Evaluation of urinary biomarkers for early detection of acute kidney injury in a rat nephropathy model.

INTRODUCTION: The implementation of novel, reliable biomarkers for the early and differential diagnosis of acute kidney injury (AKI) could greatly improve the timely treatment and prevention of disease progression, particularly since the current gold standards for detecting kidney injury such as serum creatinine (SCr) and blood urea nitrogen (BUN) lack sensitivity and specificity. We evaluated novel urinary kidney injury biomarkers focusing on early detection and better prediction of AKI with higher sensitivity and specificity. METHODS: In the rat, urinary biomarkers for kidney injury, i.e. albumin, beta-2-microglobulin (B2M), clusterin, cystatin C, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), osteopontin (OPN), and total protein (TP), were investigated in an AKI model using different hyperosmolar and high-dose solutions, i.e. mannitol, sucrose, and contrast medium (CM), as acute single insults leading to kidney injury. Additionally, dose-dependency of sucrose was investigated and effects were compared to the sucrose- and iron-containing marketed drug Venofer®. RESULTS: Levels of excreted urinary biomarkers correlated with severity of AKI, exhibited a dose-dependent response to sucrose treatment, and demonstrated evidence of recovery from kidney injury with transient and reversible changes. The exceptions were KIM-1 and NGAL, which showed later responses following CM and iron-induced renal injury. All biomarkers outperformed plasma creatinine (PCr), BUN, and histopathology, with regard to practicability and/or detection of proximal tubular injury. DISCUSSION: The use of a panel of urinary kidney injury biomarkers emerged as an early, sensitive, and predictive tool to detect AKI showing enhanced sensitivity compared to current state-of-the-art markers.

The Coagulation Factor XIIa Inhibitor rHA-Infestin-4 Improves Outcome after Cerebral Ischemia/Reperfusion Injury in Rats.

BACKGROUND AND PURPOSE: Ischemic stroke provokes severe brain damage and remains a predominant disease in industrialized countries. The coagulation factor XII (FXII)-driven contact activation system plays a central, but not yet fully defined pathogenic role in stroke development. Here, we investigated the efficacy of the FXIIa inhibitor rHA-Infestin-4 in a rat model of ischemic stroke using both a prophylactic and a therapeutic approach. METHODS: For prophylactic treatment, animals were treated intravenously with 100 mg/kg rHA-Infestin-4 or an equal volume of saline 15 min prior to transient middle cerebral artery occlusion (tMCAO) of 90 min. For therapeutic treatment, 100 mg/kg rHA-Infestin-4, or an equal volume of saline, was administered directly after the start of reperfusion. At 24 h after tMCAO, rats were tested for neurological deficits and blood was drawn for coagulation assays. Finally, brains were removed and analyzed for infarct area and edema formation. RESULTS: Within prophylactic rHA-Infestin-4 treatment, infarct areas and brain edema formation were reduced accompanied by better neurological scores and survival compared to controls. Following therapeutic treatment, neurological outcome and survival were still improved although overall effects were less pronounced compared to prophylaxis. CONCLUSIONS: With regard to the central role of the FXII-driven contact activation system in ischemic stroke, inhibition of FXIIa may represent a new and promising treatment approach to prevent cerebral ischemia/reperfusion injury.

Correlation of coagulation markers and 4F-PCC-mediated reversal of rivaroxaban in a rabbit model of acute bleeding.

INTRODUCTION: Rivaroxaban is an oral, selective direct factor Xa inhibitor approved for several indications in patients at risk of thrombotic events. One limitation of its clinical use is the lack of data pertaining to its reversal in situations where urgent response is critical (e.g. acute bleeding events or emergency surgery). MATERIALS AND METHODS: This study assessed the effectiveness of a four-factor prothrombin complex concentrate (4F-PCC; Beriplex(®)/Kcentra(®)) for the reversal of rivaroxaban-associated bleeding in an in vivo rabbit model, and evaluated the correlations between in vitro coagulation parameters and haemostasis in vivo. RESULTS: Administration of single intravenous doses of rivaroxaban (150-450 µg/kg) resulted in increased and prolonged bleeding following standardised kidney incision. Pre-incision treatment with 4F-PCC (25-100 IU/kg) resulted in a dose-dependent reversal of rivaroxaban (150 and 300 µg/kg)-associated increases in time to haemostasis and blood loss; no reversal was seen at the highest rivaroxaban dose (450 µg/kg). Of the in vitro biomarkers tested, thrombin generation and whole-blood clotting time correlated well with in vivo measures of 4F-PCC-mediated effects. Thrombin generation was highly reagent-dependent, with the assay initiated using the phospholipid-only reagent being the most predictive of effective haemostasis in vivo. CONCLUSIONS: In summary, in a rabbit model of acute bleeding, treatment with 4F-PCC reduced bleeding to control levels following rivaroxaban 150 µg/kg and 300 µg/kg administration.

