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 γ-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; γ-ENaC (two different epitopes within the inhibitory peptide tract), α-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 α-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, ~90-100 kDa full-length γ-ENaC was detected while no full-length γ-ENaC but 75, 60, and 37 kDa variants dominated in voided urine exosomes. There was no difference in γ-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 α-ENaC subunit while γ-subunit appeared predominantly in cleaved form independently of conditions and with a significant contribution from post-renal cleavage.

The epithelial sodium channel γ-subunit is processed proteolytically in human kidney.

The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the γ-subunit and putative release of a 43-amino acid inhibitory tract from the γ-subunit ectodomain. We hypothesized that proteolytic processing of γENaC occurs in the human kidney under physiologic conditions and that proteinuria contributes to aberrant proteolytic activation. Here, we used monoclonal antibodies (mAbs) with specificity to the human 43-mer inhibitory tract (N and C termini, mAbinhibit, and mAb4C11) and the neoepitope generated after proteolytic cleavage at the prostasin/kallikrein cleavage site (K181-V182 and mAbprostasin) to examine human nephrectomy specimens. By immunoblotting, kidney cortex homogenate from patients treated with angiotensin II type 1 receptor antagonists (n=6) or angiotensin-converting enzyme inhibitors (n=6) exhibited no significant difference in the amount of full-length or furin-cleaved γENaC or the furin-cleaved-to-full-length ratio of γENaC compared with homogenate from patients on no medication (n=5). Patients treated with diuretics (n=4) displayed higher abundance of full-length and furin-cleaved γENaC, with no significant change in the furin-cleaved-to-full-length γENaC ratio. In patients with proteinuria (n=6), the inhibitory tract was detected only in full-length γENaC by mAbinhibit. Prostasin/kallikrein-cleaved γENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney γENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin/kallikrein site and removal of the inhibitory tract within γENaC.

A novel mutation affecting the arginine-137 residue of AVPR2 in dizygous twins leads to nephrogenic diabetes insipidus and attenuated urine exosome aquaporin-2.

Mutations in the vasopressin V2 receptor gene AVPR2 may cause X-linked nephrogenic diabetes insipidus by defective apical insertion of aquaporin-2 in the renal collecting duct principal cell. Substitution mutations with exchange of arginine at codon 137 can cause nephrogenic syndrome of inappropriate antidiuresis or congenital X-linked nephrogenic diabetes insipidus. We present a novel mutation in codon 137 within AVPR2 with substitution of glycine for arginine in male dizygotic twins. Nephrogenic diabetes insipidus was demonstrated by water deprivation test and resistance to vasopressin administration. While a similar urine exosome release rate was shown between probands and controls by western blotting for the marker ALIX, there was a selective decrease in exosome aquaporin-2 versus aquaporin-1 protein in probands compared to controls.