Figure 8

Effect of glucose on the expression of TIMPs in HK-2 cells. A, Total RNA was isolated from cells cultured in 5 mm (L) or 25 mm (H) glucose. Twenty micrograms of each sample were probed with DIG-labeled cDNA for TIMP-1 or TIMP-2 and rehybridized with a cDNA probe for GAPDH, as internal control, to verify RNA loads. C, Aliquots of equal amounts of conditioned media based on cell numbers from cells cultured in 5 mm (L) or 25 mm (H) glucose were concentrated and analyzed on 10% SDS-polyacrylamide gel under reducing conditions. Electrophoretically transferred proteins were immunoblotted using primary antibodies against TIMP-1 and TIMP-2. Results from both Northern and Western blottings were analyzed by densitometry (B and D, respectively). Expression in 5 mm glucose was plotted as 100%. Each bar represents the mean ± sd of three independent experiments. Differences were significant at p< 0.05.