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Aim: In rodent models of nephrotic syndrome (NS), edema formation was prevented by blockade of the epithelial sodium channel ENaC with amiloride. However, apart from case reports, there is no evidence favoring ENaC blockade in patients with NS. Methods: The monocentric randomized controlled AMILOR study investigated the antiedematous effect of amiloride (starting dose 5 mg/day, max. 15 mg/day) in comparison to standard therapy with the loop diuretic furosemide (40 mg/day, max. 120 mg/day) over 16 days. Overhydration (OH) was measured by bioimpedance spectroscopy (BCM, Fresenius). Depending on the OH response, diuretic dose was adjusted on days 2, 5, 8 and 12, and if necessary, hydrochlorothiazide (HCT) was added from d8 (12.5 mg/day, max. 25 mg/day). The primary endpoint was the decrease in OH on d8. The study was terminated prematurely due to insufficient recruitment and a low statistical power due to a low actual effect size. Results: Median baseline OH was +26.4 (interquartile range 15.5–35.1)% extracellular water (ECW) in the amiloride arm and + 27.9 (24.1–29.4)% ECW in the furosemide arm and decreased by 1.95 (0.80–6.40) and 5.15 (0.90–8.30)% ECW after 8 days, respectively, and by 10.10 (1.30–14.40) and 7.40 (2.80–10.10)% ECW after 16 days, respectively. OH decrease on d8 and d16 was not significantly different between both arms. Conclusion: The AMILOR study is the first randomized controlled pilot study suggesting a similar antiedematous effect as furosemide. Further studies are required to better define the role of amiloride in NS (EudraCT 2019‐002607‐18). [ABSTRACT FROM AUTHOR]

Proteolytic activation of the renal epithelial Na+ channel (ENaC) involves cleavage events in its α- and γ-subunits and is thought to mediate Na+ retention in nephrotic syndrome (NS). However, the detection of proteolytically processed ENaC in kidney tissue from nephrotic mice has been elusive so far. We used a refined Western blot technique to reliably discriminate full-length α-ENaC and γ-ENaC and their cleavage products after proteolysis at their proximal and distal cleavage sites (designated from the NH2-terminus), respectively. Proteolytic ENaC activation was investigated in kidneys from mice with experimental NS induced by doxorubicin or inducible podocin deficiency with or without treatment with the serine protease inhibitor aprotinin. Nephrotic mice developed Na+ retention and increased expression of fragments of α-ENaC and γ-ENaC cleaved at both the proximal cleavage site and, more prominently, the distal cleavage site, respectively. Treatment with aprotinin but not with the mineralocorticoid receptor antagonist canrenoate prevented Na+ retention and upregulation of the cleavage products in nephrotic mice. Increased expression of cleavage products of α-ENaC and γ-ENaC was similarly found in healthy mice treated with a low-salt diet, sensitive to mineralocorticoid receptor blockade. In human nephrectomy specimens, γ-ENaC was found in the full-length form and predominantly cleaved at its distal cleavage site. In conclusion, murine experimental NS leads to aprotinin-sensitive proteolytic activation of ENaC at both proximal and, more prominently, distal cleavage sites of its α- and γ-subunit, most likely by urinary serine protease activity or proteasuria. [ABSTRACT FROM AUTHOR]

Aim: The serine protease prostasin (Prss8) is expressed in the distal tubule and stimulates proteolytic activation of the epithelial sodium channel (ENaC) in co‐expression experiments in vitro. The aim of this study was to explore the role of prostasin in proteolytic ENaC activation in the kidney in vivo. Methods: We used genetically modified knockin mice carrying a Prss8 mutation abolishing proteolytic activity (Prss8‐S238A) or a mutation leading to a zymogen‐locked state (Prss8‐R44Q). Mice were challenged with low sodium diet and diuretics. Regulation of ENaC activity by Prss8‐S238A and Prss8‐R44Q was studied in vitro using the Xenopus laevis oocyte expression system. Results: Co‐expression of murine ENaC with Prss8‐wt or Prss8‐S238A in oocytes caused maximal proteolytic ENaC activation, whereas ENaC was activated only partially in oocytes co‐expressing Prss8‐R44Q. This was paralleled by a reduced proteolytic activity at the cell surface of Prss8‐R44Q expressing oocytes. Sodium conservation under low sodium diet was preserved in Prss8‐S238A and Prss8‐R44Q mice but with higher plasma aldosterone concentrations in Prss8‐R44Q mice. Treatment with the ENaC inhibitor triamterene over four days was tolerated in Prss8‐wt and Prss8‐S238A mice, whereas Prss8‐R44Q mice developed salt wasting and severe weight loss associated with hyperkalemia and acidosis consistent with impaired ENaC function and renal failure. Conclusion: Unlike proteolytically inactive Prss8‐S238A, zymogen‐locked Prss8‐R44Q produces incomplete proteolytic ENaC activation in vitro and causes a severe renal phenotype in mice treated with the ENaC inhibitor triamterene. This indicates that Prss8 plays a role in proteolytic ENaC activation and renal function independent of its proteolytic activity. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aim: In nephrotic syndrome, aberrantly filtered plasminogen (plg) is converted to active plasmin by tubular urokinase‐type plasminogen activator (uPA) and thought to lead to sodium retention by proteolytic activation of the epithelial sodium channel (ENaC). This concept predicts that uPA is an important factor for sodium retention and that inhibition of uPA might be protective in nephrotic syndrome. Methods: Activation of amiloride‐sensitive currents by uPA and plg were studied in Xenopus laevis oocytes expressing murine ENaC. In doxorubicin‐induced nephrotic mice, uPA was inhibited pharmacologically by amiloride and genetically by the use of uPA‐deficient mice (uPA−/−). Results: Experiments in Xenopus laevis oocytes expressing murine ENaC confirmed proteolytic ENaC activation by a combination of plg and uPA which stimulated amiloride‐sensitive currents with concomitant cleavage of the ENaC γ‐subunit at the cell surface. Treatment of nephrotic wild‐type mice with amiloride inhibited urinary uPA activity, prevented urinary plasmin formation and sodium retention. In nephrotic mice lacking uPA (uPA−/−), urinary plasmin formation from plg was suppressed and urinary uPA activity absent. However, in nephrotic uPA−/− mice, sodium retention was not reduced compared to nephrotic uPA+/+ mice. Amiloride prevented sodium retention in nephrotic uPA−/− mice which confirmed the critical role of ENaC in sodium retention. Conclusion: uPA is responsible for the conversion of aberrantly filtered plasminogen to plasmin in the tubular lumen in vivo. However, uPA‐dependent plasmin generation is not essential for ENaC‐mediated sodium retention in experimental nephrotic syndrome. [ABSTRACT FROM AUTHOR]