Combining streptozotocin and unilateral nephrectomy is an effective method for inducing experimental diabetic nephropathy in the ‘resistant’ C57Bl/6J mouse strain

Figures

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  Figure 1. Albumin and glucose excretion in urine. (A) Albumin excretion in 24 hours urine was measured by ELISA. CSU mice showed increased urinary albumin levels. (B) Glucose was measured by an enzymatic glucose kit. CSU mice showed a large increase in urinary glucose compared to C, and WS and WSU mice had increased urinary glucose excretion compared to WU mice. Data are represented as mean ± SEM. *p < 0.05.

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  Figure 2. Glomerular lesions on PAS-D stainings. (A) Glomerular injury score was calculated by the sum of mesangial proliferation, mesangial matrix expansion and mesangiolysis. (B) Mesangial matrix expansion was scored as absent or present and results are shown as the percentage of glomeruli that showed mesangial matrix expansion. (C) The circumference of glomeruli was determined as a measure for glomerular hypertrophy. CSU mice show increased glomerular injury, mesangial expansion and glomerular hypertrophy. W mice show elevated glomerular hypertrophy when compared to C mice. Representative photographs (magnification 40x) of the PAS-D stainings are shown for C mice (D), CS mice (E), and CSU mice (F). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.0001, #p < 0.0001 in C vs. W comparison.

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  Figure 3. Glomerular collagen expression. (A) Quantification of glomerular collagen by a picrosirius red (PSR) staining. No significant differences were found between the groups, however, CSU and all western diet groups tend to have increased collagen deposition in the glomeruli. (B–D) Representative photographs (magnification 80x) of glomerular PSR-positive area in C, CS, and CSU mice, respectively. (E) Quantification of glomerular immunostaining of collagen IV. CS mice have elevated glomerular collagen IV deposition and also W mice show increased collagen IV compared to C mice. (F–H) Representative photographs (magnification 80x) of collagen IV staining from C, CS, and CSU mice respectively. Data are represented as mean ± SEM. **p < 0.01, #p < 0.05 in C vs. W comparison.

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  Figure 4. Renal mRNA expression. mRNA expression in renal tissue was determined by qPCR. (A) Connective tissue growth factor (cTGF) was upregulated in the CSU mice. (B) Transforming growth factor-β1 (TGF-β1) was upregulated in CS and CSU mice, but also in W mice compared to C mice. (C) Collagen IV was upregulated in CSU mice only. Data are represented as mean ± SEM *p < 0.05, #p < 0.05 in C vs. W comparison.

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  Figure 5. Tubular injury score and cortical collagen expression. (A) Tubular injury was scored based on tubular dilatation and atrophy, and the presence of the brush border. Tubular injury was increased in CSU mice. (B) Cortical collagen expression was determined in picrosirius red (PSR)-stained renal sections. Tubulo-interstitial collagen deposition was elevated in both CSU mice and W mice. (C) Cortical collagen IV was quantified in immunostained sections. CS mice showed increased collagen IV deposition in the tubulo-interstitial area. (D–F) Representative photographs (magnification 40x) of cortical PSR sections in C, CS, and CSU mice, respectively. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, #p < 0.01 in C vs. W comparison.

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  Figure 6. GBM thickness. (A) Glomerular basement membrane (GBM) thickness was measured by electron microscopy. CSU mice showed increased GBM thickness compared to C mice. (B–D) Representative EM photographs (magnification 18500x) of C, CS and CSU mouse kidney, respectively. Data are represented as mean ± SEM and the scale bar shows 2 μm. *p < 0.05, P = podocyte.