Hexosamine induction of oxidative stress, hypertrophy and laminin expression in renal mesangial cells: effect of the anti‐oxidant α‐lipoic acid

Abstract

We have previously shown that one of the potential mediators of the deleterious effects of high glucose on extracellular matrix protein (ECM) expression in renal mesangial cells is its metabolic flux through the hexosamine biosynthesis pathway (HBP). Here, we investigate further whether the hexosamines induce oxidative stress, cell-cycle arrest and ECM expression using SV-40-transformed rat mesangial (MES) cells and whether the anti-oxidant alpha-lipoic acid will reverse some of these effects. Culturing renal MES cells with high glucose (HG, 25 mM) or glucosamine (GlcN, 1.5 mM) for 48 h stimulates laminin gamma1 subunit expression significantly approximately 1.5 +/- 0.2- and 1.9 +/- 0.3-fold, respectively, when compared to low glucose (LG, 5 mM). Similarly, HG and GlcN increase the level of G0/G1 cell-cycle progression factor cyclin D1 significantly approximately 1.7 +/- 0.2- and 1.4 +/- 0.04-fold, respectively, versus LG (p < 0.01 for both). Azaserine, an inhibitor of glutamine:fruc-6-PO(4) amidotransferase (GFAT) in the HBP, blocks the HG-induced expression of laminin gamma1 and cyclin D1, but not GlcN's effect because it exerts its metabolic function distal to GFAT. HG and GlcN also elevate reactive oxygen species (ROS) generation, pro-apoptotic caspase-3 activity, and lead to mesangial cell death as revealed by TUNEL and Live/Dead assays. FACS analysis of cell-cycle progression shows that the cells are arrested at G1 phase; however, they undergo cell growth and hypertrophy as the RNA/DNA ratio is significantly (p < 0.05) increased in HG or GlcN-treated cells relative to LG. The anti-oxidant alpha-lipoic acid (150 microM) reverses ROS generation and mesangial cell death induced by HG and GlcN. Alpha-lipoic acid also reduces HG and GlcN-induced laminin gamma1 and cyclin D1 expression in MES cells. In addition, induction of diabetes in rats by streptozotocin (STZ) increases both laminin gamma1 and cyclin D1 expression in the renal cortex and treatment of the diabetic rats with alpha-lipoic acid (400 mg kg(-1) body weight) reduces the level of both proteins significantly (p < 0.05) when compared to untreated diabetic rats. These results support the hypothesis that the hexosamine pathway mediates mesangial cell oxidative stress, ECM expression and apoptosis. Anti-oxidant alpha-lipoic acid reverses the effects of high glucose, hexosamine and diabetes on oxidative stress and ECM expression in mesangial cells and rat kidney.

The hexosamine biosynthesis inhibitor azaserine prevents endothelial inflammation and dysfunction under hyperglycemic condition through antioxidant effects

“…The relationship between the HBP pathway and oxidative stress seems obscure. It has been reported that oxidative stress stimulates the HBP pathway on one hand (4) and that the HBP pathway is also able to induce oxidative stress on the other hand (19,36). Moreover, azaserine is frequently used as an inhibitor of the HBP pathway to delineate its functional roles based on the effect of the compound on cellular O-GlcNAc levels.…”

Protein O-Glycosylation Induces Collagen Expression and Contributes to Diabetic Cardiomyopathy in Rat Cardiac Fibroblasts

“…cultures such as rat mesangial cells and 3T3-L1 adipocytes (16,17). In order to induce effects, neonatal rat cardiomyocytes were treated with 15 mM glucosamine (18), suggesting that the effects of glucosamine on HBP activation may be cell-and context-specific.…”

p38 Mitogen-Activated Protein Kinase Is Required for Glucosamine-Induced Endothelial Nitric Oxide Synthase Uncoupling and Plasminogen-Activator Inhibitor Expression

“…42 The pro-oxidative effect of GlcN has also been shown in renal mesangial cells, pancreas β-cells. 43, 44 The present study showed that the enhanced O2 .-generation by GlcN in endothelial cells is attributed to eNOS uncoupling, because the increased O2 .-generation by GlcN is abolished by the eNOS inhibitor, L-NAME. Importantly, in situ production of O2 .-in isolated mouse aortas caused by GlcN is inhibited and NO production restored by p38 mapk inhibitor, which is paralleled with restoration of endothelium-dependent relaxation in isolated mouse aortas.…”