Effects of Allicin on Pathophysiological Mechanisms during the Progression of Nephropathy Associated to Diabetes

Abstract

This study aimed to assess the impact of allicin on the course of diabetic nephropathy. Study groups included control, diabetes, and diabetes-treated rats. Allicin treatment (16 mg/kg day/p.o.) started after 1 month of diabetes onset and was administered for 30 days. In the diabetes group, the systolic blood pressure (SBP) increased, also, the oxidative stress and hypoxia in the kidney cortex were evidenced by alterations in the total antioxidant capacity as well as the expression of nuclear factor (erythroid-derived 2)-like 2/Kelch ECH associating protein 1 (Nrf2/Keap1), hypoxia-inducible factor 1-alpha (HIF-1α), vascular endothelial growth factor (VEGF), erythropoietin (Epo) and its receptor (Epo-R). Moreover, diabetes increased nephrin, and kidney injury molecule-1 (KIM-1) expression that correlated with mesangial matrix, the fibrosis index and with the expression of connective tissue growth factor (CTGF), transforming growth factor-β1 (TGF-β1), and α-smooth muscle actin (α-SMA). The insulin levels and glucose transporter protein type-4 (GLUT4) expression were decreased; otherwise, insulin receptor substrates 1 and 2 (IRS-1 and IRS-2) expression was increased. Allicin increased Nrf2 expression and decreased SBP, Keap1, HIF-1α, and VEGF expression. Concurrently, nephrin, KIM-1, the mesangial matrix, fibrosis index, and the fibrotic proteins were decreased. Additionally, allicin decreased hyperglycemia, improved insulin levels, and prevented changes in (GLUT4) and IRSs expression induced by diabetes. In conclusion, our results demonstrate that allicin has the potential to help in the treatment of diabetic nephropathy. The cellular mechanisms underlying its effects mainly rely on the regulation of antioxidant, antifibrotic, and antidiabetic mechanisms, which can contribute towards delay in the progression of renal disease.

Keywords: allicin; diabetes; fibrosis; hypoxia; nephropathy; oxidative stress.

Figure 1

Experimental design to assess the effects of allicin on progression of diabetic nephropathy. After one month of follow-up, the treatments were started and given for one month.

Figure 2

Effects of allicin on systolic blood pressure. Ctrl: control; DM: diabetic; DA: diabetic treated with allicin. mmHg; millimeters of mercury. Values represent mean ± SEM of at least 6 animals from each experimental group. * p < 0.05 vs. Ctrl; ⁺ p < 0.05 vs. DM; ^α p < 0.05 vs. DM day 30.

Figure 3

Effects of allicin on glomerular and tubular markers of kidney injury in cortex. (a) Nephrin and (b) kidney injury molecule-1 (KIM-1). Ctrl: control; DM: diabetes; DA: diabetes treated with allicin; a.u: arbitrary units. For representative Western blotting, 3 randomly selected samples per group were analyzed. Results are presented as mean ± SEM and analyzed by one-way ANOVA. Statistical significance was established as * p < 0.05 vs. Ctrl, ⁺ p < 0.05 vs. DM.

Figure 4

Mesangial matrix and quantitative analysis of rat kidneys. Representative photomicrographs of one kidney cortex section of each experimental group. Control (Ctrl) with no changes in glomeruli mesangium seen in light purple (arrows). Diabetes (DM) illustrates expansion of the mesangium (arrows), which is thicker and denser than the one of control group. Diabetes + Allicin (DA) depicts a mesangium (arrows) with characteristics between the control and the DM groups. Periodic acid-Schiff (PAS stain), 200× original magnification. Graph shows quantitative analysis of mesangial matrix index. Values are expressed as mean ± SEM of n = 5 * p < 0.001 vs. Ctrl; ⁺ p < 0.001 vs. DM.

