Apixaban Inhibits Progression of Experimental Diabetic Nephropathy by Blocking Advanced Glycation End Product-Receptor Axis

Abstract

Diabetes is associated with an increased risk of thromboembolism. However, the effects of apixaban, a factor Xa inhibitor on diabetic nephropathy, remain unknown. Six-week-old Wistar rats received a single 60 mg/kg intraperitoneal injection of streptozotocin to produce a model of type 1 diabetes. Type 1 diabetic and non-diabetic control rats were treated with or without apixaban orally for 8 weeks, and blood and kidneys were obtained for biochemical, real-time reverse transcription-polymerase chain reaction (RT-PCR) and morphological analyses. Although apixaban treatment did not affect glycemic or lipid parameters, it significantly (p < 0.01) inhibited the increases in advanced glycation end products (AGEs), the receptor for AGEs (RAGE) mRNA and protein levels, 8-hydroxy-2'-deoxyguanosine (8-OHdG), and NADPH oxidase-driven superoxide generation in diabetic rats at 14 weeks old. Compared with non-diabetic rats, gene and protein expression levels of monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), transforming growth factor-β (TGF-β), connective tissue growth factor (CTGF), and fibronectin were increased in 14-week-old diabetic rats, which were associated with enhanced renal expression of kidney injury molecule-1 (KIM-1) and Mac-3, increased extracellular matrix accumulation in the kidneys, and elevated urinary excretion levels of protein and KIM-1, all of which were significantly inhibited by the treatment with apixaban. Urine KIM-1 levels were significantly (p < 0.01) and positively correlated with AGEs (r = 0.690) and 8-OHdG (r = 0.793) in the kidneys and serum 8-OHdG levels (r = 0.823). Our present findings suggest that apixaban could ameliorate renal injury in streptozotocin-induced type 1 diabetic rats partly by blocking the AGE-RAGE-oxidative stress axis in diabetic kidneys.

Keywords: AGEs; RAGE; apixaban; diabetic nephropathy.

Figure 1

Effects of apixaban (Apx) on AGE-RAGE-oxidative stress axis in the kidneys of streptozotocin-induced diabetic rats. Levels of AGEs (a), RAGE protein (b), RAGE mRNA (c), 8-OHdG (d), NADPH oxidase-driven superoxide generation (e), and gene expression levels of components of NADPH oxidase (f). (a,b,d) Each left panel shows representative immunostainings of AGEs (a), RAGE protein (b), and 8-OHdG (d) in the kidneys. Each right panel shows the quantitative data. * and **, p < 0.05 and p < 0.01 compared with 14-week-old non-diabetic rats (Con), respectively. # and ##, p < 0.05 and p < 0.01 compared with 14-week-old diabetic rats (Stz), respectively. AGEs: advanced glycation end products, RAGE: receptor for AGEs, 8-OHdG: 8-hydroxy-2′-deoxyguanosine. Con: N = 6, Con + Apx: N = 5, Stz: N = 5, Stz+Apx: N = 4. Data except for Nox1 mRNA (f) were analyzed by ANOVA with Tukey–Kramer test. Data of Nox1 mRNA (f) were analyzed by ANOVA with Steel–Dwass test.

Figure 2

Effects of apixaban (Apx) on inflammatory reactions in the kidneys of streptozotocin-induced diabetic rats. Levels of MCP-1 protein (a), MCP-1 mRNA (b), VCAM-1 protein (c), VCAM-1 mRNA (d), and Mac-3 protein (e). (a,c,e) Each left panel shows representative immunostainings of MCP-1 protein (a), VCAM-1 protein (c), and Mac-3 (e) in the kidneys. Each right panel shows the quantitative data. # and ##, p < 0.05 and p < 0.01 compared with 14-week-old diabetic rats (Stz), respectively. Con: 14-week-old non-diabetic rats. MCP-1: monocyte chemoattractant protein-1, VCAM-1: vascular cell adhesion molecule-1. Con: N = 6, Con + Apx: N = 5, Stz: N = 5, Stz + Apx: N = 4. Data except for MCP-1 mRNA (b) and VCAM-1 mRNA (d) were analyzed by ANOVA with Tukey–Kramer test. Data of MCP-1 mRNA (b) and VCAM-1 mRNA (d) were analyzed by ANOVA with Steel–Dwass test.

