Research Letter: Is the 129S1/SvImJ Mouse Strain More Suitable to Study Anticoagulant-Related Nephropathy Than the C57BL/6 Strain?

Background: We have previously demonstrated that excessive anticoagulation with warfarin or dabigatran may result in acute kidney injury with red blood cell (RBC) tubular casts in some patients with chronic kidney disease, and this condition was named anticoagulant-related nephropathy (ARN). 5/6 nephrectomy (5/6NE) rats treated with warfarin or dabigatran reproduce the main pathologic features of human ARN. We had reported that 5/6NE C57BL/6 mice only partially develop ARN with increased serum creatinine and hematuria but no RBC tubular casts in the kidney. Objectives: The aim of this study was to investigate whether ARN can develop in 5/6NE 129S1/SvImJ mice. Methods: 5/6NE was performed in 129S1/SvImJ mice. Three weeks after 5/6NE, mice were treated with warfarin (1.0 and 1.5 mg/kg/day) or vehicle for 7 days. Serum creatinine, hematuria, and prothrombin time (PT) were monitored daily. Renal morphology was evaluated at the end of the studies. Results: Treatment with warfarin resulted in PT elevation 2 to 3 folds from baseline (1.0 mg/kg/day warfarin) and 4 to 5 folds from baseline (1.5 mg/kg/day warfarin) by day 7. Serum creatinine and hematuria elevated by day 7 in a dose-dependent manner. Histologically, 2 of 8 (25%) 5/6NE mice had RBCs in the tubules, and there was acute tubular epithelial cell injury in all warfarin-treated 5/6NE 129S1/SvImJ mice. Conclusions: Our findings suggest that 129S1/SvImJ mouse strain is a more suitable murine model to study ARN than C57BL/6 mouse strain.

Contexte: Nous avons précédemment démontré qu'un traitement anticoagulant excessif par warfarine ou dabigatran pouvait entraîner une insuffisance rénale aiguë avec formation de cylindres urinaires avec globules rouges (GR) chez certains patients atteints d'insuffisance rénale chronique. Cette affection a été nommée « néphropathie liée à un anticoagulant ¼ (NLA). Les rats 5/6NE (néphrectomie 5/6) traités par warfarine ou dabigatran reproduisent les principales caractéristiques pathologiques d'une NLA chez l'humain. Nous avions rapporté que les souris 5/6NE C57BL/6 ne développaient qu'une NLA partielle, présentant une augmentation de la créatinine sérique et de l'hématurie, mais aucune formation de cylindres urinaires avec GR dans les reins. Objectif: Vérifier si une NLA peut se développer chez les souris 5/6 NE 129S1/SvImJ. Méthodologie: Une 5/6NE a été réalisée chez des souris 129S1/SvImJ. Trois semaines après l'intervention, les souris ont été traitées avec de la warfarine (1,0 mg/kg/jour et 1,5 mg/kg/jour) ou un placebo pendant 7 jours. La créatinine sérique, l'hématurie et le temps de prothrombine (TP) ont été surveillés quotidiennement. La morphologie rénale a été évaluée à la fin des études. Résultats: Au jour 7, le traitement par warfarine avait entraîné une augmentation du TP de 2 à 3 fois par rapport à la mesure initiale pour le groupe traité avec 1,0 mg/kg/jour, et de 4 à 5 fois pour le groupe traité avec 1,5 mg/kg/jour. Les taux de créatinine sérique et l'hématurie s'étaient élevés en fonction de la dose. Sur le plan histologique, 2 souris 5/6NE sur 8 (25 %) avaient des globules rouges dans les tubules, et toutes les souris 5/6NE 129S1/SvImJ traitées avec la warfarine présentaient une atteinte aiguë des cellules tubulaires épithéliales. Conclusion: Nos résultats suggèrent que la souche de souris 129S1/SvImJ serait un modèle murin plus approprié que la souche C57BL/6 pour étudier la néphropathie liée à un anticoagulant.

N-acetylcysteine ameliorates hematuria-associated tubulointerstitial injury in 5/6 nephrectomy mice.

