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

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Abstract
We investigated the effects of hyperosmolality on survival and proliferation of subconfluent cultures of mIMCD3 mouse renal collecting duct cells. High NaCl and/or urea (but not glycerol) reduces the number of viable cells, as measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Raising osmolality from a normal level (300 mosmol/kg) to 550–1,000 mosmol/kg by adding NaCl and/or urea greatly increases the proportion of cells in the G2M 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 at 550 mosmol/kg the effect reverses and most cells are in G1. Flow cytometry with 5-bromodeoxyuridine (BrdU) pulse-chase demonstrates that movement through the S phase of the cell cycle slows, depending on the concentrations of NaCl and/or urea, and that the duration of G2M increases greatly (from 2.5 h at 300 mosmol/kg to more than 16 h at the higher osmolalities). Addition of NaCl and/or urea to total osmolality of 550 mosmol/kg or more also 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 activated caspase-3 peaks at 8–12 h. Caspase-3 activation occurs in all phases of the cell cycle, but to a disproportionate degree in G0/G1and S phases. We conclude that elevated NaCl and/or urea reduces the number of proliferating mIMCD3 cells by slowing the transit through the S phase, by cell cycle delay in the G2M and G1, and by inducing apoptotic cell death.