Recovery from glycerol-induced acute kidney injury is accelerated by suramin

Abstract

Acute kidney injury (AKI) is a common and potentially life-threatening complication after ischemia/reperfusion and exposure to nephrotoxic agents. In this study, we examined the efficacy and mechanism(s) of suramin in promoting recovery from glycerol-induced AKI, a model of rhabdomyolysis-induced AKI. After intramuscular glycerol injection (10 ml of 50% glycerol per kilogram) into male Sprague-Dawley rats, serum creatinine maximally increased at 24 to 72 h and then decreased at 120 h. Creatinine clearance (CrCl) decreased 75% at 24 to 72 h and increased at 120 h. Suramin (1 mg/kg i.v.) administered 24 h after glycerol accelerated recovery of renal function as demonstrated by increased CrCl, decreased renal kidney injury molecule-1, and improved histopathology 72 h after glycerol injection. Suramin treatment decreased interleukin-1β (IL-1β) mRNA, transforming growth factor-β(1) (TGF-β(1)), phospho-p65 of nuclear factor-κB (NF-κB), and cleaved caspase-3 at 48 h compared with glycerol alone. Suramin treatment also decreased glycerol-induced activation of intracellular adhesion molecule-1 (ICAM-1) and leukocyte infiltration at 72 h. Urinary/renal neutrophil gelatinase-associated lipocalin 2 (NGAL) levels, hemeoxygenase-1 expression, and renal cell proliferation were increased by suramin compared with glycerol alone at 72 h. Mechanistically, suramin decreases early glycerol-induced proinflammatory (IL-1β and NF-κB) and growth inhibitory (TGF-β(1)) mediators, resulting in the prevention of late downstream inflammatory effects (ICAM-1 and leukocyte infiltration) and increasing compensatory nephrogenic repair. These results support the hypothesis that delayed administration of suramin is effective in abrogating apoptosis, attenuating inflammation, and enhancing nephrogenic repair after glycerol-induced AKI.

Fig. 1.

Effect of delayed administration of suramin on renal dysfunction in rats subjected to glycerol-induced AKI. A, serum creatinine in groups of male Sprague-Dawley rats (n = 5) that were given intramuscular injections of 10 ml of 50% glycerol per kilogram, with or without 1 mg of suramin per kilogram administered intravenously at 24 h after glycerol treatment. One more group did not receive any treatment. Data are expressed as mean ± S.E. (n = 5). *, statistically significant from respective 0 h controls. #, statistically significant from rats receiving glycerol treatment alone at the corresponding time point (p ≤ 0.05). B, urine creatinine in groups of male Sprague-Dawley rats (n = 5) that were injected intramuscularly with 10 ml of 50% glycerol per kilogram, with or without 1 mg of suramin per kilogram administered intravenously at 24 h after glycerol treatment. One more group did not receive any treatment. Data are expressed as mean ± S.E. (n = 5). *, statistically significant from respective 0 h controls (p ≤ 0.05). C, urine volume in groups of male Sprague-Dawley rats (n = 5) that were given intramuscular injections of 10 ml of 50% glycerol per kilogram, with or without 1 mg of suramin per kilogram administered intravenously at 24 h after glycerol treatment. One more group did not receive any treatment. Data are expressed as mean ± S.E. (n = 5). *, statistically significant from respective 0 h controls. (p ≤ 0.05). D, CrCl in groups of male Sprague-Dawley rats (n = 5) that were injected intramuscularly with 10 ml of 50% glycerol per kilogram, with or without 1 mg of suramin per kilogram administered intravenously at 24 h after glycerol treatment. One more group did not receive any treatment. Data are expressed as mean ± S.E. (n = 5). *, statistically significant from respective 0 h controls. #, statistically significant from rats receiving glycerol treatment alone at the corresponding time point (p ≤ 0.05).

Fig. 2.

Effect of delayed administration of suramin on renal injury in rats subjected to glycerol-induced AKI. A–C, representative photomicrographs of hematoxylin and eosin-stained kidney sections from rats that were untreated (A), treated with glycerol alone at 72 h (B), and subjected to suramin intervention after glycerol treatment at 72 h (C). *, denotes necrotic tubules. #, denotes tubules that are lined with regenerating cells. All of the fields were chosen from the OSOM. Original magnification, 200×.