Figure 5

Interstitial fibrosis and quantitative analysis of rat kidneys. Sirius Red for collagen staining (fibrosis) in red of kidney cortex sections, representative of each experimental group. Control (Ctrl) shows very thin red lines (arrows) separating the cortex tubules in a normal interstitium. Diabetes (DM) depicts a clear modest widening of the interstitium (arrows) in comparison with the control. Diabetes + Allicin (DA) photomicrograph allows the visualization of a near normal interstitium, with a few areas of a thicker space between tubules (arrows). Original magnification 200×. Graph shows quantitative analysis of interstitial fibrosis. Values are expressed as mean ± SEM of n = 5 * p < 0.001 vs. Ctrl; ⁺ p < 0.001 vs. DM.

Figure 5

Interstitial fibrosis and quantitative analysis of rat kidneys. Sirius Red for collagen staining (fibrosis) in red of kidney cortex sections, representative of each experimental group. Control (Ctrl) shows very thin red lines (arrows) separating the cortex tubules in a normal interstitium. Diabetes (DM) depicts a clear modest widening of the interstitium (arrows) in comparison with the control. Diabetes + Allicin (DA) photomicrograph allows the visualization of a near normal interstitium, with a few areas of a thicker space between tubules (arrows). Original magnification 200×. Graph shows quantitative analysis of interstitial fibrosis. Values are expressed as mean ± SEM of n = 5 * p < 0.001 vs. Ctrl; ⁺ p < 0.001 vs. DM.

Figure 6

Effects of allicin on fibrotic markers in kidney. (a) Connective tissue growth factor (CTGF), (b) transforming growth factor-β1 (TGF-β1) and (c) alpha-smooth muscle actin (α-SMA). Ctrl: control; DM: diabetes; DA: diabetes treated with allicin; a.u: arbitrary units. For representative Western blotting, 3 randomly selected samples per group were analyzed. Results are presented as mean ± SEM and analyzed by one-way ANOVA. Statistical significance was established as * p < 0.05 vs. Ctrl, ⁺ p < 0.05 vs. DM.

Figure 7

Evaluation of oxidative stress. (a) Total antioxidant status in plasma, (b) nuclear factor erythroid 2-related factor 2 (Nrf2) expression in kidney cortex, and (c) kelch-like ECH associated-protein 1 (Keap1) expression in kidney cortex. Ctrl: control; DM: diabetes; DA: diabetes treated with allicin; a.u: arbitrary units. For representative Western blotting, 3 randomly selected samples per group were analyzed. Results are presented as mean ± SEM and analyzed by one-way ANOVA. Statistical significance was established as * p < 0.05 vs. Ctrl, ⁺ p < 0.05 vs. DM.

Figure 8

Effects of allicin on hypoxic factors induced by oxidative stress. (a) Hypoxia-inducible factor 1-alpha (HIF-1α), (b) vascular endothelial growth factor (VEGF), (c) erythropoietin (Epo), and (d) erythropoietin receptor (Epo-R). Ctrl: control; DM: diabetes; DA: diabetes treated with allicin; a.u: arbitrary units. For representative Western blotting, 3 randomly selected samples per group were analyzed. Results are presented as mean ± SEM and analyzed by one-way ANOVA. Statistical significance was established as * p < 0.05 vs. Ctrl, ⁺ p < 0.05 vs. DM.

Figure 9

Effects of allicin on glucose homeostasis of rats. (a) Blood glucose and (b) plasma insulin levels; (c) glucose transporter expression and (d) insulin receptor substrate expression were assessed in skeletal muscle homogenate. Ctrl: control; DM: diabetic; DA: diabetic treated with allicin; a.u: arbitrary units. For representative Western blotting, 3 randomly selected samples per group were analyzed. Results are presented as mean ± SEM and analyzed by one-way ANOVA. Statistical significance was established as * p < 0.05 vs. Ctrl, ⁺ p < 0.05 vs. DM, ^α p < 0.05 vs. DM day 30, ^β p < 0.05 vs. DA day 30.

Figure 10

Effects of allicin on pathophysiological mechanisms during progression of nephropathy associated to diabetes. Structure of allicin (diallyl thiosulfinate): red spheres, oxygen; yellow spheres, sulfur; gray spheres, carbon; white spheres, hydrogen.