Cell cycle delay and apoptosis are induced by high salt and urea in renal medullary cells

We investigated the effects of hyperosmolality on survival and proliferatio n of subconfluent cultures of mIMCD3 mouse renal collecting duct cells. Hig h NaCl and/or urea (but not glycerol) reduces the number of viable cells, a s measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromi de (MTT). Raising osmolality from a normal level (300 mosmol/kg) to 550-1,0 00 mosmol/kg by adding NaCl and/or urea greatly increases the proportion of cells in the G(2)M phase of the cell cycle within 8 h, as measured by flow cytometry. Up to 600 mosmol/kg the effect is only transient, and by 12 h a t 550 mosmol/kg the effect reverses and most cells are in G(1). Flow cytome try with 5-bromodeoxyuridine (BrdU) pulse-chase demonstrates that movement through the 8 phase of the cell cycle slows, depending on the concentration s of NaCl and/or urea, and that the duration of G(2)M increases greatly (fr om 2.5 h at 300 mosmol/kg to more than 16 h at the higher osmolalities). Ad dition of NaCl and/or urea to total osmolality of 550 mosmol/kg or more als o induces apoptosis, as demonstrated by characteristic electron microscopic morphological changes, appearance of a subdiploid peak in flow cytometry, and caspase-3 activation. The number of cells with subdiploid DNA and activ ated caspase-3 peaks at 8-12 h. Caspase-3 activation occurs in all phases o f the cell cycle, but to a disproportionate degree in G(0)/G(1) and 8 phase s. We conclude that elevated NaCl and/or urea reduces the number of prolife rating mIMCD3 cells by slowing the transit through the 8 phase, by cell cyc le delay in the G(2)M and G(1), and by inducing apoptotic cell death.