Brodifacoum induces early hemoglobinuria and late hematuria in rats: novel rapid biomarkers of poisoning.

INTRODUCTION: Brodifacoum (BDF) is a superwarfarin that is used primarily as a rodenticide. There have been increasing numbers of reports of human cases of accidental or intentional BDF ingestion with high mortality rate. Its broad availability and high lethality suggest that BDF should be considered a potential chemical threat. Currently, there is no biomarker for early detection of BDF ingestion in humans; patients typically present with severe coagulopathy. Since we demonstrated earlier that warfarin can induce acute kidney injury with hematuria, we tested whether BDF would also lead to change in urinary biomarkers. MATERIAL AND METHODS: BDF was administered to Sprague Dawley rats via oral gavage. N-acetylcysteine (NAC) was given per os in drinking water 24 h prior to BDF. Urinalysis was performed at different times after BDF administration. Anticoagulation and serum creatinine levels were analyzed in the blood. RESULTS: We observed that within a few hours the animals developed BDF-dose-dependent transient hemoglobinuria, which ceased within 24 h. This was accompanied by a transient decrease in hematocrit, gross hemolysis and an increase in free hemoglobin in the serum. At later times, animals developed true hematuria with red blood cells in the urine, which was associated with BDF anticoagulation. NAC prevented early hemoglobinuria, but not late hematuria associated with BDF. CONCLUSIONS: We propose that transient early hemoglobinuria (associated with oxidative stress) with consecutive late hematuria (associated with anticoagulation) are novel biomarkers of BDF poisoning, and they can be used in clinical setting or in mass casualty with BDF to identify poisoned patients.

Warfarin-related nephropathy is the tip of the iceberg: direct thrombin inhibitor dabigatran induces glomerular hemorrhage with acute kidney injury in rats.

BACKGROUND: Excessive anticoagulation with warfarin can result in acute kidney injury (AKI) by causing glomerular hemorrhage and renal tubular obstruction by red blood cell (RBC) casts in some patients, especially in those with chronic kidney disease (CKD). This condition was described as warfarin-related nephropathy (WRN). Recent evidence suggests that WRN-like syndromes are not confined to anticoagulation with warfarin, but may be seen with other anticoagulants, such as dabigatran. The aim of this study was to investigate dabigatran effects on kidney function in an animal model of CKD and possible pathogenic mechanisms of AKI. METHODS: Control and 5/6 nephrectomy rats were treated with different doses of dabigatran and protease-activated receptor 1 (PAR-1) inhibitor SCH79797. RESULTS: Dabigatran resulted in changes in coagulation in rats similar to those in humans at 50 mg/kg/day. Dabigatran resulted in a dose-dependent increase in serum creatinine (Scr) and hematuria in both control and 5/6 nephrectomy rats. SCH79797 also increased Scr and hematuria, more prominent in animals with CKD. Morphologically, numerous RBC tubular casts were seen in 5/6 nephrectomy rats treated with either dabigatran or SCH79797 and only occasional RBC casts in control rats. CONCLUSIONS: Our data indicate that WRN represents part of a broader syndrome, anticoagulant-related nephropathy (ARN). ARN, at least partially, is mediated via PAR-1. Our findings suggest that not only CKD patients, but other patients as well, are at high risk of developing AKI if the therapeutic range of anticoagulation with dabigatran is exceeded. Close monitoring of kidney function in patients on dabigatran therapy is warranted.

N-acetylcysteine ameliorates acute kidney injury but not glomerular hemorrhage in an animal model of warfarin-related nephropathy.

Warfarin-related nephropathy (WRN) occurs under conditions of overanticoagulation with warfarin. WRN is characterized by glomerular hemorrhage with occlusive tubular red blood cell (RBC) casts and acute kidney injury (AKI). Herein we test the hypothesis that oxidative stress plays a role in the AKI of WRN. 5/6 Nephrectomy rats were treated with either warfarin (0.04 mg·kg⁻¹·day⁻¹) alone or with four different doses of the antioxidant N-acetylcysteine (NAC). Also tested was the ability of our NAC regimen to mitigate AKI in a standard ischemia-reperfusion model in the rat. Warfarin resulted in a threefold or greater increase in prothrombin time in each experimental group. Serum creatinine (Scr) increased progressively in animals receiving only warfarin + vehicle. However, in animals receiving warfarin + NAC, the increase in Scr was lessened, starting at 40 mg·kg⁻¹·day⁻¹ NAC, and completely prevented at 80 mg·kg⁻¹·day⁻¹ NAC. NAC did not decrease hematuria or obstructive RBC casts, but mitigated acute tubular injury. Oxidative stress in the kidney was increased in animals with WRN and it was decreased by NAC. The NAC regimen used in the WRN model preserved kidney function in the ischemia-reperfusion model. Treatment with deferoxamine (iron chelator) did not affect WRN. No iron was detected in tubular epithelial cells. In conclusion, this work taken together with our previous works in WRN shows that glomerular hematuria is a necessary but not sufficient explanation for the AKI in WRN. The dominant mechanism of the AKI of WRN is tubular obstruction by RBC casts with increased oxidative stress in the kidney.

