Mohrmann M, Pauli A, Ritzer M, Schönfeld B, Seifert B, Brandis M
Kinderklinik, Albert-Ludwigs-Universität Freiburg, BRD.
Ren Physiol Biochem. 1992 Nov-Dec;15(6):289-301. doi: 10.1159/000173465.
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) induced 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 l-alanine and D-glucose in LLC-PK1 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 mumol/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 mumol/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 mumol/l. Sodium-coupled glucose and alanine transport is inhibited by acrolein at concentrations higher than 50 mumol/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 amino-peptidase, 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.
异环磷酰胺（IF）是一种源自氮芥的细胞毒性烷化剂。除了其众所周知的泌尿毒性作用（出血性膀胱炎）外，已有多例IF治疗后发生范科尼综合征的病例报道。关于这种肾小管毒性的发病机制尚无相关信息。我们使用永生化肾上皮细胞系LLC-PK1来研究IF的主要代谢产物（即4-羟基异环磷酰胺、丙烯醛和氯乙醛）是否会诱导在IF体内治疗后最常检测到的转运缺陷。传代162 - 177代、在塑料培养皿中生长至汇合状态的LLC-PK1细胞用于实验。通过标准技术测定L-[3H]丙氨酸和D-[3H]葡萄糖的钠依赖性和非依赖性通量。在细胞匀浆中测定顶端和基底外侧膜、线粒体和内质网的标记酶活性。IF本身对LLC-PK1细胞中L-丙氨酸和D-葡萄糖的通量没有可检测到的影响。在细胞暴露于100μmol/L的4-羟基异环磷酰胺24小时后，IF代谢产物4-羟基异环磷酰胺对两种底物的钠依赖性通量均有明显抑制作用。氯乙醛对L-丙氨酸的钠依赖性通量诱导双相反应，在低浓度（<200μmol/L）且孵育时间短时摄取率增加，而较高浓度和长时间细胞暴露则导致钠偶联转运减少。葡萄糖转运受到类似影响，然而，与丙氨酸转运不同的是，氯乙醛还刺激葡萄糖的非钠依赖性通量。丙烯醛是所测试的毒性最大的物质。在浓度超过75μmol/L时，它会严重破坏细胞单层。在浓度高于50μmol/L时，丙烯醛会抑制钠偶联的葡萄糖和丙氨酸转运。钠偶联的葡萄糖转运对所有测试代谢产物比丙氨酸转运更敏感。虽然丙烯醛强烈影响两种转运系统，但顶端质膜的标记酶，即碱性磷酸酶和亮氨酸氨肽酶，未受到明显抑制，这表明毒性作用对转运蛋白具有特异性。我们得出结论，LLC-PK1细胞是进一步研究IF治疗后范科尼综合征发病机制的良好模型。钠依赖性转运系统对丙烯醛比其他细胞表面蛋白更敏感。
