title: High glucose-induced oxidative stress causes apoptosis in proximal tubular epithelial cells and is mediated by multiple caspases.
abstract: Diabetic nephropathy is the leading cause of end-stage renal disease in the Western world. Poor glycemic control contributes to the development of diabetic nephropathy, but the mechanisms underlying high glucose-induced tissue injury are not fully understood. In the present study, the effect of high glucose on a proximal tubular epithelial cell (PTEC) line was investigated. Reactive oxygen species (ROS) were detected using the fluorescent probes dichlorofluorescein diacetate, dihydrorhodamine 123, and 2,3-diaminonapthalene. Peroxynitrite (ONOO-) generation and nitrite concentrations were increased after 24 h of high glucose treatment (P<0.05). LLC-PK1 cells exposed to high D-glucose (25 mM) for up to 48 h had increased DNA fragmentation (P<0.01), caspase-3 activity (P<0.001), and annexin-V staining (P<0.05) as well as decreased expression of XIAP when compared with controls (5 mM D-glucose). The ONOO- scavenger ebselen reduced DNA fragmentation and caspase-3 activity as well as the high glucose-induced nitrite production and DCF fluorescence. High glucose-induced DNA fragmentation was completely prevented by an inhibitor of caspase-3 (P<0.01) and a pan-caspase inhibitor (P<0.001). Caspase inhibition did not affect ROS generation. This study, in a PTEC line, demonstrates that high glucose causes the generation of ONOO-, leading to caspase-mediated apoptosis. Ebselen and a caspase-3 inhibitor provided significant protection against high glucose-mediated apoptosis, implicating ONOO- as a proapoptotic ROS in early diabetic nephropathy.
authors.affiliation: Department of Experimental Medicine and Nephrology, William Harvey Research Institute, St Bartholomew's and Royal London School of Medicine and Dentistry, Queen Mary, University of London, UK. d.a.allen@qmul.ac.uk
authors.forename: David A
authors.initials: DA
authors.lastname: Allen
authors.forename: Steven
authors.initials: S
authors.lastname: Harwood
authors.forename: Mira
authors.initials: M
authors.lastname: Varagunam
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Raftery
authors.forename: Muhammad M
authors.initials: MM
authors.lastname: Yaqoob
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D001393:Azoles / Q000494:pharmacology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology*; D054833:Isoindoles; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D016566:Organoselenium Compounds / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects*; D030421:Peroxynitrous Acid / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D000975:Antioxidants; D001393:Azoles; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D054833:Isoindoles; D009573:Nitrites; D009842:Oligopeptides; D016566:Organoselenium Compounds; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; C093260:benzyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene; D030421:Peroxynitrous Acid; D001224:Aspartic Acid; C042986:ebselen; D020169:Caspases; D005947:Glucose