Effect of BSA-induced ER stress on SGLT protein expression levels and alpha-MG uptake in renal proximal tubule cells.
Recent studies demonstrated that endoplasmic reticulum (ER) stress regulates glucose homeostasis and that ER stress preconditioning which induces an adaptive, protective unfolded protein response (UPR) offers cytoprotection against nephrotoxins. Thus the aim of the present study was to use renal proximal tubule cells (PTCs) to further elucidate the link between the BSA-induced ER stress and alpha-methyl-d-glucopyranoside (alpha-MG) uptake and to identify related signaling pathways. Among ER stress inducers such as high glucose, BSA, H2O2, or tumicamycin, BSA pretreatment ameliorated the reduction of Na(+)-glucose cotransporter (SGLT) expression and alpha-MG uptake by gentamicin or cyclosporine A. Immunofluorescence studies revealed that BSA (10 mg/ml) stimulated the expression of glucose-regulated protein 78 (GRP78), an ER stress biomarker. In addition, BSA increased levels of GRP78 protein expression and eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation in a time-dependent manner. Furthermore, transfection with a GRP78-specific small interfering RNA (siRNA) inhibited BSA-stimulated SGLT expression and alpha-MG uptake. In experiments designed to unravel the mechanisms underlying BSA-induced ER stress, BSA stimulated the production of cellular reactive oxygen species (ROS), and antioxidants such as ascorbic acid or N-acetylcysteine (NAC) blocked BSA-induced increases in GRP78 activation, eIF2alpha phosphorylation, SGLT expression, and alpha-MG uptake. Moreover, the cells upregulated peroxisome proliferator-activated receptor-gamma (PPARgamma) mRNA levels in response to BSA or troglitazone (a PPARgamma agonist), but BSA was ineffective in the presence of GW9662 (a PPARgamma antagonist). In addition, both BSA and troglitazone stimulated GRP78 and eIF2alpha activation, SGLT expression, and alpha-MG uptake, whereas GW9662 inhibited the effects of BSA. BSA also stimulated phosphorylation of JNK and NF-kappaB, and GW9662 or GRP78 siRNA attenuated this response. Moreover, SP600125 or SN50 effectively blocked SGLT expression and alpha-MG uptake in BSA- or PPARgamma agonists (troglitazone or PGJ2)-treated PTCs. We conclude that BSA induces ER stress through ROS production and PPARgamma activation, which subsequently activates JNK/NF-kappaB signaling to enhance glucose uptake in renal PTCs.
Lee
Yu Jin
YJ
Department. of Veterinary Physiology, College of Veterinary Medicine, Chonnam National Univ., Gwangju 500-757, Korea.
Suh
Han Na
HN
Han
Ho Jae
HJ
D000818:Animals; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000187:drug effects*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005786:Gene Expression Regulation / Q000502:physiology; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000378:metabolism; D048670:MAP Kinase Kinase 4 / Q000378:metabolism; D008297:Male; D008757:Methylglucosides / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D047495:PPAR gamma / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D017382:Reactive Oxygen Species; D012710:Serum Albumin, Bovine / Q000633:toxicity*; D015398:Signal Transduction / Q000187:drug effects; D051247:Sodium-Glucose Transport Proteins / Q000235:genetics / Q000378:metabolism*
D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D008757:Methylglucosides; D018832:Molecular Chaperones; D016328:NF-kappa B; D047495:PPAR gamma; D017382:Reactive Oxygen Species; D051247:Sodium-Glucose Transport Proteins; D012710:Serum Albumin, Bovine; C027020:methylglucoside; D017346:Protein Serine-Threonine Kinases; D048670:MAP Kinase Kinase 4