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

Abstract

<p>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 <i>et al.</i>, 2003).</p> <p>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.</p> <p>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.</p> <p>In all regions and under both stress and non-stress conditions the TBARS and lactate concentration showed a similar pattern with 12 weeks old < 36 weeks old < 5 weeks old and < 60 weeks old. The greatest concentration was measured in the 60 weeks rat under stress condition with the superficial cortex greater than the outer and inner medulla.</p> <p>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.</p> <p>mRNA was extracted from the superficial cortex, outer medulla and inner medulla. The expression of <i>Gpx1</i>, <i>Gpx4</i> and <i>Hsp-70</i> was analysed by quantitative reverse transcribed polymerase chain reaction (QRT-PCR).</p> <p>Gene expression of <i>Gpx1</i> was highest in the cortex, with a trend to decrease with age, after reaching a maximum at 12 weeks. By contrast, <i>Gpx4</i> 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 <i>Gpx</i> and <i>Hsp70</i> was striking although there is no data on protein expression of HSP70.</p> <p>Thus, in stark contrast with the expression of both <i>Gpx1</i> and <i>4</i>, gene expression of stress protein <i>Hsp-70</i> was elevated in the inner medulla, and was increased overall in the ageing rats at 60 weeks.</p> <p>Overall then, the thesis provides general support for the free radical theory of ageing particularly as this pertains to kidney function.</p>