Participation of Aquaporin-2 during apoptosis in renal cells.

MP. FLAMENCO; L. GALIZIA; V. RIVAROLA; P. FORD; C. CAPURRO

Montevideo, Uruguay

Congreso; 5th Southern Cone Biophysical Congress; 2007

Biophysical Society and SAB

The earliest event in apoptosis is the efflux of K+ from the cell which generates an osmotic gradient that draws water out causing cell shrinkage (apoptotic volume decrease; AVD). Despite the importance of AVD, little is known about the pathways involved in water efflux. The aim of this work was to investigate the putative participation of aquaporins (AQPs) during apoptosis. Two collecting duct cell models were used: WT-RCCD1 not expressing AQPs and AQP2-RCCD1 which have a constitutive expression of AQP2. Apoptosis was induced either by incubating cells up to 24 h with a hypertonic mannitol solution or up to 7 h with 10 ìg.ml-1cycloheximide (CHX). Apoptosis induction was evaluated by DNA ladder analysis, fluorescence stain and flow cytometry. The osmotic water permeability (Pf) was measured by fluorescence videomicroscopy. Results show that in the presence of apoptotic stimuli the number of cells with nuclear condensation was significantly higher in cells expressing AQP2. The time-dependence of the apoptotic rate was then evaluated. WT cells exposition to mannitol gradually increases apoptosis in a time and concentration-dependent manner. Contrary, in AQP2 cells the rate of apoptosis abruptly increases. Even more the % of apoptosis was significantly higher in AQP2 cells, compared to WT cells, both at each evaluated time and concentration. CHX incubation significantly increased the rate of apoptosis rising up to the maximum at 5 h in both cell lines. However, the maximal apoptotic rate was higher in AQP2 cells (AQP2 vs. WT: 169 ± 14 vs. 109 ± 8, p < 0.01, n=6). Furthermore, in AQP2 cells CHX decrease Pf reaching values similar to those of WT cells at 5 h. Conversely, no differences were observed in Pf values of WT cells, at the different times exposition to CHX. We conclude that the high values of Pf in AQP2 cells would be necessary for a cell to rapidly undergo the AVD. Even more, the possibility to decrease Pf, by inactivation of AQP2, may be critical to allow intracellular K+ to be reduced to levels necessary to activate the apoptotic enzymatic cascade.