Amiloride versus furosemide for the treatment of edema in patients with nephrotic syndrome: A pilot study (AMILOR).

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).

SGLT2 Inhibitors Decrease Overhydration and Proteasuria in Patients with Chronic Kidney Disease: A Longitudinal Observational Study.

INTRODUCTION: SGLT2 inhibitors are used to reduce the risk of progression of chronic kidney disease (CKD). In patients with type 2 diabetes, they have been found to reduce extracellular volume. Given the high prevalence of extracellular volume expansion and overhydration (OH) in CKD, we investigated whether SGLT2 inhibitors might correct these disturbances in CKD patients. METHODS: CKD patients who started treatment with an SGLT2 inhibitor were investigated in this prospective observational study for 6 months. Body composition and fluid status were measured by bioimpedance spectroscopy. In addition, spot urine samples were analyzed for albuminuria, glucosuria, and urinary aprotinin-sensitive serine protease activity. RESULTS: Forty-two patients (29% with diabetic/hypertensive CKD, 31% with IgA nephropathy; 88% dapagliflozin 10 mg, 10% dapagliflozin 5 mg, 2% empagliflozin 20 mg; median eGFR 46 mL/min/1.73 m2 and albuminuria 1,911 mg/g creatinine) participated in the study. Median glucosuria increased to 14 (10-19) g/g creatinine. At baseline, patients displayed OH with +0.4 (-0.2 to 2.2) L/1.73 m2, which decreased by 0.5 (0.1-1.2) L/1.73 m2 after 6 months. Decrease of OH correlated with higher OH at BL, decrease of albuminuria, glucosuria, and urinary aprotinin-sensitive protease activity. Adipose tissue mass was not significantly reduced after 6 months. CONCLUSION: SGLT2 inhibitors reduce OH in patients with CKD, which is pronounced in the presence of high albuminuria, glucosuria, and urinary aprotinin-sensitive protease activity.

Proteolytic Activity against the Distal Polybasic Tract of the Gamma Subunit of the Epithelial Sodium Channel ENaC in Nephrotic Urine.

BACKGROUND: Experimental nephrotic syndrome in mice leads to proteolytic activation of the epithelial sodium channel ENaC, possibly involving the distal polybasic tract of its γ-subunit (183RKRK). OBJECTIVE: We sought to determine if urine samples from both nephrotic mice and a cohort of patients with acute nephrotic syndrome contain a specific proteolytic activity against this region of γ-ENaC. METHODS: A peptide substrate consisting of amino acids 180-194 of murine γ-ENaC was N-terminally coupled to a fluorophore, yielding AMCA-FTGRKRKISGKIIHK. The substrate was incubated with nephrotic urine samples from mice as well as patients with or without the serine protease inhibitor, aprotinin. The digested peptides were separated on a reverse phase HPLC and detected with a fluorescence detector (350/450 nm). Peptide masses of the peaks were determined with a MALDI-TOF mass spectrometer. In addition, urinary proteolytic activity was quantitated using AMC-coupled substrates reflecting different cleavage sites within the polybasic tract. RESULTS: No significant proteolytic activity against the substrate was found in the urine of healthy humans or mice. Incubation with urine samples of nephrotic patients (n = 8) or mice subjected to three different models of experimental nephrotic syndrome (n = 4 each) led to cleavage of the substrate within the polybasic tract prevented by the serine protease inhibitor aprotinin. The most dominant cleavage product was FTGRKR in both species, which was confirmed using quantitative measurements with FTGRKR- AMC. CONCLUSION: Nephrotic urine from both humans and mice contains aprotinin-sensitive proteolytic activity against the distal polybasic tract of γ-ENaC, reflecting excretion of active proteases in the urine or proteasuria.

Sodium retention in nephrotic syndrome is independent of the activation of the membrane-anchored serine protease prostasin (CAP1/PRSS8) and its enzymatic activity.

Experimental nephrotic syndrome leads to activation of the epithelial sodium channel (ENaC) by proteolysis and promotes renal sodium retention. The membrane-anchored serine protease prostasin (CAP1/PRSS8) is expressed in the distal nephron and participates in proteolytic ENaC regulation by serving as a scaffold for other serine proteases. However, it is unknown whether prostasin is also involved in ENaC-mediated sodium retention of experimental nephrotic syndrome. In this study, we used genetically modified knock-in mice with Prss8 mutations abolishing its proteolytic activity (Prss8-S238A) or prostasin activation (Prss8-R44Q) to investigate the development of sodium retention in doxorubicin-induced nephrotic syndrome. Healthy Prss8-S238A and Prss8-R44Q mice had normal ENaC activity as reflected by the natriuretic response to the ENaC blocker triamterene. After doxorubicin injection, all genotypes developed similar proteinuria. In all genotypes, urinary prostasin excretion increased while renal expression was not altered. In nephrotic mice of all genotypes, triamterene response was similarly increased, consistent with ENaC activation. As a consequence, urinary sodium excretion dropped in all genotypes and mice similarly gained body weight by + 25 ± 3% in Prss8-wt, + 20 ± 2% in Prss8-S238A and + 28 ± 3% in Prss8-R44Q mice (p = 0.16). In Western blots, expression of fully cleaved α- and γ-ENaC was similarly increased in nephrotic mice of all genotypes. In conclusion, proteolytic ENaC activation and sodium retention in experimental nephrotic syndrome are independent of the activation of prostasin and its enzymatic activity and are consistent with the action of aberrantly filtered serine proteases or proteasuria.

