Oxidative stress is determined by the balance between the generation of reactive oxygen species (ROS) such as superoxide anion (O₂^–) and the antioxidant defence systems such as superoxide dismutase (SOD). O₂^– is produced in activated phagocytes by the enzyme NADPH oxidase. NAD(P)H oxidase has also been implicated in O₂^– production in the vasculature and in the kidney cortex and medulla. A high salt intake enhances oxidative stress in rat skeletal muscle arterioles and vessels and increases blood pressure, protein excretion and renal fibrosis and worsens renal function in several models of chronic renal failure and accelerates the decline of renal function in patients with chronic renal failure (1). It was recently shown that a high salt diet induces oxidative stress in the rat kidney. Furthermore quantitative-RT-PCR experiments showed an increased expression of mRNA for the NAD(P)H oxidase components gp91phox and p47phox and decreased expression of mRNA for the Cu/Zn and Mn isoforms of SOD in the renal cortex (2). To expand on these findings, our aim in this study was to determine the effects of a high salt diet on protein expression levels of the gp91phox subunit of NADPH oxidase and the Cu/Zn and Mn isoforms of SOD in both the renal cortex and medulla using Western blotting. Three month old male Wistar-Kyoto rats were assigned to two groups (n = 3) and were placed on either normal (0.03% Na⁺) or high salt (0.9% Na⁺) rat chow diet for 14 days. The animals were then killed with an overdose of anaesthetic (chloralose/urethane) and kidneys were immediately removed. Following dissection of cortex and medulla, tissue samples were homogenised in radio-immunoprecipitation (RIPA) buffer and following centrifugation, protein concentrations of supernatants were determined by Bradford assay. Protein samples (20 μg) were resolved by 12% SDS-polyacrylamide gel electrophoresis, electro-transferred to nitrocellulose membranes and probed with polyclonal antibodies specific for Cu/Zn SOD, Mn SOD, gp91phox and actin (loading control). Following detection of antibody binding by chemiluminescence, band intensities were quantified by densitometry. To correct for variations in protein loading, Cu/Zn SOD, Mn SOD, gp91phox band intensities were divided by corresponding actin band intensities. Means±SEMs were calculated for the high salt and normal salt groups and comparisons were undertaken using unpaired Student’s t test. Significance was taken when P < 0.05. Our result show that cortical tissue from normal and high salt fed rats contained levels of gp91phox at a relative band intensities of 0.31 ± 0.05 and 0.67 ± 0.04, respectively, a 2.2-fold increase (n=3, P < 0.05). Similarly, medullary tissue from normal and high salt fed rats contained levels of gp91phox at a relative band intensities of 0.42 ± 0.09 and 0.76 ± 0.03, respectively, a 1.75-fold increase (n=3, P < 0.05). In contrast to previous findings (2), there was no significant difference between the cortical and medullary levels of both isoforms of SOD from high-salt and normal diet fed rats. Cortical Cu/Zn SOD levels from normal and high salt groups were 2.16 ± 0.17 and 2.65 ± 0.48, respectively (n=3). Cortical Mn SOD levels from normal and high salt groups were 1.70 ± 0.11 and 1.69 ± 0.03, respectively (n=3). Medullary Cu/Zn SOD levels from normal and high salt groups were 1.19 ± 0.23 and 1.02 ± 0.07, respectively (n=3). Medullary Mn SOD levels from normal and high salt groups were 1.11 ± 0.01 and 1.03 ± 0.09, respectively (n=3).