title: Urinary plasmin inhibits TRPV5 in nephrotic-range proteinuria.
abstract: Urinary proteins that leak through the abnormal glomerulus in nephrotic syndrome may affect tubular transport by interacting with membrane transporters on the luminal side of tubular epithelial cells. Patients with nephrotic syndrome can develop nephrocalcinosis, which animal models suggest may develop from impaired transcellular Ca(2+) reabsorption via TRPV5 in the distal convoluted tubule (DCT). In nephrotic-range proteinuria, filtered plasminogen reaches the luminal side of DCT, where it is cleaved into active plasmin by urokinase. In this study, we found that plasmin purified from the urine of patients with nephrotic-range proteinuria inhibits Ca(2+) uptake in TRPV5-expressing human embryonic kidney 293 cells through the activation of protease-activated receptor-1 (PAR-1). Preincubation with a plasmin inhibitor, a PAR-1 antagonist, or a protein kinase C (PKC) inhibitor abolished the effect of plasmin on TRPV5. In addition, ablation of the PKC phosphorylation site S144 rendered TRPV5 resistant to the action of plasmin. Patch-clamp experiments showed that a decreased TRPV5 pore size and a reduced open probability accompany the plasmin-mediated reduction in Ca(2+) uptake. Furthermore, high-resolution nuclear magnetic resonance spectroscopy demonstrated specific interactions between calmodulin and residues 133-154 of the N-terminus of TRPV5 for both wild-type and phosphorylated (S144pS) peptides. In summary, PAR-1 activation by plasmin induces PKC-mediated phosphorylation of TRPV5, thereby altering calmodulin-TRPV5 binding, resulting in decreased channel activity. These results indicate that urinary plasmin could contribute to the downstream effects of proteinuria on the tubulointerstitium by negatively modulating TRPV5.
authors.affiliation: Department of Physiology, Nijmegen Centre for Molecular Life Sciences, Radboud University, Nijmegen Medical Centre, Nijmegen, The Netherlands.
authors.forename: Kukiat
authors.initials: K
authors.lastname: Tudpor
authors.forename: Sergio
authors.initials: S
authors.lastname: Laínez
authors.forename: Arjan J
authors.initials: AJ
authors.lastname: Kwakernaak
authors.forename: Nadezda V
authors.initials: NV
authors.lastname: Kovalevskaya
authors.forename: Sjoerd
authors.initials: S
authors.lastname: Verkaart
authors.forename: Siebe
authors.initials: S
authors.lastname: van Genesen
authors.forename: Annemiete
authors.initials: A
authors.lastname: van der Kemp
authors.forename: Gerjan
authors.initials: G
authors.lastname: Navis
authors.forename: René J M
authors.initials: RJ
authors.lastname: Bindels
authors.forename: Joost G J
authors.initials: JG
authors.lastname: Hoenderop
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002147:Calmodulin / Q000378:metabolism; D005341:Fibrinolysin / Q000494:pharmacology* / Q000652:urine*; D057809:HEK293 Cells; D006801:Humans; D007686:Kidney Tubules, Distal / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009404:Nephrotic Syndrome / Q000652:urine*; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011507:Proteinuria / Q000652:urine*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D044463:Receptor, PAR-1 / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D050916:TRPV Cation Channels / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002147:Calmodulin; D012333:RNA, Messenger; D044463:Receptor, PAR-1; D050916:TRPV Cation Channels; C492239:TRPV5 protein, human; D012694:Serine; D011493:Protein Kinase C; D005341:Fibrinolysin; D002118:Calcium