Figure 2

Effects of apixaban (Apx) on inflammatory reactions in the kidneys of streptozotocin-induced diabetic rats. Levels of MCP-1 protein (a), MCP-1 mRNA (b), VCAM-1 protein (c), VCAM-1 mRNA (d), and Mac-3 protein (e). (a,c,e) Each left panel shows representative immunostainings of MCP-1 protein (a), VCAM-1 protein (c), and Mac-3 (e) in the kidneys. Each right panel shows the quantitative data. # and ##, p < 0.05 and p < 0.01 compared with 14-week-old diabetic rats (Stz), respectively. Con: 14-week-old non-diabetic rats. MCP-1: monocyte chemoattractant protein-1, VCAM-1: vascular cell adhesion molecule-1. Con: N = 6, Con + Apx: N = 5, Stz: N = 5, Stz + Apx: N = 4. Data except for MCP-1 mRNA (b) and VCAM-1 mRNA (d) were analyzed by ANOVA with Tukey–Kramer test. Data of MCP-1 mRNA (b) and VCAM-1 mRNA (d) were analyzed by ANOVA with Steel–Dwass test.

Figure 3

Effects of apixaban (Apx) on fibrotic reactions in the kidneys of streptozotocin-induced diabetic rats. Levels of TGF-β protein (a), TGF-β mRNA (b), CTGF protein (c), CTGF mRNA (d), fibronectin protein (e), fibronectin mRNA (f), glomerular extracellular matrix accumulation (g), and KIM-1 protein (h). (a,c,e,g,h) Each left panel shows representative immunostainings of TGF-β protein (a), CTGF protein (c), fibronectin protein (e), and KIM-1 protein (h), and glomerular extracellular matrix accumulation (g) in the kidneys. (g) Masson’s trichrome-stained sections. Each right panel shows the quantitative data. Correlation of urinary excretion levels of KIM-1 with kidney AGEs (i), kidney 8-OHdG (j), and serum 8-OHdG levels (k). # and ##, p < 0.05 and p < 0.01 compared with 14-week-old diabetic rats (Stz), respectively. Con: 14-week-old non-diabetic rats, TGF-β: transforming growth factor-β, CTGF: connective tissue growth factor, ECM: extracellular matrix, KIM-1: kidney injury molecule-1, AGEs: advanced glycation end products, 8-OHdG: 8-hydroxy-2′-deoxyguanosine. Con: N = 6, Con + Apx: N = 5, Stz: N = 5, Stz + Apx: N = 4. Data (a–h) were analyzed by ANOVA with Tukey–Kramer test. Data (i–k) were analyzed by Pearson’s correlation test.

Figure 3

Effects of apixaban (Apx) on fibrotic reactions in the kidneys of streptozotocin-induced diabetic rats. Levels of TGF-β protein (a), TGF-β mRNA (b), CTGF protein (c), CTGF mRNA (d), fibronectin protein (e), fibronectin mRNA (f), glomerular extracellular matrix accumulation (g), and KIM-1 protein (h). (a,c,e,g,h) Each left panel shows representative immunostainings of TGF-β protein (a), CTGF protein (c), fibronectin protein (e), and KIM-1 protein (h), and glomerular extracellular matrix accumulation (g) in the kidneys. (g) Masson’s trichrome-stained sections. Each right panel shows the quantitative data. Correlation of urinary excretion levels of KIM-1 with kidney AGEs (i), kidney 8-OHdG (j), and serum 8-OHdG levels (k). # and ##, p < 0.05 and p < 0.01 compared with 14-week-old diabetic rats (Stz), respectively. Con: 14-week-old non-diabetic rats, TGF-β: transforming growth factor-β, CTGF: connective tissue growth factor, ECM: extracellular matrix, KIM-1: kidney injury molecule-1, AGEs: advanced glycation end products, 8-OHdG: 8-hydroxy-2′-deoxyguanosine. Con: N = 6, Con + Apx: N = 5, Stz: N = 5, Stz + Apx: N = 4. Data (a–h) were analyzed by ANOVA with Tukey–Kramer test. Data (i–k) were analyzed by Pearson’s correlation test.

Figure 4

Effects of apixaban (Apx) on PAR-1 and PAR-2 protein and mRNA levels in the kidneys of streptozotocin-induced diabetic rats. Levels of PAR-1 protein (a), PAR-1 mRNA (b), PAR-2 protein (c), and PAR-2 mRNA (d). (a,c) Each left panel shows representative immunostainings of PAR-1 protein (a) and PAR-2 protein (c) in the kidneys. Each right panel shows the quantitative data. # and ##, p < 0.05 and p < 0.01 compared with 14-week-old diabetic rats (Stz), respectively. Con: 14-week-old non-diabetic rats, PAR-1: protease-activated receptor-1, PAR-2: protease-activated receptor-2. Con: N = 6, Con + Apx: N = 5, Stz: N = 5, Stz + Apx: N = 4. Data were analyzed by ANOVA with Tukey–Kramer test.