Oxidant-induced alterations in glucose and phosphate transport in LLC-PK1 cells: mechanisms of injury.
To determine the effects of oxidant injury on specialized functions of proximal tubular epithelial cells, we determined sodium-dependent uptake of glucose ([alpha-14C]methylglucoside) and phosphate (32Pi) in LLC-PK1 cells after exposure to 0-500 microM hydrogen peroxide. Oxidant stress resulted in significant (P < 0.01) inhibition of glucose and phosphate transport. Decreased transport of glucose and phosphate was associated with marked ATP depletion, decreased activity of the sodium pump as determined by 86Rb uptake, direct inhibition of Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) activity, and an increase in intracellular sodium content, whereas intracellular potassium content declined. Decreased glucose and phosphate transport, inhibition of 86Rb uptake and Na(+)-K(+)-ATPase activity, and altered intracellular ion content were prevented by catalase and partially prevented by the membrane-permeable iron chelator phenathroline, whereas the slowly membrane-permeable iron chelator deferoxamine had little or no effect. To determine whether oxidant injury could also inhibit transporter function at the membrane level, plasma membrane vesicles were isolated from LLC-PK1 cells exposed to 500 microM hydrogen peroxide. Such membrane vesicles exhibited decreased sodium-dependent glucose transport, whereas sodium-dependent phosphate transport was not altered. We conclude that oxidant injury results in ATP depletion and inactivation of Na(+)-K(+)-ATPase which leads to disruption of the normal ion gradients sufficient to interfere with glucose and phosphate transport. Glucose transport is also inhibited by disruption of transporter activity within the plasma membrane. These alterations are mediated in part by the intracellular generation of an iron-dependent radical.
Andreoli
S P
SP
Department of Pediatrics, Indiana University Medical Center, Indianapolis 46202.
McAteer
J A
JA
Seifert
S A
SA
Kempson
S A
SA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D016166:Free Radical Scavengers; D005947:Glucose / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000378:metabolism; D007501:Iron / Q000502:physiology; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D010042:Ouabain / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism*; D011188:Potassium / Q000378:metabolism; D012413:Rubidium / Q000493:pharmacokinetics; D012964:Sodium / Q000378:metabolism
D016166:Free Radical Scavengers; D010710:Phosphates; D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D006861:Hydrogen Peroxide; D007501:Iron; D005947:Glucose; D012413:Rubidium; D011188:Potassium