title: Cell cycle delay and apoptosis are induced by high salt and urea in renal medullary cells.
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 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 at 550 mosmol/kg the effect reverses and most cells are in G(1). 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 G(2)M 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 G(0)/G(1) and 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 G(2)M and G(1), and by inducing apoptotic cell death.
authors.affiliation: Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892-1603, USA. michael@gwgate.nhlbi.nih.gov
authors.forename: L
authors.initials: L
authors.lastname: Michea
authors.forename: D R
authors.initials: DR
authors.lastname: Ferguson
authors.forename: E M
authors.initials: EM
authors.lastname: Peters
authors.forename: P M
authors.initials: PM
authors.lastname: Andrews
authors.forename: M R
authors.initials: MR
authors.lastname: Kirby
authors.forename: M B
authors.initials: MB
authors.lastname: Burg
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002452:Cell Count / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D016195:G2 Phase / Q000187:drug effects*; D006801:Humans; D007679:Kidney Medulla; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects*; D051379:Mice; D012965:Sodium Chloride / Q000494:pharmacology*; D014508:Urea / Q000494:pharmacology*
chemical_list: D012965:Sodium Chloride; D014508:Urea