Figure 1.

Plasmin in nephrotic urine inhibits TRPV5-mediated Ca²⁺ influx in HEK293 cells. (A) One-hour incubation of 10 nM commercial plasmin was effective to inhibit Ca²⁺ influx and was reversed by 50 nM α₂-antiplasmin (α₂), specific plasmin inhibitor. Ruthenium red (RR), 10 μM, was administered to determine TRPV5-mediated Ca²⁺ influx. Cells were pretreated with 25 μM BAPTA-AM for 30 minutes. (B) Dose-response curve of Ca²⁺ uptake. (C) Plasmin purified from nephrotic urine inhibited Ca²⁺ influx, and this effect was abolished by a heat inactivation (5 minutes at 80°C) and α₂. (D) Plasmin expression in urine samples of five nephrotic patients was shown on immunoblot (C, control urine sample; P, plasmin-added sample). Plasmin activities of the commercial plasmin, nephrotic urine–purified, and urine from healthy person are shown in the unit of relative fluorescence unit (RFU) under control (black), heat-inactivated (striped), and α₂-antiplasmin (white), respectively. ^§P<0.05 versus mock. ^†P<0.05 versus respective control.