MO061PLASMINOGEN DEFIENCY DOES NOT PROTECT MICE FROM SODIUM RETENTION IN EXPERIMENTAL NEPHROTIC SYNDROME
Authors: Xiao Mengyun , Bohnert Bernhard Nikolaus , Woern Matthias , Plow Edward , Huber Tobias B , Grahammer Florian , Artunc Ferruh 
Published: June 2020

Abstract

Background and Aims
Sodium retention and edema formation are the hallmarks of nephrotic syndrome and thought to be mediated by proteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases. Plasmin is highly abundant in nephrotic urine and has been proposed to be the principal serine protease responsible for ENaC activation in nephrotic syndrome. However, there is not enough evidence to demonstrate the essential role of plasmin in mediating sodium retention in an experimental nephrotic model.

Method
We investigated sodium retention and edema formation in a mouse model of nephrotic syndrome based on an inducible podocyte-specific podocin knockout (Bl6-Nphs2tm3.1Antc or Δipod * Tg(Nphs1-rtTA*3G8Jhm)* Tg(tetO-cre) 1Jaw). To generate an inducible podocin knockout with plasminogen deficient model (Nphs2Δipod * Plg-/-, hereafter referred to as Plg-/-), plasminogen deficient mice (Bl6-Plgtm1Jld or -/-) were intercrossed with Nphs2Δipod mice. Nephrotic syndrome was induced after oral doxycycline treatment for 14 days. Body weight, urinary protein, urinary sodium excretion, as well as urinary plasmin activity were daily determined 14 days after end of induction. To determine if sodium retention can be prevented by serine protease inhibitor aprotinin in Plg+/+ and Plg-/- mice after induction of nephrotic syndrome, sustained-release pellets containing aprotinin (2 mg per day) or placebo pellets were implanted to either Plg+/+ or Plg-/- mice (n=4 per group).

Results
Uninduced Plg+/+ (n=6-13) and Plg-/- mice (n=6-14) had normal kidney function and sodium handling. After end of doxycycline induction, there was no significant differencein proteinuria increase between Plg+/+ (from 2 ± 0 to 161 ± 16 mg/mg creatinine, p&lt;0.05) and Plg-/- mice (from 9 ± 8 to 146 ± 44 mg/mg creatinine, p&lt;0.05) leading to similar hypoalbuminemia. In urine samples from Plg+/+ mice, Western blot revealed urinary excretion of plasminogen/plasmin which was completely absent in Plg-/- mice. Accordingly, urinary plasmin activity was only detectable in Plg+/+ mice using a chromogenic substrate. After onset of proteinuria, amiloride-sensitive natriuresis was increased compared to uninduced mice indicating ENaC activation. While urinary sodium excretion dropped in both genotypes indicating sodium retention (from 193 ± 16 to 16 ± 6 µmol/24h in Plg+/+ mice, p&lt;0.05; from 229 ± 11 to 26 ± 7 µmol/24h in Plg-/-mice, p&lt;0.05). As a consequence, body weight maximum increased in both genotypes 21 ± 1% in Plg+/+ and 17 ± 2% in Plg-/- mice (p=0.616) and was paralleled by development of ascites. Urinary amidolytic activity were completely prevented by the presence of aprotinin, as well as sodium retention and ascites in both Plg+/+and Plg-/- mice.

Conclusion
This study shows for the first time that mice lacking urinary plasmin are not protected from ENaC-mediated sodium retention in experimental nephrotic syndrome, however it can be prevented by aprotinin. These findings point to an essential role of other hitherto unknown serine proteases excreted in nephrotic urine.