High glucose-induced oxidative stress inhibits Na⁺/glucose cotransporter activity in renal proximal tubule cells

Oxidative stress plays an important role in the pathogenesis of renal diseases such as diabetic nephropathy. The metabolism of excessive intracellular glucose may involve a number of processes. One consequence of excessive intracellular glucose levels is an increased rate of oxidative phosphorylation under hyperglycemic conditions, whereas another consequence is an increase in the metabolism of glucose to sorbitol by aldose reductase. In addition, hyperglycemia may result in the activation of NADPH oxidase, the production of superoxide anion, and hydrogen peroxide (H₂O₂). In this report, we investigate the mechanisms responsible for the H₂O ₂ production that occurs as the consequence of hyperglycemia and the effect of H₂O₂ on the activity of the Na ⁺/glucose cotransport system (SGLT) in primary cultures of renal proximal tubule cells (PTCs). When primary PTCs were cultured in the presence of high glucose, one consequence was that the Na⁺/glucose cotransport system was inhibited, as indicated by uptake studies utilizing α-methyl-D-glucoside (α-MG), a nonmetabolizable analog of D-glucose. Pretreatment of the cultures with either 1) aminoguanidine or pyridoxamine [inhibitors of the accumulation of advanced glycation end products (AGEs)], 2) rotenone (an inhibitor of the mitochondrial electron transport chain), or 3) apocynin or diphenylene iodonium (DPI; inhibitors of NADPH oxidase) blocked the observed changes that occurred as a consequence of the incubation of the PTCs with high glucose. Included among these changes were the observed increase in H₂O₂ levels, as well as an increase in lipid peroxide production, and a decrease both in the activity of catalase and in the level of glutathione (GSH), endogenous antioxidants. The high glucose-induced decrease in the level of the Na⁺/glucose cotransporter was similarly prevented by either aminoguanidine, rotenone, or apocynin. Thus the inhibitory effect of high glucose on both the level of the Na⁺/glucose cotransport system and the activity of the Na₊/ glucose cotransport system can be explained, at least in part, as being due to the effects of the H ₂O₂, the consequent formation of AGEs, the increase in mitochondrial metabolism, and in NADPH oxidase activity in the PTCs. Other related changes observed in the PTCs that could be reversed by treatment with either aminoguanidine, pyridoxamine, rotenone, apocynin, or DPI included an increase in transforming growth factor-β₁ secretion and the activation of the NF-κB signal transduction pathway.