Increased phosphorylated MLKL expression in urinary exosomes of women with pre-eclampsia compared to normotensive pregnancies.

Abstract

Aim To determine differential expression in necroptotic markers expressed by urinary exosomes of women with pre-eclampsia (PE) compared to women with normotensive pregnancies (NP).

Methods A cross-sectional study was undertaken whereby urine samples were collected from 22 women with PE and NP, matched to gestation. Exosomes were isolated using differential ultracentrifugation. Expression of necroptotic antibodies mixed lineage domain-like pseudokinase (MLKL) and phosphorylated MLKL (pMLKL) were analysed using western blot. CD9 and tumor susceptibility gene 101 (TSG101) antibodies were used to confirm presence of exosomes. Placental ALP (PALP) and sodium cotransporter NKCC2 expression were examined to determine whether extracellular vesicles originated from the kidney or placenta.

Results There was no difference in mean gestation between the PE (32.92  2.70 weeks) and NP groups (33.10  3.63 weeks) (p=0.854). Women in the PE group had a higher BMI (34.06  7.09 versus 28.9  6.55 kg/m2) (p=0.02). All but two PE samples had exosome markers present, despite MLKL and pMLKL expression in both. These samples were excluded from subsequent analysis. There was increased expression of pMLKL in PE compared with NP samples (20% versus 0%, p=0.043). A trend towards increased total MLKL expression in the PE group was noted (50% versus 22.73%), although this finding was not statistically different (p=0.107). One NP and two PE samples expressed PALP, suggesting some EVs captured in urine may be of placental origin.

Conclusion This study found increased expression of pMLKL in urinary exosomes of women with PE compared to NP, and a trend towards increased MLKL. These findings suggest necroptosis may play a role in the kidney injury seen in PE.

Impact An improved understanding of the mechanisms by which PE affects maternal organs, including the kidney, is essential to improving our ability to predict the onset of PE and finding a targeted treatment.