Plasmin in nephrotic urine activates the epithelial sodium channel

Abstract

Proteinuria and increased renal reabsorption of NaCl characterize the nephrotic syndrome. Here, we show that protein-rich urine from nephrotic rats and from patients with nephrotic syndrome activate the epithelial sodium channel (ENaC) in cultured M-1 mouse collecting duct cells and in Xenopus laevis oocytes heterologously expressing ENaC. The activation depended on urinary serine protease activity. We identified plasmin as a urinary serine protease by matrix-assisted laser desorption/ionization time of-flight mass spectrometry. Purified plasmin activated ENaC currents, and inhibitors of plasmin abolished urinary protease activity and the ability to activate ENaC. In nephrotic syndrome, tubular urokinase-type plasminogen activator likely converts filtered plasminogen to plasmin. Consistent with this, the combined application of urokinase-type plasminogen activator and plasminogen stimulated amiloride-sensitive transepithelial sodium transport in M-1 cells and increased amiloride-sensitive whole-cell currents in Xenopus laevis oocytes heterologously expressing ENaC. Activation of ENaC by plasmin involved cleavage and release of an inhibitory peptide from the ENaC gamma subunit ectodomain. These data suggest that a defective glomerular filtration barrier allows passage of proteolytic enzymes that have the ability to activate ENaC.

Figure 1.

Sodium retention in PAN nephrotic syndrome in rats involves a primary increase in renal ENaC activity. (A) Daily sodium balance and urinary protein excretion after PAN injection based on measurements of daily sodium intake, fecal sodium output, and urinary sodium output of rats in metabolic cages (Supplemental Figure 1). Nephrotic rats display negative cumulative sodium balance (shaded area) from days 0 to 3.6 after PAN injection and positive sodium balance thereafter. Nephrotic rats accumulated 2217 ± 167 μmol/100 g body wt sodium (n = 8) from days 0 through 8 compared with 1096 ± 70 μmol/100 g body wt in controls (n = 11; P < 0.0005). The proteinuria in nephrotic rats was significant from days 2 through 8. Arrow indicates time of PAN injection. *P < 0.05; ***P < 0.001. (B) Parallel changes in plasma renin concentrations and hematocrit values of nephrotic rats indicate a shift from volume underfilling to overfilling from days 2 through 5. Renin: *P < 0.05 between days 2 and 8 (t test). Hematocrit: *P < 0.05 versus control (Dunnett test). GU, Goldblatt units. (C) Effect of amiloride treatment on daily urinary sodium output. In PAN nephrotic rats, amiloride (2 mg/d per kg body wt) increases daily urinary sodium excretion more than in controls (n = 6). Arrow indicates time of PAN injection. *P < 0.05.

Figure 2.

Serine proteases in nephrotic urine stimulates ENaC activity. (A) Traces obtained with whole-cell patch-clamp technique on a single M-1 cell showing baseline current (black) before addition of nephrotic urine (day 5) in the presence of 2 μM amiloride (gray). Subsequently, amiloride was washed away and the same cell was stimulated with the corresponding urine sample (black). The voltage was clamped to −60 mV and then stepped to −150 mV for 200 ms. The traces shown are recorded approximately 30 to 60 s after addition of the various substances. (B) Mean values from four different patch-clamp experiments showing that addition of aprotinin (700 μg/ml) to nephrotic urine samples or the presence of amiloride (2 μM) prevents the activation of currents in single M-1 cells. *P < 0.05 versus control (n = 4, Dunnett test). (C) Preincubation of ENaC-expressing Xenopus laevis oocytes with nephrotic urine samples increased the amiloride-sensitive ENaC whole-cell current to a similar extent as preincubation with chymotrypsin. Control oocytes were preincubated in standard ND96 solution, which was also used as bath solution for all other whole-cell current measurements. The stimulatory effect of nephrotic urine samples was essentially abolished by heat-inactivating the urine samples. The nephrotic urine samples were from three different rats; the non-nephrotic urine sample was from a rat that was given PAN injection and did not develop nephrotic syndrome probably because of an insufficient injection. Numbers of individual oocytes measured in each group are shown above each column. Columns represent normalized mean values ± SEM from six different batches of oocytes (**P < 0.01; ***P < 0.001). (D) Gelatinase activity of PAN nephrotic urine is abolished when zymograms are developed in the presence of aprotinin (1 mg/ml). (E) Serine protease activity in nephrotic urine is high compared with urine from control rats. Activity in plasma is low in both. ***P < 0.001.

