Oral Presentation Society of Obstetric Medicine of Australia and New Zealand ASM 2023

Necroptotic markers in urinary extracellular vesicles of women with pre-eclampsia compared with normotensive pregnancies (#8)

Vanessa C Heron 1 2 3 , Marina Katerelos 1 , Aspasia Pefanis 4 5 , Mia Leung 1 3 , Suet-Wan Choy 1 2 6 , Peter Mount 1 3 , Kathy Paizis 1 2 7
  1. Department of Nephrology, Austin Health, Heidelberg, Victoria, Australia
  2. Department of Obstetrics, Mercy Hospital for Women, Heidelberg, Victoria, Australia
  3. Department of Medicine, University of Melbourne, Parkville, Victoria, Australia
  4. Immunology Research Centre, St Vincent’s Hospital, Melbourne, Victoria, Australia
  5. Department of Nephrology, St Vincent’s Hospital, Melbourne, Victoria, Australia
  6. Department of Renal Medicine and Department of Obstetric Medicine, Eastern Health, Box Hill, Victoria, Australia
  7. Joan Kirner Women’s and Children’s Hospital, Western Health, St Albans, Victoria, Australia

Background:  Urinary extracellular vesicles (uEVs) carry molecular cargo between cells and their analysis can help evaluate the physiologic state of their parent cell.(1) Necroptosis is a regulated process of cell death and has been found to contribute to kidney damage in several diseases.(2) While proteinuria and acute kidney injury are potential renal manifestations of pre-eclampsia (PE), the mechanisms by which these changes occur are incompletely understood.

 

Aim:  To determine whether there is differential expression in necroptotic markers from uEVs of women with PE compared to women with normotensive pregnancies (NP).

 

Methods:  A cross-sectional study was undertaken whereby urine samples were collected from 10 women with PE and 10 women with NP, matched to gestation. uEVs were isolated using ultracentrifuge as described by Wolley et al.(3)

Western Blot was used to confirm the presence of uEVs and necroptotic markers. Necroptotic antibodies analysed included mixed lineage domain-like pseudokinase (MLKL), phosphorylated MLKL (phospho-MLKL), total receptor-interacting protein kinase-3 (RIPK3) and phosphorylated RIPK3 (phospho-RIPK3). Human kidney lysate was used as a positive control.

 

Results:  Women in the PE group tended to be older (mean age 34.29 ± 4.00 years versus 32.16 ± 4.38 years) and have a higher BMI (33.52 ± 6.96 kg/m2 versus 27.93 ± 6.54 kg/m2), however, these differences were not statistically significant (p = 0.272 and p = 0.081 respectively). There was no difference between the mean gestation of the PE group (33.46 ± 2.85 weeks) and NP group (33.97 ±  2.82 weeks) (p = 0.690).

There was increased expression of phospho-MLKL in PE compared with NP samples (70% versus 10%, p = 0.004). However, there was no difference in MLKL expression between groups (20% in each group, p = 1). There was no difference in the presence of RIPK3 or phospho-RIPK3, with both antibodies being detected in a single PE sample and no NP samples (p = 0.331). All necroptotic antibodies tested were identified in the human kidney lysate control.

 

Conclusions:  This small study found altered expression of phospho-MLKL in the uEVs of pre-eclamptic women compared to those with unaffected pregnancies, but no difference in the expression of other necroptotic markers. Phospho-MLKL is a specific marker of necroptosis and these findings suggest the necroptotic pathway may regulate the kidney injury seen in women with pre-eclampsia. These findings need to be further investigated and validated in a larger cohort.

  1. van Balkom BWM, Pisitkun T, Verhaar MC, Knepper MA. Exosomes and the kidney: prospects for diagnosis and therapy of renal diseases. Kidney Int. 2011;80(11):1138–45.
  2. Sanz AB, Sanchez-Niño MD, Ramos AM, et al. Regulated cell death pathways in kidney disease. Nat Rev Nephrol. 2023;19:281–99.
  3. Wolley MJ, Wu A, Xu S, Gordon RD, Fenton RA, Stowasser M. In Primary Aldosteronism, Mineralocorticoids Influence Exosomal Sodium-Chloride Cotransporter Abundance. J Am Soc Nephrol. 2017;28(1):56-63.