This DN to DP transition was also observed by in vitro culture, and was dependent on Lck

zoledronic acid, after which viability was measured. Each data point represents the mean value SD of 4 experiments, performed on cell cultures originating from 4 different kidney specimens. During each of the four experiments at least 4 monolayers/conditions were used. Effect of zoledronic acid incubation on the viability of confluent monolayers of primary human tubular cells on the Acacetin site longer term. Confluent monolayers of primary human tubular cells were incubated with different concentrations of zoledronic acid for 2 hours and cellular viability was measured 4, 24 and 48 hours later. Confluent monolayers of primary human tubular cells were incubated with different concentrations of zoledronic acid for 4, 24 or 48 hours after which cellular viability was measured. Each data point represents the mean value SD of 2 experiments, performed on cell cultures originating from 2 different kidney specimens. During each of the 2 experiments at least 4 monolayers/condition were used. p<0.05 vs 0 M. doi:10.1371/journal.pone.0121861.g001 7 / 19 Renal Handling of Zoledronic Acid Fig 2. Confluent monolayers of primary human tubular kidney cells were incubated for 1 hour with AF647 or FAM labeled zoledronic acid formalin fixed and counterstained with Hoechst resulting in blue stained cellular nuclei. Monolayer A and B were incubated at 37C, PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19763871 Cand D at 4C. doi:10.1371/journal.pone.0121861.g002 check for a common uptake pathway of both bisphosphonate molecules. Since intracellular levels of zoledronic acid did not change in the presence of an excess of the organic anion transporter substrates PAH and E-3S, neither did the administration of pamidronate exert any effect on the intracellular levels of zoledronic acid no arguments were obtained for zoledronic acid uptake by organic anion transporters nor for a common uptake route of both bisphosphonates. Transepithelial transport of zoledronic acid We next investigated whether cellular accumulation of zoledronic acid went along with net transcellular zoledronic acid fluxes. Fluxes directed from either the apical to the basolateral side or the basolateral to the apical side were measured in monolayers originating from 4 different kidney specimens. Results showed fluxes PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19761586 in both directions to be almost equal to each other, suggesting that zoledronic acid transport takes place by the paracellular route. This was further confirmed by the finding that the transepithelial fluxes of zoledronic acid were not higher than those of mannitol. Concentration dependence of tubular handling of zoledronic acid To check the possible effect of the zoledronic acid concentration on its transport and intracellular accumulation cell cultures were incubated with different concentrations of the compound ranging from 0.25 to 100 M. Results in Fig. 10A show that fluxes from the apical to the basolateral side and from the basolateral to the apical side did not differ from 8 / 19 Renal Handling of Zoledronic Acid Fig 3. A confluent monolayer of primary human tubular kidney cells was incubated for 1 hour with AF 647- labeled zoledronic acid at 37C, formalin fixed, immunostained for the proximal tubular membrane marker leucine aminopeptidase and counterstained with Hoechst. doi:10.1371/journal.pone.0121861.g003 each other over the whole concentration range. Moreover, the finding that no plateau is reached, provides an additional indication that the transport exclusively takes place by the paracellular way. The effect of the zoledronic acid con