title: Diabetic nephropathy is associated with increased urine excretion of proteases plasmin, prostasin and urokinase and activation of amiloride-sensitive current in collecting duct cells.
abstract: BACKGROUND
  Diabetic nephropathy (DN) is associated with hypertension, expanded extracellular volume and impaired renal Na(+) excretion. It was hypothesized that aberrant glomerular filtration of serine proteases in DN causes proteolytic activation of the epithelial sodium channel (ENaC) in the kidney by excision of an inhibitory peptide tract from the γ subunit.
  
  
  METHODS
  In a cross-sectional design, urine, plasma and clinical data were collected from type 1 diabetic patients with DN (n = 19) and matched normoalbuminuric type 1 diabetics (controls, n = 20). Urine was examined for proteases by western immunoblotting, patch clamp and ELISA. Urine exosomes were isolated to elucidate potential cleavage of γENaC by a monoclonal antibody directed against the 'inhibitory' peptide tract.
  
  
  RESULTS
  Compared with control, DN patients displayed significantly higher blood pressure and urinary excretion of plasmin(ogen), prostasin and urokinase that correlated directly with urine albumin. Western blotting confirmed plasmin, prostasin and urokinase in urine from the DN group predominantly. Urine from DN evoked a significantly larger amiloride-sensitive inward current in single collecting duct cells compared with controls. Immunoblotting of urine exosomes showed aquaporin 2 in all patient samples. Exosomes displayed a virtual absence of intact γENaC while moieties compatible with cleavage by furin only, were shown in both groups. Proteolytic cleavage by the extracellular serine proteases plasmin or prostasin was observed in DN samples predominantly.
  
  
  CONCLUSIONS
  DN is associated with increased urinary excretion of plasmin, prostasin and urokinase and proteolytic activation of ENaC that might contribute to impaired renal Na(+) excretion and hypertension.
authors.affiliation: Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense C, Denmark Department of Endocrinology, Odense University Hospital, Odense C, Denmark.
authors.forename: Henrik
authors.initials: H
authors.lastname: Andersen
authors.affiliation: Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense C, Denmark.
authors.forename: Ulla G
authors.initials: UG
authors.lastname: Friis
authors.affiliation: Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense C, Denmark.
authors.forename: Pernille B L
authors.initials: PB
authors.lastname: Hansen
authors.affiliation: Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense C, Denmark.
authors.forename: Per
authors.initials: P
authors.lastname: Svenningsen
authors.affiliation: Department of Endocrinology, Odense University Hospital, Odense C, Denmark.
authors.forename: Jan Erik
authors.initials: JE
authors.lastname: Henriksen
authors.affiliation: Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense C, Denmark.
authors.forename: Boye L
authors.initials: BL
authors.lastname: Jensen
mesh_terms: D000368:Aged; D000584:Amiloride / Q000737:chemistry*; D003430:Cross-Sectional Studies; D003922:Diabetes Mellitus, Type 1 / Q000652:urine; D003928:Diabetic Nephropathies / Q000652:urine*; D004797:Enzyme-Linked Immunosorbent Assay; D053503:Epithelial Sodium Channels / Q000378:metabolism; D005260:Female; D005341:Fibrinolysin / Q000652:urine*; D006801:Humans; D006973:Hypertension / Q000503:physiopathology; D007668:Kidney / Q000503:physiopathology; D007685:Kidney Tubules, Collecting / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D012697:Serine Endopeptidases / Q000652:urine*; D012964:Sodium / Q000652:urine; D014568:Urokinase-Type Plasminogen Activator / Q000652:urine*; D000099271:Prostasin
chemical_list: D000584:Amiloride; D053503:Epithelial Sodium Channels; D005341:Fibrinolysin; D012697:Serine Endopeptidases; D012964:Sodium; D014568:Urokinase-Type Plasminogen Activator; D000099271:Prostasin
keywords: albuminuria; exosome; hypertension; proteinuria; sodium