C1-esterase inhibitor treatment: preclinical safety aspects on the potential prothrombotic risk.

Human plasma-derived C1-esterase inhibitor (C1-INH) is an efficacious and safe treatment for hereditary angioedema. However, thrombotic events in subjects treated with C1-INH at recommended or off-label, high doses have been reported. In this study, we addressed the potential prothrombotic risk of C1-INH treatment in high doses using a non-clinical rabbit model. Following intravenous infusion of C1-INH to rabbits at doses up to 800 IU/kg, the exposure and the pharmacodynamic efficacy of C1-INH in rabbits were confirmed by activity measurements of C1-esterase, and coagulation factors XIa and XIIa, respectively. Potential prothrombotic effects were assessed following induction of venous and arterial thrombosis using in vivo models of venous and arterial stasis, complemented by various in vitro assays of coagulation markers. Administration of C1-INH at doses up to 800 IU/kg did not potentiate thrombus formation during venous stasis. In contrast, inhibition of arterial occlusion was observed upon C1-INH administration when compared with isotonic saline treatment, indicating antithrombotic rather than prothrombotic activity of high dose C1-INH treatment in vivo. This was further confirmed in vitro by decreased thrombin generation, increased activated partial thromboplastin time, clotting time and clot formation time, and inhibition of platelet aggregation. No relevant changes in fibrinolysis or in the levels of thrombin-antithrombin complexes, and prothrombin fragment 1+2 were observed upon high dose C1-INH treatment. The data suggest that treatment of healthy rabbits with high doses of C1-INH could potentially inhibit coagulation and thrombus formation rather than induce a prothrombotic risk.

Prothrombin complex concentrates as reversal agents for new oral anticoagulants: lessons from preclinical studies with Beriplex.

Although new oral anticoagulants (NOACs) represent an advance in anticoagulant therapy over vitamin K antagonists (VKAs), they nevertheless have a low, but significant risk for bleeding complications. Reversal agents for VKAs, such as prothrombin complex concentrates (PCCs), are currently being evaluated in preclinical studies for NOAC reversal. This article reviews the preclinical data for the most extensively studied PCC for NOAC reversal, Beriplex, a 4-factor PCC. The results from the Beriplex studies are also compared with those obtained with other reversal agents, including different nonactivated PCCs, activated PCCs, and recombinant activated factor VII.

Prothrombin complex concentrate versus recombinant factor VIIa for reversal of hemodilutional coagulopathy in a porcine trauma model.

BACKGROUND: Fluid resuscitation after traumatic injury may necessitate coagulation factor replacement to prevent bleeding complications of dilutional coagulopathy. Recombinant activated factor VII (rFVIIa) is being widely investigated as a hemostatic agent in trauma. Multicomponent therapy with prothrombin complex concentrate (PCC) containing coagulation factors II, VII, IX, and X might offer potential advantages. METHODS: Anesthetized mildly hypothermic normotensive pigs were hemodiluted by substituting 65% to 70% of total blood volume in phases with hydroxyethyl starch and red cells. Thereafter, animals received 12.5 mL . kg isotonic saline placebo, 35 IU . kg PCC, or 180 microg x kg rFVIIa. Immediately afterward, a standardized spleen injury was inflicted, and prothrombin time (PT) and hemostasis were assessed. Thrombin generation was also determined. RESULTS: Hemodilution depleted levels of factors II, VII, IX, and X markedly, prolonged PT and decreased thrombin formation. PCC and rFVIIa both fully normalized the hemodilution-induced lengthening of PT. In PCC recipients, peak thrombin generation was greater by a median of 60.7 nM (confidence interval 56.4-64.9 nM) compared with the rFVIIa group (p = 0.008). After spleen trauma, time to hemostasis was shortened to a median of 35 minutes in animals treated with PCC versus 94 minutes with rFVIIa (p = 0.016). CONCLUSIONS: In a pilot study involving an in vivo large-animal model of spleen trauma, PCC accelerated hemostasis and augmented thrombin generation compared with rFVIIa. Further investigations are warranted on PCC as a hemostatic agent in trauma.