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 µmol/ l) and with short incubation (2h), while a 24-hour exposure of cells to 100 µmol/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 µmol/l) and inhibition at higher concentrations. Chloracetaldehyde reduces the activity of succinate-cytochrome coxidoreductase, 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 P0₄ to very low levels at 150 µmol/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.

CONTRIBUTION OF CYP3A5 TO HEPATIC AND RENAL IFOSFAMIDEN-DECHLOROETHYLATION

“…Ifosfamide itself is not cytotoxic in cultured renal epithelial cells or isolated perfused rat kidneys exposed to ifosfamide concentrations that have been observed in cancer patients receiving ifosfamide (i.e., 160 -650 M) (Kurowski and Wagner, 1993;Mohrmann et al, 1993;Zamlauski-Tucker et al, 1994;Springate et al, 1999;Boddy and Yule, 2000). This raises the possibility that ifosfamide metabolites are nephrotoxic.…”

“…In parallel, ifosfamide is also metabolized by N-dechloroethylation to 2-dechloroethylifosfamide (2-DCEI), 3-dechloroethylifosfamide (3-DCEI), and an equivalent molar amount of chloroacetaldehyde. Both acrolein and chloroacetaldehyde cause toxicity in LLC-PK1 cells (Mohrmann et al, 1992(Mohrmann et al, , 1993; however, acrolein does not impair the function of isolated perfused rat kidneys or after long-term exposure in animal models (Parent et al, 1992;Zamlauski-Tucker et al, 1994). Chloroacetaldehyde has consistently shown a concentration-dependent cytotoxic effect in several in vitro models (i.e., porcine or rabbit cultured renal tubules and isolated perfused rat kidneys) with a minimum toxic concentration that ranges from 12.5 to 64 M (Mohrmann et al, 1993;Springate, 1997;Springate et al, 1999).…”

“…Both acrolein and chloroacetaldehyde cause toxicity in LLC-PK1 cells (Mohrmann et al, 1992(Mohrmann et al, , 1993; however, acrolein does not impair the function of isolated perfused rat kidneys or after long-term exposure in animal models (Parent et al, 1992;Zamlauski-Tucker et al, 1994). Chloroacetaldehyde has consistently shown a concentration-dependent cytotoxic effect in several in vitro models (i.e., porcine or rabbit cultured renal tubules and isolated perfused rat kidneys) with a minimum toxic concentration that ranges from 12.5 to 64 M (Mohrmann et al, 1993;Springate, 1997;Springate et al, 1999). In cultured primary human proximal tubules cells, the minimum chloroacetaldehyde concentration for toxicity was reported to be 500 M, which is well above the observed peak plasma concentrations (i.e., 0.5-109 M) in patients receiving standard doses of ifosfamide (Goren et al, 1986;Kurowski and Wagner, 1993;Dubourg et al, 2001).…”

The Nephrotoxic Ifosfamide-Metabolite Chloroacetaldehyde Interferes with Renal Extracellular Matrix Homeostasis

“…Among IFO and its metabolites, CAA is most probably responsible for chronic renal disease [11,12,13,14]. Cell culture studies could show impairment of several transport mechanisms by CAA in renal proximal tubule cells and a concentration-dependent necrotic rather than apoptotic cell death [15,16,17,18,19]. Recently we could provide evidence for cAMP-dependent alterations in Ca 2+ -signaling by CAA in human proximal tubule cells in primary culture [20].…”

Carboplatin-induced Fanconi-like syndrome in rats: Amelioration by pentoxifylline

“…Fanconi syndrome is often observed in patients undergoing treatment with nephrotoxic drugs and other chemical agents. Exposure to some drugs and chemicals like outdated tetracyclines (Hawkins and Brewer, 1993), streptozotocin (Foreman and Roth, 1989), gentamicin (Melnick et al, 1994), cystine (Ben-Nun et al, 1993), valproic acid (Lande et al, 1993), 4-pentenoate (Pouliot et al, 1992), heavy metals (Fleck et al, 2001), ifosfamide (Burk et al, 1990;Mohrmann et al, 1993), maleic acid (Harrison and Harrison, 1954), and succinyl acetone (Wyss et al, 1992) lead to Fanconi-like condition. The characteristic feature of Fanconi syndrome is dysfunction of amino acid reabsorption which resembles the early phase of drug-induced nephrotoxicity (Burk et al, 1990).…”