e apical or basolateral side with 5-FAM/AF647 labeled zoledronic acid for 1 hour at either 37 or 4C. Monolayers were then formalin fixed and counterstained with Hoechst 33258. This experiment was performed on monolayers originating from two different kidney specimens. 3 / 19 Renal Handling of Zoledronic Acid In order to check for the fact that the zoledronic acid signal indeed was localized inside the cells, the cell membrane of proximal tubular cells was labeled following incubation with zoledronic acid and fomalin fixation. For this purpose the monolayers were incubated for 20 min with normal donkey serum, and for 2 hours with a mouse monoclonal antibody to human leucine aminopeptidase, a specific proximal tubular cell marker. Subsequently an AF488-labeled donkey anti mouse secondary antibody was used and monolayers were counterstained with Hoechst 33258. Fluorescent signals were evaluated using confocal microscopy and application of Volocity software. Identification of zoledronic acid-containing intracellular vesicles In order to investigate whether the cellular uptake of zoledronic acid took place by fluid phase endocytosis and/or receptor mediated endocytosis, confluent monolayers were co-incubated with AF647-labeled zoledronic acid and FITC-labeled dextran PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19763407 or FITC-labeled albumin, at either the apical or basolateral PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19763871 side at 37C for 1 hour. FITC- labeled dextran and FITC-labeled albumin are established markers of fluid phase and receptor-mediated endocytosis, respectively. This experiment was performed on monolayers originating from two different kidney specimens. Furthermore, confluent monolayers of human tubular kidney cells were incubated with cytochalasin B before exposing cells to AF647-labeled zoledronic acid, or FITClabeled dextran or FITC-labeled albumin. Cytochalasin B, a cell-permeable mycotoxin that strongly inhibits network formation by actin filaments, in general is recognized as an inhibitor of endocytosis. However, cytochalasin B does not affect the uptake of FITC-dextran by fluid phase endocytosis. Fluorescent signals were evaluated using confocal microscopy and application of Volocity software. Measurement of intracellular levels of radiolabeled zoledronic acid The intracellular concentration of zoledronic acid was quantified after creating steady state conditions. Steady state conditions were obtained as described previously, by 
adding the same concentrations of zoledronic acid in pre-warmed Krebs buffer to both the apical and basolateral side of the polarized cell cultures. After 1 hour of incubation, 14C labeled zoledronic acid was added at a concentration of 1Ci/ml to the cell culture medium, at either the apical or basolateral side of the monolayer. After an additional incubation period of 1 hour the permeable supports were cut out and the number of disintegrations per minute was measured in a scintillation counter. Intracellular levels of zoledronic acid are presented as pmoles/cm2. The experiment was performed 4 times in monolayers originating from 4 different kidney specimens. For each experiment at least 5 monolayers/condition were used. Comparison of intracellular radiolabeled zoledronic levels acid to intracellular radiolabeled 1235481-90-9 web mannitol levels On monolayers of 1 kidney, intracellular levels of zoledronic acid were compared to those of mannitol, a molecule which is not internalized by tubular cells. Cellular accumulation of mannitol was investigated by performing an experiment identical to that described
