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

Abstract

Ifosfamide (IF) is an alkylating cytostatic with urotoxic and tubulotoxic side effects which may result in the development of Fanconi syndrome in children. While the urotoxicity of IF is effectively prevented by the uroprotective thiol compound sodium-2-mercaptoethanesulfonate (Mesna), tubulotoxicity of IF may occur even in the presence of Mesna and in the absence of any signs of urotoxicity. Using the renal tubular cell line LLC-PK₁, we investigated whether there is a protective effect of Mesna or of its major dimeric metabolite Dimesna against metabolites of IF with respect to the Na/H exchanger activity. We tested the major IF metabolites 4-hydroperoxy-IF (4-OOH-IF), chloroacetaldehydc (CAA), and acrolein. All metabolites significantly inhibit the Na/H exchanger activity. Half-maximal inhibition of transport occurs at concentrations of 120 µmol/l (4-OOH-IF), 80 (CAA), and 60 µmol/l (acrolein) after 2 h of incubation. The onset of the inhibitory effect of all three metabolites is rapid. Complete inhibition of Na/H exchange by acrolein and CAA is present after a 6-hour exposure to 100 µmol/l of the respective metabolite, while 100 µmol/l 4-OOH-IF causes only 50% inhibition after 24 h of incubation. Dimesna, which the proximal tubular cell has to reduce to Mesna at the expense of intracellular glutathione before it exerts a uroprotective effect, has no protective effect in LLC-PK₁ cells. Dimesna (0.3 mmol/l) displaces the dose-response curve for acrolein to the left, indicating an increased toxicity of the combination of acrolein plus Dimesna. Mesna (0.3 mmol/l) has a complete protective effect with respect to acrolein and CAA, while the protective effect versus 100 µmol/l of 4-OOH-IF is incomplete. We conclude that the function of the Na/H exchanger in LLC-PK₁ cells is altered by metabolites of IF. The incomplete protection against the toxic effect of 4-OOH-IF by Mesna may explain the pathomechanism by which IF causes tubulotoxicity in the absence of urotoxicity.

Ifosfamide toxicity in cultured proximal renal tubule cells

“…This chemical is highly reactive, forming damaging adducts with sulfhydryl groups of proteins at low concentrations. Mohrmann et al [20,21] have shown that exposure of LLC-PK 1 cells to CAA significantly impairs several sodium-dependent transport systems as well as the sodium/proton antiport system. Recent work by Benesic et al [22] has demonstrated that CAA produces sustained elevations of intracellular free calcium in proximal tubule cells, by inhibiting sodium/calcium exchanger activity.…”

Toxicity of ifosfamide and its metabolite chloroacetaldehyde in cultured renal tubule cells

“…Both mesna and dimesna are cleared frmn the circulation by glomerular filtration. Dimesna is reabsorbed in the proximal nephron, reduced to mesna and secreted back into the urine where it combines with reactive ifosfamide metabolites to form stable nontoxic thioether compounds, thus preventing hemorrhagic cystitis, The fact that mesna prevents CAA nephrotoxicity in vitro but not in vivo suggests that the intracellular concentration of this uroprotectant is not large enough to neutralize toxic metabolites within renal tubule cells (7,10,11,18). Mohrmann et al (11) have shown that LLC-PKa cells are unable to convert dimesna to mesna.…”

End-Stage Renal Interstitial Fibrosis in an Adult Ten Years after Ifosfamide Therapy

“…Concomitant treatment with Mesna reduces the incidence of hemorrhagic cystitis following ifosfamide therapy [2], however it may be noted that Mesna has an incomplete protective effect against nephrotoxicity in isolated renal cells [19]. Recent studies have however suggested a renoprotective effect of glycine in rats receiving intraperitoneal ifosfamide [20].…”