Proteolytic activation of the epithelial sodium channel (ENaC) by factor VII activating protease (FSAP) and its relevance for sodium retention in nephrotic mice.

Proteolytic activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases is thought to contribute to renal sodium retention in nephrotic syndrome. However, the identity of the responsible proteases remains elusive. This study evaluated factor VII activating protease (FSAP) as a candidate in this context. We analyzed FSAP in the urine of patients with nephrotic syndrome and nephrotic mice and investigated its ability to activate human ENaC expressed in Xenopus laevis oocytes. Moreover, we studied sodium retention in FSAP-deficient mice (Habp2-/-) with experimental nephrotic syndrome induced by doxorubicin. In urine samples from nephrotic humans, high concentrations of FSAP were detected both as zymogen and in its active state. Recombinant serine protease domain of FSAP stimulated ENaC-mediated whole-cell currents in a time- and concentration-dependent manner. Mutating the putative prostasin cleavage site in γ-ENaC (γRKRK178AAAA) prevented channel stimulation by the serine protease domain of FSAP. In a mouse model for nephrotic syndrome, active FSAP was present in nephrotic urine of Habp2+/+ but not of Habp2-/- mice. However, Habp2-/- mice were not protected from sodium retention compared to nephrotic Habp2+/+ mice. Western blot analysis revealed that in nephrotic Habp2-/- mice, proteolytic cleavage of α- and γ-ENaC was similar to that in nephrotic Habp2+/+ animals. In conclusion, active FSAP is excreted in the urine of nephrotic patients and mice and activates ENaC in vitro involving the putative prostasin cleavage site of γ-ENaC. However, endogenous FSAP is not essential for sodium retention in nephrotic mice.

Renal effects of the serine protease inhibitor aprotinin in healthy conscious mice.

Treatment with aprotinin, a broad-spectrum serine protease inhibitor with a molecular weight of 6512 Da, was associated with acute kidney injury, which was one of the reasons for withdrawal from the market in 2007. Inhibition of renal serine proteases regulating the epithelial sodium channel ENaC could be a possible mechanism. Herein, we studied the effect of aprotinin in wild-type 129S1/SvImJ mice on sodium handling, tubular function, and integrity under a control and low-salt diet. Mice were studied in metabolic cages, and aprotinin was delivered by subcutaneously implanted sustained release pellets (2 mg/day over 10 days). Mean urinary aprotinin concentration ranged between 642 ± 135 (day 2) and 127 ± 16 (day 8) µg/mL . Aprotinin caused impaired sodium preservation under a low-salt diet while stimulating excessive hyperaldosteronism and unexpectedly, proteolytic activation of ENaC. Aprotinin inhibited proximal tubular function leading to glucosuria and proteinuria. Plasma urea and cystatin C concentration increased significantly under aprotinin treatment. Kidney tissues from aprotinin-treated mice showed accumulation of intracellular aprotinin and expression of the kidney injury molecule 1 (KIM-1). In electron microscopy, electron-dense deposits were observed. There was no evidence for kidney injury in mice treated with a lower aprotinin dose (0.5 mg/day). In conclusion, high doses of aprotinin exert nephrotoxic effects by accumulation in the tubular system of healthy mice, leading to inhibition of proximal tubular function and counterregulatory stimulation of ENaC-mediated sodium transport.

Proteinuric chronic kidney disease is associated with altered red blood cell lifespan, deformability and metabolism.

Anemia is a common complication of chronic kidney disease, affecting the quality of life of patients. Among various factors, such as iron and erythropoietin deficiency, reduced red blood cell (RBC) lifespan has been implicated in the pathogenesis of anemia. However, mechanistic data on in vivo RBC dysfunction in kidney disease are lacking. Herein, we describe the development of chronic kidney disease-associated anemia in mice with proteinuric kidney disease resulting from either administration of doxorubicin or an inducible podocin deficiency. In both experimental models, anemia manifested at day 10 and progressed at day 30 despite increased circulating erythropoietin levels and erythropoiesis in the bone marrow and spleen. Circulating RBCs in both mouse models displayed altered morphology and diminished osmotic-sensitive deformability together with increased phosphatidylserine externalization on the outer plasma membrane, a hallmark of RBC death. Fluorescence-labelling of RBCs at day 20 of mice with doxorubicin-induced kidney disease revealed premature clearance from the circulation. Metabolomic analyses of RBCs from both mouse models demonstrated temporal changes in redox recycling pathways and Lands' cycle, a membrane lipid remodeling process. Anemic patients with proteinuric kidney disease had an increased proportion of circulating phosphatidylserine-positive RBCs. Thus, our observations suggest that reduced RBC lifespan, mediated by altered RBC metabolism, reduced RBC deformability, and enhanced cell death contribute to the development of anemia in proteinuric kidney disease.

