Antioxidant Properties of Alpha-Lipoic (Thioctic) Acid Treatment on Renal and Heart Parenchyma in a Rat Model of Hypertension

Abstract

Renal and cardiac impairments are frequent events in the presence of hypertension. Organ damage is mainly linked to oxidative stress due to high blood pressure and may be reduced by antioxidant supplementation. Alpha-lipoic acid (ALA) is one of most effective antioxidants. It is widely used as a nutritional supplement in a racemic mixture (+/-), even though the (+)-enantiomer is biologically active. This study was designed to investigate the effect of treatment with (+/-)-ALA and its enantiomers on renal and heart parenchyma in spontaneously hypertensive rats (SHR), using immunochemical and immunohistochemical techniques. The results confirmed that the oxidative mechanisms of organ alterations, due to hypertension, and characterized by glomerular and tubular lesions, left ventricular hypertrophy, and fibrosis but not by apoptosis were accompanied by proteins' and nucleic acids' oxidation. We found greater effectiveness of (+)-ALA compared to (+/-)-ALA in reducing oxidative stress, cardiac and renal damages in SHR. To conclude, these data propose (+)-ALA as one of the more appropriate antioxidant molecules to prevent renal and cardiac alterations associated with hypertension.

Keywords: alpha-lipoic acid; heart; hypertension; kidney; parenchyma.

Figure 1

Oxidative stress in the kidney. (a) Lysates of kidney from WKY C, SHR C, SHR (+/−)-alpha-lipoic acid (ALA) 250 µmol/kg/day, SHR + (+/−) ALA 125 µmol/kg/day, SHR + (+) ALA 125 µmol/kg/day, SHR + (−) ALA 125 µmol/kg/day were immunoblotted using the Oxyblot kit; (b) The bar graph reports the level of oxidized proteins measured in optical density unit (ODU); (c) Sections of kidney processed for the 8-oxo-dG immunohistochemistry (IHC). Arrow: positive nucleus. Calibration bar 25 µm; (d) The bar graph shows the quantification of the number of positive cells per high-power field (HPF) (40×). Data are mean ± S.E.M. * p < 0.05 vs. WKY C; # p < 0.05 vs. SHR C.

Figure 2

Apoptosis in the kidney. (a) Lysates of kidney from WKY C, SHR C, SHR + (+/−) alpha-lipoic acid (ALA) 250 µmol/kg/day, SHR + (+/−) ALA 125 µmol/kg/day, SHR + (+) ALA 125 µmol/kg/day, SHR + (−) ALA 125 µmol/kg/day were immunoblotted using specific anti caspase-3; (b) The bar graph indicates the ratio of densitometric analysis of bands and GAPDH levels, used as loading control, considering the WKY C group as reference. Blots are representative of one of three separate experiments; (c) Sections of kidney from WKY C and SHR C processed for TUNEL staining. Arrow: apoptotic nucleus. Calibration bar 25 µm; (d) Bar graph shows the quantification of the number of positive cells per high-power field (HPF) (40×). Data are mean ± S.E.M.

Figure 3

Renal glomerular morphology. (a) Renal tissue of WKY C, SHR C, SHR + (+/−) alpha lipoic acid (ALA) 250 µmol/kg/day, SHR + (+/−) ALA 125 µmol/kg/day, SHR + (+) ALA 125 µmol/kg/day, SHR + (−) ALA 125 µmol/kg/day were stained using periodic acid-Schiff (PAS) technique. Arrow: damaged glomerulus. Calibration bar: 50 µm; (b) The bar graph shows the quantification of the intensity of PAS staining measured in optical density unit (ODU). Data are mean ± S.E.M. * p < 0.05 vs. WKY C; # p < 0.05 vs. SHR C.

Figure 4

Renal glomerular morphology. (a) Renal tissue of WKY C, SHR C, SHR + (+/−) alpha lipoic acid (ALA) 250 µmol/kg/day, SHR + (+/−) ALA 125 µmol/kg/day, SHR + (+) ALA 125 µmol/kg/day, SHR + (−) ALA 125 µmol/kg/day were stained using Masson’s trichrome technique. Arrow: damaged glomerulus. Calibration bar: 50 µm; (b) Morphometric analysis to assess the glomerular injury score. Data are mean ± S.E.M. * p < 0.05 vs. WKY C; # p < 0.05 vs. SHR C.

Figure 5

Renal tubules morphology. (a) Renal tissue of WKY C, SHR C, SHR + (+/−) alpha lipoic acid (ALA) 250 µmol/kg/day, SHR + (+/−) ALA 125 µmol/kg/day, SHR + (+) ALA 125 µmol/kg/day, SHR + (−) ALA 125 µmol/kg/day were stained using Masson’s trichrome technique. Arrow: damaged tubules. Calibration bar: 25 µm; (b) Morphometric analysis to assess the tubular injury score. Data are mean ± S.E.M. * p < 0.05 vs. WKY C; # p < 0.05 vs. SHR C.

Figure 6

Oxidative stress in heart. (a) Lysates of heart from WKY C, SHR C, SHR + (+/−) alpha lipoic acid (ALA) 250 µmol/kg/day, SHR + (+/−) ALA 125 µmol/kg/day, SHR + (+) ALA 125 µmol/kg/day, SHR + (−) ALA 125 µmol/kg/day were immunoblotted using the Oxyblot kit; (b) The bar graph reports the level of oxidized proteins measured in optical density unit (ODU); (c) Sections of heart processed for the 8-oxo-dG immunohistochemistry (IHC). Arrow: positive nucleus. Calibration bar 25 µm; (d) The bar graph shows the quantification of the number of positive cells per high-power field (HPF) (40×). Data are mean ± S.E.M. * p < 0.05 vs. WKY C; # p < 0.05 vs. SHR C.

Figure 7

Apoptosis in heart. (a) Lysates of heart from WKY C, SHR C, SHR + (+/−) alpha lipoic acid (ALA) 250 µmol/kg/day, SHR + (+/−) ALA 125 µmol/kg/day, SHR + (+) ALA 125 µmol/kg/day, SHR + (−) ALA 125 µmol/kg/day were immunoblotted using specific anti caspase-3; (b) The bar graph indicates the ratio of densitometric analysis of bands and GAPDH levels used as loading control, considering the WKY C group as reference. Blots are representative of one of three separate experiments; (c) Sections of heart from WKY C and SHR C processed for TUNEL staining. Arrow: apoptotic nucleus. Calibration bar 50 µm; (d) The bar graph shows the quantification of the number of positive cells per high-power field (HPF) (20×). Data are mean ± S.E.M.

Figure 8

Heart morphology. (a) Cardiac tissue of WKY C, SHR C, SHR + (+/−) alpha lipoic acid (ALA) 250 µmol/kg/day, SHR + (+/−) ALA 125 µmol/kg/day, SHR + (+) ALA 125 µmol/kg/day, SHR + (−) ALA 125 µmol/kg/day were stained using Masson’s trichrome technique. Arrow: damaged cardiomyocytes. Calibration bar: 50 µm; (b,c) Morphometric analysis to evaluate the areas of cardiomyocytes and cardiac fibrosis, respectively. Data are mean ± S.E.M. * p < 0.05 vs. WKY C; # p < 0.05 vs. SHR C.