Establishment of a diabetic mouse model with progressive diabetic nephropathy

Abstract

Although diabetic animal models exist, no single animal model develops renal changes identical to those seen in humans. Here we show that transgenic mice that overexpress inducible cAMP early repressor (ICER Igamma) in pancreatic beta cells are a good model to study the pathogenesis of diabetic nephropathy. Although ICER Igamma transgenic mice exhibit extremely high blood glucose levels throughout their lives, they survive long enough to develop diabetic nephropathy. Using this model we followed the progress of diabetic renal changes compared to those seen in humans. By 8 weeks of age, the glomerular filtration rate (GFR) was already increased, and glomerular hypertrophy was prominent. At 20 weeks, GFR reached its peak, and urine albumin excretion rate was elevated. Finally, at 40 weeks, diffuse glomerular sclerotic lesions were prominently accompanied by increased expression of collagen type IV and laminin and reduced expression of matrix metalloproteinase-2. Nodular lesions were absent, but glomerular basement membrane thickening was prominent. At this point, GFR declined and urinary albumin excretion rate increased, causing a nephrotic state with lower serum albumin and higher serum total cholesterol. Thus, similar to human diabetic nephropathy, ICER Igamma transgenic mice exhibit a stable and progressive phenotype of diabetic kidney disease due solely to chronic hyperglycemia without other modulating factors.

Figure 1

ICER Tg mice exhibited sustainable hyperglycemia due to reduced insulin secretion. A: Fed plasma insulin of mice at 1 week and 12 weeks of age was determined by enzyme-linked immunosorbent assay kit. B: Fed blood glucose levels at indicated weeks were determined by an enzyme-electrode method. Results are expressed as mean ± SEM. *P < 0.01. N = 6 for each group.

Figure 2

Glomerular hypertrophy in ICER Tg mice. A: Representative glomeruli from 8-, 20-, and 40-week-old WT and Tg mice are shown. Kidneys were fixed in methyl Carnoy’s solution, embedded in paraffin, sectioned, and stained with PAS. B: Glomerular surface area in PASM-stained sections was measured by Image-Pro Plus. For each mouse, 50 glomeruli were analyzed. Data are mean ± SEM. *P < 0.01. N = 6 per each group. Original magnifications, ×200.

Figure 3

Sclerotic lesions in ICER Tg mice. At 40 weeks of age, kidney sections were stained with PASM. Representative glomeruli from WT (A) and Tg (B) mice are shown. C: PASM-positive area was measured by Image-Pro Plus in 50 glomeruli for each mouse. PASM-positive area fraction was calculated by the ratio of PASM-positive area to total glomerular surface area. Data are mean ± SEM. *P < 0.01. N = 6 per each group. Original magnifications, ×400.

Figure 4

GBM thickening in ICER Tg mice. At 40 weeks of age compared to WT (A), Tg (B) mouse glomeruli have thicker GBM and increased mesangial area. C: Thickness of GBM at 40 weeks of age was measured by the orthogonal intercept method. Data are mean ± SEM. *P < 0.01. N = 4 per each group. Original magnifications: ×3000 (A and B, top); ×7000 (A and B, bottom).

Figure 5

The expression of extracellular matrix in glomeruli of ICER Tg mice. Immunohistochemical staining for collagen type IV (A, B) and laminin (C, D) at 40 weeks of age in WT (A, C) and Tg mice (B, D). MMP-2, which is important for degradation of type IV collagen, was also stained. Reduced MMP-2-positive cells at 40 weeks of age in Tg (F) compared to WT mice (E). Scale bar, 25 μm.

Figure 6

Hemodynamic change in ICER Tg mice. A, B: The GFR, calculated as described in Materials and Methods, was increased at 8, 20, and 40 weeks of age in Tg mice. C: Serum creatinine was measured by high performance liquid chromatography. D: Kidney weight/body weight was increased at both 8 and 40 weeks of age in Tg mice. All data are mean ± SEM. *P < 0.01. N = 6 per each group.

Figure 7

Functional change in ICER Tg mice. A: Urinary albumin excretion rate progressively increased with age. B: Serum albumin was decreased in Tg mice at 40 weeks of age. C: Total serum cholesterol was increased at 40 weeks of age. All data are mean ± SEM. *P < 0.01. N = 6 per each group.