4.8. Assessment of Renal Tissue Oxidative Stress Status

The right kidney was excised and rinsed in 0.1 M Tris-HCl buffer (pH 7.4). Ten percent (10%) tissue homogenate was prepared in the same buffer and centrifuged at 1000× g for 20 min using a refrigerated centrifuge (Eppendorf, Taufkirchen, Germany). The supernatant was collected for determination of SOD, CAT, GPx, GST and GR activities, GSH and malondialdehyde levels, and TAC. The results were normalized with protein concentrations of each sample as determined using a commercially available protein assay kit (BioAssay Systems, Hayward, CA, USA). Kidney LDH activity was determined using a commercially available LDH colorimetric assay kit (BioAssay Systems, Hayward, CA, USA).

The activity of SOD was assessed using nitro tetrazolium blue reduction method as previously described [29]. The activity of CAT was assessed using hydrogen peroxide substrate, which forms a yellowish complex with molybdate upon decomposition [30]. The activity of GPx was assessed following a method that is based on glutathione oxidation by hydrogen peroxide [31]. The activity of GR was determined using a method that is based on glutathione disulfide reduction in the presence of NADPH, which is later oxidized to NADP⁺ [32]. The activity of GST was assessed using a method based on GSH conjugation with 1-chloro-2,4-dinitrobenzene as substrate [33]. Total GSH level was determined using a method based on 5-thio-2-nitrobenzoic acid reduction. In this assay, 5,5-dithiobis-2-nitrobenzoic acid reacts with the sulfhydryl group of GSH to form 5-thio-2-nitrobenzoic acid [34].

Kidney homogenate malondialdehyde level was measured using a previously described method, with tetra ethoxy propane as standard [35], while total antioxidant capacity was measured using a method that is based on reduction in thiobarbituric acid reactive substance formation [36].