A new, easily generated mouse model of diabetic kidney fibrosis

Figures

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  Figure 1. Akita+/− Ren+/− mice develop significant hyperglycemia, hypertension, and albuminuria over the course of 26 weeks. (a) Capillary blood glucose levels and (b) systolic blood pressure were measured monthly, beginning at 6 weeks of age. Urinary albumin excretion was measured just prior to sacrifice at 26 weeks of age as (c) daily urine albumin excretion rate and (d) a spot urinary albumin:creatinine ratio. A one-way ANOVA with a post-hoc Fisher’s least significant difference analysis was performed. *p < 0.05.

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  Figure 2. Akita+/− Ren+/− mice develop significant glomerular injury. (a) Kidney sections were stained with Periodic Acid-Schiff stain, and glomerular area was quantified. (b) Kidney sections were stained with picrosirius red to identify fibrillar collagen, and the degree of glomerulosclerosis was quantified. A one-way ANOVA with a post-hoc Fisher’s least significant difference analysis was performed. *p < 0.05. Scale bar: 40 μm. Abbreviations: AU, arbitrary units.

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  Figure 3. Interstitial myofibroblasts are increased in Akita+/− Ren+/− mice. Kidney sections were stained with antibodies specific either to: (a) alpha-smooth muscle actin (alpha-SMA) or (b) vimentin and digital quantification of antibody staining was performed. Representative images and quantification using both stains are shown. A one-way ANOVA with a post-hoc Fisher’s least significant difference analysis was performed. *p < 0.05. Scale bar: 40 μm. Abbreviations: AU, arbitrary units.

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  Figure 4. Akita+/− Ren+/− mice exhibit significant interstitial fibrosis. Kidney sections were stained with (a) picrosirius red to identify fibrillar collagen or (b) Masson’s Trichrome. Representative images and quantification of interstitial fibrosis using both stains are shown. A one-way ANOVA with a post-hoc Fisher’s least significant difference analysis was performed. *p < 0.05. Scale bar: 20 μm. Abbreviations: AU, arbitrary units.

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  Figure 5. Collagen deposition is increased in Akita+/− Ren+/− mice. Kidney sections were stained with antibodies targeting (a) type I collagen or (b) type III collagen and digital quantification of antibody staining was performed. A one-way ANOVA with a post-hoc Fisher’s least significant difference analysis was performed. *p < 0.05. Scale bar: 20 μm. Abbreviations: AU, arbitrary units.

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  Figure 6. The expression of fibrosis-associated genes is increased in Akita+/− Ren+/− mice. (a) CTGF levels. (b) VIM (vimentin) levels. (c) COL1A1 levels. (d) COL3A1 levels. Levels of each transcript are presented relative to GAPDH. A one-way ANOVA with a post-hoc Fisher’s least significant difference analysis was performed. *p < 0.05. Abbreviations: AU, arbitrary units.

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  Figure 7. Akita+/− Ren+/− kidneys exhibit higher levels and nuclear localization of the pro-fibrotic proteins YAP and TAZ. (a) Equal amounts of kidney lysates (20 μg/lysate/membrane) were separated by SDS-PAGE and transferred on to two different membranes, with one membrane immunoblotted with an antibody directed against YAP (to detect total YAP), and a paired membrane immunoblotted with an antibody directed against YAP and TAZ (to detect total TAZ). Each of these membranes was then stripped and probed with an antibody directed against GAPDH. (b) Kidney sections were stained with an antibody targeting both YAP and TAZ, and counterstained with hematoxylin. Representative images are shown. The percentage of (c) interstitial cells and (d) tubular epithelial cells with primarily nuclear YAP/TAZ staining was quantified. Black arrows depict examples of interstitial cells with primarily nuclear YAP/TAZ staining. A one-way ANOVA with a post-hoc Fisher’s least significant difference analysis was performed. *p < 0.05. Scale bar: 20 μm.