Experimental nephrotic syndrome leads to proteolytic activation of the epithelial Na+ channel in the mouse kidney.

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.NEW & NOTEWORTHY This study demonstrates that murine experimental nephrotic syndrome leads to aprotinin-sensitive proteolytic activation of the epithelial Na+ channel at both the α- and γ-subunit, most likely by urinary serine protease activity or proteasuria.

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

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.

Proteasuria in nephrotic syndrome-quantification and proteomic profiling.

Nephrotic syndrome is characterized by urinary excretion of plasma proteases or proteasuria. There is a lack of data on the quantity, activity status and identity of these aberrantly filtered proteases. We established a fluorescence-based substrate assay to quantify protease activity in urine samples from healthy and nephrotic humans and mice. Protease class activity was determined after addition of specific inhibitors. Individual proteases were identified by tandem mass spectrometry (MS/MS). In spot urine samples from 10 patients with acute nephrotic syndrome of various etiology, urinary protease activity was significantly increased compared to that of healthy persons (753 ±â€¯178 vs. 244 ±â€¯65 relative units, p < 0.05). The corresponding proteases were highly sensitive to inhibition by the serine protease inhibitors AEBSF (reduction by 85 ±â€¯6% and 72 ±â€¯8%, respectively) and aprotinin (83 ±â€¯9% vs. 25 ±â€¯6%, p < 0.05). MS/MS of all urinary proteins or after AEBSF purification showed that most of them were active serine proteases from the coagulation and complement cascade. These findings were recapitulated in mice, pointing to a similar pathophysiology. In conclusion, nephrotic syndrome leads to increased urinary excretion of active plasma proteases which can be termed proteasuria. Serine proteases account for the vast majority of urinary protease activity in health and nephrotic syndrome. SIGNIFICANCE STATEMENT: In this study, we found that nephrotic urine samples of humans and mice have a significantly increased protease activity compared to healthy urine samples, using a universal pentapeptide substrate library. This was driven by increased excretion of aprotinin-sensitive serine proteases. With tandem mass spectrometry, we provide a comprehensive and systematic overview of all urinary proteases or the "urine proteasome". We identified renally expressed proteases in health and addition of proteases from the coagulation and complement cascade in the nephrotic state. These results set the basis to study the role of urinary proteases at both health and nephrotic syndrome to find diagnostic markers of renal disease as well as possible therapeutic targets.

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

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.

Urokinase-type plasminogen activator (uPA) is not essential for epithelial sodium channel (ENaC)-mediated sodium retention in experimental nephrotic syndrome.

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.

Proteasuria-The impact of active urinary proteases on sodium retention in nephrotic syndrome.

Sodium retention and extracellular volume expansion are typical features of patients with nephrotic syndrome. In recent years, from in vitro data, endoluminal activation of the epithelial sodium channel (ENaC) by aberrantly filtered serine proteases has been proposed as an underlying mechanism. Recently, this concept was supported in vivo in nephrotic mice that were protected from proteolytic ENaC activation and sodium retention by the use of aprotinin for the pharmacological inhibition of urinary serine protease activity. These and other findings from studies in both rodents and humans highlight the impact of active proteases in the urine, or proteasuria, on ENaC-mediated sodium retention and edema formation in nephrotic syndrome. Targeting proteasuria could become a therapeutic approach to treat patients with nephrotic syndrome. However, pathophysiologically relevant proteases remain to be identified. In this review, we introduce the concept of proteasuria to explain tubular sodium avidity and conclude that proteasuria can be considered as a key mechanism of sodium retention in patients with nephrotic syndrome.

Aprotinin prevents proteolytic epithelial sodium channel (ENaC) activation and volume retention in nephrotic syndrome.

Volume retention in nephrotic syndrome has been linked to activation of the epithelial sodium channel (ENaC) by proteolysis of its γ-subunit following urinary excretion of serine proteases such as plasmin. Here we tested whether pharmacological inhibition of urinary serine protease activity might protect from ENaC activation and volume retention in nephrotic syndrome. Urine from both nephrotic mice (induced by doxorubicin injection) and nephrotic patients exhibited high aprotinin-sensitive serine protease activity. Treatment of nephrotic mice with the serine protease inhibitor aprotinin by means of subcutaneous sustained-release pellets normalized urinary serine protease activity and prevented sodium retention, as did treatment with the ENaC inhibitor amiloride. In the kidney cortex from nephrotic mice, immunofluorescence revealed increased apical γ-ENaC staining, normalized by aprotinin treatment. In Xenopus laevis oocytes heterologously expressing murine ENaC, aprotinin had no direct inhibitory effect on channel activity but prevented proteolytic channel activation. Thus, our study shows that volume retention in experimental nephrotic syndrome is related to proteolytic ENaC activation by proteasuria and can be prevented by treatment with aprotinin. Hence, inhibition of urinary serine protease activity might become a therapeutic approach to treat patients with nephrotic-range proteinuria.