Response of IMCD3 cells to hypertonic challenges as analyzed by electron microscopy.

This work defines the ultrastructural responses of immortalized cells from the inner medullary collecting duct cells (IMCD3 cells) to hypertonic challenges. The cultured cells were either acutely exposed to hypertonic medium (550 mOsm/kgH₂O) for 24-72 h or gradually adapted to 600 or 900 mOsm/kgH₂O media with sodium chloride. After short (24 h) hypertonic challenges, there was an expansion of the Golgi apparatus with distinct expression of the γ subunit of Na,K-ATPase. The frequency of active caspase-3-positive cells was unchanged as was also the measured activity of caspase-3. Immunoelectron microscopy showed that active caspase-3 in the positive cells was localized in cytoplasmic bodies 0.5-1 μm in diameter but not in other structures. Apoptotic bodies with the nuclei were only rarely observed following acute hypertonicity for 24 to 72 h. Following prolonged hypertonic challenges, some cells showed condensation of the chromatin but still few apoptotic bodies. Gradual hypertonicity to 900 mOsm/kgH₂O led to a decrease of microvilli, dilated cisternae of the endoplasmic reticulum (ER), increased abundance of free ribosomes and longitudinal mitochondrial cristae. Virus particles were present inside and outside the cells in all experimental conditions and appeared unrelated to the apoptotic process. The results suggest that cultured IMCD3 cells are resistant to short hypertonic challenge or gradual adaptation to moderate hypertonicity and only rarely exhibit more ultrastructural apoptotic changes than control cells. The presence of caspase-3-containing bodies is a novel finding, and we suggest that they arise from the ER and are involved in the apoptotic signaling system.