Chronic kidney disease (CKD) is characterized by increased interstitial fibrosis and tubular atrophy (IFTA) in the kidney. Chronic hematuria is a hallmark of several human kidney diseases and often is seen in patients on anticoagulation therapy. We had previously demonstrated that chronic hematuria associated with warfarin increases IFTA in 5/6 nephrectomy (5/6NE) rats, and such treatment increases reactive oxygen species (ROS) in the kidney. The goal of this study was to evaluate the effects of the antioxidant N-acetylcysteine (NAC) on the progression of IFTA in 5/6NE mice. 5/6NE C57BL/6 and 5/6NE 129S1/SvImJ mice were treated with warfarin alone or with warfarin and NAC for 23 weeks. Serum creatinine (SCr), hematuria, blood pressure (BP), and ROSs in the kidney were measured; kidney morphology was evaluated. Warfarin doses were titrated to achieve prothrombin time (PT) increase to the levels seen with therapeutic human doses. Warfarin treatment resulted in an increased SCr, systolic BP, hematuria, expression of TGF-ß and ROS in the kidney in both mouse strains. Tumor necrosis factor alpha (TNF-ɑ) levels in the serum were increased in 5/6NE mice treated with warfarin. IFTA was increased as compared with control 5/6NE mice, and this increase in IFTA was more prominent in 129S1/SvImJ than in C57BL/6 mice. NAC ameliorated the warfarin-associated increase in SCr and BP but not hematuria. IFTA, TGF-ß, and ROS in the kidney as well as TNF-ɑ levels in the serum were reduced in mice treated with NAC and warfarin as compared to mice treated with warfarin alone. NAC mitigates the increase in SCr and IFTA in mice with chronic hematuria by reducing oxidative stress in the kidney. This data open novel possibilities for treatments in CKD patients.

Role of protease-activated receptor-1 (PAR-1) in the glomerular filtration barrier integrity.

Protease-activated receptors (PAR) play an important role in the regulation of cellular function by the coagulation system, and they are activated by thrombin. PAR-1 is expressed in both endothelial cells and podocytes in the kidney. The role of PAR1 in the maintenance of the glomerular filtration barrier is not clear. Anticoagulant-related nephropathy (ARN) is a kidney disease with glomerular hematuria and red blood cell tubular casts. We validated 5/6 nephrectomy (5/6NE) in rats as a model of ARN and had demonstrated that direct thrombin inhibitor (dabigatran) induces ARN. The aim of this study was to investigate the role of PAR-1 in the ARN pathogenesis. 5/6NE rats were treated with dabigatran (150 mg/kg/day), PAR-1 inhibitor SCH79797 (1 and 3 mg/kg/day) and PAR-1 agonist TFLLR-NH2 (0.25 and 0.50 µmol/kg/day) for 7 days. Serum creatinine and hematuria were assessed daily. Kidney morphology was evaluated at the end of the study. In 5/6NE rats treated with either dabigatran or combination with a PAR-1 modulator, there was an elevation in serum creatinine, glomerular hematuria, red blood casts in the tubules, and acute tubular epithelial cell injury. Interestingly, both PAR-1 modulators in a dose-depended manner had similar effects on the serum creatinine levels and hematuria as those of dabigatran. Dabigatran-induced increase in the systolic blood pressure was not affected by PAR-1 modulators. In conclusion, the normal function of PAR-1 is crucial to maintain the glomerular filtration barrier integrity. Either activation or blockage of PAR-1 leads to glomerular hematuria and subsequent acute tubular epithelial cell injury.

Role of glomerular filtration rate-modifying drugs in the development of anticoagulant-related nephropathy.

