Inhibition of Sodium-Dependent Transport Systems in LLC-PK₁ Cells by Metabolites of Ifosfamide

Abstract

Ifosfamide (IF) is an alkylating cytostatic derived from nitrogen mustard. In addition to its well-known urotoxic effects (hemorrhagic cystitis), several cases of Fanconi syndrome following IF therapy have been reported. No information is available concerning the pathomechanisms of this tubulotoxicity. We used the permanent renal epithelial cell line LLC-PK₁ in order to investigate whether major metabolites of IF (i.e. 4-OH-IF, acrolein and chloracetyldehyde) induce the transport defects most frequently detected after IF therapy in vivo. LLC-PK₁ cells of passages 162-177, grown in plastic culture dishes, were used in a confluent state. Sodium-dependent and independent fluxes of l-[³H]alanine and of D-[³H] glucose 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 itself has no detectable effect on fluxes of /-alanine and D-glucose in LLC-PK, cells. The IF metabolite 4-OOH-IF induces a clear inhibition of sodium-dependent fluxes of both substrates after a 24-hour exposure of cells to 100 µmol/l of 4-OOH-IF. Chloracetaldehyde induces a biphasic response of sodium-dependent fluxes of l-alanine with increased uptake rates at low concentrations ( < 200 µmol/l) and with a short incubation time, while higher concentrations and long exposure of the cells leads to a reduction in sodium coupled transport. Glucose transport is affected in a comparable way, however, in contrast to alanine transport, chloracetaldehyde also stimulates sodium-independent fluxes of glucose. Acrolein is the most toxic substance tested. It severely damages cell monolayers at concentrations beyond 75 µmol/l. Sodium-coupled glucose and alanine transport is inhibited by acrolein at concentrations higher than 50 µmol/l. Sodium-coupled glucose transport is more sensitive to all metabolites tested than alanine transport. While acrolein strongly affects both transport systems, marker enzymes of the apical plasma membrane, i.e. alkaline phosphatase and leucine aminopeptidase, are not significantly inhibited, suggesting a specificity of the toxic effect for the transport proteins. We conclude that LLC-PK₁ cells represent a good model for further investigation of the pathogenesis of Fanconi syndrome after IF therapy. Sodium-dependent transport systems are more sensitive to acrolein than other cell surface proteins.

CONTRIBUTION OF CYP3A5 TO HEPATIC AND RENAL IFOSFAMIDEN-DECHLOROETHYLATION

“…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).…”

Effect of Ifosfamide Metabolites on Sodium-Dependent Phosphate Transport in a Model of Proximal Tubular Cells (LLC-PK₁) in Culture

“…In a previous paper we could show that the IF metabolites 4-hydroxyifosfamide (4-OH-IF). chloracétaldéhyde (CAA), and acrolein lead to a reduction of sodium-dependent transport rates of L-alanine and Dglucose in renal epithelial cells in culture with proximal tubular properties (LLC-PK,) [15]. While hyperaminoaciduria and glucosuria in patients with tubular transport defects usually have little clinical importance, renal losses of phosphate can result in the development of rickets which has been observed in children after IF therapy [11].…”

Ifosfamide and Mesna: Effects on the Na/H Exchanger Activity in Renal Epithelial Cells in Culture (LLC-PK₁)

“…In our previous work, we have shown that the activities o f the sodiumdependent transport systems for ¿-alanine, Dglucose [7], and phosphate [X] are altered bv all metabolites tested. However, the dose-response curves for the different transport systems show major differences, and the Na/H exchange clearly has the highest sensitivity to 4-OH-IFthe central metabolite o f IF and to CAA which is a major metabolite o f IF but not of its structural isomer CP.…”

“…Another important issue is the role of the uroprotectant Mesna which prevents hemorrhagic cystitis, while tubular damage may still develop. In order to investigate the pathomechanism of IF-induced tubular toxicity, we established an in vitro model of Fanconi syndrome in renal tubular cells in culture (LLC-PK,) [6][7][8][9]. We could show that metabo lites of IF have cytotoxic effects in LLC-PK.)…”