Figure 3.

Identification of plasmin as an ENaC-activating protease in nephrotic urine. (A) After aprotinin affinity precipitation of nephrotic urine, there is reduced ability of the supernatant to stimulate sodium currents in M-1 cells and serine protease activity (▪), whereas the ability of the precipitate to stimulate sodium current and serine protease activity is increased (□). *P < 0.05 (n = 4). (B) Chromatogram of ion exchange chromatography of the aprotinin-affinity precipitate and corresponding serine protease activities (solid line, A_280nm; dotted line, conductance of the elution buffer; ▪, serine protease activity). #Fractions that were pooled, precipitated with aprotinin, and separated by gel electrophoresis to give bands with sizes of 80 and 40 kD (Supplemental Figure 3A). (C) MALDI-TOF mass spectrometry identified the 80-kD band resulting from the procedure in B as plasmin(ogen) (•). Trypsin autolysis peptides (○) were used for internal calibration. (D) Knockdown of γENaC mRNA and protein in M-1 cells. Cells were transfected with γENaC siRNA, and γENaC mRNA level was assessed the next day using reverse transcriptase-quantitative PCR (upper panel) or after 2 d at the protein level using immunofluorescence (lower panels, n = 4). (E) Stimulation of whole-cell inward currents in M-1 cells by native rat plasmin (10 μg/ml). The stimulatory effect of plasmin was prevented when plasmin was applied in the presence of amiloride (2 μM) or by knockdown of γENaC by siRNA (n = 4). *P < 0.05 versus control (t test).

Figure 4.

Plasmin is the dominant ENaC-activating protease in PAN nephrotic urine. (A) Whole-cell current traces recorded from single M-1 cells showing baseline current (gray traces) before and after (black traces) exposure to nephrotic urine containing vehicle, Pefabloc PL (10 μM), or α₂-antiplasmin (5 μM). The stimulatory effect of nephrotic urine (top traces) on the sodium inward current was prevented by the selective plasmin inhibitors Pefabloc PL (middle traces) and by α₂-antiplasmin (bottom traces). (B) Mean values from similar experiments as shown in A. **P < 0.01 versus control (t test, n = 4).

Figure 5.

Tubular activation of filtered plasminogen to plasmin by uPA. (A, top) Western blot showing appearance of plasminogen and plasmin in urine at day 3 after induction of PAN nephrosis. (Bottom) Western blot on plasma demonstrating presence of plasminogen but not plasmin in plasma from control and nephrotic rats. Plasminogen and Plasmin bands are detected at approximately 90 and 80 kD, respectively. (B, left) Sections of rat kidney demonstrating uPA immunoreactivity in CCD. (Right) Western blots of control and PAN nephrotic rat kidney tissue demonstrate presence of uPA in the renal cortex. Bar = 50 μm. (C) Western blot showing inhibition of conversion of plasminogen to plasmin after treatment with amiloride. Low dosage: 400 μg/d per kg body wt; high dosage: 2 mg/d per kg body wt. (D) Zymogram of urine from control and nephrotic (NS) rats showing reduced gelatinase activity with high-dosage amiloride.

Figure 6.

