Figure 5
Effect of hydralazine mediated transglycation on kidney proteins.
(A) Kidney AGE levels were measured by ELISA. Each well of a 96-well microtiter plate coated with 0.1 ml of 20 μg/ml of kidney proteins, was blocked, subsequent reactions were performed with 0.1 ml of anti-AGE antibody (0.5 pg/ml), secondary antibody conjugated to alkaline phosphatase and PNPP (p-nitro phenyl phosphate) substrate respectively. The absorbance of each well was recorded at 415 nm with a microplate absorbance reader. Mean of A415 were used to calculate the relative AGE levels in the kidney. The bar graph depicts decrease in relative AGE levels upon treatment with hydralazine and aminoguanidine. (The values represent mean ± SE, n = 6, P- value < 0.05). (B) 50 μg of kidney proteins were separated on 12% SDS-PAGE and transferred onto the PVDF membrane. AGE modification was probed using AGE antibodies by western blot analysis. Hydralazine decrease the AGE modification of kidney proteins (Lane DIAB-HYD) compared to STZ induced diabetic kidney proteins (Lane DIAB). (C) Western blot analysis of 20 μg of kidney proteins indicating the expression of RAGE, NOX and SOD. Hydralazine and aminoguanidine treatment decreased the expression of these proteins (Lanes DIAB-HYD and DIAB-AMG) as shown by densitometric analysis in the bar graph.
