Garlic decreases liver and kidney receptor for advanced glycation end products expression in experimental diabetes  Khaled K. Al-Qattan, Mohamed H. Mansour, Martha Thomson, Muslim Ali  Pathophysiology  Volume 23, Issue 2, Pages 135-145 (June 2016) DOI: 10.1016/j.pathophys.2016.02.003 Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 1 GC–MS chromatograms of fresh garlic aqueous extract (A) and garlic aqueous extract stored frozen at −20°C for 8 weeks (B). The IUPAC name of S-allyl cysteine is 2-amino-3-prop-2-enylsulfanyl propanoic acid. The IUPAC names of the 2 vinyldithiins show in the figure are: 1,2 vinyl dithiin: 2-ethenyl-4-H-1,2-dithiin and 1,3 vinyl dithiin: 2-ethenyl-4-H-1,3-dithiin. Pathophysiology 2016 23, 135-145DOI: (10.1016/j.pathophys.2016.02.003) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 2 Immunohistochemical localization of RAGE expression in the renal cortex of normal (A), control diabetic (B) and garlic-treated diabetic (C) rats. Renal sections were treated with the polyclonal anti-RAGE antibody and counter-stained with hematoxylin as detailed in Materials and methods. In (A), positive labeling was detected with mild intensity in glomeruli and was uniformly diffused in the apical side of proximal convoluted tubules epithelia. In (B), RAGE labeling with mild intensity was observed in the epithelial lining of the distal convoluted tubules, but was of high intensity among glomeruli and the endothelial lining of the afferent arteriolar branches of interlobular arteries as well as the inter-tubular branches of efferent arterioles (arrows). In (C), mild/moderate intensity of RAGE labeling was confined to glomeruli and appeared as faded patches in the apical microvilli of the epithelial lining of proximal convoluted tubules. No specific labeling was observed in renal sections labeled with the anti-RAGE antibody preabsorbed with a RAGE peptide/BSA complex (D). Images are representative of four different samples for each rat group. (d): distal convoluted tubule, (g): glomerulus, (p): proximal convoluted tubule. Bar=100μm, ×100. Pathophysiology 2016 23, 135-145DOI: (10.1016/j.pathophys.2016.02.003) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 3 Quantification of the RAGE expressed in the kidney and liver of normal (Nr), control diabetic (D-cont) and garlic-treated diabetic (D-Gar) rats. Tissue sections were quantitatively examined by using the Image-Pro Plus 5.1 software program (Media Cybernetics, Silver Spring, MD) as described in Materials and methods. Values are means, with standard errors (n=4) represented by vertical bars. *p<0.05 vs. Garlic-treated diabetic rats and normal rats. Pathophysiology 2016 23, 135-145DOI: (10.1016/j.pathophys.2016.02.003) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 4 RAGE labeling patterns among glomeruli. (A) Normal glomerulus with mild RAGE labeling confined to mesangial cells. (B) Nodular glomerulus showing RAGE labeling of moderate intensity within the expanded mesangium. Poorly-organized glomeruli with either segmental (C) or global (D) glomerulosclerosis showing RAGE labeling of high intensity within the extensive mesangium. Sections were counter-stained with hematoxylin. Images are representative of four different samples for each rat group. (Bc): Bowman’s capsule, (Bs): Bowman’s space, (bm): capillary endothelial basement membrane, (c): capillary lumen, (m): mesangial cell, (p): podocyte. Magnification, x400. Pathophysiology 2016 23, 135-145DOI: (10.1016/j.pathophys.2016.02.003) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 5 Comparison of the percentage of glomeruli labeled with different intensities with the polyclonal anti-RAGE antibody in the kidney of normal (Nr), control diabetic (D-cont) and garlic-treated diabetic (D-Gar) rats. (L. intensity): mild intensity, (M. intensity): moderate intensity, (H. intensity): high intensity. Values are means, with standard errors (n=4) represented by vertical bars. *p<0.05 vs. Garlic-treated diabetic rats and normal rats. Pathophysiology 2016 23, 135-145DOI: (10.1016/j.pathophys.2016.02.003) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 6 Immunohistochemical localization of RAGE expression in the liver of normal (A), control diabetic (B) and garlic-treated diabetic (C) rats. Liver sections were treated with the polyclonal anti-RAGE antibody and counter-stained with hematoxylin. In both (A) and (C), mild RAGE labeling was observed among hepatocytes arranged around the central vein (V) within hepatic lobules. In (B), RAGE labeling with higher intensity was observed among hepatocytes as well as bile ducts and vessels in portal tracts (arrows). No specific labeling was observed in liver sections labeled with the anti-RAGE antibody preabsorbed with a RAGE peptide/BSA complex (D). Images are representative of four different samples for each rat group. Bar=100μm, X100. Pathophysiology 2016 23, 135-145DOI: (10.1016/j.pathophys.2016.02.003) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 7 Two-dimensional SDS-PAGE analysis of RAGE isoforms expressed in the kidney of normal (A), control diabetic (B) and garlic-treated diabetic (C) rats. Kidney proteins were individually solubilized and lysates of equal protein content (120μg) resolved by 2D-SDS-PAGE, Western blotted and probed by polyclonal anti-RAGE antibody at a dilution of 1:500. Positions of 2D-SDS-PAGE isoelectric points as well as molecular weight (Mr×10−3) standards are shown. Estimated molecular weights of RAGE isoforms are indicated by arrowheads. Pathophysiology 2016 23, 135-145DOI: (10.1016/j.pathophys.2016.02.003) Copyright © 2016 Elsevier B.V. Terms and Conditions

Fig. 8 Two-dimensional SDS-PAGE analysis of RAGE isoforms expressed in the liver of normal (A), control diabetic (B) and garlic-treated diabetic (C) rats. Liver proteins were individually solubilized and lysates of equal protein content (120μg) resolved by 2D-SDS-PAGE, Western blotted and probed by polyclonal anti-RAGE antibody at a dilution of 1:500. Positions of 2D-SDS-PAGE isoelectric points as well as molecular weight (Mr×10−3) standards are shown. Estimated molecular weights of RAGE isoforms are indicated by arrowheads. Pathophysiology 2016 23, 135-145DOI: (10.1016/j.pathophys.2016.02.003) Copyright © 2016 Elsevier B.V. Terms and Conditions