Methods to Evaluate the Effect of Ethanol on the Folate Analogue: Fluorescein Methotrexate Uptake in Human Proximal Tubular Cells

Ethanol-induced folate deficiency is due to effects of ethanol on folate metabolism and absorption. We have already shown by using different methods that ethanol interferes with reabsorption of folate from the proximal tubule. In this study, we have used the folate analogue, the fluorescein methotrexate (FL-MTX), in order to evaluate effects of ethanol on FL-MTX uptake by the human proximal tubular (HPT) cells by using a confocal microscope and fluoroskan microplate reader. Since endothelins (ETs) play a major role in a number of diseases and also in the damage induced by a variety of chemicals, we have used endothelin-B (ET-B) and protein kinase-C (PKC) inhibitors to evaluate the role of endothelin in ethanol-mediated FL-MTX uptake by using fluoroskan microplate reader. Confocal microscope and fluoroskan studies reveal that cellular absorption of FL-MTX is concentration-dependent. Moreover, ethanol concentration has an impact on FL-MTX uptake. Fluoroskan studies reveal that the ethanol-induced decrease in FL-MTX uptake is reversed by adding the ET-B receptor antagonist (RES-701-1) or PKC-selective inhibitor (BIM). Thus, we can conclude that ethanol may act via ET and ET in turn may act via ET-B receptor and the PKC signaling pathway to impair FL-MTX transport

Aluminum citrate blocks toxicity of calcium oxalate crystals by preventing binding with cell membrane phospholipids

BACKGROUND/AIMS: Renal damage from ethylene glycol and primary hyperoxaluria is linked to accumulation of calcium oxalate monohydrate (COM) crystals in the renal proximal tubule (PT). In vitro studies have shown that aluminum citrate (AC), uniquely among citrate salts, blocks COM cytotoxicity to tubular cells. These studies were designed to evaluate the interaction of COM with membrane phospholipids and the ability of AC to reduce COM toxicity by interfering with this interaction. METHODS: Interaction of COM with phospholipids was assessed using differential scanning calorimetric analysis of structural changes in specific liposomes. Interaction of COM with cell membranes was studied by measuring binding of radiolabeled crystals by human PT (HPT) cells. RESULTS: Analysis of liposomes prepared from phosphatidylserine (PS) or phosphatidylcholine (PC) showed that COM interfered with the gel-liquid transition of PS liposomes, but not that of PC liposomes. AC reversed the COM-induced changes in liposomal structure. AC inhibited the binding of [(14)C]-COM by HPT cells in a concentration-dependent manner. AC blocked COM binding by interacting with the crystal surface and not the cell membrane. CONCLUSION: These results indicate that AC blocks the binding of COM by PT cells, and consequently its cytotoxicity, by attaching to the surface of the crystal. Thus, AC, or a related compound that works by the same mechanism, could be a useful adjunct therapy to reduce the renal damage produced by severe hyperoxaluria

Aluminum Citrate Blocks Toxicity of Calcium Oxalate Crystals by Preventing Binding with Cell Membrane Phospholipids

BACKGROUND/AIMS: Renal damage from ethylene glycol and primary hyperoxaluria is linked to accumulation of calcium oxalate monohydrate (COM) crystals in the renal proximal tubule (PT). In vitro studies have shown that aluminum citrate (AC), uniquely among citrate salts, blocks COM cytotoxicity to tubular cells. These studies were designed to evaluate the interaction of COM with membrane phospholipids and the ability of AC to reduce COM toxicity by interfering with this interaction. METHODS: Interaction of COM with phospholipids was assessed using differential scanning calorimetric analysis of structural changes in specific liposomes. Interaction of COM with cell membranes was studied by measuring binding of radiolabeled crystals by human PT (HPT) cells. RESULTS: Analysis of liposomes prepared from phosphatidylserine (PS) or phosphatidylcholine (PC) showed that COM interfered with the gel-liquid transition of PS liposomes, but not that of PC liposomes. AC reversed the COM-induced changes in liposomal structure. AC inhibited the binding of [(14)C]-COM by HPT cells in a concentration-dependent manner. AC blocked COM binding by interacting with the crystal surface and not the cell membrane. CONCLUSION: These results indicate that AC blocks the binding of COM by PT cells, and consequently its cytotoxicity, by attaching to the surface of the crystal. Thus, AC, or a related compound that works by the same mechanism, could be a useful adjunct therapy to reduce the renal damage produced by severe hyperoxaluria