Effect of ifosfamide metabolites on sodium-dependent phosphate transport in a model of proximal tubular cells (LLC-PK1) in culture

Abstract

Ifosfamide (IF) is an alkylating cytostatic drug with urotoxic (hemorrhagic cystitis) and nephrotoxic side effects. Several cases of Fanconi syndrome in children following therapy with IF were reported. Little information is available concerning the pathomechanisms of transport inhibition by IF. We used a permanent renal epithelial cell line with proximal tubular characteristics (LLC-PK1) in order to investigate the effects of IF and some of its major metabolites (4-OH-IF, chloracetaldehyde, and acrolein). LLC-PK1 cells were used in a confluent state. Sodium-dependent and sodium-independent fluxes of 32PO4 were determined by standard techniques. Activities of marker enzymes of apical and basolateral membranes, of mitochondria, and of endoplasmic reticulum were determined in cell homogenates. IF induces a moderate stimulation of PO4 transport. 4-OH-IF also has a stimulatory effect on transport at low concentrations (up to 200 mumol/l) and with short incubation (2h), while a 24-hour exposure of cells to 100 mumol/l of 4-OH-IF has an inhibitory effect of PO4 transport. Concentrations of 4-OH-IF which inhibit transport also reduce the activity of Na(+)-K(+)-ATPase. Chloracetaldehyde, like 4-OH-IF, induces a biphasic response of PO4 transport with stimulation in the low concentration range (up to 75 mumol/l) and inhibition at higher concentrations. Chloracetaldehyde reduces the activity of succinate-cytochrome c oxidoreductase, suggesting that a defect in ATP generation might play a role in the pathogenesis of Fanconi syndrome induced by IF. Acrolein strongly damages monolayers and reduces sodium-dependent transport of PO4 to very low levels at 150 mumol/l. It reduces the activities of both Na(+)-K+ ATPase and succinate-cytochrome c oxidoreductase. Acrolein also is the only metabolite with a moderate effect on alkaline phosphatase. We conclude that sodium-dependent transport of PO4 is highly sensitive to IF metabolites. In addition to direct toxic effects of IF metabolites on transport proteins within the apical plasma membrane, damage to mitochondrial enzymes and to Na(+)-K+ ATPase which generates the electrochemical gradients for secondary active PO4 transport may play an important role in the pathogenesis of Fanconi syndrome induced by IF.