis of septic circulatory failure [
1]. The contribution of these single mechanisms to the overall dysregulation of vascular tone may vary during the course of the disease.
Experiments in isolated perfused kidneys, used as a model of organ vascular resistance, revealed that the sensitivity of vascular AT1 receptors to angiotensin II was reduced in the Arap1-/- kidneys, which is consistent with previous
in vitro data indicating that Arap1 enhances the membrane surface expression of the AT1 receptor [
17,
26]. The basal renal vascular resistance, that is, in the absence of angiotensin II, was indistinguishable between the isolated kidneys from the Arap1-/- and the wildtype mice, indicating that the angiotensin II-independent components of the vascular tone were unaltered in Arap1-deficient kidneys. The dose-response curve of vascular resistance vs. angiotensin II concentration in the isolated perfused kidney closely mimics the
in vivo situation. Thus, the steepest slope of the dose-response curve was observed in the angiotensin II concentration range of 30 to 100 pM, similar to concentrations
in vivo, which have been estimated to be in the range of 50 to 100 pM for the mouse [
27].
Despite the different vascular AT1 receptor sensitivities in the Arap1-/- and wildtype mice
in vitro, the baseline blood pressure in the Arap1-/- mice was inconspicuous, suggesting that corresponding changes in systemic vasopressor systems compensated for the deviation of vascular sensitivity to angiotensin II. In fact, the PRC was increased by 60% in the Arap1-/- mice compared with that in the wildtype mice, which apparently allowed for full restoration of the total vascular resistance to wildtype levels, at least under baseline conditions. When the Arap1-/- mice were treated with an ACE inhibitor, the systolic blood pressure decreased to a larger extent than in the wildtype mice, suggesting again that blood pressure maintenance in the Arap1-/- mice requires an activated RAS. A compensatory stimulation of RAS has been shown for several models of compromised AT1 receptor function, such as AT1
a,b receptor-deficient mice [
28]. In the situation of complete AT1 deficiency, the basal PRC is elevated 6- to 10-fold compared with that in control animals; this increase in PRC has been attributed to a disinhibition of renin secretion because of an interruption of the direct negative feedback loop of angiotensin II on the renin-secreting cells and to systemic effects, such as low arterial pressure. Activation of the RAS in the Arap1-/- mice, however, appears to be related to the systemic rather than direct effects of angiotensin II on renin-producing cells because the relative suppression of renin secretion by angiotensin II was preserved in the isolated perfused mouse model. These functional data are consistent with the results of a recent localization study that suggested that renin-producing cells of the afferent arterioles do not express Arap1 [
18].
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KMe, FS, KH, OY and HC designed the study. KMe, VK, KMi and OY performed the animal experiments and analyzed the data. FS carried out the isolated perfused kidney experiments. ED performed the in vitro studies. KMe and HC drafted the manuscript. All authors participated in revising the manuscript. HC finalized the manuscript. All authors read and approved the final manuscript.