Urinary Plasmin Activates Collecting Duct ENaC Current in Preeclampsia

Abstract

In nephrotic syndrome, plasminogen is aberrantly filtered from plasma to the urinary space and activated along the tubular system. In vitro, plasmin increases ENaC current by proteolytic cleavage of the γ-subunit. It was hypothesized that preeclampsia is associated with plasmin-dependent ability of tubular fluid to activate ENaC. Urine was sampled from 16 preeclamptic (PE) patients and 17 normotensive pregnant women (Ctrl). Urine was analyzed for plasmin(ogen), creatinine, albumin, aldosterone, Na + , K + , proteolytic activity, and for its effect on inward current in cortical collecting duct cells (M1 cells) by whole-cell patch clamp. In PE, urine plasmin(ogen): creatinine ratio was elevated 40-fold (geometric mean, 160 versus 4 µg/g; P <0.0001) and urine aldosterone: creatinine ratio was suppressed to 25% of Ctrl (geometric mean, 27 versus 109 µg/g; P <0.001). A significant negative correlation was found in PE between urinary plasmin(ogen) and aldosterone ( P <0.05). In PE, proteolytic activity was detected at 90 to 75 kD by gelatin zymography in 14 of 16 patients and confirmed by serine protease assay. Immunoblotting showed active plasmin in PE urine. Whole-cell inward current increased in M1 cells on exposure to urine from PE (173±21%; n=6; P <0.001). The increase in current was abolished by amiloride (2 μmol/L; P <0.001), α 2 -antiplasmin (1 μmol/L; P <0.001), and heat denaturation ( P <0.001). Preeclampsia is associated with urinary excretion of plasmin(ogen) and plasmin-dependent activation of ENaC by urine. Proteolytic activation of ENaC by plasmin may contribute to Na + retention and hypertension in preeclampsia.

Association of Plasminuria with Overhydration in Patients with CKD

“…Because of the high collinearity between proteinuria and urinary plasminogen as well as plasmin excretion, these parameters could replace each other in multivariable models and could not be separated by using more sophisticated statistical approaches. Other studies in the field, such as the ones by Buhl et al (5,19), which showed an activating effect of protein-rich urine on ENaC activity in vitro, were also associative. Nevertheless, the data provide strong evidence that proteinuria exerts a strong influence on hypervolemia in a continuous relationship.…”

“…Nevertheless, the data provide strong evidence that proteinuria exerts a strong influence on hypervolemia in a continuous relationship. Thus far, studies on plasminuria have concentrated on a limited number of patients with single-disease etiologies (preeclampsia [5] or diabetes mellitus [18,19]). The results of this large study were derived from an unselected CKD cohort with different etiologies, making the results highly generalizable.…”

“…Recent evidence points to the crucial role of proteinuria in promoting sodium retention through activation of the epithelial sodium channel (ENaC) (2), which is known to be an important player in sodium homeostasis and BP control (3). Studies with protein-rich urine from both nephrotic rats and patients have shown a stimulation of ENaC currents in vitro that was attributed to occurrence of the serine protease plasmin in the urine (4,5). It is an emerging concept that serine proteases contribute to ENaC regulation by cleaving specific sites in the extracellular domains of the a-and g-subunits (6)(7)(8)(9)(10), resulting in the release of inhibitory peptides and activation of the channel (11)(12)(13).…”

“…Plasmin-induced ENaC activation and sodium retention may occur in patients with preeclampsia (5) and diabetic patients who are proteinuric (18,19). Recently, immunohistochemical studies in human nephrectomy specimens indicated that the g-subunit of ENaC was processed proteolytically exclusively in patients with proteinuria (20).…”

The Epithelial Sodium Channel γ-Subunit Is Processed Proteolytically in Human Kidney

“…9,14,16,28 We and other groups have shown that, in pathologic conditions with proteinuria, urine contains aberrantly filtered plasmin that activates ENaC current in vitro-either directly or in low concentrations, through prostasin. 5,[29][30][31] However, it is not known whether human kidney ENaC is cleaved under physiologic conditions and if the proteolysis is augmented in conditions with proteinuria. In this study, it was hypothesized that human kidney displays physiologic proteolytic processing of gENaC by furin and prostasin/kallikrein, that the degree of proteolysis differs between conditions with expected differences in aldosterone, and that proteolysis is amplified by proteinuria.…”