A combination of uPA and plasminogen stimulates ENaC in Xenopus laevis oocytes and in confluent polarized M-1 collecting duct cells. (A) ENaC-expressing oocytes were preincubated for 5 min in control ND96 solution, in ND96 solution containing chymotrypsin (2 μg/ml), in a combination of plasminogen (1 mg/ml) and uPA (150 U/ml), in uPA (150 U/ml) alone, or in plasminogen (1 mg/ml) alone. Preincubation was followed by the assessment of ΔI_ami at −60 mV by two-electrode voltage clamp. Numbers of individual oocytes measured in each group are given above each column. Columns represent normalized means ± SEM from 10 different batches of oocytes (***P < 0.001 versus control group preincubated in ND96). (B) C-terminally V5-tagged wild-type γENaC was coexpressed with wild-type α and βENaC in X laevis oocytes (αβγ-V5). Oocytes were treated for 5 min with chymotrypsin, trypsin, or urokinase/plasminogen as indicated. The γENaC subunit of biotinylated cell surface ENaC was detected with a monoclonal V5 antibody. Exposure to a combination of uPA and plasminogen results in the appearance of a 67-kD band similar to that observed after exposure to trypsin or chymotrypsin, which is not seen in the nontreated oocytes where the major γENaC band is detected at approximately 76 kD. A band of approximately 87 kD corresponding to full-length ENaC can be seen in the control blot on the right side using membrane fractions from whole-cell lysate of oocytes coexpressing αβγENaC or γENaC alone. As expected, the full-length band is the predominant band in oocytes expressing γENaC alone (γ-V5), whereas cleavage products can be detected in addition to full-length ENaC in oocytes coexpressing all three ENaC subunits (αβγ-V5). The positions of the 72- and 95-kD size markers are indicated. n.i., noninjected oocytes. (C) Representative equivalent short-circuit current (I_SC) recording is shown from confluent M-1 cells grown on permeable support. Urokinase (uPA) in a concentration of 150 U/ml and plasminogen in a concentration of 1 mg/ml were added sequentially to the apical bath solution as indicated by the and ▪, respectively. At the end of the experiment, amiloride (ami; 100 μM) was added apically to confirm that the stimulated I_SC was mediated by ENaC. (D) Summary of results from 12 similar experiments as shown in B. Columns represent mean I_SC values (±SEM) measured under baseline conditions before the apical addition of uPA (□), 5 min after application of uPA (), 30 min after subsequent application of plasminogen (▪), and in the presence of amiloride (). **P < 0.01, ***P < 0.001.

Figure 7.

Plasmin stimulation leads to release of a peptide from the extracellular domain of γENaC. (Left) Schematic diagram of the γENaC subunit showing the putative cleavage sites for furin and prostasin and the localization of the inserted hexahistidine tag, which can be visualized with the fluorophore NTA-Atto550. (Right) M-1 cells expressing the hexahistidine-tagged γENaC subunit are labeled by NTA-Atto550 when treated with vehicle or control urine, whereas cells treated with plasmin or nephrotic urine are unlabeled (n = 4).

Figure 8.

Urine from patients with nephrotic syndrome confirms critical findings from the animal study. (A) Whole-cell current traces recorded before (gray) and after (black) exposure of a single M-1 cell to human nephrotic urine. (B) Control urine fails to stimulate M-1 whole-cell currents, and the stimulatory effect of human nephrotic urine is prevented by amiloride and α₂-antiplasmin added to the nephrotic urine before flushing the cells with the urine sample (n = 4 in each group). Data are means ± SEM. *P < 0.05. See Supplemental Figure 5 for values from each patient. (C) Zymogram of human urine showing gelatinase activity in urine from patients with nephrotic syndrome but not in urine controls. (D) Inhibition of serine protease activity in human nephrotic urine by α₂-antiplasmin and by the plasmin inhibitor Pefabloc PL (n = 4). *P < 0.05. (E) Western blot showing plasmin immunoreactivity in urine from patients with nephrotic syndrome but not in urine controls. (F) Immunohistochemical staining of uPA in sections of human nephrectomy specimens showing uPA immunoreactivity in CCD (top). Western blot of human nephrectomy specimens demonstrating presence of uPA in the renal cortex (bottom). Bar = 50 μm.