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. Patchclamp 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. 23: 182423: -183423: , 201223: . doi: 10.1681 In the kidney, the fine regulation of Ca 2+ balance occurs through the activity of the epithelial Ca 2+ channel TRPV5. 1 TRPV5 is mostly expressed in the distal convoluted tubule (DCT) and connecting tubule of the nephron, where it constitutes the apical entry mechanism for transcellular Ca 2+ reabsorption. TRPV5 is a constitutively active ion channel that bears unique electrophysiologic characteristics, including calmodulin (CaM) and Ca 2+ -dependent inactivation and high selectivity for Ca 2+ . 2,3 The activity of TRPV5 is tightly controlled at multiple levels by an array of different factors, including parathyroid hormone and the serine protease tissue kallikrein. Both parathyroid hormone and tissue kallikrein initiate the phosphorylation of TRPV5 through the cAMP/protein kinase A (PKA) and phospholipase C (PLC)/ diacylglycerol (DAG)/protein kinase C (PKC) signaling cascades, respectively. 4,5
J Am Soc Nephrol