Fig. 3.

Effect of delayed administration of suramin on KIM-1 in rats subjected to glycerol-induced AKI. A, representative Western blot showing KIM-1 protein expression in kidneys of untreated and glycerol-treated rats (n = 4) with or without suramin over a time course. B, densitometric analysis of renal KIM-1 protein expression in untreated, glycerol-treated, and suramin intervention after glycerol treatment groups over the time course. Data were normalized to GAPDH, which served as an internal control. Data are expressed as mean ± S.E. (n = 4). *, statistically significant from respective 0 h controls. #, statistically significant from rats receiving glycerol treatment alone at the corresponding time point (p ≤ 0.05). U, untreated; G, glycerol treatment alone group; GS, glycerol + suramin treatment group. C–E, representative photomicrographs of KIM-1-stained kidney sections from rats that were untreated (C), treated with glycerol alone at 72 h (D), and subjected to suramin intervention after glycerol treatment at 72 h (E). KIM-1-positive staining was observed on the proximal tubular epithelial cells and damaged tubules (arrows). All of the fields were chosen from the OSOM. Original magnification, 200×.

Fig. 4.

Effect of delayed administration of suramin on NGAL in rats subjected to glycerol-induced AKI. A, enzyme-linked immunosorbent assay of urinary NGAL levels of untreated and glycerol-treated rats (n = 5) with or without suramin over a time course. B, representative Western blot showing NGAL protein expression in kidneys of untreated and glycerol-treated rats (n = 4) with or without suramin over a time course. C, densitometric analysis of renal NGAL protein expression in untreated, glycerol-treated, and suramin intervention after glycerol treatment groups over the time course. Data were normalized to GAPDH, which served as an internal control. Data are expressed as mean ± S.E. (n = 4). *, statistically significant from respective 0 h controls. #, statistically significant from rats receiving glycerol treatment alone at the corresponding time point (p ≤ 0.05). D–F, representative photomicrographs of NGAL-stained kidney sections from rats that were untreated (D), treated with glycerol alone at 48 h (E), and subjected to suramin intervention after glycerol treatment at 48 h (F). NGAL-positive staining was observed on the proximal tubular epithelial cells (arrows). All of the fields were chosen from the OSOM. Original magnification, 200×.

Fig. 5.

Effect of delayed administration of suramin on renal cleaved caspase-3 in rats subjected to glycerol-induced AKI. A, representative Western blot showing cleaved caspase-3 protein expression in kidneys of untreated and glycerol-treated rats (n = 4) with or without suramin over a time course. B, densitometric analysis of renal cleaved caspase-3 protein expression in untreated, glycerol-treated, and suramin intervention after glycerol treatment groups over the time course. Data were normalized to GAPDH, which served as an internal control. Data are expressed as mean ± S.E. (n = 4). *, statistically significant from respective 0 h controls. #, statistically significant from glycerol- and suramin-treated rats at the corresponding time point (p ≤ 0.05).

Fig. 6.

Effect of delayed administration of suramin on renal leukocyte infiltration in rats subjected to glycerol-induced AKI. A–C, representative photomicrographs of neutrophil and monocyte staining assessed by the formation of a stable pinkish-red complex (arrows) of free naphthol and diazonium salts after the incubation of kidney sections from rats that were untreated (A), treated with glycerol alone at 72 h (B), and subjected to suramin intervention after glycerol treatment at 72 h (C) in naphthol AS-D chloroacetate reagent. All of the fields were chosen from the OSOM. Original magnification, 200×. D, quantitative analysis of renal leukocyte infiltration assessed by the number of pink dots in a total of 25 fields in the OSOM region of kidney sections. Data are expressed as mean ± S.E. (n = 4). *, statistically significant from respective 0 h controls. #, statistically significant from rats receiving glycerol treatment alone at the corresponding time point (p ≤ 0.05).

Fig. 7.

