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.

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 hemorrhageswere 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 ismore pronounced with vitamin K antagonists.

Brodifacoum (BDF) is a superwarfarin that is used primarily as a rodenticide. By reducing vitamin K levels, BDF acts as a potent anticoagulant. Since its’ introduction as a rodenticide, there have been increasing number of reports of human cases of accidental or intentional BDF ingestion with high mortality rate. Currently, there is no biomarker for early detection of BDF ingestion in humans. 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. BDF was administered to Sprague Dawley rats via oral gavage. Urinalysis was performed at different times after BDF administration. We observed that within a few hours the animals developed BDF-dose-dependent transient hemoglobinuria, which ceased within 24 hours. 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. Early hemoglobinuria but not late hematuria was diminished or prevented by treatment with N-acetylcysteine (antioxidant). We propose that transient early hemoglobinuria with consecutive late hematuria are novel biomarkers of BDF poisoning.

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.

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.

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.

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.

•We analyzed changes in endothelial microparticles (EMPs) after kidney transplantation.•EMPs change after kidney transplantation in selected patient population.•EMPs show different dynamics in peritubular capillary C4d positive or negative biopsies.

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.

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.

Formulation of nutritionally complete and cost efficient diets for yellow perch (Perca flavescens) is a prerequisite for successful intensive culture of this species. One of the objectives of this study was to determine the optimum diet for the grow-out phase of juvenile yellow perch. Fish at the size of 12.9 +/- 4 similar to g were individually marked with passive integrated transponders (PIT)-tags and randomly distributed into six 400 similar to L tanks, 45 fish per tank. This experiment included lysine-deficient [(-) Lys] and lysine-supplemented [(+) Lys] wheat-gluten-based diets in triplicate groups. Our experiment showed that the mean weight of fish fed (+) Lys diet (83.9 +/- 1.5 similar to g) was significantly larger than fish fed (-) Lys diet (68.6 +/- 5.2 similar to g) (P similar to<similar to 0.05). This experiment also showed that the blood plasma concentration of free lysine in (+) Lys group was significantly higher than in (-) Lys group (P similar to<similar to 0.05) and the same trend appeared also in methionine concentrations. The concentration of Lys in deficient group of fish, 3 similar to h after a meal was lower compared with levels of Lys 24 similar to h post-feeding (P similar to<similar to 0.05). Lysine deficiency in diet resulted in significantly higher level of serine, and a similar trend occurred in small and large fish.

This study addressed the over-ripening process of walleye eggs that is critical during gamete collection and artificial propagation. We evaluated the quality of eggs (1) stored for varying periods of time, (2) supplemented with Moore's extender or (3) stored with added infertile “large” eggs prior to fertilization. Three separate experiments were carried out at three different egg collection periods. We sought to determine the biochemical indicators of the fertilizing ability of eggs and the changes in these indicators during over-ripening. Our results showed that survival rate at eyed stage embryo decreased from 73% to 21% when the duration of storage increased from 8 to 42h. Extended time of storage caused eggs to release higher amounts of protein and survival rate correlated inversely with the concentration of protein released into the ovarian fluid (P<0.05). Survival rates decreased with the presence of infertile “large” eggs in both cases (P<0.01). The amount of ovarian fluid positively influenced egg viability after 42h storage (P<0.01). Eggs stored in Moore's extender survived better than those stored without extender (P<0.05). Survival of embryos decreased with the addition of infertile eggs (P=0.05). In all experiments protein concentration increased with increasing storage period and resulted in increasing turbidity of ovarian fluid. Our results indicated that protein concentration and turbidity of ovarian fluid can be used as indicators to evaluate the fertilizing ability of walleye eggs. It is also suggested that walleye eggs be stored in an extender to decrease the negative effect of over-ripening on egg viability.

Abstract There is continuous interest in experimental, semipurified diet formulations and in the formulation of practical, inexpensive diets for grow out of sunshine bass (female white bass Morone chrysops × male striped bass M. saxatilis). The effects of four diets on growth, survival, and body composition of sunshine bass after a feeding period (51 d) and a subsequent fasting period (14 d) were examined. To our knowledge, no previous sunshine bass feeding experiments have incorporated a fasting period to simulate the handling and acclimation conditions experienced by farmed fish that are stocked into new environments. Diets consisted of a high-nutrient commercial diet, a wheat-gluten-based practical diet, a semipurified diet (meeting standard nutrient requirements), and a frozen natural diet (chironomids). Fish were hand fed three times per day at 4% of body weight. At the end of the fasting period, samples were collected for histological analysis of the liver and posterior intestine. Survival was signi...

Avoiding the transmission of viral disease and securing the health of fish populations are vital for hatchery-based stocking programs in regions affected by viral hemorrhagic septicemia (VHS). Iodine compounds are nonselective antiviral substances that are widely used as disinfectants, but their effects on survival and growth during early life stages of fish require further study. Two groups of walleyes Sander vitreus were collected from the Maumee River (Perrysburg, Ohio) in March 2009. Group 1 (6 females and 3 males) was collected midmorning, and group 2 (10 females and 4 males) was collected that evening. Gametes were collected on site for group 1; group 2 fish were held in tanks overnight, and their gametes were collected the next morning. Gametes were transported (3-4 h) to the laboratory, where eggs from individual females were fertilized with the combined sperm of three to four males. Directly after fertilization and tannic acid treatment, embryos were exposed to iodine at 0 mg/L (control) or 100 mg/L (triplicate) for 30 min. Survival to the eyed embryo stage was estimated for both groups. Only larvae from group 1 were sampled prior to first feeding (live nauplii of brine shrimp Artemia spp.) at 9 d posthatch (dph) and approximately every 10 d thereafter until 48 dph to assess growth. Samples of eggs, ovarian fluid, larvae, and juveniles were collected for VHS testing. Survival to the eyed embryo stage for group 1 was not affected by treatment, but iodine treatment significantly decreased survival for group 2. Growth was unaffected by iodine treatment, and all samples that were tested for VHS were negative. Histological analysis of swim bladder morphology revealed that larvae with noninflated swim bladders had features of an undeveloped organ with hyperplastic epithelium consisting of vacuolarized cells. Walleye eggs that are collected according to the procedures used for group 1 can be treated with 100-mg/L iodine for 30 min without sacrificing survival or growth in regions affected by VHS.

