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 upt ake of glucose ([alpha-C-14]methylglucoside) and phosphate (P-32i) in LLC-PK1-cells after exposure to 0-500 muM hydrogen peroxide. Oxidant s tress resulted in significant (P < 0.01) inhibition of glucose and pho sphate transport. Decreased transport of glucose and phosphate was ass ociated with marked ATP depletion, decreased activity of the sodium pu mp as determined by Rb-86 uptake, direct inhibition of Na+-K+-adenosin etriphosphatase (Na+-K+-ATPase) activity, and an increase in intracell ular sodium content, whereas intracellular potassium content declined. Decreased glucose and phosphate transport, inhibition of Rb-86 uptake and Na+-K+-ATPase activity, and altered intracellular ion content wer e prevented by catalase and partially prevented by the membrane-permea ble iron chelator phenathroline, whereas the slowly membrane-permeable iron chelator deferoxamine had little or no effect. To determine whet her oxidant injury could also inhibit transporter function at the memb rane level, plasma membrane vesicles were isolated from LLC-PK1 cells exposed to 500 muM hydrogen peroxide. Such membrane vesicles exhibited decreased sodium-dependent glucose transport, whereas sodium-dependen t phosphate transport was not altered. We conclude that oxidant injury results in ATP depletion and inactivation of Na+-K+-ATPase which lead s to disruption of the normal ion gradients sufficient to interfere wi th glucose and phosphate transport. Glucose transport is also inhibite d by disruption of transporter activity within the plasma membrane. Th ese alterations are mediated in part by the intracellular generation o f an iron-dependent radical.