Effect of ageing and oxidative stress on the glutathione concentration in different kidney regions

Oxidative stress has been implicated in ageing and the pathogenesis of chronic kidney disease. Glutathione (GSH) is the major intracellular thiol in the kidney and an indicator of this organ's redox status. The aim of this study was to investigate how the GSH concentration is affected by age and oxidative stress in different kidney regions. Kidneys were dissected from Male Wistar rats of 5, 12, 36 and 60 weeks-old. Slices of superficial cortex, outer or inner medulla were incubated for 30 min ± 0.2 mM H₂O₂ prior to homogenisation and centrifugation. GSH concentrations were measured colormetrically using a kit. Data are presented in nmol/mg protein, are means ± SE of n = 5 and statistical comparisons were carried out using ANOVA with an appropriate post-test. In all regions and all conditions the GSH concentration showed a similar pattern with 12 weeks-old > 36 weeks-old > 60 weeks-old and 5 weeks-old. The greatest concentration was measured in the 12 weeks-old with the superficial cortex (1509.16 ± 27.34) significantly greater than outer (851.67 ± 47.39,

Effect of ageing and oxidative stress on antioxidant enzyme activity in different regions of the rat kidney

Oxidative stress has been implicated in ageing and the pathogenesis of chronic kidney disease. We examined levels of antioxidant enzymes glutathione peroxidase, glutathione reductase, glutathione S-transferase, catalase and superoxide dismutase as modulated by age and oxidative stress in different regions of the kidney. Antioxidant enzymes were examined in different regions of the kidney in male Wistar rats. Kidneys from rats of different ages (5, 12, 36 and 60 weeks) were dissected into cortex, outer medulla and inner medulla. Tissues were incubated for 30 min with or without 0.2 mM H2O2 to induce oxidative stress. Antioxidant enzyme activities progressively decreased with age under both control and stress conditions (

Effect of ageing on metabolite and oxidant concentrations in different regions of rat kidney under normal and stress conditions

Metabolic and oxidative stresses have been implicated in ageing and the pathogenesis of chronic kidney disease. In this study, we investigated the glutathione (GSH), thiobarbituric acid reactive substances (TBARS) and lactate concentrations in different kidney regions under control conditions and after exposure to oxidative stress invoked by 0.2 mM H₂O₂. Slices of superficial cortex, outer or inner medulla were dissected from kidneys of male Wistar rats of 5-, 12-, 36- and 60-week old. Samples were incubated for 30 min ± 0.2 mM H2O2 prior to homogenisation and centrifugation. The concentrations of GSH, TBARS and lactate were measured by colorimetry. Each metabolite showed a distinctive pattern. For GSH, this was 12 weeks[ 36 weeks[60 weeks and 5 weeks with the highest concentration measured in the superficial cortex at 12 weeks. For TBARS and lactate, the pattern was for the lowest concentration at 12 weeks and the highest at 60 and 5 weeks. The highest lactate and TBARS concentrations were measured under oxidative stress conditions, particularly at 5 and 60 weeks. These results suggest that GSH in different kidney regions peaks at maturity and then reduces with increasing age

The effect of ageing on antioxidant status in different regions of the rat kidney

Under control physiological conditions, the use of oxygen by cells of aerobic organisms generates potentially toxic reactive oxygen metabolites. A chronic state of oxidative stress may exist in cells because of an imbalance between pro-oxidants and antioxidants. The amount of oxidative damage increases as an organism ages and this is postulated to be a major causal factor of senescence (Sohal & Weindruch, 1996). Oxidative stress probably plays a major role in the progressive compromise in the ability to maintain homeostasis characteristic of the ageing process. Abnormally high levels of free radicals and the simultaneous decline of antioxidant defence mechanisms can lead to damage of cellular organelles, enzymes, as well as increased lipid peroxidation and altered protein and gene expression (Maritim et al., 2003). The aim of this study was to investigate how the antioxidant status is affected by age and oxidative stress (±0.2 mM H2O2) in different functional regions of the rat kidney. Antioxidant enzyme activities were shown to be attenuated with age under both control and stress conditions after peaking at 12 weeks old (young adult). Antioxidant enzyme activities were higher in the cortex by comparison with the outer and inner medulla respectively. GSH concentrations followed a similar pattern to the levels of antioxidant enzymes. In all regions and under both stress and non-stress conditions the TBARS and lactate concentration showed a similar pattern with 12 weeks old Protein expression of GPX1 detected by Western blot was shown to decrease with age under both control and stress conditions after peaking at 12 weeks (young adult). GPX1 expression was greater in the cortex by comparison with the outer and inner medulla respectively. On the other hand, GPX4 expression did not show much variation across the different regions of the kidney under control or stress conditions, although it did show a significant increase in expression in the inner medulla at 12 weeks under control. GPX2 expression was not detected across the different regions of the kidney under control or stress conditions. mRNA was extracted from the superficial cortex, outer medulla and inner medulla. The expression of Gpx1, Gpx4 and Hsp-70 was analysed by quantitative reverse transcribed polymerase chain reaction (QRT-PCR). Gene expression of Gpx1 was highest in the cortex, with a trend to decrease with age, after reaching a maximum at 12 weeks. By contrast, Gpx4 gene expression did not vary in a consistently significant fashion across the different regions of the kidney in all age groups, with the one exception of a significantly higher level of expression in the inner medulla at 12 weeks. In general there was a good correlation between enzyme activity, protein expression and gene expression for the antioxidant enzymes studied. The negative correlation between gene expression of Gpx and Hsp70 was striking although there is no data on protein expression of HSP70. Thus, in stark contrast with the expression of both Gpx1 and 4, gene expression of stress protein Hsp-70 was elevated in the inner medulla, and was increased overall in the ageing rats at 60 weeks. Overall then, the thesis provides general support for the free radical theory of ageing particularly as this pertains to kidney function

Age-related protein and mRNA expression of glutathione peroxidases (GPx) and Hsp-70 in different regions of rat kidney with and without stressor

Small molecular weight oxygen free radical species (ROS) involved in oxidative stress can cause damage to cellular macromolecules including proteins, DNA and lipids. One of the most important enzymes involved in ROS detoxification is glutathione peroxidase (GPx). Here we study the age-related expression of GPx isoenzymes in various parts of the rat kidney with and without exposure to external oxidative stress. These results are correlated to the age dependent changes in the expression of the chaperone, 'Hsp-70'. Protein and mRNA expression of GPx1 and GPx4 was studied in different regions of the kidney in ageing rats in the presence and absence of the external stressor 0.2 mM H₂O₂. Protein levels were examined by Western blot analysis following detection with appropriate antibodies and mRNA levels were analysed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using appropriate primer sequences. mRNA expression for the chaperone 'Hsp70' was investigated in parallel. After reaching a peak at maturity (12 weeks), GPx1 protein and mRNA levels decreased with age under both control and stress conditions, and were higher in the cortex than in the outer and inner medulla. GPx4 protein and mRNA levels showed few comparable age-related changes. By contrast with the observed age-related decrease in GPx1 expression, chaperone 'Hsp-70' mRNA expression greatly increased with age. These findings suggest that the age-related decline in GPx1 expression in the cortex may be partly offset by a reciprocal change in 'Hsp-70' expression. These results are consistent with the oxidative stress theory of ageing