5/6 nephrectomy as a validated rat model mimicking human warfarin-related nephropathy

Abstract

Background/aims: We previously reported that patients with chronic kidney disease (CKD) receiving warfarin therapy and whose international normalized ratio increases to >3.0 may develop acute kidney injury (AKI) as a result of glomerular hemorrhage and formation of obstructive red blood cell (RBC) casts. We named this condition warfarin-related nephropathy (WRN). We also previously reported that acute excessive anticoagulation with brodifacoum (superwarfarin) induces AKI in 5/6 nephrectomy (5/6NE) rats. Limitations of the brodifacoum model precluded a careful assessment of dose-response relationships.

Methods: Warfarin treatment was used in 5/6NE.

Results: Herein we report that warfarin treatment of 5/6NE rats resulted in a dose-dependent increase in serum creatinine (SC). The increase in SC following warfarin treatment was greater at 3 and 19 weeks after the ablative surgery, than that observed 8 weeks after the ablative surgery. The SC increase was correlated with the prothrombin time increase. Morphologically, 5/6NE, but not control rats, had acute tubular injury with RBC and RBC casts in the tubules. Treatment with vitamin K prevented SC increase and morphologic changes in the kidney associated with warfarin treatment. A single episode of WRN did not affect the progression of CKD in 5/6NE.

Conclusion: (1) The 5/6NE model of CKD is an appropriate animal model to study the pathogenesis of WRN. (2) The pharmacokinetics of warfarin is better suited to the study of WRN than that of brodifacoum. (3) The more advanced stages of 5/6NE are more susceptible to WRN than the earlier stages. (4) Vitamin K treatment prevents WRN.

Fig. 1

Changes in prothrombin time (PT) and hematuria in animals treated with warfarin. A Warfarin was administered per os (doses and time are shown in rectangles on the x-axis). This protocol resulted in a gradual increase in PT (sINR is shown) in all experimental groups. B Hematuria was measured using DiaScreen (Chronimed Inc., Minnetonka, Minn., USA) reagent strips in control (solid squares, n = 11) and 5/6 nephrectomy rats at 3 weeks (5/6NE 3W, inverted triangles, n = 7), 8 weeks (5/6NE 8W, solid circles, n = 10) and 19 weeks (5/6NE 19W, upright triangles, n = 6) after the ablative surgery. Hematuria was graded using a semiquantitative scale of 0–3+. Score 0 was designated for negative hematuria, score 1+ for mild hematuria, score 2+ for moderate hematuria and score 3+ for large hematuria. * p < 0.05 compared to control.

Fig. 2

Changes in serum creatinine (SC) levels in animals treated with warfarin. A Control (solid squares, n = 11), B 5/6 nephrectomy rats 3 weeks after the ablative surgery (5/6NE 3W, n = 7), C 5/6 nephrectomy rats 8 weeks after the ablative surgery (5/6NE 8W, n = 10) and D 5/6 nephrectomy rats 19 weeks after the ablative surgery (5/6NE 19W, n = 6) were treated with increasing doses of warfarin (doses and time are shown in rectangles on the x-axis). Changes in SC levels in animals treated with warfarin (+ warfarin) (solid line, solid squares, A–D) were compared with SC levels in animals treated with vehicle (+ vehicle) (dotted line, solid circles, B–D) at the same time after the ablative surgery as the warfarin-treated animals. * p < 0.05 compared to vehicle-treated animals.

Fig. 3

Morphologic findings in the kidneys obtained from 5/6 nephrectomy animals treated with warfarin. A Occasional glomeruli in 5/6 nephrectomy rats treated with warfarin had red blood cells (RBC) in the Bowman's space (arrow). The tubules associated with those glomeruli had RBC. B Numerous RBC occlusive casts were present in the tubules. Magnification 200×. Hematoxylin-eosin stain.

Fig. 4

Changes in serum creatinine (SC) levels in animals treated with warfarin and vitamin K. Warfarin (0.75 mg/kg/24 h) results in increased sINR above 4 within 1 week. Co-treatment with warfarin and vitamin K (vit K) (40 mg/kg/24 h) prevented sINR increase (A). SC was increased in 5/6 nephrectomy rats treated with warfarin only, but not with warfarin and vitamin K together (B). A Control and 5/6 nephrectomy animals 3 weeks (5/6NE 3W) and 9 weeks (5/6NE 9W) after the ablative surgery were treated with warfarin per os (0.75 mg/kg) and either vehicle (+ vehicle) or vitamin K (intraperitoneally 40 mg/kg/24 h, + vit K). Animals received warfarin and vehicle had progressive increase in PT (shown as sINR), whereas co-treatment with vitamin K prevented sINR increase. Changes in SC levels in control (B), 5/6 nephrectomy rats 3 weeks after the ablative surgery (5/6NE 3W; C), 5/6 nephrectomy rats 9 weeks after the ablative surgery (5/6NE 9W; D) treated with warfarin (0.75 mg/kg/24 h) and either vehicle (n = 6 in each group) or vitamin K (40 mg/kg/24 h) (n = 6 in each group). * p < 0.05 compared to vehicle-treated animals. Serum creatinine was measured before the surgery and weekly thereafter until treatment with warfarin and vitamin K began.

Fig. 5

Changes in serum creatinine (SC) levels in 5/6 nephrectomy rats after a single episode of warfarin-related nephropathy. A 5/6 nephrectomy rats 8 weeks after the ablative surgery were treated with warfarin (0.75 mg/kg/24 h) for 8 days. This resulted in a rapid prothrombin time (PT) (shown as sINR) increase of more that 4-fold. Treatment with warfarin was stopped and vitamin K (40 mg/kg) was administered. This resulted in a rapid PT decline. B, C SC was increased with PT increase in 5/6 nephrectomy rats treated with warfarin 8 weeks after the ablative surgery (solid circles, n = 6; B) or 3 weeks after the ablative surgery (solid circles, n = 6; C). The time of the treatment with warfarin is scaled differently; the duration of warfarin treatment is shown as a solid line. After the warfarin treatment cessation and PT normalization, SC levels returned to the levels comparable with 5/6 nephrectomy rats treated with vehicle (no warfarin) 3 and 8 weeks after the ablative surgery (solid squares, n = 9). SC levels were monitored daily in both experimental groups until 22 weeks after the ablative surgery.