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.