Placental programmed cell death: insights into the role of aquaporins.

SZPILBARG NATALIA; CASTRO-PARODI MAURICIO; REPPETTI, JULIETA; REPETTO, MARISA; MASKIN BERNARDO; MARTINEZ, NORA; DAMIANO, ALICIA E

MOLECULAR HUMAN REPRODUCTION.

OXFORD UNIV PRESS

Lugar: Oxford; Año: 2016 vol. 22 p. 46 - 56

1360-9947

Study hypothesis: Are the placental AQPs involved in the apoptosis of trophoblast? Study finding: The general blocking of placental AQPs with HgCl2, and in particular, the blocking of AQP3 activity with CuSO4, abrogated the apoptotic events of the trophoblast cells. What is known already: Although apoptosis of trophoblast cells is a natural event involved in the normal development of the placenta, it is exacerbated in pathological processes as preeclampsia, where we found an abnormal expression and functionality of placental AQPs without alterations in the feto-maternal water flux. Furthermore, fluctuations in O2 tension are proposed to be a potent inducer of placental apoptotic changes and, in explants exposed to hypoxia/reoxygenation, transcellular water transport mediated by AQPs was undetectable. This suggests that AQPs might be involved in other processes besides water transport, such as apoptosis.Study design, samples/materials, methods: This study was approved by the ethics committee of the Hospital Nacional Dr. Prof. Alejandro Posadas, Argentina and written consent was obtained from the patients. We cultured explants from normal term placentas (n=15) in normoxia, hypoxia, and hypoxia/reoxygenation (H/R). MTT incorporation was used to determine cell viability. For the general or specific inhibition of AQPs, 0.3 mM HgCl2, 5 mM CuSO4, 0.3 mM tetraethylammonium chloride or 0.5 mM phloretin, were added to the culture medium before explants were exposed to each treatment. Oxidative stress parameters and apoptotic indexes were evaluated in the presence or absence of AQPs blockers. AQP3 expression was confirmed by Western blot and immunohistochemistry.Main results and the role of chance: First, we observed that, in H/R treatments, cell viability decreased 20.16 ± 5.73 % compared to those explants cultured in normoxia (P = 0.009; n=7). Hypoxia treatment did not modify cell viability significantly. Then, we observed that hypoxia and H/R conditions induced oxidative stress. Spontaneous chemiluminescence and TBARS levels were significantly increased in explants exposed to hypoxia and H/R conditions compared to those cultured in normoxia.Regarding apoptosis, we found that H/R is a more potent inducer of trophoblast apoptosis than hypoxia alone. Bax expression and the number of apoptotic nuclei were significantly higher in explants cultured in H/R compared to normoxia and hypoxia conditions (n = 12, P = 0.0135 and P = 0.001 respectively). Laddering of DNA was only observed in H/R and the highest activity of caspase-3 was found in explants cultured in H/R (n = 12, P = 0.0001).The sterically blocking of AQPs´ activity with HgCl2 showed that DNA degradation was undetectable. In addition, the number of apoptotic nuclei (n = 12, P = 0.001), Bax expression and caspase-3 activity were drastically reduced (n = 12, P = 0.0146 and P = 0.0001, respectively). Similar results were observed after blocking AQP3 with CuSO4. DNA degradation was undetectable, the number of apoptotic nuclei and caspase-3 activity were significantly decreased (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 and localization of AQP3 in explants exposed to H/R.Limitations, reasons for caution: These results explored the role of placental AQPs in the apoptosis events induced by H/R in an in vitro model of explants culture. Our studies are limited by the number of experimental conditions tested, which do not fully capture variability in oxygen levels, duration of exposure, and alternating patterns of oxygen in vivo. Wider implications of the findings: Our results suggest that any alteration in placental AQPs expression might disturb the equilibrium of the normal apoptotic events and may be the underlying cause in the pathophysiology of placental gestational disorders such as preeclampsia. Even more, the dysregulation of placental AQPs may be one of the crucial factors in triggering the clinical manifestations of preeclampsia. Large scale data: n/aStudy funding and competing interest(s): This study was supported by UBACyT 20020090200025 and 20020110200207 grants and PIP-CONICET 11220110100561 grant and the authors have no conflict of interest to declare.