Warfarin-related nephropathy modeled by nephron reduction and excessive anticoagulation.

An acute increase in international normalized ratio (INR) to >3.0 in patients with chronic kidney disease (CKD) can associate with an unexplained acute increase in serum creatinine and accelerated progression of CKD. A subset of these patients have renal tubular obstruction by casts of red blood cells, presumably the dominant mechanism of the acute kidney injury described as warfarin-related nephropathy. Here, we developed an animal model of this acute kidney injury that is based on the 5/6-nephrectomy model to aid future investigation of the pathogenesis of this condition. We found that acute excessive anticoagulation with brodifacoum ("superwarfarin") increased serum creatinine levels and hematuria in 5/6-nephrectomized rats but not in controls. In addition, morphologic findings in 5/6-nephrectomized rats included glomerular hemorrhage, occlusive red blood cell casts, and acute tubular injury, similar to the biopsy findings among affected patients. Furthermore, in the rat model, we observed an increase in apoptosis of glomerular endothelial cells. In summary, the 5/6-nephrectomy model combined with excessive anticoagulation may be a useful tool to study the pathogenesis of warfarin-related nephropathy.

Hypertension and the Kidney: Reduced Kidney Mass Is Bad for Both Normotensive and Hypertensive Rats.

BACKGROUND: Hypertension is a leading cause of chronic kidney disease worldwide. Early studies demonstrated the short-term effects of hypertension on kidney function and morphology in ablative nephropathy. The aim of this study was to investigate the long-term consequences of hypertension in 5/6 nephrectomy (5/6NE) model. METHODS: Reduction of the kidney mass by 5/6NE was created in spontaneous hypertensive rats (SHR) and genetically similar normotensive Wistar Kyoto (WKY) rats. Blood pressure, serum creatinine (SCr), hematuria, and proteinuria were monitored weekly for 23 weeks. Kidney morphology was assessed at the end of the study. Sham-operated rats from both strains were used as controls. RESULTS: Rats with 5/6NE had increased SCr, blood pressure, hematuria, and proteinuria in both SHR and WKY. Even though the SCr levels and blood pressure were greater in 5/6NE SHR as compared with 5/6NE WKY rats, absolute changes from sham-operated rats were not statistically significant between these 2 groups. 5/6NE SHR had earlier onset and higher proteinuria than 5/6NE WKY rats. Hematuria was similar in 5/6NE SHR and 5/6NE WKY rats. However, 5/6NE SHR had enlarged glomeruli, increased interstitial fibrosis, and prominent intimal thickening in the small arteries/arterioles as compared with 5/6NE WKY rats. CONCLUSIONS: The long-term severity of kidney injury correlated with higher blood pressure. Reduction of the kidney mass increases SCr, hematuria, proteinuria, and blood pressure in both normotensive and hypertensive rats. Histological assessment provides better information about underlying chronic kidney injury than actual changes in SCr and urinalysis.

The Severity of Intracranial Hemorrhages Measured by Free Hemoglobin in the Brain Depends on the Anticoagulant Class: Experimental Data.

Background and Purpose. Anticoagulant therapy is broadly used to prevent thromboembolic events. Intracranial hemorrhages are serious complications of anticoagulation, especially with warfarin. Direct oral anticoagulants reduce but do not eliminate the risk of intracranial hemorrhages. The aim of this study is to determine the degree of intracranial hemorrhage after application of anticoagulants without additional triggers. Methods. Rats were treated with different anticoagulant classes (vitamin K antagonists, heparin, direct thrombin inhibitor, and factor Xa inhibitor). Brain hemorrhages were assessed by the free hemoglobin concentration in the brain parenchyma. Results. Vitamin K antagonists (warfarin and brodifacoum) significantly increased free hemoglobin in the brain. Among direct oral anticoagulants, thrombin inhibitor dabigatran also significantly increased free hemoglobin in the brain, whereas treatment with factor Xa inhibitor rivaroxaban did not have significant effect on the free hemoglobin concentration. Conclusions. Our data indicates that the severity of brain hemorrhages depends on the anticoagulant class and it is more pronounced with vitamin K antagonists.

