Plasminogen deficiency does not prevent sodium retention in a genetic mouse model of experimental nephrotic syndrome

Abstract

Aim Sodium retention is the hallmark of nephrotic syndrome (NS) and mediated by the 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 NS. However, a proof of the essential role of plasmin in experimental NS is lacking. Methods We used a genetic mouse model of NS based on an inducible podocin knockout (Bl6‐Nphs2tm3.1Antc*Tg(Nphs1‐rtTA*3G)8Jhm*Tg(tetO‐cre)1Jaw or nphs2Δipod). These mice were crossed with plasminogen deficient mice (Bl6‐Plgtm1Jld or plg−/−) to generate double knockout mice (nphs2Δipod*plg−/−). NS was induced after oral doxycycline treatment for 14 days and mice were followed for subsequent 14 days. Results Uninduced nphs2Δipod*plg−/− mice had normal kidney function and sodium handling. After induction, proteinuria increased similarly in both nphs2Δipod*plg+/+ and nphs2Δipod*plg−/− mice. Western blot revealed the urinary excretion of plasminogen and plasmin in nphs2Δipod*plg+/+ mice which were absent in nphs2Δipod*plg−/− mice. After the onset of proteinuria, amiloride‐sensitive natriuresis was increased compared to the uninduced state in both genotypes. Subsequently, urinary sodium excretion dropped in both genotypes leading to an increase in body weight and development of ascites. Treatment with the serine protease inhibitor aprotinin prevented sodium retention in both genotypes. Conclusions This study shows that mice lacking urinary plasminogen are not protected from ENaC‐mediated sodium retention in experimental NS. This points to an essential role of other urinary serine proteases in the absence of plasminogen.

Overhydration Measured by Bioimpedance Spectroscopy and Urinary Serine Protease Activity Are Risk Factors for Progression of Chronic Kidney Disease

“…Albuminuria represents glomerular proteinuria in the KDIGO staging of CKD, but other urinary proteins and their mechanistic value for progression of CKD should also be considered. Proteasuria has lately been investigated as a mechanism for edema formation and OH in proteinuric kidney disease [3,12,20]. Serine proteases that reach the urine with proteinuria have been shown to cleave the gamma unit of the epithelial sodium channel and lead to HRs for continuous variables are per increase of 1 SD.…”

“…As a surrogate for proteasuria, aprotinin-sensitive proteolytic activity in urine samples was measured manually using a chromogenic tripeptide substrate (S-2302, H-D-Pro-Phe-Arg-pNA•2HCl, 2 mM; Haemochrom Diagnostica, Essen, Germany) as difference of absorption without and with addition of the broad-spectrum serine protease inhibitor aprotinin (1 mg/ mL; Loxo, Heidelberg, Germany) in vitro. In nephrotic mice, aprotinin has prevented sodium retention pointing to an essential role of trypsin-like serine proteases excreted in the urine [6,12]. Reactions took place in a total volume of 63 μL (10-µL urine +50-μL S-2302 + 3-μL aprotinin or Ampuwa) at 37°C, and absorption was measured at 405 nm after an incubation time of 8 h (EL800 Microplate Reader; BioTek Instruments Inc.,Winooski, VT, USA).…”

Zymogen‐locked mutant prostasin (Prss8) leads to incomplete proteolytic activation of the epithelial sodium channel (ENaC) and severely compromises triamterene tolerance in mice

“…Homogenization was performed using a Dounce homogenizer in 1 mL lysis buffer containing 250 mmol/L sucrose, 10 mmol/L triethanolamine HCl, 1.6 mmol/L ethanolamine and 0.5 EDTA at pH 7.4 (all Sigma). 46,47 During all preparation steps, aprotinin (40 µg mL −1 ) and a protease inhibitor cocktail (final concentration 0.1 × stock; minicomplete, Roche) was present to avoid ENaC cleavage in vitro. Homogenates were centrifuged at 1000 g for removal of the nuclei.…”

Renal failure in focus

“…Since plasminogendeficient mice also showed ENaC activity, which in turn is then prevented by aprotenin. 10 In order to maintain optimal sodium balances salt sensitivity is related to blood pressure regulation. As described for NS, ENaC activation and sodium retention lead to hypertension.…”

“…However, another paper highlighted the role of other serine proteases than plasminogen. Since plasminogen‐deficient mice also showed ENaC activity, which in turn is then prevented by aprotenin 10 …”