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.

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.

Proteolytic activation of the renal epithelial sodium channel ENaC involves cleavage events in its α- and γ-subunits and is thought to mediate sodium retention in nephrotic syndrome (NS). However, 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- and γ-ENaC and their cleavage products after proteolysis at their proximal and distal cleavage sites (designated from the N-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 sodium retention and increased expression of fragments of α- and γ-ENaC cleaved at both the proximal and more prominently at the distal cleavage site, respectively. Treatment with aprotinin but not with the mineralocorticoid receptor antagonist canrenoate prevented sodium retention and upregulation of the cleavage products in nephrotic mice. Increased expression of cleavage products of α- 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.

Susceptibility to doxorubicin-induced nephropathy (DIN), a toxic model for the induction of proteinurie in mice, is related to the single nucleotide polymorphism (SNP) C6418T of the prkdc gene encoding for the DNA repair enzyme DNA-PKcs. In addition, plasminogen (plg) has been reported to play a role in glomerular damage. Here, we investigated the interdependence of both factors for the development of DIN. Genotyping confirmed the SNP of the prkdc gene in C57BL/6 (prkdcC6418/C6418) and 129S1/SvImJ (prkdcT6418/T6418) mice. Intercross of heterozygous 129SB6F1 mice led to 129SB6F2 hybrids with Mendelian inheritance of the SNP. After doxorubicin injection, only homozygous F2 mice with prkdcT6418/T6418 developed proteinuria. Genetic deficiency of plg (plg−/-) in otherwise susceptible 129S1/SvImJ mice led to resistance to DIN. Immunohistochemistry revealed glomerular binding of plg in plg+/+ mice after doxorubicin injection involving histone H2B as plg receptor. In doxorubicin resistant C57BL/6 mice, plg binding was absent. In conclusion, susceptibility to DIN in 129S1/SvImJ mice is determined by a hierarchical two hit process requiring the C6418T SNP in the prdkc gene and subsequent glomerular binding of plasminogen.

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