Effect of delayed administration of suramin on renal ICAM-1 in rats subjected to glycerol-induced AKI. A, representative Western blot showing ICAM-1 protein expression in kidneys of untreated and glycerol-treated rats (n = 4) with or without suramin over a time course. B, densitometric analysis of renal ICAM-1 protein expression in untreated, glycerol-treated, and suramin intervention after glycerol treatment groups over the time course. Data were normalized to GAPDH, which served as an internal control. Data are expressed as mean ± S.E. (n = 4). *, statistically significant from respective 0 h controls. #, statistically significant from glycerol- and suramin-treated rats at the corresponding time point (p ≤ 0.05). C–E, representative photomicrographs of ICAM-1-stained kidney sections from rats that were untreated (C), treated with glycerol alone at 72 h (D), and subjected to suramin intervention after glycerol treatment at 72 h (E). ICAM-1-positive staining was observed on the basement membranes of proximal tubules (arrows). All of the fields were chosen from the OSOM. Original magnification, 200×.

Fig. 8.

Effect of delayed administration of suramin on renal proinflammatory cytokines in rats subjected to glycerol-induced AKI. A–D, the mRNA expression profiles in kidneys of untreated and glycerol-treated rats (n = 4) with or without suramin over a time course, and quantitative polymerase reactions were performed using primers for TNF-α (A), IL-6 (B), IL-1β (C), and MCP-1 (D). Tubulin was used as the internal control. Data are expressed as mean ± S.E. (n = 4). *, statistically significant from respective 0 h controls. #, statistically significant from rats receiving glycerol treatment alone at the corresponding time point (p ≤ 0.05).

Fig. 9.

Effect of delayed administration of suramin on renal phospho-p65 in rats subjected to glycerol-induced AKI. A, representative Western blot showing phospho-p65 protein expression in the kidneys of untreated and glycerol-treated rats (n = 4) with or without suramin over a time course. B, densitometric analysis of renal phospho-p65 protein expression in untreated, glycerol-treated, and suramin intervention after glycerol treatment groups over the time course. Data were normalized to GAPDH, which served as an internal control. Data are expressed as mean ± S.E. (n = 4). *, statistically significant from respective 0 h controls. #, statistically significant from rats receiving glycerol treatment alone at the corresponding time point (p ≤ 0.05).

Fig. 10.

Effect of delayed administration of suramin on renal HO-1 and TGF-β₁ in rats subjected to glycerol-induced AKI. A and C, representative Western blot showing HO-1 (A) and TGF-β₁ (C) protein expression in the kidneys of untreated and glycerol-treated rats (n = 4) with or without suramin over a time course. B and D, densitometric analysis of renal HO-1 (B) and TGF-β₁ (D) protein expression in untreated, glycerol-treated, and suramin intervention after glycerol treatment groups over the time course. Data were normalized to GAPDH, which served as an internal control. Data are expressed as mean ± S.E. (n = 4). *, statistically significant from respective 0 h controls. @, statistically significant from glycerol- and suramin-treated rats at the corresponding time point. #, statistically significant from rats receiving glycerol treatment alone at the corresponding time point (p ≤ 0.05).

Fig. 11.

Effect of delayed administration of suramin on renal proximal convoluted tubular epithelial cell PCNA in rats subjected to glycerol-induced AKI. A, representative Western blot showing PCNA protein expression in the kidneys of untreated and glycerol-treated rats (n = 4) with or without suramin over a time course. B, densitometric analysis of renal PCNA protein expression in untreated, glycerol-treated, and suramin intervention after glycerol treatment groups over the time course. Data were normalized to GAPDH, which served as an internal control. Data are expressed as mean ± S.E. (n = 4). *, statistically significant from respective 0 h controls. #, statistically significant from rats receiving glycerol treatment alone at the corresponding time point (p ≤ 0.05). C–E, representative photomicrographs of PCNA-stained kidney sections from rats that were untreated (C), treated with glycerol alone at 72 h (D), and subjected to suramin intervention after glycerol treatment at 72 h (E). PCNA-positive staining (intense brown) was observed in cells that are lining the proximal tubules (arrows). All of the fields were chosen from the OSOM. Original magnification, 200×.