Introduction: Preeclamptic placentas are associated to an abnormal trophoblast invasion of the spiral arteries resulting in an inadequate utero-placental blood flow. Consequently, an intermittent perfusion is established which leads to an ischemia/reperfusion injury in the syncytiotrophoblast. Fluctuations in O2 tensions may increase the generation of reactive nitrogen species (RNS) by promoting the formation of peroxynitrite (ONOO-) which can modify tyrosine groups of proteins giving rise to the formation of 3-nitrotyrosine (3-NTp). This, can damage lipids,
proteins and DNA. Previously we reported that the expression of AQP9 significantly increased in preeclamptic placentas and in normal placental explants exposed to hypoxia/reoxygenation. In both cases, the protein was not functional for water transport. However, the mechanisms involved in the increase of this protein remain unknown. Objective: Our aim was to evaluate the effect of nitrosative stress on placental AQP9 expression and the formation of the 3-NTp-AQP9 in normal and preeclamptic placentas. Methods: This study was approved by the ethics committee of the Hospital Nacional Dr. Prof. A. Posadas. Placental explants were cultured in complete DMEM-F12. In normal placenta explants, nitrosative stress was induced by 100 μM ONOO-. Lipid peroxidation (TBARS) was determined. Expression of superoxide dismutase 1 (SOD-1), as a measure of the antioxidant enzyme defense system, and AQP9 were also studied. The protein level of AQP9 with the 3-NTp post-translational modification was quantified in the trophoblast tissues from preeclamptic placenta and explants exposed to ONOO-. An immunoprecipitation assay, with 3-Nytrotyrosine antibody attached to the column was used to immunoprecipitate the total nitrated proteins. The levels of AQP9 were analyzed in the eluted samples by western blot. Results: We found that the abundance of nitrated AQP9 increased 1.5-fold in preeclamptic placentas compared with normal placentas. In normal placental explants culture in the presence of 100 μM of ONOOto induced nitrosative stress, the expression of AQP9 also significantly increased 75.42 ± 0.75% compared to control (n=7; p<0.05). In this condition, TBARS increased 3-fold (n=5, p<0.002), while SOD-1 showed a 70.35 ± 16.51% decrease (n=5, p<0.02). Conclusions: Our results show that the production of RNS could be the responsible of the increase of AQP9 expression in preeclamptic placenta, due to the formation of 3-NTp-AQP9. However, further studies are required to elucidate whether the high nitric oxide levels could result in a non-functional protein and the implications in the pathogenesis of preeclampsia.