Interference With Coagulation Cascade as a Novel Approach to Counteract Cisplatin-Induced Acute Tubular Necrosis; an Experimental Study in Rats

Abstract

Coagulation system activation plays an important role in the pathophysiology of different diseases. In spite of massive research regarding cisplatin-induced nephrotoxicity, the role of coagulation cascade in such toxicity is still questionable. Here, we aim to investigate the role of activation of coagulation system in the initiation of cisplatin-induced acute renal tubular necrosis. Moreover, the role of the anticoagulant rivaroxaban against such toxicity was investigated. Briefly, animals were classified into seven groups, eight rats each. Group 1 served as normal control group, groups (2-7) received i.p. single doses of cisplatin (6 mg/kg b.w), groups (6-7) were treated with rivaroxaban (5 and 7 mg/kg b.w, p.o., respectively) 7 days before cisplatin injection and completed for 4 days. Animals in groups (2, 3, and 4) were sacrificed after 1, 2 and 3 days of cisplatin injection, respectively, while groups (1, 5, 6, and 7) were sacrificed after 4 days of cisplatin injection. Serum cystatin-c, urea, creatinine and γ-glutamyl transferase, urinary Lipocaline-2, and KIM-1 protein densities, as well as glomerular filtration rate (GFR) were assessed. Immunofluorescence examination of glomeruli fibrin and tissue factor (TF) was also performed coupled with a histopathological study. Cisplatin administration increased expression of fibrin and TF starting 24 h of cisplatin injection even before renal failure markers elevated. Leukocytosis, thrombocytopenia, and increased prothrombin time were also observed. Cisplatin also induced tubular damage evidenced by increased serum cystatin-c, urea, and creatinine with significant decrease in GFR and Gamma glutamyl transferase (GGT) activity. Rivaroxaban significantly decreased elevation of fibrin and TF with significant reduction in serum creatinine, BUN and cystatin-c levels. Rivaroxaban also significantly improved hematological markers and histological features as well. This study showed that activation of coagulation system plays an important role in the pathophysiology of cisplatin-induced acute renal tubular damage. Interference with coagulation cascade may be a promising nephroprotective strategy against chemical nephrotoxicity.

Keywords: cisplatin; coagulation cascade; fibrin; nephrotoxicity; rivaroxaban; tissue factor.

FIGURE 1

Effect of single i.p. dose of cisplatin (CP) 6 mg/kg b.w after 1, 2, 3 and 4 days with or without rivaroxaban 5 mg /kg b.w and 7 mg/kg b.w on renal function tests including; serum cystatin-c (A), urinary lipocaline-2 (B) and urinary KIM-1 (C). Data are expressed as mean ± S.E. Multiple comparisons were done using one way ANOVA followed by Tukey-Kramer as a post-ANOVA test (a) Significantly different from control group at p < 0.05. (b) Significantly different from CP 1 day group at p < 0.05. (c) Significantly different from CP 2 day group at p < 0.05. (d) Significantly different from CP 3 day group at p < 0.05. (e) Significantly different from CP 4 day group at p < 0.05.

FIGURE 2

Effect of cisplatin (CP) with or without rivaroxaban (Riva) on fibrin expression: (A) Immunofluorescence staining of renal glomeruli of fibrin in normal and CP (6 mg/kg b.w) treated rats after 1, 2, 3 and 4 days, showing moderate to highly expression of fibrin in the glomerular cells after 3 and 4 days, respectively (yellow arrow) as compared to normal control. (B) Graphical presentation of fibrin fluorescence intensity in normal and cisplatin treated animals from day 1 to day 4 where (a) Significantly different from control group at p ≤ 0.01 (b) Significantly different from CP 1 day at p ≤ 0.01. (c) Significantly different from CP 2 day at p ≤ 0.01. (d) Significantly different from CP 3 day at p ≤ 0.01. (C) Immunofluorescence staining of renal tubular cells in normal and cisplatin (6 mg/kg) treated rats after 1, 2, 3, and 4 days in absence or presence of rivaroxaban, showing gradual increase in fibrin expression in renal tubular cells after 1 day till 4 days (yellow arrow). Basal expression of fibrin in the renal tubular cells of cisplatin pretreated with rivaroxaban. (D) Graphical histogram showing fluorescence intensity of fibrin in renal tubular cells of normal group and rats treated with cisplatin in absence or presence of rivaroxaban.

FIGURE 3

Effect of cisplatin (CP) with or without rivaroxaban (Riva) on tissue factor (TF) expression: (A) Immunofluorescence staining of renal tubular cells in normal rats and cisplatin (6 mg/kg) treated rats after 1, 2, 3 and 4 days in absence or presence of rivaroxaban. Gradual increase in TF expression in renal tubular cells staring from 1 day till 4 days which showed the highest expression of TF protein (white arrow). A significant low expression of TF in the renal tubular cells of cisplatin pretreated with rivaroxaban. (B) Histogram showing the fluorescence cells of normal group and rats treated with cisplatin in absence or presence of rivaroxaban.

FIGURE 4

Photomicrograph of kidney sections of (A) normal rats showed normal histologic structure of glomeruli and renal tubules (black arrow). (B) Cisplatin treated rats after 1 day showed more or less normal histological structure of renal glomeruli and lining epithelium lining the renal tubules (black arrows). (C) 2 days after injection of cisplatin showed mild degenerative changes of renal epithelial degeneration associated with mild desquamation of tubular epithelial cells (white arrows). (D) Cisplatin treated rats after 3 days showed moderate epithelial degeneration of lining epithelium Additionally, glomeruli tufft are suffering from mild to moderate glomerulonephrosis (black arrow). focal leukocytic infiltration could be detected in the interstitial area (black head). (E) Cisplatin after 4 days showed severe degenerative changes and necrosis associated with desquamation of renal lining epitheliums (yellow arrow). Moreover glomeruli tuft are suffering from severe degenerative changes (black arrow). (F) Rats treated with cisplatin for 4 days with rivaroxaban 5 mg/kg showed moderate degenerative changes of renal epithelium (arrow head) associated with glomerulonephrosis (arrow). (G) Rats treated with cisplatin for 4 days with rivaroxaban 7 mg/kg showed moderate degenerative changes of renal epithelium (arrow head) associated with mild glomerulonephrosis (arrow). (HE X100).