Platinum complex toxicity in cultured renal epithelia.
BACKGROUND
Cisplatin is a potent antitumor drug but its clinical use is limited by nephrotoxic side effects. We have found recently, that nephrotoxicity of platinum complexes is related to basolateral organic cation transport. In this study, effects of cell culture conditions on platinum complex toxicity and organic cation transport were investigated by an in vitro system that utilizes the high TransEpithelial Electrical Resistance (TEER) of the C7-clone of the MDCK-(Madin-Darby-Canine-Kidney) cells.


METHODS
TEER and caspase-3 activity of cells in microfilter membrane cups were measured after exposure of apical or basolateral membranes to 100 microM cis-, oxali-, or carboplatin. Caspase-3 activity after platinum complex exposure and uptake of the cation ASP+ (4-(4-(diethylamino)styryl)-N-methylpyridinium) of cells on filter membranes and impermeable supports (e.g. culture flasks) were compared. Atomic Force Microscopy (AFM) was used to depict morphometric differences between both culture conditions.


RESULTS
In cells on filter membranes, cis-, oxali- and carboplatin induced loss of epithelial monolayer integrity by apoptosis via activation of caspase-3 to different extents. Basolateral application of platinum complexes enhanced toxicity dramatically and uptake of ASP+ from the basolateral side was higher than from the apical medium compartment. Intracellular accumulation of ASP+ was less distinct in cells grown on impermeable supports. Only cisplatin, the most lipophilic investigated complex, induced activation of caspase-3 in these cells. AFM disclosed more prominent cell-cell contacts in cells grown on filter membranes.


CONCLUSIONS
We conclude that toxicity of hydrophilic substances can be underestimated in cells grown on solid supports, if basolateral transport mechanisms are involved. We suggest that unhindered access to basolateral transporters is responsible for higher levels of organic cation uptake and apoptosis in cells on filter membranes, even though more prominent cell-cell contacts indicate a better barrier function.
Ludwig
Thomas
T
Institut für Physiologie II, Nanolab, Westfälische Wilhelms-Universität Münster, Germany. thomas.ludwig@uni-muenster.de
Oberleithner
Hans
H
D000818:Animals; D000970:Antineoplastic Agents / Q000633:toxicity*; D001692:Biological Transport / Q000502:physiology; D053148:Caspase 3; D020169:Caspases / Q000032:analysis / Q000378:metabolism; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002945:Cisplatin / Q000633:toxicity; D004285:Dogs; D017097:Electric Impedance; D004847:Epithelial Cells / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure; D007684:Kidney Tubules / Q000166:cytology / Q000187:drug effects*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D018625:Microscopy, Atomic Force; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D017671:Platinum Compounds / Q000633:toxicity*; D062826:Zonula Occludens-1 Protein
D000970:Antineoplastic Agents; D008565:Membrane Proteins; D010750:Phosphoproteins; D017671:Platinum Compounds; D062826:Zonula Occludens-1 Protein; D053148:Caspase 3; D020169:Caspases; D002945:Cisplatin