Distinct Functions of Activated Protein C Differentially Attenuate Acute Kidney Injury

Administration of activated protein C (APC) protects from renal dysfunction, but the underlying mechanism is unknown. APC exerts both antithrombotic and cytoprotective properties, the latter via modulation of protease-activated receptor-1 (PAR-1) signaling. We generated APC variants to study the relative importance of the two functions of APC in a model of LPS-induced renal microvascular dysfunction. Compared with wild-type APC, the K193E variant exhibited impaired anticoagulant activity but retained the ability to mediate PAR-1-dependent signaling. In contrast, the L8W variant retained anticoagulant activity but lost its ability to modulate PAR-1. By administering wild-type APC or these mutants in a rat model of LPS-induced injury, we found that the PAR-1 agonism, but not the anticoagulant function of APC, reversed LPS-induced systemic hypotension. In contrast, both functions of APC played a role in reversing LPS-induced decreases in renal blood flow and volume, although the effects on PAR-1-dependent signaling were more potent. Regarding potential mechanisms for these findings, APC-mediated PAR-1 agonism suppressed LPS-induced increases in the vasoactive peptide adrenomedullin and infiltration of iNOS-positive leukocytes into renal tissue. However, the anticoagulant function of APC was responsible for suppressing LPS-induced stimulation of the proinflammatory mediators ACE-1, IL-6, and IL-18, perhaps accounting for its ability to modulate renal hemodynamics. Both variants reduced active caspase-3 and abrogated LPS-induced renal dysfunction and pathology. We conclude that although PAR-1 agonism is solely responsible for APC-mediated improvement in systemic hemodynamics, both functions of APC play distinct roles in attenuating the response to injury in the kidney.Acute kidney injury (AKI) leading to renal failure is a devastating disorder,¹ with a prevalence varying from 30 to 50% in the intensive care unit.² AKI during sepsis results in significant morbidity, and is an independent risk factor for mortality.^3,4 In patients with severe sepsis or shock, the reported incidence ranges from 23 to 51%^5–7 with mortality as high as 70% versus 45% among patients with AKI alone.^1,8The pathogenesis of AKI during sepsis involves hemodynamic alterations along with microvascular impairment.⁴ Although many factors change during sepsis, suppression of the plasma serine protease, protein C (PC), has been shown to be predictive of early death in sepsis models,⁹ and clinically has been associated with early death resulting from refractory shock and multiple organ failure in severe sepsis.¹⁰ Moreover, low levels of PC have been highly associated with renal dysfunction and pathology in models of AKI.¹¹ During vascular insult, PC becomes activated by the endothelial thrombin-thrombomodulin complex, and the activated protein C (APC) exhibits both antithrombotic and cytoprotective properties. We have previously demonstrated that APC administration protects from renal dysfunction during cecal ligation and puncture and after endotoxin challenge.^11,12 In addition, recombinant human APC [drotrecogin alfa (activated)] has been shown to reduce mortality in patients with severe sepsis at high risk of death.¹³ Although the ability of APC to protect from organ injury in vivo is well documented,^11,14,15 the precise mechanism mediating the response has not been ascertained.APC exerts anticoagulant properties via feedback inhibition of thrombin by cleavage of factors Va and VIIIa.¹⁶ However, APC bound to the endothelial protein C receptor (EPCR) can also exhibit direct potent cytoprotective properties by cleaving protease-activated receptor-1 (PAR-1).¹⁷ Various cell culture studies have demonstrated that the direct modulation of PAR-1 by APC results in cytoprotection by several mechanisms, including suppression of apoptosis,^18,19 leukocyte adhesion,^19,20 inflammatory activation,²¹ and suppression of endothelial barrier disruption.^22,23 In vivo, the importance of the antithrombotic activity of APC is well established in model systems^24,25 and in humans.²⁶ However, the importance of PAR-1-mediated effects of APC also has been clearly defined in protection from ischemic brain injury²⁷ and in sepsis models.²⁸ Hence, there has been significant debate whether the in vivo efficacy of APC is attributed primarily to its anticoagulant (inhibition of thrombin generation) or cytoprotective (PAR-1-mediated) properties.^17,29The same active site of APC is responsible for inhibition of thrombin generation by the cleavage of factor Va and for PAR-1 agonism. Therefore, we sought to generate point mutations that would not affect catalytic activity, but would alter substrate recognition to distinguish the two functions. Using these variants, we examined the relative role of the two known functions of APC in a model of LPS-induced renal microvascular dysfunction.