Deletion of the receptor for advanced glycation end products reduces glomerulosclerosis and preserves renal function in the diabetic OVE26 mouse

Abstract

Objective: Previous studies showed that genetic deletion or pharmacological blockade of the receptor for advanced glycation end products (RAGE) prevents the early structural changes in the glomerulus associated with diabetic nephropathy. To overcome limitations of mouse models that lack the progressive glomerulosclerosis observed in humans, we studied the contribution of RAGE to diabetic nephropathy in the OVE26 type 1 mouse, a model of progressive glomerulosclerosis and decline of renal function.

Research design and methods: We bred OVE26 mice with homozygous RAGE knockout (RKO) mice and examined structural changes associated with diabetic nephropathy and used inulin clearance studies and albumin:creatinine measurements to assess renal function. Transcriptional changes in the Tgf-beta1 and plasminogen activator inhibitor 1 gene products were measured to investigate mechanisms underlying accumulation of mesangial matrix in OVE26 mice.

Results: Deletion of RAGE in OVE26 mice reduced nephromegaly, mesangial sclerosis, cast formation, glomerular basement membrane thickening, podocyte effacement, and albuminuria. The significant 29% reduction in glomerular filtration rate observed in OVE26 mice was completely prevented by deletion of RAGE. Increased transcription of the genes for plasminogen activator inhibitor 1, Tgf-beta1, Tgf-beta-induced, and alpha1-(IV) collagen observed in OVE26 renal cortex was significantly reduced in OVE26 RKO kidney cortex. ROCK1 activity was significantly lower in OVE26 RKO compared with OVE26 kidney cortex.

Conclusions: These data provide compelling evidence for critical roles for RAGE in the pathogenesis of diabetic nephropathy and suggest that strategies targeting RAGE in long-term diabetes may prevent loss of renal function.

FIG. 1.

A: RAGE is expressed in OVE26 glomeruli: age 1 month. Original magnification: 1,000×. B: Blood glucose levels (###P < 0.0001) (n = 12–15/group, except OVE26 RKO, n = 6). C: A1C levels were measured at age 7 months on randomly selected mice (n = 4, 3, 9, and 6) used in the GFR studies in groups FVB, FVB RKO, OVE26, and OVE26 RKO, respectively (##P < 0.0005, ###P < 0.0001). D: Levels of methylglyoxal (MG) and in the kidney cortex of FVB, FVB RKO, OVE26, and OVE26 RKO mice at age 7 months (*P < 0.05). n = 6 per group. E: Real-time PCR for glyoxalase 1 was performed, normalized to 18s transcript levels, and expressed as fold-change compared with the OVE26 group (*P < 0.05). n = 6 per group. F: Glyoxalase 1 protein levels measured by Western blot, normalized to actin levels, and expressed as fold-change compared with the OVE26 group (*P < 0.05) (n = 3 per group). G: Glyoxalase 1 staining in OVE26 and OVE26 RKO glomeruli: age 7 months. Original magnification: 1,000×. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 2.

Histology and ultrastructural pathology in diabetic OVE mice: age 7 months. By light microscopy, the glomeruli of 7-month-old male OVE26 mice display global mesangial sclerosis with nodularity (A), with progression in some glomeruli to segmental glomerulosclerosis (C) and global glomerulosclerosis (D). There is focal tubular atrophy, interstitial fibrosis, and chronic inflammation (B). By electron microscopy, the mesangial areas are expanded by increased matrix and electron dense hyaline material, consistent with insuded plasma proteins (E). There is thickening of glomerular basement membranes with overlying effacement of foot processes (F). Some mesangial areas have marked mesangial sclerosis with a nodular aspect (G). In areas of severe mesangial sclerosis, the glomerular capillary lumina are narrowed and focally occluded by the mesangial encroachment (H, L = lumen) (arrow indicates focal lumenal occlusion). Original magnifications are marked above each image. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Deletion of RAGE in OVE26 mice imparts partial protection from the structural abnormalities of diabetic nephropathy at age 7 months. A: No histologic abnormalities were detected in FVB RKO mice (not shown). By contrast, OVE26 mice display well-developed features of diabetic nephropathy including diffuse and global mesangial sclerosis and focal hyaline casts (B). OVE26 RKO mice are markedly protected from the development of mesangial sclerosis and tubular cast formation (C). There were significant differences between OVE26 mice and OVE26 RKO mice with respect to percent cortical area occupied by casts (D) and the severity of mesangial sclerosis (E), where 0 = no mesangial sclerosis; 1 = mild; 2 = moderate; 3 = severe (*P < 0.05). Original magnifications are marked above each image. Semi-quantitative scoring (D and E) was performed on n = 7 OVE26 RKO and n = 13 OVE26 mice. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Deletion of RAGE in OVE26 mice imparts partial protection from the ultrastructural abnormalities of diabetic nephropathy at age 7 months. Electron microscopy was performed on 7-month-old male kidney cortex samples. FVB mice display normal glomerular ultrastructural features with well-preserved foot processes and glomerular basement membranes of normal thickness (A). By comparison, OVE26 glomeruli display increased mesangial matrix forming focal nodules, thickened glomerular basement membranes, and prominent foot process effacement (B). OVE26 RKO mice were partially protected from these changes, leading to less mesangial sclerosis and partial restoration of foot processes (C). (D). Semi-quantitative scoring of podocyte effacement (n = 5) (E) Measurements of GBM thickness (n = 5) (***P < 0.005, ###P < 0.0001). Original magnifications are marked above each image.

FIG. 5.

Deletion of RAGE in OVE26 mice imparts partial protection from functional abnormalities of diabetic nephropathy at age 7 months. Albumin-to-creatinine levels were measured in male FVB, FVB RKO, OVE26, and OVE26 RKO mouse urine retrieved from metabolic cages at 7 months of age (***P < 0.005, ###P < 0.0001). n = 7–13 per group.

FIG. 6.

Deletion of RAGE in OVE26 mice preserves GFR in OVE26 mice at age 7 months. Body weight and glomerular function at 7 months are shown. A: Weights of both kidneys from 7-month-old male mice. B: Ratios of kidney weight to body weight of 7-month-old mice. C–E: Clearance of inulin (CIN) expressed as CIN per mouse (C), CIN per 100 g body weight (D), and CIN per g kidney weight (E) (**P < 0.01, ***P < 0.005, ##P < 0.0005, ###P < 0.0001). n = 6–10 per group, as indicated in Table 1.

FIG. 7.

PAI-1 (Serpine1), Tgf-β1, Tgf-β–induced, and α1-(IV) collagen mRNA transcripts and ROCK1 activity are lower in OVE26 RKO kidney cortex than in OVE26 kidney cortex levels at age 7 months. Real-time PCR for PAI-1 (A), Tgf-β1 (B), Tgf-β1–induced (C), and α1-(IV) collagen (D) gene products was performed, normalized to 18s transcript levels, and expressed as fold-change compared with the FVB or OVE26 group (**P < 0.01, ***P < 0.005, ##P < 0.0005, ###P < 0.0001). n = 6 per group. E: ROCK1 activity was measured as the amount of phosphorylated MYPT1 compared with total ROCK1 levels by Western blot. Relative activity is expressed as fold-change compared with the OVE26 group (***P < 0.005) (n = 3 per group).