The emerging threat of superwarfarins: history, detection, mechanisms, and countermeasures.

Superwarfarins were developed following the emergence of warfarin resistance in rodents. Compared to warfarin, superwarfarins have much longer half-lives and stronger affinity to vitamin K epoxide reductase and therefore can cause death in warfarin-resistant rodents. By the mid-1970s, the superwarfarins brodifacoum and difenacoum were the most widely used rodenticides throughout the world. Unfortunately, increased use was accompanied by a rise in accidental poisonings, reaching >16,000 per year in the United States. Risk of exposure has become a concern since large quantities, up to hundreds of kilograms of rodent bait, are applied by aerial dispersion over regions with rodent infestations. Reports of intentional use of superwarfarins in civilian and military scenarios raise the specter of larger incidents or mass casualties. Unlike warfarin overdose, for which 1-2 days of treatment with vitamin K is effective, treatment of superwarfarin poisoning with vitamin K is limited by extremely high cost and can require daily treatment for a year or longer. Furthermore, superwarfarins have actions that are independent of their anticoagulant effects, including both vitamin K-dependent and -independent effects, which are not mitigated by vitamin K therapy. In this review, we summarize superwarfarin development, biology and pathophysiology, their threat as weapons, and possible therapeutic approaches.

Oral warfarin and the thrombin inhibitor dabigatran increase blood pressure in rats: hidden danger of anticoagulants?

BACKGROUND: Hypertension is a common comorbidity in patients with chronic kidney disease (CKD). We reported earlier that oral anticoagulants, including warfarin and dabigatran, may induce acute kidney injury. No effects of oral anticoagulants on blood pressure (BP) have been previously reported. The aim of this study was to examine in detail the relationship of anticoagulant therapy and BP in rats. METHODS: Sham-operated and 5/6 nephrectomy rats were treated with different doses of oral anticoagulants (warfarin and dabigatran), superoxide scavenger N-acetylcysteine (NAC), vitamin K, and protease activated receptor 1 (PAR-1) inhibitor SCH79797. BP was measured by a tail cuff daily. RESULTS: Warfarin and dabigatran both increased systolic BP in sham-operated and 5/6 nephrectomy rats in a dose-dependent manner. SCH79797 also increased systolic BP in a dose-dependent manner. Vitamin K prevented warfarin-induced increase in BP but did not affect BP when administered alone. NAC delayed the warfarin-associated increase in BP. Warfarin effects on BP were similar in 5/6 nephrectomy rats with different CKD stages. CONCLUSIONS: Both warfarin and dabigatran increase systolic BP in rats. The mechanism of this effect is not clear, but our data suggest that it is related to decreased thrombin activity associated with anticoagulant treatment. The superoxide scavenger NAC delayed, but did not prevent, warfarin-induced hypertension.

Early posttransplant changes in circulating endothelial microparticles in patients with kidney transplantation.

BACKGROUND: Endothelial microparticles (EMPs) are membrane vesicles shed from endothelial cell in response to injury, activation or apoptosis. Kidney transplantation (KTx) is the treatment of choice for patients with end stage kidney disease (ESKD). The aim of this study was to analyze changes in EMP and serum creatinine (SCr) in patients following KTx. METHODS: Blood was periodically collected from patients before (pre-KTx) and after KTx for two months. EMPs were identified as CD31(+)/CD42b(-) microparticles and quantified by fluorescence-activated cell scanning. RESULTS: This study included 213 KTx, 14 kidney/pancreas (KPTx) recipients and 60 healthy donors prior to donation. The recipients were divided into 5 groups based on the cause of ESKD. No differences in the quantity of circulating EMP were seen in the pre-KPTx or KTx recipient sera and healthy donor sera. Patients with ESKD secondary to diabetes mellitus, obstructive/inherited kidney disease and autoimmune disease had a decrease in both circulating EMP and SCr by day 60 after KTx. CONCLUSION: Reduction in both circulating EMP and SCr was seen after kidney KTx in patients with selective ESKD.