Phosphorylated MLKL is increased in urinary extracellular vesicles of women with preeclampsia.

OBJECTIVES: This study investigated whether necroptosis-associated proteins are elevated in urinary extracellular vesicles (uEVs) from women with preeclampsia (PE) compared with normotensive pregnancies (NP), and explored uEV origin. STUDY DESIGN: Urine was collected from 22 women with PE and 22 gestation-matched NP controls (Cohort A). A separate cohort of four PE samples with heavy proteinuria, gestation-matched to NP controls, was examined (Cohort B). uEVs were isolated using differential ultracentrifugation. MAIN OUTCOME MEASURES: Western blot determined presence of necroptotic markers, total mixed lineage kinase domain-like protein (MLKL) and phosphorylated MLKL (pMLKL), together with small EV markers CD9 and TSG101. Placental alkaline phosphatase (PALP) and the renal cotransporter NKCC2 were used to assess EV organ origin. Nanoparticle tracking analysis (NTA) characterised uEVs. RESULTS: uEV pMLKL was detected more frequently in the PE group than NP controls (6/22 versus 0/22, P = 0.021). There was a non-significant increase in total MLKL in PE (10/22 versus 5/22, P = 0.203). PALP was detected in 2/12 PE and 1/12 NP samples, suggesting a placental contribution to uEVs in some participants. NTA confirmed uEVs in a subset of samples, with no significant differences in size or concentration between groups. In heavily proteinuric PE subjects (Cohort B), pMLKL was detected in 3/4 samples. CONCLUSIONS: pMLKL was detected more frequently in uEVs from women with PE. Detection of PALP in a subset of uEVs suggests possible placental contribution to the uEV pool. These findings are consistent with increased necroptosis-related signalling in the urinary tract/kidney in women with PE.