title: Hyperglycemia inhibits the uptake of dehydroascorbate in tubular epithelial cell.
abstract: OBJECTIVE
  Oxidative stress has been considered to be a common pathogenetic factor of diabetic nephropathy. But the reason why renal cells are susceptible to oxidative injury in diabetes is not clear. Vitamin C plays a central role in the antioxidant defense system and exists in two major forms. The charged form, ascorbate, is taken up into cells via sodium-dependent facilitated transport. The uncharged form, dehydroascorbate, enters cells via glucose transporter and is then converted back to ascorbate within these cells. Because dehydroascorbate and glucose compete for glucose transporters, hyperglycemia will exclude vitamin C from the cell and resulted in a decreased antioxidant capacity in some cell type that is dehydroascorbate dependent. As such, we hypothesized that some renal cells were dehydroascorbate dependent and the susceptibility of renal cells to glucose-induced injury was mediated by hyperglycemic exclusion of dehydroascorbate uptake through competing for glucose transporter. The aims of the present study were to determine whether tubular epithelial cell was dehydroascorbate dependent and the effect of dehydroascorbate on the production of reactive oxygen species in cells incubated by high glucose.
  
  
  METHODS
  Tubular epithelial cell was cultured in RPMI-1640 medium containing 10% newborn calf serum. Intracellular ascorbate and dehydroascorbate contents were measured with vitamin C assay system. The intracellular formation of reactive oxygen species was detected with the fluorescent probe CM-H(2)DCFDA by using confocal microscopy.
  
  
  RESULTS
  Ascorbate entry into the cells was not significantly different from background noise. In contrast, we observed a significant increase in the uptake of dehydroascorbate in tubular cell. At a dehydroascorbate concentration of 1 mM, increasing concentrations of glucose competitively inhibited dehydroascorbate entry into the cells such that the accumulation of dehydroascorbate was smaller than half maximal at about 22 mM glucose. Cytochalasin B, a kind of hexose transporter inhibitor, inhibited dehydroascorbate entry into the cells. At a glucose concentration of 25 mM, increasing concentrations of dehydroascorbate reduced reactive oxygen species generation in a dose-dependent manner when dehydroascorbate concentration was smaller than 4 mM. However, the inhibitory effect was not observed at 8 mM of dehydroascorbate.
  
  
  CONCLUSIONS
  Tubular epithelial cells are dehydroascorbate dependent. Vitamin C exclusion from tubular epithelial cells through competition of glucose and dehydroascorbate for common transport mechanism in diabetes will deprive the cells of antioxidant ability and could lead to reactive oxygen species accumulation.
authors.affiliation: Division of Nephrology, Renmin Hospital of Wuhan University, 238 Jiefang Road, Wuhan 430060, China.
authors.forename: Ling
authors.initials: L
authors.lastname: Chen
authors.forename: Ru-han
authors.initials: RH
authors.lastname: Jia
authors.forename: Chang-jian
authors.initials: CJ
authors.lastname: Qiu
authors.forename: Guohua
authors.initials: G
authors.lastname: Ding
mesh_terms: D000818:Animals; D002460:Cell Line; D003683:Dehydroascorbic Acid / Q000037:antagonists & inhibitors / Q000493:pharmacokinetics*; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005947:Glucose / Q000008:administration & dosage / Q000494:pharmacology; D051246:Glucose Transport Proteins, Facilitative / Q000378:metabolism; D006943:Hyperglycemia / Q000378:metabolism*; D007684:Kidney Tubules / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D014815:Vitamins / Q000493:pharmacokinetics*
chemical_list: D051246:Glucose Transport Proteins, Facilitative; D017382:Reactive Oxygen Species; D014815:Vitamins; D005947:Glucose; D003683:Dehydroascorbic Acid