Alagebrium reduces glomerular fibrogenesis and inflammation beyond preventing RAGE activation in diabetic apolipoprotein E knockout mice

Abstract

Advanced glycation end products (AGEs) are important mediators of diabetic nephropathy that act through the receptor for AGEs (RAGE), as well as other mechanisms, to promote renal inflammation and glomerulosclerosis. The relative contribution of RAGE-dependent and RAGE-independent signaling pathways has not been previously studied in vivo. In this study, diabetic RAGE apoE double-knockout (KO) mice with streptozotocin-induced diabetes were treated with the AGE inhibitor, alagebrium (1 mg/kg/day), or the ACE inhibitor, quinapril (30 mg/kg/day), for 20 weeks, and renal parameters were assessed. RAGE deletion attenuated mesangial expansion, glomerular matrix accumulation, and renal oxidative stress associated with 20 weeks of diabetes. By contrast, inflammation and AGE accumulation associated with diabetes was not prevented. However, treatment with alagebrium in diabetic RAGE apoE KO mice reduced renal AGE levels and further reduced glomerular matrix accumulation. In addition, even in the absence of RAGE expression, alagebrium attenuated cortical inflammation, as denoted by the reduced expression of monocyte chemoattractant protein-1, intracellular adhesion molecule-1, and the macrophage marker cluster of differentiation molecule 11b. These novel findings confirm the presence of important RAGE-independent as well as RAGE-dependent signaling pathways that may be activated in the kidney by AGEs. This has important implications for the design of optimal therapeutic strategies for the prevention of diabetic nephropathy.

FIG. 1.

PAS staining in control (C) and diabetic (D) apoE KO and RAGE apoE double-KO mice, with and without treatment: apo E KO (A); RAGE apoE KO (B); diabetic apoE KO (C); diabetic RAGE apoE KO (D); RAGE apoE KO + alagebrium (A) 1 mg/kg/day (E); and diabetic RAGE apoE KO + quinapril (Q) 30 mg/kg/day (F). Scale bar in a = 100 μm. Digital quantification of mesangial area (G) for n = 6–8 per group. *P < 0.05 vs. apoE KO. §P < 0.05 vs. diabetic apoE KO. (A high-quality color representation of this figure is available in the online issue.)

FIG. 2.

Immunostaining for collagen IV (a–f) and fibronectin (h–m) in nondiabetic control (C) and diabetic (D) apoE KO and RAGE apoE double-KO mice, with and without treatment. ApoE C (a, h); RAGE apoE KO (b, i); apoE D (c, j); diabetic RAGE apoE KO (d, k); diabetic RAGE apoE KO alagebrium (A) 1 mg/kg/day (e, l); diabetic RAGE/apoE KO quinapril (Q) 30 mg/kg/day (f, m). Scale bar = 20 μm. Digital quantification of glomerular staining for collagen IV (g) and fibronectin (n) for n = 6–10 per group. *P < 0.05 vs. apoE KO. §P < 0.05 vs. diabetic apoE. ¶P < 0.05 vs. RAGE apoE KO. #P < 0.05 diabetic RAGE apoE KO vs. diabetic RAGE apoE KO + quinapril. †P < 0.05 diabetic RAGE apoE KO vs. diabetic RAGE apoE KO + alagebrium. (A high-quality color representation of this figure is available in the online issue.)

FIG. 3.

Gene and protein expression of inflammation markers CD11b and ICAM-1 in glomeruli from nondiabetic control (C) and diabetic (D) apoE KO and RAGE apoE double-KO mice, with and without treatment, as measured by RT-PCR and ELISA. Gene expression of CD11b (A), relative protein content of CD11b (B), gene expression of ICAM-1 (C), and relative protein expression of ICAM-1 (D) for n = 6–10 per group. *P < 0.05 vs. apoE KO. §P < 0.05 vs. diabetic apoE. ¶P < 0.05 vs. RAGE apoE KO. #P < 0.05 diabetic RAGE apoE KO vs. diabetic RAGE apoE KO + quinapril. †P < 0.05 diabetic RAGE apoE KO vs. diabetic RAGE apoE KO + alagebrium.

FIG. 4.

Immunostaining for MCP-1 in nondiabetic control (C) and diabetic (D) apoE KO and RAGE apoE double-KO mice, with and without treatment. ApoE KO (A); RAGE/apoE KO (B); diabetic apoE KO (C); diabetic RAGE apoE KO (D); diabetic RAGE apoE KO + alagebrium (A) 1 mg/kg/day (E); diabetic RAGE apoE KO + quinapril (Q) 30 mg/kg/day (F). Renal cortical MCP-1 expression by RT-PCR (G) and ELISA (H) for n = 6–8 per group. Scale bar = 20 μm. *P < 0.05 vs. apoE KO. §P < 0.05 vs. diabetic apoE KO. ¶P < 0.05 vs. RAGE apoE KO. #P < 0.05 diabetic RAGE apoE KO vs. diabetic RAGE apoE KO + quinapril. †P < 0.05 diabetic RAGE apoE KO vs. diabetic RAGE apoE KO+ alagebrium. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

Immunostaining for the marker of oxidative stress, nitrotyrosine, in nondiabetic control (C) and diabetic (D) apoE KO and RAGE apoE double-KO mice, with and without treatment. ApoE KO (a); RAGE apoE KO (b); diabetic apoE KO (c); diabetic RAGE apoE KO (d); diabetic RAGE apoE KO + alagebrium (A) 1 mg/kg/day (e); diabetic RAGE apoE KO + quinapril (Q) 30 mg/kg/day (f). Digital quantification (g) for n = 6–10 per group. *P < 0.05 vs. apoE KO. §P < 0.05 vs. diabetic apoE KO. ¶P < 0.05 vs. RAGE apoE KO. (A high-quality color representation of this figure is available in the online issue.)

FIG. 6.

Immunostaining for AGEs in nondiabetic control (C) and diabetic (D) apoE KO and RAGE apoE double-KO mice, with and without treatment. ApoE KO (a); RAGE apoE KO (b); diabetic apoE KO (c); diabetic RAGE apoE KO (d); diabetic RAGE apoE KO + alagebrium (A) (1 mg/kg/day) (e); diabetic RAGE apoE KO + quinapril (Q) (30 mg/kg/day) (f). Renal cortical AGE content by ELISA (g) using the same antibody as used for immunohistochemistry for n = 5–6 per group. Data are presented as the geometric mean ± tolerance factor. P < 0.05 (n = 6–10 per group). *P < 0.05 vs. apoE KO. §P < 0.05 vs. diabetic apoE KO. ¶P < 0.05 vs. RAGE apoE KO. #P < 0.05 diabetic RAGE apoE KO vs. diabetic RAGE apoE KO + quinapril. †P < 0.05 diabetic RAGE apoE KO vs. diabetic RAGE apoE KO+ alagebrium. (A high-quality color representation of this figure is available in the online issue.)