Amelioration of Diabetic Mouse Nephropathy by Catalpol Correlates with Down-Regulation of Grb10 Expression and Activation of Insulin-Like Growth Factor 1 / Insulin-Like Growth Factor 1 Receptor Signaling

Abstract

Growth factor receptor-bound protein 10 (Grb10) is an adaptor protein that can negatively regulate the insulin-like growth factor 1 receptor (IGF-1R). The IGF1-1R pathway is critical for cell growth and apoptosis and has been implicated in kidney diseases; however, it is still unknown whether Grb10 expression is up-regulated and plays a role in diabetic nephropathy. Catalpol, a major active ingredient of a traditional Chinese medicine, Rehmannia, has been reported to possess anti-inflammatory and anti-aging activities and then used to treat diabetes. Herein, we aimed to assess the therapeutic effect of catalpol on a mouse model diabetic nephropathy and the potential role of Grb10 in the pathogenesis of this diabetes-associated complication. Our results showed that catalpol treatment improved diabetes-associated impaired renal functions and ameliorated pathological changes in kidneys of diabetic mice. We also found that Grb10 expression was significantly elevated in kidneys of diabetic mice as compared with that in non-diabetic mice, while treatment with catalpol significantly abrogated the elevated Grb10 expression in diabetic kidneys. On the contrary, IGF-1 mRNA levels and IGF-1R phosphorylation were significantly higher in kidneys of catalpol-treated diabetic mice than those in non-treated diabetic mice. Our results suggest that elevated Grb10 expression may play an important role in the pathogenesis of diabetic nephropathy through suppressing IGF-1/IGF-1R signaling pathway, which might be a potential molecular target of catalpol for the treatment of this diabetic complication.

Fig 1. Quantitative real-time PCR analysis of Grb10 and IGF-1 mRNA expressions in kidneys.

The levels of Growth factor receptor bound protein 10(Grb10) (A) and insulin-like growth factor 1(IGF- 1) (B) mRNA expressions were measured using qPCR. The average levels were shown in the graphs. The data were shown as the mean ± SEM (A, n = 3; B, n = 5). (*, P < 0.05; **, p < 0.01). Abbreviations: Con: normal control group; DM: diabetes mellitus group; DM + Cat: diabetes mellitus treated with catalpol group.

Fig 2. The expression levels of Grb10 and IGF-1R proteins in kidneys.

The levels of Growth factor receptor bound protein 10 (Grb10) (A) and Insulin-like growth factor 1 receptor (IGF- 1R) (B) proteins were measured by Western Blotting. Statistical analysis was done using factorial ANOVA with Fisher’s multiple comparisons post (*, P < 0.05; **, P < 0.01). The data were shown as the mean ± SEM (A, n = 3; B, n = 5). Abbreviations: Con: normal control group; DM: diabetes mellitus group; DM + Cat: diabetes mellitus treated with catalpol group.

Fig 3. Periodic acid-Schiff staining of kidney tissues in each group.

The kidney tissues were stained with Periodic acid-Schiff. Cells with a positive reaction were stained purple (magnification, ×400). In picture B, fuchsia positive cells were more than that of the other two groups. A (Con): normal control group; B (DM): diabetes mellitus group; C (DM + Cat): diabetes mellitus treated with catalpol group.

Fig 4. Masson staining of kidney tissues in each group.

The topically microscopic images of the kidney tissues were stained with ponceau and aniline blue dye. Positive reaction is represented by blue staining (magnification, x400). In picture B, blue stained areas were more than that of the other two groups, indicating more severe fibrosis. A (Con): normal control group; B (DM): diabetes mellitus group; C (DM + Cat): diabetes mellitus treated with catalpol group.

Fig 5. Immunohistochemical study on the expression of Grb10 and caspase-3 proteins in kidneys.

The distribution and expression levels of growth factor receptor-bound protein 10 (Grb10) (A) and caspase-3 (B) were determined by immunohistochemical staining. Images shown at magnifications of ×400. Brown granules represent positive results. Average optical density values were measured by Image-Pro Plus, version 6.0, and expressed as the mean ± SEM (n = 4) of two independent experiments. Statistical analysis was performed using factorial analysis of variance with Fisher’s multiple comparisons. (*, p < 0.05; **, p < 0.01). Con: normal control group; DM: diabetes mellitus group; DM + Cat: diabetes mellitus treated with catalpol group.