INTRODUCTION: Anticoagulant-related nephropathy (ARN), that was described in humans first as warfarin-related nephropathy, is characterized by acute kidney injury and red blood cell (RBC) tubular casts in the kidney. 5/6 nephrectomy (5/7NE) rats treated with warfarin or dabigatran show changes in kidney function and morphology that are similar to human disease. The role of glomerular filtration rate (GFR) in the pathogenesis of ARN is not clear. The aim of these studies was to elucidate the role of GFR in the pathogenesis of dabigatran-induced ARN in 5/6NE rats. METHODS: 5/6NE rats were treated per os with 150 mg/kg/day dabigatran alone or with drugs that lower (enalapril, 1.5 mg/kg/day) or increase (albuterol, 4.0 mg/kg/day) GFR for 7 days. Changes in coagulation and kidney function were recorded daily. Kidney morphology was evaluated on day 7 after the treatment. RESULTS: Dabigatran resulted in activated partial thromboplastin time increase that was not affected by GFR-modifying drugs. Blood pressure was significantly lower in 5/6NE rats treated with enalapril and dabigatran as compared to dabigatran alone. The GFR was decreased by 35% in enalapril/dabigatran- and increased by 26% in albuterol/dabigatran-treated animals. There were no changes in serum creatinine, hematuria or urinary kidney injury molecule (KIM-1) levels when GFR-modifying drugs were added to dabigatran. All dabigatran-treated animals had RBC casts in the kidney regardless of the GFR modification. CONCLUSIONS: GFR does not play a significant role in the dabigatran-induced acute kidney injury in 5/6 nephrectomy model in rats. Based in these data, modification of GFR in patients with ARN is not warranted.

Chronic Hematuria Increases Chronic Kidney Injury and Epithelial-Mesenchymal Transition in 5/6 Nephrectomy Rats.

Chronic kidney disease (CKD) is a common outcome of many kidney diseases. Interstitial fibrosis and tubular atrophy (IFTA) is a histologic hallmark of CKD. Hematuria is a common symptom in many human kidney diseases. Free hemoglobin may affect tubular epithelial cells by generating reactive oxygen species (ROS). Epithelial-mesenchymal transition (EMT) of the tubular epithelial cells has been shown to play an important role in the IFTA development. The aim of this study was to determine the effects of chronic hematuria on the CKD progression in 5/6 nephrectomy (5/6NE) rat model of CKD. 5/6 NE rats were treated with oral warfarin (0.5 mg/kg/day) or vehicle (control). The animals were monitored for 26 weeks, while prothrombin time (PT), serum creatinine (SCr), and hematuria were measured weekly. Staining for iron, trichrome, and EMT (vimentin, E-cadherin, smooth muscle actin) markers was performed on the remnant kidneys. ROS were detected in the kidneys by protein carbonyl assay and immunohistochemistry for heme oxygenase 1 (HMOX1), at the end of the study. Apoptosis was detected by TUNEL assay. Warfarin treatment resulted in a PT increase 1.5-2.5 times from control and an increase in hematuria and SCr. Histologically, warfarin-treated animals had more iron-positive tubular epithelial cells and increased IFTA as compared to control (42.9 ± 17% vs. 18.3 ± 2.6%). ROS were increased in the kidney in warfarin-treated rats. The number of tubules that show evidence of EMT was significantly higher in warfarin-treated 5/6NE as compared to control 5/6NE rats. The number of apoptotic tubular epithelial cells was higher in warfarin-treated 5/6 NE rats. Chronic hematuria results in increased iron-positive tubular epithelial cells, EMT, apoptosis, and more prominent IFTA in CKD rats. Our data suggest an important role of chronic hematuria in the progression of CKD.

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.

Anticoagulant Related Nephropathy Only Partially Develops in C57BL/6 Mice: Hematuria Is Not Accompanied by Red Blood Cell Casts in the Kidney.

Anticoagulant-related nephropathy (ARN) may develop in patients that are on anticoagulation therapy. Rats with 5/6 nephrectomy treated with different anticoagulants showed acute kidney injury (AKI) and red blood cell (RBC) casts in the tubules similar to ARN in humans. The aim of the current study was to investigate the feasibility of inducing ARN in mice. C57BL/6 5/6 nephrectomy mice were treated with warfarin and dabigatran 3 weeks after ablative surgery for 7 days. Two doses of each anticoagulant were used. All anticoagulants resulted in serum creatinine and hematuria increase. Mortality was 63% in 5.0 mg/kg/day of warfarin but only 13% in 2.5 mg/kg/day of warfarin or in 400 mg/kg/day of dabigatran and 0% in 200 mg/kg/day of dabigatran. In spite of increasing hematuria, RBC tubular casts were not seen in mice treated with any anticoagulant. The 5/6 nephrectomy murine model in C57BL/6 mice only partially reproduced ARN in terms of increasing serum creatinine and hematuria, but there were no RBC tubular casts in the remnant kidney.