Lactate is an ideal non-invasive marker for evaluating temporal alterations in cell stress and toxicity in repeat dose testing regimes

Technological developments are driving in vitro methods towards integrated "omic" strategies. However, there is still an over reliance on classical viability assays for dose range finding. Such assays are not readily suited to the investigation of subtle alterations in cell function and most require termination of the experiment, which makes it difficult to monitor temporal alterations in repeat-dose long term exposure experiments. To this end, we investigated the use of lactate production as a marker of cell stress in long term repeat dose experiments. We conducted daily exposures to eight compounds at five concentrations for 14 days on human renal proximal tubular cells (RPTEC/TERT1), human hepatoma cells (HepaRG) and mouse fibroblasts (BALB-3T3) cells. Compounds were chosen from a training set used in the 7th EU Framework project Predict-IV and consisted of amiodarone, diclofenac, troglitazone, cadmium chloride, cephaloridine, cidofovir, cyclosporine A and buflomedil. At days 1, 3, 7 and 14, lactate was measured in the supernatant medium. At day 14, cells were assayed for resazurin reduction capability and subsequently lysed in methanol for ATP determination. Compound-induced loss of viability was comparable across all cell lines. For all cell types, when cell viability was compromised at day 14, lactate production was induced during the treatment period. In some situations, lactate also fell below control values, indicating cell death. Thus, temporal alterations in supernatant lactate provides information on the time and concentration of stress induction and the time and concentration where cell death becomes the dominant factor. Supernatant lactate production is a simple, cheap and non-invasive parameter. Since many molecular pathways converge on the glycolytic pathway, enhanced lactate production may be considered as a global marker of sub-lethal injury and thus an ideal marker for investigating temporal alterations in long term repeat dose testing in vitro regimes

Uromodulin facilitates neutrophil migration across renal epithelial monolayers

The glycosylated protein uromodulin is exclusively found in the thick ascending limb cells (TAL) of the kidney, where it is produced on mass and apically targeted, eventually being secreted into the urine. Recently, there has been a renewed interest in this protein due to its ability to interact with the immune system, implicating this protein as a renal inflammatory molecule. Here we investigated the potential role of membrane bound uromodulin on neutrophil adhesion and trans-epithelial migration. The renal tubular epithelial cell line, LLC-PK1, stably transfected with human uromodulin was used to investigate the influence of uromodulin on neutrophil adherence and migration. Uromodulin expression resulted in a significant increase of neutrophil adherence and trans-epithelial migration, in both the apical to basolateral and the basolateral to apical direction. Although uromodulin is GPI anchored and targeted to the apical membrane, we could also observe expression in the basal and lateral membranes domains, which may be responsible for basolateral to apical migration. Furthermore we show that uromodulin binds both the heavy and light chain of IgG, and that IgG enhances neutrophil migration. This study demonstrates that uromodulin can facilitate neutrophil trans-epithelial migration and that this migration can be amplified by co-factors such as IgG