We quantified thiamine in eggs of Lake Erie walleye to determine if differences exist between spawning stocks within the Maumee and Sandusky rivers, both of which drain into the Western Basin. In spring 2004, eggs of walleye were collected in the Maumee River at three occasions (early, peak and late of the spawning run) and in the Sandusky River during the spawning run. After collection, eggs were fertilized with a known amount of milt, incubated and embryo survival was determined at the pigmented eyed stage. Thiamine and its derivatives were analyzed using high performance liquid chromatography. Thiamine pyrophosphate (TPP) was the most abundant form of vitamin B1 present in eggs (60–95% of the total vitamin B1 concentration). Total thiamine concentrations in walleye eggs from the Maumee and Sandusky rivers at the peak of the spawning run averaged 6.1 ± 1.6 nmol g−1 and 5.0 ± 2.9 nmol g−1, respectively. Our results also indicated that Maumee River stock survival to the eyed stage embryo declined as the spawning season progressed (72%, 59% and 37% in the early, middle and late of spawning run, respectively) as well as the total thiamine and TPP concentrations. At the peak of the spawning run, survival to the eyed stage embryo did not differ significantly between stocks 59% versus 65% in the Maumee and Sandusky rivers, respectively, and thiamine concentrations were not significantly different between sites.

Securing health and avoiding viral disease transmission are of paramount importance for stocking and management of walleyes Sander vitreus. Iodine compounds are widely used as nonselective antiviral substances; however, their adverse effects on embryo survival are less known. Our experiments investigated how maternal attributes influence the relationship between toxicity of iodine (expressed as concentrations and duration of treatment) and survival of walleyes after fertilization and during early larval growth. Eleven female and four male walleyes were collected in April 2007 from the Maumee River, Perrysburg, Ohio, and the gametes were transported unfertilized (2 h) to the laboratory. After fertilization and tannic acid treatment, embryos were exposed for 15, 30, and 180 min to iodine concentrations of 0 (control), 50, 100, 200, 400, and 800 mg/L. The calculated survival of 50% of the embryos at the eyed stage corresponded to an iodine concentration of 380 mg/L for the exposure duration of 15 min (i.e., the LD50; linear relationship). An increase in exposure time (to 30 or 180 min) significantly decreased embryo survival. Progeny from individual females varied in sensitivity to iodine treatment, particularly with respect to the high concentration (800 mg/L), but high survival in controls did not correspond to better resistance to iodine treatments. No significant differences were observed in larval walleye size or swim bladder inflation rates among various iodine exposure groups within 15 d after treatment.

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.

Abstract Abstract 819 Recently we reported that an excessive anticoagulation with warfarin (INR>3.0) can result in acute kidney injury (AKI). Morphologic findings included glomerular hemorrhage and renal tubular obstruction by red blood cell (RBC) casts. The clinical outcome in these patients was unfavorable; more than half of them did not recover from acute kidney injury even after normalization of INR. Later we analyzed serum creatinine (SC) and INR in patients with chronic kidney disease (CKD) on warfarin therapy. We found that 46% of patients had increase in SC levels >0.3 mg/dl associated with INR>3.0. SC remained elevated above baseline after the first episode of abnormal INR. The slope of the following SC increase was higher after this abnormal INR episode. We called this condition warfarin related nephropathy (WRN). The current study is based on medical records of 4059 consecutive patients who were on warfarin therapy at the Ohio State Medical Center for a 5-year period. Of these, 838 (21%) experienced an increase in SC>0.3 mg/dl within 1 week after INR>3.0 (WRN group). The remaining 3221 patients (79%) were designated no-WRN. The WRN group had a 5-year mortality rate of 42%, as compared to 27% for the no-WRN group (p<.001). The highest risk of death in the WRN cohort occurred within the 1st month after INR>3.0 (hazard ratio =2.15). For both WRN and no-WRN groups, the 5-year mortality rate was consistently higher in those with CKD compared to those with no-CKD (50.8% vs. 37.0% for the WRN cohort; 39.7% vs. 24.5% for the no-WRN cohort; p<.0001). Compared to no-WRN patients, WRN patients tended to be older (63.7±14.7 years vs. 61.7±15.6 years, p=.025), diabetic (47% vs 37%, p<.0001), hypertensive (82% vs 72%, p<.001) and had a history of heart failure (62% vs 42%, p<.001). Preliminary models indicate that WRN still is a significant predictor of death even after adjusting for these factors. We conclude that WRN is associated with increased mortality rate in the elderly, the diabetic, and those with CKD and cardiovascular diseases. The possible pathophysiologic mechanisms may be glomerular hematuria and formation of occlusive RBC casts. Physicians, involved in the clinical care of patients on systemic anticoagulation therapy, should be aware of this serious renal complication of warfarin overdose and carefully monitor the kidney function and coagulation parameters in these patients Disclosures: No relevant conflicts of interest to declare.