Flavocoxid, a Natural Antioxidant, Protects Mouse Kidney from Cadmium-Induced Toxicity

Abstract

Background: Cadmium (Cd), a diffused environmental pollutant, has adverse effects on urinary apparatus. The role of flavocoxid, a natural flavonoid with antioxidant activity, on the morphological and biochemical changes induced in vivo by Cd in mice kidney was evaluated.

Methods: C57 BL/6J mice received 0.9% NaCl alone, flavocoxid (20 mg/kg/day i.p.) alone, Cd chloride (CdCl₂) (2 mg/kg/day i.p.) alone, or CdCl₂ plus flavocoxid (2 mg/kg/day i.p. plus 20 mg/kg/day i.p.) for 14 days. The kidneys were processed for biochemical, structural, ultrastructural, and morphometric evaluation.

Results: Cd treatment alone significantly increased urea nitrogen and creatinine, iNOS, MMP-9, and pERK 1/2 expression and protein carbonyl; reduced GSH, GR, and GPx; and induced structural and ultrastructural changes in the glomeruli and in the tubular epithelium. After 14 days of treatment, flavocoxid administration reduced urea nitrogen and creatinine, iNOS, MMP-9, and pERK 1/2 expression and protein carbonyl; increased GSH, GR, and GPx; and showed an evident preservation of the glomerular and tubular structure and ultrastructure.

Conclusions: A protective role of flavocoxid against Cd-induced oxidative damages in mouse kidney was demonstrated for the first time. Flavocoxid may have a promising antioxidant role against environmental Cd harmful effects on glomerular and tubular lesions.

Figure 1

Representative Western blot analysis of iNOS (a), pERK 1/2 (b), and MMP-9 (c) of kidneys from mice of control plus vehicle (0.9% NaCl), control plus flavocoxid (20 mg/kg/day i.p.), CdCl₂ (2 mg/kg/day i.p.) plus vehicle, and CdCl₂ plus flavocoxid groups. ^∗p < 0.05 versus both controls and ^†p < 0.05 versus CdCl₂ plus vehicle. Bars represent the mean ± SE of seven experiments.

Figure 2

Structural organization of glomeruli and tubules of kidneys from mice of control plus vehicle (0.9% NaCl, 1 ml/kg/day i.p.), control plus flavocoxid (20 mg/kg/day i.p.), CdCl₂ (2 mg/kg/day i.p.) plus vehicle, and CdCl₂ plus flavocoxid groups (hematoxylin and eosin stain). (a, b) In both control groups, glomeruli and tubules show normal architecture. (c) In CdCl₂-challenged mice, glomeruli present enlarged Bowman's space (arrow), while tubules show epithelial lesions (arrowhead). A mild interstitial edema is also present (∗). (d) In CdCl₂-challenged mice administered with flavocoxid, glomerular and tubular morphology is normal. (e) Glomerular area evaluation. ^∗p < 0.05 versus both controls and ^†p < 0.05 versus CdCl₂ plus vehicle (scale bar: 50 μm).

Figure 3

Tubular brush border of kidney sections from mice of control plus vehicle (0.9% NaCl, 1 ml/kg/day i.p.), control plus flavocoxid (20 mg/kg/day i.p.), CdCl₂ (2 mg/kg/day i.p.) plus vehicle, and CdCl₂ plus flavocoxid groups (periodic acid-Schiff stain). (a, b) In both control plus vehicle and control plus flavocoxid-treated mice, the proximal tubules show a regular and well-stained brush border. (c) In CdCl₂-challenged mice, the brush border is particularly thin or absent (arrowhead) and the tubular epithelium shows structural changes (arrow), with mild interstitial edema. (d) In CdCl₂-challenged mice administered with flavocoxid, the brush border has normal organization. (e) Tubular damage evaluation indicated by the brush border behavior. ^∗p < 0.05 versus both controls and ^†p < 0.05 versus CdCl₂ plus vehicle (scale bar: 50 μm).

Figure 4

Structural organization of the interstitial connective tissue of kidneys from mice of control plus vehicle (0.9% NaCl, 1 ml/kg/day i.p.), control plus flavocoxid (20 mg/kg/day i.p.), CdCl₂ (2 mg/kg/day i.p.) plus vehicle, and CdCl₂ plus flavocoxid groups (Sirius red stain). (a, b) In both control plus vehicle and control plus flavocoxid-treated mice, the normal presence of collagen fibers is evident in the interstitial tissue. (c) In CdCl₂-challenged mice, SR stain is less evident around the glomerular capsule and the tubules. (d) In CdCl₂ plus flavocoxid-treated mice, no apparent difference with normal specimens is present. (e) Quantitative evaluation of the SR-positive areas. ^∗p < 0.05 versus both controls and ^†p < 0.05 versus CdCl₂ plus vehicle (scale bar: 50 μm).

Figure 5

Ultrastructural organization of the glomeruli of kidneys from mice of control plus vehicle (0.9% NaCl, 1 ml/kg/day i.p.), control plus flavocoxid (20 mg/kg/day i.p.), CdCl₂ (2 mg/kg/day i.p.) plus vehicle, and CdCl₂ plus flavocoxid groups. (a, b) In both control plus vehicle and control plus flavocoxid-treated mice, glomeruli show normal morphology of either the podocytes or the endothelial cells. (c) In CdCl₂-challenged mice, podocytes are elongated and fewer, and their contacts with the capillaries are lacking (arrow). (d) In CdCl₂ plus flavocoxid-treated mice, glomerular morphology was superimposable to controls (scale bar: 4 μm).

Figure 6

Ultrastructural organization of the proximal tubules of kidneys from mice of control plus vehicle (0.9% NaCl, 1 ml/kg/day i.p.), control plus flavocoxid (20 mg/kg/day i.p.), CdCl₂ (2 mg/kg/day i.p.) plus vehicle, and CdCl₂ plus flavocoxid groups. (a, b) In both control plus vehicle and control plus flavocoxid-treated mice, the epithelium of the proximal tubules shows a well-preserved brush border, normal intercellular junctions, and elongated mitochondria. (c) In CdCl₂-challenged mice, apical microvilli are short, few, or sometimes absent (arrow), the intercellular spaces are wide (full arrowhead), and the tubular cells show round or swollen mitochondria and cytoplasmic vacuoles (empty arrowhead). (d) In CdCl₂ plus flavocoxid-treated mice, no apparent tubular lesions are present (scale bar: 4 μm).

Figure 7

Graphical model indicating the different effects of CdCl₂ alone and CdCl₂ plus flavocoxid on the mouse kidney. CdCl₂ = cadmium chloride; iNOS = inducible nitric oxide synthase; pERK 1/2 = phosphorylated extracellular signal-regulated protein kinase 1/2; MMP-9 = matrix metalloproteinase 9; GSH = reduced glutathione; GR = glutathione reductase; GPx = glutathione peroxidase.