Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C021

Oral Communications

Induced nephrotic range glomerular proteinuria in mice is associated with amiloride-sensitive urinary plasmin formation and sodium retention

G. R. Hinrichs1, W. Kathrin2, U. Friis1, P. Svenningsen1, R. Nielsen2, G. Mollet3,4, C. Antignac3,4,5, C. Bistrup6,7, B. L. Jensen1, H. Birn2,8

1. Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, Odense, Denmark. 2. Department of Biomedicine, Aarhus University, Aarhus, Denmark. 3. Laboratory of Hereditary Kidney Diseases, Inserm, U1116, Imagine Institute, Paris, France. 4. Paris Descartes-Sorbonne Paris Cité University, Paris, France. 5. Department of Genetics, Necker Hospital, Assistance Publique-Hôpitaux de Paris, Paris, France. 6. Department of Nephrology, Odense University Hospital, Odense, Denmark. 7. Department of Clinical Research, University of Southern Denmark, Odense, Denmark. 8. Department of Renal Medicine, Aarhus University Hospital, Aarhus, Denmark.

Introduction: In several proteinuric conditions, aberrantly filtered serine proteases, e.g. plasmin, can activate the epithelial sodium channel (ENaC) through proteolytic interaction with the gamma subunit. This may lead to increased sodium reabsorption and subsequent expansion of the extracellular volume. This supports that nephrotic edema is a primary "overfill" situation founded in an intrarenal mechanism that mediates sodium and water retention, independently of systemic factors. Aim: The study characterizes sodium balance in a murine model of pure glomerular nephrotic syndrome and tests the hypothesis that nephrotic range glomerular proteinuria in mice leads to plasmin-mediated sodium and water retention abolished by amiloride. Methods: We used a previously established mouse model of focal and segmental glomerulosclerosis (FSGS) by inducible conditional inactivation of the podocin gene in adult mice creating podocin knock out mice (Pod KO, Nph2lox,lox;Cre+). Wild type (WT, Nph2lox,lox;Cre-) littermates served as controls. Gene knockout was induced by intraperitoneal injection with tamoxifen at a dose of 0.05 mg/g BW x 1/day for 5 days during a short anesthesia with isoflurane. Beginning 10 days after induction, urine and feces were collected in metabolic cages for a total of 10 days. Daily body weight was measured and sodium balance was calculated. Urine plasminogen was analyzed by ELISA and western blotting, and serine protease activity was identified by zymography and the ability to evoke inward current by whole-cell patch clamp of single M-1 cells. In a separate series, Pod KO mice were treated with amiloride (2.5 mg/kg for 2 days followed by 10 mg/kg for 2 days) or vehicle by intraperitoneal injection twice daily beginning at day 13 after induction. Results: From day 12 after induction Pod KO mice revealed significantly increased urinary albumin excretion and body weight when compared to WT, which was associated with ascites and suppressed plasma renin. Zymography demonstrated protease activity that co-migrated with active plasmin. Plasminogen was identified in the urine from Pod KO mice and correlated with albumin excretion. Compared to WT, the urine from Pod KO mice induced a greater and amiloride-sensitive inward current in M1 cells. Administration of amiloride promoted significant weight loss, increased Na excretion, normalized sodium balance, and prevented activation of plasminogen to plasmin in urine. Conclusion: Proteinuria in a mouse model of FSGS is associated with ENaC-dependent sodium accumulation and suppression of renin which supports an overfill model of nephrotic syndrome in mice.

Where applicable, experiments conform with Society ethical requirements