Urokinase‐type plasminogen activator contributes to amiloride‐sensitive sodium retention in nephrotic range glomerular proteinuria in mice

Abstract

Aim: Activation of sodium reabsorption by urinary proteases has been implicated in sodium retention associated with nephrotic syndrome. The study was designed to test the hypothesis that nephrotic proteinuria in mice after conditional deletion of podocin leads to urokinase-dependent, amiloride-sensitive plasmin-mediated sodium and water retention. Methods: Ten days after podocin knockout, urine and faeces were collected for 10 days in metabolic cages and analysed for electrolytes, plasminogen, protease activity and ability to activate γENaC by patch clamp and western blot. Mice were treated with amiloride (2.5 mg kg −1 for 2 days and 10 mg kg −1 for 2 days) or an antiurokinase-type plasminogen activator (uPA) targeting antibody (120 mg kg −1 /24 h) and compared to controls. Results: Twelve days after deletion, podocin-deficient mice developed significant protein and albuminuria associated with increased body wt, ascites, sodium accumulation and suppressed plasma renin. This was associated with increased urinary excretion of plasmin and plasminogen that correlated with albumin excretion, urine protease activity co-migrating with active plasmin, and the ability of urine to induce an amiloride-sensitive inward current in M1 cells in vitro. Amiloride treatment in podocin-deficient mice resulted in weight loss, increased sodium excretion, normalization of sodium balance and prevention of the activation of plasminogen to plasmin in

Rebuttal to editorial: Sodium retention by uPA in nephrotic syndrome?

“…In our reply, we first comment on some technical aspects discussed by Prof. Ehmke as possible explanations for the discrepant conclusions reached in the two studies. In addition, we highlight some in vivo data reported by Hinrichs et al which in our view do not oppose but rather support our conclusion that urokinase, also known as urokinase‐type plasminogen activator (uPA), is not essential for sodium retention in nephrotic syndrome.…”

“…Doxorubicin‐induced nephropathy is primarily a toxic model inducing podocyte ablation without inflammation and resembles human focal segmental glomerulosclerosis . The kinetics of the onset of proteinuria is very similar to that seen in the inducible model used by Hinrichs et al in which the podocyte foot process protein podocin is specifically deleted. Both models lead to global glomerulosclerosis, interstitial fibrosis, tubular atrophy and finally progressive renal failure leading to death of the nephrotic mice after 40 days .…”

“…We welcome the opportunity to reply to the elegant editorial of Prof. Ehmke in which he highlights contradicting conclusions reached by Hinrichs et al and by us in two recently published articles in Acta Physiologica. In our reply, we first comment on some technical aspects discussed by Prof. Ehmke as possible explanations for the discrepant conclusions reached in the two studies.…”

“…As pointed out by Prof. Ehmke, different experimental approaches were used in the two studies to test the hypothesis that uPA‐mediated plasmin generation from aberrantly filtered plasminogen is a key mechanism responsible for proteolytic ENaC activation and increased renal sodium absorption in nephrotic syndrome . Hinrichs et al used a monoclonal antibody to inhibit uPA (uPAab) acutely in mice with nephrotic syndrome due to an inducible podocin knockout (Pod KO mice). In contrast, we used a constitutive uPA knockout mouse model in which we induced nephrotic syndrome with doxorubicin.…”

“…Therefore, it has to be kept in mind that in addition to plasmin nephrotic urine may contain several other serine proteases with the ability to mediate proteolytic ENaC activation. In this context the question raised by Prof. Ehmke, whether the antibody against uPA may inhibit other serine proteases, is obviously relevant for the interpretation of the in vivo data reported by Hinrichs et al…”