Calcium Dobesilate Restores Autophagy by Inhibiting the VEGF/PI3K/AKT/mTOR Signaling Pathway

Figures

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  Figure 1. Calcium dobesilate (CaD) restored hyperglycemia-induced renal histological changes. (A) Body weight and blood glucose measured after 12 weeks of CaD treatment. (B) The measurement of kidney samples of the mice compared to unified size markers. HE, PAS, and Masson staining of kidney samples. (C) The results of MMA, MGV, and Masson staining analysis. (D, E) The immunohistochemical quantification and analysis of nephrin and podocin in kidney samples. Original magnification of 400×. The scale bars in the right lower corner represent 100 µm. The error bars represent the SEM. The arrows indicate deposited glomerular mesangial matrix in the PAS-stained tissue, glomerular and tubulointerstitial fibrosis in Masson-stained tissue, and positive staining in immunohistochemical-stained tissue. HE, hematoxylin/eosin; PAS, periodic acid-Schiff; MGA, mean glomerular area; MGV, mean glomerular volume, MGV = β/K x (MGA)3/2; NK/NC, normal control, n = 15; STZ, high-fat diet- and streptozotocin-treated, n = 15; CaD, calcium dobesilate, n = 15. *p < 0.05 vs. control group; # p < 0.05 vs. DKD (diabetic kidney disease) group.

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  Figure 2. Calcium dobesilate upregulated autophagy in DKD mice. (A) Representative western blot analysis of Beclin 1, Atg5, p62, and LC3 in mice. (B) The densitometric results of Beclin 1, Atg5, P62, and LC3 expression, as determined by western blotting. The error bars represent the SEM. (C, D) The immunohistochemical quantification and analysis of LC3 and p62 in kidney samples. The scale bars in the right lower corner represent 100 µm. The arrows indicate positive staining. NK/NC: normal control, n = 15; STZ, high-fat diet- and streptozotocin-treated, n = 15; CaD: calcium dobesilate, n = 15. *p < 0.05 vs. control group; # p < 0.05 vs. DKD group.

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  Figure 3. Calcium dobesilate decreased VEGF and VEGFR2. (A) Western blots of VEGF for each group. (B) Densitometric results of VEGF as determined by western blot. Error bars represent SEM. (C) Western blots of VEGFR2 for each group. (D) The densitometric results of VEGFR2 expression, as determined by western blot. The error bars represent the SEM. VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2; NK/NC: normal control, n = 15; STZ, high-fat diet- and streptozotocin-treated, n = 15; CaD: calcium dobesilate, n = 15. *p < 0.05 vs. control group; # p < 0.05 vs. DKD group.

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  Figure 4. Calcium dobesilate inhibited the downstream of VEGF/VEGFR2/PI3K/AKT/mTOR signaling pathway. (A) Representative western blots of p-PI3K and t-PI3K. (B) The densitometric results of p-PI3K and t-PI3K expression, as determined by western blot. The error bars represent the SEM. (C) Representative western blots of p-Akt and t-Akt. (D) The densitometric results of p-Akt and t-Akt expression, as determined by western blot. The error bars represent the SEM. (E) Representative western blots of p-mTOR and t-mTOR. (F) The densitometric results of p-mTOR and t-mTOR expression, as determined by western blot. The error bars represent the SEM. p, phosphorylated; t, total; NK/NC: normal control, n = 15; STZ, high-fat diet- and streptozotocin-treated, n = 15; CaD: calcium dobesilate, n = 15. *p < 0.05 vs. control group; # p < 0.05 vs. DKD group.

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  Figure 5. Hypothetical pathway between autophagy and VEGF. A presentation of the interplay between the implicated signaling factors under hyperglycemia and CaD therapy conditions. VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor 2; Tyr-P, phosphorylated tyrosinase; CaD, calcium dobesilate.