EFFECT OF IFOSFAMIDE METABOLITES ON SODIUM-DEPENDENT PHOSPHATE-TRANSPORT IN A MODEL OF PROXIMAL TUBULAR CELLS (LLC-PK1) IN CULTURE

Ifosfamide (IF) is an alkylating cytostatic drug with urotoxic (hemorr hagic ctstitis) 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 i nhibition by IF. We used permanent renal epithelial cell line with pro ximal tubular characteristics(LLC-PK1) in order to investigate the eff ects of IF and some of its major metabolites (4-OH-IF, chloracetaldehy de, and acrolein). LLC-PK1 cells were used in a confluent state. Sodiu m-dependent and sodium-independent fluxes of (PO4)-P-32 were determine d by standard techniques. Activities of marker enzymes of apical and b asolateral membranes, of mitochondria, and of endoplasmic reticulum we re determined in cell homogenates. IF induces a moderate stimulation o f PO4 transport. 4-OH-IF also has a stimulatory effect on transport at low concentrations (up to 200 mu mol/l) and with short incubation (2 h), while a 24-hour exposure of cells to 100 mu mol/l 4-OH-IF has an i nhibitory effect of PO4 transport. Concentrations of 4-OH-IF which inh ibit transport also reduce the activity of Na+-K+-ATPase. Chloracetald ehyde, like 4-OK-IF, induces a biphasic response of PO4 transport with stimulation in the low concentration range (up to 75 mu mol/l) and in hibition at higher concentrations. Chloracetaldehyde reduces the activ ity of succinate-cytochrome c oxidoreductase, suggesting that a defect in ATP generation might play a role in the pathogenesis of Fanconi sy ndrome induced by IF. Acrolein strongly damages monolayers and reduces sodium-dependent transport of PO4 to very low levels at It reduces th e activities of both Na+-K+ ATPase and succinate-cytochrome c oxidored uctase. 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 pla sma membrane, damage to mitochondrial enzymes and to Na+-K+ ATPase whi ch generates the electrochemical gradients for secondary active PO4 tr ansport may play an important role in the pathogenesis of Fanconi synd rome induced by IF.