Urine exosomes from healthy and hypertensive pregnancies display elevated level of α-subunit and cleaved α- and γ-subunits of the epithelial sodium channel-ENaC

Preeclampsia is characterized by hypertension, proteinuria, suppression of plasma renin-angiotensin-aldosterone, and impaired urine sodium excretion. Aberrantly filtered plasmin in urine may activate proteolytically the gamma-subunit of the epithelial sodium channel (ENaC) and promote Na+ reabsorption and urine K+ loss. Plasma and urine was sampled from patients with preeclampsia, healthy pregnant controls and non-pregnant women, and from patients with nephrostomy catheters. Aldosterone concentration, urine plasminogen, and protein were determined. Exosomes were isolated by ultracentrifugation. Immunoblotting was used to detect exosome markers; gamma-ENaC (two different epitopes within the inhibitory peptide tract), alpha-ENaC, and renal outer medullary K-channel (ROMK) and compared with human kidney cortex homogenate. Urine total plasmin(ogen) was significantly increased in preeclampsia, plasma and urine aldosterone was higher in pregnancy compared to non-pregnancy, and the urine Na/K ratio was lower in preeclampsia compared to healthy pregnancy. Exosome markers ALIX and AQP-2 were stably associated with exosomes across groups. Exosomal alpha-ENaC-subunit migrated at 75 kDa and dominantly at 50 kDa and was significantly elevated in pregnancy. In human kidney cortex tissue and two of four pelvis catheter urine, similar to 90-100 kDa full-length gamma-ENaC was detected while no full-length gamma-ENaC but 75, 60, and 37 kDa variants dominated in voided urine exosomes. There was no difference in gamma-ENaC protein abundances between healthy pregnancy and preeclampsia. ROMK was detected inconsistently in urine exosomes. Pregnancy and preeclampsia were associated with increased abundance of furin-cleaved alpha-ENaC subunit while gamma-subunit appeared predominantly in cleaved form independently of conditions and with a significant contribution from post-renal cleavage.