Preeclampsia is associated with increased oxidative and nitrative stress, resulting in elevated protein nitration and potential functional impairment. Previously, we found an increased expression of AQP9 protein with a loss of function in preeclamptic placentas. However, the link between nitrative stress and AQP9 has not yet been explored. Here, we aimed to evaluate the effect of nitrative stress on placental AQP9 and its role in the pathogenesis of preeclampsia. In silico analysis was conducted on the amino acid sequences of AQP9 to identify potential nitration sites. Levels of 3NyT-AQP9 were assessed by immunoprecipitation in normal and preeclamptic placentas. AQP9 expression and function were evaluated by culturing normal placental explants with 0, 25, 50, 100, and 200 μM ONOO- to induce nitrative stress. Viability and integrity of the explants and stress markers were determined. Water uptake and utilization of lactate mediated by AQP9 were studied along with the molecular expression of AQP9 and 3-NyT-AQP9. The in silico analysis showed that AQP9 is more susceptible to nitration than other AQPs. The abundance of nitrated AQP9 significantly increased in preeclamptic placentas compared to normal ones (n = 4
p <
0.05). Peroxynitrite treatment also increased AQP9 protein expression without altering its gene expression and impaired the transport of water and lactate mediated by this protein. Our findings provide evidence that nitrative stress induces the nitration of AQP9 protein, leading to the accumulation of a non-functional protein in the syncytiotrophoblasts. Therefore, this altered protein may play a pivotal role in the pathogenesis of preeclampsia by disrupting cellular homeostasis.