PLACENTAL PROGRAMMED CELL DEATH: INSIGHTS INTO THE ROLE OF AQUAPORINS

SZPILBARG N.; CASTRO PARODI M.; REPPETTI J.; REPETTO M.; MASKIN B.; MARTÍNEZ N.; DAMIANO A.

MOLECULAR HUMAN REPRODUCTION.

OXFORD UNIV PRESS

Lugar: Oxford; Año: 2015 vol. 1 p. 46 - 56

1360-9947

ABSTRACTStudy hypothesis: Are the placental aquaporins (AQPs) involved in the apoptosisof human trophoblast? Study finding: The general blocking of placental AQPswith HgCl2 and, in particular, the blocking of AQP3 activity with CuSO4abrogated the apoptotic events of human trophoblast cells. What is knownalready: Although apoptosis of trophoblast cells is a natural event involved inthe normal development of the placenta, it is exacerbated in pathologicalprocesses, such as pre-eclampsia, where an abnormal expression andfunctionality of placental AQPs occurs without alterations in the feto-maternalwater flux. Furthermore, fluctuations in O2 tension are proposed to be a potentinducer of placental apoptotic changes and, in explants exposed to hypoxia/reoxygenation(H/R), transcellular water transport mediated by AQPs was undetectable. Thissuggests that AQPs might be involved in processes other than water transport,such as apoptosis. Study design, samples/materials, methods: Explants fromnormal term placentas were maintained in culture under conditions of normoxia,hypoxia, and H/R. Cell viability was determined by assessing3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide incorporation.For the general or specific inhibition of AQPs, 0.3 mM HgCl2, 5 mM CuSO4, 0.3mM tetraethylammonium chloride (TEA) or 0.5 mM phloretin were added to theculture medium before explants were exposed to each treatment. Oxidative stressparameters and apoptotic indexes were evaluated in the presence or absence ofAQPs blockers. AQP3 expression was confirmed by Western blot andimmunohistochemistry. Main results and the role of chance: First, we observedthat in H/R treatments cell viability decreased by 20.16 ± 5.73 % compared tothose explants cultured in normoxia (P = 0.009; n=7). Hypoxia did not modifycell viability significantly. Both hypoxia and H/R conditions induced oxidativestress. Spontaneous chemiluminescence and thiobarbituric acid reactivesubstance levels were significantly increased in explants exposed to hypoxia(n= 6 per group, P = 0.0316 and P = 0.0009, respectively) and H/R conditions(n= 6 per group, P = 0,0281 and P = 0,0001, respectively) compared to thosecultured in normoxia. Regarding apoptosis, H/R was a more potent inducer oftrophoblast apoptosis than hypoxia alone. Bax expression and the number ofapoptotic nuclei were significantly higher in explants cultured in H/R comparedto normoxia and hypoxia conditions (n = 12, P = 0.0135 and P = 0.001,respectively). DNA fragmentation was only observed in H/R and, compared tonormoxia and hypoxia, the activity of caspase-3 was highest in explantscultured in H/R (n = 12, P = 0.0001). In explants exposed to H/R, stericblocking of AQP activity with HgCl2 showed that DNA degradation was undetectable(n = 12, P = 0.001). Bax expression and caspase-3 activity were drasticallyreduced (n = 12, P = 0.0146 and P = 0.0001, respectively) compared to explantscultured in H/R but not treated with HgCl2. Similar results were observed inexplants exposed to H/R when we blocked AQP3 activity with CuSO4. DNAdegradation was undetectable and the number of apoptotic nuclei and caspase-3activity were significantly decreased compared to explants cultured in H/R butnot treated with CuSO4 (n = 12, P = 0.001 and P = 0.0001, respectively).However, TEA and phloretin treatments, to block AQP1/4 or AQP9 respectively,failed in abrogate apoptosis. In addition, we confirmed the expression andlocalization of AQP3 in explants exposed to H/R. Limitations, reasons for caution:Our studies are limited by the number of experimental conditions tested, whichdo not fully capture the variability in oxygen levels, duration of exposure,and alternating patterns of oxygen seen in vivo. Wider implications of thefindings: Our results suggest that any alteration in placental AQP expressionmight disturb the equilibrium of the normal apoptotic events and may be anunderlying cause in the pathophysiology of placental gestational disorders suchas preeclampsia. Furthermore, the dysregulation of placental AQPs may be one ofthe crucial factors in triggering the clinical manifestations of pre-eclampsia.